U.S. patent application number 12/070053 was filed with the patent office on 2008-09-11 for pancreatic cancer secreted targets and uses thereof.
This patent application is currently assigned to APPLERA CORPORATION. Invention is credited to Kim Alving, Bruno Domon, Ian McCaffery, Steve Ruben.
Application Number | 20080219992 12/070053 |
Document ID | / |
Family ID | 39281564 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080219992 |
Kind Code |
A1 |
Alving; Kim ; et
al. |
September 11, 2008 |
Pancreatic cancer secreted targets and uses thereof
Abstract
The present invention provides a method for diagnosing and
detecting diseases associated with pancreas. The present invention
provides one or more proteins or fragments thereof, peptides or
nucleic acid molecules differentially expressed in pancreatic
diseases (PCAST) and antibodies binds to PCAST. The present
invention provides that PCAST is used as targets for screening
agents that modulates the PCAST activities. Further the present
invention provides methods for treating diseases associated with
pancreas.
Inventors: |
Alving; Kim; (Rockville,
MD) ; Ruben; Steve; (Brookville, MD) ;
McCaffery; Ian; (Moorpark, CA) ; Domon; Bruno;
(Zurich, CH) |
Correspondence
Address: |
CELERA, AN APPLERA BUSINESS UNIT
1401 HARBOR BAY PARKWAY
ALAMEDA
CA
94502
US
|
Assignee: |
APPLERA CORPORATION
Norwalk
CT
|
Family ID: |
39281564 |
Appl. No.: |
12/070053 |
Filed: |
February 13, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11606949 |
Dec 1, 2006 |
7358231 |
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12070053 |
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60781305 |
Mar 13, 2006 |
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60741041 |
Dec 1, 2005 |
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Current U.S.
Class: |
424/139.1 ;
435/331; 435/375; 435/6.16; 435/7.1; 514/1.1; 530/387.3; 530/387.9;
536/23.5 |
Current CPC
Class: |
G01N 33/57438 20130101;
C07K 14/4748 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/139.1 ;
514/12; 536/23.5; 530/387.9; 530/387.3; 435/331; 435/375; 435/6;
435/7.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 35/00 20060101 A61P035/00; C07K 14/47 20060101
C07K014/47; A61K 38/17 20060101 A61K038/17; C12N 15/12 20060101
C12N015/12; C12Q 1/68 20060101 C12Q001/68; G01N 33/566 20060101
G01N033/566; C07K 16/18 20060101 C07K016/18; C12N 5/16 20060101
C12N005/16; C12N 5/10 20060101 C12N005/10 |
Claims
1. An isolated protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NOS:1-23 and 40-51.
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:24-39; b) nucleotide sequences that encode a protein comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOS:1-23 and 40-51; and c) nucleotide sequences that are completely
complementary to the nucleotide sequences of a) or b).
4. An isolated RNAi or antisense nucleic acid molecule that
selectively binds to the nucleic acid molecule of claim 3.
5. An isolated antibody that selectively binds to the protein of
claim 1.
6. The antibody of claim 5, wherein the antibody is at least one of
a monoclonal, polyclonal, fully human, humanized, chimeric,
single-chain, or anti-idiotypic antibody.
7. A cell line, hybridoma, phage, or transgenic organism that
produces the antibody of claim 5.
8. The antibody of claim 5, wherein the antibody is coupled to a
composition selected from the group consisting of detectable
substances and therapeutic agents.
9. A composition comprising the antibody of claim 5 and a
pharmaceutically acceptable carrier.
10. An isolated antibody fragment of the antibody of claim 5,
wherein the antibody fragment comprises a fragment selected from
the group consisting of: a) an Fab fragment; b) an F(ab').sub.2
fragment; and c) an Fv fragment.
11. A method of modulating cell proliferation or apoptosis, the
method comprising contacting a cell with the antibody of claim
5.
12. The method of claim 11, wherein the method comprises either
inhibiting proliferation of pancreatic cancer cells or stimulating
apoptosis of pancreatic cancer cells.
13. A method of modulating cell proliferation or apoptosis, the
method comprising contacting a cell with the RNAi or antisense
nucleic acid molecule of claim 4.
14. A method of detecting the protein of claim 1 in a sample, the
method comprising contacting the sample with an isolated antibody
that selectively binds to the protein and determining whether the
antibody binds to the protein.
15. A method of detecting the nucleic acid molecule of claim 3 in a
sample, the method comprising contacting the sample with an
oligonucleotide that specifically hybridizes to the nucleic acid
molecule and determining whether the oligonucleotide binds to the
nucleic acid molecule.
16. A method of diagnosing, prognosing, or determining risk of
pancreatic cancer in a subject, the method comprising detecting at
least one molecule in a sample, wherein the presence or abundance
of the molecule is indicative of pancreatic cancer, and wherein the
molecule is selected from the group consisting of: a) proteins
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOS:1-23 and 40-51; 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:24-39 and nucleotide sequences that encode the
protein of a); and d) nucleic acid molecules comprising a
nucleotide sequence that is completely complementary to the nucleic
acid molecule of c).
17. A method of treating pancreatic cancer, the method comprising
administering a therapeutically effective amount of the antibody of
claim 5 to a subject.
18. A method of screening agents, the method comprising contacting
the protein of claim 1 or a cell that expresses the protein with an
agent, and assaying for whether the agent binds to the protein or
modulates the function, activity, or expression of the protein.
19. A composition comprising the agent identified by the method of
claim 18 and a pharmaceutically acceptable carrier.
20. A method of determining or predicting the effectiveness of a
treatment or selecting a treatment for administration to a subject
having pancreatic cancer, the method comprising detecting the
presence, abundance, or activity of the protein of claim 1 in a
sample and determining or predicting the effectiveness of the
treatment or selecting the treatment for administration based on
the presence, abundance, or activity of the protein.
Description
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 cancers.
BACKGROUND OF THE INVENTION
[0002] Cancer currently constitutes the second most common cause of
death in the United States. Carcinomas of the pancreas are the
eighth most prevalent form of cancer and fourth among the most
common causes of cancer deaths in this country.
[0003] The prognosis for pancreatic carcinoma is, at present, very
poor, it displays the lowest five-year survival rate among all
cancers. Such prognosis results primarily from delayed diagnosis,
due in part to the fact that the early symptoms are shared with
other more common abdominal ailments. Despite the advances in
diagnostic imaging methods like ultrasonography (US), endoscopic
ultrasonography (EUS), dualphase spiral computer tomography (CT),
magnetic resonance imaging (MRT), endoscopic retrograde
cholangiopancreatography (ERCP) and transcutaneous or EUS-guided
fine-needle aspiration (FNA), distinguishing pancreatic carcinoma
from benign pancreatic diseases, especially chronic pancreatitis,
is difficult because of the similarities in radiological and
imaging features and the lack of specific clinical symptoms for
pancreatic carcinoma.
[0004] Substantial efforts have been directed to developing tools
useful for early diagnosis of pancreatic carcinomas. Nonetheless, a
definitive diagnosis is often dependent on exploratory surgery
which is inevitably performed after the disease has advanced past
the point when early treatment may be effected.
[0005] 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.
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.
Secreted Proteins
[0006] Many human proteins serve as pharmaceutically active
compounds. Several classes of human proteins that serve as such
active compounds include hormones, cytokines, cell growth factors,
and cell differentiation factors. Most proteins that can be used as
a pharmaceutically active compound fall within the family of
secreted proteins. It is, therefore, important in developing new
pharmaceutical compounds to identify secreted proteins that can be
tested for activity in a variety of animal models. The present
invention advances the state of the art by providing many novel
human secreted proteins.
[0007] Secreted proteins are generally produced within cells at
rough endoplasmic reticulum, are then exported to the golgi
complex, and then move to secretory vesicles or granules, where
they are secreted to the exterior of the cell via exocytosis.
[0008] Secreted proteins are particularly useful as diagnostic
markers. Many secreted proteins are found, and can easily be
measured, in serum. For example, a `signal sequence trap` technique
can often be utilized because many secreted proteins, such as
certain secretory breast cancer proteins, contain a molecular
signal sequence for cellular export. Additionally, antibodies
against particular secreted serum proteins can serve as potential
diagnostic agents, such as for diagnosing cancer.
[0009] Secreted proteins play a critical role in a wide array of
important biological processes in humans and have numerous
utilities; several illustrative examples are discussed herein. For
example, fibroblast secreted proteins participate in extracellular
matrix formation. Extracellular matrix affects growth factor
action, cell adhesion, and cell growth. Structural and quantitative
characteristics of fibroblast secreted proteins are modified during
the course of cellular aging and such aging related modifications
may lead to increased inhibition of cell adhesion, inhibited cell
stimulation by growth factors, and inhibited cell proliferative
ability (Eleftheriou et al., Mutat Res 1991 March-November;
256(2-6): 127-38).
[0010] The secreted form of amyloid beta/A4 protein precursor (APP)
functions as a growth and/or differentiation factor. The secreted
form of APP can stimulate neurite extension of cultured
neuroblastoma cells, presumably through binding to a cell surface
receptor and thereby triggering intracellular transduction
mechanisms. (Roch et al., Ann N Y Acad Sci 1993 Sep. 24;
695:149-57). Secreted APPs modulate neuronal excitability,
counteract effects of glutamate on growth cone behaviors, and
increase synaptic complexity. The prominent effects of secreted
APPs on synaptogenesis and neuronal survival suggest that secreted
APPs play a major role in the process of natural cell death and,
furthermore, may play a role in the development of a wide variety
of neurological disorders, such as stroke, epilepsy, and
Alzheimer's disease (Mattson et al., Perspect Dev Neurobiol 1998;
5(4):337-52).
[0011] Breast cancer cells secrete a 52K estrogen-regulated protein
(see Rochefort et al., Ann N Y Acad Sci 1986; 464:190-201). This
secreted protein is therefore useful in breast cancer
diagnosis.
[0012] Two secreted proteins released by platelets, platelet factor
4 (PF4) and beta-thromboglobulin (betaTG), are accurate indicators
of platelet involvement in hemostasis and thrombosis and assays
that measure these secreted proteins are useful for studying the
pathogenesis and course of thromboembolic disorders (Kaplan, Adv
Exp Med Biol 1978; 102:105-19).
[0013] Vascular endothelial growth factor (VEGF) is another example
of a naturally secreted protein. VEGF binds to cell-surface heparan
sulfates, is generated by hypoxic endothelial cells, reduces
apoptosis, and binds to high-affinity receptors that are
up-regulated by hypoxia (Asahara et al., Semin Interv Cardiol 1996
September; 1(3):225-32). Many critical components of the immune
system are secreted proteins, such as antibodies, and many
important functions of the immune system are dependent upon the
action of secreted proteins. For example, Saxon et al., Biochem Soc
Trans 1997 May; 25(2):383-7, discusses secreted IgE proteins. For a
further review of secreted proteins, see Nilsen-Hamilton et al.,
Cell Biol Int Rep 1982 September; 6(9):815-36.
[0014] Secreted proteins, particularly members of the pancreatic
cancer associated secreted proteins, are a major target for drug
action and development. Accordingly, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown members of this subfamily of secreted proteins. The present
invention advances the state of the art by providing previously
unidentified human secreted proteins that have homology to members
of the secreted protein.
SUMMARY OF THE INVENTION
[0015] The present invention is based on the identification of
secreted proteins that are differentially expressed in pancreatic
cancer. A malignant cell often differs from a normal cell by a
differential expression of one or more proteins. These
differentially expressed proteins, and the fragments thereof, are
important markers for the diagnosis of pancreatic disease such as
cancer. The differentially expressed proteins of the present
invention and the nucleic acids encoding said proteins and the
fragments of said proteins are referred to herein as pancreatic
cancer associated secreted target, PCAST proteins or PCAST nucleic
acids or PCAST peptides, respectively.
[0016] The present invention provides peptides and proteins
differentially expressed in pancreatic diseases (hereinafter
PCAST). Based on the site of protein localization, e.g., blood or
other body fluids, and protein characterization, e.g. hormone,
enzyme, specific uses of these PCASTs are provided. Some of the
PCASTs of the present invention serve as targets for one or more
classes of therapeutic agents, while others may be suitable for
antibody therapeutics.
[0017] Accordingly, the present invention provides a method for
diagnosing or detecting pancreatic disease in a subject comprising:
determining the level of one or more PCAST proteins, or any
fragment(s) thereof, in a test sample from said subject, wherein
said PCAST protein comprises a sequence selected from a group
consisting of SEQ ID NOS:1-23; wherein a differential level of said
PCAST protein(s) or fragment(s) in said sample relative to the
level of said protein(s) or fragment(s) in a test sample from a
healthy subject, or the level established for a healthy subject, is
indicative of pancreatic disease.
[0018] The present invention also provides a method for detecting
pancreatic cancer in a subject comprising: determining the level of
one or more PCAST peptide(s) comprising a peptide sequence selected
from a group consisting of SEQ ID NOS:40-51 in a test sample from
said subject, wherein a differential level of said PCAST peptide(s)
in said sample to the level of said PCAST peptide(s) in a test
sample from a healthy subject, or the level of said PCAST
peptide(s) established for a healthy subject, is indicative of
pancreatic disease.
[0019] The present invention further provides a method for
detecting pancreatic disease in a subject comprising: determining
the level of one or more PCAST nucleic acid(s), or any fragment(s)
thereof, in a test sample from said subject, wherein said PCAST
nucleic acid(s) encode a PCAST protein sequence selected from a
group consisting of SEQ ID NOS:1-23; wherein a differential level
of said PCAST 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 pancreatic disease.
[0020] The invention also provides methods for detecting the PCAST
peptides, genes or mRNAs 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.
[0021] 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:1-23.
[0022] The present invention further provides a composition
comprising an antibody that binds to a protein selected from a
group consisting of SEQ ID NOS:1-23, and an acceptable carrier.
[0023] The present invention further provides a method for treating
pancreatic disease, comprising administering to a patient in need
of said treatment a therapeutically effective amount of one or more
antibody(ies) of this invention.
[0024] The present invention further provides a method for treating
pancreatic disease comprising (i) identifying a subject having
pancreatic disease and (ii) administering to a said patient a
therapeutically effective amount of one or more antibody(ies) of
this invention.
[0025] The present invention further provides a method to screen
for agents that modulate PCAST protein activity, comprising the
steps of (i) contacting a test agent with a PCAST protein and (ii)
assaying for PCAST protein activity, wherein a change in said
activity in the presence of said agent relative to PCAST protein
activity in the absence of said agent indicates said agent
modulates said PCAST protein activity.
[0026] The present invention further provides a method to screen
for agents that bind to PCAST protein, comprising the steps of (i)
contacting a test agent with a PCAST protein and (ii) measuring the
level of binding of agent to said PCAST protein.
[0027] The invention also provides diagnostic methods for human
disease, in particular for pancreatic diseases or cancers, its
metastatic stage, and therapeutic potential.
[0028] 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.
[0029] The invention also provides a method for monitoring the
disease progression and the treatment progress.
[0030] The invention further provide a method of diagnosis by an
array, wherein the array is immobilized with two or more PCAST
proteins, peptides or nucleic acid molecules. The proteins,
peptides or nucleic acid molecules include but are not limited to
the SEQ ID NOS:1-23.
[0031] The invention also provides monoclonal or polyclonal
antibodies and composition thereof reactive with antigentic portion
of PCAST protein, peptides or fragments thereof in a form for use
in pancreatic diseases diagnosis.
[0032] The invention further provides an immunogenic antibody for
treating pancreatic disease or diseases associated with pancreatic
diseases.
[0033] The present invention provides a method for screening agents
that modulate PCAST activity, comprising the steps of (a)
contacting a sample comprising PCAST with an agent; and (b)
assaying for PCAST activity, wherein a change in said PCAST
activity in the presence of said agent relative to PCAST activity
in the absence of said compound indicates said agent modulates
PCAST. The agents include but are not limited to protein, peptide,
antibody, nucleic acid such as antisense RNA, RNAi fragments, small
molecules.
[0034] The present invention further provides a method for treating
pancreatic diseases, comprising: administering to a patient one or
more agents in a therapeutically effective amount to treat
pancreatic diseases.
[0035] The present invention provides a method for treating
pancreatic diseases, comprising: identifying a subject having
pancreatic diseases; and administering to a patient to one or more
antibodies in a therapeutically effective amount to treat
pancreatic diseases.
[0036] 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
CL001658CDR
[0037] The CD-R named CL001658CDR contains the following two text
(ASCII) files:
1) File SEQLIST.sub.--1658txt provides the Sequence Listing. The
Sequence Listing provides the protein sequences (SEQ ID NOS:1-23);
transcript sequences (SEQ ID NOS:24-39) and peptide sequences (SEQ
ID NOS:40-51) as shown in Table 1. File SEQLIST.sub.--1658.txt is
132 KB in size. 2) File TABLE1.sub.--1658.txt provides Table 1,
which is 10 KB in size.
[0038] The material contained on the CD-R labeled CL001658CDR is
hereby incorporated by reference pursuant to 37 CFR 1.77(b)(4).
Description of Table 1
[0039] Table 1 (provided on the CD-R) discloses the PCAST proteins,
transcripts, and peptides (each protein, transcript, and peptide is
represented by a SEQ ID NO, and the corresponding sequence is
provided in the Sequence Listing; the range of numbers in
parentheses following each peptide SEQ ID NO represent the amino
acid residues of the location of the peptide within its
corresponding protein), the pancreatic cancer cell lines or
tissues, and the expression ratio ("ratio") compared to the control
sample.
[0040] The expression ratio 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 a 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.
[0041] The protein/gene/transcript information provided for each
target includes any or all of the information selected from: [0042]
a protein SEQ ID NO [0043] a Celera internal identification number
for the protein (hCP and/or UID) [0044] a public protein accession
number (Genbank, e.g., RefSeq NP number, Swiss-prot, or Derwent)
for the protein [0045] a protein name recognized in the art [0046]
a Celera internal identification number for the gene (hCG and/or
UID) [0047] an art-recognized gene symbol [0048] Celera genomic
axis position (indicating start nucleotide position-stop nucleotide
position) [0049] the chromosome number of the chromosome on which
the gene is located [0050] 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, and alternative gene/protein name(s) and/or symbol(s) in
the OMIM entry [0051] a transcript SEQ ID NO [0052] a Celera
internal identification number for the transcript (hCT and/or UID)
[0053] a public transcript accession number (Genbank, e.g., RefSeq
NM number, or Derwent)
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
General Description
[0054] 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 pancreatic disease by determining the level of one or
more PCAST protein(s) or any fragment(s) thereof is wherein the
level of PCAST protein(s) are determined by contacting one or more
antibody(ies) that specifically bind to the antigenic regions of
the PCAST 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.
[0055] A preferred method for detecting pancreatic disease by
determining the level of one or more PCAST peptide(s) is wherein
the level of PCAST peptides(s) are determined by contacting one or
more antibody(ies) that specifically bind to the antigenic regions
of the PCAST 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.
[0056] A preferred method for detecting pancreatic disease by
determining the level of one or more PCAST nucleic acid(s) is
wherein the level of said PCAST 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.
[0057] The methods for detecting pancreatic disease provided by the
present invention may be used for diagnosing the presence of
disease in a patent, monitoring the presence of pancreatic disease
in patients undergoing treatment and testing for the reoccurrence
of pancreatic disease in patients that were successfully treated
for pancreatic disease; preferably wherein the pancreatic disease
is pancreatic cancer. The test sample may be, but is not limited
to, a biological sample such as tissue, blood, serum or biological
fluid.
[0058] The present invention is based on the discovery of
protein(s) and peptide(s) that are differentially expressed in
pancreatic disease samples for example cancer sample versus normal
pancreatic samples. These proteins and peptide, and the encoding
nucleic acid molecules are associated with pancreatic diseases,
hereinafter the PCAST protein, peptide or nucleic acids.
[0059] The discovery of disease specific target proteins is base on
discoveries made using proteomics techniques. The method uses on
MALDI-TOF TOF LC/MS analyses platform to generate protein
expression profiles from pancreatic diseases tissues or cell lines
in an effort to discover and identify novel molecules associated
with the disease.
[0060] Based on these discoveries, the present invention provides
proteins, peptides, nucleic acids that are differential in
pancreatic diseases, as well as antibodies binds to the proteins or
peptides. The present invention also provides methods for
detection, monitoring, diagnosis, prognosis, preventive and
treatment of pancreatic diseases. The present invention provides a
detection reagent, markers for pancreatic diseases at various
stages, comprises PCAST sequences isolated from human pancreatic
diseases tissue, sera, cell lines, blood or biological fluids.
[0061] The present invention provides a method for treating
pancreatic diseases targeting at PCAST. The treatment includes
administration of a therapeutically effective amount of composition
comprise, but not limit to, an antibody, an immunogenic peptide
which induces T cell response, a small molecule, a protein or a
nucleic acid molecule. The composition further comprises an agonist
or antigonist to PCAST. A "Panceratic disease" includes pancreatic
cancer, pancreatic tumor (exocrine or endocrine), pancreatic cysts,
acute pancreatitis, chronic pancreatitis, diabetes (type I and II)
as well as pancreatic trauma, preferably pancreatic cancer.
[0062] 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.
[0063] The present invention further provides the target for
screening an agent for PCAST, wherein the agent is compounds of
small molecules, proteins, peptides, nucleic acids, antibodies or
other agonists or antigonists.
PCAST Peptide/Proteins and Peptides
[0064] The present invention provides isolated PCAST peptide and
protein molecules that consisting of, consisting essentially of, or
comprising the amino acid sequences of the PCAST peptides and
proteins disclosed in the 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.
[0065] In one embodiment PCAST peptides include, but are not
limited to, the amino acid sequence of SEQ ID NOS:40-51 and
variants thereof. A PCAST protein includes, but is not limited to,
the amino acid sequence of SEQ ID NOS: 1-23 and variants thereof.
PCAST proteins may be differentially expressed in pancreatic cell
line, blood, tissue, serum or body fluids.
[0066] 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.
[0067] The PCAST 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
are discussed below).
[0068] As used herein, a "peptide" is defined as amino acid
sequences between 5-20 amino acids derived from PCAST proteins such
as SEQ ID NOS:1-23 or variants thereof. The peptide differentially
expressed in either pancreatic diseases cell line, blood, tissue,
serum or body fluids. In one embodiment peptides include, but are
not limited to, the amino acid sequence of SEQ ID NOS:40-51, or
variants thereof.
[0069] As used herein, a "protein" is full-length protein
differentially expressed in pancreatic diseases cell line, tissue,
blood, serum or body fluids. A protein includes, but is not limited
to, the amino acid sequence of SEQ ID NOS:1-23.
[0070] 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).
[0071] 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.
[0072] 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 PCAST 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.
[0073] The isolated PCAST proteins and peptides 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
pancreatic cell lines. For example, a nucleic acid molecule
encoding the PCAST 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.
[0074] A PCAST 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.
[0075] 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 PCAST 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.
[0076] A chimeric or fusion PCAST 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 PCAST-encoding nucleic acid can be cloned into such an
expression vector such that the fusion moiety is linked in-frame to
the PCAST protein or peptide.
[0077] As mentioned above, the PCAST peptide or the PCAST protein
has obvious variants of the amino acid sequence, such as naturally
occurring mature forms of the PCAST, allelic/sequence variants of
the PCAST, non-naturally occurring recombinantly derived variants
of the PCASTs, and orthologs and paralogs of the PCAST proteins or
peptides. Such variants can readily be generated using art-known
techniques in the fields of recombinant nucleic acid technology and
protein biochemistry.
[0078] It is understood, however, that PCAST and variants exclude
any amino acid sequences disclosed prior to the invention.
[0079] 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 PCAST 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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 PCAST peptides of the present invention as
well as being encoded by the same genetic locus as the PCAST
peptide provided herein (see Table 1).
[0084] Allelic variants of a PCAST 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 PCAST peptide as well as being encoded by the same genetic
locus as the PCAST peptide provided herein. 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 PCAST peptide encoding
nucleic acid molecule under stringent conditions as more fully
described below.
[0085] Paralogs of a PCAST peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the PCAST 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 PCAST peptide encoding nucleic
acid molecule under moderate to stringent conditions as more fully
described below.
[0086] Orthologs of a PCAST peptide can readily be identified as
having some degree of significant sequence homology/identity to at
least a portion of the PCAST 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 PCAST 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.
[0087] Non-naturally occurring variants of the PCAST 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 PCAST peptide. For example, one class of substitutions is
conserved amino acid substitution. Such substitutions are those
that substitute a given amino acid in a PCAST 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).
[0088] Variant PCAST 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.
[0089] 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.
[0090] 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 PCAST 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)).
[0091] The present invention further provides fragments of the
PCASTs, 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 PCAST. Such fragments can be chosen
based on the ability to retain one or more of the biological
activities of the PCAST 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 PCAST, 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).
[0092] 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 PCASTs are described in basic
texts, detailed monographs, and the research literature, and they
are well known to those of skill in the art.
[0093] 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.
[0094] Such modifications are well known to those of skill in the
art and have been described in 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)).
[0095] Accordingly, the PCASTs 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 PCAST is fused
with another compound, such as a compound to increase the half-life
of the PCAST (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature PCAST, such as a
leader or secretory sequence or a sequence for purification of the
mature PCAST or a pro-protein sequence.
Protein/Peptide Uses
[0096] 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
PCAST-effector protein interaction or PCAST-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.
[0097] 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", 3.sup.rd, 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.
[0098] 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, PCASTs 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 PCAST.
Experimental data as provided in Table 1 indicate that the PCASTs
of the present invention are expressed at differential level in
various pancreatic cell lines, for example, SEQ ID NOS:1-23 are
overexpressed in all tested cell lines. A large percentage of
pharmaceutical agents are being developed that modulate the
activity of PCAST proteins, particularly members of the PCAST
subfamily (see Background of the Invention). The structural and
functional information provided in the Background 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 pancreatic cell lines. Such
uses can readily be determined using the information provided
herein, that which is known in the art, and routine
experimentation.
[0099] 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 PCASTs that
are related to members of the PCAST subfamily. Such assays involve
any of the known PCAST functions or activities or properties useful
for diagnosis and treatment of PCAST-related conditions that are
specific for the subfamily of PCASTs that the one of the present
invention belongs to, particularly in cells and tissues that
express the PCAST. Experimental data as provided in Table 1
indicate that the PCASTs of the present invention are expressed at
differential level in various pancreatic cell lines, for example,
SEQ ID NOS:1-23 are overexpressed in all tested cell lines.
[0100] 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 PCAST, as a biopsy or expanded in cell culture. Experimental
data as provided in Table 1 indicates expression in human
pancreatic cell lines. In an alternate embodiment, cell-based
assays involve recombinant host cells expressing the PCAST
protein.
[0101] The polypeptides can be used to identify compounds or agents
that modulate PCAST activity of the protein in its natural state or
an altered form that causes a specific disease or pathology
associated with the PCAST. Both the PCASTs 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 PCAST. These compounds can be further
screened against a functional PCAST to determine the effect of the
compound on the PCAST 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 PCAST to a desired
degree.
[0102] 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 PCAST protein and a molecule that
normally interacts with the PCAST protein, e.g. a substrate or or
an extracellular binding ligand or a component of the signal
pathway that the PCAST protein normally interacts (for example, a
cytosolic signal protein or another PCAST). Such assays typically
include the steps of combining the PCAST protein with a candidate
compound under conditions that allow the PCAST 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
PCAST 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.
[0103] 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).
[0104] One candidate compound or agent is a soluble fragment of the
PCAST that competes for substrate binding. Other candidate
compounds include mutant PCASTs or appropriate fragments containing
mutations that affect PCAST 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.
[0105] The invention further includes other end point assays to
identify compounds that modulate (stimulate or inhibit) PCAST
activity. The assays typically involve an assay of events in the
signal transduction pathway that indicate PCAST 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 PCAST protein dependent signal cascade can be
assayed.
[0106] Any of the biological or biochemical functions mediated by
the PCAST 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 PCAST can be
assayed. Experimental data as provided in Table 1 indicate that the
PCASTs of the present invention are expressed at differential level
in various pancreatic cell lines, for example, SEQ ID NOS:1-23 are
overexpressed in all tested cell lines.
[0107] Binding and/or activating compounds can also be screened by
using chimeric PCAST 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
PCAST. 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 PCAST is derived.
[0108] The proteins of the present invention are also useful in
competition binding assays in methods designed to discover
compounds that interact with the PCAST (e.g. binding partners
and/or ligands). Thus, a compound is exposed to a PCAST polypeptide
under conditions that allow the compound to bind or to otherwise
interact with the polypeptide. Soluble PCAST polypeptide is also
added to the mixture. If the test compound interacts with the
soluble PCAST polypeptide, it decreases the amount of complex
formed or activity from the PCAST. This type of assay is
particularly useful in cases in which compounds are sought that
interact with specific regions of the PCAST. Thus, the soluble
polypeptide that competes with the target PCAST region is designed
to contain peptide sequences corresponding to the region of
interest.
[0109] To perform cell free drug screening assays, it is sometimes
desirable to immobilize either the PCAST 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.
[0110] 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 PCAST-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 PCAST-binding
protein and a candidate compound are incubated in the PCAST
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 PCAST protein target molecule, or which are
reactive with PCAST protein and compete with the target molecule,
as well as PCAST-linked assays which rely on detecting an enzymatic
activity associated with the target molecule.
[0111] The following technologies can be used to detect
interactions between a protein and a compound without knowing the
biological function of the protein. A short description of three
methods is presented, including fluorescence correlation
spectroscopy, surface-enhanced laser desorption/ionization, and
biacore technologies.
[0112] Fluorescence Correlation Spectroscopy (FCS) theory was
developed in 1972 but it is only in recent years that the
technology to perform FCS became available (Madge et al. (1972)
Phys. Rev. Lett., 29: 705-708; Maiti et al. (1997) Proc. Natl.
Acad. Sci. USA, 94: 11753-11757). FCS measures the average
diffusion rate of a fluorescent molecule within a small sample
volume. The sample size can be as low as 10' fluorescent molecules
and the sample volume as low as the cytoplasm of a single
bacterium. The diffusion rate is a function of the mass of the
molecule and decreases as the mass increases. FCS can therefore be
applied to protein-ligand interaction analysis by measuring the
change in mass and therefore in diffusion rate of a molecule upon
binding. In a typical experiment, the target to be analyzed is
expressed as a recombinant protein with a sequence tag, such as a
poly-histidine sequence, inserted at the N or C-terminus. The
expression takes place in E. coli, yeast or insect cells. The
protein is purified by chromatography. For example, the
poly-histidine tag can be used to bind the expressed protein to a
metal chelate column such as Ni2+ chelated on iminodiacetic acid
agarose. The protein is then labeled with a fluorescent tag such as
carboxytetramethylrhodamine or BODIPY..RTM.. (Molecular Probes,
Eugene, Oreg.). The protein is then exposed in solution to the
potential ligand, and its diffusion rate is determined by FCS using
instrumentation available from Carl Zeiss, Inc. (Thornwood, N.Y.).
Ligand binding is determined by changes in the diffusion rate of
the protein.
[0113] Surface-Enhanced Laser Desorption/Ionization (SELDI) was
invented by Hutchens and Yip during the late 1980's (Hutchens and
Yip (1993) Rapid Commun. Mass Spectrom. 7: 576-580). When coupled
to a time-of-flight mass spectrometer (TOF), SELDI provides a mean
to rapidly analyze molecules retained on a chip. It can be applied
to ligand-protein interaction analysis by covalently binding the
target protein on the chip and analyze by MS the small molecules
that bind to this protein (Worrall et al. (1998) Anal. Biochem. 70:
750-756). In a typical experiment, the target to be analyzed is
expressed as described for FCS. The purified protein is then used
in the assay without further preparation. It is bound to the SELDI
chip either by utilizing the poly-histidine tag or by other
interaction such as ion exchange or hydrophobic interaction. The
chip thus prepared is then exposed to the potential ligand via, for
example, a delivery system capable to pipet the ligands in a
sequential manner (autosampler). The chip is then submitted to
washes of increasing stringency, for example a series of washes
with buffer solutions containing an increasing ionic strength.
After each wash, the bound material is analyzed by submitting the
chip to SELDI-TOF. Ligands that specifically bind the target will
be identified by the stringency of the wash needed to elute
them.
[0114] Biacore relies on changes in the refractive index at the
surface layer upon binding of a ligand to a protein immobilized on
the layer. In this system, a collection of small ligands is
injected sequentially in a 2-5 ul cell with the immobilized
protein. Binding is detected by surface plasmon resonance (SPR) by
recording laser light refracting from the surface. In general, the
refractive index change for a given change of mass concentration at
the surface layer, is practically the same for all proteins and
peptides, allowing a single method to be applicable for any protein
(Liedberg et al. (1983) Sensors Actuators 4: 299-304; Malmquist
(1993) Nature, 361: 186-187). In a typical experiment, the target
to be analyzed is expressed as described for FCS. The purified
protein is then used in the assay without further preparation. It
is bound to the Biacore chip either by utilizing the poly-histidine
tag or by other interaction such as ion exchange or hydrophobic
interaction. The chip thus prepared is then exposed to the
potential ligand via the delivery system incorporated in the
instruments sold by Biacore (Uppsala, Sweden) to pipet the ligands
in a sequential manner (autosampler). The SPR signal on the chip is
recorded and changes in the refractive index indicate an
interaction between the immobilized target and the ligand. Analysis
of the signal kinetics on rate and off rate allows the
discrimination between non-specific and specific interaction.
[0115] Agents that modulate one of the PCASTs 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.
[0116] Modulators of PCAST protein activity identified according to
these drug screening assays can be used to treat a subject with a
disorder mediated by the PCAST pathway, by treating cells or
tissues that express the PCAST. Experimental data as provided in
Table 1 indicates expression in human pancreatic cell lines. These
methods of treatment include the steps of administering a modulator
of PCAST activity in a pharmaceutical composition to a subject in
need of such treatment, the modulator being identified as described
herein.
[0117] In yet another aspect of the invention, the PCAST 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
PCAST and are involved in PCAST activity. Such PCAST-binding
proteins are also likely to be involved in the propagation of
signals by the PCAST proteins or PCAST targets as, for example,
downstream elements of a PCAST-mediated signaling pathway.
Alternatively, such PCAST-binding proteins are likely to be PCAST
inhibitors.
[0118] 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 PCAST
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 PCAST-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 PCAST protein.
[0119] 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 PCAST-modulating
agent, an antisense PCAST nucleic acid molecule, an PCAST-RNAi
fragment, a PCAST-specific antibody, or a PCAST-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.
[0120] The PCAST proteins of the present invention are also useful
to provide a target for diagnosing a disease or predisposition to
disease mediated by the peptide. Accordingly, the invention
provides methods for detecting the presence, or levels of, the
protein (or encoding mRNA) in a cell, tissue, or organism.
Experimental data as provided in Table 1 indicates expression in
human pancreatic cell lines. The method involves contacting a
biological sample with a compound capable of interacting with the
PCAST 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.
[0121] 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.
[0122] 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 PCAST 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.
[0123] 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.
[0124] 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
PCAST protein in which one or more of the PCAST 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 PCAST 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.
[0125] The peptides are also useful for treating a disorder
characterized by an absence of, inappropriate, or unwanted
expression of the protein. Experimental data as provided in Table 1
indicates expression in human pancreatic cell lines. Accordingly,
methods for treatment include the use of the PCAST protein or
fragments.
Antibodies
[0126] The present invention provides antibodies specifically bind
to PCAST proteins or fragments thereof, peptides, or antigenic
portion thereof.
[0127] 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.
[0128] The antibody of present invention selectively binds a target
PCAST when it binds the target domian 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.
[0129] 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.
[0130] 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).
[0131] 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.
[0132] 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.
[0133] "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).
[0134] 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
variants 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.
[0135] 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 complementary 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] The present invention further provides monoclonal antibody,
polyclonal antibody as well as humanized antibody. In general, to
generate antibodies, an isolated peptide is used as an immunogen
and is administered to a mammalian organism, such as a rat, rabbit
or mouse. The full-length protein, an antigenic peptide fragment or
a fusion protein of the PCAST 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).
[0140] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to conventional
(polyclonal) antibody preparations, which typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In additional to their specificity, the
monoclonal antibodies are advantageous in that they are synthesized
by the hybridoma culture, uncontaminated by other immunoglobulins.
The modifier "monoclonal" antibody indicates the character of the
antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method first described by
Kohler and Milstein, Nature 256, 495 (1975), or may be made by
recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.
The monoclonal antibodies for use with the present invention may
also be isolated from phage antibody libraries using the techniques
described in Clackson et al. Nature 352: 624-628 (1991), as well as
in Marks et al., J. Mol. Biol. 222: 581-597 (1991). For detailed
procedure for making a monoclonal antibody, see the Example
below.
[0141] "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).
[0142] 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 PCAST
protein, peptides or fragments thereof and a carrier protein is
prepared and an animal is immunized by the complex according to the
same manner as that described with respect to the above monoclonal
antibody preparation and the description in the Example. A serum or
plasma containing the antibody against the protein is recovered
from the immunized animal and the antibody is separated and
purified. The gamma globulin fraction or the IgG antibodies can be
obtained, for example, by use of saturated ammonium sulfate or DEAE
Sephadex, or other techniques known to those skilled in the
art.
[0143] 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.
[0144] 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 PCAST
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:40-51 and fragments of SEQ ID NOS:1-23. 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
elecrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human delta.1, .delta.2 or .delta.4 heavy chains
(Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .delta.3 (Guss et
al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a CH3 domain, the Bakerbond ABX.TM.
resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin SEPHAROSE..TM..
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0150] 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
[0151] 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 PCASTs
of the present invention are expressed at differential level in
various pancreatic cell lines, for example, SEQ ID NOS:1-23 are
overexpressed in all tested cell lines. 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.
[0152] Further, the antibodies can be used to assess expression in
disease states such as in active stages of the disease or in an
individual with a predisposition toward disease related to the
protein's function. When a disorder is caused by an inappropriate
tissue distribution, developmental expression, level of expression
of the protein, or expressed/processed form, the antibody can be
prepared against the normal protein. Experimental data as provided
in Table 1 indicates expression in human pancreatic cell lines. If
a disorder is characterized by a specific mutation in the protein,
antibodies specific for this mutant protein can be used to assay
for the presence of the specific mutant protein.
[0153] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Experimental data as provided in Table 1 indicates
expression in human pancreatic cell lines. The diagnostic uses can
be applied, not only in genetic testing, but also in monitoring a
treatment modality. Accordingly, where treatment is ultimately
aimed at correcting expression level or the presence of aberrant
sequence and aberrant tissue distribution or developmental
expression, antibodies directed against the protein or relevant
fragments can be used to monitor therapeutic efficacy. More
detection and diagnosis methods are described in detail below.
[0154] 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.
[0155] The antibodies are also useful for tissue typing.
Experimental data as provided in Table 1 indicates expression in
human pancreatic cell lines. Thus, where a specific protein has
been correlated with expression in a specific tissue, antibodies
that are specific for this protein can be used to identify a tissue
type.
[0156] The antibodies are also useful for inhibiting protein
function, for example, blocking the binding of the PCAST peptide to
a binding partner such as a substrate or another antibody binding
to the PCAST. 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.
[0157] 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
[0158] The present invention further provides isolated nucleic acid
molecules that encode a PCAST 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 PCAST peptides of the present invention, an allelic variant
thereof, or an ortholog or paralog thereof. The nucleic acid
molecules and the fragements 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] The present invention further provides nucleic acid
molecules that comprise the nucleotide sequences shown in Table 1
(SEQ ID NOS:24-39), or any nucleic acid molecule that encodes a
protein provided in Table 1 (SEQ ID NOS: 1-23). 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.
[0163] 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.
[0164] 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.
[0165] As mentioned above, the isolated nucleic acid molecules
include, but are not limited to, the sequence encoding the PCAST
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.
[0166] 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).
[0167] 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 PCAST
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.
[0168] 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.
[0169] A fragment comprises a contiguous nucleotide sequence
greater than 12 or more nucleotides. Further, a fragment could at
least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length
of the fragment will be based on its intended use. For example, the
fragment can encode epitope bearing regions of the peptide, or can
be useful as DNA probes and primers. Such fragments can be isolated
using the known nucleotide sequence to synthesize an
oligonucleotide probe. A labeled probe can then be used to screen a
cDNA library, genomic DNA library, or mRNA to isolate nucleic acid
corresponding to the coding region. Further, primers can be used in
PCR reactions to clone specific regions of gene.
[0170] 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.
[0171] 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.
[0172] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a peptide at
least 60-70% homologous to each other typically remain hybridized
to each other. The conditions can be such that sequences at least
about 60%, at least about 70%, or at least about 80% or more
homologous to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent
hybridization conditions are hybridization in 6.times. sodium
chloride/sodium citrate (SSC) at about 45 C, followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 50-65 C. Examples of
moderate to low stringency hybridization conditions are well known
in the art.
Nucleic Acid Molecule Uses
[0173] 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.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] The nucleic acid molecules are also useful for expressing
antigenic portions of the proteins.
[0178] 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.
[0179] The nucleic acid molecules are also useful in making vectors
containing the gene regulatory regions of the nucleic acid
molecules of the present invention.
[0180] 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.
[0181] The nucleic acid molecules are also useful for constructing
host cells expressing a part, or all, of the nucleic acid molecules
and peptides.
[0182] The nucleic acid molecules are also useful for constructing
transgenic animals expressing all, or a part, of the nucleic acid
molecules and peptides.
[0183] 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 PCASTs of the present invention are expressed
at differential level in various pancreatic cell lines, for
example, SEQ ID NOS:1-23 are overexpressed in all tested cell
lines. 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 PCAST protein expression relative to normal
results.
[0184] 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.
[0185] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express a PCAST protein, such as
by measuring a level of a PCAST-encoding nucleic acid in a sample
of cells from a subject e.g., mRNA or genomic DNA, or determining
if a PCAST gene has been mutated. Experimental data as provided in
Table 1 indicate that the PCASTs of the present invention are
expressed at differential level in various pancreatic cell lines,
for example, SEQ ID NOS:1-23 are overexpressed in all tested cell
lines. More detection and diagnosis methods are described in detail
below.
[0186] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate PCAST nucleic acid
expression.
[0187] 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 PCAST gene, particularly biological
and pathological processes that are mediated by the PCAST in cells
and tissues that express it. Experimental data as provided in Table
1 indicates expression in human pancreatic cell lines. The method
typically includes assaying the ability of the compound to modulate
the expression of the PCAST nucleic acid and thus identifying a
compound that can be used to treat a disorder characterized by
undesired PCAST nucleic acid expression. The assays can be
performed in cell-based and cell-free systems. Cell-based assays
include cells naturally expressing the PCAST nucleic acid or
recombinant cells genetically engineered to express specific
nucleic acid sequences.
[0188] The assay for PCAST 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 PCAST 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.
[0189] Thus, modulators of PCAST 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 PCAST mRNA in the presence of the candidate compound
or agent is compared to the level of expression of PCAST 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.
[0190] 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
PCAST nucleic acid expression in cells and tissues that express the
PCAST. Experimental data as provided in Table 1 indicate that the
PCASTs of the present invention are expressed at differential level
in various pancreatic cell lines, for example, SEQ ID NOS:1-23 are
overexpressed in all tested cell lines. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or nucleic acid expression.
[0191] 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
PCAST nucleic acid expression in the cells and tissues that express
the protein. Experimental data as provided in Table 1 indicates
expression in human pancreatic cell lines.
[0192] The nucleic acid molecules are also useful for monitoring
the effectiveness of modulating compounds or agents on the
expression or activity of the PCAST 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.
[0193] The nucleic acid molecules are also useful in diagnostic
assays for qualitative changes in PCAST nucleic acid expression,
and particularly in qualitative changes that lead to pathology. The
nucleic acid molecules can be used to detect mutations in PCAST
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 PCAST 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 PCAST 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 PCAST protein.
[0194] Individuals carrying mutations in the PCAST 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.
[0195] Alternatively, mutations in a PCAST gene can be directly
identified, for example, by alterations in restriction enzyme
digestion patterns determined by gel electrophoresis.
[0196] 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.
[0197] 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 PCAST 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)).
[0198] 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.
[0199] 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 PCAST gene in an
individual in order to select an appropriate compound or dosage
regimen for treatment.
[0200] 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.
[0201] The nucleic acid molecules are thus useful as antisense
constructs to control PCAST 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 PCAST protein. An
antisense RNA or DNA nucleic acid molecule would hybridize to the
mRNA and thus block translation of mRNA into PCAST protein.
[0202] 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.
[0203] The present invention relates to isolated RNA molecules
(double-stranded; single-stranded) of from about 21 to about 25
nucleotides which mediate RNAi. As used herein, about 21 to about
25 nt includes nucleotides 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28 and 29 nucleotides in length. The isolated RNAs of the
present invention mediate degradation of mRNA, the transcriptional
product of a gene. Such mRNA is also referred to herein as mRNA to
be degraded. As used herein, the terms RNA, RNA molecule(s), RNA
segment(s) and RNA fragment(s) are used interchangeably to refer to
RNA that mediates RNA interference. These terms include
double-stranded RNA, single-stranded RNA, isolated RNA (partially
purified RNA, essentially pure RNA, synthetic RNA, recombinantly
produced RNA), as well as altered RNA that differs from naturally
occurring RNA by the addition, deletion, substitution and/or
alteration of one or more nucleotides. Such alterations can include
addition of non-nucleotide material, such as to the end(s) of the
21-25 nt RNA or internally (at one or more nucleotides of the RNA).
Nucleotides in the RNA molecules of the present invention can also
comprise non-standard nucleotides, including non-naturally
occurring nucleotides or deoxyribonucleotides. Collectively, all
such altered RNAs are referred to as analogs or analogs of
naturally-occurring RNA. RNA of 21-25 nucleotides of the present
invention need only be sufficiently similar to natural RNA that it
has the ability to mediate RNAi. As used herein the phrase
"mediates RNAi" refers to the ability to distinguish which RNAs are
to be degraded by the RNAi machinery or process. RNA that mediates
RNAi interacts with the RNAi machinery such that it directs the
degradation of particular mRNAs. Such RNA may include RNAs of
various structure, including short hairpin RNA.
[0204] In one embodiment, the present invention relates to RNA
molecules of about 21 to about 25 nucleotides that direct cleavage
of specific mRNA to which their sequence corresponds. It is not
necessary that there be perfect correspondence of the sequences,
but the correspondence must be sufficient to enable the RNA to
direct RNAi cleavage of the target mRNA (Holen et al. (2005)
Nucleic Acids Res. 33, 4704-4710). In a particular embodiment, the
21-25 nt RNA molecules of the present invention comprise a 3'
hydroxyl group.
[0205] The present invention relates to 21-25 nt RNAs of specific
genes, produced by chemical synthesis or recombinant DNA
techniques, that mediate RNAi. As used herein, the term isolated
RNA includes RNA obtained by any means, including processing or
cleavage of dsRNA; production by chemical synthetic methods; and
production by recombinant DNA techniques. The invention further
relates to uses of the 21-25 nt RNAs, such as for therapeutic or
prophylactic treatment and compositions comprising 21-25 nt RNAs
that mediate RNAi, such as pharmaceutical compositions comprising
21-25 nt RNAs and an appropriate carrier.
[0206] The present invention also relates to a method of mediating
RNA interference of genes of a patient. In one embodiment, RNA of
about 21 to about 25 nt which targets the specific mRNA to be
degraded is introduced into a patient's cells. The cells are
maintained under conditions allowing degradation of the mRNA,
resulting in RNA-mediated interference of the mRNA of the gene in
the cells of the patient. Treatment of patients with cancer with
the RNAi will inhibit the growth and spread of the cancer and
reduce the tumor. Treatment of patients using RNAi can also be in
combination with other anti-cancer compounds. The RNAi may be used
in combination with other treatment modalities, such as
chemotherapy, cryotherapy, hyperthermia, radiation therapy, and
other similar treatments. In one embodiment, a chemotherapy agent
was combined with the RNAi. In another embodiment, a chemotherapy
named Gemzar was used.
[0207] Treatment of cancer or tumors in patients requires
introduction of the RNA into the cancer or tumor cells. RNA may be
directly introduced into the cell, or introduced extracellularly
into a cavity, interstitial space, into the circulation of a
patient, or introduced orally. Methods for oral introduction
include direct mixing of the RNA with food, as well as engineered
approaches in which a species that is used as food is engineered to
express the RNA and then ingested. Physical methods of introducing
nucleic acids, for example, injection directly into the cell or
extracellular injection into the patient, may also be used.
Vascular or extravascular circulation, the blood or lymph system,
and the cerebrospinal fluid are sites where the RNA may be
introduced. RNA may be introduced into an embryonic stem cell, or
another multipotent cell derived from the patient. Physical methods
of introducing nucleic acids include injection of a solution
containing the RNA, bombardment by particles covered by the RNA,
soaking cells or tissue in a solution of the RNA, or
electroporation of cell membranes in the presence of the RNA. A
viral construct packaged into a viral particle may be used to
introduce an expression construct into the cell, with the construct
expressing RNA. Other methods known in the art for introducing
nucleic acids to cells may be used, such as lipid-mediated carrier
transport, chemical-mediated transport, and the like. Thus the RNA
may be introduced along with components that perform one or more of
the following activities: enhance RNA uptake by the cell, promote
annealing of the duplex strands, stabilize the annealed strands, or
otherwise increase inhibition of the target gene. The RNAi may be
used in combination with other treatment modalities, such as
chemotherapy, cryotherapy, hyperthermia, radiation therapy, and the
like.
[0208] The present invention may be used alone or as a component of
a kit having at least one of the reagents necessary to carry out
the in vitro or in vivo introduction of RNA to tissue or patients.
Preferred components are the dsRNA and a vehicle that promotes
introduction of the dsRNA. Such a kit may also include instructions
to allow a user of the kit to practice the invention.
[0209] Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of PCAST nucleic
acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired PCAST 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 PCAST protein, such as substrate
binding.
[0210] The nucleic acid molecules also provide vectors for gene
therapy in patients containing cells that are aberrant in PCAST
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 PCAST protein to treat the individual.
[0211] The invention also encompasses kits for detecting the
presence of a PCAST nucleic acid in a biological sample.
Experimental data as provided in Table 1 indicate that the PCASTs
of the present invention are expressed at differential level in
various pancreatic cell lines, for example, SEQ ID NOS:1-23 are
overexpressed in all tested cell lines. For example, the kit can
comprise reagents such as a labeled or labelable nucleic acid or
agent capable of detecting PCAST nucleic acid in a biological
sample; means for determining the amount of PCAST nucleic acid in
the sample; and means for comparing the amount of PCAST 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 PCAST 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. 3r. 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 methodology. Generally, the DNA sequence
that will ultimately be expressed is joined to an expression vector
by cleaving the DNA sequence and the expression vector with one or
more restriction enzymes and then ligating the fragments together.
Procedures for restriction enzyme digestion and ligation are well
known to those of ordinary skill in the art.
[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 is
difficult to achieve with multi-transmembrane domain containing
proteins such as PCASTs, 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 PCASTs, 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 PCAST protein or peptide that can be further purified
to produce desired amounts of PCAST 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 PCAST protein or PCAST protein fragments, such as
those described above as well as other formats known in the art.
Thus, a recombinant host cell expressing a native PCAST protein is
useful for assaying compounds that stimulate or inhibit PCAST
protein function.
[0240] Host cells are also useful for identifying PCAST 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 PCAST protein (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native PCAST 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 PCAST protein and identifying
and evaluating modulators of PCAST 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 PCAST
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 PCAST
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
formula 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, PCAST 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 PCAST protein function,
including substrate interaction, the effect of specific mutant
PCAST proteins on PCAST protein function and substrate interaction,
and the effect of chimeric PCAST proteins. It is also possible to
assess the effect of null mutations, that is, mutations that
substantially or completely eliminate one or more PCAST protein
functions.
Detection and Diagnosis
[0248] The present invention provides a method for detecting PCAST
nucleic acids, proteins, peptides and fragments thereof that are
differentially expressed in pancreatic diseases in a test sample,
preferably in a biological sample.
[0249] The present invention further provides a method for
diagnosing the pancreatic diseases, by detecting the nucleic acids,
proteins, peptides and fragements thereof. The further embodiment
includes but is not limited to, monintoring the disease prognosis
(recurrance), diagnosing disease stage, preventing the disease and
treating the disease.
[0250] As used herein, a "biological sample" can be collected from
tissues, blood, sera, cell lines or biological fluids such as,
plasma, interstitial fluid, urine, cerebrospinal fluid, and the
like, containing cells. In preferred embodiments, a biological
sample comprises cells or tissues suspected of having diseases
(e.g., cells obtained from a biopsy).
[0251] As used herein, a "differential level" is defined as the
level of PCAST protein or nucleic acids in a test sample either
above or below the level of the ones in control samples, wherein
the level of control samples is obtained from a control cell line,
a normal tissue or body fluids, or combination thereof, from a
healthy subject. While the protein is overexpressed, the expression
of PCAST is preferably greater than about 20%, or preferably
greater than about 30%, and most preferably greater than about 50%
or more of pancreatic disease sample, at a level that is at least
two fold, and preferably at least five fold, greater than the level
of expression in control samples, as determined using a
representative assay provided herein. While the protein is
underexpressed, the expression of PCAST is preferably less than
about 20%, or preferably less than 30%, and most preferably less
than about 50% or more of the pancreatic disease sample, at a level
that is at least 0.5 fold, and preferably at least 0.2 fold less
than the level of the expression in control samples, as determined
using a representative assay provided herein.
[0252] As used herein, a "subject" can be a mammalian subject or
non mammalian subject, preferably, a mammalian subject. A mammalian
subject can be human or non-human, preferably human. A healthy
subject is defined as a subject without detectable pancreatic
diseases or pancreatic associated diseases by using conventional
diagnostic methods.
[0253] As used herein, the "diseases" include pancreatic diseases
and pancreatic associated disease.
[0254] As used herein, "cancer" includes epithelial-cell related
cancers, for example pancreatic, lung, colon, prostate, ovarian,
breast, bladder renal, hepatocellular, pharyngeal and gastric
cancers.
Nucleic Acid Detections
[0255] The present invention is not limited to the detection
methods described above. Any suitable detection method that allows
for the specific detection of pancreatic diseases cells, tissues or
organs may be utilized. For example, in some embodiments, the
expression of RNA corresponding to a PCAST 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.).
[0256] In another embodiment, the present invention provides a
method for diagnosing or detecting pancreatic diseases in a subject
comprising: determining the level of one or more PCAST nucleic acid
molecules or any fragment(s) thereof in a test sample from said
subject, wherein said PCAST nucleic acid molecule(s) comprises a
sequence selected from a group consisting of SEQ ID NOS:24-39 and a
combination thereof; wherein a differential level of said PCAST
nucleic acid molecule(s) relative to the level of said nucleic acid
molecule(s) in a test sample from a healthy subject, or the level
established for a healthy subject, is indicative of pancreatic
diseases.
[0257] 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:24-39 and combination thereof.
[0258] In further embodiments of the present invention, the
presence of particular sequences in the genome of a subject is
detected. Such sequences include PCAST sequences associated with
abnormal expression of PCAST (e.g., overexpression or expression at
a physiological inappropriate time). These sequences include
polymorphisms, including polymorphisms in the transcribed sequence
(e.g., that effect PCAST 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
[0259] The present invention provides methods for diagnosing or
detecting the differential presence of PCAST protein. In some
embodiments (e.g., where PCASTs are overexpressed in diseased
cells), PCAST proteins are detected directly. In other embodiments
(e.g., where the presence of a PCASTs are underexpressed), PCAST to
the disease antigens are detected non-existence.
[0260] 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 PCAST may be indicative
of a specific stage or sub-type of the same cancer or disease.
[0261] The information obtained is also used to determine prognosis
and appropriate course of treatment. For example, it is
contemplated that individuals with a specific PCAST expression or
stage of pancreatic diseases may respond differently to a given
treatment that individuals lacking the PCAST expression. The
information obtained from the diagnostic methods of the present
invention thus provides for the personalization of diagnosis and
treatment.
[0262] In one embodiment, the present invention provides a method
for monitoring pancreatic diseases treatment in a subject
comprising: determining the level of one or more PCAST proteins or
any fragment(s) or peptide(s) thereof in a test sample from said
subject, wherein said PCAST protein(s) comprises a sequence
selected from a group consisting of SEQ ID NOS:1-23, SEQ ID
NOS:40-51 and a combination thereof; wherein an level of said PCAST
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.
[0263] In another embodiment, the present invention provides a
method for diagnosing recurrence of pancreatic diseases following
successful treatment in a subject comprising: determining the level
of one or more PCAST proteins or any fragment(s) or peptide(s)
thereof in a test sample from said subject, wherein said PCAST
protein(s) comprises a sequence selected from a group consisting of
SEQ ID NOS:1-23, SEQ ID NOS:40-51 or a combination thereof; wherein
a changed level of said PCAST protein(s) relative to the level of
said protein(s) in a test sample from a healthy subject, or the
level established for a healthy subject, is indicative of
recurrence of pancreatic diseases.
[0264] In yet another embodiment, the presend invention provides a
method for diagnosing or detecting pancreatic diseases in a subject
comprising: determining the level of one or more PCAST proteins or
any fragment(s) or peptides thereof in a test sample from said
subject, wherein said PCAST protein(s) comprises a sequence
selected from a group consisting of SEQ ID NOS:1-23, SEQ ID
NOS:40-51 and a combination thereof; wherein a differential level
of said PCAST protein(s) relative to the level of said protein(s)
in a test sample from a healthy subject, or the level established
for a healthy subject, is indicative of pancreatic diseases.
[0265] The detecting or diagnosing method comprises determining
level of differential expression of 2, 4, 8, 10, 20 or more
proteins, preferably, the proteins are selected from a group
consisting of SEQ ID NOS:1-23 and combination thereof.
[0266] Further, the detecting or diagnosing method comprises
determining level of differential expression of 5, 10, 15, 20, 40,
60, 80, 100 or more PCAST peptides, preferably the peptides are
selected from the group consisting of SEQ ID NOS:40-51 and
combination thereof.
[0267] 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.
[0268] In yet another embodiment, the present invention provides a
detection or diagonistic method of PCASTs by using LC/MS. The
proteins from cells are prepared by methods known in the art (R.
Aebersold Nature Biotechnology Volume 21 Number 6 Jun. 2003). The
differential expression of proteins in disease and healthy samples
are quantitated using Mass Spectrometry and ICAT (Isotope Coded
Affinity Tag) labeling, which is known in the art. ICAT is an
isotope label technique that allows for discrimination between two
populations of proteins, such as a healthy and a disease sample.
The LC/MS spectra are collected for the labeled samples. The raw
scans from the LC/MS instrument are subjected to peak detection and
noise reduction software. Filtered peak lists are then used to
detect `features` corresponding to specific peptides from the
original sample(s). Features are characterized by their
mass/charge, charge, retention time, isotope pattern and
intensity.
[0269] 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 PCAST protein or peptide are similar to the
expression pattern in Table 1 in a test subject indicates the
likelihood of having pancreatic diseases or diseases associated
with pancreas.
[0270] Immunological methods for detecting and measuring complex
formation as a measure of protein expression using either specific
polyclonal or monoclonal antibodies are known in the art. Examples
of such techniques include enzyme-linked immunosorbent assays
(ELISAs), radioimmunoassays (RIAs), fluorescence-activated cell
sorting (FACS) and antibody arrays. Such immunoassays typically
involve the measurement of complex formation between the protein
and its specific antibody. These assays and their quantitation
against purified, labeled standards are well known in the art
(Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-based
immunoassay utilizing antibodies reactive to two non-interfering
epitopes is preferred, but a competitive binding assay may be
employed (Pound (1998) Immunochemical Protocols, Humana Press,
Totowa N.J.). More immunological detections are described in
section below.
Antibody Detections
[0271] Antibodies are useful to detect the presence of one of the
proteins or fragements thereof, peptides of the present invention
in cells or tissues to determine the pattern of expression of the
protein among various tissues in an organism and over the course of
normal development.
[0272] 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.
[0273] 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 PCAST
protein, peptide or fragment thereof, in specific cells, tissues,
blood, serum or body fluids.
[0274] 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:
[0275] (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.
[0276] (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.
[0277] (c) Various enzyme-substrate labels are available and U.S.
Pat. Nos. 4,275,149, 4,318,980 provides a review of some of these.
The enzyme generally catalyzes a chemical alteration of the
chromogenic substrate, which can be measured using various
techniques. For example, the enzyme may catalyze a color change in
a substrate, which can be measured spectrophotometrically.
Alternatively, the enzyme may alter the fluorescence or
chemiluminescence of the substrate. Techniques for quantifying a
change in fluorescence are described above. The chemiluminescent
substrate becomes electronically excited by a chemical reaction and
may then emit light which can be measured (using a
chemiluminometer, for example) or donates energy to a fluorescent
acceptor. Examples of enzymatic labels include luciferases (e.g.,
firefly luciferase and bacterial luciferase; U.S. Pat. No.
4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate
dehydrogenase, urease, peroxidase such as horseradish peroxidase
(HRPO), alkaline phosphatase, .beta.-galactosidase, glucoamylase,
lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose
oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic
oxidases (such as uricase and xanthine oxidase), lactoperoxidase,
microperoxidase, and the like. Techniques for conjugating enzymes
to antibodies are described in O'Sullivan et al., Methods for the
Preparation of Enzyme-Antibody Conjugates for Use in Enzyme
Immunoassay, in Methods in Enzyme. (Ed. J. Langone & H. Van
Vunakis), Academic press, New York, 73: 147-166 (1981).
[0278] 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.
[0279] In another embodiment of the invention, the antibody need
not be labeled, and the presence thereof can be detected using a
labeled antibody, which binds to the antibody.
[0280] 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).
[0281] The biological samples can then be tested directly for the
presence of PCAST 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 PCAST
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.
[0282] 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.
[0283] 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.
[0284] 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.
[0285] 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.
[0286] 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 PCAST
targets and the affinity value(Kd) is less than 1.times.10.sup.8
M.
[0287] 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.
[0288] For immunohistochemistry, the disease tissue sample may be
fresh or frozen or may be embedded in paraffin and fixed with a
preservative such as formalin (see Example). The fixed or embedded
section contains the sample are contacted with a labeled primary
antibody and secondary antibody, wherein the antibody is used to
detect the PCAST protein express in situ. The detailed procedure is
shown in the Example.
Array:
[0289] Array technologies and quantitative PCR provide the means to
explore the expression profiles of a large number of related or
unrelated genes, and proteins. When an expression profile is
examined, arrays provide a platform for examining which genes or
proteins are tissue-specific, carrying out housekeeping functions,
parts of a signaling cascade, or specifically related to a
particular genetic predisposition, condition, disease, or disorder.
The potential application of gene or protein expression profiling
is particularly relevant to improving diagnosis, prognosis, and
treatment of disease. For example, both the sequences and the
amount of expression can be compared between tissues from subjects
with different types of pancreatic diseases and cytologically
normal healthy tissue.
[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, mirrors the nucleic acid expression
profile and is produced using gel electrophoresis, mass
spectrometry, or an array and labeling moieties or antibodies which
specifically bind the protein. The nucleic acids, proteins, or
antibodies specifically binding the protein may be used in solution
or attached to a substrate, and their detection is based on methods
well known in the art.
[0292] A substrate includes but not limits 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 PCAST proteins or peptides or fragments
thereof, antibodies, which specifically bind PCAST proteins,
peptides or fragments thereof. Preferably, two or more of the
nucleic acid molecules (e.g., SEQ ID NOS:24-39), proteins (e.g.,
SEQ ID NOS:1-23) or peptides (e.g., SEQ ID NOS:40-51) are
immobilized on a substrate.
[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 after hybridization with a
sample. The transcripts of the invention may be used as elements on
an array to produce a gene expression profile. In one embodiment,
the array is used to diagnose or monitor the progression of
disease. Researchers can assess and catalog the differences in gene
expression between healthy and diseased tissues or cells.
[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 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.
Treatment
[0305] The following terms, as used in the present specification
and claims, are intended to have the meaning as defined below,
unless indicated otherwise.
[0306] "Treat," "treating" or "treatment" of a disease includes:
(1) preventing the disease, i.e. causing the clinical symptoms of
the disease not to develop in a mammal that may be exposed to or
predisposed to the disease but does not yet experience or display
symptoms of the disease, (2) inhibiting the disease, i.e.,
arresting or reducing the development of the disease or its
clinical symptoms, or (3) relieving the disease, i.e., causing
regression of the disease or its clinical symptoms.
[0307] 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.
[0308] A "panceratic disease" includes pancreatic cancer,
pancreatic tumor (exocrine or endocrine), pancreatic cysts, acute
pancreatitis, chronic pancreatitis, diabetes (type I and II) as
well as pancreatic trauma, preferably pancreatic cancer.
[0309] A "cancer" is epithelial-cell related cancers include but
not limit to pancreatic, lung, colon, prostate, ovarian, breast,
bladder renal, hepatocellular, pharyngeal and gastric cancers.
[0310] The present invention provides an application of treatment
by using antibody, immunogenic peptides as well as other PCAST
agonists or antagonists.
[0311] PCASTs are proteins differentially expressed in the
pancreatic diseases cell lines or tissues. The proteins are
secreted pancreatic proteins (see the list in Table 1). These
proteins are associated with the diseases especially pancreatic
diseases, particularly pancreatic cancer; thus, they serve as
candidate targets for the treatment of the diseases.
[0312] 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.
[0313] 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.
[0314] 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
[0315] 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 pancreatic diseases.
[0316] In general, the antibodies are also useful for inhibiting
protein function, for example, blocking the binding of the PCAST
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.
[0317] 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 PCAST can
be passively administered alone or in conjunction with other
anti-cancer therapies to a mammal afflicted-with pancreatic
diseases or cancer. Examples of anti-cancer therapies include, but
are not limited to, chemotherapy, radiation therapy, adoptive
immunotherapy therapy with TIL (Tumor Infiltration
Lymphocytes).
[0318] 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.
[0319] The mechanism for antibody therapy is that the therapeutic
antibody recognizes a cell surface protein or a cytosolic protein
or secreted protein that is overexpressed in diseased cells. By NK
cell or complement activation, conjugation of the antibody with an
immunotoxin or radiolabel, the interaction can abrogate
ligand/resceptor interaction or activation of apoptosis.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] 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.
[0324] Depending on the type and severity of the disease, about
1.mu.g/kg to 150 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. A typical daily dosage might range from
about 1.mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment is sustained
until a desired suppression of disease symptoms occurs. However,
other dosage regimens may be useful. The progress of this therapy
is easily monitored by conventional techniques and assays.
[0325] 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.
[0326] 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.
[0327] Antibodies of the present invention may also be used as
therapeutic reagents, to diminish or eliminate cancer or tumors.
For example, the antibodies may be used on their own (for instance,
to inhibit metastases) or coupled to one or more therapeutic
agents. Suitable agents in this regard include radionuclides,
differentiation inducers, drugs, toxins, and derivatives thereof.
Preferred radionuclides include .sup.90Y, .sup.123I, .sup.125I,
.sup.131I, .sup.186Re, .sup.188Re, .sup.211At, and .sup.212Bi.
Preferred drugs include methotrexate, and pyrimidine and purine
analogs. Preferred differentiation inducers include phorbol esters
and butyric acid. Preferred toxins include ricin, abrin, diptheria
toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella
toxin, and pokeweed antiviral protein
[0328] 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.
[0329] 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.
[0330] 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.
[0331] 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.).
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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.
[0336] 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.
[0337] 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.
[0338] 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.
[0339] 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 PCAST
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.
[0340] 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.
[0341] 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
[0342] The PCAST 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.
[0343] The antiserum from immunized individuals may be administered
as a prophylactic measure for individuals who are at risk of
developing pancreatic diseases or cancer. The antiserum is also
useful in treating an individual afflicted with pancreatic diseases
or cancer for post-disease prophylaxis.
[0344] Alternatively, peptides derived form the PCAST 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.).
[0345] 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.
[0346] Alternatively, amino acid sequence variants of the peptide
can be prepared by mutations in the DNA, which encodes the peptide,
or by peptide synthesis.
[0347] 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.
[0348] 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).
[0349] 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.
[0350] The peptides of the present invention are used for treatment
of the pancreatic diseases. Treatment involves administration of
the protective composition after the appearance of the disease.
[0351] 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."
[0352] The peptides are used for treating T cell-mediated
pathology. The term "T cell-mediated pathology" refers to any
condition in which an inappropriate T cell response is a component
of the pathology. The term is intended to encompass both T cell
mediated pancreatic diseases and diseases resulting from
unregulated clonal T cell replication.
[0353] Therefore, the present invention relates to peptides or
modified peptides derived from the protein sequences of the PCAST
proteins that differentially expressed in the pancreatic diseases.
By way of example, modification may include substitution, deletion
or addition of an amino acid in the given immunogenic peptide
sequence or mutation of existing amino acids within the given
immunogenic peptide sequence, or derivatization of existing amino
acids within the given immunogenic peptide sequence. Any amino acid
comprising the immunogenic peptide sequence may be modified in
accordance with this invention. In one aspect, at least one amino
acid is substituted or replaced within the given immunogenic
peptide sequence. Any amino acid may be used to substitute or
replace a given amino acid within the immunogenic peptide sequence.
Modified peptides are intended to include any immunogenic peptide
obtained from differentially expressed proteins, which has been
modified and exhibits enhanced binding to the MHC molecule with
which it associates when presented to the T-cell. These modified
peptides may be synthetically or recombinantly produced by
conventional methods.
[0354] 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.
[0355] The recombinant or natural protein, peptides, or fragment
thereof of PCAST, or modified peptides, may be used as a vaccine
either prophylactically or therapeutically. When provided
prophylactically the vaccine is provided in advance of any evidence
of pancreatic diseases, particularly, cancer. The prophylactic
administration of the pancreatic diseases vaccine should serve to
prevent or attenuate pancreatic diseases, preferably cancer, in a
mammal.
[0356] Preparation of vaccine is using recombinant protein or
peptide expression vectors comprising all or part of nucleic acid
sequence of PCAST 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:24-39 can be introduced into a mammal either prior to any
evidence of pancreatic diseases or to mediate regression of the
disease in a mammal afflicted with pancreatic diseases. Examples of
methods for administering the viral vector into the mammals
include, but are not limited to, exposure of cells to the virus ex
vivo, or injection of the retrovirus or a producer cell line of the
virus into the affected tissue or intravenous administration of the
virus. Alternatively the viral vector carrying all or part of the
PCAST 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 PCAST 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.
[0357] Alternatively all or parts thereof of a substantially or
partially purified the PCAST protein or their peptides may be
administered as a vaccine in a pharmaceutically acceptable carrier.
Ranges of the protein that may be administered are about 0.001 to
about 100 mg per patient, preferred doses are about 0.01 to about
100 mg per patient. In a preferred embodiment, the peptides or
modified peptides thereof is administered therapeutically or
prophylactically to a mammal in need of such treatment. The peptide
may be synthetically or recombinantly produced. Immunization is
repeated as necessary, until a sufficient titer of anti-immunogen
antibody or immune cells has been obtained.
[0358] 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.).
[0359] The vaccine formulation of the present invention comprises
an immunogen that induces an immune response directed against the
cancer associated antigens such as the PCASTs, 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.
[0360] Measurement of candidate disease tumor antigen or vaccine
expression in patients is the first step of the present invention.
Subsequent steps will focus on measuring immune responses to these
candidate antigens or vaccine. Sera from disease patients,
particularly cancer patients, and healthy donors will be screened
for antibodies to the candidate antigens as well as for levels of
circulating tumor derived antigens antigen. The vaccine
formulations may be evaluated first in animal models, initially
rodents
[0361] In one embodiment mammals, preferably human, at high risk
for pancreatic diseases, particularly cancer, are prophylactically
treated with the vaccines of this invention. Examples of such
mammals include, but are not limited to, humans with a family
history of pancreatic diseases, humans with a history of pancreatic
diseases, particular cancer, or humans afflicted with pancreatic
cancer previously resected and therefore at risk for reoccurrence.
When provided therapeutically, the vaccine is provided to enhance
the patient's own immune response to the diseased antigen present
on the pancreatic diseases or advanced stage of pancreatic
diseases. The vaccine, which acts as an immunogen, may be a cell,
cell lysate from cells transfected with a recombinant expression
vector, cell lysates from cells transfected with a recombinant
expression vector, or a culture supernatant containing the
expressed protein. Alternatively, the immunogen is a partially or
substantially purified recombinant protein, peptide or analog
thereof or modified peptides or analogs thereof. The proteins or
peptides may be conjugated with lipoprotein or administered in
liposomal form or with adjuvant.
[0362] 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.
[0363] 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-PCAST immune cells or anti-PCAST antibody
is produced. The presence of anti-PCAST immune cells may be
assessed by measuring the frequency of precursor CTL (cytoxic
T-lymphocytes) against PCAST 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.
[0364] The safety of the immunization procedures is determined by
looking for the effect of immunization on the general health of the
immunized animal (weight change, fever, appetite behavior etc.) and
looking for pathological changes on autopsies. After initial
testing in animals, pancreatic diseases patients can be tested.
Conventional methods would be used to evaluate the immune response
of the patient to determine the efficiency of the vaccine.
[0365] In yet another embodiment of this invention all, part, or
parts of the PCAST 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 PCAST antigen activated dendritic cells or
processed dendritic cell antigens may be used as immunogens for
vaccines or for the treatment of pancreatic diseases, particularly
pancreatic cancer. The dendritic cells should be exposed to antigen
for sufficient time to allow the antigens to be internalized and
presented on the dendritic cells surface. The resulting dendritic
cells or the dendritic cell process antigens can than be
administered to an individual in need of therapy. Such methods are
described in Steinman et al. (WO93/208185) and in Banchereau et al.
(EPO Application 0563485A1).
[0366] In yet another aspect of this invention T-cells isolated
from individuals can be exposed to the PCAST proteins, peptides or
fragment thereof, or modified peptides in vitro and then
administered to a patient in need of such treatment in a
therapeutically effective amount. Examples of where T-lymphocytes
can be isolated include but are not limited to, peripheral blood
cells lymphocytes (PBL), lymph nodes, or tumor infiltrating
lymphocytes (TIL). Such lymphocytes can be isolated from the
individual to be treated or from a donor by methods known in the
art and cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol.
142: 2453-3461). Lymphocytes are cultured in media such as RPMI or
RPMI 1640 or AIM V for 1-10 weeks. Viability is assessed by trypan
blue dye exclusion assay. Examples of how these sensitized T-cells
can be administered to the mammal include but are not limited to,
intravenously, intraperitoneally or intralesionally. Parameters
that may be assessed to determine the efficacy of these sensitized
T-lymphocytes include, but are not limited to, production of immune
cells in the mammal being treated or tumor regression. Conventional
methods are used to assess these parameters. Such treatment can be
given in conjunction with cytokines or gene modified cells
(Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 75-90;
Rosenberg, S. A. et al. (1992) Human Gene Therapy, 3: 57-73).
[0367] 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.
[0368] All examples outlined here were carried out using standard
techniques, which are well known and routine to those of skill in
the art. Routine molecular biology techniques of the following
example can be carried out as described in standard laboratory
manuals, such as Sambrook et al., Molecular Cloning: A laboratory
Manual, 3rd Ed.; Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (2001).
WORKING EXAMPLES
1. Pancreatic Cell Line Model System
[0369] Analysis of gene expression in various pancreatic cancer
cell lines as well as pancreatic duct epithelial tissue has shown
that the cell line Hs766T correlates well with normal tissue. For
this reason, this cell line is reported in the literature as being
a good surrogate for normal tissue in analyses of differential
expression between pancreatic adenomcarcinoma (and derived tumor
lines) and normal tissue (or surrogate, Hs766T). The model system
employed here involves the use of Hs766T as a "normal" reference to
which secreted expression in tumor-derived cell lines is compared.
These differentials or candidates are then validated in tissues,
pancreatic cancer and normal pancreas, to confirm that they are
differentially expressed between these tissues as well as within
the cell line model system. Details of the pancreatic tumor lines
that were used for this study, as well as the pancreatic line
Hs766T are provided below.
Cell Lines and Media:
TABLE-US-00001 [0370] Non- essential Fetal ATCC Base amino Sodium
Sodium Bovine Cell line Reference medium Glutamine acids Carbonate
Pyruvate Hepes Serum Panc-1 CRL-1469 DMEM 2 mM 1% (w/v) 0.1% 1 mM
10% (v/v) (w/v) Hs766t HTB-134 DMEM 2 mM 1% (w/v) 0.1% 1 mM 10%
(v/v) (w/v) SU.86.86 CRL-1837 DMEM 2 mM 1% (w/v) 0.1% 1 mM 10%
(v/v) (w/v) AsPC1 CRL-1682 RPMI 2 mM 1% (w/v) 0.1% 1 mM 10 mM 20%
(v/v) (w/v) HPAF II CRL-1997 DMEM 2 mM 1% (w/v) 0.1% 1 mM 10% (v/v)
(w/v) HPAC CRL-2119 DMEM 2 mM 1% (w/v) 0.1% 1 mM 10% (v/v) (w/v)
Mia-Paca-2 CRL-1420 DMEM 2 mM 1% (w/v) 0.1% 1 mM 10% (v/v) (w/v)
Mpanc-96 CRL-2380 RPMI 2 mM 1% (w/v) 0.1% 1 mM 10 mM 10% (v/v)
(w/v) BxPC-3 CRL-1687 RPMI 2 mM 1% (w/v) 0.1% 1 mM 10 mM 10% (v/v)
(w/v) Capan-2 HTB-80 DMEM 2 mM 1% (w/v) 0.1% 1 mM 10% (v/v)
(w/v)
Pancreatic Cancer Cell Line Culture
[0371] Cells are grown under routine tissue culture conditions in
490 cm.sup.2 roller bottles at an initial seeding density of
approximately 15 million cells per roller bottle. When the cells
reach .about.70-80% confluence, the culturing media was removed,
the cells were washed 3 times with D-PBS and once with CD293
protein-free media (Invitrogen cat# 11913-019), and the culturing
media was replaced with CD293 for generating conditioned media.
Cells were incubated for 72 hours in CD293 and the media was
collected for secreted protein MS analysis (30-300 ml). Cell debris
was removed from the conditioned media by centrifugation at 300 g
for 5 minutes and filtering through a 0.2 micron filter prior to MS
analysis.
[0372] To monitor cell proliferation and apoptosis, the remaining
cells were incubated with 1:100 dilution of BrdU in culturing media
for 2 hours (BrdU Flow Kit cat# 559619 BD Biosciences). Cells were
washed 3 times with D-PBS and disassociated from the flask with
versene. Cells were washed once with Flow Staining Buffer (0.5%
BSA, 0.05% NaN.sub.3 in D-PBS). Cells were incubated with 400 ul of
Cytofix/Cytoperm Buffer (BrdU Flow Kit BD Biosciences) for 15-30
minutes at 4.degree. C. Cells were washed once with Flow Staining
Buffer and resuspended in 400 ul Cytoperm Plus Buffer (BrdU Flow
Kit BD Biosciences). Cells were incubated for 10 minutes at
4.degree. C. and washed once with 1.times. Perm/Wash Buffer (BrdU
Flow Kit BD Biosciences). Cells were incubated for 1 hour at
37.degree. C. protected from light in DNAse solution (BrdU Flow Kit
BD Biosciences). Cells were washed once with 1.times. Perm/Wash
Buffer and incubated for 20 minutes at room temperature with
anti-BrdU FITC conjugated antibody (BrdU Flow Kit BD Biosciences)
and PE-conjugated active caspase 3 (BD Biosciences cat# 550821) and
PE mouse IgG2B isotype control. Cells were washed once with
1.times. Perm/Wash Buffer and resuspended in DAPI for LSR flow
cytometry analysis.
2. Cloning and Expression of Target Proteins
[0373] cDNA Retrieval
[0374] Peptide sequences were searched by BlastP against the Celera
Discovery System (CDS) and public database to identify the
corresponding full-length open reading frames (ORFs). Each ORF
sequence was then searched by BlastN against the Celera in-house
human cDNA clone collection. For each sequence of interest, up to
three clones are pulled and streaked onto LB/Ampicillin (100 ug/ml)
plates. Plasmid DNA is isolated using Qiagen spin mini-prep kit and
verified by restriction digest. Subsequently, the isolated plasmid
DNA is sequence verified against the ORF reference sequence.
Sequencing reactions are carried out using Applied Biosystems
BigDye Terminator kit followed by ethanol precipitation. Sequence
data is collected using the Applied Biosystems 3100 Genetic
Analyzer and analyzed by alignment to the reference sequence using
the Clone Manager alignment tool.
PCR
[0375] PCR primers are designed to amplify the full-length ORF as
well as any regions of the ORF that are interest for expression
(antigenic or hydrophilic regions as determined by the Clone
Manager sequence analysis tool). Primers also contain 5' and 3'
overhangs to facilitate cloning (see below). PCR reactions contain
2.5 units Platinum Taq DNA Polymerase High Fidelity (Invitrogen),
50 ng cDNA plasmid template, 1 uM forward and reverse primers, 800
uM dNTP cocktail (Applied Biosystems) and 2 mM MgSO4. After 20-30
cycles (94.degree. C. for 30 seconds, 55.degree. C. for 1 minutes
and 73.degree. C. for 2 minutes), product is verified and
quantitated by agarose gel electrophoresis.
Construction of Entry Clones
[0376] PCR products are cloned into an entry vector for use with
the Gateway recombination based cloning system (Invitrogen). These
vectors included pDonr221, pDonr201, pEntr/D-TOPO or
pEntr/SD/D-TOPO and are used as described in the cloning methods
below.
TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO
[0377] For cloning using this method, the forward PCR primer
contain a 5' overhang containing the sequence "CACC". PCR products
are generated as described above and cloned into the entry vector
using the Invitrogen TOPO cloning kit. Reactions are typically
carried out at room temperature for 10 minutes and subsequently
transformed into TOP10 chemically competent cells (Invitrogen,
Calif.). Candidate clones are picked, plasmid DNA is prepared using
Qiagen spin mini-prep kit and screened using restriction digest.
Inserts are subsequently sequence verified as described above.
Gateway Cloning into pDonr201 or pDonr221
[0378] For cloning using this method, PCR primers contain the
following overhangs:
TABLE-US-00002 Forward 5' overhang:
5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3' Reverse 5' overhang:
5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3'
[0379] PCR products are generated as described above. ORFs are
recombined into the entry vector using the Invitrogen Gateway BP
Clonase enzyme mix. Reactions are typically carried out at
25.degree. C. for 1 hour, treated with Proteinase K at 37.degree.
C. for 10 minutes and transformed into Library Efficiency
DH5.alpha. chemically competent cells (Invitrogen, Calif.).
Candidate clones are picked, plasmid DNA is prepared using Qiagen
spin mini-prep kit and screened using restriction digest. Inserts
are subsequently sequence verified as described above.
Construction of Expression Clones
[0380] ORFs are transferred from the entry construct into a series
of expression vectors using the Gateway LR Clonase enzyme mix.
Reactions are typically carried out for 1 hour at 25.degree. C.,
treated with Proteinase K at 37.degree. C. for 10 minutes and
subsequently transformed into Library Efficiency DH5a chemically
competent cells (Invitrogen). Candidate clones are picked, plasmid
DNA is prepared using Qiagen spin mini-prep kit and screened using
restriction digest. Expression vectors include but are not limited
to pDest14, pDest15, pDest17, pDest8, pDest10 and pDest20. These
vectors allow expression in systems such as E. coli and recombinant
baculovirus. Other vectors not listed here allow expression in
yeast, mammalian cells, or in vitro.
Expression of Recombinant Proteins in E. coli
[0381] Constructs are transformed into one or more of the following
host strains: BL21 S1, BL21 AI, (Invitrogen); Origami B (DE3),
Origami B (DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS,
Rosetta-Gami (DE3), Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B
(DE3) pLysS (Novagen). The transformants are grown in LB with or
without NaCl and with appropriate antibiotics, at temperatures in
the range of 20-37.degree. C., with aeration. Expression is induced
with the addition of IPTG (0.03-0.3 mM) or NaCl (75-300 mM) when
the cells are in mid-log growth. Growth is continued for one to 24
hours post-induction. Cells are harvested by centrifugation in a
Sorvall RC-3C centrifuge in a H6000A rotor for 10 minutes at 3000
rpm, at 4.degree. C. Cell pellets are stored at -80.degree. C.
Expression of Recombinant Proteins Using Baculovirus
[0382] Recombinant proteins are expressed using baculovirus in Sf21
fall army worm ovarian cells. Recombinant baculoviruses are
prepared using the Bac-to-Bac system (Invitrogen) per the
manufacturer's instructions. Proteins are expressed on the large
scale in Sf900 .mu.l serum-free medium (Invitrogen) in a 10 L
bioreactor tank (27.degree. C., 130 rpm, 50% dissolved oxygen for
48 hours).
3. Recombinant Protein Purification
[0383] Recombinant proteins are purified from E. coli and/or insect
cells using a variety of standard chromatography methods. Briefly,
cells are lysed using sonication or detergents. The insoluble
material is pelleted by centrifugation at 10,000.times.g for 15
minutes. The supernatant is applied to an appropriate affinity
column, e.g. His-tagged proteins are separated using a pre-packed
chelating sepharose column (Pharmacia) or GST-tagged proteins are
separated using a glutathione sepharose column (Pharmacia). After
using the affinity column, proteins are further separated using
various techniques, such as ion exchange chromatography (columns
from Pharmacia) to separate on the basis of electrical charge or
size exclusion chromatography (columns from Tosohaas) to separate
on the basis of molecular weight, size and shape.
[0384] Expression and purification of the protein are also achieved
using either a mammalian cell expression system or an insect cell
expression system. The pUB6/V5-His vector system (Invitrogen,
Calif.) 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.H is) sequence
for rapid purification on PROBOND resin (Invitrogen, Calif.).
Transformed cells are selected on media containing blasticidin.
[0385] Spodoptera frugiperda (Sf9) insect cells are infected with
recombinant Autographica californica nuclear polyhedrosis virus
(baculovirus). The polyhedrin gene is replaced with the cDNA by
homologous recombination and the polyhedrin promoter drives cDNA
transcription. The protein is synthesized as a fusion protein with
6.times. his which enables purification as described above.
Purified protein is used in the following activity and to make
antibodies
4. Chemical Synthesis of Peptides
[0386] Proteins or portions thereof may be produced not only by
recombinant methods, but also by using chemical methods well known
in the art. Solid phase peptide synthesis may be carried out in a
batchwise or continuous flow process, which sequentially adds
.alpha.-amino- and side chain-protected amino acid residues to an
insoluble polymeric support via a linker group. A linker group such
as methylamine-derivatized polyethylene glycol is attached to
poly(styrene-co-divinylbenzene) to form the support resin. The
amino acid residues are N-a-protected by acid labile Boc
(t-butyloxycarbonyl) or base-labile Fmoc
(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected
amino acid is coupled to the amine of the linker group to anchor
the residue to the solid phase support resin. Trifluoroacetic acid
or piperidine are used to remove the protecting group in the case
of Boc or Fmoc, respectively. Each additional amino acid is added
to the anchored residue using a coupling agent or pre-activated
amino acid derivative, and the resin is washed. The full length
peptide is synthesized by sequential deprotection, coupling of
derivitized amino acids, and washing with dichloromethane and/or
N,N-dimethylformamide. The peptide is cleaved between the peptide
carboxy terminus and the linker group to yield a peptide acid or
amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook,
San Diego Calif. pp. S1-S20). Automated synthesis may also be
carried out on machines such as the 431A peptide synthesizer (ABI).
A protein or portion thereof may be purified by preparative high
performance liquid chromatography and its composition confirmed by
amino acid analysis or by sequencing (Creighton (1984) Proteins,
Structures and Molecular Properties, W H Freeman, New York
N.Y.).
5. Antibody Development
Polyclonal Antibody Preparations:
[0387] Polyclonal antibodies against recombinant proteins are
raised in rabbits (Green Mountain Antibodies, Burlington, Vt.).
Briefly, two New Zealand rabbits are immunized with 0.1 mg of
antigen in complete Freund's adjuvant. Subsequent immunizations are
carried out using 0.05 mg of antigen in incomplete Freund's
adjuvant at days 14, 21 and 49. Bleeds are collected and screened
for recognition of the antigen by solid phase ELISA and western
blot analysis. The IgG fraction is separated by centrifugation at
20,000.times.g for 20 minutes followed by a 50% ammonium sulfate
cut. The pelleted protein is resuspended in 5 mM Tris and separated
by ion exchange chromatography. Fractions are pooled based on IgG
content. Antigen-specific antibody is affinity purified using
Pierce AminoLink resin coupled to the appropriate antigen.
Isolation of Antibody Fragments Directed Against PCASTs from a
Library of scFvs
[0388] Naturally occurring V-genes isolated from human PBLs are
constructed into a library of antibody fragments which contain
reactivities against PCAST to which the donor may or may not have
been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein
by reference in its entirety).
[0389] Rescue of the Library: A library of scFvs is constructed
from the RNA of human PBLs as described in PCT publication WO
92/01047. To rescue phage displaying antibody fragments,
approximately 109 E. coli harboring the phagemid are used to
inoculate 50 ml of 2.times.TY containing 1% glucose and 100.mu.g/ml
of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of 0.8 with
shaking. Five ml of this culture is used to innoculate 50 ml of
2.times.TY-AMP-GLU, 2.times.108 TU of delta gene 3 helper (M13
delta gene III, see PCT publication WO 92/01047) are added and the
culture incubated at 37.degree. C. for 45 minutes without shaking
and then at 37.degree. C. for 45 minutes with shaking. The culture
is centrifuged at 4000 r.p.m. for 10 min. and the pellet
resuspended in 2 liters of 2.times.TY containing 100.mu.g/ml
ampicillin and 50 ug/ml kanamycin and grown overnight. Phage are
prepared as described in PCT publication WO 92/01047.
[0390] M13 delta gene III is prepared as follows: M13 delta gene
III helper phage does not encode gene III protein, hence the
phage(mid) displaying antibody fragments have a greater avidity of
binding to antigen. Infectious M13 delta gene III particles are
made by growing the helper phage in cells harboring a pUC19
derivative supplying the wild type gene III protein during phage
morphogenesis. The culture is incubated for 1 hour at 37.degree. C.
without shaking and then for a further hour at 37.degree. C. with
shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),
resuspended in 300 ml 2.times.TY broth containing 100.mu.g
ampicillin/ml and 25.mu.g kanamycin/ml (2.times.TY-AMP-KAN) and
grown overnight, shaking at 37.degree. C. Phagre particles are
purified and concentrated from the culture medium by two
PEG-precipitations (3.sup.rd edition of Sambrook et al. (2001)),
resuspended in 2 ml PBS and passed through a 0.45.mu.m filter
(Minisart NML; Sartorius) to give a final concentration of
approximately 1013 transducing units/ml (ampicillin-resistant
clones).
[0391] Panning of the Library: Immunotubes (Nunc) are coated
overnight in PBS with 4 ml of either 100.mu.g/ml or 10.mu.g/ml of a
polypeptide of the present invention. Tubes are blocked with 2%
Marvel-PBS for 2 hours at 37.degree. C. and then washed 3 times in
PBS. Approximately 1013 TU of phage is applied to the tube and
incubated for 30 minutes at room temperature tumbling on an over
and under turntable and then left to stand for another 1.5 hours.
Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with
PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and
rotating 15 minutes on an under and over turntable after which the
solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl,
pH 7.4. Phages are then used to infect 10 ml of mid-log E. coli TG1
by incubating eluted phage with bacteria for 30 minutes at
37.degree. C. The E. coli are then plated on TYE plates containing
1% glucose and 100.mu.g/ml ampicillin. The resulting bacterial
library is then rescued with delta gene 3 helper phage as described
above to prepare phage for a subsequent round of selection. This
process is then repeated for a total of 4 rounds of affinity
purification with tube-washing increased to 20 times with PBS, 0.1%
Tween-20 and 20 times with PBS for rounds 3 and 4.
[0392] Characterization of Binders: Eluted phage from the 3rd and
4th rounds of selection are used to infect E. coli HB 2151 and
soluble scFv is produced (Marks, et al., 1991) from single colonies
for assay. ELISAs are performed with microtitre plates coated with
either 10.mu.g/ml of the polypeptide of the present invention in 50
mM bicarbonate pH 9.6. Clones positive in ELISA are further
characterized by PCR fingerprinting (see, e.g., PCT publication WO
92/01047) and then by sequencing.
Monoclonal Antibody Generation
[0393] i) Materials: 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. 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. 3) The
L-glutamine (200 mM, 100.times. solution), which is stored at
-20.degree. C. freezer, is thawed and warmed until completely in
solution. The L-glutamin is dispensed into media to supplement
growth. L-glutamin is added to 2 mM for myelomas, and 4 mM for
hybridoma media. Further the Penicillin, Streptomycin, Amphotericin
(antibacterial-antifungal stored at -20.degree. C.) is thawed and
added to Cell Mab Media to 1%. 4) Myeloma growth media is Cell Mab
Media (Cell Mab Media, Quantum Yield from BD is stored in the
refrigerator at 4.degree. C. in the dark) which are added
L-glntamine to 2 mM and antibiotic/antimycotic solution to 1% and
is called CMNS. 5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.). 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). 7)
Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1.times.)
stored in the refrigerator at 4.degree. C. 8) Clonal cell medium D
(Stemcell, Vancouver) contains HAT and methyl cellulose for
semi-solid direct cloning from the fusion. This comes in 90 ml
bottles with a CoA and must be "melted at 37.degree. C. in a
waterbath in the morning of the day of the fusion. Loosen the cap
and leave in CO2 incubator to sufficiently gas the medium D and
bring the pH down. 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. 10) Origen HCF can be obtained directly from Igen and
is a cell supernatant produced from a macrophage-like cell-line. It
can be thawed and aliqouted to 15 ml tubes at 5 ml per tube and
stored frozen at -20.degree. C. Positive Hybridomas are fed HCF
through the first subcloning and are gradually weaned. It is not
necessary to continue to supplement unless you have a particularly
difficult hybridoma clone. This and other additives have been shown
to be more effective in promoting new hybridoma growth than
conventional feeder layers.
[0394] ii) Procedure
[0395] 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 5 ug of antigen in PBS i.p. or
i.v.
[0396] Myeloma cells are washed with 30 ml serum free media by
centrifugation at 500 g at 4.degree. C. for 5 minutes. Viable cell
density is determined in resuspended cells using hemocytometry and
vital stains. Cells resuspended in complete growth medium are
stored at 37.degree. C. during the preparation of splenocytes.
Meanwhile, to test aminopterin sensitivity, 1.times.10.sup.6
myeloma cells are transferred to a 15 ml conical tube and
centrifuged at 500 g at 4.degree. C. for 5 minutes. The resulting
pellet is resuspended in 15 ml of HAT media and cells plated at 2
drops/well on a 96 well plate.
[0397] 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.
[0398] Cells are harvested and washed by centrifugation (at 500 g
at 4.degree. C. for 5 minutes) with 30 ml of medium. Cells are
resuspended in 10 ml of medium and the density of viable cells
determined by hemocytometry using vital stains. The splenocytes are
mixed with myeloma cells at a ratio of 5:1 (spleen cells: myeloma
cells). Both the myeloma and spleen cells are washed 2 more times
with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12 minutes.
[0399] Supernatant is removed and cells are resuspended in 5 ml of
RPMI-CMNS and are pooled to fill volume to 30 ml and spin down as
before. Then, the pellet is broken up by gently tapping on the flow
hood surface and resuspended in 1 ml of BMB REG1500 (prewarmed to
37.degree. C.) dropwise with 1 cc needle over 1 minute.
[0400] RPMI-CMNS to the PEG cells and RPMI-CMNS are added to slowly
dilute out the PEG. Cells are centrifuged and diluted in 5 ml of
Complete media and 95 ml of Clonacell Medium D (HAT) media (with 5
ml of HCF). The cells are plated out 10 ml per small petri
plate.
[0401] Myeloma/HAT control. P is prepared as follows: dilute about
1000 P3X63 Ag8.653 myeloma cells into 1 ml of medium D and transfer
into a single well of a 24 well plate. Plates are placed in
incubator, with two plates inside of a large petri plate, with an
additional petri plate full of distilled water, for 10-18 days
under 5% CO2 overlay at 37.degree. C. Clones are picked from
semisolid agarose into 96 well plates containing 150-200 ul of
CM-HT. Supernatants are screened 4 days later in ELISA, and
positive clones are moved up to 24 well plates. Heavy growth will
require changing of the media at day 8 (+/-150 ml). One should
further decrease the HCF to 0.5% (gradually--2%, then 1%, then
0.5%) in the cloning plates.
[0402] 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; 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
[0403] mRNA Expression Validation by Taqman
[0404] Expression of mRNA is quantitated by RT-PCR using
TaqMan.RTM. technology. The Taqman system couples a 5' fluorogenic
nuclease assay with PCR for real time quantitation. A probe is used
to monitor the formation of the amplification product.
[0405] Total RNA is isolated from cancer model cell lines using the
RNEasy kit.RTM. (Qiagen) per manufacturer's instructions and
included DNase treatment. Normal human tissue RNAs are acquired
from commercial vendors (Ambion, Austin, Tex.; Stratagene, La
Jolla, Calif., BioChain Institute, Newington, N.H.) as are RNAs
from matched disease/normal tissues.
[0406] Target transcript sequences are identified for the
differentially expressed peptides by searching the BlastP database.
TaqMan assays (PCR primer/probe set) specific for those transcripts
are identified by searching the Celera Discovery System.TM. (CDS)
database. The assays are designed to span exon-exon borders and do
not amplify genomic DNA.
[0407] The TaqMan primers and probe sequences are as designed by
Applied Biosystems (AB) as part of the Assays on Demand.TM. product
line or by custom design through the AB Assays by Design.sup.SM
service.
[0408] RT-PCR is accomplished using AmpliTaqGold and MultiScribe
reverse transcriptase in the One Step RT-PCR Master Mix reagent kit
(AB) according to the manufacturer's instructions. Probe and primer
concentrations are 250 nM and 900 nM, respectively, in a 15 .mu.l
reaction. For each experiment, a master mix of the above components
is made and aliquoted into each optical reaction well. Eight
nanograms of total RNA is the template. Each sample is assayed in
triplicate. Quantitative RT-PCR is performed using the ABI
Prism.RTM. 7900HT Sequence Detection System (SDS). Cycling
parameters follow: 48.degree. C. for 30 min. for one cycle;
95.degree. C. for 10 min for one cycle; 95.degree. C. for 15 sec,
60.degree. C. for 1 min. for 40 cycles.
[0409] The SDS software calculates the threshold cycle (C.sub.T)
for each reaction, and C.sub.T values are used to quantitate the
relative amount of starting template in the reaction. The C.sub.T
values for each set of three reactions are averaged for all
subsequent calculations
[0410] Data are analyzed for fold difference in expression using an
endogenous control for normalization and is expressed relative to a
normal tissue or normal cell line reference. The choice of
endogenous control is determined empirically by testing various
candidates against the cell line and tissue RNA panels and
selecting the one with the least variation in expression. Relative
changes in expression are quantitated using the
2.sup.-.DELTA..DELTA.CT Method. Livak, K. J. and Schmittgen, T. D.
(2001) Methods 25: 402-408; User bulletin #2: ABI Prism 7700
Sequence Detection System.
Protein Expression Validation by Western
[0411] Western blot analysis of target proteins is carried out
using whole cell extracts prepared from each of the pancreatic cell
lines. 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 as 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 are developed using Pierce
NBT.
Tissue Flow Cytometry Analysis
[0412] Post tissue processing, cells are sorted by flow cytometry
known in the art to enrich for epithelial cells. Alternatively,
cells isolated from pancreatic tissue are stained directly with
EpCAM (for epithelial cells) and the specific antibody to PCAST.
Cell numbers and viability are determined by PI exclusion (GUAVA)
for cells isolated from both normal and tumor pancreatic tissue. A
minimum of 0.5.times.106 cells are used for each analysis. Cells
are washed once with Flow Staining Buffer (0.5% BSA, 0.05% NaN3 in
D-PBS). To the cells, 20 ul of each antibody for PCAST are added.
An additional 5 ul of EpCAM antibody conjugated to APC are added
when unsorted cells are used in the experiment. Cells are incubated
with antibodies for 30 minutes at 4.degree. C. Cells are washed
once with Flow Staining Buffer and either analyzed immediately on
the LSR flow cytometry apparatus or fixed in 1% formaldehyde and
store at 4.degree. C. until LSR analysis. The antibodies used to
detect PCAST targets are all purchased by BD Biosciences and
PE-conjugated. The isotype control antibody used for these
experiments is PE-conjugated mouse IgG1k.
[0413] Tumors tissue from pancreatic cancer and from Xenograft
mouse pancreatic tissues are obtained and detected via Flow
Cytometry analysis. Tissue factor is shown to be overexpressed
among the pancreatic tumor tissues. Xenograft mouse is obtained by
using cultured human BXPC pancreatic cells (ATCC) that are injected
subcutaneously in BALB nude mouse. The pancreatic tumors are
obtained from the xenograft mouse for further FACS study.
7. Detection and Diagnosis of PCAST by Liquid Chromatography and
Mass Spectrometry (LC/MS)
[0414] The proteins secreted from cells can be prepared by
reduction, alkylation and cysteine-containing peptide enrichment of
concentrated conditioned media.
[0415] The differential expression of proteins in disease and
healthy samples are quantitated using Liquid Chromatography Mass
Spectrometry. The LC/MS spectra from disease and healthy (control)
samples are collected and processed using the following steps:
[0416] 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.
[0417] Similar experiments are repeated in order to increase the
confidence in detection of a peptide. These multiple acquisitions
are computationally aggregated into one experiment. The intensity
of a peptide present in both healthy and disease samples is used to
calculate the differential expression, or relative abundance, of
the peptide. The intensity of a peptide found exclusively in one
sample is used to calculate a theoretical expression ratio for that
peptide (singleton). Expression ratios are calculated for each
peptide of each replicate of the experiment
[0418] Statistical tests are performed to assess the robustness of
the data and select statistically significant differentials. To
assess general quality of the data, a) ensured that similar
features are detected in all replicates of the experiment; b)
number of matched ions between replicates; c) calculated the
overall pair wise intensity correlations between LC/MS maps of
process replicates to ensure that the expression ratios for
peptides are reproducible across the multiple replicates; d)
aggregated multiple experiments in order to compare the expression
ratio of a peptide in multiple diseases or disease samples.
8. Expression Validation by IHC in Tissue Sections
Tissue Sections
[0419] Paraffin embedded, fixed tissue sections are obtained from a
panel of normal tissues (Adrenal, Bladder, Lymphocytes, Bone
Marrow, Breast, Cerebellum, Cerebral cortex, Colon, Endothelium,
Eye, Fallopian tube, Small Intestine, Heart, Kidney [glomerulus,
tubule], Liver, Lung, Testes and Thyroid) as well as 30 tumor
samples with matched normal adjacent tissues from pancreas, lung,
colon, prostate, ovarian and breast. In addition, other tissues are
selected for testing such as bladder renal, hepatocellular,
pharyngeal and gastric tumor tissues.
[0420] Esophageal Replicate sections are also obtained from
numerous tumor types (Bladder Cancer, Lung Cancer, Breast Cancer,
Melanoma, Colon Cancer, Non-Hodgkins Lymphoma, Endometrial Cancer,
Ovarian Cancer, Head and Neck Cancer, Prostate Cancer, Leukemia
[ALL and CML] and Rectal Cancer). Sections are stained with
hemotoxylin and eosin and histologically examined to ensure
adequate representation of cell types in each tissue section.
[0421] An identical set of tissues will be obtained from frozen
sections and are used in those instances where it is not possible
to generate antibodies that are suitable for fixed sections. Frozen
tissues do not require an antigen retrieval step.
Paraffin Fixed Tissue Sections
[0422] Hemotoxylin and Eosin staining of paraffin embedded, fixed
tissue sections. Sections are deparaffinized in 3 changes of xylene
or xylene substitute for 2-5 minutes each. Sections are rinsed in 2
changes of absolute alcohol for 1-2 minutes each, in 95% alcohol
for 1 minute, followed by 80% alcohol for 1 minute. Slides are
washed well in running water and stained in Gill solution 3
hemotoxylin for 3 to 5 minutes. Following a vigorous wash in
running water for 1 minute, sections are stained in Scott's
solution for 2 minutes. Sections are washed for 1 min in running
water then conterstained in Eosin solution for 2-3 minutes
depending upon development of desired staining intensity. Following
a brief wash in 95% alcohol, sections are dehydrated in three
changes of absolute alcohol for 1 minute each and three changes of
xylene or xylene substitute for 1-2 minutes each. Slides are
coverslipped and stored for analysis.
Optimisation of Antibody Staining
[0423] For each antibody, a positive and negative control sample is
generated using data from the ICAT analysis of the pancreatic
cancer cell lines. Cell lines are selected that are known to
express low levels of a particular target as determined from the
ICAT data. This cell line is the reference normal control "Hs766T".
Similarly, a pancreatic tumour line is selected that is determined
to overexpress the target is selected.
Antigen Retrieval
[0424] Sections are deparaffinized and rehydrated by washing 3
times for 5 minutes in xylene; two times for 5 minutes in 100%
ethanol; two times for 5 minutes in 95% ethanol; and once for 5
minutes in 80% ethanol. Sections are then placed in endogenous
blocking solution (methanol+2% hydrogen peroxide) and incubated for
20 minutes at room temperature. Sections are rinsed twice for 5
minutes each in deionized water and twice for 5 minutes in
phosphate buffered saline (PBS), pH 7.4. Alternatively, where
necessary sections are deparrafinized by High Energy Antigen
Retrieval as follows: sections are washed three times for 5 minutes
in xylene; two times for 5 minutes in 100% ethanol; two times for 5
minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.
Sections are placed in a Coplin jar with dilute antigen retrieval
solution (10 mM citrate acid, pH 6). The Coplin jar containing
slides is placed in a vessel filled with water and microwaved on
high for 2-3 minutes (700 watt oven). Following cooling for 2-3
minutes, steps 3 and 4 are repeated four times (depending on
tissue), followed by cooling for 20 minutes at room temperature.
Sections are then rinsed in deionized water, two times for 5
minutes, placed in modified endogenous oxidation blocking solution
(PBS+2% hydrogen peroxide).and rinsed for 5 minutes in PBS.
Blocking and Staining
[0425] Sections are blocked with PBS/1% bovine serum albumin (PBA)
for 1 hour at room temperature followed by incubation in normal
serum diluted in PBA (2%) for 30 minutes at room temperature to
reduce non-specific binding of antibody. Incubations are performed
in a sealed humidity chamber to prevent air-drying of the tissue
sections. (The choice of blocking serum is the same as the species
of the biotinylated secondary antibody). Excess antibody is gently
removed by shaking and sections covered with primary antibody
diluted in PBA and incubated either at room temperature for 1 hour
or overnight at 4.degree. C. (Care is taken that the sections do
not touch during incubation). Sections are rinsed twice for 5
minutes in PBS, shaking gently. Excess PBS is removed by gently
shaking. The sections are covered with diluted biotinylated
secondary antibody in PBA and incubated for 30 minutes to 1 hour at
room temperature in the humidity chamber. If using a monoclonal
primary antibody, addition of 2% rat serum is used to decrease the
background on rat tissue sections. Following incubation, sections
are rinsed twice for 5 minutes in PBS, shaking gently. Excess PBS
is removed and sections incubated for 1 hour at room temperature in
Vectastain ABC reagent (as per kit instructions). The lid of the
humidity chamber is secured during all incunations to ensure a
moist environment. Sections are rinsed twice for 5 minutes in PBS,
shaking gently.
Develop and Counterstain
[0426] Sections are incubated for 2 minutes in peroxidase substrate
solution that is made up immediately prior to use as follows:
[0427] 10 mg diaminobenzidine (DAB) dissolved in 10 ml 50 mM sodium
phosphate buffer, pH 7.4. [0428] 12.5 microliters 3% CoCl2/NiCl2 in
deionized water [0429] 1.25 microliters hydrogen peroxide
[0430] Slides are rinsed well three times for 10 min in deionized
water and counterstained with 0.01% Light Green acidified with
0.01% acetic acid for 1-2 minutes depending on intensity of
counterstain desired.
[0431] Slides are rinsed three times for 5 minutes with deionized
water and dehydrated two times for 2 minutes in 95% ethanol; two
times for 2 minutes in 100% ethanol; and two times for 2 minutes in
xylene. Stained slides are mounted for visualization by
microscopy.
9. IHC Staining of Frozen Tissue Sections
[0432] 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 are stored at -80.degree. C. Blocks are
sectioned with a cryostat with care taken to avoid warming to
greater than -10.degree. C. Initially, the block is equilibrated in
the cryostat for about 5 minutes and 6-10 mm sections are cut
sequentially. Sections are allowed to dry for at least 30 minutes
at room temperature. Following drying, tissues are stored at
4.degree. C. for short term and -80.degree. C. for longterm
storage.
[0433] Sections are fixed by immersing in acetone jar for 1-2
minutes at room temperature, followed by drying at room
temperature. Primary antibody is added (diluted in 0.05 M
Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4], 2.5% serum)
directly to the sections by covering the section dropwise to cover
the tissue entirely. Binding is carried out by incubation a chamber
for 1 hour at room temperature. Without letting the sections dry
out, the secondary antibody (diluted in Tris-saline/2.5% serum) is
added in a similar manner to the primary and incubated as before
(at least 45 minutes). Following incubation, the sections are
washed gently in Tris-saline for 3-5 minutes and then in
Tris-saline/2.5% serum for another 3-5 minutes. If a biotinylated
primary antibody is used, in place of the secondary antibody
incubation, slides are covered with 100 ul of diluted alkaline
phosphatase conjugated streptavidin, incubated for 30 minutes at
room temperature and washed as above. Sections are incubated with
alkaline phosphatse substrate (1 mg/ml Fast Violet; 0.2 mg/ml
Napthol AS-MX phosphate in Tris-Saline pH 8.5) for 10-20 minutes
until the desired positive staining is achieved at which point the
reaction is stopped by washing twice with Tris-saline. Slides are
counter-stained with Mayer's hematoxylin for 30 seconds and washed
with tap water for 2-5 minutes. Sections are mounted with Mount
coverslips and mounting media.
10. Assay for Antibody Dependent Cellular Cytotoxicity
[0434] 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 being washed
three times with culture medium, cells are resuspended at
10.sup.5/ml, and 100 .mu.l/well are plated onto 96-well
round-bottom plates. A range of antibody concentrations are applied
to the wells, including an isotype control together with donor
peripheral blood mononuclear cells that are plated at a 100:1 and
50:1 ratio. After an 18-h incubation at 37.degree. C., supernatant
(30 .mu.l/well) is harvested and transferred onto Lumaplate 96
(Packard), dried, and read in a Packard Top-Count NXT .gamma.
counter. Each measurement is carried out in triplicate. Spontaneous
release is determined by cpm of tumor cells incubated with medium
and maximum release by cpm of tumor cells plus 1% Triton X-100
(Sigma). Specific lysis is defined as: % specific
lysis=[(experimental release-spontaneous release)/(maximum
release-spontaneous release)].times.100. The percent ADCC is
expressed as peak specific lysis postimmune subtracted by preimmune
percent specific lysis. A doubling of the ADCC to >20% is
considered significant.
11. Assay for Complement Dependent Cytotoxicity
[0435] Chromium release assays to assess complement-mediated
cytotoxicity are performed for each patient at various time points;
Dickler, M. N., Ragupathi, G., Liu, N. X., Musselli, C., Martino,
D. J., Miller, V. A., Kris, M. G., Brezicka, F. T., Livingston, P.
O. & Grant, S. C. (1999) Clin. Cancer Res. 5, 2773-2779.
Cultured tumor cells are washed in FCS-free media two times,
resuspended in 500 .mu.l of media, and incubated with 100 .mu.Ci
.sup.51Cr per 10 million cells for 2 h at 37.degree. C. The cells
are then shaken every 15 min for 2 h, washed 3 times in media to
achieve a concentration of approximately 20,000 cells/well, and
then plated in round-bottom plates. The plates contain either 50
.mu.l cells plus 50 .mu.l monoclonal antibody, 50 .mu.l cells plus
serum (pre- and post therapy), 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
[0436] Lipofectamine is purchased from Invitrogen (Carlsbad,
Calif.) and GeneSilencer from Gene Therapy Systems (San Diego,
Calif.). Synthetic siRNA oligonucleotides are from Dharmacon
(Lafayette, Colo.), Qiagen (Valencia, Calif.) or Ambion (Austin,
Tex.) RNeasy 96 Kit is purchased from Qiagen (Valencia, Calif.).
Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous One
Solution Cell Proliferation Assay are both purchased from Promega
(Madison, Wis.). Alamar Blue proliferation assay can be purchased
from Biosource (Camarillo, Calif.). Function blocking antibodies
are purchased from Chemicon (Temecula, Calif.), Biotrend (Cologne,
Germany) or Alexis Corporation (San Diego, Calif.). Cell invasion
assay kits from purchased from Chemicon (Temecula, Calif.).
RiboGreen RNA Quantitation Kit is purchased from Molecular probes
(Eugene, Oreg.).
RNAi
[0437] 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).
[0438] i) RNAi Transfections--Lipofectamine 2000
[0439] Transient transfections are carried out on sub-confluent
pancreatic cancer cell lines as previously described. Elbashir, S.
M. et al. (2001) Nature 411: 494-498; Caplen, N. J. et al. (2001)
Proc Natl Acad Sci USA 98: 9742-9747; Sharp, P. A. (2001) Genes and
Development 15: 485-490. Synthetic RNA to gene of interest or
scrambled negative control siRNA is transfected using lipofectamine
according to manufacturer's instructions. Cells are plated in 96
well plates in antibiotics free medium. The next day, the
transfection reagent and siRNA are prepared for transfections as
follows: Each 0.1-1 ul of lipofectamine 2000 and 10-150 mM siRNA
are resuspended 25 ul serum-free media and incubated at room
temperature for 5 minutes. After incubation, the diluted siRNA and
the lipofectamine 2000 are combined and incubated for 20 minutes at
room temperature. The cells are then washed and the combined
siRNA-Lipofectamine 2000 reagent added. After further 4 hours
incubation, 50 ul serum containing medium is added to each well. 1
and 4 days after transfection, expression of mRNA is quantitated by
RT-PCR using TaqMan technology and protein expression levels are
examined by flow cytometry. Apoptosis and proliferation assays are
performed daily using Apop-one homogeneous caspase-3/7 kit and
CellTiter 96 AQueous One Solution Cell Proliferation Assay (see
protocols below).
[0440] ii) RNAi Transfections--GeneSilencer
[0441] Transient transfections are carried out on sub-confluent
pancreatic cancer cell lines as previously described. Elbashir, S.
M. et al. (2001) Nature 411: 494-498; Caplen, N. J. et al. (2001)
Proc Natl Acad Sci USA 98: 9742-9747; Sharp, P. A. (2001) Genes and
Development 15: 485-490. Synthetic RNA to gene of interest or
scrambled negative control siRNA is transfected using GeneSilencer
according to manufacturer's instructions. Cells are plated in 96
well plates in antibiotics free medium. The next day, the
transfection reagent and the synthetic siRNA are prepared for
transfections as follows: predetermined amount of Gene Silencer is
diluted in serum-free media to a final volume of 20 ul per well.
After resuspending 10-150 mM siRNA in 20 ul serum-free media, the
reagents are combined and incubated at room temperature for 5-20
minutes. After incubation, the siRNA--Gene Silencer reagent is
added to each well and incubated in a 37.degree. C. incubator for 4
hours before an equal volume of serum containing media is added
back to the cultured cells. The cells are then incubated for 1 to 4
days before mRNA, protein expression and effects on apoptosis and
proliferation are examined.
Testing of Function Blocking Antibodies
[0442] Sub-confluent pancreatic cancer cell lines are serum-staved
overnight. The next day, serum-containing media is added back to
the cells in the presence of 5-50 ng/ml of function blocking
antibodies. After 2 or 5 days incubation at 37.degree. C. 5%
CO.sub.2, antibody binding is examined by flow cytometry and
apoptosis and proliferation are examined by using protocols
described below.
Apoptosis
[0443] Apoptosis assay is performed by using the Apop-one
homogeneous caspase-3/7 kit from Promega. Briefly, the caspase-3/7
substrate is thawed to room temperature and diluted 1:100 with
buffer. The diluted substrate is then added 1:1 to cells, control
or blank. The plates are then placed on a plate shaker for 30
minutes to 18 hours at 300-500 rpm. The fluorescence of each well
is then measured at using an excitation wavelength of 485+/-20 nm
and an emission wavelength of 530+/-25 nm.
Proliferation--MTS
[0444] Proliferation assay is performed by using the CellTiter 96
AQueous One Solution Cell Proliferation Assay kit from Promega. 20
ul of CellTiter 96 AQueous One Solution is added to 100 ul of
culture medium. The plates are then incubated for 1-4 hours at
37.degree. C. in a humidified 5% CO.sub.2 incubator. After
incubation, the change in absorbance is read at 490 nm.
Proliferation--Alamar Blue
[0445] Proliferation assay is performed by using the Alamar Blue
assay from Biosource. 10 ul of Alamar Blue reagent is added to 100
ul of cells in culture medium. The plates are then incubated for
1-4 hours at 37.degree. C. in a humidified 5% CO.sub.2 incubator.
After incubation, the change in fluorescence is measured at using
an excitation wavelength of 530 nm and an emission wavelength of
595 nm.
Cell Invasion
[0446] Cell invasion assay is performed by using the 96 well cell
invasion assay kit available from Chemicon. After the cell invasion
chamber plates are adjusted to room temperature, 100 ul serum-free
media is added to the interior of the inserts. 1-2 hours later,
cell suspensions of 1.times.10.sup.6 cells/ml are prepared. Media
is then carefully removed from the inserts and 100 ul of prepared
cells are added into the insert +/-0 to 50 ng function blocking
antibodies. The cells are pre-incubated for 15 minutes at
37.degree. C. before 150 ul of media containing 10% FBS is added to
the lower chamber. The cells are then incubated for 48 hours at
37.degree. C. After incubation, the cells from the top side of the
insert are discarded and the invasion chamber plates are then
placed on a new 96-well feeder tray containing 150 ul of pre-warmed
cell detachment solution in the wells. The plates are incubated for
30 minutes at 37.degree. C. and are periodically shaken. Lysis
buffer/dye solution (4 ul CyQuant Dye/300 ul 4.times. lysis buffer)
is prepared and added to each well of dissociation buffer/cells on
feeder tray. The plates are incubated for 15 minutes at room
temperature before 150 ul is transferred to a new 96-well plate.
Fluorescence of invading cells is then read at 480 excitation and
520 emission.
Receptor Internalization
[0447] 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 secreted target
of interest is then added at a pre-determined concentration in
prewarmed DMEM to the wells. The cells are then incubated for
various times at 37.degree. C. and reactions are stopped by
removing the media and fixing the cells in 3.7% formaldehyde/TBS
for 5 min at room temperature. The cells are then washed three
times with TBS and nonspecific binding blocked with TBS containing
1% BSA for 45 min at room temperature. The first antibody is added
at a pre-determined dilution in TBS/BSA for 1 h at room
temperature. Three washes with TBS followed, and cells are briefly
reblocked for 15 min at room temperature. Incubation with goat
anti-mouse conjugated alkaline phosphatase (Bio-Rad) diluted 1:1000
in TBS/BSA is carried out for 1 h at room temperature. The cells
are washed three times with TBS and a colorimetric alkaline
phosphatase substrate is added. When the adequate color change is
reached, 100-11 samples are taken for colorimetric readings.
mRNA Expression
[0448] Expression of mRNA is quantitated by RT-PCR using
TaqMan.RTM. technology. Total RNA is isolated from cancer model
cell lines using the RNEasy 96 kit (Qiagen) per manufacturer's
instructions and included DNase treatment. Target transcript
sequences are identified for the differentially expressed peptides
by searching the BlastP database. TaqMan assays (PCR primer/probe
set) specific for those transcripts are identified by searching the
Celera Discovery System.TM. (CDS) database. The assays are designed
to span exon-exon borders and do not amplify genomic DNA. The
TaqMan primers and probe sequences are as designed by Applied
Biosystems (AB) as part of the Assays on Demand.TM. product line or
by custom design through the AB Assays by Design.sup.SM service.
RT-PCR is accomplished using AmpliTaqGold and MultiScribe reverse
transcriptase in the One Step RT-PCR Master Mix reagent kit (AB)
according to the manufacturers instructions. Probe and primer
concentrations are 900 nM and 250 nM, respectively, in a 25 .mu.l
reaction. For each experiment, a master mix of the above components
is made and aliquoted into each optical reaction well. 5 ul of
total RNA is the template. Each sample is assayed in triplicate.
Quantitative RT-PCR is performed using the ABI Prism.RTM. 7900HT
Sequence Detection System (SDS). Cycling parameters follow:
48.degree. for 30 min. for one cycle; 95.degree. C. for 10 min for
one cycle; 95.degree. C. for 15 sec, 60.degree. C. for 1 min. for
40 cycles.
[0449] The SDS software calculates the threshold cycle (C.sub.T)
for each reaction, and C.sub.T values are used to quantitate the
relative amount of starting template in the reaction. The C.sub.T
values for each set of three reactions are averaged for all
subsequent calculations.
[0450] Total RNA is quantitated by using RiboGreen RNA Quantitation
Kit according to manufacturer's instructions and the % mRNA
expression is calculated using total RNA for normalization. %
knockdown is then calculated relative to the no addition
control.
13. In Vivo Studies by Using Antibodies
[0451] Treatment of Pancreatic Cancer Cells with Monoclonal
Antibodies.
[0452] Pancreatic cancer cells are seeded at a density of
4.times.10.sup.4 cells per well in 96-well microtiter plates and
allowed to adhere for 2 hours. The cells are then treated with
different concentrations of anti-PCAST 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 PCAST protein with
monoclonal antibodies.
[0453] NIH 3T3 Expressing PCAST Protein are Treated with Different
Concentrations of anti-PCAST MAbs. Cell Growth Inhibition is
Monitored.
In Vivo Treatment of NIH 3T3 Cells Overexpressing PCAST with
Anti-PCAST Monoclonal Antibodies.
[0454] NIH 3T3 cells transfected with etiher a PCAST expression
plasmid or the neo-DHFR vector are injected into nu/nu (athymic)
mice subcutaneously at a dose of 10.sup.6 cells in 0.1 ml of
phosphate-buffered saline. On days 0, 1, 5 and every 4 days
thereafter, 100 mug (0.1 ml in PBS) of either an irrelevant or
anti-PCAST monoclonal antibody of the IG2A subclass is injected
intraperitoneally. Tumor occurrence and size are monitored for 1
month period of treatment.
[0455] 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 1
1
51121PRTHomo sapiens 1Tyr Leu Cys Ala Ser Arg Pro Asp Gly Ser Ser
Gly Asn Thr Ile Tyr1 5 10 15Phe Gly Glu Gly Ser202346PRTHomo
sapiens 2Met Ala Met Val Ser Glu Phe Leu Lys Gln Ala Trp Phe Ile
Glu Asn1 5 10 15Glu Glu Gln Glu Tyr Val Gln Thr Val Lys Ser Ser Lys
Gly Gly Pro20 25 30Gly Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro
Ser Ser Asp Val35 40 45Ala Ala Leu His Lys Ala Ile Met Val Lys Gly
Val Asp Glu Ala Thr50 55 60Ile Ile Asp Ile Leu Thr Lys Arg Asn Asn
Ala Gln Arg Gln Gln Ile65 70 75 80Lys Ala Ala Tyr Leu Gln Glu Thr
Gly Lys Pro Leu Asp Glu Thr Leu85 90 95Lys Lys Ala Leu Thr Gly His
Leu Glu Glu Val Val Leu Ala Leu Leu100 105 110Lys Thr Pro Ala Gln
Phe Asp Ala Asp Glu Leu Arg Ala Ala Met Lys115 120 125Gly Leu Gly
Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg130 135 140Thr
Asn Lys Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu145 150
155 160Lys Arg Asp Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp
Phe165 170 175Arg Asn Ala Leu Leu Ser Leu Ala Lys Gly Asp Arg Ser
Glu Asp Phe180 185 190Gly Val Asn Glu Asp Leu Ala Asp Ser Asp Ala
Arg Ala Leu Tyr Glu195 200 205Ala Gly Glu Arg Arg Lys Gly Thr Asp
Val Asn Val Phe Asn Thr Ile210 215 220Leu Thr Thr Arg Ser Tyr Pro
Gln Leu Arg Arg Val Phe Gln Lys Tyr225 230 235 240Thr Lys Tyr Ser
Lys His Asp Met Asn Lys Val Leu Asp Leu Glu Leu245 250 255Lys Gly
Asp Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys Ala Thr260 265
270Ser Lys Pro Ala Phe Phe Ala Glu Lys Leu His Gln Ala Met Lys
Gly275 280 285Val Gly Thr Arg His Lys Ala Leu Ile Arg Ile Met Val
Ser Arg Ser290 295 300Glu Ile Asp Met Asn Asp Ile Lys Ala Phe Tyr
Gln Lys Met Tyr Gly305 310 315 320Ile Ser Leu Cys Gln Ala Ile Leu
Asp Glu Thr Lys Gly Asp Tyr Glu325 330 335Lys Ile Leu Val Ala Leu
Cys Gly Gly Asn340 3453346PRTHomo sapiens 3Met Ala Met Val Ser Glu
Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn1 5 10 15Glu Glu Gln Glu Tyr
Val Gln Thr Val Lys Ser Ser Lys Gly Gly Pro20 25 30Gly Ser Ala Val
Ser Pro Tyr Pro Thr Phe Asn Pro Ser Ser Asp Val35 40 45Ala Ala Leu
His Lys Ala Ile Met Val Lys Gly Val Asp Glu Ala Thr50 55 60Ile Ile
Asp Ile Leu Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln Ile65 70 75
80Lys Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu85
90 95Lys Lys Ala Leu Thr Gly His Leu Glu Glu Val Val Leu Ala Leu
Leu100 105 110Lys Thr Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala
Ala Met Lys115 120 125Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu
Ile Leu Ala Ser Arg130 135 140Thr Asn Lys Glu Ile Arg Asp Ile Asn
Arg Val Tyr Arg Glu Glu Leu145 150 155 160Lys Arg Asp Leu Ala Lys
Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe165 170 175Arg Asn Ala Leu
Leu Ser Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe180 185 190Gly Val
Asn Glu Asp Leu Ala Asp Ser Asp Ala Arg Ala Leu Tyr Glu195 200
205Ala Gly Glu Arg Arg Lys Gly Thr Asp Val Asn Val Phe Asn Thr
Ile210 215 220Leu Thr Thr Arg Ser Tyr Pro Gln Leu Arg Arg Val Phe
Gln Lys Tyr225 230 235 240Thr Lys Tyr Ser Lys His Asp Met Asn Lys
Val Leu Asp Leu Glu Leu245 250 255Lys Gly Asp Ile Glu Lys Cys Leu
Thr Ala Ile Val Lys Cys Ala Thr260 265 270Ser Lys Pro Ala Phe Phe
Ala Glu Lys Leu His Gln Ala Met Lys Gly275 280 285Val Gly Thr Arg
His Lys Ala Leu Ile Arg Ile Met Val Ser Arg Ser290 295 300Glu Ile
Asp Met Asn Asp Ile Lys Ala Phe Tyr Gln Lys Met Tyr Gly305 310 315
320Ile Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr Lys Gly Asp Tyr
Glu325 330 335Lys Ile Leu Val Ala Leu Cys Gly Gly Asn340
3454359PRTHomo sapiens 4Met Ser Pro Arg Met Glu Cys Ser Asp Thr Phe
Ser Lys Met Ala Met1 5 10 15Val Ser Glu Phe Leu Lys Gln Ala Trp Phe
Ile Glu Asn Glu Glu Gln20 25 30Glu Tyr Val Gln Thr Val Lys Ser Ser
Lys Gly Gly Pro Gly Ser Ala35 40 45Val Ser Pro Tyr Pro Thr Phe Asn
Pro Ser Ser Asp Val Ala Ala Leu50 55 60His Lys Ala Ile Met Val Lys
Gly Val Asp Glu Ala Thr Ile Ile Asp65 70 75 80Ile Leu Thr Lys Arg
Asn Asn Ala Gln Arg Gln Gln Ile Lys Ala Ala85 90 95Tyr Leu Gln Glu
Thr Gly Lys Pro Leu Asp Glu Thr Leu Lys Lys Ala100 105 110Leu Thr
Gly His Leu Glu Glu Val Val Leu Ala Leu Leu Lys Thr Pro115 120
125Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala Ala Met Lys Gly Leu
Gly130 135 140Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg
Thr Asn Lys145 150 155 160Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg
Glu Glu Leu Lys Arg Asp165 170 175Leu Ala Lys Asp Ile Thr Ser Asp
Thr Ser Gly Asp Phe Arg Asn Ala180 185 190Leu Leu Ser Leu Ala Lys
Gly Asp Arg Ser Glu Asp Phe Gly Val Asn195 200 205Glu Asp Leu Ala
Asp Ser Asp Ala Arg Ala Leu Tyr Glu Ala Gly Glu210 215 220Arg Arg
Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile Leu Thr Thr225 230 235
240Arg Ser Tyr Pro Gln Leu Arg Arg Val Phe Gln Lys Tyr Thr Lys
Tyr245 250 255Ser Lys His Asp Met Asn Lys Val Leu Asp Leu Glu Leu
Lys Gly Asp260 265 270Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys
Ala Thr Ser Lys Pro275 280 285Ala Phe Phe Ala Glu Lys Leu His Gln
Ala Met Lys Gly Val Gly Thr290 295 300Arg His Lys Ala Leu Ile Arg
Ile Met Val Ser Arg Ser Glu Ile Asp305 310 315 320Met Asn Asp Ile
Lys Ala Phe Tyr Gln Lys Met Tyr Gly Ile Ser Leu325 330 335Cys Gln
Ala Ile Leu Asp Glu Thr Lys Gly Asp Tyr Glu Lys Ile Leu340 345
350Val Ala Leu Cys Gly Gly Asn3555346PRTHomo sapiens 5Met Ala Met
Val Ser Glu Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn1 5 10 15Glu Glu
Gln Glu Tyr Val Gln Thr Val Lys Ser Ser Lys Gly Gly Pro20 25 30Gly
Ser Ala Val Ser Pro Tyr Pro Thr Phe Asn Pro Ser Ser Asp Val35 40
45Ala Ala Leu His Lys Ala Ile Met Val Lys Gly Val Asp Glu Ala Thr50
55 60Ile Ile Asp Ile Leu Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln
Ile65 70 75 80Lys Ala Ala Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp
Glu Thr Leu85 90 95Lys Lys Ala Leu Thr Gly His Leu Glu Glu Val Val
Leu Ala Leu Leu100 105 110Lys Thr Pro Ala Gln Phe Asp Ala Asp Glu
Leu Arg Ala Ala Met Lys115 120 125Gly Leu Gly Thr Asp Glu Asp Thr
Leu Ile Glu Ile Leu Ala Ser Arg130 135 140Thr Asn Lys Glu Ile Arg
Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu145 150 155 160Lys Arg Asp
Leu Ala Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe165 170 175Arg
Asn Ala Leu Leu Ser Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe180 185
190Gly Val Asn Glu Asp Leu Ala Asp Ser Asp Ala Arg Ala Leu Tyr
Glu195 200 205Ala Gly Glu Arg Arg Lys Gly Thr Asp Val Asn Val Phe
Asn Thr Ile210 215 220Leu Thr Thr Arg Ser Tyr Pro Gln Leu Arg Arg
Val Phe Gln Lys Tyr225 230 235 240Thr Lys Tyr Ser Lys His Asp Met
Asn Lys Val Leu Asp Leu Glu Leu245 250 255Lys Gly Asp Ile Glu Lys
Cys Leu Thr Ala Ile Val Lys Cys Ala Thr260 265 270Ser Lys Pro Ala
Phe Phe Ala Glu Lys Leu His Gln Ala Met Lys Gly275 280 285Val Gly
Thr Arg His Lys Ala Leu Ile Arg Ile Met Val Ser Arg Ser290 295
300Glu Ile Asp Met Asn Asp Ile Lys Ala Phe Tyr Gln Lys Met Tyr
Gly305 310 315 320Ile Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr Lys
Gly Asp Tyr Glu325 330 335Lys Ile Leu Val Ala Leu Cys Gly Gly
Asn340 3456346PRTHomo sapiens 6Met Ala Met Val Ser Glu Phe Leu Lys
Gln Ala Trp Phe Ile Glu Asn1 5 10 15Glu Glu Gln Glu Tyr Val Gln Thr
Val Lys Ser Ser Lys Gly Gly Pro20 25 30Gly Ser Ala Val Ser Pro Tyr
Pro Thr Phe Asn Pro Ser Ser Asp Val35 40 45Ala Ala Leu His Lys Ala
Ile Met Val Lys Gly Val Asp Glu Ala Thr50 55 60Ile Ile Asp Ile Leu
Thr Lys Arg Asn Asn Ala Gln Arg Gln Gln Ile65 70 75 80Lys Ala Ala
Tyr Leu Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu85 90 95Lys Lys
Ala Leu Thr Gly His Leu Glu Glu Val Val Leu Ala Leu Leu100 105
110Lys Thr Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala Ala Met
Lys115 120 125Gly Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu
Ala Ser Arg130 135 140Thr Asn Lys Glu Ile Arg Asp Ile Asn Arg Val
Tyr Arg Glu Glu Leu145 150 155 160Lys Arg Asp Leu Ala Lys Asp Ile
Thr Ser Asp Thr Ser Gly Asp Phe165 170 175Arg Asn Ala Leu Leu Ser
Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe180 185 190Gly Val Asn Glu
Asp Leu Ala Asp Ser Asp Ala Arg Ala Leu Tyr Glu195 200 205Ala Gly
Glu Arg Arg Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile210 215
220Leu Thr Thr Arg Ser Tyr Pro Gln Leu Arg Arg Val Phe Gln Lys
Tyr225 230 235 240Thr Lys Tyr Ser Lys His Asp Met Asn Lys Val Leu
Asp Leu Glu Leu245 250 255Lys Gly Asp Ile Glu Lys Cys Leu Thr Ala
Ile Val Lys Cys Ala Thr260 265 270Ser Lys Pro Ala Phe Phe Ala Glu
Lys Leu His Gln Ala Met Lys Gly275 280 285Val Gly Thr Arg His Lys
Ala Leu Ile Arg Ile Met Val Ser Arg Ser290 295 300Glu Ile Asp Met
Asn Asp Ile Lys Ala Phe Tyr Gln Lys Met Tyr Gly305 310 315 320Ile
Ser Leu Cys Gln Ala Ile Leu Asp Glu Thr Lys Gly Asp Tyr Glu325 330
335Lys Ile Leu Val Ala Leu Cys Gly Gly Asn340 3457357PRTHomo
sapiens 7Met Asn Leu Ile Leu Arg Tyr Thr Phe Ser Lys Met Ala Met
Val Ser1 5 10 15Glu Phe Leu Lys Gln Ala Trp Phe Ile Glu Asn Glu Glu
Gln Glu Tyr20 25 30Val Gln Thr Val Lys Ser Ser Lys Gly Gly Pro Gly
Ser Ala Val Ser35 40 45Pro Tyr Pro Thr Phe Asn Pro Ser Ser Asp Val
Ala Ala Leu His Lys50 55 60Ala Ile Met Val Lys Gly Val Asp Glu Ala
Thr Ile Ile Asp Ile Leu65 70 75 80Thr Lys Arg Asn Asn Ala Gln Arg
Gln Gln Ile Lys Ala Ala Tyr Leu85 90 95Gln Glu Thr Gly Lys Pro Leu
Asp Glu Thr Leu Lys Lys Ala Leu Thr100 105 110Gly His Leu Glu Glu
Val Val Leu Ala Leu Leu Lys Thr Pro Ala Gln115 120 125Phe Asp Ala
Asp Glu Leu Arg Ala Ala Met Lys Gly Leu Gly Thr Asp130 135 140Glu
Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg Thr Asn Lys Glu Ile145 150
155 160Arg Asp Ile Asn Arg Val Tyr Arg Glu Glu Leu Lys Arg Asp Leu
Ala165 170 175Lys Asp Ile Thr Ser Asp Thr Ser Gly Asp Phe Arg Asn
Ala Leu Leu180 185 190Ser Leu Ala Lys Gly Asp Arg Ser Glu Asp Phe
Gly Val Asn Glu Asp195 200 205Leu Ala Asp Ser Asp Ala Arg Ala Leu
Tyr Glu Ala Gly Glu Arg Arg210 215 220Lys Gly Thr Asp Val Asn Val
Phe Asn Thr Ile Leu Thr Thr Arg Ser225 230 235 240Tyr Pro Gln Leu
Arg Arg Val Phe Gln Lys Tyr Thr Lys Tyr Ser Lys245 250 255His Asp
Met Asn Lys Val Leu Asp Leu Glu Leu Lys Gly Asp Ile Glu260 265
270Lys Cys Leu Thr Ala Ile Val Lys Cys Ala Thr Ser Lys Pro Ala
Phe275 280 285Phe Ala Glu Lys Leu His Gln Ala Met Lys Gly Val Gly
Thr Arg His290 295 300Lys Ala Leu Ile Arg Ile Met Val Ser Arg Ser
Glu Ile Asp Met Asn305 310 315 320Asp Ile Lys Ala Phe Tyr Gln Lys
Met Tyr Gly Ile Ser Leu Cys Gln325 330 335Ala Ile Leu Asp Glu Thr
Lys Gly Asp Tyr Glu Lys Ile Leu Val Ala340 345 350Leu Cys Gly Gly
Asn3558345PRTHomo sapiens 8Ala Met Val Ser Glu Phe Leu Lys Gln Ala
Trp Phe Ile Glu Asn Glu1 5 10 15Glu Gln Glu Tyr Val Gln Thr Val Lys
Ser Ser Lys Gly Gly Pro Gly20 25 30Ser Ala Val Ser Pro Tyr Pro Thr
Phe Asn Pro Ser Ser Asp Val Ala35 40 45Ala Leu His Lys Ala Ile Met
Val Lys Gly Val Asp Glu Ala Thr Ile50 55 60Ile Asp Ile Leu Thr Lys
Arg Asn Asn Ala Gln Arg Gln Gln Ile Lys65 70 75 80Ala Ala Tyr Leu
Gln Glu Thr Gly Lys Pro Leu Asp Glu Thr Leu Lys85 90 95Lys Ala Leu
Thr Gly His Leu Glu Glu Val Val Leu Ala Leu Leu Lys100 105 110Thr
Pro Ala Gln Phe Asp Ala Asp Glu Leu Arg Ala Ala Met Lys Gly115 120
125Leu Gly Thr Asp Glu Asp Thr Leu Ile Glu Ile Leu Ala Ser Arg
Thr130 135 140Asn Lys Glu Ile Arg Asp Ile Asn Arg Val Tyr Arg Glu
Glu Leu Lys145 150 155 160Arg Asp Leu Ala Lys Asp Ile Thr Ser Asp
Thr Ser Gly Asp Phe Arg165 170 175Asn Ala Leu Leu Ser Leu Ala Lys
Gly Asp Arg Ser Glu Asp Phe Gly180 185 190Val Asn Glu Asp Leu Ala
Asp Ser Asp Ala Arg Ala Leu Tyr Glu Ala195 200 205Gly Glu Arg Arg
Lys Gly Thr Asp Val Asn Val Phe Asn Thr Ile Leu210 215 220Thr Thr
Arg Ser Tyr Pro Gln Leu Arg Arg Val Phe Gln Lys Tyr Thr225 230 235
240Lys Tyr Ser Lys His Asp Met Asn Lys Val Leu Asp Leu Glu Leu
Lys245 250 255Gly Asp Ile Glu Lys Cys Leu Thr Ala Ile Val Lys Cys
Ala Thr Ser260 265 270Lys Pro Ala Phe Phe Ala Glu Lys Leu His Gln
Ala Met Lys Gly Val275 280 285Gly Thr Arg His Lys Ala Leu Ile Arg
Ile Met Val Ser Arg Ser Glu290 295 300Ile Asp Met Asn Asp Ile Lys
Ala Phe Tyr Gln Lys Met Tyr Gly Ile305 310 315 320Ser Leu Cys Gln
Ala Ile Leu Asp Glu Thr Lys Gly Asp Tyr Glu Lys325 330 335Ile Leu
Val Ala Leu Cys Gly Gly Asn340 3459593PRTHomo sapiens 9Met Trp Thr
Leu Val Ser Trp Val Ala Leu Thr Ala Gly Leu Val Ala1 5 10 15Gly Thr
Arg Cys Pro Asp Gly Gln Phe Cys Pro Val Ala Cys Cys Leu20 25 30Asp
Pro Gly Gly Ala Ser Tyr Ser Cys Cys Arg Pro Leu Leu Asp Lys35 40
45Trp Pro Thr Thr Leu Ser Arg His Leu Gly Gly Pro Cys Gln Val Asp50
55 60Ala His Cys Ser Ala Gly His Ser Cys Ile Phe Thr Val Ser Gly
Thr65 70 75 80Ser Ser Cys Cys Pro Phe Pro Glu Ala Val Ala Cys Gly
Asp Gly His85 90 95His Cys Cys Pro Arg Gly Phe His Cys Ser Ala Asp
Gly Arg Ser Cys100 105 110Phe Gln Arg Ser Gly Asn Asn Ser Val Gly
Ala Ile Gln Cys Pro Asp115 120 125Ser Gln Phe Glu Cys Pro Asp Phe
Ser Thr Cys Cys Val Met Val Asp130 135 140Gly Ser Trp Gly Cys Cys
Pro Met Pro Gln Ala Ser Cys Cys Glu Asp145 150 155 160Arg Val His
Cys Cys Pro His Gly Ala Phe Cys Asp Leu Val His
Thr165 170 175Arg Cys Ile Thr Pro Thr Gly Thr His Pro Leu Ala Lys
Lys Leu Pro180 185 190Ala Gln Arg Thr Asn Arg Ala Val Ala Leu Ser
Ser Ser Val Met Cys195 200 205Pro Asp Ala Arg Ser Arg Cys Pro Asp
Gly Ser Thr Cys Cys Glu Leu210 215 220Pro Ser Gly Lys Tyr Gly Cys
Cys Pro Met Pro Asn Ala Thr Cys Cys225 230 235 240Ser Asp His Leu
His Cys Cys Pro Gln Asp Thr Val Cys Asp Leu Ile245 250 255Gln Ser
Lys Cys Leu Ser Lys Glu Asn Ala Thr Thr Asp Leu Leu Thr260 265
270Lys Leu Pro Ala His Thr Val Gly Asp Val Lys Cys Asp Met Glu
Val275 280 285Ser Cys Pro Asp Gly Tyr Thr Cys Cys Arg Leu Gln Ser
Gly Ala Trp290 295 300Gly Cys Cys Pro Phe Thr Gln Ala Val Cys Cys
Glu Asp His Ile His305 310 315 320Cys Cys Pro Ala Gly Phe Thr Cys
Asp Thr Gln Lys Gly Thr Cys Glu325 330 335Gln Gly Pro His Gln Val
Pro Trp Met Glu Lys Ala Pro Ala His Leu340 345 350Ser Leu Pro Asp
Pro Gln Ala Leu Lys Arg Asp Val Pro Cys Asp Asn355 360 365Val Ser
Ser Cys Pro Ser Ser Asp Thr Cys Cys Gln Leu Thr Ser Gly370 375
380Glu Trp Gly Cys Cys Pro Ile Pro Glu Ala Val Cys Cys Ser Asp
His385 390 395 400Gln His Cys Cys Pro Gln Gly Tyr Thr Cys Val Ala
Glu Gly Gln Cys405 410 415Gln Arg Gly Ser Glu Ile Val Ala Gly Leu
Glu Lys Met Pro Ala Arg420 425 430Arg Ala Ser Leu Ser His Pro Arg
Asp Ile Gly Cys Asp Gln His Thr435 440 445Ser Cys Pro Val Gly Gly
Thr Cys Cys Pro Ser Leu Gly Gly Ser Trp450 455 460Ala Cys Cys Gln
Leu Pro His Ala Val Cys Cys Glu Asp Arg Gln His465 470 475 480Cys
Cys Pro Ala Gly Tyr Thr Cys Asn Val Lys Ala Arg Ser Cys Glu485 490
495Lys Glu Val Val Ser Ala Gln Pro Ala Thr Phe Leu Ala Arg Ser
Pro500 505 510His Val Gly Val Lys Asp Val Glu Cys Gly Glu Gly His
Phe Cys His515 520 525Asp Asn Gln Thr Cys Cys Arg Asp Asn Arg Gln
Gly Trp Ala Cys Cys530 535 540Pro Tyr Arg Gln Gly Val Cys Cys Ala
Asp Arg Arg His Cys Cys Pro545 550 555 560Ala Gly Phe Arg Cys Ala
Ala Arg Gly Thr Lys Cys Leu Arg Arg Glu565 570 575Ala Pro Arg Trp
Asp Ala Pro Leu Arg Asp Pro Ala Leu Arg Gln Leu580 585
590Leu10250PRTHomo sapiens 10Met Arg Ile Leu Gln Leu Ile Leu Leu
Ala Leu Ala Thr Gly Leu Val1 5 10 15Gly Gly Glu Thr Arg Ile Ile Lys
Gly Phe Glu Cys Lys Pro His Ser20 25 30Gln Pro Trp Gln Ala Ala Leu
Phe Glu Lys Thr Arg Leu Leu Cys Gly35 40 45Ala Thr Leu Ile Ala Pro
Arg Trp Leu Leu Thr Ala Ala His Cys Leu50 55 60Lys Pro Arg Tyr Ile
Val His Leu Gly Gln His Asn Leu Gln Lys Glu65 70 75 80Glu Gly Cys
Glu Gln Thr Arg Thr Ala Thr Glu Ser Phe Pro His Pro85 90 95Gly Phe
Asn Asn Ser Leu Pro Asn Lys Asp His Arg Asn Asp Ile Met100 105
110Leu Val Lys Met Ala Ser Pro Val Ser Ile Thr Trp Ala Val Arg
Pro115 120 125Leu Thr Leu Ser Ser Arg Cys Val Thr Ala Gly Thr Ser
Cys Leu Ile130 135 140Ser Gly Trp Gly Ser Thr Ser Ser Pro Gln Leu
Arg Leu Pro His Thr145 150 155 160Leu Arg Cys Ala Asn Ile Thr Ile
Ile Glu His Gln Lys Cys Glu Asn165 170 175Ala Tyr Pro Gly Asn Ile
Thr Asp Thr Met Val Cys Ala Ser Val Gln180 185 190Glu Gly Gly Lys
Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Val195 200 205Cys Asn
Gln Ser Leu Gln Gly Ile Ile Ser Trp Gly Gln Asp Pro Cys210 215
220Ala Ile Thr Arg Lys Pro Gly Val Tyr Thr Lys Val Cys Lys Tyr
Val225 230 235 240Asp Trp Ile Gln Glu Thr Met Lys Asn Asn245
25011381PRTHomo sapiens 11Met Ser Ser Arg Ile Ala Arg Ala Leu Ala
Leu Val Val Thr Leu Leu1 5 10 15His Leu Thr Arg Leu Ala Leu Ser Thr
Cys Pro Ala Ala Cys His Cys20 25 30Pro Leu Glu Ala Pro Lys Cys Ala
Pro Gly Val Gly Leu Val Arg Asp35 40 45Gly Cys Gly Cys Cys Lys Val
Cys Ala Lys Gln Leu Asn Glu Asp Cys50 55 60Ser Lys Thr Gln Pro Cys
Asp His Thr Lys Gly Leu Glu Cys Asn Phe65 70 75 80Gly Ala Ser Ser
Thr Ala Leu Lys Gly Ile Cys Arg Ala Gln Ser Glu85 90 95Gly Arg Pro
Cys Glu Tyr Asn Ser Arg Ile Tyr Gln Asn Gly Glu Ser100 105 110Phe
Gln Pro Asn Cys Lys His Gln Cys Thr Cys Ile Asp Gly Ala Val115 120
125Gly Cys Ile Pro Leu Cys Pro Gln Glu Leu Ser Leu Pro Asn Leu
Gly130 135 140Cys Pro Asn Pro Arg Leu Val Lys Val Thr Gly Gln Cys
Cys Glu Glu145 150 155 160Trp Val Cys Asp Glu Asp Ser Ile Lys Asp
Pro Met Glu Asp Gln Asp165 170 175Gly Leu Leu Gly Lys Glu Leu Gly
Phe Asp Ala Ser Glu Val Glu Leu180 185 190Thr Arg Asn Asn Glu Leu
Ile Ala Val Gly Lys Gly Ser Ser Leu Lys195 200 205Arg Leu Pro Val
Phe Gly Met Glu Pro Arg Ile Leu Tyr Asn Pro Leu210 215 220Gln Gly
Gln Lys Cys Ile Val Gln Thr Thr Ser Trp Ser Gln Cys Ser225 230 235
240Lys Thr Cys Gly Thr Gly Ile Ser Thr Arg Val Thr Asn Asp Asn
Pro245 250 255Glu Cys Arg Leu Val Lys Glu Thr Arg Ile Cys Glu Val
Arg Pro Cys260 265 270Gly Gln Pro Val Tyr Ser Ser Leu Lys Lys Gly
Lys Lys Cys Ser Lys275 280 285Thr Lys Lys Ser Pro Glu Pro Val Arg
Phe Thr Tyr Ala Gly Cys Leu290 295 300Ser Val Lys Lys Tyr Arg Pro
Lys Tyr Cys Gly Ser Cys Val Asp Gly305 310 315 320Arg Cys Cys Thr
Pro Gln Leu Thr Arg Thr Val Lys Met Arg Phe Arg325 330 335Cys Glu
Asp Gly Glu Thr Phe Ser Lys Asn Val Met Met Ile Gln Ser340 345
350Cys Lys Cys Asn Tyr Asn Cys Pro His Ala Asn Glu Ala Ala Phe
Pro355 360 365Phe Tyr Arg Leu Phe Asn Asp Ile His Lys Phe Arg
Asp370 375 38012258PRTHomo sapiens 12Met Leu Pro Leu Cys Leu Val
Ala Ala Leu Leu Leu Ala Ala Gly Pro1 5 10 15Gly Pro Ser Leu Gly Asp
Glu Ala Ile His Cys Pro Pro Cys Ser Glu20 25 30Glu Lys Leu Ala Arg
Cys Arg Pro Pro Val Gly Cys Glu Glu Leu Val35 40 45Arg Glu Pro Gly
Cys Gly Cys Cys Ala Thr Cys Ala Leu Gly Leu Gly50 55 60Met Pro Cys
Gly Val Tyr Thr Pro Arg Cys Gly Ser Gly Leu Arg Cys65 70 75 80Tyr
Pro Pro Arg Gly Val Glu Lys Pro Leu His Thr Leu Met His Gly85 90
95Gln Gly Val Cys Met Glu Leu Ala Glu Ile Glu Ala Ile Gln Glu
Ser100 105 110Leu Gln Pro Ser Asp Lys Asp Glu Gly Asp His Pro Asn
Asn Ser Phe115 120 125Ser Pro Cys Ser Ala His Asp Arg Arg Cys Leu
Gln Lys His Phe Ala130 135 140Lys Ile Arg Asp Arg Ser Thr Ser Gly
Gly Lys Met Lys Val Asn Gly145 150 155 160Ala Pro Arg Glu Asp Ala
Arg Pro Val Pro Gln Gly Ser Cys Gln Ser165 170 175Glu Leu His Arg
Ala Leu Glu Arg Leu Ala Ala Ser Gln Ser Arg Thr180 185 190His Glu
Asp Leu Tyr Ile Ile Pro Ile Pro Asn Cys Asp Arg Asn Gly195 200
205Asn Phe His Pro Lys Gln Cys His Pro Ala Leu Asp Gly Gln Arg
Gly210 215 220Lys Cys Trp Cys Val Asp Arg Lys Thr Gly Val Lys Leu
Pro Gly Gly225 230 235 240Leu Glu Pro Lys Gly Glu Leu Asp Cys His
Gln Leu Ala Asp Ser Phe245 250 255Arg Glu13258PRTHomo sapiens 13Met
Leu Pro Leu Cys Leu Val Ala Ala Leu Leu Leu Ala Ala Gly Pro1 5 10
15Gly Pro Ser Leu Gly Asp Glu Ala Ile His Cys Pro Pro Cys Ser Glu20
25 30Glu Lys Leu Ala Arg Cys Arg Pro Pro Val Gly Cys Glu Glu Leu
Val35 40 45Arg Glu Ala Gly Cys Gly Cys Cys Ala Thr Cys Ala Leu Gly
Leu Gly50 55 60Met Pro Cys Gly Val Tyr Thr Pro Arg Cys Gly Ser Gly
Leu Arg Cys65 70 75 80Tyr Pro Pro Arg Gly Val Glu Lys Pro Leu His
Thr Leu Met His Gly85 90 95Gln Gly Val Cys Met Glu Leu Ala Glu Ile
Glu Ala Ile Gln Glu Ser100 105 110Leu Gln Pro Ser Asp Lys Asp Glu
Gly Asp His Pro Asn Asn Ser Phe115 120 125Ser Pro Cys Ser Ala His
Asp Arg Arg Cys Leu Gln Lys His Phe Ala130 135 140Lys Ile Arg Asp
Arg Ser Thr Ser Gly Gly Lys Met Lys Val Asn Gly145 150 155 160Ala
Pro Arg Glu Asp Ala Arg Pro Val Pro Gln Gly Ser Cys Gln Ser165 170
175Glu Leu His Arg Ala Leu Glu Arg Leu Ala Ala Ser Gln Ser Arg
Thr180 185 190His Glu Asp Leu Tyr Phe Ile Pro Ile Pro Asn Cys Asp
Arg Asn Gly195 200 205Asn Phe His Pro Lys Gln Cys His Pro Ala Leu
Asp Gly Gln Arg Gly210 215 220Lys Cys Trp Cys Val Asp Arg Lys Thr
Gly Val Lys Leu Pro Gly Gly225 230 235 240Leu Glu Pro Lys Gly Glu
Leu Asp Cys His Gln Leu Ala Asp Ser Phe245 250 255Arg
Glu141390PRTHomo sapiens 14Met Lys Ala Pro Ala Val Leu Ala Pro Gly
Ile Leu Val Leu Leu Phe1 5 10 15Thr Leu Val Gln Arg Ser Asn Gly Glu
Cys Lys Glu Ala Leu Ala Lys20 25 30Ser Glu Met Asn Val Asn Met Lys
Tyr Gln Leu Pro Asn Phe Thr Ala35 40 45Glu Thr Pro Ile Gln Asn Val
Ile Leu His Glu His His Ile Phe Leu50 55 60Gly Ala Thr Asn Tyr Ile
Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys65 70 75 80Val Ala Glu Tyr
Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe85 90 95Pro Cys Gln
Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp100 105 110Lys
Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp115 120
125Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg
His130 135 140Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu
Val His Cys145 150 155 160Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser
Gln Cys Pro Asp Cys Val165 170 175Val Ser Ala Leu Gly Ala Lys Val
Leu Ser Ser Val Lys Asp Arg Phe180 185 190Ile Asn Phe Phe Val Gly
Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp195 200 205His Pro Leu His
Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp210 215 220Gly Phe
Met Phe Leu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu225 230 235
240Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser
Asn245 250 255Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu
Asp Ala Gln260 265 270Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser
Ile Asn Ser Gly Leu275 280 285His Ser Tyr Met Glu Met Pro Leu Glu
Cys Ile Leu Thr Glu Lys Arg290 295 300Lys Lys Arg Ser Thr Lys Lys
Glu Val Phe Asn Ile Leu Gln Ala Ala305 310 315 320Tyr Val Ser Lys
Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser325 330 335Leu Asn
Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp340 345
350Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile
Lys355 360 365Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn
Asn Val Arg370 375 380Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu
His Cys Phe Asn Arg385 390 395 400Thr Leu Leu Arg Asn Ser Ser Gly
Cys Glu Ala Arg Arg Asp Glu Tyr405 410 415Arg Thr Glu Phe Thr Thr
Ala Leu Gln Arg Val Asp Leu Phe Met Gly420 425 430Gln Phe Ser Glu
Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly435 440 445Asp Leu
Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln450 455
460Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe
Leu465 470 475 480Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val
Glu His Thr Leu485 490 495Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr
Gly Lys Lys Ile Thr Lys500 505 510Ile Pro Leu Asn Gly Leu Gly Cys
Arg His Phe Gln Ser Cys Ser Gln515 520 525Cys Leu Ser Ala Pro Pro
Phe Val Gln Cys Gly Trp Cys His Asp Lys530 535 540Cys Val Arg Ser
Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile545 550 555 560Cys
Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu565 570
575Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg
Arg580 585 590Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu
Gly Asn Glu595 600 605Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met
Asn Thr Leu Lys Cys610 615 620Thr Val Gly Pro Ala Met Asn Lys His
Phe Asn Met Ser Ile Ile Ile625 630 635 640Ser Asn Gly His Gly Thr
Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp645 650 655Pro Val Ile Thr
Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly660 665 670Thr Leu
Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg675 680
685His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser
Asn690 695 700Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser
Thr Glu Phe705 710 715 720Ala Val Lys Leu Lys Ile Asp Leu Ala Asn
Arg Glu Thr Ser Ile Phe725 730 735Ser Tyr Arg Glu Asp Pro Ile Val
Tyr Glu Ile His Pro Thr Lys Ser740 745 750Phe Ile Ser Gly Gly Ser
Thr Ile Thr Gly Val Gly Lys Asn Leu Asn755 760 765Ser Val Ser Val
Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg770 775 780Asn Phe
Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys785 790 795
800Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu
Lys805 810 815Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys
Tyr Phe Asp820 825 830Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro
Phe Glu Lys Pro Val835 840 845Met Ile Ser Met Gly Asn Glu Asn Val
Leu Glu Ile Lys Gly Asn Asp850 855 860Ile Asp Pro Glu Ala Val Lys
Gly Glu Val Leu Lys Val Gly Asn Lys865 870 875 880Ser Cys Glu Asn
Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val885 890 895Pro Asn
Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys900 905
910Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro
Asp915 920 925Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile
Ser Thr Ala930 935 940Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu
Lys Lys Arg Lys Gln945 950 955 960Ile Lys Asp Leu Gly Ser Glu Leu
Val Arg Tyr Asp Ala Arg Val His965 970 975Thr Pro His Leu Asp Arg
Leu Val Ser Ala Arg Ser Val Ser Pro Thr980 985 990Thr Glu Met Val
Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro995 1000 1005Glu Asp
Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln Val1010 1015
1020Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly Asp
Ser1025 1030 1035 1040Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val
His Ile Asp Leu Ser1045 1050 1055Ala Leu Asn Pro Glu Leu Val Gln
Ala Val Gln His Val Val Ile Gly1060 1065 1070Pro Ser
Ser Leu Ile Val His Phe Asn Glu Val Ile Gly Arg Gly His1075 1080
1085Phe Gly Cys Val Tyr His Gly Thr Leu Leu Asp Asn Asp Gly Lys
Lys1090 1095 1100Ile His Cys Ala Val Lys Ser Leu Asn Arg Ile Thr
Asp Ile Gly Glu1105 1110 1115 1120Val Ser Gln Phe Leu Thr Glu Gly
Ile Ile Met Lys Asp Phe Ser His1125 1130 1135Pro Asn Val Leu Ser
Leu Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser1140 1145 1150Pro Leu
Val Val Leu Pro Tyr Met Lys His Gly Asp Leu Arg Asn Phe1155 1160
1165Ile Arg Asn Glu Thr His Asn Pro Thr Val Lys Asp Leu Ile Gly
Phe1170 1175 1180Gly Leu Gln Val Ala Lys Gly Met Lys Tyr Leu Ala
Ser Lys Lys Phe1185 1190 1195 1200Val His Arg Asp Leu Ala Ala Arg
Asn Cys Met Leu Asp Glu Lys Phe1205 1210 1215Thr Val Lys Val Ala
Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys1220 1225 1230Glu Tyr
Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys1235 1240
1245Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys
Ser1250 1255 1260Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu
Met Thr Arg Gly1265 1270 1275 1280Ala Pro Pro Tyr Pro Asp Val Asn
Thr Phe Asp Ile Thr Val Tyr Leu1285 1290 1295Leu Gln Gly Arg Arg
Leu Leu Gln Pro Glu Tyr Cys Pro Asp Pro Leu1300 1305 1310Tyr Glu
Val Met Leu Lys Cys Trp His Pro Lys Ala Glu Met Arg Pro1315 1320
1325Ser Phe Ser Glu Leu Val Ser Arg Ile Ser Ala Ile Phe Ser Thr
Phe1330 1335 1340Ile Gly Glu His Tyr Val His Val Asn Ala Thr Tyr
Val Asn Val Lys1345 1350 1355 1360Cys Val Ala Pro Tyr Pro Ser Leu
Leu Ser Ser Glu Asp Asn Ala Asp1365 1370 1375Asp Glu Val Asp Thr
Arg Pro Ala Ser Phe Trp Glu Thr Ser1380 1385 139015240PRTHomo
sapiens 15Met Thr Pro His Arg Leu Leu Pro Pro Leu Leu Leu Leu Leu
Ala Leu1 5 10 15Leu Leu Ala Ala Ser Pro Gly Gly Ala Leu Ala Arg Cys
Pro Gly Cys20 25 30Gly Gln Gly Val Gln Ala Gly Cys Pro Gly Gly Cys
Val Glu Glu Glu35 40 45Asp Gly Gly Ser Pro Ala Glu Gly Cys Ala Glu
Ala Glu Gly Cys Leu50 55 60Arg Arg Glu Gly Gln Glu Cys Gly Val Tyr
Thr Pro Asn Cys Ala Pro65 70 75 80Gly Leu Gln Cys His Pro Pro Lys
Asp Asp Glu Ala Pro Leu Arg Ala85 90 95Leu Leu Leu Gly Arg Gly Arg
Cys Leu Pro Ala Arg Ala Pro Ala Val100 105 110Ala Glu Glu Asn Pro
Lys Glu Ser Lys Pro Gln Ala Gly Thr Ala Arg115 120 125Pro Gln Asp
Val Asn Arg Arg Asp Gln Gln Arg Asn Pro Gly Thr Ser130 135 140Thr
Thr Pro Ser Gln Pro Asn Ser Ala Gly Val Gln Asp Thr Glu Met145 150
155 160Gly Pro Cys Arg Arg His Leu Asp Ser Val Leu Gln Gln Leu Gln
Thr165 170 175Glu Val Tyr Arg Gly Ala Gln Thr Leu Tyr Val Pro Asn
Cys Asp His180 185 190Arg Gly Phe Tyr Arg Lys Arg Gln Cys Arg Ser
Ser Gln Gly Gln Arg195 200 205Arg Gly Pro Cys Trp Cys Val Asp Arg
Met Gly Lys Ser Leu Pro Gly210 215 220Ser Pro Asp Gly Asn Gly Ser
Ser Ser Cys Pro Thr Gly Ser Ser Gly225 230 235 24016266PRTHomo
sapiens 16Met Met Ala Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val
Ala Met1 5 10 15Val Ala Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val
Ser Ala Thr20 25 30Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn
Leu Pro Pro Pro35 40 45Leu Gly Gly Ala Ala Gly His Pro Gly Ser Ala
Val Ser Ala Ala Pro50 55 60Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr
Gln Thr Ile Asp Asn Tyr65 70 75 80Gln Pro Tyr Pro Cys Ala Glu Asp
Glu Glu Cys Gly Thr Asp Glu Tyr85 90 95Cys Ala Ser Pro Thr Arg Gly
Gly Asp Ala Gly Val Gln Ile Cys Leu100 105 110Ala Cys Arg Lys Arg
Arg Lys Arg Cys Met Arg His Ala Met Cys Cys115 120 125Pro Gly Asn
Tyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp Gln Asn130 135 140His
Phe Arg Gly Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn145 150
155 160Asp His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser
Ser165 170 175Lys Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys
Leu Arg Ser180 185 190Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg
His Phe Trp Ser Lys195 200 205Ile Cys Lys Pro Val Leu Lys Glu Gly
Gln Val Cys Thr Lys His Arg210 215 220Arg Lys Gly Ser His Gly Leu
Glu Ile Phe Gln Arg Cys Tyr Cys Gly225 230 235 240Glu Gly Leu Ser
Cys Arg Ile Gln Lys Asp His His Gln Ala Ser Asn245 250 255Ser Ser
Arg Leu His Thr Cys Gln Arg His260 26517266PRTHomo sapiens 17Met
Met Ala Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val Ala Met1 5 10
15Val Ala Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr20
25 30Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro
Pro35 40 45Leu Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala
Ala Pro50 55 60Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile
Asp Asn Tyr65 70 75 80Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys
Gly Thr Asp Glu Tyr85 90 95Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala
Gly Val Gln Ile Cys Leu100 105 110Ala Cys Arg Lys Arg Arg Lys Arg
Cys Met Arg His Ala Met Cys Cys115 120 125Pro Gly Asn Tyr Cys Lys
Asn Gly Ile Cys Val Ser Ser Asp Gln Asn130 135 140His Phe Arg Gly
Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn145 150 155 160Asp
His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser165 170
175Lys Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys Leu Arg
Ser180 185 190Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe
Trp Ser Lys195 200 205Ile Cys Lys Pro Val Leu Lys Glu Gly Gln Val
Cys Thr Lys His Arg210 215 220Arg Lys Gly Ser His Gly Leu Glu Ile
Phe Gln Arg Cys Tyr Cys Gly225 230 235 240Glu Gly Leu Ser Cys Arg
Ile Gln Lys Asp His His Gln Ala Ser Asn245 250 255Ser Ser Arg Leu
His Thr Cys Gln Arg His260 265181179PRTHomo sapiens 18Met Ala Ala
Cys Gly Arg Val Arg Arg Met Phe Arg Leu Ser Ala Ala1 5 10 15Leu His
Leu Leu Leu Leu Phe Ala Ala Gly Ala Glu Lys Leu Pro Gly20 25 30Gln
Gly Val His Ser Gln Gly Gln Gly Pro Gly Ala Asn Phe Val Ser35 40
45Phe Val Gly Gln Ala Gly Gly Gly Gly Pro Ala Gly Gln Gln Leu Pro50
55 60Gln Leu Pro Gln Ser Ser Gln Leu Gln Gln Gln Gln Gln Gln Gln
Gln65 70 75 80Gln Gln Gln Gln Pro Gln Pro Pro Gln Pro Pro Phe Pro
Ala Gly Gly85 90 95Pro Pro Ala Arg Arg Gly Gly Ala Gly Ala Gly Gly
Gly Trp Lys Leu100 105 110Ala Glu Glu Glu Ser Cys Arg Glu Asp Val
Thr Arg Val Cys Pro Lys115 120 125His Thr Trp Ser Asn Asn Leu Ala
Val Leu Glu Cys Leu Gln Asp Val130 135 140Arg Glu Pro Glu Asn Glu
Ile Ser Ser Asp Cys Asn His Leu Leu Trp145 150 155 160Asn Tyr Lys
Leu Asn Leu Thr Thr Asp Pro Lys Phe Glu Ser Val Ala165 170 175Arg
Glu Val Cys Lys Ser Thr Ile Thr Glu Ile Lys Glu Cys Ala Asp180 185
190Glu Pro Val Gly Lys Gly Tyr Met Val Ser Cys Leu Val Asp His
Arg195 200 205Gly Asn Ile Thr Glu Tyr Gln Cys His Gln Tyr Ile Thr
Lys Met Thr210 215 220Ala Ile Ile Phe Ser Asp Tyr Arg Leu Ile Cys
Gly Phe Met Asp Asp225 230 235 240Cys Lys Asn Asp Ile Asn Ile Leu
Lys Cys Gly Ser Ile Arg Leu Gly245 250 255Glu Lys Asp Ala His Ser
Gln Gly Glu Val Val Ser Cys Leu Glu Lys260 265 270Gly Leu Val Lys
Glu Ala Glu Glu Arg Glu Pro Lys Ile Gln Val Ser275 280 285Glu Leu
Cys Lys Lys Ala Ile Leu Arg Val Ala Glu Leu Ser Ser Asp290 295
300Asp Phe His Leu Asp Arg His Leu Tyr Phe Ala Cys Arg Asp Asp
Arg305 310 315 320Glu Arg Phe Cys Glu Asn Thr Gln Ala Gly Glu Gly
Arg Val Tyr Lys325 330 335Cys Leu Phe Asn His Lys Phe Glu Glu Ser
Met Ser Glu Lys Cys Arg340 345 350Glu Ala Leu Thr Thr Arg Gln Lys
Leu Ile Ala Gln Asp Tyr Lys Val355 360 365Ser Tyr Ser Leu Ala Lys
Ser Cys Lys Ser Asp Leu Lys Lys Tyr Arg370 375 380Cys Asn Val Glu
Asn Leu Pro Arg Ser Arg Glu Ala Arg Leu Ser Tyr385 390 395 400Leu
Leu Met Cys Leu Glu Ser Ala Val His Arg Gly Arg Gln Val Ser405 410
415Ser Glu Cys Gln Gly Glu Met Leu Asp Tyr Arg Arg Met Leu Met
Glu420 425 430Asp Phe Ser Leu Ser Pro Glu Ile Ile Leu Ser Cys Arg
Gly Glu Ile435 440 445Glu His His Cys Ser Gly Leu His Arg Lys Gly
Arg Thr Leu His Cys450 455 460Leu Met Lys Val Val Arg Gly Glu Lys
Gly Asn Leu Gly Met Asn Cys465 470 475 480Gln Gln Ala Leu Gln Thr
Leu Ile Gln Glu Thr Asp Pro Gly Ala Asp485 490 495Tyr Arg Ile Asp
Arg Ala Leu Asn Glu Ala Cys Glu Ser Val Ile Gln500 505 510Thr Ala
Cys Lys His Ile Arg Ser Gly Asp Pro Met Ile Leu Ser Cys515 520
525Leu Met Glu His Leu Tyr Thr Glu Lys Met Val Glu Asp Cys Glu
His530 535 540Arg Leu Leu Glu Leu Gln Tyr Phe Ile Ser Arg Asp Trp
Lys Leu Asp545 550 555 560Pro Val Leu Tyr Arg Lys Cys Gln Gly Asp
Ala Ser Arg Leu Cys His565 570 575Thr His Gly Trp Asn Glu Thr Ser
Glu Phe Met Pro Gln Gly Ala Val580 585 590Phe Ser Cys Leu Tyr Arg
His Ala Tyr Arg Thr Glu Glu Gln Gly Arg595 600 605Arg Leu Ser Arg
Glu Cys Arg Ala Glu Val Gln Arg Ile Leu His Gln610 615 620Arg Ala
Met Asp Val Lys Leu Asp Pro Ala Leu Gln Asp Lys Cys Leu625 630 635
640Ile Asp Leu Gly Lys Trp Cys Ser Glu Lys Thr Glu Thr Gly Gln
Glu645 650 655Leu Glu Cys Leu Gln Asp His Leu Asp Asp Leu Val Val
Glu Cys Arg660 665 670Asp Ile Val Gly Asn Leu Thr Glu Leu Glu Ser
Glu Asp Ile Gln Ile675 680 685Glu Ala Leu Leu Met Arg Ala Cys Glu
Pro Ile Ile Gln Asn Phe Cys690 695 700His Asp Val Ala Asp Asn Gln
Ile Asp Ser Gly Asp Leu Met Glu Cys705 710 715 720Leu Ile Gln Asn
Lys His Gln Lys Asp Met Asn Glu Lys Cys Ala Ile725 730 735Gly Val
Thr His Phe Gln Leu Val Gln Met Lys Asp Phe Arg Phe Ser740 745
750Tyr Lys Phe Lys Met Ala Cys Lys Glu Asp Val Leu Lys Leu Cys
Pro755 760 765Asn Ile Lys Lys Lys Val Asp Val Val Ile Cys Leu Ser
Thr Thr Val770 775 780Arg Asn Asp Thr Leu Gln Glu Ala Lys Glu His
Arg Val Ser Leu Lys785 790 795 800Cys Arg Arg Gln Leu Arg Val Glu
Glu Leu Glu Met Thr Glu Asp Ile805 810 815Arg Leu Glu Pro Asp Leu
Tyr Glu Ala Cys Lys Ser Asp Ile Lys Asn820 825 830Phe Cys Ser Ala
Val Gln Tyr Gly Asn Ala Gln Ile Ile Glu Cys Leu835 840 845Lys Glu
Asn Lys Lys Gln Leu Ser Thr Arg Cys His Gln Lys Val Phe850 855
860Lys Leu Gln Glu Thr Glu Met Met Asp Pro Glu Leu Asp Tyr Thr
Leu865 870 875 880Met Arg Val Cys Lys Gln Met Ile Lys Arg Phe Cys
Pro Glu Ala Asp885 890 895Ser Lys Thr Met Leu Gln Cys Leu Lys Gln
Asn Lys Asn Ser Glu Leu900 905 910Met Asp Pro Lys Cys Lys Gln Met
Ile Thr Lys Arg Gln Ile Thr Gln915 920 925Asn Thr Asp Tyr Arg Leu
Asn Pro Met Leu Arg Lys Ala Cys Lys Ala930 935 940Asp Ile Pro Lys
Phe Cys His Gly Ile Leu Thr Lys Ala Lys Asp Asp945 950 955 960Ser
Glu Leu Glu Gly Gln Val Ile Ser Cys Leu Lys Leu Arg Tyr Ala965 970
975Asp Gln Arg Leu Ser Ser Asp Cys Glu Asp Gln Ile Arg Ile Ile
Ile980 985 990Gln Glu Ser Ala Leu Asp Tyr Arg Leu Asp Pro Gln Leu
Gln Leu His995 1000 1005Cys Ser Asp Glu Ile Ser Ser Leu Cys Ala Glu
Glu Ala Ala Ala Gln1010 1015 1020Glu Gln Thr Gly Gln Val Glu Glu
Cys Leu Lys Val Asn Leu Leu Lys1025 1030 1035 1040Ile Lys Thr Glu
Leu Cys Lys Lys Glu Val Leu Asn Met Leu Lys Glu1045 1050 1055Ser
Lys Ala Asp Ile Phe Val Asp Pro Val Leu His Thr Ala Cys Ala1060
1065 1070Leu Asp Ile Lys His His Cys Ala Ala Ile Thr Pro Gly Arg
Gly Arg1075 1080 1085Gln Met Ser Cys Leu Met Glu Ala Leu Glu Asp
Lys Arg Val Arg Leu1090 1095 1100Gln Pro Glu Cys Lys Lys Arg Leu
Asn Asp Arg Ile Glu Met Trp Ser1105 1110 1115 1120Tyr Ala Ala Lys
Val Ala Pro Ala Asp Gly Phe Ser Asp Leu Ala Met1125 1130 1135Gln
Val Met Thr Ser Pro Ser Lys Asn Tyr Ile Leu Ser Val Ile Ser1140
1145 1150Gly Ser Ile Cys Ile Leu Phe Leu Ile Gly Leu Met Cys Gly
Arg Ile1155 1160 1165Thr Lys Arg Val Thr Arg Glu Leu Lys Asp
Arg1170 1175191179PRTHomo sapiens 19Met Ala Ala Cys Gly Arg Val Arg
Arg Met Phe Arg Leu Ser Ala Ala1 5 10 15Leu His Leu Leu Leu Leu Phe
Ala Ala Gly Ala Glu Lys Leu Pro Gly20 25 30His Gly Val His Ser Gln
Gly Gln Gly Pro Gly Ala Asn Phe Val Ser35 40 45Phe Val Gly Gln Ala
Gly Gly Gly Gly Pro Ala Gly Gln Gln Leu Pro50 55 60Gln Leu Leu Gln
Ser Ser Gln Leu Gln Gln Gln Gln Gln Gln Gln Gln65 70 75 80Gln Gln
Gln Gln Leu Gln Pro Pro Gln Pro Pro Phe Pro Ala Gly Gly85 90 95Pro
Pro Ala Arg Arg Gly Gly Ala Gly Ala Gly Gly Gly Trp Lys Leu100 105
110Ala Glu Glu Glu Ser Cys Arg Glu Asp Val Thr Arg Val Cys Pro
Lys115 120 125His Thr Trp Ser Asn Asn Leu Ala Val Leu Glu Cys Leu
Gln Asp Val130 135 140Arg Glu Pro Glu Asn Glu Ile Ser Ser Asp Cys
Asn His Leu Leu Trp145 150 155 160Asn Tyr Lys Leu Asn Leu Thr Thr
Asp Pro Lys Phe Glu Ser Val Ala165 170 175Arg Glu Val Cys Lys Ser
Thr Ile Thr Glu Ile Lys Glu Cys Ala Asp180 185 190Glu Pro Val Gly
Lys Gly Tyr Met Val Ser Cys Leu Val Asp His Arg195 200 205Gly Asn
Ile Thr Glu Tyr Gln Cys His Gln Tyr Ile Thr Lys Met Thr210 215
220Ala Ile Ile Phe Ser Asp Tyr Arg Leu Ile Cys Gly Phe Met Asp
Asp225 230 235 240Cys Lys Asn Asp Ile Asn Ile Leu Lys Cys Gly Ser
Ile Arg Leu Gly245 250 255Glu Lys Asp Ala His Ser Gln Gly Glu Val
Val Ser Cys Leu Glu Lys260 265 270Gly Leu Val Lys Glu Ala Glu Glu
Arg Glu Pro Lys Ile Gln Val Ser275 280 285Glu Leu Cys Lys Lys Ala
Ile Leu Arg Val Ala Glu Leu Ser Ser Asp290 295 300Asp Phe His Leu
Asp Arg His Leu Tyr Phe Ala Cys Arg Asp Asp Arg305 310 315 320Glu
Arg Phe Cys Glu Asn Thr Gln Ala Gly Glu Gly Arg Val Tyr Lys325 330
335Cys Leu Phe Asn His Lys Phe Glu Glu Ser Met Ser Glu Lys Cys
Arg340 345 350Glu Ala Leu Thr Thr Arg Gln Lys Leu Ile Ala Gln Asp
Tyr Lys Val355 360 365Ser
Tyr Ser Leu Ala Lys Ser Cys Lys Ser Asp Leu Lys Lys Tyr Arg370 375
380Cys Asn Val Glu Asn Leu Pro Arg Ser Arg Glu Ala Arg Leu Ser
Tyr385 390 395 400Leu Leu Met Cys Leu Glu Ser Ala Val His Arg Gly
Arg Gln Val Ser405 410 415Ser Glu Cys Gln Gly Glu Met Leu Asp Tyr
Arg Arg Met Leu Met Glu420 425 430Asp Phe Ser Leu Ser Pro Glu Ile
Ile Leu Ser Cys Arg Gly Glu Ile435 440 445Glu His His Cys Ser Gly
Leu His Arg Lys Gly Arg Thr Leu His Cys450 455 460Leu Met Lys Val
Val Arg Gly Glu Lys Gly Asn Leu Gly Met Asn Cys465 470 475 480Gln
Gln Ala Leu Gln Thr Leu Ile Gln Glu Thr Asp Pro Gly Ala Asp485 490
495Tyr Arg Ile Asp Arg Ala Leu Asn Glu Ala Cys Glu Ser Val Ile
Gln500 505 510Thr Ala Cys Lys His Ile Arg Ser Gly Asp Pro Met Ile
Leu Ser Cys515 520 525Leu Met Glu His Leu Tyr Thr Glu Lys Met Val
Glu Asp Cys Glu His530 535 540Arg Leu Leu Glu Leu Gln Tyr Phe Ile
Ser Arg Asp Trp Lys Leu Asp545 550 555 560Pro Val Leu Tyr Arg Lys
Cys Gln Gly Asp Ala Ser Arg Leu Cys His565 570 575Thr His Gly Trp
Asn Glu Thr Ser Glu Phe Met Pro Gln Gly Ala Val580 585 590Phe Ser
Cys Leu Tyr Arg His Ala Tyr Arg Thr Glu Glu Gln Gly Arg595 600
605Arg Leu Ser Arg Glu Cys Arg Ala Glu Val Gln Arg Ile Leu His
Gln610 615 620Arg Ala Met Asp Val Lys Leu Asp Pro Ala Leu Gln Asp
Lys Cys Leu625 630 635 640Ile Asp Leu Gly Lys Trp Cys Ser Glu Lys
Thr Glu Thr Gly Gln Glu645 650 655Leu Glu Cys Leu Gln Asp His Leu
Asp Asp Leu Val Val Glu Cys Arg660 665 670Asp Ile Val Gly Asn Leu
Thr Glu Leu Glu Ser Glu Asp Ile Gln Ile675 680 685Glu Ala Leu Leu
Met Arg Ala Cys Glu Pro Ile Ile Gln Asn Phe Cys690 695 700His Asp
Val Ala Asp Asn Gln Ile Asp Ser Gly Asp Leu Met Glu Cys705 710 715
720Leu Ile Gln Asn Lys His Gln Lys Asp Met Asn Glu Lys Cys Ala
Ile725 730 735Gly Val Thr His Phe Gln Leu Val Gln Met Lys Asp Phe
Arg Phe Ser740 745 750Tyr Lys Phe Lys Met Ala Cys Lys Glu Asp Val
Leu Lys Leu Cys Pro755 760 765Asn Ile Lys Lys Lys Val Asp Val Val
Ile Cys Leu Ser Thr Thr Val770 775 780Arg Asn Asp Thr Leu Gln Glu
Ala Lys Glu His Arg Val Ser Leu Lys785 790 795 800Cys Arg Arg Gln
Leu Arg Val Glu Glu Leu Glu Met Thr Glu Asp Ile805 810 815Arg Leu
Glu Pro Asp Leu Tyr Glu Ala Cys Lys Ser Asp Ile Lys Asn820 825
830Phe Cys Ser Ala Val Gln Tyr Gly Asn Ala Gln Ile Ile Glu Cys
Leu835 840 845Lys Glu Asn Lys Lys Gln Leu Ser Thr Arg Cys His Gln
Lys Val Phe850 855 860Lys Leu Gln Glu Thr Glu Met Met Asp Pro Glu
Leu Asp Tyr Thr Leu865 870 875 880Met Arg Val Cys Lys Gln Met Ile
Lys Arg Phe Cys Pro Glu Ala Asp885 890 895Ser Lys Thr Met Leu Gln
Cys Leu Lys Gln Asn Lys Asn Ser Glu Leu900 905 910Met Asp Pro Lys
Cys Lys Gln Met Ile Thr Lys Arg Gln Ile Thr Gln915 920 925Asn Thr
Asp Tyr Arg Leu Asn Pro Met Leu Arg Lys Ala Cys Lys Ala930 935
940Asp Ile Pro Lys Phe Cys His Gly Ile Leu Thr Lys Ala Lys Asp
Asp945 950 955 960Ser Glu Leu Glu Gly Gln Val Ile Ser Cys Leu Lys
Leu Arg Tyr Ala965 970 975Asp Gln Arg Leu Ser Ser Asp Cys Glu Asp
Gln Ile Arg Ile Ile Ile980 985 990Gln Glu Ser Ala Leu Asp Tyr Arg
Leu Asp Pro Gln Leu Gln Leu His995 1000 1005Cys Ser Asp Glu Ile Ser
Ser Leu Cys Ala Glu Glu Ala Ala Ala Gln1010 1015 1020Glu Gln Thr
Gly Gln Val Glu Glu Cys Leu Lys Val Asn Leu Leu Lys1025 1030 1035
1040Ile Lys Thr Glu Leu Cys Lys Lys Glu Val Leu Asn Met Leu Lys
Glu1045 1050 1055Ser Lys Ala Asp Ile Phe Val Asp Pro Val Leu His
Thr Ala Cys Ala1060 1065 1070Leu Asp Ile Lys His His Cys Ala Ala
Ile Thr Pro Gly Arg Gly Arg1075 1080 1085Gln Met Ser Cys Leu Met
Glu Ala Leu Glu Asp Lys Arg Val Arg Leu1090 1095 1100Gln Pro Glu
Cys Lys Lys Arg Leu Asn Asp Arg Ile Glu Met Trp Ser1105 1110 1115
1120Tyr Ala Ala Lys Val Ala Pro Ala Asp Gly Phe Ser Asp Leu Ala
Met1125 1130 1135Gln Val Met Thr Ser Pro Ser Lys Asn Tyr Ile Leu
Ser Val Ile Ser1140 1145 1150Gly Ser Ile Cys Ile Leu Phe Leu Ile
Gly Leu Met Cys Gly Arg Ile1155 1160 1165Thr Lys Arg Val Thr Arg
Glu Leu Lys Asp Arg1170 1175201179PRTHomo sapiens 20Met Ala Ala Cys
Gly Arg Val Arg Arg Met Phe Arg Leu Ser Ala Ala1 5 10 15Leu His Leu
Leu Leu Leu Phe Ala Ala Gly Ala Glu Lys Leu Pro Gly20 25 30Gln Gly
Val His Ser Gln Gly Gln Gly Pro Gly Ala Asn Phe Val Ser35 40 45Phe
Val Gly Gln Ala Gly Gly Gly Gly Pro Ala Gly Gln Gln Leu Pro50 55
60Gln Leu Pro Gln Ser Ser Gln Leu Gln Gln Gln Gln Gln Gln Gln Gln65
70 75 80Gln Gln Gln Gln Pro Gln Pro Pro Gln Pro Pro Phe Pro Ala Gly
Gly85 90 95Pro Pro Ala Arg Arg Gly Gly Ala Gly Ala Gly Gly Gly Trp
Lys Leu100 105 110Ala Glu Glu Glu Ser Cys Arg Glu Asp Val Thr Arg
Val Cys Pro Lys115 120 125His Thr Trp Ser Asn Asn Leu Ala Val Leu
Glu Cys Leu Gln Asp Val130 135 140Arg Glu Pro Glu Asn Glu Ile Ser
Ser Asp Cys Asn His Leu Leu Trp145 150 155 160Asn Tyr Lys Leu Asn
Leu Thr Thr Asp Pro Lys Phe Glu Ser Val Ala165 170 175Arg Glu Val
Cys Lys Ser Thr Ile Thr Glu Ile Lys Glu Cys Ala Asp180 185 190Glu
Pro Val Gly Lys Gly Tyr Met Val Ser Cys Leu Val Asp His Arg195 200
205Gly Asn Ile Thr Glu Tyr Gln Cys His Gln Tyr Ile Thr Lys Met
Thr210 215 220Ala Ile Ile Phe Ser Asp Tyr Arg Leu Ile Cys Gly Phe
Met Asp Asp225 230 235 240Cys Lys Asn Asp Ile Asn Ile Leu Lys Cys
Gly Ser Ile Arg Leu Gly245 250 255Glu Lys Asp Ala His Ser Gln Gly
Glu Val Val Ser Cys Leu Glu Lys260 265 270Gly Leu Val Lys Glu Ala
Glu Glu Arg Glu Pro Lys Ile Gln Val Ser275 280 285Glu Leu Cys Lys
Lys Ala Ile Leu Arg Val Ala Glu Leu Ser Ser Asp290 295 300Asp Phe
His Leu Asp Arg His Leu Tyr Phe Ala Cys Arg Asp Asp Arg305 310 315
320Glu Arg Phe Cys Glu Asn Thr Gln Ala Gly Glu Gly Arg Val Tyr
Lys325 330 335Cys Leu Phe Asn His Lys Phe Glu Glu Ser Met Ser Glu
Lys Cys Arg340 345 350Glu Ala Leu Thr Thr Arg Gln Lys Leu Ile Ala
Gln Asp Tyr Lys Val355 360 365Ser Tyr Ser Leu Ala Lys Ser Cys Lys
Ser Asp Leu Lys Lys Tyr Arg370 375 380Cys Asn Val Glu Asn Leu Pro
Arg Ser Arg Glu Ala Arg Leu Ser Tyr385 390 395 400Leu Leu Met Cys
Leu Glu Ser Ala Val His Arg Gly Arg Gln Val Ser405 410 415Ser Glu
Cys Gln Gly Glu Met Leu Asp Tyr Arg Arg Met Leu Met Glu420 425
430Asp Phe Ser Leu Ser Pro Glu Ile Ile Leu Ser Cys Arg Gly Glu
Ile435 440 445Glu His His Cys Ser Gly Leu His Arg Lys Gly Arg Thr
Leu His Cys450 455 460Leu Met Lys Val Val Arg Gly Glu Lys Gly Asn
Leu Gly Met Asn Cys465 470 475 480Gln Gln Ala Leu Gln Thr Leu Ile
Gln Glu Thr Asp Pro Gly Ala Asp485 490 495Tyr Arg Ile Asp Arg Ala
Leu Asn Glu Ala Cys Glu Ser Val Ile Gln500 505 510Thr Ala Cys Lys
His Ile Arg Ser Gly Asp Pro Met Ile Leu Ser Cys515 520 525Leu Met
Glu His Leu Tyr Thr Glu Lys Met Val Glu Asp Cys Glu His530 535
540Arg Leu Leu Glu Leu Gln Tyr Phe Ile Ser Arg Asp Trp Lys Leu
Asp545 550 555 560Pro Val Leu Tyr Arg Lys Cys Gln Gly Asp Ala Ser
Arg Leu Cys His565 570 575Thr His Gly Trp Asn Glu Thr Ser Glu Phe
Met Pro Gln Gly Ala Val580 585 590Phe Ser Cys Leu Tyr Arg His Ala
Tyr Arg Thr Glu Glu Gln Gly Arg595 600 605Arg Leu Ser Arg Glu Cys
Arg Ala Glu Val Gln Arg Ile Leu His Gln610 615 620Arg Ala Met Asp
Val Lys Leu Asp Pro Ala Leu Gln Asp Lys Cys Leu625 630 635 640Ile
Asp Leu Gly Lys Trp Cys Ser Glu Lys Thr Glu Thr Gly Gln Glu645 650
655Leu Glu Cys Leu Gln Asp His Leu Asp Asp Leu Val Val Glu Cys
Arg660 665 670Asp Ile Val Gly Asn Leu Thr Glu Leu Glu Ser Glu Asp
Ile Gln Ile675 680 685Glu Ala Leu Leu Met Arg Ala Cys Glu Pro Ile
Ile Gln Asn Phe Cys690 695 700His Asp Val Ala Asp Asn Gln Ile Asp
Ser Gly Asp Leu Met Glu Cys705 710 715 720Leu Ile Gln Asn Lys His
Gln Lys Asp Met Asn Glu Lys Cys Ala Ile725 730 735Gly Val Thr His
Phe Gln Leu Val Gln Met Lys Asp Phe Arg Phe Ser740 745 750Tyr Lys
Phe Lys Met Ala Cys Lys Glu Asp Val Leu Lys Leu Cys Pro755 760
765Asn Ile Lys Lys Lys Val Asp Val Val Ile Cys Leu Ser Thr Thr
Val770 775 780Arg Asn Asp Thr Leu Gln Glu Ala Lys Glu His Arg Val
Ser Leu Lys785 790 795 800Cys Arg Arg Gln Leu Arg Val Glu Glu Leu
Glu Met Thr Glu Asp Ile805 810 815Arg Leu Glu Pro Asp Leu Tyr Glu
Ala Cys Lys Ser Asp Ile Lys Asn820 825 830Phe Cys Ser Ala Val Gln
Tyr Gly Asn Ala Gln Ile Ile Glu Cys Leu835 840 845Lys Glu Asn Lys
Lys Gln Leu Ser Thr Arg Cys His Gln Lys Val Phe850 855 860Lys Leu
Gln Glu Thr Glu Met Met Asp Pro Glu Leu Asp Tyr Thr Leu865 870 875
880Met Arg Val Cys Lys Gln Met Ile Lys Arg Phe Cys Pro Glu Ala
Asp885 890 895Ser Lys Thr Met Leu Gln Cys Leu Lys Gln Asn Lys Asn
Ser Glu Leu900 905 910Met Asp Pro Lys Cys Lys Gln Met Ile Thr Lys
Arg Gln Ile Thr Gln915 920 925Asn Thr Asp Tyr Arg Leu Asn Pro Met
Leu Arg Lys Ala Cys Lys Ala930 935 940Asp Ile Pro Lys Phe Cys His
Gly Ile Leu Thr Lys Ala Lys Asp Asp945 950 955 960Ser Glu Leu Glu
Gly Gln Val Ile Ser Cys Leu Lys Leu Arg Tyr Ala965 970 975Asp Gln
Arg Leu Ser Ser Asp Cys Glu Asp Gln Ile Arg Ile Ile Ile980 985
990Gln Glu Ser Ala Leu Asp Tyr Arg Leu Asp Pro Gln Leu Gln Leu
His995 1000 1005Cys Ser Asp Glu Ile Ser Ser Leu Cys Ala Glu Glu Ala
Ala Ala Gln1010 1015 1020Glu Gln Thr Gly Gln Val Glu Glu Cys Leu
Lys Val Asn Leu Leu Lys1025 1030 1035 1040Ile Lys Thr Glu Leu Cys
Lys Lys Glu Val Leu Asn Met Leu Lys Glu1045 1050 1055Ser Lys Ala
Asp Ile Phe Val Asp Pro Val Leu His Thr Ala Cys Ala1060 1065
1070Leu Asp Ile Lys His His Cys Ala Ala Ile Thr Pro Gly Arg Gly
Arg1075 1080 1085Gln Met Ser Cys Leu Met Glu Ala Leu Glu Asp Lys
Arg Val Arg Leu1090 1095 1100Gln Pro Glu Cys Lys Lys Arg Leu Asn
Asp Arg Ile Glu Met Trp Ser1105 1110 1115 1120Tyr Ala Ala Lys Val
Ala Pro Ala Asp Gly Phe Ser Asp Leu Ala Met1125 1130 1135Gln Val
Met Thr Ser Pro Ser Lys Asn Tyr Ile Leu Ser Val Ile Ser1140 1145
1150Gly Ser Ile Cys Ile Leu Phe Leu Ile Gly Leu Met Cys Gly Arg
Ile1155 1160 1165Thr Lys Arg Val Thr Arg Glu Leu Lys Asp Arg1170
11752197PRTHomo sapiens 21Met Cys Ser Ser Leu Glu Gln Ala Leu Ala
Val Leu Val Thr Thr Phe1 5 10 15His Lys Tyr Ser Cys Gln Glu Gly Asp
Lys Phe Lys Leu Ser Lys Gly20 25 30Glu Met Lys Glu Leu Leu His Lys
Glu Leu Pro Ser Phe Val Gly Glu35 40 45Lys Val Asp Glu Glu Gly Leu
Lys Lys Leu Met Gly Ser Leu Asp Glu50 55 60Asn Ser Asp Gln Gln Val
Asp Phe Gln Glu Tyr Ala Val Phe Leu Ala65 70 75 80Leu Ile Thr Val
Met Cys Asn Asp Phe Phe Gln Gly Cys Pro Asp Arg85 90
95Pro2298PRTHomo sapiens 22Met Met Cys Ser Ser Leu Glu Gln Ala Leu
Ala Val Leu Val Thr Thr1 5 10 15Phe His Lys Tyr Ser Cys Gln Glu Gly
Asp Lys Phe Lys Leu Ser Lys20 25 30Gly Glu Met Lys Glu Leu Leu His
Lys Glu Leu Pro Ser Phe Val Gly35 40 45Glu Lys Val Asp Glu Glu Gly
Leu Lys Lys Leu Met Gly Asn Leu Asp50 55 60Glu Asn Ser Asp Gln Gln
Val Asp Phe Gln Glu Tyr Ala Val Phe Leu65 70 75 80Ala Leu Ile Thr
Val Met Cys Asn Asp Phe Phe Gln Gly Cys Pro Asp85 90 95Arg
Pro23116PRTHomo sapiens 23Met Gln Arg Leu Arg Trp Leu Arg Asp Trp
Lys Ser Ser Gly Arg Gly1 5 10 15Leu Thr Ala Ala Lys Glu Pro Gly Ala
Arg Ser Ser Pro Leu Gln Ala20 25 30Met Arg Ile Leu Gln Leu Ile Leu
Leu Ala Leu Ala Thr Gly Leu Val35 40 45Gly Gly Glu Thr Arg Ile Ile
Lys Gly Phe Glu Cys Lys Pro His Ser50 55 60Gln Pro Trp Gln Ala Ala
Leu Phe Glu Lys Thr Arg Leu Leu Cys Gly65 70 75 80Ala Thr Leu Ile
Ala Pro Arg Trp Leu Leu Thr Ala Ala His Cys Leu85 90 95Lys Pro Trp
Pro Leu His Ser Ser Pro Gly Ala Ala Gln Pro Pro Glu100 105 110Gly
Gly Gly Leu1152463DNAHomo sapiens 24tacctctgtg ccagcaggcc
ggacgggagc tctggaaaca ccatatattt tggagaggga 60agt 63251669DNAHomo
sapiens 25actttgtttt tggacatagc tgagccatgt acttcaaaca gaaggcagcc
aattactaac 60ttctggttgc taggtgtggc ttcctttaaa atcctataaa atcagaagcc
caagtctcca 120ctgccagtgt gaaatcttca gagaagaatt tctctttagt
tctttgcaag aaggtagaga 180taaagggaag gtgtgggaag gacttgtgaa
atacatattc gaggaaaaac tatgcacaag 240gccgtgcatt taaaaataaa
ctccctaagg ctggggtgaa acctgctacg gtctgcgcaa 300gttgactgtt
aatgaatttg attctcagac actttttcaa aaatggcaat ggtatcagaa
360ttcctcaagc aggcctggtt tattgaaaat gaagagcagg aatatgttca
aactgtgaag 420tcatccaaag gtggtcccgg atcagcggtg agcccctatc
ctaccttcaa tccatcctcg 480gatgtcgctg ccttgcataa ggccataatg
gttaaaggtg tggatgaagc aaccatcatt 540gacattctaa ctaagcgaaa
caatgcacag cgtcaacaga tcaaagcagc atatctccag 600gaaacaggaa
agcccctgga tgaaacactg aagaaagccc ttacaggtca ccttgaggag
660gttgttttag ctctgctaaa aactccagcg caatttgatg ctgatgaact
tcgtgctgcc 720atgaagggcc ttggaactga tgaagatact ctaattgaga
ttttggcatc aagaactaac 780aaagaaatca gagacattaa cagggtctac
agagaggaac tgaagagaga tctggccaaa 840gacataacct cagacacatc
tggagatttt cggaacgctt tgctttctct tgctaagggt 900gaccgatctg
aggactttgg tgtgaatgaa gacttggctg attcagatgc cagggccttg
960tatgaagcag gagaaaggag aaaggggaca gacgtaaacg tgttcaatac
catccttacc 1020accagaagct atccacaact tcgcagagtg tttcagaaat
acaccaagta cagtaagcat 1080gacatgaaca aagttctgga cctggagttg
aaaggtgaca ttgagaaatg cctcacagct 1140atcgtgaagt gcgccacaag
caaaccagct ttctttgcag agaagcttca tcaagccatg 1200aaaggtgttg
gaactcgcca taaggcattg atcaggatta tggtttcccg ttctgaaatt
1260gacatgaatg atatcaaagc attctatcag aagatgtatg gtatctccct
ttgccaagcc 1320atcctggatg aaaccaaagg agattatgag aaaatcctgg
tggctctttg tggaggaaac 1380taaacattcc cttgatggtc tcaagctatg
atcagaagac tttaattata tattttcatc 1440ctataagctt aaataggaaa
gtttcttcaa caggattaca gtgtagctac ctacatgctg 1500aaaaatatag
cctttaaatc atttttatat tataactctg tataatagag ataagtccat
1560tttttaaaaa tgttttcccc aaaccataaa accctataca agttgttcta
gtaacaatac 1620atgagaaaga tgtctatgta gctgaaaata aaatgacgtc
acaagacaa 1669261669DNAHomo sapiens 26actttgtttt tggacatagc
tgagccatgt acttcaaaca gaaggcagcc aattactaac 60ttctggttgc taggtgtggc
ttcctttaaa atcctataaa
atcagaagcc caagtctcca 120ctgccagtgt gaaatcttca gagaagaatt
tctctttagt tctttgcaag aaggtagaga 180taaagggaag gtgtgggaag
gacttgtgaa atacatattc gaggaaaaac tatgcacaag 240gccgtgcatt
taaaaataaa ctccctaagg ctggggtgaa acctgctacg gtctgcgcaa
300gttgactgtt aatgaatttg attctcagac actttttcaa aaatggcaat
ggtatcagaa 360ttcctcaagc aggcctggtt tattgaaaat gaagagcagg
aatatgttca aactgtgaag 420tcatccaaag gtggtcccgg atcagcggtg
agcccctatc ctaccttcaa tccatcctcg 480gatgtcgctg ccttgcataa
ggccataatg gttaaaggtg tggatgaagc aaccatcatt 540gacattctaa
ctaagcgaaa caatgcacag cgtcaacaga tcaaagcagc atatctccag
600gaaacaggaa agcccctgga tgaaacactg aagaaagccc ttacaggtca
ccttgaggag 660gttgttttag ctctgctaaa aactccagcg caatttgatg
ctgatgaact tcgtgctgcc 720atgaagggcc ttggaactga tgaagatact
ctaattgaga ttttggcatc aagaactaac 780aaagaaatca gagacattaa
cagggtctac agagaggaac tgaagagaga tctggccaaa 840gacataacct
cagacacatc tggagatttt cggaacgctt tgctttctct tgctaagggt
900gaccgatctg aggactttgg tgtgaatgaa gacttggctg attcagatgc
cagggccttg 960tatgaagcag gagaaaggag aaaggggaca gacgtaaacg
tgttcaatac catccttacc 1020accagaagct atccacaact tcgcagagtg
tttcagaaat acaccaagta cagtaagcat 1080gacatgaaca aagttctgga
cctggagttg aaaggtgaca ttgagaaatg cctcacagct 1140atcgtgaagt
gcgccacaag caaaccagct ttctttgcag agaagcttca tcaagccatg
1200aaaggtgttg gaactcgcca taaggcattg atcaggatta tggtttcccg
ttctgaaatt 1260gacatgaatg atatcaaagc attctatcag aagatgtatg
gtatctccct ttgccaagcc 1320atcctggatg aaaccaaagg agattatgag
aaaatcctgg tggctctttg tggaggaaac 1380taaacattcc cttgatggtc
tcaagctatg atcagaagac tttaattata tattttcatc 1440ctataagctt
aaataggaaa gtttcttcaa caggattaca gtgtagctac ctacatgctg
1500aaaaatatag cctttaaatc atttttatat tataactctg tataatagag
ataagtccat 1560tttttaaaaa tgttttcccc aaaccataaa accctataca
agttgttcta gtaacaatac 1620atgagaaaga tgtctatgta gctgaaaata
aaatgacgtc acaagacaa 1669271560DNAHomo sapiens 27actttgtttt
tggacatagc tgagccatgt acttcaaaca gaaggcagcc aattactaac 60ttctggttgc
taggtgtggc ttcctttaaa atcctataaa atcagaagcc caagtctcca
120ctgccagtgt gaaatcttca gagaagaatt tctctttagt tctttgcaag
aaggtagaga 180taaagagtct tgttatgtcg cccaggatgg agtgtagtga
cactttttca aaaatggcaa 240tggtatcaga attcctcaag caggcctggt
ttattgaaaa tgaagagcag gaatatgttc 300aaactgtgaa gtcatccaaa
ggtggtcccg gatcagcggt gagcccctat cctaccttca 360atccatcctc
ggatgtcgct gccttgcata aggccataat ggttaaaggt gtggatgaag
420caaccatcat tgacattcta actaagcgaa acaatgcaca gcgtcaacag
atcaaagcag 480catatctcca ggaaacagga aagcccctgg atgaaacact
gaagaaagcc cttacaggtc 540accttgagga ggttgtttta gctctgctaa
aaactccagc gcaatttgat gctgatgaac 600ttcgtgctgc catgaagggc
cttggaactg atgaagatac tctaattgag attttggcat 660caagaactaa
caaagaaatc agagacatta acagggtcta cagagaggaa ctgaagagag
720atctggccaa agacataacc tcagacacat ctggagattt tcggaacgct
ttgctttctc 780ttgctaaggg tgaccgatct gaggactttg gtgtgaatga
agacttggct gattcagatg 840ccagggcctt gtatgaagca ggagaaagga
gaaaggggac agacgtaaac gtgttcaata 900ccatccttac caccagaagc
tatccacaac ttcgcagagt gtttcagaaa tacaccaagt 960acagtaagca
tgacatgaac aaagttctgg acctggagtt gaaaggtgac attgagaaat
1020gcctcacagc tatcgtgaag tgcgccacaa gcaaaccagc tttctttgca
gagaagcttc 1080atcaagccat gaaaggtgtt ggaactcgcc ataaggcatt
gatcaggatt atggtttccc 1140gttctgaaat tgacatgaat gatatcaaag
cattctatca gaagatgtat ggtatctccc 1200tttgccaagc catcctggat
gaaaccaaag gagattatga gaaaatcctg gtggctcttt 1260gtggaggaaa
ctaaacattc ccttgatggt ctcaagctat gatcagaaga ctttaattat
1320atattttcat cctataagct taaataggaa agtttcttca acaggattac
agtgtagcta 1380cctacatgct gaaaaatata gcctttaaat catttttata
ttataactct gtataataga 1440gataagtcca ttttttaaaa atgttttccc
caaaccataa aaccctatac aagttgttct 1500agtaacaata catgagaaag
atgtctatgt agctgaaaat aaaatgacgt cacaagacaa 1560284079DNAHomo
sapiens 28tggaaaacct aaaaagaagg gaaatacttt ctgggtagtg tcagatacat
tatcctaggg 60gaatgtcagt gaggccctcc aacagctatt ccacttgatt gttgcatgag
ctaatggcca 120taaaactcct tagaaaagca caaagcaaaa ctaaagaaga
gtatttacta gtgttggata 180tatttgtaaa agtgagatta caaatcatgt
atctggctat ttttttctta aacatgttcc 240ttcaagaatt tttctgttcg
ttcattttaa atatttatta aatgttctga tttcttatgt 300tcactgctag
ctaattaaca aggatggaat ttttcttgcc ttggttatat ctaaaagatt
360gtaaaaactt tgagaaagca atgttgccct ctttccacag gagtattttg
gtagctgtaa 420gagaatgcac attgcaaatg actcaaatgt ggtaaaatgt
tggtttcata attctgaaat 480ggcctcttcc ccaaaagtga cagtaacacc
ctagctccag gctcaaccac atccagcaca 540tagccaacat ttaacagatg
ttgacaaaat ggttaataat aatattatta aggaaccagc 600cagagtttca
tgcttattaa atactttttc aaccagaagg tctgcaaagg ttgatttctg
660aatatgacgt tagctctctc tagacctatt aatttacgac atttcaaatc
agaggtaaac 720cagcagacct attatttttc aatgataaac tataaacagt
tttttgaagt caactatttc 780cttttccttt aaaattggac agaatttcat
caggaagctc agaacaatcc aatttatata 840atgccttcct tcattatgct
aattttcttc atcttcatct acttgaagta atatttaaga 900ttggcacttc
agagctgtaa atggatctta gatgataatt acttcaacat tcttatttta
960taaacgaaga ccctctggag aagctaaatg tttttcttga ggtcagatcg
ccagataatc 1020aagaaccagt atacttcaat ttgtatttta tatcctatag
ttgtgggtag aaaatgttca 1080ttaaattagt gtagatatgt tttttaaaat
attagcaatg atacatgata ttcattcaat 1140aatacttatt aacatcctaa
attccaggca ttttgctagg gattggaagt acatacataa 1200tgaatattat
tatttgtatg ttagcacggg cctcatggcc tgctcctggg gaatataaac
1260aagccaatgg aaagtgataa tacagaaagg tggggctggt gtaggggtga
agtcaggatg 1320ctttgggaga gcatggaagg tcaccgaatc cagtgctgag
gaactaatga agggtttctg 1380aagaacgtga tgagatcaat gctgatgagt
cacttagaag tagcaattag ttaggcaaag 1440ggaagtgaat gtggaggagg
aacaagcatt ccaggcaaga agaacaccct atcgaaaagc 1500ctggaagcaa
aacattagtg aggctacctt tcataaattg ctttctgtaa gtcatgccat
1560tgtgtagtct taattgcttt ctctcaccag ggaaggtgtg ggaaggactt
gtgaaataca 1620tattcgagga aaaactatgc acaaggccgt gcatttaaaa
ataaactccc taaggctggg 1680gtgaaacctg ctacggtctg cgcaagttga
ctgttaatga atttgattct caggtgtgag 1740tgattaaaag aacactgatc
atgtcatttt ctttttggtc actaattccc tccctccctt 1800ctctttcttt
tcttttttct tttcttttct ttttctttct ttcttcccga cagaagaaag
1860actccatctc aaaaaaaaaa aaaaaaaaaa aagcaatcat taagcttttc
atctagccat 1920tacttttgta caggctctca ttaattccac atagatggtc
taatggtcta ctaaatagta 1980ttctaatcct ccatcttgtc tatctcttag
ccattctcca tgataaagga tgaatggtct 2040ttcggaaaca taaatgaaac
catgctgctt taaaaaaaca aacaaacaaa aagaaaccca 2100ttaatgtttt
cccaaagctt tgtggacaca ttgaagcttc ttgtttcagc ttctgccaac
2160atttctcgct ccagcaaact ttcttctgct tcccaaatat gacaagcttc
ttctttaact 2220tttctgccag gttaatcttg actcatttca agagaggctt
gggtcagcca gggcctactc 2280taagaagttt ctttagccct tatgttttcc
tatggagagc acacactagc tttaagtcct 2340gagggtactt aagatgatat
ttcaatgttt ttgtactctt agtgcttggc atatggtggg 2400tggtctacaa
atttagaaca atgtagtgaa ccacaaactc ctctcttcag cctgtgtgct
2460tacttgtagt agttgtctga tctctaaaat ttagaactga atatgccaag
aaagagttca 2520atttcagaga ggagggtatg gtttcatttt aagtataaaa
gcttccttta aaaaaaatta 2580cgtcttacat tttgttatgc tcctaagtgt
aattgatcct ggaaagtaag cgcaaggcta 2640ctctctaatg ctaactctta
tgtatgtaag aggtgaggaa ggagaggttg tgtgtggtgc 2700atttgttttg
taaaagcatc taactgtttt ctttccagac actttttcaa aaatggcaat
2760ggtatcagaa ttcctcaagc aggcctggtt tattgaaaat gaagagcagg
aatatgttca 2820aactgtgaag tcatccaaag gtggtcccgg atcagcggtg
agcccctatc ctaccttcaa 2880tccatcctcg gatgtcgctg ccttgcataa
ggccataatg gttaaaggtg tggatgaagc 2940aaccatcatt gacattctaa
ctaagcgaaa caatgcacag cgtcaacaga tcaaagcagc 3000atatctccag
gaaacaggaa agcccctgga tgaaacactg aagaaagccc ttacaggtca
3060ccttgaggag gttgttttag ctctgctaaa aactccagcg caatttgatg
ctgatgaact 3120tcgtgctgcc atgaagggcc ttggaactga tgaagatact
ctaattgaga ttttggcatc 3180aagaactaac aaagaaatca gagacattaa
cagggtctac agagaggaac tgaagagaga 3240tctggccaaa gacataacct
cagacacatc tggagatttt cggaacgctt tgctttctct 3300tgctaagggt
gaccgatctg aggactttgg tgtgaatgaa gacttggctg attcagatgc
3360cagggccttg tatgaagcag gagaaaggag aaaggggaca gacgtaaacg
tgttcaatac 3420catccttacc accagaagct atccacaact tcgcagagtg
tttcagaaat acaccaagta 3480cagtaagcat gacatgaaca aagttctgga
cctggagttg aaaggtgaca ttgagaaatg 3540cctcacagct atcgtgaagt
gcgccacaag caaaccagct ttctttgcag agaagcttca 3600tcaagccatg
aaaggtgttg gaactcgcca taaggcattg atcaggatta tggtttcccg
3660ttctgaaatt gacatgaatg atatcaaagc attctatcag aagatgtatg
gtatctccct 3720ttgccaagcc atcctggatg aaaccaaagg agattatgag
aaaatcctgg tggctctttg 3780tggaggaaac taaacattcc cttgatggtc
tcaagctatg atcagaagac tttaattata 3840tattttcatc ctataagctt
aaataggaaa gtttcttcaa caggattaca gtgtagctac 3900ctacatgctg
aaaaatatag cctttaaatc atttttatat tataactctg tataatagag
3960ataagtccat tttttaaaaa tgttttcccc aaaccataaa accctataca
agttgttcta 4020gtaacaatac atgagaaaga tgtctatgta gctgaaaata
aaatgacgtc acaagacaa 4079291669DNAHomo sapiens 29actttgtttt
tggacatagc tgagccatgt acttcaaaca gaaggcagcc aattactaac 60ttctggttgc
taggtgtggc ttcctttaaa atcctataaa atcagaagcc caagtctcca
120ctgccagtgt gaaatcttca gagaagaatt tctctttagt tctttgcaag
aaggtagaga 180taaagggaag gtgtgggaag gacttgtgaa atacatattc
gaggaaaaac tatgcacaag 240gccgtgcatt taaaaataaa ctccctaagg
ctggggtgaa acctgctacg gtctgcgcaa 300gttgactgtt aatgaatttg
attctcagac actttttcaa aaatggcaat ggtatcagaa 360ttcctcaagc
aggcctggtt tattgaaaat gaagagcagg aatatgttca aactgtgaag
420tcatccaaag gtggtcccgg atcagcggtg agcccctatc ctaccttcaa
tccatcctcg 480gatgtcgctg ccttgcataa ggccataatg gttaaaggtg
tggatgaagc aaccatcatt 540gacattctaa ctaagcgaaa caatgcacag
cgtcaacaga tcaaagcagc atatctccag 600gaaacaggaa agcccctgga
tgaaacactg aagaaagccc ttacaggtca ccttgaggag 660gttgttttag
ctctgctaaa aactccagcg caatttgatg ctgatgaact tcgtgctgcc
720atgaagggcc ttggaactga tgaagatact ctaattgaga ttttggcatc
aagaactaac 780aaagaaatca gagacattaa cagggtctac agagaggaac
tgaagagaga tctggccaaa 840gacataacct cagacacatc tggagatttt
cggaacgctt tgctttctct tgctaagggt 900gaccgatctg aggactttgg
tgtgaatgaa gacttggctg attcagatgc cagggccttg 960tatgaagcag
gagaaaggag aaaggggaca gacgtaaacg tgttcaatac catccttacc
1020accagaagct atccacaact tcgcagagtg tttcagaaat acaccaagta
cagtaagcat 1080gacatgaaca aagttctgga cctggagttg aaaggtgaca
ttgagaaatg cctcacagct 1140atcgtgaagt gcgccacaag caaaccagct
ttctttgcag agaagcttca tcaagccatg 1200aaaggtgttg gaactcgcca
taaggcattg atcaggatta tggtttcccg ttctgaaatt 1260gacatgaatg
atatcaaagc attctatcag aagatgtatg gtatctccct ttgccaagcc
1320atcctggatg aaaccaaagg agattatgag aaaatcctgg tggctctttg
tggaggaaac 1380taaacattcc cttgatggtc tcaagctatg atcagaagac
tttaattata tattttcatc 1440ctataagctt aaataggaaa gtttcttcaa
caggattaca gtgtagctac ctacatgctg 1500aaaaatatag cctttaaatc
atttttatat tataactctg tataatagag ataagtccat 1560tttttaaaaa
tgttttcccc aaaccataaa accctataca agttgttcta gtaacaatac
1620atgagaaaga tgtctatgta gctgaaaata aaatgacgtc acaagacaa
1669301671DNAHomo sapiens 30actttgtttt tggacatagc tgagccatgt
acttcaaaca gaaggcagcc aattactaac 60ttctggttgc taggtgtggc ttcctttaaa
atcctataaa atcagaagcc caagtctcca 120ctgccagtgt gaaatcttca
gagaagaatt tctctttagt tctttgcaag aaggtagaga 180taaagggaag
gtgtgggaag gacttgtgaa atacatattc gaggaaaaac tatgcacaag
240gccgtgcatt taaaaataaa ctccctaagg ctggggtgaa acctgctacg
gtctgcgcaa 300gttgactgtt aatgaatttg attctcaggt acactttttc
aaaaatggca atggtatcag 360aattcctcaa gcaggcctgg tttattgaaa
atgaagagca ggaatatgtt caaactgtga 420agtcatccaa aggtggtccc
ggatcagcgg tgagccccta tcctaccttc aatccatcct 480cggatgtcgc
tgccttgcat aaggccataa tggttaaagg tgtggatgaa gcaaccatca
540ttgacattct aactaagcga aacaatgcac agcgtcaaca gatcaaagca
gcatatctcc 600aggaaacagg aaagcccctg gatgaaacac tgaagaaagc
ccttacaggt caccttgagg 660aggttgtttt agctctgcta aaaactccag
cgcaatttga tgctgatgaa cttcgtgctg 720ccatgaaggg ccttggaact
gatgaagata ctctaattga gattttggca tcaagaacta 780acaaagaaat
cagagacatt aacagggtct acagagagga actgaagaga gatctggcca
840aagacataac ctcagacaca tctggagatt ttcggaacgc tttgctttct
cttgctaagg 900gtgaccgatc tgaggacttt ggtgtgaatg aagacttggc
tgattcagat gccagggcct 960tgtatgaagc aggagaaagg agaaagggga
cagacgtaaa cgtgttcaat accatcctta 1020ccaccagaag ctatccacaa
cttcgcagag tgtttcagaa atacaccaag tacagtaagc 1080atgacatgaa
caaagttctg gacctggagt tgaaaggtga cattgagaaa tgcctcacag
1140ctatcgtgaa gtgcgccaca agcaaaccag ctttctttgc agagaagctt
catcaagcca 1200tgaaaggtgt tggaactcgc cataaggcat tgatcaggat
tatggtttcc cgttctgaaa 1260ttgacatgaa tgatatcaaa gcattctatc
agaagatgta tggtatctcc ctttgccaag 1320ccatcctgga tgaaaccaaa
ggagattatg agaaaatcct ggtggctctt tgtggaggaa 1380actaaacatt
cccttgatgg tctcaagcta tgatcagaag actttaatta tatattttca
1440tcctataagc ttaaatagga aagtttcttc aacaggatta cagtgtagct
acctacatgc 1500tgaaaaatat agcctttaaa tcatttttat attataactc
tgtataatag agataagtcc 1560attttttaaa aatgttttcc ccaaaccata
aaaccctata caagttgttc tagtaacaat 1620acatgagaaa gatgtctatg
tagctgaaaa taaaatgacg tcacaagaca a 1671311204DNAHomo sapiens
31gttccgcaga tgcagaggtt gaggtggctg cgggactgga agtcatcggg cagaggtctc
60acagcagcca aggaacctgg ggcccgctcc tcccccctcc aggccatgag gattctgcag
120ttaatcctgc ttgctctggc aacagggctt gtagggggag agaccaggat
catcaagggg 180ttcgagtgca agcctcactc ccagccctgg caggcagccc
tgttcgagaa gacgcggcta 240ctctgtgggg cgacgctcat cgcccccaga
tggctcctga cagcagccca ctgcctcaag 300ccccgctaca tagttcacct
ggggcagcac aacctccaga aggaggaggg ctgtgagcag 360acccggacag
ccactgagtc cttcccccac cccggcttca acaacagcct ccccaacaaa
420gaccaccgca atgacatcat gctggtgaag atggcatcgc cagtctccat
cacctgggct 480gtgcgacccc tcaccctctc ctcacgctgt gtcactgctg
gcaccagctg cctcatttcc 540ggctggggca gcacgtccag cccccagtta
cgcctgcctc acaccttgcg atgcgccaac 600atcaccatca ttgagcacca
gaagtgtgag aacgcctacc ccggcaacat cacagacacc 660atggtgtgtg
ccagcgtgca ggaagggggc aaggactcct gccagggtga ctccgggggc
720cctctggtct gtaaccagtc tcttcaaggc attatctcct ggggccagga
tccgtgtgcg 780atcacccgaa agcctggtgt ctacacgaaa gtctgcaaat
atgtggactg gatccaggag 840acgatgaaga acaattagac tggacccacc
caccacagcc catcaccctc catttccact 900tggtgtttgg ttcctgttca
ctctgttaat aagaaaccct aagccaagac cctctacgaa 960cattctttgg
gcctcctgga ctacaggaga tgctgtcact taataatcaa cctggggttc
1020gaaatcagtg agacctggat tcaaattctg ccttgaaata ttgtgactct
gggaatgaca 1080acacctggtt tgttctctgt tgtatcccca gccccaaaga
cagctcctgg ccatatatca 1140aggtttcaat aaatatttgc taaatgaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200aaaa 1204322246DNAHomo sapiens
32aaagtccggg ggagccggtc ccgggcagcc gctcagcccc ctgcccctcg ccgcccgccg
60cctgcctggg ccgggccgag gatgcggcgc agcgcctcgg cggccaggct tgctcccctc
120cggcacgcct gctaacttcc cccgctacgt ccccgttcgc ccgccgggcc
gccccgtctc 180cccgcgccct ccgggtcggg tcctccagga gcgccaggcg
ctgccgccgt gtgccctccg 240ccgctcgccc gcgcgcccgc gctccccgcc
tgcgcccagc gccccgcgcc cgcgcccagt 300cctcgggcgg tcatgctgcc
cctctgcctc gtggccgccc tgctgctggc cgccgggccc 360gggccgagcc
tgggcgacga agccatccac tgcccgccct gctccgagga gaagctggcg
420cgctgccgcc cccccgtggg ctgcgaggag ctggtgcgag agccgggctg
cggctgttgc 480gccacttgcg ccctgggctt ggggatgccc tgcggggtgt
acaccccccg ttgcggctcg 540ggcctgcgct gctacccgcc ccgaggggtg
gagaagcccc tgcacacact gatgcacggg 600caaggcgtgt gcatggagct
ggcggagatc gaggccatcc aggaaagcct gcagccctct 660gacaaggacg
agggtgacca ccccaacaac agcttcagcc cctgtagcgc ccatgaccgc
720aggtgcctgc agaagcactt cgccaaaatt cgagaccgga gcaccagtgg
gggcaagatg 780aaggtcaatg gggcgccccg ggaggatgcc cggcctgtgc
cccagggctc ctgccagagc 840gagctgcacc gggcgctgga gcggctggcc
gcttcacaga gccgcaccca cgaggacctc 900tacatcatcc ccatccccaa
ctgcgaccgc aacggcaact tccaccccaa gcagtgtcac 960ccagctctgg
atgggcagcg tggcaagtgc tggtgtgtgg accggaagac gggggtgaag
1020cttccggggg gcctggagcc aaagggggag ctggactgcc accagctggc
tgacagcttt 1080cgagagtgag gcctgccagc aggccaggga ctcagcgtcc
cctgctactc ctgtgctctg 1140gaggctgcag agctgaccca gagtggagtc
tgagtctgag tcctgtctct gcctgcggcc 1200cagaagtttc cctcaaatgc
gcgtgtgcac gtgtgcgtgt gcgtgcgtgt gtgtgtgttt 1260gtgagcatgg
gtgtgccctt ggggtaagcc agagcctggg gtgttctctt tggtgttaca
1320cagcccaaga ggactgagac tggcacttag cccaagaggt ctgagccctg
gtgtgtttcc 1380agatcgatcc tggattcact cactcactca ttccttcact
catccagcca cctaaaaaca 1440tttactgacc atgtactacg tgccagctct
agttttcagc cttgggaggt tttattctga 1500cttcctctga ttttggcatg
tggagacact cctataagga gagttcaagc ctgtgggagt 1560agaaaaatct
cattcccaga gtcagaggag aagagacatg taccttgacc atcgtccttc
1620ctctcaagct agccagaggg tgggagccta aggaagcgtg gggtagcaga
tggagtaatg 1680gtcacgaggt ccagacccac tcccaaagct cagacttgcc
aggctccctt tctcttcttc 1740cccaggtcct tcctttaggt ctggttgttg
caccatctgc ttggttggct ggcagctgag 1800agccctgctg tgggagagcg
aagggggtca aaggaagact tgaagcacag agggctaggg 1860aggtggggta
catttctctg agcagtcagg gtgggaagaa agaatgcaag agtggactga
1920atgtgcctaa tggagaagac ccacgtgcta ggggatgagg ggcttcctgg
gtcctgttcc 1980ctaccccatt tgtggtcaca gccatgaagt caccgggatg
aacctatcct tccagtggct 2040cgctccctgt agctctgcct ccctctccat
atctccttcc cctacacctc cctccccaca 2100cctccctact cccctgggca
tcttctggct tgactggatg gaaggagact taggaaccta 2160ccagttggcc
atgatgtctt ttcttctttt tctttttttt aacaaaacag aacaaaacca
2220aaaaatgtcc agatgaaaaa aaaaaa 2246334576DNAHomo sapiens
33ggccctcgcc gcccgcggcg ccccgagcgc tttgtgagca gatgcggagc cgagtggagg
60gcgcgagcca gatgcggggc gacagctgac ttgctgagag gaggcgggga ggcgcggagc
120gcgcgtgtgg tccttgcgcc gctgacttct ccactggttc ctgggcaccg
aaagataaac 180ctctcataat gaaggccccc gctgtgcttg cacctggcat
cctcgtgctc ctgtttacct 240tggtgcagag gagcaatggg gagtgtaaag
aggcactagc aaagtccgag atgaatgtga 300atatgaagta tcagcttccc
aacttcaccg cggaaacacc catccagaat gtcattctac 360atgagcatca
cattttcctt ggtgccacta actacattta tgttttaaat gaggaagacc
420ttcagaaggt tgctgagtac aagactgggc ctgtgctgga acacccagat
tgtttcccat 480gtcaggactg cagcagcaaa gccaatttat caggaggtgt
ttggaaagat aacatcaaca 540tggctctagt tgtcgacacc tactatgatg
atcaactcat tagctgtggc agcgtcaaca 600gagggacctg ccagcgacat
gtctttcccc acaatcatac tgctgacata cagtcggagg 660ttcactgcat
attctcccca cagatagaag agcccagcca gtgtcctgac tgtgtggtga
720gcgccctggg agccaaagtc ctttcatctg taaaggaccg gttcatcaac
ttctttgtag 780gcaataccat aaattcttct
tatttcccag atcatccatt gcattcgata tcagtgagaa 840ggctaaagga
aacgaaagat ggttttatgt ttttgacgga ccagtcctac attgatgttt
900tacctgagtt cagagattct taccccatta agtatgtcca tgcctttgaa
agcaacaatt 960ttatttactt cttgacggtc caaagggaaa ctctagatgc
tcagactttt cacacaagaa 1020taatcaggtt ctgttccata aactctggat
tgcattccta catggaaatg cctctggagt 1080gtattctcac agaaaagaga
aaaaagagat ccacaaagaa ggaagtgttt aatatacttc 1140aggctgcgta
tgtcagcaag cctggggccc agcttgctag acaaatagga gccagcctga
1200atgatgacat tcttttcggg gtgttcgcac aaagcaagcc agattctgcc
gaaccaatgg 1260atcgatctgc catgtgtgca ttccctatca aatatgtcaa
cgacttcttc aacaagatcg 1320tcaacaaaaa caatgtgaga tgtctccagc
atttttacgg acccaatcat gagcactgct 1380ttaataggac acttctgaga
aattcatcag gctgtgaagc gcgccgtgat gaatatcgaa 1440cagagtttac
cacagctttg cagcgcgttg acttattcat gggtcaattc agcgaagtcc
1500tcttaacatc tatatccacc ttcattaaag gagacctcac catagctaat
cttgggacat 1560cagagggtcg cttcatgcag gttgtggttt ctcgatcagg
accatcaacc cctcatgtga 1620attttctcct ggactcccat ccagtgtctc
cagaagtgat tgtggagcat acattaaacc 1680aaaatggcta cacactggtt
atcactggga agaagatcac gaagatccca ttgaatggct 1740tgggctgcag
acatttccag tcctgcagtc aatgcctctc tgccccaccc tttgttcagt
1800gtggctggtg ccacgacaaa tgtgtgcgat cggaggaatg cctgagcggg
acatggactc 1860aacagatctg tctgcctgca atctacaagg ttttcccaaa
tagtgcaccc cttgaaggag 1920ggacaaggct gaccatatgt ggctgggact
ttggatttcg gaggaataat aaatttgatt 1980taaagaaaac tagagttctc
cttggaaatg agagctgcac cttgacttta agtgagagca 2040cgatgaatac
attgaaatgc acagttggtc ctgccatgaa taagcatttc aatatgtcca
2100taattatttc aaatggccac gggacaacac aatacagtac attctcctat
gtggatcctg 2160taataacaag tatttcgccg aaatacggtc ctatggctgg
tggcacttta cttactttaa 2220ctggaaatta cctaaacagt gggaattcta
gacacatttc aattggtgga aaaacatgta 2280ctttaaaaag tgtgtcaaac
agtattcttg aatgttatac cccagcccaa accatttcaa 2340ctgagtttgc
tgttaaattg aaaattgact tagccaaccg agagacaagc atcttcagtt
2400accgtgaaga tcccattgtc tatgaaattc atccaaccaa atcttttatt
agtggtggga 2460gcacaataac aggtgttggg aaaaacctga attcagttag
tgtcccgaga atggtcataa 2520atgtgcatga agcaggaagg aactttacag
tggcatgtca acatcgctct aattcagaga 2580taatctgttg taccactcct
tccctgcaac agctgaatct gcaactcccc ctgaaaacca 2640aagccttttt
catgttagat gggatccttt ccaaatactt tgatctcatt tatgtacata
2700atcctgtgtt taagcctttt gaaaagccag tgatgatctc aatgggcaat
gaaaatgtac 2760tggaaattaa gggaaatgat attgaccctg aagcagttaa
aggtgaagtg ttaaaagttg 2820gaaataagag ctgtgagaat atacacttac
attctgaagc cgttttatgc acggtcccca 2880atgacctgct gaaattgaac
agcgagctaa atatagagtg gaagcaagca atttcttcaa 2940ccgtccttgg
aaaagtaata gttcaaccag atcagaattt cacaggattg attgctggtg
3000ttgtctcaat atcaacagca ctgttattac tacttgggtt tttcctgtgg
ctgaaaaaga 3060gaaagcaaat taaagatctg ggcagtgaat tagttcgcta
cgatgcaaga gtacacactc 3120ctcatttgga taggcttgta agtgcccgaa
gtgtaagccc aactacagaa atggtttcaa 3180atgaatctgt agactaccga
gctacttttc cagaagatca gtttcctaat tcatctcaga 3240acggttcatg
ccgacaagtg cagtatcctc tgacagacat gtcccccatc ctaactagtg
3300gggactctga tatatccagt ccattactgc aaaatactgt ccacattgac
ctcagtgctc 3360taaatccaga gctggtccag gcagtgcagc atgtagtgat
tgggcccagt agcctgattg 3420tgcatttcaa tgaagtcata ggaagagggc
attttggttg tgtatatcat gggactttgt 3480tggacaatga tggcaagaaa
attcactgtg ctgtgaaatc cttgaacaga atcactgaca 3540taggagaagt
ttcccaattt ctgaccgagg gaatcatcat gaaagatttt agtcatccca
3600atgtcctctc gctcctggga atctgcctgc gaagtgaagg gtctccgctg
gtggtcctac 3660catacatgaa acatggagat cttcgaaatt tcattcgaaa
tgagactcat aatccaactg 3720taaaagatct tattggcttt ggtcttcaag
tagccaaagg catgaaatat cttgcaagca 3780aaaagtttgt ccacagagac
ttggctgcaa gaaactgtat gctggatgaa aaattcacag 3840tcaaggttgc
tgattttggt cttgccagag acatgtatga taaagaatac tatagtgtac
3900acaacaaaac aggtgcaaag ctgccagtga agtggatggc tttggaaagt
ctgcaaactc 3960aaaagtttac caccaagtca gatgtgtggt cctttggcgt
gctcctctgg gagctgatga 4020caagaggagc cccaccttat cctgacgtaa
acacctttga tataactgtt tacttgttgc 4080aagggagaag actcctacaa
cccgaatact gcccagaccc cttatatgaa gtaatgctaa 4140aatgctggca
ccctaaagcc gaaatgcgcc catccttttc tgaactggtg tcccggatat
4200cagcaatctt ctctactttc attggggagc actatgtcca tgtgaacgct
acttatgtga 4260acgtaaaatg tgtcgctcca tatccttctc tgttgtcatc
agaagataac gctgatgatg 4320aggtggacac acgaccagcc tccttctggg
agacatcata gtgctagtac tatgtcaaag 4380caacagtcca cactttgtcc
aatggttttt tcactgcctg acctttaaaa ggccatcgat 4440attctttgct
acttgtatat acattcttga gaacactgca atgtgaaaat cacgtttgct
4500atttataaac ttgtccttag attaatgtgt ctggacagat tgtgggagta
agtgattctt 4560ctaagaatta gatact 457634720DNAHomo sapiens
34atgacccccc acaggctgct gccaccgctg ctgctgctgc tagctctgct gctcgctgcc
60agcccaggag gcgccttggc gcggtgccca ggctgcgggc aaggggtgca ggcgggttgt
120ccagggggct gcgtggagga ggaggatggg gggtcgccag ccgagggctg
cgcggaagct 180gagggctgtc tcaggaggga ggggcaggag tgcggggtct
acacccctaa ctgcgcccca 240ggactgcagt gccatccgcc caaggacgac
gaggcgcctt tgcgggcgct gctgctcggc 300cgaggccgct gccttccggc
ccgcgcgcct gctgttgcag aggagaatcc taaggagagt 360aaaccccaag
caggcactgc ccgcccacag gatgtgaacc gcagagacca acagaggaat
420ccaggcacct ctaccacgcc ctcccagccc aattctgcgg gtgtccaaga
cactgagatg 480ggcccatgcc gtagacatct ggactcagtg ctgcagcaac
tccagactga ggtctaccga 540ggggctcaaa cactctacgt gcccaattgt
gaccatcgag gcttctaccg gaagcggcag 600tgccgctcct cccaggggca
gcgccgaggt ccctgctggt gtgtggatcg gatgggcaag 660tccctgccag
ggtctccaga tggcaatgga agctcctcct gccccactgg gagtagcggc
720351933DNAHomo sapiens 35cggtataaag gcagccgcgg tggcggtggc
ggcgcagagc tctgtgctcc ctgcagtcag 60gactctggga ccgcaggggg ctcccggacc
ctgactctgc agccgaaccg gcacggtttc 120gtggggaccc aggcttgcaa
agtgacggtc attttctctt tctttctccc tcttgagtcc 180ttctgagatg
atggctctgg gcgcagcggg agctacccgg gtctttgtcg cgatggtagc
240ggcggctctc ggcggccacc ctctgctggg agtgagcgcc accttgaact
cggttctcaa 300ttccaacgct atcaagaacc tgcccccacc gctgggcggc
gctgcggggc acccaggctc 360tgcagtcagc gccgcgccgg gaatcctgta
cccgggcggg aataagtacc agaccattga 420caactaccag ccgtacccgt
gcgcagagga cgaggagtgc ggcactgatg agtactgcgc 480tagtcccacc
cgcggagggg acgcgggcgt gcaaatctgt ctcgcctgca ggaagcgccg
540aaaacgctgc atgcgtcacg ctatgtgctg ccccgggaat tactgcaaaa
atggaatatg 600tgtgtcttct gatcaaaatc atttccgagg agaaattgag
gaaaccatca ctgaaagctt 660tggtaatgat catagcacct tggatgggta
ttccagaaga accaccttgt cttcaaaaat 720gtatcacacc aaaggacaag
aaggttctgt ttgtctccgg tcatcagact gtgcctcagg 780attgtgttgt
gctagacact tctggtccaa gatctgtaaa cctgtcctga aagaaggtca
840agtgtgtacc aagcatagga gaaaaggctc tcatggacta gaaatattcc
agcgttgtta 900ctgtggagaa ggtctgtctt gccggataca gaaagatcac
catcaagcca gtaattcttc 960taggcttcac acttgtcaga gacactaaac
cagctatcca aatgcagtga actcctttta 1020tataatagat gctatgaaaa
ccttttatga ccttcatcaa ctcaatccta aggatataca 1080agttctgtgg
tttcagttaa gcattccaat aacaccttcc aaaaacctgg agtgtaagag
1140ctttgtttct ttatggaact cccctgtgat tgcagtaaat tactgtattg
taaattctca 1200gtgtggcact tacctgtaaa tgcaatgaaa cttttaatta
tttttctaaa ggtgctgcac 1260tgcctatttt tcctcttgtt atgtaaattt
ttgtacacat tgattgttat cttgactgac 1320aaatattcta tattgaactg
aagtaaatca tttcagctta tagttcttaa aagcataacc 1380ctttacccca
tttaattcta gagtctagaa cgcaaggatc tcttggaatg acaaatgata
1440ggtacctaaa atgtaacatg aaaatactag cttattttct gaaatgtact
atcttaatgc 1500ttaaattata tttcccttta ggctgtgata gtttttgaaa
taaaatttaa catttaatat 1560catgaaatgt tataagtaga catacatttt
gggattgtga tcttagaggt ttgtgtgtgt 1620gtacgtatgt gtgtgttcta
caagaacgga agtgtgatat gtttaaagat gatcagagaa 1680aagacagtgt
ctaaatataa gacaatattg atcagctcta gaataacttt aaagaaagac
1740gtgttctgca ttgataaact caaatgatca tggcagaatg agagtgaatc
ttacattact 1800actttcaaaa atagtttcca ataaattaat aatacctacc
taaatggtca atatttttcg 1860gacaaggaag aaaatcatcc acaaaaataa
tactccaaag tacttggtga ttggcaggaa 1920caggatgtgt gcc
1933365191DNAHomo sapiens 36ggcgagtgcg tcgagctcgc cgcggactca
agatggcggc gtgtggacgt gtacggagga 60tgttccgctt gtcggcggcg ctgcatctgc
tgctgctatt cgcggccggg gccgagaaac 120tccccggcca gggcgtccac
agccagggcc agggtcccgg ggccaacttt gtgtccttcg 180tagggcaggc
cggaggcggc ggcccggcgg gtcagcagct gccccagctg cctcagtcat
240cgcagcttca gcagcaacag cagcagcagc aacagcaaca gcagcctcag
ccgccgcagc 300cgcctttccc ggcgggtggg cctccggccc ggcggggagg
agcgggggct ggtgggggct 360ggaagctggc ggaggaagag tcctgcaggg
aggacgtgac ccgcgtgtgc cctaagcaca 420cctggagcaa caacctggcg
gtgctcgagt gcctgcagga tgtgagggag cctgaaaatg 480aaatttcttc
agactgcaat catttgttgt ggaattataa gctgaaccta actacagatc
540ccaaatttga atctgtggcc agagaggttt gcaaatctac tataacagag
attaaagaat 600gtgctgatga accggttgga aaaggttaca tggtttcctg
cttggtggat caccgaggca 660acatcactga gtatcagtgt caccagtaca
ttaccaagat gacggccatc atttttagtg 720attaccgttt aatctgtggc
ttcatggatg actgcaaaaa tgacatcaac attctgaaat 780gtggcagtat
tcggcttgga gaaaaggatg cacattcaca aggtgaggtg gtatcatgct
840tggagaaagg cctggtgaaa gaagcagaag aaagagaacc caagattcaa
gtttctgaac 900tctgcaagaa agccattctc cgggtggctg agctgtcatc
ggatgacttt cacttagacc 960ggcatttata ttttgcttgc cgagatgatc
gggagcgttt ttgtgaaaat acacaagctg 1020gtgagggcag agtgtataag
tgcctcttta accataaatt tgaagaatcc atgagtgaaa 1080agtgtcgaga
agcacttaca acccgccaaa agctgattgc ccaggattat aaagtcagtt
1140attcattggc caaatcctgt aaaagtgact tgaagaaata ccggtgcaat
gtggaaaacc 1200ttccgcgatc gcgtgaagcc aggctctcct acttgttaat
gtgcctggag tcagctgtac 1260acagagggcg acaagtcagc agtgagtgcc
agggggagat gctggattac cgacgcatgt 1320tgatggaaga cttttctctg
agccctgaga tcatcctaag ctgtcggggg gagattgaac 1380accattgttc
cggattacat cgaaaagggc ggaccctaca ctgtctgatg aaagtagttc
1440gaggggagaa ggggaacctt ggaatgaact gccagcaggc gcttcaaaca
ctgattcagg 1500agactgaccc tggtgcagat taccgcattg atcgagcttt
gaatgaagct tgtgaatctg 1560taatccagac agcctgcaaa catataagat
ctggagaccc aatgatcttg tcgtgcctga 1620tggaacattt atacacagag
aagatggtag aagactgtga acaccgtctc ttagagctgc 1680agtatttcat
ctcccgggat tggaagctgg accctgtcct gtaccgcaag tgccagggag
1740acgcttctcg tctttgccac acccacggtt ggaatgagac cagtgaattt
atgcctcagg 1800gagctgtgtt ctcttgttta tacagacacg cctaccgcac
tgaggaacag ggaaggaggc 1860tctcacggga gtgccgagct gaagtccaaa
ggatcctaca ccagcgtgcc atggatgtca 1920agctggatcc tgccctccag
gataagtgcc tgattgatct gggaaaatgg tgcagtgaga 1980aaacagagac
tggacaggag ctggagtgcc ttcaggacca tctggatgac ttggtggtgg
2040agtgtagaga tatagttggc aacctcactg agttagaatc agaggatatt
caaatagaag 2100ccttgctgat gagagcctgt gagcccataa ttcagaactt
ctgccacgat gtggcagata 2160accagataga ctctggggac ctgatggagt
gtctgataca gaacaaacac cagaaggaca 2220tgaacgagaa gtgtgccatc
ggagttaccc acttccagct ggtgcagatg aaggattttc 2280ggttttctta
caagtttaaa atggcctgca aggaggacgt gttgaagctt tgcccaaaca
2340taaaaaagaa ggtggacgtg gtgatctgcc tgagcacgac cgtgcgcaat
gacactctgc 2400aggaagccaa ggagcacagg gtgtccctga agtgccgcag
gcagctccgt gtggaggagc 2460tggagatgac ggaggacatc cgcttggagc
cagatctata cgaagcctgc aagagtgaca 2520tcaaaaactt ctgttccgct
gtgcaatatg gcaacgctca gattatcgaa tgtctgaaag 2580aaaacaagaa
gcagctaagc acccgctgcc accaaaaagt atttaagctg caggagacag
2640agatgatgga cccagagcta gactacaccc tcatgagggt ctgcaagcag
atgataaaga 2700ggttctgtcc ggaagcagat tctaaaacca tgttgcagtg
cttgaagcaa aataaaaaca 2760gtgaattgat ggatcccaaa tgcaaacaga
tgataaccaa gcgccagatc acccagaaca 2820cagattaccg cttaaacccc
atgttaagaa aagcctgtaa agctgacatt cctaaattct 2880gtcacggtat
cctgactaag gccaaggatg attcagaatt agaaggacaa gtcatctctt
2940gcctgaagct gagatatgct gaccagcgcc tgtcttcaga ctgtgaagac
cagatccgaa 3000tcattatcca ggagtccgcc ctggactacc gcctggatcc
tcagctccag ctgcactgct 3060cagacgagat ctccagtcta tgtgctgaag
aagcagcagc ccaagagcag acaggtcagg 3120tggaggagtg cctcaaggtc
aacctgctca agatcaaaac agaattgtgt aaaaaggaag 3180tgctaaacat
gctgaaggaa agcaaagcag acatctttgt tgacccggta cttcatactg
3240cttgtgccct ggacattaaa caccactgcg cagccatcac ccctggccgc
gggcgtcaaa 3300tgtcctgtct catggaagca ctggaggata agcgggtgag
gttacagccc gagtgcaaaa 3360agcgcctcaa tgaccggatt gagatgtgga
gttacgcagc aaaggtggcc ccagcagatg 3420gcttctctga tcttgccatg
caagtaatga cgtctccatc taagaactac attctctctg 3480tgatcagtgg
gagcatctgt atattgttcc tgattggcct gatgtgtgga cggatcacca
3540agcgagtgac acgagagctc aaggacaggt agagccacct tgaccaccaa
aggaactacc 3600tatccagtgc ccagtttgta cagccctctt gtatagcatc
cccactcacc tcgctcttct 3660cagaagtgac accaaccccg tgttagagca
ttagcagatg tccactgcgt tgtcccatcc 3720agcctccact cgtgtccatg
gtgtcctcct cctcctcacc gtgcagcagc agcagctggt 3780cgctggggtt
actgcctttg tttggcaaac ttgggtttac ctgcctgtag acaagtctct
3840ctcataccaa cagaacttcc ggtacttcca gaaccaactc acctgacctg
caactcaaag 3900gcttttttaa gaaaaccacc aaaaaaaaaa atttttttaa
agaaaaaaat gtatatagta 3960acgcatctcc tccaggcttg atttgggcaa
tggggttatg tctttcatat gactgtgtaa 4020aacaaagaca ggacttggag
gggaagcaca ccacccagtg tgccatgact gaggtgtctc 4080gttcatctct
cagaagcacc ttggggcctc gccagggccg tggtcttcac cgaggcgtgg
4140gtgggcagcc gttccccagg ctgtgtgggg tcctgctttc ttctgctgag
acagtgacgc 4200tttccagttt ccaccctaat cagccactgc tggtcacagc
cccacagcca tgggtatttc 4260tgtggtctcc tcgcttcatt gaagcaaagc
atgagccttc ctagacaagg gcagctgggg 4320aggggaaggg accggaagtt
tgtgaagttg aacagtccat ccatctgcac tgagaggctg 4380gatcctgagt
cccggggcag caggatccca ggaaccttcc tcctccaggg cagcacagga
4440ctcagccatg tctggaccgg ccctgctgag gctacagtca ctctggaagc
tctgcgcttc 4500atcaggaggc aggactgtgg cgggaggggt ccttgaagat
gggtgtgggg agcagtgggt 4560caggaagtgg gagccagagg tttgactcac
tttgctttat ttttcaggct acaatacagg 4620tcagagacaa tggcttataa
aggtttagtg tggtctcagg atgtgacagg cagtccagcc 4680tgacctttct
gcacactcca gacaaacttc ccagacaagc tcctttgtgc ctctacgtgg
4740agagggtgtg gaaagttatc acattaaaag atggaggatt tgctctgttt
tttttctttc 4800tgtccatttg ctgcgtgtac ccactctagt aggcattggc
taaatgttgt attttggcga 4860ttcatcaacc tttgcagaat atgggcttta
tagaagcaat attcttggcc atcccgcctc 4920attcctccag tgtggagatg
acaagtctgg gtgtgagagg gaggggtccg ggcatcatgg 4980ttcagcgtgg
cactcctttg gttgagtttg gggcatgaga tcacagtggc tgcacaagag
5040agcagtgtgt acagtaggag agacatttat gtaatatata ttttattaac
ctgttagatg 5100tccacaaagt attataaatc acgtgcctaa aactgtccat
gtagaccaag gcctgccctc 5160ggcgcccccc actcttgcct ctgctctgca c
5191373901DNAHomo sapiens 37aagatggcgg cgtgtggacg tgtacggagg
atgttccgct tgtcggcggc gctgcatctg 60ctgctgctat tcgcggccgg ggccgagaaa
ctccccggcc atggcgtcca cagccagggc 120cagggtcccg gggccaactt
tgtgtccttc gtagggcagg ccggaggcgg cggcccggcg 180ggtcagcagc
tgccccagct gcttcagtca tcgcagcttc agcagcaaca gcagcagcag
240caacagcaac agcagcttca gccgccgcag ccgcctttcc cggcgggtgg
gcctccggcc 300cggcggggag gagcgggggc tggtgggggc tggaagctgg
cggaggaaga gtcctgcagg 360gaggacgtga cccgcgtgtg ccctaagcac
acctggagca acaacctggc ggtgctcgag 420tgcctgcagg atgtgaggga
gcctgaaaat gaaatttctt cagactgcaa tcatttgttg 480tggaattata
agctgaacct aactacagat cccaaatttg aatctgtggc cagagaggtt
540tgcaaatcta ctataacaga gattaaagaa tgtgctgatg aaccggttgg
aaaaggttac 600atggtttcct gcttagtgga tcaccgaggc aacatcactg
agtatcagtg tcaccagtac 660attaccaaga tgacggccat catttttagt
gattaccgtt taatctgtgg cttcatggat 720gactgcaaaa atgacatcaa
cattctgaaa tgtggcagta ttcggcttgg agaaaaggat 780gcacattcac
aaggtgaggt ggtatcatgc ttggagaaag gcctggtgaa agaagcagaa
840gaaagagaac ccaagattca agtttctgaa ctctgcaaga aagccattct
ccgggtggct 900gagctgtcat cggatgactt tcacttagac cggcatttat
attttgcttg ccgagatgat 960cgggagcgtt tttgtgaaaa tacacaagct
ggtgagggca gagtgtataa gtgcctcttt 1020aaccataaat ttgaagaatc
catgagtgaa aagtgtcgag aagcacttac aacccgccaa 1080aagctgattg
cccaggatta taaagtcagt tattcattgg ccaaatcctg taaaagtgac
1140ttgaagaaat accggtgcaa tgtggaaaac cttccgcgat cgcgtgaagc
caggctctcc 1200tacttgttaa tgtgcctgga gtcagctgta cacagagggc
gacaagtcag cagtgagtgc 1260cagggggaga tgctggatta ccgacgcatg
ttgatggaag acttttctct gagccctgag 1320atcatcctaa gctgtcgggg
ggagattgaa caccattgtt ccggattaca tcgaaaaggg 1380cggaccctac
actgtctgat gaaagtagtt cgaggggaga aggggaacct tggaatgaac
1440tgccagcagg cgcttcaaac actgattcag gagactgacc ctggtgcaga
ttaccgcatt 1500gatcgagctt tgaatgaagc ttgtgaatct gtaatccaga
cagcctgcaa acatataaga 1560tctggagacc caatgatctt gtcgtgcctg
atggaacatt tatacacaga gaagatggta 1620gaagactgtg aacaccgtct
cttagagctg cagtatttca tctcccggga ttggaagctg 1680gaccctgtcc
tgtaccgcaa gtgccaggga gacgcttctc gtctttgcca cacccacggt
1740tggaatgaga ccagtgaatt tatgcctcag ggagctgtgt tctcttgttt
atacagacac 1800gcctaccgca ctgaggaaca gggaaggagg ctctcacggg
agtgccgagc tgaagtccaa 1860aggatcctac accagcgtgc catggatgtc
aagctggatc ctgccctcca ggataagtgc 1920ctgattgatc tgggaaaatg
gtgcagtgag aaaacagaga ctggacagga gctggagtgc 1980cttcaggacc
atctggatga cttggtggtg gagtgtagag atatagttgg caacctcact
2040gagttagaat cagaggatat tcaaatagaa gccttgctga tgagagcctg
tgagcccata 2100attcagaact tctgccacga tgtggcagat aaccagatag
actctgggga cctgatggag 2160tgtctgatac agaacaaaca ccagaaggac
atgaacgaga agtgtgccat cggagttacc 2220cacttccagc tggtgcagat
gaaggatttt cggttttctt acaagtttaa aatggcctgc 2280aaggaggacg
tgttgaagct ttgcccaaac ataaaaaaga aggtggacgt ggtgatctgc
2340ctgagcacga ccgtgcgcaa tgacactctg caggaagcca aggagcacag
ggtgtccctg 2400aagtgccgca ggcagctccg tgtggaggag ctggagatga
cggaggacat ccgcttggag 2460ccagatctat acgaagcctg caagagtgac
atcaaaaact tctgttccgc tgtgcaatat 2520ggcaacgctc agattatcga
atgtctgaaa gaaaacaaga agcagctaag cacccgctgc 2580caccaaaaag
tatttaagct gcaggagaca gagatgatgg acccagagct agactacacc
2640ctcatgaggg tctgcaagca gatgataaag aggttctgtc cggaagcaga
ttctaaaacc 2700atgttgcagt gcttgaagca aaataaaaac agtgaattga
tggatcccaa atgcaaacag 2760atgataacca agcgccagat cacccagaac
acagattacc gcttaaaccc catgttaaga 2820aaagcctgta aagctgacat
tcctaaattc tgtcacggta tcctgactaa ggccaaggat 2880gattcagaat
tagaaggaca agtcatctct tgcctgaagc tgagatatgc tgaccagcgc
2940ctgtcttcag actgtgaaga ccagatccga atcattatcc aggagtccgc
cctggactac 3000cgcctggatc ctcagctcca gctgcactgc tcagacgaga
tctccagtct atgtgctgaa 3060gaagcagcag cccaagagca gacaggtcag
gtggaggagt gcctcaaggt caacctgctc 3120aagatcaaaa cagaattgtg
taaaaaggaa gtgctaaaca tgctgaagga aagcaaagca 3180gacatctttg
ttgacccggt acttcatact gcttgtgccc tggacattaa acaccactgc
3240gcagccatca cccctggccg cgggcgtcaa atgtcctgtc tcatggaagc
actggaggat 3300aagcgggtga ggttacagcc cgagtgcaaa aagcgcctca
atgaccggat tgagatgtgg 3360agttacgcag caaaggtggc cccagcagat
ggcttctctg atcttgccat gcaagtaatg 3420acgtctccat ctaagaacta
cattctctct gtgatcagtg ggagcatctg tatattgttc 3480ctgattggcc
tgatgtgtgg acggatcacc aagcgagtga cacgagagct caaggacagg
3540tagagccacc ttgaccacca aaggaactac ctatccagtg cccagtttgt
acagccctct 3600tgtatagcat ccccactcac ctcgctcttc tcagaagtga
caccaacccc gtgttagagc 3660attagcagat gtccactgcg ttgtcccatc
cagcctccac tcgtgtccat ggtgtcctcc 3720tcctcctcac cgtgcagcag
cagcagctgg tcgctggggt tactgccttt gtttggcaaa 3780cttgggttta
cctgcctgta gacaagtctc tctcatacca acagaacttc cggtacttcc
3840agaaccaact cacctgacct gcaactcaaa ggctttttta agaaaaccac
caaaaaaaaa 3900a 3901385191DNAHomo sapiens 38ggcgagtgcg tcgagctcgc
cgcggactca agatggcggc gtgtggacgt gtacggagga 60tgttccgctt gtcggcggcg
ctgcatctgc tgctgctatt cgcggccggg gccgagaaac 120tccccggcca
gggcgtccac agccagggcc agggtcccgg ggccaacttt gtgtccttcg
180tagggcaggc cggaggcggc ggcccggcgg gtcagcagct gccccagctg
cctcagtcat 240cgcagcttca gcagcaacag cagcagcagc aacagcaaca
gcagcctcag ccgccgcagc 300cgcctttccc ggcgggtggg cctccggccc
ggcggggagg agcgggggct ggtgggggct 360ggaagctggc ggaggaagag
tcctgcaggg aggacgtgac ccgcgtgtgc cctaagcaca 420cctggagcaa
caacctggcg gtgctcgagt gcctgcagga tgtgagggag cctgaaaatg
480aaatttcttc agactgcaat catttgttgt ggaattataa gctgaaccta
actacagatc 540ccaaatttga atctgtggcc agagaggttt gcaaatctac
tataacagag attaaagaat 600gtgctgatga accggttgga aaaggttaca
tggtttcctg cttggtggat caccgaggca 660acatcactga gtatcagtgt
caccagtaca ttaccaagat gacggccatc atttttagtg 720attaccgttt
aatctgtggc ttcatggatg actgcaaaaa tgacatcaac attctgaaat
780gtggcagtat tcggcttgga gaaaaggatg cacattcaca aggtgaggtg
gtatcatgct 840tggagaaagg cctggtgaaa gaagcagaag aaagagaacc
caagattcaa gtttctgaac 900tctgcaagaa agccattctc cgggtggctg
agctgtcatc ggatgacttt cacttagacc 960ggcatttata ttttgcttgc
cgagatgatc gggagcgttt ttgtgaaaat acacaagctg 1020gtgagggcag
agtgtataag tgcctcttta accataaatt tgaagaatcc atgagtgaaa
1080agtgtcgaga agcacttaca acccgccaaa agctgattgc ccaggattat
aaagtcagtt 1140attcattggc caaatcctgt aaaagtgact tgaagaaata
ccggtgcaat gtggaaaacc 1200ttccgcgatc gcgtgaagcc aggctctcct
acttgttaat gtgcctggag tcagctgtac 1260acagagggcg acaagtcagc
agtgagtgcc agggggagat gctggattac cgacgcatgt 1320tgatggaaga
cttttctctg agccctgaga tcatcctaag ctgtcggggg gagattgaac
1380accattgttc cggattacat cgaaaagggc ggaccctaca ctgtctgatg
aaagtagttc 1440gaggggagaa ggggaacctt ggaatgaact gccagcaggc
gcttcaaaca ctgattcagg 1500agactgaccc tggtgcagat taccgcattg
atcgagcttt gaatgaagct tgtgaatctg 1560taatccagac agcctgcaaa
catataagat ctggagaccc aatgatcttg tcgtgcctga 1620tggaacattt
atacacagag aagatggtag aagactgtga acaccgtctc ttagagctgc
1680agtatttcat ctcccgggat tggaagctgg accctgtcct gtaccgcaag
tgccagggag 1740acgcttctcg tctttgccac acccacggtt ggaatgagac
cagtgaattt atgcctcagg 1800gagctgtgtt ctcttgttta tacagacacg
cctaccgcac tgaggaacag ggaaggaggc 1860tctcacggga gtgccgagct
gaagtccaaa ggatcctaca ccagcgtgcc atggatgtca 1920agctggatcc
tgccctccag gataagtgcc tgattgatct gggaaaatgg tgcagtgaga
1980aaacagagac tggacaggag ctggagtgcc ttcaggacca tctggatgac
ttggtggtgg 2040agtgtagaga tatagttggc aacctcactg agttagaatc
agaggatatt caaatagaag 2100ccttgctgat gagagcctgt gagcccataa
ttcagaactt ctgccacgat gtggcagata 2160accagataga ctctggggac
ctgatggagt gtctgataca gaacaaacac cagaaggaca 2220tgaacgagaa
gtgtgccatc ggagttaccc acttccagct ggtgcagatg aaggattttc
2280ggttttctta caagtttaaa atggcctgca aggaggacgt gttgaagctt
tgcccaaaca 2340taaaaaagaa ggtggacgtg gtgatctgcc tgagcacgac
cgtgcgcaat gacactctgc 2400aggaagccaa ggagcacagg gtgtccctga
agtgccgcag gcagctccgt gtggaggagc 2460tggagatgac ggaggacatc
cgcttggagc cagatctata cgaagcctgc aagagtgaca 2520tcaaaaactt
ctgttccgct gtgcaatatg gcaacgctca gattatcgaa tgtctgaaag
2580aaaacaagaa gcagctaagc acccgctgcc accaaaaagt atttaagctg
caggagacag 2640agatgatgga cccagagcta gactacaccc tcatgagggt
ctgcaagcag atgataaaga 2700ggttctgtcc ggaagcagat tctaaaacca
tgttgcagtg cttgaagcaa aataaaaaca 2760gtgaattgat ggatcccaaa
tgcaaacaga tgataaccaa gcgccagatc acccagaaca 2820cagattaccg
cttaaacccc atgttaagaa aagcctgtaa agctgacatt cctaaattct
2880gtcacggtat cctgactaag gccaaggatg attcagaatt agaaggacaa
gtcatctctt 2940gcctgaagct gagatatgct gaccagcgcc tgtcttcaga
ctgtgaagac cagatccgaa 3000tcattatcca ggagtccgcc ctggactacc
gcctggatcc tcagctccag ctgcactgct 3060cagacgagat ctccagtcta
tgtgctgaag aagcagcagc ccaagagcag acaggtcagg 3120tggaggagtg
cctcaaggtc aacctgctca agatcaaaac agaattgtgt aaaaaggaag
3180tgctaaacat gctgaaggaa agcaaagcag acatctttgt tgacccggta
cttcatactg 3240cttgtgccct ggacattaaa caccactgcg cagccatcac
ccctggccgc gggcgtcaaa 3300tgtcctgtct catggaagca ctggaggata
agcgggtgag gttacagccc gagtgcaaaa 3360agcgcctcaa tgaccggatt
gagatgtgga gttacgcagc aaaggtggcc ccagcagatg 3420gcttctctga
tcttgccatg caagtaatga cgtctccatc taagaactac attctctctg
3480tgatcagtgg gagcatctgt atattgttcc tgattggcct gatgtgtgga
cggatcacca 3540agcgagtgac acgagagctc aaggacaggt agagccacct
tgaccaccaa aggaactacc 3600tatccagtgc ccagtttgta cagccctctt
gtatagcatc cccactcacc tcgctcttct 3660cagaagtgac accaaccccg
tgttagagca ttagcagatg tccactgcgt tgtcccatcc 3720agcctccact
cgtgtccatg gtgtcctcct cctcctcacc gtgcagcagc agcagctggt
3780cgctggggtt actgcctttg tttggcaaac ttgggtttac ctgcctgtag
acaagtctct 3840ctcataccaa cagaacttcc ggtacttcca gaaccaactc
acctgacctg caactcaaag 3900gcttttttaa gaaaaccacc aaaaaaaaaa
atttttttaa agaaaaaaat gtatatagta 3960acgcatctcc tccaggcttg
atttgggcaa tggggttatg tctttcatat gactgtgtaa 4020aacaaagaca
ggacttggag gggaagcaca ccacccagtg tgccatgact gaggtgtctc
4080gttcatctct cagaagcacc ttggggcctc gccagggccg tggtcttcac
cgaggcgtgg 4140gtgggcagcc gttccccagg ctgtgtgggg tcctgctttc
ttctgctgag acagtgacgc 4200tttccagttt ccaccctaat cagccactgc
tggtcacagc cccacagcca tgggtatttc 4260tgtggtctcc tcgcttcatt
gaagcaaagc atgagccttc ctagacaagg gcagctgggg 4320aggggaaggg
accggaagtt tgtgaagttg aacagtccat ccatctgcac tgagaggctg
4380gatcctgagt cccggggcag caggatccca ggaaccttcc tcctccaggg
cagcacagga 4440ctcagccatg tctggaccgg ccctgctgag gctacagtca
ctctggaagc tctgcgcttc 4500atcaggaggc aggactgtgg cgggaggggt
ccttgaagat gggtgtgggg agcagtgggt 4560caggaagtgg gagccagagg
tttgactcac tttgctttat ttttcaggct acaatacagg 4620tcagagacaa
tggcttataa aggtttagtg tggtctcagg atgtgacagg cagtccagcc
4680tgacctttct gcacactcca gacaaacttc ccagacaagc tcctttgtgc
ctctacgtgg 4740agagggtgtg gaaagttatc acattaaaag atggaggatt
tgctctgttt tttttctttc 4800tgtccatttg ctgcgtgtac ccactctagt
aggcattggc taaatgttgt attttggcga 4860ttcatcaacc tttgcagaat
atgggcttta tagaagcaat attcttggcc atcccgcctc 4920attcctccag
tgtggagatg acaagtctgg gtgtgagagg gaggggtccg ggcatcatgg
4980ttcagcgtgg cactcctttg gttgagtttg gggcatgaga tcacagtggc
tgcacaagag 5040agcagtgtgt acagtaggag agacatttat gtaatatata
ttttattaac ctgttagatg 5100tccacaaagt attataaatc acgtgcctaa
aactgtccat gtagaccaag gcctgccctc 5160ggcgcccccc actcttgcct
ctgctctgca c 519139989DNAHomo sapiens 39gcgcagatgc catcaaaatg
ggactctggt caccctgtca tttcccttct ggcagacact 60aaaatgggga gccctgccct
cagtggggtg tcccaagtgc catcagagga ggcttggtga 120ctcccagaca
caagggaagc tttagcgtct gccctcaggg tgaggtggag gtatcgcctc
180cggcctcagg gaaccacagt ctgaggggga gatgcagccc ctgccttccc
attcagagag 240gggttttgtg aggtggcttg ggggcatagg gcagaagtgg
atcctacagg ctgagctaag 300gccccaagag cctcagcagt gtacccatca
cctggcacct ctgcagccac agatccatga 360tgtgcagttc cctggagcag
gcgctggctg tgctggtcac taccttccac aagtactcct 420gccaagaggg
cgacaagttc aagctgagta aaggggaaat gaaggaactt ctgcacaagg
480agctgcccag ctttgtgggg gagaaagtgg atgaggaggg gctgaagaag
ctgatgggca 540acctggatga gaacagtgac cagcaggtgg acttccagga
gtatgctgtt ttcctggcgc 600tcatcactgt catgtgcaat gacttcttcc
agggctgccc agaccgaccc tgaagcagaa 660ctcttgactc cctgccatgg
atctcttggg cccaggactc tcgatgcctt tgagttttgt 720attcaataaa
ctttttttgt ctgttgataa tattttaatt gctcagtgac gttccataac
780ccgtctggct cagctggagt gctgggagat gagggcctcc tggatcctgc
tcccttctgg 840gctctgactc tcctggaaat ctctccaagg ccagagctat
gctttaggtc tcaattttgg 900aatttcaaac accagcaaaa aattggaaat
cgagataggt tgctgacttt tattttgtca 960aataaagata ttaaaaaagg caaatacca
9894012PRTHomo sapiens 40Cys Ala Thr Ser Lys Pro Ala Phe Phe Ala
Glu Lys1 5 104121PRTHomo sapiens 41Tyr Leu Cys Ala Ser Arg Pro Asp
Gly Ser Ser Gly Asn Thr Ile Tyr1 5 10 15Phe Gly Glu Gly
Ser204212PRTHomo sapiens 42Cys Ile Thr Pro Thr Gly Thr His Pro Leu
Ala Lys1 5 104310PRTHomo sapiens 43Phe Thr Tyr Ala Gly Cys Leu Ser
Val Lys1 5 104412PRTHomo sapiens 44Trp Leu Leu Thr Ala Ala His Cys
Leu Lys Pro Arg1 5 104514PRTHomo sapiens 45Gly Glu Leu Asp Cys His
Gln Leu Ala Asp Ser Phe Arg Glu1 5 104622PRTHomo sapiens 46Ser Cys
Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val1 5 10 15Pro
Asn Asp Leu Leu Lys204713PRTHomo sapiens 47Gly Ala Gln Thr Leu Tyr
Val Pro Asn Cys Asp His Arg1 5 104813PRTHomo sapiens 48Asn Gly Ile
Cys Val Ser Ser Asp Gln Asn His Phe Arg1 5 10499PRTHomo sapiens
49Glu Cys Ala Asp Glu Pro Val Gly Lys1 55011PRTHomo sapiens 50Leu
Leu Cys Gly Ala Thr Leu Ile Ala Pro Arg1 5 105110PRTHomo sapiens
51Tyr Ser Cys Gln Glu Gly Asp Lys Phe Lys1 5 10
* * * * *