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CA 02392757 2002-05-24
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Nucleic Acids, Proteins, and Antibodies
[001] This application refers to a "Sequence Listing" that is provided only on
electronic media in computer readable form pursuant to Administrative
Instructions
Section 801(a)(i). The Sequence Listing forms a part of this description
pursuant to
Rule 5.2 and Administrative Instructions Sections 801 to 806, and is hereby
incorporated in its entirety.
[002] The Sequence Listing is provided as an electronic file (PA112
seqList.txt,
78,041 bytes in size, created on January 12, 2001) on four identical compact
discs
(CD-R), labeled "COPY 1," "COPY 2," "COPY 3," and "CRF." The Sequence
Listing complies with Annex C of the Administrative Instructions, and may be
viewed,
for example, on an IBM-PC machine running the MS-Windows operating system by
using the V viewer software, version 2000 (see World Wide Web URL:
http : //www. fileviewer. com).
Field of the Inventiosz
[003] The present invention relates to novel pancreatic related
polynucleotides, the
polypeptides encoded by these polynucleotides herein collectively referred to
as
"pancreatic antigens," and antibodies that immunospecifically bind these
polypeptides,
and the use of such pancreatic polynucleotides, antigens, and antibodies for
detecting,
treating, preventing and/or prognosing disorders of the pancreas, including,
but not
limited to, the presence of pancreatic cancer and pancreatic cancer
metastases. More
specifically, isolated pancreatic nucleic acid molecules are provided encoding
novel
CA 02392757 2002-05-24
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pancreatic polypeptides. Novel pancreatic polypeptides and antibodies that
bind to
these polypeptides are provided. Also provided are vectors, host cells, and
recombinant and synthetic methods for producing human pancreatic
polynucleotides,
polypeptides, and/or antibodies. The invention further relates to diagnostic
and
therapeutic methods useful for diagnosing, treating, preventing and/or
prognosing
disorders related to the pancreas, including pancreatic cancer, and
therapeutic methods
for treating such disorders. The invention further relates to screening
methods for
identifying agonists and antagonists of polynucleotides and polypeptides of
the
invention. The invention further relates to methods and/or compositions for
inhibiting
or promoting the production and/or function of the polypeptides of the
invention.
Baehground of the Invention
[004] The pancreas is comprised of cells with two types of functions: 1)
exocrine, in
the secretion of digestive enzymes into the small intestine, and 2) endocrine,
in the
secretion of the hormones insulin and glucagon, which regulate blood glucose
levels.
Accordingly, the pancreas plays important roles in the Endocrine and
Gastrointestinal
Systems.
[005] Inflammation of the pancreas, or pancreatitis, is probably the most
common
disease of this organ. The disorder may be confined to either singular or
repeated acute
episodes, or it may become a chronic disease. There are many factors
associated with
the onset of pancreatitis, including direct injury, certain drugs, viral
infections,
heredity, hyperlipidemia (increased levels of blood fats), and congenital
derangements
of the ductal system. Localized, severe abdominal and midback pain resulting
from
enzyme leakage, tissue damage, and nerve irritation is the most common symptom
of
acute pancreatitis. In severe cases, respiratory failure, shock, and even
death may
occur. Chronic pancreatitis rarely follows repeated acute attacks. It seems
instead to be
a separate disorder that results in mucus plugs and precipitation of calcium
salts in the
smaller pancreatic ducts. Cystic fibrosis is inherited, but it is not
expressed unless both
members of a pair of hemologous, or corresponding, chromosomes carry the
trait. The
major functional abnormality in persons with the disease appears to be the
elaboration
by mucous glands throughout the body of secretions containing greater than
normal
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concentrations of protein and calcium. This imbalance leads to increased
viscosity of
the secretions and precipitation of mucus and organic constituents in gland
ducts. The
resulting plugging process in the pancreas almost invariably causes
destruction and
scarring of the acinar tissue, usually without damaging the islets of
Langerhans. A
similar process in the hepatic biliary system produces foci of fibrosis and
bile duct
proliferation, a singular form of cirrhosis.
[006] Islet cells of the pancreas synthesize and secrete insulin, the primary
hormone
responsible for controlling the uptake, utilization, and storage of cellular
nutrients. One
of the most common diseases of the endocrine system is diabetes mellitus. Most
patients can be classified as having either insulin-dependent diabetes
mellitus (type I
diabetes) or non-insulin-dependent diabetes mellitus (type II diabetes).
Virtually all
forms of diabetes mellitus are due to a decrease in the circulating
concentration of
insulin and a decrease in the response of peripheral tissues to insulin. These
abnormalities lead to alterations in the metabolism of charbohydrates, lipids,
ketones,
and amino acids; the central feature of the syndrome is hyperglycemia.
[007] Pancreatic cancer is the fourth leading cause of cancer death in the
United
States. According to the American Cancer Society, approximately 28,200 people
will
die of pancreatic cancer in the United States in 2000. The pancreas is a
tongue-shaped
glandular organ composed of both endocrine and exocrine gland portions, as
well as
ducts that connect the pancreas to the bile duct and small intestine. The
endocrine
portion of the pancreas secretes hormones, such as insulin and glucagon which
are
involved in blood sugar regulation, into the bloodstream. The exocrine portion
of the
pancreas produces pancreatic enzymes involved in the digestion of fats and
proteins;
these enzymes are delivered to the bile duct and into the small intestine.
[008] Carcinoma of the pancreas arises primarily from the ductal system. The
exocrine cells and endocrine cells of the pancreas form completely different
types of
tumors. These tumors can be either benign (noncancerous) or malignant
(cancerous).
Exocrine cells of the pancreas can form benign tumors, although these are much
less
common than cancers. Most of these benign tumors are cystadenomas. About 95%
of
cancers of the exocrine pancreas are adenocarcinomas. These adenocarcinornas
usually
begin in the ducts of the pancreas, but sometimes may develop from the acinar
cells of
the exocrine glands (the cells that actually produce the pancreatic enzymes).
Less
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common cancers of the exocrine pancreas include adenosquamous carcinomas,
squamous cell carcinomas, and giant cell carcinomas. These types are
distinguished
from one another based on their appearance under the microscope. Treatment of
an
exocrine pancreatic cancer is rn. ostly based on how far it metastasized,
however, and
not its exact type. Tumors of the endocrine pancreas are much less common. As
a
group, they are known as neuroendocrine tumors, or more specifically, islet
cell
tumors. There are several subtypes of islet cell tumors that are named
according to the
type of hormone they produce. Islet cell tumors that produce insulin are known
as
insulinomas, and tumors that produce glucagon are called glucagonomas. Less
often,
islet cell tumors may produce other hormones. Most islet cell tumors are
benign. Some
are malignant and are called islet cell cancers or islet cell carcinomas.
[009] The incidence of carcinoma of the pancreas has increased slightly
(somewhat
more in men than in woman) and now exceeds cancer of the stomach. No certain
risk
factors have been identified, although suggestions have been made that
pancreatic
cancer occurs at increased rates among diabetics and,persons with chronic
pancreatitis.
Pancreatic cancer is one of the most dangerous cancers, killing half its
victims within 6
weelcs and having a 5-year survival rate of only 4%. The diagnosis of
pancreatic
carcinoma is often associated with a poor prognosis, because. most patients
already
have advanced disease. Radiation and chemotherapy have shown some promise as
therapeutic agents if they are started promptly in the course of the disease
and
continued for long periods. Despite the many advances reported during the past
few
years, pancreatic cancer remains a profound therapeutic challenge. It is hoped
that the
increasing knowledge of the molecular biology of pancreatic carcinoma will
lead to
improvements in diagnosing, staging, and treating pancreatic adenocarcinoma
(Brand
et al., Curr Opin Oncol 10:362-6 (1998)).
[010] There is a need, therefore, for identification and characterization of
factors that
modulate activation and differentiation of pancreatic cells, both normally and
in
disease states. In particular, there is a need to isolate and characterize
additional
molecules that mediate apoptosis, DNA repair, tumor-mediated angiogenesis,
genetic
imprinting, immune responses to tumors and tumor antigens and, among other
things,
that can play a role in detecting, preventing, ameliorating or correcting
dysfunctions or
diseases related to the pancreas.
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[011] The discovery of new human pancreatic associated polynucleotides, the
polypeptides encoded by them, and antibodies that immunospecifically bind
these
polypeptides, satisfies a need in the art by providing new compositions which
are
useful in the diagnosis, treatment, prevention and/or prognosis of disorders
of the
pancreas, including, but not limited to, diabetes mellitus, diabetes
insipidus, congenital
pancreatic agenesis, pancreatic cancers (e.g., benign or malignant forms of
pancreatic
cancer, as well as any particular type of cancer arising from cells of the
pancreas (e.g.,
duct cell carcinoma, acinar cell carcinoma, papillary carcinoma, adenosquamous
carcinoma, undifferentiated carcinoma, mucinous carcinoma, giant cell
carcinoma,
mixed type pancreatic cancer, small cell carcinoma, cystadenocarcinoma,
unclassified
pancreatic cancers, pancreatoblastoma, adenocarcinoma, islet-cell tumors,
cystic
neoplasms, and papillary-cyctic neoplasm and the like), as well as any stage
of such
cancers (e.g., stages I to IV in severity)), cystic fibrosis, cyst (e.g.,
pancreatic
pseudocyst), pancreatic fistula, insufficiency, pancreatic dysplasia,
pancreatitis (e.g.,
chronic pancreatitis, acute pancreatitis, acute necrotizing pancreatitis,
alcoholic
pancreatitis, and pancreatic abscesses associated with pancreatic
inflammation), and/or
those disorders as described under "Endocrine Disorders" and/or
"Gastrointestinal
Disorders" below.
Summary of the Ihvehtion
[012] The present invention relates to novel pancreatic related
polynucleotides, the
polypeptides encoded by these polynucleotides herein collectively referred to
as
"pancreatic antigens," and antibodies that immunospecifically bind these
polypeptides,
and the use of such pancreatic polynucleotides, antigens, and antibodies for
detecting,
treating, preventing and/or prognosing disorders of the pancreas, including,
but not
limited to, the presence of pancreatic cancer and pancreatic cancer
metastases. More
specifically, isolated pancreatic nucleic acid molecules are provided encoding
novel
pancreatic polypeptides. Novel pancreatic polypeptides and antibodies that
bind to
these polypeptides are provided. Also provided are vectors, host cells, and
recombinant and synthetic methods for producing human pancreatic
polynucleotides,
S
CA 02392757 2002-05-24
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polypeptides, and/or antibodies. The invention further relates to diagnostic
and
therapeutic methods useful for diagnosing, treating, preventing and/or
prognosing
disorders related to the pancreas, including pancreatic cancer, and
therapeutic methods
for treating such disorders. The invention further relates to screening
methods for
identifying agonists and antagonists of polynucleotides and polypeptides of
the
invention. The invention further relates to methods and/or compositions for
inhibiting
or promoting the production and/or function of the polypeptides of the
invention.
Detailed Desc~iptioh
Tables
[013] Table 1A summarizes some of the polynucleotides encompassed by the
invention (including cDNA clones related to the sequences (Clone ID NO:Z),
contig
sequences (contig identifier (Contig ID:) and contig nucleotide sequence
identifier
(SEQ ID NO:X)) and further summarizes certain characteristics of these
polynucleotides and the polypeptides encoded thereby. The first column
provides a
unique clone identifier, "Clone ID NO:Z", for a cDNA plasmid related to each
pancreatic associated contig sequence disclosed in Table 1A. The second column
provides a unique contig identifier, "Contig ID:" for each of the contig
sequences
disclosed in Table 1A. The third column provides the sequence identifier, "SEQ
ID
NO:X", for each of the contig polynucleotide sequences disclosed in Table 1A.
The
fourth column, "ORF (From-To)", provides the location (i.e., nucleotide
position
numbers) within the polynucleotide sequence of SEQ ID NO:X that delineate the
preferred open reading frame (ORF) shown in the sequence listing and
referenced in
Table 1A as SEQ ID NO:Y (column 5). Columm 6 lists residues comprising
predicted
epitopes contained in the polypeptides encoded by each of the preferred ORFs
(SEQ
ID NO:Y). Identification of potential immunogenic regions was performed
according
to the method of Jameson and Wolf (CABIOS, 4:181-186 (1988)); specifically,
the
Genetics Computer Group (GCG) implementation of this algorithm, embodied in
the
program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer
Group (GCG), Madison, Wisc.). This method returns a measure of the probability
that
a given residue is found on the surface of the protein. Regions where the
antigenic
index score is greater than 0.9 over at least 6 amino acids are indicated in
Table 1A as
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"Predicted Epitopes." In particular embodiments, pancreatic associated
polypeptides of
the invention comprise, or alternatively consist of, one, two, three, four,
five or more
of the predicted epitopes described in Table 1A. It will be appreciated that
depending
on the analytical criteria used to predict antigenic determinants, the exact
address of
the detemninant may vary slightly. Column 7, "Tissue Distribution" shows the
expression profile of tissue, cells, and/or cell line libraries which express
the
polynucleotides of the invention. The first number in column 7 (preceding the
colon),
represents the tissue/cell source identifier code corresponding to the code
and
description provided in Table 4. Expression of these polynucleotides was not
observed
in the other tissues and/or cell libraries tested. For those identifier codes
in which the
first two letters are not "AR", the second number in column 7 (following the
colon),
represents the number of times a sequence corresponding to the reference
polynucleotide sequence (e.g., SEQ ID NO:X) was identified in the tissue/cell
source.
Those tissue/cell source identifier codes in which the first two letters are
"AR"
designate information generated using DNA array technology. Utilizing this
technology, cDNAs were amplified by PCR and then transferred, in duplicate,
onto the
array. Gene expression was assayed through hybridization of first strand cDNA
probes
to the DNA array. cDNA probes were generated from total RNA extracted from a
variety of different tissues and cell lines. Probe synthesis was performed in
the
presence of 33P dCTP, using oligo(dT) to prime reverse transcription. After
hybridization, high stringency washing conditions were employed to remove non-
specific hybrids from the array. The remaining signal, emanating from each
gene
target, was measured using a Phosphorimager. Gene expression was reported as
Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor
signal
generated from the probe hybridized to each of the gene targets represented on
the
array. A local background signal subtraction was performed before the total
signal
generated from each array was used to normalize gene expression between the
different hybridizations. The value presented after "[array code] :"
represents the mean
of the duplicate values, following background subtraction and probe
normalization.
One of skill in the art could routinely use this information to identify
normal and/or
diseased tissues) which show a predominant expression pattern of the
corresponding
polynucleotide of the invention or to identify polynucleotides which show
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predominant and/or specific tissue and/or cell expression. Column 8,
"Cytologic
Band," provides the chromosomal location of polynucleotides corresponding to
SEQ
ID NO:X. Chromosomal location was determined by finding exact matches to EST
and cDNA sequences contained in the NCBI (National Center for Biotechnology
Information) UniGene database. Given a presumptive chromosomal location,
disease
locus association was determined by comparison with the Morbid Map, derived
from
Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man,
OMIMTM. McI~usick-Nathans Institute for Genetic Medicine, Johns Hopkins
University (Baltimore, MD) and National Center for Biotechnology Information,
National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of
the
Query overlapped with the chromosomal location of a Morbid Map entry, an OMIM
identification number is provided in column 9 labeled "OMIM Disease
References)".
A key to the OMIM reference identification numbers is provided in Table 5.
[014] Table 1B summarizes additional polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone ID NO:Z), contig
sequences
(contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID
NO:X)),
and genomic sequences (SEQ ID NO:B). The first column provides a unique clone
identifier, "Clone ID NO:Z", for a cDNA clone related to each contig sequence.
The
second column provides the sequence identifier, "SEQ ID NO:X", for each contig
sequence. The third column provides a unique contig identifier, "Contig ID:"
for each
contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A"
for
the BAC clone referenced in the corresponding row of the table. The fifth
column
provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of
the
BAC clone identified in column four of the corresponding row of the table. The
sixth
column, "Exon From-To", provides the location (i.e., nucleotide position
numbers)
within the polynucleotide sequence of SEQ ID NO:B which delineate certain
polynucleotides of the invention that are also exemplary members of
polynucleotide
sequences that encode polypeptides of the invention (e.g., polypeptides
containing
amino acid sequences encoded by the polynucleotide sequences delineated in
column
six, and fragments and variants thereof).
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[015] Table 2 summarizes homology and features of some of the polypeptides of
the
invention. The first column provides a unique clone identifier, "Clone ID
NO:Z",
corresponding to a cDNA disclosed in Table IA. The second column provides the
unique contig identifier, "Contig ID:" corresponding to contigs in Table 1A
and
allowing for correlation with the information in Table 1A. The third column
provides
the sequence identifier, "SEQ ID NO:X", for the contig polynucleotide
sequences.
The fourth column provides the analysis method by which the homology/identity
disclosed in the row was determined. Comparisons were made between
polypeptides
encoded by the polynucleotides of the invention and either a non-redundant
protein
database (herein referred to as "NR"), or a database of protein families
(herein referred
to as "PFAM") as further described below. The fifth column provides a
description of
PFAM/NR hits having significant matches to a polypeptide of the invention.
Column
six provides the accession number of the PFAM/NR hit disclosed in the fifth
column.
Column seven, "Score/Percent Identity", provides a quality score or the
percent
identity, of the hit disclosed in column five. Columns 8 and 9, "NT From" and
"NT
To" respectively, delineate the polynucleotides in "SEQ ID NO:X" that encode a
polypeptide having a significant match to the PFAM/NR database as disclosed in
the
fifth column. In specific embodiments, polypeptides of the invention comprise,
or
alternatively consist of, an amino acid sequence encoded by the
polynucleotides in
SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants
thereof.
[016] Table 3 provides polynucleotide sequences that may be disclaimed
according
to certain embodiments of the invention. The first column provides a unique
clone
identifier, "Clone ID NO:Z", for a cDNA clone related to pancreatic associated
contig
sequences disclosed in Table 1A. The second column provides the sequence
identifier,
"SEQ ID NO:X", for contig polynucleotide sequences disclosed in Table 1A. The
third column provides the unique contig identifier, "Contig ID", for contigs
disclosed
in Table 1A. The fourth column provides a unique integer 'a' where 'a' is any
integer
between 1 and the final nucleotide minus 15 of SEQ ID NO:X, represented as
"Range
of a", and the fifth column provides a unique integer 'b' where 'b' is any
integer
between 15 and the final nucleotide of SEQ ID NO:X, represented as "Range of
b",
where both a and b correspond to the positions of nucleotide residues shown in
SEQ
ID NO:X, and where b is greater than or equal to a + 14. For each of the
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polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be
substituted into the general formula of a~b, and used to describe
polynucleotides which
may be preferably excluded from the invention. In certain embodiments,
preferably
excluded from the polynucleotides of the invention (including polynucleotide
fragments and variants as described herein and diagnostic and/or therapeutic
uses
based on these polynucleotides) are at least one, two, three, four, five, ten,
or more of
the polynucleotide sequences) having the accession numbers) disclosed in the
sixth
column of this Table (including for example, published sequence in connection
with a
particular BAC clone). In further embodiments, preferably excluded from the
invention are the specific polynucleotide sequences) contained in the clones
corresponding to at least one, two, three, four, five, ten, or more of the
available
material having the accession numbers identified in the sixth column of this
Table
(including for example, the actual sequence contained in an identified BAC
clone).
[017] Table 4 provides a key to the tissue/cell source identifier code
disclosed in
Table 1A, column 7. Column 1 provides the key to the tissue/cell source
identifier
code disclosed in Table 1A, Column 7. Columns 2-5 provide a description of the
tissue
or cell source. Codes corresponding to diseased tissues are indicated in
column 6 with
the word "disease". The use of the word "disease" in column 6 is non-limiting.
The
tissue or cell source may be specific (e.g. a neoplasm), or may be disease-
associated
(e.g., a tissue sample from a normal portion of a diseased organ).
Furthermore, tissues
and/or cells lacking the "disease" designation may still be derived from
sources
directly or indirectly involved in a disease state or disorder, and therefore
may have a
further utility in that disease state or disorder. In numerous cases where the
tissue/cell
source is a library, column 7 identifies the vector used to generate the
library.
[018] Table 5 provides a key to the OMIMTM reference identification numbers
disclosed in Table 1A, column 9. OMIM reference identification numbers (Column
1)
were derived from Online Mendelian Inheritance in Man (Online Mendelian
Inheritance in Man, OMIMTM. McI~usick-Nathans Institute for Genetic Medicine,
Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology
Information, National Library of Medicine, (Bethesda, MD) 2000. World Wide Web
URL: http://www.ncbi.nlm.nih.gov/omim~. Column 2 provides diseases associated
with the cytologic band disclosed in Table 1A, column S, as determined from
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Morbid Map database.
[019] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation
numbers of deposits made With the ATCC in connection with the present
application.
[020] Table 7 shows the cDNA libraries sequenced, tissue source description,
vector
information and ATCC designation numbers relating to these cDNA libraries.
[021] Table S provides a physical characterization of clones encompassed by
the
invention. The first column provides the unique clone identifier, "Clone ID
NO:Z",
for certain cDNA clones of the invention, as described in Table 1A. The second
column provides the size of the cDNA insert contained in the corresponding
cDNA
clone.
Definitions
[022] The following definitions are provided to facilitate understanding of
certain
terms used throughout this specification.
[023] In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide. The
term "isolated" does not refer to genomic or ~cDNA libraries, whole cell total
or mRNA
preparations, genomic DNA preparations (including those separated by
electrophoresis
and transferred onto blots), sheared whole cell genomic DNA preparations or
other
compositions where the art demonstrates no distinguishing features of the
polynucleotide sequences of the present invention.
[024] As used herein, a "polynucleotide" refers to a molecule having a nucleic
acid
sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid
sequence contained in SEQ ID NO:X (as described in column 3 of Table 1A) or
the
complement thereof; or cDNA sequence contained in Clone ID NO:Z (as described
in
column 1 of Table 1A and contained within a library deposited with the ATCC);
a
nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence
in
SEQ ID NO:B as defined in column 6 of Table 1B or a fragment or variant
thereof; or
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a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1B
or
the complement thereof. For example, the polynucleotide can contain the
nucleotide
sequence of the full length cDNA sequence, including the 5' and 3'
untranslated
sequences, the coding region, as well as fragments, epitopes, domains, and
variants of
the nucleic acid sequence. Moreover, as used herein, a "polypeptide" refers to
a
molecule having an amino acid sequence encoded by a polynucleotide of the
invention
as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine
peptide
sequences which result from translation of a polyA tail of a sequence
corresponding to
a cDNA).
[025] As used herein, a "pancreatic antigen" refers collectively to any
polynucleotide
disclosed herein (e.g., a nucleic acid sequence contained in SEQ ID NO:X or
the
complement thereof, or cDNA sequence contained in Clone ID NO:Z, or a
nucleotide
sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID
NO:B as defined in column 6 of Table 1B, or a nucleotide coding sequence in
SEQ ID
NO:B as defined in column 6 of Table 1B or the complement thereof and
fragments or
variants thereof as described herein) or any polypeptide disclosed herein
(e.g., an
amino acid sequence contained in SEQ ID NO:Y, an amino acid sequence encoded
by
SEQ ID NO:X, or the complement thereof, an amino acid sequence encoded by the
cDNA sequence contained in Clone ID NO:Z, an amino acid sequence encoded by
SEQ ID NO:B, or the complement thereof, and fragments or variants thereof as
described herein). These pancreatic antigens have been determined to be
predominantly expressed in pancreatic tissues, including normal or diseased
tissues (as
shown in Table 1A column 7 and Table 4).
[026] In the present invention, "SEQ ID NO:X" was often generated by
overlapping
sequences contained in multiple clones (contig analysis). A representative
clone
containing all or most of the sequence for SEQ ID NO:X is deposited at Human
Genome Sciences, Inc. (HGS) in a catalogued and archived library. As shown,
for
example, in column 1 of Table 1A, each clone is identified by a cDNA Clone ID
(identifier generally referred to herein as Clone ID NO:Z). Each Clone ID is
unique to
an individual clone and the Clone ID is all the information needed to retrieve
a given
clone from the HGS library. Furthermore, certain clones disclosed in this
application
have been deposited with the ATCC on October 5, 2000, having the ATCC
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designation numbers PTA 2574 and PTA 2575; and on January 5, 2001, having the
depositor reference numbers TS-1, TS-2, AC-l, and AC-2. In addition to the
individual cDNA clone deposits, most of the cDNA libraries from which the
clones
were derived were deposited at the American Type Culture Collection
(hereinafter
"ATCC"). Table 7 provides a list of the deposited cDNA libraries. One can use
the
Clone ID NO:Z to determine the library source by reference to Tables 6 and 7.
Table
7 lists the deposited cDNA libraries by name and links each library to an ATCC
Deposit. Library names contain four characters, for example, "HTWE." The name
of a
cDNA clone (Clone ID NO:Z) isolated from that library begins with the same
four
characters, for example "HTWEP07". As mentioned below, Table 1A correlates the
Clone ID NO:Z names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one
can use Tables l, 6 and 7 to determine the corresponding Clone ID NO:Z, which
library it came from and which ATCC deposit the library is contained in.
Furthermore,
it is possible to retrieve a given cDNA clone from the source library by
techniques
known in the art and described elsewhere herein. The ATCC is located at 10801
University Boulevard, Manassas, Virginia 20110-2209, USA. The ATCC deposits
were made pursuant to the terms of the Budapest Treaty on the international
recognition
[027] In specific embodiments, the polynucleotides of the invention are at
least 15, at
least 30, at least 50, at least 100, at least 125, at least 500, or at least
1000 continuous
nucleotides but are less than or equal to 300 lcb, 200 kb, .100 kb, 50 kb, 15
kb, 10 kb,
7.5 kb, 5 lcb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment,
polynucleotides of the invention comprise a portion of the coding sequences,
as
disclosed herein, but do not comprise all or a portion of any intron. In
another
embodiment, the polynucleotides comprising coding sequences do not contain
coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest
in the
genome). In other embodiments, the polynucleotides of the invention do not
contain
the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,
4, 3, 2, or
1 genomic flanking gene(s).
[028] A "polynucleotide" of the present invention also includes those
polynucleotides
capable of hybridizing, under stringent hybridization conditions, to sequences
contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of
any
13
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WO 01/55327 PCT/USO1/01352
one, two, three, four, or more of the polynucleotide fragments described
herein), the
polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the
complement
thereof, and/or cDNA sequences contained in Clone ID NO:Z (e.g., the
complement of
any one, two, three, four, or more of the polynucleotide fragments, or the
cDNA clone
within the pool of cDNA clones deposited with the ATCC, described herein)
and/or
the polynucleotide sequence delineated in column 6 of Table 1B or the
complement
thereof. "Stringent hybridization conditions" refers to an overnight
incubation at 42
degree C in a solution comprising 50% fonnamide~ Sx SSC (750 mM NaCI, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx Denhardt's solution,
10%
dextran sulfate, and 20 ~.g/ml denatured, sheared salmon sperm DNA, followed
by
washing the filters in O.lx SSC at about 65 degree C.
[029] Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency), salt conditions, or
temperature. For example, lower stringency conditions include an overnight
incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M
NaCI;
0.2M NaH2P04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE, 0.1%
SDS. In addition, to achieve even lower stringency, washes performed following
stringent hybridization can be done at higher salt concentrations (e.g. 5X
SSC).
[030] Note that variations in the above conditions may be accomplished through
the
inclusion and/or substitution of alternate blocking reagents used to suppress
background in hybridization experiments. Typical blocking reagents include
Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and
commercially available proprietary formulations. The inclusion of specific
blocking
reagents may require modification of the hybridization conditions described
above,
due to problems with compatibility.
[031] Of course, a polynucleotide which hybridizes only to polyA+ sequences
(such
as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or
to a
complementary stretch of T (or U) residues, would not be included in the
definition of
14
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WO 01/55327 PCT/USO1/01352
"polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid
molecule containing a poly (A) stretch or the complement thereof (e.g.,
practically any
double-stranded cDNA clone generated using oligo dT as a primer).
[032] The polynucleotide of the present invention can be composed of any
polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or
DNA or modified RNA or DNA. For example, polynucleotides can be composed of
single- and double-stranded DNA, DNA that is a mixture of single- and double-
stranded regions, single- and double-stranded RNA, and RNA that is mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and RNA
that
may be single-stranded or, more typically, double-stranded or a mixture of
single- and
double-stranded regions. In addition, the polynucleotide can be composed of
triple-
stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide
may also contain one or more modified bases or DNA or RNA backbones modified
for
stability or for other reasons. "Modified" bases include, for example,
tritylated bases
and unusual bases such as inosine. A variety of modifications can be made to
DNA
and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or
metabolically modified forms.
[033] The polypeptide of the present invention can be composed of amino acids
joined to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres,
and may contain amino acids other than the 20 gene-encoded amino acids. The
polypeptides may be modified by either natural processes, such as
posttranslational
processing, or by chemical modification techniques which are well known in the
art.
Such modifications are well described in basic texts and in more detailed
monographs,
as well as in a voluminous research literature. Modifications can occur
anywhere in a
polypeptide, including the peptide backbone, the amino acid side-chains and
the amino
or carboxyl termini. It will be appreciated that the same type of modification
may be
present in the same or varying degrees at several sites in a given
polypeptide. Also, a
given polypeptide may contain many types of modifications. Polypeptides may be
branched, for example, as a result of ubiquitination, and they may be cyclic,
with or
without branching. Cyclic, branched, and branched cyclic polypeptides may
result
from posttranslation natural processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation,
covalent
CA 02392757 2002-05-24
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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 cross-links, formation of
cysteine,
formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI
anchor formation, hydroxylation, iodination, methylation, myristoylation,
oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins
such as arginylation, and ubiquitination. (See, for instance, PROTEINS -
STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New Yorlc,
pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et
al.,
Ann. N.Y. Acad. Sci. 663:48-62 (1992).)
[034] "SEQ ID NO:X" refers to a polynucleotide sequence described, for
example, in
Tables 1 or 2, while "SEQ ID NO:Y" refers to a polypeptide sequence described
in
column 5 of Table 1A. SEQ ID NO:X is identified by an integer specified in
column 3
of Table 1A. The polypeptide sequence SEQ ID NO:Y is a translated open reading
frame (ORF) encoded by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a
cDNA clone described in column 1 of Table 1A.
[035] "A polypeptide having biological activity" refers to a polypeptide
exhibiting
activity similar to, but not necessarily identical to, an activity of a
polypeptide of the
present invention, including mature forms, as measured in a particular
biological
assay, with or without dose dependency. In the case where dose dependency does
exist, it need not be identical to that of the polypeptide, but rather
substantially similar
to the dose-dependence in a given activity as compared to the polypeptide of
the
present invention (i.e., the candidate polypeptide will exhibit greater
activity or not
more than about 25-fold less and, preferably, not more than about tenfold less
activity,
and most preferably, not more than about three-fold less activity relative to
the
polypeptide of the present invention).
[036] Table 1A summarizes some of the polynucleotides encompassed by the
invention (including contig sequences (SEQ ID NO:X) and clones (Clone ID NO:Z)
16
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WO 01/55327 PCT/USO1/01352
and further summarizes certain characteristics of these polynucleotides and
the
polypeptides encoded thereby.
~ptides
Polynucleotides and Pol
TABLE 1A
17
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
OA ~
,
..
bA .~
0
y, Pa
v
r", ,--~ ~ ..o ,-;,--,--,-;
o ~ o . ~ .~0000.. oo Ov
x. l~ - ool~l~~~ d' M
tV d1
V ~ p , V1M M Ol ~
C~ ,
lfl .-a
G~ f,~"~~ ~ I~ ~ o ~ l~ ~ r~
O ~ O
~ ~ ..o ~ o M ,~ ,..d
V ~ 0 00 ~ '~"0,~
0
Od~
ed ~ N l~ c~'i N N ..-~c ,--i,~ ,--i
.. . ,-~~
e~ y
.~ ~ ~ O ~ O
Q ran N ~ ~ ~ V'i ~ ~ Ov
a O~
~~
, ~ O ~Oo~o~ O 0 0 0 0~ ~ O
, .
~ r~
O
M
i O
O
~~
V O ~ O
O
O d.
N
G7
N N N N N N N N
N
z
N ~ ~ N ~ ~ N N N
Q ~ ~ y n
~ ~ N M N ~
1 ~
0
~ N M dw n ~ ~ oo a1 O
~z
~ N
N m ~ ~
M l~M V~ V7O~Old" ~7 O
O
v
a o ~ ~ 0 0 0
~ N ~qw H ~ ~ H w ~ w w
00 d ~ d
Uz x ~ ~ ~ ~ ~ ~ ~ x x
1$
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
[037] The first column in Table 1A provides a unique "Clone ID NO:Z" for a
cDNA
clone related to each contig sequence disclosed in Table 1A. This clone ID
references
the cDNA clone which contains at least the 5' most sequence of the assembled
contig,
and at least a portion of SEQ ID NO:X was determined by directly sequencing
the
referenced clone. The reference clone may have more sequence than described in
the
sequence listing or the clone may have less. In the vast majority of cases,
however, the
clone is believed to encode a full-length polypeptide. In the case where a
clone is not
full-length, a full-length cDNA can be obtained by methods known in the art
and/or as
described elsewhere herein.
[038] The second column in Table 1A provides a unique "Contig ID"
identification
for each contig sequence. The third column provides the "SEQ ID NO:X"
identifier
for each of the pancreatic associated contig polynucleotide sequences
disclosed in
Table 1A. The fourth column, "ORF (From-To)", provides the location (i.e.,
nucleotide position numbers) within the polynucleotide sequence "SEQ ID NO:X"
that
delineate the preferred open reading frame (ORF) shown in the sequence listing
and
referenced in Table 1A, column 5, as SEQ ID NO:Y. Where the nucleotide
position
number "To" is lower than the nucleotide position number "From", the preferred
ORF
is the reverse complement of the referenced polynucleotide sequence.
[039] The fifth column in Table 1A provides the corresponding SEQ ID NO:Y for
the polypeptide sequence encoded by the preferred ORF delineated in column 4.
In
one embodiment, the invention provides an amino acid sequence comprising, or
alternatively consisting of, a polypeptide encoded by the portion of SEQ ID
NO:X
delineated by "ORF (From-To)". Also provided are polynucleotides encoding such
amino acid sequences and the complementary strand thereto.
[040] Column 6 in Table 1A lists residues comprising epitopes contained in the
polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using
the
algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The
Jameson-Wolf antigenic analysis was performed using the computer program
PROTEAN (Version 3.11 for the Power Macintosh, DNASTAR, Inc., 1228 South
Park Street Madison, WI). In specific embodiments, polypeptides of the
invention
comprise, or alternatively consist of, at least one, two, three, four, five or
more of the
19
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
predicted epitopes as described in Table 1A. It will be appreciated that
depending on
the analytical criteria used to predict antigenic determinants, the exact
address of the
determinant may vary slightly.
[041] Column 7 in Table 1A provides an expression profile and library code:
count
for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which
can
routinely be combined with the information provided in Table 4 and used to
determine
the normal or diseased tissues, cells, and/or cell line libraries which
predominantly
express the polynucleotides of the invention. The first number in column 7
(preceding
the colon), represents the tissue/cell source identifier code corresponding to
the code
and description provided in Table 4. For those identifier codes in which the
first two
letters are not "AR", the second number in column 7 (following the colon)
represents
the number of times a sequence corresponding to the reference polynucleotide
sequence was identified in the tissue/cell source. Those tissue/cell source
identifier
codes in which the first two letters are "AR" designate information generated
using
DNA array technology. Utilizing this technology, cDNAs were amplified by PCR
and
then transferred, in duplicate, onto the array. Gene expression was assayed
through
hybridization of first strand cDNA probes to the DNA array. cDNA probes were
generated from total RNA extracted from a variety of different tissues and
cell lines.
Probe synthesis was performed in the presence of 33P dCTP; using oligo(dT) to
prime
reverse transcription. After hybridization, high stringency washing conditions
were
employed to remove non-specific hybrids from the aiTay. The remaining signal,
emanating from each gene target, was measured using a Phosphorimager. Gene
expression was reported as Phosphor Stimulating Luminescence (PSL) which
reflects
the level of phosphor signal generated from the probe hybridized to each of
the gene
targets represented on the array. A local background signal subtraction was
performed
before the total signal generated from each array was used to normalize gene
expression between the different hybridizations. The value presented after
"[array
code]:" represents the mean of the duplicate values, following background
subtraction
and probe normalization. One of skill in the art could routinely use this
information to
identify normal and/or diseased tissues) which show a predominant expression
pattern
of the corresponding polynucleotide of the invention or to identify
polynucleotides
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
which show predominant andlor specific tissue and/or cell expression. The
sequences
disclosed herein have been determined to be predominantly expressed in
pancreatic
tissues, including normal and diseased pancreatic tissues (See Table 1A,
column 7 and
Table 4).
[042] Column 8 in Table 1A provides a chromosomal map location for certain
polynucleotides of the invention. Chromosomal location was determined by
finding
exact matches to EST and cDNA sequences contained in the NCBI (National Center
for Biotechnology Information) UniGene database. Each sequence in the UniGene
database is assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a
cluster are
believed to be derived from a single gene. Chromosomal mapping data is often
available for one or more sequences) in a UniGene cluster; this data (if
consistent) is
then applied to the cluster as a whole. Thus, it is possible to infer the
chromosomal
location of a new polynucleotide sequence by determining its identity with a
mapped
UniGene cluster.
[043] A modified version of the computer program BLASTN (Altshul et al., J.
Mol.
Biol. 215:403-410 (1990), and Gish et al., Nat. Genet. 3:266-272 (1993)) was
used to
search the UniGene database for EST or cDNA sequences that contain exact or
near-
exact matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence
from the UniGene database (the 'Subject') was said to be an exact match if it
contained a segment of 50 nucleotides in length such that 48 of those
nucleotides were
in the same order as found in the Query sequence. If all of the matches that
met this
criteria were in the same UniGene cluster, and mapping data was available for
this
cluster, it is indicated in Table 1A under the heading "Cytologic Band". Where
a
cluster had been further localized to a distinct cytologic band, that band is
disclosed;
where no banding information was available, but the gene had been localized to
a
single chromosome, the chromosome is disclosed.
[044] Once a presumptive chromosomal location was determined for a
polynucleotide of the invention, an associated disease locus was identified by
comparison with a database of diseases which have been experimentally
associated
with genetic loci. The database used was the Morbid Map, derived from OMIMTM
(supra). If the putative chromosomal location of a polynucleotide of the
invention
21
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(Query sequence) was associated with a disease in the Morbid Map database, an
OMIM reference identification number was noted in column 9, Table 1A, labeled
"OMIM Disease References)". Table 5 is a key to the OMIM reference
identification
numbers (column 1), and provides a description of the associated disease in
Column
2.
TABLE 1B
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: NO:B From-To
HVAET61 13 965365 AC009524 31 I-151
HVAET61 13 965365 AC025036 32 1-148
HVAET61 13 965365 AC027300 33 1-158
HVAET61 13 965365 AC022232 34 1-152
HVAET61 13 965365 AC016327 35 1-165
HVAET61 13 965365 AC048342 36 1-130
HVAET61 13 965365 AC016327 37 1-195
HVAET61 13 965365 AC048342 38 1-118
HVAFD06 14 933527 AL118505 39 1-1166
HVAHA.06 15 933531 AC011290 40 1-145
552-865
1223-1358
3157-3930
3934-4305
4523-6836
HVAHA06 15 933531 AC011290 41 1-393
HVAETO1 16 913958 AL133243 42 1-428
HVAETOI 16 913958 AL133243 43 1-218
1713-1892
2799-2896
3039-3242
4416-4668
5011-5516
6251-6667
7174-7305
1
HVAETO1 16 913958 AL133243 44 1-502
891-997
3296-3521
5357-6137
7436-7920
8616-8765
9182-9311
22
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10530-11068
11184-11626
12025-14398
14692-15155
15250-15383
15828-15994
17708-19731
19976-20476
21720-21860
HVACY04 18 926473 AC073253 45 1-478
568-1003
HVACY04 18 926473 AC073253 46 ~ 1-547
~
[045] Table 1B summarizes additional polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone ID NO:Z), contig
sequences
(contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID
NO:X)),
and genomic sequences (SEQ ID NO:B). The first column provides a unique clone
identifier, "Clone TD NO:Z", for a cDNA clone related to each contig sequence.
The
second column provides the sequence identifier, "SEQ ID NO:X", for each contig
sequence. The third column provides a unique contig identifier, "Contig ID:"
for each
contig sequence. The fourth column, provides a BAC identifier "BAC ID NO:A"
for
the BAC clone referenced in the corresponding row of the table. The fifth
column
provides the nucleotide sequence identifier, "SEQ ID NO:B" for a fragment of
the
BAC clone identified in column four of the corresponding row of the table. The
sixth
column, "Exon From-To", provides the location (i.e., nucleotide position
numbers)
within the polynucleotide sequence of SEQ ID NO:B which delineate certain
polynucleotides of the invention that are also exemplary members of
polynucleotide
sequences that encode polypeptides of the invention (e.g., polypeptides
containing
amino acid sequences encoded by the polynucleotide sequences delineated in
column
six, and fragments and variants thereof).
TABLE 2
23
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
o ~n
H o,
E~
z
M
O
O
zw
N
N
d'
~G7 ~ +;
x, V
H
C~
O
."
m
c~
O
O
wz
0
;~ m
A
U
P~
~~' o
d' ~ x
a
M
O
U A
~N
U ~
24
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
[046j Table 2 further characterizes certain encoded polypeptides of the
invention, by
providing the results of comparisons to protein and protein family databases.
The first
column provides a unique clone identifier, "Clone ID NO:", corresponding to a
cDNA
clone disclosed in Table 1A. The second column provides the unique contig
indentifier, "Contig ID:" which allows correlation with the information in
Table 1A.
The third column provides the sequence identifier, "SEQ ID NO:X", for the
contig
polynucleotide sequences. The fourth column provides the analysis method by
which
the homology/identity disclosed in the row was determined. The fifth column
provides a description of PFam/NR hits having significant matches identified
by each
analysis. Column six provides the accession number of the PFam/NR hit
disclosed in
the fifth column. Column seven, "Score/Percent Identity", provides a quality
score or
the percent identity, of the hit disclosed in column five. Comparisons were
made
between polypeptides encoded by polynucleotides of the invention and a non-
redundant protein database (herein referred to as "NR"), or a database of
protein
families (herein referred to as "PFam"), as described below.
[047j The NR database, which comprises the NBRF PIR database, the NCBI
GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-
redundant using the computer program nrdb2 (Warren Gish, Washington University
in
Saint Louis). Each of the polynucleotides shown in Table 1A, column 3 (e.g.,
SEQ ID
NO:X or the 'Query' sequence) was used to search against the NR database. The
computer program BLASTX was used to compare a 6-frame translation of the Query
sequence to the NR database (for information about the BLASTX algorithm please
see
Altshul et al., J. Mol. Biol. 215:403-410 (1990), and Gish et al., Nat. Genet.
3:266-272
(1993)). A description of the sequence that is most similar to the Query
sequence (the
highest scoring 'Subject') is shown in column five of Table 2 and the database
accession number for that sequence is provided in column six. The highest
scoring
'Subject' is reported in Table 2 if (a) the estimated probability that the
match occurred
by chance alone is less than 1.0e-07, and (b) the match was not to a known
repetitive
element. BLASTX returns alignments of short polypeptide segments of the Query
and
Subject sequences which share a high degree of similarity; these segments are
known
as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of
similarity
CA 02392757 2002-05-24
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between the Query and the Subject for each HSP as a percent identity in Column
7.
The percent identity is determined by dividing the number of exact matches
between
the two aligned sequences in the HSP, dividing by the number of Query amino
acids in
the HSP and multiplying by 100. The polynucleotides of SEQ ID NO:X which
encode
the polypeptide sequence that generates an HSP axe delineated by columns 8 and
9 of
Table 2.
[048) The PFam database, PFam version 5.2, (Sonnhammer et al., Nucl. Acids
Res.,
26:320-322, (1998)) consists of a series of multiple sequence alignments; one
alignment for each protein family. Each multiple sequence alignment is
converted into
a probability model called a Hidden Markov Model, or HMM, that represents the
position-specific variation among the sequences that make up the multiple
sequence
alignment (see, e.g., R. Durbin et al., Biological seque~rce afaalysis:
probabilistic
models of proteins anal faucleic acids, Cambridge University Press, 1998 for
the theory
of HMMs). The program HMMER version 1.8 (Sean Eddy, Washington University in
Saint Louis) was used to compare the predicted protein sequence for each Query
sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs derived from PFam
version 5.2. A HMM derived from PFam version 5.2 was said to be a significant
match to a polypeptide of the invention if the score returned by HMMER 1.8 was
greater than 0.8 times the HMMER 1.8 score obtained with the most distantly
related
known member of that protein family. The description of the PFam family which
shares a significant match with a polypeptide of the invention is listed in
column 5 of
Table 2, and the database accession number of the PFam hit is provided in
column 6.
Column 7 provides the score returned by HMMER version 1.8 for the alignment.
Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the
polypeptide sequence which shows a significant match to a PFam protein family.
[049] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT To",
delineate
the polynucleotides of "SEQ ID NO:X" that encode a polypeptide having a
significant
match to the PFam/NR database as disclosed in the fifth column of Table 2. In
one
embodiment, the invention provides a protein comprising, or alternatively
consisting
of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in
26
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such
proteins, and the complementary strand thereto.
[050] The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are
sufficiently accurate and otherwise suitable for a variety of uses well known
in the art
and described further below. For instance, the nucleotide sequences of SEQ ID
NO:X
are useful for designing nucleic acid hybridization probes that will detect
nucleic acid
sequences contained in SEQ ID NO:X or the cDNA contained in Clone ID NO:Z.
These probes will also hybridize to nucleic acid molecules in biological
samples,
thereby enabling immediate applications in chromosome mapping, linkage
analysis,
tissue identification and/or typing, and a variety of forensic and diagnostic
methods of
the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used
to
generate antibodies which bind specifically to these polypeptides, or
fragments
thereof, and/or to the polypeptides encoded by the cDNA clones identified in,
for
example, Table 1A.
[051] Nevertheless, DNA sequences generated by sequencing reactions can
contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or
deletions of nucleotides in the generated DNA sequence. The erroneously
inserted or
deleted nucleotides cause frame shifts in the reading frames of the predicted
amino
acid sequence. In these cases, the predicted amino acid sequence diverges from
the
actual amino acid sequence, even though the generated DNA sequence may be
greater
than 99.9% identical to the actual DNA sequence (for example, one base
insertion or
deletion in an open reading frame of over 1000 bases). .
[052] Accordingly, for those applications requiring precision in the
nucleotide
sequence or the amino acid sequence, the present invention provides not only
the
generated nucleotide sequence identified as SEQ ID NO:X, and a predicted
translated
amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid
DNA
containing cDNA Clone ID NO:Z (deposited with the ATCC on October 5, 2000, and
receiving ATCC designation numbers PTA 2574 and PTA 2575; deposited with the
ATCC on January 5, 2001, having the depositor reference numbers TS-1, TS-2, AC-
1,
and AC-2; and/or as set forth, fox example, in Table 1A, 6 and 7). The
nucleotide
sequence of each deposited clone can readily be determined by sequencing the
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deposited clone in accordance with known methods. Further, techniques known in
the
art can be used to verify the nucleotide sequences of SEQ ID NO:X.
[053] The predicted amino acid sequence can then be verified from such
deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone
can
also be directly determined by peptide sequencing or by expressing the protein
in a
suitable host cell containing the deposited human cDNA, collecting the
protein, and
determining its sequence.
RACE Py~otocol For Recovery of Full-Length Genes
[054] Partial cDNA clones can be made full-length by utilizing the rapid
amplification of cDNA ends (RACE) procedure described in Frohman, M.A., et
al.,
Proc. Nat'1. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clone missing either
the
5' or 3' end can be reconstructed to include the absent base pairs extending
to the
translational start or stop codon, respectively. In some cases, cDNAs are
missing the
start codon of translation. The following briefly describes a modification of
this
original 5' RACE procedure. Poly A+ or total RNA is reverse transcribed with
Superscript II (Gibco/BRL) and an antisense or complementary primer specific
to the
cDNA sequence. The primer is removed from the reaction with a Microcon
Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and
terminal
deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced
which is needed for PCR amplification. The second strand is synthesized from
the
dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT
primer containing three adjacent restriction sites (XhoI, SaII and CIaI) at
the 5' end and
a primer containing just these restriction sites. This double-stranded cDNA is
PCR
amplified for 40 cycles with the same primers as well as a nested cDNA-
specific
antisense primer. The PCR products are size-separated on an ethidium bromide-
agarose gel and the region of gel containing cDNA products the predicted size
of
missing protein-coding DNA is removed. cDNA is purified from the agarose with
the
Magic PCR Prep kit (Promega), restriction digested with XhoI or SaII, and
ligated to a
plasmid such as pBluescript SKII (Stratagene) at XhoI and EcoRV sites. This
DNA is
transformed into bacteria and the plasmid clones sequenced to identify the
correct
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protein-coding inserts. Correct 5' ends are confirmed by comparing this
sequence with
the putatively identified homologue and overlap with the partial cDNA clone.
Similar
methods known in the art and/or commercial kits are used to amplify and
recover 3'
ends.
[055] Several quality-controlled kits are commercially available for purchase.
Similar reagents and methods to those above are supplied in kit form from
Gibco/BRL
for both 5' and 3' RACE for recovery of full length genes. A second lcit is
available
from Clontech which is a modification of a related technique, SLIC (single-
stranded
ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids
Res.,
19:5227-32 (1991). The major differences in procedure are that the RNA is
alkaline
hydrolyzed after reverse transcription and RNA ligase is used to join a
restriction site-
containing anchor primer to the first-strand cDNA. This obviates the necessity
for the
dA-tailing reaction which results in a polyT stretch that is difficult to
sequence past.
[056] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library
double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is
synthesized with an antisense cDNA-specific primer and a plasmid-anchored
primer.
These primers are removed and a symmetric PCR reaction is performed with a
nested
cDNA-specific antisense primer and the plasmid-anchored primer.
RNA Ligase Protocol Fog GetZe~atif2g The 5' or 3' End Sequences To Obtain Full
Length
Genes
[057] Once a gene of interest is identified, several methods are available for
the
identification of the 5' or 3' portions of the gene which may not be present
in the
original cDNA plasmid. These methods include, but are not limited to, filter
probing,
clone enrichment using specific probes and protocols similar and identical to
5' and 3'
RACE. While the full length gene may be present in the library and can be
identified
by probing, a useful method for generating the 5' or 3' end is to use the
existing
sequence information from the original cDNA to generate the missing
information. A
method similar to 5' RACE is available for generating the missing 5' end of a
desired
full-length gene. (This method was published by Fromont-Racine et al., Nucleic
Acids
Res., 21(7):1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is
ligated to
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the 5' ends of a population of RNA presumably containing full-length gene RNA
transcript. A primer set containing a primer specific to the ligated RNA
oligonucleotide and a primer specific to a known sequence of the gene of
interest, is
used to PCR amplify the 5' portion of the desired full length gene which may
then be
sequenced and used to generate the full length gene. This method starts with
total
RNA isolated from the desired source, poly A RNA may be used but is not a
prerequisite for this procedure. The RNA preparation may then be treated with
phosphatase if necessary to eliminate 5' phosphate groups on degraded or
damaged
RNA, which may interfere with the later RNA ligase step. The phosphatase, if
used, is
then inactivated and the RNA is treated with tobacco acid pyrophosphatase in
order to
remove the cap structure present at the 5' ends of messenger RNAs. This
reaction
leaves a 5' phosphate group at the 5' end of the cap cleaved RNA which can
then be
ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA
preparation can then be used as a template for first strand cDNA synthesis
using a
gene specific oligonucleotide. The first strand synthesis reaction can then be
used as a
template for PCR amplification of the desired 5' end using a primer specific
to the
ligated RNA oligonucleotide and a primer specific to the known sequence of the
pancreatic antigen of interest. The resultant product is then sequenced and
analyzed to
confirm that the 5' end sequence belongs to the relevant pancreatic antigen.
[058] The present invention also relates to vectors or plasmids, which include
such
DNA sequences, as well as the use of the DNA sequences. The material deposited
with the ATCC (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January
5, 2001, having the depositor reference numbers TS-l, TS-2, AC-1, and AC-2;
and/or
as set forth, for example, in Table 1A, 6 and 7) is a mixture of cDNA clones
derived
from a variety of human tissue and cloned in either a plasmid vector or a
phage vector,
as shown, for example, in Table 7. These deposits are referred to as "the
deposits"
herein. The tissues from which some of the clones were derived are listed in
Table 7,
and the vector in which the corresponding cDNA is contained is also indicated
in
Table 7. The deposited material includes cDNA clones corresponding to SEQ ID
NO:X described, for example, in Table 1A (Clone ID NO:Z). A clone which is
CA 02392757 2002-05-24
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isolatable from the ATCC Deposits by use of a sequence listed as SEQ ID NO:X,
may
include the entire coding region of a human gene or in other cases such clone
may
include a substantial portion of the coding region of a human gene.
Furthermore,
although the sequence listing may in some instances list only a portion of the
DNA
sequence in a clone included in the ATCC Deposits, it is well within the
ability of one
skilled in the art to sequence the DNA included in a clone contained in the
ATCC
Deposits by use of a sequence (or portion thereof) described in, for example
Tables 1A
or 2 by procedures hereinafter further described, and others apparent to those
skilled in
the art.
[059] Also provided in Table 7 is the name of the vector which contains the
cDNA
clone. Each vector is routinely used in the art. The following additional
information
is provided for convenience.
[060] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap
XR
(U.S. Patent Nos. 5,128,256 and 5,286,636), Zap Express (LT.S. Patent Nos.
5,128,256
and 5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res.
16.7583-
7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17: 9494
(1989))
and pBK (Alting-Mees, M. A. et al., StJ°ategies 5:58-61 (1992)) are
commercially
available from Stratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road,
La
Jolla, CA, 92037. pBS contains an ampicillin resistance gene and pBK contains
a
neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and
Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express
vector.
Both phagemids may be transformed into E. coli strain XL-1 Blue, also
available from
Stratagene.
[061) Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were
obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
All
Sport vectors contain an ampicillin resistance gene and may be transformed
into E.
coli strain DH10B, also available from Life Technologies. See, for instance,
Gruber,
C. E., et al., Focus 15:59- (1993). Vector lafmid BA (Bento Soares, Columbia
University, New York, NY) contains an ampicillin resistance gene and can be
transformed into E. coli strain XL-1 Blue. Vector pCR~2.1, which is available
from
Invitrogen, 1600 Faraday Avenue, Carlsbad, CA 92008, contains an ampicillin
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resistance gene and may be transformed into E. coli strain DHlOB, available
from Life
Technologies. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686
(1988)
and Mead, D. et al., Bio/Technology 9: (1991).
[062] The present invention also relates to the genes corresponding to SEQ ID
NO:X,
SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding
gene
can be isolated in accordance with known methods using the sequence
information
disclosed herein. Such methods include preparing probes or primers from the
disclosed sequence and identifying or amplifying the corresponding gene from
appropriate sources of genomic material.
[063] Also provided in the present invention are allelic variants, orthologs,
and/or
species homologs. Procedures known in the art can be used to obtain full-
length genes,
allelic variants, splice variants, full-length coding portions, orthologs,
and/or species
homologs of pancreatic associated genes corresponding to SEQ ID NO:X or the
complement thereof, polypeptides encoded by SEQ ID NO:X or the complement
thereof, and/or the cDNA contained in Clone ID NO:Z, using information from
the
sequences disclosed herein or the clones deposited with the ATCC. For example,
allelic variants and/or species homologs may be isolated and identifted by
making
suitable probes or primers from the sequences provided herein and screening a
suitable
nucleic acid source for allelic variants and/or the desired homologue.
[064] The polypeptides of the invention can be prepared in any suitable
manner.
Such polypeptides include isolated naturally occurring polypeptides,
recombinantly
produced polypeptides, synthetically produced polypeptides, or polypeptides
produced
by a combination of these methods. Means for preparing such polypeptides are
well
understood in the art.
[065] The polypeptides may be in the form of the secreted protein, including
the
mature form, or may be a part of a larger protein, such as a fusion protein
(see below).
It is often advantageous to include an additional amino acid sequence which
contains
secretory or leader sequences, pro-sequences, sequences which aid in
purification,
such as multiple histidine residues, or an additional sequence for stability
during
recombinant production.
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[066] The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. A recombinantly produced
version of
a polypeptide, including the secreted polypeptide, can be substantially
purified using
techniques described herein or otherwise known in the art, such as, for
example, by the
one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
Polypeptides of the invention also can be purified from natural, synthetic or
recombinant sources using techniques described herein or otherwise known in
the art,
such as, for example, antibodies of the invention raised against the
pancreatic
polypeptides of the present invention in methods which are well known in the
art.
[067] The present invention provides a polynucleotide comprising, or
alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence
contained in Clone ID NO:Z. The present invention also provides a polypeptide
comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y,
a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide
encoded by the cDNA contained in Clone ID NO:Z, and/or the polypeptide
sequence
encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of
Table
1B. Polynucleotides encoding a polypeptide comprising, or alternatively
consisting of
the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X,
a polypeptide encoded by the cDNA contained in Clone ID NO:Z and/or a
polypeptide
sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column
6
of Table 1B are also encompassed by the invention. The present invention
further
encompasses a polynucleotide comprising, or alternatively consisting of, the
complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid
sequence
encoding a polypeptide encoded by the complement of the nucleic acid sequence
of
SEQ ID NO:X, and/or the cDNA contained in Clone ID NO:Z.
[068] Moreover, representative examples of polynucleotides of the invention
comprise, or alternatively consist of, one, two, three, four, five, six,
seven, eight, nine,
ten, or more of the sequences delineated in Table 1B column 6, or any
combination
thereof. Additional, representative examples of polynucleotides of the
invention
comprise, or alternatively consist of, one, two, three, four, five, six,
seven, eight, nine,
ten, or more of the complementary strands) of the sequences delineated in
Table 1B
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column 6, or any combination thereof. In further embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences
delineated in Table 1B, column 6, and have a nucleic acid sequence which is
different
from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B
(see
Table 1B, column 5). In additional embodiments, the above-described
polynucleotides
of the invention comprise, or alternatively consist of, sequences delineated
in Table
1B, column 6, and have a nucleic acid sequence which is different from that
published
fox the BAC clone identified as BAC ID NO:A (see Table 1 B, column 4). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or alternatively consist of, sequences delineated in Table 1B,
column 6, and
have a nucleic acid sequence which is different from that contained in the BAC
clone
identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides encoded by
these
polynucleotides, other polynucleotides that encode these polypeptides, and
antibodies
that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides and polypeptides
are
also encompassed by the invention.
[069] Further, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, eve, six, seven, eight,
nine, ten, or
more of the sequences delineated in column 6 of Table 1B which correspond to
the
same Clone ID NO:Z (see Table 1B, column 1), or any combination thereof.
Additional, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
of the complementary strands) of the sequences delineated in column 6 of Table
1B
which correspond to the same Clone ID NO:Z (see Table 1B, column 1), or any
combination thereof. In further embodiments, the above-described
polynucleotides of
the invention comprise, or alternatively consist of, sequences delineated in
column 6
of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B, column
1)
and have a nucleic acid sequence which is different from that of the BAC
fragment
having the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or alternatively consist of, sequences delineated in column 6 of
Table 1B
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which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a
nucleic acid sequence which is different from that published for the BAC clone
identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments,
the above-described polynucleotides of the invention comprise, or
alternatively consist
of, sequences delineated in column 6 of Table 1B which correspond to the same
Clone
ID NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is
different
from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B,
column 4). Polypeptides encoded by these polynucleotides, other
polynucleotides that
encode these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[070] Further, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or
more of the sequences delineated in column 6 of Table 1B which correspond to
the
same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any
combination thereof. Additional, representative examples of polynucleotides of
the
invention comprise, or alternatively consist of, one, two, three, four, five,
six, seven,
eight, nine, ten, or more of the complementary strands) of the sequences
delineated in
column 6 of Table 1B which correspond to the same contig sequence identifer
SEQ ID
NO:X (see Table 1B, column 2), or any combination thereof. In further
embodiments,
the above-described polynucleotides of the invention comprise, or
alternatively consist
of, sequences delineated in column 6 of Table 1B which correspond to the same
contig
sequence identifer SEQ ID NO:X (see Table IB, column 2) and have a nucleic
acid
sequence which is different from that of the BAC fragment having the sequence
disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional embodiments,
the
above-described polynucleotides of the invention comprise, or alternatively
consist of,
sequences delineated in column 6 of Table 1B which correspond to the same
contig
sequence identifer SEQ ID NO:X (see Table 1B, column 2) and have a nucleic
acid
sequence which is different from that published for the BAC clone identified
as BAC
ID NO:A (see Table 1B, column 4). In additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences
CA 02392757 2002-05-24
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delineated in column 6 of Table 1B which correspond to the same contig
sequence
identifer SEQ ID NO:X (see Table 1B, column 2) and have a nucleic acid
sequence
which is different from that contained in the BAC clone identified as BAC ID
NO:A
(See Table 1B, column 4). Polypeptides encoded by these polynucleotides, other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides are also encompassed by the invention. Additionally, fragments
and
variants of the above-described polynucleotides and polypeptides are also
encompassed by the invention.
[071] Moreover, representative examples of polynucleotides of the invention
comprise, or alternatively consist of, one, two, three, four, five, six,
seven, eight, nine,
ten, or more of the sequences delineated in the same row of Table 1B column 6,
or any
combination thereof. Additional, representative examples of polynucleotides of
the
invention comprise, or alternatively consist of, one, two, three, four, five,
six, seven,
eight, nine, ten, or more of the complementary strands) of the sequences
delineated in
the same row of Table 1B column 6, or any combination thereof. In preferred
embodiments, the polynucleotides of the invention comprise, or alternatively
consist
of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the
complementary strands) of the sequences delineated in the same row of Table 1B
column 6, wherein sequentially delineated sequences in the table (i.e.
corresponding to
those exons located closest to each other) are directly contiguous in a 5' to
3'
orientation. In further embodiments, above-described polynucleotides of the
invention
comprise, or alternatively consist of, sequences delineated in the same row of
Table
1B, column 6, and have a nucleic acid sequence which is different from that of
the
BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B,
column
5). In additional embodiments, the above-described polynucleotides of the
invention
comprise, or alternatively consist of, sequences delineated in the same row of
Table
1B, column 6, and have a nucleic acid sequence which is different from that
published
for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or alternatively consist of, sequences delineated in the same row of
Table
1B, column 6, and have a nucleic acid sequence which is different from that
contained
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in the BAC clone identified as BAC ID NO.:A (see Table 1B, column 4).
Polypeptides
encoded by these polynucleotides, other polynucleotides that encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention.
[072] In additional specific embodiments, polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or
more of the sequences delineated in column 6 of Table 1B, and the
polynucleotide
sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments
or
variants thereof. Polypeptides .encoded by these polynucleotides, other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides are also encompassed by the invention.
[073] In additional specific embodiments, polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or
more of the sequences delineated in column 6 of Table 1B which correspond to
the
same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence
of
SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants
thereof. In
preferred embodiments, the delineated sequences) and polynucleotide sequence
of
SEQ ID NO:X correspond to the same Clone ID NO:Z. Polypeptides encoded by
these polynucleotides, other polynucleotides that encode these polypeptides,
and
antibodies that bind these polypeptides are also encompassed by the invention.
[074] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
of the sequences delineated in the same row of column 6 of Table 1B, and the
polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or
fragments or variants thereof. In preferred embodiments, the delineated
sequences)
and polynucleotide sequence of SEQ ID NO:X correspond to the same row of
column
6 of Table 1B. Polypeptides encoded by these polynucleotides, other
polynucleotides
that encode these polypeptides, and antibodies that bind these polypeptides
are also
encompassed by the invention.
[075] In additional specific embodiments, polynucleotides of the invention
comprise,
or alternatively consist of a polynucleotide sequence in which the 3' 10
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polynucleotides of one of the sequences delineated in column 6 of Table 1B and
the 5'
polynucleotides of the sequence of SEQ ID NO:X are directly contiguous.
Nucleic
acids which hybridize to the complement of these 20 contiguous polynucleotides
under
stringent hybridization conditions or alternatively, under lower stringency
conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids that
encode these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[076] In additional specific embodiments, polynucleotides of the invention
comprise,
or alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one of the sequences delineated in column 6 of Table 1B and
the 5'
10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are
directly contiguous Nucleic acids which hybridize to the complement of these
20
contiguous polynucleotides under stringent hybridization conditions or
alternatively,
under lower stringency conditions, are also encompassed by the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies that
bind these polypeptides are also encompassed by the invention. Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
[077] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides
of the sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence
of one
of the sequences delineated in column 6 of Table 1B are directly contiguous.
Nucleic
acids which hybridize to the complement of these 20 contiguous polynucleotides
under
stringent hybridization conditions or alternatively, under lower stringency
conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids
encoding these polypeptides, and antibodies that bind these polypeptides are
also
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encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[078] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides
of a fragment or variant of the sequence of SEQ ID NO:X and the 5' 10
polynucleotides of the sequence of one of the sequences delineated in column 6
of
Table 1B are directly contiguous. Nucleic acids which hybridize to the
complement of
these 20 contiguous polynucleotides under stringent hybridization conditions
or
alternatively, under lower stringency conditions, are also encompassed by the
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids,
other
polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies that
bind these polypeptides are also encompassed by the invention. Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides, are also encompassed by the invention.
[079] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides
of one of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another sequence in column 6 are directly contiguous.
Nucleic
acids which hybridize to the complement of these 20 contiguous polynucleotides
under
stringent hybridization conditions or alternatively, under lower stringency
conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids
encoding these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[080] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides
of one of the sequences delineated in column 6 of Table IB and the 5' IO
polynucleotides of another sequence in column 6 corresponding to the same
Clone ID
39
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
NO:Z (see Table 1B, column 1) are directly contiguous. Nucleic acids which
hybridize to the complement of these 20 lower stringency conditions, are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides,
and antibodies that bind these polypeptides are also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic
acids, and polypeptides are also encompassed by the invention.
[081] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides
of one sequence in column 6 corresponding to the same contig sequence
identifer
SEQ ID NO:X (see Table 1B, column 2) are directly contiguous. Nucleic acids
which
hybridize to the complement of these 20 contiguous polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides,
and antibodies that bind these polypeptides are also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic
acids, and polypeptides are also encompassed by the invention.
[082] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides
of one of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another sequence in column 6 corresponding to the same row
are
directly contiguous. In preferred embodiments, the 3' 10 polynucleotides of
one of the
sequences delineated in column 6 of Table 1B is directly contiguous with the
5' 10
polynucleotides of the next sequential exon delineated in Table 1B, column 6.
Nucleic
acids which hybridize to the complement of these 20 contiguous polynucleotides
under
stringent hybridization conditions or alternatively, under lower stringency
conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids
encoding these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[083] Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases and may have been publicly available
prior
to conception of the present invention. Preferably, such related
polynucleotides are
specifically excluded from the scope of the present invention. Accordingly,
for each
contig sequence (SEQ ID NO:X) listed in the third column of Table 1A,
preferably
excluded are one or more polynucleotides comprising a nucleotide sequence
described
by the general formula of a-b, where a is any integer between 1 and the final
nucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the final
nucleotide of
SEQ ID NO:X, where both a and b correspond to the positions of nucleotide
residues
shown in SEQ ID NO:X, and where b is greater than or equal to a + 14. More
specifically, preferably excluded are one or more polynucleotides comprising a
nucleotide sequence described by the general formula of a-b, where a and b are
integers as defined in columns 4 and 5, respectively, of Table 3. In specific
embodiments, the polynucleotides of the invention do not consist of at least
one, two,
three, four, five, ten, or more of the specific polynucleotide sequences
referenced by
the Genbanlc Accession No. as disclosed in column 6 of Table 3 (including for
example, published sequence in connection with a particular BAC clone). In
further
embodiments, preferably excluded from the invention are the specific
polynucleotide
sequences) contained in the clones corresponding to at least one, two, three,
four,
five, ten, or more of the available material having the accession numbers
identified in
the sixth column of this Table (including for example, the actual sequence
contained in
an identified BAC clone). In no way is this listing meant to encompass all of
the
sequences which may be excluded by the general formula, it is just a
representative
example. All references available through these accessions are hereby
incorporated by
reference in their entirety.
TABLE 3
4I
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
~ Os M ~ ~ OMO pip "' N v0 I~ Os ~ ~t N ~
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42
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
vo ~ ~"
M ~ N M
lp M o0 V~1 00 r; ~ O 0~1' 'd' ~ ,-i ~ ,-i
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xx
43
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
TABLE 4
Code Description Tissue Or:~an ell LineDiseaseVector
AR022 a_Heart a_Heart
AR023 a_Liver a_Liver
AR024 a_mamm land a_mammar
land
AR025 a_Prostate a_Prostate
AR026 a_small intestinea_small intestine
AR027 a_Stomach a_Stomach
AR028 Blood B cellsBlood B cells
AR029 Blood B cellsBlood B cells
activated activated
AR030 Blood B cellsBlood B cells
resting resting
AR031 Blood T cellsBlood T cells
activated activated
AR032 Blood T cellsBlood T cells
resting restin
AR033 brain brain
AR034 breast breast
AR035 breast cancerbreast cancer
AR036 Cell Line Cell Line
CAOV3 CAOV3
AR037 cell line cell line
PA-1 PA-1
AR038 cell line cell line
transformed transformed
AR039 colon colon
AR040 colon (9808co65R)colon (9808co65R)
AR041 colon (9809co15)colon (9809co15)'
AR042 colon cancercolon cancer
AR043 colon cancercolon cancer
(9808co64R) (9808co64R)
AR044 colon cancercolon cancer
9809co14 .
9809co14
AR045 corn clone corn clone
5 5
AR046 corn clone corn clone
6 6
AR047 corn clone2 com clone2
AR048 corn clone3 corn clone3
AR049 Corn Clone4 Corn Clone4
AR050 Donor II Donor II
B Cells B Cells
24hrs 24hrs
AR051 Donor II Donor II
B Cells B Cells
72hrs 72hrs
AR052 Donor II Donor II
B-Cells B-Cells
24 24
hrs. hrs.
AR053 Donor II Donor II
B-Cells B-Cells
72hrs 72hrs
AR054 Donor II Donor II
Resting Resting
B B
Cells Cells
AR055 Heart Heart
AR056 Human Lung Human Lung
(clonetech) (clonetech)
AR057 Human MammaryHuman Mammary
(clontech) (clontech)
AR058 Human ThymusHuman Thymus
(clonetech) (clonetech)
AR059 Jurkat (unstimulated)Jurkat
(unstimulated)
AR060 Kidne Kidne
AR061 Liver ~ Liver
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
AR062Liver (Clontech)Liver (Clontech)
AR063Lymphocytes Lymphocytes
chronic
lymphocytic chronic lymphocytic
leukaemia leukaemia
AR064Lymphocytes Lymphocytes
diffuse
large B cell diffuse large
B cell
1 m homa 1 m homa
AR065Lymphocytes Lymphocytes
follicular follicular
lym homa I m homa
AR066normal breastnormal breast
AR067Normal0varianNormal Ovarian
(4004901) (4004901)
AR068Normal Ovary Normal Ovary
95086045 95086045
AR069Normal Ovary Normal Ovary
97016208 97016208
AR070Normal Ovary Normal Ovary
98066005 98066005
AR071Ovarian CancerOvarian Cancer
AR072Ovarian CancerOvarian Cancer
(97026001) (97026001)
AR073Ovarian CancerOvarian Cancer
(97076029) (97076029)
AR074Ovarian CancerOvarian Cancer
(98046011) (98046011)
AR075Ovarian CancerOvarian Cancer
(98066019) (98066019)
AR076Ovarian CancerOvarian Cancer
(98076017) (98076017)
.
AR077Ovarian CancerOvarian Cancer
(98096001) (98096001)
AR078ovarian cancerovarian cancer
15799
15799
AR079Ovarian CancerOvarian Cancer
17717AID 17717AID
AR080Ovarian CancerOvarian Cancer
400466481 400466481
AR081Ovarian CancerOvarian Cancer
4005315A1 4005315A1
AR082ovarian cancerovarian cancer
94127303 94127303
AR083Ovarian CancerOvarian Cancer
_
96069304 96069304
AR084Ovarian CancerOvarian Cancer
97076029 97076029
AR085Ovarian CancerOvarian Cancer
98076045 98076045
AR086ovarian cancerovarian cancer
98096001 98096001
AR087Ovarian CancerOvarian Cancer
9905C032RC 9905C032RC
AR088Ovarian cancerOvarian cancer
9907
C00 3rd 9907 C00
3rd
AR089Prostate Prostate
AR090Prostate (clonetech)Prostate
(clonetech)
AR091rostate cancerrostate cancer
AR092prostate cancerprostate
cancer
#15176 #15176
AR093prostate cancerrostate cancer
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
#15509 #15509
AR094prostate cancerprostate
cancer
#15673 #15673
AR095Small IntestineSmall Intestine
(Clontech) (Clontech)
AR096S Teen S Teen
AR097Thymus T cellsThymus T
cells
activated activated
AR098Thymus T cellsThymus T
cells
restin restin
AR099Tonsil Tonsil
AR100Tonsil geminalTonsil geminal
center
centroblast center centroblast
AR101Tonsil germinalTonsil germinal
center B cellcenter B
cell
AR102Tonsil lymph Tonsil lymph
node node
AR103Tonsil memoryTonsil memory
B cell B
cell
AR104Whole Brain Whole Brain
AR105Xeno raft Xeno raft
ES-2 ES-2
AR106Xenograft Xenograft
SW626 SW626
H0270HPAS (human Human PancreasPancreas Uni-ZAP
ancreas, subtracted) XR
S0378Pancreas normalPancreas pSportl
Normal
PCA4 No PCA4 No
L0439Soares infant whole Lafmid
brain brain
1NIB BA
L0581Stratagene liver pBluescri
liver
(#937224) pt SK
L0599Stratagene lung pBluescri
lung
(#937210) t SK-
L0663NCI_CGAP_Ut2 moderately- uterus pCMV-
differentiated ~ SPORT6
endometrial
adenocarcino
L07485oares fetal Liver pT7T3D
liver and
spleen 1NFLS Spleen (Pharmaci
a) with
a
modified
of linker
L0776NCI_CGAP_Lu5 carcinoid lung pTTT3D-
Pac
(Pharmaci
a) with
a
modified
oI linker
L0789NCI_CGAP_Sub3 pTTT3D-
Pac
(Pharmaci
a) with
a
modified
of linker
L0805NCI_CGAP_Lu24carcinoid lung pT7T3D-
Pac
(Pharmaci
a) with
a
modified
of linker
L0809~ NCI CGAP_Pr28 prostate~ ~ pT7T3D-
46
CA 02392757 2002-05-24
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Pac
(Pharmaci
a) with
a
modified
olylinker
TABLE 5
OMIM Description
Reference
No Entries
Polyyzucleotide azzd Polypeptide Tlariazzts
[084] The present invention is also directed to variants of the pancreatic
associated
polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand
thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the
nucleotide sequence of SEQ ID NO:X encoding the polypeptide sequence as
defined
in column 6 of Table 1A, nucleotide sequences encoding the polypeptide as
defined in
column 6 of Table 1A, the nucleotide sequence as defined in columns 8 and 9 of
Table 2, nucleotide sequences encoding the polypeptide encoded by the
nucleotide
sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as
defined
in column 6 of Table 1B, nucleotide sequences encoding the polypeptide encoded
by
the nucleotide sequence as defined in column 6 of Table 1B, the cDNA sequence
contained in Clone ID NO:Z, and/or nucleotide sequences encoding a polypeptide
encoded by the cDNA sequence contained in Clone ID NO:Z.
[085] The present invention also encompasses variants of the polypeptide
sequence
disclosed in SEQ ID NO:Y, a polypeptide sequence as defined in column 6 of
Table
1A, a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID
NO:X,
a polypeptide sequence encoded by the nucleotide sequence as defined in
columns 8
and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as
defined in column 6 of Table 1B, a polypeptide sequence encoded by the
complement
47
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
of the polynucleotide sequence in SEQ ID NO:X, and/or a polypeptide sequence
encoded by the cDNA sequence contained in Clone ID NO:Z.
[086] "Variant" refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but retaining
essential
properties thereof. Generally, variants are overall closely similar, and, in
many
regions, identical to the polynucleotide or polypeptide of the present
invention.
[087] Thus, one aspect of the invention provides an isolated nucleic acid
molecule
comprising, or alternatively consisting of, a polynucleotide having a
nucleotide
sequence selected from the group consisting of: (a) a nucleotide sequence
described in
SEQ ID NO:X or contained in the cDNA sequence of Clone ID NO:Z; (b) a
nucleotide sequence in SEQ ID NO:X or the cDNA in Clone ID NO:Z which encodes
a mature pancreatic associated polypeptide; (c) a nucleotide sequence in SEQ
ID
NO:X or the cDNA sequence of Clone ID NO:Z, which encodes a biologically
active
fragment of a pancreatic associated polypeptide; (d) a nucleotide sequence in
SEQ ID
NO:X or the cDNA sequence of Clone ID NO:Z, which encodes an antigenic
fragment of a pancreatic associated polypeptide; (e) a nucleotide sequence
encoding a
pancreatic associated polypeptide having the complete amino acid sequence of
SEQ ID
NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z;
(f) a nucleotide sequence encoding a mature pancreatic associated polypeptide
of the
amino acid sequence of SEQ ID NO:Y or the amino acid sequence encoded by the
cDNA in Clone ID NO:Z; (g) a nucleotide sequence encoding a biologically
active
fragment of a pancreatic associated polypeptide having the complete amino acid
sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the
cDNA in Clone ID NO:Z; (h) a nucleotide sequence encoding an antigenic
fragment of
a pancreatic associated polypeptide having the complete amino acid sequence of
SEQ
ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID
NO:Z; and (i) a nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f), (g), or (h), above.
[088] The present invention is also directed to nucleic acid molecules which
comprise, or alternatively consist of, a nucleotide sequence which is at least
80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of
the
48
0
CA 02392757 2002-05-24
WO 01/55327 PCT/USO1/01352
nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above,
the nucleotide
coding sequence in SEQ ID NO:X or the complementary strand thereto, the
nucleotide
coding sequence of the cDNA contained in Clone ID NO:Z or the complementary
strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y,
a
nucleotide sequence encoding a polypeptide sequence encoded by the nucleotide
sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of
the
polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the
polypeptide encoded by the cDNA contained in Clone ID NO:Z, the nucleotide
coding
sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the
complementary strand thereto, a nucleotide sequence encoding the polypeptide
encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and
9 of
Table 2 or the complementary strand thereto, the nucleotide coding sequence in
SEQ
ID NO:B as defined in column 6 of Table 1B or the complementary strand
thereto, a
nucleotide sequence encoding the polypeptide encoded by the nucleotide
sequence in
SEQ ID NO:B as defined in column 6 of Table 1B or the complementary strand
thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide
sequence
as defined in column 6 of Table 1A or the complementary strand thereto,
nucleotide
sequences encoding a polypeptide as defined in column 6 of Table 1A or the
complementary strand thereto, and/or polynucleotide fragments of any of these
nucleic
acid molecules (e.g., those fragments described herein). Polynucleotides which
hybridize to the complement of these nucleic acid molecules under stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides
and nucleic acids.
[089] In a preferred embodiment, the invention encompasses nucleic acid
molecules
which comprise, or alternatively, consist of a polynucleotide which hybridizes
under
stringent hybridization conditions, or alternatively, under lower stringency
conditions,
to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above,
as are polypeptides
encoded by these polynucleotides. In another preferred embodiment,
polynucleotides
which hybridize to the complement of these nucleic acid molecules under
stringent
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hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
[090] In another embodiment, the invention provides a purified protein
comprising,
or alternatively consisting of, a polypeptide having an amino acid sequence
selected
from the group consisting of: (a) the complete amino acid sequence of SEQ ID
NO:Y
or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b)
the
amino acid sequence of a mature pancreatic associated polypeptide having the
amino
acid sequence of SEQ ID NO:Y or the amino acid sequence encoded by the cDNA in
Clone ID NO:Z; (c) the amino acid sequence of a biologically active fragment
of a
pancreatic associated polypeptide having the complete amino acid sequence of
SEQ ID
NO:Y or the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z;
and (d) the amino acid sequence of an antigenic fragment of a pancreatic
associated
polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the
complete amino acid sequence encoded by the cDNA in Clone ID NO:Z.
[091] The present invention is also directed to proteins which comprise, or
alternatively consist of, an amino acid sequence which is at Least 80%, 85%,
90%,
95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino
acid
sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in
SEQ ID
NO:Y, the amino acid sequence encoded by the cDNA contained in Clone ID NO:Z,
the amino acid sequence of the polypeptide encoded by the nucleotide sequence
in
SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence
of
the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined
in
column 6 of Table 1B, the amino acid sequence as defined in column 6 of Table
1A,
an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and
an
amino acid sequence encoded by the complement of the polynucleotide sequence
in
SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those
fragments described herein). Further proteins encoded by polynucleotides which
hybridize to the complement of the nucleic acid molecules encoding these amino
acid
sequences under stringent hybridization conditions or alternatively, under
lower
stringency conditions, are also encompassed by the invention, as are the
polynucleotides encoding these proteins.
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[092] By a nucleic acid having a nucleotide sequence at least, for example,
95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended
that the nucleotide sequence of the nucleic acid is identical to the reference
sequence
except that the nucleotide sequence may include up to five point mutations per
each
100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
In
other words, to obtain a nucleic acid having a nucleotide sequence at least
95%
identical to a reference nucleotide sequence, up to 5% of the nucleotides in
the
reference sequence may be deleted or substituted with another nucleotide, or a
number
of nucleotides up to 5% of the total nucleotides in the reference sequence may
be
inserted into the reference sequence. The query sequence may be an entire
sequence
referred to in Table 1A or 2 as the ORF (open reading frame), or any fragment
specified, as described herein.
[093] As a practical matter, whether any particular nucleic acid molecule or
polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
a
nucleotide sequence of the present invention can be determined conventionally
using
known computer programs. A preferred method for determining the best overall
match between a query sequence (a sequence of the present invention) and a
subject
sequence, also referred to as a global sequence alignment, can be determined
using the
FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App.
Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject
sequences
axe both DNA sequences. An RNA sequence can be compared by converting U's to
T's. The result of said global sequence alignment is expressed as percent
identity.
Preferred parameters used in a FASTDB alignment of DNA sequences to calculate
percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining
Penalty=30, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide
sequence,
whichever is shorter.
[094] If the subject sequence is shorter than the query sequence because of 5'
or 3'
deletions, not because of internal deletions, a manual correction must be made
to the
results. This is because the FASTDB program does not account for 5' and 3'
truncations of the subject sequence when calculating percent identity. For
subject
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sequences truncated at the 5' or 3' ends, relative to the query sequence, the
percent
identity is corrected by calculating the number of bases of the query sequence
that are
5' and 3' of the subject sequence, which are not matched/aligned, as a percent
of the
total bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment,
which are not matched/aligned With the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[095] For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the 5' end of
the
subject sequence and therefore, the FASTDB alignment does not show ,a
matched/alignment of the first IO bases at 5' end. The 10 unpaired bases
represent
10% of the sequence (number of bases at the 5' and 3' ends not matched/total
number
of bases in the query sequence) so 10% is subtracted from the percent identity
score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the final percent identity would be 90%. In another example, a 90 base
subject sequence is compared with a 100 base query sequence. This time the
deletions
are internal deletions so that there are no bases on the 5' or 3' of the
subject sequence
which are not matched/aligned with the query. In this case the percent
identity
calculated by FASTDB is not manually corrected. Once again, only bases 5' and
3' of
the subject sequence which are not matched/aligned with the query sequence are
manually corrected for. No other manual corrections are to be made for the
purposes
of the present invention.
[096] By a polypeptide having an amino acid sequence at least, for example,
95%
"identical" to a query amino acid sequence of the present invention, it is
intended that
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other
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words, to obtain a polypeptide having an amino acid sequence at least 95%
identical to
a query amino acid sequence, up to 5% of the amino acid residues in the
subject
sequence may be inserted, deleted, (indels) or substituted with another amino
acid.
These alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the reference amino acid sequence or anywhere between
those
terminal positions, interspersed either individually among residues in the
reference
sequence or in one or more contiguous groups within the reference sequence.
[097] As a practical matter, whether any particular polypeptide is at least
80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequence
of a polypeptide referred to in Table 1A (e.g., an amino acid sequence
identified in
columns 5 or 6) or Table 2 (e.g., the amino acid sequence of the polypeptide
encoded
by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a
fragment
thereof, the amino acid sequence of the polypeptide encoded by the
polynucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B or a fragment
thereof,
the amino acid sequence of the polypeptide encoded by the nucleotide sequence
in
SEQ ID NO:X or a fragment thereof, or an amino acid sequence of the
polypeptide
encoded by cDNA contained in Clone ID NO:Z, or a fragment thereof, can be
determined conventionally using known computer programs. A preferred method
for
determining the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a global
sequence
alignment, can be determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a
sequence
alignment the query and subject sequences are either both nucleotide sequences
or
both amino acid sequences. The result of said global sequence alignment is
expressed
as percent identity. Preferred parameters used in a FASTDB amino acid
alignment
are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=S00 or the length of the subject
amino acid sequence, whichever is shorter.
[098] If the subject sequence is shorter than the query sequence due to N- or
C-
tenninal deletions, not because of internal deletions, a manual correction
must be made
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to the results. This is because the FASTDB program does not account for N- and
C-
terminal truncations of the subject sequence when calculating global percent
identity.
For subject sequences truncated at the N- and C-termini, relative to the query
sequence, the percent identity is corrected by calculating the number of
residues of the
query sequence that are N- and C-terminal of the subject sequence, which are
not
matched/aligned with a corresponding subject residue, as a percent of the
total bases of
the query sequence. Whether a residue is matched/aligned is determined by
results of
the FASTDB sequence alignment. This percentage is then subtracted from the
percent
identity, calculated by the above FASTDB program using the specified
parameters, to
arrive at a final percent identity score. This final percent identity score is
what is used
for the purposes of the present invention. Only residues to the N- and C-
termini of the
subject sequence, which are not matched/aligned with the query sequence, are
considered for the purposes of manually adjusting the percent identity score.
That is,
only query residue positions outside the farthest N- and C- terminal residues
of the
subject sequence.
[099] For example, a 90 amino acid residue subject sequence is aligned with a
I00
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show
a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired
residues represent 10% of the sequence (number of residues at the N- and C-
termini
not matched/total number of residues in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. If the remaining
90
residues were perfectly matched the final percent identity would be 90%. In
another
example, a 90 residue subject sequence is compared with a 100 residue query
sequence. This time the deletions are internal deletions so there are no
residues at the
N- or C-termini of the subject sequence which are not matched/aligned with the
query.
In this case the percent identity calculated by FASTDB is not manually
corrected.
Once again, only residue positions outside the N- and C-terminal ends of the
subject
sequence, as displayed in the FASTDB alignment, which are not matched/aligned
with
the query sequence are manually corrected for. No other manual corrections are
to be
made for the purposes of the present invention.
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[0100] The polynucleotide variants of the invention may contain alterations in
the
coding regions, non-coding regions, or both. Especially preferred are
polynucleotide
variants containing alterations, which produce silent substitutions,
additions, or
deletions, but do not alter the properties or activities of the encoded
polypeptide.
Nucleotide variants produced by silent substitutions due to the degeneracy of
the
genetic code are preferred. Moreover, polypeptide variants in which less than
50, less
than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5,
or 1-2 amino
acids are substituted, deleted, or added in any combination are also
preferred.
Polynucleotide variants can be produced for a variety of reasons, e.g., to
optimize
codon expression for a particular host (change codons in the human mRNA to
those
preferred by a bacterial host such as E. coli).
[0101] Naturally occurring variants are called "allelic variants," and refer
to one of
several alternate forms of a gene occupying a given locus on a chromosome of
an
organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New Yorlc (1985).)
These
allelic variants can vary at either the polynucleotide and/or polypeptide
level and are
included in the present invention. Alternatively, non-naturally occurring
variants may
be produced by mutagenesis techniques or by direct synthesis.
[0102] Using known methods of protein engineering and recombinant DNA
technology, variants may be generated to improve or alter the characteristics
of the
polypeptides of the present invention. For instance, one or more amino acids
can be
deleted from the N-terminus or C-terminus of the polypeptides of the present
invention
without substantial loss of biological function. As an example, the authors of
Ron et
al., J. Biol. Chem. 268: 2984-2988 (1993), reported variant KGF proteins
having
heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino
acid
residues. Similarly, Interferon gamma exhibited up to ten times higher
activity after
deleting 8-10 amino acid residues from the carboxy terminus of this protein.
(Dobeli
et al., J. Biotechnology 7:199-216 (1988).)
[0103] Moreover, ample evidence demonstrates that variants often retain a
biological activity similar to that of the naturally occurring protein. For
example,
Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted
extensive
mutational analysis of human cytokine IL-la. They used random mutagenesis to
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generate over 3,500 individual IL-la mutants that averaged 2.5 amino acid
changes per
variant over the entire length of the molecule. Multiple mutations were
examined at
every possible amino acid position. The investigators found that "[m]ost of
the
molecule could be altered with little effect on either [binding or biological
activity]."
In fact, only 23 unique amino acid sequences, out of more than 3,500
nucleotide
sequences examined, produced a protein that significantly differed in activity
from
wild-type.
[0104] Furthermore, even if deleting one or more amino acids from the N-
terminus
or C-terminus of a polypeptide results in modification or loss of one or more
biological
functions, other biological activities may still be retained. For example, the
ability of a
deletion variant to induce and/or to bind antibodies which recognize the
secreted form
will likely be retained when less than the majority of the residues of the
secreted form
are removed from the N-terminus or C-terminus. Whether a particular
polypeptide
lacking N- or C-terminal residues of a protein retains such immunogenic
activities can
readily be determined by routine methods described herein and otherwise known
in the
art.
[0105] Thus, the invention further includes polypeptide variants which show a
functional activity (e.g., biological activity) of the polypeptides of the
invention. Such
variants include deletions, insertions, inversions, repeats, and substitutions
selected
according to general rules known in the art so as have little effect on
activity.
[0106] The present application is directed to nucleic acid molecules at least
80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid
sequences disclosed herein, (e.g., encoding a polypeptide having the amino
acid
sequence of an N and/or C terminal deletion), irrespective of whether they
encode a
polypeptide having functional activity. This is because even where a
particular nucleic
acid molecule does not encode a polypeptide having functional activity, one of
skill in
the art would still know how to use the nucleic acid molecule, fox instance,
as a
hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the
nucleic
acid molecules of the present invention that do not encode a polypeptide
having
functional activity include, inter alia, (1) isolating a gene or allelic or
splice variants
thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to
metaphase
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chromosomal spreads to provide precise chromosomal location of the gene, as
described in Verma et al., Human Chromosomes: A Manual of Basic Techniques,
Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in specific tissues (e.g., normal pancreatic or diseased pancreatic
tissues);
and (4) ih situ hybridization (e.g., histochemistry) for detecting mRNA
expression in
specific tissues (e.g., normal pancreatic or diseased pancreatic tissues).
[0107] Preferred, however, are nucleic acid molecules having sequences at
least
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid
sequences disclosed herein, which do, in fact, encode a polypeptide having
functional
activity. By a polypeptide having "functional activity" is meant, a
polypeptide capable
of displaying one or more known functional activities associated with a full-
length
(complete) protein of the invention. Such functional activities include, but
are not
limited to, biological activity, antigenicity [ability to bind (or compete
with a
polypeptide of the invention for binding) to an anti-polypeptide of the
invention
antibody], immunogenicity (ability to generate antibody which binds to a
specific
polypeptide of the invention), ability to form multimers with polypeptides of
the
invention, and ability to bind to a receptor or ligand for a polypeptide of
the invention.
[0108] The functional activity of the polypeptides, and fragments, variants
and
derivatives of the invention, can be assayed by various methods.
[0109] For example, in one embodiment where one is assaying for the ability to
bind or compete with full-length polypeptide of the present invention for
binding to an
anti-polypeptide of the invention antibody, various immunoassays known in the
art can
be used, including but not limited to, competitive and non-competitive assay
systems
using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ immunoassays (using
colloidal
gold, enzyme or radioisotope labels, for example), western blots,
precipitation
reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination
assays), complement fixation assays, immunofluorescence assays, protein A
assays,
and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is
detected by detecting a label on the primary antibody. In another embodiment,
the
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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.
[0110] In another embodiment, where a ligand is identified, or the ability of
a
polypeptide fragment, variant or derivative of the invention to multimerize is
being
evaluated, binding can be assayed, e.g., ~by means well-known in the art, such
as, for
example, reducing and non-reducing gel chromatography, protein affinity
chromatography, and affinity blotting. See generally, Phizicky et al.,
Microbiol. Rev.
59:94-123 (1995). In another embodiment, the ability of physiological
correlates of a
polypeptide of the present invention to bind to a substrates) of the
polypeptide of the
invention can be routinely assayed using techniques lcnown in the art.
[0111] In addition, assays described herein (see Examples) and otherwise known
in the art may routinely be applied to measure the ability of polypeptides of
the present
invention and fragments, variants and derivatives thereof to elicit
polypeptide related
biological activity (either ih vitro or in vivo). Other methods will be known
to the
spilled artisan and are within the scope of the invention.
[0112] Of course, due to the degeneracy of the genetic code, one of ordinary
skill
in the art will immediately recognize that a large number of the nucleic acid
molecules
having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to, for example, the nucleic acid sequence of the cDNA contained in
Clone
ID NO:Z, a nucleic acid sequence referred to in Table 1A (e.g., SEQ ID NO:X),
a
nucleic acid sequence disclosed in Table 2 (e.g., the nucleic acid sequence
delineated
in columns 8 and 9) or fragments thereof, will encode polypeptides "having
functional
activity." In fact, since degenerate variants of any of these nucleotide
sequences all
encode the same polypeptide, in many instances, this will be clear to the
skilled artisan
even without performing the above described comparison assay. It will be
further
recognized in the art that, for such nucleic acid molecules that are not
degenerate
variants, a reasonable number will also encode a polypeptide having functional
activity. This is because the slcilled artisan is fully aware of amino acid
substitutions
that are either less likely or not likely to significantly effect protein
function (e.g.,
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replacing one aliphatic amino acid with a second aliphatic amino acid), as
further
described below.
[0113] For example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Sowie et al., "Deciphering the Message
in
Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247:1306-
1310
(1990), wherein the authors indicate that there are two main strategies for
studying the
tolerance of an amino acid sequence to change.
[0114] The first strategy exploits the tolerance of amino acid substitutions
by
natural selection during the process of evolution. By comparing amino acid
sequences
in different species, conserved amino acids can be identified. These conserved
amino
acids are likely important for protein function. In contrast, the amino acid
positions
where substitutions have been tolerated by natural selection indicates that
these
positions are not critical for protein function. Thus, positions tolerating
amino acid
substitution could be modified while still maintaining biological activity of
the protein.
[0115] The second strategy uses genetic engineering to introduce amino acid
changes at specific positions of a cloned gene to identify regions critical
for protein
function. For example, site directed mutagenesis or alanine-scanning
mutagenesis
(introduction of single alanine mutations at every residue in the molecule)
can be used.
See Cunningham et al., Science 244:1081-1085 (1989). The resulting mutant
molecules can then be tested for biological activity.
(0116] As the authors state, these two strategies have revealed that proteins
are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at certain amino acid positions
in the
protein. For example, most buried (within the tertiary structure of the
protein) amino
acid residues require nonpolar side chains, whereas few features of surface
side chains
are generally conserved. Moreover, tolerated conservative amino acid
substitutions
involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu
and Ile;
replacement of the hydroxyl residues Ser and Thr; replacement of the acidic
residues
Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the
basic
residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and
Trp,
and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Besides
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conservative amino acid substitutions, variants of the present invention
include (i)
substitutions with one or more of the non-conserved amino acid residues, where
the
substituted amino acid residues may or may not be one encoded by the genetic
code, or
(ii) substitutions with one or more of the amino acid residues having a
substituent
group, or (iii) fusion of the mature polypeptide with another compound, such
as a
compound to increase the stability and/or solubility of the polypeptide (for
example,
polyethylene glycol), or (iv) fusion of the polypeptide with additional amino
acids,
such as, for example, an IgG Fc fusion region peptide, serum albumin
(preferably
human serum albumin) or a fragment or variant thereof, or leader or secretory
sequence, or a sequence facilitating purification. Such variant polypeptides
are
deemed to be within the scope of those skilled in the art from the teachings
herein.
[0117] For example, polypeptide variants containing amino acid substitutions
of
charged amino acids with other charged or neutral amino acids may produce
proteins
with improved characteristics, such as less aggregation. Aggregation of
pharmaceutical formulations both reduces activity and increases clearance due
to the
aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol.
2:331-340
(1967); Robbins et al., Diabetes .36: 838-845 (1987); Cleland et al., Crit.
Rev.
Therapeutic Drug Carrier Systems 10:307-377 (1993).
[0118] A further embodiment of the invention relates to polypeptides which
comprise the amino acid sequence of a polypeptide having an amino acid
sequence
which contains at least one amino acid substitution, but not more than 50
amino acid
substitutions, even more preferably, not more than 40 amino acid
substitutions, still
more preferably, not more than 30 amino acid substitutions, and still even
more
preferably, not more than 20 amino acid substitutions from a polypeptide
sequence
disclosed herein. Of course it is highly preferable for a polypeptide to have
an amino
acid sequence which comprises the amino acid sequence of a polypeptide of SEQ
ID
NO:Y, an amino acid sequence encoded by SEQ ID NO:X, an amino acid sequence
encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table
2, an
amino, acid sequence encoded by the complement of SEQ ID NO:X, andlor the
amino
acid sequence encoded by cDNA contained in Clone TD NO:Z which contains, in
order
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of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6,
5, 4, 3, 2 or
1 amino acid substitutions.
[0119] In specific embodiments, the polypeptides of the invention comprise, or
alternatively, consist of, fragments or variants of a reference amino acid
sequence
selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof
(e.g., the mature form andlor other fragments described herein); (b) the amino
acid
sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid
sequence
encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino
acid
sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9
of
Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA
contained in Clone ID NO:Z or fragments thereof; wherein the fragments or
variants
have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions,
substitutions, andlor deletions when compared to the reference amino acid
sequence.
In preferred embodiments, the amino acid substitutions are conservative.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Poly~cucleotide ahd Polypeptide F~agmeti.ts
[0120] The present invention is also directed to polynucleotide fragments of
the
polynucleotides (nucleic acids) of the invention. In the present invention, a
"polynucleotide fragment" refers to a polynucleotide having a nucleic acid
sequence
which, for example: is a portion of the cDNA contained in Clone ID NO:Z or the
complementary strand thereto; is a portion of the polynucleotide sequence
encoding
the polypeptide encoded by the cDNA contained in Clone ID NO:Z or the
complementary strand thereto; is a portion of a polynucleotide sequence
encoding the
amino acid sequence encoded by the region of SEQ ID NO:X as defined in columns
8
and 9 of Table 2 or the complementary strand thereto; is a portion of the
polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table
2 or
the complementary strand thereto; is a portion of the polynucleotide sequence
in SEQ
ID NO:X or the complementary strand thereto; is a polynucleotide sequence
encoding
a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence
encoding
a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide
sequence
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encoding a portion of a polypeptide encoded by the complement of the
polynucleotide
sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding
the
amino acid sequence encoded by the region of SEQ ID NO:B as defined in column
6
of Table 1B or the complementary strand thereto; or is a portion of the
polynucleotide
sequence of SEQ ID NO:B as defined in column 6 of Table 1B or the
complementary
strand thereto.
[0121] The polynucleotide fragments of the invention are preferably at least
about
15 nt, and more preferably at least about 20 nt, still more preferably at
least about 30
nt, and even more preferably, at least about 40 nt, at least about 50 nt, at,
least about 75
nt, or at least about 150 nt in length. A fragment "at least 20 nt in length,"
for
example, is intended to include 20 or more contiguous bases from the cDNA
sequence
contained in Clone ID NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or
the complementary stand thereto. In this context "about" includes the
particularly
recited value or a value larger or smaller by several (5, 4, 3, 2, or 1)
nucleotides, at
either terminus or at both termini. These nucleotide fragments have uses that
include,
but are not limited to, as diagnostic probes and primers as discussed herein.
Of course,
larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000,
or 2000
nucleotides in length) are also encompassed by the invention.
[0122] Moreover, representative examples of polynucleotide fragments of the
invention, comprise, or alternatively consist of, a sequence from about
nucleotide
number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-
450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900,
901-
950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-
1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-
1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-
2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-
2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-
2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-
3050, 3051-3100, 3101-3150, 3I5I-3200, 3201-3250, 3251-3300, 3301-3350, 3351-
3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-
3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-
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4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-
4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-
4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-
5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-
5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-
5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-
6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-
6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-
6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-
7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand
thereto. In this context "about" includes the particularly recited range or a
range larger
or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at
both termini.
Preferably, these fragments encode a polypeptide, which has a functional
activity (e.g.,
biological activity). More preferably, these polynucleotides can be used as
probes or
primers as discussed herein. Polynucleotides which hybridize to one or more of
these
polynucleotides under stringent hybridization conditions or alternatively,
under Iower
stringency conditions are also encompassed by the invention, as are
polypeptides
encoded by these polynucleotides.
[0123] Further representative examples of polynucleotide fragments of the
invention, comprise, or alternatively consist of, a sequence from about
nucleotide
number 1-50, SI-100, 101-I50, 151-200, 201-250, 25I-300, 301-350, 351-400, 401-
450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900,
901-
950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-
1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-
1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-
2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-
2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-
2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-
3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-
3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-
3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-
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4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-
4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-
4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-
5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-
5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-
5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-
6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-
6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-
6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-
7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in Clone ID
NO:Z, or the complementary strand thereto. In this context "about" includes
the
particularly recited range or a range larger or smaller by several (5, 4, 3,
2, or 1)
nucleotides, at either terminus or at both termini. Preferably, these
fragments encode a
polypeptide, which has a functional activity (e.g., biological activity). More
preferably, these polynucleotides can be used as probes or primers as
discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under
stringent hybridization conditions or alternatively, under lower stringency
conditions
are also encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
[0124] Moreover, representative examples of polynucleotide fragments of the
invention comprise, or alternatively consist of, a nucleic acid sequence
comprising
one, two, three, four, five, six, seven, eight, nine, ten, or more of the
above described
polynucleotide fragments of the invention in combination with a polynucleotide
sequence delineated in Table 1B column 6. Additional, representative examples
of
polynucleotide fragments of the invention comprise, or alternatively consist
of, a
nucleic acid sequence comprising one, two, three, four, five, six, seven,
eight, nine,
ten, or more of the above described polynucleotide fragments of the invention
in
combination with a polynucleotide sequence that is the complementary strand of
a
sequence delineated in column 6 of Table 1B. In fiu-ther embodiments, the
above-
described polynucleotide fragments of the invention comprise, or alternatively
consist
of, sequences delineated in Table 1B, column 6, and have a nucleic acid
sequence
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which is different from that of the BAC fragment having the sequence disclosed
in
SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-
described polynucleotide fragments of the invention comprise, or alternatively
consist
of, sequences delineated in Table 1B, column 6, and have a nucleic acid
sequence
which is different from that published for the BAC clone identified as BAC ID
NO:A
(see Table 1B, column 4). In additional embodiments, the above-described
polynucleotides of the invention comprise, or alternatively consist of,
sequences
delineated Table 1B, column 6, and have a nucleic acid sequence which is
different
from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B,
column 4). Polypeptides encoded by these polynucleotides, other
polynucleotides that
encode these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[0125] In additional specific embodiments, polynucleotides of the invention
comprise, or alternatively consist of, one, two, three, four, five, six,
seven, eight, nine,
ten, or more fragments of the sequences delineated in column 6 of Table 1B,
and the
polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column
2) or
fragments or variants thereof. Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides are also encompassed by the invention.
[0126] In additional specific embodiments, polynucleotides of the invention
comprise, or alternatively consist of, one, two, three, four, five, six,
seven, eight, nine,
ten, or more fragments of the sequences delineated in column 6 of Table 1B
which
correspond to the same Clone ID NO:Z (see Table 1B, column 1), and the
polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or
fragments or variants thereof. Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides are also encompassed by the invention.
[0127] In further specific embodiments, polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or
more fragments of the sequences delineated in the same row of column 6 of
Table 1B,
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and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A
or 1B)
or fragments or variants thereof. Polypeptides encoded by these
polynucleotides, other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides axe also encompassed by the invention.
[0128] In additional specific embodiments, polynucleotides of the invention
comprise, or alternatively consist of a polynucleotide sequence in which the
3' 10
polynucleotides of one of the sequences delineated in column 6 of Table 1B and
the 5'
polynucleotides of the sequence of SEQ ID NO:X axe directly contiguous.
Nucleic
acids which hybridize to the complement of these 20 contiguous polynucleotides
under
stringent hybridization conditions or alternatively, under lower stringency
conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids that
encode these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[0129] In additional specific embodiments, polynucleotides of the invention
comprise, or alternatively consist of a polynucleotide sequence in which the
3' 10
polynucleotides of one of the sequences delineated in column 6 of Table 1B and
the 5'
10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X
(e.g., as
described herein) are directly contiguous Nucleic acids which hybridize to the
complement of these 20 contiguous polynucleotides under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed
by the invention. Polypeptides encoded by these polynucleotides and/or nucleic
acids,
other polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies
that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
[0130] In further specific embodiments, polynucleotides of the invention
comprise,
or alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and
the 5'
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polynucleotides of the sequence of one of the sequences delineated in column 6
of
Table 1B are directly contiguous. Nucleic acids which hybridize to the
complement of
these 20 contiguous polynucleotides under stringent hybridization conditions
or
alternatively, under lower stringency conditions, are also encompassed by the
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids,
other
polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies that
bind these polypeptides are also encompassed by the invention. Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
[0131] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides
of one of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another sequence in column 6 are directly contiguous. In
preferred
embodiments, the 3' 10 polynucleotides of one of the sequences delineated in
column
6 of Table 1B is directly contiguous with the 5' 10 polynucleotides of the
next
sequential exon delineated in Table 1B, column 6. Nucleic acids which
hybridize to
the complement of these 20 contiguous polynucleotides under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed
by the invention. Polypeptides encoded by these polynucleotides and/or nucleic
acids,
other polynucleotides andlor nucleic acids encoding these polypeptides, and
antibodies
that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
[0132] In the present invention, a "polypeptide fragment" refers to an amino
acid
sequence which is a portion of that contained in SEQ ID NO:Y, a portion of an
amino
acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8
and 9
of Table 2, a portion of an amino acid sequence encoded by the polynucleotide
sequence of SEQ ID NO:X, a portion of an amino acid sequence encoded by the
complement of the polynucleotide sequence in SEQ ID NO:X, and/or a portion of
an
amino acid sequence encoded by the cDNA contained in Clone ID NO:Z. Protein
(polypeptide) fragments may be "free-standing," or comprised within a larger
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polypeptide of which the fragment forms a part or region, most preferably as a
single
continuous region. Representative examples of polypeptide fragments of the
invention, include, for example, fragments comprising, or alternatively
consisting of,
from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-
140,
141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-
320,
321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-
500,
501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-
680,
681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-
860,
861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020,
1021-
1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-
1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-
1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or
1441
to the end of the coding region. In a preferred embodiment, polypeptide
fragments of
the invention include, for example, fragments comprising, or alternatively
consisting
of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 102-120,
121-
140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300,
301-
320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480,
481-
500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660,
661-
680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840,
841-
860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-
1020,
1021-1040, 1041-1060, 1.061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160,
1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300,
1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440,
or
1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide
fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40,
45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in
length. In
this context "about" includes the particularly recited ranges or values, or
ranges or
values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either
extreme or at
both extremes. Polynucleotides encoding these polypeptide fragments are also
encompassed by the invention.
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[0133] Even if deletion of one or more amino acids from the N-terminus of a
protein results in modification of loss of one or more biological functions of
the
protein, other functional activities (e.g., biological activities, ability to
multimerize,
ability to bind a ligand) may still be retained. For example, the ability of
shortened
muteins to induce and/or bind to antibodies which recognize the complete or
mature
forms of the polypeptides generally will be retained when less than the
majority of the
residues of the complete or mature polypeptide are removed from the N-
terminus.
Whether a particular polypeptide lacking N-terminal residues of a complete
polypeptide retains such immunologic activities can readily be determined by
routine
methods described herein and otherwise known in the art. It is not unlikely
that a
mutein with a large number of deleted N-terminal amino acid residues may
retain
some biological or immunogenic activities. In fact, peptides composed of as
few as
six amino acid residues may often evolve an immune response.
[0134] Accordingly, polypeptide fragments include the secreted protein as well
as
the mature form. Further preferred polypeptide fragments include the secreted
protein
or the mature form having a continuous series of deleted residues from the
amino or
the carboxy terminus, or both. For example, any number of amino acids, ranging
from
1-60, can be deleted from the amino terminus of either the secreted
polypeptide or the
mature form. Similarly, any number of amino acids, ranging from 1-30, can be
deleted
from the carboxy terminus of the secreted protein or mature form. Furthermore,
any
combination of the above amino and carboxy terminus deletions is preferred.
Similarly, polynucleotides encoding these polypeptide fragments are also
preferred.
[0135] The present invention further provides polypeptides having one or more
residues deleted from the amino terminus of the amino acid sequence of a
polypeptide
disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by
the
polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a
polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and
9 of
Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in
column 6
of Table 1B, and/or a polypeptide encoded by the cDNA contained in Clone ID
NO:Z). In particular, N-terminal deletions may be described by the general
formula
m-q, where q is a whole integer representing the total number of amino acid
residues
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in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID
NO:Y, or
the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8
and
9 of Table 2), and m is defined as any integer ranging from 2 to q-6.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[0136] The present invention further provides polypeptides having one or more
residues from the carboxy terminus of the amino acid sequence of a polypeptide
disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by
the
polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the
portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or a
polypeptide encoded by the cDNA contained in Clone ID NO:Z). In particular, C-
terminal deletions may be described by the general formula 1-n, where n is any
whole
integer ranging from 6 to q-1, and where n corresponds to the position of
amino acid
residue in a polypeptide of the invention. Polynucleotides encoding these
polypeptides
are also encompassed by the invention.
[0137] In addition, any of the above described N- or C-terminal deletions can
be
combined to produce a N- and C-terminal deleted polypeptide. The invention
also
provides polypeptides having one or more amino acids deleted from both the
amino
and the carboxyl termini, which may be described generally as having residues
m-n of
a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the
preferred polypeptide disclosed as SEQ ID NO:Y and the polypeptide encoded by
the
portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA
contained in Clone ID NO:Z, and/or the complement thereof, where n and m are
integers as described above. Polynucleotides encoding these polypeptides are
also
encompassed by the invention.
[0138] Also as mentioned above, even if deletion of one or more amino acids
from
the C-terminus of a protein results in modification of loss of one or more
biological
fitnctions of the protein, other functional activities (e.g., biological
activities, ability to
multimerize, ability to bind a ligand) may still be retained. For example the
ability of
the shortened mutein to induce and/or bind to antibodies which recognize the
complete
or mature forms of the polypeptide generally will be retained when less than
the
majority of the residues of the complete or mature polypeptide are removed
from the
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C-terminus. Whether a particular polypeptide lacking C-terminal residues of a
complete polypeptide retains such immunologic activities can readily be
determined
by routine methods described herein and otherwise known in the art. It is not
unlikely
that a mutein with a large number of deleted C-terminal amino acid residues
may
retain some biological or immunogenic activities. In fact, peptides composed
of as
few as six amino acid residues may often evoke an immune response.
[0139] The present application is also directed to proteins containing
polypeptides
at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide
sequence set forth herein. In preferred embodiments, the application is
directed to
proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or
99% identical to polypeptides having the amino acid sequence of the specific N-
and
C-terminal deletions. Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0140] Any polypeptide sequence encoded by, for example, the polynucleotide
sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for
example, in Tables 1A and 2), the cDNA contained in Clone ID NO:Z, or the
polynucleotide sequence as defined in column 6 of Table 1B, may be analyzed to
determine certain preferred regions of the polypeptide. For example, the amino
acid
sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X
(e.g., the polypeptide of SEQ ID NO:Y and the polypeptide encoded by the
portion of
SEQ ID NO:X as defined in columns 8 and 9 of Table 2) or the cDNA contained in
Clone ID NO:Z may be analyzed using the default parameters of the DNASTAR
computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, WI 53715 USA;
http://www.dnastar.com~.
[0141] Polypeptide regions that may be routinely obtained using the DNASTAR
computer algorithm include, but are not limited to, Gamier-Robson alpha-
regions,
beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-
regions,
and turn-regions; I~yte-Doolittle hydrophilic regions and hydrophobic regions;
Eisenberg alpha- and beta-amphipathic regions; I~arplus-Schulz flexible
regions;
Emini surface-forming regions; and Jameson-Wolf regions of high antigenic
index.
Among highly preferred polynucleotides of the invention in this regard are
those that
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encode polypeptides comprising regions that combine several structural
features, such
as several (e.g., 1, 2, 3 or 4) of the features set out above.
[0142] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic
regions,
Emini surface-forming regions, and Jameson-Wolf regions of high antigenic
index
(i.e., containing four or more contiguous amino acids having an antigenic
index of
greater than or equal to 1.5, as identified using the default parameters of
the Jameson-
Wolf program) can routinely be used to determine polypeptide regions that
exhibit a
high degree of potential for antigenicity. Regions of high antigenicity are
determined
from data by DNASTAR analysis by choosing values which represent regions of
the
polypeptide which are likely to be exposed on the surface of the polypeptide
in an
environment in which antigen recognition may occur in the process of
initiation of an
immune response.
[0143] Preferred polypeptide fragments of the invention are fragments
comprising,
or alternatively, consisting of, an amino acid sequence that displays a
functional
activity (e.g. biological activity) of the polypeptide sequence of which the
amino.acid
sequence is a fragment. By a polypeptide displaying a "functional activity" is
meant a
polypeptide capable of one or more known functional activities associated with
a full-
length protein, such as, for example, biological activity, antigenicity,
immunogenicity,
and/or multimerization, as described herein.
[0144] Other preferred polypeptide fragments are biologically active
fragments.
Biologically active fragments are those exhibiting activity similar, but not
necessarily
identical, to an activity of the polypeptide of the present invention. The
biological
activity of the fragments may include an improved desired activity, or a
decreased
undesirable activity.
[0145] In preferred embodiments, polypeptides of the invention comprise, or
alternatively consist of, one, two, three, four, five or more of the antigenic
fragments
of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides
encoding
these polypeptides are also encompassed by the invention.
[0146] The present invention encompasses polypeptides comprising, or
alternatively consisting of, an epitope of the polypeptide sequence shown in
SEQ ID
NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary
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strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X
as
defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the
portion of SEQ ID NO:B as defined in column 6 of Table 1B or the complement
thereto; the polypeptide sequence encoded by the cDNA contained in Clone ID
NO:Z;
or the polypeptide sequence encoded by a polynucleotide that hybridizes to the
sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the
complement of a portion of SEQ ID NO:X as defined in columns ~ and 9 of Table
2,
or the cDNA sequence contained in Clone ID NO:Z under stringent hybridization
conditions or alternatively, under lower stringency hybridization as defined
sups°a. The
present invention further encompasses polynucleotide sequences encoding an
epitope
of a polypeptide sequence of the invention (such as, for example, the sequence
disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of
the
complementary strand of a polynucleotide sequence encoding an epitope of the
invention, and polynucleotide sequences which hybridize to the complementary
strand
under stringent hybridization conditions or alternatively, under lower
stringency
hybridization conditions defined supra.
[0147] The term "epitopes," as used herein, refers to portions of a
polypeptide
having antigenic or immunogenic activity in an animal, preferably a mammal,
and
most preferably in a human. In a preferred embodiment, the present invention
encompasses a polypeptide comprising an epitope, as well as the polynucleotide
encoding this polypeptide. An "immunogenic epitope," as used herein, is
defined as a
portion of a protein that elicits an antibody response in an animal, as
determined by
any method known in the art, for example, by the methods for generating
antibodies
described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998- 4002 (1983)). The term "antigenic epitope," as used herein, is
defined as a
portion of a protein to which an antibody can immunospecifically bind its
antigen as
determined by any method well known in the art, for example, by the
immunoassays
described herein. Immunospecific binding excludes non-specific binding but
does not
necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
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[0148] Fragments, which function as epitopes may be produced by any
conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA
82:5131-
5135 (1985) further described in U.S. Patent No. 4,631,211.)
[0149] In the present invention, antigenic epitopes preferably contain a
sequence
of at least 4, at least 5, at least 6, at least 7, more preferably at least 8,
at least 9, at least
10, at least 1 l, at least 12, at least 13, at least 14, at least 15, at least
20, at least 25, at
least 30, at least 40, at least 50, and, most preferably, between about 15 to
about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100
amino acid residues in length. Additional non-exclusive preferred antigenic
epitopes
include the antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic
epitopes are useful, for example, to raise antibodies, including monoclonal
antibodies,
that specifically bind the epitope. Preferred antigenic epitopes include the
antigenic
epitopes disclosed herein, as well as auy combination of two, three, four,
five or more
of these antigenic epitopes. Antigenic epitopes can be used as the target
molecules in
immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984);
Sutcliffe et
al., Science 219:660-666 (1983)).
[0150] Non-limiting examples of epitopes of polypeptides that can be used to
generate antibodies of the invention include a polypeptide comprising, or
alternatively
consisting of, at least one, two, three, four, five, six or more of the
portions) of SEQ
ID NO:Y specified in column 6 of Table 1A. These polypeptide fragments have
been
determined to bear antigenic epitopes of the proteins of the invention by the
analysis of
the Jameson-Wolf antigenic index, which is included in the DNAStar suite of
computer programs. By "comprise" it is intended that a polypeptide contains at
least
one, two, three, four, five, six or more of the portions) of SEQ ID NO:Y shown
in
column 6 of Table 1A, but it may contain additional flanking residues on
either the
amino or carboxyl termini of the recited portion. Such additional flanking
sequences
are preferably sequences naturally found adjacent to the portion; i.e.,
contiguous
sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be
sequences from a heterolgous polypeptide, such as from another protein
described
herein or from a heterologous polypeptide not described herein. In particular
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embodiments, epitope portions of a polypeptide of the invention comprise one,
two,
three, or more of the portions of SEQ ID NO:Y shown in column 6 of Table 1A.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
(0151] Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. See, for instance,
Sutcliffe et
al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA
82:910-914;
and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic
epitopes include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic epitopes.
The
polypeptides comprising one or more immunogenic epitopes may be presented for
eliciting an antibody response together with a carrier protein, such as an
albumin, to an
animal system (such as rabbit or mouse), or, if the polypeptide is of
sufficient length
(at least about 25 amino acids), the polypeptide may be presented without a
carrier.
However, immunogenic epitopes comprising as few as 8 to 10 amino acids have
been
shown to be sufficient to raise antibodies capable of binding to, at the very
least, linear
epitopes in a denatured polypeptide (e.g., in Western blotting).
[0152] Epitope-bearing polypeptides of the present invention may be used to
induce antibodies according to methods well known in the art including, but
not
limited to, ih vivo immunization, in vitro immunization, and phage display
methods.
See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al.,
J. Gen. Virol.,
66:2347-2354 (1985). If ih vivo immunization is used, animals may be immunized
with free peptide; however, anti-peptide antibody titer may be boosted by
coupling the
peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KI,H)
or
tetanus toxoid. For instance, peptides containing cysteine residues may be
coupled to
a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester
(MBS), while other peptides may be coupled to carriers using a more general
linking
agent such as glutaraldehyde. Animals such as rabbits, rats and mice are
immunized
with either free or carrier- coupled peptides, for instance, by
intraperitoneal and/or
intradermal injection of emulsions containing about 100 ~g of peptide or
carrier
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune
response. Several booster injections may be needed, for instance, at intervals
of about
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two weeks, to provide a useful titer of anti-peptide antibody which can be
detected, for
example, by ELISA assay using free peptide adsorbed to a solid surface. The
titer of
anti-peptide antibodies in serum from an immunized animal may be increased by
selection of anti-peptide antibodies, for instance, by adsorption to the
peptide on a
solid support and elution of the selected antibodies according to methods well
known
in the art.
[0153] As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention (e.g., those comprising an immunogenic
or
antigenic epitope) can be fused to heterologous polypeptide sequences. For
example,
polypeptides of the present invention (including fragments or variants
thereof), may be
fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or
portions
thereof (CHl, CH2, CH3, or any combination thereof and portions thereof,
resulting in
chimeric polypeptides. By way of another non-limiting example, polypeptides
and/or
antibodies of the present invention (including fragments or variants thereof)
may be
fused with albumin (including but not limited to recombinant human serum
albumin or
fragments or variants thereof (see, e.g., U.S. Patent No. 5,876,969, issued
March 2,
1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June 16,
1998,
herein incorporated by reference in their entirety)). In a preferred
embodiment,
polypeptides and/or antibodies of the present invention (including fragments
or
variants thereof) are fused with the mature form of human serum albumin (i.e.,
amino
acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP Patent
0 322
094) which is herein incorporated by reference in its entirety. In another
preferred
embodiment, polypeptides and/or antibodies of the present invention (including
fragments or variants thereof) are fused with polypeptide fragments
comprising, or
alternatively consisting of, amino acid residues 1-z of human serum albumin,
where z
is an integer from 369 to 419, as described in U.S. Patent 5,766,883 herein
incorporated by reference in its entirety. Polypeptides and/or antibodies of
the present
invention (including fragments or variants thereof) may be fused to either the
N- or C-
terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide
or
human serum albumin polypeptide). Polynucleotides encoding fusion proteins of
the
invention are also encompassed by the invention.
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[0154] Such fusion proteins as those described above may facilitate
purification
and may increase half life izz vivo. This has been shown for chimeric proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86
(1988).
Enhanced delivery of an antigen across the epithelial barrier to the immune
system has
been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding
partner
such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO
99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure
due to
the IgG portion desulfide bonds have also been found to' be more efficient in
binding
and neutralizing other molecules than monomeric polypeptides or fragments
thereof
alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).
Nucleic
acids encoding the above epitopes can also be recombined with a gene of
interest as an
epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection
and
purification of the expressed polypeptide. For example, a system described by
Janknecht et al. allows for the ready purification of non-denatured fusion
proteins
expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci.
USA
88:8972- 897). In this system, the gene of interest is subcloned into a
vaccinia
recombination plasmid such that the open reading frame of the gene is
translationally
fused to an amino-terminal tag consisting of six histidine residues. The tag
serves as a
matrix binding domain for the fusion protein. Extracts from cells infected
with the
recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose
column and
histidine-tagged proteins can be selectively eluted with imidazole-containing
buffers.
Fusion Proteins
[0155] Any polypeptide of the present invention can be used to generate fusion
proteins. For example, the polypeptide of the present invention, when fused to
a
second protein, can be used as an antigenic tag. Antibodies raised against the
polypeptide of the present invention can be used to indirectly detect the
second protein
by binding to the polypeptide. Moreover, because secreted proteins target
cellular
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locations based on trafficking signals, polypeptides of the present invention
which are
shown to be secreted can be used as targeting molecules once fused to other
proteins.
[0156] Examples of domains that can be fused to polypeptides of the present
invention include not only heterologous signal sequences, but also other
heterologous
functional regions. The fusion does not necessarily need to be direct, but may
occur
through linker sequences.
[0157] In certain preferred embodiments, proteins of the invention are fusion
proteins comprising an amino acid sequence that is an N and/or C- terminal
deletion of
a polypeptide of the invention. In preferred embodiments, the invention is
directed to
a fusion protein comprising an amino acid sequence that is at least 80%, 85%,
90%,
95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the
invention.
Polynucleotides encoding these proteins are also encompassed by the invention.
[0158] Moreover, fusion proteins may also be engineered to improve
characteristics of the polypeptide of the present invention. For instance, a
region of
additional amino acids, particularly charged amino acids, may be added to the
N-
terminus of the polypeptide to improve stability and persistence during
purification
from the host cell or subsequent handling and storage. Also, peptide moieties
may be
added to the polypeptide to facilitate purification. Such regions may be
removed prior
to final preparation of the polypeptide. The addition of peptide moieties to
facilitate
handling of polypeptides is familiar and routine techniques in the art.
[0159] As one of skill in the art will appreciate' that, as discussed above,
polypeptides of the present invention, and epitope-bearing fragments thereof,
can be
combined with heterologous polypeptide sequences. For example, the
polypeptides of
the present invention may be fused with heterologous polypeptide sequences,
for
example, the polypeptides of the present invention may be fused with the
constant
domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CHl, CH2,
CH3, and any combination thereof, including both entire domains and portions
thereof), or albumin (including, but not limited to, native or recombinant
human
albumin or fragments or variants thereof (see, e.g., U.S. Patent No.
5,876,969, issued
March 2, 1999, EP Patent 0 413 622, and U.S. Patent No. 5,766,883, issued June
16,
1998, herein incorporated by reference in their entirety)), resulting in
chimeric
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polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant region of
immunoglobulin molecules together with another human protein or part thereof.
In
many cases, the Fc part in a fusion protein is beneficial in therapy and
diagnosis, and
thus can result in, for example, improved pharmacokinetic properties (EP-A
0232
262). Alternatively, deleting the Fc part after the fusion protein has been
expressed,
detected, and purified, Would be desired. For example, the Fc portion may
hinder
therapy and diagnosis if the fusion protein is used as an antigen for
immunizations. In
drug discovery, for example, human proteins, such as hIL-5, have been fused
with Fc
portions for the purpose of high-throughput screening assays to identify
antagonists of
hIL-5. See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K.
Johanson et
al., J. Biol. Chem. 270:9459-9471 (1995).
[0160] Moreover, the polypeptides of the present invention can be fused to
marker
sequences, such as a polypeptide, which facilitates purification of the fused
polypeptide. In preferred embodiments, the marker amino acid sequence is a
hexa-
histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc.,
9259 Eton
Avenue, Chatsworth, CA, 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824
(1989),
for instance, hexa-histidine provides for convenient purification of the
fusion protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767
(1984).) .
[0161] Additional fusion proteins of the invention may be generated through
the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"), briefly described below, and
further
described herein. DNA shuffling may be employed to modulate the activities of
polypeptides of the invention, such methods can be used to generate
polypeptides with
altered activity, as well as agonists and antagonists of the polypeptides.
See, generally,
U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458,
and
Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends
Biotechnol. 16(2):76-82 (1998); Hansson et al., J. Mol. Biol. 287:265-76
(1999); and
Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998); each of these patents
and
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publications are hereby incorporated by reference in its entirety). In a
preferred
embodiment, one or more components, motifs, sections, parts, domains,
fragments,
etc., of a polynucleotide encoding a polypeptide of the invention may be
recombined
with one or more components, motifs, sections, parts, domains, fragments,
etc., of one
or more heterologous molecules encoding a heterologous polypeptide.
[0162] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
Recombinant and Synthetic Production of Polyueptides of the Invention
[0163] The present invention also relates to vectors containing the
polynucleotide
of the present invention, host cells, and the production of polypeptides by
synthetic
and recombinant techniques. The vector may be, for example, a phage, plasmid,
viral,
or retroviral vector. Retroviral vectors may be replication competent or
replication
defective. In the latter case, viral propagation generally will occur only in
complementing host cells.
[0164] The polynucleotides of the invention may be joined to a vector
containing a
selectable marker for propagation in a host. Generally, a plasmid vector is
introduced
in a precipitate, such as a calcium phosphate precipitate, or in a complex
with a
charged lipid. If the vector is a virus, it may be packaged in vitro using an
appropriate
packaging cell line and then transduced into host cells.
[0165] The polynucleotide insert should be operatively linked to an
appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating codon at the beginning and a termination codon (UAA,
UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
[0166] As indicated, the expression vectors will preferably include at least
one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
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resistance, glutamine synthase, for eukaryotic cell culture and tetracycline,
kanamycin
or ampicillin resistance genes for culturing in E. coli and other bacteria.
Representative examples of appropriate hosts include, but are not limited to,
bacterial
cells, such as E. coli, Sty~eptomyces and Salmonella typhimu~ium cells; fungal
cells,
such as yeast cells (e.g., Saccha~omyces ce~evisiae or Piclaia
pastor°is (ATCC
Accession No. 201178)); insect cells such as D~osophila S2 and
Spodoptef°a Sf9 cells;
animal cells such as CHO, COS, 293, NSO and Bowes melanoma cells; and plant
cells. Appropriate culture mediums and conditions for the above-described host
cells
are known in the art.
[0167] Among vectors preferred for use in bacteria include pQE70, pQE60 and
pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors,
pNH8A, pNHl6a, pNHl8A, pNH46A, available from Stratagene Cloning Systems,
Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia
Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,
pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Preferred expression vectors for use in yeast
systems
include, but are not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ,
pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and
PA0815 (all available from Invitrogen, Carlsbad, CA). Other suitable vectors
will be
readily apparent to the slcilled artisan.
[0168] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers can be amplified in the presence of the drugs methionine sulphoximine
or
methotrexate, respectively. An advantage of glutamine synthase based vectors
is the
availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are
glutamine
synthase negative. Glutamine synthase expression systems can also function in
glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells)
by
providing additional inhibitor to prevent the functioning of the endogenous
gene. A
glutamine synthase expression system and components thereof are detailed in
PCT
publications: W087/04462; W086/05807; W089/01036; W089/10404; and
W091/06657 which are hereby incorporated in their entireties by reference
herein.
Additionally, glutamine synthase expression vectors can be obtained from Lonza
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Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal
antibodies using a GS expression system in murine myeloma cells is described
in
Bebbington et al., Bioltecl2yaology 10:169(1992) and in Biblia and Robinson
Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.
[0169] The present invention also relates to host cells containing the above-
described vector constructs described herein, and additionally encompasses
host cells
containing nucleotide sequences of the invention that are operably associated
with one
or more heterologous control regions (e.g., promoter and/or enhancer) using
techniques known of in the art. The host cell can be a higher eukaryotic cell,
such as a
mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such
as a yeast
cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A
host strain
may be chosen, which modulates the expression of the inserted gene sequences,
or
modifies and processes the gene product in the specific fashion desired.
Expression
from certain promoters can be elevated in the presence of certain inducers;
thus
expression of the genetically engineered polypeptide may be controlled.
Furthermore,
different host cells have characteristics and specific mechanisms for the
translational
and post-translational processing and modification (e.g., phosphorylation,
cleavage) of
proteins. Appropriate cell lines can be chosen to ensure the desired
modifications and
processing of the foreign protein expressed.
[0170] Introduction of the nucleic acids and nucleic acid constructs of the
invention into the host cell can be effected by calcium phosphate
transfection, DEAE-
dextran mediated transfection, cationic lipid-mediated transfection,
electroporation,
transduction, infection, or other methods. Such methods are described in many
standard laboratory manuals, such as Davis et al., Basic Methods In Molecular
Biology
(1986). It is specifically contemplated that the polypeptides of the present
invention
may in fact be expressed by a host cell lacking a recombinant vector.
[0171] In~addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous genetic material (e.g.,
pancreatic
antigen coding sequence), and/or to include genetic material (e.g.,
heterologous
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polynucleotide sequences) that is operably associated with pancreatic
associated
polynucleotides of the invention, and which activates, alters, and/or
amplifies
endogenous pancreatic associated polynucleotides. For example, techniques
known in
the art may be used to operably associate heterologous control regions (e.g.,
promoter
and/or enhancer) and endogenous pancreatic associated polynucleotide sequences
via
homologous recombination (see, e.g., US Patent Number 5,641,670, issued June
24,
1997; International Publication Number WO 96/29411; International Publication
Number WO 94/12650; Koller et al., P~oc. Natl. Acad. Sci. USA 86:8932-8935
(1989);
and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of
which are
incorporated by reference in their entireties).
[0172] Polypeptides of the present invention can also be recovered from:
products
purified from natural sources, including bodily fluids, tissues and cells,
whether
directly isolated or cultured; products of chemical synthetic procedures; and
products
produced by recombinant techniques from a prokaryotic or eukaryotic host,
including,
for example, bacterial, yeast, higher plant, insect, and mammalian cells.
Depending
upon the host employed in a recombinant production procedure, the polypeptides
of
the present invention may be glycosylated or may be non-glycosylated. In
addition,
polypeptides of the invention may also include an initial modified methionine
residue,
in some cases as a result of host-mediated processes. Thus, it is well known
in the art
that the N-terminal methionine encoded by the translation initiation codon
generally is
removed with high efficiency fiom any protein after translation in all
eukaryotic cells.
While the N-terminal methionine on most proteins also is efficiently removed
in most
prokaryotes, for some proteins, this prokaryotic removal process is
inefficient,
depending on the nature of the amino acid to which the N-terminal methionine
is
covalently linked.
[0173] In one embodiment, the yeast Pichia pastoris is used to express
polypeptides of the invention in a eukaryotic system. Pichia pastoris is a
methylotrophic yeast which can metabolize methanol as its sole carbon source.
A
main step in the methanol metabolization pathway is the oxidation of methanol
to
formaldehyde using 02. This reaction is catalyzed by the enzyme alcohol
oxidase. In
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order to metabolize methanol as its sole carbon source, Pichia pastoris must
generate
high levels of alcohol oxidase due, in part, to the relatively low affinity of
alcohol
oxidase for O2. Consequently, in a growth medium depending on methanol as a
main
carbon source, the promoter region of one of the two alcohol oxidase genes
(AOXl ) is
highly active. In the presence of methanol, alcohol oxidase produced from the
AOXI
gene comprises up to approximately 30% of the total soluble protein in Piclzia
pasto~is. See, Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz,
P.J, et al.,
Yeast 5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76
(1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide
of the
present invention, under the transcriptional regulation of all or part of the
AOXl
regulatory sequence is.expressed at exceptionally high levels in Pichia yeast
grown in
the presence of methanol.
[0174] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a polypeptide of the invention, as set forth herein, in a Pichea
yeast system
essentially as described in "Pichia Protocols: Methods in Molecular Biology,"
D.R.
Higgins and J. Cregg, eds. The Humana Press, Totowa, NJ, 1998. This expression
vector allows expression and secretion of a polypeptide of the invention by
virtue of
the strong AOXI promoter linked to the Pichia pasto~is alkaline phosphatase
(PHO)
secretory signal peptide (i.e., leader) located upstream of a multiple cloning
site.
[0175] Many other yeast vectors could be used in place of pPIC9K, such as,
pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,
pPIC3.5, PHIL-D2, pHIL-S 1, pPIC3.5K, and PA0815, as one slcilled in the art
would
readily appreciate, as long as the proposed expression construct provides
appropriately
located signals for transcription, translation, secretion (if desired), and
the like,
including an in-frame AUG as required.
[0176] In another embodiment, high-level expression of a heterologous coding
sequence, such as, for example, a polynucleotide of the present invention, may
be
achieved by cloning the heterologous polynucleotide of the invention into an
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expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
[0177] In addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide
sequences) that is operably associated with polynucleotides of the invention,
and
which activates, alters, and/or amplifies endogenous polynucleotides. For
example,
techniques known in the art may be used to operably associate heterologous
control
regions (e.g., promoter and/or enhancer) and endogenous polynucleotide
sequences
via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued
June 24,
1997; International Publication No. WO 96/29411, published September 26, 1996;
International Publication No. WO 94/12650, published August 4, 1994; Koller et
al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-
438 (1989), the disclosures of each of which are incorporated by reference in
their
entireties).
[0178] In addition, polypeptides of the invention can be chemically
synthesized
using techniques known in the art (e.g., see Creighton, 1983, Proteins:
Structures and
Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al.,
Nature,
310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of
a
polypeptide can be synthesized by use of a peptide synthesizer. Furthermore,
if
desired, nonclassical amino acids or chemical amino acid analogs can be
introduced as
a substitution or addition into the polypeptide sequence. Non-classical amino
acids
include, but are not limited to, to the D-isomers of the common amino acids,
2,4-
diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-
amino
butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric
acid,
3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine,
citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine,
phenylglycine,
cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as
b-
methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino
acid
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analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0179] The invention encompasses polypeptides of the present invention which
are
differentially modified during or after translation, e.g., by glycosylation,
acetylation,
phosphorylation, amidation, derivatization by lcnown protecting/blocking
groups,
proteolytic cleavage, linkage to an antibody molecule or other cellular
ligand, etc.
Any of numerous chemical modifications may be carried out by known techniques,
including but not limited, to specific chemical cleavage by cyanogen bromide,
trypsin,
chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation,
reduction; metabolic synthesis in the presence of tunicamycin; etc.
[0180] Additional post-translational modifications encompassed by the
invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic host
cell expression. The polypeptides may also be modified with a detectable
label, such
as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
isolation of the protein.
[0181] Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and avidin/biotin;
examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include iodine (121h izsh lash i3lI)~ carbon
(14C), sulfur
(35s)~ tritium (3H), indium (iliIn' 112In' 113mIn' il5mln), technetium
(99TC,99mTC),
thallium (2°1Ti), gallium (6$Ga, 67Ga), palladium (losPd), molybdenum
(99Mo), xenon
(133Xe) fluorine (18F) 153Sm 177Lu 159Gd 149pm 140La 17s~ 166Ho 90y 47SC 186Re
> > > > > > > > > > > >
issRe~ i4zPr~ ios~~ ~d ~7Ru.
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[0182] In specific embodiments, a polypeptide of the present invention or
fragment
or variant thereof is attached to macrocyclic chelators that associate with
radiometal
ions, including but not limited to, 177Lu, 9°y, i66Ho, and lsssm, to
polypeptides. In a
preferred embodiment, the radiometal ion associated with the macrocyclic
chelators is
mln. In another preferred embodiment, the radiometal ion associated with the
macrocyclic chelator is 9°Y. In specific embodiments, the macrocyclic
chelator is
1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other
specific
embodiments, DOTA is attached to an antibody of the invention or fragment
thereof
via a linker molecule. Examples of linker molecules useful for conjugating
DOTA to a
polypeptide are commonly known in the art - see, for example, DeNardo et al.,
Clin
Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-
7
(1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are
hereby incorporated by reference in their entirety.
[0183] As mentioned, the pancreatic associated proteins of the invention may
be
modified by either natural processes, such as posttranslational processing, or
by
chemical modification techniques which are well known in the art. It will be
appreciated that the same type of modification may be present in the same or
varying
degrees at several sites in a given pancreatic associated polypeptide.
pancreatic
associated polypeptides may be branched, for example, as a result of
ubiquitination,
and they may be cyclic, with or without branching. Cyclic, branched, and
branched
cyclic pancreatic associated polypeptides may result from posttranslation
natural
processes or may be made by synthetic methods. Modifications include
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 cross-links, formation of cysteine,
formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization,
selenoylation, sulfation, transfer-RNA mediated addition of amino acids to
proteins
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such as arginylation, and ubiquitination. (See, for instance, PROTEINS -
STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,
pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et
al.,
Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[0184] Also provided by the invention are chemically modified derivatives of
the
polypeptides of the invention which may provide additional advantages such as
increased solubility, stability and circulating time of the polypeptide, or
decreased
immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene
glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,
dextran,
polyvinyl alcohol and the like. The polypeptides may be modiEed at random
positions
within the molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0185] The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about
1 kDa and about 100 kDa (the term "about" indicating that in preparations of
polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic
protein or analog). For example, the polyethylene glycol may have an average
molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000,
4500,
5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500,
11,000,
11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,
16,000,
16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000,
30,000,
35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000,
85,000,
90,000, 95,000, or 100,000 kDa.
[0186] As noted above, the polyethylene glycol may have a branched structure.
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Branched polyethylene glycols are described, for example, in U.S. Patent No.
5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996);
Vorobjev et
al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al.,
Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are incorporated
herein by
reference.
[0187] The polyethylene glycol molecules (or other chemical moieties) should
be
attached to the protein with consideration of effects on functional or
antigenic domains
of the protein. There are a number of attachment methods available to those
skilled in
the art, such as, for example, the method disclosed in EP 0 401 384 (coupling
PEG to
G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol.
20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For
example, polyethylene glycol may be covalently bound through amino acid
residues
via a reactive group, such as a free amino or carboxyl group. Reactive groups
are
those to which an activated polyethylene glycol molecule may be bound. The
amino
acid residues having a free amino group may include lysine residues and the
N-terminal amino acid residues; those having a free carboxyl group may include
aspartic acid residues glutamic acid residues and the C-terminal amino acid
residue.
Sulfhydryl groups may also be used as a reactive group for attaching the
polyethylene
glycol molecules. Preferred for therapeutic purposes is attachment at an amino
group,
such as attachment at the N-terminus or lysine group.
[0188] As suggested above, polyethylene glycol may be attached to proteins
via linkage to any of a number of amino acid residues. For example,
polyethylene
glycol can be linked to proteins via covalent bonds to lysine, histidine,
aspartic acid,
glutamic acid, or cysteine residues. One or more reaction chemistries may be
employed to attach polyethylene glycol to specific amino acid residues (e.g.,
lysine,
histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to
more than one
type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic
acid, cysteine
and combinations thereof) of the protein.
[0189] One may specifically desire proteins chemically modified at the
N-terminus. Using polyethylene glycol as an illustration of the present
composition,
one may select from a variety of polyethylene glycol molecules (by molecular
weight,
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branching, etc.), the proportion of polyethylene glycol molecules to protein
(polypeptide) molecules in the reaction mix, the type of pegylation reaction
to be
performed, and the method of obtaining the selected N-terminally pegylated
protein.
The method of obtaining the N-terminally pegylated preparation (i.e.,
separating this
moiety from other monopegylated moieties if necessary) may be by purification
of the
N-terminally pegylated material from a population of pegylated protein
molecules.
Selective proteins chemically modified at the N-terminus modification may be
accomplished by reductive alkylation which exploits differential reactivity of
different
types of primary amino groups (lysine versus the N-terminal) available for
derivatization in a particular protein. Under the appropriate reaction
conditions,
substantially selective derivatization of the protein at the N-terminus with a
carbonyl
group containing polymer is achieved.
[0190] As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached to the protein either directly or by an intervening linker.
Linlcerless systems
for attaching polyethylene glycol to proteins are described in Delgado et al.,
Crit. Rev.
Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of
Hematol. 68:1-
18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058;
and
WO 98/32466, the disclosures of each of which are incorporated herein by
reference.
[0191] One system for attaching polyethylene glycol directly to amino acid
residues of proteins without an intervening linker employs tresylated MPEG,
which is
produced by the modification of monmethoxy polyethylene glycol (MPEG) using
tresylchloride (C1SOZCH2CF3). Upon reaction of protein with tresylated MPEG,
polyethylene glycol is directly attached to amine groups of the protein. Thus,
the
invention includes protein-polyethylene glycol conjugates produced by reacting
proteins of the invention with a polyethylene glycol molecule having a
2,2,2-trifluoreothane sulphonyl group.
[0192] Polyethylene glycol can also be attached to proteins using a number of
different intervening linkers. For example, U.S. Patent No. 5,612,460, the
entire
disclosure of which is incorporated herein by reference, discloses urethane
linkers for
connecting polyethylene glycol to proteins. Protein-polyethylene glycol
conjugates
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wherein the polyethylene glycol is attached to the protein by a linker can
also be
produced by reaction of proteins with compounds such as MPEG-
succinimidylsuccinate, MPEG activated with 1,1'-carbonyldiimidazole, MPEG-
2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-
succinate derivatives. A number of additional polyethylene glycol derivatives
and
reaction chemistries for attaching polyethylene glycol to proteins are
described in
International Publication No. WO 98/32466, the entire disclosure of which is
incorporated herein by reference. Pegylated protein products produced using
the
reaction chemistries set out herein are included within the scope of the
invention.
[0193] The number of polyethylene glycol moieties attached to each protein of
the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated
proteins of the invention may be linked, on average, to l, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12,
15, 17, 20, or more polyethylene glycol molecules. Similarly, the average
degree of
substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-
11, 10-12,
11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties per protein molecule. Methods for determining the degree of
substitution are
discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.
9:249-
304 (1992).
[0194] The pancreatic associated polypeptides of the invention can be
recovered
and purified from chemical synthesis and recombinant cell cultures by standard
methods which include, but are not limited to, ammonium sulfate or ethanol
precipitation, acid extraction, anion or ration exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is employed for
purification. Well known techniques for refolding protein may be employed to
regenerate active conformation when the polypeptide is denatured during
isolation
and/or purification.
[0195] Pancreatic associated polynucleotides and polypeptides may be used in
accordance with the present invention for a variety of applications,
particularly those
that make use of the chemical and biological properties of pancreatic
associated
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antigens. Among these are applications in the detection, prevention, diagnosis
and/or
treatment of diseases associated with pancreas, such as e.g., diabetes
mellitus, diabetes
insipidus, congenital pancreatic agenesis, pancreatic cancers (e.g., benign or
malignant
forms of pancreatic cancer, as well as any particular type of cancer arising
from cells
of the pancreas (e.g., duct cell carcinoma, acinar cell carcinoma, papillary
carcinoma,
adenosquamous carcinoma, undifferentiated carcinoma, mutinous carcinoma, giant
cell carcinoma, mixed type pancreatic cancer, small cell carcinoma,
cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma,
adenocarcinoma, islet-cell tumors, cystic neoplasms, and papillary-cyctic
neoplasm
and the like), as well as any stage of such cancers (e.g., stages I to IV in
severity)),
cystic fibrosis, cyst (e.g., pancreatic pseudocyst), pancreatic fistula,
insufficiency,
pancreatic dysplasia, pancreatitis (e.g., chronic pancreatitis, acute
pancreatitis, acute
necrotizing pancreatitis, alcoholic pancreatitis, and pancreatic abscesses
associated
with pancreatic inflammation), and/or those disorders as described under
"Endocrine
Disorders" and/or "Gastrointestinal Disorders" below. Additional applications
relate
to diagnosis and to treatment of disorders of cells, tissues and organisms.
These
aspects of the invention are discussed further below.
[0196] In a preferred embodiment, polynucleotides expressed in a particular
tissue
type are used to detect, diagnose, treat, prevent and/or prognose disorders
associated
with the tissue type.
[0197] The polypeptides of the invention may be in monomers or multimers
(i.e.,
dimers, trimers, tetramers and higher multimers). Accordingly, the present
invention
relates to monomers and multimers of the polypeptides of the invention, their
preparation, and compositions (preferably, Therapeutics) containing them. In
specific
embodiments, the polypeptides of the invention are monomers, dimers, trimers
or
tetramers. In additional embodiments, the multimers of the invention are at
least
dimers, at least trimers, or at least tetramers.
[0198] Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer refers to a multimer containing only
polypeptides
corresponding to a protein of the invention (e.g., the amino acid sequence of
SEQ ID
NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ
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ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as
defined in columns ~ and 9 of Table 2, and/or an amino acid sequence encoded
by
cDNA contained in Clone ID NO:Z (including fragments, variants, splice
variants, and
fusion proteins, corresponding to these as described herein)). These homomers
may
contain polypeptides having identical or different amino acid sequences. In a
specific
embodiment, a homomer of the invention is a multimer containing only
polypeptides
having an identical amino acid sequence. In another specific embodiment, a
homomer
of the invention is a multimer containing polypeptides having different amino
acid
sequences. In specific embodiments, the multimer of the invention is a
homodimer
(e.g., containing two polypeptides having identical or different amino acid
sequences)
or a homotrimer (e.g., containing three polypeptides having identical andlor
different
amino acid sequences). In additional embodiments, the homomeric multimer of
the
invention is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
[0199] As used herein, the term heteromer refers to a multimer containing two
or
more heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to
the polypeptides of the invention. In a specific embodiment, the multimer of
the
invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional
embodiments, the heteromeric multimer of the invention is at least a
heterodimer, at
least a heterotrimer, or at least a heterotetramer.
[0200] Multimers of the invention may be the result of hydrophobic,
hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked by, for
example,
liposome formation. Thus, in one embodiment, multimers of the invention, such
as,
for example, homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of
the invention, such as, for example, heterotrimers or heterotetramers, are
formed when
polypeptides of the invention contact antibodies to the polypeptides of the
invention
(including antibodies to the heterologous polypeptide sequence in a fusion
protein of
the invention) in solution. In other embodiments, multimers of the invention
are
formed by covalent associations with andlor between the polypeptides of the
invention. Such covalent associations may involve one or more amino acid
residues
contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:Y,
encoded by
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the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or
encoded
by the cDNA contained in Clone ID NO:Z). In one instance, the covalent
associations
are cross-linking between cysteine residues located within the polypeptide
sequences
which interact in the native (i.e., naturally occurring) polypeptide. In
another instance,
the covalent associations are the consequence of chemical or recombinant
manipulation. Alternatively, such covalent associations may involve one or
more
amino acid residues contained in the heterologous polypeptide sequence in a
fusion
protein. In one example, covalent associations are between the heterologous
sequence
contained in a fusion protein of the invention (see, e.g., U.S. Patent Number
5,478,925). In a specific example, the covalent associations are between the
heterologous sequence contained in a Fc fusion protein of the invention (as
described
herein). In another specific example, covalent associations of fusion proteins
of the
invention are between heterologous polypeptide sequence from another protein
that is
capable of forming covalently associated multimers, such as for example,
osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the
contents of
which are herein incorporated by reference in its entirety). In another
embodiment,
two or more polypeptides of the invention are joined through peptide linkers.
Examples include those peptide linkers described in U.S. Pat. No. 5,073,627
(hereby
incorporated by reference). Proteins comprising multiple polypeptides of the
invention
separated by peptide linkers may be produced using conventional recombinant
DNA
technology.
[0201] Another method for preparing multimer polypeptides of the invention
involves use of polypeptides of the invention fused to a leucine zipper or
isoleucine
zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are
polypeptides that promote multimerization of the proteins in which they are
found.
Leucine zippers were originally identified in several DNA-binding proteins
(Landschulz et al., Science 240:1759, (1988)), and have since been found in a
variety
of different proteins. Among the known leucine zippers are naturally occurring
peptides and derivatives thereof that dimerize or trimerize. Examples of
leucine zipper
domains suitable for producing soluble multimeric proteins of the invention
are those
described in PCT application WO 94!10308, hereby incorporated by reference.
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Recombinant fusion proteins comprising a polypeptide of the invention fused to
a
polypeptide sequence that dimerizes or trimerizes in solution are expressed in
suitable
host cells, and the resulting soluble multimeric fusion protein is recovered
from the
culture supernatant using techniques known in the art.
[0202] Trimeric polypeptides of the invention may offer the advantage of
enhanced biological activity. Preferred leucine zipper moieties and isoleucine
moieties
are those that preferentially form trimers. One example is a leucine zipper
derived
from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS
Letters
344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated by reference. Other peptides derived from naturally occurring
trimeric
proteins may be employed in preparing trimeric polypeptides of the invention.
[0203] In another example, proteins of the invention are associated by
interactions
between Flag~ polypeptide sequence contained in fusion proteins of the
invention
containing Flag~ polypeptide sequence. In a further embodiment, proteins of
the
invention are associated by interactions between heterologous polypeptide
sequence
contained in Flag~ fusion proteins of the invention and anti-Flag~ antibody.
[0204] The multimers of the invention may be generated using chemical
techniques known in the art. For example, polypeptides desired to be contained
in the
multimers of the invention may be chemically cross-linked using linker
molecules and
linker molecule length optimization techniques known in the art (see, e.g.,
U.S. Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
Additionally, multimers of the invention may be generated using techniques
known in
the art to form one or more inter-molecule cross-links between the cysteine
residues
located within the sequence of the polypeptides desired to be contained in the
multimer
(see, e.g., U.S. Patent Number 5,478,925, which is herein incorporated by
reference in
its entirety). Further, polypeptides of the invention may be routinely
modified by the
addition of cysteine or biotin to the C-terminus or N-terminus of the
polypeptide and
techniques known in the art may be applied to generate multimers containing
one or
more of these modified polypeptides (see, e.g., U.S. Patent Number 5,478,925,
which
is herein incorporated by reference in its entirety). Additionally, techniques
known in
the art may be applied to generate liposomes containing the polypeptide
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desired to be contained in the multimer of the invention (see, e.g., U.S.
Patent Number
5,478,925, which is herein incorporated by reference in its entirety).
[0205] Alternatively, multimers of the invention may be generated using
genetic
engineering techniques known in the art. In one embodiment, polypeptides
contained
in multimers of the invention are produced recombinantly using fusion protein
technology described herein or otherwise known in the art (see, e.g., U.S.
Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
In a
specific embodiment, polynucleotides coding for a homodimer of the invention
are
generated by ligating a polynucleotide sequence encoding a polypeptide of the
invention to a sequence encoding a linker polypeptide and then further to a
synthetic
polynucleotide encoding the translated product of the polypeptide in the
reverse
orientation from the original C-terminus to the N-terminus (lacking the leader
sequence) (see, e.g., U..S Patent Number 5,478,925, which is herein
incorporated by
reference in its entirety). In another embodiment, recombinant techniques
described
herein or otherwise known in the art are applied to generate recombinant
polypeptides
of the invention which contain a transmembrane domain (or hydrophobic or
signal
peptide) and which can be incorporated by membrane reconstitution techniques
into
liposomes (see, e.g., U.S. Patent Number 5,478,925, which is herein
incorporated by
reference in its entirety).
Antibodies
[0206] Further polypeptides of the invention relate to antibodies and T-cell
antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment,
or variant of the invention (e.g., a polypeptide or fragment or variant of the
amino acid
sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in
Clone
ID NO:Z, and/or an epitope, of the present invention) as determined by
immunoassays
well known in the art for assaying specific antibody-antigen binding.
Antibodies of
the invention include, but are not limited to, polyclonal, monoclonal,
multispecific,
human, humanized or chimeric antibodies, single chain antibodies, Fab
fragments,
F(ab') fragments, fragments produced by a Fab expression library, anti-
idiotypic (anti-
Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the
invention),
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intracellularly-made antibodies (i.e., intrabodies), and epitope-binding
fragments of
any of the above. The term "antibody," as used herein, refers to
immunoglobulin
molecules and immunologically active portions of immunoglobulin molecules,
i.e.,
molecules that contain an antigen binding site that immunospecifically binds
an
antigen. The immunoglobulin molecules of the invention can be of any type
(e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgA1
and
IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the
immunoglobulin molecules of the invention are IgGl. In other preferred
embodiments, the immunoglobulin molecules of the invention are IgG4.
[0207] Most preferably the antibodies are human antigen-binding antibody
fragments of the present invention and include, but are not limited to, Fab,
Fab' and
F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-
linked Fvs
(sdFv) and fragments comprising either a VL or VH domain. Antigen-binding
antibody fragments, including single-chain antibodies, may comprise the
variable
regions) alone or,:in combination with the entirety or a portion of the
following: hinge
region, CHl, CH2, and CH3 domains. Also included in the invention are antigen-
binding fragments also comprising any combination of variable regions) with a
hinge
region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from
any animal origin including birds and mammals. Preferably, the antibodies are
human,
murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or
chicken. As used herein, "human" antibodies include antibodies having the
amino
acid sequence of a human immunoglobulin and include antibodies isolated from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described
iyaf'a and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
[0208] The antibodies of the present invention may be monospecific,
bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both
a polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
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147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; I~ostelny et al., J. Immunol. 148:1547-1553 (1992).
[0209] Antibodies of the present invention may be described or specified in
terms
of the epitope(s) or portions) of a polypeptide of the present invention which
they
recognize or specifically bind. The epitope(s) .or polypeptide portions) may
be
specified as described herein, e.g., by N-terminal and C-terminal positions,
or by size
in contiguous amino acid residues, or listed in the Tables and Figures.
Preferred
epitopes of the invention include those shown in column 6 of Table 1A, as well
as
polynucleotides that encode these epitopes. Antibodies, which specifically
bind any
epitope or polypeptide of the present invention may also be excluded.
Therefore, the
present invention includes antibodies that specifically bind polypeptides of
the present
invention, and allows for the exclusion of the same.
[0210] Antibodies of the present invention may also be described or specified
in
terms of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog,
or homolog of a polypeptide of the present invention are included. Antibodies
that
bind polypeptides with at least 95%, at least 90°l0, at least 85%, at
least 80%, at least
75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%
identity
(as calculated using methods known in the art and described herein) to a
polypeptide
of the present invention are also included in the present invention. In
specific
embodiments, antibodies of the present invention cross-react with murine, rat
and/or
rabbit homologs of human proteins and the corresponding epitopes thereof.
Antibodies
that do not bind polypeptides with less than 95%, less than 90%, less than
85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less
than 55%,
and less than 50% identity (as calculated using methods known in the art and
described
herein) to a polypeptide of the present invention are also included in the
present
invention. In a specific embodiment, the above-described cross-reactivity is
with
respect to any single specific antigenic or immunogenic polypeptide, or
combinations)
of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic
polypeptides
disclosed herein. Further included in the present invention are antibodies
which bind
polypeptides encoded by polynucleotides which hybridize to a polynucleotide of
the
present invention under stringent hybridization conditions (as described
herein).
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Antibodies of the present invention may also be described or specified in
terms of their
binding affinity to a polypeptide of the invention. Preferred binding
affinities include
those with a dissociation constant or Kd less than 5 X 10-2 M, 10-2 M, 5 X 10-
3 M, 10-3
M, 5 X 10-4 M, 10-4 M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10-7 M, 107
M, 5 X
10-8 M, 10-8 M, 5 X 10-9 M, 10-9 M, 5 X 10-1° M, 10-1° M, 5 X 10-
11 M, 10-11 M, 5 X
10-12 M, 10-12 M, 5 X 10-13 M, 10-13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, or
10-15 M.
[0211] The invention also provides antibodies that competitively inhibit
binding of
an antibody to an epitope of the invention as determined by any method known
in the
art for determining competitive binding, for example, the immunoassays
described
herei-n. In preferred embodiments, the antibody competitively inhibits binding
to the
epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least
75%, at least
70%, at least 60%, or at least 50%.
[0212] Antibodies of the present invention may act as agonists or antagonists
of
the polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. Preferably, antibodies of the present
invention bind
an antigenic epitope disclosed herein, or a portion thereof. The invention
features both
receptor-specific antibodies and ligand-specific antibodies. The invention
also
features receptor-specific antibodies, which do not prevent ligand binding but
prevent
receptor activation. Receptor activation (i.e., signaling) may be determined
by
techniques described herein or otherwise known in the art. For example,
receptor
activation can be determined by detecting the phosphorylation (e.g., tyrosine
or
serine/threonine) of the receptor or its substrate by immunoprecipitation
followed by
western blot analysis (for example, as described supra). In specific
embodiments,
antibodies are provided that inhibit ligand activity or receptor activity by
at least 95%,
at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least
60%, or at
least 50% of the activity in absence of the antibody.
[0213] The invention also features receptor-specific antibodies which both
prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound
receptor or the unbound ligand. Likewise, included in the invention are
neutralizing
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antibodies which bind the ligand and prevent binding of the ligand to the
receptor, as
well as antibodies which bind the ligand, thereby preventing receptor
activation, but do
not prevent the ligand from binding the receptor. Further included in the
invention are
antibodies, which activate the receptor. These antibodies may act as receptor
agonists,
i.e., potentiate or activate either all or a subset of the biological
activities of the ligand-
mediated receptor activation, for example, by inducing dimerization of the
receptor.
The antibodies may be specified as agonists, antagonists or inverse agonists
for
biological activities comprising the specific biological activities of the
peptides of the
invention disclosed herein. The above antibody agonists can be made using
methods
known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No.
5,811,097; Deng et .al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer
Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161 (4):1786-1794 (1998);
Zhu et
al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-
3179
(1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J.
Immunol.
Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);
Carlson et al., J. Biol. Chem. 272(17):11295-11301 (I997); Taryman et al.,
Neuron
14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek
et al.,
Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in
their
entireties).
[0214] Antibodies of the present invention may be used, for example, to
purify,
detect, and target the polypeptides of the present invention, including both
ih vitro and
ih vivo diagnostic and therapeutic methods. For example, the antibodies have
utility in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides
of the present invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
incorporated by reference herein in its entirety.
[0215] As discussed in more detail below, the antibodies of the present
invention
may be used either alone or in combination with other compositions. The
antibodies
may further be recombinantly fused to a heterologous polypeptide at the N- or
C-
terminus or chemically conjugated (including covalent and non-covalent
conjugations) to polypeptides ox other compositions. For example, antibodies
of the
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present invention may be recombinantly fused or conjugated to molecules useful
as
labels in detection assays and effector molecules such as heterologous
polypeptides,
drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO
91/14438; WO 89/12624; LT.S. Patent No. 5,314,995; and EP 396,387; the
disclosures
of which are incorporated herein by reference in their entireties.
[0216] The antibodies of the invention include derivatives that are modified,
i.e.,
by the covalent attachment of any type of molecule to the antibody such that
covalent
attachment does not prevent the antibody from generating an anti-idiotypic
response.
For example, but not by way of limitation, the antibody derivatives include
antibodies
that have been modified, e.g., by glycosylation, acetylation, pegylation,
phosphylation,
amidation, derivatization by known protecting/bloclcing groups, proteolytic
cleavage,
linkage to a cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including, but not
limited to
specific chemical cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or more non-
classical
amino acids.
[0217] The antibodies of the present invention may be generated by any
suitable
method lcnown in the art. Polyclonal antibodies to an antigen-of interest can
be
produced by various procedures well known in the art. For example, a
polypeptide of
the invention can be administered to various host animals including, but not
limited to,
rabbits, mice, rats, etc. to induce the production of sera containing
polyclonal
antibodies specific for the antigen. Various adjuvants may be used to increase
the
immunological response, depending on the host species, and include but are not
limited to, Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions,
peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially
useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium
parvum. Such adjuvants are also well known in the art.
[0218] Monoclonal antibodies can be prepared using a wide variety of
techniques
known in the art including the use of hybridoma, recombinant, and phage
display
technologies, or a combination thereof. For example, monoclonal antibodies can
be
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produced using hybridoma techniques including those known in the art and
taught, for
example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring
Harbor
Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies
and
T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated
by
reference in their entireties). The term "monoclonal antibody" as used herein
is not
limited to antibodies produced through hybridoma technology. The term
"monoclonal
antibody" refers to an antibody that is derived from a single clone, including
any
eukaryotic, prokaryotic, or phage clone, and not the method by which it is
produced.
[0219] Methods for producing and screening for specific antibodies using
hybridoma technology are routine and well known in the art and are discussed
in detail
in the Examples. In a non-limiting example, mice can be immunized with a
polypeptide of the invention or a cell expressing such peptide. Once an immune
response is detected, e.g., antibodies specific for the antigen are detected
in the mouse
serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes
are
then fused by well known techniques to any suitable myeloma cells, for example
cells
from cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by
limited dilution. The hybridoma clones are then assayed by methods lcnown in
the art
for cells that secrete antibodies capable of binding a polypeptide of the
invention.
Ascites fluid, which generally contains high levels of antibodies, can be
generated by
immunizing mice with positive hybridoma clones.
[0220] Accordingly, the present invention provides methods of generating
monoclonal antibodies as well as antibodies produced by the method comprising
culturing a hybridoma cell secreting an antibody of the invention wherein,
preferably,
the hybridoma is generated by fusing splenocytes isolated from a mouse
immunized
with an antigen of the invention with myeloma cells and then screening the
hybridomas resulting from the fusion for hybridoma clones that secrete an
antibody
able to bind a polypeptide of the invention.
[0221] Another well known method for producing both polyclonal and monoclonal
human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols
for
generating EBV-transformed B cell lines are commonly known in the art, such
as, for
example, the protocol outlined in Chapter 7.22 of Current Protocols in
Immunology,
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Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby
incorporated in its
entirety by reference herein. The source of B cells for transformation is
commonly
human peripheral blood, but B cells for transformation may also be derived
from other
sources including, but not limited to, lymph nodes, tonsil, spleen, tumor
tissue, and
infected tissues. Tissues are generally made into single cell suspensions
prior to EBV
transformation. Additionally, steps may be taken to either physically remove
or
inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-
containing samples,
because T cells from individuals seropositive for anti-EBV antibodies can
suppress B
cell immortalization by EBV.
[0222] In general, the sample containing human B cells is innoculated with
EBV,
and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant
of the
B95-8 cell line (ATCC #VR-1492). Physical signs of EBV transformation can
generally be seen towards the end of the 3-4 week culture period. By phase-
contrast
microscopy, transformed cells may appear large, clear, hairy and tend to
aggregate in
tight clusters of cells. Initially, EBV lines are generally polyclonal.
However, over
prolonged periods of cell cultures, EBV lines may become monoclonal or
polyclonal
as a result of the selective outgrowth of particular B cell clones.
Alternatively,
polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution
culture)
or fused with a suitable fusion partner and plated at limiting dilution to
obtain
monoclonal B cell lines. Suitable fusion partners for EBV transformed cell
lines
include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma
cell
lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines
(e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention
also
provides a method of generating polyclonal or monoclonal human antibodies
against
polypeptides of the invention or fragments thereof, comprising EBV-
transformation of
human B cells.
[0223] Antibody fragments, which recognize specific epitopes may be generated
by known techniques. For example, Fab and F(ab')2 fragments of the invention
may
be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments).
F(ab')2 fragments contain the variable region, the light chain constant region
and the
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CHl domain of the heavy chain. For example, the antibodies of the present
invention can also be generated using various phage display methods lcnown in
the art
and as discussed in detail in the Examples (e.g., Example 10). 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
embodiment,
such phage can be utilized to display antigen binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or marine). 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.
Patent 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.
[0224] As described in the above references, after phage selection, the
antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect cells, plant
cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI
34:26-
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34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
[0225] Examples of techniques which can be used to produce single-chain Fvs
and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et
al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999
(1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including ifa
vivo use of antibodies in humans and ih vitro detection assays, it may be
preferable to
use chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule in
which different portions of the antibody are derived from different animal
species,
such as antibodies having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entirety. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted
with
the corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known
in the art, e.g., by modeling of the interactions of the CDR and framework
residues to
identify framework residues important for antigen binding and sequence
comparison to
identify unusual framework residues at particular positions. (See, e.g., Queen
et al.,
U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are
incorporated herein by reference in their entireties.) Antibodies can be
humanized
using a variety of techniques known in the art including, for example, CDR-
grafting
(EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539;
5,530,101;
and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan,
Molecular
Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-
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814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling
(U.S.
Patent No. 5,565,332).
[0226] Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is incorporated herein by reference in its entirety.
[0227] Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express human immunoglobulin genes. For example, the human heavy and light
chain
immunoglobulin gene complexes may be introduced randomly or by homologous
recombination into mouse embryonic stem cells. Alternatively, the human
variable
region, constant region, and diversity region may be introduced into mouse
embryonic
stem cells in addition to the human heavy and light chain genes. The mouse
heavy and
light chain immunoglobulin genes may be rendered non-functional separately or
simultaneously with the introduction of human immunoglobulin loci by
homologous
recombination. In particular, homozygous deletion of the JH region prevents
endogenous antibody production. The modified embryonic stem cells are expanded
and microinjected into blastocysts to produce chimeric mice. The chimeric mice
are
then bred to produce homozygous offspring, which express human antibodies. The
transgenic mice are immunized in the normal fashion with a selected antigen,
e.g., all
or a portion of a polypeptide of the invention. Monoclonal antibodies directed
against
the antigen can be obtained from the immunized, transgenic mice using
conventional
hybridoma technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and subsequently
undergo class
switching and somatic mutation. Thus, using such a technique, it is possible
to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of
this technology for producing human antibodies, see Lonberg and Huszar, Int.
Rev.
Immunol. 13:65-93 (1995). For a detailed discussion of this technology for
producing
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human antibodies and human monoclonal antibodies and protocols for producing
such
antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096;
WO 96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;
5,916,771; 5,939,598; 6,075,181 and 6,114,598, which are incorporated by
reference
herein in their entirety. In addition, companies such as Abgenix, Inc.
(Freemont, CA)
and Genpharm (San Jose, CA) can be engaged to provide human antibodies
directed
against a selected antigen using technology similar to that described above.
[0228] Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
selected non-human monoclonal antibody, e.g., a mouse antibody, is used to
guide the
selection of a completely human antibody recognizing the same epitope.
(Jespers et
al., Biotechnology 12:899-903 (1988)).
[0229] Further, antibodies to the polypeptides of the invention can, in turn,
be
utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the
invention
using techniques well known to those skilled in the art. (See, e.g., Greenspan
& Bona,
FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438
(1991)). For example, antibodies which bind to and competitively inhibit
polypeptide
multimerization and/or binding of a polypeptide of the invention to a ligand
can be
used to generate anti-idiotypes that "mimic" the polypeptide multimerization
and/or
binding domain and, as a consequence, bind to and neutralize polypeptide
and/or its
ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-
idiotypes can be
used in therapeutic regimens to neutralize polypeptide ligand/receptor. For
example,
such anti-idiotypic antibodies can be used to bind a polypeptide of the
invention and/or
to bind its ligand(s)/receptor(s), and thereby block its biological activity.
Alternatively, antibodies which bind to and enhance polypeptide
multimerization
and/or binding,. and/or receptor/ligand multimerization, binding and/or
signaling can
be used to generate anti-idiotypes that function as agonists of a polypeptide
of the
invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab
fragments of
such anti-idiotypes can be used in therapeutic regimens as agonists of the
polypeptides
of the invention or its ligand(s)/receptor(s). For example, such anti-
idiotypic antibodies
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can be used to bind a polypeptide of the invention and/or to bind its
ligand(s)/receptor(s), and thereby promote or enhance its biological activity.
[0230] Intrabodies of the invention can be produced using methods known in the
art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther.
5:595-601
(1994); Marasco, W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu.
Rev.
Microbiol. 51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998);
Cohen
et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-
1128
(1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe,
Protein
Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999);
and
references cited therein.
Polynucleotides Eyacodihg Af2tibodies
[0231] The invention further provides polynucleotides comprising a nucleotide
sequence encoding an antibody of the invention and fragments thereof. The
invention
also encompasses polynucleotides that hybridize under stringent or
alternatively, under
lower stringency hybridization conditions, e.g., as defined supra, to
polynucleotides
that encode an antibody, preferably, that specifically binds to a polypeptide
of the
invention, preferably, an antibody that binds to a polypeptide having the
amino acid
sequence of SEQ ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X
as
defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the
cDNA
contained in Clone ID NO:Z.
[0232] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described in
Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the
synthesis
of overlapping oligonucleotides containing portions of the sequence encoding
the
antibody, annealing and ligating of those oligonucleotides, and then
amplification of
the ligated oligonucleotides by PCR.
[0233] Alternatively, a polynucleotide encoding an antibody may be generated
from nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding
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a particular antibody is not available, but the sequence of the antibody
molecule is
known, a nucleic acid encoding the immunoglobulin may be chemically
synthesized
or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA
library
generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any
tissue or
cells expressing the antibody, such as hybridoma cells selected to express an
antibody
of the invention) by PCR amplification using synthetic primers hybridizable to
the 3'
and 5' ends of the sequence or by cloning using an oligonucleotide probe
specific for
the particular gene sequence to identify, e.g., a cDNA clone from a cDNA
library that
encodes the antibody. Amplified nucleic acids generated by PCR may then be
cloned
into replicable cloning vectors using any method well known in the art.
[0234] Once the nucleotide sequence and corresponding amino acid sequence of
the antibody is determined, the nucleotide sequence of the antibody may be
manipulated using methods well known in the art for the manipulation of
nucleotide
sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR,
etc.
(see, for example, the techniques described in Sambrook et al., 1990,
Molecular
Cloning, A Laboratory Manual, Zd Ed., Cold Spring Harbor Laboratory, Cold
Spring
Harbor, NY and Ausubel et al., eds., 1998, Current Protocols in Molecular
Biology,
John Wiley & Sons, NY, which are both incorporated by reference herein in
their
entireties ), to generate antibodies having a different amino acid sequence,
for example
to create amino acid substitutions, deletions, and/or insertions.
[0235] In a specific embodiment, the amino acid sequence of the heavy and/or
light chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the
art, e.g., by comparison to known amino acid sequences of other heavy and
light chain
variable regions to determine the regions of sequence hypervariability. Using
routine
recombinant DNA techniques, one or more of the CDRs may be inserted within
framework regions, e.g., into human framework regions to humanize a non-human
antibody, as described supra. The framework regions may be naturally occurring
or
consensus framework regions, and preferably human framework regions (see,
e.g.,
Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human
framework
regions). Preferably, the polynucleotide generated by the combination of the
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framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
substitutions may be made within the framework regions, and, preferably, the
amino
acid substitutions improve binding of the antibody to its antigen.
Additionally, such
methods may be used to make amino acid substitutions or deletions of one or
more
variable region cysteine residues participating in an intrachain disulfide
bond to
generate antibody molecules lacking one or more intrachain disulfide bonds.
Other
alterations to the polynucleotide are encompassed by the present invention and
within
the slcill of the art.
[0236] In addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);
Neuberger et
al., Nature 312:604-608 (1984); Talceda et al., Nature 314:452-454 (1985)) by
splicing
genes from a mouse antibody molecule of appropriate antigen specificity
together
with genes from a human antibody molecule of appropriate biological activity
can be
used. As described supy~a, a chimeric antibody is a molecule in which
different
portions are derived from different animal species, such as those having a
variable
region derived from a murine mAb and a human immunoglobulin constant region,
e.g., humanized antibodies.
[0237] Alternatively, techniques described for the production of single chain
antibodies (U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988);
Huston et
al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature
334:544-
54 (1989)) can be adapted to produce single chain antibodies. Single chain
antibodies
are formed by linking the heavy and light chain fragments of the Fv region via
an
amino acid bridge, resulting in a single chain polypeptide. Techniques for the
assembly of functional Fv fragments in E. coli may also be used (Skerra et
al., Science
242:1038- 1041 (1988)).
Methods of Producing Ayitibodies
[0238] The antibodies of the invention can be produced by any method known in
the art for the synthesis of antibodies, in particular, by chemical synthesis
or
preferably, by recombinant expression techniques. Methods of producing
antibodies
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include, but are not limited to, hybridoma technology, EBV transformation, and
other
methods discussed herein as well as through the use recombinant DNA
technology, as
discussed below.
[0239] Recombinant expression of an antibody of the invention, or fragment,
derivative or analog thereof, (e.g., a heavy or light chain of an antibody of
the
invention or a single chain antibody of the invention), requires construction
of an
expression vector containing a polynucleotide that encodes the antibody. Once
a
polynucleotide encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably containing the heavy or light chain
variable
domain), of the invention has been obtained, the vector for the production of
the
antibody molecule may be produced by recombinant DNA technology using
techniques well known in the art. Thus, methods for preparing a protein by
expressing
a polynucleotide containing an antibody encoding nucleotide sequence are
described
herein. Methods which are well known to those skilled in the art can be used
to
construct expression vectors containing antibody coding sequences and
appropriate
transcriptional and translational control signals. These methods include, for
example,
i~ vitro recombinant DNA techniques, synthetic techniques, and ifz vivo
genetic
recombination. The invention, thus, provides replicable vectors comprising a
nucleotide sequence encoding an antibody molecule of the invention, or a heavy
or
light chain thereof, or a heavy or light chain variable domain, operably
linked to a
promoter. Such vectors may include the nucleotide sequence encoding the
constant
region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT
Publication WO 89/01036; and U.S, Patent No. 5,122,464) and the variable
domain of
the antibody may be cloned into such a vector for expression of the entire
heavy or
light chain.
[0240] The expression vector is transferred to a host cell by conventional
techniques and the transfected cells are then cultured by conventional
techniques to
produce an antibody of the invention. Thus, the invention includes host cells
containing a polynucleotide encoding an antibody of the invention, or a heavy
or light
chain thereof, or a single chain antibody of the invention, operably linked to
a
heterologous promoter. In preferred embodiments for the expression of double-
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chained antibodies, vectors encoding both the heavy and light chains may be co-
expressed in the host cell for expression of the entire immunoglobulin
molecule, as
detailed below.
[0241] A variety of host-expression vector systems may be utilized to express
the
antibody molecules of the invention. Such host-expression systems represent
vehicles
by which the coding sequences of interest may be produced and subsequently
purified,
but also represent cells which may, when transformed or transfected with the
appropriate nucleotide coding sequences, express an antibody molecule of the
invention in situ. These include but are not limited to microorganisms such as
bacteria
(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid
DNA or cosmid DNA expression vectors containing antibody coding sequences;
yeast
(e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression
vectors
containing antibody coding sequences; insect cell systems infected with
recombinant
virus expression vectors (e.g., baculovirus) containing antibody coding
sequences;
plant cell systems infected with recombinant virus expression vectors' (e.g.,
cauliflower
mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant
plasmid expression vectors (e.g., Ti plasmid) containing antibody coding
sequences; or
mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g.,
the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,
bacterial
cells such as~Escherichia coli, and more preferably, eukaryotic cells,
especially for the
expression of whole recombinant antibody molecule, are used for the expression
of a
recombinant antibody molecule. For example, mammalian cells such as Chinese
hamster ovary cells (CHO), in conjunction with a vector such as the major
intermediate early gene promoter element from human cytomegalovirus is an
effective
expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett
et al.,
Bio/Technology 8:2 (1990)).
[0242] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the antibody
molecule
being expressed. For example, when a large quantity of such a protein is to be
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produced, for the generation of pharmaceutical compositions of an antibody
molecule,
vectors which direct the expression of high levels o,f fusion protein products
that are
readily purified may be desirable. Such vectors include, but are not limited,
to the E.
coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which
the
antibody coding sequence may be ligated individually into the vector in frame
with the
lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye
&
Inouye, Nucleic Acids Res. 13:3101-3109 (I985); Van Heeke & Schuster, J. Biol.
Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to
express
foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
In
general, such fusion proteins are soluble and can easily be purified from
lysed cells by
adsorption and binding to matrix glutathione-agarose beads followed by elution
in the
presence of free glutathione. The pGEX vectors are designed to include
thrombin or
factor Xa protease cleavage sites so that the cloned target gene product can
be released
from the GST moiety.
[0243] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptey~a
fi~ugiperda cells. The antibody coding sequence may be cloned individually
into non-
essential regions (for example the polyhedrin gene) of the virus and placed
under
control of an AcNPV promoter (for example the polyhedrin promoter).
[0244] In mammalian host cells, a number of viral-based expression systems may
be utilized. In cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcriptionltranslation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus genome by ira vitro or ifz vivo
recombination. Insertion in a non- essential region of the viral genome (e.g.,
region E1
or E3) will result in a recombinant virus that is viable and capable of
expressing the
antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.
Acad. Sci.
USA 81:355-359 (1984)). Specific initiation signals may also be required for
efficient
translation of inserted antibody coding sequences. These signals include the
ATG
initiation codon and adjacent sequences. Furthermore, the initiation codon
must be in
phase with the reading frame of the desired coding sequence to ensure
translation of
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the entire insert. These exogenous translational control signals and
initiation codons
can be of a variety of origins, both natural and synthetic. The efficiency of
expression
may be enhanced by the inclusion of appropriate transcription enhancer
elements,
transcription terminators, etc. (see Bittner et al., Methods in Enzymol.
153:51-544
(1987)).
[0245] In addition, a host cell strain may be chosen which modulates the
expression of the inserted sequences, or modifies and processes the gene
product in
the specific fashion desired. Such modifications (e.g., glycosylation) and
processing
(e.g., cleavage) of protein products may be important for the function of the
protein.
Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins and gene products.
Appropriate
cell lines or host systems can be chosen to ensure the correct modification
and
processing of the foreign protein expressed. To this end, eukaryotic host
cells which
possess the cellular machinery for proper processing of the primary
transcript,
glycosylation, and phosphorylation of the gene product may be used. Such
mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS,
MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for
example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell
line such as, for example, CRL7030 and Hs578Bst.
[0246] For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines, which stably express the
antibody
molecule may be engineered. Rather than using expression vectors which contain
viral
origins of replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of the foreign DNA, engineered cells may be allowed
to
grow for 1-2 days in an enriched media, and then are switched to a selective
media.
The selectable marker in the recombinant plasmid confers resistance to the
selection
and allows cells to stably integrate the plasmid into their chromosomes and
grow to
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines, which express the antibody
molecule.
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Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
[0247] A number of selection systems may be used, including but not limited to
the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc.
Natl.
Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et
al.,
Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells,
respectively.
Also, antimetabolite resistance can be used as the basis of selection for the
following
genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl.
Acad. Sci.
USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981));
gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad.
Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside
G-418
Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev,
Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932
(1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIB
TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin
(Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of
recombinant DNA technology may be routinely applied to select the desired
recombinant clone, and such methods are described, for example, in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols
in Human
Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1
(1981), which are incorporated by reference herein in their entireties.
[0248] The expression levels of an antibody molecule can be increased by
vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based
on gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
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amplified region is associated with the antibody gene, production of the
antibody will
also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0249] Vectors, which use glutamine synthase (GS) or DHFR as the selectable
markers can be amplified in the presence of the drugs methionine sulphoximine
or
methotrexate, respectively. An advantage of glutamine synthase based vectors
are the
availabilty of cell lines (e.g., the murine myeloma cell line, NSO) which are
glutamine
synthase negative. Glutamine synthase expression systems can also function in
glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells)
by
providing additional inhibitor to prevent the functioning of the endogenous
gene. A
glutamine synthase expression system and components thereof are detailed in
PCT
publications: W087/04462; W086/05807; W089/01036; W089/10404; and
W091/06657, which are incorporated in their entireties by reference herein.
Additionally, glutamine synthase expression vectors that may be used according
to the
present invention are commercially available from suplliers, including, for
example
Lonza Biologics, Inc. (Portsmouth, NH). Expression and production of
monoclonal
antibodies using a GS expression system in murine myeloma cells is described
in
Bebbington et al., Biotechnology 10:169(1992) and in Biblia and Robinson
Biotechnol. Prog. 11:1 (1995) which are incorporated in their entirities by
reference
herein.
[0250] The host cell may be co-transfected with two expression vectors of the
invention, the first vector encoding a heavy chain derived polypeptide and the
second
vector encoding a light chain derived polypeptide. The two vectors may contain
identical selectable markers, which enable equal expression of heavy and light
chain
polypeptides. Alternatively, a single vector may be used which encodes, and is
capable of expressing, both heavy and light chain polypeptides. In such
situations, the
light chain should be placed before the heavy chain to avoid an excess of
toxic free
heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.
USA
77:2197 (1980)). The coding sequences for the heavy and light chains may
comprise
cDNA or genomic DNA.
[0251] Once an antibody molecule of the invention has been produced by an
animal, chemically synthesized, or recombinantly expressed, it may be purified
by any
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method known in the art for purification of an immunoglobulin molecule, for
example,
by chromatography (e.g., ion exchange, affinity, particularly by affinity for
the specific
antigen after Protein A, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of
proteins. In addition, the antibodies of the present invention or fragments
thereof can
be fused to heterologous polypeptide sequences described herein or otherwise
known
in the art, to facilitate purification.
[0252] The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations) to
a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60,
70, 80, 90
or 100 amino acids of the polypeptide) of the present invention to generate
fusion
proteins. The fusion does not necessarily need to be direct, but may occur
through
linker sequences. The antibodies may be specific for antigens other than
polypeptides
(or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or
100 amino
acids of the polypeptide) of the present invention. For example, antibodies
may be
used to target the polypeptides of the present invention to particular cell
types, either izz
vitro or i>2 vivo, by fusing or conjugating the polypeptides of the present
invention to
antibodies specific for particular cell surface receptors. Antibodies fused or
conjugated to the polypeptides of the present invention may also be used in
izz vitro
immunoassays and purification methods using methods known in the art. See
e.g.,
Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et
al.,
Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS
89:1428-
1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are
incorporated
by reference in their entireties.
[0253] The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example, the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any
combination
of whole domains or portions thereof. The polypeptides may also be fused or
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conjugated to the above antibody portions to form multimers. For example, Fc
portions fused to the polypeptides of the present invention can form dimers
through
disulfide bonding between the Fc portions. Higher multimeric forms can be made
by
fusing the polypeptides to portions of IgA and IgM. Methods for fusing or
conjugating the polypeptides of the present invention to antibody portions are
known
in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO
91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acad. Sci.
USA 89:11337- 11341 (1992) (said references incorporated by reference in their
entireties).
[0254] As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated
to the
above antibody portions to increase the ifz vivo half life of the polypeptides
or for use
in immunoassays using methods known in the art. Further, the polypeptides
corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody
portions to facilitate purification. One reported example describes chimeric
proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. See EP 394,827; Traunecker et al., Nature 331:84-86 (1988).
The
polypeptides of the present invention fused or conjugated to an antibody
having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in
binding and neutralizing other molecules, than the monomeric secreted protein
or
protein fragment alone. See, for example, Fountoulakis et al., J. Biochem.
270:3958-
3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in
therapy and
diagnosis, and thus can result in, for example, improved pharmacokinetic
properties.
See, for example, EP A 232,262. Alternatively, deleting the Fc part after the
fusion
protein has been expressed, detected, and purified, would be desired. For
example, the
Fc portion may hinder therapy and diagnosis if the fusion protein is used as
an antigen
for immunizations. In drug discovery, for example, human proteins, such as hIL-
5,
have been fused with Fc portions for the purpose of high-throughput screening
assays
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to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular
Recognition 8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
(0255] Moreover, the antibodies or fragments thereof of the present invention
can
be fused to marker sequences, such as a peptide to facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311), among others, many of which are commercially available. As described
in
Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for convenient purification of the fusion protein. Other
peptide tags
useful for purification include, but are not limited to, the "HA" tag, which
corresponds
to an epitope derived from the influenza hemagglutinin protein (Wilson et al.,
Cell
37:767 (1984)) and the "flag" tag.
[0256] The present invention further encompasses antibodies or fragments
thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given treatment
regimen. Detection can be facilitated by coupling the antibody to a detectable
substance. Examples of detectable substances include various enzymes,
prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials,
radioactive materials, positron emitting metals using various positron
emission
tomographies, and nonradioactive paramagnetic metal ions. The detectable
substance
may be coupled or conjugated either directly to the antibody (or fragment
thereof) or
indirectly, through an intermediate (such as, for example, a linker known in
the art)
using techniques known in the art. See, for example, U.S. Patent No. 4,741,900
for
metal ions which can be conjugated to antibodies for use as diagnostics
according to
the present invention.
[0257] Further, an antibody or fragment thereof may be conjugated to a
therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or a
radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells. Examples
include
paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,
mitomycin,
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etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine,
thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents (e.g., vincristine and vinblastine).
[0258] The conjugates of the invention can be used for modifying a given
biological response, the therapeutic agent or drug moiety is not to be
construed as
limited to classical chemical therapeutic agents. For example, the drug moiety
may be
a protein or polypeptide possessing a desired biological activity. Such
proteins may
include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, 13-
interferon,
nerve growth factor, platelet derived growth factor, tissue plasminogen
activator, an
apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International
Publication No.
WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas
Ligand
(Takahashi et al., Int. Immuf2ol., 6:1567-1574 (1994)), VEGI (See,
International
Publication No. WO 99/23105), a thrombotic agent or an anti- angiogenic agent,
e.g.,
angiostatin or endostatin; or, biological response modifiers such as, for
example,
lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6
("IL-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte
colony
stimulating factor ("G-CSF"), or other growth factors.
[0259] Antibodies may also be attached to solid supports, which are
particularly
useful for immunoassays or purification of the target antigen. Such solid
supports
include, but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene,
polyvinyl chloride or polypropylene.
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[0260] Techniques for conjugating such therapeutic moiety to antibodies are
well
known. See, for example, Arnon et al., "Monoclonal Antibodies For
Immunotargeting
Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et
al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.),
Robinson et
al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers
Of
Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84:
Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506
(1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use Of
Radiolabeled
Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And
Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe
et al.,
"The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
[0261] Alternatively, an antibody can be conjugated to a second antibody to
form
an antibody heteroconjugate as described by Segal in U.S. Patent No.
4,676,980,
which is incorporated herein by reference in its entirety.
[0262] An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic.
Ifnmunophenotyping
[0263] The antibodies of the invention may be utilized for immunophenotyping
of
cell lines and biological samples. Translation products of the genes of the
present
invention may be useful as cell specific markers, or more specifically as
cellular
markers that are differentially expressed at various stages of differentiation
and/or
maturation of particular cell types. Monoclonal antibodies directed against a
specific
epitope, or combination of epitopes, will allow for the screening of cellular
populations expressing the marker. Various techniques can be utilized using
monoclonal antibodies to screen for cellular populations expressing the
marker(s), and
include magnetic separation using antibody-coated magnetic beads, "panning"
with
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antibody attached to a solid matrix (i.e., plate), and flow cytometry (See,
e.g., U.S.
Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
[0264] These techniques allow for the screening of particular populations of
cells,
such as might be found with hematological malignancies (i.e. minimal residual
disease
(MRD) in acute leukemic patients) and "non-self' cells in transplantations to
prevent
Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for
the
screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation and/or differentiation, as might be found in human umbilical
cord blood.
Assays Fo>" Azztibody Bizzdizzg
[0265] The antibodies of the invention may be assayed for immunospecific
binding by any method known in the art. The immunoassays which can be used
include but are not limited to competitive and non-competitive assay systems
using
techniques such as western blots, radioimmunoassays, ELISA (enzyme linlced
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, geI diffusion precipitin reactions, immunodiffusion
assays,
agglutination assays, complement-fixation assays, immunoradiometric assays,
fluorescent immunoassays, and protein A immunoassays, to name but a few. Such
assays are routine and well known in the art (see, e.g., Ausubel et al, eds,
1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York,
which is incorporated by reference herein in its entirety). Exemplary
immunoassays
are described briefly below (but are not intended by way of limitation).
[0266] Immunoprecipitation protocols generally comprise lysing a population of
cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1%
sodium
deoxycholate, 0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1%
Trasylol) supplemented with protein phosphatase and/or protease inhibitors
(e.g.,
EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to
the cell
lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C,
adding protein A and/or
protein G sepharose beads to the cell lysate, incubating for about an hour or
more at
4° C, washing the beads in lysis buffer and resuspending the beads in
SDS/sample
buffer. The ability of the antibody of interest to immunoprecipitate a
particular
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antigen can be assessed by, e.g., western blot analysis. One of skill in the
art would be
knowledgeable as to the parameters that can be modified to increase the
binding of the
antibody to an antigen and decrease the background (e.g., pre-clearing the
cell lysate
with sepharose beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in
Molecular
Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
[0267] Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-
PAGE depending on the molecular weight of the antigen), transferring the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or
non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), bloclcing
the
membrane with primary antibody (the antibody of interest) diluted in blocking
buffer,
washing the membrane in washing buffer, blocking the membrane with a secondary
antibody (which recognizes the primary antibody, e.g., an anti-human antibody)
conjugated to an enzymatic substrate (e.g., horseradish peroxidase or
allcaline
phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking
buffer,
washing the membrane in wash buffer, and detecting the presence of the
antigen. One
of slcill in the art would be knowledgeable as to the parameters that can be
modified to
increase the signal detected and to reduce the background noise. For further
discussion regarding western blot protocols see, e.g., Ausubel et al., eds.,
(1994),
Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New
York,
section 10.8.1.
[0268] ELISAs comprise preparing antigen, coating the well of a 96 well
microtiter plate with the antigen, adding the antibody of interest conjugated
to a
detectable compound such as an enzymatic substrate (e.g., horseradish
peroxidase or
alkaline phosphatase) to the well and incubating for a period of time, and
detecting the
presence of the antigen. In ELISAs the antibody of interest does not have to
be
conjugated to a detectable compound; instead, a second antibody (which
recognizes
the antibody of interest) conjugated to a detectable compound may be added to
the
well. Further, instead of coating the well with the antigen, the antibody may
be coated
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to the well. In this case, a second antibody conjugated to a detectable
compound may
be added following the addition of the antigen of interest to the coated well.
One of
skill in the art would be knowledgeable as to the parameters that can be
modified to
increase the signal detected as well as other variations of ELISAs known in
the art.
For further discussion regarding ELISAs see, e.g., Ausubel et al., eds,
(1994), Current
Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New York,
section
11.2.1.
[0269] The binding affinity of an antibody to an antigen and the off rate of
an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody
bound to the labeled antigen. The affinity of the antibody of interest for a
particular
antigen and the binding off rates can be determined from the data by scatchard
plot
analysis. Competition with a second antibody can also be determined using
radioimmunoassays. In this case, the antigen is incubated with antibody of
interest
conjugated to a labeled compound (e.g., 3H or 125I) in the presence of
increasing
amounts of an unlabeled second antibody.
[0270] Antibodies of the invention may be characterized using
immunocytochemisty methods on cells (e.g., mammalian cells, such as CHO cells)
transfected with a vector enabling the expression of a pancreatic antigen or
with vector
alone using techniques commonly lcnown in the art. Antibodies that bind
pancreatic
antigen transfected cells, but not vector-only transfected cells, are
pancreatic antigen
specific.
Therapeutic Uses
[0271] The present invention is further directed to antibody-based therapies
which
involve administering antibodies of the invention to an animal, preferably a
mammal,
and most preferably a human, patient for treating one or more of the disclosed
diseases, disorders, or conditions. Therapeutic compounds of the invention
include,
but are not limited to, antibodies of the invention (including fragments,
analogs and
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derivatives thereof as described herein) and nucleic acids encoding antibodies
of the
invention (including fragments, analogs and derivatives thereof and anti-
idiotypic
antibodies as described herein). The antibodies of the invention can be used
to treat,
inhibit or prevent diseases, disorders or conditions associated with aberrant
expression
and/or activity of a polypeptide of the invention, including, but not limited
to, any one
or more of the diseases, disorders, or conditions described herein. The
treatment
and/or prevention of diseases, disorders, or conditions associated with
aberrant
expression and/or activity of a polypeptide of the invention includes, but is
not limited
to, alleviating symptoms associated with those diseases, disorders or
conditions.
Antibodies of the invention may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
[0272] In a specific and preferred embodiment, the present invention is
directed to
antibody-based therapies which involve administering antibodies of the
invention to an
animal, preferably a mammal, and most preferably a human, patient for treating
one or
more of the diseases, disorders, or conditions of the pancreas, including, but
not
limited to, diabetes mellitus, diabetes insipidus, congenital pancreatic
agenesis,
pancreatic cancers (e.g., benign or malignant forms of pancreatic cancer, as
well as any
particular type of cancer arising from cells of the pancreas (e.g., duct cell
carcinoma,
acinar cell carcinoma, papillary carcinoma, adenosquamous carcinoma,
undifferentiated carcinoma, mutinous carcinoma, giant cell carcinoma, mixed
type
pancreatic cancer, small cell carcinoma, cystadenocarcinoma, unclassified
pancreatic
Lancers, pancreatoblastoma, adenocarcinoma, islet-cell tumors, cystic
neoplasms, and
papillary-cyctic neoplasm and the like), as well as any stage of such cancers
.(e.g.,
stages I to IV in severity)), cystic fibrosis, cyst (e.g., pancreatic
pseudocyst),
pancreatic fistula, insufficiency, pancreatic dysplasia, pancreatitis (e.g.,
chronic
pancreatitis, acute pancreatitis, acute necrotizing pancreatitis, alcoholic
pancreatitis,
and pancreatic abscesses associated with pancreatic inflammation), and/or
those
disorders as described under "Endocrine Disorders" and/or "Gastrointestinal
Disorders" below.Therapeutic compounds of the invention include, but are not
limited
to, antibodies of the invention (e.g., antibodies directed to the full length
protein
expressed on the cell surface of a mammalian cell; antibodies directed to an
epitope of
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a pancreatic associated polypeptide of the invention (such as, a linear
epitope (shown
in Table 1A, column 6) or a conformational epitope), including fragments,
analogs and
derivatives thereof as described herein) and nucleic acids encoding antibodies
of the
invention (including fragments, analogs and derivatives thereof and anti-
idiotypic
antibodies as described herein). The antibodies of the invention can be used
to treat,
inhibit or prevent diseases, disorders or conditions associated with aberrant
expression
and/or activity of a polypeptide of the invention, including, but not limited
to, any one
or more of the diseases, disorders, or conditions of the pancreas described
herein. The
treatment and/or prevention of diseases, disorders, or conditions of the
pancreatic
associated with aberrant expression and/or activity of a polypeptide of the
invention
includes, but is not limited to, alleviating symptoms associated with those
diseases,
disorders or conditions. Antibodies of the invention may be provided in
pharmaceutically acceptable compositions as known in the art or as described
herein.
[0273] A summary of the ways in which the antibodies of the present invention
may be used therapeutically includes binding polynucleotides or polypeptides
of the
present invention locally or systemically imthe body or by direct cytotoxicity
of the
antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC).
Some
of these approaches are described in more detail below. Armed with the
teachings
provided herein, one of ordinary skill in the art will know how to use the
antibodies of
the present invention for diagnostic, monitoring or therapeutic purposes
without undue
experimentation.
[0274] The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for
example, which
serve to increase the number or activity of effector cells which interact with
the
antibodies.
[0275] The antibodies of the invention may be administered alone or in
combination with other types of treatments (e.g., radiation therapy,
chemotherapy,
hormonal therapy, immunotherapy and anti-tumor agents). Generally,
administration
of products of a species origin or species reactivity (in the case of
antibodies) that is
the same species as that of the patient is preferred. Thus, in a preferred
embodiment,
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human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered
to a human patient for therapy or prophylaxis.
[0276] It is preferred to use high affinity and/or potent if2 vivo inhibiting
and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy
of disorders related to polynucleotides or polypeptides, including fragments
thereof, of
the present invention. Such antibodies, fragments, or regions, will preferably
have an
affinity for polynucleotides or polypeptides of the invention, including
fragments
thereof. Preferred binding affinities include those with a dissociation
constant or Kd
less than 5 X 10-2 M, 10-2 M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5 X 10-5
M, 10-5
M, 5 X 10-6 M, 10-6 M, 5 X 10-7 M, 10-7 M, 5 X 10-$ M, 10-8 M, 5 X 10-9 M, 10-
9 M, 5
X 10-1° M, 10-1° M, 5 X 10-11 M, 10-11 M, 5 X 10-is M, 10-12 M,
5 X 10-13 M, 10-13 M,
X 10-14 M, 10-14 M, 5 X 10-15 M, and 10-15 M.
Gene Therapy
[0277] In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
prevent a disease or disorder associated with aberrant expression and/or
activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy refers to
therapy
performed by the administration to a subject of an expressed or expressible
nucleic
acid. In this embodiment of the invention, the nucleic acids produce their
encoded
protein that mediates a therapeutic effect.
[0278] Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
[0279] For general reviews of the methods of gene therapy, see Goldspiel et
al.,
Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993); May, TIBTECH 11(5):I55-215 (1993). Methods commonly known in the art
of recombinant DNA technology which can be used are described in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
and
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Kxiegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990).
[0280] In a preferred embodiment, the compound comprises nucleic acid
sequences encoding an antibody, said nucleic acid sequences being part of
expression
vectors that express the antibody or fragments or chimeric proteins or heavy
or light
chains thereof in a suitable host. In particular, such nucleic acid sequences
have
promoters operably linked to the antibody coding region, said promoter being
inducible or constitutive, and, optionally, tissue-specific. In another
particular
embodiment, nucleic acid molecules are used in which the antibody coding
sequences
and any other desired sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal
expression of the antibody encoding nucleic acids (Koller and Smithies, Proc.
Natl.
Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438
(1989). In
specific embodiments, the expressed antibody molecule is a single chain
antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy
and light chains, or fragments thereof, of the antibody.
[0281] Delivery of the nucleic acids into a patient may be either direct, in
which
case the patient is directly exposed to the nucleic acid or nucleic acid-
carrying vectors,
or indirect, in which case, cells are first transformed with the nucleic acids
ih vitro,
then transplanted into the patient. These two approaches are known,
respectively, as in
vivo or ex vivo gene therapy.
[0282] In a specific embodiment, the nucleic acid sequences are directly
administered ifz vivo, where it is expressed to produce the encoded product.
This can
be accomplished by any of numerous methods known in the art, e.g., by
constructing
them as part of an appropriate nucleic acid expression vector and
administering it so
that they become intracellular, e.g., by infection using defective or
attenuated
retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by
direct injection
of naked DNA, or by use of microparticle bombardment (e.g., a gene gun;
Biolistic,
Dupont), or coating with lipids or cell-surface receptors or transfecting
agents,
encapsulation in liposomes, microparticles, or microcapsules, or by
administering
them in linkage to a peptide which is known to enter the nucleus, by
administering it
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in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu,
J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types
specifically expressing the receptors), etc. In another embodiment, nucleic
acid-ligand
complexes can be formed in which the ligand comprises a fusogenic viral
peptide to
disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
In yet
another embodiment, the nucleic acid can be targeted ifs vivo for cell
specific uptake
and expression, by targeting a specific receptor (see, e.g., PCT Publications
WO
92/06180; WO 92/22635; W092/20316; W093/14188, WO 93/20221).
Alternatively, the nucleic acid can be introduced intracellularly and
incorporated
within host cell DNA for expression, by homologous recombination (Koller and
Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al.,
Nature
342:435-438 (1989)).
[0283] In a specific embodiment, viral vectors that contains nucleic acid
sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can
be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These
retroviral
vectors contain the components necessary for the correct paclcaging of the
viral
genome and integration into the host cell DNA. The nucleic acid sequences
encoding
the antibody to be used in gene therapy are cloned into one or more vectors,
which
facilitates delivery of the gene into a patient. More detail about retroviral
vectors can
be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the
use of a
retroviral vector to deliver the mdrl gene to hematopoietic stem cells in
order to make
the stem cells more resistant to chemotherapy. Other references illustrating
the use of
retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-
651
(1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human
Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in
Genetics
and Devel. 3:110-114 (1993).
[0284] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery systems are liver, the
central
nervous system, endothelial cells, and muscle. Adenoviruses have the advantage
of
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being capable of infecting non-dividing cells. Kozarsky and Wilson, Current
Opinion
in Genetics and Development 3:499-503 (1993) present a review of adenovirus-
based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the
use
of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus
monkeys.
Other instances of the use of adenoviruses in gene therapy can be found in
Rosenfeld
et a1., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143- 155 (1992);
Mastrangeli et al., J. Clin, Invest. 91:225-234 (1993); PCT Publication
W094/12649;
and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment,
adenovirus vectors are used.
[0285] Adeno-associated virus (AAV) has also been proposed for use in gene
therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S.
Patent No.
5,436,146).
[0286] Another approach to gene therapy involves transferring a gene to cells
in
tissue culture by such methods as electroporation, lipofection, calcium
phosphate
mediated transfection, or viral infection. Usually, the method of transfer
includes the
transfer of a selectable marker to the cells. The cells are then placed under
selection
to isolate those cells that have taken up and are expressing the transferred
gene. Those
cells are then delivered to a patient.
[0287] In this embodiment, the nucleic acid is introduced into a cell prior to
administration irz vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method hcnown in the art, including but not limited to
transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector
containing the nucleic acid sequences, cell fusion, chromosome-mediated gene
transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous
techniques are known in the art for the introduction of foreign genes into
cells (see,
e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al.,
Meth.
Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92m (1985) and may be
used in accordance with the present invention, provided that the necessary
developmental and physiological functions of the recipient cells are not
disrupted. The
technique should provide for the stable transfer of the nucleic acid to the
cell, so that
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the nucleic acid is expressible by the cell and preferably heritable and
expressible by
its cell progeny.
[0288] The resulting recombinant cells can be delivered to a patient by
various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired effect, patient state, etc., and can
be
determined by one skilled in the art.
[0289] Cells into which a nucleic acid can be introduced for purposes of gene
therapy encompass any desired, available cell type, and include but are not
limited to
epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes;
blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained
from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
[0290] In a preferred embodiment, the cell used for gene therapy is autologous
to
the patient.
[0291] In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they are expressible by the cells or their progeny, and the recombinant cells
are then
administered in. vivo for therapeutic effect. In a specific embodiment, stem
or
progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and
maintained in vitro can potentially be used in accordance with this embodiment
of the
present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson,
Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and
Pittelkow
and Scott, Mayo Clinic Proc. 61:771 (1986)).
[0292] In a specific embodiment, the nucleic acid to be introduced for
purposes of
gene therapy comprises an inducible promoter operably linked to the coding
region,
such that expression of the nucleic acid is controllable by the presence or
absence of an
appropriate inducer of transcription.
Demonst~atioh of Therapeutic or Prophylactic Activity
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[0293] The compounds or pharmaceutical compositions of the invention are
preferably tested in vitro, and then in vivo for the desired therapeutic or
prophylactic
activity, prior to use in humans. For example, if2 vitro assays to demonstrate
the
therapeutic or prophylactic utility of a compound or pharmaceutical
composition
include, the effect of a compound on a cell line or a patient tissue sample.
The effect
of the compound or composition on the cell line and/or tissue sample can be
determined utilizing techniques known to those of slcill in the art including,
but not
limited to, rosette formation assays and cell lysis assays. In accordance with
the
invention, i~ vitro assays which can be used to determine whether
administration of a
specific compound is indicated, include in vitro cell culture assays in which
a patient
tissue sample is grown in culture, and exposed to or otherwise administered a
compound, and the effect of such compound upon the tissue sample is observed.
Thes°apeuticlP~ophylactic Administration a~cd Composition
[0294] The invention provides methods of treatment, inhibition and prophylaxis
by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the invention, preferably a polypeptide or antibody of the
invention. In
a preferred embodiment, the compound is substantially purified (e.g.,
substantially
free from substances that limit its effect or produce undesired side-effects).
The
subject is preferably an animal, including but not limited to animals such as
cows,
pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most
preferably human.
[0295] Formulations and methods of administration that can be employed when
the compound comprises a nucleic acid or an immunoglobulin are described
above;
additional appropriate formulations and routes of administration can be
selected from
among those described herein below.
[0296] Various delivery systems are known and can be used to administer a
compound of the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the compound, receptor-
mediated endocytosis (see, e.g., VJu and Wu, J. Biol. Chem. 262:4429-4432
(197)),
construction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of
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introduction include but are not limited to intradermal, intramuscular,
intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes. The
compounds or
compositions may be administered by any convenient route, for example by
infusion
or bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g., oral
mucosa, rectal and intestinal mucosa, etc.) and may be administered together
with
other biologically active agents. Administration can be systemic or local. In
addition,
it may be desirable to introduce the pharmaceutical compounds or compositions
of the
invention into the central nervous system by any suitable route, including
intraventricular and intrathecal injection; intraventricular injection may be
facilitated
by an intraventricular catheter, for example, attached to a reservoir, such as
an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
(0297] In a specific embodiment, it may be desirable to administer the
pharmaceutical compounds or compositions of the invention locally to the area
in need
of treatment; this may be achieved by, for example, and not by way of
limitation, local
infusion during surgery, topical application, e.g., in conjunction with a
wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by
means of an implant, said implant being of a porous, non-porous, or gelatinous
material, including membranes, such as sialastic membranes, or fibers.
Preferably,
when administering a protein, including an antibody, of the invention, care
must be
taken to use materials to which the protein does not absorb.
[0298] In another embodiment, the compound or composition can be delivered in
a
vesicle, in particular a Iiposome (see Langer, Science 249:1527-1533 (1990);
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
[0299] In yet another embodiment, the compound or composition can be delivered
in a controlled release system. In one embodiment, a pump may be used (see
Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled
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Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);
Controlled
Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball
(eds.),
Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol.
Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et
al., Ann.
Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105 (1989)). In yet
another
embodiment, a controlled release system can be placed in proximity of the
therapeutic
target, e.g., the brain, thus requiring only a fraction of the systemic dose
(see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-
138
(1984)).
[0300] Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
[0301] In a specific embodiment where the compound of the invention is a
nucleic
acid encoding a protein, the nucleic acid can be administered ih vivo to
promote
expression of its encoded protein, by constructing it as part of an
appropriate nucleic
acid expression vector and administering it so that it becomes intracellular,
e.g., by use
of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct
injection, or by use
of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with
lipids or cell-surface receptors or transfecting agents, or by administering
it in linkage
to a homeobox- like peptide which is known to enter the nucleus (see e.g.,
Joliot et al.,
Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic
acid
can be introduced intracellularly and incorporated within host cell DNA for
expression, by homologous recombination.
[0302] The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal
or a state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids, such as
water and
oils, including those of petroleum, animal, vegetable or synthetic origin,
such as
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peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred
carrier when the pharmaceutical composition is administered intravenously.
Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical
excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. The composition, if
desired,
can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
agents. These compositions can take the form of solutions, suspensions,
emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. The
composition can be formulated as a suppository, with traditional binders and
carriers
such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical
carriers are described in "Remington's Pharmaceutical Sciences" by E.W.
Martin.
Such compositions will contain a therapeutically effective amount of the
compound,
preferably in purified form, together with a suitable amount of carrier so as
to provide
the form for proper administration to the patient. The formulation should suit
the
mode of administration.
(0303] In a preferred embodiment, the composition is formulated in accordance
with routine procedures as a pharmaceutical composition adapted for
intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition may also include a solubilizing agent and a local anesthetic such
as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such as
an ampoule or sachette indicating the quantity of active agent. Where the
composition
is to be administered by infusion, it can be dispensed with an infusion bottle
containing
sterile pharmaceutical grade water or saline. Where the composition is
administered
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by injection, an ampoule of sterile water for injection or saline can be
provided so that
the ingredients may be mixed prior to administration.
[0304] The compounds of the invention can be formulated as neutral or salt
forms.
Pharmaceutically acceptable salts include those formed with anions such as
those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
and those
formed with nations such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
[0305] The amount of the compound of the invention, which will be effective in
the treatment, inhibition and prevention of a disease or disorder associated
with
aberrant expression and/or activity of a polypeptide of the invention can be
determined
by standard clinical techniques. In addition, irr vitro assays may optionally
be
employed to help identify optimal dosage ranges. The precise dose to be
employed in
the formulation will also depend on the route of administration, and the
seriousness of
the disease or disorder, and should be decided according to the judgment of
the
practitioner and each patient's circumstances. Effective doses may be
extrapolated
from dose-response curves derived from in vitro or animal model test systems.
[0306] For antibodies, the dosage administered to a patient is typically 0.1
mg/kg
to 100 mg/kg of the patient's body weight. Preferably, the dosage administered
to a
patient is between 0.1 mg%kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half life within the human body than antibodies from
other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages
of human antibodies and less frequent administration is often possible.
Further, the
dosage and frequency of administration of antibodies of the invention may be
reduced
by enhancing uptake and tissue penetration (e.g., into the brain) of the
antibodies by
modifications such as, for example, lipidation.
[0307] The invention also provides a pharmaceutical pack or kit comprising one
or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
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use or sale of pharmaceuticals or biological products, which notice reflects
approval by
the agency of manufacture, use or sale for human administration.
Diagnosi,r and Irrtaging
[0308] Labeled antibodies, and derivatives and analogs thereof, which
specifically
bind to a polypeptide of interest can be used for diagnostic purposes to
detect,
diagnose, or monitor diseases, disorders, and/or conditions associated with
the aberrant
expression and/or activity of a polypeptide of the invention. The invention
provides
for the detection of aberrant expression of a polypeptide of interest,
comprising (a)
assaying the expression of the polypeptide of interest in cells or body fluid
of an
individual using one or more antibodies specific to the polypeptide interest
and (b)
comparing the level of gene expression with a standard gene expression level,
whereby
an increase or decrease in the assayed polypeptide gene expression level
compared to
the standard expression level is indicative of aberrant expression.
[0309] The invention provides a diagnostic assay for diagnosing a pancreatic
disorder, comprising (a) assaying the expression of the polypeptide of
interest in cells
or body fluid of an individual using one or more antibodies specific to the
polypeptide
interest and (b) comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed polypeptide
gene
expression level compared to the standard expression level is indicative of a
particular
disorder. With respect to cancer, the presence of a relatively high amount of
transcript
in biopsied tissue from an individual may indicate a predisposition for the
development of the disease, or may provide a means for detecting the disease
prior to
the appearance of actual clinical symptoms. A more definitive diagnosis of
this type
may allow health professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further progression of
the
cancer.
[0310] Antibodies of the invention can be used to assay protein levels in a
biological sample using classical immunohistological methods known to those of
skill
in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985);
Jalkanen et al., J.
Cell . Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for
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detecting protein gene expression include immunoassays, such as the enzyme
linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucose
oxidase;
radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S),
tritium (3H),
indium (112In), and technetium (99Tc); luminescent labels, such as luminol;
and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
[0311] One facet of the invention is the detection and diagnosis of a disease
or
disorder associated with aberrant expression of a polypeptide of interest in
an animal,
preferably a mammal and most preferably a human. A preferred embodiment of the
invention is the detection and diagnosis of a disease or disorder of the
pancreas
associated with aberrant expression of a pancreatic antigen in an animal,
preferably a
mammal and most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a
subject an effective amount of a labeled molecule which specifically binds to
the
polypeptide of interest; b) waiting for a time interval following the
administering for
permitting the labeled molecule to preferentially concentrate at sites in the
subject
where the polypeptide is expressed (and for unbound labeled molecule to be
cleared to
baclcground level); c) determining background level; and d) detecting the
labeled
molecule in the subject, such that detection of labeled molecule above the
background
level indicates that the subject has a particular disease or disorder
associated with
aberrant expression of the polypeptide of interest. Background level can be
determined by various methods including, comparing the amount of labeled
molecule
detected to a standard value previously determined for a particular system.
[0312] It will be understood in the art that the size of the subject and the
imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of
99mTc. The labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain the specific protein. I~
vivo tumor
imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The
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Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
Publishing Inc. (1982)).
[0313] Depending on several variables, including the type of label used and
the
mode of administration, the time interval following the administration for
permitting
the labeled molecule to preferentially concentrate at sites in the subject and
for
unbound labeled molecule to be cleared to background level is 6 to 48 hours or
6 to 24
hours or 6 to 12 hours. In another embodiment the time interval following
administration is 5 to 20 days or 5 to 10 days.
[0314] In an embodiment, monitoring of the disease or disorder is carried out
by
repeating the method for diagnosing the disease or disorder, for example, one
month
after initial diagnosis, six months after initial diagnosis, one year after
initial diagnosis,
etc.
[0315] Presence of the labeled molecule can be detected in the patient using
methods known in the art for in vivo scanning. These methods depend upon the
type
of label used. Skilled artisans will be able to determine the appropriate
method for
detecting a particular label. Methods and devices that may be used in the
diagnostic
methods of the invention include, but are not limited to, computed tomography
(CT),
whole body scan such as position emission tomography (PET), magnetic resonance
imaging (MRI), and sonography.
[0316] In a specific embodiment, the molecule is labeled with a radioisotope
and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al.,
U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled
with a
fluorescent compound and is detected in the patient using a fluorescence
responsive
scanning instrument. In another embodiment, the molecule is labeled with a
positron
emitting metal and is detected in the patent using positron emission-
tomography. In
yet another embodiment, the molecule is labeled with a paramagnetic label and
is
detected in a patient using magnetic resonance imaging (MRI).
Kits
[0317] The present invention provides kits that can be used in the above
methods.
In one embodiment, a kit comprises an antibody of the invention, preferably a
purified
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antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits
of the present invention further comprise a control antibody which does not
react with
the polypeptide of interest. In another specific embodiment, the kits of the
present
invention contain a means for detecting the binding of an antibody to a
polypeptide of
interest (e.g., the antibody may be conjugated to a detectable substrate such
as a
fluorescent compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the first antibody
may
be conjugated to a detectable substrate).
[0318] In another specific embodiment of the present invention, the kit is a
diagnostic kit for use in screening serum containing antibodies specific
against
proliferative and/or cancerous polynucleotides and polypeptides. Such a kit
may
include a control antibody that does not react with the polypeptide of
interest. Such a
kit may include a substantially isolated polypeptide antigen comprising an
epitope,
which is specifically immunoreactive with at least one anti-polypeptide
antigen
antibody. Further, such a kit includes means for detecting the binding of said
antibody
to the antigen (e.g., the antibody may be conjugated to a fluorescent compound
such as
fluorescein or rhodamine, which can be detected by flow cytometry). In
specific
embodiments, the kit may include a recombinantly produced or chemically
synthesized
polypeptide antigen. The polypeptide antigen of the kit may also be attached
to a solid
support.
[0319] In a more specific embodiment the detecting means of the above-
described
kit includes a solid support to which said polypeptide antigen is attached.
Such a kit
may also include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding of the antibody to the polypeptide antigen can be detected
by
binding of the said reporter-labeled antibody.
[0320] In an additional embodiment, the invention includes a diagnostic kit
for use
in screening serum containing antigens of the polypeptide of the invention.
The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive
with polypeptide or polynucleotide antigens, and means for detecting the
binding of
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the polynucleotide or polypeptide antigen to the antibody. In one embodiment,
the
antibody is attached to a solid support. In a specific embodiment, the
antibody may be
a monoclonal antibody. The detecting means of the kit may include a second,
labeled
monoclonal antibody. Alternatively, or in addition, the detecting means may
include a
labeled, competing antigen.
[0321] In one diagnostic configuration, test serum is reacted with a solid
phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing
unbound serum components by washing, the reagent is reacted with reporter-
labeled
anti-human antibody to bind reporter to the reagent in proportion to the
amount of
bound anti-antigen antibody on the solid support. The reagent is again washed
to
remove unbound labeled antibody, and the amount of reporter associated with
the
reagent is determined. Typically, the reporter is an enzyme, which is detected
by
incubating the solid phase in the presence of a suitable fluorometric,
luminescent or
colorimetric substrate (Sigma, St. Louis, MO).
[0322] The solid surface reagent in the above assay is prepared by lcnown
techniques for attaching protein material to solid support material, such as
polymeric
beads, dip sticks, 96-well plate or filter material. These attachment methods
generally
include non-specific adsorption of the protein to the support or covalent
attachment of
the protein, typically through a free amine group, to a chemically reactive
group on the
solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with
biotinylated
antigen(s).
[0323] Thus, the invention provides an assay system or kit for carrying out
this
diagnostic method. The kit generally includes a support with surface- bound
recombinant antigens, and a reporter-labeled anti-human antibody for detecting
surface-bound anti-antigen antibody.
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Uses of the Polynucleotides
[0324] Each of the polynucleotides identified herein can be used in numerous
ways
as reagents. The following description should be considered exemplary and
utilizes
known techniques.
[0325] The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
since few chromosome marlcing reagents, based on actual sequence data (repeat
polymorphisms), are presently available. Each sequence is specifically
targeted to and
can hybridize with a particular location on an individual human chromosome,
thus
each polynucleotide of the present invention can routinely be used as a
chromosome
marker using techniques known in the art. Table 1A, column 8 provides the
chromosome location of some of the polynucleotides of the invention.
[0326] Briefly, sequences can be mapped to chromosomes by preparing PCR
primers (preferably at least 15 by (e.g., 15-25 bp) from the sequences shown
in SEQ
ID NO:X. Primers can optionally be selected using computer analysis so that
primers
do not span more than one predicted exon in the genomic DNA. These primers are
then used for PCR screening of somatic cell hybrids containing individual
human
chromosomes. Only those hybrids containing the human gene corresponding to SEQ
ID NO:X will yield an amplified fragment.
[0327] Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal cycler. Moreover, sublocalization of the
polynucleotides
can be achieved with panels of specific chromosome fragments. Other gene
mapping
strategies that can be used include in situ hybridization, prescreening with
labeled
flow-sorted chromosomes, preselection by hybridization to construct chromosome
specific-cDNA libraries, and computer mapping techniques (See, e.g., Shuler,
Trends
Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its
entirety).
[0328] Precise chromosomal location of the polynucleotides can also be
achieved
using fluorescence in situ hybridization (FISH) of a metaphase chromosomal
spread.
This technique uses polynucleotides as short as 500 or 600 bases; however,
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polynucleotides 2,000-4,000 by are preferred. For a review of this technique,
see
Verma et al., "Human Chromosomes: a Manual of Basic Techniques," Pergamon
Press, New York (1988).
[0329] For chromosome mapping, the polynucleotides can be used individually
(to
mark a single chromosome or a single site on that chromosome) or in panels
(for
marking multiple sites and/or multiple chromosomes).
[0330] Thus, the present invention also provides a method for chromosomal
localization which involves (a) preparing PCR primers from the polynucleotide
sequences in Table 1A and/or Table 2 and SEQ ID NO:X and (b) screening somatic
cell hybrids containing individual chromosomes.
[0331] The polynucleotides of the present invention would lilcewise be useful
for
radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
For a
review of these techniques and others known in the art, see, e.g. Dear,
"Genome
Mapping: A Practical Approach," IRL Press at Oxford University Press, London
(I997); Aydin, J. MoI. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry
3:483-
492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al.,
Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999),
each of
which is hereby incorporated by reference in its entirety.
[0332] Once a polynucleotide has been mapped to a precise chromosomal
location,
the physical position of the polynucleotide can be used in linkage analysis.
Linlcage
analysis establishes coinheritance between a chromosomal location and
presentation of
a particular disease. (Disease mapping data are found, for example, in V.
McKusick,
Mendelian Inheritance in Man (available on line through Johns Hopkins
University
Welch Medical Library).) Column 9 of Table 1A provides an OMIM reference
identification number of diseases associated with the cytologic band disclosed
in
column 8 of Table 1A, as determined using techniques described herein and by
reference to Table 5. Assuming 1 megabase mapping resolution and one gene per
20
kb, a cDNA precisely localized to a chromosomal region associated with the
disease
could be one.of 50-500 potential causative genes.
[0333] Thus, once coinheritance is established, differences in a
polynucleotide of
the invention and the corresponding gene between affected and unaffected
individuals
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can be examined. First, visible structural alterations in the chromosomes,
such as
deletions or translocations, are examined in chromosome spreads or by PCR. If
no
structural alterations exist, the presence of point mutations are ascertained.
Mutations
observed in some or all affected individuals, but not in normal individuals,
indicate
that the mutation may cause the disease. However, complete sequencing of the
polypeptide and the corresponding gene from several normal individuals is
required to
distinguish the mutation from a polymorphism. If a new polymorphism is
identified,
this polymorphic polypeptide can be used for further linkage analysis.
[0334] Furthermore, increased or decreased expression of the gene in affected
individuals as compared to unaffected individuals can be assessed using the
polynucleotides of the invention. Any of these alterations (altered
expression,
chromosomal rearrangement, or mutation) can be used as a diagnostic or
prognostic
marker. Diagnostic and prognostic methods, lcits and reagents encompassed by
the
present invention are briefly described below and more thoroughly elsewhere
herein
(see e.g., the sections labeled "Antibodies", "Diagnostic Assays", and
"Methods for
Detecting Pancreatic Disease, Including Cancer").
[0335] Thus, the invention also provides a diagnostic method useful during
diagnosis of a disorder, involving measuring the expression level of
polynucleotides of
the present invention in cells or body fluid from an individual and comparing
the
measured gene expression level with a standard level of polynucleotide
expression
level, whereby an increase or decrease in the gene expression level compared
to the
standard is indicative of a disorder. Additional non-limiting examples of
diagnostic
methods encompassed by the present invention are more thoroughly described
elsewhere herein (see, e.g., Example 12).
[0336] In still another embodiment, the invention includes a kit for analyzing
samples for the presence of proliferative and/or cancerous polynucleotides
derived
from a test subject, as further described herein. In a general embodiment, the
kit
includes at least one polynucleotide probe containing a nucleotide sequence
that will
specifically hybridize with a polynucleotide of the invention and a suitable
container.
In a specific embodiment, the kit includes two polynucleotide probes defining
an
internal region of the polynucleotide of the invention, where each probe has
one strand
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containing a 31'mer-end .internal to the region. In a further embodiment, the
probes
may be useful as primers for polymerase chain reaction amplif cation.
[0337] Where a diagnosis of a related disorder, including, for example,
diagnosis
of a tumor, has already been made according to conventional methods, the
present
invention is useful as a prognostic indicator, whereby patients exhibiting
enhanced or
depressed polynucleotide of the invention expression will experience a worse
clinical
outcome relative to patients expressing the gene at a level nearer the
standard level.
[0338] By "measuring the expression level of polynucleotides of the invention"
is
intended qualitatively or quantitatively measuring or estimating the level of
the
polypeptide of the invention or the level of the mRNA encoding the polypeptide
of the
invention in a first biological sample either directly (e.g., by determining
or estimating
absolute protein level or mRNA level) or relatively (e.g., by comparing to the
polypeptide level or mRNA level in a second biological sample). Preferably,
the
polypeptide level or mRNA Ievel in the Brst biological sample is measured or
estimated and compared to a standard polypeptide level or mRNA level, the
standard
being taken from a second biological sample obtained from an individual not
having
the related disorder or being determined by averaging levels from a population
of
individuals not having a related disorder. As will be appreciated in the art,
once a
standard polypeptide level or mRNA level is known, it can be used repeatedly
as a
standard for comparison.
[0339] By "biological sample" is intended any biological sample obtained from
an
individual, body fluid, cell line, tissue culture, or other source which
contains
polypeptide of the present invention or the corresponding mRNA. As indicated,
biological samples include body fluids (such as semen, lymph, vaginal pool,
sera,
plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide
of the
present invention, and tissue sources found to express the polypeptide of the
present
invention. Methods for obtaining tissue biopsies and body fluids from mammals
are
well known in the art. Where the biological sample is to include mRNA, a
tissue
biopsy is the preferred source.
[0340] The methods) provided above may preferably be applied in a diagnostic
method and/or kits in which polynucleotides and/or polypeptides of the
invention are
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attached to a solid support. In one exemplary method, the support may be a
"gene
chip" or a "biological chip" as described in U.S. Patents 5,837,832,
5,874,219, and
5,856,174. Further, such a gene chip with polynucleotides of the invention
attached
may be used to identify polymorphisms between the isolated polynucleotide
sequences
of the invention, with polynucleotides isolated from a test subject. The
knowledge of
such polymorphisms (i.e., their location, as well as, their existence) would
be
beneficial in identifying disease loci for many disorders, such as for
example, in neural
disorders, immune system disorders, muscular disorders, reproductive
disorders,
gastrointestinal disorders, pulmonary disorders, digestive disorders,
cardiovascular
disorders, renal disorders, proliferative disorders, and/or cancerous diseases
and
conditions. Such a method is described in U.S. Patents 5,858,659 and
5,856,104. The
U.S. Patents referenced supf~a are hereby incorporated by reference in their
entirety
herein.
[0341] The present invention encompasses polynucleotides of the present
invention that are chemically synthesized, or reproduced as peptide nucleic
acids
(PNA), or according to other methods known in the art. The use of PNAs would
serve
as the preferred form if the polynucleotides of the invention are incorporated
onto a
solid support, or gene chip. For the purposes of the present invention, a
peptide nucleic
acid (PNA) is a polyamide type of DNA analog and the monomeric units for
adenine,
guanine, thymine and cytosine are available commercially (Perceptive
Biosystems).
Certain components of DNA, such as phosphorus, phosphorus oxides, or
deoxyribose
derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science
254:1497
(1991); and Egholm et al., Nature 365:666 (1993), PNAs bind specifically and
tightly
to complementary DNA strands and are not degraded by nucleases. In fact, PNA
binds
more strongly to DNA than DNA itself does. This is probably because there is
no
electrostatic repulsion between the two strands, and also the polyamide
backbone is
more flexible. Because of this, PNA/DNA duplexes bind under a wider range of
stringency conditions than DNA/DNA duplexes, making it easier to perform
multiplex
hybridization. Smaller probes can be used than with DNA due to the strong
binding. In
addition, it is more likely that single base mismatches can be determined with
PNA/DNA hybridization because a single mismatch in a PNAIDNA 15-mer lowers the
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melting point (Tm) by 8°-20° C, vs. 4°-16° C
for the DNA/DNA 15-mer duplex.
Also, the absence of charge groups in PNA means that hybridization can be done
at
low ionic strengths and reduce possible interference by salt during the
analysis.
[0342] The compounds of the present invention have uses which include, but are
not limited to, detecting cancer in mammals. In particular the invention is
useful
during diagnosis of pathological cell proliferative neoplasias which include,
but are not
limited to: acute myelogenous leukemias including acute monocytic leukemia,
acute
myeloblastic leukemia, acute promyelocytic leukemia, acute
my°elomonocytic
leukemia, acute erythroleukemia, acute megakaryocytic leulcemia, and acute
undifferentiated leukemia, etc.; and chronic myelogenous leukemias including
chronic
myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals
include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans.
Particularly preferred are humans.
[0343] The compounds of the present invention have preferred uses which
include,
but are not limited to, detecting pancreatic. cancer in mammals. In particular
the
invention is useful during diagnosis of pathological cell proliferative
neoplasias, which
include, but are not limited to: pancreatic cancers (e.g., benign or malignant
forms of
pancreatic cancer, as well as any particular type of cancer arising from cells
of the
pancreas (e.g., duct cell carcinoma, acinar cell carcinoma, papillary
carcinoma,
adenosquamous carcinoma, undifferentiated carcinoma, mucinous carcinoma, giant
cell carcinoma, mixed type pancreatic cancer, small cell carcinoma,
cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma,
adenocarcinoma, islet-cell tumors, cystic neoplasms, and papillary-cyctic
neoplasm
and the lilce), as well as any stage of such cancers (e.g., stages I to IV in
severity)),
cystic fibrosis, cyst (e.g., pancreatic pseudocyst), pancreatic fistula,
insufficiency,
pancreatic dysplasia, pancreatitis (e.g., chronic pancreatitis, acute
pancreatitis, acute
necrotizing pancreatitis, alcoholic pancreatitis, and pancreatic abscesses
associated
with pancreatic inflammation), and/or those disorders as described under
"Endocrine
Disorders" and/or "Gastrointestinal Disorders" below. Preferred mammals
include
monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans.
Particularly
preferred are humans.
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[0344] Pathological cell proliferative disorders are often associated with
inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., "The
Etiology of
Acute Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic Diseases
of
the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are
now
believed to result from the qualitative alteration of a normal cellular gene
product, or
from the quantitative modification of gene expression by insertion into the
chromosome of a viral sequence, by chromosomal translocation of a gene to a
more
actively transcribed region, or by some other mechanism. (Gehnann et al.,
supra) It is
Iilcely that mutated or altered expression of specific genes is involved in
the
pathogenesis of some leukemias, among other tissues and cell types. (Gelmann
et al.,
supra) Indeed, the human counterparts of the oncogenes involved in some animal
neoplasias have been amplified or translocated in some cases of human
leulcemia and
carcinoma. (Gelmann et al., supra)
[0345] For example, c-myc expression is highly amplified in the non-
lymphocytic
leukemia cell line HL-60. When HL-60 cells are chemically induced to stop
proliferation, the level of c-myc is found to be downregulated. (International
Publication Number WO 91/15580). However, it has been shown that exposure of
HL-
60 cells to a DNA construct that is complementary to the 5' end of c-myc or c-
myb
blocks translation of the corresponding mRNAs which downregulates expression
of
the c-myc or c-myb proteins and causes arrest of cell proliferation and
differentiation
of the treated cells. (International Publication Number WO 91/15580;
Wiclcstrom et
al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad.
Sci.
86:3379 (I989)). However, the skilled artisan would appreciate the present
invention's
usefulness is not be limited to treatment, prevention, diagnosis and/or
prognosis, of
proliferative disorders of cells and tissues of hematopoietic origin, in light
of the
numerous cells and cell types of varying origins which are known to exhibit
proliferative phenotypes. In preferred embodiments, the compounds and/or
methods
of the invention are used to treat, prevent, diagnose, and/or prognose,
proliferative
disorders of pancreatic cells and tissues.
[0346] In addition to the foregoing, a polynucleotide of the present invention
can
be used to control gene expression through triple helix formation or through
antisense
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DNA or RNA. Antisense techniques are discussed, for example, in Okano, J.
Neurochem. 56: 560 (1991); "Oligodeoxynucleotides as Antisense Inhibitors of
Gene
Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is
discussed iri,
for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al.,
Science
241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods
rely on
binding of the polynucleotide to a complementary DNA or RNA. For these
techniques, preferred polynucleotides are usually oligonucleotides 20 to 40
bases in
length and complementary to either the region of the gene involved in
transcription
(triple helix - see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al.,
Science
241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA
itself
(antisense - Okano; J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as
Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).)
Triple
helix formation optimally results in a shut-off of RNA transcription from DNA,
while
antisense RNA hybridization blocks translation of an mRNA molecule into
polypeptide. The oligonucleotide described above can also be delivered to
cells such
that the antisense RNA or DNA may be expressed ira vivo to inhibit production
of
polypeptide of the present invention antigens. Both techniques are effective
in model
systems, and the information disclosed herein can be used to design antisense
or triple
helix polynucleotides in an effort to treat disease, and in particular, for
the treatment of
proliferative diseases and/or conditions. Non-limiting antisense and triple
helix
methods encompassed by the present invention are more thoroughly described
elsewhere herein (see, e.g., the section labeled "Antisense and Ribozyme
(Antagonists)").
[0347] Polynucleotides of the present invention are also useful in gene
therapy.
One goal of gene therapy is to insert a normal gene into an organism having a
defective gene, in an effort to correct the genetic defect. The
polynucleotides
disclosed in the present invention offer a means of targeting such genetic
defects in a
highly accurate manner. Another goal is to insert a new gene that was not
present in
the host genome, thereby producing a new trait in the host cell. Additional
non-
limiting examples of gene therapy methods encompassed by the present invention
are
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more thoroughly described elsewhere herein (see, e.g., the sections labeled
"Gene
Therapy Methods" and Examples 16, 17 and 18).
[0348] The polynucleotides are also useful for identifying individuals from
minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel.
In this technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique bands for
identifying personnel. This method does not suffer from the current
limitations of
"Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The polynucleotides of the present invention can be used as
additional DNA
markers for RFLP.
[0349] The polynucleotides of the present invention can also be used as an
alternative to RFLP, by determining the actual base-by-base DNA sequence of
selected portions of an individual's genome. These sequences can be used to
prepare
PCR primers for amplifying and isolating such selected DNA, which can then be
sequenced. Using this technique, individuals can be identified because each
individual
will have a unique set of DNA sequences. Once an unique ID database is
established
for an individual, positive identification of that individual, living or dead,
can be made
from extremely small tissue samples.
[0350] Forensic biology also benefits from using DNA-based identification
techniques as disclosed herein. DNA sequences taken from very small biological
samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, semen,
synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or
surfactant,
urine, fecal matter, etc., can be amplified using PCR. In one prior art
technique, gene
sequences amplified from polymorphic loci, such as DQa class II HLA gene, are
used
in forensic biology to identify individuals. (Erlich, H., PCR Technology,
Freeman and
Co. (1992).) Once these specific polymorphic loci are amplified, they are
digested
with one or more restriction enzymes, yielding an identifying set of bands on
a
Southern blot probed with DNA corresponding to the DQa class II HLA gene.
Similarly, polynucleotides of the present invention can be used as polymorphic
markers for forensic purposes.
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(0351] There is also a need for reagents capable of identifying the source of
a
particular tissue. Such need arises, for example, in forensics when presented
with
tissue of unknown origin. Appropriate reagents can comprise, for example, DNA
probes or primers prepared from the sequences of the present invention,
specific to
tissues, including but not limited to, those sequences referred to in Table
1A. Panels
of such reagents can identify tissue by species and/or by organ type. In a
similar
fashion, these reagents can be used to screen tissue cultures for
contamination.
Additional non-limiting examples of such uses are further described herein.
[0352] Because pancreatic antigens are found expressed in the pancreas, the
polynucleotides of the present invention are also useful as hybridization
probes for
differential identification of the tissues) or cell types) present in a
biological sample.
Similarly, polypeptides and antibodies directed to polypeptides of the present
invention are useful to provide immunological probes for differential
identification of
the tissues) (e.g., immunohistochemistry assays) or cell types) (e.g.,
immunocytochemistry assays). In a specific embodiment, the polynucleotides of
the
present invention are also useful as hybridization probes for differential
identification
of pancreatic tissues) or cell types) present in a biological sample.
Similarly,
polypeptides and antibodies directed to polypeptides of the present invention
are useful
to provide immunological probes for differential identification of pancreatic
tissues)
(e.g., immunohistochemistry assays) or .cell types) (e.g., immunocytochemistry
assays). In addition, for a number of disorders of the above tissues or cells,
significantly higher or lower levels of gene expression of the
polynucleotides/polypeptides of the present invention may be detected in
certain
tissues (e.g., tissues expressing polypeptides and/or polynucleotides of the
present
invention, for example, normal pancreatic or diseased pancreatic tissues,
and/or those
tissues/cells corresponding to the library source relating to a polynucleotide
sequence
of the invention as disclosed in column 7 of Table IA, and/or cancerous and/or
wounded tissues) or bodily fluids (e.g., semen, lymph, vaginal pool, serum,
plasma,
urine, synovial fluid or spinal fluid) taken from an individual having such a
disorder,
relative to a "standard" gene expression level, i.e., the expression level in
healthy
tissue from an individual not having the disorder.
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[0353] Thus, the invention provides a diagnostic method of a disorder, which
involves: (a) assaying gene expression level in cells or body fluid of an
individual; (b)
comparing the gene expression level with a standard gene expression level,
whereby
an increase or decrease in the assayed gene expression level compared to the
standard
expression level is indicative of a disorder.
[0354] In the very least, the polynucleotides of the present invention can be
used
as molecular weight markers on Southern gels, as diagnostic probes for the
presence of
a specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences in the process of discovering novel polynucleotides, for selecting
and
making oligomers for attachment to a "gene chip" or other support, to raise
anti-DNA
antibodies using DNA immunization techniques, and as an antigen to elicit an
immune
response.
Uses of the Pol~~pe~tides
[0355] Each of the polypeptides identified herein can be used in numerous
ways.
The following description should be considered exemplary and utilizes known
techniques.
[0356] Polypeptides and antibodies directed to polypeptides of the present
invention are useful to provide immunological probes for differential
identification of
the tissues) (e.g., immunohistochemistry assays such as, for example, ABC
immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) or
cell
types) (e.g., immunocytochemistry assays).
[0357] Antibodies can be used to assay levels of polypeptides encoded by
polynucleotides of the invention in a biological sample using classical
immunohistological methods known to those of skill in the art (see, e.g.,
Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jallcanen, et al., J. Cell. Biol.
105:3087-3096
(1987)). Other antibody-based methods useful for detecting protein gene
expression
include immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and
the radioimmunoassay (RIA). Suitable antibody assay labels are known in the
art and
include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine
(1311,
lzsh 123I' lall)~ carbon (14C), sulfur (35S), tritium (3H), indium (115mIn'
113mIn' 112In,
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lllln), and technetium (99Tc, 9smTc), thallium (ZOITi), gallium (68Ga, 67Ga),
palladium
lo3Pd mol bdenum 99Mo xenon (lssXe) fluorine (18F) ls3Sm 177Lu lsaGd l4aPm
( )~ Y ( )> > > > > > >
l4oLa mss I66H0 90Y 47SC 186Re 188Re 142Pr 105 97Ru. luminescent labels such
> > > > > > > > > > >
as luminol; and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0358] In addition to assaying levels of polypeptide of the present invention
in a
biological sample, proteins can also be detected ifs vivo by imaging. Antibody
labels
or marlcers for irr vivo imaging of protein include those detectable by X-
radiography,
NMR or ESR. For X-radiography, suitable labels include radioisotopes such as
barium or cesium, which emit detectable radiation but are not overtly harmful
to the
subject. Suitable markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated into the
antibody by
labeling of nutrients for the relevant hybridoma.
[0359] A pancreatic antigen-specific antibody or antibody fragment which has
been labeled with an appropriate detectable imaging moiety, such as a
radioisotope
for exam 1e 1311 llaln 99mTc (lsll lasl lzsl lull) carbon (14C) sulfur (35S)
tritium
( p > > > > > > > > > >
(3H), indium (lls"'In, llsmln, llaln, 111In), and technetium (99Tc, 99mTc),
thallium (201Ti),
gallium (6gGa, 67Ga), palladium (losPd), molybdenum (99Mo), xenon (ls3Xe),
fluorine
(18F 153Sm 177IJu 159Gd 149Pm 140IJa 175yb 166Ho 90Y 47s~ 186Re 188Re 142Pr
> > > > > a > > > > > > >
losl~~ 97Ru), a radio-opaque substance, or a material detectable by nuclear
magnetic
resonance, is introduced (for ~ example, parenterally, subcutaneously or
intraperitoneally) into the mammal to be examined for pancreatic disorder. It
will be
understood in the art that the size of the subject and the imaging system used
will
determine the quantity of imaging moiety needed to produce diagnostic images.
In the
case of a radioisotope moiety, for a human subject, the quantity of
radioactivity
injected will normally range from about 5 to 20 millicuries of 9amTc. The
labeled
antibody or antibody fragment will then preferentially accumulate at the
location of
cells which express the polypeptide encoded by a polynucleotide of the
invention. Ih
vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumof~ Imaging:
The
Radiochemical Detection of Cahce~, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
Publishing Inc. (1982)).
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[0360] In one embodiment, the invention provides a method for the specific
delivery of compositions of the invention to cells by administering
polypeptides of the
invention (e.g., polypeptides encoded by polynucleotides of the invention
and/or
antibodies) that are associated with heterologous polypeptides or nucleic
acids. In one
example, the invention provides a method for delivering a therapeutic protein
into the
targeted cell. In another example, the invention provides a method for
delivering a
single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded
nucleic
acid (e.g., DNA that can integrate into the cell's genome or replicate
episomally and
that can be transcribed) into the targeted cell.
[0361] In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides
of the invention in association with toxins or cytotoxic prodrugs.
[0362] In a preferred embodiment, the invention provides a method for the
specific
destruction of pancreatic cells (e.g., aberrant pancreatic cells, pancreatic
neoplasm) by
administering polypeptides of the invention (e.g., polypeptides encoded by
polynucleotides of the invention and/or antibodies) in association with toxins
or
cytotoxic prodrugs. In another preferred embodiment the invention provides a
method
for the specific destruction of tissues/cells corresponding to the library
source relating
to a polynucleotide sequence of the invention as disclosed in column 7 of
Table 1A by
administering polypeptides of the invention in association with toxins or
cytotoxic
prodrugs.
[0363] By "toxin" is meant one or more compounds that bind and activate
endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified
toxins,
catalytic subunits of toxins, or any molecules or enzymes not normally present
in or on
the surface of a cell that under defined conditions cause the cell's death.
Toxins that
may be used according to the methods of the invention include, but are not
limited to,
radioisotopes known in the art, compounds such as, for example, antibodies (or
complement fixing containing portions thereof) that bind an inherent or
induced
endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse,
alpha
toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin,
momordin,
gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. "Toxin"
also
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includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive
metal ion,
e.g., alpha-emitters such as, for example, 213$i, or other radioisotopes such
as, for
exam 1e lo3Pd 133Xe 1311 111In 68Ge 57Co 65zn 85Sr 32P 35S 90Y 153Sm 153Gd
p > > > > > > > > > > > > > >
1691,b~ slCr~ s4Mn~ 7sSe, 113Sn~ 9oYttrium, 117Tin, 186Rhenium, l6sHolmiurn,
and
188Rhenium; luminescent labels, such as luminol; and fluorescent labels, such
as
fluorescein and rhodamine, and biotin.
[0364] In a specific embodiment, the invention provides a method for the
specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides
of the invention or antibodies of the invention in association with the
radioisotope 9oY.
In another specific embodiment, the invention provides a method for the
specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides
of the invention or antibodies of the invention in association with the
radioisotope
111In. In a further specific embodiment, the invention provides a method for
the
specific destruction of cells (e.g., the destruction of tumor cells) by
administering
polypeptides of the invention or antibodies of the invention in association
with the
radioisotope 1311.
[0365] Techniques known in the art may be applied to label polypeptides of the
invention (including antibodies). Such techniques include, but are not limited
to, the
use of bifunctional conjugating agents (see e.g., U.S. Patent Nos. 5,756,065;
5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139;
5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which
are'
hereby incorporated by reference in its entirety).
[0366] Thus, the invention provides a diagnostic method of a disorder, which
involves (a) assaying the expression level of a polypeptide of the present
invention in
cells or body fluid of an individual; and (b) comparing the assayed
polypeptide
expression Ievel with a standard polypeptide expression level, whereby an
increase or
decrease in the assayed polypeptide expression level compared to the standard
expression level is indicative of a disorder. With respect to cancer, the
presence of a
relatively high amount of transcript in biopsied tissue from an individual may
indicate
a predisposition for the development of the disease, or may provide a means
for
detecting the disease prior to the appearance of actual clinical symptoms. A
more
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definitive diagnosis of this type may allow health professionals to employ
preventative
measures or aggressive treatment earlier thereby preventing the development or
further
progression of the cancer.
[0367] Moreover, polypeptides of the present invention can be used to treat or
prevent diseases or conditions of the pancreas such as, for example, diabetes
mellitus,
diabetes insipidus, congenital pancreatic agenesis, pancreatic cancers (e.g.,
benign or
malignant forms of pancreatic cancer, as well as any particular type of cancer
arising
from cells of the pancreas (e.g., duct cell carcinoma, acinar cell carcinoma,
papillary
carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, mutinous
carcinoma, giant cell carcinoma, mixed type pancreatic cancer, small cell
carcinoma,
cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma,
adenocarcinoma, islet-cell tumors, cystic neoplasms, and papillary-cyctic
neoplasm
and the like), as well as any stage of such cancers (e.g., stages I to IV in
severity)),
cystic fibrosis, cyst (e.g., pancreatic pseudocyst), pancreatic fistula,
insufficiency,
pancreatic dysplasia, pancreatitis (e.g., chronic pancreatitis, acute
pancreatitis, acute
necrotizing pancreatitis, alcoholic pancreatitis, and pancreatic abscesses
associated
With pancreatic inflammation), and/or those disorders as described under
"Endocrine
Disorders" and/or "Gastrointestinal Disorders" below. In preferred
embodiments,
polynucleotides expressed in a particular tissue type (see, e.g., Table 1A,
column 7)
are used to diagnose, detect, prevent, treat and/or prognose disorders
associated with
the tissue type. For example, patients can be administered a polypeptide of
the present
invention in an effort to replace absent or decreased levels of the
polypeptide (e.g.,
insulin), to supplement absent or decreased levels of a different polypeptide
(e.g.,
hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit
the
activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate
the activity
of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a
membrane
bound receptor by competing with it for free ligand (e.g., soluble TNF
receptors used
in reducing inflammation), or to bring about a desired response (e.g., blood
vessel
growth inhibition, enhancement of the immune response to proliferative cells
or
tissues).
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[0368] Similarly, antibodies directed to a polypeptide of the present
invention can
also be used to treat disease (as described supra, and elsewhere herein). For
example,
administration of an antibody directed to a polypeptide of the present
invention can
bind, and/or neutralize the polypeptide, and/or reduce overproduction of the
polypeptide. Similarly, administration of an antibody can activate the
polypeptide,
such as by binding to a polypeptide bound to a membrane (receptor).
[0369] At the very least, the polypeptides of the present invention can be
used as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of slcill in the art. Polypeptides
can also
be used to raise antibodies, which in turn are used to measure protein
expression from
a recombinant cell, as a way of assessing transformation of the host cell.
Moreover,
the polypeptides of the present invention can be used to test the biological
activities
described herein.
Diagnostic Asssays
[0370] The compounds of the present invention are useful for diagnosis,
treatment,
prevention and/or prognosis of various pancreatic related disorders in
mammals,
preferably humans. Such disorders include, but are not limited to, diabetes
mellitus,
diabetes insipidus, congenital pancreatic agenesis, pancreatic cancers (e.g.,
benign or
malignant forms of pancreatic cancer, as well as any particular type.of cancer
arising
from cells of the pancreas (e.g., duct cell carcinoma, acinar cell carcinoma,
papillary
carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, mucinous
carcinoma, giant cell carcinoma, mixed type pancreatic cancer, small cell
carcinoma,
cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma,
adenocarcinoma, islet-cell tumors, cystic neoplasms, and papillary-cyctic
neoplasm
and the like), as well as any stage of such cancers (e.g., stages I to IV in
severity)),
cystic fibrosis, cyst (e.g., pancreatic pseudocyst), pancreatic fistula,
insufficiency,
pancreatic dysplasia, pancreatitis (e.g., chronic pancreatitis, acute
pancreatitis, acute
necrotizing pancreatitis, alcoholic pancreatitis, and pancreatic abscesses
associated
with pancreatic inflammation), and/or those disorders as described under
"Endocrine
Disorders" and/or "Gastrointestinal Disorders" below. In preferred
embodiments,
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polynucleotides expressed in a particular tissue type (see, e.g., Table 1A,
column 7)
are used to diagnose, detect, prevent, treat and/or prognose disorders
associated with
the tissue type.
[0371] Pancreatic antigens axe expressed in the pancreas, with an increased
expression level in pancreatic tissues. For a number of pancreatic-related
disorders,
substantially altered (increased or decreased) levels of pancreatic antigen
gene
expression can be detected in pancreatic tissue or other cells or bodily
fluids (e.g., sera,
plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual
having
such a disorder, relative to a "standard" pancreatic antigen gene expression
level, that
is, the pancreatic antigen expression level in pancreatic tissues or bodily
fluids from an
individual not having the pancreatic disorder. Thus, the invention provides a
diagnostic method useful during diagnosis of a pancreatic disorder, which
involves
measuring the expression level of the gene encoding the pancreatic associated
polypeptide in pancreatic tissue or other cells or body fluid from an
individual and
comparing the measured gene expression level with a standard pancreatic
antigens
gene expression level, whereby an increase or decrease in the gene expression
levels)
compared to the standard is indicative of an pancreatic disorder.
[0372] In specific embodiments, the invention provides a diagnostic method
useful
during diagnosis of a disorder of a normal or diseased tissue/cell source
corresponding
to column 7 of Table 1A, which involves measuring the expression level of the
coding
sequence of a polynucleotide sequence associated with this tissue/cell source
as
disclosed in Table 1A in the tissue/cell source or other cells or body fluid
from an
individual and comparing the expression level of the coding sequence with a
standard
expression level of the coding sequence of a polynucleotide sequence, whereby
an
increase or decrease in the gene expression levels) compared to the standard
is
indicative of a disorder of a normal or diseased tissue/cell source
corresponding to
column 7 of Table 1A.
[0373] In particular, it is believed that certain tissues in mammals with
cancer of
cells or tissue of the pancreas express signif cantly enhanced or reduced
levels of
normal or altered pancreatic antigen expression and mRNA encoding the
pancreatic
associated polypeptide when compared to a corresponding "standard" level.
Further, it
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is believed that enhanced or depressed levels of the pancreatic associated
polypeptide
can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal
fluid) or
cells or tissue from mammals with such a cancer when compared to sera from
mammals of the same species not having the cancer.
[0374] For example, as disclosed herein, pancreatic associated polypeptides of
the
invention are expressed in the pancreas. Accordingly, polynucleotides of the
invention
(e.g., polynucleotide sequences complementary to all or a portion of a
pancreatic
antigen mRNA nucleotide sequence of SEQ ID NO:X, nucleotide sequence encoding
SEQ ID NO:Y, nucleotide sequence encoding a polypeptide encoded by SEQ ID
NO:X and/or a nucleotide sequence delineated by columns 8 and 9 of Table 2)
and
antibodies (and antibody fragments) directed against the polypeptides of the
invention
may be used to quantitate or qualitate concentrations of cells of the pancreas
expressing pancreatic antigens, preferrably on their cell surfaces. These
polynucleotides and antibodies additionally have diagnostic applications in
detecting
abnormalities in the level of pancreatic antigens gene expression, or
abnormalities in
the structure and/or temporal, tissue, cellular, or subcellular location of
pancreatic
antigens. These diagnostic assays may be performed ih vivo or in vitro, such
as, for
example, on blood samples, biopsy tissue or autopsy tissue. In specific
embodiments,
polynucleotides and antibodies of the invention are used to quantitate or
qualitate
tissues/cells corresponding to the library source disclosed in column 7 of
Table 1A
expressing the corresponding pancreatic sequence disclosed in the same row of
Table
1A, preferrably on their cell surface.
[0375] Thus, the invention provides a diagnostic method useful during
diagnosis of
a pancreatic disorder, including cancers, which involves measuring the
expression
level of the gene encoding the pancreatic antigen polypeptide in pancreatic
tissue or
other cells or body fluid from an individual and comparing the measured gene
expression level with a standard pancreatic antigen gene expression level,
whereby an
increase or decrease in the gene expression level compared to the standard is
indicative
of a pancreatic disorder. In specific embodiments, polynucleotides and
antibodies of
the invention are used to quantitate or qualitate tissues/cells corresponding
to the
library source disclosed in column 7 of Table 1A expressing the corresponding
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pancreatic sequence disclosed in the same row of Table 1A, preferrably on
their cell
surface.
[0376] Where a diagnosis of a disorder in the pancreas, including diagnosis of
a
tumor, has already been made according to conventional methods, the present
invention is useful as a prognostic indicator, whereby patients exhibiting
enhanced or
depressed pancreatic antigen gene expression will experience a worse clinical
outcome
relative to patients expressing the gene at a level nearer the standard level.
[0377] By "assaying the expression level of the gene encoding the pancreatic
associated polypeptide" is intended qualitatively or quantitatively measuring
or
estimating the level of the pancreatic antigen polypeptide or the level of the
mRNA
encoding the pancreatic antigen polypeptide in a first biological sample
either directly
(e.g., by determining or estimating absolute protein level or mRNA level) or
relatively
(e.g., by comparing to the pancreatic associated polypeptide level or mRNA
level in a
second biological sample). Preferably, the pancreatic antigen polypeptide
expression
level or mRNA level in the first biological sample is measured or estimated
and
compared to a standard pancreatic antigen polypeptide level or mRNA level, the
standard being taken from a second biological sample obtained from an
individual not
having the disorder or being determined by averaging levels from a population
of
individuals not having a disorder of the pancreas. As will be appreciated in
the art,
once a standard pancreatic antigen polypeptide level or mRNA level is known,
it can
be used repeatedly as a standard fox comparison.
[0378] By "biological sample" is intended any biological sample obtained from
an
individual, cell line, tissue culture, or other source containing pancreatic
antigen
polypeptides (including portions thereof) or rnRNA. As indicated, biological
samples
include body fluids (such as sera, plasma, urine, synovial fluid and spinal
fluid) which
contain cells expressing pancreatic antigen polypeptides, pancreatic tissue,
and other
tissue sources found to express the full length or fragments thereof of a
pancreatic
antigen. Methods for obtaining tissue biopsies and body fluids from mammals
are
well known in the art. Where the biological sample is to include mRNA, a
tissue
biopsy is the preferred source.
[0379] Total cellular RNA can be isolated from a biological sample using any
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suitable technique such as the single-step guanidinium-thiocyanate-phenol-
chloroform
method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-1S9 (1987).
Levels of mRNA encoding the pancreatic antigen polypeptides are then assayed
using
any appropriate method. These include Northern blot analysis, S 1 nuclease
mapping,
the polymerase chain reaction (PCR), reverse transcription in combination with
the
polymerase chain reaction (RT-PCR), and reverse transcription in combination
with
the ligase chain reaction (RT-LCR).
[0380] The present invention also relates to diagnostic assays such as
quantitative
and diagnostic assays for detecting levels of pancreatic antigen polypeptides,
in a
biological sample (e.g., cells and tissues), including determination of normal
and
abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in
accordance
with the invention for detecting over-expression of pancreatic antigens
compared to
normal control tissue samples may be used to detect the presence of tumors.
Assay
techniques that can be used to determine levels of a polypeptide, such as a
pancreatic
antigen polypeptide of the present invention in a sample derived from a host
are well-
known to those of skill in the art. Such assay methods include
radioimmunoassays,
competitive-binding assays, Western Blot analysis and ELISA assays. Assaying
pancreatic antigen polypeptide levels in a biological sample can occur using
any art-
known method.
[0381] Assaying pancreatic antigen polypeptide levels in a biological sample
can
occur using antibody-based techniques. For example, pancreatic antigen
polypeptide
expression in tissues can be studied with classical immunohistological methods
(Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J.
Cell . Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for detecting
pancreatic
antigen polypeptide gene expression include immunoassays, such as the enzyme
linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucose
oxidase,
and radioisotopes, such as iodine (1251, lzll), carbon (~4C), sulfur (35S),
tritium (3H),
indium (llzIn), and technetium (9smTc), and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
[0382] The tissue or cell type to be analyzed will generally include those
which are
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known, or suspected, to express the pancreatic antigen gene (such as, for
example,
cells of the pancreas or pancreatic cancer). The protein isolation methods
employed
herein may, for example, be such as those described in Harlow and Lane
(Harlow, E.
and Lane, D., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New Yorlc), which is incorporated herein
by
reference in its entirety. The isolated cells can be derived from cell culture
or from a
patient. The analysis of cells taken from culture may be a necessary step in
the
assessment of cells that could be used as part of a cell-based gene therapy
technique
or, alternatively, to test the effect of compounds on the expression of the
pancreatic
antigen gene.
[0383] For example, antibodies, or fragments of antibodies, such as those
described herein, may be used to quantitatively or qualitatively detect the
presence of
pancreatic antigen gene products or conserved variants or peptide fragments
thereof.
This can be accomplished, for example, by immunofluorescence techniques
employing
a fluorescently labeled antibody coupled with light microscopic, flow
cytometric, or
fluorimetric detection.
(0384] In a preferred embodiment, antibodies, or fragments of antibodies
directed
to any one or all of the predicted epitope domains of the pancreatic antigen
polypeptides (Shown in Table 1A, column 6) may be used to quantitatively or
qualitatively detect the presence of pancreatic antigen gene products or
conserved
variants or peptide fragments- thereof. This can be accomplished, for example,
by
immunofluorescence techniques employing a fluorescently labeled antibody
coupled
with light microscopic, flow cytometric, or fluorimetric detection.
(0385] In an additional preferred embodiment, antibodies, or fragments of
antibodies directed to a conformational epitope of a pancreatic antigen may be
used to
quantitatively or qualitatively detect the presence of pancreatic antigen gene
products
or conserved variants or peptide fragments thereof. This can be accomplished,
for
example, by immunofluorescence techniques employing a fluorescently labeled
antibody coupled with light microscopic, flow cytometric, or fluorimetric
detection.
(0386] The antibodies (or fragments thereof), and/or pancreatic antigen
polypeptides of the present invention may, additionally, be employed
histologically, as
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in immunofluorescence, immunoelectron microscopy or non-immunological assays,
for in situ detection of pancreatic antigen gene products or conserved
variants or
peptide fragments thereof. In situ detection may be accomplished by removing a
histological specimen from a patient, and applying thereto a labeled antibody
or
pancreatic antigen polypeptide of the present invention. The antibody (or
fragment
thereof) or pancreatic antigen polypeptide is preferably applied by overlaying
the
labeled antibody (or fragment) onto a biological sample. Through the use of
such a
procedure, it is possible to determine not only the presence of the pancreatic
antigen
gene product, or conserved variants or peptide fragments, or pancreatic
antigen
polypeptide binding, but also its distribution in the examined tissue. Using
the present
invention, those of ordinary skill will readily perceive that any of a wide
variety of
histological methods (such as staining procedures) can be modified in order to
achieve
such in situ detection.
[0387] Immunoassays and non-immunoassays for pancreatic antigen 'gene products
or conserved variants or peptide fragments thereof will typically comprise
incubating a
sample, such as a biological fluid, a tissue extract, freshly harvested cells,
or lysates of
cells which have been incubated in cell culture, in the presence of a
detectably labeled
antibody capable of binding pancreatic antigen gene products or conserved
variants or
peptide fragments thereof, and detecting the bound antibody by any of a number
of
techniques well-known in the art.
[0388] The biological sample may be brought in contact with and immobilized
onto a solid phase support or carrier such as nitrocellulose, or other solid
support
which is capable of immobilizing cells, cell particles or soluble proteins.
The support
may then be washed with suitable buffers followed by treatment with the
detectably
labeled anti-pancreatic antigen antibody or detectable pancreatic antigen
polypeptide.
The solid phase support may then be washed with the buffer a second time to
remove
unbound antibody or polypeptide. Optionally the antibody is subsequently
labeled.
The amount of bound label on solid support may then be detected by
conventional
means.
[0389] By "solid phase support or carrier" is intended any support capable of
binding an antigen or an antibody. Well-known supports or carriers include
glass,
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polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural
and
modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the
carrier can be either soluble to some extent or insoluble for the purposes of
the present
invention. The support material may have virtually any possible structural
configuration so long as the coupled molecule is capable of binding to an
antigen or
antibody. Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the external surface
of a rod.
Alternatively, the surface may be flat such as a sheet, test strip, etc.
Preferred supports
include polystyrene beads. Those skilled in the art will know many other
suitable
carriers for binding antibody or antigen, or will be able to ascertain the
same by use of
routine experimentation.
[0390] The binding activity of a given lot of anti-pancreatic antigen antibody
or
pancreatic antigen polypeptide may be determined according to well known
methods.
Those skilled in the art will be able to determine operative and optimal assay
conditions for each determination by employing routine experimentation.
[0391] In addition to assaying pancreatic antigen polypeptide levels or
polynucleotide levels in a biological sample obtained from an individual,
pancreatic
antigen polypeptide or polynucleotide can also be detected in vivo by imaging.
For
example, in one embodiment of the invention, pancreatic antigen polypeptide
and/or
anti-pancreatic antigen antibodies are used to image pancreatic diseased
cells, such as
neoplasms. In another embodiment, pancreatic antigen polynucleotides of the
invention (e.g., polynucleotides complementary to all or a portion of
pancreatic
antigen mRNA) and/or anti-pancreatic antigen antibodies (e.g., antibodies
directed to
any one or a combination of the epitopes of pancreatic antigens, antibodies
directed to
a conformational epitope of pancreatic antigens, antibodies directed to the
full length
polypeptide expressed on the cell surface of a mammalian cell) are used to
image
diseased or neoplastic cells of the pancreas.
[0392] Antibody labels or markers for izz vivo imaging of pancreatic antigen
polypeptides include those detectable by X-radiography, NMR, MRI, CAT-scans or
ESR. For X-radiography, suitable labels include radioisotopes such as barium
or
cesium, which emit detectable radiation but are not overtly harmful to the
subject.
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Suitable markers for NMR and ESR include those with a detectable
characteristic spin,
such as deuterium, which may be incorporated into the antibody by labeling of
nutrients for the relevant hybridoma. Where i~a vivo imaging is used to detect
enhanced
levels of pancreatic antigen polypeptides for diagnosis in humans, it may be
preferable
to use human antibodies or "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using techniques described herein or otherwise
known in
the art. For example methods for producing chimeric antibodies are known in
the art.
See, for review, Morrison, Sciezzce 229:1202 (1985); Oi et al., BioTechniques
4:214
(1986); Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP
171496;
Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO
8702671; Boulianne et al., Natuz°e 312:643 (1984); Neuberger et al.,
Natu>~e 314:268
(1985).
[0393] Additionally, any pancreatic antigen polypeptides whose presence can be
detected, can be administered. For example, pancreatic antigen polypeptides
labeled
with a radio-opaque or other appropriate compound can be administered and
visualized
in vivo, as discussed, above for labeled antibodies. Further such pancreatic
antigen
polypeptides can be utilized for in vitro diagnostic procedures.
[0394] A pancreatic antigen polypeptide-specific antibody or antibody fragment
which has been labeled with an appropriate detectable imaging moiety, such as
a
radioisotope (for example, 1311, llzln, ~~"'Tc), a radio-opaque substance, or
a material
detectable by nuclear magnetic resonance, is introduced (for example,
parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined for a
pancreatic
disorder. It will be understood in the art that the size of the subject and
the imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of
99mTC. The labeled antibody or antibody fragment will then preferentially
accumulate
at the location of cells which contain pancreatic antigen protein. In vivo
tumor
imaging is described in S.W. Burchiel et aL, "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
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Publishing Inc. (1982)).
[0395] With respect to antibodies, one of the ways in which the anti-
pancreatic
antigen antibody can be detectably labeled is by linking the same to an enzyme
and
using the linked product in an enzyme immunoassay (EIA) (Volley, A., "The
Enzyme
Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,
Microbiological Associates Quarterly Publication, Walkersville, MD); Volley et
al., J.
Clirz. Pathol. 31:507-520 (1978); Butler, J.E., Metlz. Efzzymol. 73:482-523
(1981);
Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,;
Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, I~gaku Shoin, Tokyo).
The
enzyme, which is bound to the antibody will react with an appropriate
substrate,
preferably a chromogenic substrate, in such a manner as to produce a chemical
moiety
which can be detected, for example, by spectrophotometric, fluorimetric or by
visual
means. Enzymes which can be used to detectably label the antibody include, but
are
not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase,
triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,
catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
Additionally, the detection can be accomplished by colorimetric methods which
employ a chromogenic substrate for the enzyme. Detection may also be
accomplished
by visual comparison of the extent of enzymatic reaction of a substrate in
comparison
with similarly prepared standards.
[0396] Detection may also be accomplished using any of a variety of other
immunoassays. For example, by radioactively labeling the antibodies or
antibody
fragments, it is possible to detect pancreatic antigens through the use of a
radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of
Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques,
The
Endocrine Society, March, 1986, which is incorporated by reference herein).
The
radioactive isotope can be detected by means including, but not limited to, a
gamma
counter, a scintillation counter, or autoradiography.
[0397] It is also possible to label the antibody with a fluorescent compound.
When
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the fluorescently labeled antibody is exposed to light of the proper wave
length, its
presence can then be detected due to fluorescence. Among the most commonly
used
fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
[0398] The antibody can also be detectably labeled using fluorescence emitting
metals such as lszEu, or others of the lanthanide series. These metals can be
attached
to the antibody using such metal chelating groups as
diethylenetriaminepentacetic acid
(DTPA) ar ethylenediaminetetraacetic acid (EDTA).
[0399] The antibody also can be detectably labeled by coupling it to a
chemiluminescent compound. The presence of the chemiluminescent-tagged
antibody
is then determined by detecting the presence of luminescence that arises
during the
course of a chemical reaction. Examples of particularly useful
chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium ester,
imidazole,
acridinium salt and oxalate ester.
[0400] Likewise, a bioluminescent compound may be used to label the antibody
of
the present invention. Bioluminescence is a type of chemiluminescence found in
biological systems in, which a catalytic protein increases the efficiency of
the
chemiluminescent reaction. The presence of a bioluminescent protein is
determined
by detecting the presence of luminescence. Important bioluminescent compounds
for
purposes of labeling are luciferin, luciferase and aequorin.
Methods for Detecting Pancreatic Disease, Including Cancer
[0401] In general, a pancreatic disease or cancer may be detected in a patient
based
on the presence of one or more pancreatic antigen proteins of the invention
and/or
polynucleotides encoding such proteins in a biological sample (for example,
blood,
sera, urine, and/or tumor biopsies) obtained from the patient. In other words,
such
proteins and/or polynucleotides may be used as markers to indicate the
presence or
absence of a pancreatic disease or disorder, including cancer. Cancers that
may be
diagnosed, and/or prognosed using the compositions of the invention include
but are
not limited to, pancreatic cancer. In addition, such proteins and/or
polynucleotides may
be useful for the detection of other diseases and cancers, including cancers
of
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tissues/cells corresponding to the library source disclosed in column 7 of
Table 1A
expressing the corresponding pancreatic sequence disclosed in the same row of
Table
1A. The binding agents provided herein generally permit detection of the level
of
antigen that binds to the agent in the biological sample. Polynucleotide
primers and
probes may be used to detect the level of mRNA encoding pancreatic antigen
polypeptides, which is also indicative of the presence or absence of a
pancreatic
disease or disorder, including cancer. In general, pancreatic antigen
polypeptides
should be present at a level that is at least three fold higher in diseased
tissue than in
normal tissue.
[0402] There are a variety of assay formats known to those of ordinary skill
in the
art for using a binding agent to detect polypeptide markers in a sample. See,
e.g.,
Harlow and Lane, s~pr~a. In general, the presence or absence of a pancreatic
disease in
a patient may be determined by (a) contacting a biological sample obtained
from a
patient with a binding agent; (b) detecting in the sample a level of
polypeptide that
binds to the binding agent; and (c) comparing the level of polypeptide with a
predetermined cut-off value.
[0403] In a preferred embodiment, the assay involves the use of binding agent
immobilized on a solid support to bind to and remove the pancreatic antigen
polypeptide of the invention from the remainder of the sample. The bound
polypeptide
may then be detected using a detection reagent that contains a reporter group
and
specifically binds to the binding agent/polypeptide complex. Such detection
reagents
may comprise, for example, a binding agent that specifically binds to the
polypeptide
or an antibody or other agent that specifically binds to the binding agent,
such as an
anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a
competitive
assay may be utilized, in which a polypeptide is labeled with a reporter group
and
allowed to bind to the immobilized binding agent after incubation of the
binding agent
with the sample. The extent to which components of the sample inhibit the
binding of
the labeled polypeptide to the binding agent is indicative of the reactivity
of the sample
with the immobilized binding agent. Suitable polypeptides for use within such
assays
include pancreatic antigen polypeptides and portions thereof, or antibodies,
to which
the binding agent binds, as described above.
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[0404] The solid support may be any material known to those of skill in the
art to
which pancreatic antigen polypeptides of the invention may be attached. For
example,
the solid support may be a test well in a microtiter plate or a nitrocellulose
or other
suitable membrane. Alternatively, the support may be a bead or disc, such as
glass
fiberglass, latex or a plastic material such as polystyrene or
polyvinylchloride. The
support may also be a magnetic particle or a fiber optic sensor, such as those
disclosed,
for example, in U.S. Patent No. 5,359,61. The binding agent may be immobilized
on
the solid support using a variety of techniques known to those of skill in the
art, which
are amply described in the patent and scientific literature. In the context of
the present
invention, the term "immobilization" refers to both noncovalent association,
such as
adsorption, and covalent attachment (which may be a direct linkage between the
agent
and functional groups on the support or may be a linkage by way of a cross-
linking
agent). Immobilization by adsorption to a well in a microtiter plate or to a
membrane is
preferred. In such cases, adsorption may be achieved by contacting the binding
agent,
in a suitable buffer, with the solid support for the suitable amount of time.
The contact
time varies with temperature, but is typically between about 1 hour and about
1 day. In
general, contacting a well of plastic microtiter plate (such as polystyrene or
polyvinylchloride) with an amount of binding agent ranging from about 10 ng to
about
ug, and preferably about 100 ng to about 1 ug, is sufficient to immobilize an
adequate amount of binding agent.
[0405] Covalent attachment of binding agent to a solid support may generally
be
achieved by first reacting the support with a bifunctional reagent that will
react with
both the support and a functional group, such as a hydroxyl or amino group, on
the
binding agent. For example, the binding agent may be covalently attached to
supports
having an appropriate polymer coating using benzoquinone or by condensation of
an
aldehyde group on the support with an amine and an active hydrogen on the
binding
partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-
A13).
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Gene Therapy Methods
[0406] Also encompassed by the present invention are gene therapy methods for
treating or preventing disorders, diseases and conditions. The gene therapy
methods
relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA)
sequences into an animal to achieve expression of a pancreatic antigen of the
present
invention. This method requires a polynucleotide, which codes for a
polypeptide of the
present invention operatively linked to a promoter and any other genetic
elements
necessary for the expression of the polypeptide by the target tissue. Such
gene therapy
and delivery techniques are known in the art, see, for example, W090/11092,
which is
herein incorporated by reference.
[0407] Thus, for example, cells from a patient may be engineered with a
polynucleotide (DNA or RNA) comprising a promoter operably linked to a
polynucleotide of the present invention ex vivo, with the engineered cells
then being
provided to a patient to be treated with the polypeptide of the present
invention. Such
methods are well-known in the art. For example, see Belldegrun, A., et al., J.
Natl.
Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53:
1107-1112
(1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); I~aido,
T., et al.,
Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-
5106
(1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996);
Santodonato, L.,
et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene
Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one
embodiment, the cells, which are engineered are arterial cells. The arterial
cells may
be reintroduced into the patient through direct injection to the artery, the
tissues
surrounding the artery, or through catheter injection.
[0408] As discussed in more detail below, the polynucleotide constructs can be
delivered by any method that delivers injectable materials to the cells of an
animal,
such as, injection into the interstitial space of tissues (heart, muscle,
skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically
acceptable liquid or aqueous carrier.
[0409] In one embodiment, the polynucleotide of the present invention is
delivered
as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers
to
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sequences that are free from any delivery vehicle that acts to assist, promote
or
facilitate entry into the cell, including viral sequences, viral particles,
liposome
formulations, lipofectin or precipitating agents and the like. However, the
polynucleotide of the present invention can also be delivered in liposome
formulations
and lipofectin formulations and the like can be prepared by methods well known
to
those skilled in the art. Such methods are described, for example, in U.S.
Patent Nos.
5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by
reference.
[0410] The polynucleotide vector constructs used in the gene therapy method
are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Appropriate vectors include pWLNEO,
pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVI~3, pBPV, pMSG
and pSVL available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2
available from Invitrogen. Other suitable vectors will be readily apparent to
the skilled
artisan.
[0411] Any strong promoter lrnown to those skilled in the art can be used for
driving the expression of the polynucleotide sequence. Suitable promoters
include
adenoviral promoters, such as the adenoviral major late promoter; or
heterologous
promoters, such as the cytomegalovirus (CMV) promoter; the respiratory
syncytial
virus (RSV) promoter; inducible promoters, such as the MMT promoter, the
metallothionein promoter; heat shock promoters; the albumin promoter; the
ApoAI
promoter; human globin promoters; viral thymidine kinase promoters, such as
the
Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin
promoter; and
human growth hormone promoters. The promoter also may be the native promoter
for
the polynucleotide of the present invention.
[0412] Unlike other gene therapy techniques, one major advantage of
introducing
naked nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis in the cells. Studies have shown that non-replicating
DNA
sequences can be introduced into cells to provide production of the desired
polypeptide
for periods of up to six months.
[0413] The polynucleotide construct can be delivered to the interstitial space
of
tissues within the an animal, including of muscle, skin, brain, lung, liver,
spleen, bone
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marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall
bladder,
stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,
and
connective tissue. Interstitial space of the tissues comprises the
intercellular, fluid,
mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic
fibers in
the walls of vessels or chambers, collagen fibers of fibrous tissues, or that
same matrix
within connective tissue ensheathing muscle cells or in the lacunae of bone.
It is
similarly the space occupied by the plasma of the circulation and the lymph
fluid of the
lymphatic channels. Delivery to the interstitial space of muscle tissue is
preferred for
the reasons discussed below. They may be conveniently delivered by injection
into the
tissues comprising these cells. They are preferably delivered to and expressed
in
persistent, non-dividing cells which are differentiated, although delivery and
expression
may be achieved in non-differentiated 'or less completely differentiated
cells, such as,
for example, stem cells of blood or skin fibroblasts. hz vivo muscle cells are
particularly competent in their ability to take up and express
polynucleotides.
[0414] For the naked nucleic acid sequence injection, an effective dosage
amount of
DNA or RNA will be in the range of from about 0.05 mglkg body weight to about
50
mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to
about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course,
as the
artisan of ordinary slcill will appreciate, this dosage will vary according to
the tissue site
of injection. The appropriate and effective dosage of nucleic acid sequence
can readily
be determined by those of ordinary skill in the art and may depend on the
condition
being treated and the route of administration.
[0415] The preferred route of administration is by the parenteral route of
injection
into the interstitial space of tissues. However, other parenteral routes may
also be
used, such as, inhalation of an aerosol formulation particularly for delivery
to lungs or
bronchial tissues, throat or mucous membranes of the nose. In addition, naked
DNA
constructs can be delivered to arteries during angioplasty by the catheter
used in the
procedure.
[0416] The naked polynucleotides are delivered by any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
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injection, topical administration, catheter infusion, and so-called "gene
guns". These
delivery methods are known in the art.
[0417] The constructs may also be delivered with delivery vehicles such as
viral
sequences, viral particles, liposome formulations, lipofectin, precipitating
agents, etc.
Such methods of delivery are known in the art.
[0418] In certain embodiments, the polynucleotide constructs are complexed in
a
liposome preparation. Liposomal preparations for use in the instant invention
include
cationic (positively charged), anionic (negatively charged) and neutral
preparations.
However, cationic liposomes are particularly preferred because a tight charge
complex
can be formed between the cationic liposome and the polyanionic nucleic acid.
Cationic liposomes have been shown to mediate intracellular delivery of
plasmid DNA
(Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is
herein
incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA
(1989)
86:6077-6081, which is herein incorporated by reference); and purified
transcription
factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein
incorporated by reference), in functional form.
[0419] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
particularly useful and are available under the trademark Lipofectin, from
GIBCO
BRL, Grand Island, N.Y., (see, also, Felgner et al., Proc. Natl Acad. Sci. USA
(1987)
84:7413-7416, which is herein incorporated by reference). Other commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
[0420] Other cationic liposomes can be prepared from readily available
materials
using techniques well known in the art. See, e.g. PCT Publication No. WO
90/11092
(which is herein incorporated by reference) for a description of the synthesis
of
DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation
of
DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al.,
Proc. Natl.
Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference.
Similar
methods can be used to prepare liposomes from other cationic lipid materials.
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[0421] Similarly, anionic and neutral liposomes are readily available, such as
from
Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using
readily
available materials. Such materials include phosphatidyl choline, cholesterol,
phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE),
among others. These materials can also be mixed with the DOTMA and DOTAP
starting materials in appropriate ratios. Methods for making liposomes using
these
materials are well known in the art.
[0422] For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE) can be used in various combinations to make conventional liposomes,
with or
without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can
be
prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas
into a sonication vial. The sample is placed under a vacuum pump overnight and
is
hydrated the following day with deionized water. The sample is then sonicated
for 2
hours in a capped vial, using a Heat Systems model 350 sonicator equipped with
an
inverted cup (bath type) probe at the maximum setting while the bath is
circulated at
15EC. Alternatively, negatively charged vesicles can be prepared without
sonication to
produce multilamellar vesicles or by extrusion through nucleopore membranes to
produce unilamellar vesicles of discrete size. Other methods are known and
available
to those of skill in the art.
(0423] The liposomes can comprise multilamellar vesicles (MLVs), small
unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs
being
preferred. The various liposome-nucleic acid complexes are prepared using
methods
well known in the art. See, e.g., Straubinger et al., Methods of Immunology
(1983),
101:512-527, which is herein incorporated by reference. For example, MLVs
containing nucleic acid can be prepared by depositing a thin film of
phospholipid on
the walls of a glass tube and subsequently hydrating with a solution of the
material to
be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a
homogeneous population of unilamellar liposomes. The material to be entrapped
is
added to a suspension of preformed MLVs and then sonicated. When using
liposomes
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containing cationic lipids, the dried lipid film is resuspended in an
appropriate solution
such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCI,
sonicated, and then the preformed liposomes are mixed directly with the DNA.
The
liposome and DNA form a very stable complex due to binding of the positively
charged liposomes to the cationic DNA. SUVs find use with small nucleic acid
fragments. LUVs are prepared by a number of methods, well known in the art.
Commonly used methods include Ca2+-EDTA chelation (Papahadjopoulos et al.,
Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979); ether
injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976);
Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al.,
Proc. Natl.
Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and
Strittmatter, P.,
Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV)
(Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka et al., Proc. Natl.
Acad. Sci.
USA 75:145 (1978); Schaefer-Ridder et al., Science ZIS:I66 (1982)), which are
herein incorporated by reference.
[0424] Generally, the ratio of DNA to liposomes will be from about 10:1 to
about
1:10. Preferably, the ration will be from about 5:1 to about 1:5. More
preferably, the
ration will be about 3:1 to about 1:3. Still more preferably, the ratio will
be about 1:1.
[0425] U.S. Patent No. 5,676,954 (which is herein incorporated by reference)
reports on the injection of genetic material, complexed with cationic
liposomes
carriers, into mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386,
5,459,127,
5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no.
WO
94/9469 (which are herein incorporated by reference) provide cationic lipids
for use in
transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466,
5,693,622,
5,580,859, 5,703,055, and International Publication No. WO 94/9469 provide
methods
for delivering DNA-cationic lipid complexes to mammals.
[0426] In certain embodiments, cells are engineered, ex vivo or in vivo, using
a
retroviral particle containing RNA, which comprises a sequence encoding a
polypeptide of the present invention. Retroviruses from which the retroviral
plasmid
vectors may be derived include, but are not limited to, Moloney Murine
Leukemia
Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian
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leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus,
Myeloproliferative Sarcoma Virus, and mammary tumor virus.
[0427] The retroviral plasmid vector is employed to transduce packaging cell
lines
to form producer cell lines. Examples of paclcaging cells which may be
transfected
include, but are not limited to, the PE501, PA317, R-2, R-AM, PAl2, T19-14X,
VT-
19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described
in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by
reference in its entirety. The vector may transduce the packaging cells
through any
means known in the art. Such means include, but are not limited to,
electroporation,
the use of liposomes, and CaP04 precipitation. In one alternative, the
retroviral
plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and
then
administered to a host.
[0428] The producer cell line generates infectious retroviral vector
particles, which
include polynucleotide encoding a polypeptide of the present invention. Such
retroviral vector particles then may be employed, to transduce eukaryotic
cells, either
iia vitf°o or in vivo. The transduced eulcaryotic cells will express a
polypeptide of the
present invention.
[0429] In certain other embodiments, cells are engineered, ex vivo or i~ vivo,
with
polynucleotide contained in an adenovirus vector. Adenovirus can be
manipulated
such that it encodes and expresses a polypeptide of the present invention, and
at the
same time is inactivated in terms of its ability to replicate in a normal
lytic viral life
cycle. Adenovirus expression is achieved without integration of the viral DNA
into the
host cell chromosome, thereby alleviating concerns about insertional
mutagenesis.
Furthermore, adenoviruses have been used as live enteric vaccines for many
years with
an excellent safety profile (Schwartz, et al., Am. Rev. Respir. Dis.109:233-
238
(1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a
number of instances including transfer of alpha-1-antitrypsin and CFTR to the
lungs of
cotton rats (Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al.,
Cell
68:143-155 (1991)). Furthermore, extensive studies to attempt to establish
adenovirus
as a causative agent in human cancer were uniformly negative (Green et al.,
Proc. Natl.
Acad. Sci. USA 76:6606 (1979)).
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[0430] Suitable adenoviral vectors useful in the present invention are
described,
for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503
(1993);
Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther. 4:759-
769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature
365:691-692 (1993); and U.S. Patent No. 5,652,224, which are herein
incorporated by
reference. For example, the adenovirus vector Ad2 is useful and can be grown
in
human 293 cells. These cells contain the E1 region of adenovirus and
constitutively
express Ela and Elb, which complement the defective adenoviruses by providing
the
products of the genes deleted from the vector. In addition to Ad2, other
varieties of
adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the present invention.
[0431] Preferably, the adenoviruses used in the present invention are
replication
deficient. Replication deficient adenoviruses require the aid of a helper
virus and/or
paclcaging cell line to form infectious particles. The resulting virus is
capable of
infecting cells and can express a polynucleotide of interest which is operably
linked to
a promoter, but cannot replicate in most cells. Replication deficient
adenoviruses may
be deleted in one or more of all or a portion of the following genes: EIa,
Elb, E3', E4,
E2a, or L1 through L5.
[0432] In certain other embodiments, the cells are engineered, ex vivo or i~
vivo,
using an adeno-associated virus (AAV). AAVs are naturally occurring defective
viruses that require helper viruses to produce infectious particles (Muzyczka,
N., Curr.
Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few
viruses that
may integrate its DNA into non-dividing cells. Vectors containing as little as
300 base
pairs of AAV can be packaged and can integrate, but space for exogenous DNA is
limited to about 4.5 kb. Methods for producing and using such AAVs are known
in
the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
[0433] Fox example, an appropriate AAV vector for use in the present invention
will include all the sequences necessary for DNA replication, encapsidation,
and host-
cell integration. The polynucleotide construct is inserted into the AAV vector
using
standard cloning methods, such as those found in Sambrook et al., Molecular
Cloning:
A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV
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vector is then transfected into packaging cells Which are infected with a
helper virus,
using any standard technique, including lipofection, electroporation, calcium
phosphate precipitation, etc. Appropriate helper viruses include adenoviruses,
cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging
cells are
transfected and infected, they will produce infectious AAV viral particles,
which
contain the polynucleotide construct. These viral particles are then used to
transduce
eukaryotic cells, either ex vivo or if2 vivo. The transduced cells will
contain the
polynucleotide construct integrated into its genome, and will express a
polypeptide of
the invention.
[0434] Another method of gene therapy involves operably associating
heterologous control regions and endogenous pancreatic antigen polynucleotide
sequences (e.g., encoding a pancreatic antigen polypeptide of the present
invention)
via homologous recombination (see, e.g., U.S. Patent No. 5,641,670, issued
June 24,
1997; International Publication No. WO 96/29411, published September 26, 1996;
International Publication No. WO 94/12650, published August 4, 1994; Koller et
al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438 (1989), which are herein incorporated by reference. This method
involves
the activation of a gene which is present in the target cells, but which is
not normally
expressed in the cells, or is expressed at a lower level than desired.
[0435] Polynucleotide constructs are made, using standard techniques known in
the art, which contain the promoter with targeting sequences flanking the
promoter.
Suitable promoters are described herein. The targeting sequence is
sufficiently
complementary to an endogenous sequence to permit homologous recombination of
the promoter-targeting sequence with the endogenous sequence. The targeting
sequence will be sufficiently near the 5' end of the desired endogenous
polynucleotide
sequence so the promoter will be operably linked to the endogenous sequence
upon
homologous recombination.
[0436] The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the 5'
and 3' ends. Preferably, the 3' end of the first targeting sequence contains
the same
restriction enzyme site as the 5' end of the amplified promoter and the 5' end
of the
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second targeting sequence contains the same restriction site as the 3' end of
the
amplified promoter. The amplified promoter and targeting sequences are
digested and
ligated together.
[0437] The promoter-targeting sequence construct is delivered to the cells,
either
as naked polynucleotide, or in conjunction with transfection-facilitating
agents, such as
liposomes, viral sequences, viral particles, whole viruses, lipofection,
precipitating
agents, etc., described in more detail above. The P promoter-targeting
sequence can be
delivered by any method, included direct needle injection, intravenous
injection,
topical administration, catheter infusion, particle accelerators, etc. The
methods are
described in more detail below.
[0438] The promoter-targeting sequence construct is talcen up by cells.
Homologous recombination between the construct and the endogenous sequence
takes
place, such that an endogenous sequence is placed under the control of the
promoter.
The promoter then drives the expression of the endogenous sequence.
[0439] The polynucleotide encoding a polypeptide of the present invention may
contain a secretory signal sequence that facilitates secretion of the protein.
Typically,
the signal sequence is positioned in the coding region of the polynucleotide
to be
expressed towards or at the 5' end of the coding region. The signal sequence
may be
homologous or heterologous to the pancreatic antigen polynucleotide of
interest and
may be homologous or heterologous to the cells to be transfected.
Additionally, the
signal sequence may be chemically synthesized using methods known in the art.
[0440] Any mode of administration of any of the above-described
polynucleotides
constructs can be used so long as the mode results in the expression of one or
more
molecules in an amount sufficient to provide a therapeutic effect. This
includes direct
needle injection, systemic injection, catheter infusion, biolistic injectors,
particle
accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially
available
depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial
solid
(tablet or pill) pharmaceutical formulations, and decanting or topical
applications
during surgery. For example, direct injection of naked calcium phosphate-
precipitated
plasmid into rat liver and rat spleen or a protein-coated plasmid into the
portal vein has
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resulted in gene expression of the foreign gene in the rat livers (Kaneda et
al., Science
243:375 (1989)).
[0441] A preferred method of local administration is by direct injection.
Preferably, a recombinant molecule of the present invention complexed with a
delivery
vehicle is administered by direct injection into or locally within the area of
arteries.
Administration of a composition locally within the area of arteries refers to
injecting
the composition centimeters and preferably, millimeters within arteries.
[0442] Another method of local administration is to contact a polynucleotide
construct of the present invention in or around a surgical wound. For example,
a
patient can undergo surgery and the polynucleotide construct can be coated on
the
surface of tissue inside the wound or the construct can be injected into areas
of tissue
inside the wound.
[0443] Therapeutic compositions useful in systemic administration, include
recombinant molecules of the present invention complexed to a targeted
delivery
vehicle of the present invention. Suitable delivery vehicles for use with
systemic
administration comprise liposomes comprising ligands for targeting the vehicle
to a
particular site. In specific embodiments, suitable delivery vehicles for use
with
systemic administration comprise liposomes comprising polypeptides of the
invention
for targeting the vehicle to a particular site.
[0444] Preferred methods of systemic administration, include intravenous
inj ection, aerosol, oral and percutaneous (topical) delivery. Intravenous inj
ections can
be performed using methods standard in the art. Aerosol delivery can also be
performed using methods standard in the art (see, for example, Stribling et
al., Proc.
Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by
reference). Oral delivery can be performed by complexing a polynucleotide
construct
of the present invention to a carrier capable of withstanding degradation by
digestive
enzymes in the gut of an animal. Examples of such carriers, include plastic
capsules or
tablets, such as those known in the art. Topical delivery can be performed by
mixing a
polynucleotide construct of the present invention with a lipophilic reagent
(e.g.,
DMSO) that is capable of passing into the skin.
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[0445] Determining an effective amount of substance to be delivered can depend
upon a number of factors including, for example, the chemical structure and
biological
activity of the substance, the age and weight of the animal, the precise
condition
requiring treatment and its severity, and the route of administration. The
frequency of
treatments depends upon a number of factors, such as the amount of
polynucleotide
constructs administered per dose, as well as the health and history of the
subject. The
precise amount, number of doses, and timing of doses will be determined by the
attending physician or veterinarian.
[0446] Therapeutic compositions of the present invention can be administered
to
any animal, preferably to mammals and birds. Preferred mammals include humans,
dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humans
being
particularly preferred.
Biological Activities
[0447] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, can be used in assays to test for one or more biological
activities. If these
polynucleotides or polypeptides, or agonists or antagonists of the present
invention, do
exhibit activity in a particular assay, it is lil~ely that these molecules may
be involved
in the diseases associated with the biological activity. Thus, the
polynucleotides and
polypeptides, and agonists or antagonists could be used to treat, prevent
diagnose
and/or prognose the associated disease.
[0448] The pancreatic antigen polynucleotides and polypeptides of the
invention
are predicted to have predominant expression in pancreatic tissues.
[0449] Thus, the pancreatic antigens of the invention may be useful as
therapeutic
molecules. Each would be useful for diagnosis, detection, treatment and/or
prevention
of diseases or disorders of the pancreas, including but not limited to
diabetes mellitus,
diabetes insipidus, congenital pancreatic agenesis, pancreatic cancers (e.g.,
benign or
malignant forms of pancreatic cancer, as well as any particular type of cancer
arising
from cells of the pancreas (e.g., duct cell carcinoma, acinar cell carcinoma,
papillary
carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, mutinous
carcinoma, giant cell carcinoma, mixed type pancreatic cancer, small cell
carcinoma,
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cystadenocarcinoma, unclassified pancreatic cancers, pancreatoblastoma,
adenocarcinoma, islet-cell tumors, cystic neoplasms, and papillary-cyctic
neoplasm
and the like), as well as any stage of such cancers (e.g., stages I to IV in
severity)),
cystic fibrosis, cyst (e.g., pancreatic pseudocyst), pancreatic fistula,
insufficiency,
pancreatic dysplasia, pancreatitis (e.g., chronic pancreatitis, acute
pancreatitis, acute
necrotizing pancreatitis, alcoholic pancreatitis, and pancreatic abscesses
associated
with pancreatic inflammation), and/or those disorders as described under
"Endocrine
Disorders" and/or "Gastrointestinal Disorders" below.
[0450] In a preferred embodiment, polynucleotides of the invention (e.g., a
nucleic
acid sequence of SEQ ID NO:X or the complement thereof; or the cDNA sequence
contained in Clone ID NO:Z, or fragments or variants thereof) and/or
polypeptides of
the invention (e.g., an amino acid sequence contained in SEQ ID NO:Y, an amino
acid
sequence encoded by SEQ ID NO:X, or the complement threof, an amino acid
sequence encoded by the cDNA sequence contained in Clone ID NO:Z and fragments
or variants thereof as described herein) are useful for the diagnosis,
detection,
treatement, and/or prevention of diseases or disorders of the tissues/cells
corresponding to the library source disclosed in column 7 of Table 1A
expressing the
corresponding pancreatic sequence disclosed in the same row of Table 1A. In
certain
embodiments, a polypeptide of the invention, or polynucleotides, antibodies,
agonists,
or antagonists corresponding to that polypeptide, may be used to diagnose
and/or
prognose diseases and/or disorders associated with the tissues) in which the
polypeptide of the invention is expressed, including one, two, three, four,
Eve, or more
tissues disclosed in Table 1A, column 7 (Tissue Distribution Library Code).
[0451] Particularly, the pancreatic antigens may be a useful therapeutic for
pancreatic cancer. Treatment, diagnosis, detection, and/or prevention of
pancreatic
disorders could be carried out using a pancreatic antigen or soluble form of a
pancreatic antigen, a pancreatic antigen ligand, gene therapy, or ex vivo
applications.
Moreover, inhibitors of a pancreatic antigen, either blocking antibodies or
mutant
forms, could modulate the expression of the pancreatic antigen. These
inhibitors may
be useful to treat, diagnose, detect, and/or prevent diseases associated with
the
misregulation of a pancreatic antigen.
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[0452] In one embodiment, the invention provides a method for the specific
delivery of compositions of the invention to cells (e.g., normal or diseased
pancreatic
cells) by administering polypeptides of the invention (e.g., pancreatic
antigen
polypeptides or anti-pancreatic antigen antibodies) that are associated with
heterologous polypeptides or nucleic acids. In one example, the invention
provides a
method for delivering a therapeutic protein into the targeted cell (e.g., an
aberrant
pancreatic cell or pancreatic cancer cell). In another example, the invention
provides a
method for delivering a single stranded nucleic acid (e.g., antisense or
ribozymes) or
double stranded nucleic acid (e.g., DNA that can integrate into the cell's
genome or
replicate episomally and that can be transcribed) into the targeted cell.
[0453] In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of aberrant pancreatic cells,
including, but not
limited to, pancreatic tumor cells) by administering polypeptides of the
invention
(e.g., pancreatic antigen polypeptides or fragments thereof, or anti-
pancreatic antigen
antibodies) in association with toxins or cytotoxic prodrugs.
[0454] By "toxin" is meant compounds that bind and activate endogenous
cytotoxic effector systems, radioisotopes, holotoxins, modified toxins,
catalytic
subunits of toxins, cytotoxins (cytotoxic agents), or any molecules or enzymes
not
normally present in or on the surface of a cell that under defined conditions
cause the
cell's death. Toxins that may be used according to the methods of the
invention
include, but are not limited to, radioisotopes known in the art, compounds
such as, for
example, antibodies (or complement fixing containing portions thereof) that
bind an
inherent or induced endogenous cytotoxic effeetor system, thymidine kinase,
endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomofaas exotoxin A,
diphtheria
toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin
and
cholera toxin. "Toxin" also includes a cytostatic or cytocidal agent, a
therapeutic agent
or a radioactive metal ion, e.g., alpha-emitters such as, for example, zi3Bi,
or other
radioisoto es such as for exam 1e losPd 133Xe 1311 6aGe s7Co ssZn assr sap sss
p ~ P > > > > > > > > > >
90~, isssm is3Gd 169Yb siCr s4Mn 7sse assn 9oyttrium 117Tin 186Rheniurn
> > > > > > > > > > >
issHolmium, and 188Rhenium; luminescent labels, such as luminol; and
fluorescent
labels, such as fluorescein and rhodamine, and biotin.
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[0455] Techniques known in the art may be applied to label antibodies of the
invention. Such techniques include, but are not limited to, the use of
bifunctional
conjugating agents (see e.g., U.S. Patent Nos. 5,756,065; 5,714,631;
5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560; and 5,808,003; the contents of each of which are hereby
incorporated by
reference in its entirety). A cytotoxin or cytotoxic agent includes any agent
that is
detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin
D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate,
6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine
(BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin,
mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin),
anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).
[0456] By "cytotoxic prodrug" is meant a non-toxic compound that is converted
by
an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic
prodrugs that may be used according to the methods of the invention include,
but are
not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent,
phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,
daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
[0457] It will be appreciated that conditions caused by a decrease in the
standard
or normal level of a pancreatic antigen activity in an individual,
particularly disorders
of the pancreas, can be treated by administration of a pancreatic antigen
polypeptide
(e.g., such as, for example, the complete pancreatic antigen polypeptide, the
soluble
form of the extracellular domain of a pancreatic antigen polypeptide, or cells
expressing the complete protein) or agonist. Thus, the invention also provides
a
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method of treatment of an individual in need of an increased level of
pancreatic
antigen activity comprising administering to such an individual a
pharmaceutical
composition comprising an amount of an isolated pancreatic antigen polypeptide
of the
invention, or agonist thereof (e.g., an agonistic anti-pancreatic antigen
antibody),
effective to increase the pancreatic antigen activity level in such an
individual.
[0458] It will also be appreciated that conditions caused by a increase in the
standard or normal level of pancreatic antigen activity in an individual,
particularly
disorders of the pancreas, can be treated by administration of pancreatic
antigen
polypeptides (e.g., such as, for example, the complete pancreatic antigen
polypeptide,
the soluble form of the extracellular domain of a pancreatic antigen
polypeptide, or
cells expressing the complete protein) or antagonist (e.g., an antagonistic
pancreatic
antigen antibody). Thus, the invention also provides a method of treatment of
an
individual in need of an decreased level of pancreatic antigen activity
comprising
administering to such an individual a pharmaceutical composition comprising an
amount of an isolated pancreatic antigen polypeptide of the invention, or
antagonist
thereof (e.g., an antagonistic anti-pancreatic antigen antibody), effective to
decrease
the pancreatic antigen activity level in such an individual.
[0459] More generally, polynucleotides, translation products and antibodies
corresponding to this gene may be useful for the diagnosis, prognosis,
prevention,
and/or treatment of diseases and/or disorders associated with the following
systems.
Endocrine Disorders
[0460] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or prognose disorders
and/or
diseases related to hormone imbalance, and/or disorders or diseases of the
endocrine
system.
[0461] Hormones secreted by the glands of the endocrine system control
physical
growth, sexual function, metabolism, and other functions. Disorders may be
classified
in two ways: disturbances in the production of hormones, and the inability of
tissues to
respond to hormones. The etiology of these hormone imbalance or endocrine
system
diseases, disorders or conditions may be genetic, somatic, such as cancer and
some
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autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or
infectious.
Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention can be used as a marker or detector of a particular
disease or
disorder related to the endocrine system and/or hormone imbalance.
[0462] Endocrine system and/or hormone imbalance and/or diseases encompass
disorders of uterine motility including, but not limited to: complications
with
pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous
abortion,
and slow or stopped labor); and disorders and/or diseases of the menstrual
cycle (e.g.,
dysmenorrhea and endometriosis).
[0463] Endocrine system and/or hormone imbalance disorders and/or diseases
include disorders and/or diseases of the pancreas, such as, for example,
diabetes
mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma-
-islet
cell tumor syndrome; disorders and/or diseases of the adrenal glands such as,
for
example, Addison's Disease, corticosteroid deficiency, virilizing disease,
hirsutism,
Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or
diseases of the pituitary gland, such as, for example, hyperpituitarism,
hypopituitarism,
pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly,
gigantism;
disorders and/or diseases of the thyroid, including but not limited to,
hyperthyroidism,
hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter),
toxic
nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous
thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome,
myxedema,
cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia,
Hurthle
cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary
thyroid
carcinoma; disorders and/or diseases of the parathyroid, such as, for example,
hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the
hypothalamus.
[0464] In addition, endocrine system and/or hormone imbalance disorders and/or
diseases may also include disorders and/or diseases of the testes or ovaries,
including
cancer. Other disorders and/or diseases of the testes or ovaries further
include, for
example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome,
vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's
cells,
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cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma
of
the testis (benign), neoplasias of the testis and neo-testis.
[0465] Moreover, endocrine system and/or hormone imbalance disorders and/or
diseases may also include disorders and/or diseases such as, for example,
polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple
Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
[0466] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders
associated with the tissues) in which the polypeptide of the invention is
expressed,
including one, two, three, four, five, or more tissues disclosed in Table 1A,
column 7
(Tissue Distribution Library Code).
Immune Activity
[0467] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in treating, preventing, diagnosing and/or
prognosing diseases, disorders, and/or conditions of the immune system, by,
for
example, activating or inhibiting the proliferation, differentiation, or
mobilization
(chemotaxis) of immune cells. Immune cells develop through a process called
hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and
macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem
cells.
The etiology of these immune diseases, disorders, and/or conditions may be
genetic,
somatic, such as cancer and some autoimmune diseases, acquired (e.g., by
chemotherapy or toxins), or infectious. Moreover, polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention can be
used as a
marker or detector of a particular immune system disease or disorder.
[0468] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
treat diseases and disorders of the immune system and/or to inhibit or enhance
an
immune response generated by cells associated with the tissues) in which the
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polypeptide of the invention is expressed, including one, two, three, four,
five, or more
tissues disclosed in Table 1A, column 7 (Tissue Distribution Library Code).
[0469] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in treating, preventing, diagnosing,
and/or
prognosing immunodeficiencies, including both congenital and acquired
immunodeficiencies. Examples of B cell immunodeficiencies in which
immunoglobulin levels B cell function and/or B cell numbers are decreased
include:
X-linked agammaglobulinemia (Bruton's disease), X-linked infantile
agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked
immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP),
agammaglobulinemia including congenital and acquired agammaglobulinemia, adult
onset agammaglobulinemia, late-onset agamrriaglobulinemia,
dysgammaglobulinemia,
hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive
agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA
deficiency,
selective IgG subclass deficiencies, IgG subclass deficiency (with or without
IgA
deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with
increased
IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain
deletions, kappa
chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable
immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired),
and transient hypogammaglobulinemia of infancy.
[0470] In specific embodiments, ataxia-telangiectasia or conditions associated
with
ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing
using the
polypeptides or polynucleotides of the invention, and/or agonists or
antagonists
thereof.
[0471] Examples of congenital immunodeficiencies in which T cell and/or B cell
function and/or number is decreased include, but are not limited to: DiGeorge
anomaly, severe combined immunodeficiencies (SLID) (including, but not limited
to,
X-linked SCID, autosomal recessive SLID, adenosine deaminase deficiency,
purine
nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare
lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia),
thymic
hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion,
chronic
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mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic
CD4+ T-
lymphocytopenia, immunodeficiency with predominant T cell defect
(unspecified),
and unspecified immunodeficiency of cell mediated immunity.
[0472] In specific embodiments, DiGeorge anomaly or conditions associated with
DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using
polypeptides or polynucleotides of the invention, or antagonists or agonists
thereof.
[0473] Other immunodeficiencies that may be treated, prevented, diagnosed,
and/or prognosed using polypeptides or polynucleotides of the invention,
and/or
agonists or antagonists thereof, include, but are not limited to, chronic
granulomatous
disease, Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte
glucose-
6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome
(XLP), leukocyte adhesion deficiency, complement component deficiencies
(including
Cl, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular
dysgenesis,~thymic
alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital
leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short
limbed
dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
[0474] In a preferred embodiment, the immunodeficiencies and/or conditions
associated with the immunodeficiencies recited above are treated, prevented,
diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention.
[0475] In a preferred embodiment polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention could be used as an
agent to
boost immunoresponsiveness among immunodeficient individuals. In specific
embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention could be used as an agent to boost
immunoresponsiveness
among B cell and/or T cell immunodeficient individuals.
[0476] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be useful in treating, preventing,
diagnosing
and/or prognosing autoimmune disorders. Many autoimmune disorders result from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to the
destruction of
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the host tissue. Therefore, the administration of polynucleotides and
polypeptides of
the invention that can inhibit an immune response, particularly the
proliferation,
differentiation, or chemotaxis of T-cells, may be~ an effective therapy in
preventing
autoimmune disorders.
[0477] Autoimmune diseases or disorders that may be treated, prevented,
diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention include, but are not limited
to, one or
more of the following: systemic lupus erythematosus, rheumatoid arthritis,
ankylosing
spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's
thyroiditis,
autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoirnmune
thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic
thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura),
autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia
gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes
mellitus.
[0478] Additional disorders that are likely to have an autoimmune component
that
may be treated, prevented, and/or diagnosed with the compositions of the
invention
include, but are not limited to, type II collagen-induced arthritis,
antiphospholipid
syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing
polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia,
polyendocrinopathies, Reiter's Disease, Stiff Man Syndrome, autoimmune
pulmonary
inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes
mellitus,
and autoimmune inflammatory eye disorders.
[0479] Additional disorders that are likely to have an autoimmune component
that
may be treated, prevented, diagnosed and/or prognosed with the compositions of
the
invention include, but are not limited to, scleroderma with anti-collagen
antibodies
(often characterized, e.g., by nucleolar and other nuclear antibodies), mixed
connective
tissue disease (often characterized, e.g., by antibodies to extractable
nuclear antigens
(e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by
nonhistone
ANA), pernicious anemia (often characterized, e.g., by antiparietal cell,
microsomes,
and intrinsic factor antibodies), idiopathic Addison's disease (often
characterized, e.g.,
by humoral and cell-mediated adrenal cytotoxicity, infertility (often
characterized, e.g.,
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by antispermatozoal antibodies), glomerulonephritis (often characterized,
e.g., by
glomerular basement membrane antibodies or immune complexes), bullous
pemphigoid (often characterized, e.g., by IgG and complement in basement
membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue
antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often
characterized, e.g., by cell-mediated and humoral islet cell antibodies), and
adrenergic
drug resistance (including adrenergic drug resistance with asthma or cystic
fibrosis)
(often characterized, e.g., by beta-adrenergic receptor antibodies).
[0480] Additional disorders that may have an autoimmune component that may be
treated, prevented, diagnosed and/or prognosed with the compositions of the
invention
include, but are not limited to, chronic active hepatitis (often
characterized, e.g., by
smooth muscle antibodies), primary biliary cirrhosis (often characterized,
e.g., by
mitochondria antibodies), other endocrine gland failure (often characterized,
e.g., by
specific tissue antibodies in some cases), vitiligo (often characterized,
e.g., by
melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and
complement in
vessel walls and/or low serum complement), post-MI (often characterized, e.g.,
by
myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by
myocardial
antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies
to IgE),
atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to
IgE), asthma
(often characterized, e.g., by IgG and IgM antibodies to IgE), and many other
inflammatory, granulomatous, degenerative, and atrophic disorders.
[0481] In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
prevented, diagnosed and/or prognosed using for example, antagonists or
agonists,
polypeptides or polynucleotides, or antibodies of the present invention. In a
specific
preferred embodiment, rheumatoid arthritis is treated, prevented, and/or
diagnosed
using polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention.
[0482] In another specific preferred embodiment, systemic Iupus erythematosus
is
treated, prevented, and/or diagnosed using polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention. In another specific
preferred
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embodiment, idiopathic thrombocytopenia purpura is treated, prevented, and/or
diagnosed using polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention.
[0483] In another specific preferred embodiment IgA nephropathy is treated,
prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention.
[0484] In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
prevented, diagnosed and/or prognosed using polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention.
[0485] In preferred embodiments, polypeptides, antibodies, polynucleotides
and/or
agonists or antagonists of the present invention are used as a
immunosuppressive
agent(s).
[0486] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in treating, preventing, prognosing,
and/or
diagnosing diseases, disorders, and/or conditions of hematopoietic cells.
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention could be used to increase differentiation and proliferation of
hematopoietic
cells, including the pluripotent stem cells, in an effort to treat or prevent
those diseases,
disorders, and/or conditions associated with a decrease in certain (or many)
types
hematopoietic cells, including but not limited to, leukopenia, neutropenia,
anemia, and
thrombocytopenia. Alternatively, Polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention could be used to increase
differentiation and proliferation of hematopoietic cells, including the
pluripotent stem
cells, in an effort to treat or prevent those diseases, disorders, and/or
conditions
associated with an increase in certain (or many) types of hematopoietic cells,
including
but not limited to, histiocytosis.
[0487] Allergic reactions and conditions, such as asthma (particularly
allergic
asthma) or other respiratory problems, may also be treated, prevented,
diagnosed
and/or prognosed using polypeptides, antibodies, or polynucleotides of the
invention,
and/or agonists or antagonists thereof. Moreover, these molecules can be used
to treat,
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prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an
antigenic
molecule, or blood group incompatibility.
[0488] Additionally, polypeptides or polynucleotides of the invention, and/or
agonists or antagonists thereof, may be used to treat, prevent, diagnose
and/or
prognose IgE-mediated allergic reactions. Such allergic reactions include, but
are not
limited to, asthma, rhinitis, and eczema. In specific embodiments,
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may
be used to modulate IgE concentrations in vitro or in vivo.
[0489] Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention have uses in the diagnosis, prognosis,
prevention,
and/or treatment of inflammatory conditions. For example, since polypeptides,
antibodies, or polynucleotides of the invention, and/or agonists or
antagonists of the
invention may inhibit the activation, proliferation and/or differentiation of
cells
involved in an inflammatory response, these molecules can be used to prevent
and/or
treat chronic and acute inflammatory conditions. Such inflammatory conditions
include, but are not limited to, for example, inflammation associated with
infection
(e.g., septic shock, sepsis, or systemic inflammatory response syndrome),
ischemia-
reperfusion injury, endotoxin lethality, complement-mediated hyperacute
rejection,
nephritis, cytokine or chemokine induced lung injury, inflammatory bowel
disease,
Crohn's disease, over production of cytokines (e.g., TNF or IL-1.),
respiratory
disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g.,
inflammatory
bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and
breast); CNS
disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke,
traumatic brain
injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's
disease); AIDS-related dementia; and prion disease); cardiovascular disorders
(e.g.,
atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary
bypass
complications); as well as many additional diseases, conditions, and disorders
that are
characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout,
trauma,
pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury,
Grave's
disease, systemic lupus erythematosus, diabetes mellitus, and allogenic
transplant
rej ection).
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[0490] Because inflammation is a fundamental defense mechanism, inflammatory
disorders can effect virtually any tissue of the body. Accordingly,
polynucleotides,
polypeptides, and antibodies of the invention, as well as agonists or
antagonists
thereof, have uses in the treatment of tissue-specific inflammatory disorders,
including,
but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis,
balanitis,
blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis,
cholecystitis, chorditis,
cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis,
encephalitis,
endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis,
gastroenteritis,
gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis,
lymphangitis,
mastitis, media otitis, meningitis, metritis, mucitis, myocarditis,
myosititis, myringitis,
nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis,
peritendonitis,
peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis,
retinitis, rhinitis,
salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis,
steatitis,
stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and
vaginitis.
[0491] In specific embodiments, polypeptides, antibodies, or polynucleotides
of
the invention, and/or agonists or antagonists thereof, are useful to diagnose,
prognose,
prevent, and/or treat organ transplant rejections and graft-versus-host
disease. Organ
rejection occurs by host immune cell destruction of the transplanted tissue
through an
immune response. Similarly, an immune response is also involved in GVHD, but,
in
this case, the foreign transplanted immune cells destroy the host tissues.
Polypeptides,
antibodies, or polynucleotides of the invention, and/or agonists or
antagonists thereof,
that inhibit . an immune response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective therapy in
preventing
organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or
polynucleotides of the invention, and/or agonists or antagonists thereof, that
inhibit an
immune response, particularly the activation, proliferation, differentiation,
or
chemotaxis of T-cells, may be an effective therapy in preventing experimental
allergic
and hyperacute xenograft rejection.
[0492] In other embodiments, polypeptides, antibodies, or polynucleotides of
the
invention, and/or agonists or antagonists thereof, are useful to diagnose,
prognose,
prevent, and/or treat immune complex diseases, including, but not limited to,
serum
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sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and
immune
complex-induced vasculitis.
[0493] Polypeptides, antibodies, polynucleotides andlor agonists or
antagonists of
the invention can be used to treat, detect, and/or prevent infectious agents.
For
example, by increasing the immune response, particularly increasing the
proliferation
activation and/or differentiation of B and/or T cells, infectious diseases may
be treated,
detected, and/or prevented. The immune response may be increased by either
enhancing an existing immune response, or by initiating a new immune response.
Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention may also directly inhibit the infectious agent (refer
to section
of application listing infectious agents, etc), without necessarily eliciting
an immune
response.
[0494] In another embodiment, polypeptides, antibodies, polynucleotides andlor
agonists or antagonists of the present invention are used as a vaccine
adjuvant that
enhances immune responsiveness to an antigen. In a specific embodiment,
polypeptides, antibodies, polynucleotides andlor agonists or antagonists of
the present
invention are used as an adjuvant to enhance tumor-specific immune responses.
[0495] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an
adjuvant to
enhance anti-viral immune responses. Anti-viral immune responses that may be
enhanced using the compositions of the invention as an adjuvant, include virus
and
virus associated diseases or symptoms described herein or otherwise known in
the art.
In specific embodiments, the compositions of the invention are used as an
adjuvant to
enhance an immune response to a virus, disease, or symptom selected from the
group
consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis
B). In
another pecific embodiment, the compositions of the invention are used as an
adjuvant to enhance an immune response to a virus, disease, or symptom
selected from
the group consisting of: HIV/ATDS, respiratory syncytial virus, Dengue,
rotavirus,
Japanese B encephalitis, influenza A and B, parainfluenza, measles,
cytomegalovirus,
rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow
fever.
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[0496] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an
adjuvant to
enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-
fungal
immune responses that may be enhanced using the compositions of the invention
as an
adjuvant, include bacteria or fungus and bacteria or fungus associated
diseases or
symptoms described herein or otherwise known in the art. In specific
embodiments,
the compositions of the invention are used as an adjuvant to enhance an immune
response to a bacteria or fungus, disease, or symptom selected from the group
consisting of: tetanus, Diphtheria, botulism, and meningitis type B.
[0497] In another specific embodiment, the compositions of the invention are
used
as an adjuvant to enhance an immune response to a bacteria or fungus, disease,
or
symptom selected from the group consisting of: Vibf°io
cholef°ae, Mycobactef°ium
leprae, Salmonella typhi, Salmoyaella pa~atyphi, Meisseria meningitidis,
Stj°eptococcus
pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Esche~ichia
coli,
Enterohemorrhagic E. coli, and Bo~relia bu~gdorfer~i.
[0498] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an
adjuvant to
enhance anti-parasitic immune responses. Anti-parasitic immune responses that
may
be enhanced using the compositions of the invention as an adjuvant, include
parasite
and parasite associated diseases or symptoms described herein or otherwise
known in
the art. In specific embodiments, the compositions of the invention are used
as an
adjuvant to enhance an immune response to a parasite. In another specific
embodiment, the compositions of the invention are used as an adjuvant to
enhance an
immune response to Plasmodium (malaria) or Leishmania.
[0499] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention may also be employed
to treat
infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary
fibrosis;
for example, by preventing the recruitment and activation of mononuclear
phagocytes.
[0500] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an antigen
for the
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generation of antibodies to inhibit or enhance immune mediated responses
against
polypeptides of the invention.
[0501] In one embodiment, polypeptides, antibodies, polynucleotides and/or
agonists or antagonists of the present invention are administered to an animal
(e.g.,
mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel,
goat, horse,
cow, sheep, dog, cat, non-human primate, and human, most preferably human) to
boost the immune system to produce increased quantities of one or more
antibodies
(e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production
and
immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to
increase an
immune response.
[0502] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a
stimulator of B
cell responsiveness to pathogens.
[0503] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an
activator of T
cells.
[0504] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
that
elevates the immune status of an individual prior to their receipt of
immunosuppressive therapies.
[0505] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to induce
higher affinity antibodies.
[0506] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to increase
serum immunoglobulin concentrations.
[0507] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to
accelerate recovery of immunocompromised individuals.
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(0508] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to boost
immunoresponsiveness among aged populations and/or neonates.
[0509] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an immune
system
enhancer prior to, during, or after bone marrow transplant and/or other
transplants
(e.g., allogeneic or xenogeneic organ transplantation). With respect to
transplantation,
compositions of the invention may be administered prior to, concomitant with,
and/or
after transplantation. In a specific embodiment, compositions of the invention
are
administered after transplantation, prior to the beginning of recovery of T-
cell
populations. In another specific embodiment, compositions of the invention are
first
administered after transplantation after the beginning of recovery of T cell
populations,
but prior to full recovery of B cell populations.
[0510] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to boost
immunoresponsiveness among individuals having an acquired loss of B cell
function.
Conditions resulting in an acquired loss of B cell function that may be
ameliorated or
treated by administering the polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists thereof, include, but are not limited to, HIV Infection, AIDS,
bone
marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
[0511] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to boost
immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated
or
treated by administering the polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists thereof, include, but are not limited to, recovery from viral
infections
(e.g., influenza), conditions associated with malnutrition, recovery from
infectious
mononucleosis, or conditions associated with stress, recovery from measles,
recovery
from blood transfusion, and recovery from surgery.
[0512] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a
regulator of
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antigen presentation by monocytes, dendritic cells, and/or B-cells. In one
embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention enhance antigen presentation or antagonizes antigen
presentation in vitro or in vivo. Moreover, in related embodiments, said
enhancement
or antagonism of antigen presentation may be useful as an anti-tumor treatment
or to
modulate the immune system.
[0513] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to direct an
individual's immune system towards development of a humoral response (i.e.
TH2) as
opposed to a THl cellular response.
[0514] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a means to
induce
tumor proliferation and thus make it more susceptible to anti-neoplastic
agents. For
example, multiple myeloma is a slowly dividing disease and is thus refractory
to
virtually all anti-neoplastic regimens. If these cells were forced to
proliferate more
rapidly.their susceptibility profile would likely change.
[0515] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a
stimulator of B
cell production in pathologies such as AIDS, chronic lymphocyte disorder
and/or
Common Variable Immunodificiency.
[0516] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a therapy
for
generation and/or regeneration of lymphoid tissues following surgery, trauma
or
genetic defect. In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present invention are
used in the
pretreatment of bone marrow samples prior to transplant.
[0517] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a gene-
based
therapy for genetically inherited disorders resulting in immuno-
incompetence/immunodeficiency such as observed among SCID patients.
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[0518] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a means of
activating monocytes/macrophages to defend against parasitic diseases that
effect
monocytes such as Leishmania.
[0519] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a means of
regulating secreted cytokines that are elicited by polypeptides of the
invention.
[0520] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists or antagonists of the present invention are used in one or more of
the
applications decribed herein, as they may apply to veterinary medicine.
[0521] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a means of
blocking
various aspects of immune responses to foreign agents or self. Examples of
diseases
or conditions in which blocking of certain aspects of immune responses may be
desired include autoimmune disorders such as lupus, and arthritis, as well as
immunoresponsiveness to skin allergies, inflammation, bowel disease, injury
and
diseases/disorders associated with pathogens.
[0522] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a therapy
for
preventing the B cell proliferation and Ig secretion associated with
autoimmune
diseases such as idiopathic thrombocytopenic purpura, systemic lupus
erythematosus
and multiple sclerosis.
[0523] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a
inhibitor of B
and/or T cell migration in endothelial cells. This activity disrupts tissue
architecture or
cognate responses and is useful, for example in disrupting immune responses,
and
blocking sepsis.
[0524] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a therapy
for chronic
hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of
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undetermined significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonal gammopathies, and plasmacytomas.
[0525] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention may be employed for
instance
to inhibit polypeptide chemotaxis and activation of macrophages and their
precursors,
and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g.,
activated
and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and
chronic
inflammatory and infective diseases. Examples of autoimmune diseases are
described
herein and include multiple sclerosis, and insulin-dependent diabetes.
[0526] The polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the present invention may also be employed to treat idiopathic
hyper-
eosinophilic syndrome by, for example, preventing eosinophil production and
migration.
[0527] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used to enhance or
inhibit
complement mediated cell lysis.
[0528] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used to enhance or
inhibit
antibody dependent cellular cytotoxicity.
[0529] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention may also be employed
for
treating atherosclerosis, for example, by preventing monocyte infiltration in
the artery
wall.
[0530] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention may be employed to
treat adult
respiratory distress syndrome CARDS).
[0531] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention may be useful for
stimulating
wound and tissue repair, stimulating angiogenesis, and/or stimulating the
repair of
vascular or lymphatic diseases or disorders. Additionally, agonists and
antagonists of
the invention may be used to stimulate the regeneration of mucosal surfaces.
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[0532] In a specific embodiment, polynucleotides or polypeptides, and/or
agonists
thereof are used to diagnose, prognose, treat, and/or prevent a disorder
characterized
by primary or acquired immunodeficiency, deficient serum immunoglobulin
production, recurrent infections, and/or immune system dysfunction. Moreover,
polynucleotides or polypeptides, and/or agonists thereof may be used to treat
or
prevent infections of the joints, bones, skin, and/or parotid glands, blood-
borne
infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),
autoimmune
diseases (e.g., those disclosed herein), inflammatory disorders, and
malignancies,
and/or any disease or disorder or condition associated with these infections,
diseases,
disorders and/or malignancies) including, but not limited to, CVID, other
primary
immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis
media,
conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe
herpes
zoster), and/or pneumocystis carnii. Other diseases and disorders that may be
prevented, diagnosed, prognosed, and/or treated with polynucleotides or
polypeptides,
and/or agonists of the present invention include, but are not limited to, HIV
infection,
HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia,
thrombocytopenia, and hemoglobinuria.
[0533] In another embodiment, polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention are used to treat, and/or
diagnose an
individual having common variable immunodeficiency disease ("CVID"; also known
as "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of this disease.
[0534] In a specific embodiment, polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be used to diagnose,
prognose,
prevent, and/or treat cancers or neoplasms including immune cell or immune
tissue-
related cancers or neoplasms. Examples of cancers or neoplasms that may be
prevented, diagnosed, or treated by polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention include, but are not limited
to, acute
myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-
Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte
leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed
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diseases, and/or diseases and disorders described in the section entitled
"Hyperproliferative Disorders" elsewhere herein.
[0535] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a therapy
for
decreasing cellular proliferation of Large B-cell Lymphomas.
[0536] In another specific embodiment, polypeptides, antibodies,
polynucleotides
and/or agonists or antagonists of the present invention are used as a means of
decreasing the involvement of B cells and Ig associated with Chronic
Myelogenous
Leukemia.
[0537] In specific embodiments, the compositions of the invention are used as
an
agent to boost immunoresponsiveness among B cell immunodeficient individuals,
such
as, for example, an individual who has undergone a partial or complete
splenectomy.
[0538] Antagonists of the invention include, for example, binding and/or
inhibitory
antibodies, antisense nucleic acids, ribozymes or soluble forms of the
polypeptides of
the present invention (e.g., Fc fusion protein; see, e.g., Example 9).
Agonists of the
invention include, for example, binding or stimulatory antibodies, and soluble
forms of
the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9).
Polypeptides,
antibodies, polynucleotides and/or agonists or antagonists of the present
invention may
be employed in a composition with a pharmaceutically acceptable carrier, e.g.,
as
described herein.
[0539] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists or antagonists of the present invention are administered to an animal
(including, but not limited to, those listed above, and also including
transgenic
animals) incapable of producing functional endogenous antibody molecules or
having
an otherwise compromised endogenous immune system, but which is capable of
producing human immunoglobulin molecules by means of a reconstituted or
partially
reconstituted immune system from another animal (see, e.g., published PCT
Application Nos. W098124893, WO/9634096, WO/9633735, and WO/9110741).
Administration of polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the present invention to such animals is useful for the
generation of
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monoclonal antibodies against the polypeptides, antibodies, polynucleotides
and/or
agonists or antagonists of the present invention.
Blood-Related Disorders
[0540] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be used to modulate hemostatic (the
stopping
of bleeding) or thrombolytic (clot dissolving) activity. For example, by
increasing
hemostatic or thrombolytic activity, polynucleotides or polypeptides, and/or
agonists
or antagonists of the present invention could be used to treat or prevent
blood
coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia,
factor
deficiencies, hemophilia), blood platelet diseases, disorders, and/or
conditions (e.g.,
thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention that can decrease hemostatic or thrombolytic activity
could be
used to inhibit or dissolve clotting. These molecules could be important in
the
treatment or prevention of heart attacks (infarction), strokes, or scarring.
[0541] In specific embodiments, the polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention may be used to
prevent,
diagnose, prognose, and/or treat thrombosis, arterial thrombosis, venous
thrombosis,
thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction,
transient ischemic attaclc, unstable angina. ~ In specific embodiments, the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention may be used for the prevention of occulsion of saphenous grafts, for
reducing the risk of periprocedural thrombosis as might accompany angioplasty
procedures, for reducing the risk of stroke in patients with atrial
fibrillation including
nonrheumatic atrial fibrillation, for reducing the risk of embolism associated
with
mechanical heart valves and or mitral valves disease. Other uses for the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention, include, but are not limited to, the prevention of occlusions in
extrcorporeal
devices (e.g., intravascular canulas, vascular access shunts in hemodialysis
patients,
hemodialysis machines, and cardiopulmonary bypass machines).
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[0542] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
prevent, diagnose, prognose, and/or treat diseases and disorders of the blood
and/or
blood forming organs associated with the tissues) in which the polypeptide of
the
invention is expressed, including one, two, three, four, five, or more tissues
disclosed
in Table 1A, column 7 (Tissue Distribution Library Code).
[0543] The polynucleotides, polypeptides, antibodies, and/or agonists 'or
antagonists of the present invention may be used to modulate hematopoietic
activity
(the formation of blood cells). For example, the polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention may be
used to
increase the quantity of all or subsets of blood cells, such as, for example,
erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils,
eosinophils,
neutrophils, mast cells, macrophages) and platelets. The ability to decrease
the
quantity of blood cells or subsets of blood cells may be useful in the
prevention,
detection, diagnosis and/or treatment of anemias and leulcopenias described
below.
Alternatively, the polynucleotides, polypeptides, antibodies, and/or agonists
or
antagonists of the present invention may be used to decrease the quantity of
all or
subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or
T cells),
myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells,
macrophages) and
platelets.. The ability to decrease the quantity of blood cells or subsets of
blood cells
may be useful in the prevention, detection, diagnosis and/or treatment of
leukocytoses,
such as, for example eosinophilia.
[0544] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be used to prevent, treat, or
diagnose blood
dyscrasia.
[0545] Anemias are conditions in which the number of red blood cells or amount
of hemoglobin (the protein that carries oxygen) in them is below normal.
Anemia may
be caused by excessive bleeding, decreased red blood cell production, or
increased red
blood cell destruction (hemolysis). The polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention may be useful in
treating,
preventing, and/or diagnosing anemias. Anemias that may be treated prevented
or
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diagnosed by the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention include iron deficiency anemia,
hypochromic
anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia,
idiopathic
acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g.,
pernicious
anemia, (vitamin B 12 deficiency) and folic acid deficiency anemia), aplastic
anemia,
hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic
hemolytic
anemia, and paroxysmal nocturnal hemoglobinuria). The polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may
be useful in treating, preventing, and/or diagnosing anemias associated with
diseases
including but not limited to, anemias associated with systemic lupus
erythematosus,
cancers, lymphomas, chronic renal disease, and enlarged spleens. The
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may
be useful in treating, preventing, and/or diagnosing anemias arising from drug
treatments such as anemias associated with methyldopa, dapsone, and/or
sulfadrugs.
Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists
or
antagonists of the present invention may be useful in treating, preventing,
and/or
diagnosing anemias associated with abnormal red blood cell architecture
including but
not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-
phosphate
dehydrogenase deficiency, and sickle cell anemia.
[0546] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be useful in treating, preventing,
and/or
diagnosing hemoglobin abnormalities, (e.g., those associated with sickle cell
anemia,
hemoglobin C disease, hemoglobin S-C disease, and hemoglobin E disease).
Additionally, the polynucleotides, polypeptides, antibodies, andlor agonists
or
antagonists of the present invention may be useful in diagnosing, prognosing,
preventing, and/or treating thalassemias, including, but not limited to major
and minor
forms of alpha-thalassemia and beta-thalassemia.
[0547] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention may be useful in
diagnosing,
prognosing, preventing, and/or treating bleeding disorders including, but not
limited
to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and
thrombotic
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thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet
disorders
(e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak
syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier
syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or
Factor
VII deficiency and Christmas disease or Factor IX deficiency, Hereditary
Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic
purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
[0548] The effect of the polynucleotides, polypeptides, antibodies, and/or
agonists
or antagonists of the present invention on the clotting time of blood may be
monitored
using any of the clotting tests lcnown in the art including, but not limited
to, whole
blood partial thromboplastin time (PTT), the activated partial thromboplastin
time
(aPTT), the activated clotting time (ACT), the recalcified activated clotting
time, or the
Lee-White Clotting time.
[0549] Several diseases and a variety of drugs can cause platelet dysfunction.
Thus, in a specific embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing, preventing, and/or treating acquired platelet dysfunction such as
platelet
dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis
of
the liver, and systemic lupus erythematosus as well as platelet dysfunction
associated
with drug treatments, including treatment with aspirin, ticlopidine,
nonsteroidal anti-
inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in
high doses.
[0550] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention may be useful in
diagnosing,
prognosing, preventing, and/or treating diseases and disorders characterized
by or
associated with increased or decreased numbers of white blood cells.
Leukopenia
occurs when the number of white blood cells decreases below normal.
Leukopenias
include, but are not limited to, neutropenia and lymphocytopenia. An increase
in the
number of white blood cells compared to normal is known as leukocytosis. The
body
generates increased numbers of white blood cells during infection. Thus,
leukocytosis
may simply be a normal physiological parameter that reflects infection.
Alternatively,
leukocytosis may be an indicator of injury or other disease such as cancer.
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Leokocytoses, include but are not limited to, eosinophilia, and accumulations
of
macrophages. In specific embodiments, the polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention may be useful in
diagnosing,
prognosing, preventing, and/or treating leukopenia. In other specific
embodiments, the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention may be useful in diagnosing, prognosing, preventing, and/or treating
leulcocytosis
[0551] Leukopenia may be a generalized decreased in all types of white blood
cells, or may be a specific depletion of particular types of white blood
cells. Thus, in
specific embodiments, the polynucleotides, polypeptides, antibodies, and/or
agonists
or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating decreases in neutrophil numbers, known as
neutropenia.
Neutropenias that may be diagnosed, prognosed, prevented, and/or treated by
the
polynucleotides, polypeptides, antibodies, andlor agonists or antagonists of
the present
invention include, but are not limited to, infantile genetic agranulocytosis,
familial
neutropenia, cyclic neutropenia, neutropenias resulting from or associated
with dietary
deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency),
neutropenias
resulting from or associated with drug treatments (e.g., antibiotic regimens
such as
penicillin treatment, sulfonamide treatment, anticoagulant treatment,
anticonvulsant
drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias
resulting from
increased neutrophil destruction that may occur in association with some
bacterial or
viral infections, allergic disorders, autoimmune diseases, conditions in which
an
individual has an enlarged spleen (e.g., Felty syndrome, malaria and
sarcoidosis), and
some drug treatment regimens.
[0552] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be useful in diagnosing, prognosing,
preventing, and/or treating lymphocytopenias (decreased numbers of B and/or T
lymphocytes), including, but not limited lymphocytopenias resulting from or
associated with stress, drug treatments (e.g., drug treatment with
corticosteroids,
cancer chemotherapies, and/or radiation therapies), AIDS infection and/or
other
diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus
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erythematosus, chronic infections, some viral infections and/or hereditary
disorders
(e.g., DiGeorge syndrome, Wiskott-Aldricli Syndome, severe combined
immunodeficiency, ataxia telangiectsia).
[0553] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be useful in diagnosing, prognosing,
preventing, and/or treating diseases and disorders associated with macrophage
numbers and/or macrophage function including, but not limited to, Gaucher's
disease,
Niemann-Piclc disease, Letterer-Siwe disease and Hand-Schuller-Christian
disease.
[0554] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention may be useful in
diagnosing,
prognosing, preventing, and/or treating diseases and disorders associated with
eosinophil numbers and/or eosinophil function including, but not limited to,
idiopathic
hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-
Christian disease.
[0555] In yet another embodiment, the polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention may be useful in
diagnosing,
prognosing, preventing, and/or treating leukemias and lymphomas including, but
not
limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid
(myelocytic, myelogenous, myeloblastic, or myelomonocytic) leulcemia, chronic
lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary
syndrome, and
Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or
granulocytic)
leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and
mycosis fungoides.
[0556] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing, preventing, and/or treating diseases and disorders of plasma .
cells
including, but not limited to, plasma cell dyscrasias, monoclonal
gammaopathies,
monoclonal gammopathies of undetermined significance, multiple myeloma,
macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and
Raynaud's phenomenon.
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[0557] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in treating,
preventing,
and/or diagnosing myeloproliferative disorders, including but not limited to,
polycythemia vera, relative polycythemia, secondary polycythemia,
myelofibrosis,
acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including
both
primary and seconday thrombocythemia) and chronic myelocytic leukemia.
[0558] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as a treatment
prior to
surgery, to increase blood cell production.
[0559] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
enhance
the migration, phagocytosis, superoxide production, antibody dependent
cellular
cytotoxicity of neutrophils, eosionophils and macrophages.
[0560] ~ In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
increase
the number of stem cells in circulation prior to stem cells pheresis. In
another specific
embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention may be useful as an agent to increase the
number
of stem cells in circulation prior to platelet pheresis.
(0561] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
increase
cytolcine pr oduction.
[0562] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in preventing,
diagnosing, and/or treating primary hematopoietic disorders.
Hyperproliferative Disorders
[0563] Pancreatic associated polynucleotides or polypeptides, or agonists or
antagonists thereof, can be used to treat, prevent, diagnose and/or prognose
hyperproliferative diseases, disorders, and/or conditions, including
neoplasms.
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[0564] In a specific embodiment, pancreatic associated polynucleotides or
polypeptides, or agonists or antagonists thereof, can be used to treat,
prevent, and/or
diagnose hyperproliferative diseases, disorders, and/or conditions of the
pancreas.
[0565] In a preferred embodiment, pancreatic associated polynucleotides or
polypeptides, or agonists or antagonists thereof, can be used to treat,
prevent, and/or
diagnose pancreatic neoplasms.
[0566] Pancreatic associated polynucleotides or polypeptides, or agonists or
antagonists of the invention, may inhibit the proliferation of the disorder
through direct
or indirect interactions. Alternatively, pancreatic associated polynucleotides
or
polypeptides, or agonists or antagonists thereof, may proliferate other cells,
which can
inhibit the hyperproliferative disorder.
[0567] For example, by increasing an immune response, particularly increasing
antigenic qualities of the hyperproliferative disorder or by proliferating,
differentiating,
or mobilizing T-cells, hyperproliferative diseases, disorders, and/or
conditions can be
treated, prevented, and/or diagnosed. This immune response may be increased by
either enhancing an existing immune response, or by initiating a new immune
response. Alternatively, decreasing an immune response may also be a method of
treating, preventing, and/or diagnosing hyperproliferative diseases,
disorders, and/or
conditions, such as a chemotherapeutic agent.
[0568] Examples of hyperproliferative diseases, disorders, and/or conditions
that
can be treated, prevented, and/or diagnosed by pancreatic associated
polynucleotides
or polypeptides, or agonists or antagonists thereof, include, but are not
limited to
neoplasms located in the: prostate, colon, abdomen, bone, breast, digestive
system,
liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary, testicles,
ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral),
lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.
[0569] Similarly, other hyperproliferative disorders can also be treated or
detected
by polynucleotides or polypeptides, or agonists or antagonists of the present
invention.
Examples of such hyperproliferative disorders include, but are not limited to:
Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult
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(Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute
Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's
Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related
Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct
Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast
Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System
(Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral
Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer,
Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood
Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial
Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic
Leukemia, Childhood Medulloblastoma, Childhood Non-Hodglcin's Lymphoma,
Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors,
Childhood
Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue
Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous
T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,
Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related
Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal
Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast
Cancer,
Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal
Carcinoid
Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic
Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer,
Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia,
Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell
Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer,
Laryngeal
Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,
Lymphoproliferative
Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma,
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Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult
Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,
Metastatic Squamous Neclc Cancer, Multiple Myeloma, Multiple Myeloma/Plasma
Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid
Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus
Cancer,
Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During
Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary
Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-!Malignant
Fibrous
Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant
Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell
Tumor,
Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,
Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor,
Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System
Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell
Cancer,
Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary
Gland
Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer,
Stomach Cancer, Supratentorial Primitive Neuroectodennal and Pineal Tumors, T-
Cell
Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer
of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer,
Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethra! Cancer,
Uterine
Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic
Glioma,
Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other
hyperproliferative disease, besides neoplasia, located in an organ system
listed above.
[0570] In another preferred embodiment, polynucleotides or polypeptides, or
agonists or antagonists of the present invention are used to diagnose,
prognose,
prevent, and/or treat premalignant conditions and to prevent progression to a
neoplastic or malignant state, including but not limited to those disorders
described
above. Such uses are indicated in conditions known or suspected of preceding
progression to neoplasia or cancer, in particular, where non-neoplastic cell
growth
consisting of hyperplasia, metaplasia, or most particularly, dysplasia has
occurred (for
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review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic
Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.)
[0571] Hyperplasia is a form of controlled cell proliferation, involving an
increase
in cell number in a tissue or organ, without significant alteration in
structure or
function. Hyperplastic disorders which can be diagnosed, prognosed, prevented,
and/or
treated with compositions of the invention (including polynucleotides,
polypeptides,
agonists or antagonists) include, but are not limited to, angiofollicular
mediastinal
lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical
melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node
hyperplasia,
cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous
hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture
hyperplasia,
ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal
epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous
hyperplasia,
inflammatory papillary hyperplasia, intravascular papillary endothelial
hyperplasia,
nodular hyperplasia of prostate, nodular regenerative hyperplasia,
pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous
hyperplasia.
[0572] Metaplasia is a form of controlled cell growth in which one type of
adult or
fully differentiated cell substitutes for another type of adult cell.
Metaplastic disorders
which can be diagnosed, prognosed, prevented, and/or treated with compositions
of the
invention (including polynucleotides, polypeptides, agonists or antagonists)
include,
but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia,
atypical
metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia,
epithelial
metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic
ossification,
metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary
myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and
symptomatic myeloid metaplasia.
[0573] Dysplasia is frequently a forerunner of cancer, and is found mainly in
the
epithelia; it is the most disorderly form of non-neoplastic cell growth,
involving a loss
in individual cell uniformity and in the architectural orientation of cells.
Dysplastic
cells often have abnormally large, deeply stained nuclei, and exhibit
pleomorphism.
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Dysplasia characteristically occurs where there exists chronic irritation or
inflammation. Dysplastic disorders which can be diagnosed, prognosed,
prevented,
and/or treated with compositions of the invention (including polynucleotides,
polypeptides, agonists or antagonists) include, but are not limited to,
anhidrotic
ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia,
cervical
dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital
ectodermal
dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial
dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia,
enamel
dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial
dysplasia,
faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial
white folded
dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia,
hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,
hypohidrotic
ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia,
mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,
monostotic
fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral
dysplasia, odontogenic dysplasia, ophthahnomandibulomelic dysplasia,
periapical
cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic
spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,
spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
[0574] Additional pre-neoplastic disorders which can be diagnosed, prognosed,
prevented, and/or treated with compositions of the invention (including
polynucleotides, polypeptides, agonists or antagonists) include, but are not
limited to,
benign dysproliferative disorders (e.g., benign tumors, fibrocystic
conditions, tissue
hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia),
leukoplakia,
keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar
keratosis.
[0575] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose disorders associated with the tissues) in which the
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polypeptide of the invention is expressed, including one, two, three, four,
five, or more
tissues disclosed in Table 1A, 7 (Tissue Distribution Library Code).
[0576] In another embodiment, polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention conjugated to a toxin or a
radioactive
isotope, as described herein, may be used to treat cancers and neoplasms,
including,
but not limited to those described herein. In a further preferred embodiment,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention conjugated to a toxin or a radioactive isotope, as described herein,
may be
used to treat acute myelogenous leukemia.
[0577] Additionally, polynucleotides, polypeptides, and/or agonists or
antagonists
of the invention may affect apoptosis, and therefore, would be useful in
treating a
number of diseases associated with increased cell survival or the inhibition
of
apoptosis. For example, diseases associated with increased cell survival or
the
inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or
treated
by polynucleotides, polypeptides, and/or agonists or antagonists of the
invention,
include cancers (such as follicular lymphomas, carcinomas with p53 mutations,
and
hormone-dependent tumors, including, but not limited to colon cancer, cardiac
tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer,
intestinal
cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's
disease, polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and rheumatoid arthritis) and viral infections (such as
herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host disease,
acute graft
rejection, and chronic graft rejection.
[0578] In preferred embodiments, polynucleotides, polypeptides, and/or
agonists
or antagonists of the invention are used to inhibit growth, progression,
and/or
metastasis of cancers, in particular those listed above.
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[0579] Additional diseases or conditions associated with increased cell
survival
that could be diagnosed, prognosed, prevented, and/or treated by
polynucleotides,
polypeptides, and/or agonists or antagonists of the invention, include, but
are not
limited to, progression, and/or metastases of malignancies and related
disorders such
as leulcemia (including acute leukemias (e.g., acute lymphocytic leukemia,
acute
myelocytic leulcemia (including myeloblastic, promyelocytic, myelomonocytic,
monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic
myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera,
lymphomas (e.g., Hodgkin's disease and non-Hodglcin's disease), multiple
myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors
including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous
cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
sebaceous
gland ~ carcinoma, papillary carcinoma, papillary adenocarcinomas,
cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell
carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma,
small cell
lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma,
acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and
retinoblastoma.
[0580] Diseases associated with increased apoptosis that could be diagnosed,
prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or
agonists
or antagonists of the invention, include AIDS; neurodegenerative disorders
(such as
Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,
retinitis
pigmentosa, cerebellar degeneration and brain tumor or prior associated
disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,
Hashimoto's
thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic
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lupus erythematosus and immune-related glomerulonephritis and rheumatoid
arthritis)
myelodysplastic syndromes (such as aplastic anemia), graft v. host disease,
ischemic
injury (such as that caused by myocardial infarction, stroke and reperfusion
injury),
liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion
injury, cholestosis
(bile duct injury) and liver cancer); toxin-induced liver disease (such as
that caused by
alcohol), septic shock, cachexia and anorexia.
[0581] Hyperproliferative diseases and/or disorders that could be diagnosed,
prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or
agonists
or antagonists of the invention, include, but are not limited to, neoplasms
located in the
liver, abdomen, bone, breast, digestive system, pancreas, peritoneum,
endocrine glands
(adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye,
head and neck,
nervous system (central and peripheral), lymphatic system, pelvis, skin, soft
tissue,
spleen, thorax, and urogenital tract.
(0582] Similarly, other hyperproliferative disorders can also be diagnosed,
prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or
agonists
or antagonists of the invention. Examples of such hyperproliferative disorders
include,
but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's
macroglobulinemia, Gaucher's Disease, histiocytosis, and any other
hyperproliferative
disease, besides neoplasia, located in an organ system listed above.
[0583] One preferred embodiment utilizes polynucleotides of the present
invention
to inhibit aberrant cellular division, by gene therapy using the present
invention, and/or
protein fusions or fragments thereof.
[0584] Thus, the present invention provides a method for treating cell
proliferative
diseases, disorders, and/or conditions by inserting into an abnormally
proliferating cell
a polynucleotide of the present invention, wherein said polynucleotide
represses said
cell proliferation, disease, disorder, and/or condition.
[0585] In a preferred embodiment, the present invention provides a method for
treating cell proliferative diseases, disorders and/or conditions of the
pancreas by
inserting into a cell, a polynucleotide of the present invention, wherein said
polynucleotide represses said cell proliferation, disease andlor disorder.
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[0586] Another embodiment of the present invention provides a method of
treating
cell-proliferative diseases, disorders, and/or conditions in individuals
comprising
administration of one or more active gene copies of the present invention to
an
abnormally proliferating cell or cells. In a preferred embodiment,
polynucleotides of
the present invention is a DNA construct comprising a recombinant expression
vector
effective in expressing a DNA sequence encoding said polynucleotides. In
another
preferred embodiment of the present invention, the DNA construct encoding the
polynucleotides of the present invention is inserted into cells to be treated
utilizing a
retrovirus, or more preferably an adenoviral vector (see, e.g., G J. Nabel,
et. al., PNAS
96: 324-326 (1999), which is hereby incorporated by reference). In a most
preferred
embodiment, the viral vector is defective and will not transform non-
proliferating
cells, only proliferating cells. Moreover, in a preferred embodiment, the
polynucleotides of the present invention inserted into proliferating cells
either alone, or
in combination with or fused to other polynucleotides, can then be modulated
via an
external stimulus (i.e., magnetic, specific small molecule, chemical, or drug
administration, etc.), which acts upon the promoter upstream of said
polynucleotides to
induce expression of the encoded protein product. As such the beneficial
therapeutic
affect of the present invention may be expressly modulated (i.e., to increase,
decrease,
or inhibit expression of the present invention) based upon said external
stimulus.
[0587] Polynucleotides of the present invention may be useful in repressing
expression of oncogenic genes or antigens. By "repressing expression of the
oncogenic genes " is intended the suppression of the transcription of the
gene, the
degradation of the gene transcript (pre-message RNA), the inhibition of
splicing, the
destruction of the messenger RNA, the prevention of the post-translational
modifications of the protein, the destruction of the protein, or the
inhibition of the
normal function of the protein.
[0588] For local administration to abnormally proliferating cells,
polynucleotides
of the present invention may be administered by any method known to those of
skill in
the art including, but not limited to transfection, electroporation,
microinjection of
cells, or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any
other method described throughout the specification. The polynucleotide of the
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present invention may be delivered by known gene delivery systems such as, but
not
limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature
320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014),
vaccinia virus
system (Chalcrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient
DNA
delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled
in the
art. These references are exemplary only and are hereby incorporated by
reference. In
order to specifically deliver or transfect cells which are abnormally
proliferating and
spare non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as
described in the art and elsewhere herein) delivery system known to those of
skill in
the art. Since host DNA replication is required for retroviral DNA to
integrate and the
retrovirus will be unable to self replicate due to the lack of the retrovirus
genes needed
for its life cycle. Utilizing such a retroviral delivery system for
polynucleotides of the
present invention will target said gene and constntcts to abnormally
proliferating cells
and will spare the non-dividing normal cells.
[0589] ' The polynucleotides of the present invention may be delivered
directly to
cell proliferative disorder/disease sites in internal organs, body cavities
and the like by
use of imaging devices used to guide an injecting needle directly to the
disease site.
The polynucleotides of the present invention may also be administered to
disease sites
at the time of surgical intervention.
[0590] By "cell proliferative disease" is meant any human or animal disease or
disorder, affecting any one or any combination of organs, cavities, or body
parts,
which is characterized by single or multiple local abnormal proliferations of
cells,
groups of cells, or tissues, whether benign or malignant.
[0591] Any amount of the polynucleotides of the present invention may be
administered as long as it has a biologically inhibiting effect on the
proliferation of the
treated cells. Moreover, it is possible to administer more than one of the
polynucleotide of the present invention simultaneously to the same site. By
"biologically inhibiting" is meant partial or total growth inhibition as well
as decreases
in the rate of proliferation or growth of the cells. The biologically
inhibitory dose may
be determined by assessing the effects of the polynucleotides of the present
invention
on target malignant or abnormally proliferating cell growth in tissue culture,
tumor
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growth in animals and cell cultures, or any other method known to one of
ordinary
skill in the art.
[0592] The present invention is further directed to antibody-based therapies
which
involve administering of anti-polypeptides and anti-polynucleotide antibodies
to a
mammalian, preferably human, patient for treating one or more of the described
diseases, disorders, and/or conditions. Methods for producing anti-
polypeptides and
anti-polynucleotide antibodies polyclonal and monoclonal antibodies are
described in
detail elsewhere herein. Such antibodies may be provided in pharmaceutically
acceptable compositions as known in the art or as described herein.
[0593] A summary of the ways in which the antibodies of the present invention
may be used therapeutically includes binding polynucleotides or polypeptides
of the
present invention locally or systemically in the body or by direct
cytotoxicity of the
antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC).
Some
of these approaches are described in more detail below. Armed with the
teachings
provided herein, one of ordinary skill in the art will know how to use the
antibodies of
the present invention for diagnostic, monitoring or therapeutic purposes
without undue
experimentation.
[0594] In particular, the antibodies, fragments and derivatives of the present
invention are useful for treating a subject having or developing cell
proliferative and/or
differentiation diseases, disorders, and/or conditions as described herein.
Such
treatment comprises administering a single or multiple doses of the antibody,
or a
fragment, derivative, or a conjugate thereof.
[0595] The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors, for example, which serve to increase the number
or
activity of effector cells which interact with the antibodies.
[0596] It is preferred to use high affinity andlor potent in vivo inhibiting
and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention, fragments or regions thereof, for both immunoassays directed to and
therapy
of diseases, disorders, and/or conditions related to polynucleotides or
polypeptides,
including fragments thereof, of the present invention. Such antibodies,
fragments, or
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regions, will preferably have an affinity for polynucleotides or polypeptides,
including
fragments thereof. Preferred binding affinities include those with a
dissociation
constant or Kd less than SX10-6M, 10-6M, 5X10-7M, 10-7M, SX10-$M, 10-gM,.5X10-
9M, 10-~M, SXIO-1°M, 10-1°M, SX10-11M, 10-11M, SX10-12M, 10-12M,
SX10-13M, 10-
13M, SX10-14M, 10-14M, SX10-15M, and 10-15M.
[0597] Moreover, pancreatic antigen polypeptides of the present invention or
fragments thereof, are useful in inhibiting the angiogenesis of proliferative
cells or
tissues, either alone, as a protein fusion, or in combination with other
polypeptides
directly or indirectly, as described elsewhere herein. In a most preferred
embodiment,
said anti-angiogenesis effect may be achieved indirectly, for example, through
the
inhibition of hematopoietic, tumor-specific cells, such as tumor-associated
macrophages (see, e.g., Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53
(1998),
which is hereby incorporated by reference). Antibodies directed to
polypeptides or .
polynucleotides of the present invention may also result in inhibition of
angiogenesis
directly, or indirectly (see, e.g., Witte L, et al., Cancer Metastasis Rev.
17(2):155-61
(1998), which is hereby incorporated by reference)).
[0598) Polypeptides, including protein fusions, of the present invention, or
fragments thereof may be useful in inhibiting proliferative cells or tissues
through the
induction of apoptosis. Said polypeptides may act either directly, or
indirectly to
induce apoptosis of proliferative cells and tissues, for example in the
activation of a
death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95
(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-
related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (see, e.g.,
Schulze-
Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998), which is hereby
incorporated
by reference). Moreover, in another preferred embodiment of the present
invention,
said polypeptides may induce apoptosis through other mechanisms, such as in
the
activation of other proteins which will activate apoptosis, or through
stimulating the
expression of said proteins, either alone or in combination with small
molecule drugs
or adjuvants, such as apoptonin, galectins, thioredoxins, antiinflammatory
proteins
(See for example, Mutat. Res. 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-
33
(1998), Chem. Biol. Interact. Apr 24;111-112:23-34 (1998), J. Mo. Med.
76(6):402-
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12 (1998), Int. J. Tissue React. 20(1):3-15 (1998), which are all hereby
incorporated
by reference).
[0599] Polypeptides, including protein fusions to, or fragments thereof, of
the
present invention are useful in inhibiting the metastasis of proliferative
cells or tissues.
Inhibition may occur as a direct result of administering polypeptides, or
antibodies
directed to said polypeptides as described elsewhere herein, or indirectly,
such as
activating the expression of proteins known to inhibit metastasis, for example
alpha 4
integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is
hereby
incorporated by reference). Such therapeutic affects of the present invention
may be
achieved either alone, or in combination with small molecule drugs or
adjuvants.
[0600] In another embodiment, the invention provides a method of delivering
compositions containing the polypeptides of the invention (e.g., compositions
containing polypeptides or anti-pancreatic antigen polypeptide antibodies
associated
with heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs) to
targeted cells expressing the polypeptide of the present invention. pancreatic
antigen
polypeptides or anti-pancreatic antigen polypeptide antibodies of the
invention may be
associated with heterologous polypeptides, heterologous nucleic acids, toxins,
or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
[0601] Polypeptides, protein fusions to, or fragments thereof, of the present
invention are useful in enhancing the immunogenicity and/or antigenicity of
proliferating cells or tissues, either directly, such as would occur if the
polypeptides of
the present invention 'vaccinated' the immune response to respond to
proliferative
antigens and immunogens, or indirectly, such as in activating the expression
of
proteins known to enhance the immune response (e.g. chemokines), to said
antigens
and immunogens.
Urinary System Disorders
[0602] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention, may be used to treat, prevent, diagnose, and/or
prognose
disorders of the urinary system, including but not limited to disorders of the
renal
system, bladder, ureters, and urethra. Renal disorders include, but are not
limited to,
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kidney failure, nephritis, blood vessel disorders of kidney, metabolic and
congenital
kidney disorders, urinary disorders of the kidney, autoimmune disorders,
sclerosis and
necrosis, electrolyte imbalance, and kidney cancers.
[0603] Kidney failure diseases include, but are not limited to, acute kidney
failure,
chronic kidney failure, atheroembolic renal failure, and end-stage renal
disease.
Inflammatory diseases of the kidney include acute glomerulonephritis,
postinfectious
glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic
syndrome,
membranous glomerulonephritis, familial nephrotic syndrome,
membranoproliferative
glomerulonephritis I and II, mesangial proliferative glomerulonephritis,
chronic
glomerulonephritis, acute tubulointerstitial nephritis, chronic
tubulointerstitial
nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis,
lupus
nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal
glomerulonephritis.
[0604] Blood vessel disorders of the kidneys include, but are not limited to,
kidney
infarction, atheroembolic kidney disease, cortical necrosis, malignant
nephrosclerosis,
renal vein thrombosis, renal underperfusion, renal ischemia-reperfusion, renal
artery
embolism, and renal artery stenosis. Kidney disorders resulting form urinary
tract
problems include, but are not limited to, pyelonephritis, hydronephrosis,
urolithiasis
(renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract
infections, urinary
retention, and acute or chronic unilateral obstructive uropathy.
[0605] Metabolic and congenital disorders of the kidneys include, but are not
limited to, renal tubular acidosis, renal glycosuria, nephrogenic diabetes
insipidus,
cystinuria, Fanconi's syndrome, vitamin D-resistant rickets, Hartnup disease,
Bartter's
syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic
disease,
medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital
nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy,
nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and
membranous nephropathy, Kidney disorders resulting from an autoimmune response
include, but are not limited to, systemic lupus erythematosus (SLE),
Goodpasture
syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis.
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[0606] Sclerotic or necrotic disorders of the kidney include, but are not
limited to,
glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis
(FSGS),
necrotizing glomerulonephritis, and renal papillary necrosis. Kidneys may also
develop carcinomas, including, but not limited to, hypernephroma,
nephroblastoma,
renal cell cancer, transitional cell cancer, squamous cell cancer, and Wilm's
tumor.
[0607] Kidney disorders may also result in electrolyte imbalances, including,
but
not limited to, nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria,
hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia,
hypercalcemia, hypophosphatemia, and hyperphosphatemia.
[060x) Bladder disorders include, but are not limited to, benign prostatic
hyperplasia (BPH), interstitial cystitis (IC), prostatitis, proteinuria,
urinary tract
infections, urinary incontinence, urinary retention. Disorders of the ureters
and urethra
include, but are not limited to, acute or chronic unilateral obstructive
uropathy. The
bladder, ureters, and urethra may also develop carcinomas, including, but not
limited
to, superficial bladder canccer, invasive bladder cancer, carcinoma of the
ureter, and
urethra cancers.
[0609] Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
topical applications during surgery, aerosol delivery. Such methods are known
in the
art. Polypeptides may be administered as part of a Therapeutic, described in
more
detail below. Methods of delivering polynucleotides are described in more
detail
herein.
Cardiovascular Disorders
[0610] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or prognose
cardiovascular
disorders, including, but not limited to, peripheral artery disease, such as
limb
ischemia.
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[0611] Cardiovascular disorders include cardiovascular abnormalities, such as
arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous
malformations,
congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital
heart
defects include aortic coarctation, cor triatriatum, coronary vessel
anomalies,
crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly,
Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of
fallot,
transposition of great vessels, double outlet right ventricle, tricuspid
atresia, persistent
truncus arteriosus, total anomalous pulmonary venous connection, hypoplastic
left
heart syndrome, and heart septal defects, such as aortopulmonary septal
defect,
endocardial cushion defects, Lutembacher's Syndrome, atrioventricular canal
defect,
trilogy of Fallot, ventricular heart septal defects.
[0612] Cardiovascular disorders also include heart disease, such as
arrhythmias,
carcinoid heart disease, high cardiac output, low cardiac output, cardiac
tamponade,
endocarditis (including bacterial), heart aneurysm, cardiac arrest, sudden
cardiac death,
congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea,
cardiac
edema, heart hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy,
right ventricular hypertrophy, post-infarction heart rupture, ventricular
septal rupture,
heart valve diseases, myocardial diseases, myocardial ischemia, pericardial
effusion,
pericarditis (including constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease,
ventricular dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar
Syndrome, diastolic dysfunction, enlarged heart, heart block, J-curve
phenomenon,
rheumatic heart disease, Marfan syndrome, cardiovascular syphilis, and
cardiovascular
tuberculosis.
[0613] Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial
flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial
block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-
type pre-excitation syndrome, Wolff Parkinson-White syndrome, sick sinus
syndrome,
tachycardias, and. ventricular fibrillation. Tachycardias include paroxysmal
tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm,
atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,
ectopic functional
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tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades
de
Pointes, and ventricular tachycardia.
[0614] Heart valve disease include aortic valve insufficiency, aortic valve
stenosis,
heart murmurs, aortic valve prolapse, mural valve prolapse, tricuspid valve
prolapse,
mural valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary
valve
insuff ciency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve
insuff ciency,
tricuspid valve stenosis; and bicuspid aortic valve.
[0615] Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
pulmonary
subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy,
endocardial
fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, Barth syndrome,
myocardial reperfusion injury, and myocarditis.
[0616] Myocardial ischemias include coronary disease, such as angina pectoris,
Prinzmetal's angina, unstable angina, coronary aneurysm, coronary
arteriosclerosis,
coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial
stunning.
[0617] Cardiovascular diseases also include vascular diseases such as
aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-
Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic
diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive diseases,
arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders,
diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia,
hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension
(shock),
ischemia, peripheral vascular diseases, phlebitis, superficial phlebitis,
pulmonary
veno-occlusive disease, chronic obstructive pulmonary disease, Buerger's
disease,
Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome,
superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary
hemorrhagic telangiectasia, deep vein thrombosis, varicocele, varicose veins,
varicose
ulcer,.vasculitis, and venous insufficiency.
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[0618] Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
[0619] Arterial occlusive diseases include arteriosclerosis,
arteriolosclerosis,
atherosclerosis, intermittent claudication, carotid stenosis, fibromuscular
dysplasias,
mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction,
retinal
artery occlusion, and thromboangiitis obliterans.
[0620] Cerebrovascular disorders include carotid artery diseases, cerebral
amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformation, cerebral artery diseases, cerebral embolism and
thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's
syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid
hemorrhage, cerebral infarction, cerebral ischemia (including transient),
subclavian
steal syndrome, periventricular leukomalacia, vascular headache, cluster
headache,
migraine, and vertebrobasilar insufficiency.
[0621] Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, deep vein thrombosis, retinal vein occlusion, carotid artery
thrombosis,
sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
[0622] Ischemia includes cerebral ischemia, ischemic colitis, silent ischemia,
compartment syndromes, anterior compartment syndrome, myocardial ischemia,
reperfusion injuries, and peripheral limb ischemia. Vasculitis includes
aortitis,
arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node
syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-
Henoch
purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
[0623] Cardiovascular diseases can also occur due to electrolyte imbalances
that
include, but are not limited to hyponatremia, hypernatremia, hypokalemia,
hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphophatemia. Neoplasm and/or cancers of the cardiovascular system
include,
but are not limited to, myxomas, fibromas, and rhabdomyomas.
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(0624] Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
topical applications during surgery, aerosol delivery. Such methods are known
in the
art. Polypeptides may be administered as part of a Therapeutic, described in
more
detail. below. Methods of delivering polynucleotides are described in more
detail
herein.
Respiratory Disorders
[0625] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention may be used to treat, prevent, diagnose, and/or prognose diseases
and/or
disorders of the respiratory system.
[0626] Diseases and disorders of the respiratory system include, but are not
limited
to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic
rhinitis, atrophic
rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile
angiofibromas, cancer
of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's
nodules),
contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral
and bacterial),
tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis,
laryngoceles, and
throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx
cancer), lung
cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large
cell
carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia,
hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic
interstitial
pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary
aspergillosis,
asthma, Wegener's granulomatosis (granulomatous vasculitis), Goodpasture's
syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus
pneumoniae
(pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia),
Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and PseudonZas
spp.),
Mycoplasma pneumoraiae pneumonia, Hemophilus infZuenzae pneumonia, Legiohella
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pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)),
and viral
pneumonia (e.g., influenza, chickenpox (varicella).
[0627] Additional diseases and disorders of the respiratory system include,
but are
not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory
syncytial viral
infection, mumps, erythema infectiosum (fifth disease), roseola infantum,
progressive
rubella panencephalitis, german measles, and subacute sclerosing
panencephalitis),
fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis,
fungal
infections in people with severely suppressed immune systems (e.g.,
cryptococcosis,
caused by Cyyptococcus neoformahs; aspergillosis, caused by Aspergillus spp.;
candidiasis, caused by Candida; and mucormycosis)), Pneumocystis cariyaii
(pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlarnydia
spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical
pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy,
pleural
effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated
spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases
(e.g.,
asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or
acute
bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal
workers'
pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis,
and benign
pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g.,
fibrosing
alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis,
desquamative
interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X
(e.g., Letterer-
Siwe disease, Hand-Schiiller-Christian disease, eosinophilic granuloma),
idiopathic
pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis),
Acute
respiratory distress syndrome (also called, e.g., adult respiratory distress
syndrome),
edema, pulmonary embolism, bronchitis (e.g., viral, bacterial),
bronchiectasis,
atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or
Legioraella
pfieumophila), and cystic fibrosis.
Anti-An~io~enesis Activity
[0628] The naturally occurring balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
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Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which
neovascularization occurs under normal physiological conditions, such as wound
healing, organ regeneration, embryonic development, and female reproductive
processes, angiogenesis is stringently regulated and spatially and temporally
delimited.
Under conditions of pathological angiogenesis such as that characterizing
solid tumor
growth, these regulatory controls fail. Unregulated angiogenesis becomes
pathologic
and sustains progression of many neoplastic and non-neoplastic diseases. A
number of
serious diseases are dominated by abnormal neovascularization including solid
tumor
growth and metastases, arthritis, some types of eye disorders, and psoriasis.
See, e.g.,
reviews by Moses et al., Biotech. 9:630-634 (1991); Follcman et al., N. Engl.
J. Med.,
333:1757-1763 (1995); Auerbach et al., J. Micf°ovasc. Res. 29:401-411
(1985);
Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic
Press,
New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and
Folkman et al., Science 221:719-725 (1983). In a number of pathological
conditions,
the process of angiogenesis contributes to the disease state. For example,
significant
data have accumulated which suggest that the growth of solid tumors is
dependent on
angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
[0629] The present invention provides for treatment of diseases or disorders
associated with neovascularization by administration of the polynucleotides
and/or
polypeptides of the invention, as well as agonists or antagonists of the
present
invention. Malignant and metastatic conditions which can be treated with the
polynucleotides and polypeptides, or agonists or antagonists of the invention
include,
but are not limited to, malignancies, solid tumors, and cancers described
herein and
otherwise known in the art (fox a review of such disorders, see Fishman et
al.,
Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the
present
invention provides a method of treating an angiogenesis-related disease and/or
disorder, comprising administration to an individual in need thereof a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
of the
invention. For example, polynucleotides, polypeptides, antagonists and/or
agonists
may be utilized in a variety of additional methods in order to therapeutically
treat a
cancer or tumor. Cancers which may be treated with polynucleotides,
polypeptides,
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antagonists and/or agonists include, but are not limited to solid tumors,
including
prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes,
liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,
bladder,
thyroid cancer; primary tumors and metastases; melanomas; glioblastoma;
I~aposi's
sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer;
advanced
malignancies; and blood born tumors such as leukemias. For example,
polynucleotides, polypeptides, antagonists and/or agonists may be delivered
topically,
in order to treat cancers such as skin cancer, head and neck tumors, breast
tumors, and
I~aposi's sarcoma.
[0630] Within yet other aspects, polynucleotides, polypeptides, antagonists
and/or
agonists may be utilized to treat superficial forms of bladder cancer by, for
example,
intravesical administration. Polynucleotides, polypeptides, antagonists and/or
agonists
may be delivered directly into the tumor, or near the tumor site, via
injection or a
catheter. Of course, as the artisan of ordinary slcill will appreciate, the
appropriate
mode of administration will vary according to the cancer to be treated. Other
modes of
delivery are discussed herein.
[0631] Polynucleotides, polypeptides, antagonists and/or agonists may be
useful in
treating other disorders, besides cancers, which involve angiogenesis. These
disorders
include, but are not limited to: benign tumors, for example hemangiomas,
acoustic
neuromas, neurofibromas, trachomas, arid pyogenic granulomas; artheroscleric
plaques; ocular angiogenic diseases, for example, diabetic retinopathy,
retinopathy of
prematurity, macular degeneration, corneal graft rejection; neovascular
glaucoma,
retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia
(abnormal blood
vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound
healing;
endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids);
nonunion
fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis;
coronary collaterals; cerebral collaterals; arteriovenous malformations;
ischemic limb
angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia;
hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation;
Crohn's disease; and atherosclerosis.
[0632] For example, within one aspect of the present invention methods are
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provided for treating hypertrophic scars and keloids, comprising the step of
administering a polynucleotide, polypeptide, antagonist and/or agonist of the
invention
to a hypertrophic scar or keloid.
[0633] Within one embodiment of the present invention polynucleotides,
polypeptides, antagonists and/or agonists of the invention are directly
injected into a
hypertrophic scar or keloid, in order to prevent the progression of these
lesions. This
therapy is of particular value in the prophylactic treatment of conditions
which are
known to result in the development of hypertrophic scars and keloids (e.g.,
burns), and
is preferably initiated after the proliferative phase has had time to progress
(approximately 14 days after the initial injury), but before hypertrophic scar
or keloid
development. As noted above, the present invention also provides methods for
treating neovascular diseases of the eye, including for example, corneal
neovascularization, neovascular glaucoma, proliferative diabetic retinopathy,
retrolental fibroplasia and macular degeneration.
[0634] Moreover, ocular disorders associated with neovascularization which can
be treated with the polynucleotides and polypeptides of the present invention
(including agonists and/or antagonists) include, but are not limited to:
neovascular
glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia,
uveitis,
retinopathy of prematurity macular degeneration, corneal graft
neovascularization, as
well as other eye inflammatory diseases, ocular tumors and diseases associated
with
choroidal or iris neovascularization. See, e.g., reviews by Waltman et al.,
Am. J.
Ophthal. 85:704-710 (1978) and Gartner et al., Sure. Op7~thal. 22:291-312
(1978).
[0635] Thus, within one aspect of the present invention methods are provided
for
treating neovascular diseases of the eye such as corneal neovascularization
(including
corneal graft neovascularization), comprising the step of administering to a
patient a
therapeutically effective amount of a compound (as described above) to the
cornea,
such that the formation of blood vessels is inhibited. Briefly, the cornea is
a tissue,
which normally lacks blood vessels. In certain pathological conditions
however,
capillaries may extend into the cornea from the pericorneal vascular plexus of
the
limbus. When the cornea becomes vascularized, it also becomes clouded,
resulting in
a decline in the patient's visual acuity. Visual loss may become complete if
the cornea
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completely opacitates. A wide variety of disorders can result in corneal
neovascularization, including for example, corneal infections (e.g., trachoma,
herpes
simplex keratrtis, leishmaniasis and onchocerciasis), immunological processes
(e.g.,
graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma,
inflammation
(of any cause), toxic and nutritional deficiency states, and as a complication
of wearing
contact lenses.
[0636] Within particularly preferred embodiments of the invention, may be
prepared for topical administration in saline (combined with any of the
preservatives
and antimicrobial agents commonly used in ocular preparations), and
administered in
eyedrop form. The solution or suspension may be prepared in its pure form and
administered several times daily. Alternatively, anti-angiogenic compositions,
prepared as described above, may also be administered directly to the cornea.
Within
preferred embodiments, the anti-angiogenic composition is prepared with a muco-
adhesive polymer, which binds to cornea. Within further embodiments, the anti-
angiogenic factors or anti-angiogenic compositions may be utilized as an
adjunct to
conventional steroid therapy. Topical therapy may also be useful
prophylactically in
corneal lesions which are known to have a high probability of inducing an
angiogenic
response (such as chemical burns). .In these instances the treatment, likely
in
combination with steroids, may be instituted immediately to help prevent
subsequent
complications.
[0637] Within other embodiments, the compounds described above may be
injected directly into the corneal stroma by an ophthalmologist under
microscopic
guidance. The preferred site of injection may vary with the morphology of the
individual lesion, but the goal of the administration would be to place the
composition
at the advancing front of the vasculature (i.e., interspersed between the
blood vessels
and the normal cornea). In most cases this would involve perilimbic corneal
injection
to "protect" the cornea from the advancing blood vessels. This method may also
be
utilized shortly after a corneal insult in order to prophylactically prevent
corneal
neovascularization. In this situation, the material could be injected in the
perilimbic
cornea interspersed between the corneal lesion and its undesired potential
limbic blood
supply. Such methods may also be utilized in a similar fashion to prevent
capillary
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invasion of transplanted corneas. In a sustained-release form, injections
might only be
required 2-3 times per year. A steroid could also be added to the injection
solution to
reduce inflammation resulting from the injection itself.
[0638] Within another aspect of the present invention, methods are provided
for
treating neovascular glaucoma, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. In
one
embodiment, the compound may be administered topically to the eye in order to
treat
early forms of neovascular glaucoma. Within other embodiments, the compound
may
be implanted by injection into the region of the anterior chamber angle.
Within other
embodiments, the compound may also be placed in any location such that the
compound is continuously released into the aqueous humor. Within another
aspect of
the present invention, methods are provided for treating proliferative
diabetic
retinopathy, comprising the step of administering to a patient a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
to the
eyes, such that the formation of blood vessels is inhibited.
[0639] Within particularly preferred embodiments of the invention,
proliferative
diabetic retinopathy may be treated by injection into the aqueous humor or the
vitreous, in order to increase the local concentration of the polynucleotide,
polypeptide, antagonist and/or agonist in the retina. Preferably, this
treatment should
be initiated prior to the acquisition of severe disease requiring
photocoagulation.
[0640] Within another aspect of the present invention, methods are provided
for
treating retrolental fibroplasia, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. The
compound may be administered topically, via intravitreous inj ection and/or
via
intraocular implants.
[0641] Additionally, disorders which can be treated with the polynucleotides,
polypeptides, agonists and/or agonists include, but are not limited to,
hemangioma,
arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound
healing,
granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-
Weber
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syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
[0642] Moreover, disorders and/or states, which can be treated, prevented,
diagnosed and/or prognosed with the polynucleotides, polypeptides, agonists
and/or
agonists of the invention include, but are not limited to, solid tumors, blood
born
tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors,
for
example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for
example,
diabetic retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft
rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, and
uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations,
hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma,
vascular
adhesions, myocardial angiogenesis, coronary collaterals, cerebral
collaterals,
arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber
Syndrome,
plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma
fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth
control agent by preventing vascularization required for embryo implantation
controlling menstruation, diseases that have angiogenesis as a pathologic
consequence
such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter
pylori),
Bartonellosis and bacillary angiomatosis.
[0643] In one aspect of the birth control method, an amount of the compound
sufficient to block embryo implantation is administered before or after
intercourse and
fertilization have occurred, thus providing an effective method of birth
control,
possibly a "morning after" method. Polynucleotides, polypeptides, agonists
and/or
agonists may also be used in controlling menstruation or administered as
either a
peritoneal lavage fluid or for peritoneal implantation in the treatment of
endometriosis.
[0644] Polynucleotides, polypeptides, agonists and/or agonists of the present
invention may be incorporated into surgical sutures in order to prevent stitch
granulomas.
[0645] Polynucleotides, polypeptides, agonists and/or agonists may be utilized
in a
wide variety of surgical procedures. For example, within one aspect of the
present
invention a compositions (in the form of, for example, a spray or film) may be
utilized
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to coat or spray an area prior to removal of a tumor, in order to isolate
normal
surrounding tissues from malignant tissue, and/or to prevent the spread of
disease to
surrounding tissues. Within other aspects of the present invention,
compositions (e.g.,
in the form of a spray) may be delivered via endoscopic procedures in order to
coat
tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects
of the
present invention, surgical meshes, which have been coated with anti-
angiogenic
compositions of the present invention may be utilized in any procedure wherein
a
surgical mesh might be utilized. For example, within one embodiment of the
invention
a surgical mesh laden with an anti-angiogenic composition may be utilized
during
abdominal cancer resection surgery (e.g., subsequent to colon resection) in
order to
provide support to the structure, and to release an amount of the anti-
angiogenic factor.
[0646] Within further aspects of the present invention, methods are provided
for
treating tumor excision sites, comprising administering a polynucleotide,
polypeptide,
agonist and/or agonist to the resection margins of a tumor subsequent to
excision, such
that the local recurrence of cancer and the formation of new blood vessels at
the site is
inhibited. Within one embodiment of the invention, the anti-angiogenic
compound is
administered directly to the tumor excision site (e.g., applied by swabbing,
brushing or
otherwise coating the resection margins of the tumor with the anti-angiogenic
compound). Alternatively, the anti-angiogenic compounds may be incorporated
into
known surgical pastes prior to administration. Within particularly preferred
embodiments of the invention, the anti-angiogenic compounds are applied after
hepatic
resections for malignancy, and after neurosurgical operations.
[0647] Within one aspect of the present invention, polynucleotides,
polypeptides,
agonists and/or agonists may be administered to the resection margin of a wide
variety
of tumors, including for example, breast, colon, brain and hepatic tumors. For
example, within one embodiment of the invention, anti-angiogenic compounds may
be
administered to the site of a neurological tumor subsequent to excision, such
that the
formation of new blood vessels at the site are inhibited.
[0648] The polynucleotides, polypeptides, agonists and/or agonists of the
present
invention may also be administered along with other anti-angiogenic factors.
Representative examples of other anti-angiogenic factors include: Anti-
Invasive
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Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of
Metalloproteinase-l, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the
lighter "d
group" transition metals.
[0649] Lighter "d group" transition metals include, for example, vanadium,
molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition
metal
species may form transition metal complexes. Suitable complexes of the above-
mentioned transition metal species include oxo transition metal complexes.
[0650] Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include metavanadate and orthovanadate complexes such as, for example,
ammonium
metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl
complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate
including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
[0651] Representative examples of tungsten and molybdenum complexes also
include oxo complexes. Suitable oxo tungsten complexes include tungstate and
tungsten oxide complexes. Suitable tungstate complexes include ammonium
tungstate,
calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum
complexes include molybdate, molybdenum oxide, and molybdenyl complexes.
Suitable molybdate complexes include ammonium molybdate and its hydrates,
sodium
molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable
molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and
molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl
acetylacetonate. Other suitable tungsten and molybdenum complexes include
hydroxo
derivatives derived from, for example, glycerol, tartaric acid, and sugars.
[0652] A wide variety of other anti-angiogenic factors may also be utilized
within
the context of the present invention. Representative examples include platelet
factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab
shells),
(Murata et al., Cancer Res. 51:22-26 (1991)); Sulphated Polysaccharide
Peptidoglycan
Complex (SP- PG) (the function of this compound may be enhanced by the
presence
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of steroids such as estrogen, and tamoxifen citrate); Staurosporine;
modulators of
matrix metabolism, including for example, proline analogs, cishydroxyproline,
d,L-
3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile
fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin;
Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem.
267:17321-17326 (1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480
(1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin
(Ingber et al., Nature 348:555-557 (1990)); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446 (1987)); anticollagenase-
serum;
alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664 (1987));
Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-
316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminohnidazole;
and metalloproteinase inhibitors such as BB94.
Musculoskeletal System Disorders
[0653] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention, may be used to treat, prevent, diagnose, and/or
prognose
disorders of the musculoskeletal system, including but not limited to,
disorders of the
bone, joints, ligaments, tendons, bursa, muscle, and/or neoplasms and cancers
associated with musculoskeletal tissue.
[0654] Diseases or disorders of the bone include, but are not limited to,
Albers-
Schonberg disease, bowlegs, heel spurs, I~ohler's bone disease, knock-knees,
Legg-
Calve-Perthes disease, Marfan's syndrome, mucopolysaccharidoses, Osgood-
Schlatter
disease, osteochondroses, osteochondrodysplasia, osteomyelitis, osteopetroses,
osteoporosis (postmenopausal, senile, and juvenile), Paget's disease,
Scheuermann's
disease, scoliosis, Severs disease, and patellofemoral stress syndrome.
[0655] Joint diseases or disorders include, but are not limited to, ankylosing
spondylitis, Beh~et's syndrome, CREST syndrome, Ehlers-Danlos syndrome,
infectious arthritis, discoid lupus erythematosus, systemic lupus
erythematosus, Lyme
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disease, osteoarthritis, psoriatic arthritis, relapsing polychondrites,
Reiter's syndrome,
rheumatoid arthritis (adult and juvenile), scleroderma, and Still's disease.
[0656] Diseases or disorders affecting ligaments, tendons, or bursa include,
but are
not limited to, ankle sprain, bursitis, posterior Achilles tendon bursitis
(Haglund's
deformity), anterior Achilles tendon bursitis (Albert's disease), tendinitis,
tenosynovitis, poplieus tendinitis, Achilles tendinitis, medial or lateral
epicondylitis,
rotator cuff tendinitis, spasmodic torticollis, and fibromyalgia syndrome.
[0657] Muscle diseases or disorders include, but are not limited to, Becker's
muscular dystrophy, Duchenne's muscular dystrophy, Landouzy-Dejerine muscular
dystrophy, Leyden-Mobius muscular dystrophy, Erb's muscular dystrophy,
Charcot's
joints, dermatomyositis, gout, pseudogout, glycogen storage diseases, Pompe's
disease, mitochondrial myopathy, periodic paralysis, polymyalgia rheumatics,
polymyositis, Steinert's disease, Thomsen's disease, anterolateral and
posteromedial
shin splints, posterior femoral muscle strain, and fibromyositis.
[0658] Musculoskeletal tissue may also develop cancers and/or neoplasms that
include, but are not limited to, osteochondroma, benign chondroma,
chondroblastoma,
chondromyxoid fibroma, osteoid osteoma, giant cell tumor, multiple myeloma,
' osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma,
Ewing's tumor, and malignant lymphoma of bone.
Neural Activitv and Neurological Diseases
[0659] The polynucleotides, polypeptides and agonists or antagonists of the
invention may be used for the diagnosis and/or treatment of diseases,
disorders,
damage or injury of the brain and/or nervous system. Nervous system disorders
that
can be treated with the compositions of the invention (e.g., polypeptides,
polynucleotides, and/or agonists or antagonists), include, but are not limited
to,
nervous system injuries, and diseases or disorders which result in either a
disconnection of axons, a diminution or degeneration of neurons, or
demyelination.
Nervous system lesions which may be treated in a patient (including human and
non-
human mammalian patients) according to the methods of the invention, include
but are
' not limited to, the following lesions of either the central (including
spinal cord, brain)
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or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen
in a
portion of the nervous system results in neuronal injury or death, including
cerebral
infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic
lesions,
including lesions caused by physical injury or associated with surgery, for
example,
lesions which sever a portion of the nervous system, or compression injuries;
(3)
malignant lesions, in which a portion of the nervous system is destroyed or
injured by
malignant tissue which is either a nervous system associated malignancy or a
malignancy derived from non-nervous system tissue; (4) infectious lesions, in
which a
portion of the nervous system is destroyed or injured as a result of
infection, for
example, by an abscess or associated with infection by , human
immunodeficiency
virus, herpes zoster, or herpes simplex virus or with Lyme disease,
tuberculosis, or
syphilis; (5) degenerative lesions, in which a portion of the nervous system
is
destroyed or injured as a result of a degenerative process including but not
limited to,
degeneration associated with Parkinson's disease, Alzheimer's disease,
Huntington's
chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with
nutritional
diseases or disorders, in which a portion of the nervous system is destroyed
or injured
by a nutritional disorder or disorder of metabolism including, but not limited
to,
vitamin B 12 deficiency, folic acid deficiency, Wernicke disease, tobacco-
alcohol
amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus
callosum), and alcoholic cerebellar degeneration; (7) neurological lesions
associated
with systemic diseases including, but not limited to, diabetes (diabetic
neuropathy,
Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8)
lesions
caused by toxic substances including alcohol, lead, or particular neurotoxins;
and (9)
demyelinated lesions in which a portion of the nervous system is destroyed or
injured
by a demyelinating disease including, but not limited to, multiple sclerosis,
human
immunodeficiency virus-associated myelopathy, transverse myelopathy or various
etiologies, progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
[0660j In one embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to protect neural cells from the
damaging effects
of hypoxia. In a further preferred embodiment, the polypeptides,
polynucleotides, or
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agonists or antagonists of the invention are used to protect neural cells from
the
damaging effects of cerebral hypoxia. According to this embodiment, the
compositions of the invention are used to treat or prevent neural cell injury
associated
with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the
polypeptides, polynucleotides, or agonists or antagonists of the invention,
are used to
treat or prevent neural cell injury associated with cerebral ischemia. In
another non-
exclusive aspect of this embodiment, the polypeptides, polynucleotides, or
agonists or
antagonists of the invention are used to treat or prevent neural cell injury
associated
with cerebral infarction.
[0661] In another preferred embodiment, the polypeptides, polynucleotides, or
agonists or antagonists of the invention are used to treat or prevent neural
cell injury
associated with a stroke. In a specific embodiment, the polypeptides,
polynucleotides,
or agonists or antagonists of the invention are used to treat or prevent
cerebral neural
cell injury associated with a stroke.
[0662] In another preferred embodiment, the polypeptides, polynucleotides, or
agonists or antagonists of the invention are used to treat or prevent neural
cell injury
associated with a heart attack. In a specific embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are used to treat
or prevent
cerebral neural cell injury associated with a heart attack.
[0663] The compositions of the invention which are useful for treating or
preventing a nervous system disorder may be selected by testing for biological
activity
in promoting the survival or differentiation of neurons. For example, and not
by way
of limitation, compositions of the invention which elicit any of the following
effects
may be useful according to the invention: (1) increased survival time of
neurons in
culture either in the presence or absence of hypoxia or hypoxic conditions;
(2)
increased sprouting of neurons in culture or i~c vivo; (3) increased
production of a
neuron-associated molecule in culture or i~ vivo, e.g., choline
acetyltransferase or
acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms
of
neuron dysfunction in vivo. Such effects may be measured by any method known
in
the art. In preferred, non-limiting embodiments, increased survival of neurons
may
routinely be measured using a method set forth herein or otherwise known in
the art,
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such as, for example, in Zhang et al., Pr~oc Natl Acad Sci USA 97:3637-42
(2000) or in
Arakawa et al., J. Neu~osci., 10:3507-15 (1990); increased sprouting of
neurons may
be detected by methods known in the art, such as, for example, the methods set
forth in
Pestronk et al., Exp. Neu~ol., 70:65-82 (1980), or Brown et al., Ayah. Rev.
Neu~osci.,
4:17-42 (1981); increased production of neuron-associated molecules may be
measured by bioassay, enzymatic assay, antibody binding, Northern blot assay,
etc.,
using techniques known in the art and depending on the molecule to be
measured; and
motor neuron dysfunction may be measured by assessing the physical
manifestation of
motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or
functional disability.
[0664] In specific embodiments, motor neuron disorders that may be treated
according to the invention include, but are not limited to, disorders such as
infarction,
infection, exposure to toxin, trauma, surgical damage, degenerative disease or
malignancy that may affect motor neurons as well as other components of the
nervous
system, as well as disorders that selectively affect neurons such as
amyotrophic lateral
sclerosis, and including, but not limited to, progressive spinal muscular
atrophy,
progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile
muscular
atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary Motorsensory
Neuropathy
(Charcot-Marie-Tooth Disease).
[0665] Further, polypeptides or polynucleotides of the invention may play a
role in
neuronal survival; synapse formation; conductance; neural differentiation,
etc. Thus,
compositions of the invention (including polynucleotides, polypeptides, and
agonists
or antagonists) may be used to diagnose and/or treat or prevent diseases or
disorders
associated with these roles, including, but not limited to, learning and/or
cognition
disorders. The compositions of the invention may also be useful in the
treatment or
prevention of neurodegenerative disease states and/or behavioural disorders.
Such
neurodegenerative disease states and/or behavioral disorders include, but are
not
limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette
Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder,
panic disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
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including disorders in feeding, sleep patterns, balance, and perception. In
addition,
compositions of the invention may also play a role in the treatment,
prevention and/or
detection of developmental disorders associated with the developing embryo, or
sexually-linked disorders.
[0666] Additionally, polypeptides, polynucleotides and/or agonists or
antagonists
of the invention, may be useful in protecting neural cells from diseases,
damage,
disorders, or injury, associated with cerebrovascular disorders including, but
not
limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid
stenosis, or
Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral
anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,
cerebral artery
diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis,
sinus
thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or
subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral
ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or
vertebrobasilar insufficiency), vascular dementia (e.g., mufti-infarct),
leukomalacia,
periventricular, and vascular headache (e.g., cluster headache or migraines).
[0667] In accordance with yet a further aspect of the present invention, there
is
provided a process for utilizing polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, for therapeutic purposes, for example,
to stimulate
neurological cell proliferation and/or differentiation. Therefore,
polynucleotides,
polypeptides, agonists and/or antagonists of the invention may be used to
treat and/or
detect neurologic diseases. Moreover, polynucleotides or polypeptides, or
agonists or
antagonists of the invention, can be used as a marker or detector of a
particular
nervous system disease or disorder.
[0668] Examples of neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include brain diseases, such as metabolic brain diseases which includes
phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase
deficiency,
pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain
edema, brain neoplasms such as cerebellar neoplasms which include
infratentorial
neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms,
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hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar
diseases such as cerebellar ataxia which include spinocerebellar degeneration
such as
ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-
Joseph
Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as
infratentorial
neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis,
globoid cell
leukodystrophy, metachromatic leukodystrophy and subacute sclerosing
panencephalitis.
[0669] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include cerebrovascular disorders (such as carotid artery diseases which
include
carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral
amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformations, cerebral artery diseases, cerebral embolism and
thrombosis such as carotid artery thrombosis, sinus thrombosis and
Wallenberg's
Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and
subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as
transient
cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar
insufficiency, '
vascular dementia such as mufti-infarct dementia, periventricular
leukomalacia,
vascular headache such as cluster headache and migraine.
[0670] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include dementia such as AIDS Dementia Complex, presenile dementia such as
Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as
Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such
as
mufti-infarct dementia, encephalitis vc~hich include encephalitis periaxialis,
viral
encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis
Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated
encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic
syndrome,
Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis,
encephalomalacia such as periventricular leukomalacia, epilepsy such as
generalized
epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy
which
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includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as
complex
partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-
traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua, and
Hallervorden-
Spatz Syndrome.
[0671] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, andlor antagonists of the present
invention
include hydrocephalus such as Dandy-Walker Syndrome and normal pressure
hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral
malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri
pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral
toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central
nervous
system infections such as AIDS Dementia Complex, Srain Abscess, subdural
empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine
Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and
cerebral
malaria.
[0672] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, andlor antagonists of the present
invention
include meningitis such as arachnoiditis, aseptic meningtitis such as viral
meningtitis
which includes lymphocytic choriomeningitis, Bacterial meningtitis which
includes
Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such
as
Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal
tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural
effusion,
meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as
transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which
includes
bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as
Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-
Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.
[0673] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include central nervous system neoplasms such as brain neoplasms that include
cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle
neoplasms
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such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial .
neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural
neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral
sceloris
which includes adrenoleulcodystrophy, encephalitis periaxialis, globoid cell
leukodystrophy, diffuse cerebral sclerosis such as metachromatic
leukodystrophy,
allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis,
progressive
multifocal leukoencephalopathy, multiple sclerosis, central pontine
myelinolysis,
transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue
Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord
diseases
such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular
atrophy
such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord
neoplasms
such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff Man Syndrome,
mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's
Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff
Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Lawrence-Moon-
Biedl
Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, rnucolipidosis such
as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome,
phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett
Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome,
nervous system abnormalities such as holoprosencephaly, neural tube defects
such as
anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,
encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina
bifida cystica and spina bifida occulta.
[0674] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include hereditary motor and sensory neuropathies which include Charcot-Marie
Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic
paraplegia,
Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such
as
Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations
(such as
agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia,
apraxia, neurogenic bladder, cataplexy, communicative disorders such as
hearing
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disorders that includes deafness, partial hearing loss, loudness recruitment
and tinnitus,
language disorders such as aphasia which include agraphia, anomia, broca
aphasia, and
Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development
disorders, speech disorders such as aphasia which includes anomia, broca
aphasia and
Wernicke Aphasia, articulation disorders, communicative disorders such as
speech
disorders which include dysarthria, echolalia, mutism and stuttering, voice
disorders
such as aphonia and hoarseness, decerebrate state, delirium, fasciculation,
hallucinations, meningism, movement disorders such as angelman syndrome,
ataxia,
athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic,
torticollis
and tremor, muscle hypertonia such as muscle rigidity such as stiff man
syndrome,
muscle spasticity, paralysis such as facial paralysis which includes Herpes
Zoster
Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's
Syndrome, Homer's Syndrome, Chronic progressive external ophthalmoplegia such
as
Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia
such as
Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal cord
paralysis, paresis, phantom limb, taste disorders such as ageusia and
dysgeusia, vision
disorders such as amblyopia, blindness, color vision defects, diplopia,
hemianopsia,
scotoma and subnormal vision, sleep disorders such as hypersomnia which
includes
Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus,
unconsciousness such as coma, persistent vegetative state and syncope and
vertigo,
neuromuscular diseases such as amyotonia congenita, amyotrophic lateral
sclerosis,
Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such
as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann
Disease,
Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia
Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis,
Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff Man Syndrome,
peripheral nervous system diseases such as acrodynia, amyloid neuropathies,
autonomic nervous system diseases such as Adie's Syndrome, Barre-Lieou
Syndrome,
Familial Dysautonomia, Homer's Syndrome, Reflex Sympathetic Dystrophy and Shy-
Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such
as
Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases
such as
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Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders
which
includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia
such as
Duane's Syndrome, Homer's Syndrome, Chronic Progressive External
Ophthalmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia
and
Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic
Atrophy
which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis
such as
Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis,
Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic
neuropathies such as diabetic foot.
[0675] ~ Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include nerve compression syndromes such as carpal tunnel syndrome, tarsal
tunnel
syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve
compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia,
facial
neuralgia and trigeminal neuralgia, neuritis such as experimental allergic
neuritis, optic
neuritis, polyneuritis, polyradiculoneuritis and radiculities such as
polyradiculitis,
hereditary motor and sensory neuropathies such as Charcot-Marie Disease,
Hereditary
Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-
Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include
Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica,
Gustatory Sweating and Tetany).
Gastrointestinal Disorders
[0676] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or prognose
gastrointestinal
disorders, including inflammatory diseases and/or conditions, infections,
cancers (e.g.,
intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's
lymphoma
of the small intestine, small bowl lymphoma)), and ulcers, such as peptic
ulcers.
[0677] Gastrointestinal disorders include dysphagia, odynophagia, inflammation
of
the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and
stricturing,
Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers,
adeoncarcinomas,
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gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach
cancers,
polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric
stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic
erosive,
atrophic, plasma cell, and Menetrier's), and peritoneal diseases (e.g.,
chyloperioneum,
hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular
occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic
abscess).
[0678] Gastrointestinal disorders also include disorders associated with the
small
intestine, such as malabsorption syndromes, distension, irritable bowel
syndrome,
sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical
sprue,
Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis,
obstructions of the ileum, Meckel's diverticulum, multiple diverticula,
failure, of
complete rotation of the small and large intestine, lymphoma, and bacterial
and
parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid,
cholera,
infection by Roundworms (Asca~iasis lumbnicoides), Hookworms (Ancylostorna
duodenale), Threadwonns (Entenobius ve~micula~is), Tapeworms (Taenia saginata,
Eehinococcus g~anulosus, Diphyllobothrium spp., and T. solium).
[0679] Liver diseases and/or disorders include intrahepatic cholestasis
(alagille
syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye
syndrome),
hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly,
hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension
(esophageal
and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis
(alcoholic,
biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis,
cirrhosis),
parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess),
jaundice
(hemolytic, hepatocellular, and cholestatic), cholestasis, portal
hypertension, liver
enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis,
chronic hepatitis
(autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic
hepatitis, viral
human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis
E),
Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis,
hepatic
encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary
biliary
cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma,
hemangiomas, bile
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stones, liver failure (hepatic encephalopathy, acute liver failure), and liver
neoplasms
(angiomyolipoma, calcified liver metastases, cystic liver metastases,
epithelial tumors,
fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma,
hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma,
liver
cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors
of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts
[Simple
cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst],
Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma,
Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor,
Miscellaneous],
Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct
adenoma),
Hepatocyte ~ (Adenoma, Focal nodular hyperplasia, Nodular regenerative
hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma,
hepatocellular
carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors
of
blood vessels, angiosarcoma, I~arposi's sarcoma, hemangioendothelioma, other
tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma,
carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]),
peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute
intermittent
porphyria, porphyria cutanea tarda), Zellweger syndrome).
[0680] Pancreatic diseases and/or disorders include acute pancreatitis,
chronic
pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis),
neoplasms
(adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma,
and
glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other
pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst,
pancreatic fistula,
insufficiency)).
[0681] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the
gallbladder,
acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
[0682] Diseases and/or disorders of the large intestine include antibiotic-
associated
colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses,
fungal and
bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids),
colonic diseases
(colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g.,
villous
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adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic
diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid
diseases
[proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea
(infantile
diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal
obstruction,
duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, deal
diseases
(ileal neoplasms, ileitis), immunoproliferative small intestinal disease,
inflammatory
bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia,
parasitic diseases
(anisalciasis, balantidiasis, blastocystis infections, cryptosporidiosis,
dientamoebiasis,
amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal
neoplasms
(cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms,
intestinal
polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent
loop
syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction
[cecal
volvulus], intussusception), intestinal perforation, intestinal polyps
(colonic polyps,
gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal
neoplasms),
malabsorption syndromes (blind loop syndrome, celiac disease, lactose
intolerance,
short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular
occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies
(intestinal
lymphagiectasis), rectal diseases (anus diseases, fecal incontinence,
hemorrhoids,
proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal
ulcer, peptic
esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison
syndrome),
postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g.,
achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular
ectasia, gastric
fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic),
gastroparesis,
stomach dilatation, stomach diverticulum, stomach rieoplasms (gastric cancer,
gastric
polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture,
stomach
ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g.,
hematemesis,
hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic
colitis.
[0683] Further diseases and/or disorders of the gastrointestinal system
include
biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary
fistula, esophageal
fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms
(e.g., biliary tract
neoplasms, esophageal neoplasms,, such as adenocarcinoma of the esophagus,
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esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic
neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of
the
pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal
neoplasms),
esophageal disease (e.g., bullous diseases, candidiasis, glycogenic
acanthosis,
ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g.,
Zenker's
diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders
(e.g., CREST
syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux),
neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome),
stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia);
gastrointestinal
diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus
infection),
hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach
neoplasms
(gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)),
hernia (e.g.,
congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator
hernia,
umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal
diseases
(appendicitis, cecal neoplasms)).
Reproductive System Disorders
[0684] The polynucleotides or polypeptides, or agonists or antagonists of the
invention may be used for the diagnosis, treatment, or prevention of diseases
and/or
disorders of the reproductive system. Reproductive system disorders that can
be
treated by the compositions of the invention, include, but are not limited to,
reproductive system injuries, infections, neoplastic disorders, congenital
defects, and
diseases or disorders which result in infertility, complications with
pregnancy, labor,
or parturition, and postpartum difficulties.
[0685] Reproductive system disorders and/or diseases include diseases and/or
disorders of the testes, including, but not limited to, testicular atrophy,
testicular
feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic
testis,
epididymitis and orchitis (typically resulting from infections such as, for
example,
gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis
nodosa, germ
cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas,
choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g.,
Leydig cell
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tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and
disorders of sperm production (e.g., immotile cilia syndrome, aspermia,
asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).
[0686] Reproductive system disorders also include, but are not limited to,
disorders of the prostate gland, such as acute non-bacterial prostatitis,
chronic non-
bacterial prostatitis, acute bacterial prostatitis, chronic bacterial
prostatitis,
prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign
prostatic
hypertrophy or hyperplasia, and prostate neoplastic disorders, including
adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous
cell
carcinomas.
[0687] Additionally, the compositions of the invention may be useful in the
diagnosis, treatment, and/or prevention of disorders or diseases of the penis
and
urethra, including, but not limited to, inflammatory disorders, such as
balanoposthitis,
balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes
simplex virus,
gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV,
AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly
penile papules; urethral abnormalities, such as hypospadias, epispadias, and
phimosis;
premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease,
Bowenoid
paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma;
penile
cancers, including squamous cell carcinomas, carcinoma in situ, verrucous
carcinoma,
and disseminated penile carcinoma; urethral neoplastic disorders, including
penile
urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral
carcinoma; and erectile disorders, such as priapism, Peyronie's disease,
erectile
dysfunction, and impotence.
[0688] Moreover, diseases and/or disorders of the vas deferens include, but
are not
limited to, vasculititis and CBAVD (congenital bilateral absence of the vas
deferens);
additionally, the polynucleotides, polypeptides, and agonists or antagonists
of the
present invention may be used in the diagnosis, treatment, and/or prevention
of
diseases and/or disorders of the seminal vesicles, including but not limited
to, hydatid
disease, congenital chloride diarrhea, and polycystic kidney disease.
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[0689] Other disorders and/or diseases of the male reproductive system that
may
be diagnosed, treated, and/or prevented with the compositions of the invention
include,
but are not limited to, Klinefelter's syndrome, Young's syndrome, premature
ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high
fever,
multiple sclerosis, and gynecomastia.
[0690] Further, the polynucleotides, polypeptides, and agonists or antagonists
of
the present invention may be used in the diagnosis, treatment, and/or
prevention of
diseases and/or disorders of the vagina and vulva, including, but not limited
to,
bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid,
granuloma
inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal
trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas
vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic
vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic
shock
syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic
disorders,
such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma,
melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.
[0691] Disorders and/or diseases of the uterus that may be diagnosed, treated,
and/or prevented with the compositions of the invention include, but are not
limited to,
dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis,
anovulatory
bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's
syndrome, premature menopause, precocious puberty, uterine polyps,
dysfunctional
uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic
disorders,
such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the
polypeptides, polynucleotides, or agonists or antagonists of the invention may
be
useful as a marker or detector of, as well as in the diagnosis, treatment,
and/or
prevention of congenital uterine abnormalities, such as bicornuate uterus,
septate
uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary
rudimentary
horn, unicornuate uterus with a non-communicating cavitary rudimentary horn,
unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine
didelfus, and T-shaped uterus.
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