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17 Human Secreted Proteins
Field of the hzvezztiozz
This invention relates to newly identified polynucleotides, polypeptides
encoded by these polynucleotides, antibodies that bind these polypeptides,
uses of
such polynucleotides, polypeptides, and antibodies, and their production.
Backgrouszd of the Invefztio~z
Unlike bacterium, which exist as a single compartment surrounded by a
membrane, human cells and other eucaxyotes are subdivided by membranes into
many
functionally distinct comparhnents. Each membrane-bounded compartment, or
organelle, contains different proteins essential for the function of the
organelle. The
cell uses "sorting signals," which are amino acid motifs located within the
protein, to
target proteins to particular cellular organelles.
One type of sorting signal, called a signal sequence, a signal peptide, or a
leader sequence, directs a class of proteins to an organelle called the
endoplasmic
reticulum (ER). The ER separates the membrane-bounded proteins from all other
types of proteins. Once localized to the ER, both groups of proteins can be
further
directed to another organelle called the Golgi apparatus. Here, the Golgi
distributes
the proteins to vesicles, including secretory vesicles, the cell membrane,
lysosomes,
and the other organelles.
Proteins targeted to the ER by a signal sequence can be released into the
extracellular space as a secreted protein. For example, vesicles containing
secreted
proteins can fuse with the cell membrane and release their contents into the
extracellular space - a process called exocytosis. Exocytosis can occur
constitutively
or after receipt of a triggering signal. In the latter case, the proteins are
stored in
secretory vesicles (or secretory granules) until exocytosis is triggered.
Similarly,
proteins residing on the cell membrane can also be secreted into the
extracellular
space by proteolytic cleavage of a "linker" holding the protein to the
membrane.
Despite the great progress made in recent years, only a small number of genes
encoding human secreted proteins have been identified. These secreted proteins
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include the commercially valuable human insulin, interferon, Factor VIII,
human
growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in
light of
the pervasive role of secreted proteins in human physiology, a need exists for
identifying and characterizing novel human secreted proteins and the genes
that
encode them. This knowledge will allow one to detect, to treat, and to prevent
medical diseases, disorders, and/or conditions by using secreted proteins or
the genes
that encode them.
Susnmary of tlae hZVentio~z
The present invention relates to novel polynucleotides and the encoded
polypeptides. Moreover, the present invention relates to vectors, host cells,
antibodies, and recombinant and synthetic methods for producing the
polypeptides
and polynucleotides. Also provided are diagnostic methods for detecting
diseases,
disorders, and/or conditions related to the polypeptides and polynucleotides,
and
therapeutic methods for treating such diseases, disorders, and/or conditions.
The
invention further relates to screening methods for identifying binding
partners of the
polypeptides.
Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide. The
term "isolated" does not refer to genomic or cDNA libraries, whole cell total
or
mRNA preparations, genomic DNA preparations (including those separated by
electrophoresis and transferred onto blots), sheared whole cell genomic DNA
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preparations or other compositions where the art demonstrates no
distinguishing
features of the polynucleotide/sequences of the present invention.
In the present invention, a "secreted" protein refers to those proteins
capable
of being directed to the ER, secretory vesicles, or the extracellular space as
a result of
a signal sequence, as well as those proteins released into the extracellular
space
without necessarily containing a signal sequence. If the secreted protein is
released
into the extracellular space, the secreted protein can undergo extracellular
processing
to produce a "mature" protein. Release into the extracellular space can occur
by many
mechanisms, including exocytosis and proteolytic cleavage.
In specific embodiments, the polynucleotides of the invention are at least 15,
at least 30, at least 50, at least 100, at least 125, at least 500, or at
least 1000
continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb,
50 kb, 15
kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further
embodiment,
polynucleotides of the invention comprise a portion of the coding sequences,
as
disclosed herein, but do not comprise all or a portion of any intron. In
another
embodiment, the polynucleotides comprising coding sequences do not contain
coding
sequences of a genomic flanking gene (i.e., 5' or 3' to the gene of interest
in the
genome). In other embodiments, the polynucleotides of the invention do not
contain
the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5,
4, 3, 2, or
1 genomic flanking gene(s).
As used herein, a "polynucleotide" refers to a molecule having a nucleic acid
sequence contained in SEQ ID NO:X or the cDNA contained within the clone
deposited with the ATCC. 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, with or without the signal
sequence, the
secreted protein 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 the translated amino acid sequence generated from the
polynucleotide as broadly defined.
In the present invention, the full length sequence identified as SEQ ID NO:X
was often generated by overlapping sequences contained in multiple clones
(contig
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4
analysis). A representative clone containing all or most of the sequence for
SEQ ID
NO:X was deposited with the American Type Culture Collection ("ATCC"). As
shown in Table l, each clone is identified by a cDNA Clone ID (Identifier) and
the
ATCC Deposit Number. The ATCC is located at 10801 University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the
terms of the Budapest Treaty on the international recognition of the deposit
of
microorganisms for purposes of patent procedure.
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, the complement thereof, or the cDNA within
the clone deposited with the ATCC. "Stringent hybridization conditions" refers
to an
overnight incubation at 42 degree C in a solution comprising 50% formamide, Sx
SSC
{750 xnM 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.
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
NaCl;
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).
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
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reagents may require modification of the hybridization conditions described
above,
due to problems with compatibility.
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).
The polynucleotide of the present invention can be composed of any
polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or
DNA or modified RNA or DNA. For example, polynucleotides can be composed of
single- and double-stranded DNA, DNA that is a mixture of single- and double-
stranded regions, single- and double-stranded RNA, and RNA that is mixture of
single- and double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded or a mixture
of single-
and double-stranded regions. In addition, the polynucleotide can be composed
of
triple-stranded regions comprising RNA or DNA or both RNA and DNA. A
polynucleotide may also contain one or more modified bases or DNA or RNA
backbones modified for stability or for other reasons. "Modified" bases
include, for
example, tritylated bases and unusual bases such as inosine. A variety of
modifications can be made to DNA and RNA; thus, "polynucleotide" embraces
chemically, enzymatically, or metabolically modified forms.
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
CA 02420705 2003-02-26
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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, demethylatiori,
formation of
covalent cross-links, formation of cysteine, formation of pyroglutamate,
formylation,
gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, pegylation, proteolytic
processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA
mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES,
2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et a1., Meth
Enzyrnol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
"SEQ 1D NO:X" refers to a polynucleotide sequence while "SEQ 1D NO:Y"
refers to a polypeptide sequence, both sequences identified by an integer
specified in
Table 1.
"A polypeptide having biological activity" refers to pohypeptides exhibiting
activity similar, but not necessarily identical to, an activity of a
polypeptide of the
present invention, including mature forms, as measured in a particular
biological
assay, with or without dose dependency. In the case where dose dependency does
exist, it need not be identical to that of the polypeptide, but rather
substantially similar
to the dose-dependence in a given activity as compared to the polypeptide of
the
present invention (i.e., the candidate polypeptide will exhibit greater
activity or not
more than about 25-fold less and, preferably, not more than about tenfold less
activity, and most preferably, not more than about three-fold less activity
relative to
the polypeptide of the present invention.)
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Polynucleotides and Polypeptides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
The translation product of this gene shares sequence homology with rat alpha-
2,6-sialyltransferase (see GenBank accession AAC42086; all references
available
through this accession are hereby incorporated by reference herein), an enzyme
which
attaches sialic acid to carbohydrate groups on glycoproteins and glycolipids.
Sialyltransferases have been linked to a number of disease states including
metastatic
tumors, rheumatoid arthritis, acute hepatitis, and the acute-phase
inflammatory
response. Based upon this homology it is anticipated that translation products
of this
gene will share some biological functions.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
REQKLELHRGGGRSRTSGSPGLQEFGTSCRGTSLQPAPRTAPDPGAPAARWQ
EGRRSAMACIL,KRKSVIAVSFIAAFLFLLVVRLVNEVNFPLLLNCFGQPGTKW
IPFSYTYRRPLRTHYGYINVKTQEPLQLDCDLCAIVSNSGQMVGQKVGNEIDR
SSCI'v~'I~VINNAPTKGYEEDVGRMTMIRVVSHTSVPLLLKNPDYFFKEANTTIY
VIWGPFRNMRKDGNGIVYNMLKKTVGIYPNAQIYVTTEKRMSYCDGVFKKE
TGKDRVQSGSYLSTGWFTFILAMDACYGIHVYGMINDTYCKTEGYRKVPYH
YYEQGRDECDEYFLHEHAPYGGHRFITEKKVFAKWAKKHRTIFTHPNWTLS
(SEQ ID NO: 89). Moreover, fragments and variants of this polypeptide (such
as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridize, under stringent conditions, to the
polynucleotide
encoding this polypeptide are encompassed by the invention. Antibodies that
bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding this polypeptide are also encompassed by the invention.
This gene is expressed in human early stage embryo, synovium, melanocytes,
neuroepithelium, neurons, and the pineal gland.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
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and for diagnosis of diseases and conditions which include but are not limited
to:
developmental, skeletal, neurological, inflammatory, and proliferative
disorders.
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
developing embryo, nervous system, and skeletal system, expression of this
gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., embryonic, neural, cancerous and wounded tissues) or bodily
fluids (e.g.,
lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred polypeptides of the present
invention
comprise, or altenlatively consist of, one or more immunogenic epitopes shown
in
SEQ ID NO: 50 as residues: Tyr-52 to Gly-63, Lys-68 to Leu-73, Met-107 to Asp-
118, Pro-157 to Asn-166, Thr-189 to Tyr-195, Phe-200 to Ser-211, Asp-240 to
Lys-
250, His-254 to Asp-264. Polynucleotides encoding said polypeptides are also
encompassed by the invention.
The homology to the rat alpha-2,6-sialyltransferase indicates that this gene
may be involved in sialic acid metabolism. Therefore, polynucleotides and
polypeptides corresponding to this gene, as well as antibodies directed
against
polypeptides of this gene, may be useful for the detection, prevention, andlor
treatment of metastatic tumors and inflammatory disorders (such as, for
example,
rheumatoid arthritis, inflammatory bowel disease, and asthma). More generally,
the
expression of this gene product in synovium would suggest a role in the
detection and
treatment of disorders and conditions afflicting the skeletal system, in
particular
osteoporosis, bone cancer, connective tissue disorders (e.g. arthritis,
trauma,
tendonitis, chrondomalacia and inflammation). The protein is also useful in
the
diagnosis or treatment of various autoimmune disorders (i.e., rheumatoid
arthritis,
lupus, scleroderma, and dermatomyositis), dwarfism, spinal deformation, joint
abnormalities, and chondrodysplasias (i.e. spandyloepiphyseal dysplasia
congenita,
familial osteoarthritis, Atelosteogenesis type II, metaphyseal
chondrodysplasia type
Schmid, etc.). Furthermore, the distribution in embryonic and neural tissues
suggests
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that this gene plays a role in development and nervous system function.
Therefore,
polynucleotides and polypeptides corresponding to this gene, as well as
antibodies
directed against polypeptides of this gene, may be useful for the detection,
prevention,
and/or treatment of development and neurological disorders, such as
Alzheimer's
Disease, Parkinson's Disease, Huntington's Disease, Tourette's Syndrome,
meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia,
trauma,
congenital malformations, spinal cord injuries, ischemia and infarction,
aneurysms,
hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, depression, panic disorder, learning disabilities, ALS, psychoses,
autism,
and altered behaviors, including disorders in feeding, sleep patterns,
balance, and
perception. In addition, elevated expression of this gene product in regions
of the
brain indicates it plays a role in normal neural function. Potentially, this
gene product
is involved in synapse formation, neurotransmission, learning, cognition,
homeostasis, or neuronal differentiation or survival. The protein may also be
used to
determine biological activity, to raise antibodies, as tissue markers, to
isolate cognate
ligands or receptors, to identify agents that modulate their interactions, in
addition to
its use as a nutritional supplement. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or irmnunotherapy targets for
the
above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:11 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 3175 of SEQ B7 NO:
l l, b
is an integer of 15 to 3189, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m NO:1 l, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
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The translation product of this gene shares sequence homology with
ixmnunoglobulin superfamily members (see, for example, Genbank accession
CAA48104; all references available through this accession are hereby
incorporated by
reference herein). Immunoglobulin superfamily members are usually secreted or
5 membrane bound surface molecules. The known functions are: immune functions,
such as antibodies, and histocompatibility. The potential uses of the proteins
in this
family can be surface markers for cell differentiation, secreted forms as
cytokines,
antibodies, hormones or their receptors. Some examples in this family include
irmnunoglobulins or MHC.
10 In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
MKDLWFLLLVVAAPTWVLSQVRLQESGPGLVSPSQTLSLTCSVSG1NIGGGK
YYWAWVRQRPGEGPEWVGYISYTGVADYNPSLRGRLTISLGESNSFSLTLTS
MTAADAAVYYCATDRWTSAWGNYQS QGYFMD V W GRGTAV V V S SASTKGP
SVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTQTYTCl\fVNHKPSNTKVDKRVELKTPLGDTTHTCP
RCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKT
KPREEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTT
PPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG
K (SEQ ZD NO: 90). Moreover, fragments and variants of this polypeptide (such
as,
for example, fragments as described herein, polypeptides at least 80%, 85%,
90%,
95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides
encoded by the polynucleotide which hybridize, under stringent conditions, to
the
polynucleotide encoding this polypeptide are encompassed by the invention.
Antibodies that bind polypeptides of the invention axe also encompassed by the
invention. Polynucleotides encoding this polypeptide are also encompassed by
the
invention.
This gene is expressed primarily in fetal and infant brain, pituitary, and
spinal
cord, and to a lesser extent in cancers derived from a number of tissue
sources,
including lung, prostate, colon, adrenal gland, breast, and ovary.
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11
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
neurological and proliferative disorders, including cancer. Similarly,
polypeptides
and antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the nervous system,
expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
tissues or cell types (e.g., neural, cancerous and wounded tissues) or bodily
fluids
(e.g., serum, plasma, urine, synovial fluid and spinal fluid) or another
tissue or sample
taken from an individual having such a disorder, relative to the standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred polypeptides of the present
invention
comprise, or alternatively consist of, one or more immunogenic epitopes shown
in
SEQ )D NO: 51 as residues: Arg-59 to Glu-67, Tyr-80 to Arg-85. Polynucleotides
encoding said polypeptides are also encompassed by the invention.
The tissue distribution and homology to immunoglobulin superfamily
members indicates that polynucleotides and polypeptides corresponding to this
gene
are useful for the detection, treatment, andlor prevention of neurological
disorders,
such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette's
Syndrome, meningitis, encephalitis, demyelinating diseases, peripheral
neuropathies,
neoplasia, trauma, congenital malformations, spinal cord injuries, ischemia
and
infarction, aneurysms, hemorrhages, schizophrenia, mania, dementia, paranoia,
obsessive compulsive disorder, depression, panic disorder, learning
disabilities, ALS,
psychoses, autism, and altered behaviors, including disorders in feeding,
sleep
patterns, balance, and perception. More generally, the gene product of this
gene may
be used in (i) modulation of cellular signal transduction, either in vitro or
in vivo; (ii)
regulation of communication between cells; (iii) regulation of gene
transcription in a
cell involved in development or differentiation, either in vitro or in vivo
(e.g.
induction of cell differentiation); (iv) regulation of cellular proliferation,
either in
vitro or in vivo (e.g. induction of cellular proliferation or inhibition of
proliferation as
in the case of suppression of tumorigenesis; (v) formation and maintenance of
ordered
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12
spatial arrangements of differentiated tissues in vertebrates, both adult and
embryonc
(e.g. induction of head formation during vertebrate development or maintenance
of
hematopoietic progenitor cells); (vi) modulation of cell death, such as
stimulation of
cell survival; (vii) regulating cell migration, and/or (viii) immune
modulation. e.g.
treating hyperproliferative diseases such as neoplastic and hyperplastic
disease, e.g.
various cancers and leukemias, and fibroproliferative disorders, malignancy,
premalignant and benign conditions, to stimulate tissue repair or wound
healing or
treat spinal cord injuries, brain injuries, lesions associated with surgery,
ischemic
lesions, malignant lesions, infectious lesions, degenerative lesions,
demyelinating
diseases (multiple sclerosis, human immunodeficiency associated myelopathy,
pontine myelinolysis), motor neuron injuries, progressive spinal muscular
atrophy,
progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile
muscular
atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis, and hereditary motorsensory neuropathy. Furthermore, the
protein may
also be used to determine biological activity, to raise antibodies, as tissue
markers, to
isolate cognate ligands or receptors, to identify agents that modulate their
interactions,
in addition to its use as a nutritional supplement. Protein, as well as,
antibodies
directed against the protein rnay show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ m N0:12 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 415 of SEQ ID
N0:12, b
is an integer of 15 to 429, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:12, and where b is greater than or equal
to a
+ 14.
FEATURES OF PROTEIN ENCODED SY GENE NO: 3
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13
Translation products of this gene share sequence homology with the rat
Multidrug Resistance Protein-3 (MRP3; see Genbanlc accession AAC25416), an
ABC-type transporter protein thought to be involved in cellular drug
absorption and
liver function. Based upon this homology it is anticipated that translation
products of
this gene will share some biological activities.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
MAVYLLWQELGPAVLAGVAVLVFVIPINALAATKIKKLI~VSLATLCVYFLLD
EGNILTATKVFTSMSLFNILRIPLFELPTVISAVVQTKISLGRLEDFLNTEELLPQ
SIETNYTGDHAIGFTDASFSWDKTGMPVLKEALWLMFLSRPGFRIAFCI~KTFS
LAPS (SEQ ID NO: 91). Moreover, fragments and variants of this polypeptide
(such
as, for example, fragments as described herein, polypeptides at least 80%,
85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides
encoded by the polynucleotide which hybridize, under stringent conditions, to
the
polynucleotide encoding this polypeptide are encompassed by the invention.
Antibodies that bind polypeptides of the invention are also encompassed by the
invention. Polynucleotides encoding this polypeptide are also encompassed by
the
invention.
The polypeptide of this gene has been determined to have transmembrane
domains at about amino acid positions 14 - 30 and 140 - 156 of the amino acid
sequence referenced in Table 1 fox this gene. Based upon these
characteristics, it is
believed that the protein product of this gene shares structural features to
type IIIb
membrane proteins.
This gene is expressed primarily in colon cancer and a fetal liver / spleen
library.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to: liver
diseases, disorders related to or involving drug resistance or chemical
hypersensitivity, and cancer. Similarly, polypeptides and antibodies directed
to these
polypeptides axe useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
CA 02420705 2003-02-26
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14
tissues or cells, particularly of the liver, expression of this gene at
significantly higher
or lower levels may be routinely detected in certain tissues or cell types
(e.g., hepatic,
cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum,
plasma,
urine, synovial fluid and spinal fluid) or another tissue or sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
Preferred polypeptides of the present invention comprise, or alternatively
consist of, one or more irnmunogenic epitopes shown in SEQ ID NO: 52 as
residues:
Ile-110 to Gly-116, Phe-127 to Thr-132. Polynucleotides encoding said
polypeptides
are also encompassed by the invention.
The distribution in fetal liver / spleen and homology to the rat MRP3 gene
indicates that this gene may be involved in liver functions, such as lipid
metabolism
and bile production. Therefore, polynucleotides and polypeptides corresponding
to
this gene, as well as antibodies directed against polypeptides of this gene,
are useful
for the control of lipid metabolism in such conditions as
hypercholesterolemia,
Tangier disease, and lipidosis. More generally, polynucleotides and
polypeptides
corresponding to this gene, as well as antibodies directed against
polypeptides of this
gene, may be useful in the detection, prevention, and/or treatment of liver
disorders,
such as Dubin-Johnson syndrome, Rotor's syndrome, Ritter's disease, and
cirrhosis.
In addition, the expression of this gene in colon cancer tissue suggests a
role in the
progression and treatment of proliferative disorders. In particular,
translation
products of this gene may confer chemotherapeutic drug resistance to cancerous
cells.
Therefore, antibodies directed to translation products of this gene may
potentiate the
effects of chemotherapeutic drugs. Furthermore, the protein may also be used
to
determine biological activity, to raise antibodies, as tissue markers, to
isolate cognate
ligands or receptors, to identify agents that modulate their interactions, in
addition to
its use as a nutritional supplement. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
CA 02420705 2003-02-26
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related to SEQ ID N0:13 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
5 are one or more polynucleotides comprising a nucleotide sequence described
by the
general formula of a-b, where a is any integer between 1 to 793 of SEQ 11?
N0:13, b
is an integer of 15 to 807, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:13, and where b is greater than or equal
to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 4
This gene is expressed primarily in neutrophils and to a lesser extent in
synovial sarcoma.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
immunological diseases and cancer. Similarly, polypeptides and antibodies
directed
to these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the immune system, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or sample
taken from
an individual having such a disorder, relative to the standard gene expression
level,
i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
The expression of this gene in neutrophils indicates that polynucleotides and
polypeptides corresponding to this gene, as well as antibodies directed
against
translation products of this gene, are useful for detection, prevention,
and/or treatment
of immunological disorders such as arthritis, asthma, AmS, leukemia,
rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne,
neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated
CA 02420705 2003-02-26
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16
cytotoxicity; immune reactions to transplanted organs and tissues, such as
host-
versus-graft and graft-versus-host diseases, or autoimmunity disorders, such
as
autoimmune infertility, lens tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma, neutropenia, neutrophilia, respiratory distress
syndrome,
emphysema, and megaloblastic anemia. Furthermore, expression of this gene in
cancerous tissue suggests that the protein, as well as antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues. In addition, the protein may be used to determine
biological
activity, raise antibodies, as tissue markers, to isolate cognate ligands or
receptors, to
identify agents that modulate their interactions, in addition to its use as a
nutritional
supplement.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:14 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 64~ of SEQ >D
NO:14, b
is an integer of 15 to 662, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:14, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
This gene is expressed primarily in testis tumors.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
male reproductive disorders, including testicular cancer. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
CA 02420705 2003-02-26
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17
disorders of the above tissues or cells, particularly of the male reproductive
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., testicular, cancerous and
wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and
spinal fluid) or
another tissue or sample taken from an individual having such a disorder,
relative to
the standard gene expression level, i.e., the expression Level in healthy
tissue or bodily
fluid from an individual not having the disorder. Preferred polypeptides of
the
present invention comprise, or alternatively consist of, one or more
immunogenic
epitopes shown in SEQ ID NO: 54 as residues: Pro-41 to Ser-49. Polynucleotides
encoding said polypeptides are also encompassed by the invention.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of male
reproductive
disorders, including impotence, infertility, testis tumors (such as
choriocarcinoma,
leydig cell tumors, seminoma, Sertoli cell tumors and teratoma), atrophy of
testis, and
inflammation of the testis.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:15 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 2947 of SEQ ID
N0:15, b
is an integer of 15 to 2961, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:15, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 6
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
MWSSSWDHRITTPRLANFFFFFFFFFFVEMGFRYVGQAGLI~LLASSNLPALAS
QSAGITGVSHHVWLGGLIKTPILSLTPRVSG (SEQ ID NO: 55). Moreover,
CA 02420705 2003-02-26
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18
fragments and variants of this polypeptide (such as, for example, fragments as
described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% identical to these polypeptides and polypeptides encoded by the
polynucleotide
which hybridize, under stringent conditions, to the polynucleotide encoding
this
polypeptide are encompassed by the invention. Antibodies that bind
polypeptides of
the invention are also encompassed by the invention. Polynucleotides encoding
this
polypeptide are also encompassed by the invention.
This gene is expressed primarily in testes.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
male reproductive disorders, including testicular cancer. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the male reproductive
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., testis, cancerous and wounded
tissues) or
bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or
another
tissue or sample taken from an individual having such a disorder, relative to
the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of male
reproductive
disorders, such as infertility, impotence, testicular tumors (including
choriocarcinoma,
leydig cell tumors, seminoma, Sertoli cell tumors and teratoma), atrophy of
testis, and
inflammation of testis. Furthermore, the protein may also be used to determine
biological activity, raise antibodies, as tissue markers, to isolate cognate
ligands or
receptors, to identify agents that modulate their interactions, in addition to
its use as a
nutritional supplement. Protein, as well as, antibodies directed against the
protein
may show utility as a tumor marker and/or immunotherapy targets for the above
listed
tissues.
CA 02420705 2003-02-26
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19
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:16 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 964 of SEQ ID
N0:16, b
is an integer of 15 to 978, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:16, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
In one embodiment, polypeptides comprising the amino acid sequence of the
open reading frame upstream of the predicted signal peptide are contemplated
by the
present invention. In specific embodiments, polypeptides of the invention
comprise,
or alternatively consists of, the following amino acid sequence:
NGLVWNPHYRVSWYAXRYRSGIPGGKGLAMLHVTRGVWGSRVRVWPLLP
ALLGPPRALSSLAAI~MGEYRKMWNPREPRDWAQQYRERFIPFSKEQLLRLLI
QALYDPINPDRETLDQPSLTDPQRLSNEQEVLRALEPLLAQANFSPLSEDTLA
YALVVHHPQDEVQVTVNLDQYVYIHFWALGQPSXADAPEVQRGLQACLLSP
KLPLRERRYFKRVVLASPDQNGDTWDLKKFSXTPPLGKAWEXLLXGT (SEQ
ID NO: 92). Moreover, fragments and variants of this polypeptide (such as, for
example, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded
by
the polynucleotide which hybridize, under stringent conditions, to the
polynucleotide
encoding this polypeptide are encompassed by the invention. Antibodies that
bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding this polypeptide are also encompassed by the invention.
This gene is expressed primarily in TF-1 cells and neutrophils.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
CA 02420705 2003-02-26
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and for diagnosis of diseases and conditions which include but are not limited
to:
disorders of hematopoiesis and disorders involving neutrophil related
functions (e.g.
neutropenia). Similarly, polypeptides and antibodies directed to these
polypeptides
are useful in providing immunological probes for differential identification
of the
5 tissues) or cell type(s). For a number of disorders of the above tissues or
cells,
particularly of the hematopoietic and immune systems, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., lymph,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or sample
taken from
10 an individual having such a disorder, relative to the standard gene
expression level,
i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
Preferred polypeptides of the present invention comprise, or alternatively
consist of, one or more immunogenic epitopes shown in SEQ ID NO: 56 as
residues:
15 Gly-37 to Met-42, Pro-45 to Trp-51, Gln-54 to Phe-59, Ile-79 to Asp-87, Pro-
89 to
Glu-102, Pro-178 to Arg-183, Ser-203 to Pro-208, Pro-288 to Gly-296.
Polynucleotides encoding said polypeptides are also encompassed by the
invention.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for diagnosis and treatment of
hematopoietic
20 disorders such as anemia, pancytopeiua, leukopenia, thrombocytopenia or
leukemia;
as well as imrnunological disorders such as arthritis, asthma, AIDS, leukemia,
rheumatoid arthritis, granulomatous disease, inflammatory bowel disease,
sepsis,
acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated
cytotoxicity; immune reactions to transplanted organs and tissues, such as
host-
versus-graft and graft-versus-host diseases, or autoimmunity disorders, such
as
autoimmune infertility, lens tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Furthermore, the receptor of the present invention
is also
useful as a tool for screening for the natural ligand of the receptor, the
discovery of
which may enable a further understanding of the processes mediated by this
receptor
and its ligand. Additionally, translation products corresponding to this gene,
as well
CA 02420705 2003-02-26
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21
as antibodies directed against these translation products, may show utility as
a tumor
marker and/or immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:17 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1196 of SEQ ID
N0:17, b
is an integer of 15 to 1210, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:17, and Where b is greater than or
equal to a
+ 14.
FEATiTRES OF PROTEIN ENCODED BY GENE NO: 8
The translation product of this gene shares sequence homology with a novel
glucose transporter, GLUTTX1.
This gene is expressed primarily in synovial sarcoma, testis, and neutrophils.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to: type
I and type II diabetes, synovial sarcoma, neutropenia, neutrophilia,
respiratory distress
syndrome, emphysema, inflammatory diseases, megaloblastic anemia. Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s).
For a number of disorders of the above tissues or cells, particularly of the
immune
system, expression of this gene at significantly higher or lower levels may be
routinely detected in certain tissues or cell types (e.g., cancerous and
wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and
spinal fluid) or
another tissue or sample taken from an individual having such a disorder,
relative to
the standard gene expression level, i.e., the expression level in healthy
tissue or bodily
fluid from an individual not having the disorder. Preferred polypeptides of
the
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22
present invention comprise, or alternatively consist of, one or more
immunogenic
epitopes shown in SEQ DJ NO: 57 as residues: Val-31 to Gly-49. Polynucleotides
encoding said polypeptides are also encompassed by the invention.
The tissue distribution and homology to glucose transporter, GLUTTX1,
indicates that polynucleotides and polypeptides corresponding to this gene are
useful
for diagnosis and treatment of type I and type II diabetes, synovial sarcoma,
neutropenia, neutrophilia, respiratory distress syndrome, emphysema,
inflammatory
diseases, megaloblastic anemia.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:18 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 520 of SEQ ID
N0:18, b
is an integer of 15 to 534, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:18, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED SY GENE NO: 9
This gene shares sequence homology with human RABP-3 (see International
Patent Application WO 9909182; all references available through this accession
are
hereby incorporated by reference herein), a member of the RAB family~of genes
which are thought to be involved in intracellular vesicle transport. In
another
embodiment, polypeptides comprising the amino acid sequence of the open
reading
frame upstream of the predicted signal peptide are contemplated by the present
invention. In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
IIHYCLLDPTTQYHQLLVSVDQKHLFEARSGILSILHMIMSSVTLLWSILHQAD
S SEKMTIAASASLTT1NLGATKNLRQQILELLGPISMNHGVHFMAAIAF V WNE
RRQNKTTTRTKVCI (SEQ ID NO: 93). Moreover, fragments and variants of this
CA 02420705 2003-02-26
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23
polypeptide (such as, for example, fragments as described herein, polypeptides
at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these
polypeptides
and polypeptides encoded by the polynucleotide which hybridize, under
stringent
conditions, to the polynucleotide encoding this polypeptide are encompassed by
the
invention. Antibodies that bind polypeptides of the invention are also
encompassed by
the invention. Polynucleotides encoding this polypeptide are also encompassed
by the
invention.
This gene is expressed in human whole embryo.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
developmental disorders, and disorders of vesicle transport (such as
neurotransmission). Similarly, polypeptides and antibodies directed to these
polypeptides are useful in providing immunological probes for differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
tissues or cells, particularly of the nervous system and developing embryo,
expression
of this gene at significantly higher or lower levels may be routinely detected
in certain
tissues or cell types (e.g., neural, embryonic, cancerous and wounded tissues)
or
bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or
another
tissue or sample taken from an individual having such a disorder, relative to
the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder.
Preferred polypeptides of the present invention comprise, or alternatively
consist of, one or more immunogenic epitopes shown in SEQ m NO: 58 as
residues:
Glu-70 to Arg-79. Polynucleotides encoding said polypeptides are also
encompassed
by the invention.
The homology to a human RASP-3 indicates that this translation products of
this gene may be involved in intracellular vesicle transport. Therefore,
polynucleotides and polypeptides corresponding to this gene are useful for the
treatment of disorders involving abnormal vesicular trafficking. For example,
translation products of this gene, or antibodies directed against translation
products of
this gene, may be useful for the treatment of central nervous system
disorders.
CA 02420705 2003-02-26
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24
Briefly, such uses include, but are not limited to the detection, treatment,
and/or
prevention of Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette's Syndrome, meningitis, encephalitis, demyelinating diseases,
peripheral
neuropathies, neoplasia, trauma, congenital malformations, spinal cord
injuries,
ischemia and infarction, aneurysms, hemorrhages, schizophrenia, mania,
dementia,
paranoia, obsessive compulsive disorder, depression, panic disorder, learning
disabilities, ALS, psychoses, autism, and altered behaviors, including
disorders in
,,j
feeding, sleep patterns, balance, and perception. In addition, elevated
expression of
this gene product in human embryonic tissues indicates it plays a role in
normal
development. Potentially, this gene product is involved in synapse formation,
neurotransmission, learning, cognition, homeostasis, or neuronal
differentiation or
survival. Furthermore, the protein may also be used to determine biological
activity,
to raise antibodies, as tissue markers, to isolate cognate ligands or
receptors, to
identify agents that modulate their interactions, in addition to its use as a
nutritional
supplement. Protein, as well as, antibodies directed against the protein may
show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ m N0:19 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
~general formula of a-b, where a is any integer between 1 to 907 of SEQ ID
N0:19, b
is an integer of 15 to 921, where both a and b correspond to the positions of
nucleotide residues shown in SEQ )D N0:19, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
This gene is expressed primarily in neutrophils.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
CA 02420705 2003-02-26
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and for diagnosis of diseases and conditions which include but are not limited
to:
immunological disorders and diseases (particularly those involving
neutrophils).
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
5 type(s). For a number of disorders of the above tissues or cells,
particularly of the
immune system, expression of this gene at significantly higher or lower levels
may be
routinely detected in certain tissues or cell types (e.g., cancerous and
wounded
tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid
and spinal
fluid) or another tissue or sample taken from an individual having such a
disorder,
10 relative to the standard gene expression level, i.e., the expression level
in healthy
tissue or bodily fluid from an individual not having the disorder. Preferred
polypeptides of the present invention comprise, or alternatively consist of,
one or
more immunogenic epitopes shown in SEQ m NO: 59 as residues: Met-1 to Trp-9.
Polynucleotides encoding said polypeptides are also encompassed by the
invention.
15 The expression in neutrophils indicates that polynucleotides and
polypeptides
corresponding to this gene, as well as antibodies directed against translation
products
of this gene, are useful for the detection, prevention, and/or treatment of
immunological disorders such as neutropenia, neutrophilia, respiratory
distress
syndrome, emphysema, megaloblastic anemia, astlnna, immunodeficiency diseases
20 such~as AmS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory
bowel disease, sepsis, acne, psoriasis, host-versus-graft and graft-versus-
host
diseases, systemic lupus erythematosis, rheumatoid arthritis, Sjogren's
disease, and
scleroderma. Furthermore, the protein may also be used to determine biological
activity, raise antibodies, as tissue markers, to isolate cognate ligands or
receptors, to
25 identify agents that modulate their interactions, in addition to its use as
a nutritional
supplement. Protein, as well as, antibodies directed against the protein may
show
utility as a tumor marker and/or immunotherapy targets for the above listed
tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ m N0:20 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. To list every related
sequence
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26
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 507 of SEQ ID
N0:20, b
is an integer of 15 to 521, where both a and b correspond to the positions of
nucleotide residues shown in SEQ 1D N0:20, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
This gene is expressed primarily in ovarian cancer.
Polynucleotides and polypeptides of the invention are useful as reagents fox
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
reproductive and proliferative disease, including cancer. Similarly,
polypeptides and
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the reproductive
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., ovarian, cancerous and
wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid)
or another
tissue or sample taken from an individual having such a disorder, relative to
the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene, as well as antibodies directed to polypeptides of
this gene,
are useful for diagnosis and treatment of ovarian cancers, including Brenner
tumor,
choriocarcinoma, dysgerminoma and Krukenberg's tumor. More generally,
polynucleotides, polypeptides, and antibodies directed to polypeptides of this
gene
may be useful for the treatment of female reproductive disorders such as
infertility,
ectopic pregnancy, and preterm labor. Furthermore, the protein may also be
used to
determine biological activity, raise antibodies, as tissue markers, to isolate
cognate
ligands or receptors, to identify agents that modulate their interactions, in
addition to
its use as a nutritional supplement. Protein, as well as, antibodies directed
against the
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27
protein may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:21 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 654 of SEQ ID
N0:21, b
is an integer of 15 to 668, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:21, and where b is greater than or
equal to a
+ 14:
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
MLVAWCLAPGDLLLLVIITLPRKEVTGSMSTVCQCEAQPAMLPKGHFTHHSP
KAARKAQEGTRKARWVALEDSAPFHPSPGWGLILQLHPQPMNCSQSAWKHC
CWKNCEEP (SEQ ID NO: 98). Moreover, fragments and variants of this
polypeptide (such as, for example, fragments as described herein, polypeptides
at
least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these
polypeptides
and polypeptides encoded by the polynucleotide which hybridize, under
stringent
conditions, to the polynucleotide encoding this polypeptide are encompassed by
the
invention. Antibodies that bind polypeptides of the invention axe also
encompassed
by the invention. Polynucleotides encoding this polypeptide are also
encompassed by
the invention.
This gene is expressed primarily in a prostate cancer line.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
male reproductive disorders, including prostate cancer. Similarly,
polypeptides and
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28
antibodies directed to these polypeptides are useful in providing
immunological
probes for differential identification of the tissues) or cell type(s). For a
number of
disorders of the above tissues or cells, particularly of the male reproductive
system,
expression of this gene at significantly higher or lower levels may be
routinely
detected in certain tissues or cell types (e.g., prostate, cancerous and
wounded tissues)
or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid)
or another
tissue or sample taken from an individual having such a disorder, relative to
the
standard gene expression level, i.e., the expression level in healthy tissue
or bodily
fluid from an individual not having the disorder. Preferred polypeptides of
the
present invention comprise, or alternatively consist of, one or more
immunogenic
epitopes shown in SEQ ID NO: 61 as residues: Pro-21 to Gly-27, Ser-51 to Ala-
65,
His-89 to Asn-94. Polynucleotides encoding said polypeptides are also
encompassed
by the invention.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for detection, prevention, and/or
treatment of
male reproductive disorders, including infertility, impotence, prostate
cancers,
hypertrophy of the prostate, and inflammation of the prostate. Furthermore,
the
protein may also be used to determine biological activity, to raise
antibodies, as tissue
markers, to isolate cognate ligands or receptors, to identify agents that
modulate their
interactions, in addition to its use as a nutritional supplement. Protein, as
well as,
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:22 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 662 of SEQ m
N0:22, b
is an integer of 15 to 676, where both a and b correspond to the positions of
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29
nucleotide residues shown in SEQ m N0:22, and where b is greater than or equal
to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
The translation product of this gene shares sequence homology with a human
breast cancer-related protein.
This gene is expressed primarily in prostate cancer and neutrophils, and to a
lesser extent in testis and a variety of human tissues.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
prostate cancer, breast cancer, neutropenia, neutrophilia, respiratory
distress
syndrome, emphysema, megaloblastic anemia, asthma, immunodeficiency diseases
such as AmS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory
bowel disease, sepsis, acne, psoriasis, host-versus-graft and graft-versus-
host
diseases, systemic lupus erythematosis, rheumatoid arthritis, Sjogren's
disease, and
scleroderma. Similarly, polypeptides and antibodies directed to these
polypeptides
are useful in providing immunological probes for differential identification
of the
tissues) or cell type(s). For a number of disorders of the above tissues or
cells,
particularly of the immune and reproductive systems, expression of this gene
at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma,
urine, synovial fluid and spinal fluid) or another tissue or sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder. Preferred polypeptides of the present invention comprise, or
alternatively
consist of, one or more immunogenic epitopes shown in SEQ ID NO: 62 as
residues:
Pro-13 to Ser-18, Gly-52 to Ala-58, Ser-67 to Trp-74. Polynucleotides encoding
said
polypeptides are also encompassed by the invention.
The tissue distribution and homology to a breast cancer related gene indicates
that polynucleotides and polypeptides corresponding to this gene are useful
for
prostate cancer, breast cancer, neutropenia, neutrophilia, respiratory
distress
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syndrome, emphysema, megaloblastic anemia, asthma, immunodeficiency diseases
such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease,
inflammatory
bowel disease, sepsis, acne, psoriasis, host-versus-graft and graft-versus-
host
diseases, systemic lupus erythematosis, rheumatoid arthritis, Sjogren's
disease, and
5 scleroderma.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ D? N0:23 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
10 excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 558 of SEQ >D
N0:23, b
is an integer of 15 to 572, where both a and b correspond to tie positions of
15 nucleotide residues shown in SEQ ID N0:23, and where b is greater than or
equal to a
+ 14.
FEATTJRES OF PROTEIN ENCODED SY GENE NO: I4
The polypeptide of this gene has been determined to have transmembrane
20 domains at about amino acid positions 246 - 262, 340 - 356, 400 - 416, 370 -
386, 3I0
- 326, 276 - 292, 217 - 233, I85 - 201, and 49 - 65 of the amino acid sequence
referenced in Table 1 for this gene. Based upon these characteristics, it is
believed
that the protein product of this gene shares structural features to type IIIa
membrane
proteins.
25 In another embodiment, polypeptides comprising the amino acid sequence of
the open reading frame upstream of the predicted signal peptide are
contemplated by
the present invention. Specifically, polypeptides of the invention comprise
the
following amino acid sequence:
VRGRGRGAGRLRGLTGPLMVPPRRHRGAGRPGVLSSSPPFRLRSAKFSGIALE
30 DLRRALKTRLQMVCVFVl~I7VRMNSQNSGFTQRRRMALGIXILLLVDVIWVAS
SELTSYVFTQYNKPFFSTFAKTSMFVLYLLGFIIWKPWRQQCTRGLRGKHAAF
FADAEGYFAACTTDTTMNSSLSEPLYVPVKFHDLPSEKPEXTNIDTEKTPKKS
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RVRFSNIME1RQLPSSHALEAKLSRMSYPVKEQESILKTVGKLTATQVAKISFF
FCFVWFLANLSYQEALSDTQVAIVNILSSTSGLFTLILAAVFPSNSGDRFTLSK
LLAVILSIGGVVLVNLAGSEKPAGRDTVGSIWSLAGAMLYAVYIVMII~RKVD
REDKLDIPMFFGFVGLFNLLLLWPGFFLLHYTGFEDFEFPNKVVLMCIIINGLI
GTVLSEFLWLWGCFLTSSLIGTLALSLTIPLSIIADMCMQKVQFSWLFFAGAIP
VFFSFFIVTLLCHYNNWDPVMVGIRRIFAFICRKHRIQRVPEDSEQCESLISMH
SVSQEDGAS (SEQ ID NO: 94) and
MVPPRRHRGAGRPGV LS S SPPFRLRSAKFS GIALEDLRRALKTRLQMV C VF V
MNRMNS QNS GFTQRRRMALGIXILLLVD VIWVAS SELTSYVFTQYNKPFFSTF
IO AKTSMFVLYLLGFIIWKPWRQQCTRGLRGKHAAFFADAEGYFAACTTDTTM
NSSLSEPLYVPVKFHDLPSEKPEXTNIDTEKTPKKSRVRFSNIMEIRQLPSSHAL
EAKLSRMSYPVKEQESILKTVGKLTATQVAKISFFFCFVWFLANLSYQEALSD
TQVAIVNILSSTSGLFTLILAAVFPSNSGDRFTLSKLLAVILSIGGVVLVNLAGS
EKPAGRDTVGSIWSLAGAMLYAVYIVMIZ~RKVDREDKLDIPMFFGFVGLFNL
LLLWPGFFLLHYTGFEDFEFPNKVVLMCIIINGLIGTVLSEFLWLWGCFLTSS'LI
GTLALSLTIPLSIIADMCMQKVQFSWLFFAGAIPVFFSFFIVTLLCHYNNWDPV
MVGIRRIFAFICRKHRIQRVPEDSEQCESLISMHSVSQEDGAS (SEQ ID NO:
95). Moreover, fragments and variants of these polypeptides (such as, for
example,
fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by
the
polynucleotide which hybridizes, under stringent conditions, to the
polynucleotide
encoding these polypeptides ) are encompassed by the invention. Antibodies
that bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in fetal heart and to a lesser extent in
osteoclastoma, colon carcinoma, and pancreas tumor.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
cardiovascular disorders and cancer. Similarly, polypeptides and antibodies
directed
to these polypeptides are useful in providing immunological probes for
differential
identification of the tissues) or cell type(s). For a number of disorders of
the above
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32
tissues or cells, particularly of the cardiovascular system, expression of
this gene at
significantly higher or lower levels may be routinely detected in certain
tissues or cell
types (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum,
plasma,
urine, synovial fluid and spinal fluid) or another tissue or sample taken from
an
individual having such a disorder, relative to the standard gene expression
level, i.e.,
the expression level in healthy tissue or bodily fluid from an individual not
having the
disorder.
Preferred polypeptides of the present invention comprise, or alternatively
consist of, one or more immunogenic epitopes shown in SEQ ID NO: 63 as
residues:
Arg-4 to Leu-12, Arg-148 to Gln-161, Val-171 to Gly-176. Polynucleotides
encoding
said polypeptides are also encompassed by the invention.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for detection, prevention, and/or
treatment of
cardiovascular diseases such as angina, angiosarcoma of the heart, congestive
heart
failure, hypertrophy of the heart, ischemia, lipoma of the heart, papillary
fibroelastoma of the heart, rheumatoid heart disease. Furthermore, the protein
may
also be used to determine biological activity, raise antibodies, as tissue
markers, to
isolate cognate ligands or receptors, to identify agents that modulate their
interactions,
in addition to its use as a nutritional supplement. Protein, as well as,
antibodies
directed against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:24 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. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 2253 of SEQ )D
N0:24, b
is an integer of 15 to 2267, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:24, and where b is greater than or equal
to a
+ 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 15
The translation product of this gene shares sequence homology with the
human microfibril-associated glycoprotein-3 (MFA3; see GenBank accession
AAC41753), an extracellular matrix protein which is thought to be involved in
connective tissue disease. The polypeptide of this gene has been determined to
have a
transmembrane domain at about amino acid position 151 - 167 of the amino acid
sequence referenced in Table 1 for this gene. Moreover, a cytoplasmic tail
encompassing amino acids 168 - 331 of this protein has also been determined.
Based
upon these characteristics, it is believed that the protein product of this
gene shares
structural features to type Ia membrane proteins.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
MDRLKSHLTVCFLPSVPFLILVSTLATAKSVTNSTLNGTNVVLGSVPVIIARTD
HIIVKEGNSAL1NCSVYGIPDPQFKWYNSIGKLLKEEEDEKERGGGKWQMHD
S GLLNITKV SFSDRGKYTCVASNIYGTVNNTV TLRVIFT S GDMGVYYMV V CL
VAFTIVMVLNITRLCMMSSHLKKTEKAINEFFRTEGAEKLQKAFEIAKRIPIITS
AKTLELAKVTQFKTMEFARYIEELARSVPLPPLIMNCRTIMEEIMEVVGLEEQ
GQNFVRHTPEGQEAADRDEVYTIPNSLKRSDSPAADSDASSLHEQPQQIAIKV
SVHPQSKKEHADDQEGGQFEVKDVEETELSAEHSPETAEPSTDVTSTELTSEE
PTPVEVPDKVLPPAYLEATEPAVTHDKNTCIIYESHV (SEQ ID NO: 96).
Moreover, fragments and variants of this polypeptide (such as, for example,
fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by
the
2S polynucleotide which hybridize, under stringent conditions, to the
polynucleotide
encoding this polypeptide are encompassed by the invention. Antibodies that
bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding this polypeptide are also encompassed by the invention.
This gene is expressed primarily in testis and to a lesser extent in human
endometrial tumor and neutrophils.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
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34
and for diagnosis of diseases and conditions which include but are not limited
to:
reproductive, immunological, proliferative and connective tissue diseases.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s).
For a number of disorders of the above tissues or cells, particularly of the
immune and
reproductive systems and connective tissues, expression of this gene at
significantly
higher or lower levels may be routinely detected in certain tissues or cell
types (e.g.,
immune, reproductive, connective, cancerous and wounded tissues) or bodily
fluids
(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or
another tissue or
sample taken from an individual having such a disorder, relative to the
standard gene
expression level, i.e., the expression level in healthy tissue or bodily fluid
from an
individual not having the disorder. Preferred polypeptides of the present
invention
comprise, or alternatively consist of, one or more immunogenic epitopes shown
in
SEQ )D NO: 64 as residues: Met-1 to Ser-6, Leu-88 to Gly-99, Ser-118 to Tyr-
123,
His-178 to Ala-185, Glu-193 to Gln-199. Polynucleotides encoding said
polypeptides
are also encompassed by the invention.
The homology to the human MFA3 protein suggests that this gene may be
involved in connective tissue diseases, such as rheumatoid arthritis,
scleroderna, and
systemic lupus erythematosus. In addition, the distribution in endometrial
tumor
tissue and homology to the human MFA3 extracellular matrix protein suggests
that
this gene may also be involved in the initiation and/or proliferation of
cancerous
tissues. Therefore polynucleotides, polypeptides, and antibodies corresponding
to this
gene may have uses in the detection, prevention, and/or treatment of cancer.
Furthermore, expression of this gene in testis and endometrium-derived tissues
suggests potential uses in the treatment of reproductive disorders such as
infertility,
impotence, and preterm labor. Furthermore, the protein may also be used to
determine biological activity, raise antibodies, as tissue markers, to isolate
cognate
ligands or receptors, to identify agents that modulate their interactions, in
addition to
its use as a nutritional supplement. Protein, as well as, antibodies directed
against the
protein may show utility as a tumor marker and/or immunotherapy targets for
the
above listed tissues.
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Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:25 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
5 excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 2623 of SEQ ID
NO:25, b
is an integer of 15 to 2637, where both a and b correspond to the positions of
10 nucleotide residues shown in SEQ ID N0:25, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16
The translation product of this gene shares sequence homology with the
15 human CTL1 protein (see GenBank accession CAB75541; all references
available
through this accession are hereby incorporated by reference herein), which is
though
to be involved in choline transport into cells. Based on the sequence
similarity, the
translation product of this clone is expected to share at least some
biological activities
with transporter proteins. The polypeptide of this gene has been determined to
have
20 transmembrane domains at about amino acid positions 156 - 172, 185 - 201,
64 - 80,
205 - 221, and 15 - 31 of the amino acid sequence referenced in Table 1 for
this gene.
Based upon these characteristics, it is believed that the protein product of
this gene
shares structural features to type IIIa membrane proteins.
In another embodiment, polypeptides comprising the amino acid sequence of
25 the open reading frame upstream of the predicted signal peptide are
contemplated by
the present invention. In specific embodiments, polypeptides of the invention
comprise, or alternatively consists of, the following amino acid sequence::
PVEGAPLSGQDMTLKKHVFFMNSCNLEVKGTQLNRMGLCVSNCPEEQLDSL
EEVQFFANTSGSFLCVYSLNSFNYTHSPKADSLCPRLPVPPSKSFPLFNRCVPQ
30 TPECYSLFASVLINDVDTLHRILSGIMSGRDTILGLCILALALSLAMMFTFRFIT
TLLVHIFISLVILGLLFVCGVLWWLYYDYTNDLSIELDTERENMKCVLGFAIV
STGITAVLLVLIFVLRKRIKLTVELFQITNKAISSAPFLLFQPLWTFAILIFFWVL
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WVAVLLSLGTAGAAQVMEGGQVEYKPLSGIRYMWSYHLIGLIWTSEFILACQ
QMTIAGAVVTCYFNRSKNDPPDHPILSSLSILFFYHQGTIVKGSFLISVVXIPRII
VMYMQNALKEQQHGALSRYLFRCCYCCFWCLDKYLLHLNQNAYTTTA1NG
TDFCTSAKDAFKILSKNSSHFTS1NCXGDFIIFLGKVLVVCFTVFGGLMAFNYN
R.AFQVWAVPLLLVAFFAYLVAHSFLSVFETVLDALFLCFAVDLETNDGSSEK
PYFMDQEFLSFVKRSNKLNNARAQQDKHSLRNEEGTELQAIVR (SEQ ID NO:
97). Moreover, fragments and variants of this polypeptide (such as, for
example,
fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%,
97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by
the
polynucleotide which hybridize, under stringent conditions, to the
polynucleotide
encoding this polypeptide are encompassed by the invention. Antibodies that
bind
polypeptides of the invention are also encompassed by the invention.
Polynucleotides
encoding this polypeptide are also encompassed by the invention.
This gene is expressed in colon, corpus callosum, cerebellum, fetal tissue,
and
in tumors derived from a number of tissue sources, including prostate, kidney,
and
palate.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
gastrointestinal, neurological, and proliferative disorders (including
cancer).
Similarly, polypeptides and antibodies directed to these polypeptides are
useful in
providing immunological probes for differential identification of the tissues)
or cell
type(s). For a number of disorders of the above tissues or cells, particularly
of the
gastrointestinal and central nervous systems, expression of this gene at
significantly
higher or lower levels may be routinely detected in certain tissues or cell
types (e.g.,
neural, digestive, cancerous and wounded tissues) or bodily fluids (e.g.,
serum,
plasma, urine, synovial fluid and spinal fluid) or another tissue or sample
taken from
an individual having such a disorder, relative to the standard gene expression
level,
i.e., the expression level in healthy tissue or bodily fluid from an
individual not
having the disorder.
Preferred polypeptides of the present invention comprise, or alternatively
consist of, one or more immunogenic epitopes shown in SEQ ID NO: 65 as
residues:
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37
Phe-33 to Asp-42, Glu-224 to Tyr-234, Lys-245 to Asn-251, Arg-254 to His-260,
Leu-262 to Thr-268. Polynucleotides encoding said polypeptides are also
encompassed by the invention.
The distribution in central nervous system tissues (i.e. corpus callosum and
cerebellum) and homology to the human CTL1 protein indicates that this gene
may be
involved in choline transport into neurons and/or glial cells. Choline is a
major
cellular metabolite known to be involved in membrane formation, as well as a
precursor to the neurotransmitter acetylcholine. Therefore, polynucleotides
and
polypeptides corresponding to this gene, as well as antibodies directed to
translation
products of this gene, are useful for the detection, prevention, and/or
treatment of
neurological disorders such as epilepsy, myasthenia gravis, Alzheimer's
Disease,
Parkinson's Disease, Huntington's Disease, Tourette's Syndrome, meningitis,
encephalitis, demyelinating diseases, peripheral neuropathies, neoplasia,
trauma,
congenital malformations, spinal cord injuries, ischemia and infarction,
aneurysms,
hemorrhages, schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder, depression, panic disorder, learning disabilities, ALS, psychoses,
autism,
and altered behaviors, including disorders in feeding, sleep patterns,
balance, and
perception. Alternatively, the expression of this gene in colon tissue and
homology to
a transporter-like protein suggests that translation products of this gene may
play a
role in digestive functions such as food absorption and intestinal motility.
In addition,
the expression of this gene in highly proliferative tissue sources such as the
fetus and
tumors suggests that translation products of this gene, as well as antibodies
directed
against translation products of this gene, may show utility as tumor markers
and/or
immunotherapy targets for the above listed tissues. Furthermore, the protein
may also
be used to determine biological activity, to raise antibodies, as tissue
markers, to
isolate cognate ligands or receptors, to identify agents that modulate their
interactions,
in addition to its use as a nutritional supplement.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:26 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. To list every related
sequence
CA 02420705 2003-02-26
WO 02/22654 PCT/USO1/01385
38
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1011 of SEQ ID
N0:26, b
is an integer of 1 S to 1025, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID N0:26, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED SY GENE NO: 17
The translation product of this gene shares sequence homology with the rat
peptide/histidine transporter protein (see Genbank accession BAA20489; all
references available through this accession are hereby incorporated by
reference
herein), which is thought to be involved in active transport of signaling
peptides
across the cell membrane. The polypeptide of this gene has been determined to
have
transmembrane domains at about amino acid position S3 - 69 and 3 - 19 of the
amino
acid sequence referenced in Table 1 for this gene. Based upon these
characteristics, it
is believed that the protein product of this gene shares structural features
to type IIIa
membrane proteins.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the following amino acid sequence:
MCPRAGRREVSPARGFPARGHRQLPGAGEDLARHGDPGALLDGLLPDAVHL
CPAGSXPPHPKHFPSQPGQHLCGPESPGQQLHGVLEMERLHYIHHNETVSQQI
GEVLYNAAPLSIWWQIPQYLLIGISEIFASIPGLEFAYSEAPRSMQGAIMGIFFC
LSGVGSLLGSSLVALLSLPGGWLHCPKDFGNINNCRMDLYFFLLAGIQAVTA
LLFVWIAGRYERASQGPASHSRFSRDRG (SEQ m NO: 99). Moreover,
2S fragments and variants of this polypeptide (such as, for example, fragments
as
described herein, polypeptides at least 80%, 8S%, 90%, 9S%, 96%, 97%, 98%, or
99% identical to these polypeptides and polypeptides encoded by the
polynucleotide
which hybridize, under stringent conditions, to the polynucleotide encoding
this
polypeptide are encompassed by-the invention. Antibodies that bind
polypeptides of
the invention are also encompassed by the invention. Polynucleotides encoding
this
polypeptide are also encompassed by the invention.
CA 02420705 2003-02-26
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39
This gene is expressed in immune cells (macrophages, B-cells, T-cells,
neutrophils, dendritic cells), cancerous cells (colon, lung, and breast
cancers, B-cell
lymphoma), schizophrenic hypothalamus, and the spinal cord.
Polynucleotides and polypeptides of the invention are useful as reagents for
differential identification of the tissues) or cell types) present in a
biological sample
and for diagnosis of diseases and conditions which include but are not limited
to:
immunological, neurological, and proliferative diseases, including cancer.
Similarly,
polypeptides and antibodies directed to these polypeptides are useful in
providing
immunological probes for differential identification of the tissues) or cell
type(s).
For a number of disorders of the above tissues or cells, particularly of the
immune and
central nervous systems, expression of this gene at significantly higher or
lower levels
may be routinely detected in certain tissues or cell types (e.g., neural,
immune,
cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine,
synovial
fluid and spinal fluid) or another tissue or sample taken from an individual
having
such a disorder, relative to the standard gene expression level, i.e., the
expression
level in healthy tissue or bodily fluid from an individual not having the
disorder.
Preferred polypeptides of the present invention comprise, or alternatively
consist of,
one or more immunogenic epitopes shown in SEQ )D NO: 66 as residues: Pro-37 to
Arg-47, Arg-72 to Ser-82, Ser-84 to Gly-91. Polynucleotides encoding said
polypeptides are also encompassed by the invention.
The tissue distribution in a number of immune system cells and homology to
the rat peptide/histidine transporter indicates that polynucleotides and
polypeptides
corresponding to this gene are useful for the detection, prevention, and/or
treatment of
immunological disorders such as arthritis, asthma, AIDS, leukemia, rheumatoid
arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne,
neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell
mediated
cytotoxicity; immune reactions to transplanted organs and tissues, such as
host-
versus-graft and graft-versus-host diseases, or autoimmunity disorders, such
as
autoimmune infertility, lens tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. In addition, the expression of this transporter in
neural
tissues suggests possible uses in the detection, prevention, and/or treatment
of
CA 02420705 2003-02-26
WO 02/22654 PCT/USO1/01385
neurological disorders, such as Alzheimer's Disease, Parkinson's Disease,
Huntington's Disease, Tourette's Syndrome, meningitis, encephalitis,
demyelinating
diseases, peripheral neuropathies, neoplasia, trauma, congenital
malformations, spinal
cord injuries, ischemia and infarction, aneurysms, hemorrhages, schizophrenia,
5 mania, dementia, paranoia, obsessive compulsive disorder, depression, panic
disorder,
learning disabilities, ALS, psychoses, autism, and altered behaviors,
including
disorders in feeding, sleep patterns, balance, and perception. Furthermore,
expression
in a number of cancerous tissues suggests that this gene may be involved in
the
initiation and/or proliferation of cancers. Therefore, protein, as well as
antibodies
10 directed against the protein may show utility as a tumor marker and/or
immunotherapy targets for the above listed tissues. Furthermore, the protein
may also
be used to determine biological activity, to raise antibodies, as tissue
markers, to
isolate cognate ligands or receptors, to identify agents that modulate their
interactions,
in addition to its use as a nutritional supplement.
15 Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ >D N0:27 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. To list every related
sequence
20 would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 921 of SEQ ID
N0:27, b
is an integer of 15 to 935, where both a and b correspond to the positions of
nucleotide residues shown in SEQ >Z? N0:27, and where b is greater than or
equal to a
25 + 14.
CA 02420705 2003-02-26
WO 02/22654 4~ PCT/USO1/01385
O M ~ ~ ~ \ ~ O
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CA 02420705 2003-02-26
WO 02/22654 PCT/USO1/01385
42
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CA 02420705 2003-02-26
WO 02/22654 PCT/USO1/01385
43
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CA 02420705 2003-02-26
WO 02/22654 PCT/USO1/01385
44
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CA 02420705 2003-02-26
WO 02/22654 PCT/USO1/01385
Table 1 summarizes the information corresponding to each "Gene No."
described above. The nucleotide sequence identified as "NT SEQ ID NO:X" was
assembled from partially homologous ("overlapping") sequences obtained from
the
"cDNA clone m" identified in Table 1 and, in some cases, from additional
related
5 DNA clones. The overlapping sequences were assembled into a single
contiguous
sequence of high redundancy (usually three to five overlapping sequences at
each
nucleotide position), resulting in a final sequence identified as SEQ m NO:X.
The cDNA Clone ID was deposited on the date and given the corresponding
deposit number listed in "ATCC Deposit No:Z and Date." Some of the deposits
10 contain multiple different clones corresponding to the same gene. "Vector"
refers to
the type of vector contained in the cDNA Clone ID.
"Total NT Seq." refers to the total number of nucleotides in the contig
identified by "Gene No." The deposited clone may contain all or most of these
sequences, reflected by the nucleotide position indicated as "5' NT of Clone
Seq."
15 and the "3' NT of Clone Seq." of SEQ ID NO:X. The nucleotide position of
SEQ ID
NO:X of the putative start colon (methionine) is identified as "5' NT of Start
Colon."
Similarly , the nucleotide position of SEQ ID NO:X of the predicted signal
sequence
is identified as "5' NT of First AA of Signal Pep."
The translated amino acid sequence, beginning with the methionine, is
20 identified as "AA SEQ ID NO:Y," although other reading frames can also be
easily
translated using known molecular biology techniques. The polypeptides produced
by
these alternative open reading frames are specifically contemplated by the
present
invention.
The first and last amino acid position of SEQ m NO:Y of the predicted signal
25 peptide is identified as "First AA of Sig Pep" and "Last AA of Sig Pep."
The
predicted first amino acid position of SEQ m NO:Y of the secreted portion is
identified as "Predicted First AA of Secreted Portion." Finally, the amino
acid
position of SEQ ID NO:Y of the last amino acid in the open reading frame is
identified as "Last AA of ORF."
30 SEQ ID NO:X (where X may be any of the polynucleotide sequences
disclosed in the sequence listing) and the translated SEQ m NO:Y (where Y may
be
any of the polypeptide sequences disclosed in the sequence listing) are
sufficiently
CA 02420705 2003-02-26
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46
accurate and otherwise suitable for a variety of uses well known in the art
and
described further below. For instance, SEQ m NO:X is useful for designing
nucleic
acid hybridization probes that will detect nucleic acid sequences contained in
SEQ ID
NO:X or the cDNA contained in the deposited clone. These probes will also
hybridize to nucleic acid molecules in biological samples, thereby enabling a
variety
of forensic and diagnostic methods of the invention. Similarly, polypeptides
identified from SEQ m NO:Y may be used, for example, to generate antibodies
which bind specif tally to proteins containing the polypeptides and the
secreted
proteins encoded by the cDNA clones identified in Table 1.
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).
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 m NO:X and the predicted
translated amino acid sequence identified as SEQ m NO:Y, but also a sample of
plasmid DNA containing a human cDNA of the invention deposited with the ATCC,
as set forth in Table 1. The nucleotide sequence of each deposited clone can
readily
be determined by sequencing the deposited clone in accordance with known
methods.
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.
The present invention also relates to the genes corresponding to SEQ m
NO:X, SEQ m NO:Y, or the deposited clone. The corresponding gene can be
isolated in accordance with known methods using the sequence information
disclosed
CA 02420705 2003-02-26
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47
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.
Also provided in the present invention are allelic variants, orthologs, and/or
species homologs. Procedures knomn 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, SEQ ID NO:Y, or a
deposited clone, 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.
Table 2 provides predicted epitopes contained in certain embodiments of the
invention and polynucleotide sequences that may be disclaimed according to
certain
embodiments of the invention. The first column refers to each "Gene #"
described
above in Table 1. The second column provides the sequence identifier, "NT SEQ
ID
NO:X", for polynucleotide sequences disclosed in Table 1. The third column
provides
the sequence identif er, "AA SEQ m NO:Y", for polypeptide sequences disclosed
in
Table 1. The fourth column provides a unique integer "ntA" where "ntA" is any
integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the
fifth
column provides a unique integer "ntB" where "ntB" is any integer between 15
and
the fnal nucleotide of SEQ ID NO:X, where both ntA and ntB correspond to the
positions of nucleotide residues shown in SEQ ID NO:X, and where ntB 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.
Column 6 lists residues comprising predicted epitopes contained in
the~polypeptides
encoded by each of the preferred ORFs (SEQ ID NO:Y). Identification of
potential
immunogenic regions was performed according to the method of Jameson and Wolf
((1988) CABIOS, 4; 181-186); 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
CA 02420705 2003-02-26
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48
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 2 as "Predicted epitopes".
Polypeptides of the invention may possess one, two, three, four, five or more
antigenic epitopes comprising residues described in Table 2. It will be
appreciated
that depending on the analytical criteria used to predict antigenic
determinants, the
exact address of the determinant may vary slightly.
Table 3 summarizes the expression profile of polynucleotides corresponding
to the clones disclosed in Table 1. The first column provides a unique clone
identifier, "Clone ID", for a cDNA clone related to each contig sequence
disclosed in
Table 1. Column 2, "Library Code(s)" shows the expression profile of tissue
and/or
cell line libraries which express the polynucleotides of the invention. Each
Library
Code in column 2 represents a tissue/cell source identifier code corresponding
to the
Library Code and Library description provided in Table 4. Expression of these
polynucleotides was not observed in the other tissues and/or cell libraries
tested. One
of skill in the art could routinely use this information to identify 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
expression.
Table 4 provides a key to the Library Code disclosed in Table 3. Column 1
provides the Library Code disclosed in Table 3, column 2. Column 2 provides a
description of the tissue or cell source from which the corresponding library
was
derived. Library codes corresponding to diseased Tissues are indicated in
column. 3
with the word "disease".
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49
Table 2
Gene NT SEQ ID AA SEQ nt A nt B Predicted Epitopes
# ID
NO: X NO: Y
1 11 50 1 - 15 - Tyr-52 to Gly-63
3175 3189
Lys-68 to Leu-73
Met-107 to Asp-118
Pro-157to Asn-166
Thr-189 to Tyr-195
Phe-200 to Ser-211
Asp-240 to Lys-250
His-254 to As -264.
1 28 67 1 - 15 - Tyr-52 to Gly-63
2561 2575
Lys-68 to Leu-73
Met-107 to Asp-118
Pro-157 to Asn-166
Thr-189 to Tyr-195
Phe-200 to Ser-211
Asp-240 to Lys-250
His-254 to As -264.
1 29 68 1 - 15 - Tyr-52 to Gly-63
711 725
Lys-68 to Leu-73
Met-107 to As -118.
1 30 69 1 - 15 -
603 617
2 12 51 1 - 15 - Arg-59 to Glu-67
415 429
Tyr-80 to Ar -85.
2 31 70 1 - 15 - Pro-39 to Pro-46
2133 2147
Pro-72 to Pro-85
Val-131 to Asn-139
Gly-158 to Gly-164.
3 13 52 1 - 15 - Ile-110 to Gly-116
793 807
Phe-127 to Thr-132.
3 32 71 1 - 15 - Ser-28 to Ser-33.
650 664
4 14 53 1 - 15 -
648 662
4 33 72 1-1002 15-1016
4 34 73 1 - 15 -
231 245
4 35 74 1 - 15 -
708 722
15 54 1 - 15 - Pro-41 to Ser-49.
2947 2961
5 36 75 1 - 15 - Pro-41 to Ser-49.
725 739
6 16 55 1 - 15 -
964 978
7 17 56 1 - 15 - Gly-37 to Met-42
1196 1210
Pro-45 to Trp-51
Gln-54 to Phe-59
Ile-79 to Asp-87
Pro-89 to Glu-102
Pro-178 to Arg-183
Ser-203 to Pro-208
Pro-288 to Gly-296.
7 37 76 1 - 15 - Gly-37 to Met-42
816 830
Pro-45 to Trp-51
Gln-54 to Phe-59
Ile-79 to Asp-87
Pro-89 to Glu-102.
7 38 77 1 - 15 -
664 678
18 57 1 - ~ 15 Val-31 to Gly-49.
520 - 534
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9 19 58 1 - 15 - Glu-70 to Ar -79.
907 921
9 39 78 1 - 15 - Glu-70 to Ar -79.
736 750
10 20 59 1 - 15 - Met-1 to T -9.
507 521
10 40 79 1 - 15 - Met-1 to Trp-9
528 542
Pro-121 to His-131.
11 21 60 1 - 15 -
654 668
11 41 80 1 - 15 -
584 598
12 22 61 1 - 15 - Pro-21 to Gly-27
662 676
Ser-51 to Ala-65
His-89 to Asn-94.
12 42 81 1 - 15 - Arg-19 to Tyr-25
592 606
Gly-32 to Cys-41.
13 23 62 1 - 15 - Pro-13 to Ser-18
558 572
Gly-52 to Ala-58
Ser-67 to Trp-74.
13 43 82 1 - 15 - Arg-20 to Leu-34
246 260
Glu-36 to Ser-44.
14 24 63 1 - 15 - Arg-4 to Leu-12
2253 2267
Arg-148 to Gln-161
Val-171 to Gly-176.
14 44 83 1 - 15 - Met-1 to Arg-12.
2255 2269
14 45 84 1 - 15 - Arg-4 to Leu-12
2255 2269
Arg-148 to Gin-161
Val-171 to Gly-176.
15 25 64 1 - 15 - Met-1 to Ser-6
2623 2637
Leu-88 to Gly-99
Ser-118 to Tyr-123
His-178 to Ala-185
Glu-193 to Gln-199.
15 46 85 1 - 15 - Met-7 to Gln-13
635 649
Arg-43 to Pro-56.
16 26 65 1 - 15 - Phe-33 to Asp-42
1011 1025
Glu-224 to Tyr-234
Lys-245 to Asn-251
Arg-254 to His-260
Leu-262 to Thr-268.
16 47 86 1 - 15 - Phe-33 to As -42.
1907 1921
17 27 66 1 - 15 - Pro-37 to Arg-47
921 935
Arg-72 to Ser-82
Ser-84 to Gly-91.
17 48 87 1 - 15 - Pro-37 to Arg-47
1133 1147
Arg-72 to Ser-82
Ser-84 to Gly-91.
17 49 88 1 - 15 - Pro-23 to Arg-33
425 439
Arg-58 to Ser-68
Ser-70 to Gly-77.
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S1
Table 3
Clone ID Librar Codes)
HE9PR39 H0013 H0144 H0163 H0171 HOS47 H0624 L1290 50003
T0006
HIPBA31 H0038 H0039 H0042 H0090 HO1S6 H016S H0204 H0318
H0393 H0424
HOS6S HOS87 H0618 H0647 H06S8 H0660 H0661 H0664
L1290 S0010
50045 50046 50116 50260 S0360 50380 50434 S0464
T0067
HCQDE22 HOS96 L1290
HNGNI2S L1290 50428
HTTJK27 H0634 S0136
HTLIV 19 H0618
HTFOE85 H0271 50424
HTLGE31 H0618 L1290 S0053
HE8NQ42 H0013
HNGOQ44 50428
HODHE60 H061S
HPJDM47 50152
HPJEC20 H0069 H0083 H0144 H0179 H0271 HOSSO HOSS6 H0618
H0634 H0673
H0674 S0031 S0040 SOOS2 SOOS3 50116 SO1S2 50216
50428 T0006
HOSDWS8 H0031 H0036 H0038 H0039 HOOS6 H0373 H043S HOS21
HOS43 HOS7S
HOS80 H0619 H0623 H0644 H0672 L1290 S0003 S0028
SO1S2 S0212 50278
S03S4 S03S6 S03S8 53014 T0067
HNHLA36 H0046 L1290 SOOS2 S0216 50420 S0428
HE8QD31 H0013 H0046 HOOS2 H0144 H0171 H0327 L1290 S0328
S03S4 50432 50444
T0040
HNHOD23 H027I H0521 HOS4S H0576 HOS83 HOS94 H0641 H0650
H06S6 H0663
L1290 S0142 50144 50216 50260 S0278 S03S8 S0376
50388
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Table 4
Librar Code Librar Descri tion Disease
H0013 Human 8 Week Whole Embryo
H0031 Human Placenta
H0036 Human Adult Small Intestine
H0038 Human Testes
H0039 Human Pancreas Tumor disease
H0042 Human Adult Pulmonary
H0046 Human Endometrial Tumor disease
H0052 Human Cerebellum
H0056 Human Umbilical Vein, Endo. remake
H0069 Human Activated T-Cells
H0083 HUMAN :ftJRKAT MEMBRANE BOUND POLYSOMES
H0090 Human T-Cell Lym homa disease
H0144 Nine Week Old Early Stage Human
H0156 Human Adrenal Gland Tumor disease
H0163 Human Synovium
H0165 Human Prostate Cancer, Sta a B2 disease
H0171 12 Week Old Early Stage Human, II
H0179 Human Neutro hil
H0204 Human Colon Cancer, subtracted
H0271 Human Neutro hil, Activated
H0318 HUMAN B CELL LYMPHOMA disease
H0327 human co us colosum
H0373 Human Heart
H0393 Fetal Liver, subtraction II
H0424 Human Pituitary, subt IX
H0435 Ovarian Tumor 10-3-95
H0521 Primary Dendritic Cells, lib 1
H0543 T cell hel er II
H0545 Human endometrial stromal cells-treated
with ro esterone
H0547 NTERA2 teratocarcinoma cell line+retinoic
acid (14 days)
H0550 H. Epididiymus, cauda
H0556 Activated T-cell(12h)/Thiouridine-re-excision
H0565 Human Fetal Brain, normalized 100024F
H0575 Human Adult Pulmonary,re-excision
H0576 Resting T-Cell, re-excision
H0580 Dendritic cells, ooled
H0583 B Cell lym homa disease
H0587 Healin groin wound, 7.5 hours post disease
incision
H0594 Human Lun Cancer,re-excision disease
H0596 Human Colon Cancer,re-excision
H0615 Human Ovarian Cancer Reexcision disease
H0618 Human Adult Testes, Large Inserts,
Reexcision
H0619 Fetal Heart
H0623 Human Umbilical Vein, Reexcision
H0624 12 Week Early Stage Human II, Reexcision
H0634 Human Testes Tumor, re-excision disease
H0641 LPS activated derived dendritic cells
H0644 Human Placenta (re-excision)
H0647 Lung, Cancer (4005163 B7): Invasive, disease
Poorly Diff.
Adenocarcinoma, Metastatic
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H0650 B-Cells
H0656 B-cells unstimulated)
H0658 Ovary, Cancer (9809C332): Poorly differentiateddisease
adenocarcinoma
H0660 Ovary, Cancer: 15799A1F) Poorly differentiateddisease
carcinoma
H0661 Breast, Cancer: (4004943 AS) disease
H0663 Breast, Cancer: (4005522 A2) disease
H0664 Breast, Cancer: (9806C012R) disease
H0672 Ovary, Cancer: (4004576 A8)
H0673 Human Prostate Cancer, Stage B2, re-excision
H0674 Human Prostate Cancer, Stage C, re-excission
L1290 Stratagene lun (#937210)
50003 Human Osteoclastoma disease
50010 Human Amygdala
50028 Smooth muscle,control
50031 S final cord
50040 Adi oc es
50045 Endothelial cells-control
50046 Endothelial-induced
50052 neutro hits control
50053 Neutro hils IL-1 and LPS induced
S0116 Bone marrow
S0136 PERM TF274
50142 Macrophage-oxLDL
S0144 Macrophage (GM-CSF treated)
S0152 PC3 Prostate cell line
50212 Bone Marrow Stromal Cell, untreated
50216 Neutro hils IL-1 and LPS induced
50260 S final Cord, re-excision
S0278 H Macro ha a (GM-CSF treated), re-excision
S0328 Palate carcinoma disease
50354 Colon Normal II
50356 Colon Carcinoma disease
50358 Colon Normal III
50360 Colon Tumor II disease
50376 Colon Tumor disease
50380 Pancreas Tumor PCA4 Tu disease
S0388 Human Hypothalamus,schizophrenia, re-excisiondisease
S0420 CHME Cell Line,untreated
50424 TF-1 Cell Line GM-CSF Treated
50428 Neutro hils control, re-excision
S0432 Sinus iriformis Tumour
S0434 Stomach Normal disease
S0444 Colon Tumor disease
50464 Larynx Normal
53014 Smooth muscle, senun induced,re-exc
T0006 Human Pineal Gland
T0040 HSC172 cells
T0067 Human Thyroid
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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.
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.
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 secreted
protein.
The present invention provides a polynucleotide comprising, or alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or a cDNA
contained
in ATCC deposit Z. The present invention also provides a polypeptide
comprising, or
alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y andlor a
polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides
encoding a polypeptide comprising, or alternatively consisting of the
polypeptide
sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA
contained in ATCC deposit Z are also encompassed by the invention.
Signal Seguences
The present invention also encompasses mature forms of the polypeptide
having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence
encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature
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forms (such as, for example, the polynucleotide sequence in SEQ TD NO:X and/or
the
polynucleotide sequence contained in the cDNA of a deposited clone) are also
encompassed by the invention. According to the signal hypothesis, proteins
secreted ,
by mammalian cells have a signal or secretary leader sequence which is cleaved
from
5 the mature protein once export of the growing protein chain across the rough
endoplasmic reticulum has been initiated. Most mammalian cells and even insect
cells cleave secreted proteins with the same specificity. However, in some
cases,
cleavage of a secreted protein is not entirely uniform, which results in two
or more
mature species of the protein. Further, it has long been known that cleavage
10 specificity of a secreted protein is ultimately determined by the primary
structure of
the complete protein, that is, it is inherent in the amino acid sequence of
the
polypeptide.
Methods for predicting whether a protein has a signal sequence, as well as the
cleavage point for that sequence, are available. For instance, the method of
15 McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-
terminal
charged region and a subsequent uncharged region of the complete (uncleaved)
protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses
the
information from the residues surrounding the cleavage site, typically
residues -13 to
+2, where +1 indicates the amino terminus of the secreted protein. The
accuracy of
20 predicting the cleavage points of known mammalian secretory proteins for
each of
these methods is in the range of 75-80%. (von Heinj e, supra.) However, the
two
methods do not always produce the same predicted cleavage points) for a given
protein.
In the present case, the deduced amino acid sequence of the secreted
25 polypeptide was analyzed by a computer program called SignalP (Henrik
Nielsen et
al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location
of a
protein based on the amino acid sequence. As part of this computational
prediction of
localization, the methods of McGeoch and von Heinje are incorporated. The
analysis
of the amino acid sequences of the secreted proteins described herein by this
program
30 provided the results shown in Table 1.
As one of ordinary skill would appreciate, however, cleavage sites sometimes
vary from organism to organism and cannot be predicted with absolute
certainty.
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Accordingly, the present invention provides secreted polypeptides having a
sequence
shown in SEQ m NO:Y which have an N-terminus beginning within 5 residues
(i.e.,
+ or - 5 residues) of the predicted cleavage point. Similarly, it is also
recognized that
in some cases, cleavage of the signal sequence from a secreted protein is not
entirely
uniform, resulting in more than one secreted species. These polypeptides, and
the
polynucleotides encoding such polypeptides, are contemplated by the present
invention.
Moreover, the signal sequence identified by the above analysis may not
necessarily predict the naturally occurring signal sequence. For example, the
naturally occurring signal sequence may be further upstream from the predicted
signal
sequence. However, it is likely that the predicted signal sequence will be
capable of
directing the secreted protein to the ER. Nonetheless, the present invention
provides
tile mature protein produced by expression of the polynucleotide sequence of
SEQ m
NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited
clone, in a mammalian cell (e.g., COS cells, as desribed below). These
polypeptides,
and the polynucleotides encoding such polypeptides, are contemplated by the
present
invention.
Polyuucleotide and Polypeptide Variants
The present invention is directed to variants of the polynucleotide sequence
disclosed in SEQ m NO:X, the complementary strand thereto, and/or the cDNA
sequence contained in a deposited clone.
The present invention also encompasses variants of the polypeptide sequence
disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.
"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.
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% or 99% identical to, for example, the nucleotide
coding sequence in SEQ m NO:X or the complementary strand thereto, the
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nucleotide coding sequence contained in a deposited cDNA clone or the
complementary strand thereto, a nucleotide sequence encoding the polypeptide
of
SEQ ID NO:Y, a nucleotide sequence encoding the polypeptide encoded by the
cDNA contained in a deposited clone, and/or polynucleotide fragments of any of
these nucleic acid molecules (e.g., those fragments described herein).
Polynucleotides which hybridize to these nucleic acid molecules under
stringent
hybridization conditions or lower stringency conditions are also encompassed
by the
invention, as are polypeptides encoded by these polynucleotides.
The present invention is also directed to polypeptides which comprise, or
alternatively consist of, an amino acid sequence which is at least 80%, 85%,
90%,
95%, 96%; 97%, 98%, 99% identical to, for example, the polypeptide sequence
shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained
in a deposited clone, and/or polypeptide fragments of any of these
polypeptides (e.g.,
those fragments described herein).
By a nucleic acid having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended
that the nucleotide sequence of the nucleic acid is identical to the reference
sequence
except that the nucleotide sequence may include up to five point mutations per
each
100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
In
other words, to obtain a nucleic acid having a nucleotide sequence at least
95%
identical to a reference nucleotide sequence, up to 5% of the nucleotides in
the
reference sequence may be deleted or substituted with another nucleotide, or a
number of nucleotides up to 5% of the total nucleotides in the reference
sequence may
be inserted into the reference sequence. The query sequence may be an entire
sequence shown inTable 1, the ORF (open reading frame), or any fragment
specified
as described herein.
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 presence 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
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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 in
percent
identity. Preferred parameters used in a FASTDB alignment of DNA sequences to
calculate percent identiy 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 lenght of the subject
nucleotide sequence, whichever is shorter.
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 subj ect sequence when calculating percent identity. For
subj ect
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. ~nly bases
outside the
5' and 3' bases of the subj ect 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.
For example, a 90 base subject sequence is aligned to a 100 base query
sequence to determine percent identity. The deletions occur at the 5' end of
the
subject sequence and therefore, the FASTDB alignment does not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent
10% of the sequence (number of bases at the 5' and 3' ends not matched/total
number
of bases in the query sequence) so 10% is subtracted from the percent identity
score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the final percent identity would be 90%. In another example, a 90 base
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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 made for the
purposes
of the present invention.
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.
As a practical matter, whether any particular polypeptide is at least 80%,
85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, an amino acid
sequences shown in Table 1 (SEQ ID NO:Y) or to the amino acid sequence encoded
by cDNA contained in a deposited clone can be determined conventionally using
known computer programs. A preferred method for determing 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 aligmnent the query and subject
sequences
are either both nucleotide sequences or both amino acid sequences. The result
of said
global sequence alignment is in 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,
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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.
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
5 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 subj ect 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
10 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 arnve at a final percent identity score. This final percent
identity score
15 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 subj ect sequence.
20 For example, a 90 amino acid residue subj ect sequence is aligned with a
100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subj ect sequence and therefore, the FASTDB alignment does not
show a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the sequence (number of residues at the N-
and C-
25 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
30 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
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61
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.
The variants 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, variants in which 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).
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.
. 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 secreted protein without
substantial
loss of biological function. The authors of Ron et al., J. Biol. Chem. 268:
2984-2988
(1993), reported variant I~GF 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).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity similar to that of the naturally occurring protein. For example,
Gayle arid
coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive
mutational
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62
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]." (See,
Abstract.) 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.
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.
Thus, the invention further includes polypeptide variants which show
substantial biological activity. 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. For example, guidance concerning how
to make
phenotypically silent amino acid substitutions is provided in Bowie et al.,
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.
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.
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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.
(Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant
molecules can then be tested for biological activity.
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 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) substitution with one or more of amino acid residues
having a
substituent group, or (iii) fusion of the mature polypeptide with another
compound,
such as a compound to increase the stability and/or solubility of the
polypeptide (for
example, polyethylene glycol), or (iv) fusion of the polypeptide with
additional amino
acids, such as, for example, an IgG Fc fusion region peptide, 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.
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64
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. (Pinckard et al., Clin. Exp. Imtnunol. 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).)
A further embodiment of the invention relates to a polypeptide which
comprises the amino acid sequence of the present invention 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. Of
course, in
order of ever-increasing preference, it is highly preferable for a peptide or
polypeptide
to have an amino acid sequence which comprises the amino acid sequence of the
present invention, which contains at least one, but not more than 10, 9, 8, 7,
6, 5, 4, 3,
2 or 1 amino acid substitutions. In specific embodiments, the number of
additions,
substitutions, and/or deletions in the amino acid sequence of the present
invention or
fragments thereof (e.g., the mature form and/or other fragments described
herein), is
1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions
are
preferable.
Polynucleotide and Polypeptide Fragments
The present invention is also directed to polynucleotide fragments of the
polynucleotides of the invention. .
In the present invention, a "polynucleotide fragment" refers to a short
polynucleotide having a nucleic acid sequence which: is a portion of that
contained in
a deposited clone, or encoding the polypeptide encoded by the cDNA in a
deposited
clone; is a portion of that shown in SEQ m NO:X or the complementary strand
thereto, or is a portion of a polynucleotide sequence encoding the polypeptide
of SEQ
m NO:Y. The nucleotide 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,
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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 a deposited clone or the nucleotide sequence shown in
SEQ ID
5 NO:X. In this context "about" includes the particularly recited value, 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.,
50, 150, 500, 600, 2000 nucleotides) are preferred.
10 Moreover, representative examples of polynucleotide fragments of the
invention, include, for example, fragments comprising, or alternatively
consisting of,
a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-
250,
251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-
750,
751-800, 800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150,
15 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, or 2001 to the end of SEQ m NO:X, or the
complementary strand thereto, or the cDNA contained in a deposited clone. In
this
context "about" includes the particularly recited ranges, and ranges larger or
smaller
20 by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both
termini.
Preferably, these fragments encode a polypeptide which has biological
activity. More
preferably, these polynucleotides can be used as probes or primers as
discussed
herein. Polynucleotides which hybridize to these nucleic acid molecules under
stringent hybridization conditions or lower stringency conditions are also
25 encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
In the present invention, a "polypeptide fragment" refers to an amino acid
sequence which is a portion of that contained in SEQ m NO:Y or encoded by the
cDNA contained in a deposited clone. Protein (polypeptide) fragments may be
"free-
standing," or comprised within a larger polypeptide of which the fragment
forms a
30 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,
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66
41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 to the end of the
coding
region. Moreover, polypeptide fragments can be about 20, 30, 40, 50, 60, 70,
80, 90,
100, 110, 120, 130, 140, or 150 amino acids in length. In this context "about"
includes the particularly recited ranges or values, and 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 polypeptides are also encompassed by the
invention.
Preferred 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.
Also preferred are polypeptide and polynucleotide fragments characterized by
structural or functional domains, such as fragments that comprise alpha-helix
and
alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn
and turn-
forming regions, coil and coil-forming regions, hydrophilic regions,
hydrophobic
regions, alpha amphipathic regions, beta amphipathic regions, flexible
regions,
surface-forming regions, substrate binding region, and high antigenic index
regions.
Polypeptide fragments of SEQ m NO:Y falling within conserved domains are
specifically contemplated by the present invention. Moreover, polynucleotides
encoding these domains are also contemplated.
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. Polynucleotides encoding these polypeptide fragments are
also
encompassed by the invention.
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67
Preferably, the polynucleotide fragments of the invention encode a
polypeptide which demonstrates a functional activity. By a polypeptide
demonstrating a "functional activity" is meant, a polypeptide capable of
displaying
one or more known functional activities associated with a full-length
(complete)
polypeptide of invention protein. 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 antibody to the polypeptide of
the
invention], immunogenicity (ability to generate antibody which binds to a
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.
The functional activity of polypeptides of the invention, and fragments,
variants derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind
or compete with full-length polypeptide of the invention for binding to an
antibody of
the polypeptide of the invention, 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 irmnunosorbent
assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ immunoassays (using
colloidal gold, enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays), complement fixation assays, immunofluorescence
assays,
protein A assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary antibody. In
another
embodiment, the 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.
In another embodiment, where a ligand for a polypeptide of the invention
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
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68
chromatography, protein affinity chromatography, and affinity blotting. See
generally, Phizicky, E., et al., 1995, Microbiol. Rev. 59:94-123. In another
embodiment, physiological correlates of binding of a polypeptide of the
invention to
its substrates (signal transduction) can be assayed.
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
invention and fragments, variants derivatives and analogs thereof to elicit
related
biological activity related to that of the polypeptide of the invention
(either in vitro or
in vivo). Other methods will be known to the skilled artisan and are within
the scope
of the invention.
Epitopes and Antibodies
The present invention encompasses polypeptides comprising, or alternatively
consisting of, an epitope of the polypeptide having an amino acid sequence of
SEQ m
I S NO:Y, or an epitope of the polypeptide sequence encoded by a
polynucleotide
sequence contained in ATCC deposit No. Z or encoded by a polynucleotide that
hybridizes to the complement of the sequence of SEQ m NO:X or contained in
ATCC deposit No. Z under stringent hybridization conditions or lower
stringency
hybridization conditions 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 m NO:X),
polynucleotide sequences of the complementary strand of a polynucleotide
sequence
encoding an epitope of the invention, and p~lynucleotide sequences which
hybridize
to the complementary strand under stringent hybridization conditions or lower
stringency hybridization conditions defined supra.
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 fox generating
antibodies
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69
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. hnmunospecific binding excludes non-specific binding but
does not
necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985),
further described in U.S. Patent No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at Least 15, at Least 20,
at Least 25, at
least 30, at least 40, at least 50, and, most preferably, between about 15 to
about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100
amino acid residues in length. Additional non-exclusive preferred antigenic
epitopes
include the antigenic epitopes disclosed herein, as well as portions thereof.
Antigenic
epitopes are useful, for example, to raise antibodies, including monoclonal
antibodies,
that specifically bind the epitope. Preferred antigenic epitopes include the
antigenic
epitopes disclosed herein, as well as any combination of two, three, four,
five or more
of these antigenic epitopes. Antigenic epitopes can be used as the target
molecules in
immunoassays. (See, for instance, Wilson et al., Cel137:767-778 (1984);
Sutcliffe et
al., Science 219:660-666 (1983)).
Similarly, immunogenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe
et al., supra; Wilson et al., supra; Chow et aL, Proc. Natl. Acad. Sci. USA
82:910-
914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred
immunogenic
epitopes include the immunogenic epitopes disclosed herein, as well as any
combination of two, three, four, five or more of these immunogenic epitopes.
The
polypeptides comprising one or more immunogenic epitopes may be presented for
eliciting an antibody response together with a carrier protein, such as an
albumin, to
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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
Garner. 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
5 least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
Epitope-bearing polypeptides of the present invention may be used to induce
antibodies according to methods well known in the art including, but not
limited to,
in vivo immunization, W vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-
10 2354 (1985). If in vivo immunization is used, animals may be irnlnunized
with free
peptide; however, anti-peptide antibody titer may be boosted by coupling the
peptide
to a macromolecular Garner, 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),
15 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
20 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
25 peptide on a solid support and elution of the selected antibodies according
to methods
well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention (e.g., those comprising an immunogenic
or
antigenic epitope) can be fused to heterologous polypeptide sequences. For
example,
30 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,
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71
resulting in chimeric polypeptides. By way of another non-limiting example,
polypeptides and/or antibodies of the present invention (including fragments
or
variants thereof) may be fused with albumin (including but not limited to
recombinant
human serum albumin or fragments or variants thereof (see, e.g., U.S. Patent
No.
5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their
entirety)).
In a preferred embodiment, polypeptides and/or antibodies of the present
invention
(including fragments or variants thereof) are fused with the mature form of
human
serum albumin (i.e., amino acids 1- 585 of human serum albumin as shown in
Figures 1 and 2 of EP Patent 0 322 094) which is herein incorporated by
reference in
its entirety. In another preferred embodiment, polypeptides and/or antibodies
of the
present invention (including fragments or variants thereof) are fused with
polypeptide
fragments comprising, or alternatively consisting of, amino acid residues 1-z
of
human serum albumin, where z is an integer from 369 to 419, as described in
U.S.
Patent 5,766,883 herein incorporated by reference in its entirety.
Polypeptides and/or
antibodies of the present invention (including fragments or variants thereof)
may be
fused to either the N- or C-terminal end of the heterologous protein (e.g.,
immunoglobulin Fc polypeptide or human serum albumin polypeptide).
Polynucleotides encoding fusion proteins of the invention are also encompassed
by
the invention.
Such fusion proteins may facilitate purification and may increase half life in
vivo. This has been shown for chimeric proteins consisting of the first two
domains
of the human CD4-polypeptide and various domains of the constant regions of
the
heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen
across
the epithelial barner 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 disulfde-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
monorneric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J.
Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can
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72
also be recombined with a gene of interest as an epitope tag (e.g., the
hemagglutinin
("HA") tag or flag tag) to aid in detection and purification of the expressed
polypeptide. For example, a system described by Janknecht et al. allows for
the
ready purification of non-denatured fusion proteins expressed in human cell
lines
(Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this
system, the
gene of interest is subcloned into a vaccinia recombination plasmid such that
the
open reading frame of the gene is translationally fused to an amino-terminal
tag
consisting of six histidine residues. The tag serves as a matrix binding
domain fox 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.
Additional fusion proteins of the invention may be generated through the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). 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
rnutagenesis 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.
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Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment,
or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying specif c
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), and epitope-binding fragments of
any of
the above. The term "antibody," as used herein, refers to imrnunoglobulin
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.
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,
CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable regions) with a hinge
region,
CH1, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
marine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or
chicken. As used herein, "human" antibodies include antibodies having the
amino
acid sequence of a human ixnmunoglobulin and include antibodies isolated from
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74
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described
infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
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).
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,
by size in
contiguous amino acid residues, or listed in the Tables and Figures.
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.
Antibodies of the present invention may also be described or specified in
terms of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least
85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least
55%, and at
least 50% identity (as calculated using methods known in the art and described
herein) to a polypeptide of the present invention are also included in the
present
invention. In specific embodiments, antibodies of the present invention cross-
react
with marine, 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
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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
10 constant or Kd less than 5 X 10-2 M, 10-2 M, 5 X 10-3 M, 10-3 M, 5 X 10'4
M, 10-4 M,
5 X 10-5 M, 10'5 M, 5 X 10-6 M, 10-6M, 5 X 10-~ M, 10' M, 5 X 10-8 M, 10-8 M,
5 X
10'9 M, 10-~ M, 5 X 10-1° M, 10'1° M, 5 X 10-11 M, 10-11 M, 5 X
10-lZ M, lo-la M, 5 X
10-13 M, 10-i s M, 5 X 10-14 M, 10-14 M, 5 X 10-i s M, or 10-15 M.
The invention also provides antibodies that competitively inhibit binding of
an
15 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 least 90%, at least 85 %, at least 80%, at least
75%, at least
70%, at least 60%, or at least 50%.
20 Antibodies of the present invention may act as agonists or antagonists of
the
polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. Preferrably, antibodies of the present
invention
bind an antigenic epitope disclosed herein, or a portion thereof. The
invention
25 features both receptor-specific antibodies and ligand-specific antibodies.
The
invention also features receptor-specific antibodies which do not prevent
ligand
binding but prevent receptor activation. Receptor activation (i.e., signaling)
may be
determined by techniques described herein or otherwise known in the art. For
example, receptor activation can be determined by detecting the
phosphorylation
30 (e.g., tyrosine or serine/tbreonine) 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
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76
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.
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).
Antibodies of the present invention may be used, for example, but not limited
to, to purify, detect, and target the polypeptides of the present invention,
including
both in vitro and in vivo diagnostic and therapeutic methods. For example, the
antibodies have use in immunoassays for qualitatively and quantitatively
measuring
levels of the polypeptides of the present invention in biological samples.
See, e.g.,
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Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
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 covalently and non-covalently
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 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 protectinglblocking 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.
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,
dinitrophenol, and
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potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. Such adjuvants are also well known in the art.
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.
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 (e.g., Example 16). 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.
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
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hybridomas resulting from the fusion for hybridoma clones that secrete an
antibody
able to bind a polypeptide of the invention.
Antibody fragments which recognize specific epitopes may be generated by
known techniques. For example, Fab and F(ab')2 fragments of the invention may
be
produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such
as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain constant region
and the
CH1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated
using various phage display methods known in the art. 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 92118619; 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.
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,
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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
5 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).
Examples of techniques which can be used to produce single-chain Fvs and
10 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
15 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.
20 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.
25 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
30 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
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81
humanized using a variety of techniques known in the art including, for
example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, Molecular Irmnunology 28(4/5):489-498 (1991); Studnicka et
al.,
Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973
(1994)),
and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO
98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of
which is incorporated herein by reference in its entirety.
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.
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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. linmunol. 13:65-93 (1995). For a
detailed discussion of this technology for producing human antibodies and
human
S 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 S98 877; U.S. Patent Nos. 5,413,923; S,62S,126; 5,633,425;
S,S69,825;
5,661,016; S,S45,806; 5,814,318; S,88S,793; 5,916,771; and 5,939,598, which
are
incorporated by reference herein in their entirety. In addition, companies
such as
Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to
provide human antibodies directed against a selected antigen using technology
similar
to that described above.
Completely human antibodies which recognize a selected epitope can be
generated using a technique referred to as "guided selection." In this
approach a
1 S 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)).
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(S):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 Iigand
can be
used to generate anti-idiotypes that "mimic" the polypeptide multimerization
and/or
2S 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. For example,
such
anti-idiotypic antibodies can be used to bind a polypeptide of the invention
and/or to
bind its Iigands/receptors, and thereby block its biological activity.
Polynucleotides Efacoding Afztibodies
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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
lower
stringency hybridization conditions, e.g., as defined supra, to
polynucleotides that
encode an antibody, preferably, that specifically binds to a polypeptide of
the
invention, preferably, an antibody that binds to a polypeptide having the
amino acid
sequence of SEQ ID NO:Y.
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.
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.
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
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example, the techniques described in Sambroolc 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.
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.
In addition, techniques developed for the production of "chimeric antibodies"
(Mornson et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature
312:604-608 (1984); Takeda~ et al., Nature 314:452-454 (1985)) by splicing
genes
from a mouse antibody molecule of appropriate antigen specificity together
with
genes from a human antibody molecule of appropriate biological activity can be
used.
As described supra, a chimeric antibody is a molecule in which different
portions are
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derived from different animal species, such as those having a variable region
derived
from a marine mAb and a human immunoglobulin constant region, e.g., humanized
antibodies.
Alternatively, techniques described for the production of single chain
5 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
10 assembly of functional Fv fragments in E. coli may also be used (Skerra et
al.,
Science 242:1038- 1041 (1988)).
Methods of P~~oducifzg Antibodies
The antibodies of the invention can be produced by any method known in the
15 art for the synthesis of antibodies, in particular, by chemical synthesis
or preferably,
by recombinant expression techniques.
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
20 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
25 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,
30 for example, in vitro recombinant DNA techniques, synthetic techniques, and
in vivo
genetic recombination. The invention, thus, provides replicable vectors
comprising a
nucleotide sequence encoding an antibody molecule of the invention, or a heavy
or
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86
light chain thereof, or a heavy or light chain variable domain, operably
linlced to a
promoter. Such vectors may include the nucleotide sequence encoding the
constant
region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT
Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable
domain of
the antibody may be cloned into such a vector for expression of the entire
heavy or
light chain.
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 linl~ed 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.
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
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87
vaccinia virus 7.SK promoter). Preferably, bacterial cells such as Escherichia
coli,
and more preferably, eukaryotic cells, especially for the expression of whole
recombinant antibody molecule, are used for the expression of a recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
S 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)).
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
1S 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; p1N vectors (Inouye & Inouye,
Nucleic
Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-
SS09 (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
2S GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
SpodopteYa fr-ugiperda cells. The antibody coding sequence rnay 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).
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In marmnalian 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 in vivo
recombination. Insertion in a non- essential region of the viral genome (e.g.,
region
E1 or E3) will result in a recombinant virus that is viable and capable of
expressing
the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.
Acad.
Sci. 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 acid 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)).
In addition, a host cell strain may be chosen which modulates the expression
of the inserted sequences, or modifies and processes the gene product in the
specific
fashion desired. Such modifications (e.g., glycosylation) and processing
(e.g.,
cleavage) of protein products may be important for the function of the
protein.
Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins and gene products.
Appropriate
cell lines or host systems can be chosen to ensure the correct modification
and
processing of the foreign protein expressed. To this end, eukaryotic host
cells which
possess the cellular machinery for proper processing of the primary
transcript,
glycosylation, and phosphorylation of the gene product may be used. Such
mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS,
MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for
example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell
line such as, for example, CRL7030 and Hs578Bst.
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For long-term, high-yield production of recombinant proteins, stable
expression is preferred. For example, cell lines which stably express the
antibody
molecule may be engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with DNA
controlled by
appropriate expression control elements (e.g., promoter, enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker.
Following the introduction of the foreign DNA, engineered cells may be allowed
to
grow for 1-2 days in an enriched media, and then are switched to a selective
media.
The selectable marker in the recombinant plasmid confers resistance to the
selection
and allows cells to stably integrate the plasmid into their chromosomes and
grow to
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines which express the antibody
molecule.
Such engineered cell lines may be particularly useful in screening and
evaluation of
compounds that interact directly or indirectly with the antibody molecule.
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); 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);
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Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols
in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.
Biol. 150:1 (1981), which are incorporated by reference herein in their
entireties.
5 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
10 culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).
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
15 vector encoding a light chain derived polypeptide. The two vectors may
contain
identical selectable markers which enable equal expression of heavy and light
chain
polypeptides. Alternatively, a single vector may be used which encodes, and is
capable of expressing, both heavy and light chain polypeptides. In such
situations,
the light chain should be placed before the heavy chain to avoid an excess of
toxic
20 free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl.
Acad. Sci.
USA 77:2197 (1980)). The coding sequences for the heavy and light chains rnay
comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal,
chemically synthesized, or recombinantly expressed, it may be purified by any
2S method known in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity, particularly by
affinity for
the specific antigen after Protein A, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard technique
for the
purification of proteins. In addition, the antibodies of the present invention
or
30 fragments thereof can be fused to heterologous polypeptide sequences
described
herein or otherwise known in the art, to facilitate purification.
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The present invention encompasses antibodies recombinantly fused or
chemically conjugated (including both covalently and non-covalently
conjugations)
to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50,
60, 70, 80,
90 or 100 amino acids of the polypeptide) of the present invention to generate
fusion
proteins. The fusion does not necessarily need to be direct, but may occur
through
linker sequences. The antibodies may be specific for antigens other than
polypeptides
(or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or
100 amino
acids of the polypeptide) of the present invention. For example, antibodies
may be
used to target the polypeptides of the present invention to particular cell
types, either
in vitro or in vivo, by fusing or conjugating the polypeptides of the present
invention
to antibodies specific for particular cell surface receptors. Antibodies fused
or
conjugated to the polypeptides of the present invention may also be used in 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.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example, the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any
combination of whole domains or portions thereof. The polypeptides may also be
fused or conjugated to the above antibody portions to form multimers. For
example,
Fc portions fused to the polypeptides of the present invention can form dimers
through disulfide bonding between the Fc portions. Higher multimeric forms can
be
made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing
or
conjugating the polypeptides of the present invention to antibody portions are
known
in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046;
5,349,053;
5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO
91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991);
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92
Zheng et al., J. hnmunol. 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).
As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated
to
the above antibody portions to increase the in vivo half life of the
polypeptides or for
use in immunoassays using methods known in the art. Further, the polypeptides
corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody
portions to facilitate purification. One reported example describes chimeric
proteins
consisting of the first two domains of the human CD4-polypeptide and various
domains of the constant regions of the heavy or light chains of mammalian
immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The
polypeptides of the present invention fused or conjugated to an antibody
having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in
binding and neutralizing other molecules, than the monomeric secreted protein
or
protein fragment alone. (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. (EP A
232,262). Altenlatively, 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).
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
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useful for purification include, but are not limited to, the "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson
et al., Cell 37:767 (1984)) and the "flag" tag.
The present invention further encompasses antibodies or fragments thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically to, for example, monitor the development or progression of a
tumor as
part of a clinical testing procedure to, e.g., determine the efficacy of a
given
treatment regimen. Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include various
enzymes,
prosthetic groups, fluorescent materials, luminescent materials,
bioluminescent
materials, radioactive materials, positron emitting metals using various
positron
emission tomographies, and nonradioactive paramagnetic metal ions. The
detectable
substance may be coupled or conjugated either directly to the antibody (or
fragment
thereof) or indirectly, through an intermediate (such as, for example, a
linker known
in the art) using techniques known in the art. See, for example, U.S. Patent
No.
4,741,900 for metal ions which can be conjugated to antibodies for use as
diagnostics
according to the present invention. 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.
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-
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dehydrotestosterone, glucocorticoids, procaine, tetracaine, Iidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine,
thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(fornlerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents (e.g., vincristine and vinblastine).
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 0
classical chemical therapeutic agents. For example, the drug moiety may be a
protein
or polypeptide possessing a desired biological activity. Such proteins may
include,
for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria
toxin; a protein such as tumor necrosis factor, a-interferon,13-interferon,
nerve growth
factor, platelet derived growth factor, tissue plasminogen activator, an
apoptotic
agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
97/33899), AIM II (See, International Publication No. WO 97134911), Fas Ligand
(Takahashi et al., Int. Irnmunol., 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.
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.
Techniques for conjugating such therapeutic moiety to antibodies are well
known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld
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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 Delcker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic
Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And
5 Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results,
And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy,
Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al.,
"The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
10 Rev. 62:119-58 (1982).
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.
An antibody, with or without a therapeutic moiety conjugated to it,
15 administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic.
Ifyzmuhophey~otypifag
The antibodies of the invention may be utilized for immunophenotyping of
20 cell lines and biological samples. The translation product of the gene of
the present
invention may be useful as a cell specific marker, or more specifically as a
cellular
marker that is 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
25 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 Mornson et al., Cell, 96:737-49 (1999)).
30 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
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prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques
allow for
the screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation and/or differentiation, as might be found in human umbilical
cord blood.
Assays Fo~° Antibody Bihdifzg
The antibodies of the invention may be assayed for immunospecific binding
by any method known in the art. The immunoassays which can be used include but
are not limited to competitive and non-competitive assay systems using
techniques
such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin
reactions,
gel diffusion precipitin reactions, immunodiffixsion assays, agglutination
assays,
complement-fixation assays, immunoradiometric assays, fluorescent
immunoassays,
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).
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 NaCl, 0.01 M sodium phosphate at pH 7.2, 1
Trasylol) supplemented with protein phosphatase and/or protease inhibitoxs
(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
immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
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Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-
PAGE depending on the molecular weight of the antigen), transferring the
protein
sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF
or
nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or
non-
fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking
the membrane with primary antibody (the antibody of interest) diluted in
blocking
buffer, washing the membrane in washing buffer, blocking the membrane with a
secondary antibody (which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase
or
alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in
blocking
buffer, washing the membrane in wash buffer, and detecting the presence of the
antigen. One of skill in the art would be knowledgeable as to the parameters
that can
be modified to increase the signal detected and to reduce the background
noise. For
fiu-ther 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
at 10.8.1.
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. W this case, a second antibody conjugated to a detectable
compound may be added following the addition of the antigen of interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that
can be modified to increase the signal detected as well as other variations of
ELISAs
known in the art. For further discussion regarding ELISAs see, e.g., Ausubel
et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons,
Inc.,
New York at 11.2.1.
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The binding aff nity 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.
Therapeutic Uses
The present invention is further directed to antibody-based therapies which
involve administering antibodies of the invention to an animal, preferably a
mammal,
and most preferably a human, patient for treating one or more of the disclosed
diseases, disorders, or conditions. Therapeutic compounds of the invention
include,
but are not limited to, antibodies of the invention (including fragments,
analogs and
derivatives thereof as described herein) and nucleic acids. encoding
antibodies of the
invention (including fragments, analogs and derivatives thereof and anti-
idiotypic
antibodies as described herein). The antibodies of the invention can be used
to treat,
inhibit or prevent diseases, disorders or conditions associated with aberrant
expression
and/or activity of a polypeptide of the invention, including, but not limited
to, any
one or more of the diseases, disorders, or conditions described herein. The
treatment
and/or prevention of diseases, disorders, or conditions associated with
aberrant
expression and/or activity of a polypeptide of the invention includes, but is
not
limited 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.
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
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99
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.
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.
The antibodies of the invention may be administered alone or in combination
with other types of treatments (e.g., radiation therapy, chemotherapy,
hormonal
therapy, irmnunotherapy 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.
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-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, 10-6 M, 5 X 10-~ M, 10-~ M, 5 X
10-8 M,
10-8 M, 5 X 10-9 M, 10-9 M, 5 X 10-1° M, 10-1° M, 5 X 10-11 M,
10-11 M, 5 X 10-12 M,
10-12 M, 5 X 10-13 M, 10-13 M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, and 10-15 M.
Gene TlaeYapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
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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.
Any of the methods for gene therapy available in the art can be used according
to the present invention. Exemplary methods are described below.
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 wluch 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).
In a preferred aspect, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors
that express the antibody or fragments or chimeric proteins or heavy or light
chains
thereof in a suitable host. In particular, such nucleic acid sequences have
promoters
operably linked to the antibody coding region, said promoter being inducible
or
constitutive, and, optionally, tissue- specific. In another particular
embodiment,
nucleic acid molecules are used in which the antibody coding sequences and any
other
desired sequences are flanked by regions that promote homologous recombination
at a
desired site in the genome, thus providing for intrachromosomal expression of
the
antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci.
USA
86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific
embodiments, the expressed antibody molecule is a single chain antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy
and light chains, or fragments thereof, of the antibody.
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
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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
viva or ex vivo gene therapy.
hi a specific embodiment, the nucleic acid sequences are directly
achninistered
~ 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
achninistering 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 subj ect 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. W yet another embodiment, the nucleic acid can be targeted in
vivo for
cell specific uptake and expression, by targeting a specific receptor (see,
e.g., PCT
Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, WO
93/20221). Alternatively, the nucleic acid can be introduced intracellularly
and
incorporated within host cell DNA for expression, by homologous recombination
(Roller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al.,
Nature 342:435-438 (1989)).
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
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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).
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.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy
(Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,43 6,146).
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.
In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be
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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.
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.
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 Tlymphocytes, Blymphocytes, 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.
In a preferred embodiment, the cell used for gene therapy is autologous to the
patient.
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 axe 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
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the present invention (see e.g. PCT Publication WO 94/08598; Stemple and
A~iderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980);
and
Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
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 controlling the
presence or
absence of the appropriate inducer of transcription.
Def~aoyzst~ation of Therapeutic or P~oplaylactic Activity
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.
TlZerapeuticlPf-ophylactic Administration and Conapositioh
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 an antibody of the invention. In a
preferred
aspect, the compound is substantially purified (e.g., substantially free from
substances that limit its effect or produce undesired side-effects). The
subject is
preferably an animal, including but not limited to animals such as cows, pigs,
horses,
chickens, cats, dogs, etc., and is preferably a mammal, and most preferably
human.
Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
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additional appropriate formulations and routes of administration can be
selected from
among those described herein below.
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 Ormnaya reservoir. Pulmonary administration can also be employed, e.g.,
by
use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
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 inj ection, 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.
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
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and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein,
ibid., pp.
3I7-327; see generally ibid.)
Tn yet another embodiment, the compound or composition can be delivered in
a controlled release system. hi one embodiment, a pump may be used (see
Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al.,
Surgery
88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida
(1974);
Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen
and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci.
Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985);
During
et al., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105
(1989)). In yet
another embodiment, a controlled release system can be placed in proximity of
the
therapeutic target, i.e., 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)).
Other controlled release systems are discussed in the review by Langer
(Science 249:1527-1533 (1990)).
In a specific embodiment where the compound of the invention is a nucleic
acid encoding a protein, the nucleic acid can be administered in 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.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable Garner. In a specific embodiment, the term
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"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
Garner 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
Garner
so as to provide the form for proper administration to the patient. The
formulation
should suit the mode of administration.
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
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lyophilized powder or water free concentrate in a hermetically sealed
container such
as an ampoule or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed with an
infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition
is administered by injection, an ampoule of sterile water for injection or
saline can be
provided so that the ingredients may be mixed prior to admiiustration.
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.
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.
For antibodies, the dosage administered to a patient is typically 0.1 mglkg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a
patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half life within the human body than antibodies from
other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages
of human antibodies and less frequent administration is often possible.
Further, the
dosage and frequency of administration of antibodies of the invention may be
reduced
by enhancing uptake and tissue penetration (e.g., into the brain) of the
antibodies by
modifications such as, for example, lipidation.
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The invention also provides a pharmaceutical paclc or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval
by the agency of manufacture, use or sale for human administration.
Diagnosis ayad In2aging
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.
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.
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
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(e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et
al., J. Cell .
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting
protein gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay labels are known in the art and include enzyme labels, such as, glucose
oxidase;
radioisotopes, such as iodine (125I, 1211), 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.
One aspect 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.
It will be understood in the art that the size of the subj ect 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 specif c protein. In
vivo tumor
imaging is described in S.W. Burchiel et al., "Tinmunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
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The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson Publishing Inc. (1982).
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 4S houxs or 6 to 24
hours or
6 to 12 hours. In another embodiment the time interval following
administration is 5
to 20 days or 5 to 10 days.
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.
Presence of the labeled molecule can be detected in the patient using methods
known in the art for in vivo scanning. These methods depend upon the type of
label
used. Skilled artisans will be able to determine the appropriate method for
detecting a
particular label. Methods and devices that may be used in the diagnostic.
methods of
the invention include, but are not limited to, computed tomography (CT), whole
body
scan such as position emission tomography (PET), magnetic resonance imaging
(MRl), and sonography.
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
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
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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 fox 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).
In another specific embodiment of the present invention, the kit is a
diagnostic
kit for use in screening serum containing antibodies specific against
proliferative
and/or cancerous polynucleotides and polypeptides. Such a kit may include a
control
antibody that does not react with the polypeptide of interest. Such a kit may
include a
substantially isolated polypeptide antigen comprising an epitope which is
specifically
immunoreactive with at least one anti-polypeptide antigen antibody. Further,
such a
kit includes means for detecting the binding of said antibody to the antigen
(e.g., the
antibody may be conjugated to a fluorescent compound such as fluorescein or
rhodamine Which can be detected by flow cytometry). In specific embodiments,
the
kit may include a recombinantly produced or chemically synthesized polypeptide
antigen. The polypeptide antigen of the kit may also be attached to a solid
support.
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
deflected by
binding of the said reporter-labeled antibody.
In an additional embodiment, the invention includes a diagnostic kit for use
in
screening serum containing antigens of the polypeptide of the invention. The
diagnostic kit includes a substantially isolated antibody specifically
immunoreactive
with polypeptide or polynucleotide antigens, and means for detecting the
binding of
the polynucleotide or polypeptide antigen to the antibody. In one embodiment,
the
antibody is attached to a solid support. In a specific embodiment, the
antibody may be
a monoclonal antibody. The detecting means of the kit may include a second,
labeled
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monoclonal antibody. Alternatively, or in addition, the detecting means may
include
a labeled, competing antigen.
h1 one diagnostic configuration, test serum is reacted with a solid phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing
unbound serum components by washing, the reagent is reacted with reporter-
labeled
anti-human antibody to bind reporter to the reagent in proportion to the
amount of
bound anti-antigen antibody on the solid support. The reagent is again washed
to
remove unbound labeled antibody, and the amount of reporter associated with
the
reagent is determined. Typically, the reporter is an enzyme which is detected
by
incubating the solid phase in the presence of a suitable fluorometric,
luminescent or
colorimetric substrate (Sigma, St. Louis, MO).
The solid surface reagent in the above assay is prepared by knov~nn 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).
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.
Fusion Proteins
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
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cellular locations based on trafficking signals, the polypeptides of the
present
invention can be used as targeting molecules once fused to other proteins.
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.
Moreover, fusion proteins may also be engineered to improve characteristics
of the polypeptide of the present invention. For instance, a region of
additional amino
acids, particularly charged amino acids, may be added to the N-terminus of the
polypeptide to improve stability and persistence during purification from the
host cell
or subsequent handling and storage. Also, peptide moieties may be added to the
polypeptide to facilitate purification. Such regions may be removed prior to
final
preparation of the polypeptide. The addition of peptide moieties to facilitate
handling
of polypeptides are familiar and routine techniques in the art.
Moreover, polypeptides of the present invention, including fragments, and
specifically epitopes, can be combined with parts of 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),
resulting in
chimeric polypeptides. These fusion proteins facilitate purification and show
an
increased half life in vivo. 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. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).)
Fusion proteins having disulfide-linked dimeric structures (due to the IgG)
can also be
more efficient in binding and neutralizing other molecules, than the monomeric
secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem.
270:3958-3964 (1995).) Polynucleotides comprising or alternatively consisting
of
nucleic acids which encode these fusion proteins are also encompassed by the
invention.
Similarly, 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
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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 antagoiusts 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).)
Moreover, the polypeptides of the present invention can be fused to marker
sequences, such as a peptide 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, W c., 9259 Eton
Avenue,
Chatsworth, CA, 91311), among others, many of which are commercially
available.
25 As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989),
for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
(1984).)
Thus, any of these above fusions can be engineered using the polynucleotides
or the polypeptides of the present invention.
Vectors, Host Cells, and Protein Production
The present invention also relates to vectors containing the polynucleotide of
the present invention, host cells, and the production of polypeptides by
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.
The polynucleotides 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
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vector is a virus, it may be paclcaged in vitro using an appropriate packaging
cell line
and then transduced into host cells.
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 Iate promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the transcripts expressed by the constructs will preferably include
a
translation initiating codon at the beginning and a termination codon (UAA,
UGA or
UAG) appropriately positioned at the end of the polypeptide to be translated.
As indicated, the expression vectors will preferably include at least one
selectable marker. Such markers include dihydrofolate reductase, 6418 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 Salmonella 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 Sf~ 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.
Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors,
pNHBA,
pNHl6a, pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and
ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. Preferred expression vectors for use in yeast systems include,
but are
not limited to pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ,pGAPZ, pGAPZalph,
pPIC9, pPIC3.5, PHIL-D2, PHIL-S 1, pPIC3.5K, pPIC9K, and PA0815 (all available
from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent
to the
skilled artisan.
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Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transduction, infection, or other methods. Such
methods
are described in many standard laboratory manuals, such as Davis et al., Basic
Methods In Molecular Biology (1986). It is specifically contemplated that the
polypeptides of the present invention may in fact be expressed by a host cell
lacking a
recombinant vector.
A polypeptide of this 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.
Polypeptides of the present invention, and preferably the secreted form, can
also be recovered from: products purified from natural sources, including
bodily
fluids, tissues and cells, whether directly isolated or cultured; products of
chemical
synthetic procedures; and products produced by recombinant techniques from a
prokaryotic or eukaryotic host, including, for example, bacterial, yeast,
higher plant,
insect, and mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may be
glycosylated
or may be non-glycosylated. In addition, polypeptides of the invention may
also
include an initial modified methionine residue, in some cases as a result of
host-
mediated processes. Thus, it is well known in the art that the N-terminal
methionine
encoded by the translation initiation colon 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.
In one embodiment, the yeast Pichia pasto~is is used to express the
polypeptide of the present invention in a eukaryotic system. Pichia pastaris
is a
methylotrophic yeast which can metabolize methanol as its sole carbon source.
A
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main step in the methanol metabolization pathway is the oxidation of methanol
to
formaldehyde using Oa. This reaction is catalyzed by the enzyme alcohol
oxidase. In
order to metabolize methanol as its sole carbon source, Piclaia pastor~is must
generate
high levels of alcohol oxidase due, in part, to the relatively low affinity of
alcohol
oxidase for 02. Consequently, in a growth medium depending on methanol as a
main
carbon source, the promoter region of one of the two alcohol oxidase genes
(AOXI) is
highly active. In the presence of methanol, alcohol oxidase produced from the
AOXI
gene comprises up to approximately 30% of the total soluble protein in Pichia
pastoYis. 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 Piclaia yeast
grown in
the presence of methanol.
~ 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 protein of the invention by virtue of the strong
AOXI
promoter linked to the Piclzia pasto~is alkaline phosphatase (PHO) secretory
signal
peptide (i.e., leader) located upstream of a multiple cloning site.
Many other yeast vectors could be used in place of pPIC9K, such as, pYES2,
pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5,
pHIL-D2, pHIL-SI, 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.
In another embodiment, high-level expression of a heterologous coding
sequence, such as, for example, a polynucleotide of the present invention, may
be
30 achieved by cloning the heterologous polynucleotide of the invention into
an
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expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
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 the polynucleotides of the
invention, and
which activates, alters, and/or amplifies endogenous polynucleotides. For
example,
techuques 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, resulting in the formation of a new
transcription unit
(see, e.g., U.S. Patent No. 5,641,670, issued June 24, 1997; U.S. Patent No.
5,733,761, issued March 31, 1998; International Publication No. WO 96/29411,
published September 26, 1996; International Publication No. WO 94/12650,
published August 4, 1994; Roller et al., Proc. Natl. Acad. Sci. USA 86:8932-
8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of
each of
which are incorporated by reference in their entireties).
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 sequence of the invention can be synthesized by use of a peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or chemical
amino acid
analogs can be introduced as a substitution or addition into the polypeptide
sequence.
Non-classical amino acids include, but are not limited to, to the D-isomers of
the
common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-
aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid,
Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
acid, t-
butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine,
fluoro-
amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl
amino
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acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore,
the
amino acid can be D (dextrorotary) or L (Ievorotary).
The invention encompasses polypeptides which are differentially modified
during or after translation, e.g., by glycosylation, acetylation,
phosphorylation,
amidation, derivatization by known protecting/blocking groups, proteolytic
cleavage,
linkage to an antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carried out by known techniques, including but
not
limited, to specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin,
papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic
synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic
host cell expression. The polypeptides may also be modified with a detectable
label,
such as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
isolation of the protein.
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 predeternzined positions within the
molecule and
may include one, two, three or more attached chemical moieties.
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,
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depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic protein or analog). For example, the polyethylene glycol may have
an
average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000,
3500,
4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,
10,000,
10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500,
15,000,
15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,
20,000,
25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,
75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
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. Biotecl2nol. 56:59-72 (1996);
Vorobjev et
al., Nucleosides Nucleotides 1&:2745-2750 (1999); and Caliceti et al.,
Bioconjug.
Chem. 10:638-646 (1999), the disclosures of each of which are incorporated
herein by
reference.
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, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG
to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting
pegylation of GM-CSF using tresyl chloride). Fox 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.
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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 a 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.
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) 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.
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.
Theca. DrZCg Ca~~ie~ Sys. 9:249-304 (1992); Francis et al., Intersz. J. of
Henaatol.
68:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO
95/06058;
and WO 98/32466, the disclosures of each of which are incorporated herein by
reference.
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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 (C1S02CHZCF3). 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-trifluoreothasle sulphonyl group.
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 l,l'-carbonyldiimidazole, MPEG-
2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-
succinate derivatives. A number additional polyethylene glycol derivatives and
reaction chemistries for attaching polyethylene glycol to proteins are
described in
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.
The number of polyethylene glycol moieties attached to each protein of the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated
proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12,
15, 17, 20, or more polyethylene glycol rriolecules. 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. Theca. Drug Carrier Sys.
9:249-
304 (1992).
The polypeptides of the invention may be in monomers or multimers (i.e.,
dimers, trimers, tetramers and higher multimers). Accordingly, the present
invention
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relates to monomers and multimers of the polypeptides of the invention, their
preparation, and compositions (preferably, Therapeutics) containing them. Tn
specific
embodiments, the polypeptides of the invention are monomers, dimers, trimers
ox
tetramers. In additional embodiments, the multimers of the invention are at
least
dimers, at least trimers, or at least tetramers.
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 the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA
contained in a deposited clone (including fragments, variants, splice
variants, and
fusion proteins, corresponding to these polypeptides as described herein).
These
homomers may contain polypeptides having identical or different amino acid
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only polypeptides having an identical amino acid sequence. In
another
specific embodiment, a homomer of the invention is a multimer containing
polypeptides having different amino acid sequences. In specific embodiments,
the
multimer of the invention is a homodimer (e.g., containing polypeptides having
identical or different amino acid sequences) or a homotrimer (e.g., containing
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.
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.
Multimers of the invention may be the result of hydrophobic, hydrophilic,
ionic and/or covalent associations and/or may be indirectly linked, by for
example,
liposome formation. Thus, in one embodiment, multimers of the invention, such
as,
for example, homodimers or homotrimers, are formed when polypeptides of the
invention contact one another in solution. In another embodiment,
heteromultimers of
the invention, such as, for example, heterotrimers or heterotetramers, are
formed
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when polypeptides of the invention contact antibodies to the polypeptides of
the
invention (including antibodies to the heterologous polypeptide sequence in a
fusion
protein of the invention) in solution. In other embodiments, multimers of the
invention are formed by covalent associations with and/or between the
polypeptides
of the invention. Such covalent associations may involve one or more amino
acid
residues contained in the polypeptide sequence ( e.g., that recited in the
sequence
listing, or contained in the polypeptide encoded by a deposited clone). In one
instance, the covalent associations are cross-linking between cysteine
residues located
within the polypeptide sequences which interact in the native (i.e., naturally
occurring) polypeptide. In another instance, the covalent associations are the
consequence of chemical or recombinant manipulation. Alternatively, such
covalent
associations may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence
contained in a 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 an 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, oseteoprotegerin (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 axe 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.
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 axe
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.,
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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 dirnerize 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.
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. 081446,922, hereby
incorporated by
reference. Other peptides derived from naturally occurnng trimeric proteins
may be
employed in preparing trimeric polypeptides of the invention.
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 seuqence. In a further embodiment, associations
proteins of the invention are associated by interactions between heterologous
polypeptide sequence contained in Flag~ fusion proteins of the invention and
anti-
Flag~ antibody.
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
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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).
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 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 hyrophobic 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).
Uses of the Polynucleotides
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.
The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers,
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since few chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are presently available. Each polynucleotide of the present
invention can be used as a chromosome marker.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can be
selected using computer analysis so that primers do not span more than one
predicted
exon in the genomic DNA. These primers are then used for PCR screening of
somatic cell hybrids containing individual human chromosomes. Only those
hybrids
containing the human gene corresponding to the SEQ ID NO:X will yield an
amplified fragment.
Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per
day using a single thermal cycler. Moreover, sublocalization of the
polynucleotides
can be achieved with panels of specific chromosome fragments. Other gene
mapping
strategies that can be used include in situ hybridization, prescreening with
labeled
flow-sorted chromosomes, preselection by hybridization to construct chromosome
specific-cDNA libraries and computer mapping techniques (See, e.g., Shuler,
Trends
Bioteclnlol 16:456-459 (1998) which is hereby incorporated by reference in its
entirety)..
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).
For chromosome mapping, the polynucleotides case be used individually (to
mark a single chromosome or a single site on that chromosome) or in panels
(for
marking multiple sites and/or multiple chromosomes).
The polynucleotides of the present invention would likewise be useful for
radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
For
a review of these techniques and others known in the art, see, e.g., Dear,
"Genome
Mapping: A Practical Approach," IRL Press at Oxford University Press, London
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(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.
Once a polynucleotide has been mapped to a precise chromosomal location,
the physical position of the polynucleotide can be used in linkage analysis.
Linkage
analysis establishes coinheritance between a chromosomal location and
presentation
of a particular disease. (Disease mapping data are found, for example, in V.
McKusick, Mendelian Inheritance in Man (available on line through Johns
Hopkins
University Welch Medical Library) .) 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.
Thus, once coinheritance is established, differences in the polynucleotide 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.
Furthermore, increased or decreased expression of the gene in affected
individuals as compared to unaffected individuals can be assessed using
polynucleotides of the present invention. Any of these alterations (altered
expression,
chromosomal rearrangement, or mutation) can be used as a diagnostic or
prognostic
marker.
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
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level, whereby an increase or decrease in the gene expression.level compared
to the
standard is indicative of a disorder.
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 present invention and a suitable container. In a
specific
embodiment, the kit includes two polynucleotide probes defining an internal
region of
the polynucleotide of the present 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 fox polymerase chain reaction amplification.
Where a diagnosis of a disorder, 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
present
invention expression will experience a worse clinical outcome relative to
patients
expressing the gene at a level nearer the standard level.
By "measuring the expression level of polynucleotide of the present
invention" is intended qualitatively or quantitatively measuring or estimating
the level
of the polypeptide of the present invention or the level of the mRNA encoding
the
polypeptide in a first biological sample either directly (e.g., by determining
or
estimating absolute protein level or mRNA level) or relatively (e.g., by
comparing to
the 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 disorder or being determined by averaging levels from a population of
individuals
not having a 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.
By "biological sample" is intended any biological sample obtained from an
individual, body fluid, cell line, tissue culture, or other source which
contains the
polypeptide of the present invention or mRNA. As indicated, biological samples
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include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid
and
spinal fluid) which contain the polypeptide of the present invention, and
other tissue
sources found to express the polypeptide of the present invention. Methods for
obtaining tissue biopsies and body fluids from mammals are well known in the
art.
Where the biological sample is to include mRNA, a tissue biopsy is the
preferred
source.
The methods) provided above may preferrably be applied in a diagnostic
method and/or kits in which polynucleotides and/or polypeptides 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 present invention
attached may
be used to identify polymorphisms between the polynucleotide sequences, 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, including 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.
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 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 P. E. Nielsen, M.
Egholm, R. H.
Berg and O. Buchardt, Science 254, 1497 (1991); and M. Egholm, O. Buchardt,
L.Christensen, C. Behrens, S. M. Freier, D. A. Driver, R. H. Berg, S. I~.
I~im, B.
Norden, and P. E. Nielsen, 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
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backbone is more flexible. Because of this, PNA/DNA duplexes bind under a
wider
range of stringency conditions than DNAIDNA duplexes, making it easier to
perform
multiplex hybridization. Smaller probes can be used than with DNA due to the
strong
binding. In addition, it is more likely that single base mismatches can be
determined
with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer
lowers the melting point (T<sub>m</sub>) 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.
The present invention is useful for detecting cancer in marmnals. In
particular
the invention is useful during diagnosis of pathological cell proliferative
neoplasias
which include, but are not limited to: acute myelogenous leukemias including
acute
monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,
acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic
leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous
leukemias including chronic myelomonocytic leukemia, chronic granulocytic
leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows,
pigs,
horses, rabbits and humans. Particularly preferred are humans.
Pathological cell proliferative diseases, disorders, and/or conditions 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.
(Gelinann
et al., supra) It is likely that mutated or altered expression of specific
genes is
involved in the pathogenesis of some leukemias, among other tissues and cell
types.
(Gelmann et al., supra) Indeed, the human counterparts of the oncogenes
involved in
some animal neoplasias have been amplified or translocated in some cases of
human
leukemia and carcinoma. (Gelmann et al., supra)
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For example, c-myc expression is highly amplified in the non-lymphocytic
leukemia cell line HL-60. When HL-60 cells are chemically induced to stop
proliferation, the level of c-myc is found to be downregulated. (International
Publication Number WO 91/15580) However, it has been shown that exposure of
HL-60 cells to a DNA construct that is complementary to the 5' end of c-myc or
c-
myb blocks translation of the corresponding mRNAs which downregulates
expression
of the c-myc or c-myb proteins and causes arrest of cell proliferation and
differentiation of the treated cells. (International Publication Number WO
91/15580;
Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc.
Natl.
Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the
present invention's usefulness would not be limited to treatment of
proliferative
diseases, disorders, and/or conditions of hematopoietic cells and tissues, in
light of the
numerous cells and cell types of varying origins which are known to exhibit
proliferative phenotypes.
In addition to the foregoing, a polynucleotide can be used to control gene
expression through triple helix formation or antisense DNA or RNA. Antisense
techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRCPress,
Boca
Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et
al.,
Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988);
and
Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques, preferred
polynucleotides are usually oligonucleotides 20 to 40 bases in length and
complementary to either the region of the gene involved in transcription
(triple helix -
see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456
(1988); and Dervan et al., Science 251:1360 (1991) ) or to the mRNA itself
(antisense
- Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).) Triple 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. Both techniques are effective in model systems, and the
information
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disclosed herein can be used to design antisense or triple helix
polynucleotides in an
effort to treat or prevent disease.
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.
The polynucleotides are also useful for identifying individuals from minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel.
In this technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique bands for
identifying personnel. This method does not suffer from the current
limitations of
"Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The polynucleotides of the present invention can be used as
additional DNA
markers for RFLP.
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.
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
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Technology, Freeman and Co. (1992).) Once these specific polyrnorphic 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 pohymorphic markers for forensic purposes.
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 specific to particular tissue prepared from the sequences of
the
present invention. 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.
In the very least, the polynucheotides of the present invention can be used as
molecular weight markers on Southern gels, as diagnostic probes for the
presence of a
specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences
in the process of discovering novel polynucleotides, for selecting and making
ohigomers 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
Each of the pohypeptides identified herein can be used in numerous ways. The
following description should be considered exemplary and utilizes known
techniques.
A polypeptide of the present invention can be used to assay protein levels in
a
biological sample using antibody-based techniques. For example, protein
expression
in tissues can be studied with classical immunohistological methods.
(Jalkanen, M.,
et al., J. Cehh. Biol. 101:976-985 (1985); Jalkanen, M., et a1., 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 habels are
known
in the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium
(112In), and
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technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
b10t1I1.
In addition to assaying secreted protein levels in a biological sample,
proteins
can also be detected in vivo by imaging. Antibody labels or markers for in
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 subj ect.
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.
A protein-specific antibody or antibody fragment which has been labeled with
an appropriate detectable imaging moiety, such as a radioisotope (for example,
131I,
112In, 99mTc), a radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally, subcutaneously,
or
intraperitoneally) into the mammal. It will be understood in the art that the
size of the
subj ect 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.
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. (192).)
Thus, the invention provides a diagnostic method of a disorder, which
involves (a) assaying the expression of a polypeptide of the present invention
in cells
or body fluid of an individual; (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 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
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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.
Moreover, polypeptides of the present invention can be used to treat, prevent,
and/or diagnose disease. 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).
Similarly, antibodies directed to a polypeptide of the present invention can
also be used to treat, prevent, and/or diagnose disease. For example,
administration of
an antibody directed to a polypeptide of the present invention can bind and
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).
At the very least, the polypeptides of the present invention can be used as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns using methods well known to those of skill in the art. Polypeptides
can also
be used to raise antibodies, which in turn are used to measure protein
expression from
a recombinant cell, as a way of assessing transformation of the host cell.
Moreover,
the polypeptides of the present invention can be used to test the following
biological
activities.
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Gene Therauy Methods
Another aspect of the present invention is to gene therapy methods for
treatingor preventing disorders, diseases and conditions. The gene therapy
methods
relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA)
sequences into an animal to achieve expression of a polypeptide of the present
invention. This method requires a polynucleotide which codes for a polypeptide
of the
invention that operatively linked to a promoter and any other genetic elements
necessary for the expression of the polypeptide by the target tissue. Such
gene therapy
and delivery techniques are known in the art, see, for example, W090/11092,
which
is herein incorporated by reference.
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 invention ex vivo, with the engineered cells then being
provided
to a patient to be treated with the polypeptide. Such methods are well-known
in the
art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993);
Ferrantini et al., Cancer Research, 53:107-1112 (1993); Ferrantini et al., J.
Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-
229
(1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et
al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-
1255
(1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 (1996)), which are
herein
incorporated by reference. In one embodiment, the cells which are engineered
are
arterial cells. The arterial cells may be reintroduced into the patient
through direct
injection to the artery, the tissues surrounding the artery, or through
catheter injection.
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.
In one embodiment, the polynucleotide of the invention is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences
that are free from any delivery vehicle that acts to assist, promote or
facilitate entry
into the cell, including viral sequences, viral particles, liposome
formulations,
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lipofectin or precipitating agents and the like. However, the polynucleotides
of the
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.
The polynucleotide vector constructs of the invention used in the gene therapy
method are preferably constructs that will not integrate into the host genome
nor will
they contain sequences that allow for replication. Appropriate vectors include
pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3,
pBPV, pMSG and pSVL available from Pharmacia; and pEFl/V5, pcDNA3.1, and
pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily
apparent
to the skilled artisan.
Any strong promoter lmown to those skilled.in the art can be used for driving
the expression of polynucleotide sequence of the invention. 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 polynucleotides of the invention.
Unlike other gene therapy techniques, one major advantage of introducing
naked nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis in the cells. Studies have shov~m that non-
replicating DNA
sequences can be introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
The polynucleotide construct of the invention 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,
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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
inj ection 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.
For the nakedzzucleic 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 S mg/kg. Of course,
as
the artisan of ordinary skill will appreciate, this dosage will vary according
to the
tissue site of injection. The appropriate and effective dosage of nucleic acid
sequence
can readily be determined by those of ordinary skill in the art and may depend
on the
condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of injection
into the interstitial space of tissues. However, other parenteral routes may
also be
used, such as, inhalation of an aerosol formulation particularly for delivery
to lungs or
bronchial tissues, throat or mucous membranes of the nose. In addition, naked
DNA
constructs can be delivered to arteries during angioplasty by the catheter
used in the
procedure.
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.
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.
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In certain embodiments, the polynucleotide constructs of the invention 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 ,
84:7413-7416 (1987), which is herein incorporated by reference); mRNA (Malone
et
al., Proc. Natl. Acad. Sci. USA , 86:6077-6081 (1989), which is herein
incorporated
I O by reference); and purified transcription factors (Debs et aL, J. Biol.
Chem.,
265:10189-10192 (1990), which is herein incorporated by reference), in
functional
form.
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
,
84:7413-7416 (1987), which is herein incorporated by reference). Other
commercially
available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE
(Boehringer).
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., Felgner et al.,
Proc.
Natl. Acad. Sci. USA, 84:7413-7417, which is herein incorporated by reference.
Similar methods can be used to prepare liposomes from other cationic lipid
materials.
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
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starting materials in appropriate ratios. Methods for making liposomes using
these
materials are well known in the art.
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 ovenlight 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.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (SWs), 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 , 101:512-527
(1983),
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.
SLTVs 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 such as 10 mM Tris/NaCl,
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. LIJVs
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,
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394:483 (1975); Wilson et al., Cell , 17:77 (1979)); ether injection (Deamer
et al.,
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 et al., Proc. Natl. Acad. Sci. USA , 76:145 (1979));
and
reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem., 255:10431
(1980);
Szoka et al., Proc. Natl. Acad. Sci. USA , 75:145 (1978); Schaefer-Ridder et
al.,
Science, 215:166 (1982)), which are herein incorporated by reference.
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.
U.S. Patent NO: 5,676,954 (which is herein incorporated by reference) reports
on the injection of genetic material, complexed with cationic liposomes
Garners, 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 (which are
herein incorporated by reference) provide methods for delivering DNA-cationic
lipid
complexes to mammals.
In certain embodiments, cells are engineered, ex vivo or in vivo, using a
retroviral particle containing RNA which comprises a sequence encoding
polypeptides of the 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.
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
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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.
The producer cell line generates infectious retroviral vector particles which
include polynucleotide encoding polypeptides of the invention. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or i~
vivo. The transduced eukaryotic cells will express polypeptides of the
invention.
W certain other embodiments, cells are engineered, ex vivo or ih vivo, with
polynucleotides of the invention contained in an adenovirus vector. Adenovirus
can
be manipulated such that it encodes and expresses polypeptides of the
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 (Schwartzet al., Am. Rev. Respir.
Dis.,
109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of alpha-1-
antitrypsin and
CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434
(1991);
Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensive studies to
attempt
to establish adenovirus as a causative agent in human cancer were uniformly
negative
(Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)).
Suitable adenoviral vectors useful in the present invention are described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-503 (1993);
Rosenfeld et al., Cell , 68:143-155 (1992); Engelhardt et al., Human Genet.
Ther.,
4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al.,
Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2 is useful
and can
be grown in human 293 cells. These cells contain the E1 region of adenovirus
and
constitutively express Ela and Elb, which complement the defective
adenoviruses by
providing the products of the genes deleted from the vector. In addition to
Ad2, other
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varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the
present
invention.
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: E1
a, Elb,
E3, E4, E2a, or Ll through L5.
In certain other embodiments, the cells are engineered, ex vivo or in vivo,
using an adeno-associated virus (AAV). AAVs are naturally occurnng defective
viruses that require helper viruses to produce infectious particles (Muzyczka,
Curr.
Topics in Microbiol. Immunol., 158:97 (1992)). It is also one of the few
viruses that
may integrate its DNA into non-dividing cells. Vectors containing as little as
300 base
pairs of AAV can be packaged and can integrate, but space for exogenous DNA is
limited to about 4.5 kb. Methods for producing and using such AAVs are known
in
the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678,
5,436,146, 5,474,935, 5,478,745, and 5,589,377.
For example, an appropriate AAV vector for use in the present invention will
include aII the sequences necessary for DNA replication, encapsidation, and
host-cell
integration. The polynucleotide construct containing polynucleotides of the
invention
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
Iipofection, 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 of the
invention.
These viral particles are then used to transduce eukaryotic cells, either ex
vivo or ih
vivo. The transduced cells will contain the polynucleotide construct
integrated into its
genome, and will express the desired gene product.
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Another method of gene therapy involves operably associating heterologous
control regions and endogenous polynucleotide sequences (e.g. encoding the
polypeptide sequence of interest) via homologous recombination (see, e.g.,
U.S.
Patent NO: 5,641,670, issued June 24, 1997; International Publication NO: WO
96/29411, published September 26, 1996; International Publication NO: WO
94/12650, published August 4, 1994; Roller et al., Proc. Natl. Acad. Sci. USA,
86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). 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.
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.
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.
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.
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The promoter-targeting sequence construct is taken up by cells. Homologous
recombination between the construct and the endogenous sequence takes place,
such
that an endogenous sequence is placed under the control of the promoter. The
promoter then drives the expression of the endogenous sequence.
The polynucleotides encoding polypeptides of the present invention may be
administered along with other polynucleotides encoding other angiongenic
proteins.
Angiogenic proteins include, but are not limited to, acidic and basic
fibroblast growth
factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3 (VEGF-B), epidermal growth factor
alpha and beta, platelet-derived endothelial cell growth factor, platelet-
derived growth
factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin like
growth
factor, colony stimulating factor, macrophage colony stimulating factor,
granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.
Preferably, the polynucleotide encoding a polypeptide of the invention
contains a secretory signal sequence that facilitates secretion of the
protein.
Typically, the signal sequence is positioned in the coding region of the
polynucleotide
to be expressed towards or at the 5' end of the coding region. The signal
sequence
may be homologous or heterologous to the 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. .
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 (199)).
A preferred method of local administration is by direct injection. Preferably,
a
recombinant molecule of the present invention complexed with a delivery
vehicle is
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administered by direct injection into or locally within the area of arteries.
Aclininistration of a composition locally within the area of arteries refers
to injecting
the composition centimeters and preferably, millimeters within arteries.
Another method of local administration is to contact a polymcleotide
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.
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.
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 skin.
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. Therapeutic
compositions of
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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
Siolo~ical Activities
The 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 and polypeptides 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 or polypeptides, or agonists or
antagonists could
be used to treat the associated disease.
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.
Immune Activity
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.
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
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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 1, column 8 (Tissue Distribution Library
Code).
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may be useful in treating, preventing, diagnosing, and/or
prognosing
irmnunodeficiencies, including both congenital and acquired
immunodeficiencies.
Examples of B cell immunodeficiencies in which immunoglobulin levels B cell
function and/or B cell numbers are decreased include: X-linked
agammaglobulinemia
(Bruton's disease), X-linked infantile agammaglobulinemia, X-linked
immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM,
X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including
congeutal 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.
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.
Examples of congenital irmnunodeficiencies in which T cell and/or B cell
function and/or number is decreased include, but are not limited to: DiGeorge
anomaly, severe combined immunodeficiencies (SLID) (including, but not limited
to,
X-linked SCID, autosomal recessive SLID, adenosine deaminase deficiency,
purine
nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare
lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia),
thymic
hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion,
chronic
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mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic
CD4+ T-
lymphocytopenia, immunodeficiency with predominant T cell defect
(unspecified),
and unspecified immunodeficiency of cell mediated immunity.
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.
Other immunodeficiencies that may be treated, prevented, diagnosed, and/or
prognosed using polypeptides or polynucleotides of the invention, and/or
agonists or
antagonists thereof, include, but are not limited to, chronic granulomatous
disease,
Chediak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-
phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome
(XLP),
leukocyte adhesion deficiency, complement component deficiencies (including
Cl,
C2, C3, C4, C5, C6, C7, C~ and/or C9 deficiencies), reticular dysgenesis,
thyrnic
alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital
leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short
limbed
dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
In a preferred embodiment, the irnmunodeficiencies 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.
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. hi 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.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present invention may be useful in treating, preventing, diagnosing
and/or
prognosing autoimmune disorders. Many autoimmune disorders result from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate recognition results in an innnune response leading to the
destruction of
the host tissue. Therefore, the administration of polynucleotides and
polypeptidas of
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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.
Autoimmune diseases or disorders that may be treated, prevented, diagnosed
and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists
or
antagonists of the present invention include, but are not limited to, one or
more of the
following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing
spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's
thyroiditis,
autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, 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.
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 ophthalinia,
polyendocrinopathies, Reiter's Disease, Stiff Man Syndrome, autoimmune
pulmonary
inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes
mellitus,
and autoimmune inflammatory eye disorders.
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
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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 astluna or cystic
fibrosis)
(often characterized, e.g., by beta-adrenergic receptor antibodies).
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.
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.
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.
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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.
In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated,
prevented, diagnosed and/or prognosed using polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention
In preferred embodiments, polypeptides, antibodies, polynucleotides and/or
agonists or antagonists of the present invention are used as a
immunosuppressive
agent(s).
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.
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.
Additionally, polypeptides or polynucleotides of the invention, and/or
agonists
or antagonists thereof, may be used to treat, prevent, diagnose and/or
prognose
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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.
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-l.),
respiratory
disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g.,
inflammatory
bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and
breast); CNS
disorders (e.g., multiple sclerosis; ischemic brain injury and/or stroke,
traumatic brain
injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's
disease); AIDS-related dementia; and prion disease); cardiovascular disorders
(e.g.,
atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary
bypass
complications); as well as many additional diseases, conditions, and disorders
that are
characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout,
trauma,
pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury,
Grave's
disease, systemic lupus erythematosus, diabetes mellitus, and allogenic
transplant
rej ection).
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,
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balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis,
cervicitis, cholecystitis,
chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis,
diverticulitis,
encephalitis, endocarditis, esophagitis, eustachitis, fibrositis,
folliculitis, gastritis,
gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis,
labyrinthitis, laryngitis,
lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis,
myocarditis,
myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis,
otitis, pericarditis,
peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis,
prostatitis, pulpitis,
retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis,
sinusitis, spondylitis,
steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis,
urethritis, and vaginitis.
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
~15 this case, the foreign transplanted immune cells destroy the host tissues.
Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists
or
antagonists thereof, that inhibit an immune response, particularly the
activation,
proliferation, differentiation, or chemotaxis of T-cells, may be an effective
therapy in
preventing organ rejection or GVHD. In specific embodiments, polypeptides,
antibodies, or polynucleotides of the invention, and/or agonists or
antagonists thereof,
that inhibit an immune response, particularly the activation, proliferation,
differentiation, or chemotaxis of T-cells, may be an effective therapy in
preventing
experimental allergic and hyperacute xenograft rejection.
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.
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
and/or differentiation of B and/or T cells, infectious diseases may be
treated, detected,
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and/or prevented. The immune response may be increased by either enhancing an
existing immune response, or by initiating a new immune response.
Alternatively,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may also directly inhibit the infectious agent (refer to
section of
application listing infectious agents, etc), without necessarily eliciting an
immune
response.
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.
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: AmS, 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.
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
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response to a bacteria or fungus, disease, or symptom selected from the group
consisting of tetanus, Diphtheria, botulism, and meningitis type B.
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: Viby~io cholerae, Mycobacterium
leprae, Salmonella typhi, Salmonella pa~atyphi, Meisse~ia meniyagitidis,
StYeptococcus pneufraoniae, Group B streptococcus, Shigella spp.,
Enterotoxigenic
Eschef°ichia coli, Enterohemorrhagic E. coli, and Boy-~elia
burgdorfeYi.
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.
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.
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.
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
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immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to
increase an
immune response.
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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as an
activator of T
cells.
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.
W 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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to increase
serum immunoglobulin concentrations.
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.
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.
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,
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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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to boost
immunoresponsiveness among individuals having an acquired loss of B cell
function.
Conditions resulting in an acquired loss of B cell function that may be
ameliorated or
treated by administering the polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists thereof, include, but are not limited to, HTV Infection, ADDS,
bone
marrow transplant, and B cell chronic lymphocytic leukemia (CLL).
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to boost
immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated
or
treated by administering the polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists thereof, include, but are not limited to, recovery from viral
infections
(e.g., influenza), conditions associated with malnutrition, recovery from
infectious
mononucleosis, or conditions associated with stress, recovery from measles,
recovery
from blood transfusion, and recovery from surgery.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or 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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to direct
an individual's immune system towards development of a humoral response (i.e.
TH2) as opposed to a THl cellular response.
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In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as a means to
induce
tumor proliferation and thus make it more susceptible to anti-neoplastic
agents. For
example, multiple myeloma is a slowly dividing disease and is thus refractory
to
virtually all anti-neoplastic regimens. If these cells were forced to
proliferate more
rapidly their susceptibility profile would likely change.
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.
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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as a gene-
based
therapy for genetically inherited disorders resulting in immuno-
incompetencelimmunodeficiency such as observed among SCID patients.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as a means of
activating monocytes/macrophages to defend against parasitic diseases that
effect
monocytes such as Leishmania.
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.
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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as a means of
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blocking various aspects of immune responses to foreign agents or self.
Examples of
diseases or conditions in which blocking of certain aspects of immune
responses may
be desired include autoimmune disorders such as lupus, and arthritis, as well
as
immunoresponsiveness to skin allergies, inflammation, bowel disease, injury
and
diseases/disorders associated with pathogens.
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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as a
inhibitor of B
and/or T cell migration in endothelial cells. This activity disrupts tissue
architecture
or cognate responses and is useful, for example in disrupting immune
responses, and
blocking sepsis.
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
gannnopathy of undetermined significance (MGUS), Waldenstrom's disease,
related
idiopathic monoclonal gamrnopathies, and plasmacytomas.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention may be employed for
instance
to inhibit polypeptide chemotaxis and activation of macrophages and their
precursors,
and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g.,
activated
and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and
chroiuc
inflammatory and infective diseases. Examples of autoimmune diseases are
described
herein and include multiple sclerosis, and insulin-dependent diabetes.
The polypeptides, antibodies, polynucleotides and/or agonists or antagonists
of the present invention may also be employed to treat idiopathic hyper-
eosinophilic
syndrome by, for example, preventing eosinophil production and migration.
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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.
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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention may also be employed
fox
treating atherosclerosis, for example, by preventing monocyte infiltration in
the artery
wall.
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).
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.
In a specific embodiment, polynucleotides or polypeptides, and/or agonists
thereof are used to diagnose, prognose, treat, and/or prevent a disorder
characterized
by primary or acquired immunodeficiency, deficient serum immunoglobulin
production, recurrent infections, and/or immune system dysfunction. Moreover,
polynucleotides or polypeptides, and/or agonists thereof may be used to treat
or
prevent infections of the joints, bones, skin, and/or parotid glands, blood-
borne
infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis),
autoimmune
diseases (e.g., those disclosed herein), inflammatory disorders, and
malignancies,
and/or any disease or disorder or condition associated with these infections,
diseases,
disorders and/or malignancies) including, but not limited to, CVID, other
primary
immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis
media,
conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe
herpes
zoster), and/or pneumocystis carnii. Other diseases and disorders that may be
prevented, diagnosed, prognosed, and/or treated with polynucleotides or
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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.
In another embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention are used to treat, andlor
diagnose an
individual having common variable immunodeficiency disease ("CVID"; also lmown
as "acquired agammaglobulinemia" and "acquired hypogammaglobulinemia") or a
subset of this disease.
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.
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.
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.
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.
Antagonists of the invention include, for example, binding and/or inhibitory
antibodies, antisense nucleic acids, ribozymes or soluble forms of the
polypeptides of
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the present invention (e.g., Fc fusion protein; see, e.g., Example 9).
Agonists of the
invention include, for example, binding or stimulatory antibodies, and soluble
forms
of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9).
polypeptides,
antibodies, polynucleotides and/or agonists or antagonists of the present
invention
may be employed in a composition with a pharmaceutically acceptable carrier,
e.g., as
described herein.
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 in an organ system listed
above.
Blood-Related Disorders
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present invention may be used to modulate hemostatic (the stopping of
bleeding) or thrombolytic (clot dissolving) activity. For example, by
increasing
hemostatic or thrombolytic activity, polynucleotides or polypeptides, and/or
agonists
or antagonists of the present invention could be used to treat or prevent
blood
coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia,
factor
deficiencies, hemophilia), blood platelet diseases, disorders, and/or
conditions (e.g.,
thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention that can decrease hemostatic or
thrombolytic
activity could be used to inhibit or dissolve clotting. These molecules could
be
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important in the treatment or prevention of heart attacks (infarction),
strokes, or
scarring.
In specific embodiments, the polymcleotides, 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, 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 mural 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).
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, andlor 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 1, column 8 (Tissue Distribution Library Code).
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present invention may be used to modulate hematopoietic activity (the
formation of blood cells). For example, the polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention may be used to
increase the
quantity of all or subsets of blood cells, such as, for example, erythrocytes,
lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils,
neutrophils,
mast cells, macrophages) and platelets. The ability to decrease the quantity
of blood
cells or subsets of blood cells may be useful in the prevention, detection,
diagnosis
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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.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
20 of the present invention may be used to prevent, treat, or diagnose blood
dyscrasia.
Anemias are conditions in which the number of red blood cells or amount of
hemoglobin (the protein that carnes 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 B 12 deficiency) and folic acid deficiency anemia), aplastic
anemia,
hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic
hemolytic
anemia, and paroxysmal nocturnal hemoglobinuria). The polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may
be useful in treating, preventing, and/or diagnosing anemias associated with
diseases
including but not limited to, anemias associated with systemic lupus
erythematosus,
cancers, lymphomas, chronic renal disease, and enlarged spleens. The
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may be useful in treating, preventing, andlor 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,
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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.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present invention may be useful in treating, preventing, and/or
diagnosing
hemoglobin abnormalities, (e.g., those associated with sickle cell anemia,
hemoglobin
C disease, hemoglobin S-C disease, and hemoglobin E disease). Additionally,
the
polynucleotides, polypeptides, antibodies, 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.
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-Pudlak
syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier
syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or
Factor
VII deficiency and Christmas disease or Factor IX deficiency, Hereditary
Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic
purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.
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 lmown 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.
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, andlor treating acquired platelet dysfunction such as platelet
dysfunction
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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.
In another embodiment, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing, preventing, and/or treating diseases and disorders characterized
by or
associated with increased or decreased numbers of white blood cells.
Leukopenia
occurs when the number of white blood cells decreases below normal.
Leukopenias
include, but are not limited to, neutropenia and lymphocytopenia. An increase
in the
number of white blood cells compared to normal is known as leukocytosis. The
body
generates increased numbers of white blood cells during infection. Thus,
leukocytosis
may simply be a normal physiological parameter that reflects infection.
Alternatively,
leukocytosis may be an indicator of injury or other disease such as cancer.
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.
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 drug treatments (e.g.,
antibiotic
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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.
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), AmS infection and/or other diseases such as, for
example,
cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic
infections, some
viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-
1S Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).
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.
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,
2S idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and
Hand-
Schuller-Christian disease.
In yet another embodiment, the polynucleotides, polypeptides, antibodies,
and/or agonists or antagonists of the present invention may be useful in
diagnosing,
prognosing, preventing, andlor 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
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Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or
granulocytic)
leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burlcitt's lymphoma, and
mycosis fungoides.
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 rnacroglobulinemia, cryoglobulinemia, and
Raynaud's phenomenon.
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.
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.
In other embodiments, the polynucleotides, polypeptides, antibodies, andlor
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.
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.
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.
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In other embodiments, the pol5mucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in preventing,
diagnosing, and/or treating primary hematopoietic disorders.
Hyperproliferative Disorders
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.
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 irmnune response may be increased by either enhancing an existing immune
response, or by initiating a new immune response. Alternatively, decreasing an
immune response may also be a method of treating hyperproliferative disorders,
such
as a chemotherapeutic agent.
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.
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
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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 Uxeter, 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 Neuroectodermal Tumors,
Childhood
Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue
Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic
Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous
T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,
Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related
Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal
Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast
Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
Gastrointestinal
Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational
Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular
Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia,
Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell
Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer,
Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,
Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,
Malignant
Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma,
Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous
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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-lMalignant 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 Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease,
besides
neoplasia, located in an organ system listed above.
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.)
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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.
Metaplasia is a form of controlled cell growth in which one type of adult or
fully differentiated cell substitutes for another type of adult cell.
Metaplastic disorders
which can be diagnosed, prognosed, prevented, and/or treated with compositions
of
the invention (including polynucleotides, polypeptides, agonists or
antagonists)
include, but are not limited to, agnogenic myeloid metaplasia, apocrine
metaplasia,
atypical metaplasia, autoparenchymatous metaplasia, connective tissue
metaplasia,
epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic
ossification, metaplastic polyps, myeloid metaplasia, primary myeloid
metaplasia,
secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of
amnion, and symptomatic myeloid metaplasia.
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 irntation 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
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ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia,
cervical
dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital
ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,
ectodermal
dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia
epiphysialis
hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata,
epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia
of jaws,
familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of
bone,
florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic
ectodermal
dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia,
mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini
dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple
epiphysial
dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia,
oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic
dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia,
pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-
optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
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.
In another embodiment, a polypeptide of the invention, or polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose disorders associated with the tissues) in which the
polypeptide of the invention is expressed, including one, two, three, four,
five, or
more tissues disclosed in Table 1, column 8 (Tissue Distribution Library
Code).
In another embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention conjugated to a toxin or a
radioactive
isotope, as described herein, may be used to treat cancers and neoplasms,
including,
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but not limited to those described herein. In a further preferred embodiment,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention conjugated to a toxin or a radioactive isotope, as described
herein,
may be used to treat acute myelogenous leukemia.
Additionally, polynucleotides, polypeptides, and/or agonists or antagonists of
the invention may affect apoptosis, and therefore, would be useful in treating
a
number of diseases associated with increased cell survival or the inhibition
of
apoptosis. For example, diseases associated with increased cell survival or
the
inhibition of apoptosis that could be diagnosed, prognosed, prevented, and/or
treated
by polynucleotides, polypeptides, and/or agonists or antagonists of the
invention,
include cancers (such as follicular lymphomas, carcinomas with p53 mutations,
and
hormone-dependent tumors, including, but not limited to colon cancer, cardiac
tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer,
intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma,
myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's
disease, polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and rheumatoid arthritis) and viral infections (such as
herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host disease,
acute graft
rej ection, and chronic graft rej ection.
Tn preferred embodiments, polynucleotides, polypeptides, and/or agonists or
antagonists of the invention are used to inhibit growth, progression, and/or
metastasis
of cancers, in particular those listed above.
Additional diseases or conditions associated with increased cell survival that
could be diagnosed, prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or agonists or antagonists of the invention, include, but
are not
limited to, progression, and/or metastases of malignancies and related
disorders such
as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia,
acute
myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic,
monocytic, and erythroleukemia)) and chronic leukemias (e.g.,. chronic
myelocytic
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(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera,
lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple
myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors
including,
but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lyrnphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular
tumor,
lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be diagnosed,
prognosed, prevented, andlor treated by polynucleotides, polypeptides, and/or
agonists or antagonists of the invention, include AIDS; neurodegenerative
disorders
(such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral
sclerosis,
retinitis pigmentosa, cerebellar degeneration and brain tumor or prior
associated
disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome,
Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis
and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia),
graft
v. host disease, ischemic injury (such as that caused by myocardial
infarction, stroke
and reperfusion injury), liver injury (e.g., hepatitis related liver injury,
ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer);
toxin
induced liver disease (such as that caused by alcohol), septic shock, cachexia
and
anorexia.
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Hyperproliferative diseases and/or disorders that could be diagnosed,
prognosed, prevented, and/or treated by polynucleotides, polypeptides, and/or
agonists or antagonists of the invention, include, but are not limited to,
neoplasms
located in the liver, abdomen, bone, breast, digestive system, pancreas,
peritoneum,
endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus,
thyroid),
eye, head and neck, nervous system (central and peripheral), lymphatic system,
pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.
Similarly, other hyperproliferative disorders can also be diagnosed,
prognosed,
prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or
antagonists of the invention. Examples of such hyperproliferative disorders
include,
but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders,
paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's
macroglobulinemia, Gaucher's Disease, histiocytosis, and any other
hyperproliferative disease, besides neoplasia, located in an organ system
listed above.
Another preferred embodiment utilizes polynucleotides of the present
invention to inhibit aberrant cellular division, by gene therapy using the
present
invention, and/or protein fusions or fragments thereof.
Thus, the present invention provides a method for treating cell proliferative
disorders by inserting into an abnormally proliferating cell a polynucleotide
of the
present invention, wherein said polynucleotide represses said expression.
Another embodiment of the present invention provides a method of treating
cell-proliferative disorders in individuals comprising administration of one
or more
active gene copies of the present invention to an abnormally proliferating
cell or cells.
In a preferred embodiment, polynucleotides of the present invention is a DNA
construct comprising a recombinant expression vector effective in expressing a
DNA
sequence encoding said polynucleotides. In another preferred embodiment of the
present invention, the DNA construct encoding the poynucleotides of the
present
invention is inserted into cells to be treated utilizing a retrovirus, or more
preferably
an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is
hereby incorporated by reference). In a most preferred embodiment, the viral
vector
is defective and will not transform non-proliferating cells, only
proliferating cells.
Moreover, in a preferred embodiment, the polynucleotides of the present
invention
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inserted into proliferating cells either alone, or in combination with or
fused to other
polynucleotides, can then be modulated via an external stimulus (i.e.
magnetic,
specific small molecule, chemical, or drug achninistration, etc.), which acts
upon the
promoter upstream of said polynucleotides to induce expression of the encoded
protein product. As such the beneficial therapeutic affect of the present
invention
may be expressly modulated (i.e. to increase, decrease, or inhibit expression
of the
present invention) based upon said external stimulus.
Polynucleotides of the present invention may be useful in repressing
expression of oncogeiuc genes or antigens. By "repressing expression of the
oncogenic genes " is intended the suppression of the transcription of the
gene, the
degradation of the gene transcript (pre-message RNA), the inhibition of
splicing, the
destruction of the messenger RNA, the prevention of the post-translational
modifications of the protein, the destruction of the protein, or the
inhibition of the
normal function of the protein.
1 S For local administration to abnormally proliferating cells,
polynucleotides of
the present invention may be administered by any method known to those of
skill in
the art including, but not limited to transfection, electroporation, microinj
ection of
cells, or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any
other method described throughout the specification. The polynucleotide of the
present invention may be delivered by known gene delivery systems such as, but
not
limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke,
Nature
320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014),
vaccinia virus
system (Chakrabarty et al., Mol. Cell Biol. 5:3403 (1985) or other efficient
DNA
delivery systems (Pates et al., Nature 313:812 (1985)) known to those skilled
in the
art. These references are exemplary only and are hereby incorporated by
reference.
In order to specifically deliver or transfect cells which are abnormally
proliferating
and spare non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as
described in the art and elsewhere herein) delivery system known to those of
skill in
the art. Since host DNA replication is required for retroviral DNA to
integrate and
the retrovirus will be unable to self replicate due to the lack of the
retrovirus genes
needed for its life cycle. Utilizing such a retroviral delivery system for
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polynucleotides of the present invention will target said gene and constructs
to
abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell
proliferative disorder/disease sites in internal organs, body cavities and the
like by use
of imaging devices used to guide an injecting needle directly to the disease
site. The
polynucleotides of the present invention may also be administered to disease
sites at
the time of surgical intervention.
By "cell proliferative disease" is meant any human or animal disease or
disorder, affecting any one or any combination of organs, cavities, or body
parts,
which is characterized by single or multiple local abnormal proliferations of
cells,
groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be
administered as long as it has a biologically inhibiting effect on the
proliferation of
the treated cells. Moreover, it is possible to administer more than one of the
polynucleotide of the present invention simultaneously to the same site. By
"biologically inhibiting" is meant partial or total growth inhibition as well
as
decreases in the rate of proliferation or growth of the cells. The
biologically
inhibitory dose may be determined by assessing the effects of the
polynucleotides of
the present invention on target malignant or abnormally proliferating cell
growth in
tissue culture, tumor growth in animals and cell cultures, or any other method
known
to one of ordinary skill in the art.
The present invention is further directed to antibody-based therapies which
involve administering of anti-polypeptides and anti-polynucleotide antibodies
to a
mammalian, preferably human, patient for treating one or more of the described
disorders. Methods for producing anti-polypeptides and anti-polynucleotide
antibodies polyclonal and monoclonal antibodies are described in detail
elsewhere
herein. Such antibodies may be provided in pharmaceutically acceptable
compositions as known in the art or as described herein.
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
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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.
In particular, the antibodies, fragments and derivatives of the present
invention
are useful for treating a subject having or developing cell proliferative
and/or
differentiation disorders as described herein. Such treatment comprises
administering
a single or multiple doses of the antibody, or a fragment, derivative, or a
conjugate
thereof.
The antibodies of this invention may be advantageously utilized in
combination with other monoclonal or chimeric antibodies, or with lymphokines
or
hematopoietic growth factors, for example., which serve to increase the number
or
activity of effector cells which interact with the antibodies.
It is preferred to use high affinity and/or potent ira 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
fragements
thereof, of the present invention. Such antibodies, fragments, or regions,
will
preferably have an affinity for polynucleotides or polypeptides, including
fragements
thereof. Preferred binding affinities include those with a dissociation
constant or Kd
Iess than SX10-6M, 10-6M, SX10-~M, IO-~M, SX10-$M, 10-$M; SXIO-9M, I0-9M,
SX10-1°M, 10-1°M, SX10-11M, 10-11M, SX10-12M, 10-12M, SX10-13M,
10-13M, SX10-
14M, 10-I4M, SX10-iSM, and 10-15M.
Moreover, polypeptides of the present invention are useful in inhibiting the
angiogenesis of proliferative cells or tissues, either alone, as a protein
fusion, or in
combination with other polypeptides directly or indirectly, as described
elsewhere
herein. In a most preferred embodiment, said anti-angiogenesis effect may be
achieved indirectly, for example, through the inhibition of hematopoietic,
tumor-
specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J
Natl
Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by
reference).
Antibodies directed to polypeptides or polynucleotides of the present
invention may
also result in inhibition of angiogenesis directly, or indirectly (See Witte
L, et al.,
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Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by
reference)).
Polypeptides, including protein fusions, of the present invention, or
fragments
thereof may be useful in inhibiting proliferative cells or tissues through the
induction
of apoptosis. Said polypeptides may act either directly, or indirectly to
induce
apoptosis of proliferative cells and tissues, fox example in the activation of
a death-
domain receptor, such as tumor necrosis factor (TNF) receptor-I, CD95 (Fas/APO-
1),
TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K,
et.al.,
Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by
reference).
Moreover, in another preferred embodiment of the present invention, said
polypeptides may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or through
stimulating the
expression of said proteins, either alone or in combination with small
molecule drugs
or adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory
proteins
(See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-
33
(1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-
12
(1998), Int J Tissue React;20(1):3-15 (1998), which are all hereby
incorporated by
reference).
Polypeptides, including protein fusions to, or fragments thereof, of the
present
invention are useful in inhibiting the metastasis of proliferative cells or
tissues.
Inhibition may occur as a direct result of administering polypeptides, or
antibodies
directed to said polypeptides as described elsewere herein, or indirectly,
such as
activating the expression of proteins known to inhibit metastasis; for example
alpha 4
integrins, (See, e.g., Curr Top Microbiol Immunol 1998;231:125-41, which is
hereby
incorporated by reference). Such thereapeutic affects of the present invention
may be
achieved either alone, or in combination with small molecule drugs or
adjuvants.
In another embodiment, the invention provides a method of delivering
compositions containing the polypeptides of the invention (e.g., compositions
containing polypeptides or polypeptide antibodes associated with heterologous
polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted
cells
expressing the polypeptide of the present invention. Polypeptides or
polypeptide
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antibodes of the invention may be associated with with heterologous
polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic,
ionic
and/or covalent interactions.
Polypeptides, protein fusions to, or fragments thereof, of the present
invention
are useful in enhancing the immunogenicity and/or antigenicity of
proliferating cells
or tissues, either directly, such as would occur if the polypeptides of the
present
invention 'vaccinated' the irmnune response to respond to proliferative
antigens and
immunogens, or indirectly, such as in activating the expression of proteins
known to
enhance the immune response (e.g. chemokines), to said antigens and
immunogens.
Renal Disorders
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention, may be used to treat, prevent, diagnose, and/or prognose
disorders
of the renal system. Renal disorders which can be diagnosed, prognosed,
prevented,
and/or treated with compositions of the invention include, but are not limited
to,
kidney failure, nephritis, blood vessel disorders of kidney, metabolic and
congenital
kidney disorders, urinary disorders of the kidney, autoimmune disorders,
sclerosis and
necrosis, electrolyte imbalance, and kidney cancers.
Kidney diseases which can be diagnosed, prognosed, prevented, and/or treated
with compositions of the invention include, but are not limited to, acute
kidney
failure, chronic kidney failure, atheroembolic renal failure, end-stage renal
disease,
inflammatory diseases of the kidney (e.g., acute glomerulonephritis,
postinfectious
glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic
syndrome,
membranous glomerulonephritis, familial nephrotic syndrome,
membranoproliferative
glomerulonephritis I and II, mesangial proliferative glomerulonephritis,
chronic
glomerulonephritis, acute tubulointerstitial nephritis, chronic
tubulointerstitial
nephritis, acute post-streptococcal glomerulonephritis (PSGl~, pyelonephritis,
lupus
nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal
glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney
infarction,
atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis,
renal vein
thrombosis, renal underperfusion, renal retinopathy, renal ischemia-
reperfusion, renal
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artery embolism, and renal artery stenosis), and kidney disorders resulting
form
urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis
(renal lithiasis,
nephrolithiasis), reflux nephropathy, urinary tract infections, urinary
retention, and
acute or chronic unilateral obstructive uropathy.)
In addition, compositions of the invention can be used to diagnose, prognose,
prevent, and/or treat metabolic and congenital disorders of the kidney (e.g.,
uremia,
renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal
glycosuria,
nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal
fibrocystic
osteosis (renal rickets), Hannup disease, Banter's syndrome, Liddle's
syndrome,
polycystic kidney disease, medullary cystic disease, medullary sponge kidney,
Alpon's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH
syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes
insipidus,
analgesic nephropathy, kidney stones, and membranous nephropathy), and
autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE),
Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative
glomerulonephritis).
Compositions of the invention can also be used to diagnose, prognose,
prevent, andlor treat sclerotic or necrotic disorders of the kidney (e.g.,
glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis
(FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers
of the
kidney (e.g., nephroma, hypernephroma, nephroblastorna, renal cell cancer,
transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and
Wilm's
tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema,
hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia,
hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphosphatemia).
Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
panicle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
topical applications during surgery, aerosol delivery. Such methods are known
in the
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art. Polypeptides may be administered as part of a Therapeutic, described in
more
detail below. Methods of delivering polynucleotides are described in more
detail
herein.
Cardiovascular Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present
invention, may be used to treat, prevent, diagnose, and/or prognose
cardiovascular
disorders, including, but not limited to, peripheral artery disease, such as
limb
ischemia.
Cardiovascular disorders include, but are not limited to, cardiovascular
abnormalities, such as arterio-arterial fistula, arteriovenous fistula,
cerebral
arteriovenous malformations, congenital heart defects, pulmonary atresia, and
Scimitar Syndrome. Congenital heart defects include, but are not limited to,
aortic
coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart,
dextrocardia,
patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic
left
heart syndrome, levocardia, tetralogy of fallot, transposition of great
vessels, double
outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and
heart septal
defects, such as aortopulinonary septal defect, endocardial cushion defects,
Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
Cardiovascular disorders also include, but are not limited to, heart disease,
such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac
output,
cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac
arrest,
congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea,
cardiac
edema, heart hypertrophy, congestive cardiomyopathy, left ventricular
hypertrophy,
right ventricular hypertrophy, post-infarction heart rupture, ventricular
septal rupture,
heart valve diseases, myocardial diseases, myocardial ischemia, pericardial
effusion,
pericarditis (including constrictive and tuberculous), pneumopericardium,
postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease,
ventricular dysfunction, hyperemia, cardiovascular pregnancy complications,
Scimitar
Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
Arrhythmias include, but are not limited to, sinus arrhythmia, atrial
fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes
Syndrome, bundle-
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branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-
Levine
Syndrome, Mahaim-type pre-excitation syndrome, Wolff Parkinson-White syndrome,
sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias
include
paroxysmal tachycardia, supraventricular tachycardia, accelerated
idioventricular
S rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial
tachycardia, ectopic
functional tachycardia, sinoatrial nodal reentry tachycardia, sinus
taclycardia,
Torsades de Pointes, and ventricular tachycardia.
Heart valve diseases include, but are not limited to, aortic valve
insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve
prolapse,
tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis,
pulmonary
atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid
atresia,
tricuspid valve insufficiency, and tricuspid valve stenosis.
Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular
stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiornyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns
Syndrome, myocardial reperfusion injury, and myocarditis.
Myocardial ischemias include, but are not limited to, coronary disease, such
as
angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary
thrombosis,
coronary vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema,
aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial
occlusive
diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular
disorders, diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia,
hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension,
ischemia,
peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease,
Raynaud's
disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior
vena
cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and
venous
insufficiency.
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.Aneurysms include, but are not limited to, dissecting aneurysms, false
aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral
aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include, but are not limited to, arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular dysplasias,
mesenteric
vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery
occlusion, and thromboangiitis obliterans.
Cerebrovascular disorders include, but are not limited to, carotid artery
diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia,
cerebral
arteriosclerosis, cerebral arteriovenous malformation, cerebral artery
diseases,
cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis,
Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural
hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia
(including transient), subclavian steal syndrome, periventricular
Ieukomalacia,
vascular headache, cluster headache, migraine, and vertebrobasilar
insufficiency.
Embolisms include, but are not limited to, air embolisms, amniotic fluid
embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms, and thromoboembolisms. Thrombosis include, but are not limited to,
coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid
artery
thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
Ischemic disorders include, but are not limited to, cerebral ischemia,
ischemic
colitis, compartment syndromes, anterior compartment syndrome, myocardial
ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis
includes, but
is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss
Syndrome,
mucocutaneous lymph node syndrome, thromboangiitis obliterans,
hypersensitivity
vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and
Wegener's
granulomatosis.
Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle inj ection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
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topical applications during surgery, aerosol delivery. Such methods are known
in the
art. Polypeptides may be administered as part of a Therapeutic, described in
more
detail below. Methods of delivering polynucleotides are described in more
detail
herein.
Respiratory Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present
invention may be used to treat, prevent, diagnose, and/or prognose diseases
and/or
disorders of the respiratory system.
Diseases and disorders of the respiratory system include, but are not limited
to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic
rhinitis, atrophic
rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile
angiofibromas,
cancer of the nose and juvenile papillomas, vocal cord polyps, nodules
(singer's
nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis
(e.g., viral and
bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess,
laryngitis,
laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil
cancer, larynx
cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell)
carcinoma,
large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic
pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis,
allergic
interstitial pneumonitis, organic dust pneumoconiosis, allergic
bronchopulmonary
aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis),
Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g.,
Streptococcus
pfZeumofaiae (pneumoncoccal pneumonia), Staplzylococcus auYeus (staphylococcal
pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and
Pseudomas spp.), Mycoplasma ptaeumoniae pneumonia, Hemoplailus if~flueyazae
pneumonia, Legioraella p~ceumophila (Legionnaires' disease), and Chlar~zydia
psittaci
(Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).
Additional diseases and disorders of the respiratory system include, but are
not
limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial
viral
infection, mumps, erythema infectiosum (fifth disease), roseola infantum,
progressive
rubella panencephalitis, german measles, and subacute sclerosing
panencephalitis),
fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis,
fungal
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infections in people with severely suppressed immune systems (e.g.,
cryptococcosis,
caused by Cfyptococcus neofor~mans; aspergillosis, caused byAspergillus spp.;
candidiasis, caused by Candida; and mucormycosis)), Pfzeumocystis carifaii
(pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydia
spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical
pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy,
pleural
effusion, and pneumothorax (e.g., simple spontaneous pneurnothorax,
complicated
spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases
(e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema,
chronic or
acute bronchitis), occupational lung diseases (e.g., silicosis, black lung
(coal workers'
pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis,
and
benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis
(e.g.,
fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary
fibrosis,
desquamative interstitial pneumonia, lymphoid interstitial pneumonia,
histiocytosis X
(e.g., Letterer-Siwe disease, Hand-Schiiller-Christian disease, eosinophilic
granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary
alveolar
proteinosis), Acute respiratory distress syndrome (also called, e.g., adult
respiratory
distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral,
bacterial),
bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus
aureus or
Legionella praeumoplzila), and cystic fibrosis.
Anti-An~io~enesis Activity
The naturally occurring balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et al., Cell 56:345-355 (1989). In those rare instances iri which
neovascularization occurs under normal physiological conditions, such as wound
healing, organ regeneration, embryonic development, and female reproductive
processes, angiogenesis is stringently regulated and spatially and temporally
delimited. Under conditions of pathological angiogenesis such as that
characterizing
solid tumor growth, these regulatory controls fail. Unregulated angiogenesis
becomes
pathologic and sustains progression of many neoplastic and non-neoplastic
diseases.
A number of serious diseases are dominated by abnormal neovascularization
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including solid tumor growth and metastases, arthritis, some types of eye
disorders,
and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991);
Folkman
et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc.
Res.
29:401-411 (1985); Folkman, Advances in Cancer Research; eds. Klein and
Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J.
Opthalnaol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983).
In a
number of pathological conditions, the process of angiogenesis contributes to
the
disease state. For example, significant data have accumulated which suggest
that the
growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun,
Science 235:442-447 (1987).
The present invention provides for treatment of diseases or disorders
associated with neovascularization by administration of the polynucleotides
and/or
polypeptides of the invention, as well as agonists or antagonists of the
present
invention. Malignant and metastatic conditions which can be treated with the
polynucleotides and polypeptides, or agonists or antagonists of the invention
include,
but are not limited to, malignancies, solid tumors, and cancers described
herein and
otherwise known in the art (for a review of such disorders, see Fishman et
al.,
Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).Thus, the present
invention provides a method of treating an angiogenesis-related disease and/or
disorder, comprising administering to an individual in need thereof a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
of the
invention. For example, polynucleotides, polypeptides, antagonists and/or
agonists
may be utilized in a variety of additional methods in order to therapeutically
treat a
cancer or tumor. Cancers which may be treated with polynucleotides,
polypeptides,
antagonists and/or agonists include, but are not limited to solid tumors,
including
prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes,
liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,
bladder,
thyroid cancer; primary tumors and metastases; melanomas; glioblastoma;
Kaposi's
sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer;
advanced
malignancies; and blood born tumors such as leukemias. For example,
polynucleotides, polypeptides, antagonists and/or agonists may be delivered
topically,
in order to treat cancers such as skin cancer, head and neck tumors, breast
tumors, and
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Kaposi's sarcoma.
Within yet other aspects, polynucleotides, polypeptides, antagonists and/or
agonists may be utilized to treat superficial forms of bladder cancer by, for
example,
intravesical administration. Polynucleotides, polypeptides, antagonists and/or
agonists
may be delivered directly into the tumor, or near the tumor site, via
injection or a
catheter. Of course, as the artisan of ordinary skill will appreciate, the
appropriate
mode of administration will vary according to the cancer to be treated. Other
modes
of delivery are discussed herein.
Polynucleotides, polypeptides, antagonists and/or agonists may be useful in
treating other disorders, besides cancers, which involve angiogenesis. These
disorders include, but are not limited to: benign tumors, for example
hemangiomas,
acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example, diabetic
retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular
glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis;
delayed
wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial
angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous
malformations;
ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia;
wound
granulation; Crohn's disease; and atherosclerosis.
For example, within one aspect of the present invention methods are provided
for treating hypertrophic scars and keloids, comprising the step of
administering a
polynucleotide, polypeptide, antagonist and/or agonist of the invention to a
hypertrophic scar or keloid.
Within one embodiment of the present invention polynucleotides,
polypeptides, antagonists and/or agonists of the invention are directly
injected into a
hypertrophic scar or keloid, in order to prevent the progression of these
lesions. This
therapy is of particular value in the prophylactic treatment of conditions
which are
known to xesult in the development of hypertrophic scars and keloids (e.g.,
burns),
and is preferably initiated after the proliferative phase has had time to
progress
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(approximately 14 days after the initial injury), but before hypertrophic scar
or keloid
development. As noted above, the present invention also provides methods for
treating neovascular diseases of the eye, including for example, corneal
neovascularization, neovascular glaucoma, proliferative diabetic retinopathy,
retrolental fibroplasia and macular degeneration.
Moreover, Ocular disorders associated with neovascularization which cam be
treated with the polynucleotides and polypeptides of the present invention
(including
agonists and/or antagonists) include, but are not limited to: neovascular
glaucoma,
diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,
retinopathy of
I O prematurity macular degeneration, corneal graft neovascularization, as
well as other
eye inflammatory diseases, ocular tumors and diseases associated with
choroidal or
iris neovascularization. See, e.g., reviews by Waltman et al., Am. J.
OplZtlaal. 85:704-
710 (1978) and Gaxtner et al., Su~v. Oplathal. 22:291-312 (1978).
Thus, within one aspect of the present invention methods are provided for
treating neovascular diseases of the eye such as corneal neovascularization
(including
corneal graft neovascularization), comprising the step of administering to a
patient a
therapeutically effective amount of a compound (as described above) to the
cornea,
such that the formation of blood vessels is inhibited. Briefly, the cornea is
a tissue
which normally lacks blood vessels. In certain pathological conditions
however,
capillaries may extend into the cornea from the pericorneal vascular plexus of
the
limbus. When the cornea becomes vascularized, it also becomes clouded,
resulting in
a decline in the patient's visual acuity. Visual loss may become complete if
the
cornea completely opacitates. A wide variety of disorders can result in
corneal
neovascularization, including for example, corneal infections (e.g., trachoma,
herpes
simplex keratitis, leishmaniasis and onchocerciasis), immunological processes
(e.g.,
graft rej ection and Stevens-Johnson's syndrome), alkali burns, trauma,
inflammation
(of any cause), toxic and nutritional deficiency states, and as a complication
of
wearing contact lenses.
Within particularly preferred embodiments of the invention, may be prepared
for topical administration in saline (combined with any of the preservatives
and
antimicrobial agents commonly used in ocular preparations), and administered
in
eyedrop form. The solution or suspension may be prepared in its pure form and
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administered several times daily. Alternatively, anti-angiogenic compositions,
prepared as described above, may also be administered directly to the cornea.
Within
preferred embodiments, the anti-angiogenic composition is prepared with a
rnuco-
adhesive polymer which binds to cornea. Within further embodiments, the anti-
s angiogenic factors or anti-angiogenic compositions may be utilized as an
adjunct to
conventional steroid therapy. Topical therapy may also be useful
prophylactically in
corneal lesions which are known to have a high probability of inducing an
angiogenic
response (such as chemical burns). In these instances the treatment, likely in
combination with steroids, may be instituted immediately to help prevent
subsequent
complications.
Within other embodiments, the compounds described above may be injected
directly into the corneal stroma by an ophthalmologist under microscopic
guidance.
The preferred site of injection may vary with the morphology of the individual
lesion,
but the goal of the administration would be to place the composition at the
advancing
front of the vasculature (i.e., interspersed between the blood vessels and the
normal
cornea). In most cases this would involve perilimbic corneal injection to
"protect" the
cornea from the advancing blood vessels. This method may also be utilized
shortly
after a corneal insult in order to prophylactically prevent corneal
neovascularization.
In this situation the material could be injected in the perilimbic cornea
interspersed
between the corneal lesion and its undesired potential limbic blood supply.
Such
methods may also be utilized in a similar fashion to prevent capillary
invasion of
transplanted corneas. In a sustained-release form injections might only be
xequired 2-
3 times per year. A steroid could also be added to the injection solution to
reduce
inflammation resulting from the injection itself.
Within another aspect of the present invention, methods are provided for
treating neovascular glaucoma, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. In
one
embodiment, the compound may be administered topically to the eye in order to
treat
early forms of neovascular glaucoma. Within other embodiments, the compound
may
be implanted by injection into the region of the anterior chamber angle.
Within other
embodiments, the compound may also be placed in any location such that the
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compound is continuously released into the aqueous humor. Within another
aspect of
the present invention, methods are provided for treating proliferative
diabetic
retinopathy, comprising the step of administering to a patient a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
to the
eyes, such that the formation of blood vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative
diabetic retinopathy may be treated by injection into the aqueous humor or the
vitreous, in order to increase the local concentration of the polynucleotide,
polypeptide, antagonist and/or agonist in the retina. Preferably, this
treatment should
be initiated prior to the acquisition of severe disease requiring
photocoagulation.
Within another aspect of the present invention, methods are provided for
treating retrolental fibroplasia, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. The
compound may be administered topically, via intravitreous injection and/or via
intraocular implants.
Additionally, disorders which can be treated with the polynucleotides,
polypeptides, agonists and/or agonists include, but are not limited to,
hemangioma,
arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound
healing,
granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-
Weber
syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be treated, prevented, diagnosed,
and/or prognosed with the the polynucleotides, polypeptides, agonists and/or
agonists
of the invention include, but are not limited to, solid tumors, blood born
tumors such
as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for
example,
diabetic retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft
rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,
retinoblastoma, and
uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations,
hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma,
vascular
adhesions, myocardial angiogenesis, coronary collaterals, cerebral
collaterals,
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arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber
Syndrome,
plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma
fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth
control agent by preventing vascularization required for embryo implantation
controlling menstruation, diseases that have angiogenesis as a pathologic
consequence
such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter
pylori),
Bartonellosis and bacillary angiomatosis.
In one aspect of the birth control method, an amount of the compound
sufficient to block embryo implantation is administered before or after
intercourse and
fertilization have occurred, thus providing an effective method of birth
control,
possibly a "morning after" method. Polynucleotides, polypeptides, agonists
and/or
agonists may also be used in controlling menstruation or administered as
either a
peritoneal lavage fluid or for peritoneal implantation in the treatment of
endometriosis.
Polynucleotides, polypeptides, agonists and/or agonists of the present
invention may be incorporated into surgical sutures in order to prevent stitch
granulomas.
Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a
Wide variety of surgical procedures. For example, within one aspect of the
present
invention a compositions (in the form of, for example, a spray or film) may be
utilized
to coat or spray an area prior to removal of a tumor, in order to isolate
normal
surrounding tissues from malignant tissue, and/or to prevent the spread of
disease to
surrounding tissues. Within other aspects of the present invention,
compositions (e.g.,
in the form of a spray) may be delivered via endoscopic procedures in order to
coat
tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects
of the
present invention, surgical meshes which have been coated with anti-
angiogenic
compositions of the present invention may be utilized in any procedure wherein
a
surgical mesh might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition may be
utilized
during abdominal cancer resection surgery (e.g., subsequent to colon
resection) in
order to provide support to the structure, and to release an amount of the
anti-
angiogenic factor.
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Within further aspects of the present invention, methods are provided for
treating tumor excision sites, comprising administering a polynucleotide,
polypeptide,
agonist and/or agonist to the resection margins of a tumor subsequent to
excision,
such that the local recurrence of cancer and the formation of new blood
vessels at the
site is inhibited. Within one embodiment of the invention, the anti-angiogenic
compound is administered directly to the tumor excision site (e.g., applied by
swabbing, brushing or otherwise coating the resection margins of the tumor
with the
anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be
incorporated into known surgical pastes prior to administration. Within
particularly
preferred embodiments of the invention, the anti-angiogenic compounds are
applied
after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of the present invention, polynucleotides, polypeptides,
agonists and/or agonists may be administered to the resection margin of a wide
variety of tumors, including for example, breast, colon, brain and hepatic
tumors. For
example, within one embodiment of the invention, anti-angiogenic compounds may
be administered to the site of a neurological tumor subsequent to excision,
such that
the formation of new blood vessels at the site are inhibited.
The polynucleotides, polypeptides, agonists and/or agonists of the present
invention may also be administered along with other anti-angiogenic factors.
Representative examples of other anti-angiogenic factors include: Anti-
Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of
Metalloproteinase-l, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the
lighter "d
group" transition metals.
Lighter "d group" transition metals include, for example, vanadium,
molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition
metal species may form transition metal complexes. Suitable complexes of the
above-mentioned transition metal species include oxo transition metal
complexes.
Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include metavanadate and orthovanadate complexes such as, for example,
ammonium
metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl
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complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate
including vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
Representative examples of tungsten and molybdenum complexes also include
oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide complexes. Suitable tungstate complexes include ammonium tungstate,
calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl
complexes. Suitable molybdate complexes include ammonium molybdate and its
hydrates, sodium molybdate and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (V1' oxide, molybdenum
(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
molybdenyl acetylacetonate. Other suitable tmigsten and molybdenum complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and
sugars.
A wide variety of other anti-angiogenic factors may also be utilized within
the
context of the present invention. Representative examples include platelet
factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab
shells),
(Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide
Peptidoglycan
Complex (SP- PG) (the function of this compound may be enhanced by the
presence
of steroids such as estrogen, and tamoxifen citrate); Staurosporine;
modulators of
matrix metabolism, including for example, proline analogs, cishydroxyproline,
d,L-
3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile
fumarate;
4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin;
Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem.
267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480,
1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin
(W gber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST' ;
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-
serum;
alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987);
Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
chloroanthronlic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-
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316, 1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole;
and metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level
Diseases associated with increased cell survival or the inhibition of
apoptosis
that could be treated, prevented, diagnosed, and/or prognosed using
polynucleotides
or polypeptides, as well as antagonists or agonists of the present invention,
include
cancers (such as follicular lymphomas, carcinomas with p53 mutations, and
hormone-
dependent tumors, including, but not limited to colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer,
intestinal
cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,
lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, I~aposi's sarcoma and
ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's
syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's
disease, polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis and rheumatoid arthritis) and viral infections (such as
herpes
viruses, pox viruses and adenoviruses), inflammation, graft v. host disease,
acute graft
rej ection, and chronic graft rej ection.
In preferred embodiments, polynucleotides, polypeptides, and/or antagonists
of the invention are used to inhibit growth, progression, and/or metasis of
cancers, in
particular those listed above.
Additional diseases or conditions associated with increased cell survival that
could be treated or detected by polynucleotides or polypeptides, or agonists
or
antagonists of the present invention include, but are not limited to,
progression, and/or
metastases of malignancies and related disorders such as leukemia (including
acute
leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia))
and
chronic leukemias (e.g., chronic rnyelocytic (granulocytic) leukemia and
chronic
Iymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease
and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia,
heavy
chain disease, and solid tumors including, but not limited to, sarcomas and
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carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic
cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, basal
cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,
cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma,
bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated, prevented,
diagnosed, and/or prognesed using polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention, include, but are not limited
to, AmS;
neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
and
brain tumor or prior associated disease); autoimmune disorders (such as,
multiple
sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis,
Behcet's
disease, Crohn's disease, polymyositis, systemic lupus erythematosus and
immune-
related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes
(such
as aplastic anemia), graft v. host disease, ischemic injury (such as that
caused by
myocardial infarction, stroke and reperfusion injury), liver injury (e.g.,
hepatitis
related liver injury, ischemia/reperfusion injury, cholestosis (bile duct
injury) and
liver cancer); toxin-induced liver disease (such as that caused by alcohol),
septic
shock, cachexia and anorexia.
Wound Healing and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, for therapeutic purposes, for example,
to
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stimulate epithelial cell proliferation and basal keratinocytes for the
purpose of wound
healing, and to stimulate hair follicle production and healing of dermal
wounds.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, may be clinically useful in stimulating wound healing including
surgical
wounds, excisional wounds, deep wounds involving damage of the dermis and
epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds,
diabetic
ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers,
burns
resulting from heat exposure or chemicals, and other abnormal wound healing
conditions such as uremia, malnutrition, vitamin deficiencies and
complications
associated with systemic treatment with steroids, radiation therapy and
antineoplastic
drugs and antimetabolites. Polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, could be used to promote dermal
reestablishment
subsequent to dermal loss
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention, could be used to increase the adherence of skin grafts to a
wound
bed and to stimulate re-epithelialization from the wound bed. The following
are types
of grafts that polynucleotides or polypeptides, agonists or antagonists of the
present
invention, could be used to increase adherence to a wound bed: autografts,
artificial
skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts,
Blair-Brown
grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic
graft,
epidermic graft, fascia graft, full thickness graft, heterologous graft,
xenograft,
homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal
graft, Ollier-
Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft,
split skin
graft, thick split graft. Polynucleotides or polypeptides, as well as agonists
or
antagonists of the present invention, can be used to promote skin strength and
to
improve the appearance of aged skin.
It is believed that polynucleotides or polypeptides, as well as agonists or
antagonists of the present invention, will also produce changes in hepatocyte
proliferation, and epithelial cell proliferation in the lung, breast,
pancreas, stomach,
small intestine, and large intestine. Polynucleotides or polypeptides, as well
as
agonists or antagonists of the present invention, could promote proliferation
of
epithelial cells such as sebocytes, hair follicles, hepatocytes, type II
pneumocytes,
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mucin-producing goblet cells, and other epithelial cells and their progenitors
contained within the skin, lung, liver, and gastrointestinal tract.
Polynucleotides or
polypeptides, agonists or antagonists of the present invention, may promote
proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention, could also be used to reduce the side effects of gut
toxicity that
result from radiation, chemotherapy treatments or viral infections.
Polynucleotides or
polypeptides, as well as agonists or antagonists of the present invention, may
have a
cytoprotective effect on the small intestine mucosa. Polynucleotides or
polypeptides,
as well as agonists or antagonists of the present invention, may also
stimulate healing
of mucositis (mouth ulcers) that result from chemotherapy and vixal
infections.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention, could further be used in full regeneration of skin in full
and partial
thickness skin defects, including burns, (i.e., repopulation of hair
follicles, sweat
glands, and sebaceous glands), treatment of other skin defects such as
psoriasis.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, could be used to treat epidermolysis bullosa, a defect in adherence
of the
epidermis to the underlying dermis Which results in frequent, open and painful
blisters
by accelerating reepithelialization of these lesions. Polynucleotides or
polypeptides,
as well as agonists or antagonists of the present invention, could also be
used to treat
gastric and doudenal ulcers and help heal by scar formation of the mucosal
lining and
regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis,
are
diseases which result in destruction of the mucosal surface of the small or
large
intestine, respectively. Thus, polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, could be used to promote the resurfacing
of the
mucosal surface to aid more rapid healing and to prevent progression of
inflammatory
bowel disease. Treatment with polynucleotides or polypeptides, agonists or
antagonists of the present invention, is expected to have a significant effect
on the
production of mucus throughout the gastrointestinal tract and could be used to
protect
the intestinal mucosa from injurious substances that are ingested or following
surgery.
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Polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, could be used to treat diseases associate with the under
expression.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, could be used to prevent and heal damage to the
lungs due to
various pathological states. Polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, which could stimulate proliferation and
differentiation and promote the repair of alveoli and brochiolar epithelium to
prevent
or treat acute or chronic lung damage. For example, emphysema, which results
in the
progressive loss of aveoli, and inhalation injuries, i.e., resulting from
smoke
inhalation and burns, that cause necrosis of the bronchiolar epithelium and
alveoli
could be effectively treated using polynucleotides or polypeptides, agonists
or
antagonists of the present invention. Also, polynucleotides or polypeptides,
as well as
agonists or antagonists of the present invention, could be used to stimulate
the
proliferation of and differentiation of type II pneumocytes, which may help
treat or
prevent disease such as hyaline membrane diseases, such as infant respiratory
distress
syndrome and bronchopulmonary displasia, in premature infants.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention, could stimulate the proliferation and differentiation of
hepatocytes
and, thus, could be used to alleviate or treat liver diseases and pathologies
such as
fulminant liver failure caused by cirrhosis, liver damage caused by viral
hepatitis and
toxic substances (i.e., acetaminophen, carbon tetraholoride and other
hepatotoxins
known in the art).
In addition, polynucleotides or polypeptides, as well as agonists or
antagonists
of the present invention, could be used treat or prevent the onset of diabetes
mellitus.
In patients with newly diagnosed Types I and II diabetes, where some islet
cell
function remains, polynucleotides or polypeptides, as well as agonists or
antagonists
of the present invention, could be used to maintain the islet function so as
to alleviate,
delay or prevent permanent manifestation of the disease. Also, polynucleotides
or
polypeptides, as well as agonists or antagonists of the present invention,
could be used
as an auxiliary in islet cell transplantation to improve or promote islet cell
function.
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Neural Activity and Neurological Diseases
The polynucleotides, polypeptides and agonists or antagonists of the invention
may be used for the diagnosis and/or treatment of diseases, disorders, damage
or
injury of the brain and/or nervous system. Nervous system disorders that can
be
treated with the compositions of the invention (e.g., polypeptides,
polynucleotides,
and/or agonists or antagonists), include, but are not limited to, nervous
system
injuries, and diseases or disorders which result in either a disconnection of
axons, a
diminution or degeneration of neurons, or demyelination. Nervous system
lesions
which may be treated in a patient (including human and non-human mammalian
patients) according to the methods of the invention, include but are not
limited to, the
following lesions of either the central (including spinal cord, brain) or
peripheral
nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion
of the
nervous system results in neuronal injury or death, including cerebral
infarction or
ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions,
including
lesions caused by physical injury or associated with surgery, for example,
lesions
which sever a portion of the nervous system, or compression injuries; (3)
malignant
lesions, in which a portion of the nervous system is destroyed or injured by
malignant
tissue which is either a nervous system associated malignancy or a malignancy
derived from non-nervous system tissue; (4) infectious lesions, in which a
portion of
the nervous system is destroyed or injured as a result of infection, for
example, by an
abscess or associated with infection by human immunodeficiency virus, herpes
zoster,
or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5)
degenerative lesions, in which a portion of the nervous system is destroyed or
injured
as a result of a degenerative process including but not limited to,
degeneration
associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea,
or
amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional
diseases
or disorders, in which a portion of the nervous system is destroyed or injured
by a
nutritional disorder or disorder of metabolism including, but not limited to,
vitamin
B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol
amblyopia,
Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and
alcoholic cerebellar degeneration; (7) neurological lesions associated with
systemic
diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's
palsy),
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systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by
toxic
substances including alcohol, lead, or particular neurotoxins; and (9)
demyelinated
lesions in which a portion of the nervous system is destroyed or injured by a
demyelinating disease including, but not limited to, multiple sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy or various
etiologies, progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
In one embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to protect neural cells from the
damaging effects
of hypoxia. In a further preferred embodiment, the polypeptides,
polynucleotides, or
agonists or antagonists of the invention are used to protect neural cells from
the
damaging effects of cerebral hypoxia. According to this embodiment, the
compositions of the invention are used to treat or prevent neural cell injury
associated
with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the
polypeptides, polynucleotides, or agonists or antagonists of the invention,
are used to
treat or prevent neural cell injury associated with cerebral ischemia. In
another non-
exclusive aspect of this embodiment, the polypeptides, polynucleotides, or
agonists or
antagonists of the invention are used to treat or prevent neural cell injury
associated
with cerebral infarction.
In another preferred embodiment, the polypeptides, polynucleotides, or
agonists or antagonists of the invention are used to treat or prevent neural
cell injury
associated with a stroke. In a specific embodiment, the polypeptides,
polynucleotides,
or agonists or antagonists of the invention are used to treat or prevent
cerebral neural
cell injury associated with a stroke.
In another preferred embodiment, the polypeptides, polynucleotides, or
agonists or antagonists of the invention are used to treat or prevent neural
cell injury
associated with a heart attack. In a specific embodiment, the polypeptides,
polynucleotides, or agonists or antagonists of the invention are used to treat
or prevent
cerebral neural cell injury associated with a heart attack.
The compositions of the invention which are useful for treating or preventing
a nervous system disorder may be selected by testing for biological activity
in
promoting the survival or differentiation of neurons. For example, and not by
way of
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limitation, compositions of the invention which elicit any of the following
effects may
be useful according to the invention: (1) increased survival time of neurons
in culture
either in the presence or absence of hypoxia or hypoxic conditions; (2)
increased
sprouting of neurons in culture or in vivo; (3) increased production of a
neuron-
s associated molecule in culture or in vivo, e.g., choline acetyltransferase
or
acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms
of
neuron dysfunction in vivo. Such effects may be measured by any method known
in
the art. In preferred, non-limiting embodiments, increased survival of neurons
may
routinely be measured using a method set forth herein or otherwise known in
the art,
such as, for example, in Zhang et al., P~oc Natl Acad Sci USA 97:3637-42
(2000) or
in Arakawa et al., J. NeuYOSCi., 10:3507-15 (1990); increased sprouting of
neurons
may be detected by methods known in the art, such as, for example, the methods
set
forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al.,
Ahya. Rev.
Neu~osci., 4:17-42 (1981); increased production of neuron-associated molecules
may
be measured by bioassay, enzymatic assay, antibody binding, Northern blot
assay,
etc., using techniques known in the art and depending on the molecule to be
measured; and motor neuron dysfunction may be measured by assessing the
physical
manifestation of motor neuron disorder, e.g., weakness, motor neuron
conduction
velocity, or functional disability.
In specific embodiments, motor neuron disorders that may be treated
according to the invention include, but are not limited to, disorders such as
infarction,
infection, exposure to toxin, trauma, surgical damage, degenerative disease or
malignancy that may affect motor neurons as well as other components of the
nervous
system, as well as disorders that selectively affect neurons such as
amyotrophic lateral
sclerosis, and including, but not limited to, progressive spinal muscular
atrophy,
progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile
muscular
atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome),
poliomyelitis and the post polio syndrome, and Hereditary Motorsensory
Neuropathy
(Charcot-Marie-Tooth Disease).
Further, polypeptides or polynucleotides of the invention may play a role in
neuronal survival; synapse formation; conductance; neural differentiation,
etc. Thus,
compositions of the invention (including polynucleotides, polypeptides, and
agonists
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or antagonists) may be used to diagnose andlor treat or prevent diseases or
disorders
associated with these roles, including, but not limited to, learning and/or
cognition
disorders. The compositions of the invention may also be useful in the
treatment or
prevention of neurodegenerative disease states andlor behavioural disorders.
Such
neurodegenerative disease states and/or behavioral disorders include, but are
not
limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette
Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder,
panic disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
including disorders in feeding, sleep patterns, balance, and perception. In
addition,
compositions of the invention may also play a role in the treatment,
prevention and/or
detection of developmental disorders associated with the developing embryo, or
sexually-linked disorders.
Additionally, polypeptides, polynucleotides and/or agonists or antagonists of
the invention, may be useful in protecting neural cells from diseases, damage,
disorders, or injury, associated with cerebrovascular disorders including, but
not
limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid
stenosis, or
Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral
anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,
cerebral artery
diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis,
sinus
thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or
subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral
ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or
vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct),
leukomalacia,
periventricular, and vascular headache (e.g., cluster headache or migraines).
In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, for therapeutic purposes, for example,
to
stimulate neurological cell proliferation and/or differentiation. Therefore,
polynucleotides, polypeptides, agonists and/or antagonists of the invention
may be
used to treat and/or detect neurologic diseases. Moreover, polynucleotides or
polypeptides, or agonists or antagonists of the invention, can be used as a
marker or
detector of a particular nervous system disease or disorder. °
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Examples of neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include brain diseases, such as metabolic brain diseases which includes
phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase
deficiency,
pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain
edema, brain neoplasms such as cerebellar neoplasms which include
infratentorial
neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms,
hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar
diseases such as cerebellar ataxia which include spinocerebellar degeneration
such as
ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-
Joseph
Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as
infratentorial
neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis,
globoid cell
leukodystrophy, metachromatic leukodystrophy and subacute sclerosing
panencephalitis.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include cerebrovascular disorders (such as carotid artery diseases which
include
carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral
amyloid
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformations, cerebral artery diseases, cerebral embolism and
thrombosis such as carotid artery thrombosis, sinus thrombosis and
Wallenberg's
Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and
subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as
transient
cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar
insufficiency,
vascular dementia such as multi-infarct dementia, periventricular
leukomalacia,
vasculax headache such as cluster headache and migraine.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include dementia such as AIDS Dementia Complex, presenile dementia such as
Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as
Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such
as
mufti-infarct dementia, encephalitis which include encephalitis periaxialis,
viral
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encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis
Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated
encephalomyelitis, meningoencephalitis such as uveorneningoencephalitic
syndrome,
Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis,
encephalomalacia such as periventricular leukomalacia, epilepsy such as
generalized
epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy
which
includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as
complex
partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-
traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua, and
Hallervorden-
Spatz Syndrome.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include hydrocephalus such as Dandy-Walker Syndrome and normal pressure
hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral
malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri
pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral
toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central
nervous
system infections such as AIDS Dementia Complex, Brain Abscess, subdural
empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine
Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and
cerebral
malaria.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include meningitis such as arachnoiditis, aseptic meningtitis such as viral
meningtitis
which includes lymphocytic choriomeningitis, Bacterial meningtitis which
includes
Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such
as
Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal
tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural
effusion,
meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as
transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which
includes
bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as
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Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-
Straussler Syndrome, I~uru, Scrapie), and cerebral toxoplasmosis.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include central nervous system neoplasms such as brain neoplasms that include
cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle
neoplasms
such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial
neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural
neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral
sceloris which includes adrenoleukodystrophy, encephalitis periaxialis,
globoid cell
leukodystrophy, diffuse cerebral sclerosis such as metachromatic
leukodystrophy,
allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis,
progressive
multifocal leukoencephalopathy, multiple sclerosis, central pontine
myelinolysis,
transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue
Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord
diseases
such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular
atrophy
such as Werdnig-Hoffinann Disease, spinal cord compression, spinal cord
neoplasms
such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff Man Syndrome,
mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's
Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(Ml), Sandhoff
Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-
Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis
such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal
syndrome,
phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett
Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome,
nervous system abnormalities such as holoprosencephaly, neural tube defects
such as
anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,
encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spine
bifida cystica and spine bifida occulta.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include hereditary motor and sensory neuropathies which include Charcot-Marie
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Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic
paraplegia,
Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such
as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations
(such
as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde
amnesia,
apraxia, neurogenic bladder, cataplexy, communicative disorders such as
hearing
disorders that includes deafness, partial hearing loss, loudness recruitment
and
tinnitus, language disorders such as aphasia which include agraphia, anomie,
broca
aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language
development disorders, speech disorders such as aphasia which includes anomie,
broca aphasia and Wernicke Aphasia, articulation disorders, communicative
disorders
such as speech disorders which include dysarthria, echolalia, mutism and
stuttering,
voice disorders such as aphonia and hoarseness, decerebrate state, deliriwn,
fasciculation, hallucinations, meningism, movement disorders such as angelman
syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia,
myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle
rigidity such
as stiff man syndrome, muscle spasticity, paralysis such as facial paralysis
which
includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such
as
diplopia, Duane's Syndrome, Homer's Syndrome, Chronic progressive external
ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic
Paxaparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia,
respiratory
paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders
such as
ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color
vision
defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders
such as
hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism,
spasm such as trismus, unconsciousness such as coma, persistent vegetative
state and
syncope and vertigo, neuromuscular diseases such as amyotonia congenita,
amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron
disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie
Disease
and Werdnig-Hoffinann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy,
Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy,
Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic
Paraparesis
and Stiff Man Syndrome, peripheral nervous system diseases such as acrodynia,
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amyloid neuropathies, autonomic nervous system diseases such as Adie's
Syndrome,
Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex
Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as
Acoustic Nerve Diseases such as Acoustic Neuroma wluch includes
Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-
Rosenthal Syndrome, ocular motility disorders which includes amblyopia,
nystagmus,
oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Homer's
Syndrome, Chronic Progressive External Ophthalmoplegia which includes Learns
Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve
Paralysis,
Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic
Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica,
Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating
Diseases
such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as
diabetic foot.
Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention
include nerve compression syndromes such as carpal tunnel syndrome, tarsal
tunnel
syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve
compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia,
facial
neuralgia and trigeminal neuralgia, neuritis such as experimental allergic
neuritis,
optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as
polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-
Marie
Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic
Paraplegia
and Werdnig-Hoffinann Disease, Hereditary Sensory and Autonomic Neuropathies
which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome,
Sciatica, Gustatory Sweating and Tetany).
Endocrine Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present
invention, may be used to treat, prevent, diagnose, and/or prognose disorders
andlor
diseases related to hormone imbalance, and/or disorders or diseases of the
endocrine
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system.
Hormones secreted by the glands of the endocrine system control physical
growth, sexual function, metabolism, and other functions. Disorders may be
classified
in two ways: disturbances in the production of hormones, and the inability of
tissues
to respond to hormones. The etiology of these hormone imbalance or endocrine
system diseases, disorders or conditions may be genetic, somatic, such as
cancer and
some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins),
or
infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention can be used as a marker or detector of a
particular
disease or disorder related to the endocrine system and/or hormone imbalance.
Endocrine system and/or hormone imbalance and/or diseases encompass
disorders of uterine motility including, but not limited to: complications
with
pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous
abortion, and slow or stopped labor); and disorders and/or diseases of the
menstrual
cycle.(e.g., dysmenorrhea and endometriosis).
Endocrine system andlor hormone imbalance disorders and/or diseases include
disorders and/or diseases of the pancreas, such as, for example, diabetes
mellitus,
diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma--islet
cell
tumor syndrome; disorders and/or diseases of the adrenal glands such as, for
example,
Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism,
Cushing's
Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of
the
pituitary gland, such as, for example, hyperpituitarism, hypopituitarism,
pituitary
dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism;
disorders
and/or diseases of the thyroid, including but not limited to, hyperthyroidism,
hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter),
toxic
nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous
thyroiditis, and silent Iyrnphocytic thyroiditis), Pendred's syndrome,
myxedema,
cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia,
Hurthle
cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary
thyroid
carcinoma; disorders and/or diseases of the parathyroid, such as, for example,
hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the
hypothalamus.
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In addition, endocrine system andlor hormone imbalance disorders and/or
diseases may also include disorders and/or diseases of the testes or ovaries,
including
cancer. Other disorders and/or diseases of the testes or ovaries further
include, for
example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome,
vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's
cells,
cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma
of
the testis (benign), neoplasias of the testis and neo-testis.
Moreover, endocrine system and/or hormone imbalance disorders and/or
diseases may also include disorders and/or diseases such as, for example,
polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple
Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.
In another embodiment, a polypeptide of the invention, or polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders
associated with the tissues) in which the polypeptide of the invention is
expressed,
including one, two, three, four, five, or more tissues disclosed in Table 1,
column 8
(Tissue Distribution Libraxy Code).
Reproductive System Disorders
The polynucleotides or polypeptides, or agonists or antagonists of the
invention may be used for the diagnosis, treatment, or prevention of diseases
and/or
disorders of the reproductive system. Reproductive system disorders that can
be
treated by the compositions of the invention, include, but are not limited to,
reproductive system injuries, infections, neoplastic disorders, congenital
defects, and
diseases or disorders which result in infertility, complications with
pregnancy, labor,
or parturition, and postpartum difficulties.
Reproductive system disorders and/or diseases include diseases and/or
disorders of the testes, including testicular atrophy, testicular
feminization,
cryptorchism (unilateral and bilateral), anorchia, ectopic testis,
epididymitis and
orchitis (typically resulting from infections such as, for example, gonorrhea,
mumps,
tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell
tumors (e.g.,
seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk
sac
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tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele,
hematocele, varicocele, spennatocele, inguinal hernia, and disorders of sperm
production (e.g., immotile cilia syndrome, aspennia, asthenozoospennia,
azoospennia, oligospennia, and teratozoospennia).
Reproductive system disorders also include disorders of the prostate gland,
such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis,
acute bacterial
prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis,
granulomatous
prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and
prostate
neoplastic disorders, including adenocarcinomas, transitional cell carcinomas,
ductal
carcinomas, and squamous cell carcinomas.
Additionally, the compositions of the invention may be useful in the
diagnosis,
treatment, and/or prevention of disorders or diseases of the penis and
urethra,
including inflammatory disorders, such as balanoposthitis, balanitis xerotica
obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea,
non-
gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's
syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules;
urethral abnormalities, such as hypospadias, epispadias, and phimosis;
premalignant
lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid
paplosis,
giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers,
including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma,
and
disseminated penile carcinoma; urethral neoplastic disorders, including penile
urethral
carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral
carcinoma;
and erectile disorders, such as priapism, Peyronie's disease, erectile
dysfunction, and
impotence.
Moreover, diseases and/or disorders of the vas deferens include vasculititis
and CBAVD (congenital bilateral absence of the vas deferens); additionally,
the
polynucleotides, polypeptides, and agonists or antagonists of the present
invention
may be used in the diagnosis, treatment, and/or prevention of diseases and/or
disorders of the seminal vesicles, including hydatid disease, congenital
chloride
diarrhea, and polycystic kidney disease.
Other disorders and/or diseases of the male reproductive system include, for
example, Klinefelter's syndrome, Young's syndrome, premature ejaculation,
diabetes
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mellitus, cystic fibrosis, I~artagener's syndrome, high fever, multiple
sclerosis, and
gynecomastia.
Further, the polynucleotides, polypeptides, and agonists or antagonists of the
present invention may be used in the diagnosis, treatment, and/or prevention
of
diseases and/or disorders of the vagina and vulva, including bacterial
vaginosis,
candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale,
lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma,
vulvar
trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis,
condyloma
acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's
disease,
lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus,
vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as
squamous cell
hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of
Bartholin's gland, and vulvar intraepithelial neoplasia.
Disorders and/or diseases of the uterus include dysmenorrhea, retroverted
uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding,
amenorrhea,
Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature
menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding
(e.g.,
due to aberrant hormonal signals), and neoplastic disorders, such as
adenocarcinomas,
keiomyosarcomas, and sarcomas. Additionally, the polypeptides,
polynucleotides, or
agonists or antagonists of the invention may be useful as a marker or detector
of, as
well as in the diagnosis, treatment, and/or prevention of congenital uterine
abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate
uterus,
unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus
with a
non-communicating cavitary rudimentary horn, unicornuate uterus with a
communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped
uterus.
Ovarian diseases and/or disorders include anovulation, polycystic ovary
syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction,
ovarian
insensitivity to gonadotropins, ovarian overproduction of androgens, right
ovarian
vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not
limited
to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod
carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian
mucinous
.adenocarcinoma, and Ovarian Krukenberg tumors).
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Cervical diseases and/or disorders include cervicitis, chronic cervicitis,
mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts,
cervical
erosion, cervical incompetence, and cervical neoplasms (including, for
example,
cervical carcinoma, squamous metaplasia, squamous cell carcinoma,
adenosquamous
cell neoplasia, and columnar cell neoplasia).
Additionally, diseases and/or disorders of the reproductive system include
disorders and/or diseases of pregnancy, including miscarnage and stillbirth,
such as
early abortion, late abortion, spontaneous abortion, induced abortion,
therapeutic
abortion, threatened abortion, missed abortion, incomplete abortion, complete
abortion, habitual abortion, missed abortion, and septic abortion; ectopic
pregnancy,
anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational
diabetes,
intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio
placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes
gestationis,
and urticaria of pregnancy. Additionally, the polynucleotides, polypeptides,
and
agonists or antagonists of the present invention may be used in the diagnosis,
treatment, and/or prevention of diseases that can complicate pregnancy,
including
heart disease, heart failure, rheumatic heart disease, congenital heart
disease, mitral
valve prolapse, high blood pressure, anemia, kidney disease, infectious
disease (e.g.,
rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV,
AIDS,
and genital herpes), diabetes mellitus, Graves' disease, thyroiditis,
hypothyroidism,
Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver,
primary biliary
cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis,
myasthenia
gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts,
gallbladder
disorders,and obstruction of the intestine.
Complications associated with labor and parturition include premature rupture
of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor
that
progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or
maternal),
breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed
umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.
Further, diseases and/or disorders of the postdelivery period, including
endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis,
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pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis,
mastitis, cystitis, postpartum hemorrhage, and inverted uterus.
Other disorders and/or diseases of the female reproductive system that may be
diagnosed, treated, and/or prevented by the polynucleotides, polypeptides, and
agonists or antagonists of the present invention include, for example,
Turner's
syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory
disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia,
dyspareunia, ruptured fallopian tube, and Mittelschmerz.
Infectious Disease
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention can be used to treat or detect infectious agents. For
example, by
increasing the immune response, particularly increasing the proliferation and
differentiation of B and/or T cells, infectious diseases may be treated. The
immune
response may be increased by either enhancing an existing immune response, or
by
initiating a new immune response. Alternatively, polynucleotides or
polypeptides, as
well as agonists or antagonists of the present invention may also directly
inlubit the
infectious agent, without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated ox detected by a polynucleotide or polypeptide
and/or
agonist or antagonist of the present invention. Examples of viruses, include,
but are
not limited to Examples of viruses, include, but are not limited to the
following DNA
and RNA viruses and viral families: Arbovirus, Adenovinidae, Arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,
Coronaviridae,
Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as,
Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,
Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,
Influenza
A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,
Parvoviridae,
Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus). Viruses falling within these families can cause a variety of
diseases or
symptoms, including, but not limited to: arthritis, bronchiollitis,
respiratory syncytial
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virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic
fatigue
syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B
encephalitis,
Jmiin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic
infections (e.g., A)DS), pneumonia, Burkitt's Lymphoma, chickenpox,
hemorrhagic
fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio,
leukemia,
Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),
and
viremia. polynucleotides or polypeptides, or agonists or antagonists of the
invention,
can be used to treat or detect any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g.,
hepatitis
B). In an additional specific embodiment polynucleotides, polypeptides, or
agonists
or antagonists of the invention are used to treat patients nonresponsive to
one or more
other commercially available hepatitis vaccines. In a further specific
embodiment
polynucleotides, polypeptides, or agonists or antagonists of the invention are
used to
treat AmS.
Similarly, bacterial and fungal agents that can cause disease or symptoms and
that can be treated or detected by a polynucleotide or polypeptide and/or
agonist or
antagonist of the present invention include, but not limited to, the following
Gram-
Negative and Gram-positive bacteria, bacterial families, and fungi:
Actinomyces
(e.g., Norcardia), Acinetobacter, Cryptococcus zzeoformans, Aspergillus,
Bacillaceae
(e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis),
Blastomycosis,
Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candidia,
Campylobacter,
Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium difzcile,
Clostridium peyf°ingens, Clostridiuyzz tetazzi), Coccidioides,
Corynebacterium (e.g.,
Cofynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g.,
Enterotoxigenic E. coli and Enterohemorrhagic E, coli), Enterobacter (e.g.
Enterobacter aerogehes), Enterobacteriaceae (I~lebsiella, Salmonella (e.g.,
Salmonella typhi, Salmonella enteritidis, .Salmonella typhi), Serratia,
Yersinia,
Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B),
Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria
(e.g.,
Listeria monocytogefzes), Mycoplasma, Mycobacterium (e.g., Mycobacterium
leprae
and Mycobacterium tuberculosis), Vibrio (e.g., Yibrio cholerae), Neisseriaceae
(e.g.,
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Neisser~ia gonorr°laea, Neisseria rneniragitidis), Pasteurellacea,
Proteus, Pseudomonas
(e.g., Pseudornoraas aef°ugiraosa), Rickettsiaceae, Spirochetes (e.g.,
Treponema spp.,
Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g.,
Staphylococcus
au~eus), Meningiococcus, Pneumococcus and Streptococcus (e.g., StYeptococcus
pn.eumoraiae and Groups A, B, and C Streptococci), and Ureaplasmas. These
bacterial, parasitic, and fungal families can cause diseases or symptoms,
including,
but not limited to: antibiotic-resistant infections, bacteremia, endocarditis,
septicemia,
eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis,
bacterial diarrhea,
opportunistic infections (e.g., AmS related infections), paronychia,
prosthesis-related
infections, dental caries, Reiter's Disease, respiratory tract infections,
such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease,
dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and
acute
inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea,
meningitis
(e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy,
brucellosis,
peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung
infections, ear infections, deafness, blindness, lethargy, malaise, vomiting,
chronic
diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory
diseases,
candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene,
tetanus,
impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin
diseases
(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound
infections,
noscomial infections. Polynucleotides or polypeptides, agonists or antagonists
of the
invention, can be used to treat or detect any of these symptoms or diseases.
In
specific embodiments, polynucleotides, polypeptides, agonists or antagonists
of the
invention are used to treat: tetanus, diptheria, botulism, and/or meningitis
type B.
Moreover, parasitic agents causing disease or symptoms that can be treated,
prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist
or
antagonist of the present invention include, but not limited to, the following
families
or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis,
Dientamoebiasis,
Dourine, Ectoparasitic, Giardias, Helininthiasis, Leishmaniasis, Schistisoma,
Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans
(e.g., Plasmodium vir~ax, Plasmodium falciparium, Plasmodiuyra rrralat-iae and
Plasmodium ovale). These parasites can cause a variety of diseases
or.symptoms,
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including, but not limited to: Scabies, Trombiculiasis, eye infections,
intestinal
disease (e.g., dysentery, giardiasis), liver disease, lung disease,
opportunistic
infections (e.g., AmS related), malaria, pregnancy complications, and
toxoplasmosis.
polynucleotides or polypeptides, or agonists or antagonists of the invention,
can be
used to treat, prevent, and/or diagnose any of these symptoms or diseases. In
specific
embodiments, polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat, prevent, and/or diagnose malaria.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention of the present invention could either be by administering an
effective amount of a polypeptide to the patient, or by removing cells from
the
patient, supplying the cells with a polynucleotide of the present invention,
and
returning the engineered cells to the patient (ex vivo therapy). Moreover, the
polypeptide or polynucleotide of the present invention can be used as an
antigen in a
vaccine to raise an immune response against infectious disease.
Regeneration
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention can be used to differentiate, proliferate, and attract
cells, leading to
the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration
of
tissues could be used to repair, replace, or protect tissue damaged by
congenital
defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g.
osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery, including
cosmetic plastic
surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal
or cardiac), vasculature (including vascular and lymphatics), nervous,
hematopoietic,
and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,
regeneration
occurs without or decreased scarnng. Regeneration also may include
angiogenesis.
Moreover, polynucleotides or polypeptides, as well as agonists or antagonists
of the present invention, may increase regeneration of tissues difficult to
heal. For
example, increased tendon/ligament regeneration would quicken recovery time
after
damage. Polynucleotides or polypeptides, as well as agonists or antagonists of
the
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present invention could also be used prophylactically in an effort to avoid
damage.
Specific diseases that could be treated include of tendinitis, carpal tunnel
syndrome,
and other tendon or ligament defects. A further example of tissue regeneration
of
non-healing wounds includes pressure ulcers, ulcers associated with vascular
insufficiency, surgical, and traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using
polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention, to proliferate and differentiate nerve cells. Diseases that could
be treated
using this method include central and peripheral nervous system diseases,
neuropathies, or mechanical and traumatic disorders (e.g., spinal cord
disorders, head
trauma, cerebrovascular disease, and stoke). Specifically, diseases associated
with
peripheral nerve injuries, peripheral neuropathy (e.g., resulting from
chemotherapy or
other medical therapies), localized neuropathies, and central nervous system
diseases
(e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic
lateral sclerosis, and Shy-Drager syndrome), could all be treated using the
polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention.
Gastrointestinal Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the present
invention, may be used to treat, prevent, diagnose, and/or prognose
gastrointestinal
disorders, including inflammatory diseases and/or conditions, infections,
cancers
(e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-
Hodgkin's
lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as
peptic
ulcers.
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, gastriclstomach
cancers,
polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric
stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic
erosive,
atrophic, plasma cell, and Menetrier's), and peritoneal diseases (e.g.,
chyloperioneum,
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hemoperitoneum, mesenteric cyst, mesenteric Iymphadenitis, mesenteric vascular
occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic
abscess,).
Gastrointestinal disorders also include disorders associated with the small
intestine, such as malabsorption syndromes, distension, irntable bowel
syndrome,
sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical
sprue,
Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis,
obstructions of the ileum, Meckel's diverticulum, multiple diverticula,
failure of
complete rotation of the small and large intestine, lymphoma, and bacterial
and
parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid,
cholera,
infection by Roundworms (Asca~iasis lumb~icoides), Hookworms (Aracylostoma
duodenale), Threadworms (Enterobius veYmicularis), Tapeworms (Taenia saginata,
Echinococcus g~anulosus, Diphylloboth~iuna spp., and T. soliuna).
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 Iiver failure), and liver
neoplasms
(angiomyolipoma, calcified liver metastases, cystic liver metastases,
epithelial
tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic
adenoma,
hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma,
liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal
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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).
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)).
Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the
gallbladder,
acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.
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
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(ileal neoplasms, ileitis), immunoproliferative small intestinal disease,
inflammatory
bowel disease (ulcerative 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 neoplasms
(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.
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
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diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders
(e.g.,
CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal
reflux),
neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome),
stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia);
gastrointestinal
diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus
infection),
hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach
neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach
cancer)),
hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal
hernia,
obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases
(e.g., cecal
diseases (appendicitis, cecal neoplasms)).
Chemotaxis
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention may have chemotaxis activity. A chemotaxic molecule attracts
or
mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast
cells,
eosinophils, epithelial and/or endothelial cells) to a particular site in the
body, such as
inflammation, infection, or site of hyperproliferation. The mobilized cells
can then
fight off and/or heal the particular trauma or abnormality.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention may increase chemotaxic activity of particular cells. These
chemotactic molecules can then be used to treat inflammation, infection,
hyperproliferative disorders, or any immune system disorder by increasing the
number of cells targeted to a particular location in the body. For example,
chemotaxic molecules can be used to treat wounds and other trauma to tissues
by
attracting immune cells to the injured location. Chemotactic molecules of the
present
invention can also attract fibroblasts, which can be used to treat wounds.
It is also contemplated that polynucleotides or polypeptides, as well as
agonists or antagonists of the present invention may inhibit chemotactic
activity.
These molecules could also be used to treat disorders. Thus, polynucleotides
or
polypeptides, as well as agonists or antagonists of the present invention
could be used
as an inhibitor of chemotaxis.
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Binding Activity
A polypeptide of the present invention may be used to screen for molecules
that bind to the polypeptide or for molecules to which the polypeptide binds.
The
binding of the polypeptide and the molecule may activate (agonist), increase,
inhibit
(antagonist), or decrease activity of the polypeptide or the molecule bound.
Examples
of such molecules include antibodies, oligonucleotides, proteins (e.g.,
receptors),or
small molecules.
Preferably, the molecule is closely related to the natural ligand of the
polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand,
a structural
or functional mimetic. (See, Coligan et al., Current Protocols in hnxnunology
1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the
natural
receptor to which the polypeptide binds, or at least, a fragment of the
receptor capable
of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using l~nown techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the polypeptide. Preferred cells include cells from
mammals,
yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell
membrane
containing the expressed polypeptide) are then preferably contacted with a
test
compound potentially containing the molecule to observe binding, stimulation,
or
inhibition of activity of either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the
polypeptide, wherein binding is detected by a label, or in an assay involving
competition with a labeled competitor. Further, the assay may test whether the
candidate compound results in a signal generated by binding to the
polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide, measuring
polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule activity or
binding to a
standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
The
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antibody can measure polypeptide level or activity by either binding, directly
or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
Additionally, the receptor to which the polypeptide of the present invention
binds can be identified by numerous methods known to those of skill in the
art, for
example, ligand panning and FACS sorting (Coligan, et al., Current Protocols
in
Immun., 1(2), Chapter 5, (I991)). For example, expression cloning is employed
wherein polyadenylated RNA is prepared from a cell responsive to the
polypeptides,
for example, NIH3T3 cells which are known to contain multiple receptors fox
the
FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA
is
divided into pools and used to transfect COS cells or other cells that are not
responsive to the polypeptides. Transfected cells which are grown on glass
slides are
exposed to the polypeptide of the present invention, after they have been
labeled. The
polypeptides can be labeled by a variety of means including iodination or
inclusion of
a recognition site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-
radiograpluc analysis. Positive pools are identified and sub-pools are
prepared and re-
transfected using an iterative sub-pooling and re-screening process,
eventually
yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides
can be photoaffinity linked with cell membrane or extract preparations that
express
the receptor molecule. Cross-linked material is resolved by PAGE analysis and
exposed to X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and subjected to
protein
microsequencing. The amino acid sequence obtained from microsequencing would
be used to design a set of degenerate oligonucleotide probes to screen a cDNA
library
to identify the genes encoding the putative receptors.
Moreover, the techniques of gene-shuffling, motif shuffling, exon-shuffling,
and/or codon-shuffling (collectively referred to as "DNA shuffling") may be
employed to modulate the activities of the polypeptide of the present
invention
thereby effectively generating agonists and antagonists of the polypeptide of
the
present invention. See generally, U.S. Patent Nos. 5,605,793, 5,811,238,
5,830,721,
5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opiniofa
Bioteclanol. 8:724-
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33 (1997); Harayama, S. TrefZCls Biotechf~.ol. 16(2):76-82 (1998); Hansson, L.
O., et
al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R.
Biotechfziques 24(2):308-13 (1998); each of these patents and publications are
hereby
incorporated by reference). In one embodiment, alteration of polynucleotides
and
corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling
involves the assembly of two or more DNA segments into a desired molecule by
homologous, or site-specific, recombination. In another embodiment,
polynucleotides
and corresponding polypeptides may be altered by being subj ected 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 the polypeptide of the present invention
may be
recombined with one or more components, motifs, sections, parts, domains,
fragments, etc. of one or more heterologous molecules. In preferred
embodiments,
the heterologous molecules are family members. In further preferred
embodiments,
the heterologous molecule is a growth factor such as, for example, platelet-
derived
growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth
factor
(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF),
TGF-
beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7,
activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth
differentiation
factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3,
TGF-
beta5, and filial-derived neurotrophic factor (GDNF).
Other preferred fragments are biologically active fragments of the polypeptide
of the present invention. 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.
Additionally, this invention provides a method of screening compounds to
identify those which modulate the action of the polypeptide of the present
invention.
An example of such an assay comprises combining a mammalian fibroblast cell, a
the
polypeptide of the present invention, the compound to be screened and 3[H]
thymidine under cell culture conditions where the fibroblast cell would
normally
proliferate. A control assay may be performed in the absence of the compound
to be
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screened and compared to the amount of fibroblast proliferation in the
presence of the
compound to determine if the compound stimulates proliferation by determining
the
uptake of 3[H] thymidine in each case. The amount of fibroblast cell
proliferation is
measured by liquid scintillation chromatography which measures the
incorporation of
3 [H] thymidine. Both agonist and antagonist compounds may be identified by
this
procedure.
In another method, a mammalian cell or membrane preparation expressing a
receptor for a polypeptide of the present invention is incubated with a
labeled
polypeptide of the present invention in the presence of the compound. The
ability of
the compound to enhance or block this interaction could then be measured.
Alternatively, the response of a known second messenger system following
interaction of a compound to be screened and the receptor is measured and the
ability
of the compound to bind to the receptor and elicit a second messenger response
is
measured to determine if the compound is a potential agonist or antagonist.
Such
second messenger systems include but are not limited to, CAMP guanylate
cyclase,
ion channels or phosphoinositide hydrolysis.
All of these above assays can be used as diagnostic or prognostic markers.
The molecules discovered using these assays can be used to treat disease or to
bring
about a particular result in a patient (e.g., blood vessel growth) by
activating or
inhibiting the polypeptide/molecule. Moreover, the assays can discover agents
which
may inhibit or enhance the production of the polypeptides of the invention
from
suitably manipulated cells or tissues.
Therefore, the invention includes a method of identifying compounds which
bind to a polypeptide of the invention comprising the steps of (a) incubating
a
candidate binding compound with a polypeptide of the present invention, and
(b)
determining if binding has occurred. Moreover, the invention includes a method
of
identifying agonists/antagonists comprising the steps of: (a) incubating a
candidate
compound with a polypeptide of the present invention, (b) assaying a
biological
activity, and (b) determining if a biological activity of the polypeptide has
been
altered.
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Targeted Delivery
In another embodiment, the invention provides a method of delivering
compositions to targeted cells expressing a receptor for a polypeptide of the
invention,
or cells expressing a cell bound form of a polypeptide of the invention.
As discussed herein, polypeptides or antibodies of the invention may be
associated with heterologous polypeptides, heterologous nucleic acids, toxins,
or
prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. 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
(including 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.
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 (e.g., polypeptides of the invention or antibodies of the
invention) in
association with toxins or cytotoxic prodrugs.
By "toxin" is meant compounds that bind and activate endogenous cytotoxic
effector systems, radioisotopes, holotoxins, modified toxins, catalytic
subunits of
toxins, or any molecules or enzymes not normally present in or on the surface
of a cell
that under defined conditions cause the cell's death. Toxins that may be used
according to the methods of the invention include, but are not limited to,
radioisotopes
known in the art, compounds such as, for example, antibodies (or complement
fixing
containing portions thereof) that bind an inherent or induced endogenous
cytotoxic
effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin,
abrin,
Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed
antiviral protein, alpha-sarcin and cholera toxin. By "cytotoxic prodrug" is
meant a
non-toxic compound that is converted by an enzyme, normally present in the
cell, into
a cytotoxic compound. Cytotoxic prodrugs that may be used according to the
methods of the invention include, but are not limited to, glutamyl derivatives
of
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benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or
mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives
of doxorubicin.
Drug Screening
Further contemplated is the use of the polypeptides of the present invention,
or
the polynucleotides encoding these polypeptides, to screen for molecules which
modify the activities of the polypeptides of the present invention. Such a
method
would include contacting the polypeptide of the present invention with a
selected
compounds) suspected of having antagonist or agonist activity, and assaying
the
activity of these polypeptides following binding.
This invention is particularly useful for screening therapeutic compounds by
using the polypeptides of the present invention, or binding fragments thereof,
in any
of a variety of drug screening techniques. The polypeptide or fragment
employed in
such a test may be affixed to a solid support, expressed on a cell surface,
free in
solution, or located inixacellularly. One method of drug screening utilizes
eukaryotic
or prokaryotic host cells which are stably transformed with recombinant
nucleic acids
expressing the polypeptide or fragment. Drugs are screened against such
transformed
cells in competitive binding assays. One may measure, for example, the
formulation
of complexes between the agent being tested and a polypeptide of the present
invention.
Thus, the present invention provides methods of screening for drugs or any
other agents which affect activities mediated by the polypeptides of the
present
invention. These methods comprise contacting such an agent with a polypeptide
of the
present invention or a fragment thereof and assaying for the presence of a
complex
between the agent and the polypeptide or a fragment thereof, by methods well
known
in the art. In such a competitive binding assay, the agents to screen are
typically
labeled. Following incubation, free agent is separated from that present in
bound
form, and the amount of free or uncomplexed label is a measure of the ability
of a
particular agent to bind to the polypeptides of the present invention.
Another technique for drug screening provides high throughput screening for
compounds having suitable binding affinity to the polypeptides of the present
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invention, and is described in great detail in European Patent Application
84/03564,
published on September 13, 1984, which is incorporated herein by reference
herein.
Briefly stated, large numbers of different small peptide test compounds are
synthesized on a solid substrate, such as plastic pins or some other surface.
The
S peptide test compounds are reacted with polypeptides of the present
invention and
washed. Bound polypeptides are then detected by methods well known in the art.
Purified polypeptides are coated directly onto plates for use in the
aforementioned
drug screening techniques. In addition, non-neutralizing antibodies may be
used to
capture the peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing antibodies capable of binding polypeptides of the
present
invention specifically compete with a test compound for binding to the
polypeptides
or fragments thereof. Tn this manner, the antibodies are used to detect the
presence of
any peptide which shares one or more antigenic epitopes with a polypeptide of
the
invention.
Polypeptides of the Invention Binding Peptides and Other Molecules
The invention also encompasses screening methods for identifying
polypeptides and nonpolypeptides that bind polypeptides of the invention, and
the
polypeptide of the invention binding molecules identified thereby. These
binding
molecules are useful, for example, as agonists and antagonists of the
polypeptides of
the invention. Such agonists and antagonists can be used, in accordance with
the
invention, in the therapeutic embodiments described in detail, below.
This method comprises the steps of:contacting a polypeptide of the invention
with a plurality of molecules; and identifying a molecule that binds the
polypeptide of
the invention.
The step of contacting the polypeptide of the invention with the plurality of
molecules may be effected in a number of ways. For example, one may
contemplate
immobilizing the polypeptide of the invention on a solid support and bringing
a
solution of the plurality of molecules in contact with the immobilized
polypeptide of
the invention. Such a procedure would be akin to an affinity chromatographic
process,
with the affinity matrix being comprised of the immobilized polypeptide of the
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invention. The molecules having a selective affinity for the polypeptide of
the
invention can then be purified by affinity selection. The nature of the solid
support,
process for attachment of the polypeptide of the invention to the solid
support,
solvent, and conditions of the affinity isolation or selection are largely
conventional
and well known to those of ordinary skill in the art.
Alternatively, one may also separate a plurality of polypeptides into
substantially separate fractions comprising a subset of or individual
polypeptides. For
instance, one can separate the plurality of polypeptides by gel
electrophoresis, column
chromatography, or like method known to those of ordinary skill for the
separation of
polypeptides. The individual polypeptides can also be produced by a
transformed host
cell in such a way as to be expressed on or about its outer surface (e.g., a
recombinant
phage). Individual isolates can then be "probed" by the polypeptide of the
invention,
optionally in the presence of an inducer should one be required for
expression, to
determine if any selective affinity interaction takes place between the
polypeptide of
the invention and the individual clone. Prior to contacting the polypeptide of
the
invention with each fraction comprising individual polypeptides, the
polypeptides
could first be transferred to a solid support for additional convenience. Such
a solid
support may simply be a piece of filter membxane, such as one made of
nitrocellulose
or nylon. In this manner, positive clones could be identified from a
collection of
transformed host cells of an expression library, which harbor a DNA construct
encoding a polypeptide having a selective affinity for a polypeptide of the
invention.
Furthermore, the amino acid sequence of the polypeptide having a selective
affinity
for the polypeptide of the invention can be determined directly by
conventional means
or the coding sequence of the DNA encoding the polypeptide can frequently be
determined more conveniently. The primary sequence can then be deduced from
the
corresponding DNA sequence. If the amino acid sequence is to be determined
from
the polypeptide itself, one may use microsequencing techniques. The sequencing
technique may include mass spectroscopy.
In certain situations, it may be desirable to wash away any unbound
polypeptide of the invention, or alterntatively, unbound polypeptides, from a
mixture
of the polypeptide of the invention and the plurality of polypeptides prior to
attempting to determine or to detect the presence of a selective affiiuty
interaction.
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Such a wash step may be particularly desirable when the polypeptide of the
invention
or the plurality of polypeptides is bound to a solid support.
The plurality of molecules provided according to this method may be provided
by way of diversity libraries, such as random or combinatorial peptide or
nonpeptide
S libraries which can be screened for molecules that specifically bind to a
polypeptide
of the invention. Many libraries are known in the art that can be used, e.g.,
chemically
synthesized libraries, recombinant (e.g., phage display libraries), and in
vitro
translation-based libraries. Examples of chemically synthesized libraries are
described in Fodor et al., 1991, Science 2S 1:767-773; Houghten et al., 1991,
Nature
354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology
12:709-710;Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251;
Ohlmeyer
et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994,
Proc. Natl.
Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et
al.,
1S 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO
93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-
5383.
Examples of phage display libraries are described in Scott and Smith, 1990,
Science 249:386-390; I~evlin et al., 1990, Science, 249:404-406; Christian, R.
B., et
al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth.
152:149-157;
I~ay et al., 1993, Gene 128:59-6S; and PCT Publication No. WO 94118318 dated
Aug.
18, 1994.
In vitro translation-based libraries include but are not limited to those
described in PCT Publication No. WO 91/OSOS8 dated Apr. 18, 1991; and
Mattheakis
et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.
2S By way of examples of nonpeptide libraries, a benzodiazepine library (see
e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be
adapted for
use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-
9371)
can also be used. Another example of a library that can be used, in which the
amide
functionalities in peptides have been permethylated to generate a chemically
transformed combinatorial library, is described by Ostresh et al. (1994, Proc.
Natl.
Acad. Sci. USA 9I:1 I 138-I 1142).
The variety of non-peptide libraries that are useful in the present invention
is
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great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list
benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-
mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes,
xanthines,
aminimides, and oxazolones as among the chemical species that form the basis
of
various libraries.
a Non-peptide libraries can be classified broadly into two types: decorated
monomers and oligomers. Decorated monomer libraries employ a relatively simple
scaffold structure upon which a variety functional groups is added. Often the
scaffold
will be a molecule with a known useful pharmacological activity. For example,
the
' scaffold might be the benzodiazepine structure.
Non-peptide oligomer libraries utilize a large number of monomers that are
assembled together in ways that create new shapes that depend on the order of
the
monomers. Among the monomer units that have been used are carbamates,
pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the
side
chain is attached to the alpha amino group rather than the alpha carbon, form
the basis
of another version of non-peptide oligomer libraries. The first non-peptide
oligomer
libraries utilized a single type of monomer and thus contained a repeating
backbone.
Recent libraries have utilized more than one monomer, giving the libraries
added
flexibility.
Screening the libraries can be accomplished by any of a variety of commonly
known methods. See, e.g., the following references, which disclose screening
of
peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218;
Scott
and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques
I3:422-
427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et
al.,
1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al.,
I992,
Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-
6992;
Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat.
No.
5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo,
1993,
Science 263:671-673; and CT Publication No. WO 94/18318.
In a specific embodiment, screening to identify a molecule that binds a
polypeptide of the invention can be carried out by contacting the library
members
with a polypeptide of the invention immobilized on a solid phase and
harvesting those
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library members that bind to the polypeptide of the invention. Examples of
such
screening methods, termed "panning" techniques are described by way of example
in
Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques
13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.
S In another embodiment, the two-hybrid system for selecting interacting
proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al.,
1991,
Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules
that
specifically bind to a polypeptide of the invention.
Where the polypeptide of the invention binding molecule is a polypeptide, the
polypeptide can be conveniently selected from any peptide library, including
random
peptide libraries, combinatorial peptide libraries, or biased peptide
libraries. The term
"biased" is used herein to mean that the method of generating the library is
manipulated so as to restrict one or more parameters that govern the diversity
of the
resulting collection of molecules, in this case peptides.
Thus, a truly random peptide library would generate a collection of peptides
in
which the probability of finding a particular amino acid at a given position
of the
peptide is the same for all 20 amino acids. A bias can be introduced into the
library,
however, by specifying, for example, that a lysine occur every fifth amino
acid or that
positions 4, 8, and 9 of a decapeptide library be fixed to include only
arginine.
Clearly, many types of biases can be contemplated, and the present invention
is not
restricted to any particular bias. Furthermore, the present invention
contemplates
specific types of peptide libraries, such as phage displayed peptide libraries
and those
that utilize a DNA construct comprising a lambda phage vector with a DNA
insert.
As mentioned above, in the case of a polypeptide of the invention binding
molecule that is a polypeptide, the polypeptide may have about 6 to less than
about 60
amino acid residues, preferably about 6 to about 10 amino acid residues, and
most
preferably, about 6 to about 22 amino acids. In another embodiment, a
polypeptide of
the invention binding polypeptide has in the range of 15-100 amino acids, or
20-50
amino acids.
The selected polypeptide of the invention binding polypeptide can be obtained
by chemical synthesis or recombinant expression.
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Antisense And Ribozyme (Antagonists)
In specific embodiments, antagonists according to the present invention are
nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the
complementary strand thereof, and/or to nucleotide sequences contained a
deposited
clone. In one embodiment, antisense sequence is generated internally by the
organism, in another embodiment, the antisense sequence is separately
administered
(see, for example, O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides
as
Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
Antisense technology can be used to control gene expression through antisense
DNA
or RNA, or through triple-helix formation. Antisense techniques are discussed
for
example, in Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix
formation is discussed in, for instance, Lee et al., Nucleic Acids Research,
6:3073
(1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science,
251:1300
(1991). The methods are based on binding of a polynucleotide to a
complementary
DNA or RNA.
For example, the use of c-myc and c-myb antisense RNA constructs to inhibit
the growth of the non-lymphocytic leukemia cell line HL-60 and other cell
lines was
previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These
experiments were performed in vitro by incubating cells with the
oligoribonucleotide.
A similar procedure for in vivo use is described in WO 91/15580. Briefly, a
pair of
oligonucleotides for a given antisense RNA is produced as follows: A sequence
complimentary to the frst 15 bases of the open reading frame is flanked by an
EcoRl
site on the 5 end and a HindIII site on the 3 end. Next, the pair of
oligonucleotides is
heated at 90°C for one minute and then annealed in 2X ligation buffer
(20mM TRIS
HCl pH 7.5, l OmM MgCl2, l OMM dithiothreitol (DTT) and 0.2 mM ATP) and then
ligated to the EcoRl/Hind III site of the retroviral vector PMV7 (WO
91/15580).
For example, the 5' coding portion of a polynucleotide that encodes the mature
polypeptide of the present invention may be used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide
is designed to be complementary to a region of the gene involved in
transcription
thereby preventing transcription and the production of the receptor. The
antisense
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RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of
the
mRNA molecule into receptor polypeptide.
W one embodiment, the antisense nucleic acid of the invention is produced
intracellularly by transcription from an exogenous sequence. For example, a
vector or
a portion thereof, is transcribed, producing an antisense nucleic acid (RNA)
of the
invention. Such a vector would contain a sequence encoding the antisense
nucleic
acid of the invention. Such a vector can remain episomal or become
chromosomally
integrated, as long as it can be transcribed to produce the desired antisense
RNA.
Such vectors can be constructed by recombinant DNA technology methods standard
in the art. Vectors can be plasrnid, viral, or others known in the art, used
for
replication and expression in vertebrate cells. Expression of the sequence
encoding a
polypeptide of the invention, or fragments thereof, can be by any promoter
known in
the art to act in vertebrate, preferably human cells. Such promoters can be
inducible
or constitutive. Such promoters include, but are not limited to, the SV40
early
promoter region (Bernoist and Chambon, Nature, 29:304-310 (1981), the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et
al.,
Cell, 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc.
Natl.
Acad. Sci. U.S.A., 78:1441-1445 (1981), the regulatory sequences of the
metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)), etc.
The antisense nucleic acids of the invention comprise a sequence
complementary to at least a portion of an RNA transcript of a gene of
interest.
However, absolute complementarity, although preferred, is not required. A
sequence
"complementary to at least a portion of an RNA," referred to herein, means a
sequence having sufficient complementarity to be able to hybridize with the
RNA,
forming a stable duplex; in the case of double stranded antisense nucleic
acids of the
invention, a single strand of the duplex DNA may thus be tested, or triplex
formation
may be assayed. The ability to hybridize will depend on both the degree of
complementarity and the length of the antisense nucleic acid Generally, the
larger the
hybridizing nucleic acid, the more base mismatches with a RNA sequence of the
invention it may contain and still form a stable duplex (or triplex as the
case may be).
One skilled in the art can ascertain a tolerable degree of mismatch by use of
standard
procedures to determine the melting point of the hybridized complex.
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Oligonucleotides that are complementary to the S' end of the message, e.g.,
the S' untranslated sequence up to and including the AUG initiation codon,
should
work most efficiently at inhibiting translation. However, sequences
complementary
to the 3' untranslated sequences of mRNAs have been shown to be effective at
S inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature,
372:333-335 (1994). Thus, oligonucleotides complementary to either the S' - or
3' -
non- translated, non-coding regions of a polynucleotide sequence of the
invention
could be used in an antisense approach to inhibit translation of endogenous
mRNA.
Oligonucleotides complementary to the S' untranslated region of the mRNA
should
include the complement of the AUG start codon. Antisense oligonucleotides
complementary to mRNA coding regions are less efficient inhibitors of
translation but
could be used in accordance with the invention. Whether designed to hybridize
to the
S' -, 3' - or coding region of mRNA, antisense nucleic acids should be at
least six
nucleotides in length, and are preferably oligonucleotides ranging from 6 to
about SO
1 S nucleotides in length. In specific aspects the oligonucleotide is at least
10
nucleotides, at least 17 nucleotides, at least 2S nucleotides or at least SO
nucleotides.
The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or derivatives or modified versions thereof, single-stranded or
double-
stranded. The oligonucleotide can be modified at the base moiety, sugar
moiety, or
phosphate backbone, for example, to improve stability of the molecule,
hybridization,
etc. The oligonucleotide may include other appended groups such as peptides
(e.g.,
for targeting host cell receptors in vivo), or agents facilitating transport
across the cell
membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-
6556
(1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-6S2 (1987); PCT
Publication
2S NO: W088/09810, published December 1S, 1988) or the blood-brain barrier
(see,
e.g., PCT Publication NO: W089/10134, published April 25, 1988), hybridization-
triggered cleavage agents. (See, e.g., Krol et al., BioTechniques, 6:958-976
(1988))
or intercalating agents. (See, e.g., Zon, Pharm. Res., S:S39-S49 (1988)). To
this end,
the oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization triggered cross-linking agent, transport agent, hybridization-
triggered
cleavage agent, etc.
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The antisense oligonucleotide may comprise at least one modified base moiety
which is selected from the group including, but not limited to, 5-
fluorouracil,
S-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-
acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, S-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethyla~ninomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine, N6-isopentenyladenine, 1-methylguanine, I-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
S'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine, 2-thiocytosine, S-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-S-oxyacetic acid
(v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaaninopurine.
The antisense oligonucleotide may also comprise at least one modified sugar
moiety selected from the group including, but not limited to, arabinose,
2-fluoroarabinose, xylulose, and hexose.
In yet another embodiment, the antisense oligonucleotide comprises at least
one modified phosphate backbone selected from the group including, but not
limited
to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl
phosphotriester, and a formacetal or analog thereof.
W yet another embodiment, the antisense oligonucleotide is an a-anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
hybrids with complementary RNA in which, contrary to the usual b-units, the
strands
run parallel to each other (Gautier et al., Nucl. Acids Res., 15:6625-6641
(19f7)).
The oligonucleotide is a 2-0-methylribonucleotide (moue et al., Nucl. Acids
Res.,
15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (moue et aL, FEBS Lett.
215:327-330 (1987)).
Polynucleotides of the invention may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such as are
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commercially available from Biosearch, Applied Biosystems, etc.). As examples,
phosphorothioate oligonucleotides may be synthesized by the method of Stein et
al.
(Nucl. Acids Res., 16:3209 (1988)), methylphosphonate oligonucleotides can be
prepared by use of controlled pore glass polymer supports (Sarin et al., Proc.
Natl.
Acad. Sci. U.S.A., 85:7448-7451 (1988)), etc.
While antisense nucleotides complementary to the coding region sequence of
the invention could be used, those complementary to the transcribed
untranslated
region are most preferred.
Potential antagonists according to the invention also include catalytic RNA,
or
a ribozyme (See, e.g., PCT International Publication WO 90/11364, published
October 4, 1990; Sarver et al, Science, 247:1222-1225 (1990). While ribozymes
that
cleave mRNA at site specific recognition sequences can be used to destroy
mRNAs
corresponding to the polynucleotides of the invention, the use of hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations
dictated
by flanking regions that form complementary base pairs with the target xnRNA.
The
sole requirement is that the target mRNA have the following sequence of two
bases:
5' -UG-3' . The construction and production of hammerhead ribozymes is well
known in the art and is described more fully in Haseloff and Gerlach, Nature,
334:585-591 (1988). There are numerous potential harmnerhead ribozyme cleavage
sites within each nucleotide sequence disclosed in the sequence listing.
Preferably,
the ribozyme is engineered so that the cleavage recognition site is located
near the 5'
end of the mRNA corresponding to the polynucleotides of the invention; i.e.,
to
increase efficiency and minimize the intracellular accumulation of non-
functional
mRNA transcripts.
As in the antisense approach, the ribozymes of the invention can be composed
of modified oligonucleotides (e.g. for improved stability, targeting, etc.)
and should
be delivered to cells which express the polynucleotides of the invention in
vivo.
DNA constructs encoding the ribozyme may be introduced into the cell in the
same
manner as described above for the introduction of antisense encoding DNA. A
preferred method of delivery involves using a DNA construct "encoding" the
ribozyme under the control of a strong constitutive promoter, such as, for
example,
pol III or pol II promoter, so that transfected cells will produce sufficient
quantities of
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the ribozyme to destroy endogenous messages and inhibit translation. Since
ribozymes unlike antisense molecules, are catalytic, a lower intracellular
concentration is required for efficiency.
Antagonist/agonist compounds may be employed to inhibit the cell growth
and proliferation effects of the polypeptides of the present invention on
neoplastic
cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore,
retard or
prevent abnormal cellular growth and proliferation, for example, in tumor
formation
or growth.
The antagonist/agonist may also be employed to prevent hyper-vascular
diseases, and prevent the proliferation of epithelial lens cells after
extracapsular
cataract surgery. Prevention of the mitogenic activity of the polypeptides of
the
present invention may also be desirous in cases such as restenosis after
balloon
angioplasty.
The antagonist/agonist may also be employed to prevent the growth of scar
I S tissue during wound healing.
The antagonist/agonist may also be employed to treat, prevent, and/or
diagnose the diseases described herein.
Thus, the invention provides a method of treating or preventing diseases,
disorders, and/or conditions, including but not limited to the diseases,
disorders,
and/or conditions listed throughout this application, associated with
overexpression of
a polynucleotide of the present invention by administering to a patient (a) an
antisense
molecule directed to the polynucleotide of the present invention, andlor (b) a
ribozyme directed to the polynucleotide of the present invention.
invention, and/or (b) a ribozyrne directed to the polynucleotide of the
present
invention
Other Activities
The polypeptide of the present invention, as a result of the ability to
stimulate
vascular endothelial cell growth, may be employed in treatment for stimulating
re-
vascularization of ischemic tissues due to various disease conditions such as
thrombosis, arteriosclerosis, and other cardiovascular conditions. These
polypeptide
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may also be employed to stimulate angiogenesis and limb regeneration, as
discussed
above.
The polypeptide may also be employed for treating wounds due to injuries,
burns, post-operative tissue repair, and ulcers since they are mitogenic to
various cells
of different origins, such as fibroblast cells and skeletal muscle cells, and
therefore,
facilitate the repair or replacement of damaged or diseased tissue.
The polypeptide of the present invention may also be employed stimulate
neuronal growth and to treat, prevent, and/or diagnose neuronal damage which
occurs
in certain neuronal disorders or neuro-degenerative conditions such as
Alzheimer's
disease, Parkinson's disease, and AIDS-related complex. The polypeptide of the
invention may have the ability to stimulate chondrocyte growth, therefore,
they may
be employed to enhance bone and periodontal regeneration and aid in tissue
transplants or bone grafts.
The polypeptide of the present invention may be also be employed to prevent
skin aging due to sunburn by stimulating keratinocyte growth.
The polypeptide of the invention may also be employed for preventing hair
loss, since FGF family members activate hair-forming cells and promotes
melanocyte
growth. Along the same lines, the polypeptides of the present invention may be
employed to stimulate growth and differentiation of hematopoietic cells and
bone
marrow cells when used in combination with other cytokines.
The polypeptide of the invention may also be employed to maintain organs
before transplantation or for supporting cell culture of primary tissues.
The polypeptide of the present invention may also be employed for inducing
tissue of mesodermal origin to differentiate in early embryos.
The polypeptide or polynucleotides and/or agonist or antagonists of the
present invention may also increase or decrease the differentiation or
proliferation of
embryonic stem cells, besides, as discussed above, hematopoietic lineage.
The polypeptide or polynucleotides and/or agonist or antagonists of the
present invention may also be used to modulate mammalian characteristics, such
as
body height, weight, hair color, eye color, skin, percentage of adipose
tissue,
pigmentation, size, and shape (e.g., cosmetic surgery). Similarly,
polypeptides or
polynucleotides and/or agonist or antagonists of the present invention may be
used to
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modulate mammalian metabolism affecting catabolism, anabolism, processing,
utilization, and storage of energy.
Polypeptide or polynucleotides and/or agonist or antagonists of the present
invention may be used to change a mammal's mental state or physical state by
influencing biorhythms, caricadic rhythms, depression (including depressive
diseases,
disorders, andlor conditions), tendency for violence, tolerance for pain,
reproductive
capabilities (preferably by Activin or Inhibin-like activity), hormonal or
endocrine
levels, appetite, libido, memory, stress, or other cognitive qualities.
Polypeptide or polynucleotides and/or agonist or antagonists of the present
invention may also be used as a food additive or preservative, such as to
increase or
decrease storage capabilities, fat content, lipid, protein, caxbohydrate,
vitamins,
minerals, cofactors or other nutritional components.
Other Preferred Embodiments
Other preferred embodiments of the claimed invention include an isolated
nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical to a sequence of at least about 50 contiguous nucleotides in the
nucleotide
sequence of SEQ m NO:X wherein X is any integer as defined in Table I.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ m NO:X in the range
of
positions beginning with the nucleotide at about the position of the 5'
Nucleotide of
the Clone Sequence and ending with the nucleotide at about the position of the
3'
Nucleotide of the Clone Sequence as defined for SEQ m NO:X in Table 1.
Also preferred is a nucleic acid molecule wherein said sequence of contiguous
nucleotides is included in the nucleotide sequence of SEQ m NO:X in the range
of
positions beginning with the nucleotide at about the position of the S
° Nucleotide of
the Start Codon and ending with the nucleotide at about the position of the 3'
Nucleotide of the Clone Sequence as defined for SEQ m NO:X in Table 1.
Similarly preferred is a nucleic acid molecule wherein said sequence of
contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X
in the
range of positions beginning with the nucleotide at about the position of the
5'
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Nucleotide of the First Amino Acid of the Signal Peptide and ending with the
nucleotide at about the position of the 3' Nucleotide of the Clone Sequence as
defined for SEQ ID NO:X in Table 1.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 150
contiguous nucleotides in the nucleotide sequence of SEQ lD NO:X.
Further preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least about 500
contiguous nucleotides in the nucleotide sequence of SEQ m NO:X.
A further preferred embodiment is a nucleic acid molecule comprising a
nucleotide sequence which is at least 95% identical to the nucleotide sequence
of SEQ
ID NO:X beginning with the nucleotide at about the position of the 5'
Nucleotide of
the First Amino Acid of the Signal Peptide and ending with the nucleotide at
about
the position of the 3' Nucleotide of the Clone Sequence as defined for SEQ ID
NO:X
in Table 1.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to the
complete
nucleotide sequence of SEQ 117 NO:X.
Also preferred is an isolated nucleic acid molecule which hybridizes under
stringent hybridization conditions to a nucleic acid molecule, wherein said
nucleic
acid molecule which hybridizes does not hybridize under stringent
hybridization
conditions to a nucleic acid molecule having a nucleotide sequence consisting
of only
A residues or of only T residues.
Also preferred is a composition of matter comprising a DNA molecule which
comprises a human cDNA clone identified by a cDNA Clone Identifier in Table l,
which DNA molecule is contained in the material deposited with the American
Type
Culture Collection and given the ATCC Deposit Number shown in Table 1 for said
cDNA Clone Identifier.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to a sequence of at least 50
contiguous
nucleotides in the nucleotide sequence of a human cDNA clone identified by a
cDNA
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Clone Identifier in Table 1, which DNA molecule is contained in the deposit
given the
ATCC Deposit Ntunber shown in Table 1.
Also preferred is an isolated nucleic acid molecule, wherein said sequence of
at least 50 contiguous nucleotides is included in the nucleotide sequence of
the
complete open reading frame sequence encoded by said human cDNA clone.
Also preferred is an isolated nucleic acid molecule comprising a nucleotide
sequence which is at least 95% identical to sequence of at least 150
contiguous
nucleotides in the nucleotide sequence encoded by said human cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to sequence
of at
least 500 contiguous nucleotides in the nucleotide sequence encoded by said
human
cDNA clone.
A further preferred embodiment is an isolated nucleic acid molecule
comprising a nucleotide sequence which is at least 95% identical to the
complete
nucleotide sequence encoded by said human cDNA clone.
A fizrther preferred embodiment is a method for detecting in a biological
sample a nucleic acid molecule comprising a nucleotide sequence which is at
least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence
selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X
wherein X is any integer as defined in Table l; and a nucleotide sequence
encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1; which method comprises a step of comparing a nucleotide sequence of at
least one
nucleic acid molecule in said sample with a sequence selected from said group
and
determining whether the sequence of said nucleic acid molecule in said sample
is at
least 95% identical to said selected sequence.
Also preferred is the above method wherein said step of comparing sequences
comprises determining the extent of nucleic acid hybridization between nucleic
acid
molecules in said sample and a nucleic acid molecule comprising said sequence
selected from said group. Similarly, also preferred is the above method
wherein said
step of comparing sequences is performed by comparing the nucleotide sequence
determined from a nucleic acid molecule in said sample with said sequence
selected
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from said group. The nucleic acid molecules can comprise DNA molecules or RNA
molecules.
A further preferred embodiment is a method for identifying the species, tissue
or cell type of a biological sample which method comprises a step of detecting
nucleic
acid molecules in said sample, if any, comprising a nucleotide sequence that
is at least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence
selected from the group consisting of a nucleotide sequence of SEQ ID NO:X
wherein X is any integer as defined in Table l; and a nucleotide sequence
encoded by
a human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
The method for identifying the species, tissue or cell type of a biological
sample can comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in a panel of at least two nucleotide sequences, wherein
at least
one sequence in said panel is at least 95% identical to a sequence of at least
50
contiguous nucleotides in a sequence selected from said group.
Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a gene encoding a secreted
protein identified in Table 1, which method comprises a step of detecting in a
biological sample obtained from said subject nucleic acid molecules, if any,
comprising a nucleotide sequence that is at least 95% identical to a sequence
of at
least 50 contiguous nucleotides in a sequence selected from the group
consisting of: a
nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in
Table 1;
and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA
Clone Identifier in Table 1 and contained in the deposit with the ATCC Deposit
Number shown for said cDNA clone in Table 1.
The method for diagnosing a pathological condition can comprise a step of
detecting nucleic acid molecules comprising a nucleotide sequence in a panel
of at
least two nucleotide sequences, wherein at least one sequence in said panel is
at least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence
selected from said group.
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Also preferred is a composition of matter comprising isolated nucleic acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise
a panel of at least two nucleotide sequences, wherein at least one sequence in
said
panel is at least 95% identical to a sequence of at least 50 contiguous
nucleotides in a
sequence selected from the group consisting of: a nucleotide sequence of SEQ
ID
NO:X wherein X is any integer as defined in Table 1; and a nucleotide sequence
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1. The nucleic acid molecules can comprise DNA molecules or RNA
molecules.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 90% identical to a sequence of at least about 10 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y wherein Y is any integer as defined in
Table 1.
Also preferred is a polypeptide, wherein said sequence of contiguous amino
acids is included in the amino acid sequence of SEQ ID NO:Y in the range of
positions begimiing with the residue at about the position of the First Amino
Acid of
the Secreted Portion and ending with the residue at about the Last Amino Acid
of the
Open Reading Frame as set forth for SEQ ~ NO:Y in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of SEQ ID NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to a sequence of at least about 100 contiguous
amino
acids in the amino acid sequence of SEQ ZD NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 95% identical to the complete amino acid sequence of SEQ ID
NO:Y.
Further preferred is an isolated polypeptide comprising an amino acid
sequence at least 90% identical to a sequence of at least about 10 contiguous
amino
acids in the complete amino acid sequence of a secreted protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
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Also preferred is a polypeptide wherein said sequence of contiguous amino
acids is included in the amino acid sequence of a secreted portion of the
secreted
protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in
Table 1 and contained in the deposit with the ATCC Deposit Number shown for
said
cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at Ieast 95% identical to a sequence of at least about 30 contiguous amino
acids in the
amino acid sequence of the secreted portion of the protein encoded by a human
cDNA
clone identified by a cDNA Clone Identifier in Table 1 and contained in the
deposit
with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to a sequence of at least about 100 contiguous amino
acids in
the amino acid sequence of the secreted portion of the protein encoded by a
human
cDNA clone identified by a cDNA Clone Identifier in Table 1 and contained in
the
deposit with the ATCC Deposit Number shown for said cDNA clone in Table 1.
Also preferred is an isolated polypeptide comprising an amino acid sequence
at least 95% identical to the amino acid sequence of the secreted portion of
the protein
encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1
and contained in the deposit with the ATCC Deposit Number shown for said cDNA
clone in Table 1.
Further preferred is an isolated antibody which binds specifically to a
polypeptide comprising an amino acid sequence that is at least 90% identical
to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
group
consisting of an amino acid sequence of SEQ m NO:Y wherein Y is any integer as
defined in Table 1; and a complete amino acid sequence of a protein encoded by
a
human cDNA clone identified by a cDNA Clone Identifier in Table 1 and
contained
in the deposit with the ATCC Deposit Number shown for said cDNA clone in Table
1.
Further preferred is a method for detecting in a biological sample a
polypeptide comprising an amino acid sequence which is at least 90% identical
to a
sequence of at least 10 contiguous amino acids in a sequence selected from the
group
consisting of an amino acid sequence of SEQ ID NO:Y wherein Y is any integer
as
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