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CA 02395693 2002-06-25
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Nucleic Acids, Proteins, and Antibodies
[001] Tlus 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
(PC003PCT_seqList.txt,
2,320,231 bytes in size, created on January 12, 2001) on four identical
compact discs
(CD-R), labeled "COPY l," "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:l/www.fileviewer.com).
Field of the Invention
[003] The present invention relates to novel excretory system related
polynucleotides, the polypeptides encoded by these polynucleotides herein
collectively
referred to as "excretory system antigens," and antibodies that
immunospecifically
bind these polypeptides, and the use of such excretory system polynucleotides,
antigens, and antibodies for detecting, treating, preventing and/or prognosing
disorders
of the excretory system, including, but not limited to, the presence of cancer
of
excretory system tissues and cancer metastases. More specifically, isolated
excretory
system nucleic acid molecules are provided encoding novel excretory system
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polypeptides. Novel excretory system polypeptides and antibodies that bind to
these
polypeptides are provided. Also provided are vectors, host cells, and
recombinant and
synthetic methods for producing human excretory system polynucleotides,
polypeptides, andlor antibodies. The invention further relates to diagnostic
and
therapeutic methods useful for diagnosing, treating, preventing and/or
prognosing
disorders related to the excretory system, including cancer of excretory
system tissues,
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 andlor
compositions for inhibiting or promoting the production and/or function of the
polypeptides of the invention.
Backg~ouhd of the Ihvehtioh
[004] The Human Excretory System is comprised of specialized organs and body
tissues responsible for the removal of metabolic wastes and the retention of
the proper
amounts of water, salts, and nutrients. Components of this system include
liver, lungs,
skin, and the urinary system. All of these organs work synergistically to keep
the
body's water, salts, and nutrients level properly balanced.
[005] The urinary system is a subgroup of organs, consisting of the kidneys,
ureters,
bladder, and uretha, specialized in removing waste materials and water from
the blood.
This waste is generated from the normal breakdown of food and other energy
sources
and is translated into urine as it passes through the kidneys. The kidneys
perform a
complex chemical exchange with the blood, where waste products are filtered
out into
the urinary system while essential water and conserved molecules (e.g. sodium,
phosphorous, and potassium) are reabsorbed into the blood.
[006] Kidneys also release three important hormones into the body.
Erythropoietin,
or EPO, stimulates the production of red blood cells in bone marrow. Renin is
important for regulating blood pressure. The kidneys also release the active
form of
vitamin D, which is essential for the maintenance of calcium in the bones and
for
normal chemical balance in the body.
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[007] Once waste, in the form of urine, is released from the kidneys, it
travels down
two thin tubes called ureters to the bladder. The bladder is a hollow, balloon-
shaped
organ responsible for storing urine until the nerves in the bladder cause the
need for
urination.
[008] Excretory diseases most commonly affect the kidneys and the bladder.
Disorders range in severity from easily treatable to life threatening.
Generally, the two
most common causes of kidney disease are diabetes (diabetic nephropathy) and
hypertension. Diabetes is a disease resulting from an excess of sugar in the
bloodstream. Damage done to the nephrons of the kidney by this sugar excess is
called diabetic nephropathy. High blood pressure can damage the small blood
vessels
in the kidney impairing the waste removal function of these vessels.
[009] Damage from trauma or poisons may also lead to kidney disease. For
instance,
interstitial nephritis may occur with an allergic reaction to a drug or as a
side effect of
medications and involves inflammation of the spaces between the renal tubes
and
glomeruli. Interstitial nephritis causes reduction in kidney function
including
decreased urine output and other signs of renal failure.
[010] Kidney disease is not an exclusive result of external factors.
Hereditary factors
may also contribute to kidney disease. For example, polycystic kidney disease
(PKD),
is a genetic disorder where many cysts grow on the kidney, reducing its mass
and
function, and eventually resulting in kidney failure. Congenital nephrotic
syndrome is
an inherited disorder, primarily affecting families of Finnish origin,
characterized by
protein in the urine and swelling of the body. The disorder develops shortly
after
birth, results in infection, malnutrition, and kidney failure, and is usually
fatal with the
first year.
[011] Goodpasture's syndrome is a form of rapidly progressive
glomerulonephritis
and pulmonary bleeding characterized by deposits of antibodies in the basement
membranes of both the kidney glomerulus and the lung alveoli. It is an
autoimmune
disorder that may be triggered by a viral infection or inhalation of
hydrocarbon
solvents such as gasoline. Smoking has been found to increase the risk of
developing
this order. There can also be an inherited predisposition to Goodpasture's
syndrome.
[012] Kidney disease may also result from the malfunction of the body's basic
functions. Nephrocalcinosis is a disorder where an excess of calcium and
oxalate or
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phosphate is deposited in the renal tubules and interstitium resulting in
reduced kidney
function. Fragments of the calcium oxalate or calcium phosphate may break free
from
the kidney and form stones.
[013] IgM mesangial proliferative glomerulonephritis is caused by the
inflammation
of the glomerulus, the enlargement of certain glomerular cells, and the IgM
antibody
deposits in the mesangium layer of the glomerular capillary. The disorder is
characterized by edema and bloody or dark urine. The trigger for this
condition is
unknown but it may be the result of some type of immune response. Although IgM
mesangial proliferative glomerulonephritis is rare, it can affect both adults
and
children and may progress to renal failure.
[014] Membranoproliferative glomerulonephritis I and II are disorders caused
by an
abnormal immune response resulting in deposits of antibodies in the kidneys.
Mesangial cells are increased in number and parts of the glomerular membrane
changes in structure. These changes make the glomerulus permeable to protein
and
blood cells which may result in edema, hypertension, azotemia, or ultimately,
chronic
renal failure.
[015] Unfortunately, there are no cures for kidney disease. Currently,
treatment has
revolved around reduction of symptoms, prevention of complications, and slowed
progression of the disorder towards kidney failure, mostly through diet and
medications. However, once kidney failure ensues, only dialysis or
transplantation is
remotely effective in prolonging life.
[016] Several types of cancer can develop in the kidney. For example, the most
common adult form of kidney cancer is hypernephroma, or renal cell cancer.
This
cancer affects the filtering of blood and production of urine by developing in
the lining
of the renal tubules. A less common form, called transitional cell cancer,
develops in
the lining of the bladder, ureter, or renal pelvis. Wilm's tumor, the most
common
intraabdominal tumor in children and the most common type of kidney tumor,
develops in children usually under the age of five. The cause of this tumor is
unknown, but it probably develops in the fetal tissue.
[017] Although surgery to remove the kidney, or nephrectomy, is the most
common
treatment for kidney cancer, several other therapies are available as
alternatives.
External radiotherapy is sometimes used to kill the cancer cells and relieve
pain when
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the cancer has spread to the bone. Chemotherapy has been used infrequently to
treat
kidney cancer due to its limited effectiveness. Hormone therapy may also be
used
with patients with advanced kidney cancer to relieve pain. Immunotherapy has
recently become a more popular alternative to surgery. Currently, interleukin-
2 and
interferon are types of immunotherapy used to treat advanced kidney cancer.
[018] Bladder disorders, in which muscles in the bladder weaken, can result
from
aging, illness, or injury. Many of these disorders result in the same
symptoms, for
example, problems with urination, which complicates the accuracy of clearly
diagnosing these diseases. Failure to effectively treat bladder disorders may
result in
further damage to the urinary system, especially the kidneys, as toxin may
become
backed up in the kidneys.
[019] The most common bladder disorders are urinary incontinence and urinary
retention. Urinary incontinence affects women more often than men and results
in the
loss of bladder control. Treatments range from exercise to surgery. Urinary
retention
can have many causes and results in the inability to urinate. Common causes
can
include obstruction in the urinary system, stress, bladder muscle failure, or
nerve
damage, and treatment regimens usually depend on the cause.
[020] Other bladder disorders result in painful urination such as urinary
tract
infections, interstitial cystitis (IC), or prostatitis. Urinary tract
infections (UTI) can be
caused by bacterial infection. Several species that are known to cause urinary
tract
infections are Esche~ichia coli, Staphylococcus sap~ophyticus, Klebsiella,
P~oteus
mi~-abillis, Uf°eaplasma urealytlzicum, and Entercocci. Treatment
consists of a course
of antibiotics or flushing out the bacteria by consuming plenty of fluids.
Urinary tract
infections are more common in women than men. In interstitial cystitis, also
known as
painful bladder syndrome and frequency-urgency-dysuria syndrome, the bladder
wall
becomes inflamed and irritated, resulting in stiffening of the bladder,
decreased
bladder capacity, pinpoint bleeding, and, in rare cases, ulcers in the bladder
lining. No
cause or reliable treatment for IC has been found.
[021] Prostatitis is the inflammation of the prostate gland that results in
urinary
frequency or urgency, painful urination, and lower backJgenital area pain.
Some cases
have been shown to be caused by bacterial infection and are treatable by
antibiotics.
CA 02395693 2002-06-25
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However, in most instances, prostatitis is not associated with any known
infectious
agent and is unaffected by antibiotic treatment.
(022] The bladder can also be susceptible to cancer. About 90 percent of
bladder
cancers begin in the bladder lining and are called transitional cell
carcinomas. If the
cancer is confined to the lining of the bladder, e.g., superficial bladder
cancer, it can be
readily treated. However, the cancer can recur, and most often, it recurs as
another
superficial cancer. '
[023] Treatment of bladder cancer depends on the stage of the disease and if,
or how
deeply, the cancer has invaded the bladder wall. Surgery is the most common
treatment of bladder cancer, the extent depending on the stage of the disease.
Surgery
to remove part or all of the bladder is called cystectomy. Superficial bladder
cancer
may be treated at the time of diagnosis by a transurethral resection (TUR) to
remove
the cancer or burn away the cancer cells with an electric current, e.g.
fulguration.
[024] Alternative therapies are available to treat bladder cancer. Either
external or
internal radiotherapy may be used to kill the cancer cells. Chemotherapy can
also be
used alone or in combination with surgery andlor radiation therapy or after
TUR with
fulguration to treat superficial bladder cancer. Chemotherapy can be
administered
intravesically through a catheter directly through the urethra or can be
administered
systemically through intravenous injection.
[025] In addition, superficial bladder cancer can be treated with
immunotherapy.
Currently, BCG (bacille calmette-Guerin) vaccine, an anticancer drug, is
placed in the
bladder for several hours to stimulate the body's natural immune system. Like
chemotherapy, immunotherapy can be used alone to treat superficial bladder
cancer or
after TUR with fulguration to help prevent the cancer from recurring. Other
forms of
immunotherapy axe also being studied for other stages of bladder cancer.
[026) Cancer of the bladder can also become malignant (e.g., invasive bladder
cancer), growing through the bladder wall and spread to nearby organs. Most
commonly, the cancer cells spread to the lymph nodes surrounding the bladder
and
require more aggressive treatments. For invasive bladder cancer, a radical
cystectomy
is the most recommended treatment where the entire bladder, nearby lymph
nodes, and
any surrounding organs containing cancerous cells are removed.
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[027] Diseases affecting the ureters ultimately result in damage to the
kidneys.
Chronic (e.g., slow, progressive) and acute (e.g., sudden) unilateral
obstructive
uropathy occurs when one of ureter becomes blocked, resulting in the backup of
urine
into the kidney. These disorders do not cause kidney failure if there are two
functioning kidneys; however, they may result in permanent damage to the
kidney
with distention of the renal pelvis and calysces or may cause hypertension.
Reflux
nephropathy also causes internal kidney structure damage from the backup of
urine by
the malfunction of the valve-like mechanisms between the ureters and bladder.
The
backup of urine may expose the kidneys to pyelonephritis and may lead to
chronic
renal failure and end-stage renal disease if not properly treated. Treatments
for these
diseases may involve antibiotics (e.g., for simple reflux) or may require
ureteral
reimplantation or reconstructive repair.
[028] Carcinoma of the ureter usually manifests as either transitional cell
cancer or
squamous cell carcinoma. This cancer is closely associated with and treated as
kidney
cancer. Cancer of the urethra is a rare, mostly malignant type of cancer that
occurs
more often in women than men. Prognosis and treatment depend on the anatomical
location and the depth of invasion. Superficial tumors, located in the
anterior urethra
are generally curable; however, deeply invasive lesions or lesions in the
posterior
urethra are rarely curable by any known combinations of therapy.
[029] The discovery of novel human excretory system associated polynucleotides
and the polypeptides encoded by them satisfies a need in the art by providing
new
compositions which are useful in the diagnosis, and treatment, prevention
and/or
prognosis of diseases or disorders of the urinary system, including, but not
limited to,
renal disorders (e.g., kidney failure, nephritis, blood vessel disorders of
kidney,
metabolic and congenital kidney disorders, urinary disorders of the kidney,
autoimmune disorders, sclerosis and necrosis, and electrolyte imbalance, and
kidney
cancer), bladder disorders (e.g, urinary tract infection, bladder obstruction,
urination
disorders, and bladder cancer), urether disorders (e.g., obstruction of the
ureter and
ureter cancer), urethra disorders (e.g., obstruction of the urethra and
urethra cancers)
andlor those disorders as discribed under "Urinary System Disorders" below.
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Summary of the Invention
[030] The present invention relates to novel excretory system related
polynucleotides, the polypeptides encoded by these polynucleotides herein
collectively
referred to as "excretory system antigens," and antibodies that
immunospecifically
bind these polypeptides, and the use of such excretory system polynucleotides,
antigens, and antibodies for detecting, treating, preventing and/or prognosing
disorders
of the excretory system, including, but not limited to, the presence of cancer
of
excretory system tissues and cancer metastases. More specifically, isolated
excretory
system nucleic acid molecules are provided encoding novel excretory system
polypeptides. Novel excretory system polypeptides and antibodies that bind to
these
polypeptides are provided. Also provided are vectors, host cells, and
recombinant and
synthetic methods for producing human excretory system 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 excretory system, including cancer of excretory
system tissues,
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 Description
Tables
[031] 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
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excretory system 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). Column 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 "Predicted Epitopes." In particular embodiments,
excretory system 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 determinant 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
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
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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 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 Bioteclmology 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 Table 1A, column 9 labeled "OMIM
Disease References)". A key to the OMIM reference identification numbers is
provided in Table 5.
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[032] 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).
[033] 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 1A. 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
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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.
[034] 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 excretory system
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 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).
[035] Table 4 provides the key to the tissuelcell source identifier code
disclosed in
Table 1A, column 7. Column 1 provides a key to the tissue/cell source
identifier code
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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
fiu-ther 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.
[036] 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/omixn~. Column 2 provides diseases associated
with the cytologic band disclosed in Table 1A, column 8, as determined from
the
Morbid Map database.
[037] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation
numbers of deposits made with the ATCC in connection with the present
application.
[038] Table 7 shows the cDNA libraries sequenced, tissue source description,
vector
information and ATCC designation numbers relating to these cDNA libraries.
[039] Table 8 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
[040] The following definitions are provided to facilitate understanding of
certain
terms used throughout this specification.
13
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
[041] 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.
[042] 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, a 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
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).
[043] As used herein, an "excretory system antigen" refers collectively to any
polynucleotide disclosed herein (e.g., a nucleic acid sequence contained in
SEQ ID
NO:X or the complement therof, 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
14
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
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 excretory system antigens have been
determined to
be predominantly expressed in excretory system tissues, including normal or
diseased
tissues (as shown in Table 1A column 7 and Table 4).
[044] 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 TD 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
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 1A, 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
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
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~of the deposit of microorganisms for the purposes of patent
procedure.
[045] 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 kb, 200 kb, 100 kb, 50 kb, 15
kb, 10 kb,
7.5 lcb, 5 kb, 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).
[046] 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
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% formamide, 5x SSC (750 mM NaCI, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution,
10%
dextran sulfate, and 20 ~,glml denatured, sheared salmon sperm DNA, followed
by
washing the filters in O.lx SSC at about 65 degree C.
[047] 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
16
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
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).
[048] 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.
[049] 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
"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).
[050] 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
17
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
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.
[051] 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 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 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).)
18
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
[052] "SEQ ID NO:X" refers to a polynucleotide sequence described, for
example, in
Tables 1A 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.
[053] "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).
[054] Table 1A summarizes some of the excretory system associated
polynucleotides
encompassed by the invention (including contig sequences (SEQ ID NO:X) and
clones (Clone ID NO:Z) and further summarizes certain characteristics of these
polynucleotides and the polypeptides encoded thereby.
Polynucleotides and Polypeptides
TABLE 1A
19
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
.,
.
,
o e~
U
N
o ~ , ,-a ,--i
G , ~ d'
r-,
O O
~ ~o
aU ~ a~
M N N
N N N N N ~ N N M N N N ~ ~ N N
-i ,~ ~-iO O O O d1~ ,~ fV
~ ''~~ d' v--ir--W--~e~-~1r-i r-tM O
"~ '''~ ~ O N
V i 000000~ ~ o '~~ ~ o o 0 0 0
.r ~ N N
~
~''~
~ o O O ~ o o o 0 0
x ~x x x x x x x x x ~~x x
~ ~ ~ ~
p ~rj ~ ~ t~ N N
N ~f7 M =
d'
tli
M .~ ~ ~ i
~ i M
fir. r., . ~, ~ ' ' ~' U ~
W ' ~ ?' ~ ~
~
-1
'" a 0 0 0 ~ U ~ ' 0 0
H o o .N o '~
o .~ .~ +,~ 0 0
~ o~~n ~ 0
N ~''?N Ov ~ ~n
d' oo N
p ~ ~ ~ ~ N ~
~ p
a , v~ ,~. vaa E~ U
~~ ~ C~'U 'U
G a c~ ~
'U
~
~ N M dwn \O\O l~oo a\O ~ N M ~n
A d' ' ' ' ' ~ 'd'
' ~'
M M M M M lpM M M M d d d ~ M M
M M M M M M M M M M M M M
O ~ .--~~ ~ N ool~ O
'''
'
~ M '~'-~~ ~,01 N N ~ N M M ~
~ N N ~ d- M N '
'-'ooN ' , ' , ~ ~ ~ ~ i ~ d-
~ O~I~~noo ~ a1 ~ N ~ ~ N ~ N
N ~ ~ M ~ N N '-'
~ '
~ N M d'V1 ~.~ l~o0 01O ~ N M d'
W ~'C y
~ ,-..i,-i.-i,-r ~,,~,-~ ,~.--i.~N N N N N N
b ~ l~N N M .-iM ~ ~O O~N l~ V1l~ ~ ~O
A
~ COO V1O1M l~O O~~ d"O 'd' ~OO~
M [~V1M ~ d'al ~ 00 O d'Ol O~M ~ N
o A 4n~nooM In ~ ~ 010o d'a1N d'd' O
'
N O N ~ d' d oo vWp ind-~ M 01 O M
W ~
o r m n a\ a100 a\a1 0000~n Two ~n n
d'ood W a\ ooN t~ooN M ,~ in
00M M D ~ ~D O ~ d'~OO~
~1
~ U ~1W ~ ~ ~ U U U U
c~ .. pqpal~w C7 C7 C7~ ~ ~ ~ U
o ~
~
~-' ~ ~ r~C~I~ f~ ~ pa !~0.1G~1 U U w
Z x
x x x x x x x x x x x x x x
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
N
-
O O O O O O O O d
O N p
x x x x x ~ x x o x a
h
~O MN ~ N N M ~ N ~ tn N N N N
~
O cV(V fV N N (V N w O O N O (V N
h
N ,-i,-~ ~ .~ ,~ .-~ ,-.,'"'~ ~ N .-,
Ra N O O O O
~ l0
~
O OO O O O O O O ~ ,-
x xx x x x x x ~Ox x x x x
~
M ~ ~ ~ ~ N ~ N ~
N l~ ~ ~ M
h ~-; N o 00
~ ~
cy
'~ ~ ~' "" ~, ~ O v~
~ H ~" N ~ .s~~,
H
~~ a P-~ C7 r~ H
v~ H
00 ~ 0 , 0 0 0 0 0 0
o 0 0 0
0
O N oo ~p
d-
\pN ~, ~ N '-~ ''; ~ ~ ; 01
~ ; h N ' ' '
~
o~., ~ o ~ ~ ~ ~ ~ s.
~ ~ ~, ~. .~ .~
v~ w~ ~
x ~ x
hoo p1 O ~ N M d- V~ lU h
dW d' dw n ~n ~n ~n ~n ~n ~n ~n ~n
M MM M M M M M M M M M M
O N
'd ~M N ~ N ~ M O j ~ d
'
i N M M i i M i
i i i
~ i
~ o~o ~"' N ~ M
~O ho0 01 O ~ N M d- ~ tn ~O h
N NN N M M M M M M M M M
d' ~O O~ 01 0o Ov ~ O ~n N oo v~
O O ~ d' O O d' V 00 h
~ 1M p1 01 h M h ~ O 7
O V~00 ~ M ~ d' l~ N V) p1 V'7 V1
N MO~ M 00 d'
M NO N h ~ h N ~ V~ ~ M h
d> ~~ ~ ~ ~ d> ~ d\ ~ ~ a1
d0-O h N ~ ~ h ~ O
d' h v '' ~
- '
~ ~ Z fx ~ ','~AG f~
x
~
~ w
w
x x x x x x x x w x x
x
21
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
O N' N ~ ~ O ~ O ~ ~ 00
d'
O O O ~ O ~ O O O N ~ p
x x x o~O x ~ x o~ a
~x~ ~ x~ ~ xo
M ~--~ d- N '"' cn ~ ~ N ~ M N cn '""~ . N N N N
N N O O " cV ~ N cV ~ cV N O cV iV iV N
N N '_'i e-.j t~l .-~ .--r O ~ e-.t N V'7 00 ~ ~ .--m--t
O O ~O 10 ~ O ~p O O r~ O O ~ M M O O O O
O O O O O O ~p O O O O O ~ d- O O O O
xx~x~x~~x~ x~x~x~xxaax.xxx
os °° ' ~i M r; d'
N
N i~ ~ M dh- ~ o p d- N ,-, N
cti crS s.. '~ ~ O , ~ ~ d O ,.-
~ L7 C7 ~ ~ ~ ,.~ ~ ~, C7 ~' ~ P~ ~
p p O O O O O O p O O O 0 O O
d~-~ NdON~ M~ eon X01 O.d
r ~ r r a~ b-0 b~0 ~ _c~ ~ ~ ~ >,.; a!, _~,
G7 ~ G7
due'
M M M M M l0 M M M M M M M M M
N~ NM d0'o\O~OM1N N~o~oN
M M M M ~ N r N ,~ M
r r N N
r r ~ r ~ ~ , r r r d. r r
oNo ~ ~ cNn ~ ~ '-' r.., N N
d1 O ~ N M r V'7 ~ l~ o0 d1 O ~ N
M Cf' '~' d" d' M ~ d' d' ~f' 'Cf' 'd' V1 ~ V7
l~ ~D tn ~ N O N l0 l'~ O ~ ~ O N Ov
M O 1~ Ov N N o0 00 00 ~D d' d' N Ov
O M ~' 01 M M O~ l~ V1 N ~ O 00 N O
N N ~ d- O ~ O O~ N t~ M ~ M O
N M 00 In [~ ~O M N 00 00 \O ~ l~ M V7
01 00 00 ~O 01 d\ 01 ~ l~ dl tn V~ V~ 00
N ~n M Ov d. N ~ N d- ~n ~ ~n d~ d'
f~ ~W w ~ ~ N ~ ~ ~ Z O d~
w ~ ~ ~ ~ ww ~ ~wr..~z,
xx x x x x x x xx x xxx
22
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
~~oo~~
[~ 01 M M
O O
l~ M 41 O~
00 00
O N M M d"
d'
M
C
N
C
N
N ~ cV cV
N .-~ N ,-
N N
x x x ~
O
x a
,~
l~ ~--~-~ d' d' N l~ N N M N
~ ~ 0 ~
O (V N O O O O O O O O
M ~ ~ l~
N ~ .-i N N N N N N N N
O O O O
l~ O O l~ ~ ~D l~ ~O ~O ~O ~O
~l ,-1 ,~ ,.~
O O O O O O O O O O O
x x x x x x x x x x x
~ ~ ~ ~
N ~ ~ ~
d' . d= -i d.~ ~ ~p l~ N O
N
N N ~ M 'd' , ~ ,-~ ,-yD oo ,--,
~ ~n ,-~ ,--~ ~
~
by .- ~ i
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HR H ' a a~
H
0 0 ~ 0 0 ~ ~ 0 0 0 0 0 0 0
0 0 0 0
o .~ ~ o o
.~
.~ .~~oo ,~~,~,~,~~oO
v--WI'e-t N ~ M M M M \O y --y0 [~ ~
'd' [~ rt
N N ~ N N ~ ~ ti ~
~ ' ~ ri
~ ~
W W w ~ W a ~, ~ .
~ ' a tn ~ .
v~ a H w C7 H ~
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M 'd' V~ l~ l~ 00 01 O ~ N M
M M M M M M M M M M M
a a N ~ ~ ~ h
c c d N o
n n ' o
i r ~ i i i N i M
~ i
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a1 N N M o 00 ~ ~O M
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N ~ ~ M M N M
M '<j"~ l0 (~ 00 d1 O ~--~ N M
N N l~ I~ N t~ ~ M o0
00 l~ ~n ~O d' O oo \O a1 ~ O
l~ ~ 01 l~ oo Ov o0 0o O
M 00 d' V1 V1 ~f' d' d' ~ ~ V'7
~ O> d\ ~
d" O 00 ~ l~ 'd' V7 00
p 00 M M ~p ~ O
U ~1 ~1 f~ t~ 0.1 ~ ~ W w C7
w w w
x x x x x x x x x x x
23
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
M O O ~' ~ l~ 00 lfl ~'
dl O~ 00 \O M
d' l~ V1 O~ M M O ~ 00 ~O
lfl N -~
O O ~n .-~ N V'7 0o O~ N
l~ l~ d\ -a
00 00 ~ O O O O O ~ ~ .-~
,-~ N N
M
~D l~ ~ l0 l0 l~ l0 ~ l~
~ l~
N
iV cV O
O O
, "t~ ~d O
~
c
a
wi N M d- N N v~ M
~
~
O O p O O O O O
l~ d' t~
N N N N N N N N
O O O O O O O O
O O O
a a a
~i
~
' M M O ~
d . ~ ~ N
-~ oo '
N
~ i
i ~ ~ ~ ., cC
v~ ~ s.'.
s
a ~x Ha x ~ ~
0 0
0 0 0 0 0 0
0 0 0
N ~ ~ M N ~ 0
~ ~
N y0
~
_ ~ ~. ~ c'~~.~
~. y"
C7 ~ ~ p~ ~ E-~C7 WU
v~ E-~ v 0.
a\
M M M M M M M M
O M
N M ~ N
N M d'
, '
N ~ l~ N ~ N ~
N ,--,
M O ~D o0 l~ l~d"
M 00 d' 01 d'd' 'd'
O1 d\ d\
M 00 ~--aO l0
~ w x
w ~
w x x
x
24
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
to o ~ ~ ,d o o
[~ 01 M M O O O O
M Q1 01 00 00 00
00
O N M M d d' d' '~'
M O
M
C
N
M ~ M
'
d
x ,~~M a O
Nxa
N N ,--i N N N ~ N
d=
N .-i
O O N w ~ O O O w O
O vo
x x x ~a x x x ~x
~ aa ~
O N
N N M n
'~
o ~O o 1 o N
o N M ~ ~ t ~ o O N
a ~ o O
j ;
~' ~~~~ ~a ~ ~
~
o L7 H~~ f , U ~
0 ~ 0 0 0 0 0 0 o ~ o ~ o
0 0 0 0 o
O ~ d' ~ M ~O ~ ~ 1nO
N O M 00 N M
M M M
M d' lp ~ ~ 00 ~ ~
~ ~ 00
a-r cCi cd p b~ ~ ~, ~, v~
~ p j, y~., ~,
~ s~,
9,
_ _ _ ~~ ~ _ _
_ w~c~~ ~ ~x
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CA 02395693 2002-06-25
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CA 02395693 2002-06-25
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CA 02395693 2002-06-25
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CA 02395693 2002-06-25
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CA 02395693 2002-06-25
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CA 02395693 2002-06-25
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[055] 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.
[056] 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 excretory system 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.
[057] 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.
[058] 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
predicted epitopes as described in Table 1A. It will be appreciated that
depending on
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
the analytical criteria used to predict antigenic determinants, the exact
address of the
determinant may vary slightly.
[059] 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 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 predominant and/or specif c tissue and/or cell expression. The
sequences
disclosed herein have been determined to be predominantly expressed in
excretory
46
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
system tissues, including normal and diseased excretory system tissues (See
Table 1A,
column 7 and Table 4).
[060] 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.
[061] 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.
[062] 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
(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
47
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
numbers (column 1), and provides a description of the associated disease in
Column
2.
TABLE 1B
Clone ID SEQ CONTIG BAC ID: SEQ ID EXON
A
NO:Z ID LD: NO:B From-To
NO:X
H7MBC38 12 705707 AL035088 651 1-86
H7MBC38 12 705707 AL035088 652 1-189
HBAGK69 16 881903 AL160251 653 1-830
HBAGK69 16 881903 AC007721 654 1-805
1510-1798
4114-4607
HBAGK69 16 881903 AC007721 655 1-100
HBAGK69 16 881903 AC007721 656 1-1263
HBAMC12 18 968816 AC009554 657 1-375
HBAMC12 18 968816 AC009554 658 1-469
HBAMC47 19 854049 AL139799 659 1-507
HBAMC92 21 572947 AC023353 660 1-440
HBAMC92 21 572947 AC064832 661 1-447
HCKAA 13 22 534965 AC040919 662 1-652
HCKAA13 22 534965 AP001845 663 1-652
HCKAA13 22 534965 AP001374 664 1-652
HCKAA13 22 534965 AC040919 665 1-191
201-308
454-1086
3154-3282
3440-3857
4633-4941
HCKAA13 22 534965 AP001845 666 1-271
HCKAA13 22 534965 AP001374 667 1-308
HCKAA41 23 694397 AC018663 668 I-402
HCKAA41 23 694397 AC018663 669 1-248
HCKAA41 23 694397 AC018663 670 1-1609
HFKEA55 25 530256 AP000859 671 1-108
205-628
1117-1259
1538-1745
2172-2690
2786-3154
3246-3403
4031-4131
4234-6030
48
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
6184-7366
HFKEA55 25 530256 AC018780 672 1-108
205-627
1088-1230
1509-1716
2143-2661
2757-3125
3217-3374
4002-4102
4205-6001
6155-7337
HFKEA55 25 530256 AP001785 673 1-99
368-765
963-1141
1966-2078
2175-2597
3058-3200
3479-3686
4113-4631
4727-5095
5187-5344.
5973-6073
6176-7972
8126-9308
HFKEA55 25 530256 AP000859 674 1-428
HFKEA55 25 530256 AC018780 675 1-428
HFKEA55 25 530256 AP001785 676 1-224
HFKEA55 25 530256 AP001785 677 1-428
HFKEA65 26 932094 AC027598 678 1-638
HFKEA65 26 932094 AC002366 679 1-649
HFKEA65 26 932094 AC002366 680 1-1471
2332-2618
2700-3922
HFKEH40 27' 523591 AC021138 681 1-426
HFKEH40 27 523591 AC008737 682 1-431
HFKEH40 27 523591 AC021138 683 1-162
420-504
1469-1565
2108-2321
2421-4280
HFKEH40 27 523591 AC008737 684 1-556
568-1178
HFKEH40 27 523591 AC008737 685 1-32
757-942
3844-4463
5615-5787
5904-6600
49
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
6947-7075
7265-7475
7586-8105
8828-8996
10237-10411
10669-10753
11720-11816
12360-12573
12673-14535
HFKEJ77 29 529919 AC073640 686 1-238
HFKEJ77 29 529919 AC020928 687 1-667
HFKEJ77 29 529919 AC073266 688 1-667
HFKEJ77 29 529919 AC073640 689 1-654
HFKEJ77 29 529919 AC020928 690 1-650
HFKEJ77 29 529919 AC073266 691 1-676
HFKEJ77 29 529919 AC073266 692 1-650
HFKEM26 30 573949 AC004982 693 1-420
1344-1738
2682-2852
5545-5607
5615-5894
6449-6611
8468-8843
12028-12179
12613-13049
13304-13617
13895-14373
14981-15243
16721-17118
17305-21277
22988-23119
23726-26190
HFKEM26 30 573949 AC004982 694 1-101
HFKEM70 31 963178 AL136528 695 1-892
1133-2737
3760-4178
4229-4310
4391-4668
5176-5753
6229-6333
7984-8053
HFKEN74 32 974309 AC008878 696 1-927
HFKEN74 32 974309 AC008878 697 1-471
HFKER27 33 926744 AL157386 698 1-416
HFKER27 33 926744 AL157386 699 1-129
HFKER69 34 918290 AC013396 700 1-502
HFKER69 34 918290 AC013396 701 1-277
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
502-616
3690-3819
5390-5441
HFKEU09 35 855045 AC019214 702 1-100
482-945
2126-2202
2478-2600
3182-3343
5248-5664
6502-6587
6940-7103
7254-7391
7680-7733
7971-8806
8812-9045
9281-11575
11591-11897
12403-12621
12700-12744
HFKEU09 35 855045 AC015551 703 1-100
482-945
2100-2176
2452-2574
3116-3277
5189-5605
6442-6527
6880-7043
7194-7331
7620-7673
7911-8745
8751-8984
9220-11514
11530-11836
12342-12560
12639-12683
HFKEU09 35 855045 AC019214 704 1-167
257-354
575-718
990-1289
1379-1530
1982-2132
2584-2814
2928-3098
3416-3717
4047-4314
4517-4667
4990-5016
51
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
5147-5299
5360-6252
HFKEU09 35 855045 AC015551 705 1-167
257-354
575-672
992-1291
1381-1532
1984-2134
2586-2816
2930-3100
3431-3714
4045-4312
4515-4665
4986-5012
5143-5294
5355-6248
HFKEW23 36 854952 AL353801 706 1-727
HFKEW23 36 854952 AL353801 707 I-837
HFKFB42 39 522037 AL109839 708 1-1091
1327-1458
2095-2434
5393-5501
6605-7036
9708-9880
10516-10580
10796-11504
12943-13099
13380-13848
14050-144.20
16213-17031
18080-18301
HFKFE05 40 932306 AL049775 709 1-383
HFKFE05 40 932306 AL049775 710 1-370
HFKFE59 42 854991 AC007601 711 1-593
-
HFKFE59 42 854991 AC037438 712 1-593
HFKFE59 42 854991 AC007601 713 1-220
HFKFE59 42 854991 AC037438 714 1-220
HFKFI22 44 930982 AC013396 715 1-459
HFKFI22 44 930982 AC013396 716 1-157
HFKFI22 44. 930982 AC013396 717 1-200
HFKFN66 49 511046 AL359389 718 1-1384
1747-2350
HFKF075 50 573840 AC073619 719 1-335
HFKF075 50 573840 AL354745 720 1-335
HFKF075 50 573840 AC073619 721 1-224
HFKF075 50 573840 AL354745 722 1-176
HFKGC14 53 573785 AC019124 723 1-480
52
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HFKGC14 53 573785 AC019124 724 1-264
HFKGD10 54 968172 AC015883 725 1-480
HFKGD10 54 968172 AC015883 726 1-522
HFKGD48 55 954952 AC010368 727 1-420
HFKGD48 55 954952 AC010368 728 I-444
HFKHB46 56 965767 AC055792 729 1-47
1466-1551
1881-3009
HFKHB46 56 965767 AL035405 730 1-87
3979-4108
4445-4939
5138-5294
7382-7428
8845-8930
9260-10388
HFKHB46 56 965767 AC055792 731 1-1559
HFKHB46 56 965767 AL035405 732 1-1559
HFKHB67 57 965847 AC021857 733 1-35
232-952
HFKHD38 60 854861 AC040995 734 1-612
HFKHD38 60 854861 AC069030 735 1-612
HFKHD38 60 854861 AC069030 736 1-590
HFKHF54 62 855118 AC067943 737 1-879
3052-3359
HFKHF54 62 855118 AC067943 738 1-465
HFKHF54 62 855118 AC067943 739 1-623
HFKHG06 63 965906 AC004643 740 1-1151
1269-1388
1489-1633
2129-2539
2655-2804
3076-3506
3614-3734
3804-5071
5163-5218
5299-5402
5493-5570
5666-6156
6257-6375
6671-7129
7353-7440
7709-8
851
9077-9456
HFKHG06 63 965906 AC004643 741 1-246
HFKHG06 63 965906 AC004643 742 1-616
694-833
1064-1175
53
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
1420-2038
HFKHZ39 66 928269 AC009123 743 1-635
1708-1794
HFKHZ39 66 928269 AC009123 744 1-118
HFKID43 67 854988 AC022116 745 1-537
HFKID43 67 854988 AC022116 746 1-564
HFKID43 67 854988 AC022116 747 1-672
HFKID48 68 879771 AC018730 748 1-53
1553-1660
4422-4614
4641-5155
5981-6234
HFKID48 68 879771 AC018730 749 1-59
352-2091
HFKID48 68 879771 AC018730 750 1-140
HFKIFOl 69 914971 AC015541 751 1-210
416-623
845-1481
1820-2565
3128-3565
3626-4034
4318-4416
HFKIF01 69 914.971 AC018501 752 1-210
416-623
845-1481
1820-2565
3128-3565
3626-4034.
4318-4416
HFKIF01 69 914971 AC018816 753 1-210
416-623
845-1481
1820-2565
3128-3565
3626-4034
4318-4416
HFKIFOl 69 914971 AC015541 754 1-382
HFKIFOl 69 914971 AC018501 755 1-382
HFKIFOl 69 914971 AC018816 756 1-382
HFKIH06 71 934051 AL021878 757 1-814
1532-1652
1791-2285
2588-2900
3277-3506
4106-4504
4651-5299
5432-6325
54
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
6595-6648
7112-7229
9857-10545
11800-12045
13750-13860
14992-15089
15126-15234
16713-16887
17559-17687
21811-23565
24540-24804
25938-26263
26678-26895
27833-28312
32924-32968
34093-34274
35551-35990
36375-36727
37669-39039
39351-39826
HFKIH06 71 934051 AL021878 758 1-359
HFKIH06 71 934051 AL021878 759 1-1737
HFKII11 73 965846 AL139330 760 1-468
HFKII11 73 965846 AL139330 761 1-100
HFKIIll 73 965846 AL139330 762 1-481
HFKIP11 75 965072 AC008743 763 1-191
774-896
1248-1398
1656-1742
1855-2023
2089-2532
2895-3068
HFKIP11 75 965072 AC020909 764 1-191
774-896
1248-1398
1656-1742
1855-2023
2089-2532
2895-3068
HFKIP11 75 965072 AC008743 765 1-329
746-911
1114-1425
3498-3617
HFKIPlI 75 965072 AC020909 766 1-329
749-839
HFKIR40 76 883093 AC019071 767 1-372
414-512
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
1060-1165
1659-1928
2070-2230
2342-2438
2506-2617
2645-2849
3087-3559
3767-4021
4589-4762
5003-5323
5558-5807
6092-6922
HFKIR40 76 883093 AC019071 768 1-412
HFKIS93 77 854862 AC031984 769 1-1431
HFKIS93 77 854862 AC026539 770 1-2935
HFKIS93 77 854862 AL050318 771 1-1024
1255-1346
1550-1761
1936-2203
3305-3853
4515-4876
4908-5677
7375-7473
8813-11992
HFKIS93 77 854862 AL050318 772 1-1487
HFKIS93 77 854862 AL050318 773 1-115
HFKIV55 78 922739 AC068248 774 1-475
HFKIV 55 78 922739 AC073160 775 1-99
1373-1952
2365-2666
4560-4982
5137-5611
6290-6784
7269-7317
8500-8559
HFKIVSS 78 922739 AC068248 776 1-423
HFKIV55 78 922739 AC068248 777 1-495
HFKIV55 78 922739 AC073160 778 1-275
HFKIX76 80 854993 AC008074 779 1-88
305-539
737-973
1093-1180
1791-1973
3574-6888
HFKIX76 80 854993 AC008074 780 1-186
HFKJE03 81 959898 AC026093 781 1-482
HFKJK90 82 926511 AC022401 782 1-612
56
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HFKJK90 82 926511 AC022401 783 1-186
HFKJL17 83 854829 AC025085 784 1-699
774-815
1120-1886
1981-4592
4908-5627
HFKJL17 83 854829 AL162581 785 1-699
774-815
1120-1886
1981-2345
2586-2723
2809-4595
4911-5626
8746-9204
9575-10225
HFKJL17 83 854829 AL121969~ 786 1-699
774-815
1120-1886
1981-4592
4908-5627
8746-9204
9575-10225
HFKJL36 84 930422 AC046197 787 1-548
HFKJL36 84 930422 AC007465 788 1-547
HFKJPO8 85 918312 AC044859 789 1-365
HFKJW29 86 932310 AP001822 790 1-385
2074-2451
HFKJW29 86 932310 AP000761 791 1-385
2074-2451
HFKJW29 86 932310 AC020997 792 1-351
HFKJW29 86 932310 AP000761 793 1-75
224-447
1437-3370
HFKJW29 86 932310 AP001822 794 1-75
222-443
1435-3369
HFKKG09 87 854888 AL359926 795 1-591
HFKKG09 87 854888 AL359175 796 1-591
HFKKG09 87 854888 AL359926 797 1-266
HFKKG09 87 854888 AL359175 798 1-266
HFKKM02 88 918260 AL356781 799 1-300
HFKKM02 88 918260 AL356781 800 1-177
HFKKM61 89 855082 AC010932 801 1-533
HFKKM61 89 855082 AL133510 802 1-533
HFKKS48 92 854927 AC008749 803 1-541
HFKKS48 92 854927 AC026803 804 1-497
57
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
1331-1441
1539-2079
2914-3038
4330-4471
4831-4987
5304-5446
HFKKS48 92 854927 AC026803 805 1-240
HFKKS48 92 854927 AC026803 806 1-289
HFKLD58 95 918235 AC062037 807 1-674
985-1160
HFKLD58 95 918235 AC062037 808 1-444
HFKLD58 95 918235 AC062037 809 1-1242
HFKLJ15 96 918247 AL139260 810 1-503
HFKLJ15 96 918247 AL139260 811 1-283
HFKLJ15 96 918247 AL139260 812 1-128
HFKLL08 97 958065 AC073410 813 1-878
HFKLL08 97 958065 AC073410 814 1-248
HFKLX38 99 880220 AL136383 815 1-32
1288-1454
1561-1646
3840-4700
5482-6798
HFKLY01 100 914907 AC013480 816 1-154
1410-1729
3081-3322
3480-4064
4551-4675
5305-6348
6596-6706
6872-7414
7421-8472
8530-8709
8913-9839
HFKLY01 100 914907 AC013480 817 1-534
HKDAF84 104 781937 AC020633 818 1-30
341-7207
HKDAF84 104 781937 AC020633 819 1-254
HKDAF84 104 781937 AC020633 820 1-583
HKDBL02 107 920498 AC023124 821 1-506
HKDBL02 107 920498 AC012361 822 1-506
HKIBBO8 108 960371 AC011639 823 1-476
HKIBB08 108 960371 AC009679 824 1-641
HKIBB08 108 960371 AC016824 825 1-641
HKIBB08 108 960371 AC009679 826 1-218
HKIBB08 108 960371 AC009679 827 1-195
HKIBB08 108 960371 AC016824 828 1-151
58
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
166-224
~
HKIBB08 108 960371 AC016824 829 1-195
HKIBB81 109 525592 AL118558 830 ~ 1-339
HKIBB81 109 525592 AC022281 831 1-339
HKIBB81 109 525592 AL118558 832 1-730
HKIBB81 109 525592 AL118558 833 1-90
HKIBB81 109 525592 AC022281 834 1-90
HKIBB81 109 525592 AC022281 835 1-624
HKIMC34 110 573424 AC023906 836 1-653
HKIMC34 110 573424 AC023906 837 1-396
HKIXA37 112 735445 AC015941 838 1-1559
HKIXA37 112 735445 AC021852 839 1-1745
2030-2499
HKIXA37 112 735445 AC021852 840 1-335
HKIXD42 116 683533 AC027225 841 1-419
HKIXD42 116 683533 AF127577 842 1-418
HKIXD42 116 683533 AF127577 843 1-143
466-768
HKIXI82 119 779905 AC011448 844 1-141
2130-2301
3021-3567
3611-3875
4085-4375
4486-491.6
4930-5579
5726-5837
5916-6061
7070-7973
HKIXI82 119 779905 AC011448 845 1-102
HKIXI82 119 779905 AC011448 846 1-544
HKIXX58 124 666516 AP000941 847 1-401
HKIXX58 124 666516 AP000714 848 1-401
HKIXX58 124 666516 AP000846 849 1-400
HKIXX58 124 666516 AP000869 850 1-186
343-743
HKIXX58 124 666516 AP000941 851 1-292
HKIXX58 124 666516 AP000714 852 1-261
HKIXX58 124 666516 AP000846 853 1-292
HKIXX58 124 666516 AP000869 854 1-292
HKIYF45 126 970756 AC062010 855 1-446
HKIYF45 126 970756 282243 856 1-446
HKIYF45 126 970756 AC062010 857 1-268
HKIYF45 126 970756 AC062010 858 1-117
HKIYF45 126 970756 282243 859 1-268
HKIYF45 I26 970756 282243 860 I-II7
HKIYJ26 I27 927503 AC018984 861 1-593
59
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
2673-2789
3069-3213
HKIYJ63 128 703320 AC008807 862 1-1113
HKIYJ63 128 703320 AC026439 863 1-1113
HKIYJ63 128 703320 AC008807 864 1-849
HKIYJ63 128 703320 AC008807 865 1-128
HKIYJ63 128 703320 AC026439 866 1-115
HKIYJ63 128 703320 AC026439 867 1-847
HKIYQ89 130 625636 AL158834 868 1-728
1030-1238
15 82-1665
HKIYQ89 130 625636 AL158834 869 1-134
506-1282
HKMAA07 131 954358 AC012124 870 1-260
HKMAA07 131 954358 AF176680 871 1-261
HKMAA07 131 954358 AC062010 872 1-226
HKMAA07 131 954358 AC015871 873 1-118
HKMAA07 131 954358 AC055735 874 1-143
HKMAA07 131 954358 AC046131 875 1-238
HKMAA07 131 954358 AC044.892 876 1-168
HKMAA07 131 954358 AC036181 877 1-119
HKMAA07 131 954358 AC006451 878 1-992
1238-2766
2970-3258
4727-4925
5365-5841
6281-6790
6934-7264
8088-8627
9080-9148
10250-10345
11254-12473
12516-13371
HKMAA07 131 954358 AC006451 879 1-264
HKMBAI7 I34 534523 AC005293 880 1-301
HKMBA17 134 534523 AC005293 881 1-421
HKMLD48 136 575873 AC012626 882 1-76
83-286
575-1023
3442-3552
3568-3747
5131-5259
6073-6318
HKMLD48 136 575873 AC020816 883 1-443
HKMLD48 136 575873 AC040173 884 1-76
83-286
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
s75-1o23
3445-3736
5120-5248
6062-6307
HKMLD48 136 575873 AC012626 885 1-606
HKMLD48 136 575873 AC040173 886 1-605
HKMLS46 140 575879 AC010674 887 1-321
HKMLS46 140 575879 AC010674 888 1-I33
985-1071
1887-2686
HKMLS46 140 575879 AC010674 889 1-149
HKMMF21 142 969002 AL139154 890 1-431
598-1537
2081-2194
2838-2906
3531-3905
3964-4935
6101-6251
6329-6908
8000-8334
8505-8691
8954-9485
9569-10044
10639-11625
12043-12128
12460-12700
13146-13660
HKMMF21 142 969002 AL139154 891 1-3258
3398-3707
HKMMF2I 142 969002 AL139I54 592 I-359
HKMMG47 143 720379 AL160286 893 1-29
1089-2148
HKMMJ67 144 575750 AC013583 894 1-133
HKMMJ67 144 575750 AC015776 895 1-101
HKMMJ67 144 575750 AC008439 896 1-125
HKMMJ67 144 575750 AC021923 897 1-104
HKMMJ67 144 575750 AC020834 898 1-360
HKMMJ67 144 575750 AC021923 899 I-139
935-1002
HKMMJ67 I44 575750 AC020834 900 1-2033
HRABC50 I48 720370 AC058800 901 1-722
HRABC50 148 720370 AL390294 902 1-722
HRABC50 148 720370 AL159172 903 1-722
HRABC50 I48 720370 AC058800 904 1-709
HRABC50 I48 720370 AL390294 905 1-709
HRABC50 I48 720370 AL159172 906 1-707
61
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HRABT03 150 923842 AC010310 907 1-547
HRABT03 150 923842 AC010308 _ 1-547
908
HRABT03 150 923842 AC010358 909 1-547
HRABT03 150 923842 AC010473 910 1-547
HRABT03 150 923842 AC010310 911 1-112
HRABT03 150 923842 AC010308 912 1-112
HRABT03 150 923842 AC010473 913 1-112
HRACE09 152 625425 AL359176 914 1-1635
HRACE09 152 625425 AC026900 915 1-2078
2378-2968
3217-4094
4180-4659
4664-6770
6814-7224
HRACE09 152 625425 AC060753 916 1-3585
3715-4305
4546-5403
HRACE09 152 625425 AL109937 917 1-142
HRACE09 152 625425 AL356004 918 1-1636
HRACE09 152 625425 AL390296 919 1-142
HRACE09 152 625425 AC027209 920 1-2282
2412-3002
3469-4126
4211-6401
6472-6802
6854-7412
7505-7877
8817-9469
9531-10355
11182-11518
11841-12049
12639-12673
13026-13612
13639-15711
15745-16135
16215-16907
17307-17773
HRACE09 152 625425 AL049742 921 1-1857
1863-2229
2699-5270
5474-7109
HRACE09 152 625425 AL359176 922 1-2152
HRACE09 152 625425 AC026900 923 1-1310
HRACE09 152 625425 AC060753 924 1-292
HRACE09 152 625425 AL109937 925 1-164
HRACE09 152 625425 AL359176 926 1-1407
HRACE09 152 625425 AC026900 927 1-374
62
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HRACE09 152 625425 AL109937 928 1-666
680-1146
HRACE09 152 625425 AL356004 929 1-761
796-1915
2046-2636
2885-3700
3848-6031
6105-6435
6750-7508
8743-9098
HRACE09 152 625425 AL390296 930 1-665
683-1151
HRACE09 152 625425 AL390296 931 1-502
HRACE09 152 625425 AC027209 932 1-423
HRACE09 152 625425 AC027209 933 1-1301
HRACE09 152 625425 AL049742 934 1-1554
1998-3175
HRACE09 152 625425 AL049742 935 1-762
797-1916
2047-2637
2886-3701
3849-4328
4614-5981
6055-6385
6700-7458
8693-9048
HRACL64 155 674012 AL049780 936 1-33
2486-3028
3593-3681
3828-3889
6854-6907
7350-7545
9474-9717
9723-10077
10527-11087
11166-11594
13231-13400
14027-14327
15465-15573
16076-16238
19959-20368
21799-21859
23602-24438
24694-24797
25509-25961
26557-27070
28587-28750
63
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
28843-29088
29996-30215
30398-30446
32541-35115
HRACL64 155 674012 AC007055 937 1-33
2488-3030
3594-3682
3 829-3
890
6855-6908
7351-7546
9459-9702
9708-10062
10512-11071
11150-11578
13215-13384
14006-14311
15449-15557
16060-16222
19946-20355
21782-21842
23585-24421
25493-25945
26541-27054
28572-28735
28828-29073
29981-30200
32526-35100
HRACL64 155 674012 AL049780 938 1-133
HRACL64 155 674012 AL049780 939 1-295
HRACL64 155 674012 AC007055 940 1-266
HRACL64 155 674012 AC007055 941 1-133
HRACW03 157 923428 AC073879 942 1-490
HRACW36 159 708201 AC005746 943 1-556
HRACW36 159 708201 AC005746 944 1-383
HRACW69 160 708769 AL162424 945 1-539
HRACW69 160 708769 AL162424 946 1-117
HRADP39 166 732757 AC024729 947 1-502
HRAEC03 167 923401 AC025710 948 1-561
HRAEC03 167 923401 AL359353 949 1-561
HRAEC03 167 923401 AC025710 950 1-47
2659-3236
4025-6604
HRAEC03 167 923401 AL359353 951 1-47
2659-3236
4025-6604
HRAEC03 167 923401 AC025710 952 1-389
HRAEC03 167 923401 AL359353 953 1-390
64
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HRKAB 10 168 968272 AC018431 954 1-459
HRKAB 10 168 968272 ALl 17354 955 1-201
318-397
1689-2518
2909-3252
3296-4192
4457-4937
5239-5389
8114-8209
9102-9579
10318-10448
HRKAB 10 168 968272 AL117354 956 1-160
HRKAB10 168 968272 AL117354 957 1-121
HRKAF31 171 698347 AC068942 958 1-597
1981-2374
2510-2735
3032-3130
3193-4169
4618-4792
HRKAF31 171 698347 AC016590 959 1-976
HRKAF31 171 698347 AC012309 960 1-491
594-1632
3019-3412
3548-3774
4071-4169
4232-5207
5656-5830
HRKAF31 171 698347 AC068942 961 1-304
HRKAF31 171 698347 AC016590 962 1-99
HRKAF31 171 698347 AC016590 963 1-304
HRKAF31 171 698347 AC012309 964 1-148
HRKAF31 171 698347 AC012309 965 ~ 1-304
HRKAC62 173 742429 AC015919 966 1-293
HRKAC62 173 742429 AC015919 967 1-293
HRKAC62 173 742429 AC007763 968 1-366
376-740
HRKAC62 173 742429 AC016704 969 1-366
376-740
HRKAC62 173 742429 AC015920 970 1-366
376-668
HRKAC62 173 742429 AC007763 971 1-266
1286-1478
HRKAC62 173 742429 AC007763 972 1-87
HRKAC62 173 742429 AC016704 973 1-266
1284-1490
3464-3826
5158-5320
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
6212-6300
7623-7838
HRI~AC62 173 742429 AC016704 974 1-87
HRKAC62 173 742429 AC015920 975 1-87
HRKAA82 175 880783 AC026556 976 1-1168
HRKAA82 175 880783 AC068243 977 1-1168
HRKAA82 175 880783 AC026556 978 1-626
HRKAA82 175 880783 AC026556 979 1-294
HRI~AA82 175 880783 AC068243 980 1-626
HRKAA82 175 880783 AC068243 981 1-279
HRAED30 177 867176 AC040169 982 1-143
1045-1358
1402-2764
3033-3632
3711-4143
6385-7445
8802-9689
HRAEB43 178 714842 AP000096 983 1-2486
3072-345
8
3479-3937
5086-5458
HRAEB43 178 714842 AP000200 984 1-2486
3072-3458
3479-3937
5086-5458
HRAEB43 178 714842 AP000240 985 1-387
408-866
HRAEB43 178 714842 AP000096 986 1-89
HRAEB43 178 714842 AP000200 987 1-89
HRAEB43 178 714842 AP000240 988 1-2486
HRAEB43 178 714842 AP000240 989 1-357
HRAEB29 179 690179 AC024885 990 1-3448
HRADR43 1.83 714857 AL359997 991 1-1563
HRADR21 184 830039 AC007386 992 1-391
HRADK79 I87 774597 ACOI6400 993 1-61
960-1187
1265-1372
1519-1584
1853-2385
2428-2786
3031-4137
HRADI~79 187 774597 AC016400 994 1-337
HRADE49 I88 722595 AL136109 995 1-3030
3212-3814
9089-9408
11724-12242
66
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
12308-12959
23153-23254
24314-26013
HRADE49 188 722595 AC023147 996 1-3041
3223-3825
9096-9415
11730-12248
12314-12965
23152-23259
24319-26018
HRADFA-9 188 722595 AC022219 997 1-3030
3212-3814
9084-9403
11717-12235
12301-12952
23145-23246
24307-26006
HRADFA~9 188 722595 AL136109 998 1-344
HRADFA-9 188 722595 AL136109 999 1-174
HRADE49 188 722595 AC023147 1000 1-122
HRADE49 188 722595 AC022219 1001 1-191
HRADE49 188 722595 AC022219 1002 1-344
HRADA93 190 792001 AC010463 1003 1-56
380-483
579-900
1742-1988
HRADA93 190 792001 AC010463 1004 1-27
934-1247
2557-2685
2850-2960
HRACV73 193 764141 AC013322 1005 1-421
HRACV73 193 764141 AC006038 1006 1-421
HRACV73 193 764141 AC013322 1007 1-308
HRACV73 193 764141 AC013322 1008 1-447
HRACV73 193 764141 AC006038 1009 1-311
HRACV73 193 764141 AC006038 1010 1-447
HRACR57 196 745443 AC055863 1011 1-946
HRACR57 196 745443 AC073922 1012 1-566
HRACR57 196 745443 AC055863 1013 1-284
HRACR49 197 722524 AC009568 1014 1-297
HRACR49 197 722524 AC011633 1015 1-297
HRACR49 197 722524 AC011633 1016 1-335
HRACF04 200 615036 AL354833 1017 1-316
564-1413
1607-1800
2119-2419
67
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
3091-4057
4252-4680
7398-7790
8083-8259
8450-8642
9096-11423
11866-13435
HRACF04 200 615036 AL354833 1018 1-121
HRACD65 201 918345 AL031297 1019 1-131
679-912
1349-1654
5029-5252
9376-9493
9736-10204
10953-11302
11525-12222
16952-17243
20848-21153
21457-22062
25001-25396
26949-27023
28082-28430
29411-29548
29680-30123
30797-30981
31120-31308
31959-32425
34705-34876
35061-35527
37354-37502
37933-38190
38381-39650
39839-41256
41488-41511
41519-41916
41983-42104
42275-42855
43391-43461
43574-43656
43857-44292
44317-44421
46115-46231
47800-48512
48841-49208
50149-50247
51377-51502
52685-52984
68
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
53118-53307
56807-56996
61413-61503
63173-63224
63579-63659
64464-64659
66082-66208
66720-66871
67311-67472
70450-70513
71257-71408
72162-72215
HRACD65 201 918345 AL031297 1020 1-648
HRACC06 202 867214 AC006347 1021 1-544
HRACC06 202 867214 AC006347 1022 1-532
750-1892
HRACC06 202 867214 AC006347 1023 1-806
HRABZ67 206 751496 AC004542 1024 1-4332
44.01-10126
HRABZ67 206 751496 AC004542 1025 1-195
HRABR30 207 691157 AL161447 1026 1-611
HRABR30 207 691157 AL161447 1027 1-368
HRABR30 207 691157 AL161447 1028 1-247
HRAB060 208 871380 AL359538 1029 1-548
HRAB060 208 871380 AC013721 1030 1-155
789-836
978-1056
1143-1690
1707-1999
3621-3762
3849-4168
4886-5085
5994-6080
6441-6513
7498-8147
8651-9220
10275-10381
10687-10988
12904-13077
13332-13417
13623-14029
16366-16446
17409-17507
18127-18230
18280-18693
18709-19020
20417-20713
69
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
24618-25701
27490-28016
28378-28720
29486-29516
30017-30581
30641-31070
31505-31744
32436-32725
33178-33363
33487-33630
34031-34157
34251-34807
35077-35422
35460-35863
36979-37467
37750-3
8006
38169-39169
HRAB060 208 871380 AL359763 1031 1-155
789-836
978-1056
1143-1690
1702-1992
3622-3763
3850-4170
4888-5087
5996-6082
6443-6515
7500-8149
8653-9222
10281-103
87
10687-10994
12910-13083
13338-13423
13629-14035
16372-16452
17415-17511
18133-18236
18286-18700
18716-19027
20424-20720
24625-25708
27497-28023
28078-28194
28385-28727
29493-29523
30024-30588
30648-31077
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
31512-31751
32443-32732
33168-33347
33494-33637
34038-34164
34258-34814
35084-35429
35467-35870
36985-37473
37756-38012
38175-39175
HRAB060 208 871380 AL359538 1032 1-299
HRAB060 208 871380 AC013721 1033 1-397
HRAB060 208 871380 AC013721 1034 1-439
HRAB060 208 871380 AL359763 1035 1-439
HRAB060 208 871380 AL359763 1036 1-397
HRAB008 209 959104 AC025597 1037 1-547
HRAB008 209 959104 AL355834 1038 1-547
HRAB008 209 959104 AL161804 1039 1-547
HRAB008 209 959104 AC025597 1040 1-428
550-1154
HRAB008 209 959104 AL355834 1041 1-439
1615-2106
HRABO08 209 959104 AL355834 1042 1-428
550-1154
HRAB008 209 959104 AL161804 1043 1-428
550-1008
HRAB008 209 959104 AL161804 1044 1-439
1615-2106
HRABM45 210 875735 AC003663 1045 1-1827
HRABM45 210 875735 AC003663 1046 I-559
HRABM45 210 875735 AC003663 1047 1-250
1981-2340
2645-2782
3859-4046
4073-4241
6066-6166
6314-6452
10021-10278
10529-10697
11001-11141
11615-11751
11925-12464
12728-15037
HRABD15 211 867228 AC024944 1048 1-573
670-1471
1710-2231
71
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
_ ~ 2681-2716
HRABD15 211 867228 AC024911 1049 1-573
670-1471
1710-2231
2681-2716
HRABD15 211 867228 AC024944 1050 1-545
HRABD15 211 867228 AC024911 1051 1-315
HRABCO8 212 959124 AC026840 1052 1-503
HRABC08 212 959124 AC007897 1053 1-453
1564-2870
HRABC08 212 959124 AC022221 1054 1-1294
HRABC08 212 959124 AC007897 1055 1-129
HRABC08 212 959124 AC007897 1056 1-438
HRABC08 212 959124 AC022221 1057 1-438
HRABA59 213 867239 AC072049 1058 1-531
HRABA59 213 867239 AC072049 1059 1-357
HRAAW62 215 742631 AC034115 1060 1-615
HRAAW62 215 742631 AC036108 1061 1-1763
HRAAW62 215 742631 AC018999 1062 1-1766
HRAAW62 215 742631 AC036108 1063 1-146
HRAAW62 215 742631 AC018999 1064 1-307
HRAAM31 217 945071 AC073991 1065 1-1177
HRAAM31 217 945071 AC006230 1066 1-1177
HRAAH86 220 785398 AL139331 1067 1-534
HRAAH86 220 785398 AL157697 1068 1-534
HRAAH86 220 785398 AL157697 1069 1-648
HRAAH86 220 785398 AL139331 1070 1-648
HRAAD24 221 676567 AC006208 1071 1-442
~
5I6-699
770-945
1607-1777
1779-2240
2748-3680
4220-4397
HRAAD24 221 676567 AC024148 1072 1-148
263-423
1284-1509
1650-1931
3343-3483
3737-3879
4128-4290
4371-4503
4924-5365
5439-5622
5693-5868
6530-6700
72
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
6702-7164
7281-7394
7672-8603
9143-9320
HRAAD24 221 676567 AC006208 1073 1-141
395-537
786-948
1029-1161
HRAAD24 221 676567 AC006208 1074 1-1425
1603-1678
1783-1875
2028-3154
3258-3740
3768-4488
4764-4913
5252-5382
5827-5905
6172-6319
6397-6466
6957-7076
7473-7606
8554-8608
8714-8741
8840-8984
HKPMB95 225 795060 AL031650 1075 1-423
HKPMB95 225 795060 AC068624 1076 1-336
838-1776
1968-2081
HKPMB95 225 795060 AL121751 1077 1-423
HKPMB95 225 795060 AC068624 1078 1-256
HKPMB95 225 795060 AL031650 1079 1-1839
HKPMB95 225 795060 AC068624 1080 1-1574
HKPMB95 225 795060 AL121751 1081 1-921
1179-2159
HKMNC21 231 575816 AL035070 1082 1-445
HKMNC21 231 575816 AL035070 1083 1-589
HKMMY63 232 745145 AL031985 1084 1-703
1291-1889
HKMMY63 232 745145 AL031985 1085 ~ 1-129
402-491
5473-5683
6349-6775
HKMMX31 233 731638 AC008145 1086 1-1165
1183-1440
1929-3571
4290-4866
5620-5991
73
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
6223-7048
7820-7972
8559-9062
9224-9674
10540-10878
11255-11932
12172-12614
HKMMX31 233 731638 AC008145 1087 1-2343
HKMMX31 233 731638 AC008145 1088 1-1002
HKMMU45 234 871759 AC009869 1089 1-1230
1307-2148
2168-3029
HKMMU45 234 871759 AC009869 1090 1-507
HKMMU45 234 871759 AC009869 1091 1-2222
HKMMR58 235 736099 AL162501 1092 1-373
HKMMR58 235 736099 AL353729 1093 1-373
HKMMR58 235 736099 AL353729 1094 1-116
HKMMR58 235 736099 AL353729 1095 1-546
HKMMJ41 236 920910 AC011714 1096 1-108
1971-2651
2708-3803
HKMMJ41 236 920910 AC011714 1097 1-1481
HKMMJ41 236 920910 AC011714 1098 1-644
HKMMB27 237 575799 AC007899 1099 1-78
911-1060
1125-1525
3006-3125
5016-5230
7331-7408
HKMLY45 239 575805 AC021959 1100 1-418
HKML033 240 859903 AC027I64 1101 1-680
HKML033 240 859903 AC024388 1102 1-680
HKMLO33 240 859903 AC027164 1103 1-93
HKML033 240 859903 AC024388 1104 1-93
HKML033 240 859903 AC027164 1105 1-119
HKML033 240 859903 AC024388 1106 ~ 1-119
HKMLF28 243 705533 AC008488 1107 1-1307
1691-2527
HKMLF28 243 705533 AC026424 1108 1-374
1299-2384
3050-3444
3536-3708
4988-5557
5619-5841
5882-6842
7078-9586
74
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
_ 9970-10806
HKMLF28 243 705533 AC008587 1109 1-164
2264-2301
2816-3276
4686-4842
5583-5956
6881-7966
8617-9026
9118-9290
10569-11138
11200-11422
11463-12423
12659-15166
15550-16386
HKMLF28 243 705533 AC026424 1110 1-157
'
HKMLF28 243 705533 AC008488 1111 1-355
HKMLF28 243 705533 AC026424 1112 1-355
HKMLF28 243 705533 AC008587 1113 1-355
HKMLF28 243 705533 AC008587 1114 1-212
3498-4246
4457-4587
7496-7691
11928-12166
13527-13685
14011-14103
HKMLD21 244 671010 AC009673 1115 1-438
HKMLD21 244 671010 AC024930 1116 1-439
999-1481
HKMLD21 244 671010 AC019322 1117 1-438
HKMLD21 244 671010 AC009673 1118 1-115
HKMLD21 244 671010 AC019322 1119 1-297
HKMLC56 246 733473 AC005037 1120 1-642
HKMLC56 246 733473 AC005037 1121 1-404
HKMLC56 246 733473 AC005037 1122 1-310
HKMBC21 249 670144 AP000718 1123 .1-435
HKMBC21 249 670144 AC026851 1124 1-435
735-1154
HKMBC21 249 670144 AP001351 1125 1-435
734-1157
HKMBC21 249 670144 AP001351 1126 1-613
HKMBC21 249 670144 AP000718 1127 1-613
HKMBC21 249 670144 AC026851 1128 1-613
HKMBA94 250 793011 AC007737 1129 1-924
HKMBA94 250 793011 AC007556 1130 1-925
HKMBA94 250 793011 AC007737 1131 1-74
243-1037
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HKMBA94 250 793011 AC007556 1132 1-640
1526-1768
3142-3412
4485-4610
6381-6484
8584-9086
9503-9635
11363-11561
15129-15311
17376-17893
18236-18646
21077-21228
21777-21948
23054-23493
23662-23902
25370-25589
26807-26985
28007-28203
29232-29477
30172-30300
31261-31381
32224-32440
32729-32844
33577-33694
34720-34857
37342-37464
40796-40924
45893-46023
46989-47124
48792-48938
50432-50598
52368-52497
55908-56280
56436-56657
57473-57649
59906-60018
60603-60722
62851-62943
63788-63853
64025-64143
64688-64819
65902-66001
66584-67062
67143-67869
68004-68496
68545-69613
70712-70820
76
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
73163-73483
74310-74383
74552-75346
HKIYT69 251 843327 AL031228 1133 1-352
HKIYT69 251 843327 AL031228 1134 1-325
HKIYT69 251 843327 AL031228 1135 1-113
HKIYS82 252 779904 AC015589 1136 1-1206
1256-4224
HKIYS82 252 779904 AC010328 1137 1-82
355-795
871-998
1300-1582
1629-2248
2727-2918
4474-4729
4876-6190
6240-9439
HKIYS82 252 779904 AC015589 1138 1-299
HKIYS82 252 779904 AC010328 II39 1-179
HKIYS82 252 779904 AC010328 1140 1-570
HKIYN76 253 770080 AL161910 1141 1-1421
HKIYF03 256 924241 AC022542 1142 1-2074
HKIYF03 256 924241 AC005384 1143 1-1037
HKIYF03 256 924241 AC022542 1144 1-680
HKIYF03 256 924241 AC005384 1145 1-680
HKIYC59 257 868263 AL137066 1146 1-855
873-1230
HKIYC59 257 868263 AL137066 1147 1-243
HKIYC59 257 868263 AL137066 1148 1-525
HKIXQ80 260 775405 AC011921 1149 1-443
HKIXQ80 260 775405 AC025515 1150 1-443
HKIXQ80 260 775405 AC011921 1151 1-149
HKIXQ80 260 775405 AC025515 1152 1-149
HKIXQ47 261 720373 AC068282 1153 1-386
426-1484
1594-1896
HKIXQ47 261 720373 AC068282 1154 1-238
HKIXB 15 262 660458 AC022211 1155 1-33
1164-1278
1567-2052
3306-3473
HKIXB15 262 660458 AC022211 1156 1-252
HKIMG64 265 857190 AC013647 1157 1-405
HKIMG64 265 857190 AC010741 1158 1-413
HKIMG64 265 857190 AL161790 1159 1-413
HKIMG64 265 857190 AC010741 1160 1-638
77
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HKIMG64 265 857190 AL161790 1161 1-638
HKIMG64 265 857190 AC010741 1162 1-351
HKIMG64 265 857190 AL161790 1163 1-351
HKIBB62 267 742526 AC009509 1164 1-619
HKIBB62 267 742526 AC009509 1165 1-97
978-1034
2318-2377
4978-5150
5615-5647
HKIBB62 267 742526 AC009509 1166 1-389
HFKLK34 269 907556 AC022576 1167 1-722
HFKLK34 269 907556 AC022576 1168 1-321
HFKGB24 272 677402 AC007104 1169 1-802
2342-2695
3074-3189
5319-5633
7248-8571
HFKGB24 272 677402 AC069497 1170 1-117
2470-3367
4908-5262
5641-5756
7886-8200
9815-11138
HFKGB24 272 677402 AC007104 1171 1-453
HFKGB24 272 677402 AC069497 1172 1-453
HFKFI46 274 586801 AC005586 1173 1-337
345-870
1023-1553
2148-3037
3212-3335
HFKFI46 274 586801 AC005586 1174 1-1438
2411-2511
HFKDF03 277 960977 AC019131 1175 1-4679
4858-5322
7433-7916
7918-8276
11359-11661
13034-13322
14921-15217
19170-19416
20116-23855
HFKDF03 277 960977 AP002026 1176 1-484
HFKC051 279 725968 AC074386 1177 1-470
HFKC051 279 725968 AC004889 1178 1-470
HFKC051 279 725968 AC074386 1179 1-527
4449-4476
HFKC051 279 725968 AC004889 1180 1-527
78
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4447-4552
4726-4968
HFKCG41 283 712718 AP002784 1181 1-654
1182-2920
3315-5990
HFKCC73 284 742991 AL161451 1182 1-37
873-980
1129-1342
1521-1731
3705-3985
4040-4126
5368-5773
5825-6649
HFKCC73 284 742991 AL365502 1183 1-406
458-1282
HFKCC73 284 742991 AL365502 1184 1-245
HFKCC73 284 742991 AL161451 1185 1-245
HFKBC08 286 960526 AC025597 1186 1-966
2536-2698
3500-3602
5005-5242
HFKBC08 286 960526 AL161804 1187 1-966
2536-2698
3500-3602
5005-5242
HFKBC08 286 960526 AL355834 1188 1-966
2534-2696
3498-3600
5003-5240
HFKBC08 286 960526 AC025597 1189 1-200
HFKBCO8 286 960526 AL161804 1190 1-200
HFKBC08 286 960526 AL355834 1191 1-200
HCKAA51 287 534949 AL022325 1192 1-154
1810-1865
1947-2233
2391-2757
2828-4196
4215-4421
4990-5074
5133-5294
5676-6489
6529-7035
HCKAA51 287 534949 AL031588 1193 1-154
1810-1865
1947-2233
2391-2757
2828-4196
79
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4215-4421
4990-5074
5133-5294
5676-6489
6529-7035
HCKAA51 287 534949 AL022325 1194 1-875
HCKAA51 287 534949 AL031588 1195 1-875
HBAMD83 288 573369 AC044821 1196 1-326
HBAMD83 288 573369 AC044821 1197 1-155
HBAMD79 290 573373 AC011025 1198 1-263
HBAMD79 290 573373 AC011954 1199 1-263
HBAMD74 291 573372 AC064850 1200 1-384
HBAMD74 291 573372 AC064850 1201 1-450
HBAMD66 293 750532 AC001228 1202 1-646
HBAMD66 293 750532 AC001228 1203 1-144
HBAMD66 293 750532 AC001228 1204 1-611
HBAMD57 294 573370 AL133224 1205 1-359
HBAMD50 295 864379 AP000648 1206 1-1521
HBAMD50 295 864379 AP001769 1207 1-797
1056-2966
3062-3615
HBAMD50 295 864379 AP000827 1208 1-782
893-1689
1948-3858
3954-4507
HBAMD50 295 864379 AP001769 1209 1-70
111-298
HBAMD50 295 864379 AP000648 1210 1-781
892-1688
HBAMD50 295 864379 AP000827 1211 1-1045
HBAMD41 296 573374 AC024610 1212 1-263
HBAMD36 297 573367 AC009670 1213 1-442
HBAMD35 298 571354 AC017062 1214 1-383
HBAMD33 299 573365 AC025734 1215 1-301
HBAMD33 299 573365 AC013294 1216 1-301
HBAMD33 299 573365 AC025734 1217 1-88
HBAMD33 299 573365 AC013294 1218 1-88
HBAMD32 300 573362 AL354950 1219 1-369
HBAMC66 303 575218 AC026982 1220 1-213
HBAMC66 303 575218 AC026982 1221 1-120
HBAMC65 304 750405 AL359212 1222 1-381
HBAMC65 304 750405 AL359332 1223 1-381
HBAMC65 304 750405 AL359332 1224 1-380
HBAMC63 305 572936 AC010142 1225 1-402
HBAMC63 305 572936 AL121823 1226 1-402
HBAMC63 305 572936 AL121823 1227 1-109
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
HBAMC62 306 572927 AL354747 1228 1-361
HBAMC62 306 572927 AL354747 1229 1-705
HBAMC51 308 572929 AC027561 1230 1-741
HBAMC42 309 572938 AL133419 1231 1-150
HBAMC23 312 572946 AL163151 1232 1-451
HBAMC05 313 932234 AL356019 1233 1-795
3029-3404
5031-5269
6359-6575
6757-6902
7437-7493
9843-9935
10345-10498
11221-11501
11654-11866
12881-13014
14133-14247
16803-17149
17484-17858
HBAMC05 313 932234 AL356019 1234 1-368
HBAMC05 313 932234 AL356019 1235 1-107
HBAGW31 314 960146 AC019194 1236 1-2243
2706-3044
3147-3252
3286-3441
4820-4941
6238-6536
6929-7332
8567-9048
9388-9684
9981-10341
12293-13505
13876-13926
14318-14400
14800-15308
15715-16364
16560-16748
17222-17571
18347-18756
19156-19615
19666-19982
21112-21428
22550-22693
28745-29143
29809-31336
32109-32397
32731-33181
81
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35845-36323
39352-39743
40047-40483
40677-40972
44642-44840
HBAGW31 314 960146 AC020759 1237 1-1528
HBAGW31 314 960146 AC005632 1238 1-1528
HBAGW31 314 960146 AC020759 1239 1-399
HBAGW31 314 960146 AC020759 1240 1-289
HBAGW31 314 960146 AC005632 1241 1-289
HBAGW31 314 960146 AC005632 1242 1-399
HBAGR76 315 864389 AC013400 1243 1-476
HBAGR76 315 864389 AC013400 1244 1-760
HBAGH38 316 487933 AC020659 1245 1-671
703-3178
HBAGH38 316 487933 AC020659 1246 1-309
HBAGH38 316 487933 AC020659 1247 1-276
HBAFU01 318 916248 AL355296 1248 1-474
HBAFU01 318 916248 AL355296 1249 1-320
HBAFU01 318 916248 AL355296 1250 1-143
HBAFP29 319 690521 AL353807 1251 1-957
HBAFP29 319 690521 AL353807 1252 1-155
HBAFF55 321 753801 AC021162 1253 1-967
HBAFF55 321 753801 AC021162 1254 1-1542
HBAFF55 321 753801 AC021162 1255 1-178
HAKAD91 324 509854 AC011875 1256 1-510
HAKAD91 324 509854 AP000848 1257 1-510
HAKAD91 324 509854 AP000663 1258 1-509
HAKAD91 324 509854 AP001320 1259 1-506
HAKAD91 324 509854 AP000848 1260 1-610
HAKAD91 324 509854 AC011875 1261 1-610
HAKAD91 324 509854 AP000848 1262 1-231
HAKAD91 324 509854 AP000663 1263 1-610
HAKAD91 324 509854 AP001320 1264 1-231
HAKAD91 324 509854 AP001320 1265 1-610
HAKAD72 325 509855 AP000425 1266 ~ 1-405
HAKAD72 325 509855 AP000425 1267 1-451
[063] 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
82
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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
83
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WO 01/55313 PCT/USO1/01323
t~ d- oo N a1 ~O ~ d- d- N ~t O~ co M
N a1 ~O d- d- Ov N d' O .--~ d- M O~ O
M ~--a N N N ~ M M M M
V~ 00 0~0 ~ M o0 v0 t~ d' oho ,-N~ ~ d, d1
M .-~ .-~ .~ N ~ M ~ ~ N
zw
0 0 0 0 0 0 0 0 0 0 0 0 0
y y yyy y y v
l~ M ~n oo ~n d- d- ~ O O O M N
d- ~ a\ d- d- O~ ~W o 0o O~ ~p v0 o~
v ~
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pp ~ ~: N M V7
N l~ ~ ~ N O
O it O ~ M O due- ~ 01 01
U
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O V ~ N
O ~ .-O O O "~ ~ M o0 . ~ '-~ ~ ''d O '~ O
U ~ p N ~', ~ N ~v~,' ,.hC~ ~ ~ ,~'''~' O ~ U ~ U
A ~ 01 '~ '~ 5~., l0 O .-~' . ~ . in
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N ~ v~ ~ ~-' O ~i .Wn d- ~ ~O ,-, O
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p..me p, w a c~ vW~ .,-~ c~ ~, ~r ~ U ~r ~ p, cn ~ p., v~
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CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
a1 d' d' l~ M oo ~ .-~ l0 M ~ d' M N l~ 01
M o0 00 a, O m N M ~O ~~ N d- ~ N a1 M
M M M M ~ N N N M d' N N ~--~ N
~w O~ 01 ~ ~ ~ M 41 d' 00 M V'7 O ~--~ M 00
l~ 01 d' N ~ d' O .-1 d'
N N d- N M ~ N M d- M
o ~ ~ o o ~ o ~ ~ 0 0 0 0 0 0
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l~ ~ d' O O~ l~ l~ d' 01 \O V1 01 l0 I~ ~ d'
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N o~o ~ ~ ~ ~ M ,~-~ ,-N-~ O N
00 ~O ~ 00 01 01 N d'
N ~ N
U ~ ~ U ~ P~ P~
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N
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a\ a\ ~ ~ ~ ~ D1
x x ~ x
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
O ~n oo O N ~ O~ l~ d~ O N N O~ ~O ~ l~
O ~n O M O O~ o~ ~ M N o~ d' d- N t~ M
V7 M d' V7 ~ N N ~ N M ~ .-~ M M M
r, oo ~ d- .-~ a\ M oo v1 d' d' ~n oo N d- d-
.-a O ~p r-., .-~ [~ 01 00 01 01 d' 00
d' M M d- ~~ M N
0 0 o N o ~ 0 0 0 0 0 0 o pp o
d.
M ~ 00 ~1 ~ M d' O V1 N l~ O ~ ~
l~ o0 \O N d1 O d' ~ l~ t~ ~ V1 l~ ~ o0 M
M
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O ~ ~ ~ ~ a\ N
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N ~ ~ ~, N U
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o°d~°o ~~ ~ ~o ~°o
w ø., ~"' w ~ ,~ ,~ ,.fl w ~ w
do w wo ~., du ~a cn bn a~ a~ a-.
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N~01~ NW pMp ~OVONpO..~iU
D1 ~ ~ ~ ~ ~ ~ o~J ~ ~ Q., ~ ~ ~ O ~ w '~ 'T tn x ~ _~
O ~ W ~ ~ O ~', ~ ~~ U O ~ O ~ ~ M ~ ~ ~ ~c~
O O ~ z~~W~w ~O'~''.~ ~'d 00 0~~~"C~ +~Q'
w ~1 ~ w N ~ ~ ~ ~ ° ~ ~ ° ~ w ~'~c ~ ° w
P.m--i P-~ P-~ N 4-i ~ a ~ S~ v~ c~ ~ ~r P-~ .~ O a P-~ ''a "d
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M d' N M l~
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M N O M d' M d'
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d' ~n O M ~--~ V'1 d'
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86
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
d' ~D l~ d- ~--WO N d' o0 00 ~ M ~ ~O d- l~ o0 a1 ~n
00 l~ l~ M I~ ~n I~ ~-i M d' V1 M l~ ~O d' d' O V~
M M M M N M N N 00 N N ~ M N M M
~n o0 01 N Owe N oo m 01 N oo N ~ M O ~O oo N
~O o0 01 d' v~ N ~--~ d' d- d' O d' ~n l0
N .-~ M N ~ N ~ N ~ N
0 0 0 0 0 0 0 ~ o o ~ ~ o o ~ ~ ~ o
O \O (~ O ~ O l~ M O O1 ~n ~ ~ ~ 0o O~ d- V7 00
O ~ ~O ~ l0 d' d' ~ V'1 (~ d' ~O d' 00 00 \O v7 00 01
OM ~ ~ ~ ~ ~ ~ ,-, ~
N ~ ON1 M ~ ~ ~_ ~ ~ l~ N
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S.-i" ~ U ~ '~ ~ N ~ ~ O ~ '~'' N ~ U ' ~'
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d o ~ ~ .s~ U~ ~ ~ ~ ,~ oM, ~ o ~ o ~ ~, ~ ~
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cd cd cB c~3 cd cd cd cd cd c~ cd
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t~ a1 ~o O> O~ t~ ~ ~n ~O oo a\ a1
p '~ oho O f-Nz.yU ~~-, N °o
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CA 02395693 2002-06-25
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N ~ O oo N~ ~ N N ~ v7 M due- d- ~ ~ ~ O d'
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M oo l~ O N o0 ~ N O d- two O 01 N N d'
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ss
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
M O --~ l~ l~ 00 O M ~ V7
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l0 l~ o0
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M l~ Q1 N 01 M ~ O1
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d- 01 V7 O ~O ~O 00 N 01 \O
t~ t~ ~D
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M M ,-..iM p~ M M O
M O N N 01 ~ N .-~ I~ N
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M
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d' M \O ~ l~ l~ O M
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89
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
~f1 l0 d' O 00 V7 l0 M M
O M ~ l~ ~ 00 l~ 00 M M
N N ~ M ~ M
d1 N l~ N Do N ~-~ oo d' ~O
D1 I~ .-~ lp O d- O
N d' M
0 0 ~ ~ ~ 0 0 0 0
N ~~ O ~ ~O a1 v7 d' O
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l~
M O ~ ~ o O cn ~ cn
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N N N N
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l0 l~ l~ N
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N N N ~O
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x x x
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
[064] 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.
[065] 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
between the Query and the Subject for each HSP as a percent identity in Column
7.
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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 are delineated by columns 8 and
9 of
Table 2.
[066] 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 sequence analysis:
pf°obabilistic
models of proteins and nucleic 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.
[067] 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
columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such
proteins, and the complementary strand thereto.
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[068] 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.
[069] 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).
[070] 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-
l,
and AC-2; and/or as set forth, for example, in Table 1A, 6 and 7). The
nucleotide
sequence of each deposited clone can readily be determined by sequencing the
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.
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[071] 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 Protocol Fog Recovery of Full-Length Gehes
[072] 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. Acid. 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 polymerise (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
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
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methods known in the art and/or commercial kits are used to amplify and
recover 3'
ends.
[073] 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 kit 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.
[074] 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 For Generating The 5' or 3' End Sequences To Obtain Full
Length
Genes
[075] 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
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
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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
excretory system antigen of interest. The resultant product is then sequenced
and
analyzed to confirm that the 5' end sequence belongs to the relevant excretory
system
antigen.
[076] 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, arid 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;
andlor
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
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,
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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
slcilled in
the art.
[077] 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.
[078] 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 (IJ.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., Strategies 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.
[079] 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
resistance gene and may be transformed into E. coli strain DH10B, 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).
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[080] 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.
[081] 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, ftzll-length coding portions, orthologs,
and/or species
homologs of excretory system 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.
[082] 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.
[083] 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.
[084] The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purifted. 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).
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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 excretory
system
polypeptides of the present invention in methods which are well known in the
art.
[085] 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 TD NO:X,
a polypeptide encoded by the cDNA contained in Clone ID NO:Z andlor 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.
[086] 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
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
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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 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.
[087j 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 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
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,
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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.
[088] 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 1B, column 2) and have a nucleic
acid
sequence which is different from that of the BAG 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
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.
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[089] 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 fuxther 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
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.
[090] 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
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polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides are also encompassed by the invention.
[091] 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.
[092] 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.
[093] 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 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.
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[094] 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 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.
[095] 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
encompassed by the invention. Additionally, fragments and variants of the
above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[096] 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
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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.
[097] 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.
[098] In specific embodiments, polyriucleotides 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
Clone ID
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.
[099] 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
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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.
[0100] 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-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[0101] 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 axe 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
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WO 01/55313 PCT/USO1/01323
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 Genbank 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
107
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WO 01/55313 PCT/USO1/01323
~O l~ N dW t ~t N l~ N ~t N N O~ N ~
~ O oo cri Is .-i t~
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d' ~ M O d' 00 N lW f' 'd' O~ d' d'
~ ~ M O O~ M ~ V~ O O 00
l~ ~ l~ O M O ~ M M ~D O ~O O 01 .~
Q~ V1 01 N O I~ .~ N
O
M
'd' 'd' d' d' d' d' d' d' 'V' d' d'
'd' I~ 'G' 'd' 'd' M ~ d' d' ~f' 'd'
a
a
aa
a
aa
a
a
a
a
a
a
a
a
a
a
a
a
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a
a
a
a~aaaaaaaaa~aaaaaaaaaaaa
cn N N oo O vi ~t O O ~t ~t ~i cn O
O~ ~O N O N ~i O ~t
d' N ~O N M l~ M ~D ~ ~ l~ l~ M M ~O
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'
1 00 O M ~ N M .~ M ~ M ~ M N O ~'
M ~ ~ 00 N ~ ~O O V
h ~h 00 ~ t~ .~ ~ O ~O d' ~ N ~ O~ ~
-~ .-W O O O O M
~
O
M
'd' M d' d' d' '~1' 'd" d' d' d' d'
d' M d' d' d' d'
d' ~h '~1' '~h
O ~ O O O O O O O O O O O O O O O O
O ~ O O O O
~
~
~~ ~~a
~~~
~
a
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a
00 N ~t ~O m Vi v0 M Oi o0 ~t M ~!i
N o0 00 Qi Oi t~ Vi ,-i ~n
M M M N d' N N l~ ~ M ~O ~ M 01 Q1 ~1
'~f' a1 N ~ l~ ~7 M l~
N
0o
p ~n M N oW o ~n ~ oo t~ o~ ~ N M N ~
~ ~n
~ N
00 M 00 ~ ~ M O O ~n O O l0 ~--W ~ ~O
'd' O d' O N .~ O ~ M
Q M M M 'd' d' d' d' d' 'd' 'd' d' d'
d' M d' ~h d' ~i' d' ~ d' 'd 'd' ~h
O O O O O O O O O O O O O O O O O O
O O O O
~~~~a~~~a~a~~~~a~~~a~~~~~~~~
M
N v0 ~ N O~ ~O ,-~ .~ O~ ,-W ~ ~t N l~
~n ~ ~D N oo O~ t~ N w0 v0
~ M l~ d' l~ l0 N N ~ ~O d' ~O ~ 01
,-a ..-~ ~ .--~ e--~ M M 'd' .-w--~
h '~ l~ M 00 O l~ ~ d' M O M M ~ O .~
~ d- .~ O V7 O d' O
d' N M ~ M d' d' 'cf' M d' ~h d' d'
O d' Wit' d' 'd' d' '~' Wit' d' 'd' 'd'
d'
O ~ o o O O o o ~ o O O O O O O o O
o 0 0 0 0 0
~ a~aaaaaaaaaaaaaaaa..aaaaaa
-~aaaaaaaaaaaaaaaaaaaaaaaa
0o vi o0 00 ~i m ~i ~t lWo N cn cn N
~O cW O oo ,-i Sri ,-i ~ OW i
N M O~ M M ~D d' 00 t~ 00 W N ~O N ~
d' OWn l~ d' dW t ~ ~t
M M o0 ~!1 'ch et 00 ~O ~ O O V7 ~ M
M ~O O d' ~ l~ M I~ 01 l~
[W!7 l~ N M l~ O M V'7 ~--n ~ O ~ ~ O
-,O\ 01 N O O O l~ O\ V) 00
d' M d' d' M 'd' d' ~ Ch dw1' d' d'
d' M M d' d' d' d' M M ~ M
N O O O O O O O O O O O O O O O O O O
O O O O O O
~~~ ~~~
~
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aa
aaa
aaaa
a
00 01 ~ d' M M ~ 00 ~O l~ V1 O~ M N O o0 \O
CO d' ~ N I~ N N o0 ~D ~O ~ l~
N CO 'd' N 01 00 M l~ ~ 00 O M d' 01
l~ I~ ~ O~ M l~ M 00 01 N
O O~ ~ V7 01 ~t ~D O ~ O O d' ~ ~O l~ 00 O
V W ~O N O N M o0 O ~ O M
d" 00 l~ O ,--mt' 'd' O ~ t~ .~ ~-,
M .-.n V~ 'd' l~ .-, O O~ l~ .--n O
~O
O O ~ ~ O O O O O O O O O O O O O O O O O
O O O O O O
a N
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i,
N M'd'd'~M l
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i~ ~ ~~ i
w n ~nv~~n.~mn
CC~ --n--n~ --~n~
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v . , .
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108
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WO 01/55313 PCT/USO1/01323
M
o.~, vi oN, n o0 0
~ o ~ o ~
M ~ ,.~...~ O ~ ~ M ~ ,M-, ° _ N M M
M d' ~ ~ M ~ d' ~ O M O~ a ~ 01 ~ ~h ~ M
O O N M 00 d' v0 ~ ~ ,~ ~ ~ M ~' N ~ H N
Q,'d~-~~~Wd ~OM~~O~N~
V7 00 ~_n ~ H M Pr \p ~ Vi Ov ~ ,WO
N ~ ~ due' ~ ~ [-~ ~ ~ ~ N ~ ~ ° p ~ O O
O ~ ~ ..M-.m-i pip ~ O N ~ ~ ~ ~.,.j ~ 'd N N ~ Q
0 0 ~ ~' '"' ~r N ° '~' o U o o ~t ~ m ~ ~r
M ,.pa ~ ,.°~ ~-r V7 ~ M 00 M VN7 O ~ ~ o ~ o~o~
O O ~ ~ 00 M O [v M op .--n
'~ N ~ ~ ~ ~ N ~ ~ ~ c ~ ° A ~ ~
in Oi a ~. °~ ~ t~ ~ _
V~7 d= ° ~ M O N ~ O M ~ d- ~ N oo ~ M_
OOO~OOM~O~~~~'d N\Oh0~0~00~0~ M
o o ~--r [-~ N P~ N c'i d. ~' R~''
r~ ~ .-i M O 'd'
cVoo ~ ."~~''~''~~~~~MNovo~,-~~"'~i ~i
O O M ~ Q1 O l~ O\ 00 ~ d' 01 ~ N 01 ~ O~ M ~ 00
'd' 'V' ° CO ~O ~ ~ M ° N ~ O 00 h 00 M ~O
aa1<°.i"'~°o~o~~°~°m°~N~~~~~° r
~ o ,~ N "; .
'c1' C, M M ~, 00 O 01 M pp M
N ~ ~ ~ ,-~ ~ ~ ~ ~°.~ M oho wi ~ .-: ,~ ~ o; ~ °~°
~ ~ 00
M ~ O\ M \O ~ O ~ M '~ N O A ~ Q~, M ~O O
O O O ~ ~t N N p O ~ ~ M ~D r" N '-' N ~ M
O O ~ d' N ~n ~ N O O M~ M O1 r3
N
cn oo .-i ~ ~ Oi ~ ~ ~ ~ ~ '~ ~ t~ ~ o N m OW o vo N ~ N
~n h O ~ ~,~ N ;~ ~ ~ ~ O ~ '~ N N m ~O ~ 00
N N N d. ° 'n ~ Oi M M N N ~ h t~ ~ cn op ~ ~n ~_ O M d' ~ o~
00 l~ ~ O ~ ,--n .-, l~ 01 d~ M WO ~ N O~ ~ O d~ d- O W ~p O
OOOM~O~~~~~p~p~~lllp~pOp~p~O p M l~0 M~ O
1-~ h-~ .~ O I-I r-1 .-N ~--n ~ p\ 01 ~-
~ ~ ~ ~ ~ E-~ U ~ ~ ~ ~ A d ° ~ ~ d W
O~ O~ d' 01 d' ~O O V'1 M l~ M l~ d'
00 ~n o ~t oo to dwn ~W O N d'
M ~ d' Vii' M M M M lp N M N '~-'
~ t ~ i i ~ i ~ i ~
' ~ V'7 V~ ~ ' V'7 ~ ~ ~ ~ ~ ~ V~ ~!1
47 ~ O ~ O N ~O .-n O1 M 01 M O
d'O\Ml~WINd'MV7O M~
M ~ M d' M M M M ~O N M N
i ~ i ~ i ~ i i i i i ~
v0 Ov N I V'1 t~ ~W O d- ,-n O O\ Ov
d' O d' l0 O~ 'G' V7 01 01 M v--n 'd'
0o O '~t Ov O~ M d- N O ~n o0 O~ ~
0o d' Q1 N d' d' ~i' O N M 01 O~ M
~D ~P1 ct l~ M O~ O M M N O N l~
00 00 V1 h ~O ~ ~ 01 ~ O~ tn ~f1
00 O~ O ~ N M ~t ~W O t~ oo O~ O
~ N N N N N N N N N N M
N l~ oo N M
d' \O ~ ,-n ~ ~ ~ ~ O O
pp ~ ~p d- .~. ~ N
U U U U
~~~~xx~~~~~
109
CA 02395693 2002-06-25
WO 01/55313 PCT/USO1/01323
ti: ~ N
N
~ O N ~ '-' ~,~ ~°p _
010 O~ pNV~d.-M~ O~~ ~.
O O O . 'CJ ~ ~ ~ p ~ ,-~-n o~0 ~ \O O
d .b ~ ~ ° cMn o ~ '~; "' N ~ U
d3o~~N~U~M
'~I~n nidoodw~M~o-do
M O oo ~." ~r ~ ° oo t~ .d- N ~-' d
NOO ~~_ N~U
~ V'1 V'7 ° N
d d ~ M ~ 1-N-1 O N ~ ~ ~ ~ ~ M O
,.a d°°_'~'~dd~M~~~ O
due" .~d°~~ O
01 ~ 01 ~ rr w ,~-, vi l~ ~O N
dM' d~h' d0~' ~ ~ N ~ d d' O~ 00
l~ O O ~ ..-m--~ M V~ ~ O~O M <V ~ 'C
°UU-d o~ ~c°~~~o"~o"'o°voo~o~~ c~a
d d ~ ° d d ~ ~ c°~y ~ d
U'r d~d~N dd'~'~'°M ~r N
o; cri c~i ~; d ~c ~r .o M In
M ~M ~_~d~~o~z ~~ ~ °
~oo~ ~~ °~~~oo°oo°~oo~~ M wo
~r o 0 0 0 '~ 00 0 Wit' oNO can ~ ~ Wo 00 omp
U U U o ° ~ 3 ° ~ o°°o m °° '-'
~ ~ n °
d d ~ oo U d d °' ~ d ~ ~ ~ M o0 o c~ In -v
M [~ M r-i ,.'3 O v-r M
d't~NO ono do °o~,~doNO~~ddd~ .° N
of N In N
O ° O ~ ~ N O ~ In due' N ~' ~ N In N ~ N
d U U U ~ o d ~ d m N t~ c~ ~ oo M ~ 00 0 0°,
N
ddd ~ ~ dd~~~dd~'w~~'
p V' ~ ' M \p 'd' M M d d 'd O dN' ~ ~ t~ ~ ~ 0~1
M N o '~ o~ ~" ~ N Wo ,~ ~n oo O ~t oo .-1 ~O v0 M ,-1 dw-~ N ~
N o O M ~-~ d' p~ 'dW vp .-~ N N O ~ M N N O ~p N ~ ~p
dddd ~d ~~~d ddwwddd~~H~ d N~'
I~ O d- d- In l~ O ~t oo M ~t O ~D N N O~ ~O In d' l~ ~O
N d~ O O ~n N N N ~ N ~-1 ~O N M .-mO d~ 00 0o M o0
M ~O M ~ d' l~ V7 d- M M ~O If1 In M M M M ~O h M
1 1 1 1 1 1 1 1 1 1 1 I 1 i 1 1 1 1 1 I 1
Ir1 1!1 In V7 V1 ~ N ~ tn 1!1 V~ 4'1 h 1~ 1~ h V1 In V7 In V1
~ H H H H H H ~ H ~ ~ H H H H H H ~ H ~ H
M l0 O O .~ M ~O O 'd' O\ O v0 N ~ 00 00 V7 N '~-1 O M N
N o~ o~ d~ ~~ o .~ ~ o o ~t ~ ,~ ~ In M ~ ~ N t~
M lp N d' d' l~ V'1 d' M M lp ~ V1 M N M M ~O ~ M .~
1 1 1 1 1 I 1 1 1 1 I 1 I 1 1 « 1 1 t 1
~ H
0o O~ ~t O In N ~ In l~ ~O In .~ N N ~D l~ O ~t ~O O N
t~ O d' O~ ~Y In oo I~ M O l~ O~ N o0 00 0o v0 d~ d- d- N
~-~ M l~ N O O~ ov ~ o M d' o~ M o~ t~ ~n N ~ o 0o N
M d' ~D 00 In V' V'7 1!1 N N W t" O O ~ N l~ M ~ M O
~o t~ N ~-mn In M t~ N M oo In t~ M N ~ ~ ~ ~ ~ M
0v ov o~ o~ 00 00 0~ ~O In ov o0 0o vo a~ oWn t~ oWn In In
.~ N M 'cf' V"1 ~O l~ 00 01 O ~ N M d- ~ l0 l~ 00 01 O ~
M M M M M M M M M V' d' '~f' 'ch ~ et d' d' '~f' d' to In
O 'd' l~ ~ Q\ M 00 M N In M O~ .~. N ~t N d- In ~ vo d'
Iw0 O N ,-1 N ~t O N ~n ,~ N .d. .~ r, N ~ l~ N
~z r~r~~~wr~wwww~ ~~~~~zo
110
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err"
d-
00 M l~ ~ O~ 00 O 'd' M p 00 ~ d' ~O '-i ~!1 lw--i
N M l~ M ~f' ~D pp 00 H ~n ,-~ oo N oo d~ .,
.-i 01 ~ O 'd.~ ~ ~i' .-; p O l~ ~!1 00 00 N ~ h ~ N M .~ l~ N wj ~O N ,~ .~
o°0 v0 ~' ,~-, 'd ~ O O ~ U O N O ~ dp- O p O ° O O ~ N N ~ ~ ~
~ O O d. O
°° M ~ ~ ,'~'r, ~t d' oo U ° ov d o vo °
°° 0 0 0 o N ~ o o N ~ .-. ~ o o ,-. o o yo
o°v P; ,~ ~ ,~ a °~ o ~ d ~ ~ d U d ~ ~ ~ d ~ ~ U d ~
r°,~ ~ d N d a °o d d U U
01 a; 01 ~'' O ,--,~ ~ O ~ ''~' ~ ~ d 00 .~ O ~O ,~ rj ~O d ,~ Q1 ~ d O ~..~
V7 d ,-~ Vj d d
dM- ~ o N ~ M ,~ ~ ~.,~ d' ~ ~p ~_ w ~ M N ~ ~ ~j oNO '~' ~ ~ ~ p N ~ ~ ~n N ~
O
w~j~m~oMO~~~oOp~paO~N~ONOM°NpMN~Opm~'d OMNo°O
~ M o N ~ a ',-.~' ~ d U ~ '-' 't U ° U ° U o U a ''~ o U U a ~
~'~' U ~,.~ o~, o
~~~~~~~,.~~,~~N~dN~~d~d~dUd~oUddd~od~~~
.-. ° ~ oNO '~ t~ °° N ~' °~ '° ''' ~ "'
~''' ri ~ M d c~i '" ° ~ ~ ° p" o t~ o d
"-~ N M \O o0 ~n 00 l~ [~ 00 ~ tn N \O ,~ ""~ M <') U d v0 pp ~ .-n ~ O O\ in
01 ~ O O 00 ~O M O M ~ l~ ~ ~i' ~j M ~ N ~ ~ 00 '~ O\ O ~ M O ~ M O
N v0 ~ ~O ~IW p O N ~ °W0 dwn O ~ l~ ~n ~' ~ ~ d ~ ~O ~ ~ N ~ ~t O
N I~
[~ M V'7 ~ M ~ M M O O O U p~ O M M O O O .-~ ° ~ O O ~ ~ M ° O
° O N O 01
N ~ M ~ °o ~ U ~ d f.~, U ° o, o °pq U W U '~
° U N ~ w ° U U ° U ~ U o
dd~dd~~~~ddd°o~d°~d~ddod d
_ U
~ O ~ O ~ m ~ ° oo '-' ~ vo N ~ ~ O O ~ oho due' d o d. N ~ ~ ~ p ~ d N
~ ~ d
O~ ~ ~ ~ N ° o°o O N ~ ~ N y0 O o ~ ~ N p ~' 'd ~ ~ ~ ~ O ~
'~' ~ O ~'
O M ..-n N ~ \p N O O ~ ~O O O O p ,N-, O p O O ~ p O ,d o O ~ O p ,°~,
p O p N
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TABLE 4
Code Description Tissue Organ Cell DiseaseVector
Line
AR022a_Heart a_Heart
AR023a_Liver a_Liver
AR024a_mamm land a_mamma
land
AR025a_Prostate a_Prostate
AR026a_small intestinea_small
intestine
AR027a_Stomach a_Stomach
AR028Blood B cells Blood B
cells
AR029Blood B cells Blood B
activated cells
activated
AR030Blood B cells Blood B
resting cells
restin
AR031Blood T cells Blood T
activated cells
activated
AR032Blood T cells Blood T
resting cells
resting
AR033brain brain
AR034breast breast
AR035breast cancer breast cancer
AR036Cell Line CAOV3Cell Line
CAOV3
AR037cell line PA-1cell line
PA-1
AR038cell line transformedcell line
transformed
AR039colon colon
AR040colon (9808co65R)colon (9808co65R)
AR041colon (9809co15)colon(9809co15)
AR042colon cancer colon cancer
AR043colon cancer colon cancer
(9808co64R) (9808co64R)
AR044colon cancer colon cancer
9809co14 9809co14
AR045corn clone corn clone
5 5
AR046corn clone corn clone
6 6
AR047corn clone2 corn clone2
AR048corn clone3 corn clone3
AR049Corn Clone4 Corn Clone4
AR050Donor II B Donor II
Cells 24hrs B Cells
24hrs
AR051Donor II B Donor II
Cells 72hrs B Cells
72hrs
AR052Donor II B-CellsDonor II
24 hrs. B-Cells
24 hrs.
AR053Donor II B-CellsDonor II
72hrs B-Cells
72hrs
AR054Donor II RestingDonor II
B Cells Resting
B
Cells
AR055Heart Heart
AR056Human Lung Human Lung
(clonetech) (clonetech)
AR057Human Mammary Human Mammary
(clontech) (clontech)
AR058Human Thymus Human Thymus
(clonetech) (clonetech)
AR059Jurkat (unstimulated)Jurkat
(unstimulated)
AR060Kidne Kidne
AR061Liver Liver
AR062Liver (Clontech)Liver (Clontech)
1~5
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AR063 Lymphocytes Lymphocytes
chronic
lymphocytic chronic
leukaemia
lymphocytic
leukaemia
AR064 Lymphocytes Lymphocytes
diffuse
large B cell diffuse
lymphoma large B
cell
1 m homa
AR065 Lymphocytes Lymphocytes
follicular
lymphoma follicular
I m homa
AR066 normal breast normal breast
AR067 Normal OvarianNormal Ovarian
(4004901) (4004901)
AR068 Normal Ovary Normal Ovary
95086045 95086045
AR069 Normal Ovary Normal Ovary
97016208 97016208
AR070 Normal Ovary Normal Ovary
98066005 98066005
AR071 Ovarian CancerOvarian
Cancer
AR072 Ovarian CancerOvarian
Cancer
(97026001) (97026001)
AR073 Ovarian CancerOvarian
Cancer
(97076029) (97076029)
AR074 Ovarian CancerOvarian
Cancer
(98046011) (98046011)
AR075 Ovarian CancerOvarian
Cancer
(98066019) (98066019)
AR076 Ovarian CancerOvarian
Cancer
(98076017) (98076017)
AR077 Ovarian CancerOvarian
Cancer
(98096001) (98096001)
AR078 ovarian cancerovarian
15799 cancer
15799
AR079 Ovarian CancerOvarian
Cancer
17717AID 17717AID
AR080 Ovarian CancerOvarian
Cancer
400466481 4004664B1
AR081 Ovarian CancerOvarian
Cancer
4005315A 1 4005315A
1
AR082 ovarian cancerovarian
cancer
94127303 94127303
AR083 Ovarian CancerOvarian
Cancer
96069304 96069304
AR084 Ovarian CancerOvarian
Cancer
97076029 97076029
AR085 Ovarian CancerOvarian
Cancer
98076045 98076045
AR086 ovarian cancerovarian
cancer
98096001 98096001
AR087 Ovarian CancerOvarian
Cancer
9905C032RC 9905C032RC
AR088 Ovarian cancerOvarian
9907 cancer
C00 3rd 9907 C00
3rd
AR089 Prostate Prostate
AR090 Prostate (clonetech)Prostate
(clonetech)
AR091 rostate cancerrostate
cancer
AR092 prostate cancerprostate
#15176 cancer
#15176
AR093 prostate cancerprostate
~ #15509 ~ cancer
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#15509
AR094prostate cancerprostate
#15673 cancer
#15673
AR095Small IntestineSmall Intestine
(Clontech) (Clontech)
AR096S Teen S Teen
AR097Thymus T cellsThymus T
activated cells
activated
AR098Thymus T cellsThymus T
resting cells
restin
AR099Tonsil Tonsil
AR100Tonsil geminalTonsil geminal
center
centroblast center centroblast
AR101Tonsil germinalTonsil germinal
center B "
cell center B
cell
AR102Tonsil lym Tonsil 1
h node m h node
AR103Tonsil memory Tonsil memory
B cell B
cell
AR104Whole Brain Whole Brain
AR105Xeno raft ES-2Xeno raft
ES-2
AR106Xeno raft SW626Xeno raft
SW626
H0011Human Fetal Human FetalKidney Uni-ZAP
Kidney
Kidne XR
H0012Human Fetal Human FetalKidney Uni-ZAP
Kidney
Kidney XR
H0053Human Adult Human AdultKidney Uni-ZAP
Kidney
Kidne XR
H0119Human PediatricHuman PediatricKidney Uni-ZAP
Kidney
Kidne XR
H0239Human Kidney Human KidneyKidney diseaseUni-ZAP
Tumor
Tumor XR
H0334Kidney cancer Kidney CancerKidney diseaseUni-ZAP
H0356Human Kidney Human KidneyKidney pCMVSport
1
H0361Human rejectedHuman Rejected diseasepBluescript
kidney
Kidne
H0399Human Kidney Human Kidney Lambda
Cortex,
re-rescue Cortex ZAP II
H0408Human kidney Human Kidney pBluescript
Cortex,
subtracted Cortex
H0410H. Male bladder,H Male Bladder,Bladder pSportl
adult
Adult
H0411H Female Bladder,Human FemaleBladder pSportl
Adult Adult Bladder
H0431H. Kidney Medulla,Kidney medullaKidney pBluescript
re-
excision
H0441H. Kidney Cortex,Kidney cortexKidney pBluescript
subtracted
H0453H. Kidney Pyramid,Kidney pyramidsKidney pBluescript
,
subtracted
H0461H. Kidney Medulla,Kidney medullaKidney pBluescript
subtracted
H0555Rejected Kidney,Human RejectedKidney diseasepCMVSport
lib 4
Kidney 3.0
H0620Human Fetal Human FetalKidney Uni-ZAP
Kidney;
Reexcision Kidne XR
S0015Kidney medullaKidney medullaKidney Uni-ZAP
XR
50023Human Kidney Human Kidney
Cortex -
unam lified Cortex
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50024Human Kidney Human Kidney
Medulla
- unam lified Medulla
S0025Human Kidney Human Kidney
Pyramids - P ramids
unam lified
S02887TMCTK (Kidney)7TMCTK Brain PCRII
(Kidne )
L0005Clontech human
aorta
olyA+ mRNA
(#6572)
L0163Human heart heart
cDNA
(YNakamura)
L0366Stratagene schizophrenic Bluescript
schizo brain
S11 brain 5-11 SK-
frontal
lobe
L0369NCI_CGAP_AA1 adrenal adrenal Bluescript
adenoma
land SK-
L0373NCI_CGAP_Coll tumor colon Bluescript
SK-
L0435Infant brain, lafmid
LLNL array BA
of Dr. M. Soares
1NIB
L0438normalized total brainbrain lafmid
infant brain BA
cDNA
L0439Soares infant whole Lafmid
brain 1NIB brain BA
L0471Human fetal Lambda
heart,
Lambda ZAP ZAP
Express
Ex ress
L0475KGl-a Lambda KG1-a Lambda
Zap Zap
Express cDNA Express
library
(Strata~ene)
L0485STRATAGENE skeletal leg muscle Lambda
Human muscle
skeletal muscle ZAPII
cDNA
Libra , cat.
#936215.
L0517NCI_CGAP_Prl pAMPlO
L0518NCI_CGAP_Pr2 AMP10
L0519NCI CGAP_Pr3 AMP10
L0520NCI_CGAP_Alvl alveolar pAMPlO
rhabdomyosarcom
a
L0521NCI_CGAP Ewl Ewin "s AMP10
sarcoma
L0527NCI_CGAP_Ov2 ova ~ AMP10
L0529NCI_CGAP_Pr6 rostate AMP10
L0532NCI CGAP_Th th roid AMP10
1
L0536NCI_CGAP_Br4 normal ductalbreast pAMPlO
tissue
L0542NCI_CGAP_Prl normal prostaticprostate pAMPlO
l
a ithelial
cells
L0565Normal Human Bone Hip pBluescript
Trabecular
Bone Cells
L0591Stratagene pBluescript
HeLa cell
s3
937216 SK-
L0592Stratagene pBluescript
hNT neuron
(#937233) SK-
L0594Stratagene pBluescript
neuroepithelium 5K-
NT2RAMI 937234
L0595Stratagene neuroepithelialbrain pBluescript
NT2 neuronal
recursor 937230cells SK-
L0596Stratagene colon pBluescript
colon
(#937204)
SK-
L0597Stratagene cornea pBluescript
corneal
stroma (#937222) SK-
L0598Morton Fetal cochlea ear ~ pBluescript
Cochlea ~ -
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SK-
L0599 Stratagene lung pBluescrtpt
lung
(#937210) SK-
L0600 Weizmann Olfactoryolfactory nose pBluescript
E ithelium a ithelium SK-
L0601 Stratagene pancreas pBluescript
pancreas
(#937208) SK-
L0603 Stratagene placenta pBluescript
placenta
(#937225) SK-
L0605 Stratagene fetal spleenspleen pBluescript
fetal spleen
(#937205)
SK-
L0643 NCI_CGAP_Col9 moderately colon pCMV-
differentiated SPORT6
adenocarcinoma
L0651 NCI_CGAP_Kid8 renal cell kidney pCMV-
tumor
SPORT6
L0662 NCI_CGAP_Gas4 poorly stomach pCMV-
differentiated SPORT6
adenocarcinoma
with si
net r
L0666 NCI_CGAP_Utl well-differentiateduterus pCMV-
endometrial SPORT6
adenocarcinoma,
7
L0717 Gessler Wilms SPORT1
tumor
L0731 Soares_pregnant uterus pTTT3-Pac
uterus
NbHPU
L0740 Soares melanocytemelanocyte pTTT3D
2NbHM (Pharmacia)
with
a
modified
of linker
L0742 Soares adult brain pTTT3D
brain
N2b5HB55Y (Pharmacia)
with
a
modified
of linker
L0743 Soares breast breast pTTT3D
2NbHBst
(Pharmacia)
with
a
modified
of linker
L0744 Soares breast breast pTTT3D
3NbHBst
(Pharmacia)
with
a
modified
of linker
L0745 Soares retina retina eye pTTT3D
N2b4HR
(Pharmacia)
with
a
modified
of linker
L0746 Soares retina retina eye pTTT3D
N2b5HR
(Pharmacia)
with
a
modified
of linker
L0747 Soares fetal heart pTTT3D
heart_NbH
H19W (Pharmacia)
with
a
modified
of linker
L0748 Soares fetal Liver pTTT3D
~ liver spleen and
~
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1NFLS Spleen (Pharmacia)
with
a
modified
olylinker
L0749 Soares fetal Liver pTTT3D
liver_splee and
n_1NFLS_S1 Spleen (Pharmacia)
with
a
modified
of linker
L0750 Soares fetal lung pTTT3D
lung_NbH
L19W (Pharmacia)
with
a
modified
of linker
L0751 Soares ovary ovarian ovary pTTT3D
tumor tumor
NbHOT (Pharmacia)
with
a
modified
of linker
L0752 Soares_parathyroid_tumparathyroidparathyroid pTTT3D
tumor
or_NbHPA gland (Pharmacia)
with
a
modified
of linker
L0753 Soares_pineal_gland pineal pTTT3D
N3
HPG gland (Pharmacia)
with
a
modified
polylinker
L0754 Soares placenta placenta pTTT3D
Nb2HP
(Pharmacia)
with
a
modified
of linker
L0755 Soares_placenta placenta pTTT3D
8to9we
eks 2NbHP8to9W (Pharmacia)
with
a
modified
of linker
L0756 Soares multiplemultiple pTTT3D
sclerosi sclerosis
s_2NbHMSP lesions (Pharmacia)
with
a
modified
polylinker
V_TYPE
L0758 Soares testis_NHT pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0759 Soares total_fetus_Nb2 pTTT3D-Pac
HF8_9w (Pharmacia)
with
a
modified
of linker
L0761 NCI_CGAP_CLLl B-cell, pTTT3D-Pac
chronic
lymphotic (Pharmacia)
.
leukemia with
a
modified
of linker
L0763 NCI_CGAP_Br2 breast pTTT3D-Pac
(Pharmacia)
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with
a
modified
of linker
L0764 NCI_CGAP_Co3 colon pT7T3D-Pac
(Pharmacia)
with
a
modified
of linker
L0766 NCI_CGAP_GCB1 germinalcenterB pTTT3D-Pac
cell (Pharmacia)
with
a
modified
of linker
L0768 NCI_CGAP_GC4 pooled germ pT7T3D-Pac
cell
tumors (Pharmacia)
with
a
modified
of linker
L0769 NCI_CGAP_Brn25anaplastic brain pT7T3D-Pac
oligodendroglioma (Pharmacia)
with
a
modified
of linker
L0770 NCI_CGAP_Brn23glioblastomabrain pTTT3D-Pac
(pooled) (Pharmacia)
with
a
modified
of linker
L0774 NCI_CGAP_Kid3 kidney pT7T3D-Pac
(Pharmacia)
with
a
modified
of linker
L0775 NCI_CGAP_KidS 2 pooled kidney pT7T3D-Pac
tumors
(clear cell (Pharmacia)
type)
with
a
modified
polylinker
L0776 NCI_CGAP_Lu5 carcinoid lung pT7T3D-Pac
(Pharmacia)
with
a
modified
of linker
L0777 Soares_NhHMPu Pooled humanmixed pT7T3D-Pac
S1 (see
melanocyte,below) (Pharmacia)
fetal
heart, and with
pregnant a
modified
of linker
L0779 Soares NFL._T_GBC_S pooled pT7T3D-Pac
1 (Pharmacia)
with
a
modified
polylinker
L0783 NCI_CGAP_Pr22 normal prostateprostate pT7T3D-Pac
(Pharmacia)
with
a
modified
of linker
L0789 NCI_CGAP_Sub3 pT7T3D-Pac
(Pharmacia)
with
a
modified
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of linker
L0794NCI_CGAP_GC6 pooled germ pTTT3D-Pac
cell
tumors (Pharmacia)
with
a
modified
of linker
L0803NCI_CGAP_Kidll kidney pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0805NCI_CGAP_Lu24 carcinoid lung pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0806NCI_CGAP_Lul9 squamous lung pTTT3D-Pac
cell
carcinoma, , (Pharmacia)
poorly
differentiated with
(4 a
modified
of linker
L0809NCI_CGAP_Pr28 prostate pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
TABLE 5
OMIM Description
Reference
107777 Diabetes insi idus, ne hro enic, autosomal recessive,
222000
108725 Atherosclerosis, susce tibili to
120700 C3 deficienc
123940 White s on a nevus, 193900
133171 E throc osis, familial , 133100
136836 Fucos ltransferase-6 de~cienc
139350 E idermol tic h erkeratosis, 113800
139350 Keratoderma, almo lantar, none idermol tic
145981 H ocalciuric h ercalcemia, a II
147141 Leukemia, acute lym hoblastic
148040 Epidermolysis bullosa simplex, Koebner, bowling-Meara,
and
Weber-Cocka ne es, 131900, 131760, 131800
148041 Pach on chia con enita, Jadassohn-Lewandows
e, 167200
148043 Meesmann corneal d stro h , 122100
148070 Liver disease, susce tibili to, from he atotoxins
or viruses
164953 Li osarcoma
177070 S heroc osis, heredita , Ja anese a
177070 Hermans -Pudlak s drome, 203300
182500 Cataract, con enital
188070 Bleedin disorder due to defective thromboxane
A2 rece for
203800 Alstrom syndrome
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218000 Andermann syndrome
227220 E a color, brown
229600 Fructose intolerance
231550 Achalasia-addisonianism-alacrimia s ndrome
243500 Isovalericacidemia
264300 Pseudoherma hroditism, male, with necomastia
600194 Ichth osis bullosa of Siemens, 146800
600231 Palmo lantar keratoderma, Bothnia a
600536 M o athy, con enital
600542 Chondrosarcoma, extraskeletal m oid
600808 Enuresis, nocturnal, 2
600839 Banter s ndrome, 241200
600956 Persistent Mullerian duct s ndrome, a II, 261550
600957 Persistent Mullerian duct s ndrome, a I, 261550
601238 Cerebellar ataxia, Ca man a
601284 Heredita hemorrha is telan iectasia-2, 600376
601769 Osteo orosis, involutional
601769 Rickets, vitamin D-resistant, 277440
601800 Hair color, brown
601846 Muscular d stro h with rimmed vacuoles
601928 Monilethrix, 158000
602014 H oma esemia with seconda h ocalcemia
602088 Ne hrono hthisis, infantile
602116 Glioma
602153 Monilethrix, 158000
602216 Peutz-Je hers s ndrome, 175200
602404 Parkinson disease, a 3
602477 ~ Febrile convulsions, familial, 2
Polynucleotide and Polypeptide T~ariafzts
[0102] The present invention is also directed to variants of the excretory
system
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
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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.
[0103] 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 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.
[0104] "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.
[0105] 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 excretory system 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 an excretory system 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 an excretory system associated
polypeptide;
(e) a nucleotide sequence encoding an excretory system 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 TD NO:Z; (f) a nucleotide sequence
encoding
a mature excretory system 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 an excretory
system
associated polypeptide having the complete amino acid sequence of SEQ ID NO:Y
or
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the complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (h) a
nucleotide sequence encoding an antigenic fragment of an excretory system
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.
[0106] 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
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
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encompassed by the invention, as are polypeptides encoded by these
polynucleotides
and nucleic acids.
(0107] 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 hybridization conditions or alternatively, under
lower
stringency conditions, are also encompassed by the invention, as are
polypeptides
encoded by these polynucleotides.
[0108] 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 excretory system 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 an excretory system 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 an excretory system 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.
[0109] 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
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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.
[0110] 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 eve 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.
[0111] 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
are 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
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percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, 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.
[0112] 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
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.
[0113] 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 10 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
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manually corrected for. No other manual corrections are to made for the
purposes of
the present invention.
[0114] 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
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.
[0115] 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
identiEed 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
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alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=l, Joining
Penalty=20,
Randomization Group Length=0, Cutoff Score=l, Window Size=sequence length, Gap
Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject
amino acid sequence, whichever is shorter.
[0116] If the subject sequence is shorter than the query sequence due to N- or
C-
terminal 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 N- and
C-
tenninal 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 aligmnent. 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.
[0117] For example, a 90 amino acid residue subject sequence is aligned with a
100 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.
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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 matchedlaligned
with
the query sequence are manually corrected for. No other manual corrections are
to
made for the purposes of the present invention.
[0118] 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).
[0119] Naturally occurnng 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 York (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.
[0120] 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).)
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[0121] 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
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.
[0122] 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.
[0123] 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.
[0124] 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, for instance,
as a
hybridization probe or a polymerise chain reaction (PCR) primer. Uses of the
nucleic
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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
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 excretory system or diseased
excretory
system tissues); and (4) in situ hybridization (e.g., histochemistry) for
detecting
mRNA expression in specific tissues (e.g., normal excretory system or diseased
excretory system tissues).
[0125] 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.
[0126] The functional activity of the polypeptides, and fragments, variants
and
derivatives of the invention, can be assayed by various methods.
[0127] 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
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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
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.
[0128] 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 known in the art.
[0129] 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 ih vivo). Other,methods will be known
to the
skilled artisan and are within the scope of the invention.
[0130] 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
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activity. This is because the skilled artisan is fully aware of amino acid
substitutions
that are either less likely or not likely to significantly effect protein
function (e.g.,
replacing one aliphatic amino acid with a second aliphatic amino acid), as
further
described below.
[0131] For example, guidance concerning how to make phenotypically silent
amino acid substitutions is provided in Bowie 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.
[0132] 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.
[0133] 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.
[0134] 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,
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and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
Besides
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 andlor 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.
[0135] 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 Pinclcard 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).
[0136] 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, and/or the amino
acid sequence encoded by cDNA contained in Clone ID 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.
[0137] 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, and/or 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.
Polynucleotide ahd Polypeptide Fragments
[0138] 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
encoding a portion of a polypeptide encoded by the complement of the
polynucleotide
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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.
[0139] 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.
[0140] 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, 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-
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-
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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 lower
stringency conditions are also encompassed by the invention, as are
polypeptides
encoded by these polynucleotides.
[014I] Further 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, 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-
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-
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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.
[0142] 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 fm-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
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
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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.
[0143] 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.
[0144] 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 Glone 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.
[0145] 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,
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.
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[0146] 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 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 polynucle~tides, nucleic acids, and polypeptides are also
encompassed by
the invention.
[0147] 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.
[0148] 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'
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
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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.
[0149] 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 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.
[0150] 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, andlor 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
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 I-20, 2I-40, 4I-60, 6I-80, 8I-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,
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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, 22I-240, 241-260, 261-280, 281-300,
301-
320, 321-340, 34I-360, 361-380, 381-400, 40I-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, 76I-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 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.
[0151] 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
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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 evoke an immune response.
[0152] 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 are preferred.
Similarly, polynucleotides encoding these polypeptide fragments are also
preferred.
[0153] 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
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.
[0154] 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
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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.
[0155] 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.
[0156] 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
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
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
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.
[0157] 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
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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.
[0158] 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~.
[0159] 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; Kyte-Doolittle hydrophilic regions and hydrophobic regions;
Eisenberg alpha- and beta-amphipathic regions; Karplus-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
encode polypeptides comprising regions that combine several structural
features, such
as several (e.g., l, 2, 3 or 4) of the features set out above.
[0160] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic
regions,
Ernini 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
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environment in which antigen recognition may occur in the process of
initiation of an
immune response.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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
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 8 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
supra. The
present invention further encompasses polynucleotide sequences encoding an
epitope
of a polypeptide sequence of the invention (such as, for example, the sequence
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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.
[0165] 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.
[0166] 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.)
[0167] 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 I I, at Ieast 12, at least I3, at least 14, at least 15, at Ieast
20, at Ieast 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
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epitopes disclosed herein, as well as any 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., CeII 37:767-778 (1984);
Sutcliffe et
al., Science 219:660-666 (1983)).
[0168] 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 heterologous polypeptide, such as from another protein
described
herein or from a heterologous polypeptide not described herein. In particular
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.
[0169] 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
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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).
[0170] 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, in vivo immunization, in vitwo 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 in 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 (I~I,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
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.
[0171] 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 (CH1, 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
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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 I6,
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.
[0172] Such fusion proteins as those described above may facilitate
purification
and may increase half life in 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
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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
[0173] 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
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.
[0174] 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.
[0175] 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.
[0176] 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
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to final preparation of the polypeptide. The addition of peptide moieties to
facilitate
handling of polypeptides are familiar and routine techniques in the art.
[0177] 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 (CH1, 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
polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045$69)
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); I~.
Johanson et
al., J. Biol. Chem. 270:9459-9471 (1995).
[0178] 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.
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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).)
[0179] 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. I6(2):76-82 (I998); 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
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.
[0180] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
Recombinant and Synthetic Production of Polypentides of the Invention
[0181] 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.
[0182] 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.
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[0183] The polynuclebtide insert should be operatively linked to an
appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, tip, 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.
[0184] As indicated, the expression vectors will preferably include at least
one
selectable marker. Such markers include dihydrofolate reductase, 6418 or
neomycin
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, St~eptomyces and Salmonella typhimu~ium cells; fungal
cells,
such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC
Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera 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.
[0185] Among vectors preferred for use in bacteria include pQE70, pQE60 and
pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors,
pNHBA, 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, pTEF1/Zeo, pYES2/GS, pPICZ,
pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S 1, pPIC3.5K, pPIC9K, and
PA0815 (all available from Invitrogen, Carlsbad, CA). Other suitable vectors
will be
readily apparent to the skilled artisan.
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[0186] 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 marine 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
Biologics, Inc. (Portsmouth, NH). Expression and production of monoclonal
antibodies using a GS expression system in marine myeloma cells is described
in
Bebbington et al., Bioltech~cology 10:169(1992) and in Biblia and Robinson
Biotechnol. P~og. 11:1 (1995) which are herein incorporated by reference.
[0187] 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 rnay 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.
[0188] Introduction of the nucleic acids and nucleic acid constructs of the
invention into the host cell can be effected by calcium phosphate
transfection, DEAE-
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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.
[0189] 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.,
excretory
system antigen coding sequence), and/or to include genetic material (e.g.,
heterologous
polynucleotide sequences) that is operably associated with excretory system
associated
polynucleotides of the invention, and which activates, alters, and/or
amplifies
endogenous excretory system 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 excretory system 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., Proc. Natl. Acad.
Sci.
USA 86:8932-8935 (1989); and Zijlstra et al., Nature 34:435-438 (1989), the
disclosures of each of which are incorporated by reference in their
entireties).
[0190] 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 from any protein after translation in all
eukaryotic cells.
While the N-terminal methionine on most proteins also is efficiently removed
in most
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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.
[0191] 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
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 02. Consequently, in a growth medium depending on methanol as a
main
carbon source, the promoter region of one of the two alcohol oxidase genes
(AOXI ) is
highly active. In the presence of methanol, alcohol oxidase produced from the
AOXl
gene comprises up to approximately 30% of the total soluble protein in Pichia
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
AOXI
regulatory sequence is expressed at exceptionally high levels in Pichia yeast
grown in
the presence of methanol.
[0192] 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 pastoris alkaline phosphatase
(PHO)
secretory signal peptide (i.e., leader) located upstream of a multiple cloning
site.
[0193] 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 skilled in the art
would
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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.
[0194] 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
expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
[0195] 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; Roller 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).
[0196] 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
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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
analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L
(levorotary).
[0197] 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 known 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.
[0198] 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.
[0199] 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 Iuciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include iodine (12y, i2sh lash i3lI), carbon
(14C), sulfur
(3sS), tritium (3H), indium (111In, uzln, l3mIn, llsmIn), technetium
(99Tc,99mTc),
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thallium (Z°1Ti), gallium (68Ga, 6~Ga), palladium (lo3Pd), molybdenum
(99Mo), xenon
(is3Xe) fluorine (isF) isssm i~~Lu is9Gd i49Pm i4oLa mss m6Ho 9°Y 4~Sc
rs6Re
> > > > > > > > > > > >
lasRe~ iazPr~ ios~~ and 9~Ru.
[0200] 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, l~~Lu, 9oY, 166H0, and ls3sm, 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.
[0201] As mentioned, the excretory system 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 excretory system associated polypeptide.
excretory
system 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 excretory system 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,
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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 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)).
[0202] 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 modified at random
positions
within the molecule, or at predetermined positions within the molecule and may
include one, two, three or more attached chemical moieties.
[0203] The polymer may be of any molecular weight, and rnay 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.
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[0204] As noted above, the polyethylene glycol may have a branched structure.
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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
Linkerless 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.
[0209] 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 (CISOZCH2CF3). 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.
[0210] 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
wherein the polyethylene glycol is attached to the protein by a linker can
also be
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produced by reaction of proteins with compounds such as MPEG-
succinimidylsuccinate, MPEG activated with l, l'-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.
[0211] 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 1, 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).
[0212] The excretory system 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 cation 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.
[0213] Excretory system 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 excretory
system
associated antigens. Among these are applications in the detection,
prevention,
diagnosis andlor treatment of diseases associated with excretory system, such
as e.g.,
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excretory system cancer, tumors, renal disorders (e.g., kidney failure,
nephritis, blood
vessel disorders of kidney, metabolic and congenital kidney disorders, urinary
disorders of the kidney, autoimmune disorders, sclerosis and necrosis, and
electrolyte
imbalance, and kidney cancer), bladder disorders (e.g, urinary tract
infection, bladder
obstruction, urination disorders, and bladder cancer), urether disorders
(e.g.,
obstruction of the ureter and ureter cancer), urethra disorders (e.g.,
obstruction of the
urethra and urethra cancers) and/or those disorders as discribed under
"Urinary System
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.
[0214] 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.
[0215] 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.
[0216] 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 TD NO:X or the complement of SEQ
ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as
defined in columns 8 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
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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 and/or
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.
[0217] 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 multirner 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.
[0218] 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 and/or 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
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
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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.
[0219] 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.
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.
[0220] 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
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incorporated by reference. Other peptides derived from naturally occurring
trimeric
proteins may be employed in preparing trimeric polypeptides of the invention.
[0221] 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.
[0222] 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
components
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).
[0223] 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
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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
[0224] 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),
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, IgAl
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.
[0225] Most preferably the antibodies are human antigen-binding antibody
fragments of the present invention and include, but are not limited to, Fab,
Fab' and
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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,
marine (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
infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
[0226] The antibodies of the present invention may be monospecihc, 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.
147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4;714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0227j 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.
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[0228] 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%, 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).
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 I~d 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-~ M, 10'
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.
[0229] 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%.
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[0230] 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.
[0231] 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
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; LT.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
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(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 (1997); Taryman et al.,
Neuron
14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998);
B''artunek et al.,
Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in
their
entireties).
[0232] 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
in 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.
[0233] 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 or other compositions. For example, antibodies
of the
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; U.S. Patent No. 5,314,995; and EP 396,387; the
disclosures
of which are incorporated herein by reference in their entireties.
[0234] 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/blocking 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
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tunicamycin, etc. Additionally, the derivative may contain one or more non-
classical
amino acids.
[0235] The antibodies of the present invention may be generated by any
suitable
method known 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.
[0236] 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
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.
[0237] 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
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from cell line SP20 available from the ATCC. Hybridomas are selected and
cloned by
limited dilution. The hybridoma clones are then assayed by methods known 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.
[0238] 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.
[0239] 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,
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.
[0240] 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
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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.
[0241] 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
CH1 domain of the heavy chain. For example, the antibodies of the present
invention
can also be generated using various phage display methods known 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 murine). Phage expressing an
antigen binding domain that binds the antigen of interest can be selected or
identified
with antigen, e.g., using labeled antigen or antigen bound or captured to a
solid surface
or bead. Phage used in these methods are typically filamentous phage including
fd and
M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized
Fv
antibody domains recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make the
antibodies
of the present invention include those disclosed in Brinkman et al., J.
Immunol.
Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);
Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene
187 9-18
(1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application
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No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92101047; 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, I08; each of which is incorporated herein by reference in its entirety.
[0242] 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-
34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
[0243] 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 in
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
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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-
814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling
(U.S.
Patent No. 5,565,332).
[0244] 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.
[0245] 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
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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
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. Tn 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.
[0246] 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)).
[0247] 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
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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
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.
[0248] 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 Encoding Antibodies
[0249] 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.
[0250] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
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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.
[0251] Alternatively, a polynucleotide encoding an antibody may be generated
from nucleic acid from a suitable source. If a clone containing a nucleic acid
encoding
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.
[0252] 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, 2d 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.
[0253] 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
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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
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 skill of the art.
[0254] 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); Takeda 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 supra, 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.
[0255] 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
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assembly of functional Fv fragments in E. coli may also be used (Skerra et
al., Science
242:1038- 1041 (1988)).
Methods of P~oducihg Antibodies
[0256] 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
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.
[0257] 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 slcilled 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,
irz vitro recombinant DNA techniques, synthetic techniques, and in 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.
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[0258] 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-
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.
[0259] 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
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expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett
et al.,
Bio/Technology 8:2 (1990)).
[0260] 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
produced, for the generation of pharmaceutical compositions of an antibody
molecule,
vectors which direct the expression of high levels of 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 (1985); 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.
[0261] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera
frugiperda 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).
[0262] 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
transcription/translation
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 in 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.
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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
the entire insert. These exogenous translatiorial 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)).
[0263] 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.
[0264] 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
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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.
Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
[0265] 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.
[0266] 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
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culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
[0267] 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 marine 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 marine 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.
[0268] 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.
[0269] 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.
[0270] 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 in
vitro or in 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 ih
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.
[0271] 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
conjugated to the above antibody portions to form multimers. For example, Fc
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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).
[0272] 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 in 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
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)).
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[0273] 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.
[0274] 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.
[0275] 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,
etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,
daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
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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).
[0276] 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. Immuhol., 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.
[0277] 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.
[0278] 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.,
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"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).
[0279] 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.
[0280] An antibody, with or without a therapeutic moiety conjugated to it,
administered alone or in combination with cytotoxic factors) andlor
cytolcine(s) can
be used as a therapeutic.
Immunopl2enotyping
[0281] 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
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)).
[0282] 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
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screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation and/or differentiation, as might be found in human umbilical
cord blood.
Assays Fog Antibody Binding
[0283] 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 linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays,
precipitin reactions, gel 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).
[0284] 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
SDSlsample
buffer. The ability of the antibody of interest to immunoprecipitate a
particular
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. l, John Wiley & Sons, Inc., New York, section 10.16.1.
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[0285] 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), blocking
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 alkaline
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 skill 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.
[0286] 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
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. 1, John Wiley & Sons, Inc., New York,
section
11.2.1.
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[0287] 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.
(0288] 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 excretory sy'"stem
antigen or with
vector alone using techniques commonly known in the art. Antibodies that bind
excretory system antigen transfected cells, but not vector-only transfected
cells, are
excretory system antigen specific.
Therapeutic Uses
[0289] 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
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
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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.
[0290] 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 excretory system,
including, but
not limited to, renal disorders (e.g., kidney failure, nephritis, blood vessel
disorders of
kidney, metabolic and congenital kidney disorders, urinary disorders of the
kidney,
autoimmune disorders, sclerosis and necrosis, and electrolyte imbalance, and
kidney
cancer), bladder disorders (e.g, urinary tract infection, bladder obstruction,
urination
disorders, and bladder cancer), urether disorders (e.g., obstruction of the
ureter and
ureter cancer), urethra disorders (e.g., obstruction of the urethra and
urethra cancers)
and/or those disorders as discribed under "Urinary System 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 an excretory
system
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 excretory system
described herein.
The treatment and/or prevention of diseases, disorders, or conditions of the
excretory
system 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.
[0291] A summary of the ways in which the antibodies of the present invention
may be used therapeutically includes binding polynucleotides or polypeptides
of the
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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.
[0292] 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.
[0293] 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,
human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered to
a human patient for therapy or prophylaxis.
[0294] It is preferred to use high affinity and/or 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 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 I~d
less than 5 X 10-2 M, 10-Z M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, S X 10-5
M, 10-5
M, 5 X 10-6 M, 10-6 M, 5 X 10-~ M, 10-' M, 5 X 10-8 M, 10-g M, 5 X 10-9 M, 10-
9 M, 5
X 10-1° M, 10-1° M, 5 X 10-11 M, 10-11 1VI, 5 X 10-12 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
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[0295] 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.
[0296] Any of the methods for gene therapy available in the art can be used
according to the present invention. Exemplary methods are described below.
[0297] 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):155-215 (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
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990).
[0298] 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.
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[0299] 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
i~ vitro,
then transplanted into the patient. These two approaches are known,
respectively, as ih
vivo or ex vivo gene therapy.
[0300] In a specific embodiment, the nucleic acid sequences are directly
administered irr 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
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 ih 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)).
[0301] 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 packaging 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
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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).
[0302] 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
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 al., 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.
[0303] 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).
[0304] 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.
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[0305j In this embodiment, the nucleic acid is introduced into a cell prior to
administration ih vivo of the resulting recombinant cell. Such introduction
can be
carried out by any method,known 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
the nucleic acid is expressible by the cell and preferably heritable and
expressible by
its cell progeny.
[0306] 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.
[0307] 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.
[0308] In a preferred embodiment, the cell used for gene therapy is autologous
to
the patient.
[0309] 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
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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)).
[0310] 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.
Demonstration of Therapeutic or Prophylactic Activity
[0311] 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, in 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 skill in the art including,
but not
limited to, rosette formation assays and cell lysis assays. In accordance with
the
invention, in 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.
TherapeuticlProphylactic Administration and Composition
[0312] 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,
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pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most
preferably human.
[0313] 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. .
[0314] 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., Wu and Wu, J. Biol. Chem. 262:4429-4432
(1987)),
construction of a nucleic acid as part of a retroviral or other vector, etc.
Methods of
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.
[0315] 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.
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[0316] In another embodiment, the compound or composition can be delivered in
a
vesicle, in particular a liposome (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.)
[0317] 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
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)).
[0318] Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
[0319] 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.
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[0320] 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
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.
[0321] 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
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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
by injection, an ampoule of sterile water for injection or saline can be
provided so that
the ingredients may be mixed prior to administration.
[0322] 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 cations such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino
ethanol,
histidine, procaine, etc.
[0323] 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, in 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.
[0324] 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
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by enhancing uptake and tissue penetration (e.g., into the brain) of the
antibodies by
modifications such as, for example, lipidation.
[0325] 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,
use or sale of pharmaceuticals or biological products, which notice reflects
approval by
the agency of manufacture, use or sale for human administration.
Diagv~osis ahd Imaging
[0326] 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.
[0327] The invention provides a diagnostic assay for diagnosing an excretory
system 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
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treatment earlier thereby preventing the development or further progression of
the
cancer.
[0328] 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
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.
[0329] 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
excretory
system associated with aberrant expression of an excretory system 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 background 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.
[0330] 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
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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. 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
Publishing Inc. (1982)).
[0331] 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.
[0332] 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.
[0333] 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.
[0334] 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
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yet another embodiment, the molecule is labeled with a paramagnetic label and
is
detected in a patient using magnetic resonance imaging (MRI).
Kits
[0335] 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
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).
[0336] 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.
[0337] 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
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embodiment, binding of the antibody to the polypeptide antigen can be detected
by
binding of the said reporter-labeled antibody.
[0338] 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
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.
[0339] 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).
[0340] The solid surface reagent in the above assay is prepared by known
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).
[0341] Thus, the invention provides an assay system or kit for carrying out
this
diagnostic method. . The kit generally includes a support with surface-bound
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recombinant antigens; and a reporter-labeled anti-human antibody for detecting
surface-bound anti-antigen antibody.
Uses of the Polynucleotides
[0342] 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.
[0343] The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
since few chromosome marking 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.
[0344] 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.
[0345] 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).
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[0346] 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,
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).
[0347] 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).
[0348] 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.
[0349] The polynucleotides of the present invention would likewise 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
(1997); Aydin, J. Mol. 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); andlor Ott, J. Hered. 90:68-70 (1999),
each of
which is hereby incorporated by reference in its entirety.
[0350] Once a polynucleotide has been mapped to a precise chromosomal
location,
the physical position of the polynucleotide can be used in linkage analysis.
Linkage
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 IA 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.
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[0351] Thus, once coinheritance is established, differences in a
polynucleotide of
the invention and the corresponding gene between affected and unaffected
individuals
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,
indicates
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.
[0352] 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, kits 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 Excretory System Disease, Including Cancer").
[0353] 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).
[0354] In still another embodiment, the invention includes a kit for analyzing
samples for the presence of proliferative andlor 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
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internal region of the polynucleotide of the invention, where each probe has
one strand
containing a 31'mer-end internal to the region. In a further embodiment, the
probes
may be useful as primers for polymerase chain reaction amplification.
[0355] 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.
[0356] 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 level in the first 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.
[0357] 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.
[0358] 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 supra are hereby incorporated by reference in their
entirety
herein.
[0359] 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 PNA/DNA 15-mer lowers the
melting point (Tm) by 8°-20° C, vs. 4°-16° C
for the DNA/DNA 15-mer duplex.
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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.
[0360] 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 myelomonocytic
leukemia, acute erythroleukemia, acute megakaryocytic leukemia, 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.
[0361] The compounds of the present invention have preferred uses, which
include, but are not limited to, detecting excretory system cancer in mammals.
In
particular the invention is useful during diagnosis of pathological cell
proliferative
neoplasias which include, but are not limited to: nephroblastoma, renal cell
cancer
hypernephroma, transitional cell cancer, squamous cell cancer, Wilm's tumor,
superficial bladder cancer, invasive bladder cancer, carcinoma of the ureter,
and
urethra cancer. Preferred mammals include monkeys, apes, cats, dogs, cows,
pigs,
horses, rabbits and humans. Particularly preferred are humans.
[0362] 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. (Gelmann et al.,
supra) It is
likely 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
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neoplasias have been amplified or translocated in some cases of human leukemia
and
carcinoma. (Gelmann et al., supra)
(0363] 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; Wickstrom
et
al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad.
Sci.
86:3379 (1989)). 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 excretory system cells and tissues.
[0364] 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
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 in,
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
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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 iya 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)").
[0365] 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
more thoroughly described elsewhere herein (see, e.g., the sections labeled
"Gene
Therapy Methods" and Examples 16, 17 and 18).
[0366] 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 Iost, switched, or stolen, making positive
identification
difficult. The polynucleotides of the present invention can be used as
additional DNA
markers for RFLP.
[0367] 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
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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.
[0368] 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.
[0369] 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.
[0370] . Because excretory system antigens are found expressed in excretory
system, 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
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present invention are also useful as hybridization probes for differential
identification
of excretory system 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 excretory
system
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 excretory system or diseased excretory system
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 1A,
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.
[0371j 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.
[0372] 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.
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Uses of the Polyneptides
[0373] Each of the polypeptides identified herein can be used in numerous
ways.
The following description should be considered exemplary and utilizes known
techniques.
[0374] 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).
[0375] 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); Jalkanen, 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,
izsh lash lall)~ carbon (14C), sulfur (3sS), tritium (3H), indium (lls"'In,
113mIn~ ma~~
mIn), and technetium (99Tc, 99mTc), thallium (2°1Ti), gallium (68Ga,
6~Ga), palladium
io3Pd mol bdenum 99Mo xenon (l3sXe) fluorine (18F) lssSm l~~Lu ls9Gd ia9Pm
( )~ Y ( )> > > > > > >
i4oLa~ ms~~ 166H0~ 9oY~ a~Sc, 186Re, 188Re, 142Pr, iosRh, 9~Ru; luminescent
labels, such
as luminol; and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0376] In addition to assaying levels of polypeptide of the present invention
in a
biological sample, proteins can also be detected i~ vivo by imaging. Antibody
labels
or markers for ih 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.
[0377] An excretory system antigen-specific antibody or antibody fragment
which
has been labeled with an appropriate detectable imaging moiety, such as a
radioisotope
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for exam 1e 131I 112In 99mTc (1311 lzsl lz3l 121I) carbon (14C) sulfur (3sS)
tritium
( p > > > > > > > > > >
(3H), indium (llsmln, llsmln, llaln, 111In), and technetium (99Tc, 99mTc),
thallium (2olTi),
gallium (68Ga, 6~Ga), palladium (lo3pd), molybdenum (99Mo), xenon (133Xe),
fluorine
(18F ls3sm 177Lu is9Gd 149Pm 140La l7sYb 166H~ 90Y 47Sc 186Re 188Re 142Pr
> > > > > > > > > > > > >
lost, 9~Ru), 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 an excretory system
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 express the polypeptide encoded by a polynucleotide of the
invention. Ih
vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharrnacokinetics of
Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging. The
Radiochemical Detection of Ca~zcer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson
Publishing Inc. (1982)).
[0378] 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.
[0379] 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.
[0380] In a preferred embodiment, the invention provides a method for the
specific
destruction of excretory system cells (e.g., aberrant excretory system cells,
excretory
system neoplasm) by administering polypeptides of the invention (e.g.,
polypeptides
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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.
[0381] 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, thylilidine 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
includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive
metal ion,
e.g., alpha-emitters such as, for example, 213$1, or other radioisotopes such
as, for
exam 1e lo3Pd 133Xe 1311 111In 68Ge S~Co 65zn 85Sr 32P 35S 90y 153Sm 153Gd
p > > > > > > > > > > > > > >
169yb' SiCr' S4Mn' 75se' 113sn' 9oYttrllim, ll~Tin, 186IZhenium, 166HOlmlum,
and
188Rhenium; luminescent labels, such as luminol; and fluorescent labels, such
as
fluorescein and rhodamine, and biotin.
[0382] 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.
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[0383] 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).
[0384] 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 level 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
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. a
[0385] Moreover, polypeptides of the present invention can be used to treat or
prevent diseases or conditions of the excretory system such as, for example,
renal
disorders (e.g., kidney failure, nephritis, blood vessel disorders of kidney,
metabolic
and congenital kidney disorders, urinary disorders of the kidney, autoimmune
disorders, sclerosis and necrosis, and electrolyte imbalance, and kidney
cancer),
bladder disorders (e.g, urinary tract infection, bladder obstruction,
urination disorders,
and bladder cancer), urether disorders (e.g., obstruction of the ureter and
ureter
cancer), urethra disorders (e.g., obstruction of the urethra and urethra
cancers) and/or
those disorders as discribed under "Urinary System 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
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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).
[0386] 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).
(0387] 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 skill 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
[0388] a The compounds of the present invention are useful for diagnosis,
treatment,
prevention and/or prognosis of various excretory system related disorders in
mammals,
preferably humans. Such disorders include, but are not limited to, renal
disorders (e.g.,
kidney failure, nephritis, blood vessel disorders of kidney, metabolic and
congenital
kidney disorders, urinary disorders of the kidney, autoimmune disorders,
sclerosis and
necrosis, and electrolyte imbalance, and kidney cancer), bladder disorders
(e.g, urinary
tract infection, bladder obstruction, urination disorders, and bladder
cancer), urether
disorders (e.g., obstruction of the ureter and ureter cancer), urethra
disorders (e.g.,
obstruction of the urethra and urethra cancers) and/or those disorders as
discribed
under "Urinary System Disorders" below. In preferred embodiments,
polynucleotides
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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.
[0389] Excretory system antigens are expressed in the excretory system, with
an
increased expression level in excretory system tissues. For a number of
excretory
system-related disorders, substantially altered (increased or decreased)
levels of
excretory system antigen gene expression can be detected in excretory system
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"
excretory system antigen gene expression level, that is, the excretory system
antigen
expression level in excretory system tissues or bodily fluids from an
individual not
having the excretory system disorder. Thus, the invention provides a
diagnostic
method useful during diagnosis of an excretory system disorder, which involves
measuring the expression level of the gene encoding the excretory system
associated
polypeptide in excretory system tissue or other cells or body fluid from an
individual
and comparing the measured gene expression level with a standard excretory
system
antigens gene expression level, whereby an increase or decrease in the gene
expression
levels) compared to the standard is indicative of an excretory system
disorder.
[0390] 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.
[0391] In particular, it is believed that certain tissues in mammals with
cancer of
cells or tissue of the excretory system express significantly enhanced or
reduced levels
of normal or altered excretory system antigen expression and mRNA encoding the
excretory system associated polypeptide when compared to a corresponding
"standard"
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level. Further, it is believed that enhanced or depressed levels of the
excretory system
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.
[0392] For example, as disclosed herein, excretory system associated
polypeptides
of the invention are expressed in excretory system tissues. Accordingly,
polynucleotides of the invention (e.g., polynucleotide sequences complementary
to all
or a portion of an excretory system 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 excretory system expressing excretory system antigens,
preferrably on
their cell surfaces. These polynucleotides and antibodies additionally have
diagnostic
applications in detecting abnormalities in the level of excretory system
antigens gene
expression, or abnormalities in the structure and/or temporal, tissue,
cellular, or
subcellular location of excretory system antigens. These diagnostic assays may
be
performed i~z vivo or i~c 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
excretory
system sequence disclosed in the same row of Table 1A, preferrably on their
cell
surface.
[0393] Thus, the invention provides a diagnostic method useful during
diagnosis of
an excretory system disorder, including cancers, which involves measuring the
expression level of the gene encoding the excretory system antigen polypeptide
in
excretory system tissue or other cells or body fluid from an individual and
comparing
the measured gene expression level with a standard excretory system antigen
gene
expression level, whereby an increase or decrease in the gene expression level
compared to the standard is indicative of an excretory system 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
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Table 1A expressing the corresponding excretory system sequence disclosed in
the
same row of Table 1A, preferrably on their cell surface.
[0394] Where a diagnosis of a disorder in the excretory system, 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 excretory system antigen gene expression will experience
a
worse clinical outcome relative to patients expressing the gene at a level
nearer the
standard level.
[0395] By "assaying the expression level of the gene encoding the excretory
system associated polypeptide" is intended qualitatively or quantitatively
measuring or
estimating the level of the excretory system antigen polypeptide or the level
of the
mRNA encoding the excretory system 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 excretory system associated
polypeptide
level or mRNA level in a second biological sample). Preferably, the excretory
system
antigen polypeptide expression level or mRNA level in the first biological
sample is
measured or estimated and compared to a standard excretory system 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
excretory system. As will be appreciated in the art, once a standard excretory
system
antigen polypeptide level or mRNA level is known, it can be used repeatedly as
a
standard for comparison.
[0396] By "biological sample" is intended any biological sample obtained from
an
individual, cell line, tissue culture, or other source containing excretory
system antigen
polypeptides (including portions thereof) or mRNA. As indicated, biological
samples
include body fluids (such as sera, plasma, urine, synovial fluid and spinal
fluid) which
contain cells expressing excretory system antigen polypeptides, excretory
system
tissue, and other tissue sources found to express the full length or fragments
thereof of
an excretory system 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.
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[0397] Total cellular RNA can be isolated from a biological sample using any
suitable technique such as the single-step guanidinium-thiocyanate-phenol-
chloroform
method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987).
Levels of mRNA encoding the excretory system 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).
[0398] The present invention also relates to diagnostic assays such as
quantitative
and diagnostic assays for detecting levels of excretory system 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 excretory system 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 an excretory system 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 excretory system antigen polypeptide levels in a
biological
sample can occur using any art-known method.
[0399] Assaying excretory system antigen polypeptide levels in a biological
sample can occur using antibody-based techniques. For example, excretory
system
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 excretory system 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
(lash lall)~ carbon (14C), sulfur (35S), tritium (3H), indium (~ l2In), and
technetium
(99mTc), and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
[0400] The tissue or cell type to be analyzed will generally include those,
which
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are known, or suspected, to express the excretory system antigen gene (such
as, for
example, cells of the excretory system or cancer or excretory system tissues).
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
York), 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 excretory system antigen gene.
[0401] For example, antibodies, or fragments of antibodies, such as those
described herein, may be used to quantitatively or qualitatively detect the
presence of
excretory system 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.
[0402] In a preferred embodiment, antibodies, or fragments of antibodies
directed
to any one or all of the predicted epitope domains of the excretory system
antigen
polypeptides (Shown in Table 1A, column 6) may be used to quantitatively or
qualitatively detect the presence of excretory system 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.
[0403] In an additional preferred embodiment, antibodies, or fragments of
antibodies directed to a conformational epitope of an excretory system antigen
may be
used to quantitatively or qualitatively detect the presence of excretory
system 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.
[0404] The antibodies (or fragments thereof), and/or excretory system 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 excretory system 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
excretory system antigen polypeptide of the present invention. The antibody
(or
fragment thereof) or excretory system 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
excretory system antigen gene product, or conserved variants or peptide
fragments, or
excretory system 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.
[0405] Immunoassays and non-immunoassays for excretory system 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
detestably labeled antibody capable of binding excretory system 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.
[0406] 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
detestably
labeled anti-excretory system antigen antibody or detectable excretory system
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.
[0407] 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,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural
and
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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.
[0408] The binding activity of a given lot of anti-excretory system antigen
antibody or excretory system 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.
[0409] In addition to assaying excretory system antigen polypeptide levels or
polynucleotide levels in a biological sample obtained from an individual,
excretory
system antigen polypeptide or polynucleotide can also be detected in vivo by
imaging.
For example, in one embodiment of the invention, excretory system antigen
polypeptide and/or anti-excretory system antigen antibodies are used to image
excretory system diseased cells, such as neoplasms. In another embodiment,
excretory
system antigen polynucleotides of the invention (e.g., polynucleotides
complementary
to all or a portion of excretory system antigen mRNA) and/or anti-excretory
system
antigen antibodies (e.g., antibodies directed to any one or a combination of
the
epitopes of excretory system antigens, antibodies directed to a conformational
epitope
of excretory system 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 excretory system.
[0410] Antibody labels or markers for in vivo imaging of excretory system
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 in vivo imaging is used to detect
enhanced
levels of excretory system 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, Science 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., Nature 312:643 (1984); Neuberger et al.,
Nature
314:268 (1985).
[0411] Additionally, any excretory system antigen polypeptides whose presence
can be detected, can be administered. For example, excretory system 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 excretory system antigen polypeptides can be utilized for in
vitro
diagnostic procedures.
[0412] An excretory system antigen polypeptide-specific antibody or antibody
fragment which has been labeled with an appropriate detectable imaging moiety,
such
as a radioisotope (for example, 131h il2ln, 99mTc), 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
an excretory system 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 excretory
system
antigen protein. In vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments"
(Chapter
13 in Turnor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and
B.
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A. Rhodes, eds., Masson Publishing Inc. (1982)).
[0413] With respect to antibodies, one of the ways in which the anti-excretory
system antigen antibody can be detectably labeled is by linking the same to an
enzyme
and using the linked product in an enzyme immunoassay (EIA) (Voller, A., "The
Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,
Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et
al., J.
Clin. Pathol. 31:507-520 (1978); Butler, J.E., Meth. Enzymol. 73:482-523
(1981);
Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,;
Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku 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.
[0414] 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 excretory system 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.
[0415] It is also possible to label the antibody with a fluorescent compound.
When
the fluorescently labeled antibody is exposed to light of the proper
wavelength, its
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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.
[0416] The antibody can also be detectably labeled using fluorescence emitting
metals such as lsaEu, or others of the lanthanide series. These metals can be
attached
to the antibody using such metal chelating groups as
diethylenetriaminepentacetic acid
(DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0417] 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.
[0418] 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, Iuciferase and aequorin.
Methods for Detecting Excretory System Disease, Including Cancer
[0419] In general, an excretory system disease or cancer may be detected in a
patient based on the presence of one or more excretory system .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 an excretory system 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, cancer of excretory system tissues.
In addition,
such proteins and/or polynucleotides may be useful for the detection of other
diseases
and cancers, including cancers of tissues/cells corresponding to the library
source
disclosed in column 7 of Table 1A expressing the corresponding excretory
system
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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 excretory system antigen polypeptides, which is also
indicative of
the presence or absence of a excretory system disease or disorder, including
cancer. In
general, excretory system antigen polypeptides should be present at a level
that is at
least three fold higher in diseased tissue than in normal tissue.
[0420] 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, supra. In general, the presence or absence of an excretory
system
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.
[0421] In a'preferred embodiment, the assay involves the use of binding agent
immobilized on a solid support to bind to and remove the excretory system
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 excretory system antigen polypeptides and portions thereof, or
antibodies, to
which the binding agent binds, as described above.
[0422] The solid support may be any material known to those of skill in the
art to
which excretory system 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
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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. 5,359,681. 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 10 ug, and preferably about 100 ng to about 1 ug, is
sufficient to immobilize an adequate amount of binding agent.
[0423] 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).
Gene TheraQy Methods
[0424] 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 an excretory system antigen
of the
present invention. This method requires a polynucleotide, which codes for a
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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.
[0425] 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); Kaido, 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 inj ection.
[0426] 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.
[0427] In one embodiment, the polynucleotide of the present invention is
delivered
as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers
to
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.
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[0428] 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; pSVK3, 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.
[0429] Any strong promoter known 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; retroviial 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.
[0430] 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.
[0431] The polynucleotide construct can be delivered to the interstitial space
of
tissues within the animal, including of muscle, skin, brain, lung, liver,
spleen, bone
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
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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 frbroblasts. Ih vivo muscle cells are
particularly competent in their ability to take up and express
polynucleotides.
[0432] 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 mg/kg 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 skill 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.
[0433] 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.
[0434] 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
injection, topical administration, catheter infusion, and so-called "gene
guns". These
delivery methods are known in the art.
[0435] 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.
[0436] 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.
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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.
[0437] 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).
[0438] 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.
[0439] 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.
[0440] 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
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