Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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THAN ONE VOLUME
THIS IS VOLUME ~ , OF
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50 Human Secreted Proteins
Field of the Invention
This invention relates to newly identified polynucleotides and the
polypeptides encoded by these polynucleotides, uses of such polynucleotides
and
polypeptides, and their production.
Background of the Invention
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
extraeellular 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
include the commercially valuable human insulin, interferon, Factor VIII,
human
growth hormone, tissue plasminogen activator, and erythropoeitin. Thus, in
light of
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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 disorders by using secreted proteins or the Qenes that encode them.
Summary of the Invention
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
disorders
and conditions related to the polypeptides and polynucleotides, and
therapeutic
methods for treating such disorders and conditions. The invention further
relates to
screening methods for identifying binding partners of the polypeptides.
Detailed Description
Definitions
The following definitions are provided to facilitate understanding of certain
terms used throughout this specification.
In the present invention, "isolated" refers to material removed from its
original
environment (e.g., the natural environment if it is naturally occurring), and
thus is
altered "by the hand of man" from its natural state. For example, an isolated
polynucleotide could be part of a vector or a composition of matter, or could
be
contained within a cell, and still be "isolated" because that vector,
composition of
matter, or particular cell is not the original environment of the
polynucleotide. The
term "isolated" does not refer to genomic or cDNA libraries, whole cell total
or
mRNA preparations, genomic DNA preparations (including those separated by
electrophoresis and transferred onto blots), sheared whole cell genomic DNA
preparations or other compositions where the art demonstrates no
distinguishing
features of the polynucleotide/sequences of the present invention.
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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
analysis). A representative clone containing all or most of the sequence for
SEQ ID
NO:X was deposited with the American Type Culture Collection ("ATCC"). As
shown in Table l, each clone is identified by a cDNA Clone ID (Identifier) and
the
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d
ATCC Deposit Number. The ATCC is located at 10801 University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposit was made pursuant to the
terms of the Budapest Treaty on the international recognition of the deposit
of
microorganisms for purposes of patent procedure.
A "polynucleotide" of the present invention also includes those
polynucleotides capable of hybridizing, under stringent hybridization
conditions, to
sequences contained in SEQ ID NO:X, the complement thereof, or the cDNA within
the clone deposited with the ATCC. "Stringent hybridization conditions" refers
to an
overnight incubation at 42 degree C in a solution comprising 50% formamide, Sx
SSC
(750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx
Denhardt's solution, 10% dextrin sulfate, and 20 qg/ml denatured, sheared
salmon
sperm DNA, followed by washing the filters in O.lx SSC at about 65 degree C.
Also contemplated are nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished through the manipulation of formamide concentration (lower
percentages of formamide result in lowered stringency); salt conditions, or
temperature. For example, lower stringency conditions include an overnight
incubation at 37 degree C in a solution comprising 6X SSPE (20X SSPE = 3M
NaCI;
0.2M NaH,PO~,; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml
salmon sperm blocking DNA; followed by washes at 50 degree C with 1XSSPE,
0.1 % SDS. In addition, to achieve even lower stringency, washes performed
following stringent hybridization can be done at higher salt concentrations
(e.g. SX
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
reagents may require modification of the hybridization conditions described
above,
due to problems with compatibility.
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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 oliao 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
a given polypeptide. Also, a given polypeptide may contain many types of
modifications. Polypeptides may be branched , for example, as a result of
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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.)
Many proteins (and translated DNA sequences) contain regions where the
amino acid composition is highly biased toward a small subset of the available
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residues. For example, membrane spanning domains and signal peptides (which
are
also membrane spanning) typically contain long stretches where Leucine (L),
Valine
(V), Alanine (A), and Isoleucine (I) predominate. Poly-Adenosine tracts
(polyA) at
the end of cDNAs appear in forward translations as poly-Lysine (poly-K) and
poly-
Phenylalanine (poly-F) when the reverse complement is translated. These
regions are
often referred to as "low complexity" regions.
Such regions can cause database similarity search programs such as BLAST to
find high-scoring sequence matches that do not imply true homology. The
problem is
exacerbated by the fact that most weight matrices (used to score the
alignments
generated by BLAST) give a match between any of a group of hydrophobic amino
acids (L,V and I) that are commonly found in certain low complexity regions
almost
as high a score as for exact matches.
In order to compensate for this, BLASTX.2 (version 2.Oa5MP-WashU)
employs two filters ("seg" and "xnu") which "mask" the low complexity regions
in a
particular sequence. These filters parse the sequence for such regions, and
create a
new sequence in which the amino acids in the low complexity region have been
replaced with the character "X". This is then used as the input sequence
(sometimes
referred to herein as "Query" and/or "Q") to the BLASTX program. While this
regime helps to ensure that high-scoring matches represent true homology,
there is a
negative consequence in that the BLASTX program uses the query sequence that
has
been masked by the filters to draw alignments.
Thus, a stretch of "X"s in an alignment shown in the following application
does not necessarily indicate that either the underlying DNA sequence or the
translated protein sequence is unknown or uncertain. Nor is the presence of
such
stretches meant to indicate that the sequence is identical or not identical to
the
sequence disclosed in the alignment of the present invention. Such stretches
may
simply indicate that the BLASTX program masked amino acids in that region due
to
the detection of a low complexity region, as defined above. In all cases, the
reference
sequences) (sometimes referred to herein as "Subject", "Sbjet", and/or "S")
indicated
in the specification, sequence table (Table 1 ), and/or the deposited clone is
(are) the
definitive embodiments) of the present invention, and should not be construed
as
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limiting the present invention to the partial sequence shown in an alignment,
unless
specifically noted otherwise herein.
Polynucleotides and Polyneptides of the Invention
FEATURES OF PROTEIN ENCODED BY GENE NO: 1
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. gi1288145
(all
information available through the recited accession number is incorporated
herein by
reference). A partial alignment demonstrating the observed homology is shown
immediately below.
IS >gi~288145 put. ORF [Homo Sapiens] >pir~I38022~I38022 hypothetical protein -
human
Length = 196
Plus Strand HSPs:
Score = 145 (51.0 bits), Expect = 1.3e-14, Sum P(2) = 1.3e-14
Identities = 31/45 (68%), Positives = 34/45 (75%), Frame = +3
Query: 2286 TGVQDQPGQHGETLSLQKIQKLTRHCGMCL*SQLLRRLTQENCLN 2420
+GVQD PGQHG+ SL KIQ+L H G CL SQLLRRL QEN LN
Sbjct: 6 SGVQDYPGQHGKIPSLLKIQELAGHGGRCLQSQLLRRLRQENHLN 50
Score = 84 (29.6 bits), Expect = 1.3e-14, Sum P(2) = 1.3e-14
Identities = 17/28 (60%), Positives = 18/28 (64%), Frame = +2
3~ Query: 2414 LEPGGGGCSELRLCHCTPAWATERDSVS 2497
L GG GCSE + C PAW TE DSVS
Sbjct: 49 LNSGGRGCSEPKSHLCIPAWVTEGDSVS 76
The segments of gi1288145 that are shown as "Sbjct" above are set out as SEQ
ID NO. 111
and SEQ ID NO. 113, respectively.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 112 and/or SEQ ID NO. 114 which correspond
to
the Query sequence in the alignment shown above (gaps introduced in the
sequence by the
computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Human Whole Six Week Old Embryo and to a lesser extent
in
Early Stage Human Lung, subtracted, Human Whole Brain #2 - Oligo dT > I.SKb,
Jurkat
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T-Cell, S phase. Human Manic Depression Tissue, Human Fetal Kidney, human
ovarian
cancer, Human Umbilical Vein Endothelial Cells, uninduced, Human Fetal Dura
Mater,
Stratagene neuroepithelium (#937231), H. Frontal cortex,epileptic:re-excision,
Brain
frontal cortex. Anergic T-cell, and Soaves infant brain 1NIB.
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 2506 of SEQ ID
NO:11, b
is an integer of 15 to 2520, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:1 l, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 2
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. gi12650190
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "enoyl-CoA hydratase (fad-1)". A
partial
alignment demonstrating the observed homology is shown immediately below.
>gi~2650190 (AE001074) enoyl-CoA hydratase (fad-1) [Archaeoglobus fulgidus]
>pir~C69304~C69304 enoyl-CoA hydratase (fad-1) homolog -
Archaeoglobus fulgidus >sp1029814~029814 ENOYL-COA HYDRATASE
(FAD-1).
Length = 256
Plus Strand HSPs:
Score = 190 (66.9 bits), Expect = 7.8e-20, Sum P(2) = 7.8e-20
Identities = 47/156 (30%), Positives = 77/156 (49%), Frame = +3
Query: 234 DNGIGILTLNNPSRMNAFSGVMMLQLLEKVIELENWTEGKGLIVRGAKNTFSSGSDLNAV 413
D I + TLN P ++NA ++L E + +E + LI+ G+ F++G+D+N +
Sbjct: 10 DGEIAVATLNRPEKLNALDTKTRMELAEVIEGIEE--VARVLIITGSGKAFAAGADINEL 67
4O Query. 414 KSLGTPEDGMAVCMFMQNTLTRFMRLPLISVALVQGWALGGGAEFTTACDFRLMTPESKI 593
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+ + +R L + +A V G+ LGGG E ACD R+ + ++K
Sbjct: 68 LQRDAIK-AFEATKLGTD~.FSRIEELEIPVIAAVNGYTLGGGCELAMACDIRIASEKAKF 12'0
Query: 594 RFVHKEMGIIPSWGGTTRLVEIIGSRQALK-VLSGAL 701
+ IIP GGT RL ++G A K VL+G +
Sbjct: 127 GQPEINLAIIPGAGGTQRLPRLVGLGi~L~KKLVLTGEI 153
The segment of gi12650190 that is shown as "Sbjct" above is set out as SEQ ID
NO. 115.
Based on the structural similarity these homologous polypeptides are expected
to share at
10 least some biological activities. Such activities are known in the art and
described
elsewhere herein. Assays for determining such activities are also known in the
art some of
which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 116 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues:
Soares placenta Nb2HP and to a lesser extent in Soares_NhHMPu_S 1, Stratagene
ovarian
cancer (#937219), Soares melanocyte 2NbHM, Hodgkin's Lymphoma II, Messangial
cell,
frac 1, Human Thyroid, Early Stage Human Lung, subtracted, Fetal Liver,
subtraction II,
Stratagene lung (#937210), Human Amygdala, Monocyte activated, Stratagene
ovarian
cancer (#937219), Keratinocyte, Colorectal Tumor, Human Adult Liver,
subtracted, A1-
CELL LINE, Human (HCC) cell line liver (mouse) metastasis, remake, Activated T-
cells,
Soares retina N2b4HR, Human adult small intestine,re-excision, NTERA2
teratocarcinoma cell line+retinoic acid ( 14 days), Human Pineal Gland, B Cell
lymphoma,
Soares_parathyroid tumor NbHPA, Synovial hypoxia, Human Ovary, T-Cell PHA 16
hrs, Apoptotic T-cell, 12 Week Old Early Stage Human, II, Spinal cord, Human
Rhabdomyosarcoma, Human Adrenal Gland Tumor, NTERA2, control, Colon Tumor, 12
Week Old Early Stage Human, Smooth muscle, serum treated, H Macrophage (GM-CSF
treated), re-excision, Soares_placenta_8to9weeks_2NbHP8to9W, Human Fetal Lung
III,
Human Testes, Reexcision, Human Fetal Heart, Human Osteoclastoma, Smooth
muscle,control, Soares fetal heart NbHHI9W, Soares_parathyroid_tumor NbHPA,
Human Bone Marrow, treated, Human Endometrial Tumor, Human 8 Week Whole
Embryo, Soares fetal liver spleen 1 NFLS, and
Soares_placenta_8to9weeks_2NbHP8to9W.
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Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
62 as residues: Lys-86 to Gly-93.
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 2137 of SEQ ID
N0:12, b
is an integer of 15 to 2151, 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
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares adult brain N2b5HB55Y and to a lesser extent in
Soares fetal heart NbHH 19W, HUMAN B CELL LYMPHOMA.
Soares_parathyroid_tumor NbHPA, Soares_pregnant uterus_NbHPU, Human
Endometrial Tumor, Soares fetal liver spleen 1NFLS, H. Striatum Depression,
subt,
Stratagene ovarian cancer (#937219), Soares_fetal lung_NbHLI9W,
Soares_pregnant uterus NbHPU, Endothelial-induced, NCI CGAP_Br2,
NCI CGAP_Pr2, NCI CGAP_Kid3, Soares_fetal heart NbHHI9W, Human Fetal Brain,
normalized 100024F, Prostate-BPH subtracted II, Human colon cancer,
metaticized to
liver, subtraction, Bone Cancer, Human epithelioid sarcoma, Human OB HOS
control
fraction I, Human Colon, Adipocytes;re-excision, HSC172 cells, Smooth muscle,
control;
re-excision, Soares retina N2b4HR, Soares_placenta_8to9weeks 2NbHP8to9W, H.
Whole
Brain #2, re-excision, Human Hypothalamus,schizophrenia, re-excision,
Stratagene hNT
neuron (#937233), Human endometrial stromal cells, Spinal Cord. re-excision,
human
ovarian cancer, T-Cell PHA 24 hrs, Human Ovarian Cancer Reexcision, Human
Hypothalmus,Schizophrenia, Soares_fetal heart NbHH 19W, Human Adipose, Human
Rhabdomyosarcoma, Human Chondrosarcoma, Human Fetal Brain, Human Liver,
normal,
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Pancreas Islet Cell Tumor, Human Gall Bladder, PC3 Prostate cell line, Human
Testes
Tumor, H Macrophage (GM-CSF treated), re-excision, Normal colon, Primary
Dendritic
cells,frac 2, NCI CGAP_LuS, NCI_CGAP Prl, NCI_CGAP Colt, NCI_CGAP GCB1,
NCI_CGAP_Lei2, NCI_CGAP_Brn23, Soares_pregnant uterus_NbHPU, Colon Normal
III, Soares_senescent_fibroblasts_NbHSF, Human Microvascular Endothelial
Cells, Tract.
A, Smooth muscle,control, and Soaves placenta Nb2HP.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
63 as residues: Arg-24 to Asn-39, Ser-43 to Pro-61.
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 681 of SEQ ID
N0:13, b
is an integer of 15 to 695, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:13, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 4
It has been discovered that this gene is expressed primarily in the following
.
tissues/cDNA libraries: Soaves placenta Nb2HP and to a lesser extent in Human
Cerebellum, Soaves fetal liver spleen_1NFLS_S 1, Soaves infant brain 1NIB,
Soaves
adult brain N2b4HB55Y, NCI_CGAP_GCB l, Stratagene HeLa cell s3 937216, Human
Substantia Nigra, Human Cerebellum, subtracted, Human Pituitary, subtracted,
Soaves
retina N2b4HR, Soaves senescent_fibroblasts NbHSF, Human Whole Brain #2 -
Oligo
dT > I.SKb, Strataaene colon (#937204), NCI CGAP_KidS, NCI CGAP_Pr22, Human
Heart, Soares_pregnant uterus_NbHPU, Soaves breast 2NbHBst,
Soares_fetal heart NbHHI9W, Soaves melanocyte 2NbHM, Human Placenta, Human
Fetal Heart, Monocyte activated, Keratinocyte, Soaves testis NHT, Soaves ovary
tumor
NbHOT, Human Tongue, frac 1, Human Colon, subtraction, H. cerebellum, Enzyme
CA 02364549 2001-08-27
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13
subtracted, H. Epididiymus, cauda, Breast Lymph node cDNA library, Messangial
cell,
frac 2, Human Normal Breast, HEL cell line, Stratagene placenta (#937225),
human
corpus colosum, Soares_fetal_lung NbHLI9W, H. Kidney Cortex, subtracted, Human
Adipose Tissue, re-excision, Human Osteosarcoma, Myoloid Progenitor Cell Line,
NCI CLAP GCB 1, Soares_NhHMPu_S 1, Brain Frontal Cortex, re-excision, Human
Infant Brain, Human Chronic Synovitis, T-Cell PHA 16 hrs, Human Primary Breast
Cancer Reexcision, Human Pancreas Tumor, NCI_CGAP_AA 1, NCI CGAP_Co9,
NCI CGAP_Kid3, NCI_CGAP Brn25, Human Adult Testes, Larne Inserts, Reexcision,
Soares_pineal gland N3HPG, Human Hypothalmus,Schizophrenia, Human Pancreas
Tumor; Reexcision, Liver, Hepatoma, Soares adult brain N2b5HB55Y, Bone Marrow
Stromal Cell, untreated, Human Fetal Brain, Rejected Kidney, lib 4, H. Frontal
cortex,epileptic;re-excision, Barstead spleen HPLRB2, Gessler Wilms tumor,
Soares testis_NHT, Soares_pineal_gland N3HPG, Soares_total_fetus_Nb2HF8 9w,
Soares_parathyroid_tumor NbHPA, Soares_fetal liver_spleen_1NFLS_S1,
Soares_senescent_fibroblasts NbHSF, Smooth muscle, serum induced,re-exe,
Soares
breast 3NbHBst, Adipocytes, Dendritic cells, pooled, Human Amygdala,
Soares fetal lung_NbHLI9W, Soares_fetal heart NbHHI9W, Human Microvascular
Endothelial Cells, fract. A, Soares fetal liver spleen 1NFLS.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:14 and may have been publicly available prior to conception of the present
invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence would be cumbersome.
Accordingly,
preferably excluded from the present invention are one or more polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a is any
integer between 1 to 2296 of SEQ ID N0:14, b is an integer of 15 to 2310,
where both a
and b correspond to the positions of nucleotide residues shown in SEQ ID
N0:14, and
where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 5
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in
CA 02364549 2001-08-27
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1 ~t
NCI_CGAP_GCB 1, Macrophage-oxLDL, Human Hypothalmus,Schizophrenia, Colon
Normal II, Lung Carcinoma A549 TNFalpha activated, Bone Cancer, H. cerebellum,
Enzyme subtracted, Healing groin wound, 6.5 hours post incision, Prostate BPH,
Human
Adult Small Intestine, Human Prostate, L428, T-Cell PHA 24 hrs, Human
Hippocampus,
Human Gall Bladder, Human Eosinophils, Bone marrow, Hodgkin's Lymphoma II,
Human Cerebellum.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
65 as residues: Ala-16 to Val-35.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID NO:15 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1430 of SEQ ID
NO:15, b
is an integer of 15 to 1444, 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 BY GENE NO: 6
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares_multiple_sclerosis_2NbHMSP, Soares retina
N2b4HR and
to a lesser extent in Morton Fetal Cochlea, Soares_pregnant uterus NbHPU,
Human
Substantia Nigra, Human Hypothalmus,Schizophrenia, Human Amygdala, Human
Amygdala;re-excision, H. Whole Brain #2, re-excision, Human Infant Brain,
Spinal cord,
12 Week Old Early Stage Human, Soares melanocyte 2NbHM, Human Testes,
Reexcision, Human Testes, Soares placenta Nb2HP, Soares infant brain 1NIB,
Hypothalamus, Human Hippocampus, subtracted, Human Aortic Endothelium, Human
Cerebellum, subtracted, Frontal lobe,dementia;re-excision, Human
Hypothalamus,schizophrenia, re-excision, human corpus colosum, Glioblastoma,
Human
Ovary, Soares_NhHMPu_Sl, Soares total fetus Nb2HF8 9w, Spinal Cord, re-
excision,
CA 02364549 2001-08-27
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IS
Human Brain, Striatum, L428, HUMAN JURKAT MEMBRANE BOUND
POLYSOMES, Human Hippocampus, Human Fetal Brain, Human Liver, normal, Human
Cerebellum, and Soaves fetal liver spleen 1 NFLS.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:16 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1236 of SEQ ID
N0:16, b
is an integer of I S to 1250, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID N0:16, and where b is greater than or
equal to a
+ 14.
IS
FEATURES OF PROTEIN ENCODED BY GENE NO: 7
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. gi12618733
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "CC3". A partial alignment
demonstrating the
observed homology is shown immediately below.
>gi~2618733 CC3 [Homo sapiens] >sp~015383~015383 CC3 (CC3).
Length = 242
Plus Strand HSPs:
Score = 979 (344.6 bits), Expect = 1.9e-109, Sum P(2) = 1.9e-109
3O Identities = 187/189 (98%), Positives = 187/189 (98%), Frame = +2
Query: 212 KLTFDEEAYKNVNQEWDFEKLDDYASAFQGHDVGFCCLGTTRGKAGAEGFVRVDRDYVL 391
KLTFDEEAYKNVNQEVVDFEKLDDYASAFQGHDVGFCCLGTTRGKAGAEGFVRVDRDYVL
Sbjct: 54 KLTFDEEAYKNVNQEVVDFEKLDDYASAFQGHDVGFCCLGTTRGKAGAEGFVRVDRDYVL113
Query: 392 KSAELAKAGGCKHFNLLSSKGADKSSNFLYLQVKGEVEAKVEELKFDRYSVFRPGVLLCD571
KSAELAKAGGCKHFNLLSSKGADKSSNFLYLQVKGEVEAKVEELKFDRYSVFRPGVLLCD
Sbjct: 114 KSAELAKAGGCKHFNLLSSKGADKSSNFLYLQVKGEVEAKVEELKFDRYSVFRPGVLLCD173
Query: 572 RQESRPGEWLVRKFFGSLPDSWASGHSVPWTWRAMLNNWRPRDKQMELLENKAIHDL751
RQESRPGEWLVRK FGSLPDSWA GHSVPWTWRAMLNNWRPRDKQMELLENKAIHDL
Sbjct: 174 RQESRPGEWLVRKLFGSLPDSWARGHSVPWTVVRAMLNNWRPRDKQMELLENKAIHDL233
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16
Query: 752 GK.AHGSLKP 778
GKAHGSLKP
Sbjct: 234 GKAHGSLKP 242
Score = 129 (45.4 bits), Expect = 1.9e-109, Sum P(2) = 1.9e-109
Identities = 27/29 (93%), Positives = 27/29 (93°s), Frame = +3
Query: 126 GRHGETGRVLLKEILEQGLFSKVTLIGRR 212
lO G GETGRVLLKEILEQGLFSKVTLIGRR
Sbjct: 25 GASGETGRVLLKEILEQGLFSKVTLIGRR 53
Score = 127 (44.7 bits), Expect = 3.1e-109, Sum P(2) = 3.1e-109
Identities = 31/56 (55%), Positives = 37/56 (660), Frame = +1
Query: 55 MAETEALSKLREDFRMQNKSVFILGATEKPAECS*RKSWSRACFPKSRSLAGEAHL 222
MAETEALSKLREDFRMQNKSVFILGA+ + ++ + F K +L G L
Sbjct: 1 MAETEALSKLREDFRMQNKSVFILGASGETGRVLLKEILEQGLFSKV-TLIGRRKL 55
The segments of gi12618733 that are shown as "Sbjct" above are set out as SEQ
ID NO.
117, SEQ ID NO. 119, and SEQ ID NO. 121, respectively. Based on the structural
similarity these homologous polypeptides are expected to share at least some
biological
activities. Such activities are known in the art and described elsewhere
herein. Assays for
determining such activities are also known in the art some of which have been
described
elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 118, SEQ ID NO. 120, or SEQ ID NO. 122
which
correspond to the Query sequence in the alignments shown above (gaps
introduced in the
sequence by the computer are, of course, removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Stratagene NT2 neuronal precursor 937230 and to a
lesser extent
in Soares fetal liver spleen 1NFLS, Stratagene liver (#937224), Stratagene
endothelial cell
937223, Stratagene pancreas (#937208), Stratagene colon (#937204),
Hepatocellular
Tumor, Synovial IL-1/TNF stimulated, Human endometrial stromal cells, Liver,
Hepatoma, Human T-Cell Lymphoma, Hodgkin's Lymphoma II, and Keratinocytes.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:17 and may have been publicly available prior to conception of the present
invention.
Preferably, such related polynucleotides are specifically excluded from the
scope of the
present invention. To list every related sequence would be cumbersome.
Accordingly,
preferably excluded from the present invention are one or more polynucleotides
CA 02364549 2001-08-27
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17
comprising a nucleotide sequence described by the general formula of a-b,
where a is any
integer between I to 1363 of SEQ ID N0:17, b is an integer of 1 ~ to 1377,
where both a
and b correspond to the positions of nucleotide residues shown in SEQ ID
N0:17, and
where b is greater than or equal to a + 14
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. gi14106549
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "neuronal-specific septin". A partial
alignment
demonstrating the observed homology is shown immediately below.
>gi~4106549 (AF104411) neuronal-specific septin 3 [Mus musculus]
1S >spIG4106549~G4106549 NEURONAL-SPECIFIC SEPTIN 3.
Length = 465
Plus Strand HSPs:
2~ Score =
1118 (393.6
bits),
Expect
= 2.9e-115,
Sum P(2)
= 2.9e-115
Identities= 217/319 (680), Positives = 256/319 (80%),
Frame = +2
Query: VGDMADTPRDA-GLKQAPASRNEKAPVDFGYVGIDSILEQMRRKAMKQGFEFNIMWGQS1342
1166
+ ++ PR + P S N GY+GID+I+EQMR+K MK GF+FNIMWGQS
2S
Sbjct: MSELVPEPRPKPAVPMKPVSINSNL---LGYIGIDTIIEQMRKKTMKTGFDFNIMWGQS57
1
Query: GLGKSTLINTLFKSKISRKSVQPTSEERIPKTIEIKSITHDIEEKGVRMKLTVIDTPGFG1522
1343
GLGKSTL+NTLFKS++SRK+ EE+IPKT+EIK+I H IEE GV+MKLTVIDTPGFG
Sbjct: GLGKSTLVNTLFKSQVSRKASSWNREEKIPKTVEIKAIGHVIEEGGVKMKLTVIDTPGFG117
58
30
Query: DHINNENCWQPIMKFINDQYEKYLQEEVNINRKKRIPDTRVHCCLYFIPATGHSLRPLDI1702
1523
D INNENCW+PI K+IN+QYEK+L+EEVNI RKKRIPDTRVHCCLYFI
TGHSLRPLD+
Sbjct: DQINNENCWEPIEKYINEQYEKFLKEEVNIARKKRIPDTRVHCCLYFISPTGHSLRPLDL177
118
3S Query: EFMKRLSKVVNIVPVIAKADTLTLEERVHFKQRITADLLSNGIDWPQKEFDEDSEDRLV1882
1703
EFMK LSKVVNI+PVIAKADT+TLEE+ F L S GI YPQKEFDED
ED+
Sbjct: EFMKHLSKWNIIPVIAKADTMTLEEKSEF-------LPSEGI--YPQKEFDEDLEDKTE228
178
Query. NEKFR-EMIPFAWGSDHEYQVNGKRILGRKTKWGTIEVENTTHCEFAYLRDLLIRTHMQ2059
1883
4O N+K R E +PFAWGSD EYQVNGKR+LGRKT WG IEVEN HCEFA
LRD +IRTH+Q
Sbjct: NDKIRQESMPFAWGSDKEYQVNGKRVLGRKTPWGIIEVENLNHCEFALLRDFVIRTHLQ288
229
Query: NIKDITSSIHFEAYRVKRLNE 2122
2060
++K++T +IH+E YR KRLN+
~S Sbjct: DLKEVTHNIHYETYRAKRLND 309
289
The segment of gi14106549 that is shown as "Sbjct" above is set out as
sequence SEQ ID
NO. 123. Based on the structural similarity these homologous polypeptides are
expected
to share at least some biological activities. Such activities are known in the
art and
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1~
described elsewhere herein. Assays for determining such activities are also
known in the
art some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 124 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Stratagene pancreas (#937208) and Breast Lymph node
cDNA
library. and to a lesser extent in Soares infant brain 1NIB, Primary Dendritic
Cells, lib l,
Human Cerebellum, Human Fetal Kidney, Soares_fetal heart NbHHI9W, Soares
melanocyte 2NbHM, Soares_fetal_heart NbHH 19W, Human Fetal Heart, Spleen,
Chronic
lymphocytic leukemia, Keratinocyte, Messangial cell, frac 2, Jurkat T-Cell, S
phase,
Breast Cancer Cell line, angiogenic, Human Thymus, Human Osteoblasts II, Human
Pancreas Tumor, Human Rhabdomyosarcoma, Human Chondrosarcoma, Rejected Kidney,
lib 4, Human adult testis, large inserts, Soares senescent fibroblasts_NbHSF,
Human
Adult Pulmonary;re-excision, HUMAN B CELL LYMPHOMA, Stratagene pancreas
(#937208), Bone Marrow Cell Line (RS4;11 ), Activated T-Cells, 12hrs,
differentially
expressed, Soares ovary tumor NbHOT, Human OB MG63 treated ( 10 nM E2)
fraction I,
H. Epididiymus, caput & corpus, H. cerebellum, Enzyme subtracted, Stratagene
colon
(#937204), Stratagene pancreas (#937208), Smooth Muscle- HASTE normalized,
Stratagene ovary (#937217), Soares adult brain N2b4HB55Y, pBMC stimulated w/
poly
I/C, Breast Cancer cell line, MDA 36, Soares_senescent fibroblasts NbHSF,
Human
endometrial stromal cells-treated with progesterone, Human Osteosarcoma,
Stratagene
fetal spleen (#937205), HUMAN JURKAT MEMBRANE BOUND POLYSOMES,
Human Adult Testes, Large Inserts, Reexcision, T-Cell PHA 24 hrs,
Soares_pregnant uterus NbHPU, Stratagene NT2 neuronal precursor 937230, Liver,
Hepatoma, Human Activated Monocytes, Ulcerative Colitis, Human Fetal Brain,
Soares
breast 2NbHBst, T Cell helper I, Human Eosinophils, Dendritic cells, pooled,
Colon
Tumor II, Human Synovial Sarcoma, human tonsils, Hodgkin's Lymphoma II, Soares
fetal
liver spleen 1NFLS, K562 cells, Human left hemisphere, adult, Kidney cancer,
Activated
T-Cells, 12 tars, subtracted, Brain Amygdala Depression, Human Thymus Tumor,
subtracted, Weizmann Olfactory Epithelium, Human OB HOS treated ( 10 nM E2)
fraction
CA 02364549 2001-08-27
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19
I, Human Cerebellum. subtracted, Adipocytes;re-excision, Human Pituitary,
subtracted,
Human Fetal Bone, Human Primary Breast Cancer, Human Thyroid. H. Epididiymus,
cauda, Breast Lymph node cDNA library, Human Skin Tumor, Human adult small
intestine,re-excision, Cem cells cyclohexamide treated, Human
Hypothalamus,schizophrenia, re-excision, Synovial IL-1/TNF stimulated,
Soaves fetal_lung_NbHL 19W, H. Kidney Cortex, subtracted, Human Stomach:re-
excision, Stratagene neuroepithelium (#937231), Healing groin wound, 6.5 hours
post
incision, Stratagene neuroepithelium (#937231), Human endometrial stromal
cells, Jurkat
T-cell G1 phase, Prostate BPH, CD34 depleted Buffy Coat (Cord Blood), T-Cell
PHA 16
hrs, Stratagene NT2 neuronal precursor 937230, Human Brain, Striatum, L428,
Human
Primary Breast Cancer Reexcision, Macrophage-oxLDL, Stratagene pancreas
(#937208),
Human Adipose, Synovial Fibroblasts (control), Stratagene pancreas (#937208),
Human
Thymus, Hemangiopericytoma, Human Whole Six Week Old Embryo, Pancreas Islet
Cell
Tumor, PC3 Prostate cell line, Resting T-Cell Library,II, Human T-Cell
Lymphoma,
Soaves fetal lung NbHLI9W, Brain frontal cortex, Soaves fetal_lung NbHLI9W,
Human Fetal Lung III, Human Neutrophil, Activated, Stratagene NT2 neuronal
precursor
937230, Activated T-Cell ( l2hs)/Thiouridine labelledEco, Colon Normal III,
Anergic T-
cell, Smooth muscle,control, Monocyte activated, Soares_pregnant uterus NbHPU,
and T
cell helper II.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
68 as residues: Ser-21 to Trp-32.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:18 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 4137 of SEQ ID
N0:18, b
is an integer of 15 to 4151, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:18, and where b is greater than or
equal to a
+ 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 9
It has been discovered that thin ~ene is expressed primarily in the following
tissues:
Human Manic Depression Tissue, Stromal cell TF274, and Bone marrow.
5 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
10 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 1050 of SEQ ID
N0:19, b
is an integer of 15 to 1064, 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
15 + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 10
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
20 sequence accessible through the following database accession no. gi11373055
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "sialic-acid O-acetylesterase". A
partial alignment
demonstrating the observed homology is shown immediately below.
>gi~1373055 sialic-acid O-acetylesterase [Mus musculus] >sp~Q61044~Q61044 YOLK
SAC PROTEIN 2 (SIALIC-ACID O-ACETYLESTERASE).
Length = 541
Plus Strand HSPs:
Score = 576 (202.8 bits), Expect = 3.9e-111, Sum P(2) = 3.9e-111
Identities = 102/128 (79%), Positives = 108/128 (840), Frame = +2
Query: 2 SVLWNAMIHPLCNMTLKGVVWYQGESNINYNTDLYNCTFPALIEDWRETFHRGSQGQTER 181
3S SVLWNAMIHPL NMTLKGVVWYQGESN +yN DLY C FP LIEDWR+TFH GSQGQT+R
Sbjct: 281 SVLWNAMIHPLQNMTLKGVVWYQGESNADYNRDLYTCMFPELIEDWRQTFHYGSQGQTDR 340
Query: 182 FFPFGLVQXXXXXXXXXXXXGFPQIRWHQTADFGYVPNPKMPNTFMAVAMDLCDRDSPFG 361
FFPFG VQ GFP+IRWHQTADFG+VPNPKMPNTFMAVA+DLCDRDSPFG
4O Sbjct: 341 FFPFGFVQLSSYMLKNSSDYGFPEIRWHQTADFGHVPNPKMPNTFMAVAIDLCDRDSPFG 400
CA 02364549 2001-08-27
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21
Query: 362 SIHPRDKQ 385
SIHPRDKQ
Sbjct: 401 SIHPRDKQ 408
S
Score = 548 (192.9 bits), Expect = 3.9e-111, Sum P(2) = 3.9e-111
Identities = 100/133 (750), Positives = 115/133 (86°s), Frame = +3
Query: 384 RTVAYRLHLGARALAYGEXNLTFEGPLPEKIELLAHKGLLNLTYYQQIQVQKKDNKIFEI 563
lO +TVAYRLHLGARA+AYGE NLTF+GPLP+KIELLA GLLNLTY Q+IQVQ +DNK FEI
Sbjct: 408 QTVAYRLHLGARAVAYGEKNLTFQGPLPKKIELLASNGLLNLTYDQEIQVQMQDNKTFEI 467
Query: 564 SCCSDHRCKWLPASMNTVSTQSLTLAIDSCHGTWALRYAWTTWPCEYKQCPLYHPSSAL 743
SCCSD CKPdLPA +NT STQ+L L +++C GTWA+RYAWTTWPCEYKQC +YH SS L
IS Sbjct: 468 SCCSDRHCKWLPAPWTFSTQTLILDLNACLGTWAVRYAWTTWPCEYKQCAVYHTSSML 527
Query: 744 PAPPFIAFITDQG 782
PAPPFIA I+ +G
Sbjct: 528 PAPPFIAQISHRG 540
The segments of gi113730SS that are shown as "Sbjct" above are set out as SEQ
ID
NO. 12S and SEQ ID NO. 127, respectively. Based on the structural similarity
these
homologous polypeptides are expected to share at least some biological
activities; e.g.,
sialyl O-acetylesterase activity. Such activities are known in the art and
described
2S elsewhere herein. Assays for determining such activities are also known in
the art some of
which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 126 and/or SEQ ID NO. 128 which correspond
to
the Query sequence in the alignments shown above (gaps introduced in the
sequence by
the computer are, of course, removed, and residues shown as "X" can be
resolved by
looking to the sequences corresponding to Gene No. 10 in the Sequence
listing).
It has been discovered that this gene is expressed primarily in the following
tissues:
Soares fetal liver spleen 1NFLS and to a lesser extent in
Soares_parathyroid_tumor NbHPA, Soares adult brain N2bSHBSSY, NCI CGAP_LuS,
3S NCI CGAP_KidS, Stratagene placenta (#937225), Soares_fetal
liver_spleen_1NFLS Sl,
Soares senescent fibroblasts_NbHSF, Bone Marrow Stromal Cell, untreated,
Soares
breast 2NbHBst, NTERA2, control, Soares NhHMPu_S1,
Soares_pregnant uterus NbHPU, NCI_CGAP Co3, NCI CGAP_Co9, Soares
melanocyte 2NbHM, Soares multiple sclerosis_2NbHMSP,
Soares_multiple_sclerosis_2NbHMSP, Human Amygdala, Human Kidney Tumor, Bone
marrow stroma,treated, Human Cardiomyopathy, subtracted, Human Thyroid,
Synovial
Fibroblasts (Ill/TNF), subt, Human Bone Marrow, re-excision, Human fetal
heart,
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Lambda ZAP Express, NCI_CGAP GC4, NCI_CGAP_Kid3, NCI_CGAP_Kid6,
NCI CLAP Pr22, Human Umbilical Vein Endothelial Cells, uninduced, Human
Uterine
Cancer, Human Fetal Brain, Human Adrenal Gland Tumor, Smooth muscle, serum
treated, NCI_CGAP GCB 1, Colon Normal II, Normal colon,
Soares_parathyroid_tumor NbHPA, Colon Normal III, Human Osteoclastoma, Human
Cerebellum, and in Soares infant brain 1NIB.
FEATURES OF PROTEIN ENCODED BY GENE NO: 11
It has been discovered that this gene is expressed primarily in the following
tissues:
human leiomyeloid carcinoma tissue and in neutrophils.
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
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 1977 of SEQ ID
N0:20, b
is an integer of 15 to 1991, where both a and b correspond to the positions of
2Q nucleotide residues shown in SEQ ID N0:20, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 12
It has been discovered that this gene is expressed primarily in the following
tissues
and cDNA libraries: Osteoblasts and to a lesser extent in H. Atrophic
Endometrium,
Prostate, Soares NhHMPu_S l, STROMAL -OSTEOCLASTOMA, Soares NhHMPu_S 1,
Ea.hy.926 cell line, Human Umbilical Vein Endothelial cells, frac B, re-
excision, Human
Uterus, normal, Human adult small intestine,re-excision, Human Synovium, Human
endometrial stromal cells-treated with estradiol, Human Stomach:re-excision,
Human
Adult Small Intestine, Human Fetal Kidney, Human umbilical vein endothelial
cells, IL-4
induced, Soares_fetal heart NbHH 19W, Synovial Fibroblasts (control), PERM
TF274,
Bone Marrow Stromal Cell, untreated, H. Frontal cortex,epileptic;re-excision,
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23
Hepatocellular Tumor; re-excision, Adipocytes, Endothelial-induced,
Soares_fetal_heart NbHH 19W, Nine Week Old Early Stage Human, Soares placenta
Nb2HP.
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 3083 of SEQ ID
N0:2~, b
is an integer of 15 to 3097, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:22, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 13
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. gi12734083
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "stat-like protein". A partial
alignment
demonstrating the observed homology is shown immediately below.
>gi~2734083 stat-like protein [Homo sapiens]
Length = 710
Plus Strand HSPs:
Score = 962 (338.6 bits), Expect = 3.7e-96, P = 3.7e-96
Identities = 184/184 (100%), Positives = 184/184 (100%), Frame = +2
Query: 104 NARCLVNGLSLDHSKLVDVPFQVEFPAPKNELVQKFQVYYLGNVPVAKPVGVDVINGALE 283
NARCLVNGLSLDHSKLVDVPFQVEFPAPKNELVQKFQVYYLGNVPVAKPVGVDVINGALE
Sbjct: 50$ NARCLVNGLSLDHSKLVDVPFQVEFPAPKNELVQKFQVYYLGNVPVAKPVGVDVINGALE
567
Query: 284 SVLSSSSREQWTPSHVSVAPATLTILHQQTEAVLGECRVRFLSFLAVGRDVHTFAFIMAA
463
SVLSSSSREQWTPSHVSVAPATLTILHQQTEAVLGECRVRFLSFLAVGRDVHTFAFIMAA
Sbjct: 568 SVLSSSSREQWTPSHVSVAPATLTILHQQTEAVLGECRVRFLSFLAVGRDVHTFAFIMAA
627
4O Query: 464 GPASFCCHMFWCEPNAASLSEAVQAACMLRYQKCLDARSQASTSCLPAPPAESVARRVGW
643
GPASFCCHMFWCEPNAASLSEAVQAACMLRYQKCLDARSQASTSCLPAPPAESVARRVGW
Sbjct: 628 GPASFCCHMFWCEPNAASLSEAVQAACMLRYQKCLDARSQASTSCLPAPPAESVARRVGW
687
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24
Query: 644 T~~~RR 655
TVRR.
Sbjct: 688 TVRR 691
Score = 111 (39.1 bits), Expect = 0.019, P = 0.019
Identities = 40/104 (38%), Positives = 48/104 (460), Frame = +1
Query: 400 AFPLLPGRGQRCPHVCIHHGCRPSLLLLPHVLVRAQCCQPLRGCAGCVHASLPEVSGC-P 576
lO AF + G C H+ C P+ L V+A C LR C+ A + C P
Sbjct: 622 AFIMAAGPASFCCHM---FWCEPNAASLSEA-VQAACM--LR-YQKCLDARSQASTSCLP 674
Query: 577 FPGLHLLPPSTPC*VCGTACRVDCPQGVQSLWGSLKPKRLGAHTP 711
P P + G R +GVQSLWGSLKPKRLGAHTP
IS Sbjct: 675 AP-----PAESVARRVGWTVR----RGVQSLWGSLKPKRLGAHTP 710
The segments of gi12734083 that are shown as "Sbjct" above are set out as
sequence SEQ
ID NO. 129 and SEQ ID NO. 131, respectively. Based on the structural
similarity these
homologous polypeptides are expected to share at least some biological
activities. Such
20 activities are known in the art and described elsewhere herein. Assays for
determining
such activities are also known in the art some of which have been described
elsewhere
herein.
It has been discovered that this gene is expressed primarily in the following
tissues
and cDNA libraries: Human Cerebellum and to a lesser extent in Soares adult
brain
25 N2b4HB55Y, Early Stage Human Brain, Soares infant brain 1NIB,
Soares_pregnant uterus NbHPU, Human Infant Brain, Human Brain, Striatum, H.
Kidney
Cortex, subtracted, Human Whole Brain #2 - Oligo dT > l.SKb, Human
Hippocampus,
breast lymph node CDNA library, Monocyte activated, Human Hippocampus,
subtracted,
H. Normalized Fetal Liver, II, Human Adult Retina, Human Pituitary,
subtracted, H.
30 Epididiymus, caput & corpus, H. cerebellum, Enzyme subtracted, Human Pineal
Gland, H.
Whole Brain #2, re-excision, Human Synovium, Soares total_fetus_Nb2HF8 9w,
Alzheimers, spongy change, Amniotic Cells - Primary Culture, Human endometrial
stromal cells, Jurkat T-Cell, S phase, Synovial Fibroblasts (Il1/TNF), subt,
wilm's tumor,
Human Manic Depression Tissue, Brain Frontal Cortex, re-excision, Human
Umbilical
35 Vein Endothelial Cells, uninduced, Human Hypothalmus,Schizophrenia,
Ulcerative
Colitis, Hemanaiopericytoma, Human Fetal Brain, H. Frontal cortex,epileptic;re-
excision,
Resting T-Cell Library,II, Human Substantia Nigra, Brain frontal cortex,
Soares multiple_sclerosis 2NbHMSP, Soares melanocyte 2NbHM, Anergic T-cell,
Human Microvascular Endothelial Cells, fract. A, Smooth muscle.control.
Activated T-
40 cell(12h)/Thiouridine-re-excision, Neutrophils IL-1 and LPS induced, and
Human Testes.
WO 00/55171
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~>
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
73 as residues: Ser-l~ to Trp-24, Pro-89 to Ser-96.
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
general formula of a-b, where a is any integer between I to 1 161 of SEQ ID
N0:23, b
is an integer of 15 to 1175, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 14
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. gi129486 (all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "94 kDa B-raf protein". A partial
alignment
demonstrating the observed homology is shown immediately below.
>gi~29486 94 kDa B-raf protein [Homo Sapiens] >sp~Q13878~Q13878 94 KDA B-RAF
PROTEIN (FRAGMENT).
Length = 200
Plus Strand HSPs:
Score = 168 (59.1 bits), Expect = 1.8e-11, P = 1.8e-11
Identities = 32/32 (100%), Positives = 32/32 (100%), Frame = +2
Query: 566 VPARCGVTVRDSLKKALMMRGLIPECCAVYRI 661
VPARCGVTVRDSLKKALMMRGLIPECCAVYRI
Sbjct: 169 VPARCGVTVRDSLKKALMMRGLIPECCAVYRI 200
The segment of gi129486 that is shown as "Sbjct" above is set out as
sequences)
SEQ ID NO. 133. Based on the structural similarity these homologous
polypeptides are
expected to share at least some biological activities. Such activities are
known in the art
CA 02364549 2001-08-27
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26
and described elsewhere herein. Assays for determining such activities are
also known in
the art some of which have been described elsewhere herein.
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in
Brain-
medulloblastoma, human ovarian cancer, 12 Week Old Early Stage Human, II,
Rejected
Kidney, lib 4.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:24 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 1105 of SEQ ID N0:24, b is an integer of 15 to 1119,
where both a
and b correspond to the positions of nucleotide residues shown in SEQ ID
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. gi11872200
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "alternatively spliced product using
exon 13A". A
partial alignment demonstrating the observed homology is shown immediately
below.
>gi~1872200 alternatively spliced product using exon 13A [Homo Sapiens]
Length = 666
Plus Strand HSPs:
Score = 203 (71.5 bits), Expect = 3.1e-13, P = 3.1e-13
Identities = 45/66 (68%), Positives = 46/66 (690), Frame = +2
Query: 2 FSCLSLPSSWDYRRGPPCLDNFXIXH-----HVGQAGLELLTSGDLPASASQSAGIKRVS 166
FSCLSLP SWDYR PP NF HVGQAGLELLTSGDLPASASQSA I VS
Sbjct: 587 FSCLSLPRSWDYRHPPPRPANFEFLVETGFLHVGQAGLELLTSGDLPASASQSARITGVS 646
Query. 167 HHTRPK 184
H RP+
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27
Sbjct: 647 HRARPE 652
The segment of gi11872200 that is shown as "Sbjct" above is set out as
sequences) SEQ
ID NO. 135.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 136 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues:
Hypothalamus, Human Rhabdomyosarcoma, and Human Whole Six Week Old Embryo.
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 921 of SEQ ID
N0:25, b
is an integer of 15 to 935, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:25, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 16
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Primary Dendritic Cells, lib 1 and to a lesser extent
in Soares
melanocyte 2NbHM, Soares placenta Nb2HP, Hodgkin's Lymphoma II,
Soares_pregnant uterus NbHPU, Soares_pregnant uterus NbHPU, Colon Normal III,
Human Eosinophils, Spleen, Chronic lymphocytic leukemia, B Cell lymphoma,
Soares fetal heart NbHH 19W, NCI CGAP_GCB 1, Healing groin wound, 6.5 hours
post
incision, Apoptotic T-cell, Soares_pregnant uterus NbHPU, Endothelial cells-
control,
Soares senescent fibroblasts_NbHSF, HUMAN STOMACH, pBMC stimulated wl poly
I/C, T-Cell PHA 16 hrs, human ovarian cancer, T-Cell PHA 24 hrs, NTERA2,
control,
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28
NCI_CGAP GCB l, Hepatocellular Tumor; re-excision, Human Gall Bladder, Human T-
Cell Lymphoma, Human Substantia Niara, Soaves breast 3NbHBst. Adipocytes,
Human
Fetal Heart, Human Adult Pulmonary;re-excision, Human Bone Marrow, treated,
Soares_pregnant uterus NbHPU, Human Thymus, Human kidney Cortex, subtracted,
Human Eosinophils, Human Adult Skeletal Muscle, Palate carcinoma, Rectum
normal,
Pharynx Carcinoma, Osteoclastoma-normalized B, Larynx Tumor. CD34+ cell, I,
frac II,
Soaves_multiple_sclerosis_2NbHMSP, CD34+cells, II, FRACTION 2, Human
Leukocytes, LNCAP untreated, Human Gall Bladder, fraction II, Human Adult
Spleen,
Human Cardiomyopathy, subtracted, Human Fetal Spleen, Healing Abdomen
wound;70&90 min post incision, Activated T-cells, HSA 172 Cells, Messangial
cell, frac
2, Healing groin wound - zero hr post-incision (control), Ovarian Tumor 10-3-
95,
Pancreas Tumor PCA4 Tu, Human Hypothalamus,schizophrenia, re-excision, Human
Synovium, Soaves fetal lung_NbHLI9W, H Female Bladder, Adult, Human Stomach;re-
excision, Human Adipose Tissue, re-excision, Human Osteosarcoma, Human
Osteoclastoma, re-excision, Jurkat T-cell G 1 phase, Brain Frontal Cortex, re-
excision,
KMH2, Stratagene endothelial cell 937223, Spinal Cord, re-excision, Mo7e Cell
Line
GM-CSF treated (lng/ml), Human Bone Marrow, re-excision, Human Osteoblasts II,
12
Week Old Early Stage Human, II, HUMAN JURKAT MEMBRANE BOUND
POLYSOMES, Human Heart, Human Ovarian Cancer Reexcision, Human umbilical vein
endothelial cells, IL-4 induced, Human Activated Monocytes, Human
Chondrosarcoma,
Epithelial-TNFa and INF induced, Stratagene colon (#937204), Stratagene
fibroblast
(#937212), H. Frontal cortex,epileptic;re-excision, Resting T-Cell Library,II,
12 Week Old
Early Stage Human, Soaves_multiple_sclerosis_2NbHMSP, Colon Carcinoma, Colon
Tumor II, Normal colon, Neutrophils control; re-excision, Anergic T-cell,
Human
Osteoclastoma, Human Microvascular Endothelial Cells, fract. A, Smooth
muscle,control,
Monocyte activated, HUMAN B CELL LYMPHOMA, Bone Marrow Cell Line (RS4;11 ),
Human Endometrial Tumor, Soaves fetal lung_NbHLI9W, neutrophils control, Nine
Week Old Early Stage Human, Soaves fetal liver spleen 1NFLS.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:26 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
CA 02364549 2001-08-27
WO 00/55171 PCT/US00/06043
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 4035 of SEQ ID
N0:26, b
is an integer of 1 ~ to 4049, 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
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares_placenta_8to9weeks 2NbHP8to9W and to a lesser
extent
in Smooth muscle, ILIb induced, Stratagene placenta (#937225),
Soares_pregnant uterus_NbHPU, Soares_placenta_8to9weeks_2NbHP8to9W, Human
Placenta, Endothelial cells-control, and Soares fetal liver/spleen 1NFLS.
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 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 203 of SEQ ID N0:27, b is an integer of 15 to 2067, 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
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares placenta Nb2HP and to a lesser extent in Soares
fetal liver
spleen 1NFLS, Soares multiple sclerosis 2NbHMSP, Soares infant brain 1NIB,
Soares fetal_heart NbHHI9W, Nasal polyps, Morton Fetal Cochlea, Al-CELL LINE,
L1
Cell line, human colon cancer, Smooth muscle-ILb induced, HSA 172 Cells,
Stratagene
neuroepithelium (#937231 ), NTERA2 teratocarcinoma cell line+retinoic acid (
14 days),
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3U
Morton Fetal. Human Chronic Synovitis, HUMAN JURKAT MEMBRANE BOUND
POLYSOMES, Bone Marrow Stromal Cell, untreated, Hemangiopericytoma,
Macrophage-oxLDL; re-excision, Human Placenta, Human Testes Tumor, Human
Placenta, Human Fetal Heart, Human Testes, Human 8 Week Whole Embryo, Human
Cerebellum.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:28 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
general formula of a-b, where a is any integer between 1 to 2353 of SEQ ID
N0:28, b
is an integer of 15 to 2367, 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.
FEATURES OF PROTEIN ENCODED BY GENE NO: 19
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares placenta Nb2HP and to a lesser extent in Soares
fetal liver
spleen 1NFLS, H. Kidney Medulla, re-excision, Smooth muscle, serum treated,
Soares
ovary tumor NbHOT, H. Frontal Cortex, Epileptic, Morton Fetal Cochlea,
Synovial
hypoxia, Bone Marrow Stromal Cell, untreated, Soares_fetal heart NbHHI9W,
Soares_pregnant uterus_NbHPU, Brain frontal cortex, Soares melanocyte 2NbHM,
Human Adult Pulmonary;re-excision, Endothelial-induced, NCI_CGAP GCBI,
Soares NhHMPu_S1, Human Fetal Brain, normalized A5002F, WI 38 cells,
Colorectal
Tumor, Human Aortic Endothelium. Aorta endothelial cells + TNF-a, Human Lung,
Human Soleus, Human Adult Heart;re-excision, Human Epididymus, Synovial IL-
1/TNF
stimulated, Human endometrial stromal cells, Jurkat T-cell G 1 phase, Myoloid
Progenitor
Cell Line, Synovial Fibroblasts (Ill/TNF), subt, KMH2, Human Thymus, Human
umbilical vein endothelial cells, IL-4 induced, Human Rhabdomyosarcoma,
Synovial
Fibroblasts (control), Human Thymus, Stratagene endothelial cell 937223,
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31
Hemanaiopericytoma, H. Frontal cortex,epileptic;re-excision, T Cell helper I,
Adipocytes,
Human Fetal Lung III, Soares_multiple_sclerosis_2NbHMSP, Human Placenta, Human
Fetal Heart, Activated T-Cell (l2hs)/Thiouridine IabelledEco, Human
Microvascular
Endothelial Cells, fract. A, Monocyte activated, HUMAN B CELL LYMPHOMA,
Spleen,
Chronic lymphocytic leukemia, Human 8 Week Whole Embryo.
Preferred epitopes include those comprising a sequence shown in SEQ
ID NO. 79 as residues: Gln-24 to Thr-35.
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 3223 of SEQ ID
N0:29, b
is an integer of 15 to 3237, 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
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. gi1458692
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "homologous to mouse gene PC326". A
partial
alignment demonstrating the observed homology is shown immediately below.
>gi~458692 homologous to mouse gene PC326:GenBank Accession Number M95564
[Homo
sapiens] >sp~Q12839~Q12839 (H326).
Length = 597
Plus Strand HSPs:
Score = 270 (95.0 bits), Expect = l.Oe-48, Sum P(3) = l.Oe-48
Identities = 58/129 (44%), Positives = 87/129 (670), Frame = +1
Query: 2344 YKGHRNSRTMIKEANFWG--ANFVMSGSDCGHIFIWDRHTAEHLMLLEADNH-VVNCLQP 2514
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32
YKGHRN+ T +K NF+G + FV+SGSDCGHIF+pJ++ + + + +E D VVNCL+p
Sbjct: 428 YKGHRNNAT-VKGVNFYGPKSEFWSGSDCGHIFLTVEKSSCQIIQF;,~SEGDKGGVVNCLEp
48'0
Query: 2515 HPFDPILASSGIDYDIKIL4SPLEESRIFNRKLADEVITRNELMLEE-TRNTITVPASFML 2691
S HP p+LA+SG+D+D+KIGl+p E+ L D Vi +N+ +E + + + S ML
Sbjct: 487 HPHLPVLATSGLDHDVKh~JAPTAEASTELTGLKD-VIKKNKRERDEDSLHQTDLFDSHML 545
Query: 2692 RMLASLNHIRADR 2730
L ++H+R R
Sbjct: 546 C~TFL--MHHLRQRR 556
The segment of gi1458692 that is shown as "Sbjct" above is set out as SEQ ID
NO.
137. Based on the structural similarity these homologous polypeptides are
expected to
share at least some biological activities. Such activities are known in the
art and described
elsewhere herein. Assays for determining such activities are also known in the
art some of
which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 138 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Human Testes and to a lesser extent in Soares fetal
liver spleen
1NFLS, Brain Frontal Cortex, re-excision, Human Fibrosarcoma, Human
Cardiomyopathy, subtracted, Soares_pregnant uterus NbHPU, Stratagene
endothelial cell
937223, Human Substantia Nigra, Human Bone Marrow, treated, Hodgkin's Lymphoma
II, Primary Dendritic Cells, lib l, Soares infant brain 1NIB, Human
Cardiomyopathy, diff
exp, Human Pituitary, Human Fetal Brain, Human Pituitary, subtracted, Human
Synovium, Alzheimers, spongy change, Synovial hypoxia-RSF subtracted, Human
Frontal
Cortex, Schizophrenia, Human endometrial stromal cells, Human Colon; re-
excision,
Jurkat T-cell G 1 phase, 12 Week Old Early Stage Human, II, Human Fetal Dura
Mater,
Stromal cell TF274, Synovial Fibroblasts (control), Hemangiopericytoma, Human
Whole
Six Week Old Embryo, Human adult testis, large inserts, Human Gall Bladder,
PC3
Prostate cell line, 12 Week Old Early Stage Human, Smooth muscle, serum
treated, Colon
Normal II, Soares melanocyte 2NbHM, Soares multiple_sclerosis_2NbHMSP, Human
Fetal Heart, Endothelial cells-control, Human Osteoclastoma, Monocyte
activated,
HUMAN B CELL LYMPHOMA, Bone Marrow Cell Line (RS4;11 ), Human Endometrial
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33
Tumor, Soares_parathyroid tumor_NbHPA, Soaves_multiple_sclerosis_2NbHMSP, and
Stratagene HeLa cell s3 937216.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
80 as residues: Asp-106 to Glu-126.
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
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 3194 of SEQ ID
N0:30, b
is an integer of I S to 3208, 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.
FEATURES 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.
gnIIPIDId1021677 (all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "KIAA0381 ". A partial alignment
demonstrating
the observed homology is shown immediately below.
>gnl~PID~d1021677 (AB002379) KIAA0381 [Homo Sapiens] >sp~D1021677~D1021677
KIAA0381 PROTEIN (FRAGMENT).
Length = 864
Plus Strand HSPs:
Score = 598 (210.5 bits), Expect = 7.3e-55, P = 7.3e-55
Identities = 131/203 (64%), Positives = 141/203 (69%), Frame = +1
Query: 328 LPSVSPYSLAELEKEVGNLRRGLRAVEVELEYQRRQVREPSDKFVPVMSDFITVSSFSFS 507
3S LP + +LAELEKEVGNLRRGLRAVEVELEYQRRQVREPSDKFVPVMSDFITVSSFSFS
Sbjct: 662 LPEAAKVNLAELEKEVGNLRRGLRAVEVELEYQRRQVREPSDKFVPVMSDFITVSSFSFS 721
Query: 508 ELEDQLNEARDKFAKALMHFGEHDSKMQPDEFFGIFDTFLQAFSEARQDXXXXXXXXXXX 687
ELEDQLNEARDKFAKALMHFGEHDSKMQPDEFFGIFDTFLQAFSEARQD
4O Sbjct: 722 ELEDQLNEARDKFAKALMHFGEHDSKMQPDEFFGIFDTFLQAFSEARQDLEAMRRRKEEE 781
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3-1
Query: 688 XXXXXXXXXXXXQRERDKAGN*PSLXXXXXXXXXXXXXXN*WIPRAPG-IR:-IEGLMQAQA 864
QRER++ + + G + + L + +
Sbjct: 782 ERRARMEAMLKEQRERERWQRQRKVLAAGSSLEEGGEFDDLVSALRSGEVFDKDLCKLKR 841
Query: 865 HRKRSGSQALEVTRERAINRLNY 933
RKRSGSQALEVTRERAINRLNY
Sbjct: 842 SRKRSGSQALEVTRERAINRLNY 864
The segment of gnlIPIDId1021677 that is shown as "Sbjct" above is set out as
SEQ ID
NO. 139.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 140 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed, and residues shown as "X" can be resolved by looking to the sequences
corresponding to Gene No. 21 in the Sequence listingj.
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in
human corpus
colosum, Human Infant Brain, Human Hippocampus, Soares adult brain N2b5HB55Y,
Human Substantia Nigra, Human Fetal Heart, Soares fetal liver spleen 1 NFLS,
H. Adipose
Tissue, Human Fetal Bone, H. Epididiymus, caput & corpus, Soares adult brain
N2b4HB55Y, Human Epididymus, Soares_multiple sclerosis 2NbHMSP, H. Kidney
Cortex, subtracted, Human Whole Brain #2 - Oligo dT > l.SKb, Human Adult Small
Intestine, Human Osteoblasts II, Spinal cord, Rejected Kidney, lib 4, Colon
Normal II,
human tonsils, Soares_multiple sclerosis_2NbHMSP, Human Amygdala.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:31 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 3773 of SEQ ID
N0:31, b
is an integer of 15 to 3787, 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.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 22
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
S sequence accessible through the following database accession no. gi12734081
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "similar to oxysterol-binding
protein". A partial
alignment demonstrating the observed homology is shown immediately below.
10 >gi~2734081 (AF000195) similar to oxysterol-binding proteins
[Caenorhabditis
elegans] >sp~001686~001686 C32F10.1 PROTEIN.
Length = 676 ,
1S
Plus Strand HSPS:
Score = 839 (295.3 bits), Expect = 4.6e-83, P = 4.6e-83
Identities = 170/365 (46%), Positives = 220/365 (60%), Frame = +2
Query:185 VMEDQRSIILHLISQLKLGMDLTKWLPTFILEKRSLLEMYADFMAHPDLLLAITAGATP364
ZO V +D S+++HL+SQ+ +GMDLTKV LPTFILE+RSLLEMYADF
AHPDL +A +
Sbjct:308 VQQDHGSVLMHLLSQVSVGMDLTKVTLPTFILERRSLLEMYADFFAHPDLFIATNGNDSA367
Query:365 EERVICFVEYYLTAFHEGRKGALAKKPYNPIIGETFHCSWEVPKDRVKPKRTASRSPASC544
E+R+I V YYL +F+ RK +AKKPYN I+GETF C + VP +
K+T S P
ZS Sbjct:368 EKRMIAWRYYLNSFYAARKSGVAKKPYNAILGETFRCRYSVPNSSLSGKKTES-GP---423
Query:545 HEHPMADDPSKSYKLRFVAEQVSHHPPISCFYCECEEKRLCVNTHV:~:TKSKFMGMSVGVS724
P +D+ +L F+AEQVSHHPPIS FY E + + N H++TKS F+G+S+GV+
Sbjct:424 --WPGSDEK----QLTFIAEQVSHHPPISAFYAEMPSEGISFNAHIYTKSSFLGLSIGVA477
30
Query:725 MIGEGVLRLLEHGEE-YVFTLPSAYARSILTIPWELGGKVSINCAKTGYSATVIFHTKP901
IGEG+L L +G+E Y T PS Y RSI++ pW E GGKV + C
KTGY A + F TKP
Sbjct:478 SIGEGILTLHNYGDEQYTITFPSGYGRSIMSTPWFEFGGKVKVECEKTGYRADIEFLTKP537
3S Query:902 FYGGKVHRVTAEVKHNPTNTIVCKAHGEWNGTLEFTYNNGETKV-IDTTTLPWPKKIRP1078
F+GGK HR+ + + V GEWNGT+ NGE V +D P K+ p .
Sbjct:538 FFGGKPHRIQGSICRDGAKKPVLTLRGEWNGTMYAKTQNGEEYVFVDVKAKPEVKKECVP597
Query:1079LEKQGPMESRNLWREVTRYLRLGDIDAATEQKRHLXXXXXXXXXXXXNLRTPWKPKYF-I1255
4O + +QG ESR LWR VT L I AT KR + W p F
Sbjct:598 VMQQGKRESRRLWRHVTAALLRNRIQTATTSKRFIEQRQRKEAKERLESGEKWTPLLFDT657
Query:1256QEGDG-WW 1279
+ DG W+Y
4S Sbjct:658 TDKDGEWMY 666
The segment of gi12734081 that is shown as "Sbjct" above is set out as SEQ ID
NO. 141. Based on the structural similarity these homologous polypeptides are
expected
to share at least some biological activities. Such activities are known in the
art and
SO described elsewhere herein. Assays for determining such activities are also
known in the
art some of which have been described elsewhere herein. Preferred polypeptides
of the
CA 02364549 2001-08-27
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36
invention comprise a polypeptide having the amino acid sequence set out as SEQ
ID NO.
142 which corresponds to the Query sequence in the alignment shown above (gaps
introduced in the sequence by the computer are, of course, removed, and
residues shown
as "X" can be resolved by looking to the sequences corresponding to Gene No.
22 in the
Sequence listing).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares placenta Nb2HP and to a lesser extent in
Soares fetal_liver_spleen_1NFLS_SI, Human T-Cell Lymphoma. Spleen, Chronic
lymphocytic leukemia, Human Testes, Soares fetal liver spleen 1NFLS,
Soares total_fetus_Nb2HF8 9w, Pharynx Carcinoma, H. Epididiymus, caput &
corpus,
Human T-cell lymphoma;re-excision, STROMAL -OSTEOCLASTOMA, Human Adult
Small Intestine, Human Prostate, Soares_fetal_heart NbHHI9W,
Soares_placenta_8to9weeks_2NbHP8to9W, H. Kidney Medulla, re-excision, Human
Thymus, Stromal cell TF274, Stratagene colon (#937204), Stratagene NT2
neuronal
precursor 937230, Human Adipose, Bone Marrow Stromal Cell, untreated, Colon
Tumor,
12 Week Old Early Stage Human, Human Placenta, Human Fetal Lung III, Human
Testes,
Reexcision, Human Fetal Heart, Soares_placenta_8to9weeks_2NbHP8to9W,
Endothelial-
induced, HUMAN B CELL LYMPHOMA, and Human Endometrial Tumor.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
82 as residues: Met-1 to Trp-9.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of '~ese sequences
are
related to SEQ ID N0:32 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 2579 of SEQ ID
N0:32, b
is an integer of 15 to 2593, 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.
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37
FEATURES OF PROTEIN ENCODED BY GENE NO: 23
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. gi13170458
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "diphthamide biosynthesis protein-2".
A partial
alignment demonstrating the observed homology is shown immediately below.
>gi~3170458 (AF053003) diphthamide biosynthesis protein-2; DPH2 [Homo Sapiens]
>sp~0606231060623 DIPHTHAMIDE BIOSYNTHESIS PROTEIN-2.
Length = 489
Minus Strand HSPs:
Score = 1433 (504.4 bits), Expect = 5.1e-146, P = 5.1e-146
Identities = 275/336 (81%), Positives = 275/336 (81%), Frame = -1
Query: 1887 EPACAHALEALATLLRPRYLDLLVSSPAFPQPVGSLSPEPMPLERFGRRFPLAPGRRLEE 1708
EPACAHALEALA LLRPRYLDLLVSSPAFPQPVGSLSPEPMP ERFGRRFPLAPGRRLEE
2O Sbjct: 154 EPACAHALEALAXLLRPRYLDLLVSSPAFPQPVGSLSPEPMPXERFGRRFPLAPGRRLEE 213
Query: 1707 YGAFYVGGSKAXXXXXXXXXXXXXXXGWAPGQPFSSCCPDTGKTQXXXXXXXXXXXXXXX 1528
YGAFYVGGSKA GWAPGQPFSSCCPDTGKTQ
Sbjct: 214 YGAFWGGSKASPDPDLDPDLSRLLLGWAPGQPFSSCCPDTGKTQDEGARAGGLRARRRY 273
Query: 1527 XXXXXXXXXWGLLAGTLGVAQHREALAHLRNLTQAAGKRXWLALGRPTPAKLANFPEV 1348
WGLLAGT GVAQHREALAHLRNLTQAAGKR WLALGRPTPAKLANFPEV
Sbjct: 274 XVERARDARWGLLAGTXGVAQHREALAHLRNLTQAAGKRSYVLALGRPTPAKLANFPEV 333
3O Query: 1347 DVFVLLACPLGALAPQLSGSFFQPILAPCELEAACNPAWPPPGLAPHLTHYADLLPGSPF
1168
DVFVLLACPLGALAPQLSGSFFQPILAPCELEAACNPAWPPPGLAPHLTHYADLLPGSPF
Sbjct: 334 DVFVLLACPLGALAPQLSGSFFQPILAPCELEAACNPAWPPPGLAPHLTHYADLLPGSPF 393
Query: 1167 HVALPPPESELWETPDVSLITGDLRPPPAWKSSNDHGSLALTPRPQXXXXXXXXXXXXXX 988
HVALPPPESELWETPDVSLITGDLRPPPAWKSSNDHGSLAL PRPQ
Sbjct: 394 HVALPPPESELWETPDVSLITGDLRPPPAWKSSNDHGSLALNPRPQLXLAESSPAVSFLS 453
Query: 987 XXXWQGLEPRLGQTPVTEAVSGRRGIAIAYEDEGSG 880
WQGLEPRLGQTPVTEAVSGRRGIAIAYEDEGSG
4O Sbjct: 454 SRSWQGLEPRLGQTPVTEAVSGRRGIAIAYEDEGSG 489
The segment of gi13170458 that is shown as "Sbjct" above is set out as SEQ ID
NO. 143. Based on the structural similarity these homologous polypeptides are
expected
to share at least some biological activities. Such activities are known in the
art and
described elsewhere herein. Assays for determining such activities are also
known in the
art some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 144 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
CA 02364549 2001-08-27
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3 t;
removed, and residues shown as "X" can be resolved by looking to the sequences
corresponding to Gene No. 23 in the Sequence listing).
It has been discovered that this gene is expressed primarily in the following
tissues
and cDNA libraries: Activated T-cell( 12h)/Thiouridine-re-excision and to a
lesser extent
in NCI CGAP_GCB1, Soares fetal liver spleen 1NFLS, Soares infant brain 1NIB,
Pharynx carcinoma, Human Testes Tumor, Activated T-Cell ( l2hs)/Thiouridine.
labelledEco, Soares_fetal_heart NbHHI9W, NCI_CGAP Co3,
Soares_fetal lung NbHLI9W, Soares_testis_NHT, Soares ovary tumor NbHOT,
Soares_total_fetus_Nb2HF8 9w, Soares_testis_NHT, Raji cells, cyclohexamide
treated,
subtracted, Human B Cell 8866, Human epithelioid sarcoma, Human Aortic
Endothelium,
Human Primary Breast Cancer;re-excision. L428, Human Fetal Kidney, HUMAN
JURKAT MEMBRANE BOUND POLYSOMES, Human Chondrosarcoma, Human
Thymus, Human Testes Tumor, re-excision, Human Adrenal Gland Tumor, human
tonsils,
Human Adult Pulmonary;re-excision, Endothelial-induced, Human Amygdala, Human
Microvascular Endothelial Cells, fract. A, Human Bone Marrow, treated,
NCI CGAP_GCS, NCI CGAP_Lul, NCI CGAP_Col2, NCI CGAP_Prl2,
NCI CGAP_Brn23, Soares fetal_heart NbHHI9W, Stratagene NT2 neuronal precursor
937230.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:33 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 1906 of SEQ ID
N0:33, b
is an integer of 15 to 1920, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:33, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 24
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39
It has been discovered that this Gene is expressed primarily in the following
tissues:
Activated T-Cell (l2hs)/Thiouridine labelled Eco.
Many polynucleotide sequences, such as EST sequences. are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:34 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
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 1411 of SEQ ID
N0:34, b
is an integer of 15 to 1425, 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
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. gill 196433
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "unknown protein". A partial
alignment
demonstrating the observed homology is shown immediately below.
>gi11196433 unknown protein [Homo sapiens) >sp~Q14288~Q14288 HYPOTHETICAL
PROTEIN (FRAGMENT).
Length = 641
Plus Strand HSPs:
Score = 191 (67.2 bits), Expect = l.Oe-22, Sum P(2) = 1.0e-22
3~ Identities = 41/79 (510), Positives = 50/79 (63%), Frame = +3
Query: 987 QKIFANYSSDKGLIFRTYKDLKQLSNNNKNIQF*KWAKEPNKHL*KPDASGQQVYEKKKC 1166
+KIFA YSSDKGLI R Y +LKQ+ N KWAK+ N+H K D + + KKC
Sbjct: 403 EKIFATYSSDKGLISRIYNELKQIYKKKTNNPIKKWAKDMNRHFSKEDIYAAKKH-MKKC 461
Query: 1167 *TPLIIREMQTNTTMRYYL 1223
+ L IREMQ TTMRY+L
Sbjct: 462 SSSLAIREMQIKTTMRYHL 480
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The segment of '~i11 196433 that is shown as "Sbjct" above is set out as SEQ
ID NO. 145.
Based on the structural similarity these homologous polypeptides are expected
to share at
least some biological activities. Such activities are known in the art and
described
elsewhere herein. Assays for determining such activities are also known in the
art some of
5 which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 146 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
10 It has been discovered that this gene is expressed primarily in human colon
carcinoma.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
85 as residues: Gln-1 to Asp-6.
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 N0:35 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
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 1242 of SEQ ID
N0:35, b
is an integer of 15 to 1256, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:35, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 26
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.
gnlIPIDIe1311091 (all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "ubiquitin-conjugating enzyme". A
partial
alignment demonstrating the observed homology is shown immediately below.
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41
>gnIIPID~e1311091 ubiquitin-conjugating enzyme [Mus musculus)
>sp~0887381088738
UBIQUITIN-CONJUGATING ENZYME.
Length = 4845
Plus Strand HSPs:
Score = 286 (100.7 bits), Expect = 9.8e-20, P = 9.Se-20
Identities = 66/156 (420), Positives = 86/156 (550), Frame = +2
Query: 179 DTVDMTKIHALITGPFDTPYEGGFFLFVFRCPPDYPIHPPRVKLMTTGNNTVRFNPNFYR 358
+ +D+ K+ LITGP DTPY G F F P DYP PP V L TTG ++VRFNPN Y
Sbjct: 4592 ERLDIMKV--LITGPADTPYANGCFEFDVYFPQDYPSSPPLVNLETTGGHSVRFNPNLYN 4649
1S Query: 359 NGKVCLSILGTWTG-PA--WSPAXXXXXXXXXXXXXM--TENPYHNEPGFEQERH-PG-- 514
+GKVCLSIL TW G P W+P + PY NEPG+E+ R P
Sbjct: 4650 DGKVCLSILNTWHGRPEEKL~TNPQTSSFLQVLVSVQSLILVAEPYFNEPGYERSRGTPSGT
4709
Query: 515 -DSKNYNECIRHETIRVAVCDMMEGKCPCPEPLRGVMEKSF 634
2O S+ Y+ IR T++ A+ + + PC + V+ K F
Sbjct: 4710 QSSREYDGNIRQATVKWAMLEQIRNPSPC---FKEVIHKHF'4747
The segment of gnllPIDle 1311091 that is shown as "Sbjct" above is set out as
SEQ ID NO.
147. Based on the structural similarity these homologous polypeptides are
expected to
25 share at least some biological activities, e.g., ubiquitin conjugating
enzyme activity. Such
activities are known in the art and described elsewhere herein. Assays for
determining
such activities are also known in the art some of which have been described
elsewhere
herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
30 acid sequence set out as SEQ ID NO. 148 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed, and residues shown as "X" can be resolved by looking to the sequences
corresponding to Gene No. 26 in the Sequence listing).
It has been discovered that this gene is expressed primarily in the following
35 tissues/cDNA libraries: Snares infant brain 1NIB and to a lesser extent in
Human
Microvascular Endothelial Cells, fract. A, human tonsils, Primary Dendritic
Cells, lib 1,
Smooth muscle,control, Human Fetal Kidney, Snares fetal liver spleen 1NFLS,
Snares
placenta Nb2HP, Snares melanocyte 2NbHM, Activated T-cell( 12h)/Thiouridine-re-
excision, Snares_placenta_8to9weeks_2NbHP8to9W, Human Synovium, Smooth muscle,
40 serum induced,re-exc, Human Testes Tumor, Human Synovial Sarcoma, Monocyte
activated, neutrophils control, Nine Week Old Early Stage Human, Larynx
carcinoma II,
LNCAP untreated, Hodgkin's Lymphoma I, HSC 172 cells, Human Skin Tumor,
Resting
T-Cell; re-excision, Soares_multiple_sclerosis 2NbHMSP,
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42
Soares_fetal_lung_NbHLI9W, pBMC stimulated w/ poly I/C, Human Osteoclastoma
Stromal Cells - unamplified, Human Fetal Dura Mater, Human Activated T-Cells,
Macrophage-oxLDL. Stratagene HeLa cell s3 937216, Hemangiopericytoma, Human
Fetal
Brain, Soares_pregnant_uterus NbHPU, Human Whole Six Week Old Embryo,
Stratagene lung (#937210). Soares_preQnant uterus_NbHPU, NCI_CGAP GCB l, Human
Placenta, Soares_placenta_8to9weeks_2NbHP8to9W, Stratagene HeLa cell s3
937216,
Primary Dendritic cells,frac 2, Human Adult Pulmonary;re-excision, Activated T-
Cell
(l2hs)/Thiouridine labelledEco, Soaresfetal_lung NbHLI9W, CD34 positive cells
(Cord
Blood), HUMAN B CELL LYMPHOMA, Human Testes, Osteoblasts, Human 8 Week
Whole Embryo, Soares multiple_sclerosis_2NbHMSP, Soares_pregnant uterus NbHPU,
Human Adult Lymph Node, Rectum normal, Messangial cell, frac l, Activated T-
Cells, 8
hrs., WI 38 cells, Human Prostate BPH, re-excision, CD34+cells, II, FRACTION
2,
Human Prostate, subtracted, K562 + PMA (36 hrs),re-excision, Human (Caco-2)
cell line,
adenocarcinoma, colon, remake, Morton Fetal Cochlea,
Soares_placenta_8to9weeks_2NbHP8to9W, Human Aortic Endothelium, Human Primary
Breast Cancer;re-excision, Human Adult Retina, Human Umbilical Vein
Endothelial
Cells, fract. A, Healing Abdomen wound;70&90 min post incision, Human
Placenta,
Human Neutrophils, Activated, re-excision, Smooth muscle, control; re-
excision, Supt
Cells, cyclohexamide treated, Smooth muscle-ILb induced, H. Epididiymus,
cauda,
Human adult small intestine,re-excision, Human Normal Breast, Ovarian Tumor 10-
3-95,
Human Epididymus, Human Hypothalamus,schizophrenia, re-excision, Synovial IL-
1/TNF stimulated, Amniotic Cells - Primary Culture, Human Stomach;re-excision,
.
Salivary Gland, Lib 2, HL-60, PMA 4H, re-excision, Human endometrial stromal
cells,
Jurkat T-cell G 1 phase, Human Prostate, T-Cell PHA 16 hrs, Human Thymus,
Stratagene
fetal spleen (#937205), HUMAN JURKAT MEMBRANE BOUND POLYSOMES,
Human Uterine Cancer, Human Primary Breast Cancer Reexcision, Human Pancreas
Tumor, Human Ovarian Cancer Reexcision, Merkel Cells, Human Thymus Stromal
Cells,
Soares breast 2NbHBst, Rejected Kidney, lib 4, Macrophage (GM-CSF treated),
Macrophage-oxLDL; re-excision, Smooth muscle, serum treated, Soares breast
3NbHBst,
H Macrophage (GM-CSF treated), re-excision, Human Fetal Lung III, Human
Testes,
Reexcision, Human Neutrophil, Activated, and Human Cerebellum.
CA 02364549 2001-08-27
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43
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 2844 of SEQ ID
N0:36, b
is an integer of 15 to 2858, 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
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
25
sequence accessible through the following database accession no. gi12352277
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "MAP kinase". A partial alignment
demonstrating
the observed homology is shown immediately below.
>gi~2352277 (AF002715) MAP kinase kinase kinase [Homo Sapiens]
Length = 1607
Plus Strand HSPs:
Score = 2801 (986.0 bits), Expect = 4.9e-291, P = 4.9e-291
Identities = 529/542 (970), Positives = 529/542 (970), Frame = +1
Query: 10 ISFARKWMNYVLTKCESGRGTRPRWATQGFDFLQAIEPAFISALPEDDFLSLQALMNECI 189
ISFARKWMNYVLTKCESGRGTRPRWATQGFDFLQAIEPAFISALPEDDFLSLQALMNECI
Sbjct: 1067 ISFARKWMNYVLTKCESGRGTRPRWATQGFDFLQAIEPAFISALPEDDFLSLQALMNECI 1126
Query: 190 GHVIGKPHSPVTGLYLAIHRNSPRPMKVPRCHSDPPNPHLIIPTPEGFSTRSMPSDARSH 369
GHVIGKPHSPVTGLYLAIHRNSPRPMKVPRCHSDPPNPHLIIPTPEGFSTRSMPSDARSH
3S Sbjct: 1127 GHVIGKPHSPVTGLYLAIHRNSPRPMKVPRCHSDPPNPHLIIPTPEGFSTRSMPSDARSH
1186
Query: 370 GSPXXXXXXXXXXXXXSRPSPSGGDSVLPKSISSAHDTRGSSVPENDRLASIAAELQFRS 549
GSP SRPSPSGGDSVLPKSISSAHDTRGSSVPENDRLASIAAELQFRS
Sbjct: 1187 GSPAAAAAAAAAVAA-SRPSPSGGDSVLPKSISSAHDTRGSSVPENDRLASIAAELQFRS 1245
Query: 550 LSRHSSPTEERDEPAYPRGDSSGSTRRSWELRTLISQSKDTASKLGPIEAIQKSVRLFEE 729
LSRHSSPTEERDEPAYPRGDSSGSTRRSWELRTLISQSKDTASKLGPIEAIQKSVRLFEE
Sbjct: 1246 LSRHSSPTEERDEPAYPRGDSSGSTRRSWELRTLISQSKDTASKLGPIEAIQKSVRLFEE 1305
4S Query: 730 KRYREMRRKNIIGQVCDTPKSYDNVMHVGLRKVTFKWQRGNKIGEGQYGKVYTCISVDTG 909
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~t-1
KRYREMRRKNIIGQVCDTPKSYDNVMHVGLRKVTFKWQRGNKIGEGQYGK WTCISVDTG
Sbjct: 1306 KRYREMRRKNIIGQVCDTPh;SYDNVMHVGLRKVTFKWQRGNKIGEGQYGKWTCISVDTG 1365
Query: 910 ELMAMKEIRFQPNDHKTIh:ETADELKIFEGIKHPNLVRYFGVELHREErIYIFMEYCDEGT 1089
S ELMAMKEIRFQPNDHKTIh:ETADELKIFEGIKHPNLVRYFGVELHREEbIYIFMEYCDEGT
Sbjct: 1356 ELMAMKEIRFQPNDHKTIKETADELKIFEGIKHPNLVRYFGVELHREEMYIFMEYCDEGT 1425
Query: 1090 LEEVSRLGLQEHVIRLYSKQITIAINVLHEHGIVHRDIKGANIFLTSSGLIKLGDFGCSV 1269
LEEVSRLGLQEHVIRLYSKQITIAINVLHEHGIVHRDIKGANIFLTSSGLIKLGDFGCSV
lO Sbjct: 1426 LEEVSRLGLQEHVIRLYSKQITiAINVLHEHGIVHRDIKGANIFLTSSGLIKLGDFGCSV
1485
Query: 1270 KLKIQNAQTMPGEVNSTLGT?aYMAPEVITRAKGEGHGRAADIWSLGCWIEMVTGKRPWH 1449
KLKNNAQTMPGEVNSTLGThAYMAPEVITRAKGEGHGRAADIWSLGCWIEMVTGKRPV1II
Sbjct: 1486 KLKNNAQTMPGEWSTLGTAAYMAPEVITRAKGEGHGRAADIWSLGCWIEMVTGKRPL~TH 1545
1S
Query: 1450 EYEHNFQIMYKVGMGHKPPIPERLSPEGKDFLSHCLESDPKMRWTASQLLDHSFVKVCTD 1629
EYEHNFQIMYKVGMGHKPPIPERLSPEGKDFLSHCLESDPKMRWTASQLLDHSFVKVCTD
Sbjct: 1546 EYEHNFQIMYKVGMGHKPPIPERLSPEGKDFLSHCLESDPKMRWTASQLLDHSFVKVCTD 1605
20 Query: 1630 EE 1635
EE
Sbjct: 1606 EE 1607
The segment of gi123S2277 that is shown as "Sbjct" above is set out as SEQ ID
NO. 149.
2S Based on the structural similarity these homologous polypeptides are
expected to share at
least some biological activities. Such activities are known in the art and
described
elsewhere herein. Assays for determining such activities are also known in the
art some of
which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
30 acid sequence set out as SEQ ID NO. 1 SO which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed, and residues shown as "X" can be resolved by looking to the sequences
corresponding to Gene No. 26 in the Sequence listing)
It has been discovered that this gene is expressed primarily in the following
3S tissues/cDNA libraries: Soares_pregnant uterus_NbHPU,
Soares_placenta_8to9weeks 2NbHP8to9W, Macrophage-oxLDL; re-excision, Human
Testes and to a lesser extent in Human frontal cortex, Stratagene colon
(#937204), Human
Colon Carcinoma (HCC) cell line, Early Stage Human Lung, subtracted, Human
endometrial stromal cells, Spleen metastic melanoma, Prostate BPH, Brain
Frontal Cortex,
40 re-excision, Human Activated Monocytes, Human Rhabdomyosarcoma, Human adult
(K.Okubo), Colon Normal II, Soares breast 3NbHBst, Human Amygdala, HUMAN B
CELL LYMPHOMA, Human Endometrial Tumor, Soares fetal liver spleen 1NFLS.
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Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
87 as residues: Met-1 to Gly-9, Arg-26 to Gly-32, His-44 to Arg-56. Ser-68 to
Trp-97,
Gln-105 to Ala-110, Glu-127 to Ile-139, Thr-146 to Asp-151, Glu-173 to Lys-
178,
Gln-199 to Asp-211, Leu-310 to Gln-315. Ala-339 to Gly-345, Lys-385 to Asp-
398.
5 Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:37 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
10 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 2157 of SEQ ID
N0:37, b
is an integer of 15 to 2171, 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
15 + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 28
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
20 sequence accessible through the following database accession no.
gnIIPIDIe321606 (all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "apoptosis specific protein". An
alignment
demonstrating the observed homology is shown immediately below.
25 >gnlIPIDIe321606 apoptosis specific protein [Homo Sapiens]
>sp10608751060875
APOPTOSIS SPECIFIC PROTEIN.
Length = 275
Plus Strand HSPs:
Score = 1457 (512.9 bits), Expect = 1.5e-148, P = 1.5e-148
Identities = 267/275 (970), Positives = 267/275 (97%), Frame = +1
Query: 172 MTDDKDVLRDVWFGRIPTCFTLYQDEITEREAEPYYLLLPRVSYLTLVTDKVKKHFQKVM 351
3S MTDDKDVLRDVWFGRIPTCFTLYQDEITEREAEPYYLLLPRVSYLTLVTDKVKKHFQKVM
Sbjct: 1 MTDDKDVLRDVWFGRIPTCFTLYQDEITEREAEPYYLLLPRVSYLTLVTDKVKKHFQKVM 60
Query: 352 RQEDISEIWFEYEGTPLKWHYPIGLLFDXXXXXXXXPWNITVHFKSFPEKDLLHCPSKDA 531
RQEDISEIWFEYEGTPLKWHYPIGLLFD PWNITVHFKSFPEKDLLHCPSKDA
4O Sbjct: 61 RQEDISEIWFEYEGTPLKUIHYPIGLLFDLLASSSALPWNITVHFKSFPEKDLLHCPSKDA 120
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46
Query: 532 IEAHFMSCMKEADALKHKSQVINEMQKKDHKQLWMGLQNDRFDQFWAINR.KLMEYPAEEN 711
IEAHFMSCMREADALKHk:SQVINEI7QKKDHKQLWMGLQNDRFDQFWAINRKLMEYPAEEN
Sbjct: 121 IEAHFMSCMKEADALKHKSQVINEMQKKDHKQLVIMGLQNDRFDQFWAINRKLMEYPAEEN
180
Query: 712 GFRYIPFRIYQTTTERPFIQKLFRPVAADGQLHTLGDLLKEVCPSAIDPEDGEKKNQVMI
891
GFRYIPFRIYQTTTERPFIQKLFRPVAADGQLHTLGDLLKEVCPSAIDPEDGEKKNQVMI
Sbjct: 181 GFRYIPFRIYQTTTERPFIQKLFRPVAADGQLHTLGDLLKEVCPSAIDPEDGEKKNQVMI
240
lO Query:892 HGIEPMLETPLQWLSEHLSYPDNFLHISIIPQPTD 996
HGIEPMLETPLQWLSEHLSYPDNFLHISIIPQPTD
Sbjct: 241 HGIEPMLETPLQWLSEHLSYPDNFLHISIIPQPTD 275
The segment of gnIIPIDIe321606 that is shown as "Sbjct" above is set out as
SEQ ID NO.
151. Based on the structural similarity these homologous polypeptides are
expected to
share at least some biological activities, such as the ability to induce
apoptosis. Such
activities are known in the art and described elsewhere herein. Assays for
determining
such activities are also known in the art some of which have been described
elsewhere
herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 152 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed, and residues shown as "X" can be resolved by looking to the sequences
corresponding to Gene No. 26 in the Sequence listing).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Stromal cell TF274, Activated T-cell(12h)/Thiouridine-
re-
excision, Soares infant brain 1NIB and to a lesser extent in CD34+cells, II,
Human Colon
Carcinoma (HCC) cell line, Human T-cell lymphoma;re-excision, Cem cells
cyclohexamide treated, Human Whole Brain, re-excision, HEL cell line, Human
Osteoclastoma Stromal Cells - unamplified, H. Meningima, M1,
Soares fetal lung NbHLI9W, H. Kidney Medulla, re-excision, Stratagene fetal
spleen
(#937205), Human Hippocampus, NCI CGAP_GCB 1, Stratagene pancreas (#937208),
H.
Frontal cortex,epileptic;re-excision, Human Bone Marrow, treated, Human
Testes, T cell
helper II, Soares fetal liver spleen 1NFLS.
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
CA 02364549 2001-08-27
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47
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 1515 of SEQ ID
N0:38, b
is an integer of l~ to 1529, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:38, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 29
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. gi1861321
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "T24H7.3 gene product". A partial
alignment
demonstrating the observed homology is shown immediately below.
>gi~861321 T24H7.3 gene product [Caenorhabditis elegans]
Length = 156
Plus Strand HSPs:
Score = 168 (59.1 bits), Expect = 6.Oe-11, P = 6.Oe-11
Identities = 43/115 (37%), Positives = 68/115 (590), Frame = +1
2S Query: 157 DMKDMRLEAEAV--VNDVLFAVNNMFVSKSLRCADDVAYINVETKERNRYCLELTEAGLK 330
D++ LE EA+ V + FAVN + VS+ L + +INV T E + +C+ELT+ G +
Sbjct: 36 DVEKSTLEEEAMAAVRENAFAVNLIGVSEMLPRTSQLLFINVTTFENHTHCIELTQKGWR 95
Query: 331 WGYA-------FDQVDDHLQTPYHETWSLLDTLSPAYREAFGNALLQRLEALKRD 480
3O V F Q+D H T Y E++++LL +SP +RE FG+ L+ +L LK++
Sbjct: 96 VASNRNDCMNGDFRQLDIH--TKYFESLHTLLMDISPLFRETFGSKLISKLSELKKE 150
The segment of gi1861321 that is shown as "Sbjct" above is set out as SEQ ID
NO. 153.
Based on the structural similarity these homologous polypeptides are expected
to share at
35 least some biological activities. Such activities are known in the art and
described
elsewhere herein. Assays for determining such activities are also known in the
art some of
which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 154 which corresponds to the Query
sequence in the
CA 02364549 2001-08-27
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48
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: NCI CGAP_Co9 and to a lesser extent in
Soares_parathyroid tumor NbHPA, Snares infant brain 1NIB, Stratagene ovarian
cancer
(#937219), NCI CLAP GCB l, Soares_NhHMPu_S l, Snares melanocyte 2NbHM,
NCI CGAP_GCB 1, Colon Normal III, Human Osteoclastoma, Human Testes, Snares
fetal liver spleen 1 NFLS, Primary Dendritic Cells, lib 1, Human (HCC) cell
line liver
(mouse) metastasis, remake, Human Colon Carcinoma (HCC) cell line, Salivary
Gland,
Human adult small intestine,re-excision, Human Amygdala;re-excision, Ovarian
Tumor
10-3-95, pBMC stimulated w/ poly I/C, Human Colon: re-excision, Human
Prostate,
Human Hypothalmus,Schizophrenia, Human umbilical vein endothelial cells, IL-4
induced, Ulcerative Colitis, Human Adrenal Gland Tumor, Hepatocellular Tumor;
re-
excision, Pancreas Islet Cell Tumor, 12 Week Old Early Stage Human, Human
Placenta,
Normal colon, Bone marrow, Human Fetal Heart, CD34 depleted Buffy Coat (Cord
Blood), re-excision, NCI CGAP_Kid3, Human Cerebellum.
When tested against sensory neuron cell lines, supernatants removed from cells
containing this gene activated the EGR 1 assay. Thus, it is likely that this
gene activates
sensory neuron cells through a signal transduction pathway. Early growth
response 1
(EGR1) is a promoter associated with certain genes that induces various
tissues and cell
types upon activation, leading the cells to undergo differentiation and
proliferation.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
89 as residues: Lys-28 to Thr-33.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:39 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 2100 of SEQ ID
N0:39, b
is an integer of 15 to 2114, where both a and b correspond to the positions of
CA 02364549 2001-08-27
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-19
nucleotide residues shown in SEQ ID N0:39, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 30
It has been discovered that this Qene is expressed primarily in the following
tissues/cDNA libraries: Soares_fetal heart NbHH 19W, Stratagene pancreas
(#937208)
and to a lesser extent in HCC cell line metastisis to liver, CD34+ cell. I,
frac II, Breast
Cancer cell line, MDA 36, and Human Primary Breast Cancer Reexcision.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:40 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 429 of SEQ ID
N0:40, b
is an integer of 15 to 443, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:40, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 31
It has been discovered that this gene is expressed primarily in the following
.
tissues/cDNA libraries: Human Primary Breast Cancer Reexcision, Soares fetal
liver
spleen 1NFLS and to a lesser extent in Human Osteoblasts II, Human (HCC) cell
line liver
(mouse) metastasis, remake, Human Lung Cancer;re-excision, Glioblastoma,
Healing
groin wound, 6.5 hours post incision, Rejected Kidney, lib 4, Human Whole Six
Week Old
Embryo, and Colon Tumor.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:41 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 02364549 2001-08-27
WO 00/55171 PCT/US00/06043
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprisin~~ a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1301 of SEQ ID
N0:41, b
is an integer of 15 to 1315, where both a and b correspond to the positions of
5 nucleotide residues shown in SEQ ID N0:41, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 32
The computer algorithm BLASTX has been used to determine that the translation
10 product of this gene shares sequence homology with, as a non-limiting
example, the
sequence accessible through the following database accession no. gi1940151
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "TAFII31 ". A partial alignment
demonstrating
the observed homology is shown immediately below.
1S
>gi~940151 TAFII31 [Homo sapiens] >gi~882393 TBP-associated factor TAFII31
(Homo sapiens] >gi~841308 TAFII32 precursor [Homo sapiens]
>pir~I39141~I39141 transcription factor TFIID 32K chain TAFII32 -
human
2.0 Length = 264
Plus Strand HSPs:
Score = 1255 (441.8 bits), Expect = 3.7e-127, P = 3.7e-127
2.5 Identities = 249/249 (1000), Positives = 249/249 (100%), Frame = +1
Query: 163 MESGKTASPKSMPKDAQMMAQILKDMGITEYEPRVINQMLEFAFRYVTTILDDAKIYSSH 342
MESGKTASPKSMPKDAQMMAQILKDMGITEYEPRVINQMLEFAFRYVTTILDDAKIYSSH
Sbjct: 1 MESGKTASPKSMPKDAQMMAQILKDMGITEYEPRVINQMLEFAFRYVTTILDDAKIYSSH 60
Query: 343 AKKATVDADDVRLAIQCRADQSFTSPPPRDFLLDIARQRNQTPLPLIKPYSGPRLPPDRY 522
AKKATVDADDVRLAIQCRADQSFTSPPPRDFLLDIARQRNQTPLPLIKPYSGPRLPPDRY
Sbjct: 61 AKKATVDADDVRLAIQCRADQSFTSPPPRDFLLDIARQRNQTPLPLIKPYSGPRLPPDRY 120
3S Query: 523 CLTAPNYRLKSLQKKASTSAGRITVPRLSVGSVTSRPSTPTLGTPTPQTMSVSTKVGTPM 702
CLTAPNYRLKSLQKKASTSAGRITVPRLSVGSVTSRPSTPTLGTPTPQTMSVSTKVGTPM
Sbjct: 121 CLTAPNYRLKSLQKKASTSAGRITVPRLSVGSVTSRPSTPTLGTPTPQTMSVSTKVGTPM 180
Query: 703 SLTGQRFTVQMPTSQSPAVKASIPATSAVQNVLINPSLIGSKNILITTNMMSSQNTANES 882
4O SLTGQRFTVQMPTSQSPAVKASIPATSAVQNVLINPSLIGSKNILITTNMMSSQNTANES
Sbjct: 181 SLTGQRFTVQMPTSQSPAVKASIPATSAVQNVLINPSLIGSKNILITTNMMSSQNTANES 240
Query: 883 SNALKRKRE 909
SNALKRKRE
45 Sbjct: 241 SNALKRKRE 249
The segment of gi1940151 that is shown as "Sbjct" above is set out as
sequences) SEQ ID
NO. 155. Based on the structural similarity these homologous polypeptides are
expected
CA 02364549 2001-08-27
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51
to share at least some biological activities. Such activities are known in the
art and
described elsewhere herein. Assays for determining such activities are also
known in the
art some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 156 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares fetal liver spleen 1NFLS and to a lesser extent
in Soares
infant brain 1NIB, Human Osteoclastoma, re-excision, Human Fetal Lung III,
Soaves_senescent_fibroblasts_NbHSF, Soaves retina N2b4HR, Raji Cells,
cyclohexamide
treated, Soares_pregnant uterus NbHPU, Stratagene endothelial cell 937223,
Stratagene
ovarian cancer (#937219), Stratagene neuroepithelium NT2RAMI 937234,
Soares_pregnant uterus NbHPU, Smooth muscle, serum treated, Human Testes,
Reexcision, Bone marrow, Human Adult Pulmonary;re-excision, Soares_NhHMPu S1,
Spleen, Chronic lymphocytic leukemia, Human Testes, Bone Marrow Cell Line
(RS4;11 ),
Human Adult Spleen, fractionII, Activated T-Cells, 4 hrs, subtracted, LNCAP
untreated,
Human (HCC) cell line liver (mouse) metastasis, remake, HSC172 cells, Frontal
lobe,dementia;re-excision, Aorta endothelial cells + TNF-a, Human Thyroid,
Early Stage
Human Lung, subtracted, Human Quadriceps, B Cell lymphoma, STROMAL -
OSTEOCLASTOMA, Human endometrial stromal cells-treated with estradiol,
Salivary
Gland, Lib 2, Human Colon; re-excision, Soaves NhHMPu_S 1, Human Heart, Human
Adipose, Bone Marrow Stromal Cell, untreated, Rejected Kidney, lib 4, NTERA2,
control,
Hepatocellular Tumor; re-excision, Colon Tumor, Resting T-Cell Library,II,
Soaves breast
3NbHBst, Soaves melanocyte 2NbHM, Soaves_placenta_8to9weeks_2NbHP8to9W,
Human Osteoclastoma, NCI CGAP_GCB 1, Hodgkin's Lymphoma II, and Human
Cerebellum.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:42 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 02364549 2001-08-27
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5?
would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
Qeneral formula of a-b, where a is any integer between I to I 160 of SEQ ID
N0:42, b
is an integer of 1 S to I 174, where both a and b correspond to the positions
of
nucleotide residues shown in SEQ ID N0:42, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 33
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.
gnIIPIDle1247368 (all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "W09H I .S". A partial alignment
demonstrating
the observed homology is shown immediately below.
1S
>gnl~PID~e1247368 W09H1.5 [Caenorhabditis elegans]
Length = 344
Plus Strand HSPs:
Score = 235 (82.7 bits), Expect = 3.7e-19, P = 3.7e-19
Identities = 48/96 (50%), Positives = 67/96 (690), Frame = +2
Query: 143 ARVRALVYGHHGDPAKWELKNLELAAVRGSD-VRVKMLAAPINPSDINMIQGNYGLLPE 319
2S A R LVY + +P + ++LK + +A +D V V+ +AApINP+D+N IQG Y + p
Sbjct: 15 ASTRQLVYEGYRNPPEAIQLKTVTIADKPSADQVLVQWIAAPINPADLNQIQGWPVKPA 74
Query: 320 LPAVGGNEGVAQWAVGSNVTGLKPGDWIPANAGL 427
LPAVGGNEG +V++VGSNV+ +K GD VIP +GL
3O Sbjct: 75 LPAVGGNEGFGKVISVGSNVSSIKVGDHVIPDRSGL 110
The segment of gnllPIDle 1247368 that is shown as "Sbjct" above is set out as
SEQ ID NO.
3S 1 S7.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. IS8 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
40 It has been discovered that this gene is expressed primarily in the
following
tissues/cDNA libraries: Human Rhabdomyosarcoma and to a lesser extent in
Stratagene
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53
lung (#937210), Human adult testis, large inserts, Human Synovial Sarcoma,
Soares
placenta Nb2HP. Soaves infant brain INIB, Soaves retina N2b4HR, Soares_NhHMPu
Sl,
Soares_total_fetus_Nb2HF8 9w, Human Chondrosarcoma,
Soares_pre~~nant uterus_NbHPU, Soaves_senescent_fibroblasts_NbHSF, Soaves
fetal liver
spleen 1NFLS, CD34+cells, II, HL-60, RA 4h, Subtracted, Ovarian Tumor 10-3-95,
NTERA2 + retinoic acid, 14 days, H. Kidney Cortex, subtracted, Human Adipose
Tissue,
re-excision, Human Brain, Striatum, Apoptotic T-cell, Human Fetal Dura Mater,
Stromal
cell TF274, Human Hippocampus, Soaves breast 2NbHBst, H. Frontal
cortex,epileptic;re
excision, Pancreas Islet Cell Tumor, T Cell helper I, Human T-Cell Lymphoma,
Human
Substantia Nigra, Soaves breast 3NbHBst, Early Stage Human Brain, Human Fetal
Lung
III, Bone marrow, Human Fetal Heart, Endothelial-induced, Endothelial cells-
control,
Smooth muscle,control, Osteoblasts, Soares_parathyroid_tumor_NbHPA,
Keratinocyte,
Nine Week Old Early Stage Human, and Human Cerebellum.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
93 as residues: Asn-131 to Met-140.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:43 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 591 of SEQ ID
N0:43, b
is an integer of 15 to 605, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:43, and where b is Greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 34
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: HL-60, RA 4h, Subtracted, HUMAN STOMACH, HEL cell
line.
Human Testes Tumor, Soaves melanocyte 2NbHM, Human Microvascular Endothelial
Cells, fract. A, HUMAN B CELL LYMPHOMA, Soares_testis_NHT, T cell helper II.
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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 N0:44 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 902 of SEQ ID
N0:44, b
is an integer of 15 to 916, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:44, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 35
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares adult brain N2b4HB55Y and to a lesser extent in
Soares
infant brain 1NIB, Early Stage Human Brain, Soares adult brain N2b5HB55Y,
Soares fetal heart NbHHI9W, Soares fetal liver spleen 1NFLS, HL-60, PMA 4H, re-
excision, wilm's tumor, Hemangiopericytoma, Human Fetal Brain, Soares breast
2NbHBst, H. Frontal cortex,epileptic:re-excision, Soares_senescent fibroblasts
NbHSF,
Human Fetal Lung III, HUMAN B CELL LYMPHOMA, Stratagene endothelial cell
937223, Normal Prostate, Duodenum, HL-60, PMA 4H, Human Primary Breast Cancer,
Human Tonsils, Lib 2, B Cell lymphoma. Human Synovium, Human Prostate Cancer,
Stage C fraction, Human endometrial stromal cells-treated with progesterone,
Myoloid
Progenitor Cell Line, Human Prostate, Breast Cancer Cell line, angiogenic,
Human
Activated T-Cells, Human Hypothalmus,Schizophrenia, Human Thymus Stromal
Cells,
Human Thymus, Human Whole Six Week Old Embryo, Human T-Cell Lymphoma
Soares breast 3NbHBst, Soares fetal lung_NbHLI9W, breast lymph node CDNA
library,
H Macrophage (GM-CSF treated), re-excision, Soares melanocyte 2NbHM, Human
Synovial Sarcoma, human tonsils, Endothelial-induced, Spleen, Chronic
lymphocytic
leukemia, T cell helper II, Keratinocyte, Nine Week Old Early Stage Human,
Human
Cerebellum, Stratagene neuroepithelium NT2RAMI 937234, Primary Dendritic
Cells, lib
1.
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s5
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:45 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 937 of SEQ ID
N0:45, b
is an integer of 1 ~ to 951, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:45, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 36
It has been discovered that this gene is expressed in the following cDNA
library:
7TM Receptor enriched, lib II.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:46 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 494 of SEQ ID
N0:46, b
is an integer of 15 to 508, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:46, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 37
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in
Soares fetal liver
spleen 1NFLS, Soares multiple_sclerosis_2NbHMSP, Human Amygdala;re-excision,
Bone Marrow Stromal Cell, untreated, Primary Dendritic cells,frac 2. Human
Placenta, T
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56
cell helper II, Primary Dendritic Cells, lib 1, Human Macrophage. subtracted.
Human
osteoarthritic;fraction II, Prostate, Human osteoarthritis;fraction I. H.
Adipose Tissue,
Human Adult Retina, Adipocytes;re-excision, Human Thyroid, H. cerebellum,
Enzyme
subtracted, Early Stage Human Luna, subtracted. Soares retina N2b4HR, Human
Pineal
Gland, Messangial cell, frac 2, Human Colon Cancer;re-excision, pBMC
stimulated w/
poly I/C, Alzheimers, spongy chance, Amniotic Cells - Primary Culture, NTERA2
+
retinoic acid, 14 days, Soares NhHMPu_S l, Human Adipose Tissue, re-excision,
KMH2,
Human Infant Brain, T-Cell PHA 24 hrs, Human Activated Monocytes, Soares
breast
2NbHBst, Human Adrenal Gland Tumor, Human Eosinophils,
Soares_fetal_heart NbHHI9W, Brain frontal cortex, human tonsils, Human
Neutrophil,
Activated, Human Osteoclastoma, Monocyte activated, Hodgkin's Lymphoma II,
Stratagene lung carcinoma 937218, Stratagene neuroepithelium NT2RAMI 937234,
Osteoblasts, Human Cerebellum, and Soares placenta Nb2HP.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
97 as residues: Leu-6 to Ser-12.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:47 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 1286 of SEQ ID
N0:47, b
is an integer of 15 to 1300, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:47, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 38
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares adult brain N2b4HB55Y,
Soares multiple_sclerosis_2NbHMSP and to a lesser extent in H. Adipose Tissue,
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57
Soares_multiple_sclerosis_2NbHMSP, Spinal Cord, re-excision, and Nine Week Old
Early Stage Human.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
98 as residues: Gly-8 to Ala-13.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:48 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 976 of SEQ ID
N0:48, b
is an integer of 15 to 990, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:48, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 39
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. gi1818411
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "tubulointerstitial nephritis
antigen". A partial
alignment demonstrating the observed homology is shown immediately below.
>gi~818411 tubulointerstitial nephritis antigen (Oryctolagus cuniculusJ
>pir~A57480~A57480 tubulointerstitial nephritis antigen precursor -
rabbit >sp~Q28625~Q28625 TUBULOINTERSTITIAL NEPHRITIS ANTIGEN.
Length = 474
Plus Strand HSPs:
Score = 605 (213.0 bits), Expect = 4.7e-98, Sum P(2) = 4.7e-98
Identities = 115/239 (480), Positives = 151/239 (63%), Frame = +3
3S Query: 729 NCAGSWAFSTAAVASDRVSIHSLGHMTPVLSPQNLLSCDTHQQQGCRGGRLDGAWWFLRR 908
NCA SWAFSTA+VA+DR++I S G T LSPQNL+SC + GC G +D AWW+LR+
Sbjct: 237 NCAASWAFSTASVAADRIAIQSNGRYTANLSPQNLISCCAKNRHGCNSGSIDRAWWYLRK 296
Query: 909 EGWCLTTATPSRAVNETRLALRPPV*CTAEPWVGASARPHAH--CPNSYVNNNDIYQVTP 1082
4O G P + ++ C + HA CPN+ +N IYQ +P
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Sh
Sbjct: 297 RGLVSHACYP--LFKDQNISNNT---CAbiTSKADGRGKRHATRPCPNNIEKSNRIYQCSP 351
Query: 1083 VYRLGSNDKEIMKELMENGPVQALMEVHEDFFLYKGGIYSHTPVSLGRPERYRRHGTHSV 1262
YR+ SN+ EIMKE+M+NGPVQA+Id+VHEDFF YK GIY H + E+yR+ TH+V
S Sbjct: 352 PYRVSSNETEIMKEIMQNGPVQAII~~QVHEDFFHYKTGIYRHVISTNEESEKYRKLQTHAV
411
Query: 1263 KITGWGEETLPDGRTLKYLr,'TAA~ISi~7GPAWGERGHFRIVRGVNECDIESFVLGVWGRVGME
1442
K+TGWG G+ K+P: AANS~;G +PdGE G+FRI+RGVNE DIE ++ WG++
Sbjct: 412 KLTGL~7GTLKGARGQREKFV~;IAANS;~7GKSIaGENGYFRILRGVNESDIEKLiiAAWGQLTSS
471
Query: 1443 D 1445
D
Sbjct: 472 D 472
The segment of gi1818411 that is shown as "Sbjct" above is set out as
sequences) SEQ ID
NO. 161 Based on the structural similarity these homologous polypeptides are
expected to
share at least some biological activities. Such activities are known in the
art and described
elsewhere herein. Assays for determining such activities are also known in the
art some of
which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 162 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Endothelial cells-control and to a lesser extent in
Human
Microvascular Endothelial Cells, fract. A, Keratinocyte, Soares placenta
Nb2HP, H.
Epididiymus, cauda, Human Adipose Tissue. re-excision, Human Umbilical Vein
Endothelial Cells, uninduced, Epithelial-TNFa and INF induced, Soares breast
2NbHBst,
Colon Carcinoma, Human Placenta, Endothelial-induced.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
99 as residues: Gln-19 to Glu-26, Ala-39 to Leu-49, Gly-53 to Thr-59, Tyr-I 14
to
Gln-133, Ala-205 to Thr-21 I.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:49 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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SJ
general formula of a-b, where a is any integer between 1 to 2171 of SEQ ID
N0:49, b
is an inte~~er of 15 to 218, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:49, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 40
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Keratinocyte and to a lesser extent in
Soares_fetal heart NbHHI9W, Healing groin wound; 7.5 hours post incision,
Healing
groin wound, 6.5 hours post incision, Healing Abdomen wound;70&90 min post
incision,
Soares breast 2NbHBst, Soares_fetal_heart NbHHI9W, Healing groin wound - zero
hr
post-incision (control), Epithelial-TNFa and INF induced, Soares breast
3NbHBst, human
tonsils, Sinus piniformis Tumour, Pharynx Carcinoma, Tongue carcinoma, Human
Tongue, frac 1, Tongue Normal, Larynx Normal, Human Tonsil, Lib 3, H.
Epididiymus,
caput & corpus, Amniotic Cells - Primary Culture, Human Adipose Tissue, re-
excision,
Human Thymus, Colon Carcinoma, Human Fetal Heart.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
100 as residues: Ser-16 to Glu-31.
The tissue distribution and homology to [gene or gene family] suggests that
the protein product of this clone would be useful for [take best guesses) and
explain
rationale].
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:50 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 3138 of SEQ ID
NO:50, b
is an integer of 15 to 3152, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:50, and where b is greater than or
equal to a
+ 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 41
It has been discovered that this Qene is expressed primarily in the following
tissues
and cDNA libraries: Soares melanocyte 2NbHM and to a lesser extent in Soares
placenta
5 Nb2HP, Soaves infant brain 1NIB, NCI_CGAP_GCBI, Stratagene neuroepithelium
(#937231 ), Stratagene lung (#937210). Stratagene colon (#937204),
Soares_pregnant_uterus NbHPU, Primary Dendritic Cells, lib 1, Human
osteoarthritic;fraction II, Brain Amygdala Depression, Human Whole 6 Week Old
Embryo
(II), subt, Human osteoarthritis;fraction I, stomach cancer (human),
Soares_NhHMPu_S 1,
10 Human Adipose Tissue, re-excision, wilm's tumor, L428, HUMAN JURKAT
MEMBRANE BOUND POLYSOMES, Stromal cell TF274, Pancreas Islet Cell Tumor,
Soares_fetal_luna NbHLI9W, Soares_fetal_lung NbHLI9W, Bone marrow, Activated
T-cell( 12h)/Thiouridine-re-excision, Spleen, Chronic lymphocytic leukemia,
Human
Endometrial Tumor, Human 8 Week Whole Embryo.
15 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:51 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
excluded from the scope of the present invention. To list every related
sequence
20 would be cumbersome. Accordingly, preferably excluded from the present
invention
are one or more polynucleotides comprising a nucleotide sequence described by
the
general formula of a-b, where a is any integer between 1 to 1388 of SEQ ID
NO:51, b
is an integer of 15 to 1402, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID NO:51, and where b is greater than or
equal to a
25 + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 42
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soaves breast 2NbHBst and to a lesser extent in
30 Soares_multiple_sclerosis 2NbHMSP, Bone Marrow Cell Line (RS4;11), Human
Aortic
Endothelium, Human Adipose Tissue, re-excision, Soares_fetal heart NbHH 19W,
and
Activated Human Neutrophil.
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Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
102 as residues: Ser-49 to Lys-59.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:52 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 827 of SEQ ID
N0:52, b
is an integer of 15 to 841, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:52, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 43
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares retina N2b4HR, Soares infant brain 1NIB and to
a lesser
extent in Soares fetal liver spleen 1 NFLS, Human Old Ovary, Soares retina
N2b5HR,
Human Epididymus, NCI CLAP Br2, NCI CGAP_Co3, NCI_CGAP Lul,
NCI CLAP Col l, NCI CGAP_KidS, Human Adipose Tissue, re-excision, Human Infant
Brain, and Human Brain, Striatum.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:53 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 952 of SEQ ID
N0:53, b
is an integer of 15 to 966, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:53, and where b is greater than or
equal to a
+ 14.
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FEATURES OF PROTEIN ENCODED BY GENE NO: 44
It has been discovered that this gene is expressed primarily in Human Adipose
Tissue, re-excision.
Many polynucleotide sequences. such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:54 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 991 of SEQ ID
N0:54, b
is an integer of 15 to 1005, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:54, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 45
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Soares breast 3NbHBst and to a lesser extent in Human
Adipose,
Soares breast 2NbHBst, breast lymph node CDNA library, Human Normal Breast,
Human
Synovium, Human Adipose Tissue, re-excision, Stratagene liver (#937224), Human
Infant
Adrenal Gland, Human colon cancer, metaticized to liver, subtraction, Human
White Fat,
H. Adipose Tissue, Human Quadriceps, Human Soleus, Human Pituitary, subt IX,
Human
Chronic Synovitis, and Bone marrow.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
105 as residues: Pro-45 to Gln-51.
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:55 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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63
are one or more polynucleotides comprising a nucleotide sequence described by
the
General formula of a-b, where a is any integer between I to 450 of SEQ ID
N0:55, b
is an integer of 15 to 464, where both a and b correspond to the positions of
nucleotide residues shown in SEQ ID N0:55, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 46
It has been discovered that this gene is expressed primarily in Human Adipose
tissue.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:56 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 825 of SEQ ID N0:56, b is an integer of 15 to 839, where
both a and
b correspond to the positions of nucleotide residues shown in SEQ ID N0:56,
and where b
is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 47
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. gi11710216
(all
information available through the recited accession number is incorporated
herein by
reference) which is described therein as "unknown". A partial alignment
demonstrating
the observed homology is shown immediately below.
>gi~1710216 unknown [Homo Sapiens] >sp~Q99770IQ99770 HYPOTHETICAL 15.4 KD
PROTEIN.
3~ Length = 139
Minus Strand HSPs:
Score = 140 (49.3 bits), Expect = 4.Oe-18, Sum P(2) = 4.Oe-18
Identities = 31/45 (680), Positives = 35/45 (77%), Frame = -3
Query: 1043 RLECSGAVSAHCKLHFPGSHHSPASASRVAGI--TCARHHAWLIFLY 909
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6=l
RLECSG +SAHC L PGS +SPASASRVAG TC R A LIF++
Sbjct: 62 RLECSGTISAHCNLCLPGSSNSPASASRVAGTAGTCRR--AQLIFVF 106
The segment of gi11710216 that is shown as "Sbjct" above is set out as
sequences) SEQ
ID NO. 163 Based on the structural similarity these homologous polypeptides
are expected
to share at least some biological activities. Such activities are known in the
art and
described elsewhere herein. Assays for determining such activities are also
known in the
art some of which have been described elsewhere herein.
Preferred polypeptides of the invention comprise a polypeptide having the
amino
acid sequence set out as SEQ ID NO. 164 which corresponds to the Query
sequence in the
alignment shown above (gaps introduced in the sequence by the computer are, of
course,
removed).
It has been discovered that this gene is expressed primarily in Human Adipose.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ ID
N0:57 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 1088 of SEQ ID N0:57, b is an integer of 15 to 1102,
where both a
and b correspond to the positions of nucleotide residues shown in SEQ ID
N0:57, and
where b is greater than or equal to a + 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 48
It has been discovered that this gene is expressed primarily in the following
tissues/cDNA libraries: Supt Cells, cyclohexamide treated, Apoptotic T-cell,
re-excision,
and Human Adipose.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:58 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 02364549 2001-08-27
WO 00/55171 PCT/US00/06043
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 1098 of SEQ ID
N0:58, b
is an integer of 15 to 1 112, where both a and b correspond to the positions
of
5 nucleotide residues shown in SEQ ID N0:58, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 49
It has been discovered that this gene is expressed primarily in the following
tissues:
10 Human Adipose and Human Placenta.
When tested against sensory neuron cell lines, supernatants removed from cells
containing this gene activated the EGR1 assay. Thus, it is likely that this
gene activates
sensory neuron cells through a signal transduction pathway. Early growth
response 1
(EGR1) is a promoter associated with certain genes that induces various
tissues and cell
15 types upon activation, leading the cells to undergo differentiation and
proliferation.
Many polynucleotide sequences, such as EST sequences, are publicly
available and accessible through sequence databases. Some of these sequences
are
related to SEQ ID N0:59 and may have been publicly available prior to
conception of
the present invention. Preferably, such related polynucleotides are
specifically
20 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 2517 of SEQ ID
N0:59, b
is an integer of 15 to 2531, where both a and b correspond to the positions of
25 nucleotide residues shown in SEQ ID N0:59, and where b is greater than or
equal to a
+ 14.
FEATURES OF PROTEIN ENCODED BY GENE NO: 50
It has been discovered that this gene is expressed primarily in the following
30 tissues/cDNA libraries: Soares infant brain 1NIB and to a lesser extent in
Soares ovary
tumor NbHOT, Soares adult brain N2b4HB55Y, Glioblastoma, Hemangiopericytoma,
Soares fetal heart NbHH 19W, Early Stage Human Brain, Soares_NhHMPu_S 1, Human
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66
epithelioid sarcoma, Human Adult Pulmonary, eosinophil-ILS induced. Human
Quadriceps, Human Epididymus, Human Fetal Dura Mater, Merkel Cells, Human
Adipose, Human Thymus, Soares breast 2NbHBst, Primary Dendritic cells,frac 2,
Soares total fetus Nb2HF8_9w, Hodgkin's Lymphoma II, Osteoblasts.
Keratinocyte,
Human Cerebellum, Soares_total fetus Nb2HF8_9w, and Primary Dendritic Cells,
lib 1.
Preferred epitopes include those comprising a sequence shown in SEQ ID NO.
110 as residues: Pro-9 to Thr-14, Ser-37 to Trp-44, Gly-79 to Thr-85, ArQ-88
to Lys-
139.
Many polynucleotide sequences, such as EST sequences, are publicly available
and
accessible through sequence databases. Some of these sequences are related to
SEQ
ID N0:60 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 653 of SEQ ID N0:60, b is an
integer of
15 to 667, where both a and b correspond to the positions of nucleotide
residues
shown in SEQ ID N0:60, and where b is greater than or equal to a + 14.
CA 02364549 2001-08-27
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67
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CA 02364549 2001-08-27
WO 00/55171 PCTNS00/06043
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WO 00/55171 PCT/US00/06043
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CA 02364549 2001-08-27
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CA 02364549 2001-08-27
WO 00/55171 PCT/US00/06043
71
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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 ID" 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 ID 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 ID 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 ID 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 ID 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 ID NO:X is useful for designing
nucleic
acid hybridization probes that will detect nucleic acid sequences contained in
SEQ ID
NO:X or the cDNA contained in the deposited clone. These probes will also
hybridize to nucleic acid molecules in biological samples, thereby enabling a
variety
of forensic and diagnostic methods of the invention. Similarly, polypeptides
identified from SEQ ID 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.
Nevertheless, DNA sequences generated by sequencing reactions can contain
sequencing errors. The errors exist as misidentified nucleotides, or as
insertions or
deletions of nucleotides in the generated DNA sequence. The erroneously
inserted or
deleted nucleotides cause frame shifts in the reading frames of the predicted
amino
acid sequence. In these cases, the predicted amino acid sequence diverges from
the
actual amino acid sequence, even though the generated DNA sequence may be
greater
than 99.9% identical to the actual DNA sequence (for example, one base
insertion or
deletion in an open reading frame of over 1000 bases).
Accordingly, for those applications requiring precision in the nucleotide
sequence or the amino acid sequence, the present invention provides not only
the
generated nucleotide sequence identified as SEQ 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.
The predicted amino acid sequence can then be verified from such deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone
can
also be directly determined by peptide sequencing or by expressing the protein
in a
suitable host cell containing the deposited human cDNA, collecting the
protein, and
determining its sequence.
The present invention also relates to the genes corresponding to SEQ ID
NO:X, SEQ ID 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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74
herein. Such methods include preparing probes or primers from the disclosed
sequence and identifying or amplifying the corresponding gene from appropriate
sources of aenomic 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.
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.
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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
5 polypeptide encoded by the cDNA contained in ATCC deposit Z. Polynucleotides
encoding a polypeptide comprising, or alternatively consisting of the
polypeptide
sequence of SEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA
contained in ATCC deposit Z are also encompassed by the invention.
Signal Seciuences
10 The present invention also encompasses mature forms of the polypeptide
having the polypeptide sequence of SEQ ID NO:Y and/or the polypeptide sequence
encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature
forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or
the
polynucleotide sequence contained in the cDNA of a deposited clone) are also
15 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,
20 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, it is inherent in the amino acid sequence of
the
polypeptide.
25 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
30 information from the residues surrounding the cleavage site, typically
residues -13 to
+2, where +1 indicates the amino terminus of the secreted protein. The
accuracy of
predicting the cleavage points of known mammalian secretory proteins for each
of
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76
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 cellular location
of a
protein based on the amino acid sequence. As part of this computational
prediction of
localization, the methods of McGeoch and von Heinje are incorporated. The
analysis
of the amino acid sequences of the secreted proteins described herein by this
program
provided the results shown in Table 1.
As one of ordinary skill would appreciate, however, cleavage sites sometimes
vary from organism to organism and cannot be predicted with absolute
certainty.
Accordingly, the present invention provides secreted polypeptides having a
sequence
shown in SEQ ID NO:Y which have an N-terminus beginning within 5 residues
(i.e.,
+ or - 5 residues) of the predicted cleavage point. Similarly, it is also
recognized that
in some cases, cleavage of the signal sequence from a secreted protein is not
entirely
uniform, resulting in more than one secreted species. These polypeptides, and
the
polynucleotides encoding such polypeptides, are contemplated by the present
invention.
Moreover, the signal sequence identified by the above analysis may not
necessarily predict the naturally occurring signal sequence. For example, the
naturally occurring signal sequence may be further upstream from the predicted
signal
sequence. However, it is likely that the predicted signal sequence will be
capable of
directing the secreted protein to the ER. Nonetheless, the present invention
provides
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 desribed below). These
polypeptides,
and the polynucleotides encoding such polypeptides, are contemplated by the
present
invention.
Polynucleotide and Po>xneptide Variants
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77
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 ID NO:Y and/or encoded by a deposited clone.
"Variant" refers to a polynucleotide or polypeptide differing from the
polynucleotide or polypeptide of the present invention, but retaining
essential
properties thereof. Generally, variants are overall closely similar, and, in
many
regions, identical to the polynucleotide or polypeptide of the present
invention.
The present invention is also directed to nucleic acid molecules which
comprise, or alternatively consist of, a nucleotide sequence which is at least
80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for example, the nucleotide
coding sequence in SEQ ID 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
hybridization conditions or lower stringency conditions are also encompassed
by the
invention, as are polypeptides encoded by these polynucleotides.
The present invention is also directed to polypeptides which comprise, or
alternatively consist of, an amino acid sequence which is at least 80%, 85%,
90%,
95%, 96%, 97%, 98%, 99% identical to, for example, the polypeptide sequence
shown in SEQ ID NO:Y, the polypeptide sequence encoded by the cDNA contained
in a deposited clone, and/or polypeptide fragments of any of these
polypeptides (e.g.,
those fragments described herein).
By a nucleic acid having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended
that the nucleotide sequence of the nucleic acid is identical to the reference
sequence
except that the nucleotide sequence may include up to five point mutations per
each
100 nucleotides of the reference nucleotide sequence encoding the polypeptide.
In
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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 S~b 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=1,
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 subject sequence, which are not matched/aligned, as a percent
of the
total bases of the query sequence. Whether a nucleotide is matched/aligned is
determined by results of the FASTDB sequence alignment. This percentage is
then
subtracted from the percent identity, calculated by the above FASTDB program
using
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the specified parameters, to arrive at a final percent identity score. This
corrected
score is what is used for the purposes of the present invention. Only bases
outside the
5' and 3' bases of the subject sequence. as displayed by the FASTDB alignment,
which are not matched/alianed 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 ~' end of
the
subject sequence and therefore, the FASTDB alignment does not show a
matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases
represent
10% of the sequence (number of bases at the 5' and 3' ends not matched/total
number
of bases in the query sequence) so 10% is subtracted from the percent
identity.score
calculated by the FASTDB program. If the remaining 90 bases were perfectly
matched the final percent identity would be 90%. In another example, a 90 base
subject sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the 5' or 3' of
the subject
sequence which are not matched/aligned with the query. In this case the
percent
identity calculated by FASTDB is not manually corrected. Once again, only
bases 5'
and 3' of the subject sequence which are not matched/aligned with the query
sequence
are manually corrected for. No other manual corrections are to made for the
purposes
of the present invention.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a query amino acid sequence of the present invention, it is
intended that .
the amino acid sequence of the subject polypeptide is identical to the query
sequence
except that the subject polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the query amino acid sequence. In
other
words, to obtain a polypeptide having an amino acid sequence at least 95%
identical
to a query amino acid sequence, up to 5% of the amino acid residues in the
subject
sequence may be inserted, deleted. (indels) or substituted with another amino
acid.
These alterations of the reference sequence may occur at the amino or carboxy
terminal positions of the reference amino acid sequence or anywhere between
those
terminal positions, interspersed either individually among residues in the
reference
sequence or in one or more contiguous groups within the reference sequence.
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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 Brutlaa 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=l, 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
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.
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For example, a 90 amino acid residue subject sequence is aligned with a 100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show a matching/alignment of the first 10 residues at the N-terminus. The 10
unpaired residues represent 10% of the sequence (number of residues at the N-
and C-
termini not matched/total number of residues in the query sequence) so 10% is
subtracted from the percent identity score calculated by the FASTDB program.
If the
remaining 90 residues were perfectly matched the final percent identity would
be
90%. In another example, a 90 residue subject sequence is compared with a 100
residue query sequence. This time the deletions are internal deletions so
there are no
residues at the N- or C-termini of the subject sequence which are not
matched/aligned
with the query. In this case the percent identity calculated by FASTDB is not
manually corrected. Once again, only residue positions outside the N- and C-
terminal
ends of the subject sequence, as displayed in the FASTDB alignment. which are
not
matched/aligned with the query sequnce are manually corrected for. No other
manual
corrections are to made for the purposes of the present invention.
The variants may contain alterations in the coding regions, non-coding
regions, or both. Especially preferred are polynucleotide variants containing
alterations which produce silent substitutions, additions, or deletions, but
do not alter
the properties or activities of the encoded polypeptide. Nucleotide variants
produced
by silent substitutions due to the degeneracy of the genetic code are
preferred.
Moreover, variants in which 5-10, 1-5, or 1-2 amino acids are substituted,
deleted, or
added in any combination are also preferred. Polynucleotide variants can be
produced
for a variety of reasons, e.g., to optimize codon expression for a particular
host
(change codons in the human mRNA to those preferred by a bacterial host such
as E.
coli).
Naturally occurring variants are called "allelic variants," and refer to one
of
several alternate forms of a gene occupying a given locus on a chromosome of
an
organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York ( 1985).)
These
allelic variants can vary at either the polynucleotide and/or polypeptide
level and are
included in the present invention. Alternatively, non-naturally occurring
variants may
be produced by mutagenesis techniques or by direct synthesis.
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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-la. They used random mutagenesis to generate
over
3,500 individual IL-la mutants that averaged 2.5 amino acid changes per
variant over
the entire length of the molecule. Multiple mutations were examined at every
possible amino acid position. The investigators found that "[m]ost of the
molecule
could be altered with little effect on either [binding or biological
activity]." (See,
Abstract.) In fact, only 23 unique amino acid sequences, out of more than
3,500
nucleotide sequences examined, produced a protein that significantly differed
in
activity from wild-type.
Furthermore, even if deleting one or more amino acids from the N-terminus or
C-terminus of a polypeptide results in modification or loss of one or more
biological
functions, other biological activities may still be retained. For example, the
ability of
a deletion variant to induce and/or to bind antibodies which recognize the
secreted
form will likely be retained when less than the majority of the residues of
the secreted
form are removed from the N-terminus or C-terminus. Whether a particular
polypeptide lacking N- or C-terminal residues of a protein retains such
immunogenic
activities can readily be determined by routine methods described herein and
otherwise known in the art.
Thus, the invention further includes polypeptide variants which show
substantial biological activity. Such variants include deletions, insertions,
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b3
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 amino acid changes
at specific positions of a cloned gene to identify regions critical for
protein function.
For example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction
of single alanine mutations at every residue in the molecule) can be used.
(Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant
molecules can then be tested for biological activity.
As the authors state, these two strategies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at certain amino acid positions
in the
protein. For example, most buried (within the tertiary structure of the
protein) amino
acid residues require nonpolar side chains, whereas few features of surface
side chains
are generally conserved. Moreover, tolerated conservative amino acid
substitutions
involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu
and
Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the
acidic
residues Asp and Glu; replacement of the amide residues Asn and Gln,
replacement of
the basic residues Lys, Arg, and His; replacement of the aromatic residues
Phe, Tyr,
and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,
and Gly.
Besides conservative amino acid substitution, variants of the present
invention
include (i) substitutions with one or more of the non-conserved amino acid
residues,
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8-t
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. 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 Carrier Systems 10:307-377 (1993).)
A further embodiment of the invention relates to a polypeptide which
comprises the amino acid sequence of the present invention having an amino
acid
sequence which contains at least one amino acid substitution, but not more
than 50
amino acid substitutions, even more preferably, not more than 40 amino acid
substitutions, still more preferably, not more than 30 amino acid
substitutions, and
still even more preferably, not more than 20 amino acid substitutions. Of
course, in
order of ever-increasing preference, it is highly preferable for a peptide or
polypeptide
to have an amino acid sequence which comprises the amino acid sequence of the
present invention, which contains at least one, but not more than 10, 9, 8, 7,
6, 5, 4, 3,
2 or 1 amino acid substitutions. In specific embodiments, the number of
additions,
substitutions, and/or deletions in the amino acid sequence of the present
invention or
fragments thereof (e.g., the mature form and/or other fragments described
herein), is
1-5, 5-10, 5-2~, 5-50, 10-50 or 50-150, conservative amino acid substitutions
are
preferable.
Polynucleotide and Polypeptide Fragments
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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 ID NO:X or the complementary strand
thereto, or is a portion of a polynucleotide sequence encoding the polypeptide
of SEQ
ID 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 ID
NO:X. In this context "about" includes the particularly recited value, a value
larger
or smaller by several (5, 4, 3, 2, or 1 ) nucleotides, at either terminus or
at both
termini. These nucleotide fragments have uses that include, but are not
limited to, as
diagnostic probes and primers as discussed herein. Of course, larger fragments
(e.g.,
50, 150, 500, 600, 2000 nucleotides) are preferred.
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
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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 ID 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.
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 ID NO:Y falling within conserved domains are
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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 are
not
limited to, biological activity, antigenicity [ability to bind (or compete
with a
polypeptide of the invention for binding) to an antibody to the polypeptide of
the
invention], immunogenicity (ability to generate antibody which binds to a
polypeptide
of the invention), ability to form multimers with polypeptides of the
invention, and
ability to bind to a receptor or ligand for a polypeptide of the invention.
The functional activity of polypeptides of the invention, and fragments,
variants derivatives, and analogs thereof, can be assayed by various methods.
For example, in one embodiment where one is assaying for the ability to bind
or compete with full-length polypeptide of the invention for binding to an
antibody of
the polypeptide of the invention, various immunoassays known in the art can be
used,
including but not limited to, competitive and non-competitive assay systems
using
techniques such as radioimmunoassays, ELISA (enzyme linked 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
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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
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 ID
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 ID 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 ID NO:X),
polynucleotide sequences of the complementary strand of a polynucleotide
sequence
encoding an epitope of the invention, and polynucleotide sequences which
hybridize
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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. Immunospecific binding excludes non-specific binding but
does not
necessarily exclude cross- reactivity with other antigens. Antigenic epitopes
need not
necessarily be immunogenic.
Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985),
further described in U.S. Patent No. 4,631,211).
In the present invention, antigenic epitopes preferably contain a sequence of
at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10,
at least 11, at least 12, at least 13, at least 14, at least 15, at least 20,
at least 25, at
least 30, at least 40, at least 50, and, most preferably, between about 15 to
about 30
amino acids. Preferred polypeptides comprising immunogenic or antigenic
epitopes
are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, or 100
amino acid residues in length. Additional non-exclusive preferred antigenic
epitopes
include the antigenic epitopes disclosed herein. as well as portions thereof.
Antigenic
epitopes are useful, for example, to raise antibodies, including monoclonal
antibodies,
that specifically bind the epitope. Preferred antigenic epitopes include the
antigenic
epitopes disclosed herein, as well as any combination of two, three, four,
five or more
of these antigenic epitopes. Antigenic epitopes can be used as the target
molecules in
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WO 00/55171 PCT/US00/06043
immunoassays. (See, for instance. Wilson et al., Cell 37:767-778 (1984);
Sutcliffe et
al., Science 219:660-666 ( 1983)).
Similarly, immunoaenic epitopes can be used, for example, to induce
antibodies according to methods well known in the art. (See, for instance,
Sutcliffe
5 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 immunoaenic epitopes may be presented for
10 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
15 least, linear epitopes in a denatured polypeptide (e.g., in Western
blotting).
Epitope-bearing polypeptides of the present invention may be used to induce
antibodies according to methods well known in the art including, but not
limited to,
in vivo immunization, in vitro immunization, and phage display methods. See,
e.g.,
Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen.
Virol., 66:2347-
20 2354 ( 1985). If in vivo immunization is used, animals may be immunized
with free
peptide; however, anti-peptide antibody titer may be boosted by coupling the
peptide
to a macromolecular carrier, such as keyhole limpet hemacyanin (KL,H) or
tetanus
toxoid. For instance, peptides containing cysteine residues may be coupled to
a
carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester
(MBS),
25 while other peptides may be coupled to carriers using a more general
linking agent
such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized
with
either free or carrier- coupled peptides, for instance, by intraperitoneal
and/or
intradermal injection of emulsions containing about 100 pg of peptide or
carrier
protein and Freund's adjuvant or any other adjuvant known for stimulating an
30 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
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91
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 comprising an immunoQenic or antigenic
epitope can be fused to other polypeptide sequences. For example, the
polypeptides
of the present invention may be fused with the constant domain of
immunoglobulins
(IgA, IgE, IgG, IgM), or portions thereof (CH l, CH2, CH3, or any combination
thereof and portions thereof) resulting in chimeric polypeptides. 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 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+
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WO 00/55171 PCT/US00/06043
c~ ~
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-shufl7ing, 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 aQonists and
antagonists of the
polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721;
5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-
33
(1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J.
Mol.
Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308- 13
( 1998) (each of these patents and publications are hereby incorporated by
reference in
its entirety). In one embodiment, alteration of polynucleotides corresponding
to SEQ
ID NO:X and the polypeptides encoded by these polynucleotides may be achieved
by
DNA shuffling. DNA shuffling involves the assembly of two or more DNA
segments by homologous or site-specific recombination to generate variation in
the
polynucleotide sequence. In another embodiment, polynucleotides of the
invention,
or the encoded polypeptides, may be altered by being subjected to random
mutagenesis by error-prone PCR, random nucleotide insertion or other methods
prior
to recombination. In another embodiment, one or more components, motifs,
sections,
parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of
the
invention may be recombined with one or more components, motifs, sections,
parts,.
domains, fragments, etc. of one or more heterologous molecules.
Antibodies
Further polypeptides of the invention relate to antibodies and T-cell antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment,
or variant of SEQ ID NO:Y, and/or an epitope, of the present invention (as
determined by immunoassays well known in the art for assaying specific
antibody-
antigen binding). Antibodies of the invention include, but are not limited to,
polyclonal, monoclonal, multispecific, human, humanized or chimeric
antibodies,
single chain antibodies, Fab fragments, F(ab') fragments, fragments produced
by a
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WO 00/55171 PCT/US00/06043
)3
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
immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE,
IgM,
IgD, IgA and IgY), class (e.g., IgGI, IaG2, IgG3, IgG4, IaAl and IQA2) or
subclass
of immunoglobulin molecule.
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,
CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding
fragments also comprising any combination of variable regions) with a hinge
region,
CHI, CH2, and CH3 domains. The antibodies of the invention may be from any
animal origin including birds and mammals. Preferably, the antibodies are
human,
murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel,
horse, or
chicken. As used herein, "human" antibodies include antibodies having the
amino
acid sequence of a human immunoglobulin and include antibodies isolated from
human immunoglobulin libraries or from animals transgenic for one or more
human
immunoglobulin and that do not express endogenous immunoglobulins, as
described
infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
The antibodies of the present invention may be monospecific, bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both
a polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol.
147:60-69 ( 1991 ); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648;
5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 ( 1992).
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WO 00/55171 PCT/US00/06043
94
Antibodies of the present invention may be described or specified in terms of
the epitope(s) or portions) of a polvpeptide 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 polvpeptide of the present invention may also
be
excluded. Therefore, the present in~-ention includes antibodies that
specifically bind
polypeptides of the present invention, and allows for the exclusion of the
same.
Antibodies of the present invention may also be described or specified in
terms of their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of a polypeptide of the present invention are included.
Antibodies that bind polypeptides with at least 95%, at least 90%, at least
85%, at
least 80%, at least 75%, at least 70%. at least 65%, at least 60%, at least
55%, and at
least 50% identity (as calculated using methods known in the art and described
herein) to a polypeptide of the present invention are also included in the
present
invention. In specific embodiments. antibodies of the present invention cross-
react
with murine, rat and/or rabbit homologs of human proteins and the
corresponding
epitopes thereof. Antibodies that do not bind polypeptides with less than 95%,
less
than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less
than 65%,
less than 60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of the present
invention are also included in the present invention. In a specific
embodiment, the
above-described cross-reactivity is with respect to any single specific
antigenic or
immunogenic polypeptide, or combinations) of 2, 3, 4, 5, or more of the
specific
antigenic and/or immunogenic polypeptides disclosed herein. Further included
in the
present invention are antibodies which bind polypeptides encoded by
polynucleotides
which hybridize to a polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the present
invention
may also be described or specified in terms of their binding affinity to a
polypeptide
of the invention. Preferred binding affinities include those with a
dissociation
constant or Kd less than 5 X 10~' M. 10-' M, 5 X 10~~ M, 10-~ M, 5 X 10-~' M,
10-'~ M, 5
X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M. 5 X 10-' M, 10' M, 5 X 10~~ M, 10-~ M, 5
X 10-~
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WO 00/55171 PCT/US00/06043
M, 10-'' M, 5 X 10-'° M, 10-'° M, 5 X 10-" M, 10-" M, 5 X 10-''
M, '°-'' M, 5 X 10-''
M, 10-''M,SX 10-''M, 10-'~'M,SX 10-'~M,or 10-''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
5 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 may act as agonists or antagonists of the
10 polypeptides of the present invention. For example, the present invention
includes
antibodies which disrupt the receptor/ligand interactions with the
polypeptides of the
invention either partially or fully. Preferrably, antibodies of the present
invention
bind an antigenic epitope disclosed herein, or a portion thereof. The
invention
features both receptor-specific antibodies and ligand-specific antibodies. The
15 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
20 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.
25 The invention also features receptor-specific antibodies which both prevent
ligand binding and receptor activation as well as antibodies that recognize
the
receptor-ligand complex, and, preferably, do not specifically recognize the
unbound
receptor or the unbound ligand. Likewise, included in the invention are
neutralizing
antibodies which bind the ligand and prevent binding of the ligand to the
receptor, as
30 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
CA 02364549 2001-08-27
WO 00/55171 PCT/US00/06043
96
agonists, i.e., potentiate or activate either all or a subset of the
biological activities of
the ligand-mediated receptor activation, for example, by inducing dimerization
of the
receptor. The antibodies may be specified as agonists, antagonists or inverse
agonists
for biological activities comprising the specific biological activities of the
peptides of
the invention disclosed herein. The above antibody agonists can be made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu
et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol.
160(7):3170-
3-179 (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
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.
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97
The antibodies of the invention include derivatives that are modified, i.e, by
the covalent attachment of any type of molecule to the antibody such that
covalent
attachment does not prevent the antibody from generating an anti-idiotypic
response.
For example, but not by way of limitation, the antibody derivatives include
antibodies that have been modified, e.g., by glycosylation, acetylation,
pegylation,
phosphylation, amidation, derivatization by known protectina/blockina Groups,
proteolytic cleavage, linkage to a cellular liaand 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
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
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9S
"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
CH 1 domain of the heavy chain.
For example, the antibodies of the present invention can also be generated
using various phage display methods known in the art. In phage display
methods,
functional antibody domains are displayed on the surface of phage particles
which
carry the polynucleotide sequences encoding them. In a particular embodiment,
such
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99
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 phaQe
including fd and M 13 binding domains expressed from phage with Fab, Fv or
disulfide stabilized Fv antibody domains recombinantly fused to either the
phaae
gene III or Gene VIII protein. Examples of phage display methods that can be
used to
make the antibodies of the present invention include those disclosed in
Brinkman et
al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods
184:177-I 86 ( 1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (
1994); Persic
et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Patent Nos. 5,698,426; 5.223,409; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225;
5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by
reference in its entirety.
As described in the above references, after phage selection, the antibody
coding regions from the phage can be isolated and used to generate whole
antibodies,
including human antibodies, or any other desired antigen binding fragment, and
expressed in any desired host, including mammalian cells, insect cells, plant
cells,
yeast, and bacteria, e.g., as described in detail below. For example,
techniques to
recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed
using
methods known in the art such as those disclosed in PCT publication WO
92/22324;
Mullinax et al., BioTechniques 12(6):864-869 ( 1992); and Sawai et al., AJRI
34:26-
34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
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
CA 02364549 2001-08-27
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100
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 marine monoclonal
antibody and a human immunoglobulin constant region. Methods for producing
chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202
(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J.
Immunol.
Methods 125:191-202: U.S. Patent Nos. 5.807,715; 4,816,567; and 4,816397,
which
are incorporated herein by reference in their entirety. Humanized antibodies
are
antibody molecules from non-human species antibody that binds the desired
antigen
having one or more complementarity determining regions (CDRs) from the non-
human species and a framework regions from a human immunoglobulin molecule.
Often, framework residues in the human framework regions will be substituted
with
the corresponding residue from the CDR donor antibody to alter, preferably
improve,
antigen binding. These framework substitutions are identified by methods well
known in the art, e.g., by modeling of the interactions of the CDR and
framework
residues to identify framework residues important for antigen binding and
sequence
comparison to identify unusual framework residues at particular positions.
(See, e.g.,
Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (
1988),
which are incorporated herein by reference in their entireties.) Antibodies
can be
humanized using a variety of techniques known in the art including, for
example,
CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et
al.,
Protein Engineering 7(6):805-814 ( 1994); Roguska. et al., PNAS 91:969-973 (
1994)),
and chain shuffling (U.S. Patent No. 5,565,332).
Completely human antibodies are particularly desirable for therapeutic
treatment of human patients. Human antibodies can be made by a variety of
methods
known in the art including phage display methods described above using
antibody
libraries derived from human immunoglobulin sequences. See also, U.S. Patent
Nos.
4,444,887 and 4,716,11 l; and PCT publications WO 98/46645, WO 98/50433, WO
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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. Immunol. 13:65-93 ( 1995). For a
detailed discussion of this technology for producing human antibodies and
human
monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European
Patent No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425;
5,569,825;
5,661,016; 5,545,806; 5,814,318: 5,885,793; 5,916,771; and 5,939,598, which
are
incorporated by reference herein in their entirety. In addition, companies
such as
Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to
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provide human antibodies directed against a selected antigen Lisin~ 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 ID NO:Y.
The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the
nucleotide sequence of the antibody is known, a polynucleotide encoding the
antibody
may be assembled from chemically synthesized oligonucleotides (e.g., as
described
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103
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., 1998, Current Protocols in Molecular Biology,
John
Wiley & Sons, NY, which are both incorporated by reference herein in their
entireties ), to generate antibodies having a different amino acid sequence,
for
example to create amino acid substitutions, deletions, and/or insertions.
In a specific embodiment, the amino acid sequence of the heavy and/or light
chain variable domains may be inspected to identify the sequences of the
complementarity determining regions (CDRs) by methods that are well know in
the
art, e.g., by comparison to known amino acid sequences of other heavy and
light
chain variable regions to determine the regions of sequence hypervariability.
Using
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routine recombinant DNA techniques, one or more of the CDRs may be inserted
within framework I'eg10I1S, e.g., into human framework regions to humanize a
non-
human antibody. as described supra. The framework regions may be naturally
occurring or consensus framework regions, and preferably human framework
regions
(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of
human
framework regions). Preferably, the polynucleotide generated by the
combination of
the framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
substitutions may be made within the framework regions, and, preferably, the
amino
acid substitutions improve binding of the antibody to its antigen.
Additionally, such
methods may be used to make amino acid substitutions or deletions of one or
more
variable region cysteine residues participating in an intrachain disulfide
bond to
generate antibody molecules lacking one or more intrachain disulfide bonds.
Other
alterations to the polynucleotide are encompassed by the present invention and
within
the skill of the art.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberaer 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 (U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988);
Huston et
al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature
334:544-54 ( 1989)) can be adapted to produce single chain antibodies. Single
chain
antibodies are formed by linking the heavy and light chain fragments of the Fv
region
via an amino acid bridge, resulting in a single chain polypeptide. Techniques
for the
assembly of functional Fv fragments in E. coli may also be used (Skerra et
al.,
Science 242:1038- 1041 ( 1988)).
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IUD
Methods of Producing 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 or a single chain antibody of the invention), requires construction
of an
expression vector containing a polynucleotide that encodes the antibody. Once
a
polynucleotide encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably containing the heavy or light chain
variable
domain), of the invention has been obtained, the vector for the production of
the
antibody molecule may be produced by recombinant DNA technology using
techniques well known in the art. Thus, methods for preparing a protein by
expressing a polynucleotide containing an antibody encoding nucleotide
sequence are
described herein. Methods which are well known to those skilled in the art can
be
used to construct expression vectors containing antibody coding sequences and
appropriate transcriptional and translational control signals. These methods
include,
for example, in vitro recombinant DNA techniques, synthetic techniques, and 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
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
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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.SK promoter). Preferably, bacterial cells such as Escherichia
coli, .
and more preferably, eukaryotic cells, especially for the expression of whole
recombinant antibody molecule, are used for the expression of a recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
cells (CHO), in conjunction with a vector such as the major intermediate early
gene
promoter element from human cytomegalovirus is an effective expression system
for
antibodies (Foecking et al., Gene 45:101 ( 1986); Cockett et al.,
Bio/Technology 8:2
( 1990)).
In bacterial systems, a number of expression vectors may be advantageously
selected depending upon the use intended for the antibody molecule being
expressed.
For example, when a large quantity of such a protein is to be produced, for
the
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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 liQated individually into the vector in frame with the lac Z
coding
region so that a fusion protein is produced; pIN vectors (Inouye & Inouye,
Nucleic
Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-
5509 (1989)): and the like. pGEX vectors may also be used to express foreign
polypeptides as fusion proteins with alutathione 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 Qene 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 frugiperda cells. The antibody coding sequence may be cloned
individually into non-essential regions (for example the polyhedrin gene) of
the virus
and placed under control of an AcNPV promoter (for example the polyhedrin
promoter).
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
E1 or E3) will result in a recombinant virus that is viable and capable of
expressing
the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl.
Acad.
Sci. USA 81:355-359 ( 1984)). Specific initiation signals may also be required
for
efficient translation of inserted antibody coding sequences. These signals
include the
ATG initiation codon and adjacent sequences. Furthermore, the initiation codon
must be in phase with the reading frame of the desired coding sequence to
ensure
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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
form foci which in turn can be cloned and expanded into cell lines. This
method may
advantageously be used to engineer cell lines which express the antibody
molecule.
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l0y
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 (Wialer 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 6-
418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 ( 1993); Mulligan,
Science
260:926-932 ( 1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
(1993); May, 1993, TIB TECH 11(5):155-215); and hygro, which confers
resistance
to hygromycin (Santerre et al., Gene 30:147 ( 1984)). Methods commonly known
in
the art of recombinant DNA technology may be routinely applied to select the
desired
recombinant clone, and such methods are described, for example, in Ausubel et
al.
(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993);
Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols
in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.
Biol. 150:1 ( 1981 ), which are incorporated by reference herein in their
entireties.
The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see Bebbington and Hentschel, The use of vectors
based
on gene amplification for the expression of cloned genes in mammalian cells in
DNA
cloning, Vol.3. (Academic Press, New York, 1987)). When a marker in the vector
system expressing antibody is amplifiable, increase in the level of inhibitor
present in
culture of host cell will increase the number of copies of the marker gene.
Since the
amplified region is associated with the antibody gene, production of the
antibody will
also increase (Grouse et al., Mol. Cell. Biol. 3:257 (1983)).
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110
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 purified by any
method known in the art for purification of an immunoglobulin molecule, for
example. by chromatography (e.g.. ion exchange, affinity, particularly by
affinity for
the specific antigen after Protein A, and sizing column chromatography),
centrifugation, differential solubility, or by any other standard technique
for the
purification of proteins. In addition. the 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
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.,
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Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et
al., Immunol. Lett. 39:91-99 (1994): U.S. Patent 5,474,981; Gillies et al.,
PNAS
89:1428-1432 ( 1992); Fell et al., J. Immunol. 146:2446-2452(1991), which are
incorporated by reference in their entireties.
The present invention further includes compositions comprising the
polypeptides of the present invention fused or conjugated to antibody domains
other
than the variable regions. For example. the polypeptides of the present
invention may
be fused or conjugated to an antibody Fc region, or portion thereof. The
antibody
portion fused to a polypeptide of the present invention may comprise the
constant
region, hinge region, CH 1 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
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
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disulfide- linked dimeric structures (due to the IaG) may also be more
efficient in
bindin~~ 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
emission tomographies, and nonradioactive paramagnetic metal ions. The
detectable
substance may be coupled or conjugated either directly to the antibody (or
fragment
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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, 111In
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, vineristine, vinblastine, colchicin, doxorubicin,
daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof. Therapeutic agents include, but are
not
limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-
thioguanine,
cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine,
thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),
cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and
cis-
dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents (e.g., vincristine and vinblastine).
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
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or polypeptide possessing a desired biological activity. Such proteins may
include,
for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria
toxin; a protein such as tumor necrosis factor, a-interferon, >3-interferon.
nerve growth
factor, platelet derived growth factor. tissue plasminogen activator, an
apoptotic
agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO
97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand
(Takahashi et al., Int. Inim~rfaol., 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. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For
Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),
pp.
623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic
Agents
In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis,
Results,
And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy,
Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al.,
"The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol.
Rev. 62:119-58 (1982).
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.
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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.
Immunophenotyping
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 techniques
allow for
the screening of hematopoietic stem and progenitor cells capable of undergoing
proliferation and/or differentiation, as might be found in human umbilical
cord blood:
Assays For Antibody 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,
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
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known in the art (see, e.g.. Ausubel et al, eds, 1994. Current Protocols in
Molecular
Biology, Vol. 1, John Wiley & Sons. Inc.. New York, which is incorporated by
reference herein in its entirety). Exemplary immunoassays are described
briefly
below (but are not intended by way of limitation).
Immunoprecipitation protocols generally comprise lysing a population of cells
in a lysis buffer such as RIPA buffer ( 190 NP-40 or Triton X- 100, 19o sodium
deoxycholate, 0.1 % SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 190
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 town 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
alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in
blocking
buffer, washing the membrane in wash buffer, and detecting the presence of the
antigen. One of skill in the art would be knowledgeable as to the parameters
that can
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be modified to increase the signal detected and to reduce the background
noise. For
further discussion regarding western blot protocols see, e.g., Ausubel et al,
eds, 1994,
Current Protocols in Molecular Biology. Vol. l, John Wiley & Sons, Inc., New
York
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 further discussion regarding ELISAs see, e.g., Ausubel
et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. l, John Wiley & Sons,
Inc.,
New York at 11.2.1.
The binding affinity of an antibody to an antigen and the off-rate of an
antibody-antigen interaction can be determined by competitive binding assays.
One
example of a competitive binding assay is a radioimmunoassay comprising the
incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest
in the
presence of increasing amounts of unlabeled antigen, and the detection of the
antibody bound to the labeled antigen. The affinity of the antibody of
interest for a
particular antigen and the binding off-rates can be determined from the data
by
scatchard plot analysis. Competition with a second antibody can also be
determined
using radioimmunoassays. In this case, the antigen is incubated with antibody
of
interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence
of
increasing amounts of an unlabeled second antibody.
Therapeutic Uses
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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
andlor activity of a polypeptide of the invention, including, but not limited
to, any
one or more of the diseases, disorders, or conditions described herein. The
treatment
and/or prevention of diseases, disorders, or conditions associated with
aberrant
expression and/or activity of a polypeptide of the invention includes, but is
not
limited to, alleviating symptoms associated with those diseases, disorders or
conditions. Antibodies of the invention may be provided in pharmaceutically
acceptable compositions as known in the art or as described herein.
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 IL-7), for
example, which
serve to increase the number or activity of effector cells which interact with
the
antibodies.
The antibodies of the invention may be administered alone or in combination
with other types of treatments (e.g., radiation therapy, chemotherapy,
hormonal
therapy, immunotherapy and anti-tumor agents). Generally, administration of
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119
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-' M, 10-' M, 5 X 10-' M, 10-~ M,
5 X 10-~
M, 10-~ M, 5 X 10-' M, 10-5 M, 5 X 10-° M, 10-6 M, 5 X 10-' M, 10-' M,
5 X 10-x M,
10-R M, 5 X 10-'' M, 10~'' M, 5 X 10-'° M, 10-'° M, 5 X 10-" M,
10-" M, 5 X 10-'' M, 10-
'' M, 5 X 10-'' M, 10-'~ M, 5 X 10-'~' M, 10-'~ M, 5 X 10-'' M, and 10-'' M.
Gene 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 subject of an expressed or expressible
nucleic
acid. In this embodiment of the invention, the nucleic acids produce their
encoded
protein that mediates a therapeutic effect.
Any of the methods for gene therapy available in the art can be used according
to the present invention. Exemplary methods are described below.
For general reviews of the methods of gene therapy, see Goldspiel et al.,
Clinical Pharmacy 12:488-505 ( 1993); Wu and Wu, Biotherapy 3:87-95 ( 1991 );
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 ( 1993); Mulligan,
Science
260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217
( 1993); May, TIBTECH 11 (5):155-215 ( 1993). Methods commonly known in the
art
of recombinant DNA technology which can be used are described in Ausubel et
al.
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120
(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
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
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administering it in linkage to a ligand subject to receptor-mediated
endocytosis (see,
e.g., Wu and Wu, J. Biol. Chem. 262:4=129-4432 ( 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 fusoaenic
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 are cloned into one or more vectors,
which
facilitates delivery of the gene into a patient. More detail about retroviral
vectors can
be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the
use of a
retroviral vector to deliver the mdrl gene to hematopoietic stem cells in
order to
make the stem cells more resistant to chemotherapy. Other references
illustrating the .
use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-
651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg,
Human
Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in
Genetics
and Devel. 3:110-114 ( 1993).
Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory
epithelia. Adenoviruses naturally infect respiratory epithelia where they
cause a mild
disease. Other targets for adenovirus-based delivery systems are liver, the
central
nervous system, endothelial cells, and muscle. Adenoviruses have the advantage
of
being capable of infecting non-dividing cells. Kozarsky and Wilson, Current
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Opinion in Genetics and Development 3:499-503 (1993) present a review of
adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 ( 1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory
epithelia of rhesus monkeys. Other instances of the use of adenoviruses in
gene
therapy can be found in Rosenfeld et al., Science 252:431-434 ( 1991 ):
Rosenfeld et
al., Cell 68:143- 155 ( 1992); Mastrangeli et al., J. Clin. Invest. 91:225-234
( 1993);
PCT Publication W094/12649; and Wang, et al., Gene Therapy 2:77-783 ( 1995).
In
a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene therapy
(Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 ( 1993); U.S. Patent No.
5,436,146).
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 ( 1980 and may
be
used in accordance with the present invention, provided that the necessary
developmental and physiological functions of the recipient cells are not
disrupted.
The technique should provide for the stable transfer of the nucleic acid to
the cell, so
that the nucleic acid is expressible by the cell and preferably heritable and
expressible by its cell progeny.
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The resulting recombinant cells can be delivered to a patient by various
methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or
progenitor cells) are preferably administered intravenously. The amount of
cells
envisioned for use depends on the desired effect, patient state, etc., and can
be
determined by one skilled in the art.
Cells into which a nucleic acid can be introduced for purposes of gene therapy
encompass any desired, available cell type, and include but are not limited to
epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes;
blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages,
neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or
progenitor
cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained
from bone
marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
In a preferred embodiment, the cell used for gene therapy is autologous to the
patient.
In an embodiment in which recombinant cells are used in gene therapy,
nucleic acid sequences encoding an antibody are introduced into the cells such
that
they 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)).
In a specific embodiment, the nucleic acid to be introduced for purposes of
gene therapy comprises an inducible promoter operably linked to the coding
region,
such that expression of the nucleic acid is controllable by controlling the
presence or
absence of the appropriate inducer of transcription. Demonstration of
Therapeutic or
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
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12-~
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.
Therapeutic/Prophylactic 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
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
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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 Langer, Science 249:1527-1533 (1990);
Treat et
al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-
Berestein
and Fidler (eds.), Liss, New York, pp. 353- 365 ( 1989); Lopez-Berestein,
ibid., pp.
317-327; see generally ibid.)
In yet another embodiment, the compound or composition can be delivered in
a controlled release system. In one embodiment, a pump may be used (see
Langer,
supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 ( 1987); Buchwald et al.,
Surgery
88:507 ( 1980); Saudek et al., N. Engl. J. Med. 321:574 ( 1989)). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida
(1974);
Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen
and
Ball (eds.), Wiley, New York ( 1984); Ranger and Peppas, J., Macromol. Sci.
Rev.
Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985);
During
et al., Ann. Neurol. 25:351 ( 1989); Howard et al., J.Neurosurg. 71:10 (
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
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(see, e.g., Goodson, in Medical Applications of Controlled Release, supra,
vol. 2, pp.
115-138 (1984)).
Other controlled release systems are discussed in the review by Linger
(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. Acid. Sci. USA 88:1864-1868 (1991)), etc.
Alternatively, a
nucleic acid can be introduced intracellularly and incorporated within host
cell DNA
for expression, by homologous recombination.
The present invention also provides pharmaceutical compositions. Such
compositions comprise a therapeutically effective amount of a compound, and a
pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the
Federal
or a state government or listed in the U.S. Pharmacopeia or other generally
recognized
pharmacopeia for use in animals, and more particularly in humans. The term
"carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic.
is administered. Such pharmaceutical carriers can be sterile liquids, such as
water
and oils, including those of petroleum, animal, vegetable or synthetic origin,
such as
peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a
preferred
carrier when the pharmaceutical composition is administered intravenously.
Saline
solutions and aqueous dextrose and glycerol solutions can also be employed as
liquid
carriers, particularly for injectable solutions. Suitable pharmaceutical
excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. The composition, if
desired,
can also contain minor amounts of wetting or emulsifying agents, or pH
buffering
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agents. These compositions can take the form of solutions, suspensions,
emulsion,
tablets, pills, capsules, powders, sustained-release formulations and the
like. The
composition can be formulated as a suppository, with traditional binders and
carriers
such as triglycerides. Oral formulation can include standard carriers such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences"
by
E.W. Martin. Such compositions will contain a therapeutically effective amount
of
the compound, preferably in purified form, together with a suitable amount of
carrier
so as to provide the form for proper administration to the patient. The
formulation
should suit the mode of administration.
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 for 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
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
and those
formed with canons 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
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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 mg/kg to 10 mg/kg of the patient's body weight. Generally, human
antibodies have a longer half-life within the human body than antibodies from
other
species due to the immune response to the foreign polypeptides. Thus, lower
dosages
of human antibodies and less frequent administration is often possible.
Further, the
dosage and frequency of administration of antibodies of the invention may be
reduced by enhancing uptake and tissue penetration (e.g., into the brain) of
the
antibodies by modifications such as, for example, lipidation.
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
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 and/or disorders 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
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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, 121I), carbon (14C), sulfur (35S),
tritium (3H),
indium ( 1 l2In), and technetium (99Tc); luminescent labels, such as luminol;
and
fluorescent labels, such as fluorescein and rhodamine, and biotin.
One aspect of the invention is the detection and diagnosis of a disease or
disorder associated with aberrant expression of a polypeptide of interest in
an animal,
preferably a mammal and most preferably a human. In one embodiment, diagnosis
comprises: a) administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject an effective amount of a labeled molecule
which
specifically binds to the polypeptide of interest; b) waiting for a time
interval
following the administering for permitting the labeled molecule to
preferentially
concentrate at sites in the subject where the polypeptide is expressed (and
for
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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.
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
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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. In
one embodiment, a kit comprises an antibody of the invention, preferably a
purified
antibody, in one or more containers. In a specific embodiment, the kits of the
present
invention contain a substantially isolated polypeptide comprising an epitope
which is
specifically immunoreactive with an antibody included in the kit. Preferably,
the kits
of the present invention further comprise a control antibody which does not
react with
the polypeptide of interest. In another specific embodiment, the kits of the
present
invention contain a means for detecting the binding of an antibody to a
polypeptide of
interest (e.g., the antibody may be conjugated to a detectable substrate such
as a
fluorescent compound, an enzymatic substrate, a radioactive compound or a
luminescent compound, or a second antibody which recognizes the first antibody
may
be conjugated to a detectable substrate).
In another specific embodiment of the present invention, the kit is a
diagnostic
kit for use in screening serum containing antibodies specific against
proliferative
andlor cancerous polynucleotides and polypeptides. Such a kit may include a
control
antibody that does not react with the polypeptide of interest. Such a kit may
include a
substantially isolated polypeptide antigen comprising an epitope which is
specifically
immunoreactive with at least one anti-polypeptide antigen antibody. Further,
such a
kit includes means for detecting the binding of said antibody to the antigen
(e.g., the
antibody may be conjugated to a fluorescent compound such as fluorescein or
rhodamine which can be detected by flow cytometry). In specific embodiments,
the
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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
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.
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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
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 (CHI, CH2, CH3, and
any
combination thereof, including both entire domains and portions thereof),
resulting in
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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).)
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,
for
example, improved pharmacokinetic properties. (EP-A 0232 262.) Alternatively,
deleting the Fc part after the fusion protein has been expressed, detected,
and purified,
would be desired. For example, the Fc portion may hinder therapy and diagnosis
if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for
example, human proteins, such as hIL-5. have been fused with Fc portions for
the
purpose of high-throughput screening assays to identify antagonists of hIL-5.
(See,
D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et
al., J. Biol.
Chem. 270:9459-9471 ( 1995).)
Moreover, the polypeptides of the present invention can be fused to marker
sequences, such as a peptide which facilitates purification of the fused
polypeptide.
In preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton
Avenue,
Chatsworth, CA, 9131 I), 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).)
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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 vines, 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,
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;
insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such
as
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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, pRIT~ available from Pharmacia Biotech,
Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI
and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available
from Pharmacia. 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, transduction, infection, or other methods. Such
methods
are described in many standard laboratory manuals, such as Davis et al., Basic
Methods In Molecular Biology (1986). It is specifically contemplated that the
1 _5 polypeptides of the present invention may in fact be expressed by a host
cell lacking a
recombinant vector.
A polypeptide of this invention can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or canon 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-
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mediated processes. Thus, it is well known in the art that the N-terminal
methionine
encoded by the translation initiation colon generally is removed with high
efficiency
from any protein after translation in all eukaryotic cells. While the N-
terminal
methionine on most proteins also is efficiently removed in most prokaryotes,
for some
proteins, this prokaryotic removal process is inefficient, depending on the
nature of
the amino acid to which the N-terminal methionine is covalently linked.
In addition to encompassing host cells containing the vector constructs
discussed herein, the invention also encompasses primary, secondary, and
immortalized host cells of vertebrate origin, particularly mammalian origin,
that have
been engineered to delete or replace endogenous genetic material (e.g., coding
sequence), and/or to include genetic material (e.g., heterologous
polynucleotide
sequences) that is operably associated with the polynucleotides of the
invention, and
which activates, alters, and/or amplifies endogenous polynucleotides. For
example,
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,
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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
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, NaBH~; acetylation, formylation, oxidation, reduction;
metabolic
synthesis in the presence of tunicamycin; etc.
Additional post-translational modifications encompassed by the invention
include, for example, e.g., N-linked or O-linked carbohydrate chains,
processing of
N-terminal or C-terminal ends), attachment of chemical moieties to the amino
acid
backbone, chemical modifications of N-linked or O-linked carbohydrate chains,
and
addition or deletion of an N-terminal methionine residue as a result of
procaryotic
host cell expression. The polypeptides may also be modified with a detectable
label,
such as an enzymatic, fluorescent, isotopic or affinity label to allow for
detection and
isolation of the protein.
Also provided by the invention are chemically modified derivatives of the
polypeptides of the invention which may provide additional advantages such as
increased solubility, stability and circulating time of the polypeptide, or
decreased
immunogenicity (see U.S. Patent NO: 4,179,337). The chemical moieties for
derivitization may be selected from water soluble polymers such as
polyethylene
glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose,
dextran, polyvinyl alcohol and the like. The polypeptides may be modified at
random
positions within the molecule, or at predetermined positions within the
molecule and
may include one, two, three or more attached chemical moieties.
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The polymer may be of any molecul,.n- weight, and may be branched or
unbranched. For polyethylene Glycol, the preferred molecular weight is between
about 1 kDa and about 100 kDa (the term "about" indicating that in
preparations of
polyethylene glycol, some molecules will weigh more, some less, than the
stated
molecular weight) for ease in handling and manufacturing. Other sizes may be
used,
depending on the desired therapeutic profile (e.g., the duration of sustained
release
desired, the effects, if any on biological activity, the ease in handling, the
degree or
lack of antigenicity and other known effects of the polyethylene glycol to a
therapeutic protein or analog).
The polyethylene glycol molecules (or other chemical moieties) should be
attached to the protein with consideration of effects on functional or
antigenic
domains of the protein. There are a number of attachment methods available to
those
skilled in the art, e.g., EP 0 401 384, herein incorporated by reference
(coupling PEG
to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting
pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol
may
be covalently bound through amino acid residues via a reactive group, such as,
a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene glycol molecule may be bound. The amino acid residues having a
free
amino group may include lysine residues and the N-terminal amino acid
residues;
those having a free carboxyl group may include aspartic acid residues glutamic
acid
residues and the C-terminal amino acid residue. Sulfhydryl groups may also be
used
as a reactive group for attaching the polyethylene glycol molecules. Preferred
for
therapeutic purposes is attachment at an amino group, such as attachment at
the
N-terminus or lysine group.
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
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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.
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.
Multimers encompassed by the invention may be homomers or heteromers.
As used herein, the term homomer, refers to a multimer containing only
polypeptides
corresponding to the amino acid sequence of SEQ ID NO:Y or encoded by the cDNA
contained in a deposited clone (including fragments, variants, splice
variants, and
fusion proteins, corresponding to these polypeptides as described herein).
These
homomers may contain polypeptides having identical or different amino acid
sequences. In a specific embodiment, a homomer of the invention is a multimer
containing only polypeptides having an identical amino acid sequence. In
another
specific embodiment, a homomer of the invention is a multimer containing
polypeptides having different amino acid sequences. In specific embodiments,
the
multimer of the invention is a homodimer (e.g., containing polypeptides having
identical or different amino acid sequences) or a homotrimer (e.g., containing
polypeptides having identical and/or different amino acid sequences). In
additional
embodiments, the homomeric multimer of the invention is at least a homodimer,
at
least a homotrimer, or at least a homotetramer.
As used herein, the term heteromer refers to a multimer containing one or
more heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to
the polypeptides of the invention. In a specific embodiment, the multimer of
the
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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, heterotrimer s or heterotetramers, are
formed
when polypeptides of the invention contact antibodies to the polypeptides of
the
invention (including antibodies to the heterologous polypeptide sequence in a
fusion
protein of the invention) in solution. In other embodiments, multimers of the
invention are formed by covalent associations with and/or between the
polypeptides
of the invention. Such covalent associations may involve one or more amino
acid
residues contained in the polypeptide sequence ( e.g., that recited in the
sequence
listing, or contained in the polypeptide encoded by a deposited clone). In one
instance, the covalent associations are cross-linking between cysteine
residues located
within the polypeptide sequences which interact in the native (i.e., naturally
occurring) polypeptide. In another instance, the covalent associations are the
consequence of chemical or recombinant manipulation. Alternatively, such
covalent
associations may involve one or more amino acid residues contained in the
heterologous polypeptide sequence in a fusion protein of the invention.
In one example, covalent associations are between the heterologous sequence
contained in a fusion protein of the invention (see, e.g., US Patent Number
5,478,925). In a specific example, the covalent associations are between the
heterologous sequence contained in an Fc fusion protein of the invention (as
described herein). In another specific example, covalent associations of
fusion
proteins of the invention are between heterologous polypeptide sequence from
another protein that is capable of forming covalently associated multimers,
such as for
example, oseteoprotegerin (see, e.g., International Publication NO: WO
98/49305, the
contents of which are herein incorporated by reference in its entirety). In
another
embodiment, two or more polypeptides of the invention are joined through
peptide
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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)j, and have since been found in a variety of different
proteins. Among the known leucine zippers are naturally occurring peptides and
derivatives thereof that dimerize or trimerize. Examples of leucine zipper
domains
suitable for producing soluble multimeric proteins of the invention are those
described
in PCT application WO 94/10308, hereby incorporated by reference. Recombinant
fusion proteins comprising a polypeptide of the invention fused to a
polypeptide
sequence that dimerizes or trimerizes in solution are expressed in suitable
host cells,
and the resulting soluble multimeric fusion protein is recovered from the
culture
supernatant using techniques known in the art.
Trimeric polypeptides of the invention may offer the advantage of enhanced
biological activity. Preferred leucine zipper moieties and isoleucine moieties
are
those that preferentially form trimers. One example is a leucine zipper
derived from
lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters
344:191;
(1994)) and in U.S. patent application Ser. No. 08/446,922, hereby
incorporated by
reference. Other peptides derived from naturally occurring trimeric proteins
may be
employed in preparing trimeric polypeptides of the invention.
In another example, proteins of the invention are associated by interactions
between FlagO polypeptide sequence contained in fusion proteins of the
invention
containing FlagO polypeptide seuqence. In a further embodiment, associations
proteins of the invention are associated by interactions between heterologous
polypeptide sequence contained in FlagO fusion proteins of the invention and
anti-
Flag~ antibody.
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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 cvsteine
residues
located within the sequence of the polypeptides desired to be contained in the
multimer (see, e.g., US Patent Number 5.478,925, which is herein incorporated
by
reference in its entirety). Further, polypeptides of the invention may be
routinely
modified by the addition of cysteine or biotin to the C terminus or N-terminus
of the
polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally,
techniques known in the art may be applied to generate liposomes containing
the
polypeptide components desired to be contained in the multimer of the
invention (see,
e.g., US Patent Number 5,478,925, which is herein incorporated by reference in
its
entirety).
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
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14-l
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
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, and preselection by hybridization to construct
chromosome specific-cDNA libraries.
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
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Verma et al., "Human Chromosomes: a Manual of Basic Techniques." Peraamon
Press, New York ( I )88).
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). Preferred polynucleotides
correspond to the noncoding regions of the cDNAs because the coding sequences
are
more likely conserved within gene families, thus increasing the chance of
cross
hybridization during chromosomal mapping.
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.
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Thus, the invention also provides a diagnostic method useful during diagnosis
of a disorder, involvin~T 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 least one polynucleotide
probe
containing a nucleotide sequence that will specifically hybridize with a
polynucleotide of the present invention and a suitable container. In a
specific
embodiment, the kit includes two polynucleotide probes defining an internal
region of
the polynucleotide of the present invention, where each probe has one strand
containing a 31'mer-end internal to the region. In a further embodiment, the
probes
may be useful as primers 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
polypeptide in a first biological sample either directly (e.g., by determining
or
estimating absolute protein level or mRNA level) or relatively (e.g., by
comparing to
the polypeptide level or mRNA level in a second biological sample).
Preferably, the
polypeptide level or mRNA level in the first biological sample is measured or
estimated and compared to a standard polypeptide level or mRNA level, the
standard
being taken from a second biological sample obtained from an individual not
having
the disorder or being determined by averaging levels from a population of
individuals
not having a disorder. As will be appreciated in the art, once a standard
polypeptide
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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.
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
polynueleotides 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
2S 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,
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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 i, probably because
there is no electrostatic repulsion between the two strands, and also the
polyamide
backbone is more flexible. Because of this, PNA/DNA duplexes bind under a
wider
range of stringency conditions than DNA/DNA duplexes, making it easier to
perform
multiplex hybridization. Smaller probes can be used than with DNA due to the
strong
binding. In addition, it is more likely that single base mismatches can be
determined
with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer
lowers the melting point (T<sub>m</sub>) by 8°-20° C, vs. 4°-
16° C for the DNA/DNA 15-
mer duplex. Also, the absence of charge groups in PNA means that hybridization
can
be done at low ionic strengths and reduce possible interference by salt during
the
analysis.
The present invention is useful for detecting cancer in 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 disorders are often associated with
inappropriate
activation of proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute
Leukemia: Molecular Genetics and Viral Oncology," in Neoplastic Diseases of
the
Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now
believed to result from the qualitative alteration of a normal cellular gene
product, or
from the quantitative modification of gene expression by insertion into the
chromosome of a viral sequence, by chromosomal translocation of a gene to a
more
actively transcribed region, or by some other mechanism. (Gelmann et al.,
supra) It
is likely that mutated or altered expression of specific genes is involved in
the
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pathogenesis of some leukemias, among other tissues and cell types. (Gelmann
et al.,
supra) Indeed, the human counterparts of the oncoaenes involved in some animal
neoplasias have been amplified or translocated in some cases of human leukemia
and
carcinoma. (Gelmann et al., supra)
For example, c-myc expression is highly amplified in the non-lymphocytic
leukemia
cell line HL-60. When HL-60 cells are chemically induced to stop
proliferation, the
level of c-myc is found to be downregulated. (International Publication Number
WO
91/15580) However, it has been shown that exposure of HL-60 cells to a DNA
construct that is complementary to the 5' end of c-myc or c-myb blocks
translation of
the corresponding mRNAs which downregulates expression of the c-myc or c-myb
proteins and causes arrest of cell proliferation and differentiation of the
treated cells.
(International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl.
Acad.
Sci. 85:1028 ( 1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 ( 1989)).
However,
the skilled artisan would appreciate the present invention's usefulness would
not be
limited to treatment of proliferative disorders 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
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150
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 disease.
Polynucleotides of the present invention are also useful in Gene therapy. One
goal of gene therapy is to insert a normal gene into an organism having a
defective
gene, in an effort to correct the genetic defect. The polynucleotides
disclosed in the
present invention offer a means of targeting such genetic defects in a highly
accurate
manner. Another goal is to insert a new gene that was not present in the host
genome,
thereby producing a new trait in the host cell.
The polynucleotides are also useful for identifying individuals from minute
biological samples. The United States military, for example, is considering
the use of
restriction fragment length polymorphism (RFLP) for identification of its
personnel.
In this technique, an individual's genomic DNA is digested with one or more
restriction enzymes, and probed on a Southern blot to yield unique bands for
identifying personnel. This method does not suffer from the current
limitations of
"Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The polynucleotides of the present invention can be used as
additional DNA
markers for RFLP.
The polynucleotides of the present invention can also be used as an
alternative
to RFLP, by determining the actual base-by-base DNA sequence of selected
portions
of an individual's genome. These sequences can be used to prepare PCR primers
for.
amplifying and isolating such selected DNA, which can then be sequenced. Using
this technique, individuals can be identified because each individual will
have a
unique set of DNA sequences. Once an unique ID database is established for an
individual, positive identification of that individual, living or dead, can be
made from
extremely small tissue samples.
Forensic biology also benefits from using DNA-based identification
techniques as disclosed herein. DNA sequences taken from very small biological
samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood,
saliva, semen,
synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or
surfactant,urine,fecal matter, etc., can be amplified using PCR. In one prior
art
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technique, gene sequences amplified froth polymorphic loci, such as DQa class
II
HLA gene, are used in forensic biology to identify individuals. (Erlich, H.,
PCR
Technology, Freeman and Co. ( 1992).) Once these specific polymorphic loci are
amplified, they are digested with one or more restriction enzymes, yielding an
identifying set of bands on a Southern blot probed with DNA corresponding to
the
DQa class II HLA gene. Similarly, polynucleotides of the present invention can
be
used as polymorphic markers for forensic purposes.
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 Polv~eptides
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. 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
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in the art and include enzyme labels, such as, glucose oxidise, 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-
radiography, suitable labels include radioisotopes such as barium or cesium,
which
emit detectable radiation but are not overtly harmful to the subject. Suitable
markers
for NMR and ESR include those with a detectable characteristic spin, such as
deuterium, which may be incorporated into the antibody by labeling of
nutrients for
the relevant hybridoma.
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 l2In, 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 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).)
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
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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.
Moreover, polypeptides of the present invention can be used to treat 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 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 Ther~v Methods
Another aspect of the present invention is to Gene therapy methods for
treating
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 viva, with the engineered cells then being
provided
to a patient to be treated with the polypeptide. Such methods are well-known
in the
art. For example, see Belldegrun et al., J. Natl. Cancer Inst., 85:207-216
(1993);
Ferrantini et al., Cancer Research, 53:107-1112 ( 1993); Ferrantini et al., J.
Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-
229
( 1995); Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et
al.,
Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy 4:1246-
1255
( 1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38 ( 1996)), which are
herein
incorporated by reference. In one embodiment, the cells which are engineered
are
arterial cells. The arterial cells may be reintroduced into the patient
through direct
injection to the artery, the tissues surrounding the artery, or through
catheter injection.
As discussed in more detail below, the polynucleotide constructs can be
delivered by any method that delivers injectable materials to the cells of an
animal,
such as, injection into the interstitial space of tissues (heart, muscle,
skin, lung, liver,
and the like). The polynucleotide constructs may be delivered in a
pharmaceutically
acceptable liquid or aqueous carrier.
In one embodiment, the polynucleotide of the invention is delivered as a naked
polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences
that are free from any delivery vehicle that acts to assist, promote or
facilitate entry
into the cell, including viral sequences, viral particles, liposome
formulations,
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lipofectin or precipitating agents and the like. However, the polynucleotides
of the
invention can also be delivered in liposome formulations and lipofectin
formulations
and the like can be prepared by methods well known to those skilled in the
art. Such
methods are described, for example, in U.S. Patent Nos. 5,593,972, 5.589.466,
and
5,580,859. which are herein incorporated by reference.
The polynucleotide vector constructs of the invention used in the Gene
therapy method are preferably constructs that will not integrate into the host
genome
nor will they contain sequences that allow for replication. Appropriate
vectors
include pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene;
pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pEFI/V5,
pcDNA3.1, and pRc/CMV2 available from Invitroaen. 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 major late promoter; or
heterologous promoters, such as the cytomegalovirus (CMV) promoter; the
respiratory syncytial virus (RSV) promoter; inducible promoters, such as the
MMT
promoter, the metallothionein promoter; heat shock promoters; the albumin
promoter;
the ApoAI promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-
actin
promoter; and human growth hormone promoters. The promoter also may be the
native promoter for the polynucleotides of the invention.
Unlike other gene therapy techniques, one major advantage of introducing
naked nucleic acid sequences into target cells is the transitory nature of the
polynucleotide synthesis in the cells. Studies have 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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156
fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues,
elastic
fibers in the walls of vessels or chambers, collagen fibers of fibrous
tissues, or that
same matrix within connective tissue ensheathing muscle cells or in the
lacunae of
bone. It is similarly the space occupied by the plasma of the circulation and
the lymph
fluid of the lymphatic channels. Delivery to the interstitial space of muscle
tissue is
preferred for the reasons discussed below. They may be conveniently delivered
by
injection into the tissues comprising these cells. They are preferably
delivered to and
expressed in persistent, non-dividing cells which are differentiated, although
delivery
and expression may be achieved in non-differentiated or less completely
differentiated
cells, such as. for example, stem cells of blood or skin fibroblasts. In vivo
muscle cells
are particularly competent in their ability to take up and express
polynucleotides.
For the nakedne~cleic 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 0.00 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 injection
into the interstitial space of tissues. However, other parenteral routes may
also be
used, such as, inhalation of an aerosol formulation particularly for delivery
to lungs or
bronchial tissues, throat or mucous membranes of the nose. In addition, naked
DNA
constructs can be delivered to arteries during angioplasty by the catheter
used in the
procedure.
The naked polynucleotides are delivered by any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, and so-called "gene
guns". These
delivery methods are known in the art.
The constructs may also be delivered with delivery vehicles such as viral
sequences, viral particles, liposome formulations, lipofectin, precipitating
agents, etc.
Such methods of delivery are known in the art.
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1>7
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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15~
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 overnight and
is
hydrated the following day with deionized water. The sample is then sonicated
for 2
hours in a capped vial, using a Heat Systems model 350 sonicator equipped with
an
inverted cup (bath type) probe at the maximum setting while the bath is
circulated at
15EC. Alternatively, negatively charged vesicles can be prepared without
sonication
to produce multilamellar vesicles or by extrusion through nucleopore membranes
to
produce unilamellar vesicles of discrete size. Other methods are known and
available
to those of skill in the art.
The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar
vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred.
The various liposome-nucleic acid complexes are prepared using methods well
known
in the art. See, e.g., Straubinger et al., Methods of Immunology , 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. .
SUVs are prepared by extended sonication of MLVs to produce a homogeneous
population of unilamellar liposomes. The material to be entrapped is added to
a
suspension of preformed MLVs and then sonicated. When using liposomes
containing
cationic lipids, the dried lipid film is resuspended in an appropriate
solution such as
sterile water or an isotonic buffer solution such as 10 mM Tris/NaCI,
sonicated, and
then the preformed liposomes are mixed directly with the DNA. The liposome and
DNA form a very stable complex due to binding of the positively charged
liposomes
to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are
prepared by a number of methods, well known in the art. Commonly used methods
include Ca'+-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta,
CA 02364549 2001-08-27
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159
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., 255:1043 I (
1980);
Szoka et al., Proc. Natl. Acad. Sci. USA . 75:145 ( 1978); Sehaefer-Ridder et
al.,
Science, 215:166 (1982)), which are herein incorporated by reference.
Generally, the ratio of DNA to liposomes will be from about 10: I to about
1:10. Preferably, the ration will be from about 5: I 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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160
means known in the art. Such means include, but are not limited to,
electroporation,
the use of liposomes, and CaPO~ precipitation. In one alternative, the
retroviral
plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and
then
administered to a host.
The producer cell line generates infectious retroviral vector particles which
include polynucleotide encoding polypeptides of the invention. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or 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
mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines
for
many years with an excellent safety profile (Schwartzet al., Am. Rev. Respir.
Dis.,
109:233-238 ( 1974)). Finally, adenovirus mediated gene transfer has been
demonstrated in a number of instances including transfer of alpha-1-
antitrypsin and
CFTR to the lungs of cotton rats (Rosenfeld et al.,Science , 252:431-434
(1991);
Rosenfeld et al., Cell, 68:143-155 ( 1992)). Furthermore, extensive studies to
attempt
to establish adenovirus as a causative agent in human cancer were uniformly
negative
(Green et al. Proc. Natl. Acad. Sci. USA , 76:6606 (1979)).
Suitable adenoviral vectors useful in the present invention are described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel., 3:499-X03 ( 1993);
Rosenfeld et al., Cell , 68:143-155 ( 1992); Engelhardt et al., Human Genet.
Ther.,
4:759-769 (1993); Yang et al., Nature Genet., 7:362-369 (1994); Wilson et al.,
Nature , 365:691-692 (1993); and U.S. Patent NO: 5,652,224, which are herein
incorporated by reference. For example, the adenovirus vector Ad2 is useful
and can
be grown in human 293 cells. These cells contain the E1 region of adenovirus
and
constitutively express Ela and Elb, which complement the defective
adenoviruses by
providing the products of the genes deleted from the vector. In addition to
Ad2, other
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varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are also useful in the
present
invention.
Preferably, the adenoviruses used in the present invention are replication
deficient. Replication deficient adenoviruses require the aid of a helper
virus and/or
packaging cell line to form infectious particles. The resulting virus is
capable of
infecting cells and can express a polynucleotide of interest which is operably
linked to
a promoter, but cannot replicate in most cells. Replication deficient
adenoviruses
may be deleted in one or more of all or a portion of the following Genes: Ela,
Elb,
E3, E4, E2a, or L 1 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 in
vivo. The transduced cells will contain the polynucleotide construct
integrated into its
genome, and will express the desired gene product.
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16?
Another method of Gene therapy involves operably associating heterologous
control regions and endogenous polynucleotide sequences (e.g. encodin~ the
polypeptide sequence of interest) via homologous recombination (see, e.Q .
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 are
described in more detail below.
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The promoter-targeting sequence construct is taken up by cells. Homologous
recombination between the construct and the endogenous sequence takes place,
such
that an endogenous sequence is placed under the control of the promoter. The
promoter then drives the expression of the endogenous sequence.
The polynucleotides encoding polypeptides of the present invention may be
administered along with other polynucleotides encoding other angionaenic
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 injected into areas
of tissue
inside the wound.
Therapeutic compositions useful in systemic administration. include
recombinant molecules of the present invention complexed to a targeted
delivery
vehicle of the present invention. Suitable delivery vehicles for use with
systemic
administration comprise liposomes comprising ligands for targeting the vehicle
to a
particular site.
Preferred methods of systemic administration, include intravenous injection,
aerosol, oral and percutaneous (topical) delivery. Intravenous injections can
be
performed using methods standard in the art. Aerosol delivery can also be
performed
using methods standard in the art (see, for example, Stribling et al., Proc.
Natl. Acad.
Sci. USA , 189:11277-11281 (1992), which is incorporated herein by reference).
Oral
delivery can be performed by complexing a polynucleotide construct of the
present
invention to a carrier capable of withstanding degradation by digestive
enzymes in the
gut of an animal. Examples of such carriers, include plastic capsules or
tablets, such
as those known in the art. Topical delivery can be performed by mixing a
polynucleotide construct of the present invention with a lipophilic reagent
(e.g.,
DMSO) that is capable of passing into the skin.
Determining an effective amount of substance to be delivered can depend
upon a number of factors including, for example, the chemical structure and
biological activity of the substance, the age and weight of the animal, the
precise
condition requiring treatment and its severity, and the route of
administration. The
frequency of treatments depends upon a number of factors, such as the amount
of
polynucleotide constructs administered per dose, as well as the health and
history of
the subject. The precise amount, number of doses, and timing of doses will be
determined by the attending physician or veterinarian. Therapeutic
compositions of
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the present invention can be administered to any animal, preferably to mammals
and
birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits
sheep, cattle,
horses and pigs, with humans being particularly
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.
Immune Activity
The polynucleotides or polypeptides, or agonists or antagonists of the present
invention may be useful in treating deficiencies or disorders of the immune
system,
by 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 deficiencies or disorders may be genetic,
somatic, such
as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or
toxins),
or infectious. Moreover, a polynucleotides or polypeptides, or agonists or
antagonists
of the present invention can be used as a marker or detector of a particular
immune
system disease or disorder.
A polynucleotides or polypeptides, or agonists or antagonists of the present
invention may be useful in treating or detecting deficiencies or disorders of
hematopoietic cells. A polynucleotides or polypeptides, 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 those
disorders associated with a decrease in certain (or many) types hematopoietic
cells.
Examples of immunologic deficiency syndromes include, but are not limited to:
blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),
ataxia
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telangiectasia, common variable immunodeficiency, Digeorae Syndrome, HIV
infection. HTLV-BLV infection, leukocyte adhesion deficiency syndrome,
lymphopenia, phagocyte bactericidal dysfunction, severe combined
immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia,
or hemoglobinuria.
Moreover, a polynucleotides or polypeptides, or agonists or antagonists of the
present invention could also be used to modulate hemostatic (the stopping of
bleeding) or thrombolytic activity (clot formation). For example, by
increasing
hemostatic or thrombolytic activity, a polynucleotides or polypeptides, or
agonists or
antagonists of the present invention could be used to treat blood coagulation
disorders
(e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.a
thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
Alternatively, a polynucleotides or polypeptides, or agonists or antagonists
of the
present invention that can decrease hemostatic or thrombolytic activity could
be used
to inhibit or dissolve clotting. These molecules could be important in the
treatment of
heart attacks (infarction), strokes, or scarring.
A polynucleotides or polypeptides, or agonists or antagonists of the present
invention may also be useful in treating or detecting 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 a
polynucleotides or polypeptides, or agonists or antagonists of the present
invention .
that inhibits an immune response, particularly the proliferation,
differentiation, or
chemotaxis of T-cells, may be an effective therapy in preventing autoimmune
disorders.
Examples of autoimmune disorders that can be treated or detected by the
present invention include, but are not limited to: Addison's Disease,
hemolytic
anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic
encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves'
Disease,
Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmic, Bullous
Pemphigoid,
Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man
Syndrome,
Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary
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Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,
and
autoimmune inflammatory eye disease.
Similarly, allergic reactions and conditions, such as asthma (particularly
allergic asthma) or' other respiratory problems, may also be treated by a
polynucleotides or polypeptides, or agonists or antagonists of the present
invention.
Moreover, these molecules can be used to treat anaphylaxis, hypersensitivity
to an
antigenic molecule, or blood Group incompatibility.
A polynucleotides or polypeptides, or aaonists or antagonists of the present
invention may also be used to treat and/or prevent organ rejection or Graft-
versus-host
disease (GVHD). Organ rejection occurs by host immune cell destruction of the
transplanted tissue through an immune response. Similarly, an immune response
is
also involved in GVHD, but, in this case, the foreign transplanted immune
cells
destroy the host tissues. The administration of a polynucleotides or
polypeptides, or
agonists or antagonists of the present invention that inhibits an immune
response,
particularly the proliferation, differentiation, or chemotaxis of T-cells, may
be an
effective therapy in preventing organ rejection or GVHD.
Similarly, a polynucleotides or
polypeptides, or agonists or antagonists of the present invention may also be
used to
modulate inflammation. For example. the polypeptide or polynucleotide or
agonists
or antagonist may inhibit the proliferation and differentiation of cells
involved in an
inflammatory response. These molecules can be used to treat inflammatory
conditions, both chronic and acute conditions, including inflammation
associated with
infection (e.g., septic shock, sepsis, or systemic inflammatory response
syndrome
(SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,
complement-
mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung
injury,
inflammatory bowel disease, Crohn's disease, or resulting from over production
of
cytokines (e.g., TNF or IL-1.)
Hyperproliferative Disorders
A polynucleotides or polypeptides, or agonists or antagonists of the invention
can be used to treat or detect hyperproliferative disorders, including
neoplasms. A
polynucleotides or polypeptides, or aaonists or antagonists of the present
invention
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may inhibit the proliferation of the disorder through direct or indirect
interactions.
Alternatively, a 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 a
polynucleotides or polypeptides, or agonists or antagonists of the present
invention
include, but are not limited to neoplasms located in the: abdomen, bone,
breast,
digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid,
pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous
(central and
peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic,
and
urogenital.
Similarly, other hyperproliferative disorders can also be treated or detected
by
a polynucleotides or polypeptides, or agonists or antagonists of the present
invention.
Examples of such hyperproliferative disorders include, but are not limited to:
hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,
purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's
Disease, histiocytosis, and any other hyperproliferative disease, besides
neoplasia,
located in an organ system listed above.
One preferred embodiment utilizes polynucleotides of the present invention to
inhibit aberrant cellular division, by gene therapy using the present
invention, and/or
protein fusions or fragments thereof.
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.
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Another embodiment of the present invention provides a method of treatin~
cell-proliferative disorders in individuals comprising administration of one
or more
active Qene 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
preferrably
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
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
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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
polynucleotides of the present invention will target said gene and constructs
to
abnormally proliferating cells and will spare the non-dividing normal cells.
The polynucleotides of the present invention may be delivered directly to cell
proliferative disorder/disease sites in internal organs, body cavities and the
like by use
of imaging devices used to guide an injecting needle directly to the disease
site. The
polynucleotides of the present invention may also be administered to disease
sites at
the time of surgical intervention.
By "cell proliferative disease" is meant any human or animal disease or
disorder, affecting any one or any combination of organs, cavities, or body
parts,
which is characterized by single or multiple local abnormal proliferations of
cells,
groups of cells, or tissues, whether benign or malignant.
Any amount of the polynucleotides of the present invention may be
administered as long as it has a biologically inhibiting effect on the
proliferation of
the treated cells. Moreover, it is possible to administer more than one of the
polynucleotide of the present invention simultaneously to the same site. By
"biologically inhibiting" is meant partial or total growth inhibition as well
as
decreases in the rate of proliferation or growth of the cells. The
biologically
inhibitory dose may be determined by assessing the effects of the
polynucleotides of
the present invention on target malignant or abnormally proliferating cell
growth in
tissue culture, tumor growth in animals and cell cultures, or any other method
known
to one of ordinary skill in the art.
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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
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
1 S 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 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
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 SX 10~6M, 10-~M, SX 10-'M, 10~'M, SX 1 O~~M, 10-~M, SX 10-~M, 10-
''M, SX 10-
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'°M, 10-"'M, 5X 10-"M, 10-"M, 5X 10-'-M, 10''M, 5X 10~''M, 10-''M, 5X
10-'~M, 10-
'~'M. 5X10-'SM, and 10-'~M.
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-angioaenesis 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.,
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-1, CD95 (Fas/APO-
1),
TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related
apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K,
et.al.,
Eur J Biochem 254(3):439-59 ( 1998), which is hereby incorporated by
reference).
Moreover, in another preferred embodiment of the present invention, said
polypeptides may induce apoptosis through other mechanisms, such as in the
activation of other proteins which will activate apoptosis, or through
stimulating the
expression of said proteins, either alone or in combination with small
molecule drugs
or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory
proteins
(See for example, Mutat Res 400( 1-2):447-55 ( 1998), Med Hypotheses.50(5):423-
33
(1998), Chem Biol Interact. Apr 24;111-112:23-34 (1998), J 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.
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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
antibodes of the invention may be associated with with heterologous
polypeptides,
heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic,
ionic
and/or covalent interactions.
Polypeptides, protein fusions to, or fragments thereof, of the present
invention are
useful in enhancing the immunogenicity and/or antigenicity of proliferating
cells or
tissues, either directly, such as would occur if the polypeptides of the
present
invention 'vaccinated' the 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.
Cardiovascular Disorders
Polynucleotides or polypeptides, or agonists or antagonists of the invention
may be used to treat cardiovascular disorders, including peripheral artery
disease,
such as limb ischemia.
Cardiovascular disorders include cardiovascular abnormalities, such as arterio-
arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations,
congenital
heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart
defects
include aortic coarctation, cor triatriatum, coronary vessel anomalies,
crisscross heart,
dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger
complex,
hypoplastic left heart syndrome, levocardia, tetralogy of fallot,
transposition of great
vessels, double outlet right ventricle, tricuspid atresia, persistent truncus
arteriosus,
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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 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 sinus arrhythmia, atrial fibrillation, atrial flutter,
bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block,
sinoatrial
block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim
type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus
syndrome, tachycardias, and ventricular fibrillation. Tachycardias include
paroxysmal tachycardia, supraventricular tachycardia, accelerated
idioventricular
rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial
tachycardia, ectopic
functional tachycardia, sinoatrial nodal reentry tachycardia, sinus
tachycardia,
Torsades de Pointes, and ventricular tachycardia. .
Heart valve disease include aortic valve insufficiency, aortic valve stenosis,
hear murmurs, aortic valve prolapse, mural valve prolapse, tricuspid valve
prolapse,
mitral valve insufficiency, mural valve stenosis, pulmonary atresia, pulmonary
valve
insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve
insufficiency, and tricuspid valve stenosis.
Myocardial diseases include alcoholic cardiomyopathy, congestive
cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,
myocardial
reperfusion injury, and myocarditis.
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Myocardial ischemias include coronary disease, such as angina pectoris,
coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary
vasospasm, myocardial infarction and myocardial stunning.
Cardiovascular diseases also include vascular diseases such as aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema,
aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome. arterial
occlusive
diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular
disorders, diabetic
angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia,
hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension,
ischemia,
peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease,
Raynaud's
disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior
vena
cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic
telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and
venous
insufficiency.
Aneurysms include dissecting aneurysms, false aneurysms, infected
aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary
aneurysms, heart aneurysms, and iliac aneurysms.
Arterial occlusive diseases include arteriosclerosis, intermittent
claudication,
carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion,
Moyamoya
disease, renal artery obstruction, retinal artery occlusion, and
thromboangiitis
obliterans.
Cerebrovascular disorders include carotid artery diseases, cerebral amyloid;
angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis,
cerebral
arteriovenous malformation, cerebral artery diseases, cerebral embolism and
thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's
syndrome,
cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid
hemorrhage, cerebral infarction, cerebral ischemia (including transient),
subclavian
steal syndrome, periventricular leukomalacia, vascular headache, cluster
headache,
migraine, and vertebrobasilar insufficiency.
Embolisms include air embolisms, amniotic fluid embolisms, cholesterol
embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and
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thromoboembolisms. Thrombosis include coronary thrombosis, hepatic vein
thrombosis, retinal vein occlusion, carotid artery thrombosis. sinus
thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
Ischemia includes cerebral ischemia, ischemic colitis, compartment
syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion
injuries, and peripheral limb ischemia. Vasculitis includes aortitis,
arteritis, Behcet's
Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome,
thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch
purpura,
allergic cutaneous vasculitis, and Wegener's granulomatosis.
Polynucleotides or polypeptides, or agonists or antagonists of the invention,
are especially effective for the treatment of critical limb ischemia and
coronary
disease.
Polypeptides may be administered using any method known in the art,
including, but not limited to, direct needle injection at the delivery site,
intravenous
injection, topical administration, catheter infusion, biolistic injectors,
particle
accelerators, gelfoam sponge depots, other commercially available depot
materials,
osmotic pumps, oral or suppositorial solid pharmaceutical formulations,
decanting or
topical applications during surgery, aerosol delivery. Such methods are known
in the
art. Polypeptides of the invention may be administered as part of a
Therapeutic,
described in more detail below. Methods of delivering polynucleotides of the
invention are described in more detail herein.
Anti-Angiogenesis ActivitX
The naturally occurring balance between endogenous stimulators and
inhibitors of angiogenesis is one in which inhibitory influences predominate.
Rastinejad et al., Cell 56:345-355 ( 1989). In those rare instances 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.
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A number of serious diseases are dominated by abnormal neovascularization
including solid tumor growth and metastases, arthritis, some types of eye
disorders,
and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 ( 199 I
); Folkman
et ul., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc.
Res.
29:401-41 1 ( 1985); Folkman, Advances in Cancer Research, eds. Klein and
Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J.
Opthalmol. 94:715-743 ( 1982); and Folkman et al., Science 221:719-725 (
1983). In a
number of pathological conditions, the process of anaiogenesis contributes to
the
disease state. For example, significant data have accumulated which suggest
that the
growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun,
Science 235:442-447 ( 1987).
The present invention provides for treatment of diseases or disorders
associated with neovascularization by administration of the polynucleotides
and/or
polypeptides of the invention, as well as agonists or antagonists of the
present
invention. Malignant and metastatic conditions which can be treated with the
polynucleotides and polypeptides, or agonists or antagonists of the invention
include,
but are not limited to, malignancies, solid tumors, and cancers described
herein and
otherwise known in the art (for a review of such disorders, see Fishman et
al.,
Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia ( 1985)).Thus, the
present
invention provides a method of treating an angiogenesis-related disease and/or
disorder, comprising administering to an individual in need thereof a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
of .the
invention. For example, polynucleotides, polypeptides, antagonists and/or
agonists
may be utilized in a variety of additional methods in order to therapeutically
treat a
cancer or tumor. Cancers which may be treated with polynucleotides,
polypeptides,
antagonists and/or agonists include, but are not limited to solid tumors,
including
prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes,
liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,
bladder,
thyroid cancer; primary tumors and metastases; melanomas; glioblastoma;
Kaposi's
sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer;
advanced
malignancies; and blood born tumors such as leukemias. For example,
polynucleotides, polypeptides, antagonists and/or agonists may be delivered
topically,
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in order to treat cancers such as skin cancer, head and neck tumors, breast
tumors, and
Kaposi's sarcoma.
Within yet other aspects, polynucleotides, polypeptides, antagonists and/or
agonists may be utilized to treat superficial forms of bladder cancer by, for
example,
intravesical administration. Polynucleotides, polypeptides, antagonists and/or
agonists
may be delivered directly into the tumor, or near the tumor site, via
injection or a
catheter. Of course, as the artisan of ordinary skill will appreciate, the
appropriate
mode of administration will vary according to the cancer to be treated. Other
modes
of delivery are discussed herein.
Polynucleotides, polypeptides, antagonists and/or agonists may be useful in
treating other disorders, besides cancers, which involve angiogenesis. These
disorders include, but are not limited to: benign tumors, for example
hemangiomas,
acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;
artheroscleric plaques; ocular angiogenic diseases, for example, diabetic
retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular
glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and
Pterygia
(abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis;
delayed
wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars
(keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions;
myocardial
angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous
malformations;
ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization;
telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia;
wound
granulation; Crohn's disease; and atherosclerosis.
For example, within one aspect of the present invention methods are provided
for treating hypertrophic scars and keloids, comprising the step of
administering a
polynucleotide, polypeptide, antagonist and/or agonist of the invention to a
hypertrophic scar or keloid.
Within one embodiment of the present invention polynucleotides,
polypeptides, antagonists and/or aQonists 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
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initiated after the proliferative phase has had time to progress
(approximately 14 days
after the initial injury), but before hypertrophic scar or keloid development.
As noted
above, the present invention also provides methods for treating neovascular
diseases
of the eye, including for example. corneal neovascularization, neovascular
glaucoma,
proliferative diabetic retinopathy, retrolental fibroplasia and macular
degeneration.
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 cal., Anz. J.
Ophthal. 85:704-
710 (1978) and Gartner et nl., Smv. 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.a., trachoma,
herpes
simplex keratitis, leishmaniasis and onchocerciasis), immunological processes
(e.g.,
graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma,
inflammation
(of any cause), toxic and nutritional deficiency states, and as a complication
of
wearing contact lenses.
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-anaioUenic compositions,
prepared as described above, may also be administered directly to the cornea.
Within
preferred embodiments, the anti-anaioaenic composition is prepared with a muco-
adhesive polymer which binds to cornea. Within further embodiments, the anti-
angiogenic factors or anti-angiogenic compositions may be utilized as an
adjunct to
conventional steroid therapy. Topical therapy may also be useful
prophylactically in
corneal lesions which are known to have a high probability of inducing an
angiogenic
response (such as chemical burns). In these instances the treatment, likely in
combination with steroids, may be instituted immediately to help prevent
subsequent
complications.
Within other embodiments, the compounds described above may be injected
directly into the corneal stroma by an ophthalmologist under microscopic
guidance.
The preferred site of injection may vary with the morphology of the individual
lesion,
but the goal of the administration would be to place the composition at the
advancing
front of the vasculature (i.e., interspersed between the blood vessels and the
normal
cornea). In most cases this would involve perilimbic corneal injection to
"protect" the
cornea from the advancing blood vessels. This method may also be utilized
shortly
after a corneal insult in order to prophylactically prevent corneal
neovascularization.
In this situation the material could be injected in the perilimbic cornea
interspersed
between the corneal lesion and its undesired potential limbic blood supply.
Such
methods may also be utilized in a similar fashion to prevent capillary
invasion of
transplanted corneas. In a sustained-release form injections might only be
required.2-
3 times per year. A steroid could also be added to the injection solution to
reduce
inflammation resulting from the injection itself.
Within another aspect of the present invention, methods are provided for
treating neovascular glaucoma, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. In
one
embodiment, the compound may be administered topically to the eye in order to
treat
early forms of neovascular glaucoma. Within other embodiments, the compound
may ..
be implanted by injection into the region of the anterior chamber angle.
Within other
embodiments, the compound may also be placed in any location such that the
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compound is continuously released into the aqueous humor. Within another
aspect of
the present invention, methods are provided for treating proliferative
diabetic
retinopathy, comprising the step of administering to a patient a
therapeutically
effective amount of a polynucleotide, polypeptide, antagonist and/or agonist
to the
eyes, such that the formation of blood vessels is inhibited.
Within particularly preferred embodiments of the invention, proliferative
diabetic retinopathy may be treated by injection into the aqueous humor or the
vitreous, in order to increase the local concentration of the polynucleotide,
polypeptide, antagonist and/or agonist in the retina. Preferably, this
treatment should
be initiated prior to the acquisition of severe disease requiring
photocoagulation.
Within another aspect of the present invention, methods are provided for
treating retrolental fibroplasia, comprising the step of administering to a
patient a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or
agonist to the eye, such that the formation of blood vessels is inhibited. The
compound may be administered topically, via intravitreous injection and/or via
intraocular implants.
Additionally, disorders which can be treated with the polynucleotides,
polypeptides, agonists and/or agonists include, but are not limited to,
hemangioma,
arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound
healing,
granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-
Weber
syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
Moreover, disorders and/or states, which can be treated with be treated with
the the polynucleotides, polypeptides, agonists and/or agonists include, but
are not
limited to, solid tumors, blood born tumors such as leukemias, tumor
metastasis,
Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas,
neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis,
psoriasis,
ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of
prematurity, macular degeneration, corneal graft rejection, neovascular
glaucoma,
retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound
healing,
endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids),
nonunion
fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis,
coronary collaterals, cerebral collaterals, arteriovenous malformations,
ischemic limb
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angiogenesis, Osler-Webber Syndrome, plaque neovascularization,
telangiectasia,
hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation,
Crohn's disease, atherosclerosis, birth control anent by preventing
vascularization
required for embryo implantation controlling menstruation, diseases that have
angiogenesis as a pathologic consequence such as cat scratch disease (Rochele
minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary
angiomatosis.
In one aspect of the birth control method, an amount of the compound
sufficient to block embryo implantation is administered before or after
intercourse and
fertilization have occurred, thus providing an effective method of birth
control,
possibly a "morning after" method. Polynucleotides, polypeptides, agonists
and/or
agonists may also be used in controlling menstruation or administered as
either a
peritoneal lavage fluid or for peritoneal implantation in the treatment of
endometriosis.
Polynucleotides, polypeptides, agonists and/or agonists of the present
invention may be incorporated into surgical sutures in order to prevent stitch
granulomas.
Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a
wide variety of surgical procedures. For example, within one aspect of the
present
invention a compositions (in the form of, for example, a spray or film) may be
utilized
to coat or spray an area prior to removal of a tumor, in order to isolate
normal
surrounding tissues from malignant tissue, and/or to prevent the spread of
disease to
surrounding tissues. Within other aspects of the present invention,
compositions (e.g.,
in the form of a spray) may be delivered via endoscopic procedures in order to
coat
tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects
of the
present invention, surgical meshes which have been coated with anti-
angiogenic
compositions of the present invention may be utilized in any procedure wherein
a
surgical mesh might be utilized. For example, within one embodiment of the
invention a surgical mesh laden with an anti-angiogenic composition may be
utilized
during abdominal cancer resection surgery (e.g., subsequent to colon
resection) in
order to provide support to the structure, and to release an amount of the
anti-
angiogenic factor.
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Within further aspects of the present invention, methods are provided for
treating tumor excision sites, comprising administering a polynucleotide,
polypeptide,
agonist and/or agonist to the resection margins of a tumor subsequent to
excision,
such that the local recurrence of cancer and the formation of new blood
vessels at the
site is inhibited. Within one embodiment of the invention, the anti-angiogenic
compound is administered directly to the tumor excision site (e.g., applied by
swabbing, brushing or otherwise coating the resection margins of the tumor
with the
anti-angioQenic compound). Alternatively, the anti-angioaenic compounds may be
incorporated into known surgical pastes prior to administration. Within
particularly
preferred embodiments of the invention, the anti-angiogenic compounds are
applied
after hepatic resections for malignancy, and after neurosurgical operations.
Within one aspect of the present invention, polynucleotides, polypeptides,
agonists and/or agonists may be administered to the resection margin of a wide
variety of tumors, including for example, breast, colon, brain and hepatic
tumors. For
example, within one embodiment of the invention, anti-angiogenic compounds may
be administered to the site of a neurological tumor subsequent to excision,
such that
the formation of new blood vessels at the site are inhibited.
The polynucleotides, polypeptides, agonists and/or agonists of the present
invention may also be administered along with other anti-angiogenic factors.
Representative examples of other anti-angiogenic factors include: Anti-
Invasive
Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue
Inhibitor of
Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen
Activator
Inhibitor-l, Plasminogen Activator Inhibitor-2, and various forms of the
lighter "d
group" transition metals.
Lighter "d group" transition metals include, for example, vanadium,
molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition
metal species may form transition metal complexes. Suitable complexes of the
above-mentioned transition metal species include oxo transition metal
complexes.
Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include metavanadate and orthovanadate complexes such as, for example,
ammonium
metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl
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complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate
including vanadyl sulfate hydrates such as vanadyl sulfate mono- and
trihydrates.
Representative examples of tungsten and molybdenum complexes also include
oxo complexes. Suitable oxo tungsten complexes include tunastate and tungsten
oxide complexes. Suitable tungstate complexes include ammonium tungstate,
calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable
tungsten
oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo
molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl
complexes. Suitable molybdate complexes include ammonium molybdate and its
hydrates, sodium molybdate and its hydrates, and potassium molybdate and its
hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum
(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for
example,
molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes
include hydroxo derivatives derived from, for example, glycerol, tartaric
acid, and
sugars.
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 (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987);
Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-
carboxyphenyl-4-
chloroanthronilic acid disodium or "CCA"; Takeuchi et al., Agents Actions
36:312-
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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 or detected by the polynucleotides or polypeptides
and/or
antagonists or agonists of the invention, include cancers (such as follicular
lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors,
including, but not limited to colon cancer, cardiac tumors, pancreatic cancer,
melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular
cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast
cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune
disorders
(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis,
biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid arthritis)
and
viral infections (such as herpes viruses, pox viruses and adenoviruses),
inflammation,
graft v. host disease, acute graft rejection, and chronic graft rejection. In
preferred
embodiments, the polynucleotides or polypeptides, and/or agonists 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 the polynucleotides or polypeptides, 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 (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,
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osteogenic sarcoma, chordoma, anaiosarcoma, endotheliosarcoma,
lymphangiosarcoma. lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic
cancer, breast cancer. ovarian cancer, prostate cancer, sduamous 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 IunQ carcinoma,
bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma, ependymoma, pinealoma. hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
Diseases associated with increased apoptosis that could be treated or detected
by the polynucleotides or 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, Sjo~ren's syndrome, Hashimoto's thyroiditis,
biliary
cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid arthritis)
myelodysplastic syndromes (such as aplastic anemia), graft v. host disease,
ischemic
injury (such as that caused by myocardial infarction, stroke and reperfusion
injury),
liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion
injury, cholestosis
(bile duct injury) and liver cancer); toxin-induced liver disease (such as
that caused by
alcohol), septic shock, cachexia and anorexia.
Wound Healing; and Epithelial Cell Proliferation
In accordance with yet a further aspect of the present invention, there is
provided a process for utilizing the polynucleotides or polypeptides, and/or
agonists
or antagonists of the invention, for therapeutic purposes, for example, to
stimulate
epithelial cell proliferation and basal keratinocytes for the purpose of wound
healing,
and to stimulate hair follicle production and healing of dermal wounds.
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Polynucleotides or polypeptides, as well as agonists or antagonists of the
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 associted with systemic
treatment with steroids, radiation therapy and antineoplastic drugs and
antimetabolites. Polynucleotides or polypeptides, and/or agonists or
antagonists of
the invention, could be used to promote dermal reestablishment subsequent to
dermal
loss
The polynucleotides or polypeptides, and/or agonists or antagonists of the
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 a non-
exhaustive list of grafts that polynucleotides or polypeptides; agonists or
antagonists
of the 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. The polynucleotides or polypeptides, and/or
agonists. or
antagonists of the invention, can be used to promote skin strength and to
improve the
appearance of aged skin.
It is believed that the polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, will also produce changes in hepatocyte
proliferation,
and epithelial cell proliferation in the lung, breast, pancreas, stomach,
small intesting,
and large intestine. The polynucleotides or polypeptides, and/or agonists or
antagonists of the invention, could promote proliferation of epithelial cells
such as
sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing
goblet
cells, and other epithelial cells and their progenitors contained within the
skin, lung,
liver, and gastrointestinal tract. The polynucleotides or polypeptides, and/or
agonists
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or antagonists of the invention, may promote proliferation of endothelial
cells,
keratinocytes, and basal keratinocytes.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention, could also be used to reduce the side effects of gut toxicity that
result from
radiation, chemotherapy treatments or viral infections. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may have a
cytoprotective effect on the small intestine mucosa. The polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, may also
stimulate
healing of mucositis (mouth ulcers) that result from chemotherapy and viral
infections.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
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. The
polynucleotides or polypeptides, and/or agonists or antagonists of the
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. The polynucleotides or polypeptides,
and/or
agonists or antagonists of the 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.
Inflamamatory 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, the polynucleotides or polypeptides, and/or
agonists or
antagonists of the 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 the polynucleotides or polypeptides, and/or agonists
or
antagonists of the 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.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
invention,
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could be used to treat diseases associate with the under expression of the
polynucleotides of the invention.
Moreover, the polynucleotides or polypeptides, and/or aaonists or antagonists
of
the invention, could be used to prevent and heal damage to the lungs due to
various
pathological states. A growth factor such as the polynucleotides or
polypeptides,
and/or agonists or antagonists of the 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 the polynucleotides or
polypeptides, and/or
agonists or antagonists of the invention. Also, the polynucleotides or
polypeptides,
and/or agonists or antagonists of the 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.
The polynucleotides or polypeptides, and/or agonists or antagonists of the
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, the polynucleotides or polypeptides, and/or agonists or
antagonists
of the 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, the polynucleotides or polypeptides, and/or agonists or antagonists
of the
invention, could be used to maintain the islet function so as to alleviate,
delay or
prevent permanent manifestation of the disease. Also, the polynucleotides or
polypeptides, and/or agonists or antagonists of the invention, could be used
as an
auxiliary in islet cell transplantation to improve or promote islet cell
function.
Neurological Diseases
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Nervous system disorders, which can be treated with the compositions of the
invention (e.g., polypeptides, polynucleotides, and/or aQonists 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 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, syphilis; (5) degenerative lesions, in which a portion of the
nervous
system is destroyed or injured as a result of a degenerative process including
but not
limited to degeneration associated with Parkinson's disease, Alzheimer's
disease,
Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions
associated .
with nutritional diseases or disorders, in which a portion of the nervous
system is
destroyed or injured by a nutritional disorder or disorder of metabolism
including but
not limited to, vitamin B 12 deficiency, folic acid deficiency, Wernicke
disease,
tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration
of
the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological
lesions
associated with systemic diseases including, but not limited to, diabetes
(diabetic
neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or
sarcoidosis;
(8) lesions caused by toxic substances including alcohol, lead, or particular
neurotoxins; and (9) demyelinated lesions in which a portion of the nervous
system
is destroyed or injured by a demyelinating disease including, but not limited
to,
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multiple sclerosis, human immunodeficiency virus-associated myelopathy,
transverse
myelopathy or various etiologies, progressive multifocal leukoencephalopathy,
and
central pontine myelinolysis.
In a preferred embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the invention are used to protect neural cells from the
damaQina 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 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 aspect of this embodiment, the
polypeptides, polynucleotides, or aaonists or antagonists of the invention are
used to
treat or prevent neural cell injury associated with cerebral infarction. In
another 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
a stroke.
In a further 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 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
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;
(2) increased sprouting of neurons in culture or irZ vivo; (3) increased
production of a
neuron-associated molecule in culture or in vivo, e.g., choline
acetyltransferase or
acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms
of
neuron dysfunction in vivo. Such effects may be measured by any method known
in
the art. In preferred, non-limiting embodiments, increased survival of neurons
may
routinely be measured using a method set forth herein or otherwise known in
the art,
such as, for example, the method set forth in Arakawa et al. (J. Neurosci.
10:3507-3515 ( 1990)); increased sprouting of neurons may be detected by
methods
known in the art, such as, for example, the methods set forth in Pestronk et
al. (Exp.
Neurol. 70:65-82 ( 1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 ( 1981
));
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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).
Infectious Disease
A polypeptide or polynucleotide and/or agonist or antagonist of the present
invention can be used to treat or detect infectious agents. For example, by
increasing
the immune response, particularly increasing the proliferation and
differentiation of B
and/or T cells, infectious diseases may be treated. The immune response may be
.
increased by either enhancing an existing immune response, or by initiating a
new
immune response. Alternatively, polypeptide or polynucleotide and/or agonist
or
antagonist of the present invention may also directly inhibit the infectious
agent,
without necessarily eliciting an immune response.
Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated or detected by a polynucleotide or polypeptide
and/or
agonist or antagonist of the present invention. Examples of viruses, include,
but are
not limited to Examples of viruses, include, but are not limited to the
following DNA
and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,
Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae,
Coronaviridae,
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Dengue. EBV, HIV. Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae
(such as.
Cytomegalovirus. Herpes Simplex. Herpes Zoster). Mononegavirus (e.a.,
Paramyxoviridae. Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,
Influenza
A, Influenza B, and parainlluenza), Papiloma virus. Papovaviridae,
Parvoviridae.
Picornaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus). Viruses falling within these families can cause a variety of
diseases or
symptoms, including. but not limited to: arthritis, bronchiollitis,
respiratory syncytial
virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic
fatigue
syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B
encephalitis.
Junin, Chikungunya. Rift Valley fever, yellow fever, meningitis, opportunistic
infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox,
hemorrhagic
fever, Measles. Mumps, Parainfluenza, Rabies, the common cold, Polio,
leukemia,
Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),
and
viremia. polynucleotides or polypeptides, or agonists or antagonists of the
invention,
can be used to treat or detect any of these symptoms or diseases. In specific
embodiments, polynucleotides, polypeptides, or agonists or antagonists of the
invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g.,
hepatitis
B). In an additional specific embodiment polynucleotides, polypeptides, or
agonists
or antagonists of the invention are used to treat patients nonresponsive to
one or more
other commercially available hepatitis vaccines. In a further specific
embodiment
polynucleotides, polypeptides, or agonists or antagonists of the invention are
used to.
treat AIDS.
Similarly, bacterial or fungal agents that can cause disease or symptoms and
that can be treated or detected by a polynucleotide or polypeptide and/or
agonist or
antagonist of the present invention include, but not limited to, include, but
not limited
to, the following Gram-Negative and Gram-positive bacteria and bacterial
families
and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia),
Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax,
Clostridium),
Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia
burgdorferi),
Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,
Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic
E.
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coli), Enterobacteriaceae (Klebsiella. Salmonella (e.g., Salmonella typhi, and
Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter,
Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae,
Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal),
Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus,
Heamophilus
(e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas.
Rickettsiaceae,
Chlamydiaceae, Syphilis, ShiQella spp., Staphylococcal, Meningiococcal,
Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B
Streptococcus). These bacterial or fungal families can cause the following
diseases or
symptoms, including, but not limited to: bacteremia, endocarditis, eye
infections
(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections
(e.g., AIDS
related infections), paronychia, prosthesis-related infections, Reiter's
Disease,
respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme
Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,
Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B),
Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis,
Lupus,
Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever,
sexually
transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses),
toxemia, urinary
tract infections, wound infections. Polynucleotides or polypeptides, agonists
or
antagonists of the invention, can be used to treat or detect any of these
symptoms or
diseases. In specific embodiments, Ppolynucleotides, polypeptides, agonists or
antagonists of the invention are used to treat: tetanus, Diptheria, botulism,
and/or
meningitis type B.
Moreover, parasitic agents causing disease or symptoms that can be treated or
detected by a polynucleotide or polypeptide and/or agonist or antagonist of
the
present invention include, but not limited to, the following families or
class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,
Dourine,
Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis,
Toxoplasmosis,
Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,
Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These
parasites can cause a variety of diseases or symptoms, including, but not
limited to:
Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,
giardiasis),
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liver disease, lung disease, opportunistic infections (e.g., AIDS related),
malaria,
pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides,
or
agonists or antagonists of the invention, can be used to treat or detect any
of these
symptoms or diseases. In specific embodiments, polynucleotides, polypeptides,
or
agonists or antagonists of the invention are used to treat malaria.
Preferably, treatment using a polypeptide or polynucleotide and/or agonist or
antagonist of the present invention could either be by administering an
effective
amount of a polypeptide to the patient, or by removing cells from the patient,
supplying the cells with a polynucleotide of the present invention, and
returning the
engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide
or
polynucleotide of the present invention can be used as an antigen in a vaccine
to raise
an immune response against infectious disease.
Regeneration
l~ A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention can be used to differentiate, proliferate, and attract cells,
leading to the
regeneration of tissues. (See, Science 276:59-87 (1997j.) The regeneration of
tissues
could be used to repair, replace, or protect tissue damaged by congenital
defects,
trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal disease, liver failure), surgery, including
cosmetic plastic
surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
Tissues that could be regenerated using the present invention include organs .
(e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal
or cardiac), vasculature (including vascular and lymphatics), nervous,
hematopoietic,
and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably,
regeneration
occurs without or decreased scarring. Regeneration also may include
angiogenesis.
Moreover, a polynucleotide or polypeptide and/or agonist or antagonist of the
present invention may increase regeneration of tissues difficult to heal. For
example,
increased tendon/ligament regeneration would quicken recovery time after
damage.
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention
could also be used prophylactically in an effort to avoid damage. Specific
diseases
that could be treated include of tendinitis, carpal tunnel syndrome, and other
tendon or
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ligament defects. A further example of tissue regeneration of non-healing
wounds
includes pressure ulcers. ulcers associated with vascular insufficiency,
surgical, and
traumatic wounds.
Similarly, nerve and brain tissue could also be regenerated by using a
polynucleotide or polypeptide and/or aaonist or antagonist of the present
invention to
proliferate and differentiate nerve cells. Diseases that could be treated
using this
method include central and peripheral nervous system diseases, neuropathies,
or
mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma,
cerebrovascular disease, and stoke). Specifically, diseases associated with
peripheral
nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or
other
medical therapies), localized neuropathies, and central nervous system
diseases (e.g.,
Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic
lateral
sclerosis, and Shy-Drager syndrome), could all be treated using the
polynucleotide or
polypeptide and/or agonist or antagonist of the present invention.
Chemotaxis
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes
cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells,
eosinophils,
epithelial and/or endothelial cells) to a particular site in the body, such as
inflammation, infection, or site of hyperproliferation. The mobilized cells
can then
fight off and/or heal the particular trauma or abnormality.
A polynucleotide or polypeptide and/or agonist or antagonist of the present
invention may increase chemotaxic activity of particular cells. These
chemotactic
molecules can then be used to treat inflammation, infection,
hyperproliferative
disorders, or any immune system disorder by increasing the number of cells
targeted
to a particular location in the body. For example, chemotaxic molecules can be
used
to treat wounds and other trauma to tissues by attracting immune cells to the
injured
location. Chemotactic molecules of the present invention can also attract
fibroblasts,
which can be used to treat wounds.
It is also contemplated that a polynucleotide or polypeptide and/or agonist or
antagonist of the present invention may inhibit chemotactic activity. These
molecules
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could also be used to treat disorders. Thus, a polynucleotide or polypeptide
and/or
agonist or antagonist of the present invention could be used as an inhibitor
of
chemotaxis.
Binding Activity
A polypeptide of the present invention may be used to screen for molecules
that bind to the polypeptide or for molecules to which the polypeptide binds.
The
binding of the polypeptide and the molecule may activate (aaonist), increase,
inhibit
(antagonist), or decrease activity of the polypeptide or the molecule bound.
Examples
of such molecules include antibodies, oligonucleotides, proteins (e.g.,
receptors),or
small molecules.
Preferably, the molecule is closely related to the natural ligand of the
polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand,
a structural
or functional mimetic. (See, Coligan et al., Current Protocols in Immunology
1(2):Chapter 5 (1991).) Similarly, the molecule can be closely related to the
natural
receptor to which the polypeptide binds, or at least, a fragment of the
receptor capable
of being bound by the polypeptide (e.g., active site). In either case, the
molecule can
be rationally designed using known techniques.
Preferably, the screening for these molecules involves producing appropriate
cells which express the polypeptide, either as a secreted protein or on the
cell
membrane. Preferred cells include cells from mammals, yeast, Drosophila, or E.
coli.
Cells expressing the polypeptide (or cell membrane containing the expressed
polypeptide) are then preferably contacted with a test compound potentially
containing the molecule to observe binding, stimulation, or inhibition of
activity of
either the polypeptide or the molecule.
The assay may simply test binding of a candidate compound to the
polypeptide, wherein binding is detected by a label, or in an assay involving
competition with a labeled competitor. Further, the assay may test whether the
candidate compound results in a signal generated by binding to the
polypeptide.
Alternatively, the assay can be carried out using cell-free preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
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mixtures. The assay may also simply comprise the steps of mixing a candidate
compound with a solution containing a polypeptide, measuring
polypeptide/molecule
activity or binding, and comparing the polypeptide/molecule activity or
binding to a
standard.
Preferably, an ELISA assay can measure polypeptide level or activity in a
sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
The
antibody can measure polypeptide level or activity by either binding. directly
or
indirectly, to the polypeptide or by competing with the polypeptide for a
substrate.
Additionally, the receptor to which a polypeptide of the invention binds can
be
identified by numerous methods known to those of skill in the art, for
example, ligand
panning and FAGS sorting (Coliaan, et al., Current Protocols in Immun., 1(2),
Chapter 5, ( 1991 )). For example, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to the polypeptides, for
example, NIH3T3 cells which are known to contain multiple receptors for the
FGF
family proteins, and SC-3 cells, and a cDNA library created from this RNA is
divided
into pools and used to transfect COS cells or other cells that are not
responsive to the
polypeptides. Transfected cells which are crown on glass slides are exposed to
the
polypeptide of the present invention, after they have been labelled. The
polypeptides
can be labeled by a variety of means including iodination or inclusion of a
recognition
site for a site-specific protein kinase.
Following fixation and incubation, the slides are subjected to auto-
radiographic analysis. Positive pools are identified and sub-pools are
prepared and re-
transfected using an iterative sub-pooling and re-screening process,
eventually
yielding a single clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides
can be photoaffinity linked with cell membrane or extract preparations that
express
the receptor molecule. Cross-linked material is resolved by PAGE analysis and
exposed to X-ray film. The labeled complex containing the receptors of the
polypeptides can be excised, resolved into peptide fragments, and subjected to
protein
microsequencing. The amino acid sequence obtained from microsequencing would
be used to design a set of degenerate oligonucleotide probes to screen a cDNA
library
to identify the genes encoding the putative receptors.
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brevets
JUMBO APPLICATiONSIPATENTS
THiS SECTION OF THE APPl.ICATION/PATENT CONTAINS MORE
THAN ONE VOLUME -
THIS IS VOLUME ~ , OF
NOTE: For additional volumes please contact the Canadian Patent Office
