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CA 02393912 2002-06-21
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Nucleic Acids, Proteins, and Antibodies
[1] This application refers to a "Sequence Listing" that is provided only on
electronic
media in computer readable form pursuant to Administrative Instructions
Section 801(a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and
Administrative Instructions Sections 801 to 806, and is hereby incorporated in
its entirety.
[2] The Sequence Listing is provided as an electronic file (PJZ03 seqList.txt,
206,260 bytes in size, created on January 13, 2001) on four identical compact
discs (CD-R),
labeled "COPY 1," "COPY 2," "COPY 3," and "CRF." The Sequence Listing complies
with Annex C of the Administrative Instructions, and may be viewed, for
example, on an
IBM-PC machine running the MS-Windows operating system by using the V viewer
software, version 2000 (see World Wide Web URL: http://www.fileviewer.com).
Field of the Inheyation
[3] The present invention relates to novel proteins. More specifically,
isolated
nucleic acid molecules are provided encoding novel polypeptides. Novel
polypeptides and
antibodies that bind to these polypeptides are provided. Also provided are
vectors, host
cells, and recombinant and synthetic methods for producing human
polynucleotides and/or
polypeptides, and antibodies. The invention further relates to diagnostic and
therapeutic
methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
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these novel polypeptides. The invention further relates to screening methods
for identifying
agonists and antagonists of polynucleotides and polypeptides of the invention.
The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the present invention.
Bachg~ouhil of the Inveutioh
[4] The human digestive system is a collection of specialized organs and body
tissues that prepare food for use by hundreds of millions of body cells. Food
when eaten
cannot reach cells because it cannot pass through the intestinal walls to the
bloodstream
and, if it could would not be in a useful chemical state. The gastrointestinal
system modifies
food physically and chemically and disposes of unusable waste. Physical and
chemical
modification (digestion) depends on exocrine and endocrine secretions and
controlled
movement of food through the digestive tract.
[5] The three fundamental processes of the digestive system are: secretion
(e.g.,
delivery of enzymes, mucus, ions and the like into the lumen, and hormones
into blood),
absorption (e.g., transport of water, ions and nutrients from the lumen,
across the epithelium
and into blood), and motility (e.g., contractions of smooth muscle in the wall
of the tube
that crush, mix and propel its contents). Control of digestive function is
achieved through a
combination of electrical and hormonal messages which originate either within
the
digestive system's own nervous and endocrine systems, as well as from the
central nervous
system and from endocrine organs such as the adrenal gland.
[6] The digestive system is composed of the digestive or alimentary tube and
accessory digestive organs, which include the Mouth (e.g., tongue, taste buds,
soft palate
pharynx, salivary glands, teeth), Esophagus, Stomach, Liver, Gallbladder,
Pancreas, Small
Intestine (e.g., duodenum, jejunum, and ileum), and Large Intestine (e.g.,
caecum).
[7] Common digestive system disorders including infections, inflammations,
ulcers
and cancers of the digestive or alimentary tube and above listed accessory
digestive organs
are described in more detail below.
Diso~deys of the Esophagus
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[8] Disorders of the Esophagus include dysphagia (e.g., difficulty in
swallowing) and
odynophagia (e.g., difficulty in swallowing~accompanied by pain). Inflammatory
disorders
of the esophagus result from a variety of causes; for example, ingestion of
noxious
materials (e.g., corrosive esophagitis), lodgment of foreign bodies, or a
complex of events
associated with reflux of gastric contents from the stomach into the lower
esophagus (e.g.,
peptic esophagitis).
[9] Disorders of the motility of the esophagus tend to be either precipitated
or
aggravated at times of nervous stress. A disorder commonly due to obesity is
gastric reflux.
Persisting reflux of gastric contents with acid and digesting enzymes leads to
chemical
inflammation of the lining of the esophagus and ultimately to (peptic)
ulceration. If
inadequately treated, the process leads to submucosal fibrosis and
stricturing, and, besides
the symptoms of heartburn and regurgitation, the patient experiences pain on
eating and
swallowing.
[10] Further disorders of the esophagus include the formation of diverticula.
A serious
injury to the esophagus is spontaneous rupture. It can occur in patients who
have been
vomiting or retching and in debilitated elderly persons with chronic lung
disease. A rupture
of this type confined to the mucosa only at the junction of the linings of the
esophagus and
stomach is called a Mallory-Weiss lesion.
[1l] Benign tumors of the esophagus originate in the submucosal tissues and
principally are leiomyomas (tumors composed of smooth muscle tissue) or
lipomas (tumors
composed of adipose, or fat, tissues). Malignant tumors are either epidermal
cancers, made
up of unorganized aggregates of cells, or adenocarcinomas, in which there are
gland-like
formations. Cancers arising from squamous tissues are found at all levels of
the organ,
whereas adenocarcinomas are more common at the lower end where a number of
glands of
gastric origin are normally present. The prognosis is poor because diagnosis
is difficult and
the tumor has usually been growing for one or two years before symptoms are
apparent.
Diso~~ders of the stomach
[12j Any disorder that affects the power of coordination of the stomach
muscles is
capable of producing symptoms ranging from those that are mildly unpleasant
(e.g.,
anorexia and nausea) to others that are life-threatening. The intrinsic
muscles of the
stomach are innervated by branches of the vagus nerves, which travel along the
esophagus
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from their point of emergence in the brain stem. Severing these nerves or
altering their
function by the use of anticholinergic medication may produce temporary or
more
prolonged change in the ability of the stomach to empty itself. Gastric
retention may result
from the degeneration of the nerves to the stomach that can result from
diabetes mellitus.
Obstruction due to scarring in the area of the gastric outlet, or to tumors
encroaching on the
lumen, causes the stomach to fill up with its own secretions as well as with
partially
digested food. In these circumstances, vomiting leads to dehydration and to
electrolyte
losses, which threaten life if not corrected.
[13] Disorders of the stomach include ulcerative diseases, which involve
mucosal
breakdown either confined to the superficial layers of the mucosa (e.g., an
erosion) or
extending through the intrinsic layer of muscle of the rnucosa into the
tissues below (e.g., an
ulcer). The circumstances that contribute to mucosal injury and ulcer
formation include
physical and chemical trauma that result from hot fluids and food, aspirin and
other drugs,
irritating spices, and pickling fluids. In addition, genetic factors are
involved in the
development of ulcers. The complications of peptic ulcers are hemorrhage,
perforation, and
obstruction of the outlet of the stomach (pyloric stenosis) by scarring of the
duodenal bulb
or of the pyloric channel. A diffuse inflammation of the stomach lining,
gastritis, is usually
an acute process caused by contaminated food, alcohol abuse, or by bacterial-
or viral-
induced inflammation of the gastrointestinal tract (gastroenteritis). The
other form of
gastritis is gastric atrophy, in which the thiclcness of the mucosa is
diminished. Diffuse
gastric atrophy leads to partial loss of the glands and secreting cells
throughout the stomach
and may be associated with iron-deficiency anemia.
[14] Malignant tumors of the stomach are common and are probably a result of
both
genetic and environmental factors. Gastric cancer affects men more often than
women and
accounts for about 20 percent of all deaths from cancers of the
gastrointestinal tract in the
United States. Other malignant tumors that involve the stomach are tumors
ordinarily made
up of lymphoid and connective tissue. Benign tumors, especially leiomyomas,
are common
and may, when large, cause massive hemorrhage. Polyps of the stomach are not
common
except in the presence of gastric atrophy.
Disorders of the Duodenum and Small Ti~testine
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[l5] Primary cancer of the duodenum is an infrequent disease, however, benign
tumors of the duodenum, particularly polyps and carcinoids, are more frequent.
Cancers of
the common bile duct or of the pancreas are important causes of death. A
common disorder
of the small intestine, distension, is caused by lack of coordination of the
inner circular and
outer longitudinal muscular layers of the intestinal wall which usually
results in an
accumulation of excess contents in the lumen. The most common cause of
disturbed
motility in the small intestine is food that contains an unsuitable additive,
organism, or
component. One of the most serious problems in small intestine are motor
disturbances
which arise from an intestinal obstruction that results from an actual
encroachment on the
bowel by an adhesive band or from an internal block produced by a tumor or
gallstone. In
addition, as profound an obstruction results when a portion of the intestine
undergoes partial
necrosis, or death, from failure of ifs blood supply.
[l6] The extremely common disorder known as the irritable bowel syndrome is
probably due to a disturbance of the motility of the whole intestinal tract.
The symptoms
vary from watery diarrhea to constipation and the passage of stools with
difficulty. When
the colon is involved, an excess of mucus is often observed in the stools.
Occasionally the
irritable bowel syndrome may be due to an allergy to a particular foodstuff.
The syndrome
may develop following an infection such as bacillary dysentery, after which
the small
intestine remains irritable for many months.
[17] A further disorder, malabsorption occurs when the small intestine is
unable to
transport properly broken down products of digestive materials from the lumen
of the
intestine into the lymphatics or mesenteric veins, where they are distributed
to the rest of
the body. Defects in transport occur either because the absorptive cells of
the intestine lack
certain enzymes, whether by birth defect or by acquired disease, or because
they are
hindered in their work by other disease processes that infiltrate the tissues,
disturb motility,
permit bacteria to overpopulate the bowel, or block the pathways over which
transport
normally proceeds. A malabsorption disorder of unknown cause, tropical sprue,
is
associated with partial atrophy of the mucosa of the small intestine.
Disorders of the small
intestine also include bacterial and parasitic infections. .
[18] Appendicitis is an inflammation of the vermiform appendix that may be
caused
by infection or partial or total obstruction. Chronic inflammations of the
small intestine
include tuberculosis and regional enteritis (Crohn's disease). Celiac disease
causes damage
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to the mucosa of the small intestine, though it is not clear whether it is
caused by an
immune reaction, or an inability to break down a toxic protein, gluten, to
smaller peptide
fractions. Studies of the immune function of those with celiac disease suggest
that at least a
major part of the process is a delayed hypersensitivity reaction and that the
morphological
changes are correlated with the presence of circulating antibodies to gluten.
The mucosal
reaction results in progressive atrophy, with dwarfing, if not complete
disappearance, of the
microvilli and villi that line the intestinal tract.
Diso~des°s of the Large I~testihe
[19] A wide variety of diseases and disorders occur in the large intestine. A
disease
that is analogous to achalasia of the esophagus is an idiopathic condition
called aganglionic
megacolon, or Hirschsprung's disease. It is characterized by the absence of
ganglion cells
and normal nerve fibres from the distal (or lower) portion of the large
intestine, which
results in reduced neuromuscular transmission and ceased peristalsis. The
entire colon
slowly becomes more and more distended and thick-walled. Abscesses in the
perianal area
are common complicating features of many diseases and disorders of the large
intestine.
Fungal and bacterial infections are also common causes of large intestine
disorders.
[20] The most common form of chronic colitis, ulcerative colitis, is
idiopathic. It
varies from a mild inflammation of the mucosa of the rectum, giving rise to
excessive
mucus and some spotting of blood in the stools, to a severe, sudden, intense
illness, With
destruction of a large part of the colonic mucosa, considerable blood loss,
toxemia and, less
commonly, perforation. The most common variety affects only the rectum and
sigmoid
colon and is characterized by diarrhea and the passage of mucus. Apart from
the greater
tendency for fistulas to form and for the wall of the intestine to thicken
until the channel is
obstructed, Crohn's disease is distinguishable from ulcerative colitis by
microscopic
f°mdings. In Crohn's disease, the maximum damage occurs beneath the
mucosa, and
lymphoid conglomerations, known as granulomata, are formed in the submucosa.
Crohn's
disease attacks the perianal tissues more often than does ulcerative colitis.
Although these
two diseases are not common, they are disabling.
[21] Tumors of the colon are usually polyps or cancers. A peculiar form of
polyp is
the villous adenoma, often a slowly growing, fernlike structure that spreads
along the
surface of the colon for some distance. Cancers compress the colonic lumen to
produce
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obstruction, they attach to neighbouring structures to produce pain, and they
perforate to
give rise to peritonitis. Cancers also may metastasize to distant organs
before local
symptoms appear.
[22] Anorectal disorders related to defecation are more common in the Western
world
than elsewhere. These disorders usually take the form of fissures (cuts or
cracks in the skin
or mucous membrane) at the junction of the anal mucous membrane with the skin
between
the thighs. Anal fistulas sometimes occur as complications of serious bowel
disease, as in
tuberculosis or Crohn's disease of the bowel, or in certain parasitic
diseases. A more general
disorder is the enlargement of veins of the rectum and anus to form external
or internal
hemorrhoids. Hemorrhoids protrude, are associated with anal itching and pain,
and bleed,
especially when they come in contact with hard stools.
Disorders of the Liue~
[23] A variety of agents, including viruses, drugs, environmental pollutants,
genetic
disorders, and systemic diseases, can affect the Liver. The resulting
disorders usually affect
one of the three functional components of the liver: the hepatocyte (liver
cell) itself, the bile
secretory (cholangiolar) apparatus, or the blood vascular system. Most acute
liver diseases
are self limited, and liver functioning returns to normal once the causes are
removed or
eliminated. In some cases, however, the acute disease process destroys massive
areas of
liver tissue in a short time, leading to extensive death (necrosis) of hepatic
cells and often to
death of the patient. Hepatitis may result from viral infections or toxic
damage from drugs
or poisons. When acute hepatitis lasts for six months or more, a slow but
progressive
destruction of the surrounding liver cells and bile ducts occurs, a stage
called chronic active
hepatitis. If hepatocellular damage is severe enough to destroy entire acini
(clusters of
lobules), they are often replaced with fibrous scar tissue. Bile canaliculi
and hepatocytes
regenerate in an irregular fashion adjacent to the scar tissue and result in a
chronic condition
called cirrhosis of the liver. Where inflammatory activity continues after the
onset of
cirrhosis, the disorderly regeneration of hepatocytes and cholangioles may
lead to the
development of hepatocellular or cholangiolar cancer.
[24] Although a number of viruses affect the Liver, including the
cytomegalovirus of
infancy and childhood and the Epstein-Barr virus of infectious mononucleosis,
there are
three distinctive transmissible viruses that are specifically known to cause
acute damage to
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liver cells: hepatitis virus A (HAV), hepatitis virus B (HBV), and hepatitis
virus non-A,
non-B (NANB). The symptoms characteristic of the acute hepatitis caused by the
HAV,
HBV, and NANB viruses are essentially indistinguishable from one another.
[25] Acute hepatitis also may be caused by the overconsumption of alcohol or
other
poisons, such as commercial solvents (e.g., carbon tetrachloride),
acetaminophen, and
certain fungi. Such agents are believed to cause hepatitis when the formation
of their toxic
intermediate metabolites in the liver cell (phase I reactions) is beyond the
capacity of the
hepatocyte to conjugate, or join them with another substance for
detoxification (phase II
reactions) and excretion. Acute canalicular (cholestatic) hepatitis is most
commonly caused
by certain drugs, such as chlorpromazine, that lead to idiosyncratic reactions
or, at times, by
hepatitis viruses. Acute congestive liver disease usually results from the
sudden
engorgement of the liver by fluids after congestive heart failure.
[26] A prominent autoimmune liver disease is Wilson's disease, which is caused
by
abnormal deposits of large amounts of copper in the liver. Granulomatous
hepatitis, a
condition in which localized areas of inflammation (granulomas) appear in any
portion of
the liver lobule, is a type of inflammatory disorder associated with many
systemic diseases,
including tuberculosis, sarcoidosis, schistosomiasis, and certain drug
reactions.
Granulomatous hepatitis rarely leads to serious interference with hepatic
function, although
it is often chronic. The end result of many forms of chronic liver injury is
cirrhosis, or
scarring of liver tissue in reponse to previous acinar necrosis and irregular
regeneration of
liver nodules and bile ducts.
[27] Primary biliary cirrhosis, a widespread, though uncommon, autoimmune
inflammatory disease of bile ducts, is a disorder primarily affecting middle-
aged and older
women. Secondary biliary cirrhosis results from chronic obstruction or
recurrent infection
in the extrahepatic bile ducts caused by strictures, gallstones, or tumors.
Infestation of the
biliary tract with a liver fluke, Clono~~chis si~censis, is a cause of
secondary biliary cirrhosis
in Asia.
[28] Portal hypertension, the increased pressure in the portal vein and its
tributaries
that is the result of impediments to venous flow into the liver, is brought
about by the
scarring characteristic of the cirrhotic process. The increased pressure
causes feeders of the
portal vein to distend markedly, producing varices, or dilations of the veins.
When varices
are located in superficial tissues, they may rupture and bleed profusely. Two
such locations
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are the lower esophagus and the perianal region. The accumulation of fluid in
the
abdominal cavity, or ascites, is related to portal hypertension, significant
reduction in serum
albumin, and renal retention of sodium. When albumin levels 'in blood are
lower than
normal, there is a marked reduction in the force that holds plasma water
within the blood
vessels and normally resists the effects of the intravascular pressure. The
resulting increase
in intravascular pressure, coupled with the increased internal pressure caused
by the portal
venous obstruction in the liver, leads to massive losses of plasma water into
the abdominal
cavity. The associated reduction of blood flow to the kidneys causes increased
elaboration
of the hormone aldosterone, which, in turn, causes the retention of sodium and
water and a
reduction in urinary output. In addition, because the movement of intestinal
lymph into the
liver is blocked by the cirrhotic process in the liver, the backflow of this
fluid into the
abdominal cavity is greatly increased. A progressive reduction in kidney
function that often
occurs in persons with advanced acute or chronic liver disease, hepatorenal
syndrome,
probably results from an inadequate perfusion of blood through the cortical
(outer) portions
of the kidneys, where most removal of waste products occurs. With advanced
hepatocytic
dysfunction, a spasm of blood vessels in the renal cortex can occur, often
with good blood
flow to the rest of the kidney. This spasm results in progressive failure in
kidney function
and often leads to death.
[29] Although not uncommon, cancer originating in the liver, usually in
hepatocytes
and less frequently in cells of bile duct origin, is rare in the West and is
almost always
associated with active cirrhosis, particularly the form found in patients with
chronic
hepatitis. Long exposure to certain environmental poisons, such as vinyl
chloride or carbon
tetrachloride, has also been shown to lead to hepatic cancer. Cancers arising
elsewhere in
the body, particularly in abdominal organs, lungs, and lymphoid tissue,
commonly lead to
metastatic cancer in the liver and are by far the most frequent type of
hepatic malignancy.
Various benign types of tumors and cysts arise from certain components of the
liver, such
as the hepatocytes (adenomas) or blood vessels (hemangiomas). While the cause
of these
lesions is not always clear, hepatic adenomas are associated with the
prolonged use of
female sex hormones (estrogens). Benign cysts in the liver may occur as
congenital defects
or as the result of infections from infestation of the dog tapeworm
(Echi~ococcus
grafzulosus). Abscesses on the liver result from the spread of infection from
the biliary tract
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or from other parts of the body, especially the appendix and the pelvic
organs. Specific liver
abscesses also result from infections with the intestinal parasite Entamoeba
histolytica.
Diso~~de~s of the Bilia~y Tt~act
[30] Cholelithiasis, or the formation of gallstones in the gallbladder, is the
most
common disease of the biliary tract. There are three types of Gallstones:
stones containing
primarily calcium bilirubinate (pigment stones); stones containing 25 percent
or more of
cholesterol; and stones composed of variable mixtures of both bilirubin and
cholesterol
(mixed gallstones). Pigment stones are the result of an increased amount of
bilirubin in the
liver (due to hemolytic disease) and the consequent secretion into the biliary
tract of
increased amounts of the water-soluble conjugate, bilirubin diglucuronide, a
pigment that is
normally secreted in the urine. Cholesterol and mixed cholesterol-bilirubinate
stones occur
when the proportion of cholesterol in bile exceeds the capacity of bile acids
and lecithin to
contain the total amount of cholesterol in micellar colloidal solution.
Postcholecystectomy
syndrome comprises painful attacks, often resembling preoperative symptoms,
that
occasionally occur following the surgical removal of gallstones and the
gallbladder. These
attacks may be related. to intermittent muscular spasms of the sphincter of
Oddi or of the
bile ducts.
[31] Cancer of the biliary tract is rare but may occur in almost any area,
including the
gallbladder, the hepatic ducts, the common bile duct, or the ampulla of Vater.
In cancer of
the bile duct, congenital cysts and parasitic infections, such as liver
flukes, seem to lead to
increased risks. Persons with extensive chronic ulcerative colitis also show a
greater than
normal incidence of bile duct carcinoma.
[32] Jaundice, or yellowing of the skin, scleras, and mucous membranes, occurs
whenever the level of bilirubin in the blood is significantly above normal.
This condition is
evident in three different types of disorders including, unconjugated, or
hemolytic, jaundice;
hepatocellular jaundice; and cholestatic, or obstructive jaundice.
Unconjugated jaundice
results when the amount of bilirubin produced from hemoglobin by the
destruction of red
blood cells or muscle tissue (myoglobin) overwhelms the normal capacity of the
Iiver to
transport it or When the ability of the liver to conjugate normal amounts of
bilirubin into
bilirubin diglucuronide is significantly reduced by inadequate intracellular
transport or
enzyme systems. Hepatocellular jaundice arises when liver cells are damaged so
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that their ability to transport bilirubin diglucuronide into the biliary
system is reduced,
allowing some of this yellow pigment to regurgitate into the bloodstream.
Cholestatic
jaundice, occurs when essentially normal liver cells are unable to transport
bilirubin either
through the hepatocytic-bile capillary membrane, because of damage in that
area, or
through the biliary tract, because of anatomical obstructions (e.g., atresias,
gallstones,
cancer).
Disorders of tlae Pa~c~eas
[33] Inflammation of the pancreas, or pancreatitis, is probably the most
common
disease of this organ. The disorder may be confined to either singular or
repeated acute
episodes, or it may become a chronic disease. There are many factors
associated with the
onset of pancreatitis, including direct injury, certain drugs, viral
infections, heredity,
hyperlipidemia (increased levels of blood fats), and congenital derangements
of the ductal
system. Localized, severe abdominal and midback pain resulting from enzyme
leakage,
tissue damage, and nerve irritation is the most common symptom of acute
pancreatitis. In
severe cases, respiratory failure, shock, and even death may occur. Chronic
pancreatitis
rarely follows repeated acute attacks. It seems instead to be a separate
disorder that results
in mucus plugs and precipitation of calcium salts in the smaller pancreatic
ducts. Mucous
production and plugging of the pancreas in Cystic fibrosis patients almost
invariably causes
destruction and scarring of the acinar tissue, usually without damaging the
islets of
Langerhans. A similar process in the hepatic biliary system produces foci of
fibrosis and
bile duct proliferation, a singular form of cirrhosis.
[34] The discovery of new human digestive system associated polynucleotides,
the
polypeptides encoded by them, and antibodies that immunospecifically bind
these
polypeptides, satisfies a need in the art by providing new compositions which
are useful in
the diagnosis, treatment, prevention and/or prognosis of disorders of the
digestive system,
including, but not limited to, dysphagia, odynophagia, congenital disorders of
the
esophagus, gastric reflux, diverticula, Mallory-Weiss lesions, leiomyomas of
the esophagus,
lipoma, anorexia, nausea, ulcerative disease, pyloric stenosis,
gastroenteritis, gastritis,
gastric atropy, gastric cancer, benign tumors of the duodenum (e.g., polyps
and carcinoids),
pancreatic cancer, cancer of the bile duct, distension, irritable bowel
syndrome,
malabsorption, congenital disorders of the small intestine (e.g., Meckel's
diverticulum,
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multiple diverticula), bacterial and parasitic infection (e.g., traveler's
diarrhea, typhoid,
paratyphoid, cholera, roundworms, tapeworms, amoebae, hookworms,
strongyloides,
threadworms, and blood flukes), megacolon (e.g., Hirschsprung's disease,
aganglionic
megacolon, acquired megacolon), colitis (e.g., due to bacterial, fungal, or
parasitic
infection, ulcerative colitis), tumors of the colon (e.g., polyps or cancers),
anorectal
disorders (e.g., anal fistulas, hemorrhoids, hepatitis (e.g., acute, chronic,
persistent hepatitis,
viral (for example, hepatitis caused by hepatitis virus A (HAV), hepatitis
virus B (HBV),
and hepatitis virus non-A, non-B (NANB) infection), congenital disorders of
the liver (e.g.,
Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and
alphal-antitrypsin
deficiency), cirrhosis, portal hypertension, cholelithiasis, cancer of the
biliary tract, jaundice
(e.g., unconjugated, hemolytic, hepatocellular, cholestatic, or obstructive
jaundice).
[35] The discovery of new human gastrointestinal-associated polynucleotides,
the
polypeptides encoded by them, and antibodies that immunospecifically bind
these
polypeptides, satisfies a need in the art by providing new compositions which
are useful in
the diagnosis, treatment, prevention and/or prognosis of gastrointestinal-
specific diseases
and/or disorders described in more detail below.
Summary of the Invention
[36] The present invention relates to novel proteins. More specifically,
isolated
nucleic acid molecules are provided encoding novel polypeptides. Novel
polypeptides and
antibodies that bind to these polypeptides are provided. Also provided are
vectors, host
cells, and recombinant and synthetic methods for producing human
polynucleotides and/or
polypeptides, and antibodies. The invention further relates to diagnostic and
therapeutic
methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
these novel polypeptides. The invention further relates to screening methods
for identifying
agonists and antagonists of polynucleotides and polypeptides of the invention.
The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the present invention.
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Detailed Description
Tables
[37] 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 the gene number in the application
for each
clone identifier. The second column provides a unique clone identifier, "Clone
ID NO:Z",
for a cDNA clone related to each contig sequence disclosed in Table 1A. The
third column
provides a unique contig identifier, "Contig ID:" for each of the contig
sequences disclosed
in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for
each of the contig sequences disclosed in Table 1A. The fifth 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)
that
encodes the amino acid sequence shown in the sequence listing and referenced
in Table 1A
as SEQ ID NO:Y (column 6). Column 7 lists residues comprising predicted
epitopes
contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID
NO:Y).
Identification of potential imrnunogenic 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, 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
8, "Tissue
Distribution" shows the expression profile of tissue, cells, and/or cell line
libraries which
express the polynucleotides of the invention. The first number in column 8
(preceding the
colon), represents the tissue/cell source identifier code corresponding to the
key provided in
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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 8 (following the colon), represents the number of
times a
sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID
NO:X) was
identified in the tissue/cell source. Those tissue/cell source identifier
codes in which the
first two letters are "AR" designate information generated using DNA array
technology.
Utilizing this technology, cDNAs were amplified by PCR and then transferred,
in duplicate,
onto the array. Gene expression was assayed through hybridization of first
strand cDNA
probes to the DNA array. cDNA probes were generated from total RNA extracted
from a
variety of different tissues and cell lines. Probe synthesis was performed in
the presence of
3sP 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 9 provides the
chromosomal location
of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was
determined
by finding exact matches to EST and cDNA sequences contained in the NCBI
(National
Center for Biotechnology Information) UniGene database. Given a, presumptive
chromosomal location, disease locus association was determined by comparison
with the
Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian
Inheritance in Man, OMIMTM. McKusick-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
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overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification
number is disclosed in column 10 labeled "OMIM Disease References)". A key to
the
OMIM reference identification numbers is provided in Table 5.
[38] 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).
[39] 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 clone 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 sequence. The fourth
column
provides the analysis method by which the homology/identity disclosed in the
Table 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 the PFAM/NR hit having a
significant match 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
IS
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score or the percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9,
"NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X"
that encode a polypeptide having a significant match to the PFAM/NR database
as
disclosed in the fifth and sixth columns. In specific embodiments polypeptides
of the
invention comprise, or alternatively consist of, an amino acid sequence
encoded by a
polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments
or variants
thereof.
[40] 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", for a cDNA clone related to contig sequences disclosed in Table
1A. The
second column provides the sequence identifier, "SEQ ID NO:X", for contig
sequences
disclosed in Table 1A. The third column provides the unique contig identifier,
"Contig
ID:", for contigs disclosed in Table IA. 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, 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, 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 invention 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).
(41] Table 4 provides a key to the tissue/cell source identifier code
disclosed in Table
1A, column 8. Column 1 provides the tissue/cell source identifier code
disclosed in Table
1A, Column 8. 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
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use of the word "disease" in column 6 is non-limiting. The tissue or cell
source may be
specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue
sample from a
normal portion of a diseased organ). Furthermore, tissues and/or cells lacking
the "disease"
designation may still be derived from sources directly or indirectly involved
in a disease
state or disorder, and therefore may have a further utility in that disease
state or disorder. In
numerous cases where the tissue/cell source is a library, column 7 identifies
the vector used
to generate the library.
[42] Table 5 provides a key to the OMIM reference identification numbers
disclosed
in Table 1A, column 10. OMIM reference identification numbers (Column 1) were
derived
from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man,
OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University
(Baltimore,
MD) and National Center for Biotechnology Information, National Library of
Medicine,
(Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
Column 2 provides diseases associated with the cytologic band disclosed in
Table 1A,
column 9, as determined using the Morbid Map database.
[43] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation
numbers of deposits made with the ATCC in connection with the present
application.
[44] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers
and vector information relating to these cDNA libraries.
[45] 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
[46] The following definitions are provided to facilitate understanding of
certain terms
used throughout this specification.
[47] 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
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WO 01/55387 PCT/USO1/01310
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.
[48] 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 2
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). '
[49] 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 2
of Table
1A, each clone is identified by a cDNA Clone ID (identifier generally referred
to herein as
Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone
ID is all the
information needed to retrieve a given clone from the HGS library.
Furthermore, certain
clones disclosed in this application have been deposited with the ATCC on
October 5, 2000,
having the ATCC designation numbers PTA 2574 and PTA 2575; and on January 5,
2001,
having the depositor reference numbers TS-l, TS-2, AC-1, and AC-2. In addition
to the
individual cDNA clone deposits, most of the cDNA libraries from which the
clones were
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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)
isolated
from that library begins with the same four characters, for example "HTWEP07".
As
mentioned below, Table 1A correlates the Clone ID names with SEQ ID NO:X.
Thus,
starting with an SEQ ID NO:X, one can use Tables 1, 6 and 7 to determine the
corresponding Clone ID, which library it came from and which ATCC deposit the
library is
contained in. Furthermore, it is possible to retrieve a given cDNA clone from
the source
library by techniques known in the art and described elsewhere herein. The
ATCC is
located at 10801 University Boulevard, Manassas, Virginia 20110-2209, USA. The
ATCC
deposits were made pursuant to the terms of the Budapest Treaty on the
international
recognition of the deposit of microorganisms for the purposes of patent
procedure.
[50] 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.Skb,
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).
[51] 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
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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, Sx SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium
phosphate
(pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 ~.g/ml
denatured, sheared
salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65
degree C.
[52] Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished
through the manipulation of formamide concentration (lower percentages of
formamide
result in lowered stringency); salt conditions, or temperature. For example,
lower
stringency conditions include an overnight incubation at 37 degree C in a
solution
comprising 6X SSPE (20X SSPE = 3M NaCI; 0.2M NaH2P04; 0.02M EDTA, pH 7.4),
0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by
washes
at 50 degree C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower
stringency,
washes performed following stringent hybridization can be done at higher salt
concentrations (e.g. 5X SSC).
[53] 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
modiEcation of the
hybridization conditions described above, due to problems with compatibility.
[54] 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).
[55] 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,
CA 02393912 2002-06-21
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single- and double-stranded RNA, and RNA that is mixture of single- and double-
stranded
regions, hybrid molecules comprising DNA and RNA that may be single-stranded
or, more
typically, double-stranded or a mixture of single- and double-stranded
regions. In addition,
the polynucleotide can be composed of triple-stranded regions comprising RNA
or DNA or
both RNA and DNA. A polynucleotide may also contain one or more modified bases
or
DNA or RNA backbones modified for stability or for other reasons. "Modified"
bases
include, for example, tritylated bases and unusual bases such as inosine. A
variety of
modifications can be made to DNA and RNA; thus, "polynucleotide" embraces
chemically,
enzymatically, or metabolically modified forms.
[56] 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 -
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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)).
[57] "SEQ ID NO:X" refers to a polynucleotide sequence described, for example,
in
Tables lAor 2, while "SEQ ID NO:Y" refers to a polypeptide sequence described
in column
6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4
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 2 of Table 1A.
[58] "A polypeptide having functional activity" refers to a polypeptide
capable of
displaying one or more known functional activities associated with a full-
length (complete)
protein. Such functional activities include, but are not limited to,
biological activity,
antigenicity [ability to bind (or compete with a polypeptide for binding) to
an anti-
polypeptide 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.
[59] The polypeptides of the invention can be assayed for functional activity
(e.g.
biological activity) using or routinely modifying assays known in the art, as
well as assays
described herein. Specifically, one of skill in the art may routinely assay
gastrointestinal-
associated polypeptides (including fragments and variants) of the invention
for activity
using assays as described in Examples 12 and 54.
[60] "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 Iess activity, and most preferably,
not more than
about three-fold less activity relative to the polypeptide of the present
invention).
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WO 01/55387 PCT/USO1/01310
[61) Table 1A summarizes some of the 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.
23
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
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24
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
tii cn N O O ~ O ~ ~'~' ~ ~t M ~ ~O o0 00 ~O ~t
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CA 02393912 2002-06-21
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CA 02393912 2002-06-21
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[62] The first column in Table 1A provides the gene number in the application
corresponding to the clone identifier. The second 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
described
elsewhere herein.
[63] The third column in Table 1A provides a unique "Contig ID" identification
for
each contig sequence. The fourth column provides the "SEQ ID NO:" identifier
for each of
the contig polynucleotide sequences disclosed in Table 1A. The fifth 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 6, 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.
[64] The sixth column in Table 1A provides the corresponding SEQ ID NO:Y fox
the
polypeptide sequence encoded by the preferred ORF delineated in column 5. 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.
[65] Column 7 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 Jarneson 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.
48
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It will be appreciated that depending on the analytical criteria used to
predict antigenic
determinants, the exact address of the determinant may vary slightly.
[66) Column 8 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
tissues, cells,
and/or cell Iine libraries which predominantly express the polynucleotides of
the invention.
The first number in column 8 (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 8
(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 specific tissue and/or cell
expression.
[67) Column 9 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
49
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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.
[68j A modified version of the computer program BLASTN (Altshul et al., J.
Mol.
Biol. 215:403-410 (1990); and Gish and States, 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.
[69] 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 10,
Table 1A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference
identification numbers (column 1), and provides a description of the
associated disease in
Column 2.
CA 02393912 2002-06-21
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TABLE 1B
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z . NO:X ID: NO:B From-To
HBGMZ39 23 947112 AC008537 109 1-1186
HBGMZ39 23 947112 AC019337 110 1-1182
HBGMZ39 23 947112 AC008537 111 1-1993
2105-2385
2736-3068
4364-4489
6546-6781
7025-8165
HBGMZ39 23 947112 AC019337 112 1-1991
2103-2383
2734-3066
4360-44.85
6541-6776
7021-8159
HBGMZ39 23 947112 AC008537 113 1-734
767-1001
HBGMZ39 23 947112 AC019337 114 1-158
291-565
598-832
HNTND64 27 954871 AC025090 115 1-465
HNTND64 27 954871 AC025090 116 ~ 1-454
51
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[70] 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 axe 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).
52
CA 02393912 2002-06-21
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tcS ~, ,--~ c~ c~ ~ 00 tc3 ~ DD c~ ~, o0 c~ 00
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x
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
d- N N co r--mo ~p VW O N m l~ d' N a1 ~n d- 01
t~ l~ ~O N N M a1 O ~ l~ oo ~ O d- N LO N
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56
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
~ v7 d- M O O N ~~ Q~ I~ I~ ~ N N o0 c0 M
0o M O ~D ~O N t~ O N O O d- d- ~D ~n ~
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57
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
~n ~o M N ~ ~n ~n ~n d- in ~n O M N M
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CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
~O N
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59
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[71] 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
identifier,
"Contig ID:" which allows correlation with the information in Table 1A. The
third column
provides the sequence identifier, "SEQ ID NO:", for the contig polynucleotide
sequences.
The fourth column provides the analysis method by which the homologylidentity
disclosed
in the Table was determined. The fifth column provides a description of the
PFAM/NR hit
identified by each analysis. Column six provides the accession number of the
PFAM/NR
hit disclosed in the fifth column. Column seven, scorelpercent 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.
[72] 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 and States, 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. 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
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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.
[73] The PFAM database, PFAM version 2.1, (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.,
Durbin et al., Biological sequence analysis: probabilistic 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 2.1. A HMM derived from PFAM version 2.1
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 show a significant match to a PFAM protein family.
[74] 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 signif
cant
match to the PFAM/NR database as disclosed in the fifth column. 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.
[75] 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
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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.
[76] 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).
[77] 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, and having depositor reference numbers TS-1, TS-2, AC-1, 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.
[78] 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 Lehgth Geues
62
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[79] 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'l.
Acad.
Sci. LTSA, 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,
therefor. The following briefly describes a modification of this original 5'
RACE
procedure. Poly A+ or total RNA is reverse transcribed with Superscript II
(GibcoBRL) .
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 (GibcoBRL).
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 (Xhol, 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 SI~II
(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 methods known in the art and/or
commercial
kits are used to amplify and recover 3' ends.
[80] Several quality-controlled kits are commercially available for purchase.
Similar
reagents and methods to those above are supplied in kit form from GibcoBRL 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
63
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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.
[81] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library
double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is
synthesized with an antisense cDNA-specific primer and a plasmid-anchored
primer. These
primers are removed and a symmetric PCR reaction is performed with a nested
cDNA-
specific antisense primer and the plasmid-anchored primer.
RNA Ligase Protocol Fog Generating The S' o~ 3' End Sequences To Obtain Full
Length
Genes
(82] 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 and a primer set
containing a primer
specific to the Iigated RNA oligonucleotide and a primer specific to a known
sequence of
the gene of interest, is used to PCR amplify the 5' portion of the desired
full length gene
which may then be sequenced and used to generate the full length gene. This
method starts
with total RNA isolated from the desired source, poly A RNA may be used but is
not a
prerequisite for this procedure. The RNA preparation may then be treated with
phosphatase
if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which
may
interfere with the later RNA ligase step. The phosphatase if used is then
inactivated and the
RNA is treated with tobacco acid pyrophosphatase in order to remove the cap
structure
present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate
group at the
5' end of the cap cleaved RNA which can then be ligated to an RNA
oligonucleotide using
T4 RNA Iigase. This modified RNA preparation can then be used as a template
for first
64
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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' erid using a
primer specific to the ligated RNA oligonucleotide and a primer specific to
the known
sequence of the gene of interest. The resultant product is then sequenced and
analyzed to
confirm that the 5' end sequence belongs to the relevant gene.
[83] 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 S, 2000, and receiving ATCC
designation
numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and
receiving ATCC designation numbers TS-1, TS-2, AC-l, and AC-2; and/or as set
forth, for
example, in Table 1A, Table 6, or Table 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
described, 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
Tabla 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, 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
lAor 2 by procedures hereinafter further described, and others apparent to
those skilled in
the art.
[84] 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.
[85] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(IJ.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and
5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:
7583-7600 (1988);
Aping-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17: 9494 (1989)) and
pBK (Alting-
CA 02393912 2002-06-21
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Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from
Stratager~e
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.
[86] 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 DHlOB, available from Life Technologies. See, for instance, Clark, J.
M., Nuc. Acids
Res. 16:9677-9686 (1988) and Mead, D. et al., BiolTechnology 9: (1991).
[87] 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.
[88] Also provided in the present invention are allelic variants, orthologs,
and/or
species homologs. Procedures known in the art can be used to obtain full-
length genes,
allelic variants, splice variants, full-length coding portions, orthologs,
and/or species
homologs of genes corresponding to SEQ ID NO:X or the complement thereof,
polypeptides encoded by genes corresponding to 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 identified 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.
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[89] 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.
[90] 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.
[91] The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. A recombinantly produced
version of a
polypeptide, including the secreted polypeptide, can be substantially purified
using
techniques described herein or otherwise known in the art, such as, for
example, by the one-
step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides
of the
invention also can be purified from natural, synthetic or recombinant sources
using
techniques described herein or otherwise known in the art, such as, for
example, antibodies
of the invention raised against the polypeptides of the present invention in
methods which
are well known in the art.
[92] The present invention provides a polynucleotide comprising, or
alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence
contained in Clone ID NO:Z. The present invention also provides a polypeptide
comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y, a
polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide
encoded by
the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded
by a
nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B.
Polynucleotides
encoding a polypeptide comprising, or alternatively consisting of the
polypeptide sequence
of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by
the
cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B are also
encompassed by the
invention. The present invention further encompasses a polynucleotide
comprising, or
alternatively consisting of, the complement of the nucleic acid sequence of
SEQ ID NO:X,
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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.
[93] 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 additionah
embodiments, the above-described polynucleotides of the invention comprise, or
alternatively consist of, sequences delineated in Table 1B, column 6, and have
a nucleic
acid sequence which is different from that published 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.
[94] 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,
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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, 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.
[95] 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 BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column
5). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in column 6 of Table 1B which
correspond to
the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and
have a
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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 axe also encompassed by the invention.
[96] Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the sequences delineated in the same row of Table 1B column 6, or any
combination
thereof. Additional, representative examples of polynucleotides of the
invention comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the complementary strands) of the sequences delineated in the same row of
Table 1B
column 6, or any combination thereof. In preferred embodiments, the
polynucleotides of
the invention comprise, or alternatively consist of, one, two, three, four,
five, six, seven,
eight, nine, ten, or more of the complementary strands) of the sequences
delineated in the
same row of Table 1B column 6, wherein sequentially delineated sequences in
the table (i.e.
corresponding to those exons located closest to each other) are directly
contiguous in a 5' to
3' orientation. In further embodiments, above-described polynucleotides of the
invention
comprise, or alternatively consist of, sequences delineated in the same row of
Table 1B,
column 6, and have a nucleic acid sequence which is different from that of the
BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column
5). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in the same row of Table 1B,
column 6, and
have a nucleic acid sequence which is different from that published for the
BAC clone
identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments,
the
above-described polynucleotides of the invention comprise, or alternatively
consist of,
sequences delineated in the same row of Table 1B, column 6, and have a nucleic
acid
sequence which is different from that contained in the BAC clone identified as
BAC ID
CA 02393912 2002-06-21
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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.
[97] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B, and the polynucleotide sequence
of SEQ ID
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants
thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention.
[98] 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.
[99] 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.
[100] 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 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
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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.
[101] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of
one of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X are directly contiguous
Nucleic acids
which hybridize to the complement of these 20 contiguous polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides, and
antibodies that bind these polypeptides are , also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic acids,
and polypeptides are also encompassed by the invention.
[102] 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.
[103] 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
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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.
[104] 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.
[105] 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 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.
[106] 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
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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.
[107] 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.
[108] Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are
specifically excluded from the scope of the present invention. Accordingly,
for each contig
sequence (SEQ ID NO:X) listed in the fourth column of Table 1A, preferably
excluded are
one or more polynucleotides comprising a nucleotide sequence described by the
general
formula of a-b, where a is any integer between 1 and the final nucleotide
minus 15 of SEQ
ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where
both a and b
correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and
where b is
greater than or equal to a + 14. More specifically, preferably excluded are
one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of a-b,
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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.
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TABLE 3
SEQ
ID
Clone NO: Contig EST Disclaimer
ID
NO: Z X ID: Range Accession #'s
of a
Range
of b
HLMD095 11 10683141 - 156515 - AI392922, AW204653, AI301202,
1579
AI916192, and AI541056.
HETDT70 12 12036071 - 173515 - AI765967, AI949451, AI436791,
1749 AI969563,
AI810397, AI084325, AW024539,
AA156832, AA156726, AI452756,
AA843093, AA824232, AI963642,
AI306667, T96131, AW207447,
AW243556,
AI084332, H95977, T96213,
H95978,
AA299371, AI701335, AA321353,
U30998,
AF035268, E16580, U37591,
AF035269,
D88666, and E16577.
HNHCI32 13 12038221 - 149615 - AA585439, AA585440, AL046994,
1510
AL040768, AL044015, AL046147,
AL042245, AL049007, AL043950,
AL042700, AL037341, AL036500,
AL042712, AL040414, AL043468,
AL040571, AL046097, AL045671,
AL044771, AL134123, AL044258,
AL041577, AL046150, AL043570,
AL040856, AL043848, AL046850,
AL046914, AL041459, AL037435,
AL039924, AL045794, T24119,
AI535639,
T241 I2, AL044201, 228355,
AL0438I4,
AA585453, AL039316, AL044064,
AW013814, AL043923, AI525556,
AI541510, AI546855, AL044029,
AI525316,
AL043128, AL037335, AL048647,
AL045994, AL043201, AL079876,
AL043537, D51250, AL045341,
AI525328,
AL040252, AI541374, T02921,
AL039386,
AL040992, AL039109, AL038531,
AL037726, AL039629, AL039659,
AL039625, AL039648, AL038837,
AL039074, AL039566, AL039678,
AL039I08, AL039538, AL039564,
H00069,
AL039509, AL039156, AL043845,
AL037323, AL045991, AI556967,
AL039128, AL044407, AL037371,
D80043,
AL040329, AL036973, D80253,
AL042909,
AL045337, AL037051, AL045353,
AL047593, AL039423, D59787,
AL039410,
C15189, AL039150, AI541514,
AL038025,
AL041374, AL040082, AL038821,
AI541523, AL044530, AI526180,
AL036725, 230131, D80219,
AI546999,
AA585434, AL043422, AL043445,
AL040263, AL039338, AI541534,
D59275,
AI525306, AL038532, AA585101,
AI541317, AL042096, AL043423,
AI557807, AL043604, AL040148,
AL043627, AL041523, AI541509,
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AL041730, AL038983, AI526140,
D80240,
AL046392, AI546828, AL044272,
AI541017, AL043441, AI525431,
AL040090, T23947, AA585356,
C16300,
D80210, AI526194, D80045,
AI541535,
AL044583, AL044274, AL036196,
D51423,
AI547039, AI526196, AL037343,
D80134,
AI541365, AL037639, AL037443,
AI546945, AW451070, AL040294,
AI557799, D59619, D80391,
AI541508,
AI540967, D80227, AL040458,
AL037615,
AL042135, D80193, AL041186,
AI541307,
AI546884, T11028, AI535983,
AI557731,
AL036767, AL044187, AI546899,
D80196,
AI535783, D61254, AI142134,
AI526144,
AL039085, 829445, AL045990,
AL036117,
AI557787, AL044377, AI535660,
AW452756, AL046442, AL041635,
828735,
847228, AI546875, AL044074,
D59927,
AL037526, AL040510, AL040625,
AL045817, AL041142, AL041238,
AL041133, AL041131, AL046330,
AL040322, AL047183, AL041051,
AL041292, AL047036, AL040119,
AL047170, AL047057, AL041227,
AL047219, AL040463, AL039915,
AL043612, AL041197, AL040155,
AL041346, AL040529, AL041096,
AL047012, AL041358, AL041277,
AL041163, AL041098, AL040621,
AL043538, AL041324, AL040464,
AL044162, AL041086, AL043496,
AL041296, AL041233, AL043467,
AL041159, AL045725, AL044186,
AL041140, AL040193, AR017907,
I13349,
A91965, A38214, I56772,
I95540,
AR031374, AR031375, A58521,
AR020969,
AF112462, A49700, AR035954,
AR062871,
AR062872, AR062873, A20702,
A20700,
' A43189, A43188, A63067,
A51047, A63064,
A63072, AR068507, AR068506,
A84772,
AR028564, A84776, A84773,
A84775,
A84774, AR067731, AR067732,
A58522,
I66481, A60985, I66485,
A60990, A91750,
I66488,I66489,I66490,I66491,I66492,
I66493,I66482,I66483,I66484,
A18053,
A91754, A95051, A18050,
A23334, A75888,
I70384, A60111, A23633,
AR007512,
AR043601, E12615, AR035193,
I06859,
AR027100, A92133, I28266,
I66495, I66494,
I66487,I60241,I60242,I66498,I66497,
I66496, I66486, A83642,
A83643, A83151,
E13740, A10361, A02712,
A35537, A35536,
Al 1245, A02136, A04664,
A02135, A04663,
Ai3393, A02710, A07700,
A13392, I21869,
A70040, I08051, U87250,
A25909, A85396,
AR02S207, I05488, I61310,
A32110,
A60977, A91752, A44171,108196,
A68112,
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A68104, AR027319, A91751,
AR027318,
A06419, A21892, A23997, A68114,
AR037157, AR054109, A21895,
A05160,
A08030, A20502, A86792, A60961,
A64973,
A47368, A58524, I19516, I19517,
A58523,
A89633, A89634, I63560, AR009152,
AR009151, I63561, I63563,
A85477,
AR002333, A60987, X81969,
X68127,
E14304, I08776, A81878, A76773,
A22413,
I25027, I26929, I44515, I26928,
I26930,
I26927, I44516, E16678, I25041,
A95096,
A95106, A95105, A98767, I15353,
A93963,
A93964, A29109, A32111, I07249,
AR068508, AR068510, AR068509,
A63954,
. I91969, I58322, I58323, AR003585,
AR035975, AR035974, AR035977,
AR035976, AR035978, AF082186,
AR038762, I18371, AR036905,
AR022240,
A67220, E12584, AJ244003,
AJ244004,
A95052, I63120, A95117, AR018924,
AR018923, A48774, A48775,
A23998,
AR015960, AR000007, AR015961,
I84554,
A24783, A24782, I84553, I03343,
AR043602, AR043603, A27396,
A49045,
A82653, E16636, A93016, A64081,
A58525,
I49890, AR036903, A90655,
I01987,
AF156296, A58526, A91753,
AR000006,
I19525, AF156294, 296142,
AR038855,
X73004, I62368, AR031488,
I13521, I52048,
V00745, I44531, AF118808,
D28584,
D34614, A97211, I01992, AR008429,
M28262, AB012I17, A92636,.E16590,
AR008430, I00079, E03165,
E0222I,
E01614, E13364, A98420, A98423,
and
A98432.
HLTLAX48 14 10797001 - 655 I5 - AA496150, AI829927, AW0253
669 I7,
AW183143, W49726, C05586,
C05037,
H91924, W58746, AA837507,
N26981,
AA082202, AI056686, N36704,
D80089,
AW264285, W27235, AA496151,
AA931941, AW117255, W49652,
AA683334, AA833537, AA873601,
N24722,
AA524357, AA976379, AI886806,
AW183675, AI248973, AI149898,
AA781907, AA781135, AI081048,
AA533526, 846697, AA928275,
AA904555,
AI085493, and AI342629.
HACCH94 15 12229981 -1951 15 -1965AI093369, AW292321, AA972431,
H97311,
AA746376, N40I74, AA130392,
AA286750,
AA287684, 871586, 871568,
871587,
H03136, H03946, 871567, AI471079,
N54926, AA365025, AA451915,
C14331,
C14429, AW360811, AWI77440,
AW375405, AW375406, AW177501,
AW177511, AWI78893, T03269,
AW179328, AW352117, AW366296,
AW360844, C14389, AW377671,
AW360817, AW378534, AW179332,
78
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
D58283, AW377672, AW179023,
AW178905, D59859, D80022,
D80166,
D80195, D80193, D59927, D59467,
D51423,
D59619, D80210, D51799, D80391,
D80164,
D59275, D80240, D80253, D80043,
D59787,
D80227, D59502, AW378532,
D81030,
D81026, D80045, AW179024,
AW176467,
D80212, D80196, D80188, D80219,
AA305578, 015076, D80038,
D80269,
AW352171, D59610, D80366,
D57483,
AW377676, AA305409, D51022,
D50979,
AW352170, D50995, AW177731,
D59889,
AW178907, AW178762, AW179019,
D80024, AA514188, D80378,
AI905856,
D80248, D80522, 014014, AW177505,
AW 179020, AW 178775, AW
178909,
AW177456, D80241, D80251,
D51060,
AW 179329, AA514186, AW 178980,
AW 179004, D80133, AW 177733,
AW378528, AW178906, AW178908,
AW178754, AW179018, AW352158,
T48593, AW 178914, AW 178911,
D51097,
075259, D80439, D80268, D80302,
AW178774, D80247, 005695,
D80132,
D80134, D58253, D51103, AW360834,
AW367967, AW177723, AW178983,
D80157, AW367950, AW352174,
AA809I22, AW178986, T11417,
006015,
AW378533, AA285331, D45260,
D80314,
003092, AI525913, AW378542,
AI525923,
AF039686, AFI18670, AR034800,
AF08I916, A84916, Y17I88,
A62298,
AR018138, A62300, A82595,
Y17187,
X82626, D89785, A30438, AF058696,
A67220, A78862, X67155, AB028859,
D34614, D26022, AJ132110,
AR008278,
Y12724, A25909, D88547, AR025207,
A94995, AR008443, I50126,
I50132, I50128,
I50133, AR066488, U79457,
AR016514,
AR060138, A45456, AR066487,
A26615,
AR052274, D50010, Y09669,
AB012117,
AR060385, AB002449, I18367,
A43192,
A43190, AR038669, A85396,
D88507,
AR066482, A44171, A85477,
I19525,
A86792, U46128, D13509, AR016691,
AR016690, X93549, AR066490,
AR008408,
AR060133, A63261, A70867,
AR062872,
AR032065, AF123263, A64136,
A68321,
I14842, AR054175, and AR008382.
H7TBC95 16 8659221 - 692 15 -
706
HHFL003 17 9185831 - 697 15 - AA496150, AI829927, W49726,
711 AW025317,
AW 183143, H91924, 005586,
N26981,
005037, N36704, W27235, W58746,
AA931941, AA082202, AA084759,
AA837507, AA873601, AI056686,
D80089,
AW264285, AA496151, AW117255,
W49652, AA683334, N24722,
AA976379,
AA833537, AI886806, AI248973,
79
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
AW183675, AI149898, AA781907,
AA781135, AA533526, and
846697.
HSHBN61 18 12282991 - 172315 - AI829927, AW025317, AW183143,
1737
AI141532, AI797833, AA496150,
AI056686,
W58746, AA837507, AA496151,
AI741293,
AW264285, AW118774, AW117255,
AI911263, AA142964, AA811251,
AI085493, AI342629, C05037,
W49726,
AA683334, AI376698, AI492870,
W49652,
N27387, N40799, AA524357,
C05586,
' AI224134, N24722, AA833537,
AI141804,
AA287409, AA150630, AA976379,
AA657408, AA042960, AI374687,
AI886806, AW183675, AI07S423,
AI248973, AA781907, AI149898,
AI806022,
AA781135, AA947537, AA910834,
AI017589, AA434423, AA761915,
T94779,
H91924, AI380147, AI081048,
AA933982,
AA884442, AA743945, AW057613,
AA827074, N26981, AA533526,
846697,
. AA482972, N36704, W27235,
AA931941,
AA904555, AA082202, H99105,
AA084759,
. AA815133, AA928275, AI042627,
832760,
H91925, D80089, AA553874,
AA833554,
AA303665, AA927041, AI919444,
AA873601, H26937, AA249311,
AL043281,
AW004979, T94027, AA377312,
H28529,
AW627690, and AW630994.
HNHNP81 19 11561701 - 939 15 -
953
HFIDL68 20 11623951 - 107515 - AI805323, AI375172, AL119399,
1089
AL0394S6, AA580663, AL04S413,
AI635082, AI932794, AW089844,
AI698391, AI538850, AI638644,
AI370623,
AI584130, AL036241, AA761608,
AW 189301, AW 151974, AI250282,
AI345688, AI623941, AI859644,
AI869377,
AL043152, AI927233, AI582910,
AI457113,
AI632036, AA641818, F36033,
AI586931,
AI610714, AI923989, AW023072,
AI345010, AI648699, AI610616,
AI613144,
AW019988, AI887163, AA810605,
AI653829, AA669025, AW074651,
AI383510, AI469754, AW075382,
AW083572, AI419455, N25033,
AL119863,
AI566386, AI419826, AI250353,
AL049048,
N29277, AW245729, T66952,
AL038986,
AI698987, AI798456, AI624245,
AI758445,
AI524179, AW020397; AA456793,
AW079432, AI872104, AI469270,
AI285439, AI961414, AI590043,
AI567883,
AA805751, AI560954, AL119511,
AI440263, AL119791, AI224373,
AW022084, AI382313, W45039,
AI471909,
AI440126, AI284484, F36058,
AI349012,
AA761557, AI318603, AI954504,
AW 162194, AI096432, AI923446,
AA689584, AA056265, AW303152,
AW131165, AI673363, AW238753,
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
AI306610, AI653402, AI433500,
AI799313,
AI457661, AW129106, AI954293,
AI250646, AI282268, AI886355,
AI345778,
AA769601, AA693331, AI473471,
AW087888, AL046944, N66799,
AI621341,
AL040844, AI918554, AL047826,
AW163554, AA788861, AI307285,
AL119457, AW022808, 221709,
AW059766, AW083484, AI678773,
AW410090, AI074008, F27438,
AI587209,
AI818065, AI440239, AA417278,
AW303084, AI635634, AL048298,
AI446646, AI690887, AI349958,
AI225000,
AI868204, AL045349, AW172981,
AI590624, AI559752, AA806605,
AW078593, AI673140, AI581033,
AI493572, AA505147, AW051088,
AW195169, AI270099, AW074365,
AI345005, AI815232, AI524608,
AW020480, AW129264, AI859991,
AI872423, AI473451, AL043168,
AI619820,
AI628325, AI634805, AI624304,
AW028840, AI364639, AI824576,
AI345014, AI860027, AW044367,
AI474093, AW022093, AI587000,
AI879377, AA229532, AI702527,
AW166707, AI815855, AI688848,
AW021662, AI445505, AI819106,
AI114461, AI049733, AW303179,
AI890907, AW078776, AI815233,
AW131308, AI285514, AI918809,
AI954721, AI891125, AI401697,
AI468959,
AI002285, AW020076, AI344789,
AI884459, AI251221, AA731241,
F37323,
AI432644, AW050578, AI538637,
AA057833, AI336631, AW301513,
AW072719, AW020046, AW198144,
AI567971, AW239352, AL046261,
AI151101, AI538885, AW190297,
575997,
AF115410, AL133559, 582852,
M85164,
U72621, AL137479, AF090943,
AJ005690,
AF176651, AJ238278, AL137529,
AF132676, AF061836, I09499,
AR029490,
AL133016, X70685, AL137533,
AL133049,
AB007812, AF180525, M79462,
235309,
AF013214, AF079763, AL023657,
A60092,
A60094, AF031572, AR038854,
AL133640,
AF090900, U51123, S69407,
AF043642,
AF113694, AL122104, I25049,
A03736,
AL049276, AR020905, AF 161699,
AF067728, E04233, AL137554,
AF146568,
AL137521, AL133560, AF119336,
I28326,
Y09972, A07647, AF090896,
AL1I0269,
AL110218, S54890, X61970,
U49434,
AL080129, U49908, AFO'77051,
X66862,
AL110196, A77033, A77035,
AF113699,
X63162, AF068615, AF177401,
AL117435,
AF098162, AL137550, AL136884,
81
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
AF113677, D44497, E12580,
AL133557,
553987, I32738, AR068466,
A18777,
AF067790, M19658, Y16645,
AL080159,
AL050172, AF106697, AL133619,
E02221,
297214, AL080118, E05822,
AL117587,
E12579, AL117582, AF080622,
AF076633,
AF182215, AR068751, Y10823,
AF131821,
AF112208, X67813, AF026816,
I89947,
U76419, 213966, A08912, AF090903,
I25048, AL110280, M80340,
D83032,
A08911, AC004200, E12747,
AB016226,
AL133067, Y11254, AL122050,
AL117460,
538729, AF047716, AF061795,
A58545,
AF151685, AL137495, AR034821,
I03321,
AL137292, AF032666, AF100931,
AF113690, AL117438, I42402,
282022,
Y10080, AL137281, AL133062,
AJ010277,
AF016271, AF004162, U78525,
AL050155,
AL096751, U80919, 576508,
AF061573,
X83508, U72620, AF161418,
Y11587,
X60769, I48978, AF148129,
AL137560,
AF111849, U62807, AL117394,
AF030513,
AF002672, AL137555, X65873,
AF104032,
A18788, E02914, AF175903,
AL137478,
A49139, AL137459, AF094480,
AF126488,
AF124728, AL080140, AL136842,
U73682,
AL096728, AL137254, AL133565,
E01614,
E13364, AF102578, M92439,
X06146,
A08913, Y13350, AF118090,
AL137271,
X72624, AF111851, U86379,
X59812,
AF090886, AJ006039, AFI18558,
AF150103, AL050149, AL050116,
U57352,
AL050143, U62966, Y14634,
AL080126,
U35846, X63574, U91329, AJ004832,
I08319, and AL133665.
HNHCP79 21 5657811 - 288 15 -
302
HFKKN77 22 11522451 - 161815 - AA359504, AA338949, C14015,
1632
AW438452, AA081938, D54454,
D54589,
AA604943, AI557116, D53954,
C15197,
C14435, D52837, C14896, AA209384,
AI557706, AI189843, D51952,
AI535622,
AI536057, C14420, AA146855,
AA224482,
AA125985, AA132337, W40227,
AI985406,
AI419419, AI423034, AI942421,
D52561,
AI591163, AI214247, D52521,
C14371,
AA186474; AA147432, AI160686,
C14481,
AA115758, W28103, C14386,
AI189853,
AF180326, AC005742, U00208,
AC005402,
AF006668, U00212, AF126500,
222668,
U95182, U00186, AB025024,
AF071080,
X76456, D83147, AE000665,
AC005235,
AF178641, AC008160, D45431,
AC002324,
AF109905, AL078630, M18837,
AL136329,
AF098640, AF229843, M37278,
AF146525, .
U90355, AC003019, AC002109,
AF037352,
M9579I, AC002I08, X89147,
A26225,
AB018097, AJ230881, X55230,
AE000663,
AF154503, AF060950, AC006956,
U51000,
82
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
X58264, M57284, AF131866,
AF040755,
X69986, AC005413, AE000664,
AJ231014,
571861, M18351, X89151, 222526,
D50805,
AL136328, AP000014, U56721,
M10936,
AJ271003, AF050157, U86703,
AL049553,
E15284, AC002406, D45426,
X83735,
U02090, AF134349, J00795,
V01272,
AC007306, AF045943, AF198097,
AF 142607, AF 111102, AC007263,
AF178646, U02323, 270232,
AC004096,
U02318, 284465, AF109719,
AC002397,
I65500, AF121802, X75759,
AF064865,
AC005938, AF121351, X03694,
AF094679,
AB026572, AR055737, A51707,
AF103025,
X14061, AF178644, AC006584,
AF111103,
AF084363, AJ230226, M33962,
U62658,
M64239, AC005240, X85525,
AF055451,
L15308, AJ231008, AJ002011,
Y09411,
AB030448, AF098866, and AF089743.
HBGMZ39 23 9471121 - 588 15 - AI820661, AI791493, AA989356,
602 AI791282,
AI732537, AI792053, AW207804,
822360,
872427, AA505927, 822019,
872474,
AC008537, AC008537, AC008537,
AC019337, AC019337, and AC019337.
HEQAP17 24 11263801 - 814 15 - AI131555, AI769466, H67224,
828 AA215577,
AW190975, AA258335, AA258499,
and
S63848.
HWLLBI 25 10930011 - 772 15 - AI745636, and AA102414.
1 786
HNTEF53 26 9548521 - 234215 - AA557324, AI655577, AI696732,
2356 AI923200,
AA863360, AW262723, AI697332,
AW275990, AI436648, AW276183,
856515,
AI362521, 853456, 853457,
D62878,
AA337301, AA652746, AW264444,
856123, AA319338, D79346,
D79250,
N56346, AA886832, and AL138223.
HNTND64 27 9548711 - 392 15 - AC025090, and AC025090.
406
HFPFA83 28 12077731 - 153215 - AA359504, AA338949, 014015,
1546 AA081938,
D54454, D54589, AA604943,
AI591163,
D53954, 015197, 014435, AI189843,
D52837, 014896, AA209384,
AI557706,
D51952, AI535622, AI536057,
014420,
AA146855, AA224482, AAI25985,
AAI32337, W40227, AI985406,
AI557116,
AI419419, AI423034, AI94242I,
D52561,
AI214247, D52521, 014371,
AW438452,
AA186474, AI160686, 014481,
AA115758,
W28103, 014386, AI189853,
AF180326,
AF006668, AL078630, U95182,
AC005742,
D83147, AC005402, AC002324,
AE000665,
AC006956, AF109905, AF121351,
AF178641, U90355, D45431,
AF111102,
AC007263, M18837, AJ230881,
X76456,
AF098640, AF111103, AF146525,
M95791,
X89147, A26225, AC002109,
AC008062,
X55230, AC005992, AF060950,
U51000,
X58264, M57284, AF040755,
X69986,
AC011013, AL117606, AJ231014,
571861,
X89151, AL023877, AC005874,
AF134471,
83
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
222526, D50805, M65161,
AB018097,
AF064865, AL049833, U56721,
AF110520,
AJ271003, AC002397, U86703,
AC006949,
E15284, AC006455, D45426,
X83735,
AC007773, AF134349, J00795,
V01272,
AF045943, X96607, AB017653,
AC005835,
AF142607, AF178646, 270232,
AC004096,
AF031384, AF094679, AF131205,
AF109719, AF071080, M33962,
AC005240,
AC005296, AL049792, AF121802,
X75759,
X03694, M88300, AB026572,
AR055737,
A51707, AF103025, AC007585,
AF126500,
AF190631, AF178644, AJ230226,
AC004962, AC005290, U62658,
X85525,
AE000664, AC007306, AL136328,
AC005818, AF055451, L15308,
AJ231008,
AJ002011, Y09411, AB030448,
AF098866,
M26129, Y09902, AJ230231,
AF072903,
AF130585, 574207, J05206,
AR033870,
AC003694, AL049685, E15285,
K02138,
AJOOSS42, 284465, AE000663,
AC009287,
AL022069, AF178647, D28565,
AP000028,
AJ230904, X03152, X02002,
U45313,
U02023, and L07328.
HMWFM73 29 12126771 - 264515 - U55977, AL135160, AL037548,
2659 AW300888,
AW043869, AW338980, AI082074,
AW339061, AI885709, AI952025,
AA406015, AA543047, AI936178,
AI634351, AI523303, AA211690,
AW019874, AW019884, AA564215,
AI808091, AI570486, AI270136,
AA570695,
AA164397, AA910228, AI709408,
AW009719, AA126058, AI439941,
AI218932, W76233, AW073534,
AI083740,
AA995501, AI209168, AA887057,
W72996,
884569, AI433638, AA112578,
855887,
AW403176, AW166365, N39602,
AI752509,
AA126163, AA349630, AI149404,
AI038131, AA112577, AI539566,
AA146793, AW173568, T08515,
M86152,
AA350924, AA860178, AA063181,
243935,
AA765411, AW402004, AL121331,
AI379857, AA844241, AA761513,
AI245379, F06621, AA323649,
F30915,
T74883, N46748, AW055141,
AW027963,
AI362412, AA865652, AW055135,
N39587,
AA931529, AW044055, AI269440,
AI184567, T80229, AI343860,
AW 128844,
AI434342, F36582, AI199568,
T71100,
AA688060, AA401457, AI074747,
T80207,
AA336458, AA146792, AI290101,
AI559237, AA873110, AA642225,
855804,
AA873362, AA160149, 239996,
AA031392,
AI869167, 838716, AI092934,
AW129777,
AI955036, AI880173, T34290,
AI017016,
AA324592, AI274522, AW082796,
AA160148, F02891, AA256624,
AA876304,
H41806, T74940, AA235122,
T34S52,
84
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
H41846, AW407777, AA405945,
AA256517, AA318146, T35004,
AI281264,
AI633015, N46734, AA368339,
AA100929,
AA366331, AA031267, AA063295,
AI587599, T10453, AA405383,
AI917216,
AA306152, AW128835, AA247400,
AW169286, T08148, AL045131,
AA367702,
AW363878, AI910117, AI302935,
AI680418, AI524604, AI906669,
AI910160,
AA745S41, AA724239, AI910157,
AI908381, AA582483, AI910102,
AI910161,
AF196972, AC004084, AF053356,
AF165926, AC004887, AC005740,
AC003982, AL,031681, and
AC004031.
HE8NI24 30 11606521 - 268115 - 835286, AA883367, AW 148538,
2695 AI268373,
N62306, N77597, AA332611,
850955,
AA732890, AI283442, AI673342,
AI631153,
AI200800, and AI910962.
HOFNH30 31 11822901 - 219415 - AA446859, AW274018, AW 172356,
2208
AI432375, AI223077, AI567144,
AI288165,
AW129264, AI433157, AW059713,
AI699020, AI524654, AL045500,
AW089844, AI613038, AI627893,
AI521005, AA613907, AI611743,
AI537187,
AI281867, AI620056, AI633000,
AI9340I 1,
AL042628, AW403717, AI288285,
AI863382, AW163834, AI537617,
AI446511, AI690813, AI434223,
AI479292,
AI468959, AI689420, AA572758,
AI86883I,
AW169604, AL041S62, AL038529,
AI690946, AI636588, AI610362,
AI633125,
AI799189, AI889306, H417S9,
AA420722,
AI950664, AI682640, AA641818,
AI802542,
AI433976, AI284509, AW268220,
AL039086, AW103628, AL036631,
AI538850, AI635016, AI623941,
AI500061,
AI586931, AI824746, AI6312I6,
AI289542,
AI926794, AI554821, AI698401,
AI345612,
AW084151, AL041150, AW023338,
AW 1S 1893, AW001850, AI670009,
AI473652, AL1I9836, AI345415,
AI3454I6,
AI798456, AI340519, AI340603,
AI625926,
AI280661, AW161579, AI583085,
AAS79232, AW 105431, AL037454,
AI69901I, AI620868, AI874166,
AI909697,
AL043355, AI335426, AI419650,
AI348777,
AW083804, AA830709, AI349004,
AI682891, AW079640, AI267185,
AI312428, AI345688, AI638644,
AL036396,
AI432030, AI114703, AI335209,
AI471909,
AI635492, AI540458, AI470293,
AI954080,
AW073699, AW198090, AI884318,
AI472487, AI890907, AI612913,
AI702073,
AI624529, AI538564, AL119399,
AI241741,
AI334450, AW022682, AA494167,
AI590043, AI343059, AI698391,
AW071349, AI916419, AI554343,
AI280561, AW021373, AI249946,
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
AI590423, AI580436, AL037582,
AI950729,
AI589428, AL037602, AI344935,
AI559752,
AI537677, AI609069, AL119863,
AI683395,
AI445992, AI863014, AW084425,
AI815855, AI871709, AI921176,
AI796743,
AI436429, AI539153, AW271119,
AW166870, AL038605, AI683209,
AI969655, AI247293, AI678446,
AI823719,
AL046944, AI906328, AI950892,
AI539771,
AW152182, AI828734, AI863321,
AA908294, AW089272, AI868740,
AI885982, AI684265, AI872914,
AI636719,
AI953688, AI801044, AI701097,
AI500714,
AI866608, AI435253, AI768496,
AI870192,
AW167926, AI539800, AI582912,
AW104141, AI537273, AW020358,
AI500523, AI554344, AI917253,
AI500553,
AW198112, AW078606, AW172918,
AI500659, AI818574, AI539723,
AI873604,
AI766348, AI522052, AI637521,
AI147500,
AI274508, AL046466, AI241923,
AI252748,
AI352290, AI500706, 298446,
AA640779,
AI440284, AW150308, AA806720,
AI345224, AL047172, AI679117,
AI802244,
AI277008, AI469505, AI633009,
AW128834, AI572717, AW103928,
AI469532, AA828415, AI627988,
AI536574,
AI345737, AW262767, AI579901,
AI860885, AA572872, AI345736,
AL043152, AI432110, AW265004,
AI701890, AI554818, AI819545,
AI361701,
AFI27138, AF186380, 561953,
A08916,
A15345, 578214, X79812,
AF032666,
AL133067, AL050366, I48979,
AL049466,
E05822, E02349, AL137560,
A77033,
A77035, AL137558, Ei2747,
AC004987,
282022, I89947, AR013797,
I48978,
AF061981, AF183393, AL122050,
AL050116, 297214, AF126247,
AF118094,
AF090934, AL137488, X63410,
Y16645,
AF069506, AL137533, U75932,
S36676,
AF125948, AF097996, AF141289,
AL049452, AF102578, AF106862,
AR038854, AF026816, AL136884,
AF153205, AF159615, A23630,
AF139986,
A65341, E03671, X80340,
AL117460,
AF044323, AF057300, AF057299,
AR020905, Y11587, AF113694,
AL122093,
I66342, A65340, U42031,
AL133560,
A21103, AL117416, A$029065,
AL117457,
AL096744, AF008439, AF061943,
AL049382, AF115392, A08913,
AL122100,
U57352, AL137550, AF146568,
AL080148,
AL080124, AL049938, AL110196,
AL133075, X57084, A08912,
A08910,
AL137459, AL080060, AL050149,
A08909,
AL117587, AL122098, AL050172,
AF047716, ALI37548, Y09972,
A08908,
86
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
AL137557, AF090900, AL133062,
AL137256, L31396, AL133113,
576508,
L31397, L04849, AL137271,
AF047443,
AL133081, AF000301, AJ012755,
A86558,
AF176651, AL137648, AJ238278,
U37312,
AL050138, AF113019, AF017437,
L13297,
AF051325, AF106697, M27260,
AF090896,
AF030513, X93495, I68732,
I89931,
A41575, AL133558, 237987,
S77771,
AR034821, I89934, X81464,
I49625,
AL050015, AL122110, X82434,
AF100931,
AL133093, AF118070, U67958,
AL080159,
U95114, AL049314, AF111851,
U58996,
E12806, AL133557, AF090886,
AF158248,
AF090901, AF137367, U35846,
A03736,
AL080I63, AFl 15410, A089I
1, E07361,
A18777, A08907, 578453, I33392,
AL137530, AF058921, AL023657,
AL117435, AL122123, AL110159,
S68736,
AF038562, AL049300, Y11254,
AR009628,
X63162, AF192557, AL035458,
I09499,
AF090943, U72621, AF078844,
AL133010,
AL133049, AL110280, AB016226,
L19437,
AF145233, AL110296, I03321,
E01314,
AL133665, AF081197, AF081195,
A58524,
A58523, AF111849, AL080074,
AL137711,
X54971, 583440, AF106657,
AL137529,
AL050092, AL080154, X98066,
AF003737,
AL122118, A07588, I33391,
AF090903,
AJ003118, AL117440, U49434,
AL137495,
AF195092, AF185576, AL137521,
AL137641, AF061573, X53587,
AL096751,
AL050393, Y10080, X66862,
X06146,
AF031147, I32738, AF002672,
AJ005690,
AL133112, U89295, X72387,
AL137476,
AF091084, I00734, AF107847,
U57715, and
213966.
HWMEV63 32 11247121 - 536 15 - D13626.
550
HBXBG65 33 9327801 -'521 15 - 836281, AF094480, and AF094479.
535
HPIAT34 34 12282361 - 152915,- AI765967, AI436791, AI969563,
1543 AI949451,
AI810397, AW024539, AI084325,
AA156726, AI452756, AA824232,
AA843093, AI963642, AI306667,
AW207447, T96131, AW243556,
AI084332,
AA156832, H95977, H95978,
AI701335,
U30998, AA299371, T96213,
U37591,
AF035268, E16580, AF035269,
D88666,
and E16577.
HLWAR77 35 9474841 - 127515 - AA449919, AA449920, and AF119815.
1289
HNTAV78 36 12125281 - 372715 - AI417713, AW235714, AI537274,
3741
AL036146, AW087445, AI349772,
AL135661, AW161579, AW071349,
AW238730, AL045500, AI433157,
AL036802, AI934036, AW268253,
AI580190, AL036396, AA640779,
AI064830, AI868831, AI349645,
AI500077,
AW301409, AW117882, AI433976,
AI922901, AW150578, AL036980,
87
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
AI560012, AW162071, AL047763,
AI207510, AL079963, AL037454,
AW 129106, AA572758, AL047042,
AL036274, AI815855, AL036361,
AI920968, AI340582, AA613907,
AI469532,
AI312428, AI567351, AI521012,
AI802542,
AI475371, AI538716, AL119791,
AL041772, AI560099, AI436456,
AI608667,
AL119049, AI687376, AI687415,
AL121270, AL119863, AW071417,
AI690751, AL120854, AI349933,
AI702406,
AL036759, AI249497, AL040456,
AI247193, AL044207, AI349256,
AA528822, AL046849, AI559296,
AA427700, AI312152, AI491852,
AW302965, AI815383, AI349937,
AI500553, AW089572, AI906328,
AW166645, AL043326, AL134259,
AW 103371, AI934011, AL040243,
AW 104724, AI682841, AI349004,
AL121328, AI863014, AW080838,
AI564719, AW198090, AI648509,
AI612913, AI269205, AI349614,
AI624206,
AI679724, AW008048, AI637584,
AI873731, AW 132056, AW
132121,
AI696612, AI907070, AI349598,
AI274508,
AI890833, AI926790, AW026882,
AL048871, AI889376, AI620284,
AW074993, AI524671, AW148536,
AI921248, AI619502, AL119748,
AI783504,
AI536685, AI499393, AI702073,
AL040169,
AI590128, AI758437, AI340519,
AI969601,
AI282655, AL120736, AL036901,
AI274541, AI568870, AI343112,
AI633419,
AI537677, AI345111, AI818683,
AW303152, AL041573, AI753683,
AI309401, AI934035, AI684265,
AI954183,
AW302988, AI858827, AI682743,
AI567632, AL045266, AL038605,
AL038778, AI284517, AI348897,
AI677796,
AI635492, AA508692, AI886753,
AL121365, AI440239, AI440426,
AI584140,
AI554245, AL121463, AI627360,
AI149592,
AI687728, AI284484, AW 151485,
AI500523, AI249323, AW 163464,
AI275175, AL134830, AI281779,
AI570384,
AI857296, AI872711, AI702433,
AI224992,
AI289937, AI345131, AI282281,
AW075351, AW268251, AI673256,
AI269696, AW 148320, AI800453,
AI800433, AI886124, AI498579,
AI590120,
AA225339, AI866002, AI571909,
AI631107,
AI624859, AW169653, AW068845,
AI285735, AI610645, AI702068,
AI254042,
AI627893, AL036240, AI318280,
AI536638,
AI499285, AI345735, AI285826,
AI539771,
AW 149869, AI445025, AI866608,
AW268768, AI690835, AI796743,
88
CA 02393912 2002-06-21
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AI682106, AI281773, AW183130,
AI500659, AI815232, AI538829,
AI969567,
AI909666, AW051258, AI224027,
AI340603, AC006296, AC007392,
AC005224, AC008394, ACb05723,
AC010175, I48979, AC006115,
AL034374,
AF118070, AF090901, AF090934,
AF090943, AF090900, AF113013,
AF113691, AL133640, Y11587,
AF078844,
AF113690, S78214, AL117457,
AL122050,
I48978, AL133016, AL133557,
AF090903,
AL137550, AL050393, AF125949,
L31396,
L31397, A93016, I89947,
AL133560,
AL050146, Y16645, AF118064,
AL110196,
AL009029, AJ242859, AL133075,
AL122093, AL096744, A65341,
AL049938,
AL117460, A08916, I89931,
AF146568,
AF104032, X84990, AF177401,
AL133606,
AJ000937, U42766, 568736,
AF090896,
AF113694, AL110221, AL080060,
A08913,
AL049452, AB019565, and
AF113699.
HDPBI30 37 974711 1 - 291115 - AA714520, N78665, W15172,
2925 AL134531,
AA074818, AI251157, AI311635,
AA079403, AW130754, AI935943,
AF083955, AC005015, AL034423,
AP000030, AC002992, AC004216,
AC003013, U91321, AC003684,
AC002528,
AL117258, AL021155, AP000045,
AF053356, AL033521, AC004598,
U91326,
AL035072, AD000091, U82668,
AC012384,
L44140, AF006752, AL034350,
AC006039,
AC005756, AC005072, AL034429,
AC002352, AC005682, AC003663,
AC005049, AC007298, AC005620,
AC004887, AL117694, AC005911,
AC007688, AC006014, AC004797,
AL031186, AL031283, AC004963,
L47234,
284466, AC004125, AC005529,
AL031293,
AC006276, AL034400, AC004099,
AC005089, AL049871, AC004893,
AL080243, AC007021, AL049712,
AC007993, AC006581, AC005837,
AF139813, M13792, AC005086,
AL096791,
AJ251973, AC002301, AC006139,
AC005488, L78810, AC006115,
AC004966,
AC006538, 293244, AC004834,
AL049570,
AC004084, AP000113, AP000251,
and
AC005696.
89
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TABLE 4
Code Descri tion Tissue Organ Cell DiseaseVector
Line
AR022 a_Heart a_Heart
AR023 a_Liver a_Liver
AR024 a_mammary land a_mammary
land
AR025 a_Prostate a_Prostate
AR026 a_small intestinea_small intestine
AR027 a_Stomach a_Stomach
AR028 Blood B cells Blood B cells
AR029 Blood B cells Blood B cells
activated activated
AR030 Blood B cells Blood B cells
resting restin
AR031 Blood T cells Blood T cells
activated activated
AR032 Blood T cells Blood T cells
restin restin
AR033 brain brain
AR034 breast breast
AR035 breast cancer breast cancer
AR036 Cell Line CAOV3Cell Line
CAOV3
AR037 cell line PA-1 cell line
PA-1
AR038 cell line transformedcell line
transformed
AR039 colon colon
AR040 colon (9808co65R)colon (9808co65R)
AR041 colon (9809co15)colon (9809co15)
AR042 colon cancer colon cancer
AR043 colon cancer colon cancer
(9808co64R) (9808co64R)
AR044 colon cancer colon cancer
9809co14 9809co14
AR045 corn clone 5 corn clone
5
AR046 corn clone 6 corn clone
6
AR047 corn clone2 corn clone2
AR048 corn clone3 corn clone3
AR049 Corn Clone4 Corn Clone4
AR050 Donor II B CellsDonor II
24hrs B Cells
24hrs
AR051 Donor II B CellsDonor II
72hrs B Cells
72hrs
AR052 Donor II B-CellsDonor II
24 hrs. B-Cells
24
hrs.
AR053 Donor II B-CellsDonor II
72hrs B-Cells
72hrs
AR054 Donor II RestingDonor II
B Cells Resting
B
Cells
AR055 Heart ~ Heart
AR056 Human Lung (clonetech)Human Lung
(clonetech)
AR057 Human Mammary Human Mammary
(clontech) (clontech)
AR058 Human Thymus Human Thymus
(clonetech) (clonetech)
AR059 Jurkat (unstimulated)Jurkat
(unstimulated)
AR060 Kidney Kidne
AR061 Liver Liver
AR062 Liver (Clontech)Liver (Clontech)
AR063 Lymphocytes Lymphocytes
chronic chronic 1
1 m hoc tic m hoc tic
leukaemia
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
leukaemia
AR064 Lymphocytes Lymphocytes
diffuse large
B cell lymphomadiffuse large
B cell
I m home
AR065 Lymphocytes Lymphocytes
follicular
1 m home follicular
lym home
AR066 normal breast normal breast
AR067 Normal Ovarian Normal 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 Cancer Ovarian Cancer
AR072 Ovarian Cancer Ovarian Cancer
(97026001) (97026001)
AR073 Ovarian Cancer Ovarian Cancer
(97076029) (97076029)
AR074 Ovarian Cancer Ovarian Cancer
(98046011) (98046011)
AR075 Ovarian Cancer Ovarian Cancer
(98066019) (98066019)
AR076 Ovarian Cancer Ovarian Cancer
(98076017) (98076017)
AR077 Ovarian Cancer Ovarian Cancer
(98096001) (98096001)
AR078 ovarian cancer ovarian cancer
15799
15799
AR079 Ovarian Cancer Ovarian Cancer
17717AID 17717AID
AR080 Ovarian Cancer Ovarian Cancer
400466481 4004664B1
AR081 Ovarian Cancer Ovarian Cancer
4005315A1 4005315A1
AR082 ovarian cancer ovarian cancer
94127303
94127303
AR083 Ovarian Cancer Ovarian Cancer
96069304
96069304
AR084 Ovarian Cancer Ovarian Cancer
97076029
97076029
AR085 Ovarian Cancer Ovarian Cancer
98076045
98076045
AR086 ovarian cancer ovarian cancer
98096001
98096001
AR087 Ovarian Cancer Ovarian Cancer
9905C032RC 9905C032RC
AR088 Ovarian cancer Ovarian cancer
9907 C00 9907
3rd C00 3rd
AR089 Prostate Prostate
AR090 Prostate (clonetech)Prostate
(clonetech)
AR091 rostate cancer rostate cancer
AR092 prostate cancerprostate
#15176 cancer
#15176
AR093 prostate cancerprostate
#15509 cancer
#15509
AR094 prostate cancerprostate
#15673 cancer
#15673
AR095 Small IntestineSmall Intestine
(Clontech)
(Clontech)
AR09 ~leen Spleen
91
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AR097 Thymus T cells Thymus T
activated cells
activated
AR098 Thymus T cells Thymus T
resting cells
restin
AR099 Tonsil Tonsil
AR100 Tonsil geminal Tonsil geminal
center
centroblast center centroblast
AR101 Tonsil germinalTonsil germinal
center B
cell center B
cell
AR102 Tonsil 1 m h Tonsil lym
node h node
AR103 Tonsil memory Tonsil memory
B cell B
cell
AR104 _Whole Brain Whole Brain
AR105 Xeno raft ES-2 Xeno raft
ES-2
AR106 Xeno raft SW626Xeno raft
SW626
H0009 Human Fetal Uni-ZAP
Brain
XR
H0011 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney Kidney
XR
H0012 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney Kidney
XR
H0013 Human 8 Week Human 8 WeekEmbryo Uni-ZAP
Whole Old
Embryo Emb o XR
H0014 Human Gall BladderHuman Gall Gall Bladder Uni-ZAP
Bladder
XR
H0015 Human Gall Bladder,Human Gall Gall Bladder Uni-ZAP
Bladder
fraction II XR
H0019 Human Fetal Human Fetal Heart Bluescri
Heart Heart ~ t
H0022 Jurkat Cells Jurkat T-Cell Lambda
Line
ZAP II
H0024 Human Fetal Human Fetal Lung Uni-ZAP
Lung III Lung
XR
H0026 Namalwa Cells Namalwa B-Cell Lambda
Line, EBV ZAP II
immortalized
H0028 Human Old OvaryHuman Old Ovary pBluescript
Ovary
H0030 Human Placenta Uni-ZAP
XR
H0031 Human Placenta Human PlacentaPlacenta Uni-ZAP
XR
H0032 Human Prostate Human ProstateProstate Uni-ZAP
XR
H0035 Human Salivary Human SalivarySalivary Uni-ZAP
Gland
Gland gland XR
H0036 Human Adult Human Adult Small Uni-ZAP
Small Small Int.
Intestine Intestine XR
H0037 Human Adult Human Adult Small pBluescript
Small Small Int.
Intestine Intestine
H0038 Human Testes Human TestesTestis Uni-ZAP
XR
H0039 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor
Tumor XR
H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor
Tumor XR
H0041 Human Fetal Human Fetal Bone Uni-ZAP
Bone Bone
XR
H0042 Human Adult Human Adult Lung Uni-ZAP
Pulmonary
Pulmonar XR
H0046 Human EndometrialHuman EndometrialUterus diseaseUni-ZAP
Tumor Tumor XR
H0050 Human Fetal Human Fetal Heart Uni-ZAP
Heart Heart
XR
92
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WO 01/55387 PCT/USO1/01310
H0051 Human HippocampusHuman Brain Uni-ZAP
Hi ocam us XR
H0052 Human CerebellumHuman CerebellumBrain Uni-ZAP
XR
H0056 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein,
Endo. remake Vein Endothelialvein XR
Cells
H0057 Human Fetal Uni-ZAP
Spleen
XR
H0059 Human Uterine Human UterineUterus diseaseLambda
Cancer
Cancer ZAP II
H0061 Human Macro Human Macro Blood Cell Bluescri
ha a haoe Line t
H0063 Human Thymus Human ThymusThymus Uni-ZAP
XR
H0068 Human Skin TumorHuman Skin Skin diseaseUni-ZAP
Tumor
XR
H0069 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Cells Line
XR
H0075 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells T-Cells Line
(II) XR
H0079 Human Whole Human Whole Embryo Uni-ZAP
7 Week 7
Old Emb o (II) Week Old XR
Embryo
H0083 HUMAN JURKAT Jurkat Cells Uni-ZAP
MEMBRANE BOUND XR
POLYSOMES
H0087 Human Thymus Human Thymus pBluescript
H0090 Human T-Cell T-Cell LymphomaT-Cell diseaseUni-ZAP
Lymphoma
XR
H0098 Human Adult Human Adult Liver Uni-ZAP
Liver, Liver
subtracted XR
HO100 Human Whole Human Whole Embryo Uni-ZAP
Six Week Six
Old Emb o Week Old XR
Emb o
HOl Human Placenta,Human PlacentaPlacenta pBluescript
l
l
subtracted
H0123 Human Fetal Human Fetal Brain Uni-ZAP
Dura Mater Dura
Mater XR
H0125 Cem cells cyclohexamideCyclohexamideBlood Cell Uni-ZAP
Line
treated Treated Cem, XR
Jurkat,
Ra'i, and
Su t
H0130 LNCAP untreatedLNCAP Cell Prostate Cell Uni-ZAP
Line Line
XR
H0131 LNCAP + o.3nM LNCAP Cell Prostate Cell Uni-ZAP
81881 Line Line
XR
H0134 Raji Cells, CyclohexamideBlood Cell Uni-ZAP
cyclohexamide Line
treated Treated Cem, XR
Jurkat,
Ra'i, and
Su t
H0135 Human Synovial Human SynovialSynovium Uni-ZAP
Sarcoma
Sarcoma XR
H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early
Sta a Human Sta a Human XR
H0150 Human EpididymusEpididymis Testis Uni-ZAP
H0154 Human FibrosarcomaHuman Skin Skin diseaseUni-ZAP
Fibrosarcoma XR
H0159 Activated T-Cells,Activated Blood Cell Uni-ZAP
8 hrs., T-Cells Line
1i ation 2 XR
H0163 Human Synovium Human SynoviumSynovium Uni-ZAP
XR
H0166 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer,
Sta a B2 fractionCancer, sta XR
a B2
H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer,
Sta a C fractionCancer, sta XR
a C
93
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
H0170 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old
Human Early Sta XR
a Human
H0171 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old
Human, II Earl Sta XR
a Human
H0176 CAMAlEe Cell CAMAlEe CellBreast Cell Uni-ZAP
Line Line
Line XR
H0179 Human NeutrophilHuman NeutrophilBlood Cell Uni-ZAP
Line
XR
H0186 Activated T-CellT-Cells Blood Cell Lambda
Line
ZAP II
H0187 Resting T-Cell T-Cells Blood Cell Lambda
Line
ZAP II
H0188 Human Normal Human NormalBreast Uni-ZAP
Breast
Breast XR
H0194 Human Cerebellum,Human CerebellumBrain pBluescript
subtracted
H0196 Human Cardiomyopathy,Human Heart Uni-ZAP
subtracted Cardiomyopathy XR
H0208 Early Stage Human Fetal Lung pBluescript
Human Lung, Lung
subtracted
H0214 Raji cells, CyclohexamideBlood Cell pBluescript
cyclohexamide Line
treated, subtractedTreated Cem,
Jurkat,
Ra'i, and
Su t
H0220 Activated T-Cells,Activated Blood Cell Uni-ZAP
4 hrs, T-Cells Line
subtracted XR
H0222 Activated T-Cells,Activated Blood Cell Uni-ZAP
8 hrs, T-Cells Line
subtracted XR
H0231 Human Colon, Human Colon Bluescri
subtraction t
H0233 Human Fetal Human Fetal Heart pBluescript
Heart, Heart
Differential
(Adult-
S ecific)
H0242 Human Fetal Human Fetal Heart pBluescript
Heart, Heart
Differential
(Fetal-
S ecific)
H0250 Human ActivatedHuman Monocytes Uni-ZAP
Monoc tes XR
H0251 Human ChondrosarcomaHuman Cartilage diseaseUni-ZAP
Chondrosarcoma XR
H0252 Human OsteosarcomaHuman Bone diseaseUni-ZAP
Osteosarcoma XR
H0253 Human adult Human Adult Testis Uni-ZAP
testis, large Testis
inserts XR
H0254 Breast Lymph Breast LymphLymph Uni-ZAP
node cDNA Node Node
libra XR
H025S breast lymph Breast LymphLymph Lambda
node CDNA Node Node
libr ZAP II
H0263 human colon Human Colon Colon diseaseLambda
cancer
Cancer ZAP II
H0265 Activated T-CellT-Cells Blood Cell Uni-ZAP
Line
( l2hs)/Thiouridine XR
labelledEco
H0266 Human MicrovascularHMEC Vein Cell Lambda
Line
Endothelial ZAP II
Cells, fract.
A
H0268 Human UmbilicalHUVE Cells UmbilicalCell Lambda
Vein Line
Endothelial vein ZAP II
Cells, fract.
A
H0271 Human Neutrophil,Human NeutrophilBlood Cell Uni-ZAP
- Line
Activated Activated XR
H0272 HUMAN TONSILS, Human TonsilTonsil Uni-ZAP
FRACTION 2 XR
H0286 Human OB MG63 Human Bone Cell Uni-ZAP
treated Line
(10 nM E2) fractionOsteoblastoma XR
I
MG63 cell
line
94
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
H0288 Human OB HOS Human Bone Cell Uni-ZAP
control Line
fraction I Osteoblastoma XR
HOS
cell line
H0293 WI 38 cells Uni-ZAP
XR
H0294 Amniotic Cells Amniotic Placenta Cell . Uni-ZAP
- TNF Cells - Line
induced TNF induced XR
H0305 CD34 positive CD34 PositiveCord Blood ZAP Express
cells (Cord Cells
Blood)
H0306 CD34 depleted CD34 DepletedCord Blood ZAP Express
Buffy Coat
(Cord Blood) Buffy Coat
(Cord
Blood)
H0309 Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic
Synovitis/ XR
Osteoarthritis
H0316 HUMAN STOMACH Human StomachStomach Uni-ZAP
XR
H0318 HUMAN B CELL Human B CellLymph diseaseUni-ZAP
Node
LYMPHOMA L m homa XR
H0327 human corpus Human CorpusBrain Uni-ZAP
colosum
Callosum XR
H0328 human ovarian Ovarian CancerOvary diseaseUni-ZAP
cancer
XR
H0333 HemangiopericytomaHemangiopericytomBlood diseaseLambda
vessel
a ZAP II
H0339 Duodenum Duodenum ' Uni-ZAP
XR
H0341 Bone Marrow Bone Marrow Bone MarrowCell Uni-ZAP
Cell Line Cell Line
(RS4;11) Line RS4;11 XR
H0343 stomach cancer Stomach Cancer diseaseUni-ZAP
(human) -
5383A (human) XR
H0351 Glioblastoma GlioblastomaBrain diseaseUni-ZAP
XR
H0352 wilm"s tumor Wilm"s Tumor diseaseUni-ZAP
XR
H0354 Human LeukocytesHuman LeukocytesBlood Cell pCMVSport
Line
1
H0355 Human Liver Human Liver, pCMV Sport
normal Adult 1
H0369 H. Atrophic Atrophic Uni-ZAP
Endometrium
Endometrium XR
and
m ometrium
H0370 H. Lymph node Lymph node diseaseUni-ZAP
breast with
Cancer Met. Breast XR
Cancer
H0373 Human Heart Human Adult Heart pCMV Sport
Heart
1
H0375 Human Lung Human Lung pCMVSport
1
H0392 H. Menin ima, Human Menin brain S ortl
Ml ima
H0393 Fetal Liver, Human Fetal Liver Bluescri
subtraction Liver t
II
H0394 A-14 cell line Redd-Sternber ZAP Ex
cell ress
H0395 Al-CELL LINE Redd-Sternber ZAP Ex
cell ress
H0396 Ll Cell line Redd-Sternberg ZAP Express
cell
H0400 Human Striatum Human Brain,Brain Lambda
De ression, Striatum ZAP II
re-rescue De ression
H0402 CD34 depleted CD34 DepletedCord Blood ZAP Express
Buffy Coat
(Cord Blood), Buffy Coat
re-excision (Cord
Blood)
H0404 H. Umbilical HUVE Calls UmbilicalCell Uni-ZAP
Vein Line
endothelial vein XR
cells,
uninduced
H0409 H. Striatum Human Brain,Brain Bluescri
De ression, t
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
subtracted Striatum
De ression
H0411 H Female Bladder,Human FemaleBladder pSportl
Adult
Adult Bladder
H0412 Human umbilicalHUVE Cells UmbilicalCell pSportl
vein Line
endothelial vein
cells, IL-4
induced
H0413 Human UmbilicalHUVE Cells UmbilicalCell pSportl
Vein Line
Endothelial vein
Cells,
uninduced
H0414 Ovarian Tumor Ovarian Tumor,Ovary diseasepSportl
I, OV5232
OV5232
H0415 H. Ovarian Tumor,Ovarian Tumor,Ovary diseasepCMV
II, Sport
OV5232 OV5232 2.0
H0416 Human Neutro~hils,Human NeutrophilBlood Cell pBluescript
- Line
Activated, re-excisionActivated
H0421 Human Bone Marrow,Bone Marrow pBluescript
re-
excision
H0422 T-Cell PHA 16 T-Cells Blood Cell pSportl
hrs Line
H0423 T-Cell PHA 24 T-Cells Blood Cell S ortl
hrs Line
H0427 Human Adipose Human Adipose, pSportl
left
hi 1i oma
H0428 Human Ovary Human Ovary Ovary pSportl
Tumor
H0431 H. Kidney Medulla,Kidney medullaKidney pBluescript
re-
excision
H0432 H. Kidne P ramidKidne ramidsKidne Bluescri
t
H0435 Ovarian Tumor Ovarian Tumor,Ovary pCMV
10-3-95 Sport
OV350721 2.0
H0436 Restin T-Cell T-Cells Blood Cell S ortl
Libr ,II Line
H0438 H. Whole Brain Human Whole ZAP Express
#2, re- Brain
excision ~ #2
H0441 H. Kidney Cortex,Kidney cortexKidney pBluescript
subtracted
H0445 Spleen, ChronicHuman Spleen,Spleen diseasepSportl
CLL
1 m hocytic
leukemia
H0459 CD34+cells, CD34 positive pCMVSport
II, cells
FRACTION 2 2.0
H0478 Salivary Gland,Human SalivarySalivary pSportl
Lib 2
Gland land
H0479 Salivary Gland,Human SalivarySalivary pSportl
Lib 3
Gland land
H0483 Breast Cancer Breast Cancer pSportl
cell line, Cell
MDA 36 line, MDA
36
H0484 Breast Cancer Breast Cancer pSportl
Cell line, Cell
angiogenic line, Angiogenic,
36T3
H0485 Hodgkin"s LymphomaHodgkin"s diseasepCMVSport
I
L m homa 2.0
I
H0486 Hodgkin"s LymphomaHodgkin"s diseasepCMVSport
II
L m homa 2.0
II
H0494 Keratinocyte Keratinocyte pCMVSport
2.0
H0497 HEL cell line HEL cell HEL pSportl
line
92.1.7
H0506 Ulcerative ColitisColon Colon S ortl
H0509 Liver, HepatomaHuman Liver,Liver diseasepCMVSport
He atoma, 3.0
atient 8
H0510 Human Liver, Human Liver,Liver pCMVSport
normal
normal, Patient 3.0
# 8
H0512 Keratinocyte, Keratinocyte pCMVSport
lib 3
2.0
H0517 Nasal polyps Nasal polyps pCMVSport
96
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
2.0
H0518 pBMC stimulatedpBMC stimulated , pCMVSport
w/ poly
IIC with oly 3.0
IIC
H0519 NTERA2, controlNTERA2, pCMVSport
Teratocarcinoma 3.0
cell line
H0520 NTERA2 + retinoicNTERA2, pSportl
acid,
14 days Teratocarcinoma
cell line
HOS21 Primary DendriticPrimary Dendritic pCMVSport
Cells,
lib 1 cells 3.0
H0522 Primary DendriticPrimary Dendritic pCMVSport
cells,frac 2 cells 3.0
H0529 Myoloid ProgenitorTF-1 Cell pCMVSport
Cell Line;
Line Myoloid progenitor 3.0
cell line
HOS30 Human Dermal Human Dermal pSportl
Endothelial Endothelial
Cells;
Cells,untreateduntreated
HOS37 H. Primary DendriticPrimary Dendritic pCMVSport
Cells,lib 3 cells 2.0
H0539 Pancreas Islet Pancreas Pancreas diseasepSportl
Cell Tumor Islet Cell
Tumour
H0540 Skin, burned Skin, 1e Skin S ortl
burned
H0542 T Cell helper Helper T pCMVSport
T cell
3.0
H0543 T cell helper Helper T pCMV
II cell Sport
3.0
H0544 Human endometrialHuman endometrial pCMVSport
stromal cells stromal cells 3.0
H0545 Human endometrialHuman endometrial pCMVSport
stromal cells-treatedstromal cells-treated 3.0
with
ro esterone with ro a
H054b Human endometrialHuman endometrial pCMVSport
stromal cells-treatedstromal cells-treated 3.0
with
estradiol with estra
H0547 NTERA2 teratocarcinomaNTERA2, pSportl
cell line+retinoicTeratocarcinoma
acid (14
da s) cell line
H0549 H. Epididiymus,Human Uni-ZAP
caput &
corpus Epididiymus, XR
caput
and co us
H0550 H. Epididiymus,Human Uni-ZAP
cauda
E ididi mus, XR
cauda
H0551 Human Thymus Human Thymus pCMVSport
Stromal
Cells Stromal Cells 3.0
H0553 Human Placenta Human Placenta pCMVSport
3.0
H0555 Rejected Kidney,Human RejectedKidney diseasepCMVSport
lib 4
Kidne 3.0
H0556 Activated T- T-Cells Blood Cell Uni-ZAP
Line
cell( 12h)/Thiouridine-re- XR
excision
H0559 HL-60, PMA 4H, HL-60 Cells,Blood Cell Uni-ZAP
re- PMA Line
excision stimulated XR
4H
H0560 KMH2 KMH2 pCMVSport
3.0
H0561 L428 L428 pCMVSport
3.0
H0565 HUman Fetal Human Fetal pCMVSport
Brain, Brain
normalized 100024F 2.0
H0574 Hepatocellular HepatocellularLiver diseaseLambda
Tumor; re-
97
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
excision Tumor ZAP II
H0575 Human Adult Human Adult Lung Uni-ZAP
Pulmonar ;re-excisionPulmonary XR
H0576 Resting T-Cell;T-Cells Blood Cell Lambda
re- Line
excision ZAP II
H0580 Dendritic cells,Pooled dendritic pCMVSport
pooled
cells 3.0
H0581 Human Bone Marrow,Human Bone Bone Marrow pCMVSport
.
treated Marrow 3.0
H0583 B Cell lymphomaB Cell LymphomaB Cell diseasepCMVSport
3.0
H0586 Healing groin healing groingroin diseasepCMVSport
wound, 6.5
hours post incisionwound, 6.5 . 3.0
hours
ost incision
- 2/
H0587 Healing groin Groin-2!19/97groin diseasepCMVSport
wound; 7.5
hours ost incision 3.0
H0589 CD34 positive CD34 PositiveCord Blood ZAP Express
cells (cord Cells
blood),re-ex
H0590 Human adult Human Adult Small Uni-ZAP
small Small Int.
intestine,re-excisionIntestine XR
H0591 Human T-cell T-Cell LymphomaT-Cell diseaseUni-ZAP
1 m homa;re-excision XR
H0592 Healing groin HGS wound diseasepCMVSport
wound - healing
zero hr post-incisionproject; 3.0
abdomen
(control)
H0594 Human Lung Cancer;re-Human Lung Lung diseaseLambda
Cancer
excision ZAP II
H0596 Human Colon Human Colon Colon Lambda
Cancer;re-
excision Cancer ZAP II
H0597 Human Colon; Human Colon Lambda
re-excision
ZAP II
H0598 Human Stomach;re-Human StomachStomach Uni-ZAP
excision XR
H0599 Human Adult Human Adult Heart Uni-ZAP
Heart;re- Heart
excision XR
H0600 Healing AbdomenAbdomen diseasepCMVSport
wound;70&90 3.0
min post
incision
H0606 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast
Cancer;re-excisionBreast Cancer XR
H0608 H. Leukocytes, H.Leukocytes. pCMVSport
control
1
H0610 H. Leukocytes, H.Leukocytes pCMVSport
normalized cot 1
5A
H0611 H. Leukocytes, H.Leukocytes pCMVSport
normalized cot 1
500 B
H0612 H.Leukocytes, H.Leukocytes pCMVSport
normalized
cot 50 B 1
H0615 Human Ovarian Ovarian CancerOvary diseaseUni-ZAP
Cancer
Reexcision XR
H0616 Human Testes, Human TestesTestis Uni-ZAP
Reexcision
XR
H0617 Human Primary Human PrimaryBreast diseaseUni-ZAP
Breast
Cancer ReexcisionBreast Cancer XR
H0618 Human Adult Human Adult Testis Uni-ZAP
Testes, Testis
Lar a Inserts, XR
Reexcision
H0619 Fetal Heart Human Fetal Heart Uni-ZAP
Heart
XR
H0620 Human Fetal Human Fetal Kidney Uni-ZAP
Kidney; Kidney
Reexcision XR
H0622 Human Pancreas Human PancreasPancreas diseaseUni-ZAP
Tumor;
Reexcision Tumor XR
98
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
H0623 Human UmbilicalHuman UmbilicalUmbilical Uni-ZAP
Vein;
Reexcision Vein Endothelialvein XR
Cells
H0624 12 Week Early Twelve Week Embryo Uni-ZAP
Stage Old
Human II; ReexcisionEarly Sta XR
a Human
H0625 Ku 812F Baso Ku 812F Baso S ortl
hils Line hils
H0629 Human Leukocyte,Human Normalized pCMVSport
control
#2 leukocyte 1
H0631 Saos2, DexamethosomeSaos2 Cell pSportl
Line;
Treated Dexamethosome
Treated
H0632 Hepatocellular HepatocellularLiver Lambda
Tumor;re-
excision Tumor ZAP II
H0633 Lung Carcinoma TNFalpha diseasepSportl
A549 activated
TNFalpha activatedA549--Lung
Carcinoma
H0634 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor, re-
excision Tumor XR
H0635 Human ActivatedActivated Blood Cell Uni-ZAP
T-Cells, T-Cells Line
re-excision XR
H0637 Dendritic CellsDentritic p5portl
From cells from
CD34 Cells CD34 cells
H0638 CD40 activated CD40 activated pSportl
monocyte
dendridic cellsmonocyte
dendridic
cells
H0639 Ficolled Human Ficolled Other
Stromal Human
Cells, SFu treatedStromal Cells,
SFu
treated
H0640 Ficolled Human Ficolled Other
Stromal Human
Cells; UntreatedStromal Cells,
Untreated
H0641 LPS activated LPS activated pSportl
derived
dendritic cellsmonocyte
derived
dendritic
cells
H0642 Hep G2 Cells, Hep G2 Cells Other
lambda
libr
H0643 He G2 Cells, He G2 Cells Other
PCR Libra
H0645 Fetal Heart, Human Fetal Heart Uni-ZAP
re-excision Heart
XR
H0646 Lung, Cancer Metastatic pSportl
(4005313
A3): Invasive squamous
Poorly cell lung
Differentiated carcinoma,
Lung poorly di
Adenocarcinoma,
H0647 Lung, Cancer Invasive diseasepSportl
(4005163 poorly
B7): Invasive, differentiated ,
Poorly Diff. lung
Adenocarcinoma,adenocarcinoma
Metastatic
H0648 Ovary, Cancer: Papillary diseasepSportl
(4004562 Cstic
B6) Papillary neoplasm
Serous of low
Cystic Neoplasm,malignant
Low potentia
Mali nant Pot
H0650 B-Cells B-Cells pCMVSport
3.0
H0651 Ovary, Normal: Normal Ovary pSportl
(9805C040R)
H0656 B-cells (unstimulated)B-cells pSportl
(unstimulated)
H0657 B-cells (stimulated)B-cells (stimulated) S ortl
H0658 Ovary, Cancer 9809C332- Ovary diseasepSportl
Poorly &
(9809C332): differentiateFallopian
Poorly
differentiated Tubes
adenocarcinoma
H0659 ~ Ovary, CancerGrade II Ovary ~ disease~ pSportl
Papillary
99
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
(15395A1F): Carcinoma, ' ,
Grade II Ovary
Pa ill Carcinoma
H0660 Ovary, Cancer: Poorly differentiated' diseasepSportl
(15799A1F) Poorlycarcinoma,
ovary
differentiated
carcinoma
H0661 Breast, Cancer:Breast cancer diseasepSportl
(4004943
A5)
H0662 Breast, Normal:Normal BreastBreast pSportl
-
(4005522B2) #4005522(B2)
H0663 Breast, Cancer:Breast CancerBreast diseasepSportl
(4005522 -
A2) #4005522(A2)
H0665 Stromal cells Stromal cells S ortl
3.88 3.88
H0667 Stromal cells(HBM3.18)Stromal cell(HBM pSportl
3.18)
H0669 Breast, Cancer:Breast CancerBreast pSportl
(4005385
A2) (4005385A2)
H0670 Ovary, Cancer(4004650Ovarian Cancer pSportl
-
A3): Well-Differentiated4004650A3
Micropapillary
Serous
Carcinoma
H0672 Ovary, Cancer: Ovarian Ovary p5portl
(4004576
A8) Cancer(4004576A8)
H0673 Human Prostate Human ProstateProstate Uni-ZAP
Cancer,
Sta a B2; re-excisionCancer, sta XR
a B2
H0674 Human Prostate Human ProstateProstate Uni-ZAP
Cancer,
Sta a C; re-excissionCancer, sta XR
a C
H0676 Colon, Cancer: Colon Cancer pCMVSport
(9808C064R)-total9808C064R 3.0
RNA
H0682 Serous Papillaryserous papillary ' pCMVSport
Adenocarcinoma adenocarcinoma 3.0
(9606G3045PA3B)
H0683 Ovarian Serous Serous papillary pCMVSport
Papillary
Adenocarcinoma adenocarcinoma, 3.0
sta a 3C
(9804601
H0684 Serous PapillaryOvarian Cancer-Ovaries pCMVSport
Adenocarcinoma 98106606 3.0
H0685 Adenocarcinoma Adenocarcinoma pCMVSport
of of
Ovary, Human Ovary, Human 3.0
Cell Line, Cell
# OVCAR-3 Line, # OVCAR-
H0686 Adenocarcinoma Adenocarcinoma pCMVSport
of of
Ovary, Human Ovary, Human 3.0
Cell Line Cell
Line, # SW-626
H0687 Human normal Human normalOvary pCMVSport
ov (#96106215) ova (#96106215) 3.0
H0688 Human Ovarian Human Ovarian pCMVSport
Cancer(#98076017)cancer(#98076017), 3.0
mRNA from
Maura
Ru
H0689 Ovarian Cancer Ovarian Cancer, pCMVSport
#98066019 3.0
H0690 Ovarian Cancer,Ovarian Cancer, pCMVSport
#
97026001 #97026001 3.0
H0691 Normal Ovary, normal ovary, pCMVSport
#97106208 #97106208 3.0
N0006 Human Fetal Human Fetal
Brain Brain
50001 Brain frontal Brain frontalBrain Lambda
cortex cortex
ZAP II
50002 Monocyte activatedMonocyte-activatedblood Cell Uni-ZAP
Line
XR
50003 Human OsteoclastomaOsteoclastomabone diseaseUni-ZAP
XR
S0004 Prostate Prostate Prostate Lambda
BPH
100
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
ZAP II
50007 Early Stage Human Fetal Uni-ZAP
Human Brain Brain
XR
S0010 Human Amygdala Amygdala Uni-ZAP
XR
50011 STROMAL - Osteoclastomabone diseaseUni-ZAP
OSTEOCLASTOMA XR
S0013 Prostate Prostate prostate Uni-ZAP
XR
S0015 Kidney medulla Kidney medullaKidney Uni-ZAP
XR
50026 Stromal cell stromal cellBone Cell Uni-ZAP
TF274 marrow Line
XR
S0027 Smooth muscle, Smooth musclePulmanaryCeII Uni-ZAP
serum Line
treated artery XR
50028 Smooth muscle,control,Smooth musclePulmanaryCell Uni-ZAP
Line
arte XR
50031 Spinal cord Spinal cord spinal Uni-ZAP
cord
XR
50032 Smooth muscle-ILbSmooth musclePulmanaryCell Uni-ZAP
Line
induced arte XR
S0036 Human SubstantiaHuman Substantia Uni-ZAP
Nigra
Nigra XR
S0037 Smooth muscle, Smooth musclePulmanaryCell Uni-ZAP
ILlb Line
induced arter XR
.
S0038 Human Whole Human Whole ZAP Express
Brain #2 - Brain
Oli o dT > l.SKb#2
50040 Adipocytes Human Adipocytes Uni-ZAP
from Osteoclastoma XR
50044.Prostate BPH prostate Prostate diseaseUni-ZAP
BPH
XR
S0045 Endothelial Endothelial endothelialCell Uni-ZAP
cells-control cell Line
cell-lun XR
50046 Endothelial-inducedEndothelial endothelialCell Uni-ZAP
cell Line
cell-lun XR
S0051 Human Human diseaseUni-ZAP
Hypothalmus,SchizophrenHypothalamus, XR
is Schizo hrenia
S0052 neutrophils human neutrophilsblood Cell Uni-ZAP
control Line
XR
S0053 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line
induced induced XR
S0114 Anergic T-cell Anergic T-cell Cell Uni-ZAP
Line
XR
SO1I6 Bone marrow Bone marrow Bone Uni-ZAP
marrow
XR
S0126 Osteoblasts Osteoblasts Knee Cell Uni-ZAP
Line
XR
S0132 Epithelial-TNFaAirway Epithelial Uni-ZAP
and INF
induced XR
S0134 Apoptotic T-cellapoptotic Cell Uni-ZAP
cells Line
XR
S0142 Macrophage-oxLDLmacrophage- blood Cell Uni-ZAP
Line
oxidized XR
LDL
treated
S0144 Macrophage (GM-CSFMacrophage Uni-ZAP
(GM-
treated) CSF treated) XR
50146 prostate-editedprostate Prostate Uni-ZAP
BPH
XR
S0150 LNCAP prostate LNCAP Cell ProstateCell Uni-ZAP
cell line Line Line
XR
50152 PC3 Prostate PC3 prostate ~ ~ Uni-ZAP
cell line cell
101
CA 02393912 2002-06-21
WO 01/55387 PCT/USO1/01310
line - XR
S0176 Prostate, normal,Prostate prostate Uni-ZAP
subtraction XR
I
50190 Prostate BPH,LibHuman Prostate pSportl
2,
subtracted BPH
S0192 Synovial FibroblastsSynovial p5portl
Fibroblasts
(control)
S0194 S novial h oxiaS novial S ortl
Fibroblasts
50198 TTM-pbfd PBLS, TTM PCRII
rece for
enriched
S0206 Smooth Muscle- Smooth musclePulmanaryCell pBluescript
HASTE Line
normalized artery
S0208 Messan ial cell,Messan ial S ortl
frac 1 cell
S0210 Messan ial cell,Messan ial S ortl
frac 2 cell
S0212 Bone Marrow Bone Marrow pSportl
Stromal
Cell, untreatedStromal
Cell,untreated
S0216 Neutrophils human neutrophilblood Cell Uni-ZAP
IL-1 and LPS Line
induced induced XR
S0218 Apoptotic T-cell,apoptotic Cell Uni-ZAP
re- cells Line
excision XR
S0220 H. hypothalamus,HypothalamusBrain ZAP Express
frac
A;re-excision
S0222 H. Frontal H. Brain, Brain diseaseUni-ZAP
Frontal
cortex,epileptic;re-Cortex, Epileptic XR
excision
S0228 PSMIX PBLS, TTM PCRII
rece for
enriched
50242 Synovial FibroblastsSynovial pSportl
Fibroblasts
(Ill/TNF), subt
S0250 Human OsteoblastsHuman OsteoblastsFemur diseasepCMVSport
II
2.0
50252 TTM-PIMIX PBLS, TTM PCRII
receptor
enriched
S0260 Spinal Cord, Spinal cord spinal Uni-ZAP
re-excision cord
XR
S0264 PPMIX PPMIX (HumanPituitary PCRII
Pituita )
S0268 PRMIX PRMIX (Humanprostate PCRII
Prostate)
S0270 PTMIX PTMIX (HumanThymus PCRII
Thymus)
S0274 PCMIX PCMIX (HumanBrain PCRII
Cerebellum)
S0276 Synovial hypoxia-RSFSynovial Synovial pSportl
fobroblasts
subtracted (rheumatoid)tissue
50278 H Macrophage Macrophage Uni-ZAP
(GM-CSF (GM-
treated), re-excisionCSF treated) XR
S0280 Human Adipose Human Adipose Uni-ZAP
Tissue,
re-excision Tissue XR
50282 Brain Frontal Brain frontalBrain Lambda
Cortex, re- cortex
excision ZAP II
S0294 Larynx tumor Larynx tumorLarynx,vocal diseasep5portl
cord
S0308 S leen/normal S leen normal S ortl
S0312 Human Human diseasepSportl
osteoarthritic;fractionosteoarthritic
II
cartila a
S0316 Human Normal Human Normal pSportl
Cartila e,FractionCartila a
I
S0318 Human Normal Human Normal pSportl
Cartilage
Fraction II Cartila a
102
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S0328 Palate carcinomaPalate carcinomaUvula diseaseS ortl
S0330 Palate normal Palate normalUvula ~ S ortl
50334 Human Normal Human Normal . pSportl
Cartilage Cartilage '
Fraction III
S0342 Adipocytes;re-excisionHuman Adipocytes Uni-ZAP
from Osteoclastoma XR
50344 Macrophage-oxLDL;macrophage- blood Cell Uni-ZAP
re- oxidized Line XR
excision LDL
treated
50346 Human Amygdala;re-Amygdala Uni-ZAP
excision XR
S0348 Cheek CarcinomaCheek Carcinoma diseasepSportl
S0354 Colon Normal Colon NormalColon S ortl
II
S0356 Colon CarcinomaColon CarcinomaColon diseaseS ortl
50358 Colon Normal Colon NormalColon S ortl
III
50360 Colon Tumor Colon Tumor Colon diseaseS ortl
II
S0364 Human Quadrice Quadrice S ortl
s s muscle
S0374 Normal colon Normal colon S ortl
S0376 Colon Tumor Colon Tumor diseasep5portl
S0378 Pancreas normalPancreas p5portl
PCA4 Normal
No PCA4 No
S0380 Pancreas Tumor Pancreas ' diseasepSportl
PCA4 Tu Tumor
PCA4 Tu
50382 La nx carcinomaLar nx carcinoma diseaseS ortl
IV
S0384 Ton ue carcinomaTon ue carcinoma diseaseS ortl
S0388 Human Human diseaseUni-ZAP
Hypothalamus,schizophreHypothalamus, XR
nia,re-excisionSchizo hrenia
50392 Salivary Gland Salivary p5portl
gland;
normal
S0404 Rectum normal Rectum, normal S ortl
S0406 Rectum tumour Rectum tumour S ortl
S0408 Colon, normal Colon, normal S ortl
S0410 Colon, tumour Colon, tumour S ortl
50412 Temporal cortex-Temporal diseaseOther
Alzheizmer; cortex,
subtracted alzheimer
S0414 Hippocampus, Hippocampus, Other
Alzheimer Alzheimer
Subtracted Subtracted
S0418 CHME Cell Line;treatedCHME Cell pCMVSport
5 Line; 3.0
hrs treated
S0420 CHME Cell CHME Cell p5portl
Line,untreated line,
untreatetd
50422 Mo7e Cell Line Mo7e Cell pCMVSport
GM-CSF Line 3.0
treated (lng/ml)GM-CSF treated
(1n /ml)
S0424 TF-1 Cell Line TF-1 Cell pSportl
GM-CSF Line
Treated GM-CSF Treated
S0426 Monocyte activated;Monocyte-activatedblood Cell Uni-ZAP
re- Line XR
excision
50428 Neutrophils human neutrophilsblood Cell Uni-ZAP
control; re- Line XR
excision
S0432 Sinus piniformisSinus piniformis p5portl
Tumour Tumour
50434 Stomach Normal Stomach Normal diseaseS ortl
50436 Stomach Tumour Stomach Tumour diseaseS ortl
S0438 Liver Normal Liver Normal pSportl
MetSNo MetSNo
S0440 Liver Tumour Liver Tumour pSportl
Met 5 Tu
S0442 Colon Normal Colon Normal S ortl
50444 Colon Tumor Colon Tumour diseaseS ortl
50458 ~ Thyroid NormalThyroid normal pSportl
(SDCA2
103
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No) ~ -
S0468 Ea.hy.926 cell Ea.hy.926 ~ S ortl
line cell line
S3012 Smooth Muscle Smooth musclePulmanaryCell pBluescript
Serum Line
Treated, Norm artery
S3014 Smooth muscle, Smooth musclePulmanaryCell pBluescript
serum Line
induced,re-exc artery
S6026 Frontal Lobe, Frontal LobeBrain ~ Uni-ZAP
Dementia
dementialAlzheimer' XR
's
56028 Human Manic Human Manic Brain diseaseUni-ZAP
Depression
Tissue de ression XR
tissue
T0002 Activated T-cellsActivated Blood Cell pBluescript
T-Cell, Line
PBL fraction SK-
T0003 Human Fetal Human Fetal pBluescript
Lung Lung
SK-
T0004 Human White Human White pBluescript
Fat Fat
SK-
T0006 Human Pineal Human Pinneal pBluescript
Gland
Gland SK-
T0010 Human Infant Human Infant Other
Brain Brain
T0039 HSA 172 Cells Human HSA172 pBluescript
cell
line SK-
T0040 HSC172 cells SA172 Cells pBluescript
SK-
T0041 Jurkat T-cell Jurkat T-cell pBluescript
G1 phase
SK-
T0042 Jurkat T-Cell, Jurkat T-Cell pBluescript
S phase Line
5K-
T0048 Human Aortic Human Aortic pBluescript
Endothelium Endothilium SK-
T0049 Aorta endothelialAorta endothelial pBluescript
cells +
TNF-a cells SK-
T0060 Human White Human White p$luescript
Adipose Fat
SK-
T0068 Normal Ovary, Normal Ovary, pBluescript
Premeno ausal Premeno ausal SK-
T0069 Human Uterus, Human Uterus, pBluescript
normal
normal SK-
T0071 Human $one MarrowHuman Bone p$luescript
Marrow SK-
T0082 Human Adult Human Adult p$luescript
Retina Retina
SK-
T0109 Human (HCC) p$luescript
cell line
liver (mouse) SK-
metastasis,
remake
L0021 Human adult
(K.Okubo)
L0024 Human brain
ARSanders
L0033 Human chromosome
13 14 cDNA
L0055 Human promyelocyte
L0062 Human whole
brain
L0065 Liver He G2
cell line.
L0105 Human aorta aorta
polyA+
(TFu'iwara)
L0142 Human placenta placenta
cDNA
(TFu'iwara)
L0143 Human placenta placenta
polyA+
(TFujiwara)
L0157 Human fetal brain
brain
(TFu'iwara)
L0163 Human heart heart
cDNA
(YNakamura)
104
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L0351 Infant brain, , BA',
Bento Soares M13-
derived
.
L0352 Normalized infant BA, M13-
brain,
Bento Soares derived
L0355 P, Human foetal Bluescript
Brain
Whole tissue
L0361 Stratagene ovary ovary Bluescript
(#937217) SK
L0362 Stratagene ovarian Bluescript
cancer
(#937219) SK-
L0363 NCI_CGAP_GC2 germ cell Bluescript
tumor
SK-
L0368 NCI_CGAP_SS1 synovial Bluescript
sarcoma
SK-
L0369 NCI_CGAP_AAI adrenal adenomaadrenal Bluescript
gland
SK-
L0371 NCI_CGAP_Br3 breast tumorbreast Bluescript
SK-
L0372 NCI_CGAP_Col2 colon tumor colon Bluescript
SK-
L0373 NCI_CGAP_Coll tumor colon Bluescript
SK-
L0375 NCI_CGAP_Kid6 kidney tumorkidney Bluescript
SK-
L0376 NCI_CGAP_Larl larynx larynx Bluescript
SK-
L0379 NCI_CGAP_Lym3 lymphoma lymph Bluescript
node
SK-
L0381 NCI_CGAP_HN4 squamous pharynx Bluescript
cell
carcinoma SK-
L0382 NCI_CGAP_Pr25 epithelium prostate Bluescript
(cell line)
SK-
L0383 NCI_CGAP_Pr24 invasive prostate Bluescript
tumor (cell
line) SK-
L0384 NCI_CGAP_Pr23 prostate prostate Bluescript
tumor
SK-
L0387 NCI_CGAP_GCBO germinal tonsil Bluescript
center B-
cells SK-
L0389 NCI_CGAP_HNS normal gingiva Bluescript
(cell
line from SK-
primary
keratinoc
t
L0406 b4HB3MA Cot14.5 Lafmid
A
L0416 b4HB3MA-Cot0.38-HAP- Lafmid
BA
B
L0419 b4HB3MA- Lafmid
BA
Cot109+103+85-Bio
L0420 b4HB3MA-Cot109+103- Lafmid
BA
Bio
L0427 b4HB3MA-FT20%-Biotin Lafmid
BA
L0438 normalized infanttotal brain brain lafmid
brain BA
cDNA
L0439 Soares infant whole Lafmid
brain 1NIB brain BA
L0441 2HB3MK Lafmid
BK
L0455 Human retina retina eye lambda
cDNA gtl0
randomly primed
sublibrar
L0462 WATM1 lambda
tll
L0471 Human fetal Lambda
heart,
Lambda ZAP Ex ZAP Ex
ress ress
L0483 Human pancreatic Lambda
islet
ZAPII
L0497 NCI_CGAP_HSC4 CD34+, CD38-bone marrow pAMPl
from
1~5
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normal bone
marrow
donor
L0499 NCI_CGAP HSC2 stem cell bone marrow ~ AMP1
34+/38+
L0500 NCT_CGAP_Brn20 oligodendrogliomabrain pAMP1
L0501 NCI CGAP Brn21 oli~odendro brain AMPl
lioma
LOS10 NCI_CGAP_Ov33 borderline ovary pAMPl
ovarian
carcinoma
L0513 NCI_CGAP_Ov37 early stage ovary pAMPl
papillary
serous carcinoma
L0517 NCI_CGAP Prl AMP10
L0518 NCI_CGAP_Pr2 AMP10
L0519 NCI CGAP Pr3 AMP10
L0520 NCI_CGAP_Alvl alveolar pAMPlO
rhabdomyosarcoma
L0521 NCT_CGAP_Ewl Ewin "s sarcoma AMP10
L0522 NCI CGAP_Kidl kidney AMP10
L0526 NCI_CGAP_Prl2 metastatic pAMPlO
prostate
bone lesion
L0527 NCI_CGAP_Ov2 ovary pAMPlO
L0529 NCI_CGAP_Pr6 rostate AMP10
L0530 NCI_CGAP_Pr8 rostate AMP10
L0532 NCI_CGAP Th th roid AMP10
1
L0535 NCI_CGAP_BrS infiltratingbreast pAMPlO
ductal
carcinoma
LOS42 NCI_CGAP_Prll normal prostaticprostate pAMPlO
a ithelial
cells
LOS43 NCI_CGAP_Pr9 normal prostaticprostate pAMPlO
a ithelial
cells
L0545 NCI_CGAP_Pr4.1 prostatic prostate pAMPlO
intraepithelial
neoplasia
- high
rade
L0546 NCI_CGAP_Prl8 stroma rostate AMP10
L0565 Normal Human Bone Hip pBluescript
Trabebular Bone
Cells
LOS81 Stratagene liver liver pBluescript
(#937224)
SK
L0586 HTCDL1 pBluescript
SK(-)
L0588 Stratagene endothelial pBluescript
cell
937223 SK-
L0589 Stratagene fetal pBluescript
retina
937202 SK-
L0590 5tratagene fibroblast pBluescript
(#937212) SK-
L0591 Stratagene HeLa pBluescript
cell s3
937216 SK-
L0592 Stratagene hNT pBluescript
neuron
(#937233) SK-
L0593 Stratagene pBluescript
neuroepithelium SK-
(#93723I)
L0594 Stratagene pBluescript
neuroepithelium SK-
NT2RAMI 937234
L0595 Stratagene NT2 neuroepithelialbrain pBluescript
neuronal cells
recursor 937230 SK-
L0596 Stratagene colon colon pBluescript
(#937204)
SK-
L0597 Stratagene corneal cornea pBluescript
stroma
(#937222) SK-
L0598 Morton Fetal cochlea ear pBluescript
Cochlea
106
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SK-
L0599 Stratagene lung lung pBluescript
(#937210)
SK-
L0600 Weizmann Olfactoryolfactory nose pBluescript
epithelium
E ithelium SK-
L0601 Stratagene pancreas pancreas pBluescript
(#937208)
SK-
L0602 Pancreatic Isletpancreatic pancreas pBluescript
islet
SK-
L0603 Stratagene placenta placenta pBluescript
(#937225)
SK-
L0604 Stratagene musclemuscle skeletal pBluescript
937209
muscle SK-
L0605 Stratagene fetalfetal spleenspleen pBluescript
spleen
(#937205)
SK-
L0606 NCI_CGAP_LymS follicular lymph pBluescript
lymphoma node
SK-
L0607 NCI_CGAP_Lym6 mantle cell lymph pBluescript
node
1 m homa SK-
L0608 Stratagene lunglung carcinomalung NCI-H69 pBluescript
carcinoma
937218 SK-
L0623 HM3 pectoral pcDNAII
muscle
(after mastectomy) (Invitrogen)
L0625 NCI_CGAP_AR1 bulk alveolar pCMV-
tumor
SPORT2
L0626 NCI_CGAP_GC1 bulk germ pCMV-
cell
seminoma SPORT2
L0627 NCI_CGAP_Col bulk tumor colon pCMV-
SPORT2
L0629 NCI_CGAP_Mel3 metastatic bowel pCMV-
(skin
melanoma rima ) SPORT4
to bowel
L0635 NCI_CGAP_PNS1 dorsal root peripheral pCMV-
ganglion
nervous SPORT4
s stem
L0637 NCI_CGAP_Brn53 three pooledbrain pCMV-
menin iomas SPORT6
L0638 NCI_CGAP_Brn35 tumor, 5 brain pCMV-
pooled (see
descri lion) SPORT6
L0640 NCI_CGAP_Brl8 four pooled breast pCMV-
high-
grade tumors, SPORT6
includin
two rima
L0641 NCI_CGAP_Col7 juvenile colon pCMV-
granulosa
tumor SPORT6
L0643 NCI_CGAP_Col9 moderately colon pCMV-
differentiated SPORT6
adenocarcinoma
L0644 NCI_CGAP_Co20 moderately colon pCMV-
differentiated SPORT6
adenocarcinoma
L0646 NCI_CGAP_Col4 moderately- colon pCMV-
differentiated SPORT6
adenocarcinoma
L0647 NCI_CGAP_Sar4 five pooled connective pCMV-
sarcomas, tissue SPORT6
including
m xoid 1i
osarcoma
L0648 NCI_CGAP_Eso2 squamous esophagus pCMV-
cell
carcinoma SPORT6
L0649 NCI_CGAP_GU1 2 pooled genitourinary pCMV-
high-grade
transitionaltract SPORT6
cell
tumors
L0650 NCI_CGAP_Kidl3 2 pooled kidney pCMV-
Wilms"
tumors, one SPORT6
primary
1~7
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and one metast
L0651 NCI_CGAP_Kid8 renal cell kidney pCMV-
tumor
SPORT6
L0653 NCI_CGAP_Lu28 two pooled lung pCMV-
squamous SPORT6
cell
carcinomas
L0655 NCI_CGAP_Lyml2 lymphoma, lymph pCMV-
node
follicular SPORT6
mixed
small and
lar a cell
L0657 NCI_CGAP_Ov23 tumor, S ovary pCMV-
pooled (see
descri tion) SPORT6
L0659 NCI_CGAP_Panl adenocarcinomapancreas pCMV-
SPORT6
L0661 NCI_CGAP_Me115 malignant skin pCMV-
melanoma, SPORT6
metastatic
to lymph
node
L0662 NCI_CGAP_Gas4 poorly differentiatedstomach pCMV-
adenocarcinoma SPORT6
with si net
r ,
L0663 NCI_CGAP_Ut2 moderately- uterus pCMV-
differentiated SPORT6
endometrial
adenocarcino
L0664 NCI_CGAP_Ut3 poorly-differentiateduterus pCMV-
endometrial SPORT6
adenocarcinoma,
L0665 NCI_CGAP_Ut4 serous papillaryuterus pCMV-
carcinoma, SPORT6
high
grade, 2
pooled t
L0666 NCI_CGAP_Utl well-differentiateduterus pCMV-
endometrial SPORT6
adenocarcinoma,
7
L0667 NCI_CGAP_CMLl myeloid cells,whole pCMV-
18 blood
pooled CML SPORT6
cases,
BCR/ABL rearra
L0717 Gessler Wilms SPORT1
tumor
L0731 Soares_pregnant uterus pT7T3-Pac
uterus,
NbHPU
L0740 Soares melanocytemelanocyte pTTT3D
2NbHM (Pharmacia)
with
a
modified
of linker
L0741 Soares adult brain, pT7T3D
brain
N2b4HB55Y (Pharmacia)
with
a
modified
of linker
L0742 Soares adult brain pT7T3D
brain
N2b5HB55Y (Pharmacia)
with
a
modified
of linker
L0743 Soares breast breast pT7T3D
2NbHBst
(Pharmacia)
with
a
modified
of linker
L0744 Soares breast breast pT7T3D
3NbHBst
(Pharmacia)
with
a
modified
1~g
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olylinker
L0745 Soares retina retina eye pTTT3D
N2b4HR
(Pharmacia)
with
a
modified
01 linker
L0746 Soares retina retina eye pTTT3D
N2b5HR
(Pharmacia)
with
a
modified
olylinker
L0747 Soares fetal heart pTTT3D
heart NbHH
19W (Pharmacia)
with
a
modified
of linker
L0748 Soares fetal Liver pT7T3D
liver spleen and
1NFLS Spleen (Pharmacia)
with
a
modified
of linker
L0749 Soares_fatal Liver pT7T3D
liver_spleen and
1NFLS_S 1 Spleen (Pharmacia)
with
a
modified
of linker
L0750 Soares fetal_lung_NbHLl lung pTTT3D
9W (Pharmacia)
with
a
modified
of linker
L0751 Soares ovary ovarian tumorovary pT7T3D
tumor
NbHOT (Pharmacia)
with
a
modified
of linker
L0752 Soares_parathyroidparathyroid parathyroid pT7T3D
tumor tumor
NbHPA gland (Pharmacia)
with
a
modified
polylinker
L0754 Soares placenta placenta pTTT3D
Nb2HP
(Pharmacia)
with
a
modified
of linker
L0755 Soares_placenta_8to9wee placenta pTTT3D
ks_2NbHP8to9W (Pharmacia)
with
a
modified
of linker
L0756 Soares_multiple_sclerosismultiple pTTT3D
sclerosis
2NbHMSP lesions (Pharmacia)
with
a
modified
polylinker
V_TYPE
L0757 Soares senescentsenescent pTTT3D
fibrobla fibroblast
sts_NbHSF (Pharmacia)
with
a
modified
polylinker
V TYPE
109
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L0758 Snares testis_NHT pTTT3D-Pac
(Pharmacia)
with
a
modified
olylinker
L0759 Soares_total pTTT3D-Pac
fetus Nb2H
F8_9w (Pharmacia)
with
a
modified
of linker
L0760 Barstead aorta aorta pTTT3D-Pac
HPLRB3
(Pharmacia)
with
a
modified
of linker
L0761 NCI_CGAP_CLLl B-cell, chronic pTTT3D-Pac
lymphotic (Pharmacia)
leukemia
with
a
modified
polylinker
L0762 NCI_CGAP_Brl.l breast pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0763 NCI_CGAP_Br2 breast pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0764 NCI_CGAP_Co3 colon pTTT3D-Pac
(Pharmacia)
with
a
modified
polylinker
L0766 NCI_CGAP_GCB1 germinal pTTT3D-Pac
centerB
cell (Pharmacia)
with
a
modified
of linker
L0767 NCI_CGAP_GC3 pooled germ pT7T3D-Pac
cell
tumors (Pharmacia)
with
a
modified
of linker
L0768 NCI_CGAP_GC4 pooled germ pTTT3D-Pac
cell
tumors (Pharmacia)
with
a
modified
of linker
L0769 NCI_CGAP_Brn25 anaplastic brain pTTT3D-Pac
oligodendroglioma (Pharmacia)
with
a
modified
of linker
L0770 NCI_CGAP_Brn23 glioblastomabrain pTTT3D-Pac
(pooled) (Pharmacia)
with
a
modified
of linker
L0771 NCI_CGAP_Co8 adenocarcinomacolon pT7T3D-Pac
(Pharmacia)
with
a
modified
110
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olylinker
L0772 NCI_CGAP_ColO colon tumor colon pTTT3D-Pac
RER+
(Pharmacia)
with
a
modified
olylinker
L0773 NCI_CGAP_Co9 colon tumor colon pTTT3D-Pac
RER+
(Pharmacia)
with
a
modified
of linker
L0774 NCI_CGAP_Kid3 kidney pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0775 NCI_CGAP_KidS 2 pooled kidney pTTT3D-Pac
tumors
(clear cell (Pharmacia)
type)
with
a
modified
of linker
L0776 NCI_CGAP Lu5 carcinoid lung pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0777 Soares_NhHMPu Pooled humanmixed pTTT3D-Pac
S1 (see
melanocyte, below) (Pharmacia)
fetal
heart, and with
pregnant a
modified
of linker
L0778 Barstead pancreas pancreas pTTT3D-Pac
HPLRB 1 (Pharmacia)
with
a
modified
_ of linker
L0779 Soares_NF'L_T_GBC_S1 pooled pTTT3D-Pac
(Pharmacia)
with
a
modified
polylinker
L0782 NCI_CGAP_Pr21 normal prostateprostate pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0783 NCI_CGAP Pr22 normal prostateprostate pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0785 Barstead spleen spleen pTTT3D-Pac
HPLRB2
(Pharmacia)
with
a
modified
polylinker
L0789 NCI_CGAP_Sub3 pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0790 NCI_CGAP_Sub4 pTTT3D-Pac
(Pharmacia)
111
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with
a
modified
olylinker
L0794 NCI_CGAP_GC6 pooled germ pTTT3D-Pac
cell
tumors (Pharmacia)
with
a
modified
of linker
L0800 NCI_CGAP_Col6 colon tumor,colon pTTT3D-Pac
RER+
(Pharmacia)
with
a
modified
of linker
L0803 NCI_CGAP_Kidll kidney pTTT3D-Pac
(Pharmacia)
with
a
modified
of linker
L0804 NCI CGAP_Kidl2 2 pooled kidney pTTT3D-Pac
tumors
(clear cell (Pharmacia)
type)
with
a
modified
of linker
L0806 NCI_CGAP_LuI9 squamous lung pTTT3D-Pac
cell
carcinoma, (Pharmacia)
poorly
differentiated with
(4 a
modified
of linker
L0807 NCI_CGAP_Ovl8 fibrotheoma ovary pTTT3D-Pac
(Pharmacia)
with
a
modified
olylinker
L0809 NCI_CGAP_Pr28 prostate pTTT3D-Pac
(Pharmacia)
with
a
modified
polylinker
L2251 Human fetal Fetal lung
lung
112
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TAELE 5
OMIM Description
Reference
107280 Cerebrovascular disease, occlusive
107280 Al ha-1-antich mot sin deficienc
107400 Em h sema
107400 Em h sema-cirrhosis
122500 Transcortin deficienc
186960 Leukemia/1 m homa, T-cell
245200 Krabbe disease
601841 Protein C inhibitor deficiency
Polyhucleotide arzd Poly~ieptide T~a~iants
[109] The present invention is directed to variants of the polynucleotide
sequence
disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide
sequences
encoding the polypeptide of SEQ TD NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A,
nucleotide
sequences encoding the polypeptide as defined in column 7 of Table 1A, the
nucleotide
sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences
encoding the
polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9
of Table 2,
the nucleotide sequence as defined in column 6 of Table 1B, nucleotide
sequences encoding
the polypeptide encoded by the nucleotide sequence as defined in column 6 of
Table 1B, the
cDNA sequence contained in Clone ID NO:Z, and/or nucleotide sequences encoding
the
polypeptide encoded by the cDNA sequence contained in Clone ID NO:Z.
[110] The present invention also encompasses variants of the polypeptide
sequence
disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 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.
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[111] "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.
[112] 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 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; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in Clone ID. NO:Z
which
encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the
cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a
polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ID
NO:Z,
which encodes an antigenic fragment of a polypeptide; (f) a nucleotide
sequence encoding
a polypeptide comprising 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; (g) a
nucleotide
sequence encoding a mature 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; (h) a nucleotide
sequence encoding a biologically active fragment of a 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; (i) a nucleotide sequence encoding an antigenic
fragment of a
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 (j) a nucleotide
sequence complementary to any of the nucleotide sequences in (a), (b), (c),
(d), (e), (f), (g),
(h), or (i) above.
[113] 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), (i), or (j) 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
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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 polype~tide
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
7 of
Table 1A or the complementary strand thereto, nucleotide sequences encoding
the
polypeptide as defined in column 7 of Table 1A or the complementary strand
thereto,
and/or polynucleotide fragments of any of these nucleic acid molecules (e.g.,
those
fragments described herein). Polynucleotides which hybridize to the complement
of these
nucleic acid molecules under stringent hybridization conditions or
alternatively, under
lower stringency conditions, are also encompassed by the invention, as are
polypeptides
encoded by these polynucleotides and nucleic acids.
[114] 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.
[115] 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 form of a polypeptide having the amino acid sequence of
SEQ ID
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NO:Y or the amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the
amino
acid sequence of a biologically active fragment of a polypeptide having the
complete amino
acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by
the
cDNA in Clone ID NO:Z; and (d) the amino acid sequence of an antigenic
fragment of a
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.
[116] The present invention is also directed to proteins which comprise, or
alternatively
consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%,
99% or 100%, identical to, for example, any of the amino acid sequences in
(a), (b), (c), or
(d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid
sequence
encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the
polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in
columns 8
and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino acid
sequence as
defined in column 7 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.
[117] By a nucleic acid having a nucleotide sequence at least, for examples
95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended that the
nucleotide sequence of the nucleic acid is identical to the reference sequence
except that the
nucleotide sequence may include up to five point mutations per each 100
nucleotides of the
reference nucleotide sequence encoding the polypeptide. In other words, to
obtain a nucleic
acid having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence,
up to 5% of the nucleotides in the reference sequence may be deleted or
substituted with
another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the
reference sequence may be inserted into the reference sequence. The query
sequence may
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be an entire sequence referred to in Table 1A or 2 as the ORF (open reading
frame), or any
fragment specified as described herein.
[118] 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 percent identity are:
Matrix=Unitary,
k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group
Length=0,
Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the
length of
the subject nucleotide sequence, whichever is shorter.
[119] 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.
(120] 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
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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
manually
corrected for. No other manual corrections are to be made for the purposes of
the present
invention.
[121] 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.
[122] 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., the amino acid sequence identified
in column 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
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the amino acid sequence of the polypeptide encoded by cDNA contained in Clone
ID
NO:Z, or a fragment thereof, can be determined conventionally using known
computer
programs. A preferred method for determining the best overall match between a
query
sequence (a sequence of the present invention) and a subject sequence, also
referred to as a
global sequence alignment, can be determined using the FASTDB computer program
based
on the algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a
sequence
alignment the query and subject sequences are either both nucleotide sequences
or both
amino acid sequences. The result of said global sequence alignment is
expressed as percent
identity. Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM
0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group
Length=0,
Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size
Penalty=0.05,
Window Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
[123] 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-terminal
truncations
of the subject sequence when calculating global percent identity. For subject
sequences
truncated at the N- and C-termini, relative to the query sequence, the percent
identity is
corrected by calculating the number of residues of the query sequence that are
N- and C-
terminal of the subject sequence, which are not matched/aligned with a
corresponding
subject residue, as a percent of the total bases of the query sequence.
Whether a residue is
matched/aligned is determined by results of the FASTDB sequence alignment.
This
percentage is then subtracted from the percent identity, calculated by the
above FASTDB
program using the specified parameters, to arrive at a anal 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. .
[124] 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
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represent 10% of the sequence (number of residues at the N- and C- termini not
matched/total number of residues in the query sequence) so 10% is subtracted
from the
percent identity score calculated by the FASTDB program. If the remaining 90
residues
were perfectly matched the final percent identity would be 90%. In another
example, a 90
residue subject sequence is compared with a 100 residue query sequence. This
time the
deletions are internal deletions so there are no residues at the N- or C-
termini of the subject
sequence which are not matched/aligned with the query. In this case the
percent identity
calculated by FASTDB is not manually corrected. Once again, only residue
positions
outside the N- and C-terminal ends of the subject sequence, as displayed in
the FASTDB
alignment, which are not matched/aligned with the query sequnce are manually
corrected
for. No other manual corrections are to made for the purposes of the present
invention.
[125] 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).
[126] Naturally occurring variants are called "allelic variants," and refer to
one of
several alternate forms of a gene occupying a given locus on a chromosome of
an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New 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.
[127] 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 polypeptide of the present invention without
substantial loss
of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-
2988 (1993))
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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).)
[128] 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-1 a 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.
[129] 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.
[130] 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.
[131] 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
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the nucleic acid molecule, for instance, as a hybridization probe or a
polymerase chain
reaction (PCR) primer. Uses of the nucleic acid molecules of the present
invention that do
not encode a polypeptide having functional activity include, inter alia, (1)
isolating a gene
or allelic or splice variants thereof in a cDNA library; (2) in situ
hybridization (e.g.,
"FISH") to metaphase 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 or diseased tissues); and (4) in
situ hybridization
(e.g., histochemistry) for detecting mRNA expression in specific tissues
(e.g., normal or
diseased tissues).
[132] 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.
[133] The functional activity of the polypeptides, and fragments, variants and
derivatives of the invention, can be assayed by various methods.
[134] For example, in one embodiment where one is assaying for the ability to
bind or
compete with a full-length polypeptide of the present invention for binding to
an anti-
polypetide antibody, various immunoassays known in the art can be used,
including but not
limited to, competitive and non-competitive assay systems using techniques
such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope labels, for example), western blots, precipitation reactions,
agglutination
assays (e.g., gel agglutination assays, hemagglutination assays), complement
fixation
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assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis
assays,
etc. In one embodiment, antibody binding is detected by detecting a Iabel 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.
[135] 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.
[136] 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 i~c vitro or i~c vivo). Other methods will be known to the
skilled artisan and
are within the scope of the invention.
[137] 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, the
nucleic
acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence
disclosed
in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or
fragments
thereof, will encode polypeptides "having functional activity." In fact, since
degenerate
variants of any of these nucleotide sequences all encode the same polypeptide,
in many
instances, this will be clear to the skilled artisan even without performing
the above
described comparison assay. It will be further recognized in the art that, for
such nucleic
acid molecules that are not degenerate variants, a reasonable number will also
encode a
polypeptide having functional activity. This is because the skilled artisan is
fully aware of
amino acid substitutions that are either less likely or not likely to
significantly effect protein
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function (e.g., replacing one aliphatic amino acid with a second aliphatic
amino acid), as
further described below.
[138] 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.
[139] 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.
[140] 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 and
Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then
be tested
for biological activity.
[141] As the authors state, these two strategies have revealed that proteins
are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which amino
acid changes-are likely to be permissive at certain amino acid positions in
the protein. For
example, most buried (within the tertiary structure of the protein) amino acid
residues
require nonpolar side chains, whereas few features of surface side chains are
generally
conserved. Moreover, tolerated conservative amino acid substitutions involve
replacement
of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement
of the
hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu;
replacement of the amide residues Asn and Gln, replacement of the basic
residues Lys, Arg,
and His; replacement of the aromatic residues Phe, Tyr, and Trp, and
replacement of the
small-sized amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative
amino acid
substitution, variants of the present invention include (i) substitutions with
one or more of
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the non-conserved amino acid residues, where the substituted amino acid
residues may or
may not be one encoded by the genetic code, or (ii) substitutions with one or
more of the
amino acid residues having a substituent group, or (iii) fusion of the mature
polypeptide
with another compound, such as a compound to increase the stability and/or
solubility of
the polypeptide (for example, polyethylene glycol), (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 thereof, or leader or
secretory
sequence, or a sequence facilitating purification, or (v) fusion of the
polypeptide with
another compound, such as albumin (including but not limited to recombinant
albumin (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)).
Such variant polypeptides are deemed to be within the scope of those skilled
in the art from
the teachings.herein.
[142] For example, polypeptide variants containing amino acid substitutions of
charged
amino acids with other charged or neutral amino acids may produce proteins
with improved
characteristics, such as less aggregation. Aggregation of pharmaceutical
formulations both
reduces activity and increases clearance due to the aggregate's immunogenic
activity. See
Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al.,
Diabetes 36: 838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377
(1993).
[143] 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
columnns 8 and 9 of Table 2, an amino acid sequence encoded by the complement
of SEQ
ID NO:X, and/or an amino acid sequence encoded by cDNA contained in Clone ID
NO:Z
which contains, in order 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.
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[144] 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 and/or 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 F~agmehts
[145] 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
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
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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.
[146] 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.
[147] 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, 601-650, 651-700, 701-750, 751-800, 801-850, 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-
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,
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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.
[148] 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, 601-650, 651-700, 701-750, 751-800, 801-850, 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,
2'101-
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-
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
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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 po~ynucleotides 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.
[149] 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
further 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
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.
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[150] 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.
[151] In additional specific embodiments, polynucleotides of the invention
comprise, or ,
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in column 6 of Table 1B which correspond
to the
same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence
of SEQ
ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention.
[152] 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.
[153] 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 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
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and variants of the above-described polynucleotides, nucleic acids, and
polypeptides are
also encompassed by the invention.
[154] 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.
[155] 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
polyriucleotides 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.
[l56] 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
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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.
[157] 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 columnns 8 and 9
of Table
2, a portion of an amino acid sequence encoded by the polynucleotide sequence
of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the
polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid
sequence
encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide)
fragments may
be "free-standing," or comprised within a larger polypeptide of which the
fragment forms a
part or region, most preferably as a single continuous region. Representative
examples of
polypeptide fragments of the invention, include, for example, fragments
comprising, or
alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-
100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260,
261-280,
281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-
460, 461-
480, 481-500; 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640,
641-660,
661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-
840, 841-
860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-
1020, 1021-
1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-
1180,
1181-1200, 1201-1220, I22I-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 cDNA and SEQ ID NO: Y. 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,
101-120,
121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-
300, 301-
320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480,
481-500,
501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-
680, 681-
700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860,
861-880,
881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040,
1041-
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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.
[158] Even if deletion of one or more amino acids from the N-terminus of a
protein
results in modification of loss of one or more biological functions of the
protein, other
functional activities (e.g., biological activities, ability to multimerize,
ability to bind a
ligand) may still be retained. For example, the ability of shortened muteins
to induce and/or
bind to antibodies which recognize the complete or mature forms of the
polypeptides
generally will be retained when less than the majority of the residues of the
complete or
mature polypeptide are removed from the N-terminus. Whether a particular
polypeptide
lacking N-terminal residues of a complete polypeptide retains such immunologic
activities
can readily be determined by routine methods described herein and otherwise
known in the
art. It is not unlikely that a mutein with a large number of deleted N-
terminal amino acid
residues may retain some biological or immunogenic activities. In fact,
peptides composed
of as few as six amino acid residues may often evoke an immune response.
[159] 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.
[160] 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
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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.
[161] The present invention further provides polypeptides having one or more
residues
from the carboxy terminus of the amino acid sequence of a polypeptide
disclosed herein
(e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the
polynucleotide
sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ
ID
NO:X as defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by
the cDNA
contained in Clone ID NO:Z). In particular, C-terminal deletions may be
described by the
general formula 1-n, where n is any whole integer ranging from 6 to q-1, and
where n
corresponds to the position of amino acid residue in a polypeptide of the
invention.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
[162] 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.
[163] 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,
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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.
[164] The present application is also directed to proteins containing
polypeptides at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide
sequence set
forth herein. In preferred embodiments, the application is directed to
proteins containing
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
polypeptides having the amino acid sequence of the specific N- and C-terminal
deletions.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
[165] 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 columnns 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/).
[166] 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
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polynucleotides of the invention in this regard are those that encode
polypeptides
comprising regions that combine several structural features, such as several
(e.g., 1, 2, 3 or
4) of the features set out above.
[167] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic
regions, Emini
surface-forming regions, and Jameson-Wolf regions of high antigenic index
(i.e., containing
four or more contiguous amino acids having an antigenic index of greater than
or equal to
1.5, as identified using the default parameters of the Jameson-Wolf program)
can routinely
be used to determine polypeptide regions that exhibit a high degree of
potential for
antigenicity. Regions of high antigenicity are determined from data by DNASTAR
analysis
by choosing values which represent regions of the polypeptide which are likely
to be
exposed on the surface of the polypeptide in an environment in which antigen
recognition
may occur in the process of initiation of an immune response.
[168] 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.
[169] 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.
[170] 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.
[171] 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
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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 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.
[172] 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.
[173] 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. 4,631,21 l.)
[174] 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
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least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at
least 25, at least 30, at
Ieast 40, at least 50, and, most preferably, between about 15 to about 30
amino acids.
Preferred polypeptides comprising immunogenic or antigenic epitopes are at
least 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino
acid residues in
length. Additional non-exclusive preferred antigenic epitopes include the
antigenic
epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are
useful, for
example, to raise antibodies, including monoclonal antibodies, that
specifically bind the
epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed
herein, as well
as any combination of two, tlmee, four, five or more of these antigenic
epitopes. Antigenic
epitopes can be used as the target molecules in immunoassays. (See, for
instance, Wilson
et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)):
[175] 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 7 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 7 of Table 1A, but it
may
contain additional flanking residues on either the amino or carboxyl termini
of the recited
portion. Such additional flanking sequences are preferably sequences naturally
found
adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The
flanking
sequence may, however, be sequences from a heterolgous polypeptide, such as
from
another protein described herein or from a heterologous polypeptide not
described herein.
In particular 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 7 of Table
1A.
[176] 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
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immunogenic epitopes may be presented for eliciting an antibody response
together with a
carrier protein, 'such as an albumin, to an animal system (such as rabbit or
mouse), or, if the
polypeptide is of sufficient length (at least about 25 amino acids), the
polypeptide may be
presented without a carrier. However, immunogenic epitopes comprising as few
as 8 to 10
amino acids have been shown to be sufficient to raise antibodies capable of
binding to, at
the very least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
[177] Epitope-bearing polypeptides of the present invention may be used to
induce
antibodies according to methods well known in the art including, but not
limited to, ih vivo
immunization, in vitro immunization, and phage display methods. See, e.g.,
Sutcliffe et al.,
supf°a; Wilson et al., supra, and Bittle et al., J. Gen. Virol.,
66:2347-2354 (1985). If ih vivo
immunization is used, animals may be immunized with free peptide; however,
anti-peptide
antibody titer may be boosted by coupling the peptide to a macromolecular
carrier, such as
keyhole limpet hemacyanin (KLH) 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.
[178] As one of skill in the art will appreciate, and as discussed above, the
pblypeptides
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
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(including fragments or variants thereof) may be fused with albumin (including
but not
limited to recombinant human serum albumin or fragments or variants thereof
(see, e.g.,
U.S. Patent No. 5,876,969, issued March 2, .1999, EP Patent 0 413 622, and
U.S. Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their
entirety)). In a
preferred embodiment, polypeptides and/or antibodies of the present invention
(including
fragments or variants thereof) are fused with the mature form of human serum
albumin (i.e.,
amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0
322 094) which is herein incorporated by reference in its entirety. In another
preferred
embodiment, polypeptides and/or antibodies of the present invention (including
fragments
or variants thereof) are fused with polypeptide fragments comprising, or
alternatively
consisting of, amino acid residues 1-z of human serum albumin, where z is an
integer from
369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by
reference in its
entirety. Polypeptides and/or antibodies of the present invention (including
fragments or
variants thereof) may be fused to either the N- or C-terminal end of the
heterologous protein
(e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide).
Polynucleotides encoding fusion proteins of the invention are also encompassed
by the
invention.
[179] Such fusion proteins as those described above may facilitate
purification and may
increase half life iya 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-
tenninal 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
[180] 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.
[181] 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.
[182] 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 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.
[183] Moreover, fusion proteins may also be engineered to improve
characteristics of
the polypeptide of the present invention. For instance, a region of additional
amino acids,
particularly charged amino acids, may be added to the N-terminus of the
polypeptide to
improve stability and persistence during purification from the host cell or
subsequent
handling and storage. Also, peptide moieties may be added to the polypeptide
to facilitate
purification: Such regions may be removed prior to final preparation of the
polypeptide.
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The addition of peptide moieties to facilitate handling of polypeptides are
familiar and
routine techniques in the art.
[184] 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 2045869) discloses fusion proteins comprising
various portions
of constant region of immunoglobulin molecules together with another human
protein or
part thereof. In many cases, the Fc part in a fusion protein is beneficial in
therapy and
diagnosis, and thus can result in, for example, improved pharmacokinetic
properties (EP-A
0232 262). Alternatively, deleting the Fc part after the fusion protein has
been expressed,
detected, and purified, would be desired. For example, the Fc portion may
hinder therapy
and diagnosis if the fusion protein is used as an antigen for immunizations.
In drug
discovery, for example, human proteins, such as hIL-5, have been fused with Fc
portions
for the purpose of high-throughput screening assays to identify antagonists of
hIL-5. See,
D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et
al., J. Biol.
Chem. 270:9459-9471 (1995).
[185] Moreover, the polypeptides of the present invention can be fused to
marker
sequences, such as a polypeptide which facilitates purification of the fused
polypeptide. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
CA,
91311), among others, many of which are commercially available. As described
in Gentz et
al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for
convenient purification of the fusion protein. Another peptide tag useful for
purification,
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the "HA" tag, corresponds to an epitope derived from the influenza
hemagglutinin protein
(Wilson et al., Cell 37:767 (1984)).
[186] Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif shuffling, exon-shuffling, andlor codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to
modulate the activities of polypeptides of the invention, such methods can be
used to
generate polypeptides with altered activity, as well as agonists and
antagonists of the
polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721; 5,834,252;
and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama,
Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-
76 (1999);
and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these
patents and
publications are hereby incorporated by reference in its entirety). In one
embodiment,
alteration of polynucleotides corresponding to SEQ ID NO:X and the
polypeptides encoded
by these polynucleotides may be achieved by DNA shuffling. DNA shuffling
involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to
generate variation in the polynucleotide sequence. In another embodiment,
polynucleotides
of the invention, or the encoded polypeptides, may be altered by being
subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior to
recombination. In another 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:
[187] Thus, any of these above fusions can be engineered using the
polynucleotides or
the polypeptides of the present invention.
Recombinant and Synthetic Production of Polxpeptides of the Invention
[188] 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.
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[189] 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.
[190] The polynucleotide insert should be operatively linked to an appropriate
promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and
tac
promoters, the SV40 early and late promoters and promoters of retroviral LTRs,
to name a
few. Other suitable promoters will be known to the slcilled 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.
[191] As indicated, the expression vectors will preferably include at least
one selectable
marker. Such markers include dihydrofolate reductase, 6418, glutamine
synthase, or
neomycin resistance 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,
Streptomyces and Salinonella typhimurium 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 S~ cells; animal cells such as CHO, COS,
293, and
Bowes melanoma cells; and plant cells. Appropriate culture mediums and
conditions for
the above-described host cells are known in the art.
[192] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A,
pNHl6a,
pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available
from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred
expression vectors for use in yeast systems include, but are not limited to
pYES2, pYDl,
pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-
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Sl, pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Carlbad, CA).
Other
suitable vectors will be readily apparent to the skilled artisan.
[193] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoximine or
methotrexate,
respectively. An advantage of glutamine synthase based vectors are the
availabilty of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase
negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing
cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional
inhibitor to
prevent the functioning of the endogenous gene. A glutamine synthase
expression system
and components thereof are detailed in PCT publications: W087/04462;
W086/05807;
VV089/01036; 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 murine myeloma cells is
described
in Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson
Biotechrzol.
P~og. 11:1 (1995) which are herein incorporated by reference.
[194] The present invention also relates to host cells containing the above-
described
vector constructs described herein, and additionally encompasses host cells
containing
nucleotide sequences of the invention that are operably associated with one or
more
heterologous control regions (e.g., promoter and/or enhancer) using techniques
known of in
the art. The host cell can be a higher eukaryotic cell, such as a mammalian
cell (e.g., a
human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a
prokaryotic cell, such as a bacterial cell. A host strain may be chosen which
modulates the
expression of the inserted gene sequences, or modifies and processes the gene
product in
the specific fashion desired. Expression from certain promoters can be
elevated in the
presence of certain inducers; thus expression of the genetically engineered
polypeptide may
be controlled. Furthermore, different host cells have characteristics and
specific
mechanisms for the translational and post-translational processing and
modification (e.g.,
phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen
to ensure the
desired modifications and processing of the foreign protein expressed.
[195] Introduction of the nucleic acids and nucleic acid constructs of the
invention into
the host cell can be effected by calcium phosphate transfection, DEAE-dextran
mediated
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transfection, cationic lipid-mediated transfection, electroporatiori,
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.
[196] 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., the 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 andlor enhancer) and
endogenous
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 ~t al., P~-oc. Natl.
Acad. Sci. USA
86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the
disclosures of
each of which are incorporated by reference in their entireties).
[197] Polypeptides of the invention can be recovered and purified from
recombinant
cell cultures by well-known methods including 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.
[198] 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-
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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 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.
[199] In one embodiment, the yeast Pichia pastoris is used to express
polypeptides of
the invention in a eukaryotic system. Pichia pasto~is 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 O2. This reaction
is catalyzed
by the enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source,
Pichia pasto~is must generate high levels of alcohol oxidase due, in part, to
the relatively
low affinity of alcohol oxidase for OZ. 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 AOXI gene comprises up to approximately 30% of the total soluble protein
in Pichia
pastor°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.
[200] 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 pastof°is alkaline phosphatase (PHO)
secretory signal peptide
(i.e., leader) located upstream of a multiple cloning site.
[201] 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,
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pHIL-S 1, pPIC3.5K, and PA0815, as one skilled in the art would readily
appreciate, as
long as the proposed expression construct provides appropriately located
signals for
transcription, translation, secretion (if desired), and the like, including an
in-frame AUG as
required.
[202] 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.
[203] 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 96/2941 l,
published
September 26, 1996; International Publication No. WO 94/12650, published
August 4,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and
Zijlstra~ et al.,
Nature. 342:435-438 (1989), the disclosures of each of which are incorporated
by reference
in their entireties).
[204] In addition, polypeptides of the invention can be chemically synthesized
using
techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures
and Molecular
Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-
111
(1984)). For example, a polypeptide corresponding to a fragment of a
polypeptide can be
synthesized by use of a peptide synthesizer. Furthermore, if desired,
nonclassical amino
acids or chemical amino acid analogs can be introduced as a substitution or
addition into the
polypeptide sequence. Non-classical amino acids include, but are not limited
to, to the D-
isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric
acid, 4-
aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid, Aib,
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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)..
[205] 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, V~ protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic
synthesis in the presence of tunicamycin; etc.
[206] Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linlced 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.
[207] Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an
example of a luminescent material includes luminol; examples of bioluminescent
materials
include luciferase, luciferin, and aequorin; and examples of suitable
radioactive material
include iodine (121I, i23h lash i3i1), carbon (14C), sulfur (35S), tritium
(3H), indium (111In,
naIn m3mIn 115mIn) technetium (99Tc 99mTc) thallium (2°1Ti) allium 68Ga
6~Ga)
> > > > > > g ( > >
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palladium (losPd), molybdenum (99Mo), xenon (133xe), fluorine (18F), ls3Sm,
l~~Lu, ls9Gd,
i49Pm~ i4oLa~ ms~~ 166H~~ 90~,~ 4~Sc~ ia6Re~ iaBRe~ i4aPr~ ios~~ and 9~Ru.
[208] 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, 9°Y, ls6Ho, and ls3Sm, to
polypeptides. In a preferred
embodiment, the radiometal ion associated with the macrocyclic chelators is
11n. 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.
(209] As mentioned, the 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
polypeptide.
Polypeptides of the invention 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
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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.
182:626-6.46 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62.(1992)).
[210] 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.
[211] The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1
kDa and about 100 kDa (the term "about" indicating that in preparations of
polyethylene
glycol, some molecules will weigh more, some less, than the stated molecular
weight) for
ease in handling and manufacturing. Other sizes may be used, depending on the
desired
therapeutic profile (e.g., the duration of sustained release desired, the
effects, if any on
biological activity, the ease in handling, the degree or lack of antigenicity
and other known
effects of the polyethylene glycol to a therapeutic protein or analog). For
example, the
polyethylene glycol may have an average molecular weight of about 200, 500,
1000, 1500,
2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,
8500,
9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500,
14,000,
14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500,
19,000, 19,500,
20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000,
65,000, 70,000,
75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
[.212] 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.
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[213] 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.
[214] 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.
[215] 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, 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)
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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.
[216] 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 95106058; and WO 98/32466,
the
disclosures of each of which are incorporated herein by reference.
[217] 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
(C1S02CH2CF3). 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.
[218] 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 produced by reaction of
proteins with
compounds such as MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-
nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of
additional
polyethylene glycol derivatives and reaction chemistries for attaching
polyethylene glycol
to proteins are described in International Publication No. WO 98/32466, the
entire
disclosure of which is incorporated herein by reference. Pegylated protein
products
produced using the reaction chemistries set out herein are included within the
scope of the
invention.
[219] 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
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proteins of the invention may be linked, on average, to l, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 15, 17,
20, or more polyethylene glycol molecules. Similarly, the average degree of
substitution
within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-
13, 12-14, 13-
15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per
protein molecule.
Methods for determining the degree of substitution are discussed, for example,
in Delgado
et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[220] The 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.
[221] 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. .
[222] Multimers encompassed by the invention may be homomers or heteromers. As
used herein, the term homomer refers to a multimer containing only
polypeptides
corresponding to a protein of the invention (e.g., the amino acid sequence of
SEQ ID NO:Y,
an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ 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
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specific embodiment, a homomer of the invention is a multimer containing
polypeptides
having different amino acid sequences. In specific embodiments, the multimer
of the
invention is a homodimer (e.g., containing two polypeptides having identical
or different
amino acid sequences) or a homotrimer (e.g., containing three polypeptides
having identical
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.
[223] As used herein, the term heteromer refers to a multimer containing one
or more
heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to the
polypeptides of the invention. In a specific embodiment, the multimer of the
invention is a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the
heteromeric.multimer of the invention is at least a heterodimer, at least a
heterotrimer, or at
least a heterotetramer.
[224] Multirners of the invention may be the result of hydrophobic,
hydrophilic, ionic
and/or covalent associations and/or may be indirectly linked by, for example,
liposome
formation. Thus, in one embodiment, multimers of the invention, such as, for
example,
homodimers or homotrimers, are formed when polypeptides of the invention
contact one
another in solution. In another embodiment, heteromultimers of the invention,
such as, for
example, heterotrimers or heterotetramers, are formed when polypeptides of the
invention
contact antibodies to the polypeptides of the invention (including antibodies
to the
heterologous polypeptide sequence in a fusion protein of the invention) in
solution. In other
embodiments, multimers of the invention are formed by covalent associations
with andlor
between the polypeptides of the invention. Such covalent associations may
involve one or
more amino acid residues contained in the polypeptide sequence (e.g., that
recited in SEQ
ID NO:Y, encoded by 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 fission
protein. In
one example, covalent associations are between the heterologous sequence
contained in a
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fusion protein of the invention (see, e.g., US. Patent Number 5,478,925). In a
specific
example, the covalent associations are between the heterologous sequence
contained in a Fc
fusion protein of the invention (as described herein). In another specific
example, covalent
associations of fusion proteins of the invention are between heterologous
polypeptide
sequence from another protein that is capable of forming covalently associated
multimers,
such as for example, osteoprotegerin (see, e.g., International Publication NO:
WO
98/49305, the contents of which are herein incorporated by reference in its
entirety). In
another embodiment, two or more polypeptides of the invention are joined
through peptide
linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627
(hereby incorporated by reference). Proteins comprising multiple polypeptides:
of the
invention separated by peptide linkers may be produced using conventional
recombinant
DNA technology.
[225] 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.
[226] Trimeric polypeptides of the invention may offer the advantage of
enhanced
biological activity. Preferred leucine zipper moieties and isoleucine moieties
are those that
preferentially form trimers. One example is a leucine zipper derived from lung
surfactant
protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994))
and in U.S.
patent application Ser. No. 08/446,922, hereby incorporated by reference.
Other peptides
derived from naturally occurring trimeric proteins may be employed in
preparing trimeric
polypeptides of the invention.
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[227] 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 RO antibody.
[228] 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., US 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., US 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., US
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., US Patent Number 5,478,925, which is herein incorporated by reference in
its
entirety).
[229] 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., US Patent Number
5,478,925,
which is herein incorporated by reference in its entirety). In a specific
embodiment,
polynucleotides coding for a homodimer of the invention are generated by
ligating a
polynucleotide sequence encoding a polypeptide of the invention to a sequence
encoding a
linker polypeptide and then further to a synthetic polynucleotide encoding the
translated
product of the polypeptide in the reverse orientation from the original C-
terminus to the N-
terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925,
which is
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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., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety).
Antibodies
[230] 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.
[231] Most preferably the antibodies are human antigen-binding antibody
fragments of
the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd, ~single-
chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments
comprising either a VL or VH domain. Antigen-binding antibody fragments,
including
single-chain antibodies, may comprise the variable regions) alone or in
combination with
the entirety or a portion of the following: hinge region, CHl, CH2, and CH3
domains. Also
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included in the invention are antigen-binding fragments also comprising any
combination of
variable regions) with a hinge region, CH1, CH2, and CH3 domains. The
antibodies of the
invention may be from any animal origin including birds and mammals.
Preferably, the
antibodies are human, murine (e:g., mouse and rat), donkey, ship rabbit, goat,
guinea pig,
camel, horse, or chicken. As used herein, "human" antibodies include
antibodies having
the amino acid sequence of a human immunoglobulin and include antibodies
isolated from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immurioglobulins, as
described infra
and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
[232] The antibodies of the present invention may be monospecific, bispecific,
trispecific or of. greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both a
polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
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).
[233] 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 the
predicted epitopes shown in column 7 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.
[234] 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
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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 Kd less than 5 X 10-Z 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-
$ M, 10-8 M,
X 10-9 M, 10-9 M, 5 X 10-1° M, 10-'° M, 5 X 10-11 M, 10-11 M, 5
X 10-1z M, 10-12 M, 5 X
10-13 M, 10-13 M, 5 X 10-14 M, 10-14 M, 5 X 10-is M, or 10-15 M.
[235j 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
herein. In
preferred embodiments, the antibody competitively inhibits binding to the
epitope by at
least 95%, at Ieast 90%, at least 85 %, at least 80%, at least 75%, at Ieast
70%, at least 60%,
or at least 50%.
[236] 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 xeceptor/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 Iigand-specific antibodies. The invention
also features
receptor-specific antibodies which do not prevent ligand binding but prevent
receptor
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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.
[237] 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; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6):1981-1988
(1998); Chen et
al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.
161(4):1786-1794
(1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J.
Immunol.
160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997);
Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al.,
Neuron
14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek
et al.,
Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in
their
entireties).
[238] 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 iya vivo
diagnostic and therapeutic methods. For example, the antibodies have utility
in
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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.
[239] 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.
[240] 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 tunicamycin, etc.
Additionally, the
derivative may contain one or more non-classical amino acids.
[241] 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 imrnunological 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,
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dinitrophenol, and potentially useful human adjuvants such as BCG (bacille
Calmette-
Guerin) and corynebacterium parvum. Such adjuvants are also well known in the
art.
[242] 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.
(243] Methods for producing and screening for specific antibodies using
hybridoma
technology are routine and well known in the art and are discussed in detail.
in the
Examples. In a non-limiting example, mice can be immunized with a polypeptide
of the
invention or a cell expressing such peptide. Once an immune response is
detected, e.g.,
antibodies specific for the antigen are detected in the mouse serum, the mouse
spleen is
harvested and splenocytes isolated. The splenocytes are then fused by well
known
techniques to any suitable myeloma cells, for example cells from cell line
SP20 available
from the ATCC. Hybridomas are selected and cloned by limited dilution. The
hybridoma
clones are then assayed by methods 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.
[244] 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.
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[245] 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. 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.
[246] In general, the sample containing human B cells is innoculated with EBV,
and
cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of
the B95-8 cell
line (ATCC #VR-1492). Physical signs of EBV transformation can generally be
seen
towards the end of the 3-4 week culture period. By phase-contrast microscopy,
transformed
cells may appear large, clear, hairy and tend to aggregate in tight clusters
of cells. Initially,
EBV lines are generally polyclonal. However, over prolonged periods of cell
cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective
outgrowth of
particular B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned
(e.g., by limiting dilution culture) or fused with a suitable fusion partner
and plated at
limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners
for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-
Ag8.653),
heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and
human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the
present
invention also provides a method of generating polyclonal or monoclonal human
antibodies
against polypeptides of the invention or fragments thereof, comprising EBV-
transformation
of human B cells.
[247] 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
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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.
[248] For example, the antibodies of the present invention can also be
generated using
various phage display methods known in the art. In phage display methods,
functional
antibody domains are displayed on the surface of phage particles which carry
the
polynucleotide sequences encoding them. In a particular embodiment, such phage
can be
utilized to display antigen binding domains expressed from a repertoire or
combinatorial
antibody library (e.g., human or marine). Phage expressing an antigen binding
domain that
binds the antigen of interest can be selected or identified with antigen,
e.g., using labeled
antigen or antigen bound or captured to a solid surface or bead. Phage used in
these
methods are typically filamentous phage including fd and M13 binding domains
expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused
to either the phage gene III or gene VIII protein. Examples of phage display
methods that
can be used. to make the antibodies of the present invention include those
disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol. Methods
184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994);
PCT application No. PCTlGB91/01134; PCT publications WO 90/02809; WO 91/10737;
WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and
5,969,108; each of
which is incorporated herein by reference in its entirety.
[249] 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).
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[250] 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 in vitro detection assays, it may be preferable to
use chimeric,
humanized, or human antibodies. A chimeric antibody is a molecule in which
different
portions of the antibody are derived from different animal species, such as
antibodies
having a variable region derived from a murine monoclonal antibody and a human
immunoglobulin constant region. Methods for producing chimeric antibodies are
known in
the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques
4:214
(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Patent
Nos.
5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference
in their
entirety. Humanized antibodies are antibody molecules from non-human species
antibody
that binds the desired antigen having one or more complementarity determining
regions
(CDRs) from the non-human species and a framework regions from a human
immunoglobulin molecule. Often, framework residues in the human framework
regions
will be substituted with the corresponding residue from the CDR donor antibody
to alter,
preferably improve, antigen binding. These framework substitutions are
identified by
methods well known in the art, e.g., by modeling of the interactions of the
CDR and
framework residues to identify framework residues important for antigen
binding and
sequence comparison to identify unusual framework residues at particular
positions. (See,
e.g., Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature
332:323 (1988),
which are incorporated herein by reference in their entireties.) Antibodies
can be
humanized using a variety of techniques known in the art including, for
example, CDR-
grafting (EP 239,400; PCT publication WO 91109967; 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 (LJ.S.
Patent No. 5,565,332).
[251] 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
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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.
[252] Human antibodies can also be produced using transgenic mice which are
incapable of expressing functional endogenous immunoglobulins, but which can
express
human immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene complexes may be introduced randomly or by homologous
recombination into mouse embryonic stem cells. Alternatively, the human
variable region,
constant region, and diversity region may be introduced into mouse embryonic
stem cells
in addition to the human heavy and light chain genes. The mouse heavy and
light chain
immunoglobulin genes may be rendered non-functional separately or
simultaneously with
the introduction of human immunoglobulin loci by homologous recombination. In
particular, homozygous deletion of the JH region prevents endogenous antibody
production.
The modified embryonic stem cells are expanded and microinjected into
blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce homozygous
offspring
which express human antibodies. The transgenic mice are immunized in the
normal fashion
with a selected antigen, e.g., all or a portion of a polypeptide of the
invention. Monoclonal
antibodies directed against the antigen can be obtained from the immunized,
transgenic
mice using conventional hybridoma technology. The human immunoglobulin
transgenes
harbored by the transgenic mice rearrange during B cell differentiation, and
subsequently
undergo class switching and somatic mutation. Thus, using such a technique, it
is possible
to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an
overview of
this technology for producing human antibodies, see Lonberg and Huszar, Int.
Rev.
Immunol. 13:65-93 (1995). . For a detailed discussion of this technology for
producing
human antibodies and human monoclonal antibodies and protocols for producing
such
antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096;
WO
96/33735; European Patent No. 0 598 877; U.S. Patent Nos. 5,413,923;
5,625,126;
5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771;
5,939,598;
6,075,181; and 6,114,598, which are incorporated by reference herein in their
entirety. In
addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San
Jose, CA)
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can be engaged to provide human antibodies directed against a selected antigen
using
technology similar to that described above.
[253] 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)).
[254] Further, antibodies to the polypeptides of the invention can, in turn,
be utilized to
generate anti-idiotype antibodies that "mimic" polypeptides of the invention
using
techniques well known to those skilled in the art. (See, e.g., Greenspan &
Bona, FASEB J.
7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For
example,
antibodies which bind to and competitively inhibit polypeptide multimerization
and/or
binding of a polypeptide of the invention to a ligand can be used to generate
anti-idiotypes
that "mimic" the polypeptide multimerization and/or binding domain and, as a
consequence, bind to and neutralize polypeptide and/or its ligand. Such
neutralizing anti-
idiotypes or'Fab fragments of such anti-idiotypes can be used in therapeutic
regimens to
neutralize polypeptide ligand(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 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.
[255] 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.,
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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.
PolyfZUCleotides Encodi~rg Antibodies
[256] 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 supy~a, 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. .
[257] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody may
be assembled from chemically synthesized oligonucleotides (e.g., as described
in Kutmeier
et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis
of overlapping
oligonucleotides containing portions of the sequence encoding the antibody,
annealing and
ligating of those oligonucleotides, and then amplification of the ligated
oligonucleotides by
PCR.
[258] 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.
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[259] 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.
[260] In a specific embodiment, the amino acid sequence of the heavy and/or
light
chain variable domains may be inspected to identify the sequences of the
complementarity
determining regions (CDRs) by methods that are well know in the art, e.g., by
comparison
to known amino acid sequences of other heavy and light chain variable regions
to determine
the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one
or more of. the CDRs may be inserted. within framework regions, e.g., into
human
framework regions to humanize a non-human antibody, as described supra. The
framework
regions may be naturally occurring or consensus framework regions, and
preferably human
framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing
of human framework regions). Preferably, the polynucleotide generated by the
combination
of the 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.
[261] 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
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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.
[262] Alternatively, techniques described for the production of single chain
antibodies
(U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al.,
Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989))
can be
adapted to produce single chain antibodies. Single chain antibodies are formed
by linking
the heavy and light chain fragments of the Fv region via an amino acid bridge,
resulting in a
single chain polypeptide. Techniques for the assembly of functional Fv
fragments in E. coli
may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods of Pr~oducihg Antibodies
[263] The antibodies of the invention can be produced by any method known in
the art
fox 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.
[264] Recombinant expression of an antibody of the invention, or fragment,
derivative
or analog thereof, (e.g., a heavy or light chain of an antibody of the
invention or a single
chain antibody of the invention), requires construction of an expression
vector containing a
polynucleotide that encodes the antibody. Once a polynucleotide encoding an
antibody
molecule or a heavy or light chain of an antibody, or portion thereof
(preferably containing
the heavy or light chain variable domain), of the invention has been obtained,
the vector for
the production of the antibody molecule may be produced by recombinant DNA
technology
using techniques well known in the art. Thus, methods for preparing a protein
by
expressing a polynucleotide containing an antibody encoding nucleotide
sequence are
described herein. Methods which are well known to those skilled in the art can
be used to
construct expression vectors containing antibody coding sequences and
appropriate
transcriptional and translational control signals. These methods include, for
example, in
vitro recombinant DNA techniques, synthetic techniques, and iu vivo genetic
recombination. The invention, thus, provides replicable vectors comprising a
nucleotide
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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
fox expression of the entire heavy or light chain.
[265] 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.
[266] 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
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expression of whole recombinant antibody molecule, are used for the expression
of a
recombinant antibody molecule. For example, mammalian cells such as Chinese
hamster
ovary cells (CHO), in conjunction with a vector such as the major intermediate
early gene
promoter element from human cytomegalovirus is an effective expression system
for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2
(1990)).
[267] 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.
[268] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV)
is used as a vector to express foreign genes. The virus grows in Spodopte~a
fi~ugiperda
cells. The antibody coding sequence may be cloned individually into non-
essential regions
(for example the polyhedrin gene) of the virus and placed under control of an
AcNPV
promoter (for example the polyhedrin promoter).
[269] 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 in vitro or ira vivo
recombination. Insertion in
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a non- essential region of the viral genome (e.g., region El or E3) will
result in a
recombinant virus that is viable and capable of expressing the antibody
molecule in
infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-
359 (1984)).
Specific initiation signals may also be required for efficient translation of
inserted antibody
coding sequences. These signals include the ATG initiation codon and adjacent
sequences.
Furthermore, the initiation codon must be in phase with the reading frame of
the desired
coding sequence to ensure translation of the entire insert. These exogenous
translational
control signals and initiation codons can be of a variety of origins, both
natural and
synthetic. The efficiency of expression may be enhanced by the inclusion of
appropriate
transcription enhancer elements, transcription terminators, etc. (see Bittner
et al., Methods
in Enzymol. 153:51-544 (1987)).
[270] In addition, a host cell strain may be chosen which modulates the
expression of
the inserted sequences, or modifies arid 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 nor~rnal
mammary
gland cell line such as, for example, CRL7030 and Hs578Bst.
[271] 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
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resistance to the selection and allows cells to stably integrate the plasmid
into their
chromosomes and grow to form foci which in turn can be cloned and expanded
into cell
lines. This method may advantageously be used to engineer cell lines which
express the
antibody molecule. Such engineered cell lines may be particularly useful in
screening and
evaluation of compounds that interact directly or indirectly with the antibody
molecule.
[272] 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); May, 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); I~riegler, 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.
[273] The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based on
gene amplification for the expression of cloned genes in mammalian cells in
DNA cloning,
Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system
expressing antibody is amplifiable, increase in the level of inhibitor present
in culture of
host cell will increase the number of copies of the marker gene. Since the
amplified region
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is associated with the antibody gene, production of the antibody will also
increase (Grouse
et al., Mol. Cell. Biol. 3:257 (1983)).
[274] 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 myelorna 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., Bioltechnology 10:169(1992) and in Biblia and Robinson
Biotech~col.
Prog. 11:1 (1995) which are incorporated in their entirities by reference
herein.
[275] 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, arid 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);
I~ohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for
the heavy
and light chains may comprise cDNA or genomic DNA.
[276] Once an antibody molecule of the invention has been produced by an
animal,
chemically synthesized, or recombinantly expressed, it may be purified by any
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
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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.
[277] 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 ira 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 in 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.
[278] 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, CHl
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 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;
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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).
[279] 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 iu 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; and
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)).
[280] 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
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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.
[281] 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 linlcer 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. Examples of suitable enzymes include
horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples
of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include 125I, 131I, 11 lIn or 99Tc.
[282] 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 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),
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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).
[283] 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 97133899), AIM II (See, International
Publication
No. WO 97/34911.), Fas Ligand (Takahashi et al., Int. Immunol., 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.
[284] 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.
[285] Techniques for conjugating such therapeutic moiety to antibodies are
well
known. See, for example, Arnon et al., "Monoclonal Antibodies For
Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld et al.
(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug
Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
623-53
(Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In
Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications,
Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future
Prospective Of
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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 Thozpe et al., "The Preparation And Cytotoxic Properties Of
Antibody-
Toxin Conjugates", Immunol. Rev. 62:119-58 (1982).
[286] 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.
[287] An antibody, with or without a therapeutic moiety conjugated to it,
administered
alone or in combination with cytotoxic factors) and/or cytokine(s) can be used
as a
therapeutic.
Immunophenotypihg
[288] The antibodies of the invention may be utilized for immunophenotyping of
cell
lines and biological samples. Translation products of the gene 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)).
[289] These techniques allow for the screening of particular populations of
cells, such
as might be found with hematological malignancies (i.e. minimal residual
disease (MRD) in
acute leukemic patients) and "non-self' cells in transplantations to prevent
Graft-versus-
Host Disease (GVHD). Alternatively, these techniques allow for the screening
of
hematopoietic stem and progenitor cells capable of undergoing proliferation
and/or
differentiation, as might be found in human umbilical cord blood.
Assays For Antibody BiradifZg
[290] 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
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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).
[291] Immunoprecipitation protocols generally comprise lysing a population of
cells in
a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium
deoxycholate,
0.1% SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol)
supplemented
with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium
vanadate), adding the antibody of interest to the cell lysate, incubating for
a period of time
(e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose
beads to the cell
lysate, incubating for about an hour or more at 4° C, washing the beads
in lysis buffer and
resuspending the beads in SDS/sample buffer. The ability of the antibody of
interest to
immunoprecipitate a particular 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
immurloprecipitation protocols see, e.g., Ausubel et al., eds., (1994),
Current Protocols in
Molecular Biology, Vol. I, John Wiley & Sons, Inc., New York, section I0.
I6.1.
[292] 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.,
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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. 1,
John Wiley &
Sons, Inc., New York, section 10.8.1.
[293] 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.
[294] 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.
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[295] 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 an antigen or with vector alone using techniques
commonly
known in the art. Antibodies that bind antigen transfected cells, but not
vector-only
transfected cells, are antigen specific.
Therapeutic Uses
[296] 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 fox 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 andlor activity of
a polypeptide
of the invention, including, but not limited to, any one or more of the
diseases, disorders, or
conditions described herein. The treatment and/or prevention of diseases,
disorders, or
conditions associated with aberrant expression and/or activity of a
polypeptide of the
invention includes, but is not limited to, alleviating symptoms associated
with those
diseases, disorders or conditions. Antibodies of the invention may be provided
in
pharmaceutically acceptable compositions as known in the art or as described
herein.
[297] 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
diseases, disorders, or conditions, including but not limited to: neural
disorders, immune
system disorders, muscular disorders, reproductive disorders, gastrointestinal
disorders,
pulmonary disorders, cardiovascular disorders, renal disorders, proliferative
disorders,
andlor cancerous diseases and conditions., and/or as described elsewhere
herein.
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
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a mammalian cell; antibodies directed to an epitope of a polypeptide of the
invention (such
as, for example, a predicted linear epitope shown in column 7 of Table 1A; 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 described herein. The treatment and/or prevention of
diseases,,disorders, or
conditions associated with aberrant expression and/or activity of a
polypeptide of the
invention includes, but is not limited to, alleviating symptoms associated
with those
diseases, disorders or conditions. Antibodies of .the invention may be
provided in
pharmaceutically acceptable compositions as known in the art or as described
herein.
[298] A summary of the ways in which the antibodies of the present invention
may be
used therapeutically includes binding polynucleotides or polypeptides of the
present
invention locally or systemically in the body or by direct cytotoxicity of the
antibody, e.g.
as mediated by complement (CDC) or by effector cells (ADCC). Some of these
approaches are described in more detail below. Armed with the teachings
provided herein,
one of ordinary skill in the art will know how to use the antibodies of the
present invention
for diagnostic, monitoring or therapeutic purposes without undue
experimentation.
[299] 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.
[300] 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.
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[301] 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, 10'2
M, 5 X 10'3 M, 10'3 M, 5 X 10'4 M, 10'4 M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M,
10'6 M, 5 X
10-~ M, 10-~ M, 5 X 10'8 M, 10-$ M, 5 X 10'9 M, 10'9 M, 5 X 10'1° M, 10-
1° M, 5 X 10-" 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, and
10-1s M.
Gene They°apy
[302] 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.
[303] Any of the methods for gene therapy available in the art can be used
according to
the present invention. Exemplary methods are described below.
[304] 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).
[305] In a preferred embodiment, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors that
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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.
[306] 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 in vitro,
then transplanted
into the patient. These two approaches are known, respectively, as in vivo or
ex vivo gene
therapy.
[307] In a specific embodiment, the nucleic acid sequences are directly
administered in
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 in
vivo for cell
specific uptalce and expression, by targeting a specific receptor (see, e.g.,
PCT Publications
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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.
Aced. Sci.
USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).
[308] 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 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).
[309] 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.
[310] Adeno-associated virus (AAV) has also been proposed for use in gene
therapy
(Welsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
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[311j 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.
[312] In this embodiment, the nucleic acid is introduced into a cell prior to
administration ira 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.
[313] 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.
[314] 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.
[315] In a preferred embodiment, the cell used for gene therapy is autologous
to the
patient.
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[316] In an embodiment in which recombinant cells are used in gene therapy,
nucleic
acid sequences encoding an antibody are introduced into the cells such that
they are
expressible by the cells or their progeny, and the recombinant cells are then
administered in
vivo for therapeutic effect. In a specific embodiment, stem or progenitor
cells are used.
Any stem and/or progenitor cells which can be isolated and maintained in vitro
can
potentially be used in accordance with this embodiment of the present
invention (see e.g.
PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992);
Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo
Clinic Proc.
61:771 (1986)).
[317] 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.
Dernonst~ation of Therapeutic o~ Prophylactic Activity
[318] 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
[319] 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
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substances that limit its effect or produce undesired side-effects). The
subject is preferably
an animal, including but not limited to animals such as cows, pigs, horses,
chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
[320] 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.
[321] 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.
[322] 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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[323j 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.)
[324] 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
(I980); 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
(I984);
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)).
[325] Other controlled release systems are discussed in the review by Langer
(Science
249:1527-1533 (I990)).
[326] 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.
[327] The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
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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.
[328] In a preferred embodiment, the composition is formulated in accordance
with
routine procedures as a pharmaceutical composition adapted for intravenous
administration
to human beings. Typically, compositions for intravenous administration are
solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may also
include a
solubilizing agent and a local anesthetic such as lignocaine to ease pain at
the site of the
injection. Generally, the ingredients are supplied either separately or mixed
together in
unit dosage form, for example, as a dry lyophilized powder or water free
concentrate in a
hermetically sealed container such as an ampoule or sachette indicating the
quantity of
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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.
[329] 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.
[330] 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.
[331] 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
mg/kg of the patient's body weight. Generally, human antibodies have a longer
half life
within the human body than antibodies from other species due to the immune
response to
the foreign polypeptides. Thus, lower dosages of human antibodies and less
frequent
administration is often possible. Further, the dosage and frequency of
administration of
antibodies of the invention may be reduced by enhancing uptake and tissue
penetration
(e.g., into the brain) of the antibodies by modifications such as, for
example, lipidation.
[332] 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
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pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration.
Diagnosis ahd Irraaging
[333] 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.
[334] The invention provides a diagnostic assay for diagnosing a disorder,
comprising
(a) assaying the expression of the polypeptide of interest in cells or body
fluid of an
individual using one or more antibodies specific to the polypeptide interest
and (b)
comparing the level of gene expression with a standard gene expression level,
whereby an .
increase or decrease in the assayed polypeptide gene expression level compared
to the
standard expression level is indicative of a particular disorder. With respect
to cancer, the
presence of a relatively high amount of transcript in biopsied tissue from an
individual may
indicate a predisposition for the development of the disease, or may provide a
means for
detecting the disease prior to the appearance of actual clinical symptoms. A
more
definitive diagnosis of this type may allow health professionals to employ
preventative
measures or aggressive treatment earlier thereby preventing the development or
further
progression of the cancer.
[335] 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
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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.
[336] 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. 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.
[337] It will be understood in the art that the size of the subject and the
imaging system
used will determine the quantity of imaging moiety needed to produce
diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of
radioactivity
injected will normally range from about 5 to 20 millicuries of 99mTc. The
labeled antibody
or antibody fragment will then preferentially accumulate at the location of
cells which
contain the specific protein. Ira vivo tumor imaging is described in S.W.
Burchiel et al.,
"Imrnunopharmacokinetics 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)).
[338] 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
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hours. In another embodiment the time interval following administration is 5
to 20 days or
to 10 days.
[339] In an embodiment, monitoring of the disease or disorder is carried out
by
repeating the method for diagnosing the disease or disease, for example, one
month after
initial diagnosis, six months after initial diagnosis, one year after initial
diagnosis, etc.
[340] Presence of the labeled molecule can be detected in the patient using
methods
known in the art for iya 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.
[341] In a specific embodiment, the molecule is labeled with a radioisotope
and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al., U.S.
Patent No. 5,441,050). In another embodiment, the molecule is labeled with a
fluorescent
compound and is detected in the patient using a fluorescence responsive
scanning
instrument. In another embodiment, the molecule is labeled with a positron
emitting metal
and is detected in the patent using positron emission-tomography. In yet
another
embodiment, the molecule is labeled with a paramagnetic label and is detected
in a patient
using magnetic resonance imaging (MRI).
Kits
[342] 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).
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[343] 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 lcit 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.
[344] In a more specific embodiment the detecting means of the above-described
kit
includes a solid support to which said polypeptide antigen is attached. Such a
kit may also
include a non-attached reporter-labeled anti-human antibody. In this
embodiment, binding
of the antibody to the polypeptide antigen can be detected by binding of the
said reporter-
labeled antibody.
[345] 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 lcit may include a second, labeled monoclonal antibody.
Alternatively, or in addition, the detecting means may include a labeled,
competing antigen.
[346] 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
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enzyme which is detected by incubating the solid phase in the presence of a
suitable
fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
[347] 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).
[348] Thus, the invention provides an assay system or kit for carrying out
this
diagnostic method. The kit generally includes a support with surface- bound
recombinant
antigens, and a reporter-labeled anti-human antibody for detecting surface-
bound anti-
antigen antibody.
Uses of the Polynucleotides
[349] 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.
[350] 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 9 provides the chromosome location of some of the
polynucleotides of the invention.
[351] 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
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hybrids containing the human gene corresponding to SEQ ID NO:X will yield an
amplified
fragment.
[352] Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Threw 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).
[353] 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).
[354j For chromosome mapping, the polynucleotides can be used individually (to
marls
a single chromosome or a single site on that chromosome) or in panels (for
marking
multiple sites and/or multiple chromosomes).
[355] 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.
[356] The polynucleotides of the present invention would likewise be useful
for
radiation hybrid mapping, HAPPY mapping, and Iong 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); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby
incorporated by
reference in its entirety.
[357] 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
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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 10 of Table 1A provides ~n OMIM reference identification
number of
diseases associated with the cytologic band disclosed in column 9 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.
[358] 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.
[359] 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 Diseases").
[360] 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
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disorder. Additional non-limiting examples of diagnostic methods encompassed
by the
present invention are more thoroughly described elsewhere herein (see, e.g.,
Example 12).
[361] In still another embodiment, the invention includes a kit for analyzing
samples
for the presence of proliferative and/or cancerous polynucleotides derived
from a test
subject. In a general embodiment, the kit includes at least one polynucleotide
probe
containing a nucleotide sequence that will specifically hybridize with a
polynucleotide of
the invention and a suitable container. In a specific embodiment, the kit
includes two
polynucleotide probes defining an internal region of the polynucleotide of the
invention,
where each probe has one strand 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.
[362] 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.
[363] 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 l~nown, it can be used
repeatedly as
a standard for comparison.
[364] 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
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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 rizRNA, a tissue biopsy is the preferred source.
[365] 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
attached to a
solid support. In one exemplary method, the support may be a "gene chip" or a
"biological
chip" as described in US 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,
metabolic disorders, cardiovascular disorders, renal disorders, proliferative
disorders; andlor
cancerous diseases and conditions. Such a method is described in US Patents
5,858,659 and
5,856,104. The US Patents referenced supra are hereby incorporated by
reference in their
entirety herein.
[366] 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
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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. 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.
[367] 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
leultemia, 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.
[368] 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 countezparts of the
oncogenes
involved in some animal neoplasias have been amplified or translocated in some
cases of
human leukemia and carcinoma. (Gelmann et al., supra)
[369] 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
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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, 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.
[370] 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); Gooney et al., Science 241: 4S6 (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); Gooney et al., Science 241:456 (1988); and Dervan et
al.,
Science 251:1360 (1991)) or to the mRNA itself (antisense - Okano, J.
Neurochem. 56:560
(1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC
Press,
Boca Raton, FL (1988)). Triple helix formation optimally results in a shut-off
of RNA
transcription from DNA, while antisense RNA hybridization blocks translation
of an
mRNA molecule into polypeptide. The oligonucleotide described above can also
be
delivered to cells such that the antisense RNA or DNA may be expressed ih 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 andlor 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)").
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[371] 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).
[372] The polynucleotides are also useful for identifying individuals from
minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel. In this
technique, an individual's genomic DNA is digested with one or more
restriction enzymes,
and probed on a Southern blot to yield unique bands for identifying personnel.
This
method does not suffer from the current limitations of "Dog Tags" which can be
lost,
switched, or stolen, malting positive identification difficult. The
polynucleotides of the
present 'invention can be used as additional DNA markers for RFLP.
[373] The polynucleotides of the present invention can also be used as an
alternative to
RFLP, by determining the actual base-by-base DNA sequence of selected portions
of an
individual's genome. These sequences can be used to prepare PCR primers for
amplifying
and isolating such selected DNA, which can then be sequenced. Using this
technique,
individuals can be identified because each individual will have a unique set
of DNA
sequences. Once an unique ID database is established for an individual,
positive
identification of that individual, living or dead, can be made from extremely
small tissue
samples.
[374] 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
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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.
[375] 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 shown 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.
[376] 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 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, those disclosed in
column 8 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.
[377] 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.
[378] 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
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process of discovering novel polynucleotides, for selecting and making
oligomers for
attachment to a "gene chip" or other support, to raise anti-DNA antibodies
using DNA'
immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
[379] Each of the polypeptides identified herein can be used in numerous ways.
The
following description should be considered exemplary and utilizes known
techniques.
[380] 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).
(381] 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 (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 (1311, lzsh lz3h izll)~ carbon (14C),
sulfur (3sS), tritium
(3H), indium (lls'T'In, 113mIn, llzln, 111In), and technetium (99Tc, 99mTc),
thallium (zolTi),
gallium (68Ga, 6~Ga), palladium (1°3Pd), molybdenum (99Mo), xenon
(133Xe), fluorine (1gF),
lssSm l~~Lu lsaGd la9Pm l4oLa l~s~,b ls6Ho aoy a~Sc 186Re lBaRe l4zPr lost
9~Ru.
> > > > > > > > > > > > > >
luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
[382] In addition to assaying levels of polypeptide of the present invention
in a
biological sample, proteins can also be detected ih vivo by imaging. Antibody
labels or
markers for ira 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,
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which may be incorporated into the antibody by labeling of nutrients for the
relevant
hybridoma.
[383] A protein-specific antibody or antibody fragment which has been labeled
with an
appropriate detectable imaging moiety, such as a radioisotope (for example,
1311, 112In,
99mTC' (131I' l2sl' 123I' lall)~ carbon (14C), sulfur (35S), tritium (3H),
indium (115mIn' 113mIn,
112In, 111In), and technetium (99Tc, 99mTc), thallium (2olTi), gallium (68Ga,
6~Ga), palladium
(lo3Pd), molybdenum (99Mo), xenon (133xe), fluorine (18F, ls3Sm, l~~Lu, ls9Gd,
149Pm, laoLa,
17s~ 166H~ 90Y 475 186Re 188Re 142Pr 105 a7Ru) a radio o a ue substance or a
a > > > > > > > > - p q
material detectable by nuclear magnetic resonance, is introduced (for example,
parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined for immune
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. 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)).
[384] . 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.
[385] 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.
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[386] By "toxin" is meant one or more compounds that bind and activate
endogenous
cytotoxic effector systems, radioisotopes, holotoxins, modified toxins;
catalytic subunits of
toxins, or any molecules or enzymes not normally present in or on the surface
of a cell that
under defined conditions cause the cell's death. Toxins that may be used
according to the
methods of the invention include, but are not limited to, radioisotopes known
in the art,
compounds such as, for example, antibodies (or complement fixing containing
portions
thereof) that bind an inherent or induced endogenous cytotoxic effector
system, thymidine
kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomofzas 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$i, or
other radioisotopes
such aS for exam 1e 103Pd 133xe 1311 68Ge S~Co 65zn $SSr 32P 35S 90Y 153Sm
153Gd
p > > > > > > > > > > > > >
169Yb~ SlCr' S4Mn' ~SSe, 113Sn, gOYttrlum, ll~Tin, 186Rhenlum, 166HOlmlum, and
1$$Rhelllum;
luminescent 1'abels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin. 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 ~°Y. 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.
[387] 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).
[388] 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
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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.
[389] Moreover, polypeptides of the present invention can be used to treat or
prevent
diseases or conditions such as, for example, neural disorders, immune system
disorders,
muscular disorders, reproductive disorders, gastrointestinal disorders,
pulmonary disorders,
cardiovascular disorders, renal disorders, proliferative disorders, and/or
cancerous diseases
and conditions. For example, patients can be administered a polypeptide of the
present
invention in an effort to replace absent or decreased levels of the
polypeptide (e.g., insulin),
to supplement absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S for
hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of
a polypeptide
(e.g., an oncogene or tumor supressor), to activate the activity of a
polypeptide (e.g., by
binding to a receptor), to reduce the activity of a membrane bound receptor by
competing
with it for free ligand (e.g., soluble TNF receptors used in reducing
inflammation), or to
bring about a desired response (e.g., blood vessel growth inhibition,
enhancement of the
immune response to proliferative cells or tissues).
[390] 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).
[391] 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
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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.
Diag~zostic Assays
[392] The compounds of the present invention are useful for diagnosis,
treatment,
prevention and/or prognosis of various disorders in mammals, preferably
humans. Such
disorders include, but are not limited to, those described herein under the
section heading
"Biological Activities".
[393] For a number of disorders, substantially altered (increased or
decreased) levels of
gene expression can be detected in tissues, 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" gene expression level, that is, the expression level
in tissues or
bodily fluids from an individual not having the disorder. Thus, the invention
provides a
diagnostic method useful during diagnosis of a disorder, which involves
measuring the
expression level of the gene encoding the polypeptide in tissues, cells or
body fluid from an
individual and comparing the measured gene expression level with a standard
gene
expression level, whereby an increase or decrease in the gene expression
levels) compared
to the standard is indicative of a disorder. These diagnostic assays may be
performed in
vivo or ih vitro, such as, for example, on blood samples, biopsy tissue or
autopsy tissue.
[394] The present invention is also useful as a prognostic indicator, whereby
patients
exhibiting enhanced or depressed gene expression will experience a worse
clinical outcome
relative to patients expressing the gene at a level nearer the standard level.
[395] In certain embodiments, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose diseases and/or disorders associated with the
tissues) in which
the polypeptide of the invention is expressed, including one, two, three,
four, five, or more
tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[396] By "assaying the expression level of the gene encoding the polypeptide"
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
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level in a second biological sample). Preferably, the polypeptide expression
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 disorder or being determined by
averaging
levels from a population of individuals not having the 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.
[397] By "biological sample" is intended any biological sample obtained from
an
individual, cell line, tissue culture, or other source containing polypeptides
of the invention
(including portions thereof) or mRNA. As indicated, biological samples include
body fluids
(such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue
sources found to
express the full length or fragments thereof of a polypeptide or mRNA. 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.
[398] 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 polypeptides of the invention 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).
[399] The present invention also relates to diagnostic assays such as
quantitative and
diagnostic assays for detecting levels of polypeptides of the invention, 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 polypeptides of the invention compared to normal
control
tissue samples may be used to detect the presence of tumors. Assay techniques
that can be
used to determine levels of a polypeptide, such as a 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 polypeptide levels in a biological sample can occur
using any
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art-known method.
[400] Assaying polypeptide levels in a biological sample can occur using
antibody-based techniques. For example, 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 polypeptide gene expression include immunoassays,
such as
the enzyme linked immunosorbent assay (ELTSA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include enzyme labels,
such as,
glucose oxidase, and radioisotopes, such as iodine (1251, 12i1), carbon (14C),
sulfur (35S),
tritium (3H), indium (112In), and technetium (99mTC), and fluorescent labels,
such as
fluorescein and rhodamine, and biotin.
[401] The tissue or cell type to be analyzed will generally include those
which are
known, or suspected, to express the gene of inteest (such as, for example,
cancer). The
protein isolation methods employed herein may, for example, be such as those
described in
Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory
Manual",
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yorlc), which is
incorporated herein by reference in its entirety. The isolated cells can be
derived from cell
culture or from a patient. The analysis of cells taken from culture may be a
necessary step
in the assessment of cells that could be used as part of a cell-based gene
therapy technique
or, alternatively, to test the effect of compounds on the expression of the
gene.
[402] For example, antibodies, or fragments of antibodies, such as those
described
herein, may be used to quantitatively or qualitatively detect the presence of
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.
[403] In a preferred embodiment, antibodies, or fragments of antibodies
directed to any
one or all of the predicted epitope domains of the polypeptides of the
invention (shown in
column 7 of Table 1A) may be used to quantitatively or qualitatively detect
the presence of
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.
[404] In an additional preferred embodiment, antibodies, or fragments of
antibodies
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directed to a conformational epitope of a polypeptide of the invention may be
used to
quantitatively or qualitatively detect the presence of 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.
[405] The antibodies (or fragments thereof), and/or polypeptides of the
present
invention may, additionally, be employed histologically, as in
immunofluorescence,
immunoelectron microscopy or non-immunological assays, for in situ detection
of 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 polypeptide of the present invention. The antibody (or
fragment
thereof) or 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 gene product, or conserved variants or
peptide
fragments, or 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.
[406] Immunoassays and non-immunoassays for gene products or conserved
variants or
peptide fragments thereof will typically comprise incubating a sample, such as
a biological
fluid, a tissue extract, freshly harvested cells, or lysates of cells which
have been incubated
in cell culture, in the presence of a detectably labeled antibody capable of
binding 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.
[407] The biological sample may be brought in contact with and immobilized
onto a
solid phase support or carrier such as nitrocellulose, or other solid support
which is capable
of immobilizing cells, cell particles or soluble proteins. The support may
then be washed
with suitable buffers followed by treatment with the detectably labeled
antibody or
detectable polypeptide of the invention. 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.
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[408] By "solid phase support or carrier" is intended any support capable of
binding an
antigen or an antibody. WeII-known supports or carriers include glass,
polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and modified
celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier can be
either soluble to
some extent or insoluble for the purposes of the present invention. The
support material
may have virtually any possible structural configuration so long as the
coupled molecule is
capable of binding to an antigen or antibody. Thus, the support configuration
may be
spherical, as in a bead, or cylindrical, as in the inside surface of a test
tube, or the external
surface of a rod. Alternatively, the surface may be flat such as a sheet, test
strip, etc.
Preferred supports include polystyrene beads. Those skilled in the art will
know many
other suitable carriers for binding antibody or antigen, or will be able to
ascertain the same
by use of routine experimentation.
[409] The binding activity of a given lot of antibody or 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.
[410] In addition to assaying polypeptide levels or polynucleotide levels in a
biological
sample obtained from an individual, polypeptide or polynucleotide can also be
detected ih
vivo by imaging. For example, in one embodiment of the invention, polypeptides
and/or
antibodies of the invention are used to image diseased cells, such as
neoplasms. In another
embodiment, polynucleotides of the invention (e.g., polynucleotides
complementary to alI
or a portion o~ an mRNA) and/or antibodies (e.g., antibodies directed to any
one or a
combination of the epitopes of a polypeptide of the invention, antibodies
directed to a
conformational epitope of a polypeptide of the invention, or antibodies
directed to the full
length polypeptide expressed on the cell surface of a mammalian cell) are used
to image
diseased or neoplastic cells.
[411] Antibody labels or markers for in vivo imaging of polypeptides of the
invention
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. 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
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in vivo imaging is used to detect enhanced levels of 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).
[412] Additionally, any polypeptides of the invention whose presence can be
detected,
can be administered. For example, polypeptides of the invention labeled with a
radio-
opaque or other appropriate compound can be administered and visualized in
vivo, as
discussed, above for labeled antibodies. Further, such polypeptides can be
utilized for in
vitro diagnostic procedures.
[413] A polypeptide-specific antibody or antibody fragment which has been
labeled
with an appropriate detectable imaging moiety, such as a radioisotope (for
example, 1311,
112In' 99m.LC), a radio-opaque substance, or a material detectable by nuclear
magnetic
resonance, is introduced (for example, parenterally, subcutaneously or
intraperitoneally)
into the mammal to be examined for a 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 the antigenic 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 Cancers, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing
Inc.
(1982)).
[414] With respect to antibodies, one of the ways in which an antibody of the
present
invention can be detectably labeled is by linking the same to a reporter
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
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Associates Quarterly Publication,.Walkersville, MD); Voller et al., J. Cliyz.
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 reporter 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. Reporter enzymes which
can be used
to detestably 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 reporter enzyme.
Detection may
also be accomplished by visual comparison of the extent of enzymatic reaction
of a
substrate in comparison with similarly prepared standards.
[415] Detection may also be accomplished using any of a vaxiety of other
immunoassays. For example, by radioactively labeling the antibodies or
antibody
fragments, it is possible to detect polypeptides 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. '
[416] It is also possible to label the antibody with a fluorescent compound.
When the
fluorescently labeled antibody is exposed to light of the proper wave length,
its presence
can then be detected due to fluorescence. Among the most commonly used
fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
[417] The antibody can also be detestably 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
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ethylenediaminetetraacetic acid (EDTA).
[418] 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.
[419] Likewise, a bioluminescent compound may be used to label the antibody of
the
present invention. Bioluminescence is a type of chemiluminescence found in
biological
systems in, which a catalytic protein increases the efficiency of the
chemiluminescent
reaction. The presence of a bioluminescent protein is determined by detecting
the presence
of luminescence. Important bioluminescent compounds for purposes of labeling
are
luciferin, luciferase and aequorin.
Methods for Detecting Diseases
[420] In general, a disease may be detected in a patient based on the presence
of one or
more 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 may be used as markers to indicate the presence or
absence of a
disease or disorder, including cancer and/or as described elsewhere herein. In
addition, such
proteins may be useful for the detection of other diseases and cancers. 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 polypeptides of the invention, which is also indicative of the
presence or
absence of a disease or disorder, including cancer. In general, polypeptides
of the invention
should be present at a level that is at least three fold higher in diseased
tissue than in normal
tissue.
[421] There are a variety of assay formats known to those of ordinary skill in
the art fox
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 a 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.
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[422] In a preferred embodiment, the assay involves the use of a binding
agents)
immobilized on a solid support to bind to and remove the polypeptide of the
invention from
the remainder of the sample. The bound polypeptide may then be detected using
a detection
reagent that contains a repoxter 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 polypeptides of the invention and portions
thereof, or
antibodies, to which the binding agent binds, as described above.
[423] The solid support may be any material known to those of skill in the art
to which
polypeptides of the invention may be attached. For example, the solid support
may be a test
well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the
support may be a bead or disc, such as glass fiberglass, latex or a plastic
material such as
polystyrene or polyvinylchloride. The support may also be a magnetic particle
or a fiber
optic sensor, such as those disclosed, for example, in U.S. Patent No.
5,359,681. 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
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ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to
immobilize an
adequate amount of binding agent.
[424] 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 Therapy Methods
[425] Also encompassed by the 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 the polypeptide of the present invention. This
method
requires a polynucleotide which codes for a polypeptide of the present
invention operatively
linked to a promoter and any other genetic elements necessary for the
expression of the
polypeptide by the target tissue. Such gene therapy and delivery techniques
are known in
the art, see, for example, WO90/11092, which is herein incorporated by
reference.
[426] 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-
(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
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reintroduced into the patient through direct injection to the artery, the
tissues surrounding
the artery, or through catheter injection.
[427] 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.
[428] 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 freelfrom 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.
[429] 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, pXTI and pSG available from Stratagene; pSVI~3, pBPV, pMSG and pSVL
available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from
Invitrogen. Other suitable vectors will be readily apparent to the skilled
artisan.
[430] 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;
retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The
promoter also may be the native promoter for the polynucleotide of the present
invention.
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[431] Unlilce 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.
[432] The polynucleotide construct can be delivered to the interstitial space
of tissues
within the an animal, including of muscle, skin, brain, lung, liver, spleen,
bone marrow,
thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder,
stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and
connective tissue.
Interstitial space of the tissues comprises the intercellular, fluid,
mucopolysaccharide matrix
among the reticular fibers of organ tissues, elastic fibers in the walls of
vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within connective
tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space occupied by
the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery to the
interstitial space of
muscle tissue is preferred for the reasons discussed below. They may be
conveniently
delivered by injection into the tissues comprising these cells. They are
preferably delivered
to and expressed in persistent, non-dividing cells which are differentiated,
although delivery
and expression may be achieved in non-differentiated or less completely
differentiated cells,
such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle
cells are
particularly competent in their ability to take up and express
polynucleotides.
[433] 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 slcill will appreciate, this dosage will vary according to the tissue
site of injection.
The appropriate and effective dosage of nucleic acid sequence can readily be
determined by
those of ordinary skill in the art and may depend on the condition being
treated and the route
of administration.
[434j 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,
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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.
[435] 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.
[436] 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.
[437] In certain embodiments, the polynucleotide constructs are complexed in a
liposome preparation. Liposomal preparations for use in the instant invention
include
cationic (positively charged), anionic (negatively charged) and neutral
preparations.
However, cationic liposomes are particularly preferred because a tight charge
complex can
be formed between the cationic liposome and the polyanionic nucleic acid.
Cationic
liposomes have been shown to mediate intracellular delivery of plasmid DNA
(Felgner et
al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein
incorporated by
reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-
6081, which
is herein incorporated by reference); and purified transcription factors (Debs
et al., J. Biol.
Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in
functional
form.
[438] 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).
[439] 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.
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USA 84:7413-7417, which is herein incorporated by reference. Similar methods
can be
used to prepare liposomes from other cationic lipid materials.
[440] 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.
[441] For example, commercially dioleoylphosphatidyl choline (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE)
can be used in various combinations to make conventional liposomes, with or
without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared
by
drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a
sonication
vial. The sample is placed under a vacuum pump overnight and is hydrated the
following
day with deionized water. The sample is then sonicated for 2 hours in a capped
vial, using a
Heat Systems model 350 sonicator equipped with an inverted cup (bath type)
probe at the
maximum setting while the bath is circulated at 15EC. Alternatively,
negatively charged
vesicles can be prepared without sonication to produce multilamellar vesicles
or by
extrusion through nucleopore membranes to produce unilamellar vesicles of
discrete size.
Other methods are known and available to those of skill in the art.
[442] The liposomes can comprise multilamellar vesicles (MLVs), small
unilamellar
vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred. The
various liposome-nucleic acid complexes are prepared using methods well known
in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527,
which is herein
incorporated by reference. For example, MLVs containing nucleic acid can be
prepared by
depositing a thin film of phospholipid on the walls of a glass tube and
subsequently
hydrating with a solution of the material to be encapsulated. SUVs are
prepared by
extended sonication of MLVs to produce a homogeneous population of unilamellar
liposomes. The material to be entrapped is added to a suspension of preformed
MLVs and
then sonicated. When using liposomes containing cationic lipids, the dried
lipid film is
resuspended in an appropriate solution such as sterile water or an isotonic
buffer solution
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such as 10 mM Tris/NaCI, sonicated, and then the preformed liposomes are mixed
directly
with the DNA. The liposome and DNA form a very stable complex due to binding
of the
positively charged liposomes to the cationic DNA. SUVs find use with small
nucleic acid
fragments. LUVs are prepared by a number of methods, well known in the art.
Commonly
used methods include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim.
Biophys.
Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection
(Deamer, D. and
Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem.
Biophys.
Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348
(1979));
detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA
76:145
(1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem.
255:10431
(1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145
(1978);
Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated
by
reference.
[443] Generally, the ratio of DNA to liposomes will be from about 10:1 to
about 1:10.
Preferably, the ration will be from about 5:1 to about 1:5. More preferably,
the ration will
be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
[444] U.S. Patent No. 5,676,954 (which is herein incorporated by reference)
reports on
the injection of genetic material, complexed with cationic liposomes carriers,
into mice.
U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466,
5,693,622,
5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are
herein
incorporated by reference) provide cationic Lipids for use in transfecting DNA
into cells and
mammals. U.S. Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and
international
publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid
complexes
to mammals.
[445] In certain embodiments, cells are engineered, ex vivo or in vivo, using
a retroviral
particle containing RNA which comprises a sequence encoding a polypeptide of
the present
invention. Retroviruses from which the retroviral plasmid vectors may be
derived include,
but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus,
Rous
sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia
virus,
human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary
tumor
virus.
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[446] The retroviral plasmid vector is employed to transduce packaging cell
lines to
form producer cell lines. Examples of packaging cells which may be transfected
include,
but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-
H2,
RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller,
Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in
its
entirety. The vector may transduce the packaging cells through any means known
in the
art. ~ Such means include, but are not limited to, electroporation, the use of
liposomes, and
CaP04 precipitation. In one alternative, the retroviral plasmid vector may be
encapsulated
into a liposome, or coupled to a lipid, and then administered to a host.
[447] The producer cell line generates infectious retroviral vector particles
which
include polynucleotide encoding a polypeptide of the present invention. Such
retroviral
vector particles then may be employed, to transduce eukaryotic cells, either
in vitro or in
vivo. The transduced eukaryotic cells will express a polypeptide of the
present invention.
[448] In certain other embodiments, cells are engineered, ex vivo or in vivo,
with
polynucleotide contained in an adenovirus vector. Adenovirus can be
manipulated such
that it encodes and expresses a polypeptide of the present invention, and at
the same time is
inactivated in terms of its ability to replicate in a normal lytic viral life
cycle. Adenovirus
expression is achieved without integration of the viral DNA into the host cell
chromosome,
thereby alleviating concerns about insertional mutagenesis. Furthermore,
adenoviruses have
been used as live enteric vaccines for many years with an excellent safety
profile (Schwartz
et al. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated
gene
transfer has: been demonstrated in a number of instances including transfer of
alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et
al. (1991)
Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore,
extensive
studies to attempt to establish adenovirus as a causative agent in human
cancer were
uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA
76:6606).
[449] Suitable adenoviral vectors useful in the present invention are
described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993);
Rosenfeld
et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-
769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692
(1993); and
U.S. Patent No. 5,652,224, which are herein incorporated by reference. For
example, the
adenovirus vector Ad2 is useful and can be grown in human 293 cells. These
cells contain
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the E1 region of adenovirus and constitutively express Ela and Elb, which
complement the
defective adenoviruses by providing the products of the genes deleted from the
vector. In
addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are
also useful in
the present invention.
[450] Preferably, the adenoviruses used in the present invention are
replication
deficient. Replication deficient adenoviruses require the aid of a helper
virus and/or
packaging cell line to form infectious particles. The resulting virus is
capable of infecting
cells and can express a polynucleotide of interest which is operably linked to
a promoter,
but cannot replicate in most cells. Replication deficient adenoviruses may be
deleted in one
or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or
L1 through L5.
[451] In certain other embodiments, the cells are engineered, ex vivo or ifa
vivo, using
an adeno-associated virus (AAV). AAVs are naturally occurring defective
viruses that
require helper viruses to produce infectious particles (Muzyczka, N., Curr.
Topics in
Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may
integrate its
DNA into non-dividing cells. Vectors containing as little as 300 base pairs of
AAV can be
packaged and can integrate, but space for exogenous DNA is limited to about
4.5 kb.
Methods forproducing and using such AAVs are known in the art. See, for
example, U.S.
Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745,
and
5,589,377.
[452] For example, an appropriate AAV vector for use in the present invention
will
include all the sequences necessary for DNA replication, encapsidation, and
host-cell
integration. The polynucleotide construct is inserted into the AAV vector
using standard
cloning methods, such as those found in Sambrook et al., Molecular Cloning: A
Laboratory
Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then
transfected into packaging cells which are infected with a helper virus, using
any standard
technique, including lipofection, electroporation, calcium phosphate
precipitation, etc.
Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia
viruses, or
herpes viruses. Once the packaging cells are transfected and infected, they
will produce
infectious AAV viral particles which contain the polynucleotide construct.
These viral
particles are then used to transduce eukaryotic cells, either ex vivo or in
vivo. The
transduced cells will contain the polynucleotide construct integrated into its
genome, and
will express a polypeptide of the invention.
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[453] Another method of gene therapy involves operably associating
heterologous
control regions and endogenous polynucleotide sequences (e.g. encoding a
polypeptide of
the present invention) via homologous recombination (see, e.g., U.S. Patent
No. 5,641,670,
issued June 24, 1997; International Publication No. WO 96/29411, published
September
26, 1996; International Publication No. WO 94/12650, published August 4, 1994;
Koller et
al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,
Nature
342:435-438 (1989), which are herein encorporated by reference. This method
involves the
activation of a gene which is present in the target cells, but which is not
normally expressed
in the cells, or is expressed at a lower level than desired.
[454] Polynucleotide constructs are made, using standard techniques known in
the art,
which contain the promoter with targeting sequences flanking the promoter.
Suitable
promoters are described herein. The targeting sequence is sufficiently
complementary to an
endogenous sequence to permit homologous recombination of the promoter-
targeting
sequence with the endogenous sequence. The targeting sequence will be
sufficiently near
the 5' end of the desired endogenous polynucleotide sequence so the promoter
will be
operably linked to the endogenous sequence upon homologous recombination.
[455] The promoter and the targeting sequences can be amplified using PCR.
Preferably, the amplified promoter contains distinct restriction enzyme sites
on the 5' and 3'
ends. Preferably, the 3' end of the first targeting sequence contains the same
restriction
enzyme site as the 5' end of the amplified promoter and the 5' end of the
second targeting
sequence contains the same restriction site as the 3' end of the amplified
promoter: The
amplified promoter and targeting sequences are digested and ligated together.
[456] The promoter-targeting sequence construct is delivered to the cells,
either as
naked polynucleotide, or in conjunction with transfection-facilitating agents,
such as
liposomes, viral sequences, viral particles, whole viruses, lipofection,
precipitating agents,
etc., described in more detail above. The P promoter-targeting sequence can be
delivered
by any method, included direct needle injection, intravenous injection,
topical
administration, catheter infusion, particle accelerators, etc. The methods are
described in
more detail below.
[457] The promoter-targeting sequence construct is taken up by cells.
Homologous
recombination between the construct and the endogenous sequence takes place,
such that an
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endogenous sequence is placed under the control of the promoter. The promoter
then drives
the expression of the endogenous sequence.
[4.58] The polynucleotide encoding a polypeptide of the present invention may
contain
a secretory signal sequence that facilitates secretion of the protein.
Typically, the signal
sequence is positioned in the coding region of the polynucleotide to be
expressed towards or
at the 5' end of the coding region. The signal sequence may be homologous or
heterologous
tb the polynucleotide of interest and may be homologous or heterologous to the
cells to be
transfected. Additionally, the signal sequence may be chemically synthesized
using
methods known in the art.
[459] Any mode of administration of any of the above-described polynucleotides
constructs can be used so long as the mode results in the expression of one or
more
molecules in an amount sufficient to provide a therapeutic effect. This
includes direct
needle injection, systemic injection, catheter infusion, biolistic injectors,
particle
accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially
available depot
materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill)
pharmaceutical formulations, and decanting or topical applications during
surgery. For
example, direct injection of naked calcium phosphate-precipitated plasmid into
rat liver and
rat spleen or a protein-coated plasmid into the portal vein has resulted in
gene expression of
the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).
[460] A preferred method of local administration is by direct injection.
Preferably, a
recombinant molecule of the present invention complexed with a delivery
vehicle is
administered by direct injection into or locally within the area of arteries.
Administration of
a composition locally within the area of arteries refers to injecting the
composition
centimeters and preferably, millimeters within arteries.
[461] Another method of local administration is to contact a polynucleotide
construct
of the present invention in or around a surgical wound. For example, a patient
can undergo
surgery and the polynucleotide construct can be coated on the surface of
tissue inside the
wound or the construct can be injected into areas of tissue inside the wound.
[462] Therapeutic compositions useful in systemic administration, include
recombinant
molecules of the present invention complexed to a targeted delivery vehicle of
the present
invention. Suitable delivery vehicles for use with systemic administration
comprise
liposomes comprising ligands for targeting the vehicle to a particular site.
In specific
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embodiments, suitable delivery vehicles for use with systemic administration
comprise
liposomes comprising polypeptides of the invention for targeting the vehicle
to a particular
site.
[463] Preferred methods of systemic administration, include intravenous
injection,
aerosol, oral and percutaneous (topical) delivery. Intravenous injections can
be performed
using methods standard in the art. Aerosol delivery can also be performed
using methods
standard in the art (see, for example, Stribling et al., Proc. Natl. Acad.
Sci. USA
189:11277-11281, 1992, which is incorporated herein by reference). Oral
delivery can be
performed by complexing a polynucleotide construct of the present invention to
a carrier
capable of withstanding degradation by digestive enzymes in the gut of an
animal.
Examples of such carriers, include plastic capsules or tablets, such as those
known in the
art. Topical delivery can be performed by mixing a polynucleotide construct of
the present
invention with a lipophilic reagent (e.g., DMSO) that is capable of passing
into the slcin.
[464] Determining an effective amount of substance to be delivered can depend
upon a
number of factors including, for example, the chemical structure and
biological activity of
the substance, the age and weight of the animal, the precise condition
requiring treatment
and its severity, and the route of administration. The frequency of treatments
depends upon
a number of factors, such as the amount of polynucleotide constructs
administered per dose,
as well as the health and history of the subject. The precise amount, number
of doses, and
timing of doses will be determined by the attending physician or veterinarian.
[465] Therapeutic compositions of the present invention can be administered to
any
animal, preferably to mammals and birds. Preferred mammals include humans,
dogs, cats,
mice, rats, rabbits sheep, cattle, horses and pigs, with humans being
particularly preferred.
Biological Activities
[466] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, can be used in assays to test for one or more biological
activities. If these
polynucleotides or polypeptides, or agonists or antagonists of the present
invention do
exhibit activity in a particular assay, it is likely that these molecules may
be involved in the
diseases associated with the biological activity. Thus, the polynucleotides
and
polypeptides, and agonists or antagonists could be used to treat the
associated disease.
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[467] Gastrointestinal-associated proteins are believed to be involved in
biological
activities associated with the function and/or maintanence of the
gastrointestinal system.
Accordingly, compositions of the invention (including polynucleotides,
polypeptides and
antibodies of the invention, and fragments and variants thereof) may be used
in the
diagnosis, prognosis, prevention, and/or treatment of diseases and/or
disorders associated
with gastrointestinal system.
[468] In preferred embodiments, compositions of the invention (including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) may be used in the diagnosis, prognosis, prevention, and/or treatment
of diseases
and/or disorders relating to the gastrointestinal system (e.g., Crohn's
disease, pancreatitis,
gallstones, antibiotic-associated colitis, duodenitis, gastrointestinal
neoplasms, and/or as
described in the sections entitled "Gastrointestinal Disorders",
"Hyperproliferative
Disorders", and "Immune Activity").
[469] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose, prognose, prevent, and/or treat gastrointestinal disorders
associated with the
tissues) in which the polypeptide of the invention is expressed, including
one, two, three,
four, five, or more tissues disclosed in Table 1A, coluriln 8 ("Tissue
Distribution").
[470] More generally, polynucleotides, translation products and antibodies
corresponding to this gene may be useful for the diagnosis, prognosis,
prevention, and/or
treatment of diseases and/or disorders associated with the following systems:
Gastrointestinal Disorders
[471] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or prognose
gastrointestinal
disorders, including inflammatory diseases and/or conditions, infections,
cancers (e.g.,
intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodglcin's
lymphoma of
the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
[472j Gastrointestinal disorders include dysphagia, odynophagia, inflammation
of the
esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and
stricturing, Mallory-
Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas,
gastric retention
disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps
of the stomach,
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autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis
(bacterial, viral,
eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and
Menetrier's), and
peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst,
mesenteric
lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms,
peritonitis,
pneumoperitoneum, bubphrenic abscess,).
[473] Gastrointestinal disorders also include disorders associated with the
small
intestine, such as malabsorption syndromes, distension, irritable bowel
syndrome, sugar
intolerance, celiac disease, duodenal ulcers, duodenitis, tropical spree,
Whipple's disease,
intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of
the ileum,
Meckel's diverticulum, multiple diverticula, failure of complete rotation of
the small and
large intestine, lymphoma, and bacterial and parasitic diseases (such as
Traveler's diarrhea,
typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis
lumbricoides),
Hookworms (An.cylostoma duodenale), Threadworms (Ercterobius
vermicula~°is),
Tapeworms (Taenia saginata, Echihococcus g~anulosus, Diphyllobothrium spp.,,
and T.
solium).
[474] Liver diseases and/or disorders include intrahepatic cholestasis
(alagille
syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye
syndrome), .hepatic
vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary
syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric
varices), liver
abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and
experimental),
alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic
(hepatic echinococcosis,
fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and
cholestatic),
cholestasis, portal hypertension, liver enlargement, ascites, hepatitis
(alcoholic hepatitis,
animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C,
hepatitis D, drug
induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B,
hepatitis C,
hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis,
secondary biliary
cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic
encephalopathy,
primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular
adenoma,
hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver
failure), and
liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver
.metastases,
epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia,
hepatic
adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma,
hepatoma,
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liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal
tumors
of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts
[Simple cysts,
Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst],
Mesenchymal
tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma,
Peliosis
hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors
[Bile duct
epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma,
Focal
nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver
tumors
[hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular,
cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma,
Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma,
fibrosarcoma; leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma,
carcinoid,
squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic
porphyria,
hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda),
Zellweger
syndrome).
[475] Pancreatic diseases and/or disorders include acute pancreatitis, chronic
pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis),
neoplasms
(adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma,
and
glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other
pancreatic
diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic
fistula, insufficiency)).
[476] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis),
postcholecystectomy syndrome, diverticulosis of the gallbladder, acute
cholecystitis,
chronic cholecystitis, bile duct tumors, and mucocele.
[477] Diseases and/or disorders of the large intestine include antibiotic-
associated
colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses,
fungal and bacterial
infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic
diseases (colitis, colonic
neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma),
colon
carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis,
megacolon
[Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis,
sigmoin
neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea,
dysentery),
duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer,
duodenitis),
enteritis (enterocolitis), HIV enteropathy, ileal diseases (deal neoplasms,
ileitis),
immunoproliferative small intestinal disease, inflammatory bowel disease
(ulcerative
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colitis, Crohn's disease), intestinal atresia, parasitic diseases
(anisakiasis, balantidiasis,
blastocystis infections, cryptosporidiosis, dientamoebiasis, amebic dysentery,
giardiasis),
intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms,
colonic neoplasms,
duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms,
rectal
neoplasms), intestinal obstruction (afferent loop syndrome, duodenal
obstruction, impacted
feces, intestinal pseudo-obstruction [cecal volvulus], intussusception),
intestinal
perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-
jeghers syndrome),
jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop
syndrome, celiac
disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's
disease),
mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-
losing
enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases,
fecal incontinence,
hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic
ulcer (duodenal
ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-
Ellison
syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases
(e.g.,
achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular
ectasia, gastric
fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic),
gastroparesis, stomach
dilatation, stomach diverticulum, stomach rieoplasms (gastric cancer, gastric
polyps, gastric
adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer,
stomach
volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis,
hyperemesis
gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.
[478] Further diseases and/or disorders of the gastrointestinal system include
biliary
tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula,
esophageal fistula, gastric
fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary
tract neoplasms,
esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal
squamous
cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as
adenocarcinoma
of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic
neoplasms,
pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g.,
bullous diseases,
candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices,
atresia, cyst,
diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal
fistula), motility
disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm,
gastroesophageal
reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss
syndrome),
stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia);
gastrointestinal diseases,
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such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection),
hemorrhage (e.g.,
hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric
cancer, gastric
polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital
diaphragmatic
hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia,
ventral hernia),
and intestinal diseases (e.g., cecal diseases (appendicitis, cecal
neoplasms)).
Immune Activity
[479] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, diagnosing and/or
prognosing
diseases, disorders, and/or conditions of the immune system, by, for example,
activating or
inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of
immune cells.
Immune cells develop through a process called hematopoiesis, producing myeloid
(platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and
T
lymphocytes) cells from pluripotent stem cells. The etiology of these immune
diseases,
disorders, and/or conditions may be genetic, somatic, such as cancer and some
autoimmune
diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention can be used as a marker or detector of a particular immune system
disease or
disorder.
[480] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to treat
diseases and disorders of the immune system and/or to inhibit or enhance an
immune
response generated by cells associated with the tissues) in which the
polypeptide of the
invention is expressed, including one, two, three, four, five, or more tissues
disclosed in
Table 1A, column 8 (Tissue Distribution Library Code).
[481] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, diagnosing, and/or
prognosing
immunodeficiencies, including both congenital and acquired immunodeficiencies.
Examples of B cell ~immunodeficiencies in which immunoglobulin levels B cell
function
and/or B cell numbers are decreased include: X-linked agammaglobulinemia
(Breton's
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disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency
with hyper
IgM, non X-linked immunodeficiency with hyper IgM, X-linked
lymphoproliferative
syndrome (XLP), agammaglobulinemia including congenital and acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, unspecified
hypogammaglobulinemia,
recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective
IgA
deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with
or without
IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with
increased
IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain
deletions, kappa chain
deficiency, B cell lymphoproliferative disorder (BLPD), common variable
immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired),
and
transient hypogammaglobulinemia of infancy.
[482] In specific embodiments, ataxia-telangiectasia or conditions associated
with
ataxia-telangiectasia are treated, prevented, diagnosed, and/or prognosing
using the
polypeptides or polynucleotides of the invention, and/or agonists or
antagonists thereof.
[483] Examples of congenital immunodeficiencies in which T cell and/or B cell
function andlor number is decreased include, but are not limited to: DiGeorge
anomaly,
severe combined immunodeficiencies (SCID) (including, but not limited to, X-
linked SCID,
autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside
phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte
syndrome),
Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third
and fourth
pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous
candidiasis, natural
killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia,
immunodeficiency with
predominant T cell defect (unspecified), and unspecified immunodeficiency of
cell
mediated immunity.
[484] In specific embodiments, DiGeorge anomaly or conditions associated with
DiGeorge anomaly are treated, 'prevented, diagnosed, and/or prognosed using
polypeptides
or polynucleotides of the invention, or antagonists or agonists thereof.
[485] Other immunodeficiencies that may be treated, prevented, diagnosed,
and/or
prognosed using polypeptides or polynucleotides of the invention, and/or
agonists or
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antagonists thereof, include, but are not limited to, chronic granulomatous
disease, Chedialc-
Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate
dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP),
leukocyte
adhesion deficiency, complement component deficiencies (including C1, C2, C3,
C4, C5,
C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-
aplasia,
immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with
immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof
syndrome-
combined immunodeficiency with Igs.
[486] In a preferred embodiment, the immunodeficiencies and/or conditions
associated
with the immunodeficiencies recited above are treated, prevented, diagnosed
and/or
prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention.
[487] In a preferred embodiment polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention could be used as an agent to
boost
immunoresponsiveness among immunodeficient individuals. In specific
embodiments,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention could be used as an agent to boost immunoresponsiveness among B cell
and/or T
cell immunodeficient individuals.
[488J The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in treating, preventing, diagnosing andlor
prognosing
autoimmune disorders. Many autoimmune disorders result from inappropriate
recognition
of self as foreign material by immune cells. This inappropriate recognition
results in an
immune response leading to the destruction of the host tissue. Therefore, the
administration
of polynucleotides and polypeptides of the invention that can inhibit an
immune response,
particularly the proliferation, differentiation, or chemotaxis of T-cells, may
be an effective
therapy in preventing autoimmune disorders.
[489] Autoimmune diseases or disorders that may be treated, prevented,
diagnosed
and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists
or
antagonists of the present invention include, but are not limited to, one or
more of the
following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing
spondylitis,
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multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis,
autoimmune hemolytic
anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia
purpura,
autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura,
purpura
(e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's
syndrome,
Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and
insulin-
resistant diabetes mellitus.
[490] Additional disorders that are likely to have an autoimmune component
that may
be treated, prevented, and/or diagnosed with the compositions of the invention
include, but
are not limited to, type II collagen-induced arthritis, antiphospholipid
syndrome, dermatitis,
allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic
heart disease,
neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff
Man Syndrome,
autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin
dependent
diabetes mellitus, and autoimmune inflammatory eye disorders.
[491] Additional disorders that are likely to have an autoimmune component
that may
be treated, prevented, diagnosed and/or prognosed with the compositions of the
invention
include, but are not limited to, scleroderma with anti-collagen antibodies
(often
characterized, e.g., by nucleolar and other nuclear antibodies), mixed
connective tissue
disease (often characterized, e.g., by antibodies to extractable nuclear
antigens (e.g.,
ribonucleoprotein)), polymyositis . (often characterized, e.g., by nonhistone
ANA),
pernicious anemia (often characterized, e.g., by antiparietal cell,
microsomes, and intrinsic
factor antibodies), idiopathic Addison's disease (often characterized, e.g.,
by humoral and
cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by
antispermatozoal
antibodies), glomerulonephritis (often characterized, e.g., by glomerular
basement
membrane antibodies or immune complexes), bullous pemphigoid (often
characterized, e.g.,
by IgG and complement in basement membrane), Sjogren's syndrome (often
characterized,
e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)),
diabetes
mellitus (often characterized, e.g., by cell-mediated and humoral islet cell
antibodies), and
adrenergic drug resistance (including adrenergic drug resistance with asthma
or cystic
fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
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[492] Additional disorders that may have an autoimmune component that may be
treated, prevented, diagnosed and/or prognosed with the compositions of the
invention
include, but are not limited to, chronic active hepatitis (often
characterized, e.g., by smooth
muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by
mitochondria
antibodies), other endocrine gland failure (often characterized, e.g., by
specific tissue
antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte
antibodies),
vasculitis (often characterized, e.g., by Ig and complement in vessel walls
and/or low serum
complement), post-MI (often characterized, e.g., by myocardial antibodies),
cardiotomy
syndrome (often characterized, e.g., by myocardial antibodies), urticaria
(often
characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis
(often
characterized, e.g., by IgG and IgM antibodies to IgE); asthma (often
characterized, e.g., by
IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous,
degenerative, and atrophic disorders.
[493] In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated, prevented,
diagnosed and/or prognosed using for example, antagonists or agonists,
polypeptides or
polynucleotides, or antibodies of the present invention. In a specific
preferred embodiment,
rheumatoid arthritis is treated, prevented, and/or diagnosed using
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention.
[494] In another specific preferred embodiment, systemic lupus erythematosus
is
treated, prevented, and/or diagnosed using polynucleotides, polypeptides,
antibodies, and/or
agonists or antagonists of the present invention. In another specific
preferred embodiment,
idiopathic thrombocytopenia purpura is treated, prevented, and/or diagnosed
using
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention.
[495] In another specific preferred embodiment IgA nephropathy is treated,
prevented,
and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention.
[496] In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated, prevented,
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diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention
[497] In preferred embodiments, polypeptides, antibodies, polynucleotides
and/or
agonists or antagonists of the present invention are used as a
immunosuppressive agent(s).
[498] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, prognosing, and/or -
diagnosing
diseases, disorders, and/or conditions of hematopoietic cells.
Polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention could be
used to increase
differentiation and proliferation of hematopoietic cells, including the
pluripotent stem cells,
in an effort to treat or prevent those diseases, disorders, and/or conditions
associated with a
decrease in certain (or many) types hematopoietic cells, including but not
limited to,
leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively,
Polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention could be
used to increase differentiation and proliferation of hematopoietic cells,
including the
pluripotent stem cells, in an effort to treat or prevent those diseases,
disorders, and/or
conditions associated with an increase in certain (or many) types of
hematopoietic cells,
including but not limited to, histiocytosis.
[499] Allergic reactions and conditions, such as asthma (particularly allergic
asthma) or
other respiratory problems, may also be treated, prevented, diagnosed and/or
prognosed
using polypeptides, antibodies, or polynucleotides of the invention, and/or
agonists or
antagonists thereof. Moreover, these molecules can be used to treat, prevent,
prognose,
and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or
blood group
incompatibility.
[500] Additionally, polypeptides or polynucleotides of the invention, and/or
agonists or
antagonists thereof, may be used to treat, prevent, diagnose and/or prognose
IgE-mediated
allergic reactions. Such allergic reactions include, but are not limited to,
asthma, rhinitis,
and eczema. In specific embodiments, polynucleotides, polypeptides,
antibodies, and/or
agonists or antagonists of the present invention may be used to modulate IgE
concentrations
in vitro or in vivo.
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[501] Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention have uses in the diagnosis, prognosis,
prevention,
and/or treatment of inflammatory conditions. For example, since polypeptides,
antibodies,
or polynucleotides of the invention, and/or agonists or antagonists of the
invention may
inhibit the activation, proliferation and/or differentiation of cells involved
in an
inflammatory response, these molecules can be used to prevent and/or treat
chronic and
acute inflammatory conditions. Such inflammatory conditions include, but are
not limited
to, for example, inflammation associated with infection (e.g., septic shock,
sepsis, or
systemic inflammatory response syndrome), ischemia-reperfusion injury,
endotoxin
lethality, complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine
induced lung injury, inflammatory bowel disease, Crohn's disease, over
production of
cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and
allergy);
gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g.,
gastric, ovarian,
lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis;
ischemic brain
injury and/or stroke, traumatic brain injury, neurodegenerative disorders
(e.g., Parkinson's
disease and Alzheimer's disease); AIDS-related dementia; and prion disease);
cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular
disease, and
cardiopulmonary bypass complications); as well as many additional diseases,
conditions,
and disorders that are characterized by inflammation (e.g., hepatitis,
rheumatoid arthritis,
gout, trauma, pancreatitis, sarcoidosis, dermatitis, renal ischemia-
reperfusion injury,
Grave's disease, systemic lupus erythematosus, diabetes mellitus, and
allogenic transplant
rejection).
[502] Because inflammation is a fundamental defense mechanism, inflammatory
disorders can effect virtually any tissue of the body. Accordingly,
polynucleotides,
polypeptides, and antibodies of the invention, as well as agonists or
antagonists thereof,
have uses in the treatment of tissue-specific inflammatory disorders,
including, but not
limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis,
balanitis, blepharitis,
bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis,
chorditis, cochlitis, colitis,
conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis,
endocarditis, esophagitis,
eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis,
glossitis,
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hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis,
media otitis,
meningitis, metritis, mucitis, myocarditis, myosititis, ~myringitis,
nephritis, neuritis, orchitis,
osteochondritis, otitis, pericarditis, peritendonitis, peritonitis,
pharyngitis, phlebitis,
poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis,
scleritis, sclerochoroiditis,
scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis,
syringitis, tendonitis,
tonsillitis, urethritis, and vaginitis.
[503] In specific embodiments, polypeptides, antibodies, or polynucleotides of
the
invention, and/or agonists or antagonists thereof, are useful to diagnose,
prognose, prevent,
and/or treat organ transplant rejections and graft-versus-host disease. Organ
rejection
occurs by host immune cell destruction of the transplanted tissue through an
immune
response. Similarly, an immune response is also involved in GVHD, but, in this
case, the
foreign transplanted immune cells destroy the host tissues. ~Polypeptides,
antibodies, or
polynucleotides of the invention, andlor agonists or antagonists thereof, that
inhibit an
immune response, particularly the activation, proliferation, differentiation,
or chemotaxis of
T-cells, may be an effective therapy in preventing organ rejection or GVHD. In
specific
embodiments, polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists
or antagonists thereof, that inhibit an immune response, particularly the
activation,
proliferation, differentiation, or chemotaxis of T-cells, may be an effective
therapy in
preventing experimental allergic and hyperacute xenograft rejection.
[504] In other embodiments, polypeptides, antibodies, or polynucleotides of
the
invention, and/or agonists or antagonists thereof, are useful to diagnose,
prognose, prevent,
and/or treat immune complex diseases, including, but not limited to, serum
sickness, post
streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-
induced
vasculitis.
[505] Polypeptides, antibodies, polynucleotides and/or agonists or antagonists
of the
invention can be used to treat, detect, and/or prevent infectious agents. For
example, by
increasing the immune response, particularly increasing the proliferation
activation andlor
differentiation of B and/or T cells, infectious diseases may be treated,
detected, and/or
prevented. The immune response may be increased by either enhancing an
existing
immune response, or by initiating a new immune response. Alternatively,
polynucleotides,
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polypeptides, antibodies, and/or agonists or antagonists of the present
invention may also
directly inhibit the infectious agent (refer to section of application listing
infectious agents,
etc), without necessarily eliciting an immune response.
[506] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are used as a vaccine adjuvant that
enhances immune
responsiveness to an antigen. In a specific embodiment, polypeptides,
antibodies,
polynucleotides and/or agonists or antagonists of the present invention are
used as an
adjuvant to enhance tumor-specific immune responses.
[507] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an adjuvant to
enhance anti-viral
immune responses. Anti-viral immune responses that may be enhanced using the
compositions of the invention as an adjuvant, include virus and virus
associated diseases or
symptoms described herein or otherwise known in the art. In specific
embodiments, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a
virus, disease, . or symptom selected from the group consisting of: AIDS,
meningitis,
Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific
embodiment, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a
virus, disease, or symptom selected from the group consisting of: HIV/AIDS,
respiratory
syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and
B,
parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift
Valley Fever,
herpes simplex, and yellow fever.
[508] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an adjuvant to
enhance anti-
bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal
immune responses
that may be enhanced using the compositions of the invention as an adjuvant,
include
bacteria or fungus and bacteria or fungus associated diseases or symptoms
described herein
or otherwise known in the art. In specific embodiments, the compositions of
the invention
are used as an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or
symptom selected from the group consisting of: tetanus, Diphtheria, botulism,
and
meningitis type B.
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[509] In another specific embodiment, the compositions of the invention are
used as an
adjuvant to enhance an immune response to a bacteria or fungus, disease, or
symptom
selected from the group consisting of: Vibrio cholerae, Mycobacterium
lep~°ae, Salmonella
typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae,
Group B
streptococcus, SlZigella spp., Enterotoxigenic Eschericlzia coli,
Enterohemorrhagic E. coli,
and Borrelia burgdorfe~°i.
[510] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an adjuvant to
enhance anti-
parasitic immune responses. Anti-parasitic immune responses that may be
enhanced using
the compositions of the invention as an adjuvant, include parasite and
parasite associated
diseases or symptoms described herein or otherwise known in the art. In
specific
embodiments, the compositions of the invention are used as an adjuvant to
enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the
invention are used as an adjuvant to enhance an immune response to Plasmodium
(malaria)
or Leishmania.
[511] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may also be employed to treat
infectious
diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis;
for example, by
preventing the recruitment and activation of mononuclear phagocytes.
[512] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an antigen for
the generation of
antibodies to inhibit or enhance immune mediated responses against
polypeptides of the
invention.
[513] In one embodiment, polypeptides, antibodies, polynucleotides and/or
agonists or
antagonists of the present invention are administered to an animal (e.g.,
mouse, rat, rabbit,
hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep,
dog, cat, non-
human primate, and human, most preferably human) to boost the immune system to
produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM,
and IgE), to
induce higher affinity antibody production and immunoglobulin class switching
(e.g., IgG,
IgA, IgM, and IgE), and/or to increase an immune response.
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[514] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a stimulator of B
cell
responsiveness to pathogens.
[515] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an activator of T
cells.
[516] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent that
elevates the
immune status of an individual prior to their receipt of immunosuppressive
therapies.
[517] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
induce higher
affinity antibodies.
[518] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
increase serum
irnmunoglobulin concentrations.
[519] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
accelerate recovery
of immunocompromised individuals.
[520] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to boost
immunoresponsiveness among aged populations and/or neonates.
[521] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an immune system
enhancer
prior to, during, or after bone marrow transplant and/or other transplants
(e.g., allogeneic or
xenogeneic organ transplantation). With respect to transplantation,
compositions of the
invention may be administered prior to, concomitant with, and/or after
transplantation. In a
specific embodiment, compositions of the invention are administered after
transplantation,
prior to the beginning of recovery of T-cell populations. In another specific
embodiment,
compositions of the invention are first administered after transplantation
after the beginning
of recovery of T cell populations, but prior to full recovery of B cell
populations.
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[522] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to boost
immunoresponsiveness among individuals having an acquired Ioss of B cell
function.
Conditions resulting in an acquired loss of B cell function that may be
ameliorated or
treated by administering the polypeptides, antibodies, polynucleotides and/or
agonists or
antagonists thereof, include, but are not limited to, HIV Infection,. AIDS,
bone marrow
transplant, and B cell chronic Iymphocytic leukemia (CLL).
[523] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to boost
immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated
or treated
by administering the polypeptides, antibodies, polynucleotides and/or agonists
ox
antagonists thereof, include, but are not limited to, recovery from viral
infections (e.g.,
influenza), conditions associated with malnutrition, recovery from infectious
mononucleosis, or conditions associated with stress, recovery from measles,
recovery from
blood transfusion, and recovery from surgery.
[524] In another specific embodiment, polypaptides, antibodies,
polynucleotides andlor
agonists or antagonists of the present invention are used as a regulator of
antigen
presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention enhance antigen presentation or antagonizes antigen presentation in
vitro or in
vivo. Moreover, in related embodiments, said enhancement or antagonism of
antigen
presentation may be useful as an anti-tumor treatment or to modulate the
immune system.
[525] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
direct an individual's
immune system towards development of a humoral response (i.e. TH2) as opposed
to a TH1
cellular response.
[526] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means to induce
tumor
proliferation and thus make it more susceptible to anti-neoplastic agents. For
example,
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multiple myeloma is a slowly dividing disease and is thus refractory to
virtually all' anti-
neoplastic regimens. If these cells were forced to proliferate more rapidly
their
susceptibility profile would likely change.
[527] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a stimulator of B
cell production
in pathologies such as AIDS, chronic lymphocyte disorder and/or Common
Variable
Immunodificiency.
[528] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
generation and/or
regeneration of lymphoid tissues following surgery, trauma or genetic defect.
In another
specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists
or
antagonists of the present invention are used in the pretreatment of bone
marrow samples
prior to transplant.
[529] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention axe used as a gene-based
therapy for
genetically inherited disorders resulting in immuno-
incompetence/immunodeficiency such
as observed among SLID patients.
[530] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or'. antagonists of the present invention are used as a means of
activating
monocytes/rnacrophages to defend against parasitic diseases that effect
monocytes such as
Leishmania.
[531] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
regulating secreted
cytokines that are elicited by polypeptides of the invention.
[532] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are used in one or more of the
applications decribed
herein, as they may apply to veterinary medicine. .
[533] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
blocking various
aspects of immune responses to foreign agents or self. Examples of diseases or
conditions
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in which blocking of certain aspects of immune responses may be desired
include
autoimmune disorders such as lupus, and arthritis, as well as
immunoresponsiveness to skin
allergies, inflammation, bowel disease, injury and diseases/disorders
associated with
pathogens.
[534) In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
preventing the B
cell proliferation and Ig secretion associated with autoimmune diseases such
as idiopathic
thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
[535] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention axe used as a inhibitor of B
and/or T cell
rni.gration in endothelial cells. This activity disrupts tissue architecture
or cognate
responses and is useful, for example in disrupting immune responses, and
blocking sepsis.
[536) In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
chronic
hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of
undetermined significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonal
gammopathies, and plasmacytomas.
(537] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may be employed for instance
to iilhibit
polypeptide chemotaxis and activation of macrophages and their precursors, and
of
neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated
and CD8
cytotoxic T cells and natural killer cells, in certain autoimmune and chronic
inflammatory
and infective diseases. Examples of autoimmune diseases are described herein
and include
multiple sclerosis, and insulin-dependent diabetes.
[538] The polypeptides, antibodies, polynucleotides andlor agonists or
antagonists of
the present invention may also be employed to treat idiopathic hyper-
eosinophilic syndrome
by, for example, preventing eosinophil production and migration.
(539] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used to enhance or
inhibit complement
mediated cell lysis.
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[540] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used to enhance or
inhibit antibody
dependent cellular cytotoxicity.
[541] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may also be employed for
treating
atherosclerosis, for example, by preventing monocyte infiltration in the
artery wall.
[542] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may be employed to treat
adult respiratory
distress syndrome CARDS).
[543] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may be useful for stimulating
wound and
tissue repair, stimulating angiogenesis, and/or stimulating the repair of
vascular or
lymphatic diseases or disorders. Additionally, agonists and antagonists.of the
invention
may be used to stimulate the regeneration of mucosal surfaces.
[544] In a specific embodiment, polynucleotides or polypeptides, and/or
agonists
thereof are used to diagnose, prognose, treat, and/or prevent a disorder
characterized by
primary or acquired immunodeficiency, deficient serum immunoglobulin
production,
recurrent infections, and/or immune system dysfunction. Moreover,
polynucleotides or
polypeptides, and/or agonists thereof may be used to treat or prevent
infections of the
joints, bones, skin, and/or parotid glands, blood-borne infections (e.g.,
sepsis, meningitis,
septic arthritis, and/or osteomyelitis), autoirximune diseases (e.g., those
disclosed herein),
inflammatory disorders, and malignancies, and/or any disease or disorder or
condition
associated with these infections, diseases, disorders and/or malignancies)
including, but not
limited to, CVID, other primary immune deficiencies, HIV disease, CLL,
recurrent
bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis,
meningitis, herpes
zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other
diseases and disorders
that may be prevented, diagnosed, prognosed, and/or treated with
polynucleotides or
polypeptides, and/or agonists of the present invention include, but are not
limited to, HIV
infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction
anemia,
thrombocytopenia, and hemoglobinuria.
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[545] In another embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention are used to treat, and/or
diagnose an
individual having common variable immunodeficiency disease ("CVID"; also known
as
"acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of
this disease.
[546] In a specific embodiment, polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be used to diagnose,
prognose, prevent,
and/or treat cancers or neoplasms including immune cell or immune tissue-
related cancers
or neoplasms. Examples of cancers or neoplasms that may be prevented,
diagnosed, or
treated by polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention include, but are not limited to, acute myelogenous leukemia,
chronic
myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute
lymphocytic
anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma,
Burkitt's
lymphoma, EBV-transformed diseases, and/or diseases and disorders described in
the
section entitled "Hyperproliferative Disorders" elsewhere herein.
(547] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
decreasing cellular
proliferation of Large B-cell Lymphomas.
[548] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
decreasing the
involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
[549] In specific embodiments, the compositions of the invention are used as
an agent
to boost immunoresponsiveness among B cell immunodeficient individuals, such
as, for
example, an individual who has undergone a partial or complete splenectomy.
[550] Antagonists of the invention include, for example, binding and/or
inhibitory
antibodies, antisense nucleic acids, ribozymes or soluble forms of the
polypeptides of the
present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of
the invention
include, for example, binding or stimulatory antibodies, and soluble forms of
the
polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides,
antibodies,
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polynucleotides andlor agonists or antagonists of the present invention may be
employed in
a composition with a pharmaceutically acceptable carrier, e.g., as described
herein.
[551] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are administered to an animal
(including, but not
limited to, those listed above, and also including transgenic animals)
incapable of producing
functional endogenous antibody molecules or having an otherwise compromised
endogenous immune system, but which is capable of producing human
immunoglobulin
molecules by means of a reconstituted or partially reconstituted immune system
from
another animal (see, e.g., published PCT Application Nos. W098/24893,
WO/9634096,
WO/9633735, and WO/9110741). Administration of polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present invention to
such animals is
useful for the generation of monoclonal antibodies against the polypeptides,
antibodies,
polynucleotides and/or agonists or antagonists of the present invention.
Blood-Related Disorders
[552] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be used to modulate hemostatic (the stopping of
bleeding) or
thrombolytic (clot dissolving) activity. For example, by increasing hemostatic
or
thrombolytic activity, polynucleotides or polypeptides, andlor agonists or
antagonists of the
present invention could be used to treat or prevent blood coagulation
diseases, disorders,
and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia),
blood platelet
diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds
resulting from
trauma, surgery, or other causes. Alternatively, polynucleotides,
polypeptides, antibodies,
and/or agonists or antagonists of the present invention that can decrease
hemostatic or
thrombolytic activity could be used to inhibit or dissolve clotting. These
molecules could
be important in the treatment or prevention of heart attacks (infarction),
strokes, or scarring.
[553] In specific embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be used to prevent,
diagnose, prognose,
and/or treat thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism,
pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic
attack,
unstable angina. In specific embodiments, the polynucleotides, polypeptides,
antibodies,
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and/or agonists or antagonists of the present invention may be used for the
prevention of
occulsion of saphenous grafts, for reducing the risk of periprocedural
thrombosis as might
accompany angioplasty~procedures, for reducing the risk of stroke in patients
with atrial
fibrillation including nonrheumatic atrial fibrillation, for reducing the risk
of embolism
associated with mechanical heart valves and or mitral valves 'disease. Other
uses for the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention, include, but are not limited to, the prevention of occlusions in
extrcorporeal
devices (e.g., intravascular canulas, vascular access shunts in hemodialysis
patients,
hemodialysis machines, and cardiopulmonary bypass machines).
[554] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
prevent, diagnose, prognose, and/or treat diseases and disorders of the blood
and/or blood
forming organs associated With the tissues) in which the polypeptide of the
invention is
expressed, including one, two, three, four, five, or more tissues disclosed in
Table 1A,
column 8 (Tissue Distribution Library Code).
[555] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be used to modulate hematopoietic activity (the
formation of
blood cells). For example, the polynucleotides, polypeptides, antibodies,
andlor agonists or
antagonists of the present invention may be used to increase the quantity of
all or subsets of
blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells),
myeloid cells
(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets. The
ability to decrease the quantity of blood cells or subsets of blood cells may
be useful in the
prevention, detection, diagnosis and/or treatment of anemias and leukopenias
described
below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention may be used to decrease the quantity of
all or subsets of
blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells),
myeloid cells
(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets.. The
ability to decrease the quantity of blood cells or subsets of blood cells may
be useful in the
prevention, detection, diagnosis and/or treatment of leukocytoses, such as,
for example
eosinophilia.
[556] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be used to prevent, treat, or diagnose blood
dyscrasia.
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[557] Anemias are conditions in which the number of red blood cells or amount
of
hemoglobin (the protein that carries oxygen) in them is below normal. Anemia
may be
caused by excessive bleeding, decreased red blood cell production, or
increased red blood
cell destruction (hemolysis). The polynucleotides, polypeptides, antibodies,
and/or agonists
or antagonists of the present invention may be useful in treating, preventing,
and/or
diagnosing anemias. Anemias that may be treated prevented or diagnosed by the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention , include iron deficiency anemia, hypochromic anemia, microcytic
anemia,
chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic
anemia, red cell
aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12
deficiency) and folic
acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune
helolytic
anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal
hemoglobinuria).
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present
invention may be useful in treating, preventing, and/or diagnosing anemias
associated with
diseases including but not limited to, anemias associated with systemic lupus
erythematosus, cancers, lymphomas, chronic renal disease, and enlarged
spleens. The
polynucleotides, polypeptides, antibodies, and/or agonists ox antagonists of
the present
invention may be useful in treating, preventing, and/or diagnosing anemias
arising from
drug treatments such as anemias associated with methyldopa, dapsone, and/or
sulfadrugs.
Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists
of the present invention may be useful in treating, preventing, and/or
diagnosing anemias
associated with abnormal red blood cell architecture including but not limited
to, hereditary
spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase
deficiency,
and sickle cell anemia.
[558] The polynucleotides, polypeptides, antibodies, andlor agonists or
antagonists of
the present invention may be useful in treating, preventing, and/or diagnosing
hemoglobin
abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C
disease,
hemoglobin S-C disease, and hemoglobin E disease). Additionally, the
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may be
useful in diagnosing, prognosing, preventing, and/or treating thalassemias,
including, but
not limited to major and minor forms of alpha-thalassemia and beta-
thalassemia.
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[559] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating bleeding disorders including, but not limited to,
thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic
thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet
disorders (e.g.,
storage pool disease such as Chediak-Higashi and Hermansky-Pudlalc syndromes,
thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome),
hemolytic-
uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and
Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic
Telangiectsia, also
known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein
purpura) and
disseminated intravascular coagulation.
[560] The effect of the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention on the clotting time of blood may be
monitored using
any of the clotting tests known in the art including, but not limited to,
whole blood partial
thromboplastin time (PTT), the activated partial thromboplastin time (aPTT),
the activated
clotting time (ACT), the recalcified activated clotting time, or the Lee-White
Clotting time.
[561] Several diseases and a variety of drugs can cause platelet dysfunction.
Thus, in a
specific embodiment, the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention may be useful in diagnosing, prognosing,
preventing,
and/or treating acquired platelet dysfunction such as platelet dysfunction
accompanying
kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and
systemic lupus
erythematosus as well as platelet dysfunction associated with drug treatments,
including
treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs
(used for arthritis,
pain, and sprains), and penicillin in high doses.
[562] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders characterized by or
associated with
increased or decreased numbers of white blood cells. Leukopenia occurs when
the number
of white blood cells decreases below normal. Leukopenias include, but are not
limited to,
neutropenia and lymphocytopenia. An increase in the number of white blood
cells
compared to normal is known as leukocytosis. The body generates increased
numbers' of
white blood cells during infection. Thus, leukocytosis may simply be a normal
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physiological parameter that reflects infection. Alternatively, leukocytosis
may be an
indicator of injury or other disease such as cancer. Leokocytoses, include but
are not
limited to, eosinophilia, and accumulations of macrophages. In specific
embodiments, the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention may be useful in diagnosing, prognosing, preventing, and/or treating
leukopenia.
In other specific embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating leukocytosis.
[563] Leukopenia may be a generalized decreased in all types of white blood
cells, or
may be a specific depletion of particular types of white blood cells. Thus, in
specific
embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention may be useful in diagnosing, prognosing, preventing,
and/or
treating decreases in neutrophil numbers, known as neutropenia.- Neutropenias
that may be
diagnosed, prognosed, prevented, and/or treated by the polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention include,
but are not
limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic
neutropenia,
neutropenias resulting from or associated with dietary deficiencies (e.g.,
vitamin B 12
deficiency or folic acid deficiency), neutropenias resulting from or
associated with cli-ug .
treatments (e.g., antibiotic regimens such as penicillin treatment,
sulfonamide treatment,
anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer
chemotherapy), and neutropenias resulting from increased neutrophil
destruction that may
occur in association with some bacterial or viral infections, allergic
disorders, autoimmune
diseases, conditions in which an individual has an enlarged spleen (e.g.,
Felty syndrome,
malaria and sarcoidosis), and some drug treatment regimens.
[564] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in diagnosing, prognosing, preventing,
and/or treating
lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but
not
limited lymphocytopenias resulting from or associated with stress, drug
treatments (e.g.,
drug treatment with corticosteroids, cancer chemotherapies, and/or radiation
therapies),
AIDS infection and/or other diseases such as, for example, cancer, rheumatoid
arthritis,
systemic lupus erythematosus, chronic infections, some viral infections and/or
hereditary
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disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined
immunodeficiency, ataxia telangiectsia).
[565] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in diagnosing, prognosing, preventing,
and/or treating
diseases and disorders associated with macrophage numbers and/or macrophage
function
including, but not limited to, Gaucher's disease, Niemann-Pick disease,
Letterer-Siwe
disease and Hand-Schuller-Christian disease.
[566] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders associated with eosinophil
numbers
and/or eosinophil function including, but not limited to, idiopathic
hypereosinophilic
syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.
[567] In yet another embodiment, the polynucleotides, polypeptides,
antibodies, and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating leukemias and lymphomas including, but not limited
to, acute
lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic,
myelogenous,
myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g.,
B cell
leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia),
chronic myelocytic
(myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-
hodgkin's
lymphoma, Burkitt's lymphoma, and mycosis fungoides.
[568] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders of plasma cells including,
but not limited
to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies
of
undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's
macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.
[569] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in treating,
preventing, and/or
diagnosing myeloproliferative disorders, including but not limited to,
polycythemia vera,
relative polycythemia, secondary polycythemia, myelofibrosis, acute
myelofibrosis,
agnogenic myelod metaplasia, thrombocythemia, (including both primary and
seconday
thrombocythemia) and chronic myelocytic leukemia.
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[570] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as a treatment
prior to
surgery, to increase blood cell production.
[571] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
enhance the
migration, phagocytosis, superoxide production, antibody dependent cellular
cytotoxicity of
neutrophils, eosionophils and macrophages.
[572] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
increase the
number of stem cells in circulation prior to stem cells pheresis. In another
specific
embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention may be useful as an agent to increase the number of
stem cells in
circulation prior to platelet pheresis.
[573] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
increase
cytokine production.
[574] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists ofrthe present invention may be useful in preventing,
diagnosing,
and/or treating primary hematopoietic disorders.
Hyperproliferative Disorders
[575] In certain embodiments, polynucleotides or polypeptides, or agonists or
antagonists of the present invention can be used to treat or detect
hyperproliferative
disorders, including neoplasms. Polynucleotides or polypeptides, or agonists
or antagonists
of the present invention may inhibit the proliferation of the disorder through
direct or
indirect interactions. Alternatively, Polynucleotides or polypeptides, or
agonists or
antagonists of the present invention may proliferate other cells which can
inhibit the
hyperproliferative disorder.
[576] For example, by increasing an immune response, particularly increasing
antigenic
qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing
T-cells, hyperproliferative disorders can be treated. This immune response may
be
increased by either enhancing an existing immune response, or by initiating a
new immune
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response. Alternatively, decreasing an immune response may also be a method of
treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[577] Examples of hyperproliferative disorders that can be treated or detected
by
polynucleotides or polypeptides, or agonists or antagonists of the present
invention include,
but are not limited to neoplasms located in the: colon, abdomen, bone, breast,
digestive
system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and
peripheral),
lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital
tract.
[578] Similarly, other hyperproliferative disorders can also be treated or
detected by
polynucleotides or polypeptides, or agonists or antagonists of the present
invention.
Examples of such hyperproliferative disorders include, but are not limited to:
Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
Lymphocytic
Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia,
Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's
Lymphoma,
Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver
Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related
Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone
Cancer,
Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and
Ureter,
Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma,
Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood
(Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute
Lymphoblastic
Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma,
Childhood
Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial
Germ
Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood
Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia,
Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal
and
Supratentorial Primitive Neuroectoderrnal Tumors, Childhood Primary Liver
Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual
Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic
Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine
Pancreas
Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer,
Esophageal
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Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer,
Extracranial
Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer,
Eye
Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric
Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors,
Gestational
Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular
Cancer,
Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal
Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet
Cell
Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and
Oral
Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,
Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant
Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous
Neck
Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck
Cancer,
Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic
Syndrome,
Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity
and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-
Hodgkin's
Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung
Cancer,
Occult Primary Metastatic Squamous, Neck Cancer, Oropharyngeal Cancer, Osteo-
/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial
Cancer,
Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer,
Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer,
Pheochromocytoma,
Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central
Nervous
System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal
Cell
Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma,
Salivary
Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell
Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer,
Stomach
Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell
Lymphoma,
Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the
Renal Pelvis
and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,
Ureter and
Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma,
Vaginal
Cancer, Visual Pathway and I~ypothalamic Glioma, Vulvar Cancer, Waldenstrom's
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Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease,
besides
neoplasia, located in an organ system listed above.
[579] In another preferred embodiment, polynucleotides or polypeptides, or
agonists or
antagonists of the present invention are used to diagnose, prognose, prevent,
and/or treat
premalignant conditions and to prevent progression to a neoplastic or
malignant state,
including but not limited to those disorders described above. Such uses are
indicated in
conditions known or suspected of preceding progression to neoplasia or cancer,
in
particular, where non-neoplastic cell growth consisting of hyperplasia,
metaplasia, or most
particularly, dysplasia has occurred (for review of such abnormal growth
conditions, see
Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co.,
Philadelphia, pp.
68-79.)
[580] Hyperplasia is a form of controlled cell proliferation, involving an
increase in cell
number in a tissue or organ, without significant alteration. in structure or
function.
Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or
treated with
compositions of the invention (including polynucleotides, polypeptides,
agonists or
antagonists) include, but are not limited to, angiofollicular mediastinal
lymph node
hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic
hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia,
cementum
hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia,
.cystic
hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal
hyperplasia,
endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial
hyperplasia, gingival
hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary
hyperplasia,
intravascular papillary endothelial hyperplasia, nodular hyperplasia of
prostate, nodular
regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous
hyperplasia, and verrucous hyperplasia.
[581] Metaplasia is a form of controlled cell growth in which one type of
adult or fully
differentiated cell substitutes for another type of adult cell. Metaplastic
disorders which can
be diagnosed, prognosed, prevented, and/or treated with compositions of the
invention
(including polynucleotides, polypeptides, agonists or antagonists) include,
but are not
limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical
metaplasia,
autoparenchymatous metaplasia, connective tissue metaplasia, epithelial
metaplasia,
intestinal metaplasia, metaplastic anemia, metaplastic ossification,
metaplastic polyps,
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myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia,
squamous
metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
[582] Dysplasia is frequently a forerunner of cancer, and is found mainly in
the
epithelia; it is the most disorderly form of non-neoplastic cell growth,
involving a loss in
individual cell uniformity and in the architectural orientation of cells.
Dysplastic cells often
have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation. Dysplastic
disorders which can be diagnosed, prognosed, prevented, and/or treated with
compositions
of the invention (including polynucleotides, polypeptides, agonists or
antagonists) include,
but are not limited to, anhidrotic ectodermal dysplasia, anterofacial
dysplasia, asphyxiating
thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,
cerebral dysplasia,
cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia,
congenital
ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,
ectodermal dysplasia,
enamel dysplasia, encephalo-ophthalxnic dysplasia, dysplasia epiphysialis
hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial
dysplasia,
faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial
white folded
dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous
dysplasia,
hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia,
hypohidrotic ectodermal
dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial
dysplasia,
metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia,
mucoepithelial
dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia,
oculodentodigital
dysplasia, oculovertebral dysplasia, odontogenic dysplasia,
ophthalmomandibulomelic
dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia,
pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-
optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
[583] Additional pre-neoplastic disorders which can be diagnosed, prognosed,
prevented, and/or treated with compositions of the invention (including
polynucleotides,
polypeptides, agonists or antagonists) include, but are not limited to, benign
dysproliferative
disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,
intestinal polyps,
colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's
disease, Farmer's
Skin, solar cheilitis, and solar keratosis.
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