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CA 02403901 2002-09-23
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29 Human Secreted Proteins
Field of the Invetztion
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.
Background of the Iszvesztiou
Unlike bacterium, which exist as a single compartment surrounded by a
membrane, human cells and other eucaryotes are subdivided by membranes into
many
functionally distinct compartments. 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 VICI,
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.
Suznznazy of the Invezztion
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 occurnng), 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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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 117 (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, 5x
SSC
(750 mM NaCl, 75 mM trisodium citrate); 50 mM sodium phosphate (pH 7.6), 5x
Denhardt's solution, I O% dextran sulfate, and 20 ~,g/ml denatured, sheared
salmon
sperm DNA, followed by washing the filters in 0.1x 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 NaHZP04; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/m1
salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE,
O.I% SDS. In addition, to achieve even Iower 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
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6
a given polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched , for example, as a result of
ubiquitination, and they may be cyclic, with or without branching. Cyclic,
branched,
and branched cyclic polypeptides may result from posttranslation natural
processes or
may be made by synthetic methods. Modifications include acetylation,
acylation,
ADP-ribosylation, amidation, covalent attachment of flavin, covalent
attachment of a
heme moiety, covalent attachment of a nucleotide or nucleotide derivative,
covalent
attachment of a lipid or lipid derivative, covalent attachment of
phosphotidylinositol,
cross-linking, cyclization, disulfide bond formation, demethylation, formation
of
covalent cross-links, formation of cysteine, formation of pyroglutamate,
formylation,
gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,
iodination, methylation, myristoylation, oxidation, pegylation, proteolytic
processing,
phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-
RNA
mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES;
2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.
Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth
Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)
"SEQ ID NO:X" refers to a polynucleotide sequence while "SEQ ID NO:Y"
refers to a polypeptide sequence, both sequences identified by an integer
specified in
Table 1.
"A polypeptide having biological activity" refers to polypeptides 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 SY GENE NO: 1
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 69. 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.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Snares fetal liver spleen 1NFLS and to a lesser extent in BLyS Receptor from
Expression Cloning; Snares ovary tumor NbHOT; Snares fetallung NbHLI9W;
Human Primary Breast Cancer; NCI CGAP Panl; Human endometrial stromal cells-
treated with progesterone; Snares senescent fibroblasts NbHSF; T Cell helper
I;
Keratinocyte; NCI CGAP GCB1; Stratagene ovary (#937217); Human Whole Six
Week Old Embryo; Early Stage Human Brain; Activated T-Cell (l2hs)/Thiouridine
labelledEco; T cell helper II; Snares total fetus Nb2HF8'9w;
Soares_pregnant uterus NbHPU; Snares placenta Nb2HP; Snares testis NHT;
Breast, Normal: (4005522B2); Macrophage-oxLDL; Human Thymus; Snares breast
2NbHBst; NCI CGAP_Co3; Human Testes Tumor; B-cells (stimulated);
NCI CGAP Brn25; Osteoblasts; Snares fetal heart NbHHI9W; Pancreatic Islet;
Human heart cDNA (YNakamura); Human Synovium; Human adult (K.Okubo);
Synovial hypoxia-RSF subtracted; H. Kidney Cortex, subtracted; wilm's tumor;
Ovarian Cancer; Gessler Wilins tumor;. Stratagene ovarian cancer (#937219);
Human
Brain, Striatum; NCI CGAP Utl; Human umbilical vein endothelial cells, IL-4
induced; Human Rhabdomyosarcoma; Human Adult Testes, Large Inserts,
Reexcision; Ulcerative Colitis; Human Ovary; Macrophage-oxLDL, re-excision;
Colon Carcinoma; Snares breast 3NbHBst; Ovary, Cancer(4004650 A3): Well-
Differentiated Micropapillary Serous Carcinoma; Human Synovial Sarcoma;
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Pancreas normal PCA4 No; Human Primary Breast Cancer Reexcision;
NCI CGAP Kid3; NCI CGAP Kids; Monocyte activated;
Soares-placenta 8to9weeks 2NbHP8to9W; Human fetal heart, Lambda ZAP
Express; Soares_parathyroid tumor NbHPA; Colon Normal III; Primary Dendritic
Cells, lib l; Human Fetal Kidney; Adrenal Gland,normal; NCI CGAP_Col; Lung,
Cancer: (4005313 A3) Invasive Poorly-differentiated Metastatic lung adenoc;
Tongue
Normal; Human Tonsils, lib I; Human Adult Heart; Brain Amygdala Depression;
NCI CGAP Pr4; NCI CGAP_SS1; NCI CGAP_Ov36; Human epithelioid sarcoma;
NCI CGAP Brn35; Human Gall Bladder, fraction II; Human Primary Breast
Cancer,re-excision; stromal cell clone 2.5; NCI CGAP_Ov23; Human Placenta;
Activated T-cells; Human Thyroid; NCI CGAP_GC3; Human Normal Breast; B Cell
lymphoma; NCI CGAP_Co9; Salivary Gland, Lib 2; NCI CGAP'ColO;
NCI CGAP-CoI4; Soares adult brain N2b4HB55Y; Synovial Fibroblasts (III/TNF),
subt; Myoloid Progenitor Cell Line; Spleen metastic melanoma; NCI CGAP Ut2;
CD34 depleted Buffy Coat (Cord Blood); H. Epididiymus, caput & corpus;
NCI CGAP Pr2; Breast Cancer Cell line, angiogenic; Ovary, Cancer: (15799A1F)
Poorly differentiated carcinoma; Human Fetal Kidney; human ovarian cancer;
Soares~ineal gland N3HPG; HUMAN JURKAT MEMBRANE BOUND
POLYSOMES; Human Fetal Dura Mater; NCI CGAP Pr28; NCI CGAP-Gas4;
Stromal cell TF274; Human Pancreas Tumor; Stratagene HeLa cell s3 937216;
Liver,
Hepatoma; Soares NSF F8 9W OT PA P Sl; Hemangiopericytoma; Human
Adipose; Epithelial-TNFa and INF induced; NCI CGAP_CLL1; Bone Marrow
Stromal Cell, untreated; Macrophage (GM-CSF treated); CHME Cell Line,treated 5
hrs; Hepatocellular Tumor, re-excision; B-cells (unstimulated); Ovary, Cancer:
(4004576 A8); Smooth muscle, serum induced,re-exc; NTERA2 + retinoic acid, 14
days; Rejected Kidney, lib 4; Fetal Liver, subtraction II; Colon Tumor; Palate
carcinoma; breast lymph node CDNA library; Smooth muscle, serum treated;
NCI CGAP Kidl l; Normal colon; NCI CGAP-GC4; H Macrophage (GM-CSF
treated), re-excision; Human Fetal Kidney, Reexcision; Ovary, Cancer
(9809C332):
Poorly differentiated adenocarcinoma; Human Ovarian Cancer Reexcision;
Endothelial-induced; NCI CGAP Brn23; Spleen, Chronic Iymphocytic leukemia;
Pancreas Tumor PCA4 Tu; HM3; Human Bone Marrow, treated; normalized infant
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brain cDNA; Activated T-cell(12h)/Thiouridine-re-excision; Nine Week Old Early
Stage Human; Colon Tumor II; Soares infant brain 1NIB; NCI CGAP_GUl;
NCI CGAP CML1; NCI CGAP Co20; NCI CGAP Sub4 and NCI CGAP Subs.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Preferred polypeptides of the present invention comprise one, two, three or
all
four immunogenic epitopes shown in shown in SEQ ID NO: 40 as residues: Thr-84
to
Pro-90, Lys-144 to Arg-149, Ile-151 to Gln-159, Ser-188 to Asn-198.
Polynucleotides
encoding said polypeptides are also encompassed by the invention. Specific
embodiments of the invention also include antibodies that bind any of these
epitopes.
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 1147 of SEQ ID
NO:1 l, b
is an integer of 15 to 1161, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:11, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 70,SEQ ID NO: 71 and/or SEQ ID NO: 72. 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,
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under stringent conditions, to the polynucleotide encoding these polypeptides
) are
encompassed by the invention. Polynucleotides encoding these polypeptides are
also
encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
5 NCI CGAP GC6 and to a lesser extent in NCI CLAP Panl; Soares testis NHT;
Human adult testis, large inserts; Stratagene colon (#937204); HUMAN B CELL
LYMPHOMA; NCI CGAP Ut4; NCI CGAP Kidl2; NCI CGAP Ut3; Stratagene
HeLa cell s3 937216; NCI CGAP Br2; Human Adult Testes, Large Inserts,
Reexcision; B-cells (stimulated); NCI CGAP Brn25; Human Primary Breast Cancer
10 Reexcision; Soares NhHMPu Sl; NCI CGAP_GCB1; Soares fetal liver spleen
1NFLS; Stratagene colon HT29 (#937221); NCI CGAP Lu24; NCI CGAP Lyml2;
NCI CGAP Ut2; NCI CGAP Utl; KMH2; NCI CGAP Pr28; NCI CGAP_Co3;
NCI CGAP Brn23; Soares ovary tumor NbHOT; NCI CGAP LuS; normalized
infant brain cDNA; Soares melanocyte 2NbHM; Larynx Tumour; NCI CGAP'Col6;
Lung, Normal: (4005313 Bl); stromal cell clone 2.5; Pancreatic Islet;
Adipocytes,re-
excision; Adenocarcinoma of Ovary, Human Cell Line, # OVCAR-3; Supt Cells,
cyclohexamide treated; Aorta endothelial 'cells + TNF-a; Human Soleus; Human
Pineal Gland; Adenocarcinoma of Ovary, Human Cell Line; Human adult (K.Okubo);
Glioblastoma; NCI CGAP_ColO; Stratagene neuroepithelium (#937231); TF-1 Cell
Line GM-CSF Treated; Gessler Wilms tumor; NCI CGAP-Gas4; Stratagene
endothelial cell 937223; Human Osteoblasts H; Human Hippocampus; Human
umbilical vein endothelial cells, IL-4 induced; Spinal cord; Human Thymus;
Ulcerative Colitis; Human Fetal Brain; CHME Cell Line,treated 5 hrs; CHME Cell
Line,untreated; Fetal Heart; Colon Tumor; Human Substantia Nigra; Human Fetal
Kidney, Reexcision; Ovary, Cancer(4004650 A3): Well-Differentiated
Micropapillary
Serous Carcinoma; Human Testes, Reexcision; Activated T-Cell
(l2hs)/Thiouridine
labelledEco; Soares senescent fibroblasts NbHSF; Monocyte activated; HM3;
Human Bone Marrow, treated; T Cell helper I; Human Testes; Bone Marrow Cell
Line (RS4,11); neutrophils control; Human 8 Week Whole Embryo; Nine Week Old
Early Stage Human; Soares fetal lung NbHLI9W; Colon Tumor II; T cell helper H;
Soares total fetus Nb2HF8'9w; Human Cerebellum;
Soares~regnant uterus NbHPU; Soares fetal liver spleen 1NFLS_Sl;
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11
Soares fetal heart NbHHI9W; Soares infant brain 1NIB; NCI CGAP GU1;
NCI CGAP Lu28; NCI CGAP Lu3l; NCI CGAP-Ov39; NCI CGAP Sub3 and
NCI CGAP Brn53.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:12 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 1559 of SEQ ID
N0:12, b
is an integer of 15 to 1573, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:12, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 3
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 73. 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.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries: 12
Week Early Stage Human II, Reexcision and to a lesser extent in Human Fetal
Brain,
normalized 50021F; Human Pancreatic Langerhans; HPAS (human pancreas,
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12
subtracted); NCI CGAP_Col6; Normal Human Trabecular Bone Cells; Aorta
endothelial cells + TNF-a; Jurkat T-Cell, S phase; TF-1 Cell Line GM-CSF
Treated;
NCI CGAP Gas4; NCI CGAP Panl; Palate normal; Human T-Cell Lymphoma;
Human Fetal Lung III; NCI CGAP_GC6; Activated T-Cell (l2hs)/Tluouridine
labelledEco; Human Ovarian Cancer Reexcision; Human Testes; Activated T-
cell(12h)/Thiouridine-re-excision; Human 8 Week Whole Embryo;
Soares NFL T GBC Sl and NCI CGAP Sub4.
Expression in ovarian cancer tissue, indicates that polynucleotides and
polypeptides corresponding to this gene would be useful for the treatment,
prevention,
detection and diagnosis of tumors, especially ovarian cancer, as well as
cancers of
other tissues where expression has been indicated. The expression in ovarian
cancer
tissue may indicate the gene or its products can be used to treat, prevent,
detect and/or
diagnose disorders of the ovary, including inflammatory disorders, such as
oophoritis
(e.g., caused by viral or bacterial infection), ovarian cysts, amenorrhea,
infertility,
hirsutism, and ovarian cancer (including, but not limited to, primary and
secondary
cancerous growth, endometrioid carcinoma of the ovary, ovarian papillary
serous
adenocarcinoma, ovarian mucinous adenocarcinoma, Ovarian Krukenberg tumor).
The tissue distribution in immune cells indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as 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-
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13
versus-graft and graft-versus-host diseases, or autoimmunity disorders, such
as
autoimmune infertility, lense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
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 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
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 1665 of SEQ ID
N0:13, b
is an integer of 15 to 1679, where both a and b correspond to the positions of
nucleotide residues shown in SEQ 1D N0:13, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED SY GENE NO: 4
This gene is expressed primarily in the following tissueslcDNA libraries:
Hepatocellular Tumor,re-excision; Soares-pineal gland N3HPG; Human Testes
Tumor, re-excision; NCI CGAP Panl; Activated T-cell(12h)/Thiouridine-re-
excision.
The tissue distribution indicates the polynucleotides and polypeptides
corresponding to this gene would be useful for the diagnosis and treatment of
a
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14
variety of immune system disorders. Representative uses are described in the
"Immune Activity" and "Infectious Disease" sections below, in Example 11, 13,
14,
16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates a role
in regulating the proliferation; survival; differentiation; and/or activation
of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as 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 autoirnrnunity disorders, such
as
autoimmune infertility, lense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
in the expansion of stem cells and committed progenitors of various blood
lirieages,
and in the differentiation and/or proliferation of various cell types.
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: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
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1S
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 1113 of SEQ ID
N0:14, b
is an integer of 1 S to 1127, 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
S + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
This gene is expressed primarily in the following tissues/cDNA libraries:
Human Adult Testes, Large Inserts, Reexcision and to a lesser extent in Human
adult
testis, large inserts; Soares fetal heart NbHHI9W; Soares NhHMPu S1; Human
Primary Breast Cancer Reexcision; Soares melanocyte 2NbHM; Primary Dendritic
Cells, lib 1; Stromal Cells; Breast, Cancer: (400SS22 A2); H Macrophage (GM-
CSF
treated), re-excision; Soares senescent fibroblasts NbHSF; Soares ovary tumor
NbHOT; Human Testes; Human Cerebellum; Soares testis NHT;
1S NCI CGAP-GCB1; Human Epididymus; Synovial IL-1/TNF stimulated; human
corpus colosum; Human normal ovary(#9610621 S); Human Pituitary, subt IX;
NCI CGAP Ut2; Human Chronic Synovitis; Gessler Wilms tumor; Human
Umbilical Vein Endothelial Cells, uninduced; NGI CGAP_Gas4; Macrophage-
oxLDL; Stratagene endothelial cell 937223; Liver, Hepatoma; NCI CGAP'Co3;
Human Pancreas Tumor, Reexcision; Soares breast 3NbHBst; NCI CGAP'CoB;
NCI CGAP-GC4; Ovary, Cancer (9809C332): Poorly differentiated
adenocarcinoma; NCI CGAP Brn2S; NCI CGAP Kid3; HUMAN B CELL
LYMPHOMA; NCI CGAP LuS; Osteoblasts; Colon Tumor II; Soares infant brain
1NIB; Human Fetal Brain, normalized CSOOHE; NCI CGAP_Ov3S; Human Fetal
2S Thymus; Human White Fat; Human colorectal cancer; Human Pituitary, re-
excision;
NCI CGAP_GCS; NCI CGAP KidB; Adipocytes,re-excision; Frontal
lobe,dementia,re-excision; Aorta endothelial cells + TNF-a; Human Primary
Breast
Cancer; Human Skin Tumor; Serous Papillary Adenocarcinoma; Human adult
(K.Okubo); NCI CGAP Co9; pBMC stimulated w/ poly I/C; Human Fetal
Epithelium (Skin); Serous Papillary Adenocarcinoma; NCI CGAP Lyml2; Breast,
Cancer: (4004943 AS); Prostate BPH; Healing groin wound - zero hr post-
incision
(control); Brain Frontal Cortex, re-excision; Spinal Cord, re-excision;
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16
NCI CGAP Utl; HUMAN JURKAT MEMBRANE BOUND POLYSOMES;
Human Pancreas Tumor; NCI CGAP Br2; Human Dermal Endothelial
Cells,untreated; Human Thymus; NCI CGAP_CLLl; Ulcerative Colitis; Human
Adrenal Gland Tumor; CHME Cell Line,treated 5 hrs; Smooth muscle, serum
induced,re-exc; CHME Cell Line,untreated; Rejected I~idney,,lib 4; breast
lymph
node CDNA library; Human Substantia Nigra; Human Placenta; Bone marrow;
NTERA2, control; Human Testes, Reexcision; Human Ovarian Cancer Reexcision;
Endothelial-induced; CD34 depleted Buffy Coat (Cord Blood), re-excision; B-
cells
(stimulated); Human Microvascular Endothelial Cells, fract. A; Spleen, Chronic
lymphocytic leukemia; Pancreas Tumor PCA4 Tu; HM3; Human Bone Marrow,
treated; Human Endometrial Tumor; Keratinocyte; Soares total fetus Nb2HF8 9w;
Colon Normal III; Soares~regnant uterus NbHPU;
Soares fetal liver spleen 1NFLS_S1; NCI CGAP~Co2l; NCI CGAP Lu31 and
NCI CLAP Brn50.
I S The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Preferred polypeptides of the present invention comprise one, two, three or
all
four immunogenic epitopes shown in SEQ ID NO: 44 as residues: Pro-51 to Asp-
56,
Gly-95 to Thr-105, Val-132 to Ala-138, Pro-229 to Leu-240. Polynucleotides
encoding said polypeptides are also encompassed by the invention. Specific
embodiments of the invention also include antibodies that bind any of these
epitopes.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ D7 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 1899 of SEQ ID
NO:15, b
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17
is an integer of 15 to 1913, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:15, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED SY GENE NO: 6
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
emb~CAA56947.1 ~ (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "~ tex261 [Mus
musculus]" A partial alignment demonstrating the observed homology is shown
immediately below.
>embICAA56947.11 tex261 [Mus musculus] >pir~S47481~547481 tex261 protein -
mouse >sp~Q62302~Q62302 TESTIS EXPRESSED PROTEIN 26l (TEG-261).
Length = 196
Plus Strand HSPs:
Score = 1010 (355.5 bits), Expect = 4.4e-101, P = 4.4e-101
Identities = 195/196 (99%), Positives = 196/196 (100%), Frame = +2
Q: 74 MWFMYLLSWLSLFIQVAFITLAVAAGLWLAELIEEYTVATSRIIKYMIWFSTAVLIGLY 253
~S MWFMY+LSWLSLFIQVAFITLAVAAGLYYLAELIEEYTVATSRIIKYMIWFSTAVLIGLY
S: 1 MWFMYVLSWLSLFIQVAFITLAVAAGLYYLAELIEEYTVATSRIIKYMIWFSTAVLIGLY 60
Q: 254 VFERFPTSMIGVGLFTNLWFGLLQTFPFIMLTSPNFILSCGLVVVNHYLAFQFFAEEYY 433
VFERFPTSMIGVGLFTNLWFGLLQTFPFIMLTSPNFILSCGLWVNHYLAFQFFAEEYY
3 O S: 61 VFERFPTSMIGVGLFTNLWFGLLQTFPFIMLTSPNFILSCGLWVNHYLAFQFFAEEW 120
Q: 434 PFSEVLAYFTFCLWIIPFAFFVSLSAGENVLPSTMQPGDDWSNYFTKGKRGKRLGILW 613
PFSEVLAYFTFCLWIIPFAFFVSLSAGENVLPSTMQPGDDWSNYFTKGKRGKRLGILW
S: 121 PFSEVLAYFTFCLWIIPFAFFVSLSAGENVLPSTMQPGDDWSNYFTKGKRGKRLGILW 180
Q: 6l4 FSFIKEAILPSRQKIY 661
FSFIKEAILPSRQKIY
S: 181 FSFIKEAILPSRQKIY 196
The segment of emb~CAA56947.1~ that is shown as "S" above is set out in the
sequence listing as SEQ m NO: 74 Based on the structural similarity these
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have.
been described elsewhere herein.
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18
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 75 which corresponds to the Q sequence in the alignment shown above
(gaps introduced in a sequence by the computer are, of course, removed).
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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares MiHMPu S1; Soares infant brain 1NIB and to a lesser extent in Soares
adult
brain N2b4HB55Y; NCI CGAP_GCB1; Soares senescent fibroblasts NbHSF;
Soares placenta Nb2HP; Soares fetal liver spleen 1NFLS; TNFR degenerate oligo;
Synovial Fibroblasts (control); Soares breast 3NbHBst; Stratagene muscle
937209;
NCI CLAP Brn25; NCI CGAP Kids; Soares multiple sclerosis 2NbHMSP;
NCI CGAP LuS; Human 8 Week Whole Embryo;
Soares-parathyroid tumor NbHPA; Colon Tumor II; Human Cerebellum;
Soares testis NHT; NCI CGAP Lul9; Human Placenta; STR.ATAGENE Human
skeletal muscle cDNA library, cat. #936215.; NCI CGAP Ut4; NCI CGAP Utl;
Stratagene fetal spleen (#937205); NCI CGAP-Gas4; Ovary, Cancer (15395A1F):
Grade II Papillary Carcinoma; Soares breast 2NbHBst; NCI CLAP Panl; NTERA2
+ retinoic acid, 14 days; Stratagene colon (#937204); Human endometrial
stromal
cells-treated with progesterone; NCI CGAP_CoB; Adipocytes; Human Synovial
Sarcoma; NTERA2, control; Activated T-Cell (l2hs)/Thiouridine labelledEco;
Htunan Primary Breast Cancer Reexcision; Human Microvascular Endothelial
Cells,
fract. A; Human Adult Heart,re-excision; Activated T-cell(12h)/Thiouridine-re-
excision; NCI CGAP Brn52; Osteoclastoma-normalized A; NCI CGAP Kidl2;
STRIATUM DEPRESSION; NCI CGAP Pr24; Human OB HOS treated (1. nM E2)
fraction I; Human promyelocyte; Human White Adipose; Human Umbilical Vein
Endothelial Cells, fract. A; Human Pancreatic Carcinoma; HSC172 cells; Frontal
lobe,dementia,re-excision; Stromal cells 3.88; NCI CGAP Ut3; Messangial cell,
frac
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19
2; Apoptotic T-cell, re-excision; Stromal cells(HBM3.18); Human Tonsils, Lib
2;
Human Synovium; Breast, Cancer: (4005522 A2); Human Osteoclastoma Stromal
Cells - unamplified; Synovial hypoxia-RSF subtracted; Jurkat T-Cell, S phase;
Jurkat
T-cell Gl phase; Human Chronic Synovitis; Gessler Wilms tumor; Apoptotic T-
cell;
12 Week Old Early Stage Human, II; Stratagene pancreas (#937208);
NCI CGAP Pr28; Human Osteoblasts II; NCI CGAP Br2; Merkel Cells; Olfactory
epithelium,nasalcavity; Spinal cord; Healing groin wound, 7.5 hours post
incision;
Epithelial-TNFa and INF induced; Human Fetal Brain; Bone Marrow Stromal Cell,
untreated; Human Testes Tumor, re-excision; Human Liver, normal; B-cells
(unstimulated); NCI CGAP Co3; Ovarian Tumor 10-3-95; CHME Cell
Line,untreated; Rej ected Kidney, lib 4; Fetal Liver, subtraction II; Human
Fetal
Kidney, Reexcision; NCI CGAP_GC6; Human Adult Pulmonary,re-excision; B-cells
(stimulated); NCI CGAP Kid3; NCI CGAP Brn23; Human fetal heart, Lambda
ZAP Express; Soares melanocyte 2NbHM; Nine Week Old Early Stage Human;
Soares total fetus Nb2HF8'9w; Soares-pregnant uterus NbHPU;
Soares fetal liver spleen 1NFLS~S1; Soares NFL T GBC_S1;
Soares fetal heart NbHHI9W; Primary Dendritic Cells, lib 1 and NCI CGAP_GUl .
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflanunatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15; and 18, and elsewhere herein.
Preferred polypeptides of the present invention comprise one or both
immunogenic epitopes shown in SEQ ID NO: 45 as residues: Phe-166 to Arg-174,
Ser-191 to Tyr-196. Polynucleotides encoding said polypeptides are also
encompassed by the invention. Specific embodiments of the invention also
include
antibodies that bind any of these epitopes.
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
CA 02403901 2002-09-23
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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 3328 of SEQ m
N0:16, b
is an integer of 15 to 3342, where both a and b correspond to the positions of
5 nucleotide residues shown in SEQ m N0:16, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
This gene is expressed primarily in the following tissues/cDNA libraries:
10 Monocyte activated; Stomach cancer (human),re-excision; Human Umbilical
Vein,
Endo. remake.
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
15 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 2771 of SEQ ID
N0:17, b
20 is an integer of 15 to 2785, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ m N0:17, and where b is greater than or equal
to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 8
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
dbj ~BAA9I245.1 ~ (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "~ (AK000496)
unnamed protein product [Homo Sapiens]. " A partial alignment demonstrating
the
observed homology is shown immediately below.
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21
>dbj~BAA91205.1~ (AK000496) unnamed protein product [Homo Sapiens]
Length = 239
Minus Strand HSPs:
Score = 287 (101.0 bits), Expect = 1.8e-23, P = 1.8e-23
Identities = 56/71 (78%), Positives = 61/71 (85%), Fame = -1
Q: 2375 LRWSLTVSPRLECSGVISAHCNFRLPGSSDSPDSASRVAGTTGDRHHARLIFVFLVETGF 2196
1 O LRWSLT+SPRLECS ISAHCN RLPGSS+SP AS+VAG TG HHAR IFVFLVETGF
S: 120 LRWSLTLSPRLECSSAISAHCNLRLPGSSNSPALASQVAGITGICHHARQIFVFLVETGF 179
Q: 2195 HHIGQAGLKLL 2163
H+GQAGL+LL
S: 180 CHVGQAGLELL 190
The segment of dbj ~BAA91205.1 ~ that is shown as "S" above is set out in the
sequence listing as SEQ ID NO: 76 Based on the structural similarity these
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 77 which corresponds to the Q sequence in the alignment shown above
(gaps introduced in a sequence by the computer are, of course, removed).
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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
This gene is expressed primarily in Human Bone Marrow, treated.
The tissue distribution in Bone Marrow indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Irmnune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
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22
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent fox immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as 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, Tense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
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 m 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 2379 of SEQ m
N0:18, b
is an integer of 15 to 2393, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:18, and where b is greater than or equal
to a
+ 14.
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23
FEATURES OF PROTEIN ENCODED SY GENE NO: 9
This gene is expressed primarily in B Cell lymphoma.
The tissue distribution in B-cell lymphoma indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 1 ~, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia,
rheumatoid arthritis, granulomatous disease, inflanunatory 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, Tense tissue injury, demyelination, systemic lupus
erythernatosis, drug induced hemolytic anemia, rheumatoid artlu-itis,
Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
in the expansion of stem cells and committed progenitors of vaxious blood
lineages,
and in the differentiation and/or proliferation of various cell types.
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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24
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:19 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 2216 of SEQ ID
NO:19, b
is an integer of IS to 2230, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:19, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED SY GENE NO: 10
This gene is expressed primarily in the following tissues/cDNA libraries:
Human Neutrophil, Activated and to a lesser extent in Colon Normal III;
NCI CGAP CMLI; Neutrophils control, re-excision; Human Neutrophil; Human
Eosinophils; neutrophils control; Human Pancreatic Carcinoma; Human Fetal
Bone;
NCI CGAP Co 12; Human Tonsils, Lib 2; Human Adult Small Intestine; Human
Bone Marrow, re-excision; Human Pancreas Tumor; Liver, Hepatoma; Healing groin
wound, 6.5 hours post incision; Fetal Heart; Human Pancreas Tumor, Reexcision;
Colon Normal II; Pancreas Islet Cell Tumor; Neutrophils IL-1 and LPS induced;
Endothelial cells-control; Neutrophils IL-1 and LPS induced and Soares fetal
liver
spleen 1NFLS.
The tissue distribution in immune tissues indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
CA 02403901 2002-09-23
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lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as AmS, leukemia,
rheumatoid arthritis, granulomatous disease, inflammatory bowel disease,
sepsis,
5 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, lense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
10 disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
in the expansion of stem cells and committed progenitors of various blood
lineages,
' and in the differentiation and/or proliferation of various cell types.
Furthermore, the
15 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.
20 Preferred polypeptides of the present invention comprise one or both
immunogenic epitopes shown in SEQ >D NO: 49 as residues: Glu-32 to Arg-38, Val-
46 to Val-61. Polynucleotides encoding said polypeptides are also encompassed
by
the invention. Specific embodiments of the invention also include antibodies
that bind
any of these epitopes.
25 Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:20 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
would be cumbersome. Accordingly, preferably excluded from the present
invention
axe one or more polynucleotides comprising a.nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1179 of SEQ m
N0:20, b
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26
is an integer of 15 to 1193, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:20, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTETN ENCODED BY GENE NO: 11
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares infant brain 1NIB; Soares fetal liver spleen 1NFLS and to a lesser
extent in
Soares NhHMPu S1; NCI CGAP Brn25; Human Cerebellum; NCI CGAP Pr28;
Activated T-cell(12h)/Thiouridine-re-excision; Smooth Muscle- HASTE
normalized;
NCI CGAP Kidl l; Activated T-Cell (l2hs)/Thiouridine labelledEco;
Soares~regnant uterus NbHPU; Infant brain, LLNL array of Dr. M. Soares 1NIB;
NCI CGAP Ut4; Stratagene muscle 937209; Stratagene pancreas (#937208);
NCI CLAP Co3; Fetal Liver, subtraction II; NCI CGAP Brn23; normalized infant
brain cDNA; Soares fetal lung NbHLI9W; Soares fetal heart NbHHI9W;
b4HB3MA-Cot51.5-HAP-Ft; NCI CGAP Brn52; Dermatofibrosarcoma
Protuberance; prostate-edited; Human promyelocyte; Human Fetal Brain; Human
Placenta; NCI CGAP Ut3; NCI CGAP AA1; Apoptotic T-cell, re-excision; Stromal
cells(HBM3.18); NCI CGAP_Co9; NCI CGAP Ut2; H. Lymph node breast Cancer;
NCI CGAP Utl; Breast Cancer Cell line, angiogenic; NCI CGAP Kid6; Ovary,
Cancer: (15799A1F) Poorly differentiated carcinoma; Human Fetal Dura Mater;
NCI CGAP Br2; Olfactory epithelium,nasalcavity; Ovary, Cancer (15395A1F):
Grade II Papillary Carcinoma; Human Adipose; Bone Marrow Stromal Cell,
untreated; NCI CGAP GC4; Pancreas normal PCA4 No; Bone marrow;
NCI CGAP_GC6; Soares,senescent fibroblasts NbHSF;
Soares multiple sclerosis 2NbHMSP; Monocyte activated; Human Bone Marrow,
treated; Bone Marrow Cell Line (RS4,11); NCI CGAP LuS; Hodgkin's Lymphoma
II; Keratinocyte; Colon Tumor II and Soares total fetus Nb2HF8 9w.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
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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 JD 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 1664 of SEQ ID
N0:21, b
is an integer of 15 to 1678, where both a and b correspond to the positions of
nucleotide residues shown in SEQ )D N0:21, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
dbj ~BAA77223.1 ~ (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "~ (AB020854)
orphan
transporter short splicing variant [Bos taurus]" A partial alignment
demonstrating the
observed homology is shown immediately below.
>dbj~BAA77223.1~ (AB020854) orphan transporter short splicing variant [Bos
taurus] >sp~Q9XS59~Q9XS59 ORPHAN TRANSPORTER SHORT SPLICING
VARIANT.
Length = 729
Plus Strand HSPs:
Score = 901 (317.2 bits), Expect = 2.1e-189, Sum P(.3) = 2.1e-189
3 0 Identities = 172/188 (91%), Positives = 181/188 (96%), Frame = +2
Q: 392 NSETIMKFLKMGNISQDIIPHHINLSTVTAEDYHLVYDIIQKVKEEEFPALHLNSCKIEE 571
NSE I+K +KMGNISQDIIPHHIN S +TAEDY L+YDIIQKVKEEEFPALHLN+C+IE+
3 S S: 371 NSEMIIKLVKMGNISQDIIPHHINFSAITAEDYDLTYDIIQKVKEEEFPALHLNACQIED 430
Q: 572 ELNKAVQGTGLAFIAFTEAMTHFPASPFWSVMFFLMLVNLGLGSMFGTIEGIVTPIVDTF 751
ELNKAVQGTGLAFIAFTEAMTHFPASPFWSVMFFLMLVNLGLGSMFGTIEGI+TP+VDTF
S: 431 ELNKAVQGTGLAFIAFTEAMTHFPASPFWSVMFFLMLVNLGLGSMFGTIEGIITPVVDTF 490
4O Q: 752 KVRKEILTVICCLLAFCIGLIFVQRSGNYFVTMFDDYSATLPLLIWILENIAVCFVYGI 931
KVRKEILTVICCLLAFCIGLIFVQRSGNYFVTMFDDYSATLPLLIWILENIAV FVYGI
S: 491 KVRKEILTVICCLLAFCIGLIFVQRSGNYFVTMFDDYSATLPLLIWILENIAVSFVYGI 550
4S Q' 932 DKFMED*K 955
DKFMED K
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S: 551 DKFMEDLK 558
Score = 842 (296.4 bits), Expect = 2.1e-189, Sum P(3) = 2.1e-189
Identities = 157/174 (90%), Positives = 167/174 (95%), Frame = +1
Q: 949 LKDMLGFAPSRYYWMWKYISPLMLLSLLIASVVNMGLSPPGYNAWIEDKASEEFLSYPT 1128
LKDMLGF P+RYYWMWKYISPLMLLSLLIAS+VNMGLSPPGYNAW+EDKASE+FLSYPT
S: 557 LKDMLGFTPNRYYYYMWKYISPLMLLSLLIASIVNMGLSPPGYNAWMEDKASEKFLSYPT 616
1 O Q: 1129 WGLWCVSLWFAILPVPWFIVRRFNLIDDSSGNLASVTYKRGRVLKEPVNLEGDDTSL 1308
WG+V+C+SL+V AILP+PWFI+RR NLIDDSSGNLASWYKRGRVLKEPVNLEGDD SL
S: 617 WGMVICISLMVLAILPIPWFIIRRCNLIDDSSGNLASVTYKRGRVLKEPVNLEGDDASL 676
Q: 1309 IHGKIPSEMPSPNFGKNIYRKQSGSPTLDTAPNGRYGIGYLMADIMPDMPESDL 1470
IS IHGKI SEM SPNFGKNIYRKQSGSPTLDTAPNGRYGIGYLMAD MPDMPESDL
S: 677 IHGKISSEMSSPNFGKNIYRKQSGSPTLDTAPNGRYGIGYLMAD-MPDMPESDL 729
The segments of dbj ~BAA77223.1 ~ that are shown as "S" above are set out in
the
sequence listing as SEQ m NO: 78 and SEQ m NO: 80 Based on the structural
20 similarity these homologous polypeptides are expected to share at least
some
biological activities. Such activities are known in the art, some of which are
described
elsewhere herein. Assays for determining such activities are also known in the
art,
some of which have been described elsewhere herein.
In specific embodiments, polypeptides of the invention comprise, or
25 alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ m NO: 79 and/or SEQ m NO: 81 which correspond to the Q sequences in the
alignment shown above (gaps introduced in a sequence by the computer are, of
course, removed). Moreover, fragments and variants of these polypeptides (such
as,
for example, fragments as described herein, polypeptides at least 80%, 85%,
90%,
30 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 ) axe encompassed by the invention.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
This gene is expressed primarily in the following tissueslcDNA libraries: H.
35 Whole Brain #2, re-excision; Human Ovaxian Cancer Reexcision.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
40 11, 15, and 18, and elsewhere herein.
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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 N0:22 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 2469 of SEQ m
N0:22, b
is an integer of 15 to 2483, where both a and b correspond to the positions of
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
This gene is expressed primarily in the following tissues/cDNA libraries:
CD34 positive cells (Cord Blood).
The tissue distribution in CD34c positive cells indicates polynucleotides and
polypeptides corresponding to this gene would be useful for the detection,
treatment,
and/or prevention of immune disorders, cancer or inflammatory conditions.
Representative uses are described in the "Regeneration" , "Hyperproliferative
Disorders" , and " Immune Disorders" sections below, in Example 1 l, 15, and
18,
and elsewhere herein.
Preferred polypeptides of the present invention comprise immunogenic
epitopes shown in SEQ ID NO: 52 as residues: Arg-19 to Gly-26. Polynucleotides
encoding said polypeptides are also encompassed by the invention. Specific
embodiments of the invention also include antibodies that bind any of these
epitopes.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:23 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
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general formula of a-b, where a is any integer between 1 to 1165 of SEQ ID
N0:23, b
is an integer of 1 S to 1179, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID N0:23, and where b is greater than or
equal to a
+ 14.
S
FEATURES OF PROTEIN ENCODED SY GENE NO: 14
This gene is expressed primarily in the following tissues/cDNA libraries:
Human Endometrial Tumor and to a lesser extent in Activated T-cells, 24 hrs,re-
excision; Osteoblasts; Human Rhabdomyosarcoma; Human Pancreas Tumor,
10 Reexcision; Keratinocyte; Activated T-Cells, 24 hrs.; Soares breast
2NbHBst;
NCI CGAP Panl; Activated T-Cell (l2hs)/Thiouridine labelledEco; Human
Neutrophil, Activated; Endothelial-induced; Human Microvascular Endothelial
Cells,
fract. A; Soares breast 3NbHBst; Activated T-cell(12h)/Thiou-idine-re-
excision;
Colon Normal III; Amniotic Cells - Primary Culture; L428; NCT CGAP-Gas4;
1 S Human Pancreas Tumor; Smooth muscle, serum treated; Colon Carcinoma;
NTER.A2, control; Human Adult Pulmonary,re-excision; Human Placenta; HUMAN
B CELL LYMPHOMA; Hunan Bone Marrow, treated; Soares ovary tumor NbHOT;
neutrophils control; Soares testis NHT; Primary Dendritic Cells, lib l; Soares
infant
brain 1NIB; NCI CGAP Brl.l; pBMC stimulated w/ poly IIC; H. Lymph node
20 breast Cancer; KMH2; CHME Cell Line,treated S hrs; CHME Cell
Line,untreated;
Colon Tumor; PC3 Prostate cell line; CD34 positive cells (Cord Blood); Colon
Tumor
II; Soares~regnant uterus NbHPU; Merkel Cells; Ovary, Cancer(4004650 A3):
Well-Differentiated Micropapillary Serous Carcinoma; Human Fetal Lung III;
Soares senescent fibroblasts NbHSF; Human Adult Heart,re-excision; Bone Marrow
2S Cell Line (RS4,11); NCI CGAP GCB1; Stromal Cells; HEL cell line;
NCI CGAP Lyml2; Breast, Normal: (400SS22B2); Human Umbilical Vein,
Reexcision; Stratagene NT2 neuronal precursor 937230; Human Thymus; HUMAN
JCTRKAT MEMBRANE BOUND POLYSOMES; Human umbilical vein endothelial
cells, IL-4 induced; Human Adipose; Ulcerative Colitis; Macrophage (GM-CSF
30 treated); Human Testes Tumor, re-excision; Stratagene liver (#937224); B-
cells
(unstimulated); Ovary, Cancer: (4004576 A8); Human Fetal Kidney, Reexcision;
Human Fetal Heart; Spleen, Chronic lymphocytic leukemia; Soares placenta
Nb2HP;
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31
Soares fetal liver spleen 1NFLS; Messangial cell, frac l; NCI CGAP Lul; Human
Neutrophils, Activated, re-excision; Smooth muscle, control, re-excision;
Lung,
Cancer (4005163 B7): Invasive, Poorly Diff. Adenocarcinoma, Metastatic;
Amniotic
Cells - TNF induced; Human Skin Tumor; Messangial cell, frac 2; Stratagene
placenta (#937225); Breast, Cancer: (4005522 A2); B Cell lymphoma; Synovial
hypoxia-RSF subtracted; NCI CGAP Col4; Myoloid Progenitor Cell Line; Human
Neutrophil; TF-1 Cell Line GM-CSF Treated; Ovary, Cancer: (15799A1F) Poorly
differentiated carcinoma; Human Uterine Cancer; NCI CGAP Co3; Ovarian Tumor
10-3-95; breast lymph node CDNA library; H Macrophage (GM-CSF treated), re-
excision; Human Testes Tumor; Adipocytes; Human Ovarian Cancer Reexcision;
Human Eosinophils; Smooth muscle,control; Hodgkin's Lymphoma II; T cell helper
II; Human Cerebellum; Human Umbilical Vein Endothelial cells, frac B, re-
excision;
Human Microvascular Endothelial Cells, fract. B; CD34+ cell, I, frac II; Lung,
Normal: (4005313 B1); Colon, Cancer: (9808C064R)-total RNA; H Umbilical Vein
Endothelial Cells, frac A, re-excision; Human Kidney; HUMAN STOMACH; Human
Fetal Bone; Stromal cells 3.88; Weizmann Olfactory Epithelium; CD40 activated
monocyte dendritic cells; NCI CGAP Ut3; Stromal cells(HBM3.18); Synovial IL-
1/TNF stimulated; Human Fetal Epithelium (Skin); Human Pre-Differentiated
Adipocytes; LPS activated derived dendritic cells; NCI CGAP Ut2; Human Adult
Small Intestine; Stratagene neuroepithelium (#937231); Mo7e Cell Line GM-CSF
treated (lng/ml); NCI CGAP_Utl; Breast Cancer Cell line, angiogenic; Monocyte
activated, re-excision; Ovary, Cancer: (4004332 A2); Human Umbilical Vein
Endothelial Cells, uninduced; Human Activated T-Cells; Liver, Hepatoma;
NCI CGAP Br2; Olfactory epithelium,nasalcavity; Healing groin wound, 7.5 hours
post incision; Human Placenta (re-excision); Human adult testis, large
inserts;
Smooth muscle, serum induced,re-exc; NTERA2 + retinoic acid, 14 days; Human
Thymus Stromal Cells; Normal colon; NCI CGAP_GC4; Colon Normal II; Ovary,
Cancer (9809C332): Poorly differentiated adenocarcinoma; Dendritic cells,
pooled;
human tonsils; Endothelial cells-control; Human Primary Breast Cancer
Reexcision;
Human fetal heart, Lambda ZAP Express; T Cell helper I; normalized infant
brain
cDNA; Soares melanocyte 2NbHM; Soares fetal lung NbHLI9W; H. Leukocytes,
Kozak; H Amygdala Depression, subtracted; LNCAP, differential expression; HL-
60,
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32
unstimulated; Human Eosinophils; Namalwa Cells; Aryepiglottis Normal; Colon,
normal; CD34+ cell, I; Larynx Carcinoma; Human Pancreatic Langerhans; Tongue
Normal; Human Tonsils, lib I; NCI CLAP HN3; Normal trachea; K562 + PMA (36
hrs); Human Adult Heart; Human Fetal Liver, subtracted, neg clone; Hurnan
epidermal keratinocyte; Breast, Cancer: (9802C020E); Breast, Cancer: (4005385
A2); Tongue Tumour; Human 7 Weeks Old Embryo, subtracted; Human Prostate,
subtracted; LNCAP + o.3nM 81881; Human Tonsil, Lib 3; Human Colon Cancer,
subtracted; Human epithelioid sarcoma; Human OB HOS treated (1 nM E2) fraction
I; HL-60, RA 4h, Subtracted; NCI CGAP Larl; NCI CGAP_GCS;
NCI CLAP Pr2l; NCI CGAP Pr4.l; NCI CGAP_Col l; Human OB HOS control
fraction T; Human OB MG63 treated (10 nM E2) fraction I; Human Aortic
Endothelium; Human Colon Carcinoma (HCC) cell line; Normal Human Trabecular
Bone Cells; NCI CGAP Pr25; NCI CGAP_Ov23; Adipocytes,re-excision; Activated
T-cells; Smooth muscle-ILb induced; Human Primary Breast Cancer; Breast Lymph
node cDNA library; Hunan Quadriceps; Activated T-Cells,l2 hrs,re-excision;
NCI CLAP GC3; Cem cells cyclohexamide treated; NCI CGAP Ut4; Human
Normal Breast; Human Lung Cancer,re-excision; NTERA2 teratocarcinoma cell
line+retinoic acid (14 days); Lung, Cancer (4005313 A3): Invasive Poorly
Differentiated Lung Adenocarcinoma,; Human Tonsils, Lib 2; STROMAL -
OSTEOCLASTOMA; Hepatocellular Tumor; H Female Bladder, Adult;
Glioblastoma; Human Stomach,re-excision; NCI CGAP_ColO; Human
Osteosarcoma; HL-60, PMA 4H, re-excision; Human Colon, re-excision; LNCAP
prostate cell line; Spleen metastic melanoma; Breast, Cancer: (4004943 AS);
Prostate
BPH; Human Chronic Synovitis; CD34 depleted Buffy Coat (Cord Blood); T-Cell
PHA 16 hrs; Human Bone Marrow, re-excision; TNFR degenerate oligo;
NCI CGAP Pr22; NCI CGAP Kid6; Human Fetal Kidney; B-Cells;
NCI CGAP Pr28; Human Heart; T-Cell PHA 24 hrs; Human Activated Monocytes;
Human Osteoblasts II; Human Hippocampus; Human Dermal Endothelial
Cells,untreated; Human Activated T-Cells, re-excision; Human Adult Testes,
Large
Inserts, Reexcision; Human Chondrosarcoma; NCI CGAP_CLLl; Human Fetal
Brain; Bone Marrow Stromal Cell, untreated; Human Whole Six Week Old Embryo;
Human Gall Bladder; Human endometrial stromal cells-treated with progesterone;
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33
Human Substantia Nigra; Pancreas Islet Cell Tumor; Human Synovial Sarcoma;
Bone
marrow; Human Testes, Reexcision; Neutrophils IL-1 and LPS induced; Primary
Dendritic cells,frac 2; B-cells (stimulated); NCI CGAP Brn25;
Soares~lacenta 8to9weeks 2NbHP8to9W; Soares fetal heart NbHHI9W and
Soares NhHMPu S 1.
The tissue distribution in immune tissues indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as ATDS, 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, Tense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
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,
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34
antibodies directed against the protein may show utility as a tumor marker
and/or
immunotherapy targets for the above listed tissues.
Preferred polypeptides of the present invention comprise immunogenic
epitopes shown in SEQ m NO: 53 as residues: Lys-107 to Lys-119.
Polynucleotides
encoding said polypeptides are also encompassed by the invention. Specific
embodiments of the invention also include antibodies that bind any of these
epitopes.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ m N0:24 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 369 of SEQ m
N0:24, b
is an integer of 15 to 383, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 15
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
emb~CAA19317.1 ~ (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "~ (AL023779)
hypothetical protein [Schizosaccharomyces pombe]" A partial alignment
demonstrating the observed homology is shown immediately below.
>emb~CAA19317.1~ (AL023779) hypothetical protein [Schizosaccharomyces pombe]
>pir~T39963~T39963 hypothetical protein SPBC244.02c - fission yeast
(Schizosaccharomyces pombe) >sp~060188'060188 HYPOTHETICAL 55.6 KD
PROTEIN.
Length = 488
Plus Strand HSPs:
Score = 158 (55.6 bits), Expect = 4.7e-17, Sum P(2) = 4.7e-Z7
Identities = 35/99 (35~), Positives = 53/99 (53~), Frame = +1
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Q: 343 RVQGVFQRASAKWKDDVQLWLSYVAFCKKWATKTRLSKVFSAMLAIHSNKPALWIMAAKW 522
+V +F RA+ K+ DV LWL Y+ + +K + K+ A L H N LW++A
S: 91 KVLFLFLRATNKFFGDVTLWLDYIHYAQKIKAVNIVGKICVAALQKHPNNAELWWACDH 150
S Q: 523 EMEDRLSSESARQLFLRALRFHPECPKLYKEYFRMELMH 639
E + +AR L RALR + E P ++ YFR+EL +
S: 151 EFSINANVSAARALMNRALRLNQENPVIWAAYFRLELSY 189
Score = 140 (49.3 bits), Expect = 4.7e-17, Sum P(2) = 4.7e-17
10 Identities = 32/75 (42%), Positives = 44/75 (58%), Frame = +2
Q: 74 MAEIIQERIEDRLPELEQLERIGLFSHAEIKAIIKKASDLEYKIQRRTLFKEDFINYVQY 253
MAE +Q +E +PELE L +F+ EI TIK E K+ RR + DF++Y+QY
5: 1 MAEKVQYYMEQSVPELEDLLEKNIFNRDEINNIIKTRRVFEEKLARRQVKLNDFLSYIQY 60
1S
Q: 254 EINLLELIQRRRTRI 298
EINL L +R R+
S: 61 EINLETLRAKRHKRL 75
20 The segments of emb~CAA19317.1 ~ that are shown as "S" above are set out in
the
sequence listing as SEQ lD NO: 82 and SEQ ID NO: 84 Based on the structural
similarity these homologous polypeptides are expected to share at least some
biological activities. Such activities are known in the art, some of which are
described
elsewhere herein. Assays for determining such activities are also known in the
art,
2S some of which have been described elsewhere herein.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 83 and/or SEQ ID NO: 8S which correspond to the Q sequences in the
alignment shown above (gaps introduced in a sequence by the computer are, of
30 course, removed). Moreover, fragments and variants of these polypeptides
(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 hybridizes, under stringent conditions, to
the
polynucleotide encoding these polypeptides ) are encompassed by the invention.
3S Polynucleotides encoding these polypeptides are also encompassed by the
invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
NCI CGAP-Ovl; Human promyelocyte; Stratagene fibroblast (#937212); stromal
cell clone 2.5; Human Whole Brain, re-excision; NCI CGAP Lyml2;
NCI CGAP Utl; Ovary, Cancer: (4004562 B6) Papillary Serous Cystic Neoplasm,
Low Malignant Pot; NCI CGAP Br2; human tonsils; CD34 depleted Buffy Coat
(Cord Blood), re-excision; Monocyte activated; Primary Dendritic Cells, lib 1.
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Preferred polypeptides of the present invention comprise immunogenic
epitopes shown in SEQ ID NO: 54 as residues: Gln-53 to Asn-58. Polynucleotides
encoding said polypeptides are also encompassed by the invention. Specific
embodiments of the invention also include antibodies that bind any of these
epitopes.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
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
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 2060 of SEQ ID
N0:25, b
is an integer of 15 to 2074, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:25, and where b is greater than or equal
to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 86 . 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.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Primary Dendritic Cells, lib 1 and to a lesser extent in Human Tongue, frac l;
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Primary Dendritic cells,frac 2; Human Fetal Heart, Differential (Fetal-
Specific);
NCI CGAP Pr22; Olfactory epithelium,nasalcavity; Healing groin wound, 7.5
hours
post incision; Soares fetal liver spleen 1NFLS; Healing Abdomen Wound,21&29
days post incision; NCI CGAP Eso2; Human Liver, normal; NCI CGAP Panl;
Healing groin wound, 6.5 hours post incision; Palate normal; Palate carcinoma;
Chromosome 7 Placental cDNA Library; Larynx carcinoma II; Human Tongue, frac
2; Human Tonsils, lib I; Larynx Normal; Tongue Tumour; NCI CGAP Larl; Lung,
Normal: (4005313 Bl); Lung, Cancer (4005163 B7): Invasive, Poorly Diff.
Adenocarcinoma, Metastatic; Human Liver; Human Lung; Human Tonsils, Lib 2;
Human adult (K.Okubo); Human Fetal Epithelium (Skin); Salivary Gland, Lib 2;
. Jurkat T-cell Gl phase; NCI CGAP Ut2; NCI CGAP Pr2; NCI CGAP_CLLl;
Resting T-Cell Library,II; Human Testes Tumor; Human Fetal Heart; human
tonsils;
Spleen, Chronic lymphocytic leukemia; H. Frontal cortex,epileptic,re-excision;
Hodgkin's Lymphoma II; Human 8 Week Whole Embryo; Keratinocyte and
Soares fetal liver spleen 1NFLS_S1.
Preferred polypeptides of the present invention comprise one, two, or all
three
immunogenic epitopes shown in SEQ ID NO: 55 as residues: Ala-50 to Pro-55, Leu-
62 to Glu-69, Ser-81 to His-88. Polynucleotides encoding said polypeptides are
also
encompassed by the invention. Specific embodiments of the invention also
include
antibodies that bind any of these epitopes.
The tissue distribution in ixmnune cells (e.g., dendritic cells) indicates the
polynucleotides and polypeptides corresponding to this gene would be useful
for the
diagnosis and treatment of a variety of immune system disorders.
Representative uses
are described in the "Immune Activity" and "Infectious Disease" sections
below, in
Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the
expression indicates a role in regulating the proliferation; survival;
differentiation;
and/or activation of hematopoietic cell lineages, including blood stem cells.
Involvement in the regulation of cytokine production, antigen presentation, or
other
processes suggests a usefulness for treatment of cancer (e.g. by boosting
immune
responses). Expression in cells of lymphoid origin, indicates the natural gene
product
would be involved in immune functions. Therefore it would also be useful as an
agent for immunological disorders including arthritis, asthma,
immunodeficiency
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diseases such as A>DS, 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, tense
tissue
injury, demyelination, systemic lupus erythematosis, drug induced hemolytic
anemia,
rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the
protein may
represent a secreted factor that influences the differentiation or behavior of
other
blood cells, or that recruits hematopoietic cells to sites of injury. Thus,
this gene
product is thought to be useful in the expansion of stem cells and committed
progenitors of various blood lineages, and in the differentiation and/or
proliferation of
various cell types. 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 >D 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
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 1854 of SEQ m
N0:26, b
is an integer of 15 to 1868, 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 BY GENE NO: 17
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
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gb~AAF04571.1~AF188181_1 (all information available through the recited
accession
number is incorporated herein by reference) which is described therein as
"(AF188181) G-protein gamma 12 subunit [Homo sapiens~" A partial alignment
demonstrating the observed homology is shown immediately below.
>gb~AAF04571.1~AF188181_1 (AF188181) G-protein gamma 12 subunit [Homo Sapiens]
>gb~AAF17220.1~AF119663_1 (AF119663) G-protein gamma-l2 subunit
[Homo Sapiens] >sp~AAF04571~AAF04571 G-protein gamma 12 subunit.
>spIAAF172201AAF17220 G-protein gamma-12 subunit.
1 0 Length = 72
Plus Strand HSPs:
Score = 356 (125.3 bits), Expect = 2.3e-30, P = 2.3e-30
1 5 Identities = 72/72 (100%), Positives = 72f72 (100%), Frame = +3
Q: 207 MSSKTASTNNIAQARRTVQQLRLEASIERIKVSKASADLMSYCEEHARSDPLLIGIPTSE 386
MSSKTASTNNIAQARRTVQQLRLEASIERIKVSKASADLMSYCEEHARSDPLLIGIPTSE
S: 1 MSSKTASTNNIAQARRTVQQLRLEASIERIKVSKASADLMSYCEEHARSDPLLIGIPTSE 60
Q: 387 NPFKDKKTCIIL 422
NPFKDKKTCIIL
S: 61 NPFKDKKTCIIL 72
The segment of gb~AAF04571.1 ~AF188181_1 that is shown as "S" above is set out
in
the sequence listing as SEQ m NO: 87 Based on the structural similarity these
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ m NO: 88 which corresponds to the Q sequence in the alignment shown above
(gaps introduced in a sequence by the computer are, of course, removed).
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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
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This gene is expressed primarily in the following tissues/cDNA libraries:
Soares placenta Nb2HP and to a lesser extent in Soares melanocyte 2NbHM;
Soares fetal heart NbHHI9W; Bone Marrow Stromal Cell, untreated; normalized
infant brain cDNA; Soares fetal liver spleen 1NFLS_S1; Soares infant brain
1N1B;
5 Synovial Fibroblasts (control); CHME Cell Line,treated 5 hrs;
Soares~regnant uterus NbHPU; NCI CGAP Utl; Colon Carcinoma;
Soares senescent fibroblasts NbHSF; Soares fetal liver spleen 1NFLS;
NCI CGAP Panl; Endothelial cells-control; NCI CGAP Kids;
Soares multiple sclerosis 2NbHMSP; Soares~lacenta 8to9weeks 2NbHP8to9W;
10 Human 8 Week Whole Embryo; Human Pre-Differentiated Adipocytes; Human
Osteoblasts II; Healing groin wound, 6.5 hours post incision; Colon Normal II;
Pancreas normal PCA4 No; Human Osteoclastoma; PC3 Prostate cell line;
NCI CGAP Kid3; Spleen, Chronic lymphocytic leukemia; Human fetal heart,
Lambda ZAP Express; Osteoblasts; Soares~arathyroid tumor NbHPA; Colon
15 Tumor II; Colon Normal III; Stratagene fibroblast (#937212); NCI CGAP Ut4;
Messangial cell, frac 2; H Female Bladder, Adult; Human Colon, re-excision;
NCI CGAP Ut2; Stratagene lung carcinoma 937218; H. Kidney Medulla, re-
excision; NCI CGAP Prl; Stratagene ovarian cancer (#937219); human ovarian
cancer; NCI CGAP_Gas4; Stromal cell TF274; Human Rhabdomyosarcoma; Human
20 Adipose; NCI CGAP_Co3; CHME Cell Line,untreated; Human Pancreas Tumor,
Reexcision; NCI CGAP'Co8; Normal colon; Ovary, Cancer (9809C332): Poorly
differentiated adenocarcinoma; Human Fetal Heart; Soares ovary tumor NbHOT;
Hodgkin's Lymphoma II; Soares total fetus Nb2HF8 9w;
Soares NFL T GBC_S1; Soares NhHMPu S1; NCI CGAP Lu28; Human
25 Macrophage; Human Rejected Kidney, 704 re-excision; NCI CGAP-Ov35; Larynx
Normal; Colon Normal; Activated T-Cells, 8 hrs, subtracted; Human Prostate,
subtracted; Colon, Cancer: (9808C064R); Saos2, Dexamethosome Treated;
NCI CGAP GCS; prostate-edited; Human Aortic Endothelium;, Jia bone marrow
stroma; Normal Human Trabecular Bone Cells; SKIN; Human Umbilical Vein
30 Endothelial Cells, fract. A; Adipocytes,re-excision; Adenocarcinoma of
Ovary,
Human Cell Line, # OVCAR-3; Fetal Heart, re-excision; Smooth Muscle Serum
Treated, Norm; Aorta endothelial cells + TNF-a; Stromal cells 3.88; Stromal
Cells;
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41
Smooth Muscle- HASTE normalized; Human Normal Breast; Human adult small
intestine,re-excision; Human Synovium; Synovial IL-1/TNF stimulated; Smooth
muscle, IL,Ib induced; Human normal ovary(#9610G215); NCI CGAP~Ov2; Human
Adipose Tissue, re-excision; Synovial hypoxia; LNCAP prostate cell line;
Prostate
BPH; Healing groin wound - zero hr post-incision (control); Human Adult Small
Intestine; Stratagene neuroepithelium (#937231); TF-1 Cell Line GM-CSF
Treated;
Human Brain, Striatum; H. Epididiymus, cauda; Stratagene fetal spleen
(#937205);
Stratagene fetal retina 937202; Ovary, Cancer: (4004332 A2); 12 Week OId Early
Stage Human, II; Human Pancreas Tumor; Ovary, Cancer: (4004562 B6) Papillary
Serous Cystic Neoplasm, Low Malignant Pot; Stratagene endothelial cell 937223;
Human umbilical vein endothelial cells, IL-4 induced; Healing groin wound, 7.5
hours post incision; Human Whole Six Week Old Embryo; Hepatocellular Tumor, re-
excision; Human Ovary; Ovary, Cancer: (4004576 A8); Ovarian Tumor 10-3-95;
Smooth muscle, serum induced,re-exc; Fetal Heart; Stratagene colon (#937204);
12
IS Week Old Early Stage Human; Human endometrial stromal cells-treated with
progesterone; Human Thymus Stromal Cells; Smooth muscle, serum treated;
NCI CGAP GC4; Adipocytes; Pancreas Islet Cell Tumor; Human Placenta; Human
Fetal Lung III; NTERA2, control; Endothelial-induced; Human Adult Pulmonary,re-
excision; Human Amygdala; Stratagene lung (#937210); Human Microvascular
Endothelial Cells, fract. A; Human Placenta; Smooth muscle,control;
NCI CGAP Brn23; Pancreas Tumor PCA4 Tu; Human Testes; Keratinocyte; Nine
Week Old Early Stage Human; Soares fetal lung NbHLI9W; Soares testis NHT;
NCI CGAP HN6; NCI CGAP HN8 and NCI CGAP Ov39.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:27 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides axe
specifically
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42
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 4403 of SEQ m
N0:27, b
is an integer of 15 to 4417, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:27, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 18
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
dbj ~BAA92076.1 ~ (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "~ (AK002083)
unnamed protein product [Homo sapiens]" A partial alignment demonstrating the
observed homology is shown immediately below.
>dbj~BAA92076.1~
(AK002083)
unnamed
protein
product
[Homo
sapiens]
Length = 262
Plus
Strand
HSPS:
Score 396 (139.4 bits), Expect = 2.0e-35, P
= = 2.0e-35
Identities
= 93/205
(45%),
Positives
= 128/205
(62%),
Frame
= +3
Q: 321 VSHSVLSEMQVTEQETPVSAKSSRSQLDLFDDVGTFASGPPKYKDNPFSLGESFGSRWD-497
+SHSV S+MQ IEQE+P+ AK R D DD ++ + +Y D
P L S S WD
S: 62 ISHSVTSDMQTIEQESPIMAKPRRKYNDDSDD--SYFTSSSRYFDEPVELRSSSFSSWDD119
3 Q: 498 -TDAAWGMDRVEEKEP--EVTISSIRPISERATNRREVESRSSGLESSEARQKFAGAKAI668
O
+D+ W + ++ E + T S RP + R + VE+ + EA++KF
KAI
S: 120 SSDSYWKKETSKDTETVLKTTGYSDRPAARRKPDYEPVEN------TDEAQKKFGNVKAI173
Q: 669 SSDMFFGREVDAEYEARSRLQQLXXXXXXXXXDLFGDMDGAHGAGSVSLGNVLPTA-DIA845
3 SSDM+FGR+ A+YE R+RL++L DLF + AG+ SL +VLP
S A D+A
S: 174 SSDMYFGRQSQADYETRARLERLSASSSISSADLFEE-PRKQPAGNYSLSSVLPNAPDMA232
Q: 846 QFKQGVKSVAGKMAVLANGVMNSLQDRYGS 935
QFKQGV+SVAGK++V ANGV+ S+QDRYGS
40 S: 233 QFKQGVRSVAGKLSVFANGVVTSIQDRYGS 262
The segment of dbj ~BAA92076.1 ~ that is shown as "S" above is set out in the
sequence listing as SEQ m NO: 89 Based on the structural similarity these
homologous polypeptides are expected to share at least some biological
activities.
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43
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
When tested against H4IIe cell lines, supernatants removed from cells
containing this gene activated the ELK-1 kinase assay. Assays for activation
of Elkl
kinase are well known in the art. For example, see, e.g., Cell 5:19-27 (1975);
Curr
Biol. 1995 Oct 1;5(10):1191-2000.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 90 which corresponds to the Q sequence in the alignment shown above
(gaps introduced in a sequence by the computer are, of course, removed).
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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Human adult testis, large inserts; Soares infant brain 1NIB and to a lesser
extent in
NCI CGAP Brn25; Soares testis NHT; Soares fetal liver spleen 1NFLS_S1;
Human Adult Testes, Large Inserts, Reexcision; Human Primary Breast Cancer
Reexcision; NCI CGAP_GCBl; NCI CGAP Lul; Stratagene fetal spleen
(#937205); Soares adult brain N2b5HB55Y; breast lymph node CDNA library; Brain
frontal cortex; Soares senescent fibroblasts NbHSF; NCI CGAP Kids;
Soares_placenta 8to9weeks_2NbHP8to9W; Soares fetal lung NbHLI9W; Human
Cerebellum; Bone Cancer, re-excision; Bone Cancer; NCI CGAP Ut4; Human retina
cDNA randomly primed sublibrary; H. Whole Brain #2, re-excision; Stratagene
placenta (#937225); Human Frontal Cortex, Schizophrenia; NCI CGAP_ColO;
Soaxes adult brain N2b4HB55Y; Human Infant Brain; Gessler Wilms tumor;
NCI CGAP Utl; H. Epididiymus, cauda; Human Fetal Kidney; Human Umbilical
Vein Endothelial Cells, uninduced; NCI CGAP Gas4; Human umbilical vein
endothelial cells, IL-4 induced; NCI CGAP_Co3; NTERA2 + retinoic acid, 14
days;
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Rejected Kidney, lib 4; Stratagene colon (#937204); NCI CGAP-CoB; Human
Synovial Sarcoma; Human Fetal Lung III; HUMAN B CELL LYMPHOMA; Spleen,
Chronic lymphocytic leukemia; Soares ovary tumor NbHOT; Human Endometrial
Tumor; normalized infant brain cDNA; Keratinocyte;
Soares total fetus Nb2HF8 9w; Soares~fetal heart NbHHI9W;
Soares NhHMPu SI; Soaxes fetal liver spleen 1NFLS; NCI CGAP_Co2l; Human
Uterine Cancer, subtracted; 1-N7B; NCI CGAP Brn52; Colon, tumour; Human
Pituitary; Infant brain, LLNL array of Dr. M. Soares 1NIB; Larynx Normal;
Human
epidermal keratinocyte; NCI CGAP-Co4; NCI CGAP_Co2; NCI CGAP Eso2;
Human Colon, subtraction; Infant brain, Bento Soares; Human Colon; Human Adult
Retina; Smooth Muscle Ser~un Treated, Norm; Smooth muscle, control, re-
excision;
Human Primary Breast Cancer; Human heart cDNA (YNakamura); Human Soleus;
Cem cells cyclohexamide treated; Raji Cells, cyclohexamide treated; Human
Normal
Breast; Human Lung Cancer,re-excision; Human Tonsils, Lib 2; Hmnan normal
ovary(#96106215); Ovarian Cancer, # 97026001; NCI CLAP Prl2;
NCI CGAP Lyml2; Jurkat T-Cell, S phase; Jurkat T-cell G1 phase; Spleen
metastic
melanoma; Human Manic Depression Tissue; H. Meningima, M1; H. Lymph node
breast Cancer; Human Bone Marrow, re-excision; Human Brain, Striatum;
NCI CGAP Pr22; Human Fetal Dura Mater; Macrophage-oxLDL; Human Adipose;
NCI CGAP-CLLl; Soares breast 2NbHBst; Human Fetal Brain; Stratagene liver
(#937224); NCI CGAP Panl; B-cells (unstimulated); CHME Cell Line,untreated;
Fetal Heart; Palate normal; Human Substantia Nigra; NCI CGAP-GC4; Human Fetal
Kidney, Reexcision; Pancreas normal PCA4 No; Bone marrow; Human Testes,
Reexcision; Human Adult Pulmonary,re-excision; Anergic T-cell; Human
Microvasculax Endothelial Cells, fract. A; Human Adult Heaxt,re-excision;
Monocyte
activated; Human Testes; NCI CGAP LuS; Human 8 Week Whole Embryo;
Soares~arathyroid tumor NbHPA; Nine Week Old Early Stage Human; Colon
Tumor II; Soares NFL T GBC S 1 and NCI CGAP GUl.
The tissue distribution and activity in the Elk-I assay indicates that
polynucleotides and polypeptides corresponding to this gene would be useful
for the
detection, treatment, and/or prevention of various endocrine disorders,
metabolic
disorders, immune disorders, and cancers. Representative uses are described in
the
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"Biological Activity", "Hyperproliferative Disorders", and "Binding Activity"
sections below, in Example 1 l, 17, 18, 19, 20 and 27, and elsewhere herein.
Briefly,
the protein can be used for the detection, treatment, and/or prevention of
Addison's
disease, Cushing's Syndrome, and disorders and/or cancers of the pancreas
(e.g.
5 diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-,
hypopituitarism),
thyroid (e.g. hyper-, hypothyroidism), parathyroid (e.g. hyper-
,hypoparathyroidism) ,
hypothallamus, and testes. 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
10 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
issues.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
15 related to SEQ ID N0:28 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
20 general formula of a-b, where a is any integer between 1 to 2190 of SEQ ll~
N0:28, b
is an integer of 15 to 2204, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:28, and where b is greater than or
equal to a
+ 14.
25 FEATURES OF PROTEIN ENCODED BY GENE NO: 19
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
gb~AAC50425.1 ~ (all information available through the recited accession
number is
30 incorporated herein by reference) which is described therein as "~ similar
to the 87
kDA Torpedo acetylcholine receptor-associated protein; similar to human
dystrophin-
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related protein, PIR Accession Number 503966 [Homo Sapiens]" A partial
alignment
demonstrating the observed homology is shown immediately below.
>gb~AAC50425.1I similar to the 87 kDA Torpedo acetylcholine receptor-
associated
protein; similar to human dystrophin-related protein, PIR Accession
Number S03966 [Homo Sapiens] >spIQ13198IQ13198 DYSTROBREVIN-ZETA.
Length = 192
Plus Strand HSPS:
Score = 167 (58.8 bits), Expect = 1.3e-10, P = 1.3e-10
Identities = 33/33 (100%), Positives = 33/33 (100%), Frame = +1
Q: 382 QPPQQRSAPDISFTIDANKQQRQLIAELENKNR 480
IS QPPQQRSAPDISFTIDANKQQRQLIAELENKNR
S: 74 QPPQQRSAPDISFTIDANKQQRQLIAELENKNR 106
The segment of gb~AAC50425.1~ that is shown as "S" above is set out in the
sequence listing as SEQ m NO: 91 Based on the structural similarity these
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ m NO: 92 which corresponds to the Q sequence in the alignment shown above
(gaps introduced in a sequence by the computer are, of course, removed).
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. Polynucleotides encoding
these
polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Human T-cell lymphoma,re-excision; Human Cerebellum; Soares placenta Nb2HP;
Soares infant brain 1NIB and to a lesser extent in Human frontal cortex;
Activated T-
Cells, 4 hrs, subtracted; Larynx Normal; Human fetal lung; Alzheimers, spongy
change; Human Osteoclastoma, re-excision; Human Adipose Tissue, re-excision;
Human Neutrophil; T-Cell PHA 16 hrs; Healing groin wound, 7.5 hours post
incision;
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Hepatocellular Tumor, re-excision; NCI CLAP Kidl l; Pancreas Islet Cell Tumor;
Ovary, Cancer(4004650 A3): Well-Differentiated Micropapillary Serous
Carcinoma;
Bone marrow; H. Frontal cortex,epileptic,re-excision; normalized infant brain
cDNA
and Soares MZFiMPu S1.
The tissue distribution in immune tissues indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 1 ~, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as AmS, 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, Tense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
~25 hematopoietic cells to sites of injury. Thus, this gene product is thought
to be useful
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
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 N0:29 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 2224 of SEQ ID
N0:29, b
is an integer of 15 to 2238, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:29, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 20
This gene is expressed primarily in the following tissues/cDNA libraries:
Fetal
Heart and to a lesser extent in human tonsils; Ficolled Human Stromal Cells,
SFu
treated; Fetal Heart, re-excision; NCI CGAP Gas4; Soares breast 2NbHBst; Bone
marrow; Monocyte activated; Soares-pregnant uterus NbHPU and Soares fetal
liver
spleen 1NFLS.
Preferred polypeptides of the present invention comprise immunogenic
epitopes shown in SEQ ID NO: 59 as residues: Met-1 to Gln-7, Pro-23 to Thr-40,
Leu-85 to Lys-101. Polynucleotides encoding said polypeptides are also
encompassed
by the invention. Specific embodiments of the invention also include
antibodies that
bind any of these epitopes.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:30 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
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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 547 of SEQ m
N0:30, b
is an integer of 15 to 561, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:30, and where b is greater than or
equal to a
+ 14.
FEATITRES OF PROTEIN ENCODED BY GENE NO: 21
The computer algorithm BLASTX has been used to determine that the
translation product of this gene shares sequence homology with, as a non-
limiting
example, the sequence accessible through the following database accession no.
emb~CAB56188.1 ~ (all information available through the recited accession
number is
incorporated herein by reference) which is described therein as "~ c380A1.2.1
(novel
protein (isoform 1)) [Homo Sapiens]" A partial alignment demonstrating the
observed
homology is shown immediately below.
>emb~CAB56188.1~ c380A1.2.1 (novel protein (isoform 1)) [Homo Sapiens]
>sp~CAB56188~CAB56188 C380A1.2.1 (novel protein (isoform 1)).
Length = 293
Plus Strand HSPs:
Score = 469 (165.1 bits), Expect = 8.6e-44, P = 8.6e-44
2 5 Identities = 88/103 (85%), Positives = 88/103 (85%), Frame = +3
Q: 9 HGIIHGVTHDVELQESVITWAARVLRQTPPLFQAGRSGDQGLTSIRTPLRCGVHPGPGT 188
HGIIHGVTHDVELQESVITWAARVLRQTPPLFQAGRSGDQGLTSIRTPLRCGVHPGPGT
S: 191 HGIIHGVTHDVELQESVITWAARVLRQTPPLFQAGRSGDQGLTSIRTPLRCGVHPGPGT 250
Q: 189 FLFMGWSRFGEARLGCAPXXXXXXXXXXXXXXXHLHPCEVALH 317
FLFMGWSRFGEARLGCAP HLHPCEVALH
S: 251 FLFMGWSRFGEARLGCAPRFQEFRRAYEAARAAHLHPCEVALH 293
The segment of emb~CAB56188.I ~ that is shown as "S" above is set out in the
sequence listing as SEQ ll~ NO: 93 Based on the structural similarity these
homologous polypeptides are expected to share at least some biological
activities.
Such activities are known in the art, some of which are described elsewhere
herein.
Assays for determining such activities are also known in the art, some of
which have
been described elsewhere herein.
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In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ll~ NO: 94 which corresponds to the Q sequence in the alignment shown
above
(gaps introduced in a sequence by the computer are, of course, removed).
Moreover,
5 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. Polynucleotides encoding
these
10 polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissuesIcDNA libraries:
NCI CGAP Brn25; Human Primary Breast Cancer Reexcision and to a lesser extent
in NCI CGAP_GC6; NCI CGAP Pr28; .Human Primary Breast Cancer,re-excision;
NCI CGAP Bni23; NCI CGAP_Col6; NCI CGAP Pr22; Stratagene pancreas
15 (#937208); NCI CGAP Kidll; Soares total fetus Nb2HF8 9w; Subtracted human
retinal pigment epithelium (RPE); C7MCF7 cell line, estrogen treated;
NCI CGAP Brn52; Human Pancreatic Langerhans; STRIATUM DEPRESSION;
NCI CGAP Brn35; Human Epididymus; Human endometrial stromal cells-treated
with estradiol; Human adult (K.Okubo); Human Frontal Cortex, Schizophrenia;
20 Human Hypothalmus,Schizophrenia; Human Liver, normal; Ovary, Cancer:
(4004576
A8); Human Fetal Lung III; H. Frontal cortex,epileptic,re-excision;
Soares-parathyroid tumor NbHPA; Soares-pregnant uterus NbHPU; Soares
placenta Nb2HP; Soares M~HMPu S 1 and Soares infant brain 1N1B.
The tissue distribution indicates polynucleotides and polypeptides
25 corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Preferred polypeptides of the present invention comprise immunogenic
30 epitopes shown in SEQ ID NO: 60 as residues: Ala-23 to Gly-38.
Polynucleotides
encoding said polypeptides are also encompassed by the invention. Specific
embodiments of the invention also include antibodies that bind any of these
epitopes.
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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 NO:31 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
N0:31, b
is an integer of 15 to 429, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:31, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 22
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares fetal liver spleen 1NFLS and to a lesser extent in Colon Normal III;
NCI CGAP Kid6; Liver, Hepatoma; Hepatocellular Tumor, re-excision; Human
Fetal Kidney, Reexcision; NCI CGAP Eso2; Hepatocellular Tumor,re-excision;
Hepatocellular Tumor; H. Kidney Medulla, re-excision; Human Fetal Kidney;
Human
Uterine Cancer; Human Rhabdomyosarcoma; Stratagene liver (#937224);
NCI CGAP CoB; Colon Normal II; H Macrophage (GM-CSF treated), re-excision;
Human Placenta; Soares retina N2b4HR; NCI CGAP Kids; Human Testes;
NCI CGAP LuS; Colon Tumor II; Soares~regnant uterus NbHPU;
Soares fetal liver spleen 1NFLS S1; Soares fetal heart NbHHI9W; Stomach
Tumour; Human colorectal cancer; Colon, Cancer: (9808C064R); Barstead pancreas
HPLRB1; Human Prostate Cancer, Stage B2 fraction; NCI CGAP_GCS; Human
Colon, subtraction; Human Liver; NCI CGAP Ut4; Serous Papillary
Adenocarcinoma; Human endometrial stromal cells-treated with estradiol;
Breast,
Cancer: (4005522 A2); Stratagene ovary (#937217); NCI CGAP Col4; H.
Meningima, M1; Prostate BPH; H. Epididiymus, cauda; Spinal cord; Ulcerative
Colitis; Soares breast 2NbHBst; Macrophage (GM-CSF treated); NCI CGAP_Co3;
Human Placenta (re-excision); Human adult testis, large inserts; Ovary,
Cancer:
(4004576 A8); Rejected Kidney, lib 4; Fetal Heart; Fetal Liver, subtraction
1I; Colon
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Tumor; Human endometrial stromal cells-treated with progesterone; Human T-Cell
Lymphoma; Human Pancreas Tumor, Reexcision; Colon Carcinoma; Human Fetal
Heart; CD34 depleted Buffy Coat (Cord Blood), re-excision; NCI CGAP Kid3;
Smooth muscle,control; Soares~lacenta 8to9weeks 2NbHP8to9W; Human fetal
heart, Lambda ZAP Express; Hodgkin's Lymphoma II; Soares NFL T GBC_S1;
NCI CGAP Co20 and NCI CGAP Sub6.
The tissue distribution indicates that polynucleotides and polypeptides
corresponding to this gene are useful for treatment, prevention, detection and
diagnosis of disorders in digestive and development systems. For example, the
tissue
distribution in colon and colon cancer indicates that polynucleotides and
polypeptides
corresponding to this gene would be useful for diagnosis, treatment,
prevention and/or
detection of tumors, especially of the intestine, such as, carcinoid tumors,
lymphomas,
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis,
colorectal carcinoma, cancer of the colon, cancer of the rectum and carcinoid
tumors,
as well as cancers in other tissues where expression has been indicated. The
expression in the colon tissue may indicate the gene or its products can be
used to
treat, detect, prevent and/or diagnose disorders of the colon, including
inflammatory
disorders such as, congenital abnormalities, such as atresia and stenosis,
Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung disease;
enterocolitis,
such as diarrhea and dysentary, infectious enterocolitis, including viral
gastroenteritis,
bacterial enterocolitis, necrotizing enterocolitis, antiboitic-associated
colitis
(pseudomembranous colitis), and collagenous and lymphocytic colitis,
miscellaneous
intestinal inflammatory disorders, including parasites and protozoa, amoebic
colitis,
acquired immunodeficiency syndrome, transplantation, drug-induced intestinal
injury,
radiation enterocolitis, neutropenic colitis, diverticular colon disease
(DCD),
inflammatory colonic disease, idiopathic inflammatory bowel disease, such as
Crohn's
disease (CD), non-inflammatory bowel disease (non-IBD) colonic inflammation;
ulcerative disorders such as, ulcerative colitis (UC); eosinophilic colitis;
noncancerous tumors, such as, polyps in the colon, adenomas, leiomyomas,
lipomas,
and angiomas. 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
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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.
Preferred polypeptides of the present invention comprise one, two or all three
immunogenic epitopes shown in SEQ ID NO: 61 as residues: Glu-26 to Asn-36, Ser
43 to Arg-60, Pro-107 to Glu-116. Polynucleotides encoding said polypeptides
are
also encompassed by the invention. Specific embodiments of the invention also
include antibodies that bind any of these epitopes.
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:32 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 1008 of SEQ ID
N0:32, b
is an integer of 15 to 1022, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:32, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 23
This gene is expressed primarily in the following tissues/cDNA libraries:
Soares total fetus Nb2HF8_9w and to a lesser extent in Soares fetal liver
spleen
1NFLS; Soares fetal liver spleen 1NFLS Sl; Soares infant brain 1NIB;
Soares~regnant uterus NbHPU; Soares multiple sclerosis 2NbHMSP;
Soares fetal lung NbHLI9W; Soares NhHMPu S1; NCI CGAP-Sub3;
NCI CGAP Lu24; NCI CGAP_Col2; Stratagene fetal retina 937202;
NCI CGAP Pr28; NCI CGAP Kidl l; 12 Week Early Stage Human II, Reexcision;
Human Placenta; Human fetal heart, Lambda ZAP Express; normalized infant brain
cDNA; Soares melanocyte 2NbHM; Larynx normal #10 261-273; Normal Prostate;
NCI CLAP Kidl2; Human 8 Week Whole Embryo, subtracted; NCI CGAP_Ov36;
stromal cell clone 2.5; Stratagene neuroepithelium NT2RAMI 937234; Smooth
muscle-ILb induced; NCI CGAP Ut4; pBMC stimulated w/ poly I/C; H. Kidney
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Cortex, subtracted; Ovarian Cancer, # 97026001; Morton Fetal Cochlea; Jurkat T-
Cell, S phase; LNCAP prostate cell line; NCI CGAP Ewl; Human Prostate; T-Cell
PHA 16 hrs; H. Kidney Medulla, re-excision; Apoptotic T-cell; Ovary, Cancer:
(15799A1F) Poorly differentiated carcinoma; Ovary, Cancer: (4004332 A2); 12
Week
Old Early Stage Human, II; NCI CGAP_Gas4; Stratagene HeLa cell s3 937216;
Ovary, Cancer: (4004562 B6) Papillary Serous Cystic Neoplasm, Low Malignant
Pot;
Olfactory epithelium,nasalcavity; Hemangiopericytoma; Human Chondrosarcoma;
Human Gall Bladder; Fetal Heart; 12 Week Old Early Stage Human; Human
Substantia Nigra; NTERA2, control; Human Microvascular Endothelial Cells,
fract.
A; NCI CGAP Brn23; Soares~lacenta 8to9weeks 2NbHP8to9W; Pancreas Tumor
PCA4 Tu; Soares ovary tumor NbHOT; H. Frontal cortex,epileptic,re-excision;
Osteoblasts; Human 8 Week Whole Embryo; Soares~arathyroid tumor NbHPA;
Nine Week Old Early Stage Human; Colon Tumor II; Soares NFL T GBC_S1;
Soares fetal heart NbHHI9W; Primary Dendritic Cells, lib 1; NCI CGAP_GCB1;
NCI CGAP_Sar4; NCI CGAP_Sub4 and NCI CGAP Brn53.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
axe
related to SEQ ID N0:33 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 1438 of SEQ ID
N0:33, b
is an integer of I S to 1452, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID NO:33, and where b is greater than or
equal to a
+ 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 24
This gene is expressed primarily in the following tissues/cDNA libraries:
NCI CGAP Lu25 and to a lesser extent in NCI CGAP HSC2; Human Primary
Breast Cancer Reexcision; normalized infant brain cDNA; Soares infant brain
1NIB;
5 NCI CGAP Eso2; NCI CGAP Ut4; NCI CGAP_Col4; Human Whole Brain #2 -
Oligo dT > 1.SKb; Human Amygdala,re-excision; Human Infant Brain; Human Adult
Testes, Large Inserts, Reexcision; Human adult testis, large inserts; Human
Substantia
Nigra and Monocyte activated.
The tissue distribution indicates polynucleotides and polypeptides
10 corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly
15 available and accessible through sequence databases. Some of these
sequences are
related to SEQ ID N0:34 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
20 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 1226 of SEQ ID
N0:34, b
is an integer of 15 to 1240, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:34, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 25
This gene is expressed primarily in the following tissues/cDNA libraries:
Human Testes, Reexcision; Human Testes and to a lesser extent in Human Adult
Testes, Large Inserts, Reexcision; Human adult testis, large inserts;
Soares testis NHT; NCI CGAP GC4; H Female Bladder, Adult;
Soares NFL T GBC_S1; Human testis (C. De Smet); Testis 1 and Testis, normal.
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When tested against CTLL lines, supernatants removed from cells containing
this gene activated the GAS assay. Thus, it is likely that this gene activates
T-cells
through the Jak-STAT signal transduction pathway. The gamma activating
sequence
(GAS) is a promoter element found upstream of many genes which are involved in
the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction
pathway involved in the differentiation and proliferation of cells. Therefore,
activation of the Jak-STAT pathway, reflected by the binding of the GAS
element,
can be used to indicate proteins involved in the proliferation and
differentiation of
cells.
Moreover, the tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Preferred polypeptides of the present invention comprise one or both
immunogenic epitopes shown in SEQ 1D NO: 64 as residues: Phe-30 to Lys-37, Pro-
43 to Lys-75. Polynucleotides encoding said polypeptides are also encompassed
by
the invention. Specific embodiments of the invention also include antibodies
that bind
any of these epitopes.
The tissue distribution in immune cells and the fact that supernatants
containing the protein of the invention activates T-cells indicates the
polynucleotides
and polypeptides corresponding to this gene would be useful for the diagnosis
and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example
11,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia,
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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, lense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought
to.be useful
in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation and/or proliferation of various cell types.
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 N0:35 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 1067 of SEQ >D
N0:35, b
is an integer of 15 to 1081, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:35, and where b is greater than or equal
to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 95. Moreover, fragments and variants of these polypeptides (such
as, for
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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.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Human Eosinophils and to a lesser extent in Colon Normal III; NCI CGAP Panl;
Human 8 Week Whole Embryo; Soares fetal heart NbHHI9W; Soares testis NHT;
Soares infant brain 1NIB; Soares fetal liver spleen 1NFLS; Human Bone Marrow,
treated; Gessler Wilms tumor; Nine Week Old Eaxly Stage Human;
Soares total fetus Nb2HF8 9w; Human fetal heart, Lambda ZAP Express;
NCI CGAP Ut2; NCI CGAP Utl; NCI CGAP_Gas4; Smooth muscle,control;
Colon Tumor II; 12 Week Old Early Stage Human, II; Clontech human aorta polyA+
mRNA (#6572); Human Testes Tumor, re-excision; Healing groin wound, 6.5 hours
post incision; Fetal Heart; Human Placenta; T Cell helper I; Human Cerebellum;
Soares NhHMPu S1; Monocyte activated, re-excision; Human Chondrosarcoma;
Human Adipose; NTERA2, control; Human Fetal Heart; Monocyte activated; Human
Testes; Hodgkin's Lymphoma II; T cell helper II; Synovial Fibroblasts
(Ill/TNF),
subt; Synovial hypoxia; T-Cell PHA 16 hrs; Human T-cell lymphoma,re-excision;
human ovarian cancer; Human Uterine Cancer; Human Ovary; NCI CGAP_Co3;
Human Placenta (re-excision); Human Gall Bladder; NCI CGAP-GC4; Human
Testes Tumor; Dendritic cells, pooled; Human Fetal Lung III; Human Amygdala;
PC3 Prostate cell line; Osteoblasts; NCI CGAP Bm53; NCI CGAP Ut4; Stratagene
schizo brain 511; NCI CGAP_Col4; NCI CGAP Lyml2; H. Meningima, M1;
Breast, Cancer: (4004943 AS); Human Neutrophil; Human Prostate; KMH2;
NCI CGAP Kid6; B-Cells; Human Fetal Dura Mater; NCI CGAP Pr28; T-Cell
PHA 24 hrs; Healing groin wound, 7.5 hours post incision; PERM TF274; B-cells
(unstimulated); NTERA2 + retinoic acid, 14 days; Rej ected Kidney, lib 4;
Human T-
Cell Lymphoma; Human Pancreas Tumor, Reexcision; Soares breast 3NbHBst;
Pancreas Islet Cell Tumor; 12 Week Eaxly Stage Human II, Reexcision;
Endothelial
cells-control; Human Adult Pulmonaryre-excision; Human Microvascular
Endothelial Cells, fract. A; NCI CGAP LuS; Soares fetal lung NbHLI9W;
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Soares~regnant uterus NbHPU; Soares fetal liver spleen 1NFLS_Sl;
NCI CGAP_CMLl; NCI CGAP Kidl3; NCI CGAP Me115; NCI CGAP Brn35;
Human Fetal Brain; NCI CGAP Ov23; Pancreatic Islet; HUMAN STOMACH;
Apoptotic T-cell, re-excision; Human Synovium; Hepatocellular Tumor,re-
excision;
STROMAL -OSTEOCLASTOMA; Human Hypothalamus,schizophrenia, re-
excision; Stratagene placenta (#937225); Human adult (K.Okubo); Synovial
hypoxia-
RSF subtracted; Glioblastoma; Salivary Gland, Lib 2; Ovarian Cancer, #
97026001;
Human endometrial stromal cells; LNCAP prostate cell line; Prostate BPH;
Hippocampus, Alzheimer Subtracted; Human Infant Brain; H. Kidney Medulla, re-
excision; NCI CGAP Prl; L428; Human Pancreas Tumor; Stratagene HeLa cell s3
937216; Human Hypothalinus,Schizophrenia; Ovary, Cancer: (4004562 B6)
Papillary
Serous Cystic Neoplasm, Low Malignant Pot; Human Rhabdomyosarcoma;
NCI CGAP_CLLl; Human Adrenal Gland Tumor; Bone Marrow Stromal Cell,
untreated; Colon Tumor; 12 Week Old Early Stage Human; Resting T-Cell
Library,II;
Early Stage Human Brain; Adipocytes; Human Testes, Reexcision; Human
Neutrophil, Activated; NCI CGAP Kids; Soares multiple sclerosis 2NbHMSP;
normalized infant brain cDNA; NCI CGAP-GCBI; NCI CGAP_GUl; Human
Pancreas; NCI CGAP LiS; Human Testes; Human Eosinophils; NCI CGAP Br7;
WATM1; NCI CGAP PNS1; PCR, pBMC I/C treated; Aryepiglottis Normal; Testis,
normal; Human Umbilical Vein Endothelial cells, frac B, re-excision; brain
stem;
Salivary Gland, Lib 3; CD34+cells, II, FRACTION 2; Human Leukocytes;
NCI CGAP Lul9; Human fetal lung; LNCAP + 30nM 81881; NCI CLAP HSC1;
H. Meniingima, M6; NCI CGAP Lei2; Dermatofibrosarcoma Protuberance; Human
Adult Liver, subtracted; Human Fetal Brain, random primed; NCI CGAP LymS;
Human Gall Bladder, fraction II; Human OB HOS control fraction I; Human (HCC)
cell line liver (mouse) metastasis, remake; Human Cerebellum, subtracted; H.
Atrophic Endometrium; Human Umbilical Vein Endothelial Cells, fract. A; Human
Placenta; NCI CGAP Brl.l; Adenocarcinoma of Ovary, Human Cell Line, #
OVCAR-3; Fetal Heart, re-excision; Stratagene neuroepithelium NT2RAMI 937234;
H. cerebellum, Enzyme subtracted; Breast Lymph node cDNA library; Early Stage
Human Lung, subtracted; Stromal Cells; NCI CGAP GC3; CD34 positive cells (cord
blood),re-ex; Raji Cells, cyclohexamide treated; NCI CGAP Ut3; Lung Carcinoma
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A549 TNFalpha activated; NTERA2 teratocarcinoma cell line+retinoic acid (14
days); Human Epididymus; Lung, Cancer (4005313 A3): Invasive Poorly
Differentiated Lung Adenocarcinoma,; Human Tonsils, Lib 2; Breast, Cancer:
(4005522 A2); B Cell lymphoma; Human pancreatic islet; H. Kidney Cortex,
5 subtracted; Human Pre-Differentiated Adipocytes; Human Stomach,re-excision;
Serous Papillary Adenocarcinoma; Human Osteosarcoma; Morton Fetal Cochlea;
Human Adipose Tissue, re-excision; Myoloid Progenitor Cell Line; Jurkat T-
Cell, S
phase; wilm's tumor; Ovarian Cancer; NCI CGAP Alvl; Healing groin wound - zero
hr post-incision (control); LPS activated derived dendritic cells; H. Lymph
node
10 breast Cancer; Brain Frontal Cortex, re-excision; Stratagene lung carcinoma
937218;
NCI CLAP Pr3; Mo7e Cell Line GM-CSF treated (lng/ml); TF-1 Cell Line GM-
CSF Treated; Human Thymus; Ovary, Cancer: (15799A1F) Poorly differentiated
c~cinoma; Stratagene fetal spleen (#937205); Human Fetal Kidney; Human
Activated T-Cells; Stromal cell TF274; Macrophage-oxLDL; Ovary, Normal:
15 (9805C040R); Human Osteoblasts II; NCI CLAP Br2; Olfactory
epithelium,nasalcavity; Human Hippocampus; Spinal cord; Ovary, Cancer
(15395A1F): Grade II Papillary Carcinoma; Soares NSF F8 9W OT PA P S1;
Hemangiopericytoma; Ulcerative Colitis; Stratagene hNT neuron (#937233); Human
Whole Six Week Old Embryo; Stratagene liver (#937224); Ovarian Tumor 10-3-95;
20 Macrophage-oxLDL, re-excision; Palate carcinoma; Human Thymus Stromal
Cells;
Human Substantia Nigra; NCI CGAP Kidl l; NCI CGAP CoB; Normal colon;
Colon Normal II; Ovary, Cancer(4004650 A3): Well-Differentiated Micropapillary
Serous Carcinoma; Endothelial-induced; human tonsils; Anergic T-cell; Human
Osteoclastoma; B-cells (stimulated); NCI CGAP Brn23; CD34 positive cells (Cord
25 Blood); Soares~lacenta 8to9weeks 2NbHP8to9W; Bone Marrow Cell Line
(RS4,11); Human Endometrial Tumor; NCI CGAP_Co2l; NCI CGAP-Ov38;
NCI CGAP_Ov40; NCI CGAP Sar4; NCI CGAP Subl; NCI CGAP Sub3 and
NCI CGAP Subs.
The tissue distribution indicates polynucleotides and polypeptides
30 corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
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in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:36 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 3963 of SEQ ID
N0:36, b
is an integer of 15 to 3977, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:36, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 27
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 97 and/or SEQ ID NO: 99. 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. Polynucleotides encoding these polypeptides are also
encompassed by
the invention.
This gene is expressed primarily in the following tissues/cDNA libraries:
Prostate gland adenocarcinoma and to a lesser extent in NCI CGAP_GCB1;
Soares fetal heart NbHHI9W; Osteoblasts; Soares infant brain 1NIB;
NCI CGAP Utl; Human Microvascular Endothelial Cells, fract. A;
NCI CGAP LuS; Soares total fetus Nb2HF8 9w; Soares fetal liver spleen 1NFLS;
Human Umbilical Vein, Reexcision; NCI CGAP_Gas4; NCI CGAP Br2;
NCI CGAP Panl; Human Pancreas Tumor, Reexcision; Human Testes;
NCI CGAP Lu24; Morton Fetal Cochlea; NCI CGAP Lyml2; NCI CGAP Ut2;
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Hodgkin's Lymphoma II; Human 8 Week Whole Embryo; Colon Normal III; Primary
Dendritic Cells, lib l; NCI CGAP Ut4; human ovarian cancer; Ovary, Cancer
(15395A1F): Grade II Papillary Carcinoma; Hepatocellular Tumor, re-excision;
NCI CGAP_Co3; Human Placenta (re-excision); Palate normal; Normal colon;
Human Ovarian Cancer Reexcision; B-cells (stimulated); NCI CGAP Brn23; Spleen,
Chronic lymphocytic leukemia; Soares~arathyroid tumor NbHPA; Nine Week Old
Early Stage Human; Soares_pregnant uterus NbHPU; Hodgkin's Lymphoma I;
Stromal Cells; Synovial hypoxia-RSF subtracted; Human Adipose Tissue, re-
excision; HEL cell line; Myoloid Progenitor Cell Line; Synovial hypoxia;
Prostate
BPH; Hippocampus, Alzheimer Subtracted; Stratagene neuroepithelium (#937231);
Human Prostate; Gessler Wilms tumor; Human Adrenal Gland Tumor; Stratagene
hNT neuron (#937233); Stratagene liver (#937224); Rejected Kidney, lib 4;
Soares
breast 3NbHBst; NCI CGAP_GC4; Ovary, Cancer (9809C332): Poorly differentiated
adenocarcinoma; Pancreas Islet Cell Tumor; Endothelial cells-control; Human
Amygdala; Soares ovary tumor NbHOT; normalized infant brain cDNA;
Keratinocyte; Colon Tumor II; T cell helper II;
Soares fetal liver spleen 1NFLS_Sl; Soares NhHMPu S1; Soares testis NHT;
NCI CGAP_CMLl; NCI CGAP Lu27; Human Pancreas; 1-NIB;
NCI CGAP Mel3; human adult liver cDNA library; Kidney Pyramids; Activated T-
Cells, 4 hrs, subtracted; Pharynx Carcinoma; Human Brain; Human aorta polyA+
(TFujiwara); NCI CGAP Kidl2; H Umbilical Vein Endothelial Cells, frac A, re-
excision; NCI CGAP Br3; Whole 6 Week Old Embryo; NCI CGAP_Schl; Human
promyelocyte; Human Aortic Endothelium; Human Umbilical Vein Endothelial
Cells,
fract. A; NCI CGAP_Ov23; HUMAN STOMACH; Smooth muscle, control, re-
excision; Aorta endothelial cells + TNF-a; Human Liver; Stromal cells 3.88;
Human
Skin Tumor; CD40 activated monocyte dendridic cells; Human Pineal Gland;
NCI CGAP Ut3; Lung, Cancer (4005313 A3): Invasive Poorly Differentiated Lung
Adenocarcinoma,; Human Ovarian Cancer(#9807G017); STROMAL -
OSTEOCLASTOMA; Stratagene schizo brain 511; Human Prostate Cancer, Stage C
fraction; Human Umbilical Vein, Endo. remake; Human pancreatic islet; pBMC
stimulated w/ poly I/C; NCI CGAP_Col4; Jurkat T-Cell, S phase; LNCAP prostate
cell line; Ovarian Cancer; Healing groin wound - zero hr post-incision
(control); LPS
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activated derived dendritic cells; Human Adult Small Intestine; NCI CGAP Ewl;
H.
Kidney Medulla, re-excision; TF-1 Cell Line GM-CSF Treated; Monocyte
activated,
re-excision; Human T-cell lymphoma,re-excision; Stratagene fetal retina
937202;
L428; Human Umbilical Vein Endothelial Cells, uninduced; B-Cells; Human Fetal
Dura Mater; Stromal cell TF274; Macrophage-oxLDL; Human Pancreas Tumor;
Stratagene endothelial cell 937223; Liver, Hepatoma; Human Osteoblasts II;
Olfactory epithelium,nasalcavity; Human Rhabdomyosarcoma;
Soares NSF F8 9W OT PA P S1; Epithelial-TNFa and INF induced; B-cells
(unstimulated); Ovary, Cancer: (4004576 A8); Healing groin wound, 6.5 hours
post
10, incision; Macrophage-oxLDL, re-excision; NTERA2 + retinoic acid, 14 days;
Fetal
Heart; Fetal Liver, subtraction II; Stratagene colon (#937204); Smooth muscle,
serum
treated; Colon Carcinoma; NCI CGAP Kidl l; NCI CGAP CoB; Colon Normal II;
Ovary, Cancer(4004650 A3): Well-Differentiated Micropapillary Serous
Carcinoma;
Pancreas normal PCA4 No; Bone marrow; NTERA2, control; Primary Dendritic
cells,frac 2; Human Fetal Heart; human tonsils; Anergic T-cell;
Soares senescent fibroblasts NbHSF; Human Placenta; NCI CGAP Kids;
Soares multiple sclerosis 2NbHMSP; Human fetal heart, Lambda ZAP Express;
Human Bone Marrow, treated; Bone Marrow Cell Line (RS4,I 1); H. Frontal
cortex,epileptic,re-excision; Human Endometrial Tumor; Soares NFL T GBC_S1;
NCI CGAP GU1; NCI CGAP HNS; NCI CGAP Sub3 and NCI CGAP Me115.
The tissue distribution indicates polynucleotides and polypeptides
corresponding to this gene would be useful for the detection, treatment,
and/or
prevention of cancer or inflammatory conditions. Representative uses are
described
in the "Regeneration" and "Hyperproliferative Disorders" sections below, in
Example
11, 15, and 18, and elsewhere herein.
Preferred polypeptides of the present invention comprise one, two, or all
three
immunogenic epitopes shown in SEQ ID NO: 66 as residues: Gln-8 to Cys-13, Pro-
18
to Leu-24, His-62 to Pro-68. Polynucleotides encoding said polypeptides are
also
encompassed by the invention. Specific embodiments of the invention also
include
antibodies that bind any of these epitopes.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
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related to SEQ ID N0:37 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 3285 of SEQ ID
N0:37, b
is an integer of 15 to 3299, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:37, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 28
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 100 . 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.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
When tested against 3T3-L1 cell lines, supernatants removed from cells
containing this gene activated the ELK-1 kinase assay. Assays for activation
of Elkl
kinase are well known in the art. For example, see, e.g., Cell 5:19-27 (1975);
Curr
Biol. 1995 Oct 1;5(10):1191-2000.
This gene is expressed primarily in the following tissues/cDNA libraries:
Epithelial-TNFa and INF induced and to a lesser extent in Activated T-cells,
24
hrs,re-excision; Colon Normal III; Monocyte activated; Primary Dendritic
Cells, lib 1;
Soares fetal liver spleen 1NFLS; Stratagene lung (#937210); Keratinocyte;
Macrophage-oxLDL; T cell helper II; Soares W HMPu Sl; Human pancreatic islet;
NCI CGAP Panl; H Macrophage (GM-CSF treated), re-excision;
NCI CGAP Kids; Human OB MG63 control fraction I; NCI CGAP-Co9; LPS
activated derived dendritic cells; T-Cell PHA 16 hrs; Human T-cell lymphoma,re-
excision; Human Prostate Cancer, Stage C, re-excission; Human Pancreas Tumor;
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Macrophage (GM-CSF treated); NCI CGAP_CoB; Normal colon; Human
Endometrial Tumor; Snares infant brain 1NIB; Human Colon, subtraction; Human
OB MG63 treated (10 nM E2) fraction I; NCI CGAP KidB; Activated T-cells;
NCI CGAP Ut3; Lung Carcinoma A549 TNFalpha activated; STROMAL -
5 OSTEOCLASTOMA; Synovial hypoxia-RSF subtracted; Prostate BPH; TNFR
degenerate oligo; Monocyte activated, re-excision; Human Prostate Cancer,
Stage B2,
re-excision; Olfactory epithelium,nasalcavity; Human Activated T-Cells, re-
excision;
Ulcerative Colitis; Bone Marrow Stromal Cell, untreated; Stratagene liver
(#937224);
Hepatocellular Tumor, re-excision; NCI CGAP_Co3; Human Placenta (re-excision);
10 Human Pancreas Tumor, Reexcision; Ovary, Cancer (9809C332): Poorly
differentiated adenocarcinoma; Human Fetal Lung III; Snares retina N2b4HR;
Human Adult Pulmonary,re-excision; B-cells (stimulated); NCI CGAP Kid3;
Hodgkin's Lymphoma II; Activated T-cell(12h)/Thiouridine-re-excision; Colon
Tumor II; Soares~regnant uterus NbHPU; Snares fetal heart NbHHI9W;
15 NCI CGAP GCB1; Jurkat Cells, cyclohexamide treated, subtraction; Human
Activated T-Cells (II); Human Infant Adrenal Gland, subtracted; Kidney Cortex;
Spleen/normal; Cheek Carcinoma; Colon Normal; Activated T-Cells, 24 hrs.;
Colon,
tumour; Bone marrow stroma,treated; CD34+cells, II, FRACTION 2;
NCI CGAP Br3; NCI CGAP Larl; Lung, Normal: (4005313 B1); Resting T-Cell;
20 Larynx carcinoma III; NCI CGAP Prl l; Human promyelocyte; stomach cancer
(human); Human OB HOS treated (10 nM E2) fraction I; Jia bone marrow stroma;
Human Adult Pulmonary; Pancreatic Islet; HUMAN STOMACH; Human Thyroid;
Early Stage Human Lung, subtracted; STRATAGENE Human skeletal muscle cDNA
library, cat. #936215.; Stromal Cells; Smooth Muscle- HASTE normalized;
25 Hepatocellular Tumor,re-excision; Synovial IL-1/TNF stimulated; Human
normal
ovary(#9610G215); Human Pre-Differentiated Adipocytes; Human Osteoclastoma,
re-excision; NCI CGAP Lyml2; Ovarian Cancer; NCI CGAP Ut2; Human Adult
Small Intestine; Human Neutrophil; Human Chronic Synovitis; Breast, Normal:
(4005522B2); Human Prostate; Human Thymus; NCI CGAP Pr22; 12 Week Old
30 Early Stage Human, II; HUMAN JURKAT MEMBRANE BOUND POLYSOMES;
Stratagene pancreas (#937208); NCI CGAP Pr28; T~Cell PHA 24 hrs; Human
Chondrosarcoma; Snares breast 2NbHBst; Human Adrenal Gland Tumor; B-cells
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(unstimulated); Ovary, Cancer: (4004576 A8); Macrophage-oxLDL, re-excision;
Colon Tumor; Human endometrial stromal cells-treated with progesterone; Human
T-
Cell Lymphoma; Palate carcinoma; NCI CGAP Kidll; Colon Normal II; Human
Testes Tumor; Pancreas Tslet Cell Tumor; Dendritic cells, pooled; Human
Placenta;
Primary Dendritic cells,frac 2; human tonsils; Endothelial cells-control;
Human
Osteoclastoma; Human Primary Breast Cancer Reexcision; Smooth muscle,control;
NCI CGAP Brn23; Soares multiple sclerosis 2NbHMSP; HUMAN B CELL
LYMPHOMA; Human Bone Marrow, treated; T Cell helper I; Human Testes;
Osteoblasts; Soares~arathyroid tumor NbHPA; Soares total fetus Nb2HF8 9w;
Soares NFL T GBC_Sl; NCI CGAP Brl8; NCI CGAP_CMLl and
NCI CGAP Lu28.
The tissue distribution and activity in the Elk-1 assay indicates that
polynucleotides and polypeptides corresponding to this gene would be useful
for the
detection, treatment, and/or prevention of various endocrine disorders,
metabolic
disorders, immune disorders, and cancers. Representative uses are described in
the
"Biological Activity", "Hyperproliferative Disorders", and "Binding Activity"
sections below, in Example 11, 17, 18, 19, 20 and 27, and elsewhere herein.
Briefly,
the protein can be used for the detection, treatment, and/or prevention of
Addison's
disease, Cushing's Syndrome, and disorders and/or cancers of the pancreas
(e.g.
diabetes mellitus), adrenal cortex, ovaries, pituitary (e.g., hyper-,
hypopituitarism),
thyroid (e.g. hyper-, hypothyroidism), parathyroid (e.g. hyper-
,hypoparathyroidism) ,
hypothallamus, and testes. 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 N0:38 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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67
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 1721 of SEQ DJ
N0:38, b
is an integer of 15 to 1735, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:38, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 29
In specific embodiments, polypeptides of the invention comprise, or
alternatively consists of, the amino acid sequence set out in the sequence
listing as
SEQ ID NO: 101 and/or SEQ ID NO: 102 . 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 bythe invention. Polynucleotides encoding these polypeptides are
also
encompassed by the invention.
When tested against CTLL lines, supernatants removed from cells containing
. this gene activated the GAS assay. Thus, it is likely that this gene
activates T-cells
through the Jak-STAT signal transduction pathway. The gamma activating
sequence
(GAS) is a promoter element found upstream of many genes which are involved in
the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction
pathway involved in the differentiation and proliferation of cells. Therefore,
activation of the Jak-STAT pathway, reflected by the binding of the GAS
element,
can be used to indicate proteins involved in the proliferation and
differentiation of
cells.
This gene is expressed primarily in the following tissues/cDNA libraries:
Primary Dendritic Cells, lib 1 and to a lesser extent in Human Thymus; Human
Primary Breast Cancer Reexcision; Human Rhabdomyosarcoma; Human Pituitary,
subt IX; B-cells (stimulated); NCI CGAP Lyml2; LPS activated derived dendritic
cells; Activated T-cell(12h)/Thiouridine-re-excision; Soares placenta Nb2HP;
Gessler
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Wilms tumor; NCI CGAP Brn25; Monocyte activated; Hodgkin's Lymphoma II;
Normal Human Trabecular Bone Cells; Human Epididymus; Human normal
ovary(#9610G215); Human Whole Brain #2 - Oligo dT > l.SKb; Human endometrial
stromal cells; Human Brain, Striatum; Merkel Cells; Soares breast 2NbHBst;
Stratagene hNT neuron (#937233); NCI CGAP Paul; Smooth muscle, serum
treated; Human Pancreas Tumor, Reexcision; Colon Carcinoma; Human Adult
Pulinonary,re-excision; Stratagene lung (#937210); NCI CGAP Kid3; Human
Microvascular Endothelial Cells, fract. A; HUMAN B CELL LYMPHOMA; Human
Bone Marrow, treated; T cell helper II; Soares total fetus Nb2HF8 9w; Human
Cerebellum; Soares testis NHT; NCI CGAP GCB1; Human W fant Adrenal Gland,
subtracted; Spleen/normal; Human Fetal Liver- Enzyme subtraction; Colon,
tumour;
Larynx Tumour; Larynx Normal; Human Whole 6 Week Old Embryo (II], subt;
Stratagene corneal stroma (#937222); NCI CGAP Prl l; Infant brain, Bento
Soares;
Human Aortic Endothelium; Human Umbilical Vein Endothelial Cells, fract. A;
NCI CGAP-Ov23; Human Pituitary, subtracted; Adenocarcinoma of Ovary, Human
Cell Line, # OVCAR-3; Smooth Muscle Serum Treated, Norm; Human Neutrophils,
Activated, re-excision; Aorta endothelial cells + TNF-a; Activated T-cells;
Smooth
muscle-ILb induced; Breast Lymph node cDNA library; Stromal cells 3.88; Human
Quadriceps; Stromal Cells; NCI CGAP_GC3; Adenocarcinoma of Ovary, Human
Cell Line; NCI CGAP Ut4; NTER.A2 teratocarcinoma cell line+retinoic acid (14
days); Synovial IL-1/TNF stimulated; Human adult (K.Okubo); Breast, Cancer:
(4005522 A2); B Cell lymphoma; Stratagene schizo brain 511; Breast Cancer cell
line, MDA 36; Smooth muscle, ILlb induced; H Female Bladder, Adult; Synovial
hypoxia-RSF subtracted; NCI CGAP_ColO; HL-60, PMA 4H, re-excision; Human
Colon, re-excision; Synovial hypoxia; Jurkat T-cell Gl phase; Human Manic
Depression Tissue; Ovarian Cancer; H. Lymph node breast Cancer; Human Chronic
Synovitis; Spinal Cord, re-excision; NCI CGAP Utl; H. Epididiymus, cauda;
Stratagene pancreas (#937208); B-Cells; Human Activated Monocytes;
NCI CGAP Br2; Human umbilical vein endothelial cells, IL-4 induced; Human
blood platelets; Human Fetal Brain; Macrophage (GM-CSF treated); CHME Cell
Line,treated 5 hrs; NCI CGAP_Co3; Human Placenta (re-excision); Human Gall
Bladder; Ovarian Tumor 10-3-95; Macrophage-oxLDL, re-excision; Rej ected
Kidney,
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lib 4; Fetal Heart; Fetal Liver, subtraction II; Human Thymus Stromal Cells;
breast
lymph node CDNA library; Soares breast 3NbHBst; Normal colon; Colon Normal II;
Dendritic cells, pooled; Activated T-Cell (l2hs)/Thiouridine labelledEco;
Human
Neutrophil, Activated; Endothelial-induced; Endothelial cells-control; Human
Adult
Heart,re-excision; NCI CGAP Brn23; Soares~lacenta 8to9weeks 2NbHP8to9W; T
Cell helper I; NCI CGAP LuS; Osteoblasts; Soares~arathyroid tumor NbHPA;
Colon Tumor II; Soares-pregnant uterus NbHPU;
Soares fetal liver spleen 1NFLS Sl; Soares NhHMPu S1; Soares infant brain
1NIB; Soares fetal liver spleen 1NFLS; NCI CGAP_Col7; NCI CGAP Lu28 and
NCI CGAP Lu3l.
The tissue distribution in immune cells and the fact that supernatants
containing this protein activates T-cells indicates the polynucleotides and
polypeptides corresponding to this gene would be useful for the diagnosis and
treatment of a variety of immune system disorders. Representative uses are
described
in the "Immune Activity" and "Infectious Disease" sections below, in Example 1
l,
13, 14, 16, 18, 19, 20, and 27, and elsewhere herein. Briefly, the expression
indicates
a role in regulating the proliferation; survival; differentiation; and/or
activation of
hematopoietic cell lineages, including blood stem cells. Involvement in the
regulation
of cytokine production, antigen presentation, or other processes suggests a
usefulness
for treatment of cancer (e.g. by boosting immune responses). Expression in
cells of
lymphoid origin, indicates the natural gene product would be involved in
immune
functions. Therefore it would also be useful as an agent for immunological
disorders
including arthritis, asthma, immunodeficiency diseases such as 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, lense tissue injury, demyelination, systemic lupus
erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's
disease, and scleroderma. Moreover, the protein may represent a secreted
factor that
influences the differentiation or behavior of other blood cells, or that
recruits
hematopoietic cells to sites of injury. Thus, this gene product is thought to
be useful
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in the expansion of stem cells and committed progenitors of various blood
lineages,
and in the differentiation andlor proliferation of various cell types.
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
5 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.
Preferred polypeptides of the present invention comprise one or both
immunogenic epitopes shown in SEQ m NO: 68 as residues: His-48 to Ser-61, Ala-
10 66 to Val-72. Polynucleotides encoding said polypeptides are also
encompassed by
the invention. Specific embodiments of the invention also include antibodies
that bind
any of these epitopes.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
15 related to SEQ m N0:39 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polymucleotides 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
20 general formula of a-b, where a is any integer between 1 to 2212 of SEQ ID
N0:39, b
is an integer of 15 to 2226, where both a and b correspond to the positions of
nucleotide residues shown in SEQ m N0:39, and where b is greater than or equal
to a
+ 14.
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71
o r~
N .~-i M ON1 N ~ M N ~ ~O ,~-,
a~
N c1' O~ .~ 01 ~ V7
cn ~0 M N M M N M M
w v~ ~
4., op s.~~ r.,
M cf i m ~ c~~t c~i can
.-, ,-, ,~ ,-~ .~ r, ,~ .-. ,-~ ,~ ....
P,
o .-i N M ~ Two ~ oo a, o
d- d- ~r ~r d' ~r d' d- d- d- N V7
E ~
b0 ~ N ~ o M ~ ~ t~ ~ d,--n ~ m O
(1, N .-, .~ ~ O~ ~ N M M N .-~ 00
O
r,.T_,+,~~O N OM~d'I~ .~~--,N
N ~, O~ ~ l~ N M M N ,-.rM.,, 00
O
~-~ M M l~ M N ~ M O M o0 M
4-, 0 ~ ~_O l~ Ov N_ t~ ~ 00 Ov M O~ l 00
M O (~ U~ .~ '-, .--, .~ ~ M N N N ~ ~ N
ri H 41 ~ C
CCt
~ M U l~ M N ~ M O M 00 M
a' v0 l~ l~ N ~~ ~t o0 O\ M 01 I~ 00
O ''~ ~ ,--mn ~O ,--n O1 M t~ M N .WO ~t
V1 .--~ .-v ~ r-, ~ M N N N .~ .-~ N
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C/1 ,~L, ~ ,-n .~ .-r .~ .-n ,-, ,~ ,-n N N N
O O
O !s' P'' m
Nw
00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0
ø,~ M O M o M O M O M O M O M O M O M O M O M O M O
pp O pp O pp O pp O pp O pp O pp O p0 O pp O pp O pp O pp O
,~ N ,-, N r., N ,.~ N ,~ N r, N ,-, N r, N ,..., N ,-, N r., N ,-, N
G~ , ~ , ~ , ~
U'~ dodododododododododododo
U N E-~ n E-W E-~ n ~' n E-~ n H n E-~ n H n ~' i E'' n E-~ n E-~ n
~ o ~ o P~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o
00 0
Z~ q U ~ H ~ U ~ ~ ~ a ~ t~7
H H H ~ H ~
U x x x x
~.NMCt~~t~o00v
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WO 01/83510 PCT/USO1/13318
'"' ~ '-' ot~o °' o °° N d. yn
~O ~ 00 01 M ~ M ,~ M ~ l~
'~ fir'
N
4-i N ° ~ V7 M O l~ O~ M ~ d' ~ O
*' O V ~0 N N N M N ~ N N N N N
w rig w
~n s~.,
d' 'd' N 01 ~O o0 N d' M O Ov
N N N N N ~ N N N N
.-, .-, ,~ ,-n ,-i .~ ,--, ,--i ,~ ,--n .~ .-. ,-,
°;
O ~ S~ M ~ O\ ~~ N ~ ~O 01 N N ~_O t<1 d'
~ N N oo ~ O d~ N N N M ~t
O
Q N~o~o N'~MV~1N
U N N M O dr., N N N .d. .-r \O
O
H O Ov ~ 00 p~ d- 00.. N Ov O M
I_~ ~ h ~O V'~ O M ~ N N 'd' d' M
O U ~ ,-~ M N ~ ~ N N ~ ~' ~ .N-,
a>
~ _O ~ 00 ,-i ,~ ~yD O .-n .~ ,-, .--.i oOp ,-,
N v~ O U C/~ h N ~ M
"' Owt ~ _lwd- oo N N O
O ~ O ~ °~ O o~0 ~t N N ~ N O 'd N O
Q ~t M Wit' V7 l0 I~ 00 O\ O .~ N M d' V7
N N N N N N N M M M M M M
~r
O
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N ~t N ac ~ ~ '~ '~ ~~ O
W W U U
a i ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o
ø,~ M O M O M O M O M O M O M O M O M O M O M O M O M O
pp O pp O pp O pp O pp O pp O pp O pp O pp O pp O pp O pp O pp O
,~ N ,~ N ,.~ N r, N ,.~ N ,-, N ,_.., N r, N ,~ N r., N ,-, N r., N ,.~ N
01 O\ O~ D\ 01 O~ O\ 01 01 Q\ O\ O\ D\
U, ~odo~o~o~o~o~o~ododo~odo~o
U,N H,~H"H"H ;H~H"H ;H,~H ;H ;H~,E-i Hn
~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o ~ o p~ o ~ o ~ o ~ o
o~°om~~°oooo
~ W ~ O ~ H ~ z A
H
x x x x
M ~t W \o l~ 00 01 0 .~ N M V ~n
,U ~ ~ .-r .-, .-, .-, ,-, N N N N N N
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WO 01/83510 PCT/USO1/13318
w
o
a~o ~ '~
b ~
O ~ ~~ N due-N
O
w v
~A~ N d' N
as
p ~ M
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O
o ,~ v~ ,-. .--.
z~~
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O
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O N N ~ N
M M ~ N
O ~ C/~
o E-~ ~ ~ ~ M N
O~ N l~ N
M M ~ N
M M M M
O
~i U
U ~ U
..~'~ N
pp pp p~ pp
O O O O
i~A M M M M
p p p O
~ ~ ~ ~
N N N N
U ~ ~ ~ '~ ~
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, N N N N
. 'Z
U
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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
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
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."
and the "3' NT of Clone Seq." of SEQ ID NO:X. The nucleotide position of SEQ
ID
NO:X of the putative start codon (methionine) is identified as "5' NT of Start
Codon."
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
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
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."
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
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accurate and otherwise suitable for a variety of uses well known in the art
and
described further below. For instance, SEQ 7D NO:X is useful for designing
nucleic
acid hybridization probes that will detect nucleic acid sequences contained in
SEQ m
NO:X or the cDNA contained in the deposited clone. These probes will also
5 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 specifically to proteins containing the polypeptides and the
secreted
proteins encoded by the cDNA clones identified in Table 1.
10 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
15 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
20 generated nucleotide sequence identified as SEQ ID NO:X and the predicted
translated amino acid sequence identified as SEQ ID 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.
25 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.
30 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
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76
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 known in the art can be used to obtain full-
length
genes, allelic variants, splice variants, full-length coding portions,
orthologs, and/or
species homologs of genes corresponding to SEQ ID NO:X, 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 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 3, column 1, provides a nucleotide sequence identifier, "SEQ ID
NO:X," that matches a nucleotide SEQ ID NO:X disclosed in Table 1, column 5.
Table 3, column 2, provides the chromosomal location, "Cytologic Band or
Chromosome," of polynucleotides corresponding to SEQ ID NO:X. Chromosomal
location was determined by finding exact matches to EST and cDNA sequences
contained in the NCBI (National Center for Biotechnology Information) UniGene
database. Given a presumptive chromosomal location, disease locus association
was
determined by comparison with the Morbid Map, derived from Online Mendelian
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Inheritance in Man (Online Mendelian Inheritance in Man, OMIMTM. McKusick-
Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore,
MD)
and National Center for Biotechnology Information, National Library of
Medicine
(Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlin.nih.gov/omim/).
If the putative chromosomal location of the Query overlapped with the
chromosomal
location of a Morbid Map entry, the OMIM reference identification number of
the
morbid map entry is provided in Table 3, column 3, labelled "OMIM
Reference(s)."
A key to the OMIM reference identification numbers is provided in Table 5.
Table 4 provides a key to the Library Code disclosed in Table 2. Column 1
provides the Library Code disclosed in Table 2, 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".
Table 5 provides a key to the OMIM reference identification numbers
disclosed in Table 3, column 3. OMIM reference identification numbers (Column
1)
were derived from Online Mendelian Inheritance in Man (Online Mendelian
Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine,
Johns
Hoplcins University (Baltimore, MD) and National Center for Biotechnology
Information, National Library of Medicine, (Bethesda, MD) 2000. World Wide Web
URL: http://www.ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated
with the cytologic band disclosed in Table 3, column 2, as determined using
the
Morbid Map database.
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Table 2
Clone LibraryCodes
ID
HTTD033 H0002H0011H0012H0015H0039H0040H0083H0086H0087HO100
H0123H0124H0135H0144H0163H0181H0188H0255H0265H0306
H0352H0393H0412H0422H0423H0427H0428H0441H0444H0445
H0478H0484H0487H0494H0506H0509H0520H0521H0529H0539
H0542H0543H0545H0547H0549H0551H0555H0556H0574H0581
H0583H0606H0615H0617H0618H0620H0637H0638H0654H0656
H0657H0658H0660H0662H0668H0670H0672L12905000250007
50026S002750049SO110
50126
50132
S0142
50144
S0212
50242
50276S027850328S0344
50356
50358
S0360
50374
50376
50378
50380S040250406S0418 067
50434
50456
S0474
53014
T0002
T0
HTXCP27 H0009H0038H0041H0051H0052H0059H0090H0136H0144H0253
H0264H0265H0318H0333H0341H0351H0393H0412H0423H0424
H0435H0436H0458H0483H0484H0494H0506H0519H0521H0529
H0542H0543H0547H0553H0556H0560H0561H0580H0583H0586
H0593H0616H0617H0618H0619H0620H0634H0643H0649H0656
H0657H0667H0668H0685H0686H0702H0707H0714H0716L1290
5000250031S003650038 0126
S0040 S0152
5 50194
S0250
50270
503545035650360S0376 0406
50404 S0408
5 50418
50420
50424
504405044450450
T0006
T0040
T0049
T0060
HTXGK12 H0013H0024H0038H0090H0265H0270H0556H0563H0615H0624
L1290S033050368S0424
T0042
T0049
HTXPT57 H0556H0632L1290
HTLJC15 H0031H0038H0039H0046H0052H0068H0081H0083H0087H0150
H0156H0181H0253H0255H0263H0266H0309H0318H0327H0402
H0413H0423H0424H0436H0445H0494H0506H0509H0518H0519
H0521H0529H0530H0545~H0555H0571H0578H0581H0592H0604
H0615H0616H0617H0618H0622H0638H0653H0657H0658H0661
H0663H0670H0682H0684H0687H0725L129050036S0044
50046
50116S01265014250196 0278
50260 S0282
S S0300
50342
S0358
503605038050406S0408 0418
50410 50420
5 S0438
50444
S0474
53014T0004T0049
HUSCJ14 H0009H0013H0030H0052H0135H0144H0171H0265H0266H0268
H0290H0309H0393H0435H0488H0510H0519H0520H0521H0538
H0545H0547H0555H0556H0575H0587H0593H0599H0617H0620
H0634H0656H0657H0659H0663H0665H0667H0677H0713L1290
5002250031S0040S0106 0132
50122 S0134
S S0192
50210
50212
S0218S025050276S0360 0406
50404 50408
5 50410
S0420
50434
S043650476T0023T0040
T0041
T0042
T0060
HUVDG58 H0056H059550002
HWADV90 H0542H0581L1290
HYAAD61 H0583
HYBAQ24 H0036H0041H0271H0488H0509H0586H062250434
HTXFD86 H0013H0030H0052H0123H0178H0265H0329H0341H0370H0393
H0423H0427H0484H0486H0494H0518H0547H0553H0556H0581
H0593H0659H0660H0667H0672H0696H0707L1290S000250116
50146502065021250218 0378
50360 50440
5
HBXGL91 H0438H0615
HCWEB38 H0305
HCWEF04 H0002H0012H0014H0024H0026H0036H0039H0040H0041H0046
H0050H0051H0059H0068H0069H0081H0083H0086HO100HO101
H0124H0131H0135H0167H0179H0181H0188H0204H0207H0250
H0251H0252H0253H0254H0255H0256H0264H0265H0266H0267
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H0271H0280H0286H0290H0294H0295H0305H0306H0309H0316
H0318H0341H0351H0356H0370H0373H0406H0411H0412H0413
H0416H0421H0422H0427H0433H0435H0437H0439H0444H0445
H0449H0457H0458H0477H0484H0486H0487H0488H0492H0494
H0497H0506H0509H0518H0519H0520H0521H0522HOS29H0530
H0538HOS39HOS42HOS43H0545H0547H0551H0553H0556H0559
H0560H0561H0575H0580H0581H0583H0584H0585H0587H0593
H0594H0597H0598HOS99H0615H0616H0617H0618H0620H0622
H0623H0628H0634H0635H0638H0641H0644H0647H0652H0653
H0656H0657H0658H0660H0661H0662H0663H0665H0666H0667
H0669H0670H0671H0672H0676H0677L1290S002750028
50036
S0040S00445004550046 0116 152
50052 54126
5 50144
50150
S0
50196S0208S021050216 0276
50250 50278
5 50310
S0352
50354
5035650358S036050364 0374
50368 50376
5 50408
50418
S0420
50422504245042650430 0456
50446 53014
5 T0002
T0048
T0115
HDPAB86 H0264H0402H0521H0529H0539H0648H0657H0668L1290S0002
50386
HDPOT33 H0013H0040H0050H0081H0242H0264H0355H0375H0379H0380
H0436H0445H0478H0486H0487H0488H0494HOS10H0521H0522
H0586H0587H0593H0602H0641H0647H0649H0661H0672H0674
H0696L12905022250328 0406
S0330 50446
50358 S0448
50370
5
T0041
HE2AC74 H0013H0024H0031H0036H0038H0039H0050H0061H0124H0144
H0163H0170H0171H0188H0212H0222H0266H0268H0328H0345
H0388H0411H0412H0427H0431H0445H0486H0494H0539H0545
H0547H0553H0574H0575H0586HOS87H0590H0592H0597H0622
H0628H0631H0646H0648H0652H0653H0658H0665H0666H0672
H0675H0685H0687H0696H0716L1290500035001050026
50027
500285004450049:50126 0150
50146 S0192
S S0194
50210
S0212
S0250502805034250354 0358
50356 S0360
S S0374
50378
50380
S04185042050424S0434 0440
50436 S0442
5 S0444
50468
53012
53014T0048T0049
HE8TY46 H0009H0012H0013H0014H0024H0033H0036H0038H0041H0046
H0050H0052H0085HO100H0117H0123H0125H0134H0135H0144
H0181H0188H0194H0199H0231H0246H0253H025SH0264H0266
H0268H0288H0318H0341H0370H0381H0392H0412H0413H0419
H0421H0423H0427H0438H0444H0445H0494H0506H0519H0520
H0521H0522H0529H0539H0543H0547H0549H0550HOS55H0556
H0575H0594H0599H0613H0616H0617H0618H0619H0620H0656
H0660H0687H0690H0694H0706H0722H0728H0729H0733L1290
S0001S00025002850036 142
S0038
50049
50050
S0114
S0116
50
SO1S2S02185027850330 0360
50354 50364
S 50366
50378
S0390
S0406S04105041850420
50422
S0434
50436
50448
S04S8
53012
S301456028T0010T0041
T0042
T0082
HFADM09 H0052H0179H0220H0320H0422H0539H0574H0587H0591H0670
L1290S01165021450222 6024
50280
S0448
S
HFHDN80 H0264H0519H0619H0639H0645H0708L129050002S011650354
HHSDL07 SODS
1
HLDBL62 H0012H0031H0038H0050HOOS9H0090H0124H0166H0231H0331
H0392H0393H0402H0431H0486H0506H0509H0545H0546H0550
H0555H0574H0619H0620H0622H0632H0644H0663H0672H0675
H0684H0696H0722L1290S00445027850354 0360
50356
S0358
5
S03765040650410S0436 0444
50438
50440
5
HSJAY14 H0013H0014H0032H0046H0144H0170H0171H0244H0251H0266
H0293H0333H0366H0422H0423H0441H0494H0518H0520H0521
H0553H0593H0624H0648H0660H0666H0668H0690L129050032
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50036501265013450150
50222
S0306
S0360
S0380
50404
50438
50440T0042TO110
HTLDY30 H0051H0253H0255H0617H0618L129050002 S0036 50038
S0346
50358T0010
HTLIF12 H0038H0253H0411H0616H0618L129050398
HE8FD92 H0013H0014H0024H0030H0032H0038H0052 H0059 H0070
H0090
H0098H0134H0144H0150H0163H0170H0171 H0172 H0178
H0251
H0261H0266H0316H0333H0351H0372H0422 H0423 H0427
H0428
H0436H0457H0478H0486H0519H0583H0586 H0587 H0591
H0619
H0622H0646H0648H0650H0652H0657H0659 H0661 H0670
H0682
H0685H0689H0690H0696H0710H0714H0716 L1290 50003
50007
5001050011S002650029 0114
50036 50126
5 50136
S0152
50222
50280503585036050376 0408
50398 50414
5 50422
S0434
50444
T0010
HTFOB75 H0008H0013H0029H0032H0036H0038H0039 H0046 H0050
H0056
H0068H0123H0124H0144H0156H0169H0220 H0264 H0266
H0268
H0316H0328H0341H0349H0355H0374H0393 H0413 H0423
H0431
H0437H0445H0485H0486H0494H0497H0509 H0518 H0519
H0520
H0521H0522H0529H0539H0543H0547H0551 H0553 H0555
H0556
H0561H0574H0581H0583H0586H0591H0592 H0593 H0615
H0619
H0622H0623H0638H0641H0644H0646H0650 H0653 H0656
H0657
H0658H0659H0665H0670H0672H0688H0689 H0694 H0704
H0717
H0722L1290S001050011
50016
S0026
S0027
50044
50045
50114
50116S01265013250142 0152'50194
50150 50212
5 S0222
50250
50276502805033050344 0354
50350 S0356
S 50358
S0360
50374
50378S0390S040650408 0422
50414 S0424
S 50426
50434
50436
5043850440S044450476
T0006
T0042
T0048
T0049
HCNSFO1 H0024H0032H0036H0039H0040H0042H0046 H0063 H0075
H0083
H0090H0107H0156H0167H0171H0179H0187 H0202 H0208
H0231
H0251H0264H0284H0286H0292H0309H0316 H0318 H0343
H0422
H0423H0459H0486H0494H0506H0521H0543 H0545 H0556
H0560
H0574H0575H0584H0591H0617H0622H0628 H0632 H0633
H0635
H0644H0649H0657H0658H0662H0672H0673 H0674 H0677
H0687
H0689H0701H0710H0713L1290S0002S0003 50011 50014
50044
S012650132S0142S0144 0212
50206 50214
5 50276
50278
50298
S03085032850348S0358 0372
S0360 S0374
5 50376
50404
50406
S040850410S042650434 04420444 50476 T0002
50440 5 T0067
5
HPMKB09 H0009H0014H0052H0087H0102H0107H0124 H0150 H0246
H0250
H0254H0255H0265H0266H0268H0271H0309 H0318 H0370
H0393
H0411H0412H0416H0424H0435H0483H0486 H0521 H0538
H0539
H0542H0543H0544H0547H0550H0551H0555 H0556 H0559
H0569
H0575H0580H0581H0583H0597H0599H0617 H0619 H0622
H0625
H0641H0644H0650H0653H0657H0663H0665 H0685 H0686
H0687
H0689H0696H0702H0708H0713H0717L1290 50002 50027
S0032
5003850045S0046S0049 01440194 50196 50260
50126 S 50276
S
503085034450354S0356 03640374 50406 S0410
50360 5 50418
5
504365044050442S0444 04500474 50476 53012
S0448 5 56028
5
T0002T0041T0048T0049
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Table 3
SEQ ID Cytologic Band OMIM
or Reference(s):
NO: X Chromosome:
11 17p13 138190254210271900600179600977601202
601777
19 17p13.3 113721247200600059601545
24 1q21-q25 104770107300107670110700131210134638
135940136132145001146740146760146790
150292152445159001159440173610174000
176310179755182860186780191030191315
208250227400230800233710249270266200
600897600923600995601105601412601518
601652601863602491
26 9p13 230400250250
33 12q13 107777123940139350148040148041148043
148070231550600194600231600536600808
600956601284601769601928602116602153
36 1q12-1q21.2 104770107670110700135940145001146760
146790152445159001174000179755182860
191315230800266200600897601105601412
601652601863602491
39 5q32-q33 109690131400138491154500180071181460
222600234000272750600807601411601596
602089
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Table 4
Library Librar Descri tion Disease
Code
H0002 Human Adult Heart
H0008 Whole 6 Week Old Embryo
H0009 Human Fetal Brain
H0011 Human Fetal Kidney
H0012 Human Fetal Kidney
H0013 Human 8 Week Whole Embryo
H0014 Human Gall Bladder
H0015 Human Gall Bladder, fraction II
H0024 Human Fetal Lung III
H0026 Namalwa Cells
H0029 Human Pancreas
H0030 Human Placenta
H0031 Human Placenta -
H0032 Human Prostate
H0033 Human Pituitary
. H0036 Human Adult Small Intestine
H0038 Human Testes
H0039 Human Pancreas Tumor disease
H0040 Human Testes Tumor disease
H0041 Human Fetal Bone
H0042 Human Adult Pulmonary
H0046 Human Endometrial Tumor disease
H0050 Human Fetal Heart
H0051 Human Hippocampus
H0052 Human Cerebellum
H0056 Human Umbilical Vein, Endo. remake
H0059 Human Uterine Cancer disease
H0061 Human Macro hage
H0063 Human Thymus
H0068 Human Skin Tumor disease
H0069 Human Activated T-Cells
H0070 Human Pancreas
H0075 Human Activated T-Cells (II)
H0081 Human Fetal Epithelium (Skin)
H0083 HUMAN JURKAT MEMBRANE BOUND POLYSOMES
H0085 Human Colon
H0086 Human epithelioid sarcoma disease
H0087 Human Thymus
H0090 Human T-Cell Lymphoma disease
H0098 Human Adult Liver, subtracted
HO100 Human Whole Six Week Old Embryo
HO101 Human 7 Weeks Old Embryo, subtracted
H0102 Human Whole 6 Week Old Embryo (II), subt
H0107 Human Infant Adrenal Gland, subtracted
HOl 17 Human Uterine Cancer, subtracted
H0123 Human Fetal Dura Mater
H0124 Human Rhabdomyosarcoma disease
H0125 Cem cells cyclohexamide treated
H0131 LNCAP + o.3nM 81881
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H0134 Raji Cells, cyclohexamide treated
H0135 Human Synovial Sarcoma
H0136 Su t Cells, cyclohexamide treated
H0144 Nine Week Old Early Stage Human
HO150 Human Epididymus
H0156 Human Adrenal Gland Tumor disease
H0163 Human Synovium
H0166 Human Prostate Cancer, Stage B2 fraction disease
H0167 Activated T-Cells, 24 hrs.
H0169 Human Prostate Cancer, Stage C fraction disease
H0170 12 Week Old Early Stage Human
H0171 12 Week Old Early Stage Human, II
H0172 Human Fetal Brain, random primed
H0178 Human Fetal Brain
H0179 Human Neutrophil
H0181 Human Primary Breast Cancer disease
H0187 Resting T-Cell
H0188 Human Normal Breast
H0194 Human Cerebellum, subtracted
H0199 Human Fetal Liver, subtracted, neg clone
H0202 Jurkat Cells, cyclohexamide treated, subtraction
H0204 Human Colon Cancer, subtracted
H0207 LNCAP, differential ex ression
H0208 Early Stage Human Lung, subtracted
H0212 Human Prostate, subtracted
H0220 Activated T-Cells, 4 hrs, subtracted
H0222 Activated T-Cells, 8 hrs, subtracted
H0231 Human Colon, subtraction
H0242 Human Fetal Heart, Differential (Fetal-Specific)
H0244 Human 8 Week Whole Embryo, subtracted
H0246 Human Fetal Liver- Enzyme subtraction
H0250 Human Activated Monocytes
H0251 Human Chondrosarcoma disease
H0252 Human Osteosarcoma disease
H0253 Htunan adult testis, large inserts
H0254 Breast Lymph node cDNA library
H0255 breast lymph node CDNA library
H0256 HL-60, unstimulated
H0261 H. cerebellum, Enzyme subtracted
H0263 human colon cancer disease
H0264 human tonsils
H0265 Activated T-Cell (l2hs)/Thiouridine IabelledEco
H0266 Human Microvascular Endothelial Cells, fract.
A
H0267 Human Microvascular Endothelial Cells, fract.
B
H0268 Human Umbilical Vein Endothelial Cells, fract.
A
H0270 HPAS (human pancreas, subtracted)
H0271 Human Neutrophil, Activated
H0280 K562 + PMA (36 hrs)
H0284 Human OB MG63 control fraction I
H0286 Human OB MG63 treated (10 nM E2) fraction
I
H0288 Human OB HOS control fraction I
H0290 Human OB HOS treated (1 nM E2) fraction I
H0292 Human OB HOS treated (10 nM E2) fraction I
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H0293 WI 38 cells
H0294 Amniotic Cells - TNF induced
H0295 Amniotic Cells - Primary Culture
H0305 CD34 positive cells (Cord Blood)
H0306 CD34 depleted Buffy Coat (Cord Blood)
H0309 Human Chronic Synovitis disease
H0316 HUMAN STOMACH
H0318 HUMAN B CELL LYMPHOMA disease
H0320 Human frontal cortex
H0327 human corpus colosum
H0328 h~.unan ovarian cancer disease
H0329 Dermatofibrosarcoma Protuberance disease
H0331 Hepatocellular Tumor disease
H0333 Hemangiopericytoma disease
H0341 Bone Marrow Cell Line (RS4,11)
H0343 stomach cancer (human) disease
H0345 SKIN
H0349 human adult liver cDNA library
H0351 Glioblastoma disease
H0352 wilin's tumor disease
H0355 Human Liver
H0356 Human Kidney
H0366 L428 cell line
H0370 H. Lymph node breast Cancer disease
H0372 Human Testes
H0373 Human Heart
H0374 Human Brain
H0375 Human Lung
H0379 Human Tongue, frac 1
H0380 Human Tongue, frac 2
H0381 Bone Cancer disease
H0388 Human Rejected Kidney, 704 re-excision disease
H0392 H. Meningima, M1
H0393 Fetal Liver, subtraction II
H0402 CD34 depleted Buffy Coat (Cord Blood), re-excision
H0406 H Amygdala Depression, subtracted
H0411 H Female Bladder, Adult
H0412 Human umbilical vein endothelial cells, IL-4
induced
H0413 Human Umbilical Vein Endothelial Cells, uninduced
H0416 Human Neutrophils, Activated, re-excision
H0419 Bone Cancer, re-excision
H0421 Human Bone Marrow, re-excision
H0422 T-Cell PHA 16 hrs
H0423 T-Cell PHA 24 hrs
H0424 Human Pituitary, subt IX
H0427 Human Adipose
H0428 Human Ovary
H0431 H. Kidney Medulla, re-excision
H0433 Human Umbilical Vein Endothelial cells, frac
B, re-excision
H0435 OvarianTumor 10-3-95
H0436 Resting T-Cell Library,II
H0437 H Umbilical Vein Endothelial Cells, frac
A, re-excision
H0438 H. Whole Bxain #2, re-excision
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H0439 Human Eosinophils
H0441 H. Kidney Cortex, subtracted
H0444 Spleen metastic melanoma disease
H0445 S Teen, Chronic lymphocytic leukemia disease
H0449 CD34+ cell, I
H0457 Human Eosino hils
H0458 CD34+ cell, I, frac II
H0459 CD34+cells, II, FRACTION 2
H0477 Human Tonsil, Lib 3
H0478 Salivary Gland, Lib 2
H0483 Breast Cancer cell line, MDA 36
H0484 Breast Cancer Cell line, angiogenic
H0485 Hodgkin's Lymphoma I disease
H0486 Hodgkin's Lymphoma II disease
H0487 Human Tonsils, lib I
H0488 Human Tonsils, Lib 2
H0492 HL-60, RA 4h, Subtracted
H0494 Keratinocyte
H0497 HEL cell line
H0506 Ulcerative Colitis
H0509 Liver, Hepatoma disease
HO510 Human Liver, normal
H0518 pBMC stimulated w! poly I/C
H0519 NTERA2, control
H0520 NTERA2 + retinoic acid, 14 days
H0521 Primary Dendritic Cells, lib 1
H0522 Primary Dendritic cells,frac 2
H0529 Myoloid Progenitor Cell Line
H0530 Human Dermal Endothelial Cells,untreated
H0538 Merkel Cells
H0539 Pancreas Islet Cell Tumor disease
H0542 T Cell helper I
H0543 T cell helper II
H0544 Human endometrial stromal cells
H0545 Human endometrial stromal cells-treated with
progesterone
H0546 Human endometrial stromal cells-treated with
estradiol
H0547 NTERA2 teratocarcinoma cell line+retinoic
acid (14 days)
H0549 H. Epididiymus, caput & corpus
HO550 H. Epididiymus, cauda
HO551 Human Thymus Stromal Cells
H0553 Human Placenta
HO555 Rejected Kidney, lib 4 disease
H0556 Activated T-cell(12h)/Thiouridine-re-excision
H0559 HL-60, PMA 4H, re-excision
H0560 KMH2
H0561 L428
H0563 Human Fetal Brain, normalized 50021F
H0569 Human Fetal Brain, normalized CO
H0571 Human Fetal Brain, normalized CSOOHE
H0574 He atocellular Tumor, re-excision disease
H0575 Human Adult Puhnonary,re-excision
H0578 Human Fetal Thymus
H0580 Dendritic cells, pooled
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H0581 Human Bone Marrow, treated
H0583 B Cell lymphoma disease
H0584 Activated T-cells, 24 hrs,re-excision
H0585 Activated T-Cells,12 hrs,re-excision
H0586 Healing groin wound, 6.5 hours post incision disease
H0587 Healing groin wound, 7.5 hours post incision disease
H0590 Human adult small intestine,re-excision
H0591 Human T-cell lym homa,re-excision disease
H0592 Healing oin wound - zero hr ost-incision (control)disease
H0593 Olfactory epithelium,nasalcavity
H0594 Human Lung Cancer,re-excision disease
H0595 Stomach cancer (human),re-excision disease
H0597 Human Colon, re-excision
H0598 Human Stomach,re-excision
H0599 Human Adult Heart,re-excision
H0602 Healing Abdomen Wound,21&29 days post incisiondisease
H0604 Human Pituitary, re-excision
H0606 Human Primary Breast Cancer,re-excision disease
H0613 H.Leukocytes, normalized cot 5B
H0615 Human Ovarian Cancer Reexcision disease
H0616 Human Testes, Reexcision
H0617 Human Primary Breast Cancer Reexcision disease
H0618 Human Adult Testes, Large Inserts, Reexcision
H0619 Fetal Heart
H0620 Human Fetal Kidney, Reexcision
H0622 Human Pancreas Tumor, Reexcision disease
H0623 Human Umbilical Vein, Reexcision
H0624 12 Week Early Stage Human II, Reexcision
H0625 Ku 812F Basophils Line
H0628 Human Pre-Differentiated Adipocytes
H0631 Saos2, Dexamethosome Treated
H0632 He atocellular Tumor,re-excision
H0633 Lun Carcinoma A549 TNFalpha activated disease
H0634 Human Testes Tumor, re-excision disease
H0635 Human Activated T-Cells, re-excision
H0637 Dendritic Cells From CD34 Cells
H0638 CD40 activated monocyte dendridic cells
H0639 Ficolled Human Stromal Cells, 5Fu treated
H0641 LPS activated derived dendritic cells
H0643 Hep G2 Cells, PCR library
H0644 Human Placenta (re-excision)
H0645 Fetal Heart, re-excision
H0646 Lung, Cancer (4005313 A3): Invasive Poorly
Differentiated Lung
Adenocarcinoma,
H0647 Lung, Cancer (4005163 B7): Invasive, Poorly disease
Diff. Adenocarcinoma,
Metastatic
H0648 Ovary, Cancer: (4004562 B6) >=' disease
.
H0649 Lung, Normal: (4005313 B1)
H0650 B-Cells
H0652 Lung, Normal: (4005313 B1)
H0653 Stromal Cells
H0654 Lung, Cancer: (4005313 A3) Invasive Poorly-differentiated
Metastatic lung
adenoc
H0656 B-cells (unstimulated)
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H0657 B-cells (stimulated)
H0658 Ovary, Cancer (9809C332): Poorly differentiateddisease
adenocarcinoma
H0659 Ovary, Cancer (15395A1F): Grade II Pa illary disease
Carcinoma
H0660 Ovary, Cancer: (15799A1F) Poorly differentiateddisease
carcinoma
H0661 Breast, Cancer: (4004943 AS) disease
H0662 Breast, Normal: (400552282)
H0663 Breast, Cancer: (4005522 A2) disease
H0665 Stromal cells 3.88
H0666 Ovary, Cancer: 4004332 A2) disease
H0667 Stromal cells(HBM3.18)
H0668 stromal cell clone 2.5
H0669 Breast, Cancer: (4005385 A2)
H0670 Ovary, Cancer(4004650 A3): Well-Differentiated
Micropapillary Serous
Carcinoma
H0671 Breast, Cancer: (9802C020E)
H0672 Ovary, Cancer: (4004576 A8)
H0673 Human Prostate Cancer, Stage B2, re-excision
H0674 Human Prostate Cancer, Stage C, re-excission
H0675 Colon, Cancer: (9808C064R)
H0676 Colon, Cancer: (9808C064R)-total RNA
H0677 TNFR degenerate oligo
H0682 Ovarian cancer, Serous Papillary Adenocarcinoma
H0684 Ovarian cancer, Serous Papillary Adenocarcinoma
H0685 Adenocarcinoma of Ovary, Human Cell Line,
# OVCAR-3
H0686 Adenocarcinoma of Ovary, Human Cell Line
H0687 Human normal ovary(#96106215)
H0688 Human Ovarian Cancer(#98076017)
H0689 Ovarian Cancer
H0690 Ovarian Cancer, # 97026001
H0694 Prostate cancer (adenocarcinoma)
H0696 Prostate Adenocarcinoma
H0701 NKyaolS(control)
H0702 NK15(IL2 treated for 48 hours)
H0704 Prostate Adenocarcinoma cell line cultured
in vivo in mice
H0706 Human Adult Skeletal Muscle
H0707 Stomach Cancer(5007635)
H0708 Human Skeletal Muscle
H0710 Patient #6 Acute Myeloid Leukemia/SGAH
H0713 Adipose tissue (diabetic type I, obese) #41706
H0714 Liver (diabetic type I, obese) #41616
H0716 Adipose tissue (diabetic type II)#41689
H0717 Adi ose tissue (diabetic type II) #41661
H0722 Diabetic Liver 99-09-A281a
H0725 Normal Adipose Tissue #41838-08
H0728 Diabetic Skeletal Muscle #42352-L
H0729 Diabetic II Skeletal Muscle #42366
H0733 Diabetic Skeletal Muscle #42483
L1290 Stratagene corneal stroma (#937222)
S0001 Brain frontal cortex
50002 Monocyte activated
S0003 Human Osteoclastoma disease
S0007 Early Sta a Human Brain
S0010 Human Amygdala
50011 STROMAL -OSTEOCLASTOMA disease
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50014 Kidney Cortex
S0016 Kidney Pyramids
50022 Human Osteoclastoma Stromal Cells - unam
lifted
50026 Stromal cell TF274
50027 Smooth muscle, serum treated
50028 Smooth muscle,control
50029 brain stem
50031 Spinal cord
50032 Smooth muscle-ILb induced
50036 Human Substantia Nigra
.
S0038 Human Whole Brain #2 - Oligo dT > l.SKb
50040 Adi ocytes
S0044 Prostate BPH disease
50045 Endothelial cells-control
S0046 Endothelial-induced
50049 Human Brain, Striatum
S0050 Human Frontal Cortex, Schizophrenia disease
50051 Human Hypothalinus,Schizophrenia disease
50052 neutrophils control
50106 STRIATUM DEPRESSION disease
SO110 Brain Amygdala Depression disease
50114 Anergic T-cell
50116 Bone marrow
S0122 Osteoclastoma-normalized A disease
50126 Osteoblasts
50132 Epithelial-TNFa and INF induced
50134 Apoptotic T-cell
50136 PERM TF274
50142 Macrophage-oxLDL
50144 Macrophage (GM-CSF treated)
S0146 prostate-edited
SO150 LNCAP prostate cell line
S0152 PC3 Prostate cell line
S0192 Synovial Fibroblasts (control)
50194 Synovial h oxia
50196 Synovial IL-1/TNF stimulated
50206 Smooth Muscle- HASTE normalized
50208 Messangial cell, frac 1
50210 Messangial cell, frac 2
50212 Bone Marrow Stromal Cell, untreated
50214 Human Osteoclastoma, re-excision disease
50216 Neutrophils IL-1 and LPS induced
50218 Apoptotic T-cell, re-excision
50222 H. Frontal cortex,e ile tic,re-excision disease
50242 Synovial Fibroblasts (Ill/TNF), subt
50250 Human Osteoblasts II disease
50260 Spinal Cord, re-excision
S0270 PTMIX
50276 Synovial h oxia-RSF subtracted
50278 H Macrophage (GM-CSF treated), re-excision
50280 Human Adipose Tissue, re-excision
S0282 Brain Frontal Cortex, re-excision
S0298 Bone marrow stroma,treated
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S0300 Frontallobe,dementia,re-excision
50306 Larynx normal #10 261-273
50308 Spleen/normal
50310 Normal trachea
S0328 Palate carcinoma disease
50330 Palate normal
S0342 Adipocytes,re-excision
S0344 Macro hage-oxLDL, re-excision
50346 Human Amygdala,re-excision
S0348 Cheek Carcinoma disease
50350 Pharynx Carcinoma disease
. 50352 Larynx Carcinoma disease
50354 Colon Normal II
50356 Colon Carcinoma disease
50358 Colon Normal III
50360 Colon Tumor II disease
50364 Human Quadriceps
50366 Human Soleus
50368 Human Pancreatic Langerhans
S0370 Larynx carcinoma II disease
50372 Larynx carcinoma III disease
50374 Normal colon
S0376 Colon Tumor disease
S0378 Pancreas normal PCA4 No
50380 Pancreas Tumor PCA4 Tu disease
50386 Human Whole Brain, re-excision
S0390 Smooth muscle, control, re-excision
S0398 Testis, normal
50402 Adrenal Gland,normal
50404 Rectum normal
50406 Rectum tumour
50408 Colon, normal
50410 Colon, tumour
50414 Hippocampus, Alzheimer Subtracted
_ CHMECell Line,treated 5 hrs
_
50418
_ CHMECell Line,untreated
_
S0420
S0422 Mo7e Cell Line GM-CSF treated (lng/ml)
S0424 TF-1 Cell Line GM-CSF Treated
50426 Monocyte activated, re-excision
50430 Aryepi lottis Normal
50434 Stomach Normal disease
50436 Stomach Tumour disease
S0438 Liver Normal MetSNo
50440 Liver Tumour Met 5 Tu
50442 Colon Normal
50444 Colon Tumor disease
50446 Tongue Tumour
S0448 Larynx Normal
50450 Larynx Tumour
S0456 Tongue Normal
50458 Thyroid Normal (SDCA2 No)
50468 Ea.hy.926 cell line
50474 Human blood platelets
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50476 Epithelial-TNFa and 1NF induced
53012 Smooth Muscle Serum Treated, Norm
53014 Smooth muscle, serum induced,re-exc
S6024 Alzheimers, spongy change disease
56028 Hurnan Manic Depression Tissue disease
T0002 Activated T-cells
T0004 Human White Fat
T0006 Human Pineal Gland
T0010 Human Infant Brain
T0023 Human Pancreatic Carcinoma disease
T0040 HSC172 cells
T0041 Jurkat T-cell G1 phase
T0042 Jurkat T-Cell, S phase
T0048 Human Aortic Endothelium
T0049 Aorta endothelial cells + TNF-a
T0060 Human White Adipose
T0067 Human Thyroid
T0082 Human Adult Retina
TO110 Human colon carcinoma (HCC) cell line, remake
TO115 Human Colon Cazcinoma (HCC) cell line
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Table 5
OMIM Description
Reference
104770 Amyloidosis, secondary, susceptibility to
107300 Antithrombin III deficiency
107670 A olipoprotein A-II deficiency
107777 Diabetes insipidus, nephrogenic, autosomal
recessive, 222000
109690 Asthma, nocturnal, susceptibility to
Obesity, susceptibility to
110700 Vivax malaria, susceptibility to
113721 Breast cancer
123940 White sponge nevus, 193900
131210 Atherosclerosis, susceptibility to
131400 Eosinophilia, familial
134638 Systemic 1u us erythematosus, susceptibility,
152700
135940 Ichthyosis vulgaris, 146700
136132 [Fish-odor syndrome], 602079
138190 Diabetes mellitus, noninsulin-dependent
138491 Hyperekplexia and spastic paraparesis
Startle disease, autosomal recessive
Startle disease/hyperekplexia, autosomal dominant,
149400
139350 Epidermolytic hyperkeratosis, 113800
Keratoderma, palmoplantar, nonepidermolytic
145001 Hyperparathyroidism jaw tumor syndrome
146740 Neutropenia, alloimmune neonatal
Viral infections, recurrent
Lupus erythematosus, systemic, susceptibility,
152700
146760 [IgG receptor I, phagocytic, familial deficiency
of]
146790 Lupus nephritis, susceptibility to
148040 Epidermolysis bullosa simplex, Koebner, bowling-Meara,
and Weber-
Cockayne types, 131900, 131760, 131800
148041 Pachyonychia con enita, Jadassolm-Lewandowsky
type, 167200
148043 Meesmann corneal dystrophy, 122100
148070 Liver disease, susceptibility to, from hepatotoxins
or viruses
150292 Epidermolysis bullosa, Herlitz functional
type, 226700
152445 Erythrokeratoderma, progressive symmetric,
602036
Vohwinkel syndrome, 124500
154500 Treacher Collins mandibulofacial dysostosis
159001 Muscular dystro hy, limb-girdle, type 1B
159440 Charcot-Marie-Tooth neuropathy-1B, 118200
Dejerine-Sottas disease, myelin P-related,
145900
Hypomyelination, con enital
173610 Platelet alpha/delta stora a pool deficiency
174000 Medullary cystic kidney disease, AD
176310 Leukemia, acute pre-B-cell
179755 Renal cell carcinoma, pa illary, 1
180071 Retinitis pigmentosa, autosomal recessive
181460 Schistosoma mansoni, susceptibility/resistance
to
182860 Elliptocytosis-2
Pyropoikilocytosis
S herocytosis, recessive
186780 CD3, zeta chain, deficiency
191030 Nemaline myopathy-1, 161800
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191315 Insensitivity to ain, congenital, with anhidrosis,
256800
208250 Jacobs syndrome
222600 Achondrogenesis Ib, 600972
Atelosteogenesis II, 256050
Diastrophic dysplasia
227400 Hemorrhagic diathesis due to factor V deficiency
Thromboembolism susceptibility due to factor
V Leiden
230400 Galactosemia
230800 Gaucher disease
Gaucher disease with cardiovascular calcification
231550 Achalasia-addisonianism-alacrimia syndrome
233710 Chronic ranulomatous disease due to deficiency
of NCF-2
234000 Factor XII deEciency
247200 Miller-Dieker lissencephaly syndrome
249270 Thiamine-responsive megaloblastic anemia
250250 Cartila e-hair hypoplasia
254210 Myasthenia avis, familial infantile
266200 Anemia, hemolytic, due to PK deficiency
271900 Canavan disease
272750 GM2-gangliosidosis, AB variant
600059 Retinitis pigmentosa-13
__ Leber congenital amaurosis, type I, 204000
600179
600194 Ichthyosis bullosa of Siemens, 146800
600231 Palmoplantar keratoderma, Bothnia type
600536 Myo athy, congenital
600807 Bronchial asthma
600808 Enuresis, nocturnal, 2
600897 Cataract, zonular pulverulent-1, 116200
600923 Porphyria variegata, 176200
600956 Persistent Mullerian duct syndrome, type II,
261550
600977 Cone dystro hy, progressive
600995 Nephrotic syndrome, idiopathic, steroid-resistant
601105 Pycnodysostosis, 265800
601202 Cataract, anterior polar-2
601284 Hereditary hemorrhagic telangiectasia-2, 600376
601411 Muscular dystrophy, limb-girdle, type 2F,
601287
601412 Deafness, autosomal dominant 7
601518 Prostate cancer, hereditary, l, 176807
601545 Lissencephaly-1
601596 Charcot-Marie-Tooth neuropathy, demyelinating
601652 Glaucoma 1A, primary open angle, juvenile-onset,
137750
601769 Osteoporosis, involutional
Rickets, vitamin D-resistant, 277440
601777 Cone dystrophy, rogressive
601863 Bare lymphocyte syndrome, complementation
rou C
601928 Monilethrix, 158000
602089 Hemangioma, capillary, hereditary
602116 Glioma
602153 Monilethrix, 158000
602491 Hyperlipidemia, familial combined, 1
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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 and/or 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 axe also encompassed by the invention.
Signal Seauences and Mature Polyneptides
The present invention also encompasses mature forms of a polypeptide having
the amino acid sequence of SEQ ID NO:Y and/or the amino acid sequence encoded
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by the cDNA in a deposited clone. Polynucleotides encoding the mature forms
(such
as, for example, the polynucleotide sequence in SEQ m NO:X and/or the
polynucleotide sequence contained in the cDNA of a deposited clone) are also
encompassed by the invention. Moreover, fragments or variants of these
polypeptides
(such as, fragments as described herein, polypeptides at least 80%, 85%, 90%,
95%,
96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide that hybridizes under stringent conditions to the
complementary strand of the polynucleotide encoding these polypeptides) are
also
encompassed by the invention. In preferred embodiments, these fragments or
variants
retain one or more functional activities of the full-length or mature form of
the
polypeptide (e.g., biological activity, antigenicity (i.e., ability to bind
polypeptide
specific antibodies), immunogeriicity (i.e., ability to elicit generation of
polypeptide
specific antibodies), ability to form heteromeric or homomeric
multimers/oligomers,
and ability to bind cognate receptors or ligands). Antibodies that bind the
polypeptides of the invention, and polynucleotides encoding these polypeptides
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 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 specificity of a secreted protein is
ultimately
determined by the primary structure of the complete protein, that is, cleavage
specificity 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
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 cleaved protein. The accuracy
of
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predicting the cleavage points of known mammalian secretory proteins by each
of
these methods is in the range of 75-80%. (von Heinje, 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
polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen
et
al., Protein Engineering 10:1-6 (1997)), which predicts the sub-cellular
localization 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
10 analysis of the amino acid sequences of the secreted proteins described
herein by this
program provided the results shown in Table 1.
In specific embodiments, polypeptides of the invention comprise, or
alternatively consist of, the predicted mature form of the polypeptide as
delineated in
columns 14 and 15 of Table 1. Moreover, fragments or variants of these
polypeptides
15 (such as, fragments as described herein, polypeptides at least 80%, 85%,
90%, 95%,
96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides
encoded by a polynucleotide that hybridizes under stringent conditions to the
complementary strand of the polynucleotide encoding these polypeptides) are
also
encompassed by the invention. In preferred embodiments, these fragments or
variants
20: retain one or more functional activities of the full-length or mature form
of the
polypeptide (e.g., biological activity, antigenicity (i.e., ability to bind
polypeptide
specific antibodies), immunogenicity (i.e., ability to elicit generation of
polypeptide
specific antibodies), ability to form heteromeric or homomeric
multimers/oligomers,
and ability to bind cognate receptors or ligands). Antibodies that bind the ,
25 polypeptides of the invention, and polynucleotides encoding these
polypeptides are
also encompassed by the invention.
Polynucleotides encoding proteins comprising, or consisting of, the predicted
mature form of polypeptides of the invention (e.g., polynucleotides having the
sequence of SEQ m NO: X (Table 1, column 5), the sequence delineated in
columns
30 7 and 8 of Table 1, and a sequence encoding the mature polypeptide
delineated in
columns 14 and 15 of Table 1 (e.g., the sequence of SEQ m NO:X encoding the
mature polypeptide delineated in columns 14 and 15 of Table 1)) are also
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encompassed by the invention, as are fragments or variants of these
polynucleotides
(such as, fragments as described herein, polynucleotides at least 80%, 85%,
90%,
95%, 96%, 97%, 98%, 99%, or 100% identical to these polynucleotides, and
nucleic
acids which hybridizes under stringent conditions to the complementary strand
of the
polynucleotide).
As one of ordinary skill would appreciate, however, polypeptide signal
sequence cleavage sites cannot be predicted with absolute certainty. For
example,
within any given organism cleavage of the signal sequence is often not
entirely
uniform (resulting in more than one mature polypeptide species). Moreover,
signal
cleavage sites may also vary from organism to organism (also resulting in
multiple
species of mature polypeptides). Accordingly, the present invention provides
secreted
polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus
beginning within 15 residues of the predicted cleavage point (i.e., having 1,
2, 3, 4, 5,
6, 7, 8 , 9, 10, 11, 12, 13, 14, or 15 more or less contiguous residues of SEQ
ID NO:Y
at the N-terminus when compared to the predicted mature form of the
polypeptide
(e.g., the mature polypeptide delineated in columns 14 and 15 of Table 1).
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 fizrther 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 endoplasmic reticulum. Nonetheless, the
present
invention provides the mature protein produced by expression of the
polynucleotide
sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the
cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described
below). These polypeptides, and the~polynucleotides encoding such
polypeptides, are
contemplated by the present invention.
Mature forms of the polypeptide of the present invention also include
polypeptides processed at the carboxyl terminus (C-terminus). For example,
post-
translational processing, within any given cell type or organism can also
result in
polypeptides with truncated C-termini (as compared to the full-length or
secreted
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polypeptides delineated in columns 12 and 15 or columns 14 and 15,
respectively, of
Table 1). Therefore, in further specific embodiments, polypeptides of the
invention
comprise, or alternatively consist of, the mature form of the polypeptide
having a C-
terminus ending within 15 residues of the predicted carboxyl-terminus (i.e.,
having 1,
2, 3, 4, 5, 6, 7, 8 , 9, 10, 1 l, 12, 13, 14, or 15 C-terminal residues less
than the mature
polypeptide delineated in columns 14 and 15 of Table 1).
Thus, mature forms of polypeptides of the present invention include
polypeptides with amino-terminal and/or carboxyl=terminal truncations of the
full-
length polypeptides delineated in Table 1, columns 12 and 15. Accordingly,
these
polypeptides, polynucleotides encoding such polypeptides, and antibodies
binding
such polypeptides, are contemplated by the present invention.
Polynucleotide and Polypeptide Variants
The present invention is directed to variants of the polynucleotide sequence
disclosed in SEQ ID 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 m 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
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
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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 m 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 l, 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
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=l,
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Joining Penalty=30, Randomization Group Length=0, Cutoff Score=l, 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 subject sequence when calculating percent identity. For
subject
sequences truncated at the 5' or 3' ends, relative to the query sequence, the
percent
identity is corrected by calculating the number of bases of the query sequence
that are
5' and 3' of the subj ect 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 aligmnent. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
S' 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
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 matchedlaligned with the query
sequence
are manually corrected for. No other manual corrections are to made for the
purposes
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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 subj ect 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.
10. 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 alignment 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,
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
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
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identity. For subject sequences truncated at the N- and C-termini, relative to
the
query sequence, the percent identity is corrected by calculating the number of
residues
of the query sequence that are N- and C-terminal of the subject sequence,
which are
not matched/aligned with a corresponding subject residue, as a percent of the
total
bases of the query sequence. Whether a residue is matched/aligned is
determined by
results of the FASTDB sequence alignment. This percentage is then subtracted
from
the percent identity, calculated by the above FASTDB program using the
specified
parameters, to arrive at a final percent identity score. This final percent
identity score
is what is used for the purposes of the present invention. Only residues to
the N- and
C-termini of the subject sequence, which are not matched/aligned with the
query
sequence, are considered for the purposes of manually adjusting the percent
identity.
score. That is, only query residue positions outside the farthest N- and C-
terminal
residues of the subject sequence.
For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the sequence (number of residues at the N-
and C-
termini not matched/total number of residues in the query sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the
remaining 90 residues were perfectly matched the final percent identity would
be
90%. In another example, a 90 residue subject sequence is compared with a 100
residue query sequence. This time the deletions axe internal deletions so
there are no
residues at the N- or C-termini of tlxe subject sequence which are not
matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually corrected. Once again, only residue positions outside the N- and C-
terminal
ends of the subj ect sequence, as displayed in the FASTDB alignment, which are
not
matched/aligned with the query sequnce are manually corrected fox. 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
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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 occurnng
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 KGF proteins having heparin binding activity even
after
deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon
gamma
exhibited up to ten times higher activity after deleting 8-10 amino acid
residues from
the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-
216
(1988).)
Moreover, ample evidence demonstrates that variants often retain a biological
activity similar to that of the naturally occurring protein. For example,
Gayle and
coworkers (J. Biol. Chem 268:22105-22111 (1993)) conducted extensive
mutational
analysis of human cytokine IL-1 a. They used random mutagenesis to generate
over
3,500 individual IL-la mutants that averaged 2.5 amino acid changes per
variant over
the entire length of the molecule. Multiple mutations were examined at every
possible amino acid position. The investigators found that "[most 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
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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.
The second strategy uses genetic engineering to introduce amilio acid changes
at specific positions of a cloned gene to identify regions critical for
protein function.
Fox 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.
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As the authors state, these two strategies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors fixrther
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.
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. Immunol. 2:331-
340
(1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit.
Rev.
Therapeutic Drug Carner Systems 10:307-377 (1993).)
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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,
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 m
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
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diagnostic probes and primers as discussed herein. Of course, larger fragments
(e.g.,
50, 150, 500, 600, 2000 nucleotides) are preferred.
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,
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 ID 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
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
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
part or region, most preferably as a single continuous region. Representative
examples of polypeptide fragments of the invention, include, for example,
fragments
comprising, or alternatively consisting of, from about amino acid number 1-20,
21-40,
41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 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.
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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.
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 axe
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
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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 immunosorbent
assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitation reactions, immunodiffusion assays, in situ immunoassays (using
colloidal gold, enzyme or radioisotope labels, for example), western blots,
precipitation reactions, agglutination assays (e.g., gel agglutination assays,
hemagglutination assays), complement fixation assays, immunofluorescence
assays,
protein A assays, and immunoelectrophoresis assays, etc. In one embodiment,
antibody binding is detected by detecting a label on the primary antibody. In
another
embodiment, the 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
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
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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
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 polynucleotide 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 for generating
antibodies
described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998- 4002 (1983)). The term "antigenic epitope," as used herein, is
defined as a
portion of a protein to which an antibody can immunospecifically bind its
antigen as
determined by any method well known in the art, for example, by the
immunoassays
described herein. Irmnunospecific binding excludes non-specific binding but
does not
necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
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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., Cell 37:767-778 (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
an animal system (such as rabbit or mouse), or, if the polypeptide is of
sufficient
length (at least about 25 amino acids), the polypeptide may be presented
without a
carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino
acids
have been shown to be sufficient to raise antibodies capable of binding to, at
the very
least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
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,
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in vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Ger.
Virol., 66:2347-
2354 (1985). If in vivo immunization is used, animals may be immunized with
free
peptide; however, anti-peptide antibody titer may be boosted by coupling the
peptide
to a macromolecular earner, 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-hydroxysucciiumide ester
(MBS),
while other peptides may be coupled to earners 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
earner
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune response. Several booster injections may be needed, for instance, at
intervals of about two weeks, to provide a useful titer of anti-peptide
antibody which
can be detected, for example, by ELISA assay using free peptide adsorbed to a
solid
surface. The titer of anti-peptide antibodies in serum from an immunized
animal may
be increased by selection of anti-peptide antibodies, for instance, by
adsorption to the
peptide on a solid support and elution of the selected antibodies according to
methods
well known in the art.
As one of skill in the art will appreciate, and as discussed above, the
polypeptides of the present invention (e.g., those comprising an immunogenic
or
antigenic epitope) can be fused to heterologous polypeptide sequences. For
example,
polypeptides of the present invention (including fragments or variants
thereof), may
be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or
portions thereof (CH1, CH2, CH3, or any combination thereof and portions
thereof,
resulting in chimeric polypeptides. By way of another non-limiting example,
polypeptides and/or antibodies of the present invention (including fragments
or
variants thereof) may be fused with albumin (including but not limited to
recombinant
human serum albumin or 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
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(including fragments or variants thereof) are fused with the mature form of
human
serum albumin (i.e., amino acids I - 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.,
irmnunoglobulin 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 barrier to~the immune system has been demonstrated for antigens
(e.g.,
insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments
(see, e.g.,
PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have
a disulfide-linked dimeric structure due to the IgG portion desulfide bonds
have also
been found to be more efficient in binding and neutralizing other molecules
than
monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et
al., J.
Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can
also be recombined with a gene of interest as an epitope tag (e.g., the
hemagglutinin
("HA") tag or flag tag) to aid in detection and purification of the expressed
polypeptide. For example, a system described by Janknecht et al. allows for
the
ready purification of non-denatured fusion proteins expressed in human cell
lines
(Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897). In this
system, the
gene of interest is subcloned into a vaccinia recombination plasmid such that
the
open reading frame of the gene is translationally fused to an amino-terminal
tag
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consisting of six histidine residues. The tag serves as a matrix binding
domain for the
fusion protein. Extracts from cells infected with the recombinant vaccinia
virus are
loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged
proteins can
be selectively eluted with imidazole-containing buffers.
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
m NO:X and the polypeptides encoded by these polynucleotides may be achieved
by
DNA shuffling. DNA shuffling involves the assembly of two or more DNA
segments by homologous or site-specific recombination to generate variation in
the
polynucleotide sequence. In another embodiment, polynucleotides of the
invention,
or the encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior
to recombination. In another embodiment, one or more components, motifs,
sections,
parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of
the
invention may be recombined with one or more components, motifs, sections,
parts,
domains, fragments, etc. of one or more heterologous molecules.
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 m NO:Y, and/or an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying specific
antibody-
antigen binding). Antibodies of the invention include, but are not limited to,
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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 immunoglobulin
molecules
and immunologically active portions of immunoglobulin molecules, i.e.,
molecules
that contain an antigen binding site that immunospecifically binds an antigen.
The
irmnunoglobulin 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,
CHl, CH2, and CH3 domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable regions) with a hinge
region,
CH1, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
marine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or
chicken. As used herein, "human" antibodies include antibodies having the
amino
acid sequence of a human imrnunoglobulin and include antibodies isolated from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described
infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
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
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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. hnmunol. 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
teens of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least
85%, at
least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least
55%, and at
least 50% identity (as calculated using methods known in the art and described
herein) to a polypeptide of the present invention are also included in the
present
invention. In specific embodiments, antibodies of the present invention cross-
react
with murine, rat and/or rabbit homologs of human proteins and the
corresponding
epitopes thereof. Antibodies that do not bind polypeptides with less than 95%,
less
than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less
than 65%,
less than 60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of the present
invention are also included in the present invention. In a specific
embodiment, the
above-described cross-reactivity is with respect to any single specific
antigenic or
immunogenic polypeptide, or combinations) of 2, 3, 4, 5, or more of the
specific
antigenic and/or immunogenic polypeptides disclosed herein. Further included
in the
present invention are antibodies which bind polypeptides encoded by
polynucleotides
which hybridize to a polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the present
invention
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may also be described or specified in terms of their binding affinity to a
polypeptide
of the invention. Preferred binding affinities include those with a
dissociation
constant or Kd less than 5 X 10-Z M, 10-2 M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M,
10'4 M,
X 10'5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10'' M, 10' M, 5 X 10-g M, 10-8 M, 5
X
5 10-~ 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, i o-i a M, 5 X
10-13 M, 10-13 M, 5 X 10'14 M, 10-14 M, 5 X 10'15 M, or 10'15 M.
The invention also provides antibodies that competitively inhibit binding of
an
antibody to an epitope of the invention as determined by any method known in
the art
for determining competitive binding, for example, the immunoassays described
herein. In preferred embodiments, the antibody competitively inhibits binding
to the
epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least
75%, at least
70%, at least 60%, or at least 50%.
Antibodies of the present invention rnay act as agonists or antagonists of the
polypeptides of the present invention. For example, the present invention
includes
I5 antibodies which disrupt the receptor/Iigand 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
features both receptor-specific antibodies and ligand-specific antibodies. The
invention also features receptor.-specific antibodies which do not prevent
ligand
binding but prevent receptor activation. Receptor activation (i.e., signaling)
may be
determined by techniques described herein or otherwise known in the art. For
example, receptor activation can be determined by detecting the
phosphorylation
(e.g., tyrosine or serine/threonine) of the receptor or its substrate by
immunoprecipitation followed by western blot analysis (for example, as
described
supra). In specific embodiments, antibodies are provided that inhibit ligand
activity
or receptor activity by at least 95%, at least 90%, at least 85%, at least
80%, at least
75%, at least 70%, at least 60%, or at least 50% of the activity in absence of
the
antibody.
The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound
receptor or the unbound ligand. Likewise, included in the invention are
neutralizing
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antibodies which bind the ligand and prevent binding of the ligand to the
receptor, as
well as antibodies which bind the ligand, thereby preventing receptor
activation, but
do not prevent the ligand from binding the receptor. Further included in the
invention
are antibodies which activate the receptor. These antibodies may act as
receptor
agonists, i.e., potentiate or activate either all or a subset of the
biological activities of
the ligand-mediated receptor activation, for example, by inducing dimerization
of the
receptor. The antibodies may be specified as agonists, antagonists or inverse
agonists
for biological activities comprising the specific biological activities of the
peptides of
the invention disclosed herein. The above antibody agonists can be made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Imrnunol. 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.,
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
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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
attaclunent does not prevent the antibody from generating an anti-idiotypic
response.
For example, but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation, acetylation,
pegylation,
phosphylation, amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any
of
numerous chemical modifications may be carried out by known techniques,
including, but not limited to specific chemical cleavage, acetylation,
formylation,
metabolic synthesis of tunicamycin, etc. Additionally, the derivative may
contain
one or more non-classical amino acids.
The antibodies of the present invention may be generated by any suitable .
method known in the art. Polyclonal antibodies to an antigen-of interest can
be
produced by various procedures well known in the art. For example, a
polypeptide of
the invention can be administered to various host animals including, but not
limited
to, rabbits, mice, rats, etc. to induce the production of sera containing
polyclonal
antibodies specific for the antigen. Various adjuvants may be used to increase
the
immunological response, depending on the host species, and include but are not
limited to, Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and
potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and
corynebacterium parvum. Such adjuvants are also well known in the art.
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
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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
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
CHl domain of the heavy chain.
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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 V1II 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. Imrnunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-186 (1995); I~ettleborough et al., Eur. J. Immunol. 24:952-958 (1994);
Persic
et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;
5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect cells, plant
cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92122324;
Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI
34:26-
34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
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Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston
et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999
(1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including
in vivo use of antibodies in humans and in vitro detection assays, it may be
preferable
to use chimeric, humanized, or human antibodies. A chimeric antibody is a
molecule
in which different portions of the antibody are derived from different animal
species,
such as antibodies having a variable region derived from a murine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Mornson, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entirety. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted
with
the corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known in the art, e.g., by modeling of the interactions of the CDR and
frannework
residues to identify framework residues important for antigen binding and
sequence
comparison to identify unusual framework residues at particular positions.
(See, e.g.,
Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323
(1988),
which axe incorporated herein by reference in their entireties.) Antibodies
can be
humanized using a variety of techniques known in the art including, for
example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al.,
Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973
(1994)),
and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
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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.
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. hmnunol. 13:65-93 (1995). For a
detailed discussion of this technology for producing human antibodies and
human
monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European
Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425;
5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which
are
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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
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(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438
(1991)). For example, antibodies which bind to and competitively inhibit
polypeptide
multimerization and/or binding of a polypeptide of the invention to a ligand
can be
used to generate anti-idiotypes that "mimic" the polypeptide multimerization
and/or
binding domain and, as a consequence, bind to and neutralize polypeptide
and/or its
ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-
idiotypes can
be used in therapeutic regimens to neutralize polypeptide ligand. For example,
such
anti-idiotypic antibodies can be used to bind a polypeptide of the invention
and/or to
bind its ligands/receptors, and thereby block its biological activity.
Polynucleotides Encoding Antibodies
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 ll~ 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
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nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described
in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the
synthesis of overlapping oligonucleotides containing portions of the sequence
encoding the antibody, annealing and ligating of those oligonucleotides, and
then
amplification of the ligated oligonucleotides by PCR.
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
example, the techniques described in Sambrook et al., 1990, Molecular Cloning,
A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY and Ausubel et al., eds., 199, 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
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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
occurnng 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"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature
312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing
genes
from a mouse antibody molecule of appropriate antigen specificity together
with
genes from a human antibody molecule of appropriate biological activity can be
used.
As described supra, a chimeric antibody is a molecule in which different
portions are
derived from different animal species, such as those having a variable region
derived
from a murine mAb and a human immunoglobulin constant region, e.g., humanized
antibodies.
Alternatively, techniques described for the production of single chain
antibodies (LJ.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988);
Huston et
al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature
334:544-54 (1989)) can be adapted to produce single chain antibodies. Single
chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region
via an amino acid bridge, resulting in a single chain polypeptide. Techniques
for the
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assembly of functional Fv fragments in E. coli may also be used (Skerra et
al.,
Science 242:1038- 1041 (1988)).
Met7zods of P~oducihg Antibodies
The antibodies of the invention can be produced by any method known in the
art for the synthesis of antibodies, in particular, by chemical synthesis or
preferably,
by recombinant expression techniques.
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 ora single chain antibody of the invention), requires construction
of an
expression vector containing a polynucleotide that encodes the antibody. Once
a .
polynucleotide encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably containing the heavy or light chain
variable
domain), of the invention has been obtained, the vector for the production of
the
antibody molecule may be produced by recombinant DNA technology using
techniques well known in the art. Thus; methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding nucleotide
sequence are
described herein. Methods which are well known to those skilled in the art can
be
used to construct expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals. These methods
include,
for example, in vitro recombinant DNA techniques, synthetic techniques, and in
vivo
genetic recombination. The invention, thus, provides replicable vectors
comprising a
nucleotide sequence encoding an antibody molecule of the invention, or a heavy
or
light chain thereof, or a heavy or light chain variable domain, operably
linked to a
promoter. Such vectors may include the nucleotide sequence encoding the
constant
region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT
Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable
domain of
the antibody may be cloned into such a vector for expression of the entire
heavy or
light chain.
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
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polynucleotide encoding an antibody of the, invention, or a heavy or light
chain
thereof, or a single chain antibody of the invention, operably linked to a
heterologous
promoter. In preferred embodiments for the expression of double-chained
antibodies,
vectors encoding both the heavy and light chains may be co-expressed in the
host cell
for expression of the entire immunoglobulin molecule, as detailed below.
A variety of host-expression vector systems may be utilized to express the
antibody molecules of the invention. Such host-expression systems represent
vehicles by which the coding sequences of interest may be produced and
subsequently
purified, but also represent cells which may, when transformed or transfected
with
the appropriate nucleotide coding sequences, express an antibody molecule of
the
invention in situ. These include but are not limited to microorganisms such as
bacteria (e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing antibody coding
sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant
yeast
expression vectors containing antibody coding sequences; insect cell systems
infected with recombinant virus expression vectors (e.g., baculovirus)
containing
antibody coding sequences; plant cell systems infected with recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus,
TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid)
containing antibody coding sequences; or mammalian cell systems (e.g., COS,
CHO,
BHK, 293, 3T3 cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the
vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia
coli,
and more preferably, eukaryotic cells, especially for the expression of whole
recombinant antibody molecule, are used for the expression of a recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
cells (CHO), in conjunction with a vector such as the major intermediate early
gene
promoter element from human cytomegalovirus is an effective expression system
for
antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al.,
Bio/Technology 8:2
(1990)).
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In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the antibody molecule being
expressed.
For example, when a large quantity of such a protein is to be produced, for
the
generation of pharmaceutical compositions of an antibody molecule, vectors
which
direct the expression of high levels of fusion protein products that are
readily purified
may be desirable. Such vectors include, but are not limited, to the E. coli
expression
vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding
sequence may be ligated individually into the vector in frame with the lac Z
coding
region so that a fusion protein is produced; pIN vectors (Tnouye & Inouye,
Nucleic
Acids ~Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-
5509 (1989)); and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with glutathione S-transferase (GST). In
general, such
fusion proteins are soluble and can easily be purified from lysed cells by
adsorption
and binding to matrix glutathione-agarose beads followed by elution in the
presence
of free glutathione. The pGEX vectors are designed to include thrombin or
factor Xa
protease cleavage sites so that the cloned target gene product can be released
from the
GST moiety.
In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes. The virus grows in
Spodoptera frugipe~da cells. The antibody coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin gene) of
the virus
and placed under control of an AcNPV promoter (for example the polyhedrin
promoter).
In mammalian host cells, a number of viral-based expression systems may be
utilized. In cases where an adenovirus is used as an expression vector, the
antibody
coding sequence of interest may be ligated to an adenovirus
transcription/translation
control complex, e.g., the late promoter and tripartite leader sequence. This
chimeric
gene may then be inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non- essential region of the viral genome (e.g.,
region
El or E3) will result in a recombinant virus that is viable and capable of
expressing
the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.
Acad.
Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required
for
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efficient translation of inserted antibody coding sequences. These signals
include the
ATG initiation codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding sequence to
ensure
translation of the entire insert. These exogenous translational control
signals and
initiation codons can be of a variety of origins, both natural and synthetic.
The
efficiency of expression may be enhanced by the inclusion of appropriate
transcription enhancer elements, transcription terminators, etc. (see Bittner
et al.,
Methods in Enzymol. 153:51-544 (1987)).
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.
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
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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 (I993); 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);
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 axe incorporated by reference herein in their
entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based
on gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
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amplified region is associated with the antibody gene, production of the
antibody will
also increase (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
vector encoding a light chain derived polypeptide. The two vectors may contain
identical selectable markers which enable equal expression of heavy and light
chain
polypeptides. Alternatively, a single vector may be used which encodes, and is
capable of expressing, both heavy and light chain polypeptides. In such
situations,
the light chain should be placed before the heavy chain to avoid an excess of
toxic
free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad.
Sci.
USA 77:2197 (1980)). The coding sequences for the heavy and light chains may
comprise cDNA or genomic DNA.
Once an antibody molecule of the invention has been produced by an animal,
chemically synthesized, or recombinantly expressed, it may be purifted by any
method known in the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity, particularly by
affinity for
the specific antigen after Protein A, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard technique
for the
purification of proteins. In addition, the antibodies of the present invention
or
fragments thereof can be fused to heterologous polypeptide sequences described
herein or otherwise known in the art, to facilitate purification.
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
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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; Dillies 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);
Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl.
Acad. Sci.
USA 89:11337- 11341 (1992) (said references incorporated by reference in their
entireties).
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
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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). Alternatively, deleting the Fc part after the fusion protein has
been
expressed, detected, and purified, would be desired. For example, the Fc
portion may
hinder therapy and diagnosis if the fusion protein is used as an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have
been fused with Fc portions for the purpose of high-throughput screening
assays to
identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition
8:52-58
(1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
Moreover, the antibodies or fragments thereof of the present invention can be
fused to marker sequences, such as a peptide to facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311), among others, many of which are commercially available. As described
in
Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for convenient purification of the fusion protein. Other
peptide tags
useful for purification include, but are not limited to, the "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson
et al., Cell 37:767 (1984)) and the "flag" tag.
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
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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-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine,
thioepa chlorambucil, melphalan, carmustine (BSNL~ and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (In (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents (e.g., vincristine and vinblastine).
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The conjugates of the invention can be used for modifying a given biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to
classical chemical therapeutic agents. For example, the drug moiety may be a
protein
or polypeptide possessing a desired biological activity. Such proteins may
include,
S for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria
toxin; a protein such as tumor necrosis factor, a-interferon,13-interferon,
nerve growth
factor, platelet derived growth factor, tissue plasminogen activator, an
apoptotic
agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
'
97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand
(Takahashi et al., Iht. Imirauyaol., 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
et al. (eds.), pp. 243-S6 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),
pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic
Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological .And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis,
Results,
And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy,
Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al.,
"The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
Rev. 62:119-58 (1982).
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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,
administered alone or in combination with cytotoxic factors) and/or
cytokine(s) can
be used as a therapeutic.
Immurzophezzotypizzg
The antibodies of the invention may be utilized for immunophenotyping of
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
populations expressing the marker. Various techniques can be utilized using
monoclonal antibodies to screen for cellular populations expressing the
marker(s), and
include magnetic separation using antibody-coated magnetic beads, "panning"
with
antibody attached to a solid matrix (i.e., plate), and flow cytometry (See,
e.g., U.S.
Patent 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).
These techniques allow for the screening of particular populations of cells,
such as might be found with hematological malignancies (i.e. minimal residual
disease (MRD) in acute leukemic patients) and "non-self' cells in
transplantations to
prevent Graft-versus-Host Disease (GVHD). Alternatively, these tech~iiques
allow for
the screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation andlor differentiation, as might be found in human umbilical
cord blood.
Assays For Antibody Binding
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,
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gel diffusion precipitin reactions, immunodiffusion 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).
Itnmunoprecipitation 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 inhibitors
(e.g.,
EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to
the cell
. lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C,
adding protein A
and/or protein G sepharose beads to the cell lysate, incubating for about an
hour or
more at 4° C, washing the beads in lysis buffer and resuspending the
beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a
particular antigen can be assessed by, e.g., western blot analysis. One of
skill in the
art would be knowledgeable as to the parameters that can be modified to
increase the
binding of the antibody to an antigen and decrease the background (e.g., pre-
clearing
the cell lysate with sepharose beads). For further discussion regarding
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.
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
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alkaline phosphatase) or radioactive molecule (e.g., 32P or 1251) diluted in
blocking
buffer, washing the membrane in wash buffer, and detecting the presence of the
antigen. One of skill in the art would be knowledgeable as to the parameters
that can
be modified to increase the signal detected and to reduce the background
noise. For
further discussion regarding western blot protocols see, e.g., Ausubel et al,
eds, 1994,
Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
York
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. In this case, a second antibody conjugated to a detectable
compound may be added following the addition of the antigen of interest to the
coated well. One of skill in the art would be knowledgeable as to the
parameters that
can be modified to increase the signal detected as well as other variations of
ELISAs
known in the art. For fixrther discussion regarding ELISAs see, e.g., Ausubel
et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc.,
New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 1251) 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
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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
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 1L-7), for
example, which
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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, immunotherapy and anti-tumor agents). Generally, administration of
products of a species origin or species reactivity (in the case of antibodies)
that is the
same species as that of the patient is preferred. Thus, in a preferred
embodiment,
human antibodies, fragments derivatives, analogs, or nucleic acids, are
administered
to a human patient for therapy or prophylaxis.
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 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-S M, 5 X 10-6 M, 10-6 M, 5 X 10-~ M, 10-~ M, 5 X
10'$ M,
10-8 M, 5 X 10-9 M, 10-9 M, S 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 10-1 S
M.
Gehe Therapy
In a specific embodiment, nucleic acids comprising sequences encoding
antibodies or functional derivatives thereof, are administered to treat,
inhibit or
prevent a disease or disorder associated with aberrant expression and/or
activity of a
polypeptide of the invention, by way of gene therapy. Gene therapy refers to
therapy
performed by the administration to a subj ect 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.
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For general reviews of the methods of gene therapy, see Goldspiel et al.,
Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
~ (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art
of recombinant DNA technology which can be used are described in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
and
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990).
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
indirect, in which case, cells are first transformed with the nucleic acids in
vitro, then
transplanted into the patient. These two approaches are known, respectively,
as in
vivo or ex vivo gene therapy.
In a specific embodiment, the nucleic acid sequences are directly administered
in vivo, where it is expressed to produce the encoded product. This can be
accomplished by any of numerous methods known in the art, e.g., by
constructing
them as part of an appropriate nucleic acid expression vector and
administering it so
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that they become intracellular, e.g., by infection using defective or
attenuated
retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by
direct
injection of naked DNA, or by use of microparticle bombardment (e.g., a gene
gun;
Biolistic, Dupont), or coating with lipids or cell-surface receptors or
transfecting
agents, encapsulation in liposomes, microparticles, or microcapsules, or by
administering them in linkage to a peptide which is known to enter the
nucleus, by
administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to
target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic
acid-ligand complexes can be formed in which the ligand comprises a fusogenic
viral
peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be targeted in
vivo for
cell specific uptake and expression, by targeting a specific receptor (see,
e.g., PCT
Publications WO 92/06180; WO 92/22635; W092/20316; W093/14188, WO
93/20221). Alternatively, the nucleic acid can be introduced intracellularly
and
incorporated within host cell DNA for expression, by homologous recombination
(Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra
et al.,
Nature 342:435-438 (1989)).
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 axe cloned into one or more vectors,
which
facilitates delivery of the gene into a patient. More detail about retroviral
vectors can
be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the
use of a
retroviral vector to deliver the mdrl gene to hematopoietic stem cells in
order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the
use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-
651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg,
Human
Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in
Genetics
and Devel. 3:110-114 (1993).
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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. I~ozarsky 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 adenavirus 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
(Welsh et al., Proc. Soc. EXp. Biol. Med. 204:289-300 (1993); U.S. Patent No.
5,436,146).
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
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
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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, mnbilical cord blood, peripheral blood, fetal liver, etc.
In a,preferred embodiment, the cell used for gene therapy is autologous to the
patient.
~ Tn an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they are expressible by the cells or their progeny, and the recombinant cells
are then
administered in vivo for therapeutic effect. In a specific embodiment, stem or
progenitor cells are used. Any stem and/or progenitor cells which can be
isolated and
maintained in vitro can potentially be used in accordance with this embodiment
of
the present invention (see e.g. PCT Publication WO 94/08598; Stemple and
Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980);
and
Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).
Tn 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.
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Demonstration of Tlaer-apeutic oY Prophylactic 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.
TherapeuticlP~ophylactic Administration and Composition .
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;
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
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introduction include but are not limited to intradermal, intramuscular,
intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes. The
compounds or
compositions may be administered by any convenient route, for example by
infusion
or bolus injection, by absorption through epithelial or mucocutaneous linings
(e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be administered
together
with other biologically active agents. Administration can be systemic or
local. In
addition, it may be desirable to introduce the pharmaceutical compounds or
compositions of the invention into the central nervous system by any suitable
route,
including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such
as an Ommaya reservoir. Pulmonary administration can also be employed, e.g.,
by
use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the pharmaceutical
compounds or compositions of the invention locally to the area in need of
treatment;
this may be achieved by, for example, and not by way of limitation, local
infusion
during surgery, topical application, e.g., in conjunction with a wound
dressing after
surgery, by injection, by means of a catheter, by means of a suppository, or
by means
of an implant, said implant being of a porous, non-porous, or gelatinous
material,
including membranes, such as sialastic membranes, or fibers. Preferably, when
administering a protein, including an antibody, of the invention, care must be
taken to
use materials to which the protein does not absorb.
In another embodiment, the compound or composition can be delivered in a
vesicle, in particular a liposome (see Larger, Science 249:1527-1533 (1990);
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in
a controlled release system. In one embodiment, a pump may be used (see
Larger,
supra; Sefton, CRC Crit. Ref. Biomed. Erg. 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, Larger and Wise (eds.), CRC Pres., Boca Raton, Florida
(1974);
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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 (I984)).
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 intracellulaxly 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 carrier. hl a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal
or a state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic
is administered. Such pharmaceutical carriers can be sterile liquids, such as
water and
oils, including those of petroleum, animal, vegetable or synthetic origin,
such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred
carrier when the pharmaceutical composition is administered intravenously.
Saline
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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
earners
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
pharnaceutical carriers are described in "Remington's Pharmaceutical Sciences"
by
E.W. Martin. Such compositions will contain a therapeutically effective amount
of
the compound, preferably in purified form, together with a suitable amount of
carrier
so as to provide the form for proper administration to the patient. The
formulation
should suit the mode of administration.
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration to human beings. Typically, compositions for intravenous
administration are solutions in sterile isotonic aqueous buffer. Where
necessary, the
composition may also include a solubilizing agent and a local anesthetic such
as
lignocaine to ease pain at the site of the injection. Generally, the
ingredients are
supplied either separately or mixed together in unit dosage form, for example,
as a dry
lyophilized powder or water free concentrate in a hermetically sealed
container such
as an ampoule or sachette indicating the quantity of active agent. Where the
composition is to be administered by infusion, it can be dispensed with an
infusion
bottle containing sterile pharmaceutical grade water or saline. Where the
composition
is administered by injection, an ampoule of sterile water fox injection or
saline can be
provided so that the ingredients may be mixed prior to administration.
The compounds of the invention can be formulated as neutral or salt forms.
Pharmaceutically acceptable salts include those formed with anions such as
those
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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 mg/kg to
100 mg/kg of the patient's body weight. Preferably, the dosage administered to
a
patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more
preferably 1 mglkg 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.
The invention also provides a pharmaceutical pack or kit comprising one or
more containers filled with one or more of the ingredients of the
pharmaceutical
compositions of the invention. Optionally associated with such containers) can
be a
notice in the form prescribed by a governmental agency regulating the
manufacture,
use or sale of pharmaceuticals or biological products, which notice reflects
approval
by the agency of manufacture, use or sale for human administration.
Diagnosis and Imaging
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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
(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, 121n, carbon (14C), sulfur (355), tritium
(3H),
indium (112In), and technetium (99Tc); luminescent labels, such as luminol;
and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
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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 subject and the imaging
system used will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the
quantity of radioactivity injected will normally range from about 5 to 20
millicuries of
99mTc. The labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain the specific protein. In
vivo tumor
imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds.,
Masson Publishing Inc. (1982).
Depending on several variables, including the type of label used and the mode
of administration, the time interval following the administration for
permitting the
labeled molecule to preferentially concentrate at sites in the subject and for
unbound
labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24
hours or
6 to 12 hours. In another embodiment the time interval following
administration is 5
to 20 days or 5 to 10 days.
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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
(MRI), 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. Tn
one embodiment, a kit comprises an antibody of the invention, preferably a
purified
antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits
of the present invention further comprise a control antibody which does not
react with
the polypeptide of interest. In another specific embodiment, the kits of the
present
invention contain a means for detecting the binding of an antibody to a
polypeptide of
interest (e.g., the antibody may be conjugated to a detectable substrate such
as a
fluorescent compound, an enzymatic substrate, a radioactive compound or a
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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). W 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 detected
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
monoclonal antibody. Alternatively, or in addition, the detecting means may
include
a labeled, competing antigen.
In one diagnostic configuration, test serum is reacted with a solid phase
reagent having a surface-bound antigen obtained by the methods of the present
invention. After binding with specific antigen antibody to the reagent and
removing
unbound serum components by washing, the reagent is reacted with reporter-
labeled
anti-human antibody to bind reporter to the reagent in proportion to the
amount of
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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 known techniques
for attaching protein material to solid support material, such as polymeric
beads, dip
sticks, 96-well plate or filter material. These attachment methods generally
include
non-specific adsorption of the protein to the support or covalent attachment
of the
protein, typically through a free amine group, to a chemically reactive group
on the
solid support, such as an activated carboxyl, hydroxyl, or aldehyde group.
Alternatively, streptavidin coated plates can be used in conjunction with
biotinylated
antigen(s).
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
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
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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
fusion protein is beneficial in therapy and diagnosis, and thus can result in,
fox
example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for
example, human proteins, such as hIL-5, have been :fused with Fc portions for
the
purpose of high-throughput screening assays to identify antagonists of hIL-5.
(See,
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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.
S In preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton
Avenue,
Chatsworth, CA, 91311), among others, many of which are commercially
available.
As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989),
for
instance, hexa-histidine provides for convenient purification of the fusion
protein.
Another peptide tag useful for purification, the "HA" tag, corresponds to an
epitope
derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767
(1984).)
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
vector is a virus, it may be packaged 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 late promoters and promoters of retroviral LTRs,
to
name a few. Other suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription initiation,
termination,
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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 Sf9 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, pXT1
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-S1, pPIC3.5K, pPIC9K, and PA0815 (all available
from Invitrogen, Carlbad, CA). Other suitable vectors will be readily apparent
to the
skilled artisan.
Introduction of the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-
mediated
transfection, electroporation, transductiori, 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.
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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 codon generally is removed with high
efficiency
from any protein after translation in all eukaryotic cells. While the N-
terminal
methionine on most proteins also is efficiently removed in most prokaryotes,
for some
proteins, this prokaryotic removal process is inefficient, depending on the
nature of
the amino acid to which the N-terminal methionine is covalently linked.
In one embodiment, the yeast Pichia pastoris is used to express the
polypeptide of the present invention in a eukaryotic system. Pichia pasto~is
is a
methylotrophic yeast which can metabolize methanol as its sole carbon source.
A
main step in the methanol metabolization pathway is the oxidation of methanol
to
formaldehyde using OZ. This reaction is catalyzed by the enzyme alcohol
oxidase. In
order to metabolize methanol as its sole carbon source, Pichia pasto~is must
generate
high levels of alcohol oxidase due, in part, to the relatively low affinity of
alcohol
oxidase for O~. Consequently, in a growth medium depending on methanol as a
main
carbon source, the promoter region of one of the two alcohol oxidase genes
(AOXl ) is
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highly active. In the presence of methanol, alcohol oxidase produced from the
AOXl
gene comprises up to approximately 30% of the total soluble protein in Piclaia
pastoris. See, Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz,
P.J, et al.,
Yeast 5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76
(1987).
Thus, a heterologous coding sequence, such as, for example, a polynucleotide
of the
present invention, under the transcriptional regulation of all or part of the
AOXI
regulatory sequence is expressed at exceptionally high levels in Pichia yeast
grown in
the presence of methanol.
In one example, the plasmid vector pPIC9K is used to express DNA encoding
a polypeptide of the invention, as set forth herein, in a Piclaea 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 Piclaia pastof-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-S1, 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
achieved by cloning the heterologous polynucleotide of the invention into an
expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the
yeast culture in the absence of methanol.
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
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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,
techniques known in the art may be used to operably associate heterologous
control
regions (e.g., promoter and/or enhancer) and endogenous polynucleotide
sequences
via homologous recombination, 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; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-
8935
(1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of
each of
which are incorporated by reference in their entireties).
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
acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore,
the
amino acid can be D (dextrorotary) or L (levorotary).
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
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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
1 S increased solubility, stability and circulating time of the polypeptide,
or decreased
immunogenicity (see U.S. Patent NO: 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene
glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,
dextran, polyvinyl alcohol and the like. The polypeptides may be modified at
random
positions within the molecule, or at predetermined positions within the
molecule and
may include one, two, three or more attached chemical moieties.
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
2S polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic protein or analog). For example, the polyethylene glycol may have
an
average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000,
3500,
4000, 4500, 5000, SS00, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,
10,000,
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10,500, 11,000, 11,500, 12,000, 12,500, 13,000, I3,S00, 14,000, 14,500,
IS,000,
1S,S00, 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, SS,000, 60,000, 65,000, 70,000,
75,000,
80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
S 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,S7S; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996);
Vorobjev et
al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al.,
Bioconjug.
Claem. 10:638-646 (1999), the disclosures of each of which axe 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
1S to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992)
(reporting
pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol
may
be covalently bound through amino acid residues via a reactive group, such as,
a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene glycol molecule may be bound. The amino acid residues having a
free
amino group may include lysine residues and the N-terminal amino acid
residues;
those having a free carboxyl group may include aspartic acid residues glutamic
acid
residues and the C-terminal amino acid residue. Sulfhydryl groups may also be
used
as a reactive group for attaching the polyethylene glycol molecules. Preferred
for
therapeutic purposes is attachment at an amino group, such as attachment at
the
2S N-terminus or lysine group.
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
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type of amino acid residue (e.g., lysine, lustidine, 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.
Tlzera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., rntern. J.
ofHematol.
6~: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.
One system for attaching polyethylene glycol directly to amino acid residues
of proteins without an intervening linker employs tresylated MPEG, which is
produced by the modification of monmethoxy polyethylene glycol (MPEG) using
tresylchloride (C1SOZCH2CF3). Upon reaction of protein with tresylated MPEG,
polyethylene glycol is directly attached to amine groups of the protein. Thus,
the
invention includes protein-polyethylene glycol conjugates produced by reacting
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proteins of the invention with a polyethylene glycol molecule having a
2,2,2-trifluoreothane 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 1,1'-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 molecules. Similarly, the average
degree of
substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-
11, 10-12,
11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol
moieties per protein molecule. Methods for determining the degree of
substitution are
discussed, for example, in Delgado et al., Crit. Rev. Theca. Drug CaYYier 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
relates to monomers and multimers of the polypeptides of the invention, their
preparation, and compositions (preferably, Therapeutics) containing them. In
specific
embodiments, the polypeptides of the invention are monomers, dimers, trimers
or
tetramers. In additional embodiments, the multimers of the invention are at
least
dimers, at least trimers, or at least tetramers.
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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 1D 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
specif c 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 andlor different amino acid sequences). In
additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at
least a homotrimer, or at least a homotetramer.
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 polypaptides 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
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
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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
occurnng) 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 are joined through
peptide
linkers. Examples include those peptide linkers described in U.S. Pat. No.
5,073,627
(hereby incorporated by reference). Proteins comprising multiple polypeptides
of the
invention separated by peptide linkers may be produced using conventional
recombinant DNA technology.
Another method for preparing multimer polypeptides of the invention involves
use of polypeptides of the invention fused to a leucine zipper or isoleucine
zipper
polypeptide sequence. Leucine zipper and isoleucine zipper domains are
polypeptides
that promote multimerization of the proteins in which they are found. Leucine
zippers were originally identified in several DNA-binding proteins (Landschulz
et al.,
Science 240:1759, (1988)), and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring peptides and
derivatives thereof that dimerize or trimerize. Examples of leucine zipper
domains
suitable for producing soluble multimeric proteins of the invention are those
described
in PCT application WO 94110308, hereby incorporated by reference. Recombinant
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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. 08/446,922, hereby
incorporated by
reference. Other peptides derived from naturally occurring trirneric 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
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
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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,
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
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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
Biotechnol 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 can be used individually (to
mark a single chromosome or a single site on that chromosome) or in panels
(for
marking multiple sites and/or multiple chromosomes).
The polynucleotides of the present invention would likewise be useful for
radiation hybrid mapping, HAPPY mapping, and long range restriction mapping.
For
a review of these techniques and others known in the art, see, e.g., Dear,
"Genome
Mapping: A Practical Approach," IRL Press at Oxford University Press, London
(1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry
3:483-
492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al.,
Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999)
each of
which is hereby incorporated by reference in its entirety.
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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
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 Ieast one polynucleotide
probe
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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 for polymerise 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
IS 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 Ievel 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
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.
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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. K. Kim,
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
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 .
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with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer
lowers the melting point (Tm) by 8°-20° C, vs. 4°-
16° C for the DNA/DNA 15-
mer duplex. Also, the absence of charge groups in PNA means that hybridization
can
be done at low ionic strengths and reduce possible interference by salt during
the
analysis.
The present invention is useful for detecting cancer in mammals. In particular
the invention is useful during diagnosis of pathological cell proliferative
neoplasias
which include, but are not limited to: acute myelogenous leukemias including
acute
monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia,
acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic
leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous
leukemias including chronic myelomonocytic leukemia, chronic granulocytic
leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows,
pigs,
horses, rabbits and humans. Particularly preferred are humans.
Pathological cell proliferative diseases, disorders, and/or conditions are
often
associated with inappropriate activation of proto-oncogenes. (Gehnann, E. P.
et al.,
"The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology," in
Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182
(1985)).
Neoplasias are now believed to result from the qualitative alteration of a
normal
cellular gene product, or from the quantitative modification of gene
expression by
insertion into the chromosome of a viral sequence, by chromosomal
translocation of a
gene to a more actively transcribed region, or by some other mechanism.
(Gelmann
et al., supra) It is likely that mutated or altered expression of specific
genes is
involved in the pathogenesis of some leukemias, among other tissues and cell
types.
(Gelmann et al., supra) Indeed, the human counterparts of the oncogenes
involved in
some animal neoplasias have been amplified or translocated in some cases of
human
leukemia and carcinoma. (Gelinann et al., supra)
For example, c-myc expression is highly amplified in the non-Iymphocytic
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-
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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
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
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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 m 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 polyrnorphic loci, such as DQa class
II
HLA gene, are used in forensic biology to identify individuals. (Erlich, H.,
PCR
Technology, Freeman and Co. (1992).) Once these specific polymorphic loci are
amplified, they are digested with one or more restriction enzymes, yielding an
identifying set of bands on a Southern blot probed with DNA corresponding to
the
DQa class II HLA gene. Similarly, polynucleotides of the present invention can
be
used as polymorphic markers for forensic purposes.
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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 polynucleotides of the present invention can be used as
molecular weight markers on Southern gels, as diagnostic probes for the
presence of a
specific mRNA in a particular cell type, as a probe to "subtract-out" known
sequences
in the process of discovering novel polynucleotides, for selecting and making
oligomers for attachment to a "gene chip" or other support, to raise anti-DNA
antibodies using DNA immunization techniques, and as an antigen to elicit an
immune response.
Uses of the Polypeptides
Each of the polypeptides 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. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell .
Biol. 1053087-
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 axt and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such
as iodine (125I, 1211), carbon (14C), sulfur (35S), tritium (3H), indium
(112In), and
technetium (99mTc), and fluorescent labels, such as fluorescein and rhodamine,
and
biotin.
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-
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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,
1 121n, 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
subject and the imaging system used will determine the quantity of imaging
moiety
needed to produce diagnostic images. In the case of a radioisotope moiety, for
a
human subj ect, the quantity of radioactivity inj ected will normally range
from about 5
to 20 millicuries of 99mTc. The labeled antibody or antibody fragment will
then
preferentially accumulate at the location of cells which contain the specific
protein.
In vivo tumor imaging is described in S.W. Burchiel et al.,
"Immunopharmacokinetics
of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging:
The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., '
Masson Publishing Inc. (1982).)
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
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.
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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 Therapy 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-lmown
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. hl 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, Iiposome
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, pXT1 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 known 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 maj or 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 shown that non-replicating
DNA
sequences can be introduced into cells to provide production of the desired
polypeptide for periods of up to six months.
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
injection into the tissues comprising these cells. They are preferably
delivered to and
expressed in persistent, non-dividing cells which are differentiated, although
delivery
and expression may be achieved in non-differentiated or less completely
differentiated
cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo
muscle cell's
are particularly competent in their ability to take up and express
polynucleotides.
For the nakedsaucleic acid sequence injection, an effective dosage amount of
DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about
50
mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to
about 20
mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course,
as
the artisan of ordinary skill will appreciate, this dosage will vary according
to the
tissue site of injection. The appropriate and effective dosage of nucleic acid
sequence
can readily be determined by those of ordinary skill in the art and may depend
on the
condition being treated and the route of administration.
The preferred route of administration is by the parenteral route of inj ection
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
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 Iiposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred.
The various liposome-nucleic acid complexes are prepared using methods well
known
in the art. See, e.g., Straubinger et al., Methods of hnmunology , 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.
SWs 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. LUVs are
prepared by a number of methods, well known in the art. Commonly used methods
include Ca2+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta,
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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 Z 0:1 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
carriers, into
mice. U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,.127, 5,589,466,
5,693,622, 5,580,859, 5,703,055, and international publication NO: WO 94/9469
(which are herein incorporated by reference) provide cationic lipids for use
in
transfecting DNA into cells and mammals. U.S. Patent Nos. 5,589,466,
5,693,622,
5,580,859, 5,703,055, and international publication NO: WO 94/9469 (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.
S 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 iya
vitro or in
vivo. The transduced eukaryotic cells will express polypeptides of the
invention.
In certain other embodiments, cells are engineered, ex vivo or in 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
1 S 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 ofalpha-1-antitrypsin
and
CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434
(1991);
Rosenfeld et al., Cell, 68:143-1SS (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 I~ozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-S03 (1993);
2S Rosenfeld et al., Cell , 68:143-1SS (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: S,6S2,224, 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 occurring 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 all 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
lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate helper
viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes
viruses.
Once the packaging cells are transfected and infected, they will produce
infectious
AAV viral particles which contain the polynucleotide construct 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; Koller 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 axe
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-l, 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 (1989)).
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.
Administration 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 polynucleotide
construct of the present invention in or around a surgical wound. For example,
a
patient can undergo surgery and the polynucleotide construct can be coated on
the
surface of tissue inside the wound or the construct can be inj ected 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
Biological 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 Liboary
Code).
Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the
present invention may be useful in treating, preventing, diagnosing, and/or
prognosing
immunodeficiencies, including both congenital and acquired immunodeficiencies.
Examples of B cell immunodeficiencies in which immunoglobulin levels B cell
function and/or B cell numbers are decreased include: X-linked
agammaglobulinemia
(Bruton's disease), X-linked infantile agammaglobulinemia, X-linked
immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM,
X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including
congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia,
late-
onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,
unspecified hypogammaglobulinemi'a, 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 immunodeficiencies in which T cell and/or B cell
function and/or number is decreased include, but are not limited to: DiGeorge
anomaly, severe combined immunodeficiencies (SCID) (including, but not limited
to,
X-linked SLID, autosomal recessive SCID, adenosine deaminase deficiency,
purine
nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare
lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia),
thymic
hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion,
chronic
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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
C1,
C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis,
thymic
alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital
leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short
limbed
dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.
In a preferred embodiment, the immunodeficiencies and/or conditions
associated with the immunodeficiencies recited above are treated, prevented,
diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention.
In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention could be used as an agent to
boost
immunoresponsiveness among immunodeficient individuals. In specific
embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention could be used as an agent to boost
immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present invention may be useful in treating, preventing, diagnosing
and/or
prognosing autoimmune disorders. Many autoimmune disorders result from
inappropriate recognition of self as foreign material by immune cells. This
inappropriate recognition results in an immune response leading to the
destruction of
the host tissue. Therefore, the administration of polynucleotides and
polypeptides 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 ophthalmia,
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 asthma 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), astlnna (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 fox example, antagonists or
agonists,
polypeptides or polynucleotides, or antibodies of the present invention. hi 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 plui7potent 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, andJor 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, fibrosrtis,
folliculitrs, gastritis,
gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratrtis,
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
this case, the foreign transplanted irmnune 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, andlor 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/AmS, respiratory syncytial virus, Dengue,
rotavirus, Japanese B encephalitis, influenza A and B, paxainfluenza, 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 hibYio cholerae, Mycobacterium
lepz°ae, Salmonella typlai, Salmonella paratyphi, Meissez°ia
nzenizzgitidis,
Streptococcus pneumoniae, Group B streptococcus, Shigella spp.,
Enterotoxigenic
EscheYichia coli, Enterohemorrhagic E. coli, and Bo>"relia bu~gdoYfe~i.
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
I
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.
In another specific embodiment, polypeptides, antibodies, polynucleotides
and/or agonists or antagonists of the present invention are used as an agent
to induce
higher affinity antibodies.
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, HIV Infection, AIDS,
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 TH1 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 axe 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-
incompetence/immunodeficiency such as observed among SLID 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
gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related
idiopathic monoclonal gammopathies, 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
chronic
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
for
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 (ARDS).
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 imrnunoglobulin
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, and/or
diagnose an
individual having common variable immunodeficiency disease ("CVTD"; also known
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.
20, 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. W098/24~93, 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, andlor
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 polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be used to prevent,
diagnose,
prognose, and/or treat thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction,
transient ischemic attack, unstable angina. In speciftc 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 rnitral 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, and/or treat diseases and disorders of the blood
and/or
blood forming organs associated with the tissues) in which the polypeptide of
the
invention is expressed, including one, two, three, four, five, or more tissues
disclosed
in Table 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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andlor 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
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 carries oxygen) in them is below normal. Anemia
may
be caused by excessive bleeding, decreased red blood cell production, or
increased
red blood cell destruction (hemolysis). The polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention may be useful in
treating,
preventing, and/or diagnosing anemias. Anemias that may be treated prevented
or
diagnosed by the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention include iron deficiency anemia,
hypochromic
anemia, microcytic anemia, chlorosis, hereditary siderob;astic anemia,
idiopathic
acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g.,
pernicious
anemia, (vitamin 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 usefizl 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 Hennansky-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, andlor agonists
or
antagonists of the present invention on the clotting time of blood may be
monitored
using any of the clotting tests known in the art including, but not limited
to, whole
blood partial thromboplastin time (PTT), the activated partial thromboplastin
time
(aPTT), the activated clotting time (ACT), the recalcified activated clotting
time, or
the Lee-White Clotting time.
Several diseases and a variety of drugs can cause platelet dysfunction. Thus,
in
a specific embodiment, the polynucleotides, polypeptides, antibodies, and/or
agonists
or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating acquired platelet dysfunction such as platelet
dysfunction
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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-
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,
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, and/or treating leukemias and lymphomas including, but
not
limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid
(myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic
lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary
syndrome, and
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Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or
granulocytic)
leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and
mycosis fiuigoides.
In other embodiments, the polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing, preventing, and/or treating diseases and disorders of plasma cells
including, but not limited to, plasma cell dyscrasias, monoclonal
gammaopathies,
monoclonal gammopathies of undetermined significance, multiple myeloma,
macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and
Raynaud's phenomenon.
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, and/or
agonists or antagonists of the present invention may be useful as an agent to
enhance
the migration, phagocytosis, superoxide production, antibody dependent
cellular
cytotoxicity of neutrophils, eosionophils and macrophages.
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 polynucleotides, polypeptides, antibodies, and/or
agonists or antagonists of the present invention may be useful in preventing,
diagnosing, and/or treating primary hematopoietic disorders.
Hyperproliferative Disorders
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 immune response may be increased by either enhancing an existing immune
response, or by initiating a new immune response. Alternatively, decreasing an
immune response may also be a method of treating 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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Lyrnphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease,
Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's
Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related
Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct
Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast
Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System
(Primary)
Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral
Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer,
Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood
Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial
Genn 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-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial
Cancer,
Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer; Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer,
Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma,
Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate
Cancer,
Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer,
Retinoblastoma,
Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary
Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft
Tissue
Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive
Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer,
Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and
Ureter,
Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and
Renal
Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal
Cancer, Visual Pathway and 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 irritation or
inflammation. Dysplastic disorders which can be diagnosed, prognosed,
prevented,
and/or treated with compositions of the invention (including polynucleotides,
polypeptides, agonists or antagonists) include, but are not limited to,
anhidrotic
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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, craniocarpotaxsal 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 ectodennal 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, andlor
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, 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 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, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular
tumor,
lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,
melanoma, neuroblastoma, and retinoblastoma.
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, lyrnphoproliferative
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 administration, etc.), which acts
upon the
promoter upstream of said polynucleotides to induce expression of the encoded
protein product. As such the beneficial therapeutic affect of the present
invention
may be expressly modulated (i.e. to increase, decrease, or inhibit expression
of the
present invention) based upon said external stimulus.
Polynucleotides of the present invention may be useful in repressing
expression of oncogenic genes or antigens. By "repressing expression of the
oncogenic genes " is intended the suppression of the transcription of the
gene, the
degradation of the gene transcript (pre-message RNA), the inhibition of
splicing, the
destruction of the messenger RNA, the prevention of the post-translational
modifications of the protein, the destruction of the protein, or the
inhibition of the
normal function of the protein.
For local administration to abnormally proliferating cells, polynucleotides of
the present invention may be administered by any method known to those of
skill in
the art including, but not limited to transfection, electroporation,
microinjection of
cells, or in vehicles such as liposomes, lipofectin, or as naked
polynucleotides, or any
other method described throughout the specification. The polynucleotide of the
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 (Yates et al., Nature 313:812 (1985)) known to those skilled
in the
art. These references are exemplary only and are hereby incorporated by
reference.
In order to specifically deliver or transfect cells which are abnormally
proliferating
and spare non-dividing cells, it is preferable to utilize a retrovirus, or
adenoviral (as
described in the art and elsewhere herein) delivery system known to those of
skill in
the art. Since host DNA replication is required for retroviral DNA to
integrate and
the retrovirus will be unable to self replicate due to the lack of the
retrovirus genes
needed for its life cycle. Utilizing such a retroviral delivery system for
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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
20. tissue culture, tumor growth in animals and cell cultures, or any other
method known
to one of ordinary shill 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. Aimed 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 ih 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
less than SX10-6M, 10-6M, SX10-~M, 10-'M, 5X10-$M, 10-$M, 5X10-9M, 10-~M,
5X10-1°M, 10-1°M, 5X10-11M, 10-11M, 5X10-12M, 10-12M, SX10-13M,
10-13M, SX10-
14M, 10-14M, SX10-15M, 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, for example in the activation of
a death-
domain receptor, such as tumor necrosis factor (TNF) receptor-l, 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 I~,
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 prodr~gs 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 immune response to respond to proliferative
antigens and
immunogens, or indirectly, such as in activating the expression of proteins
known to
enhance the immune response (e.g. chemokines), to said antigens and
immunogens.
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,
lcidney 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 (PSGI~, 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), Hartnup 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, and/or 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, nephroblastoma, 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 an,
including, but not limited to, direct needle inj ection 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
Iimb
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, Eisenrnenger 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 aortopulmonary 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
rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial
tachycardia, ectopic
functional tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia,
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
cardiomyopathy, 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, tlirombosis, 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, fbromuscular 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
leukomalacia,
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 injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
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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
pneunaoniae (pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal
pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and
Pseudomas spp.), Mycoplasfna praeufnoniae pneumonia, HernoplZilus influenzae
pneumonia, Legionella pneurnophila (Legionnaires' disease), and Chlamydia
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 CYyptococcus faeoformans; aspergillosis, caused byAspefgillus spp.;
candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii
(pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasrna and Chlamydia
S spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical
pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy,
pleural
effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated
spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases
(e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema,
chronic or
acute bronchitis), occupational lung diseases (e.g., silicosis, black lung
(coal workers'
pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis,
and
benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis
(e.g.,
fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary
fibrosis,
desquamative interstitial pneumonia, lymphoid interstitial pneumonia,
histiocytosis X
1S (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 pneurnophila), and cystic fibrosis.
Anti-An~io~enesis Activity
The naturally occurnng balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
2S Rastinejad et al., Cell 56:345-3SS (199). In those rare instances in which
neovascularization occurs under normal physiological conditions, such as wound
healing, organ regeneration, embryonic development, and female reproductive
processes, angiogenesis is stringently regulated and spatially and temporally
delimited. Under conditions of pathological angiogenesis such as that
characterizing
solid tumor growth, these regulatory controls fail. Unregulated angiogenesis
becomes
pathologic and sustains progression of many neoplastic and non-neoplastic
diseases.
A number of serious diseases are dominated by abnormal neovascularization
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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. Ehgl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.
Micf°ovasc. Res.
29:401-411 (1985); Foll~man, Advances in Cancer Research, eds. Klein and
Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, A»a. J.
Opthalfsaol. 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
15. 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 rej ection,
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 hyperixophic 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 result in the development of hypertrophic scars and keloids (e.g.,
burns),
and is preferably initiated after the proliferative phase has had time to
progress
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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 can be
treated with the polynucleotides and polypeptides of the present invention
(including
agonists and/or antagonists) include, but are not limited to: neovascular
glaucoma,
diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis,
retinopathy of
prematurity macular degeneration, corneal graft neovascularization, as well as
other
eye inflammatory diseases, ocular tumors and diseases associated with
choroidal or
iris neovascularization. See, e.g., reviews by Waltman et al., Af~a. J.
Ophthal. 85:704-
710 (1978) and Gartner et al., Su~v. Ophthal. 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, leishrnaniasis and onchocerciasis), immunological processes
(e.g.,
graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma,
inflammation
(of any cause), toxic and nutritional deficiency states, and as a complication
of
wearing contact lenses.
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 muco-
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 t~ prevent capillary
invasion of
transplanted corneas. In a sustained-release form injections might only be
required 2-
3 times per year. A steroid could also be added to the injection solution to
reduce
inflammation resulting from the inj ection 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 inj ection 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 fox
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
S 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
afterhepatic 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
1 S 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-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the
lighter "d
group" transition metals.
2S 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 dehydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl
complexes. Suitable molybdate complexes include ammonium molybdate and its
hydrates, sodium molybdate and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum
(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and
sugars.
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
(Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate ("GST";
Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-
serum;
alpha2-antiplasmin (Holrnes et al., J. Biol. Chem. 262(4):1659-1664, 1987);
Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-
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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, 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.
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 myelocytic (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, saxcomas 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 ca~icer, 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, ependyrnoma, 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, 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, ischemialreperfusion 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 fiuther 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 viral
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; (~) 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 agoiusts 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 i~r vivo, e.g., choline acetyltransferase
or
acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms
of
neuron dysfunction ih 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. Neurosci., 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. Neur°ol., 70:65-82 (1980), or Brown et
al., Anfa. Rev.
Neurosci., 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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248
or antagonists) may be used to diagnose and/or treat or prevent diseases or
disorders
associated with these roles, including, but not limited to, learning and/or
cognition
disorders. The compositions of the invention may also be useful in the
treatment or
prevention of neurodegenerative disease states and/or behavioural disorders.
Such
neurodegenerative disease states and/or behavioral disorders include, but are
not
limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease,
Tourette
Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive
disorder,
panic disorder, learning disabilities, ALS, psychoses, autism, and altered
behaviors,
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 al-tery 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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249
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, Werniclce'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 mufti-infarct dementia, periventricular
leukomalacia,
vascular 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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250
encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis
Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated
encephalomyelitis, meungoencephalitis such as uveomeningoencephalitic
syndrome,
Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis,
encephalomalacia such as periventricular leukomalacia, epilepsy such as
generalized
epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy
which
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, andlor antagonists of the present
invention
include hydrocephalus such as Dandy-Walker Syndrome and normal pressure
hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral
malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri
pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral
toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central
nervous
system infections such as AIDS Dementia Complex, 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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251
Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, ~Gerstmann-
Straussler Syndrome, T~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(M1), 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 spina
bifida cystica and spina 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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