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CA 02395178 2002-06-19
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Nucleic Acids, Proteins, and Antibodies
[1] This application refers to a "Sequence Listing" that is provided only on
electronic
media in computer readable form pursuant to Administrative Instructions
Section 801 (a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and
Administrative Instructions Sections 801 to 806, and is hereby incorporated in
its entirety.
[2] The Sequence Listing is provided as an electronic file
(PTZ03PCT_seqList.txt,
67,206 bytes in size, created on January 13, 2001) on four identical compact
discs (CD-R),
labeled "COPY 1," "COPY 2," "COPY 3," and "CRF." The Sequence Listing complies
with
Annex C of the Administrative Instructions, and may be viewed, for example, on
an IBM-PC
machine running the MS-Windows operating system by using the V viewer
software, version
2000 (see World Wide Web URL: http://www.fileviewer.com).
Field of the Invention
[3] The present invention relates to novel proteins. More specifically,
isolated nucleic
acid molecules are provided encoding novel polypeptides. Novel polypeptides
and antibodies
that bind to these polypeptides are provided. Also provided are vectors, host
cells, and
recombinant and synthetic methods for producing human polynucleotides and/or
polypeptides, and antibodies. The invention further relates to diagnostic and
therapeutic
CA 02395178 2002-06-19
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methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
these novel polypeptides. The invention further relates to screening methods
for identifying
agonists and antagonists of polynucleotides and polypeptides of the invention.
The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the present invention.
Background of the Invention
[4] The ATP-binding cassette (ABC) transporter proteins comprise a large
family of
prokaryotic and eukaryotic membrane proteins involved in the energy-dependant
transport of
a wide range of substrates across membranes (Higgins, C.F. et al., Ann. Rev.
Cell Biol., 8:67-
113 (1992)). In eukaryotes, ABC transport proteins. typically consist of four
domains that
include two conserved ATP-binding domains and two transmembrane domains (Hyde
et al.,
Nature, 346:362-5 (1990)). An example of a eukaryotic ABC transport protein is
Multi-Drug-
Resistance 1 protein (MDR1), which is also referred to as P-glycoprotein, and
is implicated
in conferring upon cells resistance to a wide range of drugs, such as
chemotherapeutic drugs
(Gottesman et al., JBiol.~ Chem., 263:12163-6 (1988).
[5] Further, examples include CFTR, which is involved in cystic fibrosis
(Riordan et
al., Science, 245:1066-73 (1989)), TAP transporter proteins involved in small
peptide
presentation for Human lymphocyte antigen (HLA) class I proteins (de la Salle
et al.,
Science, 265:237-41 (1994)), and the SUR protein, defects of which are
involved in
unregulated insulin secretion in patients with Familial persistent
hyperinsulinemic
hypoglycemia of infancy (PHHI) (Thomas et al., Science, 268:426-9 ( 1995)).
[6] Defects in the transport of specific substrates mediated by members of
this family
of proteins results in many diseases, as discussed above. Thus there exists a
clear need for
identifying and exploiting novel ABC Transport Receptor polynucleotides and
polypeptides.
Although structurally related, such proteins may possess diverse and
multifaceted functions .
in a variety of cell and~tissue types. The purified ABC Transport Receptor
polypeptides of the
invention are research tools useful for the identification, characterization
and purification of
additional proteins involved in diseases and/or disorders associated with ABC
Transport
receptor polynucleotides and polypeptides, such as, for example, CFTR and
unregulated
insulin secretion. Furthermore, the identification of new ABC Transport.
Receptor
polynucleotides and polypeptides permits the development of a range of
derivatives, agonists
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and antagonists at the nucleic acid and protein levels which in turn have
applications in the
treatment and diagnosis of a range of conditions such as, for example, CFTR
and unregulated
insulin secretion, amongst many other conditions.
Summary of the Invention
[7] The. present invention relates to novel proteins. More specifically,
isolated nucleic
acid molecules are provided encoding novel polypeptides. Novel polypeptides
and antibodies
that bind to these polypeptides are provided. Also provided are vectors, host
cells, and
recombinant and synthetic methods for producing human polynucleotides and/or
polypeptides, and antibodies. The invention further relates to diagnostic and
therapeutic
methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
these novel polypeptides. The invention further relates to screening methods
for identifying
agonists and antagonists of polynucleotides~ and polypeptides of the
invention. The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the present invention.
Detailed Description
Tables
[8] Table 1A summarizes some of the polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone ID NO:Z), contig
sequences (contig
identifier (Contig ID:) and contig nucleotide sequence identifier (SEQ ID
NO:X)) and further
summarizes certain characteristics of these polynucleotides and the
polypeptides encoded
thereby. The first column provides the gene number in the application for each
clone
identifier. The second column provides a unique clone identifier, "Clone ID
NO:Z", for a
cDNA clone related to each contig sequence disclosed in Table 1A. The third
column
provides a unique contig identifier, "Contig ID:" for each of the contig
sequences disclosed
in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for each
of the contig sequences disclosed in Table 1 A. The fifth column, "ORF (From-
To)",
provides the location (i.e., nucleotide position numbers) within the
polynucleotide sequence
of SEQ ID NO:X that delineate the preferred open reading frame (ORF) that
encodes the
amino acid sequence shown in the sequence listing and referenced in Table 1A
as SEQ ID
NO:Y .(column 6). Column 7 lists residues comprising predicted epitopes
contained in the
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polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y).
Identification of
potential immunogenic regions was performed according to the method of Jameson
and Wolf
(CABIOS, 4; 181-186 (1988)); specifically, the Genetics Computer Group (GCG)
implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE
(Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wisc.). This
method
returns a measure of the probability that a given residue is found on the
surface of the protein.
Regions where the antigenic index score is greater than 0.9 over at least 6
amino acids are
indicated in Table 1A as "Predicted Epitopes". In particular embodiments,
polypeptides of
the invention comprise, or alternatively consist of, one, two, three, four,
five or more of the
predicted epitopes described in Table 1A. It will be appreciated that
depending on the
analytical criteria used to predict antigenic determinants, the exact address
of the determinant
may vary slightly. Column 8, "Tissue Distribution" shows the expression
profile of tissue,
cells, and/or cell line libraries which express the polynucleotides of the
invention. The first
number in column 8 (preceding the colon), represents the tissue/cell source
identifier code
corresponding to the key provided in Table 4. Expression of these
polynucleotides was not
observed in the other tissues and/or cell libraries tested. For those
identifier codes in which
the first two letters are not "AR", the second number in column 8 (following
the colon),
represents the number of times a sequence corresponding to the reference
polynucleotide
sequence (e.g., SEQ ID NO:X) was identified in the tissue/cell source. Those
tissue/cell
source identifier codes in which the first two letters are "AR" designate
information
generated using DNA array technology. Utilizing this technology, cDNAs were
amplified by
PCR and then transferred, in duplicate, onto the array. Gene expression was
assayed through
hybridization of first strand cDNA probes to the DNA array. cDNA probes were
generated
from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis
was performed in the presence of 33P dCTP, using oligo(dT) toprime reverse
transcription.
After hybridization, high stringency washing conditions were employed to
remove non-
specific hybrids from the array. The remaining signal, emanating from each
gene target, was
measured using a Phosphorimager. Gene expression was reported as Phosphor
Stimulating
Luminescence (PSL) which reflects the level of phosphor signal generated from
the probe
hybridized to each of the gene targets represented on the array. A local
background signal
subtraction was performed before the total signal generated from each array
was used to
normalize gene expression between the different hybridizations. The value
presented after
"[array code]:" represents the mean of the duplicate values, following
background subtraction.
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CA 02395178 2002-06-19
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and probe normalization. One of skill in the art could routinely use this
information to
identify normal and/or diseased tissues) which show a predominant expression
pattern of the
corresponding polynucleotide of the invention or to identify polynucleotides
which show
predominant and/or specific tissue and/or cell expression. Column 9 provides
the
chromosomal location of polynucleotides corresponding to SEQ ID NO:X.
Chromosomal
location was determined by finding exact matches to EST and cDNA sequences
contained in
the NCBI (National Center for Biotechnology Information) UniGene database.
Given a
presumptive chromosomal location, disease locus association was determined by
comparison
with the Morbid Map, derived from Online Mendelian Inheritance in Man (Online
Mendelian
Inheritance in Man, OMIMT"''. McKusick-Nathans Institute for Genetic Medicine,
Johns
Hopkins University (Baltimore, MD) and National Center for Biotechnology
Information,
National Library of Medicine (Bethesda, MD) 2000. World Wide Web URL:
http://www.ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of
the Query
overlaps with the chromosomal location of a Morbid Map entry, an OMIM
identification
number is disclosed in column 10 labeled "OMIM Disease References)". A key to
the
OMIM reference identification numbers is provided in Table 5.
[9J Table 1B summarizes additional polynucleotides, encompassed by the
invention
(including cDNA clones related to the sequences (Clone ID NO:Z), contig
sequences (contig
identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)),
and genomic
sequences (SEQ ID NO:B). The first column provides a unique~clone identifier,
"Clone ID
NO:Z", for a cDNA clone related to each contig sequence. The second column
provides the
sequence identifier, "SEQ ID NO:X", for each coritig sequence. The third
column provides a
unique contig identifier, "Contig ID:" for each contig sequence. The fourth
column, provides
a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the
corresponding row of
the table: The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for
a fragment of the BAC clone identified in column four of the corresponding row
of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide
position numbers)
within the polynucleotide sequence of SEQ ID NO:B which delineate certain
polynucleotides
of the invention that are also exemplary members of polynucleotide sequences
that encode
polypeptides of the-invention (e.g., polypeptides containing amino acid
sequences encoded
by the polynucleotide sequences delineated in column six, and fragments and
variants
thereof).
CA 02395178 2002-06-19
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[10] Table 2 summarizes homology and features of some of the polypeptides of
the
invention. The first column provides a unique clone identifier, "Clone ID
NO:Z",
corresponding to a cDNA clone disclosed in Table 1A. The second column
provides the
unique contig identifier, "Contig ID:" corresponding to contigs in Table 1A
and allowing for
correlation with the information in Table 1A. The third column provides the
sequence
identifier, "SEQ ID NO:X", for the contig polynucleotide sequence. The fourth
column
provides the analysis method by which the homology/identity disclosed in the
Table was
determined. Comparisons were made between polypeptides encoded by the
polynucleotides
of the invention and either a non-redundant protein database (herein referred
to as "NR"), or
a database of protein families (herein referred to as "PFAM") as further
described below.
The fifth column provides a description of the. PFAM/NR hit having a
significant match to a
polypeptide of the invention. Column six provides the accession number of the
PFAM/NR
hit disclosed in the fifth column. Column seven, "Score/Percent Identity",
provides a quality
score or the percent identity, of the hit disclosed in columns five and six.
Columns 8 and 9,
"NT From" and "NT To" respectively, delineate the polynucleotides in "SEQ ID
NO:X" that
encode a polypeptide having a significant match to the PFAM/NR database as.
disclosed in
the fifth and sixth columns. In specific embodiments polypeptides of the
invention comprise,
or alternatively consist of, an amino acid sequence encoded by a
polynucleotide in SEQ ID
NO:X as delineated in columns 8 and 9, or fragments or variants thereof.
[11] Table 3 provides polynucleotide sequences that may be disclaimed
according to
certain embodiments of the invention. The first column provides a_unique clone
identifier,
"Clone ID", for a cDNA clone related to contig sequences disclosed in Table I
A. The
second column provides the sequence identifier, "SEQ ID NO:X", for contig
sequences
disclosed in Table 1A. The third column provides the unique contig.identifier,
"Contig ID:",
for contigs disclosed in Table IA. The fourth column provides a unique integer
'a' where 'a'
is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and
the fifth
column provides a unique integer 'b' where 'b' is any integer between 15 and
the final
nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of
nucleotide
residues shown in SEQ ID NO:X, and where b is greater than or equal to a + 14.
For each of
the polynucleotides shown as SEQ ID NO:X, the uniquely defined integers can be
substituted
into the general formula of a-b, and used to describe polynucleotides which
may be
preferably excluded from the invention. In certain embodiments, preferably
excluded from
the invention are at least one, two, three, four, five, tew, or more of the
polynucleotide
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CA 02395178 2002-06-19
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sequences) having the accession numbers) disclosed in the sixth column of this
Table
(including for example, published sequence in connection with a particular BAC
clone). In
further embodiments, preferably excluded from the invention are the specific
polynucleotide
sequences) contained in the clones corresponding to at least one, two, three,
four, five, ten,
or more of the available material having the accession numbers identified in
the sixth column
of this Table (including for example, the actual sequence contained in an
identified BAC
clone).
[12] Table 4 provides a key to the tissue/cell source identifier code
disclosed in Table
1A, column 8. Column'1 provides the tissue/cell source identifier code
disclosed in Table 1A,
Column 8. Columns 2-5 provide a description of the tissue or cell source.
Codes
corresponding to diseased tissues are indicated in column 6 with the word
"disease". The use
of the word "disease" in column 6 is non-limiting. The tissue or cell source
may be specific
(e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a
normal portion
of a diseased organ). Furthermore, tissues and/or cells lacking the "disease"
designation may
still be derived from sources directly or indirectly involved in a disease
state or disorder, and
therefore may have a further utility in that disease state or disorder. In
numerous cases where
the tissue/cell source is a library, column 7 identifies the vector used to
generate the library.
[13] Table S provides a key to the OMIM reference identification numbers
disclosed in
Table IA, column 10. OMIM reference identification numbers (Column 1) were
derived
from Online Mendelian Inheritance in Man (Online Mendelian'Inheritance in Man,
OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University
(Baltimore,
MD) and National Center for Biotechnology Information, National Library of
Medicine;
(Bethesda, MD) 2000. World Wide Web URL: http://www.ncbi.nlm.nih.gov/omim/).
Column 2 provides diseases associated with the cytologic band disclosed in
Table 1A,
column 9, as determined using the Morbid Map database.
[14] Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation
numbers of deposits made with the ATCC in connection with the present
application.
[15] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers
and
vector information relating to these cDNA libraries.
[16] Table 8 provides a physical characterization of clones encompassed by the
invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for
certain cDNA clones of the invention, as described in Table 1A. The second
column provides
the size of the cDNA insert contained in the corresponding cDNA clone.
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Definitions
[17] The following definitions are provided to facilitate understanding of
certain terms
used throughout this specification.
[18] 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.
[19] As used herein, a "polynucleotide" refers to a molecule having a nucleic
acid
sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid
sequence
contained in SEQ ID NO:X (as described in column 3 of Table 1A) or the
complement
thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2
of Table
1 A and contained within a library deposited with the ATCC); a nucleotide
sequence encoding
the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in
column 6
of Table 1B or a fragment or variant thereof; or a nucleotide.coding sequence
in SEQ ID
NO:B as defined in column 6 of Table IB or the complement thereof. For
example, the
polynucleotide can contain the nucleotide sequence of the full length cDNA
sequence,
including the 5' and 3' untranslated sequences, the coding region, as well as
fragments,
epitopes, domains, and variants of the nucleic acid sequence. Moreover, as
used herein, a
"polypeptide" refers to a molecule having an amino acid sequence encoded by a
polynucleotide of the invention as broadly-defined (obviously excluding poly-
Phenylalanine
or poly-Lysine peptide sequences which result from translation of a polyA tail
of a sequence
corresponding to a cDNA).
[20J In the present invention,. "SEQ ID NO:X" was often generated by
overlapping
sequences contained in multiple clones (contig analysis). A representative
clone containing
all or most of the sequence for SEQ ID NO:X is deposited at Human Genome
Sciences, Iric.
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(HGS) in a catalogued and archived library. As shown, for example, in column 2
of Table
1 A, each clone is identified by a cDNA Clone ID (identifier generally
referred to herein as
Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone
ID is all the
information needed to retrieve a given clone from the HGS library.
Furthermore, certain
clones disclosed in this application have been deposited with the ATCC on
October 5, 2000,
having the ATCC designation numbers PTA 2574 and PTA 2575; and on January 5,
2001,
having the depositor reference numbers TS-1, TS-2, AC-1, and AC-2. In addition
to the
individual cDNA clone deposits, most of the cDNA libraries from which the
clones were
derived were deposited at the American Type Culture Collection (hereinafter
~"ATCC").
Table 7 provides a list of the deposited cDNA libraries. One can use the Clone
ID NO:Z to
determine the library source by reference to Tables 6 and 7. Table 7 lists the
deposited
cDNA libraries by name and links each library to an ATCC Deposit. Library
names contain .
four characters, for example, "HTWE." The name of a cDNA clone (Clone ID)
isolated from
that library begins with the same four characters, for example "HTWEP07". As
mentioned
below, Table 1A correlates the Clone ID names with SEQ ID NO:X. Thus, starting
with an
SEQ ID NO:X, one can use Tables 1, 6 and 7 to determine the corresponding
Clone ID,
which library it came from and which ATCC deposit the library is contained in.
Furthermore,
it is possible to retrieve a given cDNA clone from the source library by
techniques known in
the art and described elsewhere herein. The ATCC is located at 10801
University Boulevard,
Manassas, Virginia 20110-2209, USA. The ATCC deposits were made.pursuant to
the terms
of the Budapest Treaty on the international recognition of the deposit of
microorganisms for
the purposes of patent procedure.
[21] In specific embodiments, the polynucleotides of the invention are at
least 15, at
least 30, at least 50, at least 100, at least 125, at least 500, or at least
1000 continuous
nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15
kb, 10 kb, 7.Skb, 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).
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(22] A "polynucleotide" of the present invention also includes those
polynucleotides
capable of hybridizing, under stringent hybridization conditions, to sequences
contained in
SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two,
three, four,
or more of the polynucleotide fragments described herein), the polynucleotide
sequence
delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or
cDNA sequences
contained in Clone ID NO:Z (e.g., the complement of any one, two, three, four,
or more of
the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones
deposited
with the ATCC, described herein), and/or the polynucleotide sequence
delineated in column
6 of Table IB or the complement thereof. "Stringent hybridization conditions"
refers to an
overnight incubation at 42 degree C in a solution comprising 50% formamide, Sx
SSC (750
mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), Sx
Denhardt's
solution, 10% dextran sulfate, and 20 ~g/ml denatured, sheared salmon sperm
DNA,
followed by washing the filters in O.lx SSC at about 65 degree C.
[23] 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 NaHzP04; 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). .
[24] 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.
[25] 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
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
containing a poly (A) stretch or the complement thereof (e.g., practically any
double-stranded
cDNA clone generated using oligo dT as a primer).
[26] 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.
[27] The polypeptide of the present invention can be composed of amino acids
joined to
each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres, and may
contain amino acids other than the 20 gene-encoded amino acids. The
polypeptides may be
modified by either natural processes, such as posttranslational processing, or
by chemical
modification techniques which are well known in the art. Such modifications
are well
described in basic texts and in more detailed monographs, as well as in a
voluminous
research literature. Modifications can occur anywhere in a polypeptide,
including the peptide
backbone, the amino acid side-chains and the amino or carboxyl termini. It
will be
appreciated that the same type of.modification may be present in the.same or
varying degrees
at several sites in a given polypeptide. Also, a given polypeptide may contain
many types of
modifications. Polypeptides may be branched, for example, as a result of
ubiquitination, and
they may be cyclic, with or without branching. Cyclic, branched, and branched
cyclic
polypeptides may result from posttranslation natural processes or may be made
by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation,
amidation,
covalent attachment of flavin, covalent attachment of a heme moiety, covalent
attachment of
a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid
derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine, formation
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of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor
formation,
hydroxylation, iodination, methylation, myristoylation,. oxidation,
pegylation, proteolytic
processing, phosphorylatiori, prenylation, racemization, selenoylation,
sulfation, transfer-
RNA mediated addition of amino acids to proteins such as arginylation, and
ubiquitination.
(See, for instance, PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2nd
Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993);
POSTTRANSLATIONAL COVALENT.MODIFICATION OF PROTEINS, B. C. Johnson,
Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth.
Enzymol. 182:626-
646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)). '
[28] "SEQ ID NO:X" refers to a polynucleotide sequence described, for example,
in
Tables lAor 2, while "SEQ ID NO:Y" refers to a polypeptide sequence described
in column
6 of Table 1A. SEQ ID NO:X is identified by an integer specified in column 4
of Table 1A.
The polypeptide sequence~SEQ ID NO:Y is a translated open reading frame (ORF)
encoded
by polynucleotide SEQ ID NO:X. "Clone ID NO:Z" refers to a cDNA clone
described in
column 2 of Table 1A.
[29] "A polypeptide having functional activity" refers to a polypeptide
capable of
displaying one or more known functional activities associated with a full-
length (complete)
protein. Such functional activities include, but are not limited to,
biological activity,
antigenicity [ability to bind (or compete with a polypeptide for binding) to
an anti-
polypeptide antibody], immunogenicity (ability to generate antibody which
binds to a
specific polypeptide of the invention), ability'to form multimers with
polypeptides of the
invention, and ability to bind to a receptor or ligand for a polypeptide.
[30] The polypeptides of the invention can be assayed for functional activity
(e.g.
biological activity) using or routinely modifying assays known in the art, as
well as assays
described herein. Specifically, one of skill in the art may routinely assay
ABC Transport
Receptor polypeptides (including fragments and variants) of the invention for
activity using
assays as described in Examples 53 and 54.
[31] "A polypeptide having biological activity" refers to a polypeptide
exhibiting
activity similar to, but not necessarily identical to, an activity of a
polypeptide of the present
invention, including mature forms, as measured in a particular biological
assay, with or
without dose dependency. In the case where dose dependency does exist, it need
not be
identical to that of the polypeptide, but rather substantially similar to the
dose-dependence in
a given activity as compared to the polypeptide of the present invention
(i.e., the candidate
12
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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).
[32] Table 1A summarizes some of the polynucleotides encompassed by the
invention
(including contig sequences (SEQ ID NO:X) and clones (Clone .ID NO:Z) and
further
summarizes certain characteristics of these polynucleotides and the
polypeptides encoded
thereby.
13
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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16
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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Image
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
[33] The first column in Table 1A provides the gene number in the application
corresponding to the clone identifier. The second column in Table 1 A provides
a unique
"Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed in
Table 1A.
This clone ID references the cDNA clone which contains at least the 5' most
sequence of the
assembled contig and at least a portion of SEQ ID NO:X was determined by
directly
sequencing the referenced clone. The reference clone may have more sequence
than
described in the sequence listing or the clone may have less. In the vast
majority of cases,
however, the clone is believed to encode a full-length polypeptide. In the
case where a clone
is not full-length, a full-length cDNA can be obtained by methods described
elsewhere
herein.
[34] The third column in Table 1A provides a unique "Contig ID" identification
for
each contig sequence. The fourth column provides the "SEQ ID NO:" identifier
for each of
the contig polynucleotide sequences disclosed in Table 1A. The fifth column,
"ORF (From-
To)", provides the location (i.e., nucleotide position numbers) within the
polynucleotide
sequence "SEQ ID NO:X" that delineate the preferred open reading frame (ORF)
shown in
the sequence listing and referenced in Table 1A, column 6, as SEQ ID NO:Y.
Where the
nucleotide position number "To" is lower than the nucleotide position number
"From", the
preferred ORF is the reverse complement of the referenced polynucleotide
sequence.
[35] The sixth column in Table 1A provides the corresponding SEQ ID NO:Y for
the
polypeptide sequence encoded by the preferred ORF delineated in column 5. In
one
embodiment, the invention provides an amino acid sequence comprising, or
alternatively
consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated
by "ORF
(From-To)". Also provided are polynucleotides encoding such amino acid
sequences and the
complementary strand thereto.
[36] . Column 7 in Table 1A lists residues comprising epifopes contained in
the
polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using
the
algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The
Jameson-Wolf
antigenic analysis was performed using the computer program PROTEAN (Version
3.11 for
the Power Macintosh, DNASTAR, Inc., 1228 South Park Street Madison, WI). In
specific
embodiments, polypeptides of the invention comprise, or alternatively consist
of, at least one,
two, three, four, five or more of the predicted epitopes as described in Table
1A. It will be
appreciated that depending on the analytical criteria used to predict
antigenic determinants,
the exact address of the determinant may vary slightly.
19
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
[37] Column 8 in Table 1 A provides an expression profile and library code:
count for
each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which can
routinely be
combined with the information provided in Table 4 and used to determine the
tissues, cells,
and/or cell line libraries which predominantly express the polynucleotides of
the invention.
The first number in column 8 (preceding the colon), represents the tissue/cell
source
identifier code corresponding to the code and description provided in Table 4.
For those,
identifier codes in which the first two letters are not "AR", the second
number in column 8
(following the colon) represents the number of times a sequence corresponding
to the
reference polynucleotide sequence was identified in the tissue/cell source.
Those tissue/cell
source identifier codes in which the first two letters are "AR" designate
information
generated using DNA array technology. Utilizing this technology, cDNAs were
amplified by
PCR and then transferred, in duplicate, onto the array. Gene expression was
assayed through
hybridization of first strand cDNA probes to the DNA array. cDNA probes were
generated
from total RNA extracted from a variety of different tissues and cell lines.
Probe synthesis
was performed in the presence of 33P dCTP, using oligo(dT) to prime reverse
transcription.
After hybridization, high stringency washing conditions were employed to
remove non-
specific hybrids from the array. The remaining signal, emanating from each
gene target, was
measured using a Phosphorimager. Gene expression was reported as Phosphor
Stimulating
Luminescence (PSL) which reflects the level of phosphor signal generated from
the probe
hybridized to each of the gene targets represented on the array. A local
background signal
subtraction was performed before the total signal generated from each array
was used to
normalize gene expression between the different hybridizations. The value
presented after
"[array code]:" represents the mean of the duplicate values, following
background subtraction
and probe normalization. One of skill in the art could routinely use this
information to
identify normal and/or diseased tissues) which show a predominant expression
pattern of the
corresponding polynucleotide of the invention or to identify polynucleotides
which show
predominant and/or specific tissue and/or cell expression.
[38J Column 9 in Table 1 A provides, a chromosomal map location for certain
polynucleotides of the invention. Chromosomal location was determined by
finding exact
matches to EST and cDNA sequences contained in the-NCBI (National Center for
Biotechnology Information) UniGene database. Each sequence in the UniGene
database is
assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are
believed to be
derived from a single gene. Chromosomal mapping data is often available for
one or more
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
sequence() in a .UniGene cluster; this data (if consistent) is then applied to
the cluster as a
whole. .Thus, it is possible to infer the chromosomal location _of a new
polynucleotide
sequence by determining its identity with a mapped UniGene cluster.
[39] A modified version of the computer program BLASTN (Altshul et al., J.
Mol.
Biol. 215:403-410 (1990); and Gish and States, Nat. Genet. 3:266-272 (1993))
was used to
search the UniGene database for EST or cDNA sequences that contain exact or
near-exact
matches to a polynucleotide sequence of the invention (the 'Query'). A
sequence from the
UniGene database (the 'Subject') was said to be an exact match if it contained
a segment of
50 nucleotides in length such that 48 of those nucleotides were in the same
order as found in
the Query sequence. If all of the matches that met this criteria were in the
same UniGene
cluster, and mapping data was available for this cluster, it is indicated in
Table 1A under the
heading "Cytologic Band". Where a cluster had been further localized to a
distinct cytologic
band, that band is disclosed; where no banding information was available, but
the gene had
been localized to a single chromosome, the chromosome is disclosed. .
[40] Once a presumptive chromosomal location was determined for a
polynucleotide of
the invention, an associated disease locus was identified by comparison with a
database of
diseases which have been experimentally associated with genetic loci. The
database used was
the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal
location of a
polynucleotide of the invention (Query sequence) was associated with a disease
in the
Morbid Map database, an OMIM reference identification number was noted in
column 10,
Table 1 A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference
identification numbers (column 1), and provides a description of the
associated disease in
Column 2.
21
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
TABLE 1B
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: NO:B From-To
HFRBM84 16 839090 AC034250 35 1-318
HADGE44 17 864257 AC015844 36 1-127
744-865
1513-1608
2730-3005
3060-3413
3981-4150
4205-4415
5050-5210
6026-6099
6171-6422
6993-7111
7324-7465
10120-10709
12451-12856
12945-13025
13498-13788
14369-14422
14540-15439
HADGE44 17 864257 AC015844 37 1-747
HADGF.44 17 864257 AC015844 38 1-ll04
22
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WO 01/55208 PCT/USO1/01357
[41] Table 1B summarizes additional polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone ID NO:Z), contig
sequences (contig
identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)),
and genomic
sequences (SEQ ID NO:B). The first column provides a unique clone identifier,
"Clone ID
NO:Z", for a cDNA clone related to each contig sequence. The second column
provides the
sequence identifier, "SEQ ID NO:X", for each contig sequence. The third column
provides a
unique contig identifier, "Contig ID:" for each contig sequence. The fourth
column, provides
a BAC identifier "BAC ID NO:A" for the BAC clone referenced in the
corresponding row of
the table. The fifth column provides the nucleotide sequence identifier, "SEQ
ID NO:B" for
a fragment of the BAC clone identified in column four of the corresponding row
of the table.
The sixth column, "Exon From-To", provides the location (i.e., nucleotide
position numbers).
within the polynucleotide sequence of SEQ ID NO:B which delineate certain
polynucleotides
of the invention that are also exemplary members of polynucleotide sequences
that encode
polypeptides of the invention (e.g., polypeptides containing amino acid
sequences encoded
by the polynucleotide sequences delineated in column six, and fragments and
variants
thereof).
S
23
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
o ~ r
E' 0 0
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24
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
a, 00 0. d d
~n d ~O ~n d
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M .~ N N .~
0 o M o 0
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
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CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
[42] Table 2 further characterizes certain encoded polypeptides of the
invention, by
providing the results of comparisons to protein and protein family databases.
The first
column provides a unique clone identifier, "Clone ID NO:", corresponding to a
cDNA clone
disclosed in Table 1A. The second column provides the unique contig
identifier, "Contig
ID:" which allows correlation with the information in Table 1 A. The third
column provides
the sequence identifier, "SEQ ID NO:", for the contig polynucleotide
sequences. The fourth
column provides the analysis method by which the homology/identity disclosed
in the Table
was determined. The fifth column provides a description of the PFAM/NR hit
identified by
each analysis. Column six provides the accession number of the PFAM/NR hit
disclosed in
the fifth column. Column seven, score/percent identity, provides a quality
score or the
percent identity, of the hit disclosed in column five. Comparisons were made
between
polypeptides encoded by polynucleotides of the invention and a non-redundant
protein
database (herein referred to as "NR"), or a database of protein families
(herein referred to as
"PFAM"), as described below.
[43] The NR database, which comprises the NBRF PIR database, the NCBI GenPept
database, and the SIB SwissProt and TrEMBL databases, was made non-redundant
using the
computer program nrdb2 (Warren Gish, Washington University in Saint Louis).
Each of the
polynucleotides shown in Table 1A, column 3 (e.g., SEQ ID NO:X or the 'Query'
sequence)
was used to search against the NR database. The computer program BLASTX was
used to
compare a 6-frame translation of the Query sequence to the NR database (for
information
about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403-
410 (1990);
and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the
sequence that is most
similar to the Query sequence (the highest scoring 'Subject') is shown in
column five of
Table 2 and the database accession number for that sequence is provided in
column six. The
highest scoring 'Subject' is reported in Table 2 if (a) the estimated
probability that the match
occurred by chance alone is less than 1.0e-07, and (b) the match was not to a
known
repetitive element. BLASTX returns alignments of short polypeptide segments of
the Query
and Subject sequences which share a high degree of similarity; these segments
are known as
High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity
between the
Query and the Subject for each HSP as a percent identity in Column 7. The
percent identity is
determined by dividing the number of exact matches between the two aligned
sequences in
the .HSP, dividing by the number of Query amino acids in the HSP and
multiplying by 100.
31
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that
generates
an HSP are delineated by columns 8 and 9 of Table 2.
[44] The PFAM database, PFAM version 2.1, (Sonnhammer et al., Nucl. Acids
Res.,
26:320-322, 1998)) consists of a series of multiple sequence alignments; one
alignment for
each protein family. Each multiple sequence alignment is converted into a
probability model
called a Hidden Markov Model, or HMM, that represents the position-specific
variation
among the sequences that make up the multiple sequence alignment (see, e.g.,
Durbin et al.,
Biological sequence analysis: probabilistic models of proteins and nucleic
acids, Cambridge
University Press, 1998 for the theory of HMMs). The program HMMER version 1.8
(Sean
Eddy, Washington University in Saint Louis) was used to compare the predicted
protein
sequence for each Query sequence (SEQ ID NO:Y in Table 1A) to each of the HMMs
derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to
be a
significant match to a polypeptide of the invention if the score returned by
HMMER 1.8 was
greater than 0.8 times the HMMER 1.8 score obtained with the most distantly
related known
member of that protein family. The description of the PFAM family which shares
a
significant match with a polypeptide of the invention is listed in column 5 of
Table 2, and
the database accession number of the PFAM hit is provided in column 6. Column
7 provides
the score returned by HMMER version 1.8 for the alignment. Columns 8 and 9
delineate the
polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which
show a
significant match to a PFAM protein family.
[45] As mentioned, columns 8 and 9 in Table 2, "NT From" and "NT To",
delineate the
polynucleotides of "SEQ ID NO:X" that encode a polypeptide having a
significant match to
the PFAM/NR database as disclosed in the fifth column. In one embodiment, the
invention
provides a protein comprising, or alternatively consisting of, a polypeptide
encoded by the
polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also
provided
are polynucleotides encoding such proteins, and the complementary strand
thereto.
[46] The nucleotide sequence SEQ ID NO:X and the translated' SEQ ID NO:Y are
sufficiently accurate and otherwise suitable for a variety of uses well known
in the art and
described further below. For instance, the nucleotide sequences of SEQ ID NO:X
are useful
for designing nucleic acid hybridization probes that will detect nucleic acid
sequences
contained in SEQ ID NO:X or the cDNA contained in Clone ID NO:Z. These probes
will
also hybridize to nucleic acid molecules in biological samples, thereby
enabling immediate
applications in chromosome mapping, linkage analysis, tissue identification
and/or typing,
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and a variety of forensic and diagnostic methods of the invention. Similarly,
polypeptides
identified from SEQ ID NO:Y may be used to generate antibodies which bind
specifically to
these polypeptides, or fragments thereof, and/or to the polypeptides encoded
by the cDNA
clones identified in, for example, Table 1A.
[47] 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).
[48] Accordingly, for those applications requiring precision in the nucleotide
sequence
or the amino acid sequence, the presenf invention provides not only the
generated nucleotide
sequence identified as SEQ ID NO:X, and a predicted translated amino acid
sequence
identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing cDNA
Clone ID
NO:Z (deposited with the ATCC on October 5, 2000, and receiving ATCC
designation
numbers PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and
having
depositor reference numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth,
for example,
in Table 1A, 6 and 7). The nucleotide sequence of each deposited clone can
readily be
determined by sequencing the deposited clone in accordance with known methods.
Further,
techniques known in the art can be used to verify the nucleotide sequences of
SEQ ID NO:X.
[49] The predicted amino acid sequence can then be verified from such
deposits.
Moreover, the amino acid sequence of the protein encoded by a particular clone
can also be
directly determined by peptide sequencing or by expressing the protein in a
suitable host cell
containing the deposited human cDNA, collecting the protein, and determining
its sequence.
RACE Protocol For Recovery of Full-Length Genes'
[50] Partial cDNA clones can be made full-length by utilizing the rapid
amplification of
cDNA ends (RACE) procedure described in Frohman, M.A., et al., Proc. Nat'l.
Acad. Sci.
USA, 85:8998-9002 (1988). A cDNA clone missing either the 5' or 3' end can be
reconstructed to include the absent base pairs extending to the translational
start or stop
codon, respectively. In some cases, cDNAs are missing the start codon of
translation,
33
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therefor. The following briefly describes a modification of this original 5'
RACE procedure.
Poly A+ or total RNA is reverse transcribed with Superscript II (Gibco/BRL)
and an
antisense or complementary primer specific to the cDNA sequence. The primer is
removed
from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA
is then
tailed with dATP and terminal deoxynucleotide transferase (GibcoBRL). Thus, an
anchor
sequence is produced which is needed for PCR amplification. The second strand
is
synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer
Cetus), an
oligo-dT primer containing three adjacent restriction sites (XhoI, SaII and
CIaI) at the 5' end
and a primer containing just these restriction sites. This double-stranded
cDNA is PCR
amplified for 40 cycles with the same primers as well as a nested cDNA-
specific antisense
primer. The PCR products are size-separated on an ethidium bromide-agarose gel
and the
region of gel containing cDNA products the predicted size of missing protein-
coding DNA is
removed. cDNA is purified from the agarose with the Magic PCR Prep kit
(Promega),
restriction digested with XhoI or SaII, and ligated to a plasmid such as
pBluescript SKII
(Stratagene) at XhoI and EcoRV sites. This DNA is transformed into bacteria
and the
plasmid clones sequenced to identify the correct protein-coding inserts.
Correct 5' ends are
confirmed by comparing this sequence with the putatively identified homologue
and overlap
with the partial cDNA clone. Similar methods known in the art and/or
commercial kits are
used to amplify and recover 3' ends.
[51] Several quality-controlled kits are commercially available for purchase.
Similar
reagents and methods to those above are supplied in kit form from GibcoBRL for
both 5' and
3' RACE for recovery of full length genes. A second kit is available from
Clontech which i,s
a modification of a related technique, SLIC (single-stranded ligation to
single-stranded
cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The
major
differences in procedure are that the RNA is alkaline hydrolyzed after reverse
transcription
and RNA ligase is used to join a restriction site-containing anchor primer to
the first-strand
cDNA. This obviates the necessity for the dA-tailing reaction which results in
a polyT.
stretch that is difficult to sequence past.
[52] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library
double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is
synthesized
with an antisense cDNA-specific primer and a plasmid-anchored .primer. These
primers are
removed and a symmetric PCR reaction is performed with a nested cDNA-specific
antisense
primer and the plasmid-anchored primer.
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RNA Ligase Protocol For Generating The 5' or 3' End Sequences To Obtain Full
Length
Genes
[53] Once a gene of interest is identified, several methods are available for
the
identification of the 5' or 3' portions of the gene which may not be present
in the original
cDNA plasmid. These methods include, but are not limited to, filter probing,
clone
enrichment using specific probes and protocols similar and identical to 5' and
3' RACE.
While the full length gene may be present ,in the library and can be
identified by probing, a
useful method for generating the 5' or 3' end is to use the existing sequence
information from
the original cDNA to generate the missing information. A method similar to 5'
RACE is
available for generating the missing 5' end of a desired full-length gene.
(This method was
published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684
(1993)). Briefly, a
specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA
presumably
containing full-length gene RNA transcript and a primer set containing a
primer specific to,
the ligated RNA oligonucleotide and a primer specific to a known sequence of
the gene of
interest, is used to PCR amplify the 5' portion of the desired full length
gene which may then
be sequenced and used to generate the full length gene. This method starts
with total RNA
isolated from the desired source, poly A RNA may be used but is not a
prerequisite for this
procedure. The RNA preparation may then be treated with phosphatase if
necessary to
eliminate 5' phosphate groups on degraded or damaged RNA which may interfere
with the
later RNA ligase step. The phosphatase if used is then inactivated and the RNA
is treated
with tobacco acid pyrophosphatase in order to remove the cap structure present
at the 5' ends
of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of
the cap
cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA
ligase.
This modified RNA preparation can then be used as a template for first strand
cDNA
synthesis using a gene specific oligonucleotide. The first strand synthesis
reaction can then
be used as a template for PCR amplification of the desired 5' end using a
primer specific to
the ligated RNA oligonucleotide and a primer specific to the known sequence of
the gene of
interest. The resultant product is then sequenced and analyzed to confirm that
the 5' end
sequence belongs to the relevant gene.
[54] The present invention also relates to vectors or plasmids which include
such DNA
sequences, as well as the use of the DNA sequences. The material deposited
with the ATCC
(deposited with the ATCC on October 5, 2000, and receiving ATCC designation
numbers
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PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and
receiving
ATCC designation numbers TS-1, TS-2, AC-1, and AC-2; and/or as set forth, for
example, in
Table 1A, Table 6, or Table 7) is a mixture of cDNA clones derived from a
variety of human
tissue and cloned in either a plasmid vector or a phage .vector, as described,
for example, in
Table 7. These deposits are referred to as "the deposits" herein. The tissues
from which
some of the clones were derived are listed in Table 7, and the vector in which
the
corresponding cDNA is contained is also indicated in Table 7. The deposited
material
includes cDNA clones corresponding to SEQ ID NO:X described, for example, in
Table 1A
(Clone ID NO:Z). A clone which is isolatable from the ATCC Deposits by use of
a sequence
listed as SEQ ID NO:X, may include the entire coding region of a human gene or
in other
cases such clone may include a substantial portion of the coding region of a
human gene.
Furthermore, although the sequence listing may in some instances list only a
portion of the
DNA sequence in a clone included in the ATCC Deposits, it is well within the
ability of one
skilled in the art to sequence the DNA included in a clone contained in the
ATCC Deposits
by use of a sequence (or portion thereof) described in, for example Tables 1
Aor 2 by
procedures hereinafter further described, and others apparent to those skilled
in the art.
[55] Also provided in Table 7 is the name of the vector which contains the
cDNA
clone. Each vector is routinely used in the art. The following additional
information is
provided for convenience.
[56] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(U.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (U.S. Patent Nos.
5,128,256 and
5,286,636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-
7600 (1988);
Aping-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17.9494 (1989)) and pBK
(Alting-
Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from
Stratagene
Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, CA, 92037. pBS
contains an
ampicillin resistance gene and pBK contains a neomycin resistance gene.
Phagemid pBS
may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK
may be
excised from the Zap Express vector. Both phagemids may be transformed into E.
coli strain
XL-1 Blue, also available from Stratagene.
[57] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and ~pCMVSport 3.0, were
obtained from Life Technologies, Inc., P. O. Box 6009, Gaithersburg, MD 20897.
All Sport
vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain
DH10B, also available from Life Technologies. See, for instance, Gruber, C.
E., et al., Focus
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15: 59- ( 1993). Vector lafmid BA (Bento Soares, Columbia University, New
York, NY)
contains an ampicillin resistance gene and can be transformed into E. coli
strain XL-1 Blue.
Vector pCR°2.1, which is available from Invitrogen, 1600 Faraday
Avenue, Carlsbad, CA
92008, contains an ampicillin resistance gene and may be transformed into E.
coli strain
DHIOB, available from Life Technologies. See, for instance, Clark, J. M., Nuc.
Acids Res.
16: 9677-9686 ( 1988) and Mead, D. et al., BiolTechnology 9: ( 1991 ).
[58] The present invention also relates to the genes corresponding to SEQ ID
NO:X,
SEQ ID NO:Y, and/or the deposited clone (Clone ID NO:Z). The corresponding
gene can be
isolated in accordance with known methods using the sequence information
disclosed herein.
Such methods include preparing probes or primers from the disclosed sequence
and
identifying or amplifying the corresponding gene from appropriate sources of
genomic
material.
[59] Also- provided in the present invention are allelic variants, orthologs,
and/or
species homologs. Procedures known in the art can be used to obtain full-
length genes, allelic
variants, splice variants, full-length coding portions, orthologs, and/or
species homologs of
genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides
encoded by
genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA
contained in Clone ID NO:Z, using information from the sequences disclosed
herein or the
clones deposited with the ATCC. For example, allelic variants and/or species
homologs may
be isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source for allelic variants
and/or the desired
homologue. .
[60] 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. -
[61] ~ 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.
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[62] The polypeptides of the present invention are preferably provided iman
isolated
form, and preferably are substantially purified. A recombinantly produced
version of a
polypeptide, including the secreted polypeptide, can be substantially purified
using
techniques described herein or otherwise known in the art, such as, for
example, by the one-
step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides
of the
invention also can be purified from natural, synthetic or recombinant sources
using
techniques described herein or otherwise known in the art; such as, for
example, antibodies of
the invention raised against the polypeptides of the present invention in
methods which are
well known in the art.
[63J -The present invention provides a polynucleotide comprising, or
alternatively
consisting of, the nucleic acid sequence of SEQ~ ID NO:X, and/or the- cDNA
sequence
contained in Clone ID NO:Z. The present invention also provides a polypeptide
comprising,
or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a
polypeptide
encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the
cDNA
contained in Clone ID NO:Z, and/or the polypeptide sequence encoded by a
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B. Polynucleotides
encoding a
polypeptide comprising, or alternatively consisting of the polypeptide
sequence of SEQ ID
NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA
contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B are also
encompassed by the
invention. The present invention further encompasses a polynucleotide ~
comprising, or
alternatively consisting of, the complement of the nucleic acid sequence of
SEQ ID NO:X, a
nucleic acid sequence encoding a polypeptide encoded by the complement of the
nucleic acid
sequence of SEQ ID NO:X, and/or the cDNA contained in Clone ID NO:Z.
[64] Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the sequences delineated in Table 1B column 6, or any combination thereof.
Additional,-
representative examples of.polynucleotides of the invention comprise, or
alternatively consist
of, one, two; three, four, five, six; seven, eight, nine, ten, or more of the
complementary
strands) of the sequences delineated in Table 1B column 6, or any combination
thereof. In
further embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in Table 1 B, column 6, and
have a nucleic acid
sequence which is different from that of the BAC fragment having the sequence
disclosed in
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SEQ ID NO:B (see Table 1B, column S). In.additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
Table 1B, column 6, and have a nucleic acid sequence which is different from
that published
for the BAC clone identified as BAC ID NO:A (see Table 1B, column 4). In
additional
embodiments, the above-described polynucleotides of the invention comprise, or
alternatively consist of, sequences delineated in Table 1B, column 6, and have
a nucleic acid
sequence which is different from that contained in the BAC clone identified as
BAC ID
NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[65] Further, representative examples of polynucleotides of the invention
comprise; or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID NO:Z
(see Table 1 B, column I ), or any combination thereof. Additional,
representative examples
of polynucleotides of the invention comprise, or alternatively consist of,
one, two, three, four,
five, six, seven, eight, nine, ten, or more of the complementary strands) of
the sequences
delineated in column 6 of Table 1 B which correspond to the same Clone ID NO:Z
(see Table
1B, column 1), or any combination thereof. In further embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table 1B,
column
1 ) and have a nucleic acid sequence which is different from that of the BAC
fragment having
the sequence disclosed in SEQ ID NO:B (see Table 1B, column S). In additional
embodiments, the above-described polynucleotides of the invention comprise, or
alternatively consist of, sequences delineated in column 6 of Table 1B which
correspond to
the same Clone ID NO:Z (see Table 1B, column 1) and have a nucleic acid
sequence which is.
different from that published for the BAC clone identified as BAC ID NO:A (see
Table 1B,
column 4). In additional embodiments, the above-described polynucleotides of
the invention
comprise, or alternatively consist of, sequences delineated in column 6 of
Table 1B which
correspond to the same Clone ID NO:Z (see Table 1B, column 1) and have a
nucleic acid
sequence which is different from that contained in the BAC clone identified as
BAC ID
NO:A (see Table 1B, column 4). Polypeptides encoded by these~polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
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are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[66] Further, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table IB which correspond to the same
contig sequence
identifer SEQ ID NO:X (see Table 1B, column 2), or any combination thereof.
Additional,
representative examples of polynucleotides of the invention comprise, or
alternatively consist
of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the
complementary
strands) of the sequences delineated in column 6 of Table IB which correspond
to the same
contig sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any
combination
thereof. In further embodiments, the above-described polynucleotides of the
invention
comprise, or alternatively consist of, sequences delineated in column 6 of
Table IB which
correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B,
column 2)
and have a nucleic acid sequence which is different from that of the BAC
fragment having
the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional
embodiments, the above-described polynucleotides of the invention comprise, or
alternatively consist of, sequences delineated in column 6 of Table 1B which
correspond to
the same contig sequence identifer SEQ ID NO:X (see Table IB, column 2) and
have a
nucleic acid sequence which is different from that published for the BAC clone
identified as
BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
column 6 of Table 1B which correspond to the same contig sequence identifer
SEQ ID NO:X
(see Table 1B, column 2) and have a nucleic acid sequence which is different
from that
contained in the BAC clone identified as BAC ID NO:A (See Table 1B, column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention. Additionally, fragments and variants of the above-described
polynucleotides and
polypeptides are also encompassed by the invention.
[67] Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four; five, six, seven, eight,
nine, ten, or more of
the sequences delineated in the same row of Table 1 B column 6, or any
combination thereof.
Additional, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
CA 02395178 2002-06-19
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complementary strands) of the sequences delineated in the same row of Table 1B
column 6,
or any combination thereof. In preferred embodiments, the polynucleotides of
the invention
comprise, or alternatively consist bf, one, two, three, four, five, six,
seven, eight, nine, ten, or
more of the complementary strands) of the sequences delineated in the same row
of Table
1B column 6, wherein sequentially delineated sequences in the table (i.e.
corresponding to
those exons located closest to each other) are directly contiguous in a 5' to
3' orientation. In
further embodiments, above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in the same row of Table 1B,
column 6, and
have a nucleic acid sequence which is different from that of the BAC fragment
having the
sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional
embodiments,
the above-described polynucleotides of the invention comprise, or
alternatively consist of, -
sequences delineated in the same row of Table 1B, column 6, and have a nucleic
acid
sequence which is different from that published for the BAC clone identified
as BAC ID
NO:A (see Table 1B, column 4). In additional embodiments, the above-described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated in
the same row of Table 1 B, column 6, and have a nucleic acid sequence which is
different
from that contained in the BAC clone identified as BAC ID NO:A (see Table 1B,
column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention.
[68] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B, and the polynucleotide sequence
of SEQ ID
NO:X (e.g., as defined in Table 1B, column 2) or fragments or variants
thereof. Polypeptides
encoded by these polynucleotides, other polynucleotides that encode these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
[69] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID NO:Z
(see Table 1B, column 1), and the polynucleotide sequence of SEQ ID NO:X
(e.g., as defined
in Table 1A or 1B) or fragments or variants thereof. In preferred embodiments,
the
delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to
the
same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other
polynucleotides
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that encode these polypeptides, and antibodies that bind these polypeptides
are also
encompassed by the invention.
[70] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in the same row of column 6 of Table 1B, and the
polynucleotide
sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or
variants
thereof. In preferred embodiments, the delineated sequences) and
polynucleotide sequence
of SEQ ID NO:X correspond to the same row of column 6 of Table 1B.
Polypeptides
encoded by these polynucleotides, other polynucleotides that encode these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
[71] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and,the S' 10
polynucleotides of the
sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize
to the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids that encode these polypeptides, and antibodies that bind
these
polypeptides are also encompassed by the invention. Additionally, fragments
and variants of
the above-described polynucleotides, nucleic acids, and polypeptides are also
encompassed
by the invention.
[72] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6'of Table 1B and the 5' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X are directly contiguous
Nucleic acids
which hybridize to the complement of these 20 contiguous polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
42
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[73] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of the
sequence of SEQ ID NO:X and the 5' 10 polynucleotides of the sequence of one
of the
sequences delineated in column 6 of Table 1B are directly contiguous. Nucleic
acids which
hybridize to the complement of these 20 contiguous polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions,
are also
encompassed by the invention. Polypeptides encoded by these polynucleotides
and/or
nucleic acids, other polynucleotides and/or nucleic acids encoding these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
Additionally,
fragments and variants of the above-described polynucleotides, nucleic acids,
and
polypeptides are also encompassed by the invention.
[74] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X and the 5' 10
polynucleotides of the
sequence of one of the sequences delineated in column 6 of Table 1B are
directly contiguous.
Nucleic acids which hybridize to the complement of these 20 contiguous
polynucleotides
under stringent hybridization conditions or alternatively, under lower
stringency conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides
and/or nucleic acids, other polynucleotides and/or nucleic acids encoding
these polypeptides,
and antibodies that bind these polypeptides are also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic acids,
and polypeptides, are also encompassed by the invention.
[75] In further specific embodiments, polynucleo.tides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another
sequence in column 6 are directly contiguous. Nucleic acids which hybridize to
the
complement of these 20 contiguous polyriucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention. .
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids encoding these polypeptides, and antibodies that bind
these polypeptides
are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by the
invention.
43
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[76] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of another
sequence in column 6 corresponding to the same Clone ID NO:Z (see Table 1B,
column 1)
are directly contiguous. Nucleic acids which hybridize to the complement of
these 20 lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids encoding
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention. Additionally, fragments and variants of the above-described
polynucleotides,
nucleic acids, and polypeptides are also encompassed by the invention.
[77] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of,, a polynucleotide sequence in which the 3' 10
polynucleotides of one
sequence in column 6 corresponding to the same contig sequence identifer SEQ
ID NO:X
(see Table 1B, column 2) are directly contiguous. Nucleic acids which
hybridize to the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids encoding these polypeptides, and antibodies that bind
these polypeptides
are also encompassed by the invention. Additionally, fragments arid variants
of the above-
described polynucleotides, nucleic acids, and polypeptides are also
encompassed by the
invention.
[78] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucl'eotides of another
sequence in column 6 corresponding to the same row are directly contiguous. In
preferred
embodiments, the 3' 10 polynucleotides of one of the sequences delineated in
column 6 of
Table 1B is directly contiguous with the 5' 10 polynucleotides of the next
sequential exon
delineated in Table 1 B, column 6. Nucleic acids which hybridize to the
complement of these
20 contiguous polynucleotides under stringent hybridization conditions or
alternatively,
under lower stringency conditions, are also encompassed by the invention.
Polypeptides
encoded by these polynucleotides and/or nucleic acids, other polynucleotides
and/or nucleic
acids encoding these polypeptides, and antibodies that bind these polypeptides
are also
44.
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
encompassed by the invention. Additionally, fragments and variants of the
above-described
polynucleotides, nucleic acids, and polypeptides are also encompassed by the
invention.
[79J Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are specifically
excluded from the scope of the present invention. Accordingly, for each contig
sequence
(SEQ ID NO:X) listed in the fourth column of Table 1A, preferably excluded are
one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of a-b,
where a is any integer between 1 and the final nucleotide minus 15 of SEQ ID
NO:X, b is an
integer of .15 to the final nucleotide of SEQ ID NO:X, where both a and b
correspond to the
positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater
than or equal
to a + 14. More specifically, preferably excluded are one or more
polynucleotides
comprising a nucleotide sequence described by the general formula of a-b,
where a and b are
integers as defined in columns 4 and 5, respectively, of Table 3. In specific
embodiments, the
polynucleotides of the invention do not consist of at least one, two, three,
four, five, ten, or
more of the specific polynucleotide sequences referenced by the Genbank
Accession No. as
disclosed in column 6 of Table 3 (including for example, published sequence in
connection
with a particular BAC clone). In further embodiments, preferably excluded from
the
invention are the specific polynucleotide sequences) contained in the clones
corresponding
to at least one, two, three, four, five, ten, or more of the available
material having the
accession numbers identified in the sixth column of this Table (including for
example, the
actual sequence contained in an identified BAC clone). In no way is this
listing meant to
encompass all of the sequences which may be excluded by the general formula,
it is just a
representative example. All references available through these accessions are
hereby
incorporated by reference in their entirety.
CA 02395178 2002-06-19
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TABLE 3
SEQ
ID EST Disclaimer
lone NO: ontig Range
ID X ID: of a ccession #'s
NO: Z Range
of b
HTFOB57 11 1201971l - 131715 - U66685, AI126698, AI992159,
1331 AA604377,
AI355573, AA523723, AI589741,
AA045146,
AA812594, AA085954, AA085953,
AA917648, A1800531, AI684462,
AA281007,
AA719856, AA705854, AI620440,
AA977873,
W47347, AA428564, H59649,
AA515298,
AA045255, AI342347, AI028246,
880339,
AA535613, AI129155, AA862715,
AA536067,
AI864034, AI905741, AW006676,
AW404049, AA552289, AI184189,
AI346779,
AA429520, AA309643, AA846640,
AA747728, F24595, H02423,
AW088429,
H11197, H59609, AA628329,
AA335316,
AI014512, H02321, W47451,
AA991326,
AA437120, AA192275, AA335655,
AA195630, 807941, 806392,
AI271814,
T12458, AA811669, AA552246,
AA775242,
AA732918, AA281173, F25492,
AA931765,
806391, AW069632, F19391,
T59746,
831819, T85620, AW376621,
T30711,
T93959, AA317259, 831832,
W81195,
AA969767, AI245151, AW129052,
AW002298, AA926731, AA195609,
AI474712, AI189541, 829713,
W81263,
AA923760, T16101, AA317516,
F34410,
AL119457, AL119324, T59628,
AL119399,
AI073952, AL042544, AL119511,
AI371243,
AL043152, AI433157, AI648567,
AI690946,
AI554821, AW151136, AI539771,
AI432644,
AI537677, A1494201, AI500659,
AI866465,
AI815232, AI801325, AI500523,
AI538850,
AI887775, AI582932, AI284517,
AI923989,
AI872423, AI590043, AI500706,
AI491776,
AI445237, AI289791, AW151138,
AI521560,
AI889189, AI500662, AI582912,
AW 172723,
AI284509, AI539800, AI5388.85,
AI889168,
AI440263, AI927233, AI866573,
AI633493,
' AI434256, AI866469, AI434242,
AI805769,
AI888661, AI500714, AI284513,
AI888118,
AI285439, AI436429, AI859991,
AI623736,
AI889147, AI355779, AI371228,
AI581033,
AI491710, AI431307, AI440252,
AI866786,
AI610557, AI860003, AI431316,
AI242736,
AI828574, AI887499, AW151979,
AI539781,
AI702065, AI539707, AI885949;
AI285419,
AI559957, AW089557, AI521571,
AI469775,
AI866581, AI431323, AI567953,
AI815150,
AI446495, AI867068, AA603709,
AI440238,
AI567971, AW082623, AL045500;
AI866820,
AI561170, AI583611, AI890907,
AI050666,
AI371251, AI866510, AI866461,
AI923046,
AI624529, AA464646, AL039390,
AL047422,
AI608807, AL048403, AI539723,
AI274759,
46
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AI610402, AI284516, AI433976,
AI288281,
AI804505, AL042365, A1539863,
AI366900,
AI539260, AI805774, AI499483,
AI366910,
AI923446, AL043021, AI432666,
AI225248,
AW197139, AI671642, AA835966,
AI355008,
AI582926, AI697324, AI275175,
AI921438,
AL079960, AL047187, AI678411,
AI887163,
AI277008, AL042515, AI499463,
AI273179,
AL134524, AI537191, AI610362,
AI926593,
AI440239, AI521596, AI783861,
AI499508,
AI537273, AI371265, AF213381,
Y16258,
Y16257, E02756, Y16256, AL133049,
Y08769, U90884, U77594, L10353,
AF008439, AL117578, AL133014,
AL122098,
577771, AR034821, AF031147,
AJ238278,
AF026124, AF090900, AF137367,
U30290,
AL137711, S79832, AL110222,
AF022363,
AF002985, A18777, AL122049,
X66862,
J05032, I89947, I48978, AL133558;
AL049314, A08913, AF185576,
AR038854,
AL133070, AB025103, A08912,
A27171,
AR068182, A08910, AL137476,
I89931,
A08909, AL049460, AL117585,
E02221,
I49625, A08908, U96683, S76508,
AL050277,
AF 114170, X80340, I89934,122272,
126207,
109499, X66975, D83989, AB019565,
AL080060, AF012536, AJ242859,
583440,
AL122118, AL117432, E15569,
AL080124,
I17544, A08916, AL137658,
AF106862,
AF113694, AR019470, AL137530,
AL050172,
AF057300, AF057299, AF104032,
AC006371,
AL110280, A08907, A86558,
AL049466,
E01812, X93495, AL133072,
AF055917,
IOU734, AL137276, AF100931,
AJ001838,
AL137283, AF118094, 569510,
AF113676,
E00617, E00717; E00778, AF158248,
AL137660, AL122121, E15582,
AF169154,
AF067790, AL122110, A57389,
U92068,
AL133080, AL133640, AL133081,
AF176651,
AF159615, AB007812, AL080127,
AL133077,
AF044323, AF146568, AF106657,
AF002672,
AL050092, AL133565, AF081195,
X63410,
A08911, E07361, I89944, AF119336,
- AF094480, A 12297, AL 137641,
AL 137459,
AL137523, AF162270, AF042090,
E02152,
AF113019, AL137556, AF090934,
A65340,
X79812, U95114, U58996, AF210052;
X00861, AL137521, AL080126,
U91329,
AL137665, AF113689, AL133645,
AF120268,
AF125949, 568736, X72889,
561953, A21103,
536676, A65341, AL110196,
AR000496,
I33392, U39656, AL117583,
AF151109,
X55446, AF047716, 237987,
AF090886,
U87620, I30339, I30334, AL137495,
AF180525, AL133010, AB008792,
AF102578,
297214, M92439, AB008791,
AL137537,
AF111112, AL133637, E03348,
AF028823,.
AL 137526, L 19437, Y 1 I
587, AF215669,
E04233 AL137478, A83556,
E03349,
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AL080159, AL 117626, AF 111851,
E 12806,
L30117, 146765, AL117416,
AR059958,
AF017790, AL137574, U42031,
AF061795,
Y14314, AF151685, AL133016,
AL023657,
AF I 99027, A90832, AL 117440,
U49434,
AF061981, U78525, I32738,
AL080148,
AL133113, E01614, E13364,
AJ006417,
AL133112, AL122123, AC004227,
AC002467, Y10080, S63521,
Y16645,
AL 137538, X59414, U00686,
AF040751,
X72387, U72621, AL137294,
AF195092,
AL110171, AF003737, U67958,
X06146, and
X70685.
HDPVY34 12 8391781 - 132315 - U66680, AI651024, AW338289,
1337 AA099357,
T84930, AI476130, AI651541,
T92055,
AA101906, AI082697, AI695358,
AC005495,
and AC005922.
HJMBP48 13 12102861 - 106215 - U66688, AA 116008, H67248,
1076 AA357634,
AI420480, H49149, AI168486,
AW291827,-
and AF047690.
HAGGS65 14 8553111 - 467 15 - AA029504 AC005922, and AB020629.
481
HCWDRO1 15 8391041 - 465 15 -
479
HFRBM84 16 8390901 - 309 15 - AC034250.
323
HADGE44 17 8642571 - 448 15 - N63063, AL041916, AI908941,
462 AA029504,
AB020629, AC005922, AC015844,
AC015844, and AC015844.
H7UBA56 18 11238331 - 474 15 -
488
HSLHCI 19 966325I - 244 15 - AA585098, T18597, 845895,
1 258 828735,
829445, AA585325, AA585476,
829218,
828892, AA170832, 829657,
82896?,
828965, AA283326, D60765,
AA585101,
D53447, 232887, D59751, C15406,
D54897,
D53161, C16315, D60844, D57491,
C16293,
828895, D53472, AI557734,
D61254, 232822,
C15069, AA585439, 228355,
AI557262,
AI546875, AI546999, C 16294,
C 15120,
D52835, AI541383, AI557864,
AI541356,
D59436, AA585155, D61185,
C16292,
AI557763, AI547006, AI547250,
AI526140,
829179, AI541365, AI525500,
AI557740,
AI546971, AI541374, AI525306,
AI541307,
AI535660, D57186, AI546945,
D55233,
AI541205, C16300, AI525431,
AI546921,
AI526078, C16305, AI526194,
AI557731,
AI557787, AI525316, C15762,
AI526184,
AI557727, AI541535, AI547039,
AI525556,
AI540967, AI541517, AI541013,
230131,
AA585356, AI536138, AI541346,
C15737,
C 16296, AI526016, D60730,
829177,
AI557807, AI546996, 829262,
AI526191,
829172, AI546891, AI541523,
C16290,
AI557718, AI525339, AI535639,
AI557758,
AI557084, AI541527, AI547202,
AI557408,
AI557809, C.14208, AI526109,
AI525320,
AI557155, AI547196, T19407,
AI540903,
AI557602, AI541034, AI525856,
AI526073,
233559, T41289, AA514191,
AI540974,
AI526113 AI547137 D54850
AI557264
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AI541321, AI525286, AI526180,
AI556967,
AA585453, C14723, AI541510,
A.A174170,
AA585430, AI557808, AI557279,
AI526024,
T41329, AI546829, AA585117,
D59458,
AI546954, AI546831, AI546901,
AI557533,
AI526112, AI540920, AI526158,
AI526195,
AI541345, AI557039, C 14322,
AI526117,
AI524890, AI524904, AI557799,
AI541514,
AI557852, AI547189, AI541422,
C14391,
AI525656, AI540944, AI557786,
AI525332,
AI541415, AI546828, AI541515;
AI541027,
AR062871, AR038855, A25909;
Y09813,
X55486, A20702, AR062872,
AR062873,
A20700, A43189, A43188, AR017907,
AJ244005, Y16359, A98420,
A98423,
A98432, A98436, A98417, A98427,
AF082186, AR038762, 232836,
D50010,
D13509, AJ244004, AR054723,
X81969,
D78345, AJ244003, X82786,
X76012,
AR031365, AC005913, AJ244006,
AJ243486,
A98767, A93963, A93964, I63120,
AR003381,
AR031358 AR008443, and AR017826.
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TABLE 4
Code Description Tissue Organ 11 DiseaseVector
-ine
AR022a Heart a Heart
AR023a Liver a Liver
AR024a mammar land a mamma land
AR025a Prostate a Prostate
AR026a small intestinea small intestine
AR027a Stomach a Stomach
AR028Blood B cells Blood B cells
AR029Blood B cells Blood B cells
activated acti vated
AR030Blood B cells Blood B cells
resting restin
AR031Blood T cells Blood T cells
activated activated
AR032Blood T cells Blood T cells
resting restin
AR033brain brain
AR034breast breast
AR035breast cancer breast cancer
AR036Cell Line CAOV3Cell Line
CAOV3
AR037cell line PA-1 cell line
PA-1
AR038cell line transformedcell line
transformed
AR039colon colon
AR040colon (9808co65R)colon (9808co65R)
AR041colon(9809co15)colon(9809co15)
AR042colon cancer colon cancer
AR043colon cancer colon cancer
(9808co64R) (9808co64R)
AR044colon cancer colon cancer
9809co 14 9809co14
AR045com clone 5 com clone
5
AR046com clone 6 corn clone
6
AR047com clone2 . corn clone2
AR048corn clone3 com clone3
AR049Corn Clone4 Corn Clone4
AR050Donor II B CellsDonor II
24hrs B Cells
24hrs
AR051Donor II B CellsDonor II
72hrs B Cells
72hrs
AR052Donor II B-CellsDonor II
24 hrs. B-Cells
24
hrs.
AR053Donor II B-CellsDonor II
72hrs B-Cells
72hrs
AR054Donor II RestingDonor II
B Cells- Resting
B
Cells
AR055.Heart Heart
AR056Human Lung (clonetech)Human Lung
(clonetech)
AR057Human Mammary Human Mammary
(clontech) (clontech)
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AR058 Human Thymus Human Thymus
(clonetech) (clonetech)
AR059 Jurkat (unstimulated)Jurkat
(unstimulated)
AR060 Kidney Kidne
AR061 Liver Liver
AR062 Liver(Clontech)Liver(Clontech)
AR063 Lymphocytes Lymphocytes
chronic
lymphocytic chronic lymphocytic
leukaemia
leukaemia
AR064 Lymphocytes Lymphocytes
diffuse large
B cell lymphomadiffuse large
B cell
lym homa
AR065 Lymphocytes Lymphocytes
follicular
1 m homa follicular
I m homa
AR066 normal breast normal breast
AR067 Normal Ovarian Normal Ovarian
(4004901) (4004901)
AR068 Normal Ovary Normal Ovary
95086045
95086045
AR069 Normal Ovary Normal Ovary
97016208
97016208
AR070 Normal Ovary Normal Ovary
98066005
98066005
AR071 Ovarian Cancer Ovarian Cancer
AR072 Ovanan Cancer Ovarian Cancer
(97026001 ) (97026001
)
AR073 Ovarian Cancer Ovarian Cancer '
(97076029) (97076029)
AR074 Ovarian Cancer Ovarian Cancer
(98046011 ) (98046011
)
AR075 Ovarian Cancer Ovarian Cancer
(98066019) (98066019)
AR076 Ovarian Cancer Ovarian Cancer
(98076017) (98076017)
AR077 Ovarian Cancer Ovarian Cancer
(98096001 ) (98096001
)
AR078 ovarian cancer ovarian cancer
15799
15799
AR079 Ovarian Cancer Ovarian Cancer
17717AID 17717AID
AR080 Ovarian Cancer Ovarian Cancer
400466481 400466481
AR081 Ovarian Cancer Ovarian Cancer
4005315A 1 4005315A
1
AR082 ovarian cancer ovarian cancer
94127303
94127303
AR083 Ovarian Cancer Ovarian Cancer
96069304
96069304
AR084 Ovarian Cancer Ovarian Cancer
97076029
97076029
AR085 Ovarian Cancer Ovarian Cancer
98076045
98076045
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AR086 ovarian cancer ovarian cancer
98096001 98096001
AR087 Ovarian Cancer Ovarian Cancer
9905C032ItC 9905C032RC
AR088 Ovarian cancer Ovarian cancer
9907 C00 9907
3rd C00 3rd
AR089 Prostate Prostate
AR090 Prostate (clonetech)Prostate
(clonetech)
AR091 rostate cancer rostate cancer
AR092 prostate cancerprostate
# 15176 cancer
#15176
AR093 prostate cancerprostate
#15509 cancer
#15509
AR094 prostate cancerprostate
#15673 cancer
#15673
AR095 Small IntestineSmall Intestine '
(Clontech) (Clontech)
AR096 S teen S lees
AR097 Thymus T cells Thymus T
activated cells
activated
AR098 Thymus T cells Thymus T
resting cells
restin
AR099 Tonsil Tonsil
AR100 Tonsil geminal Tonsil geminal
center center centroblast
centroblast
AR101 Tonsil germinalTonsil germinal
center B center B
cell cell
AR102 Tonsil 1 m h Tonsil I
node m h node
AR103 Tonsil memory Tonsil memory
B cell B
cell
AR104 Whole Brain Whole Brain
AR105 Xeno raft ES-2 Xeno raft
ES-2
AR106 Xeno raft SW626Xeno raft
SW626
H0009 Human Fetal Uni-ZAP
Brain XR
H0031 Human Placenta Human PlacentaPlacenta Uni-ZAP
XR
H0040 Human Testes Human TestesTestis diseaseUni-ZAP
Tumor Tumor XR
H0052 Human CerebellumHuman CerebellumBrain Uni-ZAP
XR
H0063 Human Th mus Human Th Th mus Uni-ZAP
mus XR
H0124 Human Human Sk Muscle diseaseUni-ZAP
Rhabdom osarcomaRhabdom osarcoma - XR
H0130 LNCAP untreatedLNCAP Cell Prostate Cell Uni-ZAP
Line Line XR
H0144 Nine Week Old 9 Wk Old Embryo Uni-ZAP
Early Early XR
Sta a Human Sta a Human
H0150 HumanE ididymusE ididymis Testis Uni-ZAP
XR
H0156 Human Adrenal Human AdrenalAdrenal diseaseUni-ZAP
Gland Gland Tumor Gland XR
Tumor
H0169 Human Prostate Human ProstateProstate diseaseUni-ZAP
Cancer,. Cancer, sta XR
Stage C fractiona C
H0171 12 Week Old Twelve Week Embryo Uni-ZAP
Early Stage Old XR
Human, II Earl Sta
a Human
H0172 Human Fetal Human Fetal Brain ~ Lambda
Brain, Brain ZAP II
52
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WO 01/55208 PCT/USO1/01357
random rimed
H0204Human Colon Human Colon Colon pBluescript
Cancer,
subtracted Cancer
H0216Supt cells, CyclohexamideBlood Cell pBluescript
cyclohexamide Line
treated, subtractedTreated Cem,
Jurkat,
Ra~i, and
Su t
H0265Activated T-CellT-Cells Blood Cell Uni-ZAP
Line XR
( l2hs)/Thiouridine
labelledEco
H0280K562 + PMA (36 K562 Cell cell lineCell ZAP Ex
hrs) line Line ress
H0305CD34 positive CD34 PositiveCord Blood ZAP Express
cells (Cord Cells
Blood)
H0309Human Chronic Synovium, Synovium diseaseUni-ZAP
Synovitis Chronic ~ XR
Synovitis/
Osteoarthritis
H0327human corpus Human CorpusBrain Uni-ZAP
colosum XR
Callosum
H0424Human Pituita Human Pituita Bluescri
, subt IX t
H0445Spleen, ChronicHuman Spleen,Spleen diseasepSportl
CLL
I m hoc tic
leukemia -
H0457Human Eosino Human Eosino S ortl
hils - hils
H0489Crohn"s DiseaseIleum Intestine diseaseS ortl
H0506Ulcerative ColitisColon Colon S ortl
H0521Primary DendriticPrimary Dendritic pCMVSport
Cells, 3.0
lib 1 cells
H0522Primary DendriticPrimary Dendritic pCMVSport
3.0
cells,frac 2 cells
H0539Pancreas Islet Pancreas Pancreas diseasepSportl
Cell Tumor Islet Cell
Tumour
H0544Human endometrialHuman endometrial pCMV Sport
3.0
stromal cells stromal cells
H0545Human endometrialHuman endometrial pCMVSport
3.0
stromal cells-treatedstromal cells-treated
with
ro esterone with roe
H0547NTERA2 teratocarcinomaNTERA2, pSportl
cell line+retinoicTeratocarcinoma
acid ( 14
da s) cell line
H0570Human Fetal Human Fetal pCMVSport
Brain, Brain 2.0
normalized C500H
H0576Resting T-Cell;T-Cells Blood Cell Lambda
re- Line ZAP I1
excision
H0586Healing groin healing groingroin diseasepCMVSport
wound, 6.5 3.0
hours post incisionwound; 6.5
hours
ost incision
- 2/
H0589CD34 positive CD34 PositiveCord Blood ZAP Express
cells (cord Cells
blood),re-ex
H0598Human Stomach;re-Human StomachStomach Uni-ZAP
XR
excision
H0618Human Adult Human Adult Testis Uni-ZAP
Testes, Testis. XR
Lame Inserts,
Reexcision
H0625Ku 812F Baso Ku 812F Baso S ortl
hils Line hils
H0646Lung, Cancer Metastatic pSportl
(4005313
A3): Invasive s uamous
Poorl cell lun
53
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Differentiated carcinoma,
Lung poorly di
Adenocarcinoma,
H0657B-cells (stimulated)B-cells (stimulated) S ortl
H0658Ovary, Cancer 9809C332- Ovary diseasepSportl
Poorly &
(9809C332): differentiateFallopian
Poorly
differentiated Tubes
adenocarcinoma
H0670Ovary, Cancer(4004650Ovarian Cancer- pSportl
A3): Well-Differentiated4004650A3
Micropapillary
Serous
Carcinoma
H0687Human normal Human normalOvary pCMVSport
3.0
ova (#96106215)ova (#96106215)
50028Smooth muscle,controlSmooth musclePulmanaryCell Uni-ZAP
Line XR
arter
50031S final cord S final cords final Uni-ZAP
cord XR
S0050Human Frontal Human Frontal diseaseUni-ZAP
Cortex, XR
Schizophrenia Cortex,
Schizo hrenia
SO110Brain Amygdala Brain diseaseUni-ZAP
XR
De ression
SOI Bone marrow Bone marrow Bone marrow Uni-ZAP
16 XR
50274PCMIX PCMIX (HumanBrain PCRII
Cerebellum)
S0346Human Amygdala;re-Amygdala Uni-ZAP
XR
excision
S0412Temporal cortex-Temporal diseaseOther
cortex,
Alzheizmer; alzheimer
subtracted
S0420CHME Cell CHME Cell pSportl
line,
Line,untreated untreatetd
50424TF-1 Cell Line TF-I Cell pSportl
GM-CSF Line
Treated GM-CSF Treated
S0432Sinus piniformisSinus piniformis pSportl
Tumour
Tumour
T0010Human Infant Human Infant Other
Brain Braln
T0041JurkatT-cellGl JurkatT-cell Bluescri
hase tSK-
T0049Aorta endothelialAorta endothelial ~ pBluescript
cells + SK-
TNF-a cells
T0082Human Adult Human Adult Bluescri
Retina Retina t SK-
L0015Human
L0351Infant brain, BA, M13-
Bento Soares
derived
L0369NCI CGAP AA1 adrenal adenomaadrenal Bluescri
land t SK-
L0439Soares infant whole Lafmid
brain INIB brain BA
L0534Chromosome 7 brain brain pAMPlO
Fetal
Brain cDNA Libra
L0586HTCDL1 pBluescript
SK(-)
L0596Stratagene colon colon pBluescript
SK-
(#937204)
L0599Strata ene lun lun Bluescri
(#937210) t SK-
L0600Weizmann Olfactoryolfactory nose pBluescript
epithelium SK-
E ithelium
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L0601Stratagene pancreas pancreas pBluescript
SK-
(H937208)
L0604Stratagene musclemuscle skeletal pBluescript
937209 SK-
muscle
L061522 week old pBluescriptll
human fetal
liver cDNA lib SK(-)
L0622HM 1 pcDNA-II
(Invitrooen)
L0638NCI CGAP_Brn35 tumor, 5 brain pCMV-SPORT6
, pooled (see
descri lion)
L0646NCI CGAP Col4 moderately- colon pCMV-SPORT6
differentiated
adenocarcinoma
L0648NCI-CGAP_Eso2 squamous esophagus . pCMV-SPORT6
cell
carcinoma
L0655NCI-CGAP_Lym lymphoma, lymph pCMV-SPORT6
12 node
follicular
mixed
small and
lar a cell
L0657NCI_CGAP_Ov23 tumor, 5 ovary pCMV-SPORT6
pooled (see
descri tion)
L0659NCI CGAP Panl adenocarcinomaancreas CMV-SPORT6
L0731Soares_pregnant uterus pT7T3-Pac
uterus_
NbHPU
L0741Snares adult brain pT7T3D
brain
N2b4HB55Y , (Pharmacia)
with a
modified
olylinker
L0747Snares fetal heart pT7T3D
heart_NbHH
19W (Pharmacia)
with a
modified
of linker
L0748Snares fetal Liver pT7T3D
liver spleen and
1NFLS , Spleen (Pharmacia)
_ with
a modified
of linker
L0750Snares fetal lung pT7T3D
lung_NbHLI
9W (Pharmacia)
with a
modified
of linker
L0752Soares_parathyroidparathyroid parathyroid pT7T3D
tumor tumor
NbHPA gland (Pharmacia)
with a
modified
of linker
L0753Soares_pineal~land_N3H ~ pineal pT7T3D
gland
PG (Pharmacia)
with a
modified
of linker
L0754Snares placenta placenta pT7T3D
Nb2HP
(Pharmacia)
with a
modified
of linker
L0755Soares_placenta placenta pT'TI'3D
8to9wee
ks 2NbHP8to9W (Pharmacia)
CA 02395178 2002-06-19
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with a
modified
of linker
L0757Soares_senescent_fibroblasenescent,fibroblast pT7T3D
sts_NbHSF ' (Pharmacia)
with a
modified
polylinker
V TYPE
L0758Soares_testis-NHT pT7T3D-Pac
(Pharmacia)
with a
modified
of linker
L0763NCI_CGAP_Br2 breast pT7T3D-Pac
(Pharmacia)
with a
modified
of linker
L0764NCI_CGAP_Co3 colon - pT7T3D-Pac
(Pharmacia)
with a
modified
of linker
L0766NCI_CGAP_GCB germinal pT7T3D-Pac
1 center B
cell (Pharmacia)
with a
modified
olylinker
L0768NCI_CGAP_GC4 pooled germ 'pT7T3D-Pac
cell .
tumors ( Pharmacia)
with a
modified
of linker
L0771NCI CGAP_Co8 adenocarcinomacolon pT7T3D-Pac
(Pharmacia)
with a
modified
1 linker
L0774NCI_CGAP_Kid3 kidney pT7T3D-Pac
(Pharmacia)
with a
modified
of linker
L0775NCI_CGAP_KidS 2 pooled kidney pT7T3D-Pac
tumors
(clear cell (Pharmacia)
type)
with a
modified
of linker
L0777Snares NhHMPu_S1Pooled humanmixed pT7T3D-Pac
(see
melanocyte, below) (Pharmacia)
fetal
heart, and with a
pregnant modified
olylinker
L0779Snares NFL_T_GBC_S1 pooled pT7T3D-Pac
(Pharmacia)
with a
modified
of linker
L0786Snares-NbHFB whole pT7T3D-Pac
brain
( Pharmacia)
with a
modified
of linker
L0787NCI_CGAP Subl pT7T3D-Pac
(Pharmacia)
with a
modified
56
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of linker
L0794NCI_CGAP-GC6 pooled germ pT7T3D-Pac
cell
tumors (Pharmacia)
with a
modified
of linker
L0803NCl CGAP_Kidll kidney pT7T3D-Pac
(Phartnacia)
with a
modified
of linker
L0805NCI CGAP_Lu24 carcinoid lung pT7T3D-Pac
(Phartnacia)
with a
modified
of linker
L0806NCI-CGAP_Lul9 squamous lung pT7T3D-Pac
cell
carcinoma, (Pharmacia)
poorly
differentiated with a
(4 modified
of linker
L0809NCI_CGAP-Pr28 prostate pT7T3D-Pac
(Pharmacia)~
with a
modified
lylinker
57
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TABLE 5
OMIM Description. .
Reference
109270 Renal tubular acidosis, distal, 179800
109270 S heroc tosis, heredita
109270 Acanthoc tosis, one form
109270 . ~ Elli toc tosis, Mala sian-Melanesian a
109270 Hemol tic anemia due to band 3 defect
113705 Ovarian cancer
113705 Breast cancer-1
144200 E idermol tic almo lantar keratoderma
148065 White s on a nevus, 193900
148066 Epidermolysis bullosa simplex, Koebner, bowling-Meara,
and Weber-
Cocka ne es, 131900, 131760, 131800
148066 E idermol sis bullosa sim lex, recessive, 601001
148067 None idermol tic almo lantar keratoderma, 600962
148067 Pach on chia con enita, Jadassohn-Lewandowsk e,
167200
148069 Pach on chia congenita, Jackson-Lawler e, 167210
148080 E idermol tic h erkeratosis, 113800
154275 Mali ant h erthermia susce tibili 2
168610 Parkinsonism-dementia with . allido ontoni al de
eneration
171190 H ertension, essential, 145500
176705 Breast cancer, s oradic
185800 S m halan ism, roximal
200350 Ace 1-CoA carbox lase deficienc
221820 Gliosis, familial ro essive subcortical
232200 Gl co en stora a disease I
249000 Meckel s ndrome
252920 Sanfili ~ o s ndrome, a B
253250 Mulibre nanism
600119 Muscular d stro h , Duchenne-like, a 2
600119 Adhalino ath , rima-
601363 Wilms tumor, a 4
601844 Pseudoh oaldosteronism a II
Polynucleotide and Polypeptide Variants
[80] The present invention is directed to variants of the polynucleotide
sequence
disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide
sequences
encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID
NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A,
nucleotide
sequences encoding the polypeptide as defined in column 7 of Table 1A, the
nucleotide
sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences
encoding the
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encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2,
the nucleotide
sequence as defined in column 6 of Table 1B, nucleotide sequences encoding the
polypeptide
encoded by the nucleotide sequence as defined in column 6 of Table 1B, the
cDNA sequence
contained in Clone ID NO:Z, and/or nucleotide sequences encoding the
polypeptide encoded
by the cDNA sequence contained in Clone ID NO:Z.
(81] The present invention also encompasses variants of the polypeptide
sequence
disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in coluriln 7 of
Table 1A, a
polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a
polypeptide sequence encoded by the nucleotide sequence as defined in columns
8 and 9 of
Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined
in column 6
of Table IB, a polypeptide sequence encoded b.y the complement of the
polynucleotide
sequence in SEQ ID NO:X, and/or a polypeptide sequence encoded by the cDNA
sequence
contained in Clone ID NO:Z.
[82] "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.
[83] Thus, one aspect of the invention provides an isolated nucleic acid
molecule
comprising, or alternatively consisting of, a polynucleotide having a
nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence described in
SEQ ID NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence
in SEQ ID
NO:X or the cDNA in Clone ID NO:Z which encodes the complete amino acid
sequence of
SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone
ID
NO:Z; (c) a nucleotide sequence in SEQ ID NO:X or the eDNA in Clone ID NO:Z
which
encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the
cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a
polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ID
NO:Z, which
encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence
encoding a
polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the
complete
amino acid sequence encoded by the eDNA in Clone ID NO:Z; (g) a nucleotide
sequence
encoding a mature polypeptide of the amino acid sequence of SEQ ID NO:Y or the
amino
acid sequence encoded by the cDNA in Clone ID NO:Z; (h) a nucleotide sequence
encoding
a biologically active fragment of a polypeptide having the complete amino acid
sequence of
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SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in Clone
ID
NO:Z; (i) a nucleotide sequence encoding an antigenic fragment of a
polypeptide having the
complete amino acid sequence of SEQ ID NO:Y or the complete amino acid
sequence
encoded by the cDNA in Clone ID NO:Z; and (j) a nucleotide sequence
complementary to
any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or
(i) above.
[84] The present invention is also directed to nucleic acid molecules which
comprise, or
alternatively consist of, a nucleotide sequence which is at least 80%, 85%,
90%, 95%, 96%,
97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide
sequences in (a),
(b), (c), (d),~ (e), (f), (g), (h), (i), or (j) above, the nucleotide coding
sequence in SEQ ID
NO:X or the complementary strand thereto, the nucleotide coding sequence of
the cDNA
contained in Clone ID NO:Z or the complementary strand thereto, a nucleotide
sequence
encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a
polypeptide
sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide
sequence
encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a
nucleotide
sequence encoding the polypeptide encoded by the cDNA contained in Clone ID
NO:Z, the
nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of
Table 2 or the
complementary strand thereto, a nucleotide sequence encoding the polypeptide
encoded by
the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table
2 ~ or the
complementary strand thereto, the nucleotide coding sequence in SEQ ID NO:B as
defined in
column 6 of Table 1B or the complementary strand thereto, a nucleotide
sequence encoding
the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined
in column 6
of Table 1B or the_complementary strand thereto, the nucleotide sequence in
SEQ ID NO:X
encoding the polypeptide sequence as defined in column 7 of Table 1A or the
complementary
strand thereto, nucleotide sequences encoding the polypeptide as defined in
column 7 of
Table 1A or the complementary strand thereto, and/or polynucleotide fragments
of any of
these nucleic acid molecules (e.g., those fragments described herein).
Polynucleotides which
hybridize to the complement ,of these nucleic acid molecules under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed by the
invention, as are polypeptides encoded by these polynucleotides and nucleic
acids.
[85] In a preferred embodiment, the invention encompasses nucleic acid
molecules
which comprise, or alternatively, consist of a polynucleotide which hybridizes
under
stringent hybridization conditions, or alternatively, under lower stringency
conditions, to a
polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as
are polypeptides encoded
CA 02395178 2002-06-19
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by these polynucleotides. In another preferred embodiment, polynucleotides
which hybridize
to the complement of these nucleic acid molecules under stringent
hybridization conditions,
or alternatively, under lower stringency conditions, are also encompassed by
the invention, as
are polypeptides encoded by these polynucleotides.
[86] In another embodiment, the invention provides a purified protein
comprising, or
alternatively consisting of, a polypeptide having an amino acid sequence
selected from the
group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or
the complete
amino acid sequence encoded by the cDNA in Clone ID NO:Z; (b) the amino acid
sequence
of a mature form of a polypeptide having the amino acid sequence of SEQ ID
NO:Y or the
amino acid sequence encoded by the cDNA in Clone ID NO:Z; (c) the amino acid
sequence
of a biologically active fragment of a polypeptide having the complete amino
acid sequence
of SEQ ID NO:Y or the complete amino acid sequence encoded by~the cDNA in
Clone ID
NO:Z; and (d) the amino acid sequence of an antigenic fragment of a
polypeptide having the
complete amino acid sequence of SEQ ID NO:Y or the complete amino acid
sequence
encoded by the cDNA in Clone ID NO:Z.
[87] The present invention is also directed to proteins which comprise, or
alternatively
consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%,
99% or 100%, identical to, for example, any of the amino acid sequences in
(a), (b), (c), or
(d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid
sequence
encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the
polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in
columns 8
and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino. acid
sequence as
defined in column 7 of Table ~lA, an amino acid sequence encoded by the
nucleotide
sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement
of the
polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are
also
provided (e.g., those fragments described herein). Further proteins encoded by
polynucleotides which hybridize to the complement of the nucleic acid
molecules encoding
these amino acid sequences under stringent hybridization conditions or
alternatively, under
lower stringency conditions, are also encompassed by the invention, as are the
polynucleotides encoding these proteins.
[88] 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
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nucleotide sequence of the nucleic acid is identical to the reference sequence
except that the
nucleotide sequence may include up to five point mutations per each 100
nucleotides of the
reference nucleotide sequence encoding the polypeptide. In other words, to
obtain a nucleic
acid having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence,
up to 5% of the nucleotides in the reference sequence may be deleted or
substituted with
another nucleotide, or a number of nucleotides up to 5% of the total
nucleotides in the
reference sequence may be inserted into the reference sequence. The query
sequence may be
an entire sequence referred to in Table 1A or 2 as the ORF (open reading
frame), or any
fragment specified as described herein.
[89] As a practical matter, whether any particular nucleic acid molecule or
polypeptide
is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide
sequence
of the present invention can be determined conventionally using known computer
programs.
A preferred method for determining the best overall match between a query
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, can be determined using the FASTDB computer program based
on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a
sequence alignment
the query and subject sequences are both DNA sequences. An RNA sequence can be
compared by converting U's to T's. The result of said global sequence
alignment is
expressed as percent identity. Preferred parameters used in a FASTDB alignment
of DNA
sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4,
Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap
Penalty=5, Gap
Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide
sequence,
whichever is shorter.
[90] If the subject sequence is shorter than the query sequence because of 5'
or 3'
deletions, not because of internal deletions, a manual correction must be made
to the results:
This is because the FASTDB program does not account for 5' and 3' truncations
of the
subject sequence when calculating percent identity. For subject sequences
truncated at the 5'
or 3' ends, relative to the query sequence, the percent identity is corrected
by calculating the
number of bases of the query sequence that are 5' and 3' of the subject
sequence, which are
not matched/aligned, as a percent of the total bases of the query sequence.
Whether a
nucleotide is matched/aligned is determined by results of the FASTDB sequence
alignment.
This percentage is then subtracted from the percent identity, calculated by
the above
FASTDB program using the specified parameters, to arrive at a final percent
identity score.
62
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This corrected score is what is used for the purposes of the present
invention. Only bases
outside the 5' and 3' bases of the subject sequence, as displayed by the
FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of
manually adjusting the percent identity score.
[91] ~ For example, a 90 base subject sequence is aligned to a 100 base query
sequence to
determine percent identity. The deletions occur at the 5' end of the subject
sequence and
therefore, the FASTDB alignment does not show a matched/alignment of the first
10 bases at
5' end. The 10 unpaired bases represent 10% of the sequence (number of bases
at the 5' and
3' ends not matched/total number of bases in the query sequence) so 10% is
subtracted from
the percent identity score calculated by the FASTDB program. ' If the
remaining 90 bases
were perfectly matched the final percent identity would be 90%. In another
example, a 90
base subject sequence is compared with a 100 base query sequence. This time
the deletions
are internal deletions so that there are no bases on the 5' or 3' of the
subject sequence which
are not matched/aligned with the query. In this case the percent identity
calculated by
FASTDB is not manually corrected. Once again, only bases 5' and 3' of the
subject
sequence which are not matched/aligned with the query sequence are manually
corrected for.
No other manual corrections are to be made for the purposes of the present
invention.
[92] 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.
[93] As a practical matter, whether any particular polypeptide is at least
80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequence of a
polypeptide referred to in Table 1A (e.g., the amino acid sequence identified
in column 6) or
Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the
polynucleotide
sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the
amino acid ,
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sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID
NO:B as
defined in column 6 of Table 1B or a fragment thereof, the amino acid sequence
of the
polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment
thereof, or
the amino acid sequence of the polypeptide encoded by cDNA contained in Clone
ID NO:Z,
or a fragment thereof, can be determined conventionally using known computer
programs. A
preferred method for determining the best overall match between a query
sequence (a
sequence of the present invention) and a subject sequence, also referred to as
a global
sequence alignment, can be determined using the FASTDB computer program based
on the
algorithm of Brutlag et al. (Comp. App. Biosci.6:237-245 (1990)). In a
sequence alignment
the query and subject sequences are either both nucleotide sequences or both
amino acid
sequences. The result of said global sequence alignment is expressed as
percent identity.
Preferred parameters used in a FASTDB amino .acid alignment are: Matrix=PAM 0,
k-
tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0,
Cutoff
Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window
Size=500 or the length of the subject amino acid sequence, whichever is
shorter.
[94] 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 peicent 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.
[95] 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
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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.
[96] The polynucleotide variants of the invention may contain alterations in
the coding
regions, non-coding regions, or both. Especially preferred are polynucleotide
variants
containing alterations which produce silent substitutions, additions, or
deletions, but do not
alter the properties or activities of the encoded polypeptide. Nucleotide
variants produced by
silent substitutions due to the degeneracy of the genetic code are preferred.
Moreover,
polypeptide variants in which less than 50, less than 40, less than 30, less
than 20, less than
10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or
added in any
combination are also preferred. Polynucleotide variants can be produced for a
variety of
reasons, e.g., to optimize codori expression for a particular host (change
codons in the human
mRNA to those preferred by a bacterial host such as E. coli).
[97] 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 (19'85)). 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.
(98] Using known methods of protein engineering and recombinant DNA
technology,
variants may be generated to improve or alter the characteristics of the
polypeptides of the
present invention. For instance, one or more amino acids can be deleted from
the N-terminus
or C-terminus of the polypeptide of the present invention without substantial
loss of
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biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988
(1993))
reported variant IfGF 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).)
[99] Moreover, ample evidence demonstrates that variants often retain a
biological
activity similar to that of the naturally occurring protein. For example,
Gayle and coworkers
(J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational
analysis of human
cytokine IL-la. They used random mutagenesis, to generate over 3,500
individual IL-la
mutants that averaged 2.5 amino acid changes per variant over the entire
length of the
molecule. Multiple mutations were examined at every possible amino acid
position. The
investigators found that "[m]ost of the molecule could be altered with little
effect on either
[binding or biological activity]." In fact, only 23 unique amino acid
sequences, out of more
than 3,500 nucleotide sequences examined, produced a protein that
significantly differed in
activity from wild-type.
[100] 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.
[101] Thus, the invention further includes polypeptide variants which show a
functional
activity (e.g., biological activity) of the polypeptides of the invention.
Such variants include
deletions, insertions, inversions, repeats, and substitutions selected
according to general rules
known in the art so as have little effect on activity.
[102] The present application is directed to nucleic acid molecules at least
80%, 85%,
90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences
disclosed
herein, (e.g., encoding a polypeptide having the amino acid sequence of an N
and/or C
terminal deletion), irrespective of whether they encode a polypeptide having
functional
activity. This is because even where a particular nucleic acid molecule does
not encode a
polypeptide having functional activity, one of skill in the art would still
know how to use the
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nucleic acid molecule, for instance, as a hybridization probe or a polymerase,
chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present invention that
do not encode
a polypeptide having functional activity include, inter alia, (I) isolating a
gene or allelic or
splice variants thereof in a cDNA library; (2) in situ hybridization (e.g.,
"FISH") to
metaphase chromosomal spreads to provide precise chromosomal location of the
gene, as
described in Verma et al., Human Chromosomes: A Manual of Basic Techniques,
Pergamon
Press, New York (1988); (3) Northern Blot analysis for detecting mRNA
expression in
specific tissues (e.g., normal or diseased tissues); and (4) in situ
hybridization (e.g.,
histochemistry) for detecting mRNA expression in specific tissues (e.g.,
normal or diseased
tissues).
[103] Preferred, however, are nucleic acid molecules having sequences at least
80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid
sequences
disclosed herein, which do, in fact, encode a polypeptide having functional
activity. By a
polypeptide having "functional activity" is meant, a polypeptide capable of
displaying one or
more known functional activities associated with a full-length (complete)
protein of the
invention. Such functional activities include, but are not limited to,
biological activity,
antigenicity [ability to bind (or compete with a polypeptide of the invention
for binding) to
an anti-polypeptide of the invention antibody], immunogenicity (ability to
generate antibody
which binds to a specific polypeptide of the invention), ability to form
multimers with
polypeptides of the invention, and ability to bind to ~a receptor or ligand
for a polypeptide of
the invention.
(104] The functional activity of the polypeptides, and fragments, variants and
derivatives .
of the invention, can be assayed by various methods.
[105] For example, in one embodiment where one is assaying for the ability to
bind or
compete with a full-length polypeptide of the present invention for binding to
an anti-
polypetide antibody, various immunoassays known in the art can be used,
including but not
limited to, competitive and non-competitive assay systems using techniques
such as
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or
radioisotope
labels, for example), western blots, precipitation reactions, agglutination
assays (e.g., gel
agglutination assays, hemagglutination assays), complement fixation assays,
immunofluorescence assays, protein A assays, and immunoelectrophoresis assays,
etc. In one
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embodiment, antibody binding is detected by detecting a label on the primary
antibody. In
another embodiment, the primary antibody is detected by detecting binding of a
secondary
antibody or reagent to the primary antibody. In a further embodiment, the
secondary
antibody is labeled. Many means are known in the art for. detecting binding in
an
immunoassay and are within the scope of the present invention.
[106] In another embodiment, where a ligand is identified, or the ability of a
polypeptide
fragment, variant or derivative of the invention to multimerize is being
evaluated, binding
can be assayed, e.g., by means well-known in the art, such as, for example,
reducing and non-
reducing gel chromatography, protein affinity chromatography, and affinity
blotting. See
generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another
embodiment, the
ability of physiological correlates of a polypeptide of the present invention
to bind to a
substrates) of the polypeptide of the invention can be routinely assayed using
techniques
known in the art.
[107] In addition, assays described herein (see Examples) and otherwise known
in the art
may routinely be applied to measure the ability of polypeptides of the present
invention and
fragments, variants and derivatives thereof to elicit polypeptide related
biological activity
(either in vitro or in vivo). Other methods will be known to the skilled
artisan and are within
the scope of the invention.
[108] Of course, due to the degeneracy of the genetic code, one of ordinary
skill in the
art will immediately recognize that a large number of the nucleic acid
molecules having a
sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to, for
example, the nucleic acid sequence of the cDNA contained in Clone ID NO:Z, the
nucleic
acid sequence referred to in Table 1A (SEQ ID NO:X), the nucleic acid sequence
disclosed
in Table 2 (e.g,. the nucleic acid sequence delineated in columns 8 and 9) or
fragments
thereof, will encode polypeptides "having functional activity." In fact, since
degenerate
variants of any of these nucleotide sequences all encode the same polypeptide,
in many
instances, this will be clear to the skilled artisan even without performing
the above described
comparison assay. It will be further recognized in the art that, for such
nucleic acid
molecules that are not degenerate variants, a reasonable number will also
encode a
polypeptide having functional activity. This is because the skilled artisan is
fully aware of
amino acid substitutions that are either less likely or not likely to
significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second aliphatic
amino acid), as
further described below.
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[109] For example, guidance concerning how to make phenotypically silent amino
acid
substitutions is provided in Bowie et al., "Deciphering the Message in Protein
Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310 (1990), wherein
the authors
indicate that there are two main strategies for studying the tolerance of an
amino acid
sequence to change.
[110J 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.
[111] The second strategy uses genetic engineering to introduce amino acid
changes at
specific positions of a cloned gene to identify regions critical for protein
function. For
example, site directed mutagenesis or alanine-scanning mutagenesis
(introduction of single
alanine mutations at every residue in the molecule) can be used. See
Cunningham and Wells,
Science 244:1081-1085 (1989). The resulting mutant molecules can then be
tested for
biological activity.
[112] As the authors state, these two strategies have revealed that proteins
are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which~amino
acid changes are likely to be permissive at certain amino acid positions in
the protein. For
example, most buried (within the tertiary structure of the protein) amino acid
residues require
nonpolar side chains, whereas few features of surface side chains are
generally conserved.
Moreover, tolerated conservative amino acid substitutions involve replacement
of the
aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the
hydroxyl
residues Ser and Thr; replacement of the acidic residues Asp and Glu;
replacement of the
amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and
His;
replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the
small-sized
amino acids Ala, Ser, Thr, Met, and Gly. Besides conservative amino acid
substitution,
variants of the present invention include (i) substitutions with one or more
of the non-
conserved amino acid residues, where the substituted amino acid residues may
or may not be
one encoded by the genetic code, or (ii) substitutions with one or more of the
amino acid
residues having a substituent group, or (iii) fusion of the mature polypeptide
with another
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compound, such as a compound to increase the stability and/or solubility of
the polypeptide
(for example, polyethylene glycol), (iv) fusion of the polypeptide with
additional amino
acids, such as, for example, an IgG Fc fusion region peptide, serum albumin
(preferably
human serum albumin) or a fragment thereof, or leader or secretory sequence,
or a sequence
facilitating purification, or (v) fusion of the polypeptide with another
compound, such as
albumin (including but not limited to recombinant albumin (see, e.g., U.S.
Patent No.
5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
5,766,883, issued
June 16, 1998, herein incorporated by reference in their entirety)). Such
variant
polypeptides are deemed to be within the scope of those skilled in the art
from the teachings
herein.
[113] For example, polypeptide variants containing amino acid substitutions of
charged
amino acids with other charged or neutral amino acids may produce proteins
with improved
characteristics, such as less aggregation. Aggregation of pharmaceutical
formulations both
reduces activity and increases clearance due to the aggregate's immunogenic
activity. See
Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al.,
Diabetes 36: 838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carner Systems 10:307-377
(1993).
[114] A further embodiment of the invention relates to polypeptides which
comprise the
amino acid sequence of a polypeptide having an amino acid sequence which
contains at least
one amino acid substitution, but not more than 50 amino acid substitutions,
even more
preferably, not more than 40 amino acid substitutions, still more preferably,
not more than 30
amino acid substitutions, and still even more preferably, not more than 20
amino acid
substitutions from a polypeptide sequence disclosed herein. Of course it is
highly preferable
for a polypeptide to have an amino acid sequence which comprises the amino
acid sequence
of a polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID
NO:X, an
amino acid sequence encoded by the portion of SEQ ID NO:X as defined in
columnns 8 and
9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X,
and/or
an amino acid sequence encoded by cDNA contained in Clone ID NO:Z which
contains, in
order of ever-increasing preference, at least one, but not more than 10; 9, 8,
7, 6, 5, 4, 3, 2 or
1 amino acid substitutions.
[115] In specific embodiments, the polypeptides of the invention, comprise, or
alternatively, consist of, fragments or variants of a reference amino acid
sequence selected
from: (a) the amino acid sequence of SEQ ID NO:Y or fragments 'thereof (e.g.,
the mature
form and/or other fragments described herein); (b) the amino acid sequence
encoded by SEQ
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ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the
complement of
SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the
portion of
SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and
(e) the
amino acid sequence encoded by cDNA contained in Clone ID NO:Z or fragments
thereof;
wherein the fragments or variants have 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,
amino acid
residue additions, substitutions, and/or deletions when compared to the
reference amino acid
sequence. In preferred embodiments; the amino acid substitutions are
conservative.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
Polynucleotide and Polypeptide Fragments
[116] The present invention is also directed to polynucleotide fragments of
the
polynucleotides (nucleic acids) of the invention. In the present invention, a
"polynucleotide
fragment" refers to a polynucleotide having a nucleic acid sequence which, for
example: is a
portion of the cDNA contained in Clone ID NO:Z or the coriiplementary strand
thereto; is a
portion of the polynucleotide sequence encoding the polypeptide encoded by the
cDNA
contained in Clone ID NO:Z or the complementary strand thereto; is a portion
of a
polynucleotide sequence encoding the amino acid sequence encoded by the region
of SEQ ID
NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand
thereto; is a
portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8
and 9 of
Table 2 or the complementary strand thereto; is a portion of the
polynucleotide sequence in
SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence
encoding a
portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence
encoding a portion
of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding
a portion
of a polypeptide encoded by the complement of the polynucleotide sequence in
SEQ ID
NO:X; is apportion of a polynucleotide sequence encoding the.amino acid
sequence encoded
by the region of SEQ ID NO:B as defined in column 6 of Table 1B or the
complementary.
strand thereto; or is a portion of the polynucleotide sequence of SEQ ID NO:B
as defined in
column 6 of Table 1B or the complementary strand thereto.
[117] The polynucleotide fragments of the invention are preferably at least
about 15 nt,
and more preferably at least about 20 nt, still more preferably at least about
30 nt, and even
more preferably, at least about 40 nt, at least about 50 nt, at least about 75
nt, or at least about
150 nt in length. A fragment "at least 20 nt in length," for example, is
intended to include 20
or more contiguous bases from the cDNA sequence contained in Clone ID NO:Z, or
the
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nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto.
In this
context "about" includes the particularly recited value or a value larger or
smaller by several
(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These
nucleotide
fragments have uses that include, but are not limited to, as diagnostic probes
and primers as
discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180,
190, 200, 250,
500; 600, 1000, or 2000 nucleotides in length ) are also encompassed by the
invention.
[118] Moreover, representative examples of polynucleotide fragments of the
invention
comprise, or alternatively consist of, a sequence from about nucleotide number
1-50, 51-100,
101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-
550, 551-
600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000,
1001-
1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-
1400,
1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,
1751-
1800, 1801-1850; 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-
2150,
2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500,
2501-
2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-
2900,
2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250,
3251-
3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-
3650,
3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000,
4001-
4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350,4351-
4400,
4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750,
4751-
4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-
5150,
5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500,
5501-
5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-
5900,
5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250,
6251-
6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550,.6551-6600, 6601-
6650,
6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000,
7001-
7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end
of SEQ
ID NO:X, or the complementary strand thereto. In this context "about" includes
the
particularly recited range or a range larger or smaller by several (5, 4, 3,
2, or 1) nucleotides,
at either terminus or at both termini. Preferably, these fragments encode a
polypeptide which
has a functional activity (e.g., biological activity). More preferably, these
polynucleotides
can be used as probes or primers as discussed herein. Polynucleotides which
hybridize to one
or more of these polynucleotides under stringent hybridization conditions or
alternatively,
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under lower stringency conditions are also encompassed by the invention, as
are polypeptides
encoded by these polynucleotides.
[119] Further representative examples of polynucleotide fragments of the
invention
comprise, or alternatively consist of, a sequence from' about nucleotide
number 1-50, 51-100,
101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-
550, 551-
600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000,
1001-
1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-
1400,
1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,
1751-
1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-
2150,
2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400; 2401-2450, 2451-2500,
2501-
2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-
2900,
2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250,
3251-
3300, 3301-3350, 3351-3400, 3401-3450, 3451-3_500, 3501-3550, 3551-3600, .3601-
3650,
3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000,
4001-
4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-
4400,
4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750,
4751-
4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-
5150,
5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500,
5501-
5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-
5900,
5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250,
.6251-
6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-
6650,
6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000,
7001-
7050, 7051-7100, 7101-.7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the
end of the
cDNA sequence contained in Clone ID NO:Z, or the complementary strand thereto.
In this
context "about" includes the particularly recited range or a range larger or
smaller by several
(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
Preferably, these fragments
encode a polypeptide which has a functional activity (e.g., biological
activity). More
preferably, these polynucleotides can be used as probes or primers as
discussed herein.
Polynucleotides which hybridize to one or more of these polynucleotides under
stringent
hybridization conditions or alternatively, under lower stringency conditions
are also
encompassed by the invention, as are polypeptides encoded by these
polynucleotides.
[120] ' Moreover, representative examples of polynucleotide fragments of the
invention
comprise, or alternatively consist of, a nucleic acid sequence comprising one,
two, three,
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four, five, six, seven, eight, nine, ten, or more of.the above described
polynucleotide
fragments of the invention in combination with a polynucleotide sequence
delineated in
Table 1B column 6. Additional, representative examples of polynucleotide
fragments of the
invention comprise, or alternatively consist of, a nucleic acid sequence
comprising one, two,
three, four; five, six, seven, eight, nine, ten, or more of the above
described polynucleotide
fragments of the invention in combination with a polynucleotide sequence that
is the
complementary strand of a sequence delineated in column 6 of Table IB. In
further
embodiments, the above-described polynucleotide fragments of the invention
comprise, or
alternatively consist of, sequences delineated in Table 1B, column 6, and have
a nucleic acid
sequence which is different from that of the BAC fragment having the sequence
disclosed in
SEQ ID NO:B (see Table 1B, column 5). In additional embodiments, the above-
described
polynucleotide fragments of the invention comprise, or alternatively consist
of, sequences
delineated in Table 1 B, column 6, and have a nucleic acid sequence which is
different from
that published for the BAC clone identified as BAC ID NO:A (see Table 1B,
column 4).. In
additional embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated Table 1B, column 6, and have a
nucleic acid
sequence which is different from that contained in the BAC clone identified as
BAC ID'
NO:A (see Table 1B,. column 4). Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
are also encompassed by the invention. Additionally, fragments and variants of
the above-
described polynucleotides and polypeptides are also encompassed by the
invention.
[121] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in column 6 of Table 1B, and the
polynucleotide
sequence of SEQ ID NO:X (e.g., as defined in Table 1B, column 2) or fragments
or variants
thereof. Polypeptides encoded by these polynucleotides, other polynucleotides
that encode
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention.
[122] In additional specific embodiments, polynucleotides of the invention-
comprise, or
alternatively consist 'of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in column 6 of Table IB which correspond
to the same
Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ
ID NO:X
(e.g., as defined in Table 1A or 1B) or fragments or variants thereof.
Polypeptides encoded
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by these polynucleotides, other polynucleotides that encode these
polypeptides, and
antibodies that bind these polypeptides are also encompassed by the invention.
[123] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in the same row of column 6 of Table 1B,
and the
polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A or 1B) or
fragments
or variants thereof. Polypeptides encoded by these polynucleotides, other
polynucleotides
that encode these polypeptides, and antibodies that bind these polypeptides
are also
encompassed by the invention.
[124] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of the
sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize
to the
complement of these 20 contiguous polynucleotides under stringent
hybridization conditions
or alternatively, under lower stringency conditions, are also encompassed by
the invention.
Polypeptides encoded by these polynucleotides and/or nucleic acids, other
polynucleotides
and/or nucleic acids that encode these polypeptides, and antibodies that bind
these
polypeptides are also encompassed by the invention. Additionally, fragments
and variants of
the above-described polynucleotides; nucleic acids, and polypeptides are also
encompassed
by the invention.
[125] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively- consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1 B and the 5' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein)
are directly
contiguous Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower .
stringency conditions, are also encompassed by the invention.. Polypeptides
encoded by these
polyriucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids encoding
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention. Additionally, fragments and variants of the above-described
polynucleotides,
nucleic acids, and polypeptides are also encompassed by the invention.
[126] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the ~ 3' 10
polynucleotides of a
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fragment or variant of the sequence of SEQ ID NO:X and the S' 10
polynucleotides of the
sequence of one of the sequences delineated in column 6 of Table 1B are
directly contiguous.
Nucleic acids which hybridize to the complement of these 20 contiguous
polynucleotides
under stringent hybridization conditions or alternatively, under lower
stringency conditions,
are also encompassed by the invention. Polypeptides encoded by these
polynucleotides
and/or nucleic acids, other polynucleotides and/or nucleic acids encoding
these polypeptides,
and antibodies that bind these polypeptides are also encompassed by the
invention.
Additionally, fragments and variants of the above-described polynucleotides,
nucleic acids,
and polypeptides are also encompassed by the invention.
[127] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' I0
polynucleotides of another
sequence in column 6 are directly contiguous. In preferred embodiments, the 3'
10
polynucleotides of one of the sequences delineated in column 6 of Table 1B is
directly
contiguous with the 5' 10 polynucleotides of the next sequential exon
delineated in Table 1B,
column 6. Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by these
polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic
acids encoding
these polypeptides, and antibodies that bind these polypeptides are also
encompassed by the
invention. Additionally, fragments and variants of the above-described
polynucleotides,
nucleic acids, and polypeptides are also encompassed by the invention.
[128] In the present invention, a "polypeptide fragment" refers to an amino
acid
sequence which is a portion of that contained in SEQ ID NO:Y, a portion of an
amino acid
sequence encoded by the portion of SEQ fD NO:X as defined in columnns 8 and 9
of Table
2, a portion of an amino acid sequence encoded by the polynucleotide sequence
of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the
polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid
sequence
encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide)
fragments may be
"free-standing," or comprised within a larger polypeptide of which the
fragment forms a part
or region, most preferably as a single continuous region. Representative
examples of
polypeptide fragments of the, invention, include, for example, fragments
comprising, or
alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-100,
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101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-
280, 281-
300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460,
461-480,
481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640; 641-
660, 661-
680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840,
841-860,
861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020,
1021-1040,
1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180,
1181-
1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-
1340,
1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of
the coding
region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide
fragments of
the invention include, for example, fragments comprising, or alternatively
consisting of, from
about amino acid~number 1-20,, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140,
141-160,
161-180, 181-200, 201-220, 221-240, 241-260, 261-280; 281-300, 301-320, 321-
340, 341-
360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520,
521-540,
541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-
720, 721-
740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900,
901-920,
921-940, 941-9.60, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-
1080, 1081-
1100, .1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-
1240,
1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380,
1381-
1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID
NO:Y.
Moreover, polypeptide fragments of the invention may be at least about 10, 15,
20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 100, 110, 120, 130, 140, or-150
amino acids in
length. In this context "about" includes the particularly recited ranges or
values, or ranges or
values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either
extreme or at both
extremes. Polynucleotides encoding these polypeptide fragments are also
encompassed by
the invention.
[129] Even if deletion of one or more amino acids from the N-terminus of a
protein
results in modification of loss of one or more biological functions of the
protein, other
functional activities (e.g., biological activities, ability to multimerize,
ability to bind a ligand)
may still be retained. For example, the ability of shortened muteins to induce
and/or bind to
antibodies which recognize the complete or mature forms of the polypeptides
generally will
be retained when less than the majority of the residues of the complete or
mature polypeptide
are removed from the N-terminus. Whether a particular polypeptide lacking N-
terminal
residues of a complete polypeptide retains such iriimunologic activities can
readily be
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determined by routine methods described herein and otherwise known in the art.
It is not
unlikely that a mutein with a large number of deleted N-terminal amino acid
residues may
retain some biological or immunogenic activities. In fact, peptides composed
of as few as six
amino acid residues may often evoke an immune response.
[130] Accordingly, polypeptide fragments include the secreted protein as well
as the
mature form. Further preferred polypeptide fragments include the secreted
protein or the
mature form having a continuous series of deleted residues from the amino or
the carboxy
terminus, or both. For example, any number of amino acids, ranging from 1-60,
can be
deleted from the amino terminus of either the secreted polypeptide or the
mature form.
Similarly, any number of amino acids, ranging from I-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.
[131] The present invention further provides polypeptides having one or more
residues
deleted from the amino terminus of the amino acid sequence of a polypeptide
disclosed
herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the
polynucleotide
sequence contained in SEQ ID NO:X or the complement,thereof, a polypeptide
encoded by
the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a
polypeptide
encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1B,
and/oma
polypeptide encoded by the cDNA contained in Clone ID NO:Z). In particular, N-
terminal
deletions may be described ,by the general formula m-q, where q is a whole
integer
representing the total number of amino acid residues in a polypeptide of the
invention (e.g.,
the polypeptide disclosed in SEQ ID NO:Y, or the polypeptide encoded by the
portion of
SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any
integer
ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also
encompassed by
the invention.
[132] The present invention further provides polypeptides having one or more
residues
from the carboxy terminus of the amino acid sequence of a polypeptide
disclosed herein (e.g.,
a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the polynucleotide
sequence
contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X
as
defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by the
cDNA contained
in Clone ID NO:Z). In particular, C-terminal deletions may be described by the
general
formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n
corresponds to
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the position of amino acid residue in a polypeptide of the invention.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[133] In addition, any of the above described N- or C-terminal deletions can
be
combined to produce a N- and C-terminal deleted polypeptide. The invention
also provides
polypeptides having one or more amino acids deleted from both the amino and
the carboxyl
termini, which may be described generally as having residues m=n of a
polypeptide encoded
by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide
disclosed as
SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as
defined in
columns 8 and 9 of Table 2), the cDNA contained in Clone ID NO:Z, and/or the
complement
thereof, where n and m are integers as described above. Polynucleotides
encoding these
polypeptides are also encompassed by the invention.
[134] Also as mentioned above, even if deletion of one or more amino acids
from the
C-terminus of a protein results in modification of loss of one or more
biological functions of
the protein, other functional activities (e.g., biological activities, ability
to multimerize,
ability to bind a ligand) may still be retained. For example the ability of
the shortened mutein
to induce and/or bind to antibodies which recognize the complete or mature
forms of the
polypeptide generally will be retained when less than the majority of the
residues of the
complete or mature polypeptide are removed from the C-terminus. Whether a
particular
polypeptide lacking C-terminal residues of a complete polypeptide retains such
immunologic
activities can readily be determined by routine methods described herein and
otherwise
known in the art. It is not unlikely that a mutein with a large number of
deleted C-terminal
amino acid residues may retain some biological or immunogenic activities. In
fact, peptides
composed of as few as six amino acid residues may often evoke an immune
response.
[135] The present application is also directed to proteins containing
polypeptides at least
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence
set forth
herein. In preferred embodiments, the application is directed to proteins
containing
polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to
polypeptides
having the amino acid sequence of the specific N- and C-terminal deletions.
Polynucleotides
encoding these polypeptides are also encompassed by the invention.
[136] Any polypeptide sequence encoded by, for example, the polynucleotide
sequences
set forth as SEQ ID NO:X or the complement thereof, (presented, for example,
in Tables 1A
and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide sequence as
defined in
column 6 of Table 1B, may be analyzed to determine certain preferred regions,
of the
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polypeptide. For example, the amino acid sequence of a polypeptide encoded by
a
polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y
and the
polypeptide encoded by the portion of SEQ ID, NO:X as defined in columnns. 8
and 9 of
Table 2) or the cDNA contained in Clone ID NO:Z may be analyzed using the
default
parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St.,
Madison, WI 53715 USA; http://www.dnastar.com~.
[137]_ Polypeptide regions that,may be routinely obtained using the DNASTAR
computer
algorithm include., but are not limited to, Gamier-Robson alpha-regions, beta-
regions,
turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and
turn-regions;
Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha-
and
beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-
forming regions;
and Jameson-Wolf regions of high antigenic index. Among highly preferred
polynucleotides
of the invention in this regard are those that encode polypeptides comprising
regions that
combine several structural features, such as several (e.g., 1, 2, 3 or 4) of
the features set out
above.
[138] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic
regions,. Emini
surface-forming regions, and Jameson-Wolf regions of high antigenic index
(i.e., containing
four or more contiguous amino acids having an antigenic index of greater than
or equal to
1.5, as identified using the default parameters of the Jameson-Wolf program)
can routinely be
used to determine polypeptide regions that exhibit a high degree of potential
for antigenicity.
Regions of high antigenicity are determined from data by DNASTAR analysis by
choosing
values which represent regions of the polypeptide which are likely to be
exposed on the
surface of the polypeptide in an environment in which antigen recognition may
occur in the
process of initiation of an immune response.
[139] Preferred polypeptide fragments of the invention are fragments
comprising, or
alternatively, consisting of, an amino acid sequence that displays a
functional activity (e.g.
biological activity) of the polypeptide sequence of which the amino acid
sequence is a
fragment. By a polypeptide displaying a "functional activity" is meant a
polypeptide capable
of one or more known functional activities associated with a full-length
protein, such as, for
example, biological activity, antigenicity, immunogenicity, and/or
multimerization, as
described herein.
[140] Other preferred polypeptide fragments are biologically active.
fragments.
Biologically active fragments are those exhibiting activity similar, but not
necessarily
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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.
[141] In preferred embodiments, polypeptides of the invention comprise, or
alternatively
consist of, one, two, three, four, five or more of the antigenic fragments of
the polypeptide of
~SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides
are also
encompassed by the invention.
[142] The present invention encompasses polypeptides comprising, or
alternatively
consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a
polypeptide
sequence encoded by SEQ ID NO:X or the complementary strand thereto; the
polypeptide
sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9
of Table 2;
the polypeptide sequence encoded by the portion of SEQ ID NO:B as defined in
column 6 of
Table 1 B or the complement thereto; the polypeptide sequence encoded by the
cDNA
contained in Clone ID NO:Z; or the polypeptide sequence encoded by a
polynucleotide that
hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of
SEQ ID
NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9
of Table
2, or the cDNA sequence contained in Clone ID NO:Z under stringent
hybridization
conditions or alternatively, under lower stringency hybridization as defined
supra. The
present invention further encompasses polynucleotide sequences encoding an
epitope of a
polypeptide sequence of the invention (such as, for example; the sequence
disclosed in SEQ
ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary
strand of a
polynucleotide sequence encoding an epitope of the invention, and
polynucleotide sequences
which hybridize to the complementary strand under stringent hybridization
conditions or
alternatively, under lower stringency hybridization conditions defined supra.
[143] 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
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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.
[144] Fragments which function as epitopes may be produced by any conventional
means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135
(1985) further
described in U.S. Patent No. 4,631,211.) .
[145] In the present invention, antigenic epitopes preferably contain a
sequence of at
least 4, at least 5, at least 6, at least 7, more preferably at least 8, at
least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least 20, at least
25, at least 30, at least
40, at least 50, and, most preferably, between about 15 to about 30 amino
acids. Preferred
polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15,
20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues
in length.
Additional non-exclusive preferred antigenic epitopes include the antigenic
epitopes
disclosed herein, as well as portions thereof. Antigenic epitopes are useful,
for example, to
raise antibodies, including monoclonal antibodies, that specifically bind the
epitope.
Preferred antigenic epitopes include the antigenic epitopes disclosed herein,
as well as any
combination of two, three, four, five or more of these antigenic epitopes.
Antigenic epitopes
can be used as the target molecules in immunoassays. (See, for instance,
Wilson et al., Cell
37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
[146] Non-limiting examples of epitopes of polypeptides that can be used to
generate
antibodies of the invention include a polypeptide comprising, or alternatively
consisting of,
at least one, two, three, four, five, six or more of the portions) of SEQ ID
NO:Y specified in
column 7 of Table 1A. These polypeptide fragments have been determined. to
bear antigenic
epitopes of the proteins of the invention by the analysis of the Jameson-Wolf
antigenic index
which is included in the DNAStar suite of computer programs. By "comprise" it
is intended
that a, polypeptide contains at least one, two, three, four, five, six or more
of the portions) of
SEQ ID NO:Y shown in column 7 of Table 1.A, but it may contain additional
flanking
residues on either the amino or carboxyl termini of the recited portion. Such
additional
flanking sequences are preferably sequences naturally found adjacent to the
portion; i.e.,
contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however,
be
sequences from a heterolgous polypeptide, such as from another protein
described herein or
from a heterologous- polypeptide not described herein. In particular
embodiments, epitope
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portions of a polypeptide of the invention comprise one, two, three, or more
of the portions of
SEQ ID NO:Y shown in column 7 of Table 1A.
[147] Similarly, immunogenic epitopes can be used, for example, to induce
antibodies
according to methods well known in the.art. See, for instance, Sutcliffe et
al., supra; Wilson
et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle
et al., -J. Gen.
Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the
immunogenic
epitopes disclosed herein, 'as well as any combination of two, three, four,
five or more of
these immunogenic epitopes. The polypeptides comprising one or more
immunogenic
epitopes may be presented for eliciting an antibody response together with a
carrier protein,
such as an albumin, to an animal system (such as rabbit or mouse), or, if the
polypeptide is of
sufficient length (at least about 25 amino acids), the polypeptide may be
presented without a
carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino
acids have
been shown to be sufficient to raise antibodies capable of binding to, at the
very least, linear
epitopes in a denatured polypeptide (e.g., in Western blotting).
[148] 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., 66:2347-2354
(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 (KLH) or tetanus toxoid. For instance, peptides
containing
cysteine residues may be coupled to a Garner using a linker such as
maleimidobenzoyl- N-
hydroxysuccinimide ester (MBS), while other peptides may be coupled to
carriers using a
more general linking agent such as glutaraldehyde:~ Animals such as rabbits,
rats and mice
are immunized with either free or carrier- coupled peptides, for instance, by
intraperitoneal
and/or intradermal injection of emulsions containing about 100 p.g of peptide
or carrier
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune
response. Several booster injections may be needed, for instance, at intervals
of about two
weeks, to provide a useful titer of anti-peptide antibody which can be
detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The 'titer of
anti-peptide
antibodies in serum from an immunized animal may be increased by selection of
anti-peptide
antibodies, for instance, by adsorption to the peptide on a solid support and
elution of the
selected antibodies according to methods well known in the art.
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[149] As one of skill in the art will appreciate, and as discussed above, the
polypeptides
of the present invention (e.g., those comprising an immunogenic or antigenic
epitope) can be
fused to heterologous polypeptide sequences. For example, polypeptides of the
present
invention (including fragments or variants thereofj, may be fused with the
constant domain of
immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or
any
combination thereof and portions thereof, resulting in chimeric polypeptides.
By way of
another non-limiting example, polypeptides and/or antibodies of the present
invention
(including fragments or variants thereof) may be fused with albumin (including
but not
limited to. recombinant human serum albumin or fragments or variants thereof
(see, e.g., U.S.
Patent No. 5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S.
Patent No.
5,766,883, issued June 16, 1998, herein incorporated by reference in their
entirety)). In a
preferred embodiment, polypeptides and/or antibodies of the present invention
(including
fragments or variants thereof) are fused with the mature form of human serum
albumin (i.e.,
amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0 322.
094) which is herein incorporated by reference in its entirety. In another
preferred
embodiment, polypeptides and/or antibodies of the present invention (including
fragrrients or
variants thereof) are fused with polypeptide fragments comprising; or
alternatively consisting
of, amino acid residues 1-z of human serum albumin, where z is an integer from
369 to 419,
as described in U.S. Patent 5,766,883 herein incorporated by reference in its
entirety.
Polypeptides and/or antibodies of the present invention (including fragments
or variants
thereof) may be fused to either the N- or C-terminal end of the heterologous
protein (e.g.,
immunoglobulin Fc polypeptide or human serum albumin polypeptide).
Polynucleotides
encoding fusion proteins of the invention are also encompassed by the
invention.
[150]' Such fusion proteins as those described above may facilitate
purification and may
increase half life in vivo. This has been shown for chimeric proteins
consisting of the first
two domains of the human CD4-polypeptide and various domains of the constant
regions of
the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827;
Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen
across the
epithelial barrier to the immune system has been demonstrated for antigens
(e.g., insulin)
conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g.,
PCT
Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a
disulfide-
linked dimeric structure due to the IgG portion desulfide bonds have also been
found to be
more efficient in binding and neutralizing other molecules than monomeric
polypeptides or
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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+
nitriloacetic acid-agarose column and histidine-tagged proteins can be
selectively eluted with
imidazole-containing buffers.
Fusion Proteins
[151J Any polypeptide of the present invention can be used to generate fusion
proteins.
For example, the polypeptide of the present invention, when fused to a second
protein, can be
used as an antigenic tag. Antibodies raised against the polypeptide of the
present invention
can be used to indirectly detect the second protein by binding to the
polypeptide. Moreover,
because secreted proteins target cellular locations based on trafficking
signals, polypeptides
of the present invention which are shown to be secreted can be used as
targeting molecules
once fused to other proteins.
[152J 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.
[153] In certain preferred embodiments, proteins of the invention are fusion
proteins
comprising an amino acid sequence that is an N and/or C- terminal deletion of
a polypeptide
of the invention. In preferred embodiments, the invention is directed to a
fusion protein
comprising an amino acid sequence that is at least 90%,,95%, 96%, 97%, 98% or
99%
identical to a polypeptide sequence of the invention. Polynucleotides encoding
these proteins
are also encompassed by the invention.
[154J 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,
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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.
[155] . As one of skill in the art will appreciate that, as discussed above,
polypeptides of
the present invention, and epitope-bearing fragments thereof, can be combined
with
heterologous polypeptide sequences. For example, the polypeptides of the
present invention
may be fused with heterologous polypeptide sequences, for example, the
polypeptides of the
present invention may be fused with the constant domain of immunoglobulins
(IgA, IgE,
IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof,
including both
entire domains and portions thereof), or albumin (including, but not limited
to,, native or
recombinant human albumin or fragments or variants thereof (see, e.g., .U.S.
Patent No.
5,876,969, issued March 2, 1999, EP Patent 0 413 622, and U.S. Patent No.
5,766,883, issued
June 16, 1998, herein incorporated by reference in their entirety)), resulting
in chimeric
polypeptides. For example, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses
fusion proteins comprising various portions of constant region of
immunoglobulin molecules
together with another human protein or part thereof. In many cases, the Fc
part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result in, for
example, improved
pharmacokinetic properties (EP-A 0232 262). Alternatively, deleting the Fc
part after the
fusion protein has been expressed, detected, and purified, would be desired.
For example, the
Fc portion may hinder therapy and diagnosis if the fusion protein is used as
an antigen for
immunizations. In drug discovery, for example, human proteins, such as hIL-5,
have been
fused with Fc portions for the purpose of high-throughput screening assays to
identify
antagonists of hIL-5. See, D. Bennett et. al., J.. Molecular Recognition 8:52-
58 (1995); K.
Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).
[156] Moreover, the polypeptides of the present invention can be -fused to
marker
sequences, such as a polypeptide which facilitates purification of the fused
polypeptide. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
CA,
91311 ), among others, many of which are commercially available. As described
in Gentz et
al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for
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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)).
[157] Additional fusion proteins of the invention .may be generated through
the
techniques of gene-shuffling, motif shuffling, exon-shuffling, and/or codon-
shuffling
(collectively referred to as "DNA shuffling"). DNA shuffling may be employed
to modulate
the activities of polypeptides of the invention, such methods can be used to
generate
polypeptides with altered activity, as well as agonists and antagonists of the
polypeptides.
See, generally, U.S. Patent Nos: 5,605,793; 5,811,238; 5,830,721; 5,834,252;
and 5,837,458,
and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends
Biotechnol.
16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and
Lorenzo and
Blasco, Biotechniques 24(2):308- 13 (1998) (each of these patents and
publications are
hereby incorporated by reference in its entirety). In one embodiment,
alteration of
polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by
'these
polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the
assembly
of two or more DNA segments by homologous or site-specific recombination to
generate
variation in the polyriucleotide 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.
[158] Thus, any of these above fusions can be engineered using the
polynucleotides or
the polypeptides of the present invention.
Recombinant and Synthetic Production of Polypeptides of the Invention
(159] The present invention also relates to vectors containing the
polynucleotide of the
present invention, host cells, and the production of polypeptides by synthetic
and
recombinant techniques. The vector may be, for example, a phage, plasmid,
viral, or
retroviral vector. Retroviral vectors may be replication competent or
replication defective.
In the latter case, viral propagation generally will occur only in
complementing host cells.
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[160] The polynucleotides of the invention may be joined to a vector
containing a
selectable marker for propagation in a host. Generally, a plasmid vector is
introduced in a
precipitate, such as a calcium phosphate precipitate, or in a complex with a
charged lipid. If
the vector is a virus, it may be packaged in vitro using an appropriate
packaging cell line and
then transduced into host cells.
[161] 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
(CTAA, UGA or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[162] As indicated, the expression vectors will preferably include at least
one selectable
marker. Such markers include dihydrofolate reductase, 6418, glutamine
synthase, or
neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin
or ampicillin
resistance genes for culturing in E. coli and other bacteria. Representative
examples of
appropriate hosts include,, but are not limited to, bacterial cells, such as
E. coli, Streptomyces
and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g.,
Saccharomyces
cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such
as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes
melanoma
cells; and plant cells. Appropriate culture mediums and conditions for the
above-described
host cells are known in the art.
[163] , Among vectors preferred for use in bacteria include pQE70, pQE60 and
pQE-9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA,
pNHl6a,
pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-
3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among
preferred
eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from
Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred
expression vectors for use in yeast systems include, but are not limited to
pYES2, pYD 1,
pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-
S1,
pPIC3.5K, pPIC9K, and PA0815 (all available from Invitrogen, Cailbad, CA).
Other
suitable vectors will be readily apparent to the skilled artisan.
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[164] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoximine or
methotrexate,
respectively. An advantage of glutamine synthase based vectors are the
availabilty of cell
lines (e.g., the murine myeloma cell line, NSO) which are glutamine synthase
negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing
cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional
inhibitor to prevent
the functioning of the endogenous gene. A glutamine synthase expression system
and
components thereof are detailed in PCT publications: W087/04462; W086/05807;
W089/01036; W089/10404; and W091/06657, which are hereby incorporated in their
entireties by reference herein. Additionally, glutamine synthase expression
vectors can be
obtained from Lonza Biologics, Inc. (Portsmouth, NH). Expression and
production of
monoclonal antibodies using a GS expression system in murine myeloma cells is
described in
Bebbington et al., Bioltechnology 10:169(1992) and in Biblia and Robinson
Biotechnol.
Prog. 11:1 (1995) which are herein incorporated by reference.
[165] The present invention also relates to host cells containing the above-
described
vector constructs described herein, and additionally encompasses host cells
containing
nucleotide sequences of the invention that are operably associated with one or
more
heterologous control regions (e.g:, promoter and/or enhancer) using techniques
known of in
the art. The host cell can be a higher eukaryotic cell, such as a mammalian
cell (e.g., a
human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the
host cell can be a
prokaryotic cell, such as a bacterial cell. A host strain may be chosen which
modulates the
expression of the inserted gene sequences, or modifies and processes the gene
product in the
specific fashion desired. Expression from certain promoters can be elevated in
the presence
of certain inducers; thus expression of the genetically engineered polypeptide
may be
controlled. Furthermore, different host cells have characteristics and
specific mechanisms for
the translational and post-translational processing and modification (e.g.,
phosphorylation,
cleavage) of proteins. Appropriate cell lines can be chosen to ensure the
desired
modifications and processing of the foreign protein expressed.
[166] Introduction of the nucleic acids and nucleic acid constructs of the
invention into
the host cell can be effected by calcium phosphate transfection, DEAE-dextran
mediated
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
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the polypeptides of the present invention may in fact be expressed by a host
cell lacking a
recombinant vector.
[167] In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
delete or
replace endogenous genetic material (e.g., the coding,sequence), and/or to
include genetic
material (e.g., heterologous polynucleotide sequences) that is operably
associated with
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
polynucleotides. For example, techniques known in the art may be used to
operably associate
heterologous control regions (e.g., promoter and/or enhancer) and endogenous
polynucleotide
sequences via homologous recombination (see, e.g., US Patent Number 5,641,670,
issued
June 24, 1997; International Publication Number WO 96/29411; International
Publication
Number WO 94/12650; Koller 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).
[168] Polypeptides of the invention can be recovered and purified from
recombinant cell
cultures by well-known methods including ammonium sulfate or ethanol
precipitation, acid
extraction, anion or cation exchange chromatography, phosphocellulose
chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite
chromatography and lectin chromatography. Most preferably, high performance
liquid
chromatography ("HPLC") is employed for purification.
[169] Polypeptides of the present invention can also be recovered from:
products
purified from natural sources, including bodily fluids, tissues and cells,
whether directly
isolated or cultured; products of chemical synthetic procedures; and products
produced by
recombinant techniques from a prokaryotic or eukaryotic host, including, for
example,
bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon
the host
employed in a recombinant production procedure, the polypeptides of the
present invention
may be glycosylated or may be non-glycosylated. In addition, polypeptides of
the invention
may also include an initial modified methionine residue, in some cases as a
result of host-
mediated processes. Thus, it is well known in the art that the N-terminal
methionine encoded
by the translation initiation codon generally is removed with high efficiency
from any protein
after translation in all eukaryotic cells. While the N-teiminal methionine on
most proteins
also is efficiently removed in most prokaryotes, for some proteins, this
prokaryotic removal
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process is inefficient, depending on the nature of the amino acid to which the
N-terminal
methionine is covalently linked.
[170] In one embodiment, the yeast Pichia pastoris is used to express
polypeptides of the
invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast
which can
metabolize methanol as its sole carbon source. A main step in the methanol
metabolization
pathway is the oxidation of methanol to formaldehyde using OZ. This reaction
is catalyzed
by the enzyme alcohol oxidase. In order to metabolize methanol as its sole
carbon source,
Pichia pastoris must generate high levels of alcohol oxidase due, in part, to
the relatively low
affinity of alcohol oxidase for OZ. Consequently, in a growth medium depending
on
methanol as a main carbon source, the promoter region of one of the two
alcohol oxidase
genes (AOXl ) is highly active. In 'the presence of methanol, alcohol oxidase
produced from
the AOXI gene comprises up to approximately 30% of the total soluble protein
in Pichia
pastoris. See Ellis, S.B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz,
P.J, et al., Yeast
5:167-77 (1989); Tschopp, J.F., et al., Nucl. Acids Res. 15:3859-76 (1987).
Thus, a
heterologous coding sequence, such as, for example, a polynucleotide of the
present
invention, under the transcriptional regulation of all or part of the AOXI
regulatory sequence
is expressed at exceptionally high levels in Pichia yeast grown in the
presence of methanol:
[171] In one example, the plasmid vector pPIC9K is used to express DNA
encoding a
polypeptide~ of the invention, as set forth herein, in a Pichea yeast system
essentially as
described in "Pichia Protocols: Methods in Molecular Biology," D.R. Higgins
and J. Cregg,
eds. The Humana Press, Totowa, NJ, 1998. This expression vector allows
expression and
secretion of a polypeptide of the invention by virtue of the strong AOXI
promoter linked to
the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e.,
leader) located
upstream of a multiple cloning site.
[172] Many other yeast vectors could be used in place of pPIC9K, such as,
pYES2,
pYDI, pTEFI/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2;
pHIL-S 1, pPIC3.5K, and PA0815, as one skilled in the. art would readily
appreciate, as long
as the proposed expression construct provides appropriately located signals
for transcription,
translation, secretion (if desired), and the like, including an in-frame AUG
as required.
[173] In another embodiment, high-level expression of a heterologous coding
sequence, .
such as, for example, a polynucleotide of the present invention, may be
achieved by cloning
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the heterologous polynucleotide of the invention into an expression vector
such as, for
example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of
methanol.
[174] In addition to encompassing host cells containing the vector constructs
discussed
herein, the invention also encompasses primary, secondary, and immortalized
host cells of
vertebrate origin, particularly mammalian origin, that have been engineered to
.delete or
replace endogenous genetic material (e.g., coding sequence), and/or to include
genetic
material (e.g., heterologous polynucleotide sequences) that is operably
associated with
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
polynucleotides. For example, techniques known in the art may be used to
operably associate
heterologous control regions (e.g., promoter and/or enhancer) and endogenous
polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent
No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/29411,
published
September 26, 1996; International Publication No: WO 94/12650, published
August 4, 1994;
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).
[175] In addition, polypeptides of the invention can be chemically synthesized
using
techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures
and Molecular
Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-
111 (1984)).
For example, a polypeptide corresponding to a fragment of a polypeptide can be
synthesized
by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino
acids or
chemical amino acid analogs can be introduced as a substitution or addition
into the
polypeptide sequence. Non-classical amino acids include, but are not limited
to, to the D- -
isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric
acid, 4-
aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic
acid, Aib,
2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,
norvaline,
hydroxyproline, sarcosirie, 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).
[176] The invention encompasses polypeptides of the present invention which
are
differentially modified during or after translation, e.g., by glycosylation,
acetylation,
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phosphorylation, amidation, derivatization by known protecting/blocking
groups, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any
of numerous
chemical modifications may be carried out by known techniques, including but
not limited, to
specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain,
V8
protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic
synthesis in the
presence of tunicamycin; etc.
[177] 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.
[178] Examples of suitable enzymes include horseradish peroxidase, alkaline
phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic
group complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent
materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material include
iodine (lzih lz3l, izsh i3ll), carbon (14C), sulfur (3sS), tritium (3H),
indium (111In, llzln~ n3mln,
"smln), technetium (99Tc,99mTc), thallium (z°'Ti), gallium (6gGa,
67Ga), palladium (lo3pd),
mol bdenum 99Mo xenon ls3Xe , fluorine 18F ls3Sm l~~Lu Is9Gd ia9Pm ~aoLa msyb
Y ( )~ ( ) ( )> > > > > > >
166H~ 90y 47SC 186Re'' 188Re 142Pr 105 and ~9~Ru.
> > > > > >
[179] In specific embodiments, a polypeptide of the present invention or
fragment or
variant thereof is attached to macrocyclic chelators that associate with
radiometal ions,
including but not limited to, l~~Lu, 9°Y, 166Ho, and Is3Sm, to
polypeptides. In a preferred
embodiment, the radiometal iom associated with the macrocyclic chelators is
lln. In
another preferred embodiment, the radiometal ion associated with the
macrocyclic chelator is
Soy. In specific embodiments, the macrocyclic chelator is 1,4,7,10-
tetraazacyclododecane-
N,N',N",N"'-tetraacetic acid (DOT'A). In other specific embodiments, DOTA is
attached to an
antibody of the invention or fragment thereof via, a linker molecule. Examples
of linker
molecules useful for conjugating DOTA to a polypeptide are commonly known in
the art -
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see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998);
Peterson et al.,
Bioconjug. Chem. 10(4):553=7 (1999); and Zimmerman et al, Nucl. Med. Biol.
26(8):943-50
(1999); which are hereby incorporated by reference in their entirety.
[180] As mentioned, the proteins of the invention may be modified by either
natural
processes, such as posttranslational processing, or by chemical modification
techniques
which are well known in the art. It will be appreciated that the same type of
modification
may be present in the same or varying degrees at several sites in a given
polypeptide.
Polypeptides of the invention may be branched, for example, as a result of
ubiquitination, and
they may be cyclic, with or without branching. Cyclic, branched, and branched
cyclic
polypeptides may result from posttranslation natural processes or may be made
by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation,
amidation,
covalent attachment of flavor, 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, NewYork, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol.
182:626-
646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).
[181] 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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[182] The polymer may be of any molecular weight, and may be branched or
unbranched. For polyethylene glycol, the preferred molecular weight is between
about 1 kDa
and about 100 kDa (the term "about" indicating that in preparations of
polyethylene glycol,
some molecules will weigh more, some less, than the stated molecular weight)
for ease in
handling and manufacturing. Other sizes may be used, depending on the desired
therapeutic
profile (e.g., the duration of sustained release desired, the effects, if any
on biological
activity, the ease in handling, the, degree or lack of antigenicity and other
known effects of
the polyethylene glycol to a therapeutic protein or analog). For example, the
polyethylene
glycol may have an average molecular weight of about 200, 500, 1000, 1500,
2000, 2500,
3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,_9000,
9500,
10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000,
14,500, 15,000,
15,500, 16;000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500,
20,000, 25,000,
30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000,
75,000, 80,000,
85,000, 90,000, 95,000, or 100,000 kDa.
[183] As noted above, the polyethylene glycol may have a branched structure.
Branched
polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575;
Morpurgo et
al., Appl. Biochem. Biotechnol: 56:59-72 (1996); Vorobjev et al., Nucleosides
Nucleotides
18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999),
the
disclosures of each of which are incorporated herein by reference.
(184] The polyethylene glycol molecules (or other chemical moieties) should be
attached
to the protein with consideration of effects on functional or antigenic
domains of the protein.
There are a number of attachment methods available to those skilled in the
art, such as, for
example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein
incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035
(1992),
reporting pegylation of GM-CSF using tresyl chloride. For example,
polyethylene glycol
may be covalently bound through amino acid residues via a reactive group, such
as a free
amino or carboxyl group. Reactive groups are those to which an activated
polyethylene
glycol molecule may be bound. The amino acid residues having a free amino
group may
include lysine residues and the N-terminal amino acid residues; those having a
free carboxyl
group may include aspartic acid residues glutamic acid residues and the C-
terminal amino
acid residue. Sulfhydryl groups may also be used as a reacfive group for
attaching the
polyethylene glycol molecules. Preferred for therapeutic purposes is
attachment at an amino
group, such as attachment at the N-terminus or lysine group.
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[185] As suggested above, polyethylene glycol may be attached to proteins via
linkage to
any of a number of amino acid residues. For example, polyethylene glycol can
be linked to
proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic
acid, or cysteine
residues. One or more reaction chemistries may be employed to attach
polyethylene glycol
to~ specific amino acid residues (e.g., lysine, histidine, aspartic acid,
glutamic acid, or
cysteine) of the protein or to more than one type of amino acid residue (e.g.,
lysine, histidine,
aspartic acid, glutamic acid, cysteine and combinations thereof) of the
protein.
[186] One may specifically desire proteins chemically modified at the N-
terminus.
Using polyethylene glycol as an illustration of the present composition, one
may select from
a variety of polyethylene glycol molecules (by molecular weight, branching,
etc.), the
proportion of polyethylene glycol molecules to protein (polypeptide) molecules
in the
reaction mix, the type of pegylation reaction to be performed, and the method
of obtaining
the selected N-terminally pegylated protein. The method of obtaining the N-
terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties if
necessary) may be by purification of the N-terminally pegylated material from
a population
of pegylated protein molecules. Selective proteins chemically modified at the
N-terminus
modification may be accomplished by reductive alkylation which exploits
differential
reactivity of different types of primary amino groups (lysine versus the N-
terminal) available
foi 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.
(187] As indicated above, pegylation of the proteins of the invention may be
accomplished by any number of means. For example, polyethylene glycol may be
attached
to the protein either directly or by an intervening linker. Linkerless systems
for attaching
polyethylene glycol to proteins are described in Delgado et al., Crit. Rev.
Thera. Drug Carrier
Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998);
U.S. Patent No.
4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the
disclosures of
each of which are incorporated herein by reference.
[188] One system for attaching polyethylene glycol directly to amino acid
residues of
proteins without an intervening linker employs tresylated MPEG, which is
produced by the
modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride
(C1SOZCH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene
glycol is'
directly attached to amine groups of the protein. Thus, the invention includes
protein-
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polyethylene glycol conjugates produced by reacting proteins of the invention
with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[189] Polyethylene glycol can also be attached to proteins using a number of
different
intervening linkers. For example, U.S. Patent No. 5,612,460, the entire
disclosure of which is
incorporated herein by reference, discloses urethane linkers for connecting
polyethylene
glycol to proteins. Protein-polyethylene glycol conjugates wherein the
polyethylene glycol is
attached to the protein by a linker can also be produced by reaction of
proteins with
compounds such as MPEG-succinimidylsuccinate, MPEG activated with
1,1'-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-
nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of
additional
polyethylene glycol derivatives and reaction chemistries for attaching
polyethylene glycol to
proteins are described in International Publication No. WO 98/32466, the
entire disclosure of
which is incorporated herein by reference. Pegylated protein products produced
using the
reaction chemistries set out herein are included within the scope of the
invention.
[190] The number of polyethylene glycol moieties attached to each protein of
the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated proteins
of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 15, 17, 20, or
more polyethylene glycol molecules. Similarly, the average degree of
substitution within
ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-1 l, 10-12, 11-13,
12-14, 13-15, 14-16,
15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein
molecule. Methods
for determining the degree of substitution are discussed, for example, in
Delgado et al., Crit.
Rev. Thera. Drug Carner Sys. 9:249-304 (1992). .
[191] The polypeptides of the invention can be recovered and purified from
chemical
synthesis and recombinant cell cultures by standard methods which include, but
are not
limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion
or 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. Well known techniques for refolding protein may be
employed to
regenerate active conformation when the polypeptide is denatured during
isolation and/or
purification.
[192] 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
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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.
[193] Multimers encompassed by the invention may be homomers or heteromers. As
used herein, the term homomer refers to a multimer containing only
polypeptides
corresponding to a protein of the invention (e.g., the amino acid sequence of
SEQ ID NO:Y,
an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID
NO:X, the
amino acid sequence encoded by the portion of SEQ ID NO:X as defined in
columns 8 and 9
of Table 2, and/or an amino acid sequence encoded by cDNA contained in Clone
ID NO:Z
(including fragments, variants, splice variants, and fusion proteins,
corresponding to these as
described herein)). These homomers may contain polypeptides having identical
or different
amino acid sequences. In a specific embodiment, a homomer of the invention is
a multimer
containing only polypeptides having an identical amino acid sequence. In
another specific
embodiment, a homomer of the invention is a multimer containing polypeptides
having
different amino acid sequences. In specific embodiments, the multimer of the
invention is a
homodimer (e.g., containing two polypeptides having identical or different
amino acid
sequences) or a homotrimer (e.g., containing three polypeptides -having
identical and/or
different amino acid sequences). In additional embodiments, the homomeric
multimer of the
invention is at least a homodimer, at least a homotrimer, or at least a
homotetramer.
[194] As used herein, the term heteromer refers to a multimer containing one
or more
heterologous polypeptides (i.e., polypeptides of different proteins) in
addition to the
polypeptides of the invention. In a specific embodiment, the multimer of the
invention is a
heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments,
the heteromeric
multimer of the invention is at least a heterodimer, at least a heterotrimer,
or at least a
heterotetramer.
[195] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic
and/or covalent associations and/or may be indirectly linked by, for example,
liposome
formation. Thus, in one embodiment, multimers of the invention, such as, for
example,
homodimers or homotrimers, are formed when polypeptides of the invention
contact one
another in solution. In another embodiment, heteromultimers of the invention,
such as, for
example, heterotrimers or heterotetramers, are formed when polypeptides of the
invention
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contact antibodies to the polypeptides of the invention (including antibodies
to the
heterologous polypeptide sequence in a fusion protein of the invention) in
solution. In other
embodiments, multimers of the invention are formed by covalent associations
with and/or
between the polypeptides of the invention. Such covalent associations may
involve one or
more amino acid residues contained in the polypeptide sequence (e.g., that
recited in SEQ ID
NO:Y, encoded by the portion .of SEQ ID NO:X as defined in columns 8 and 9 of
Table 2,
and/or encoded by the cDNA contained in Clone ID NO:Z). In one instance, the
covalent
associations are cross-linking between cysteine residues located within the
polypeptide
sequences which interact in the native (i.e., naturally occurring)
polypeptide. In another
instance, the covalent associations are the consequence of chemical or
recombinant
manipulation. Alternatively, such covalent associations may involve one or
more amino acid
residues contained in the heterologous polypeptide sequence in a fusion
protein. In one
example, covalent associations are between the heterologous sequence contained
in a~fusion
protein of the invention (see, e.g., US Patent Number 5,478,925). In a
specific example, the
covalent associations are between the heterologous sequence contained in a Fc
fusion protein
of the invention (as described herein). In another specific example, covalent
associations of
fusion proteins of the invention are between heterologous polypeptide sequence
from another
protein that is capable of forming covalently associated multimers, such as
for example,
osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the
contents of which
are herein incorporated by reference in its entirety). In another embodiment,
two or more
polypeptides of the invention are joined through peptide linkers. Examples
include those
peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by
reference).
Proteins comprising multiple polypeptides of the invention separated by
peptide linkers may
be produced using conventional recombinant DNA technology.
[196] Another method for preparing multimer polypeptides of the invention
involves use
of polypeptides of the invention fused to a leucine zipper or isoleucine
zipper polypeptide
sequence. Leucine zipper and isoleucine zipper domains are polypeptides that
promote
multimerization of the proteins in which. they are found. Leucine zippers were
originally
identified in several DNA-binding proteins (Landschulz et al., Science
240:1759, (1988)),
and have since been found in a variety of different proteins. Among the known
leucitie
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
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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.
[197J 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.
[198] In another example, proteins of the invention are associated by
interactions
between Flag~ polypeptide sequence contained in fusion proteins of the
invention containing
Flag~ polypeptide sequence. In a further embodiment, proteins of the invention
are
associated by interactions between heterologous polypeptide sequence contained
in Flag~
fusion proteins of the invention and anti-FlagO antibody.
[199] The multimers of the invention may be generated using chemical
techniques
known in the art. For example, polypeptides desired to be contained in the
multimers of the
invention may be chemically cross-linked using linker molecules and linker
molecule length
optimization techniques known in the art (see, e.g., US Patent Number
5,478,925, which is
herein incorporated by reference in its entirety). Additionally, multimers of
the invention
may be generated using techniques known in the art to form one or more inter-
molecule
cross-links between the cysteine residues located within the sequence of the
polypeptides
desired to be contained in the multimer (see, e.g., US Patent Number
5,478,925, which is
herein incorporated by reference in its entirety). Further,. polypeptides of
the invention may
be routinely modified by the addition of cysteine or biotin to the C-terminus
or N-terminus of
the polypeptide and techniques known in the art may be applied to generate
multimers
containing one or more of these modified polypeptides (see, e.g., ~US Patent
Number
5,478,925, which is herein incorporated by reference in its entirety).
Additionally,
techniques known in the art may be applied to generate liposomes containing
the polypeptide
components desired to be contained in the multimer of the invention (see,
e.g., US Patent
Number 5,478,925, which is herein incorporated by reference in its entirety).
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[200] 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
hydrophobic or signal peptide) and which can be incorporated by membrane
reconstitution
techniques .into liposomes (see, e.g., US Patent Number 5,478,925, which is
herein
incorporated by reference in its entirety).
Antibodies
[201] Further polypeptides of the invention relate to antibodies and T-cell
antigen
receptors (TCR) which immunospecifically bind a polypeptide, polypeptide
fragment, or
variant of the invention (e.g., a polypeptide or fragment or variant of the
amino acid sequence
of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in Clone ID
No:Z, and/or
an epitope, of the present invention) as determined by immunoassays well known
in the art
for assaying specific antibody-antigen binding. Antibodies of the invention
include, but are
not limited, to, polyclo,nal, monoclonal, multispecific, human, humanized or
chimeric
antibodies, single chain antibodies, Fab fragments, F(ab') fragments,
fragments produced by
a Fab expression library, anti-idiotypic (anti-Id) antibodies (including,
e.g., anti-Id antibodies
to antibodies of the invention), intracellularly-made antibodies (i.e.,
intrabodies), and
epitope-binding fragments of any of the above. The term "antibody," as used
herein, refers to
immunoglobulin molecules and immunologically active portions of immunoglobulin
molecules, i.e.; molecules that.contain an antigen binding site that
immunospecifically binds
an antigen. The immunoglobulin molecules of the invention can be of any type
(e.g., IgG,
IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAI and
IgA2) or
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subclass of immunoglobulin molecule. In preferred embodiments, the
immunoglobulin
molecules of the invention are IgGI. In other preferred embodiments, the
immunoglobulin
molecules of the invention are IgG4.
[202] Most preferably the antibodies are human antigen-binding antibody
fragments of
the present invention and include, but are not limited to, Fab, Fab' and
F(ab')2, Fd, single-
chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and
fragments
comprising either a VL or VH domain. Antigen-binding antibody fragments,
including
single-chain antibodies, may comprise the variable regions) alone or in
combination with the
entirety or a portion of the following: hinge region, CH1, CH2, and CH3
domains. Also
included in the invention are antigen-binding fragments also comprising any
combination of
variable regions) with a hinge region, CH1, CH2, and CH3 domains. The
antibodies of the
invention may be from any animal origin including birds and mammals.
Preferably, the
antibodies are human, 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.
[203] 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 maybe 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).
[204] Antibodies of the present invention may be described or specified in
terms of the
epitope(s) or portions) of a polypeptide of the present invention which they
recognize or
specifically bind. The epitope(s) or polypeptide portions) may be specified as
described
herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous
amino acid
residues, or listed in the Tables and Figures. Preferred epitopes of the
invention include the
predicted epitopes shown in column 7 of Table 1A, as well as polynucleotides
that encode
these epitopes. Antibodies which specifically bind any epitope or polypeptide
of the present
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invention may also be excluded. Therefore, the present invention includes
antibodies that
specifically bind polypeptides of the present invention, and allows for the
exclusion of the
same.
[205] Antibodies of the present invention may also be described or specified
in terms of
their cross-reactivity. Antibodies that do not bind any other analog,
ortholog, or homolog of
a polypeptide of the present invention are included. Antibodies that bind
polypeptides with
at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least
70%, at least 65%,
at least 60%, at least 55%, and at least 50% identity (as calculated using
methods known in
the art and described herein) to a polypeptide of the present invention are
also included in the
present invention. In specific embodiments, antibodies of the present
invention cross-react
with marine, rat and/or rabbit homologs of human proteins and the
corresponding epitopes
thereof. Antibodies that do not bind polypeptides with less than 95%, less
than 90%, less than
85%, less than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than
55%, and less than 50% identity (as calculated using methods known in the art
and described
herein) to a polypeptide of the present invention are also included in the
present invention.
In a specific embodiment, the above-described cross-reactivity is with respect
to any single
specific antigenic or immunogenic polypeptide, or combinations) of 2, 3, 4, 5,
or more of the
specific antigenic and/or immunogenic polypeptides disclosed herein. Further
included in the
present invention are antibodies which bind polypeptides encoded by
polynucleotides which
hybridize to a polynucleotide of the present invention under stringent
hybridization
conditions (as described herein). Antibodies of the present invention may also
be described
or specified in terms of their binding affinity to a polypeptide of the
invention. Preferred
binding affinities include those with a dissociation constant or Kd less than
5 X 10-Z M,.10-2
M, 5 X 103 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M,
10~6M, 5 X 10-7
M, 107 M, 5 X 10-8 M, 108 M, 5 X 10-9 M, 10-9 M, 5 X 10-'° M, 10-
'° M, 5 X 10-" M, 10-"
M, 5 X 10 ~' Z M, 10-' Z M, 5 X 10-' 3 M, 10~' 3 M, 5 X 10-' 4 M, 10-' 4 M, 5
X 10-15 M, or 10-' S M.
[206] The invention also provides antibodies that competitively inhibit
binding of an
antibody to an epitope of the invention as determined by any method known in
the art for
determining competitive binding, for example, the immunoassays described
herein. In
preferred embodiments, the antibody competitively inhibits binding to the
epitope by at least
95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at
least 60%, or at
least 50%.
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[207] Antibodies of the present invention may act as agonists ~or antagonists
of the
polypeptides of the present invention. For example, the present invention
includes antibodies
which disrupt the receptor/ligand interactions with the polypeptides of the
invention either
partially or fully. Preferably, antibodies of the present invention bind an
antigenic epitope
disclosed herein, or a portion thereof. The invention features both receptor-
specific
antibodies and ligand-specific antibodies. The invention also features
receptor-specific
antibodies which do not prevent ligand binding but prevent receptor
activation. Receptor
activation (i.e., signaling) maybe determined by techniques described herein
or otherwise
known in the art. For example, receptor activation can be determined by
detecting the
phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its
substrate by
immunoprecipitation followed by western blot analysis (for example, as
described supra). In
specific embodiments, antibodies are provided that inhibit ligand activity or
receptor activity
by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at
least 70%, at least
60%, or at least 50% of the activity in absence of the antibody.
[208] The invention also features receptor-specific antibodies which both
prevent ligand
binding and receptor activation as well as antibodies that recognize the
receptor-ligand
complex, and, preferably, do not specifically recognize the unbound receptor
or the unbound
ligand. Likewise, included in the invention are neutralizing antibodies which
bind the ligand
and prevent binding of the ligand to the receptor, as well as antibodies which
bind the ligand,
thereby preventing receptor activation, but do not prevent the ligand from
binding the
receptor. Further included in the invention are antibodies which activate the
receptor. These
antibodies may act as receptor agonists, i.e., potentiate or activate either
all or a subset of the
biological activities of the ligand-mediated receptor activation, for example,
by inducing
dimerization of the receptor. The antibodies may be specified as agonists,
antagonists or
inverse agonists for biological activities comprising the specific biological
activities of the
peptides of the invention disclosed herein. The above antibody agonists can be
made using
methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent
No.
5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res.
58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998);
Zhu et al.,
Cancer Res. 58('15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-
3179 (1998);
Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.
Methods
205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson
et al., J. Biol.
Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995);
Muller
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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).
[209] Antibodies of the present invention may be used, for example, 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 utility
in
immunoassays for qualitatively and quantitatively measuring levels of the
polypeptides of the
r
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.
[210] As discussed in more detail below, the antibodies of the present
invention may be
used either alone or in combination with other compositions. The antibodies
may further be
recombinantly fused to a heterologous polypeptide at the N- or C-terminus or
chemically
conjugated (including covalent and non-covalent conjugations) to polypeptides
or other
compositions. For example, antibodies of the present invention may be
recombinantly fused
or conjugated to molecules useful as labels in detection assays and effector
molecules such as
heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT
publications WO
92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387;
the
disclosures of which are incorporated herein by reference in their entireties.
(211] The antibodies of the invention include derivatives that are modified,
i.e, by the
covalent attachment of any type of molecule to the antibody such that covalent
attachment ,
does not prevent the antibody from generating an anti-idiotypic response. For
example, but
not by way of limitation, the antibody derivatives include antibodies that
have been modified,
e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation,
derivatization by
known protecting/blocking groups, proteolytic cleavage, linkage to a cellular
ligand or other
protein, etc. Any of numerous chemical modifications may be carried out by
known
techniques, including, but not limited to specific chemical cleavage,
acetylation, formylation,
metabolic synthesis of tunicamycin, etc. Additionally, the derivative may
contain one or
more non-classical amino acids.
[212] 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.
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Various adjuvants may be used to increase the immunological response,
depending om 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.
[213] Monoclonal antibodies can be prepared using a wide variety of techniques
known
in the art including the use of hybridoma, recombinant, and phage display
technologies, or a
combination thereof. For example, monoclonal antibodies can be produced using
hybridoma
techniques including those known in the art and taught, for example, in Harlow
et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988);
Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier,
N.Y., 1981) (said references incorporated by reference in their entireties).
The term
"monoclonal antibody" as used herein is not limited to antibodies produced
through
hybridoma technology. The term "monoclonal antibody" refers to an antibody
that is derived
from a single clone, including any eukaryotic, prokaryotic, or phage clone,
and not the
method by which it is produced.
[214] Methods for producing and screening for specific antibodies using
hybridoma
technology are routine and well known in the art and are discussed in detail
in the Examples.
In a non-limiting example, mice can be immunized with a polypeptide of the
invention or a
cell expressing such peptide. Once an immune response is detected, e.g.,
antibodies specific
for the antigen are detected in the mouse serum, the mouse spleen is harvested
and
splenocytes isolated. The splenocytes are then fused by well known techniques
to any
suitable myeloma cells, for example cells from cell line SP20 available from
the ATCC.
Hybridomas are selected and cloned by limited dilution. The hybridoma clones
are then
assayed by methods known in the art for cells that secrete antibodies capable
of binding a
polypeptide of the invention. Ascites fluid, which generally contains high
levels of
antibodies, can be generated by immunizing mice with positive hybridoma
clones.
[215] 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
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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.
[216] Another well known method for producing both polyclonal and monoclonal
human
B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for
generating
EBV-transformed B cell lines are commonly known in the art, such as, for
example, the
protocol outlined in-Chapter 7.22 of Current Protocols in Immunology, Coligan
et al., Eds.,
1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by
reference. The
source of B cells for transformation is commonly human peripheral blood, but B
cells for
transformation may also be derived from other sources including, but not
limited to, lymph
nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are
generally made into
single cell suspensions prior to EBV transformation. Additionally, steps may
be taken to
either physically remove or inactivate T cells (e.g., by treatment with
cyclosporin A) in B
cell-containing samples, because T cells from individuals seropositive for
anti-EBV
antibodies can suppress B cell immortalization by EBV.
[217] In general, the sample containing human B cells is innoculated with EB
V, and
cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of
the B95-8 cell
line (ATCC #VR-1492). Physical signs of EBV transformation can generally be
seen
towards the end of the 3-4 week culture period. By phase-contrast microscopy,
transformed
cells may appear large, clear, hairy and tend to aggregate in tight clusters
of cells. Initially,
EBV lines are generally polyclonal. However, over prolonged periods of cell
cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective
outgrowth of
particular B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned
(e.g., by limiting dilution culture) or fused with a suitable fusion partner
and plated at
limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners
for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-
Ag8.653),
heteromyeloma cell lines (human x mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and
human
cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present
invention
also provides a method of generating polyclonal or monoclonal human antibodies
against
polypeptides of the invention or fragments thereof, comprising EBV-
transformation of
human B cells.
(218] 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
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produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2
fragments contain
the variable region, the light chain constant region and the CH1 domain of the
heavy chain.
[219] For example, the antibodies of the present invention can also be
generated using
various phage display methods known in the art. In phage display methods,
functional
antibody domains are displayed on the surface of phage particles which carry
the
polynucleotide sequences encoding them. In a particular embodiment, such
phage. can be
utilized to display antigen binding domains expressed from a repertoire or
combinatorial
antibody library (e.g., human or murine). Phage expressing an antigen binding
domain that
binds the antigen of interest can be selected or identified with antigen,
e.g., using labeled
antigen or antigen bound or captured to a solid surface or bead. Phage used in
these methods
are typically filamentous phage including fd and M 13 binding domains
expressed from phage
with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused
to either the
phage gene III or gene VIII protein. Examples of phage display methods that
can be used to
make the antibodies of the present invention include those disclosed in
Brinkman et al., J.
Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-
186
(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et
al., Gene 187 9-
18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/2040_1; 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.
[220] 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).
(221] 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.,
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Methods in Enzymology 203:46-88 ( 199'1 ); Shu et al., PNAS 90:7995-7999 (
1993); and
Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo
use of
antibodies in humans and in vitro detection assays, it may be preferable to
use chimeric,
humanized, or human antibodies. A chimeric antibody is a molecule in which
different
portions of the antibody are derived from different animal species, such as
antibodies having
a variable region derived from a murine monoclonal antibody and a human
immunoglobulin
constant region. Methods for producing chimeric antibodies are known in the
art. See e.g.,
Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986);
Gillies et al.,
(1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567;
and
4,816397, which are incorporated herein by reference in their entirety.
Humanized
antibodies are antibody molecules from non-human species antibody that binds
the desired
antigen having one or more complementarity determining regions (CDRs) from the
non-
human species and a framework regions from a human immunoglobulin molecule.
Often,
framework residues in the human framework regions will be substituted with the
corresponding residue from the CDR donor antibody to alter, preferably
improve, antigen
binding. These framework substitutions are identified by methods well known in
the art, e.g.,
by modeling of the interactions of the CDR and framework residues to identify
framework
residues important for antigen binding and sequence comparison to identify
unusual
framework residues at particular positions. (See, e.g., Queen et al., U.S.
Patent No.
5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by
reference in their entireties.) Antibodies can be humanized using a variety of
techniques
known in the art including, for example, CDR-grafting (EP 239,400; PCT
publication WO
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).
[222] Completely human antibodies are particularly desirable for therapeutic
treatment
of human patients. Human antibodies can be made by a variety of methods known
in the art
including phage display methods described above using antibody libraries
derived from
human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and
4,716,111; and
PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO
96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein
by
reference in its entirety.
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[223] Human antibodies can also be produced using transgenic mice which are
incapable
of expressing functional endogenous immunoglobulins, but which can express
human
immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene
complexes may be introduced randomly or by homologous recombination into mouse
embryonic stem cells. Alternatively, the human variable region, constant
region, and.
diversity region may be introduced into mouse embryonic stem cells in addition
to the human
heavy and light chain genes. The mouse heavy and light chain immunoglobulin
genes may
be rendered non-functional separately or simultaneously with the introduction
of human
immunoglobulin loci by homologous recombination. In particular, homozygous
deletion of
the JH region prevents endogenous antibody production. The modified embryonic
stem cells
are expanded and microinjected into blastocysts to produce chimeric mice. The
chimeric
mice are then bred to produce homozygous offspring which express human
antibodies. The
transgenic mice are immunized in the normal fashion with a selected antigen,
e.g., all or a
portion of a polypeptide of the invention. Monoclonal antibodies directed
against the
antigen can be obtained from the immunized, transgenic mice using conventional
hybridoma
technology. The human immunoglobulin transgenes harbored by the transgenic
mice
rearrange during B cell differentiation, and subsequently undergo ~ class
switching and
somatic mutation. Thus, using such a technique, it is possible to produce
therapeutically
useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology
for producing
human antibodies, see Lonberg and Huszar, Int: Rev. Immunol. 13:65-93 (1995).
For a
detailed discussion of this technology for producing human antibodies and
human
monoclonal antibodies and protocols for producing such antibodies, see, e.g.,
PCT
publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European
Patent
No. 0 598 877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016;
5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and
6,114,598, which are
incorporated by reference herein in their entirety. In addition, companies
such as Abgenix,
Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be engaged to provide
human
antibodies directed. against a selected antigen using technology similar to
that described
above.
[224] 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
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human antibody recognizing the same epitope. (Jespers et al., Biotechnology
12:899-903
(1988)).
[225] Further, antibodies to the polypeptides of the invention can, in turn,
be utilized to
generate anti-idiotype antibodies that "mimic" polypeptides of the invention
using techniques
well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
7(5):437-444;
(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,
antibodies
which bind to and competitively inhibit polypeptide multimerization and/or
binding of a
polypeptide of the invention to a.ligand can be used to generate anti-
idiotypes that "mimic"
the polypeptide multimerization and/or binding domain and, as a consequence,
bind to and
neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or
Fab fragments of
such anti-idiotypes can be used in therapeutic regimens to neutralize
polypeptide
ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used
to bind a
polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and
thereby block its
biological activity. Alternatively, antibodies which bind to and enhance
polypeptide
multimerization and/or binding, and/or receptor/ligand multimerization,
binding and/or
signaling can be used to generate anti-idiotypes that function as agonists of
a polypeptide of
the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab
fragments of
such anti-idiotypes can be used in therapeutic regimens as agonists of the
polypeptides of the
invention or its ligand(s)/receptor(s). For example, such anti-idiotypic
antibodies can be used
to bind a polypeptide of the invention and/or to bind its
ligand(s)/receptor(s), and thereby
promote or enhance its biological activity.
[226] Intrabodies of the invention can be produced using methods known in the
art, such
as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601
(1994); Marasco,
W.A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu. Rev. Microbiol.
51:257-283
(1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene
17:2445-2456
(1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J.
Mol. Biol.
291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu
et.al., J.
Immunol. Methods 231:207-222 (1999); and references cited therein.
Polynucleotides Encoding Antibodies .
[227] The invention further provides polynucleotides comprising a nucleotide
sequence
encoding an antibody of the invention and fragments, thereof. The invention
also
encompasses polynucleotides that hybridize under stringent or alternatively,
under lower
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stringency hybridization conditions, e.g., as defined supra, to
polynucleotides that encode an
antibody, preferably, that specifically binds to a polypeptide of the
invention, preferably, an
antibody that binds to a polypeptide having~the amino acid sequence of SEQ ID
NO:Y, to a
polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9
of Table 2,
and/or to a polypeptide encoded by the cDNA contained in Clone ID NO:Z.
[228] The polynucleotides may be obtained, and the nucleotide sequence of the
polynucleotides determined, by any method known in the art. For example, if
the nucleotide
sequence of the antibody is known, a polynucleotide encoding the antibody may
be
assembled from chemically synthesized oligonucleotides (e.g., as described in
Kutmeier et
al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of
overlapping
oligonucleotides containing portions of the sequence encoding the antibody,
annealing and
ligating of those oligonucleotides, and then amplification of the ligated
oligonucleotides by
PCR.
[229] 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.
[230] 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
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amino acid sequence, for example to create amino acid substitutions,
deletions, and/or
insertions.
[231] In a specific embodiment, the amino acid sequence of the heavy and/or
light chain
variable domains may be inspected to identify the sequences of the
complementarity
determining regions (CDRs) by methods that are well know in the art, e.g., by
comparison to
known amino acid sequences of other heavy and light chain variable regions to
determine the
regions of sequence hypervariability. Using routine recombinant DNA
techniques, one or
more of the CDRs may be inserted within framework regions, e.g., into human
framework
regions to humanize a non-human antibody, as described supra. The framework
regions may
be naturally occurring or consensus framework regions, and preferably human
framework
regions (see, e.g., Chothia et al.; J. Mol. Biol. 278: 457-479 (1998) for a
listing of human
framework regions). Preferably, the polynucleotide generated by the
combination of the
framework regions and CDRs encodes an antibody that specifically binds a
polypeptide of
the invention. Preferably, as discussed supra, one or more amino acid
substitutions may be
made within the framework regions, and, preferably, the amino acid
substitutions improve
binding of the antibody to its antigen. Additionally, such methods may bemused
to make
amino acid substitutions or deletions of one or more variable region cysteine
residues .
r
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.
[232] In addition, techniques developed for the production of "chimeric
antibodies"
(Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al.,
Nature
312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing
genes from' a
mouse antibody molecule of appropriate antigen specificity together with genes
from a
human antibody molecule of appropriate biological activity can be used. As
described supra,
a chimeric antibody is a molecule in which different portions are derived from
different
animal species, such as those having a variable region derived from a murine
mAb and a
human immunoglobulin constant region, e.g., humanized antibodies.
[233] 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
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single chain polypeptide. Techniques for the assembly of functional Fv
fragments in E. coli
may also be used (Skerra et al., Science 242:1038- 1041 ( 1988)).
Methods of Producing Antibodies
[234] The antibodies of the invention can be produced by any method known in
the art
for the synthesis of antibodies, in particular, by chemical synthesis or
preferably, by
recombinant expression techniques. Methods of producing antibodies include,
but are not
limited to, hybridoma technology, EBV transformation, and other methods
discussed herein
as well as through the use recombinant DNA technology, as discussed below.
[235] 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.
[236] The expression vector is transferred to a host cell by conventional
techniques and
the transfected cells are then cultured by conventional techniques to produce
an antibody of
the invention. Thus, the invention.includes host cells containing a
polynucleotide encoding
an antibody of the invention, or a heavy or light chain thereof, or a single
chain antibody of
the invention, operably linked to a heterologous promoter. In preferred
embodiments for the
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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.
[237] A variety of host-expression vector systems may be utilized to express
the
antibody molecules of the invention. rSuch host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide
coding sequences, express an antibody molecule of the invention in situ. These
include but
are not limited to microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia)
transformed with
recombinant yeast expression vectors containing antibody coding sequences;
insect cell
systems infected with recombinant virus expression vectors (e.g., baculovirus)
containing
antibody coding sequences; plant cell systems infected with recombinant virus
expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid) containing
antibody coding
sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells)
harboring
recombinant expression constructs containing promoters derived from the genome
of
mammalian cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably,
bacterial cells
such as Escherichia coli, and more preferably, eukaryotic cells, especially
for the expression
of whole recombinant antibody molecule, are used for the expression of a
recombinant
antibody molecule. For example, mammalian cells such as Chinese hamster ovary
cells
(CHO), in conjunction with a vector such as the major intermediate early gene
promoter
element from human cytomegalovirus. is an effective expression system for
antibodies
(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2
(1990)).
(238] In bacterial systems, a number of expression vectors may be
advantageously
selected depending upon the use intended for the antibody molecule being
expressed. For
example, when a large quantity, of such a protein is to be produced, for the
generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression
of high levels of fusion protein products that are readily purified may be
desirable. Such
vectors include, but are not limited, to the E. coli expression vector pUR278
(Ruther et al.,
EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated
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individually into the vector in frame with the lac Z coding region so that a
fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109
(1985); Van
Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may
also be used to express foreign polypeptides as fusion proteins with
glutathione S-transferase
(GST). In general, such fusion proteins are soluble and can easily be purified
from lysed
cells by adsorption and binding to matrix glutathione-agarose beads followed
by elution in
the presence of free glutathione. The pGEX vectors are designed to include
thrombin or
factor Xa protease cleavage sites so that the cloned target gene product can
be released from
the GST moiety.
[239] 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).
[240] 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
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)).
[241] 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
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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.
[242] For long-term, high-yield production of recombinant proteins, stable
expression is
preferred. For example, cell lines which stably express the antibody molecule
may be
engineered. Rather than using expression vectors which contain viral origins
of replication,
host cells can be transformed with DNA' controlled by appropriate expression
control
elements (e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation
sites, etc.), and a selectable marker. Following the introduction of the
foreign DNA,
engineered cells may be allowed to grow for 1-2 days in an enriched media, and
then are
switched to a selective media. The selectable marker in the recombinant
plasmid confers
resistance to the selection and allows cells to stably integrate the plasmid
into their
chromosomes and grow to form foci which in turn can be cloned and expanded
into cell
lines. This method may advantageously be used to engineer cell lines which
express the
antibody molecule. Such engineered cell lines may be particularly useful in
screening and
evaluation of compounds that interact directly or indirectly with the antibody
molecule.
[243] A number of selection systems may be used, including but not limited to
the herpes
simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA
48:202
(1992)), and adenine phosphoribosyltrarisferase (Lowy et al., Cell 22:817
(1980)) genes can
be employed in tk-,. hgprt- or aprt- cells, respectively. Also, antimetabolite
resistance can be
used as the basis of selection for the following genes: dhfr, which confers
resistance to
methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et
al., Proc. Natl.
Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic
acid
(Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which
confers
resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and
Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-
596 (1993);
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Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.
Biochem.
62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which
confers
resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods
commonly known
in the art of recombinant DNA technology may be routinely applied to select
the desired
recombinant clone, and such methods are described, for example, in Ausubel et
al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY (1'993);
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.
(244] 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)).
[245] Vectors which use glutamine synthase (GS) or DHFR as the selectable
markers
can be amplified in the presence of the drugs methionine sulphoximine or
methotrexate,
respectively. An advantage of glutamine synthase based vectors are the
availabilty of cell
lines (e.g.; the murine myeloma cell line, NSO) which are glutamine synthase
negative.
Glutamine synthase expression systems can also function in glutamine synthase
expressing
cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional
inhibitor to prevent
the functioning of the endogenous gene. A glutamine synthase expression system
and
components thereof are detailed in PCT publications: W087/04462; W086/05807;
W089/01036; W089/10404; and W091/06657 which are incorporated in their
entireties by
reference herein. Additionally, glutamine synthase expression vectors that may
be used
according to the present invention are commercially available from suplliers,
including, for
example Lonza Biologics, Inc.,(Portsmouth, NH). Expression and production of
monoclonal
antibodies using a GS expression system in murine myeloma cells is described
in Bebbington
et al., Bioltechnology 10:169(1992) and in Biblia and Robinson Biotechnol.
Prog. 11:1
(1995) which are incorporated in their entirities by reference herein.
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[246] 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.
[247] 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.
[248] The present invention encompasses antibodies recombinantly fused or
chemically
conjugated (including both covalently and non-covalently conjugations) to a
polypeptide (or
portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100
amino acids of the
polypeptide) of the present invention to generate fusion proteins. The fusion
does not
necessarily need to be direct, but may occur through linker sequences. The
antibodies may
be specific for antigens other than polypeptides (or portion thereof,
preferably at least 10, 20,
30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the
present invention.
For example, antibodies may be used to target the polypeptides of the present
invention to
particular cell types, either in vitro or in vivo, by fusing or conjugating
the polypeptides of
the present invention to antibodies specific for particular cell surface
receptors. Antibodies
fused or conjugated to the polypeptides of the present invention may also be
used in in vitro
immunoassays and purification methods using methods known in the art. See
e.g., Harbor et
al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al.,
Immunol. Lett.
39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al., PNAS 89:1428-1432
(1992); Fell et
al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in
their
entireties.
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[249] The present invention further-includes compositions comprising the
polypeptides
of the present invention fused or conjugated to antibody domains other than
the variable
regions. For example, the polypeptides of the present invention may be fused
or conjugated
to an antibody Fc region, or portion thereof. The antibody portion fused to a
polypeptide of
the present invention may comprise the constant region, hinge region, CH1
domain, CH2
domain, and CH3 domain or any combination of whole domains or portions
thereof. The
polypeptides may also be fused or conjugated to the above antibody portions to
form
multimers. For example, Fc portions fused to the polypeptides of the present
invention can
form dimers through disulfide bonding between the Fc portions. Higher
multimeric forms
can be made by fusing the polypeptides to portions of IgA and IgM. Methods for
fusing or
conjugating the polypeptides of the present invention to antibody portions are
known in the
art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053;
5,447,851;
5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570;
Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et
al., J.
Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA
89:11337- 11341
(1992) (said references incorporated by reference in their entireties).
[250] As discussed, supra, the polypeptides corresponding to a polypeptide,
polypeptide
fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above
antibody
portions to increase the in vivo half life of the polypeptides or for use in
immunoassays using
methods known in the art. Further, the polypeptides corresponding to SEQ ID
NO:Y may be
fused or conjugated to the above antibody portions to facilitate purification.
One reported
example describes chimeric proteins consisting of the first two domains of the
human CD4-
polypeptide and various domains of the constant regions of the heavy or light
chains of
mammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature
331:84-86
(1988). The polypeptides of the present invention fused or conjugated to an
antibody having
disulfide- linked dimeric structures (due to the IgG) may also be more
efficient in binding
and neutralizing other molecules, than the monomeric secreted protein or
protein fragment
alone. See; for example, Fountoulakis et al., J. Biochem. 270:3958-3964
(1995). In many
cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis,
and thus can result
in, for example, improved pharmacokinetic properties. See, for example, EP A
232,262.
Alternatively, deleting the Fc part after the fusion protein has been
expressed, detected, and
purified, would be desired. For example, the Fc portion may hinder therapy and
diagnosis if
the fusion protein is used as an antigen for immunizations. In drug discovery,
for example,
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human proteins, such as hIL-5, have been fused with Fc portions for the
purpose of high-
throughput screening assays to identify antagoilists of hIL-5. (See, Bennett
et al., J.
Molecular Recognition 8:52-58 ( 1995); Johanson et al., J. Biol. Chem.
270:9459-9471
(1995)).
[251] 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., 925_9 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 (Wikon et al., Cell 37:767 (1984)) and the
"flag" tag.
[252] The present invention further encompasses antibodies or fragments
thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically
to, for example, monitor the development or progression of a tumor as part of
a clinical
testing procedure to, e.g., determine the efficacy of a given treatment
regimen. Detection
can be facilitated by coupling the antibody to a detectable substance.
Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent
materials, bioluminescent materials, radioactive materials, positron emitting
metals using
various positron emission tomographies, and nonradioactive paramagnetic metal
ions. The .
detectable substance may be coupled or conjugated either directly to the
antibody (or
fragment thereof) or indirectly, through an intermediate (such as, for
example, a linker known
in the art) using techniques known in the art. See, for example, U.S. Patent
No. 4,741,900 for
metal ~ ions which can be conjugated to antibodies for use as ~ diagnostics
according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline
phosphatase, beta-galactosidase, or ~acetylcholinesterase; examples of
suitable prosthetic
group _ complexes include streptavidin/biotin and avidin/biotin; examples of
suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example
of a luminescent material includes luminol; examples of bioluminescent
materials include
luciferase, luciferin, and aequorin; and examples of suitable radioactive
material include
1251, 131I, 11 lIn or 99Tc.
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[253] Further, an antibody or fragment thereof may be conjugated to a
therapeutic
moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a
therapeutic agent or a
radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A
cytotoxin or
cytotoxic agent includes any agent that is detrimental to cells. Examples
include paclitaxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,
tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin dione,
mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or
homologs thereof.
Therapeutic agents include, but are not limited to, antimetabolites (e.g.,
methotrexate, 6-
mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine),
alkylating agents
(e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (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).
[254] The conjugates of the invention can be used for modifying a given
biological
response, the therapeutic agent or drug moiety is not to be construed as
limited to classical
chemical therapeutic agents. For example, the drug moiety may be a protein or
polypeptide
possessing a desired biological activity. Such proteins may include, for
example, a toxin
such as abrin, ricin A; pseudomonas exotoxin, or diphtheria toxin; a protein
such as tumor
necrosis factor, a-interferon,13-interferon, nerve growth factor, platelet
derived growth factor,
tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta,
AIM I (See,
International Publication No. WO 97/33899), AIM II (See, International
Publication No. WO
97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)),
VEGI (See,
International Publication No. WO 99/23105), a thrombotic agent or an anti-
angiogenic
agent, e.g., angiostatiri 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.
[255.] 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
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limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl
chloride or
polypropylene.
[256] Techniques for conjugating such therapeutic moiety to antibodies are
well known.
See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In
Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp.
243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel
Dekker, Inc.
1987); Thorpe, "Antibody Garners Of Cytotoxic Agents In Cancer Therapy: A
Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchers 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).
[257] 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.
[258J 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
[259] The antibodies of the invention may be utilized for immunophenotyping of
cell
lines and biological samples. Translation products of the gene of the present
invention may.
be useful as cell-specific markers, or more specifically as cellular markers
that are
differentially expressed at various stages of differentiation and/or
maturation of particular
cell types. Monoclonal antibodies directed against a specific epitope, or
combination of
epitopes, will allow for the screening of cellular populations expressing the
marker. Various
techniques can be utilized using monoclonal antibodies to screen for cellular
populations
expressing the marker(s), and include magnetic separation using antibody-
coated magnetic
beads, "panning" with antibody attached to a solid matrix (i.e., plate), and
flow cytometry
(See, e.g., U.S. Patent 5,985,660; and Morrison et al., Cell, 96:737-49
(1999)).
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[260] 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
[261] The antibodies of the invention may be assayed for immunospecific
binding by
any method known in the art. The immunoassays which can be used include but
are not
limited to competitive and non-competitive assay systems using techniques such
as western
blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay),
"sandwich"
immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin
reactions, immunodiffusion assays, agglutination assays, complement-fixation
assays,
immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays, to
name but a few. Such assays are routine and well known in the art (see, e.g.,
Ausubel et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. l, 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).
[262] Immunoprecipitation protocols generally comprise lysing a population of
cells in a
lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium
deoxycholate,
0.1 % SDS, 0.15 M NaCI, 0.01 M sodium phosphate at pH 7.2, 1 % Trasylol)
supplemented
with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium
vanadate), adding the antibody of interest to the cell lysate, incubating for
a period of time
(e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose
beads to the cell lysate,
incubating for about an hour or more at 4° C, washing the beads in
lysis buffer and
resuspending the beads in SDS/sample buffer. The ability of the antibody of
interest to
immunoprecipitate a particular antigen can be assessed by, e.g., western blot
analysis. One
of skill in the art would be knowledgeable as to the parameters that can be
modified to
increase the binding of the antibody to'an antigen and decrease the background
(e.g" pre-
clearing the cell lysate with sepharose beads). For further discussion
regarding
immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current
Protocols in
Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.
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[263] 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
piimary
antibody (the antibody of interest) diluted in blocking buffer, washing the
membrane in
washing buffer, blocking the membrane with a secondary antibody (which
recognizes the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I)
diluted in blocking buffer, -washing the membrane in wash buffer, and
detecting the presence
of the antigen. One of skill in the art would be knowledgeable as to the
parameters that can
be modified to increase the signal detected and to reduce the background
noise. For further
discussion regarding western blot protocols see, e.g., Ausubel et al, eds,
(1994), Current
Protocols in Molecular Biology, Vol. l, John Wiley & Sons, Inc., New York,
section 10.8.1.
[264] 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,
section 11.2.1.
[265] 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
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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 ari unlabeled second antibody.
[266] Antibodies of the invention may be characterized using
immunocytochemisty
methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a
vector
enabling the expression of an antigen or with vector alone using techniques
commonly
known in the art. Antibodies that bind antigen transfected cells, but not
vector-only
transfected cells, are antigen specific.
Therapeutic Uses
[267] The present invention is further directed to antibody-based therapies
which involve
administering antibodies of the invention to an animal, preferably a mammal,
and most
preferably a human, patient for treating one or more of the disclosed
diseases, disorders, or
conditions. Therapeutic compounds of the invention include, but are not
limited to,
antibodies of the invention (including fragments, analogs and derivatives
thereof as described
herein) and nucleic acids encoding antibodies of the invention (including
fragments, analogs
and derivatives thereof and anti-idiotypic antibodies as described herein).
The antibodies of
the invention can be used to treat, inhibit or prevent diseases, disorders or
conditions.
associated with aberrant expression and/or activity of a polypeptide of the
invention,
including, but not limited to, any one-or more of the diseases, disorders, or
conditions
described herein. The treatment and/or prevention of diseases, disorders, or
conditions
associated with aberrant expression and/or activity of a polypeptide of the
invention
includes, but is not limited to, alleviating symptoms associated with those
diseases, disorders
or conditions. Antibodies of the invention may be provided in pharmaceutically
acceptable
compositions as known in the art or as described herein.
[268] In a specific and preferred embodiment, the present invention is
directed to
antibody-based therapies which involve administering antibodies of the
invention to an
animal, preferably a mammal, and most preferably a human, patient for treating
one or more
diseases, disorders, or conditions, including but not .limited to: neural
disorders, immune
system disorders, muscular disorders, reproductive disorders, gastrointestinal
disorders,
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pulmonary disorders, cardiovascular disorders, renal disorders, proliferative
disorders, and/or
cancerous diseases and conditions., and/or as described elsewhere herein.
Therapeutic
compounds of the invention include, but are not limited to, antibodies of the
invention (e.g.,
antibodies directed to the full length protein expressed on the cell surface
of a mammalian
cell; antibodies directed to an epitope of a polypeptide of the invention
(such as, for example,
a predicted linear epitope shown in column 7 of Table 1 A; or a conformational
epitope,
including fragments, analogs and derivatives thereof as described herein) and
nucleic acids
encoding antibodies of the invention (including fragments, analogs and
derivatives thereof
and anti-idiotypic antibodies as described herein). The antibodies of the
invention can be
used to treat, inhibit or prevent diseases, disorders or conditions associated
with aberrant
expression and/or activity of a polypeptide of the invention, including, but
not limited to, any
one or more of the diseases, disorders, or conditions described herein. The
treatment and/or
prevention of diseases, disorders, or conditions associated with aberrant
expression and/or
activity of a polypeptide of the inventiowincludes, 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.
[269] 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.
[270] 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.
[271] The antibodies of the invention may be administered alone or in
combination with
other types of treatments (e.g., radiation therapy, chemotherapy, hormonal
therapy,
immunotherapy and anti-tumor agents). Generally, administration of products of
a species
origin or species reactivity (in the case of antibodies) that is the same
species as that of the
patient is preferred. Thus; in a preferred embodiment, human antibodies,
fragments
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derivatives, analogs, or nucleic acids, are administered to a human patient
for therapy or
prophylaxis.
[272] 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 102 M, 10-Z
M, 5 X 10-3 M, 10-3 M, 5 X 10-4 M, 10-4 M, 5 X 10-s M, 10-s M, 5 X 10-6 M, 10-
6 M, 5 X 10-
' M, 10-7 M, 5 X 10~$ M, 10~$ M, 5 X 10-9 M, 10-9 M, 5 X 10-'° M,
10''° M, 5 X 10-" M, 10-"
M, 5 X 10-' Z M, 10-' 2 M, 5 X 10-' 3 M, 10' ' 3 M, 5 X 10-' 4 M, 10'' 4 M, 5
X 10-'s M, and 10-'s
M.
Gene Therapy
[273] 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.
[274] Any of the methods for gene therapy available in the art can be used
according to
the present invention. Exemplary methods are described below.
[275] 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-
21 S (1993). Methods commonly known in the art of recombinant DNA technology
which can .
be used are described in Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John
Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory
Manual, Stockton Press, NY (1990).
[276] In a preferred embodiment, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors that
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express the antibody or fragments or chimeric proteins or heavy or light
chains thereof in a
suitable host. In particular, such nucleic acid sequences have promoters
operably linked to
the antibody coding region, said promoter being inducible or constitutive,
and, optionally,
tissue-specific. In another particular embodiment, nucleic acid molecules are
used in which
the antibody coding sequences and any other desired sequences are flanked by
regions that
promote homologous recombination at a desired site in the genome, thus
providing for
intrachromosomal expression of the. antibody encoding nucleic acids (Koller
and Smithies,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature
342:435-438 (1989).
In specific embodiments, the expressed antibody molecule is a single chain
antibody;
alternatively, the nucleic acid sequences include sequences encoding both the
heavy and
light chains, or fragments thereof, of the antibody.
[277] 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.
[278] In a specific embodiment, the nucleic acid sequences are directly
administered in
vivo, where it is expressed to produce the encoded product. This can be
accomplished by
any of numerous methods known in the art, e.g., by constructing them as part
of an
appropriate nucleic acid expression vector and administering it so that they
become
intracellular, e.g., by infection using defective or attenuated retrovirals or
other viral vectors
(see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by
use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating
with lipids or
cell-surface receptors or transfecting agents, encapsulation in liposomes,
microparticles, or
microcapsules, or by administering them in linkage to a peptide which is known
to enter the
nucleus, by administering it in linkage to a ligand subject to receptor-
mediated endocytosis
(see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used
to target
cell types specifically expressing the receptors), etc. In another embodiment,
nucleic acid-
ligand complexes can be formed in which the ligand comprises a fusogenic viral
peptide to
disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
In yet another
embodiment, the nucleic acid can be targeted in vivo for cell specific 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
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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)).
[279] 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 retroW ral 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).
[280] Adenoviruses are other viral vectors that can be used in gene therapy.
Adenoviruses are especially attractive vehicles for delivering genes to
respiratory epithelia.
Adenoviruses naturally infect respiratory epithelia where they cause a mild
disease. Other
targets for adenovirus-based delivery systems are liver, the central nervous
system,
endothelial cells, and muscle. Adenoviruses have the advantage of being
capable of infecting
non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development
3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et
al., Human
Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to
transfer genes to
the respiratory epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in
gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991);
Rosenfeld et al.,
Cell 68:143- 155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234
(1993); PCT
Publication W094/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a
preferred
embodiment, adenovirus vectors are used.
[281] 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).
[282] Another approach to gene therapy involves transferring a gene to cells
in tissue
culture by such methods as electroporatiori, lipofection, calcium phosphate
mediated
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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 upland are expressing the transferred gene. Those cells are
then delivered to
a patient.
[283] 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.
Then. 29:69-92m (1985) and may be used in accordance with the present
invention, provided
that the necessary developmental and physiological functions of the recipient
cells are not
disrupted. The technique should provide for the stable transfer of the nucleic
acid to the cell,
so that the nucleic acid is expressible by the cell and preferably heritable
and expressible by
its cell progeny.
[284] 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.
[285] Cells into which a nucleic acid can be introduced for purposes of gene
therapy
encompass any desired, available cell type; and include but are not limited to
epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes;
blood cells such as T
lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
megakaryocytes, granulocytes; various stem or progenitor cells, in particular
hematopoietic
stem or progenitor cells, e.g., as obtained. from bone marrow, umbilical cord
blood,
peripheral blood, fetal liver, etc.
[286] In a preferred embodiment, the cell used for gene therapy is autologous
to the
patient.
[287] 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
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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)).
[288] In a specific embodiment, the nucleic acid to be introduced for purposes
of gene
therapy comprises an inducible promoter operably linked to the coding region,
such that
expression of the nucleic acid is controllable by the presence or absence of
an appropriate
inducer of transcription.
Demonstration of Therapeutic or Prophylactic Activity
[289] The compounds or pharmaceutical compositions of the.invention are
preferably
tested in vitro, and then in vivo for the desired therapeutic or prophylactic
activity, prior to
use in humans: For example, in vitro assays to demonstrate the therapeutic or
prophylactic
utility of a compound or pharmaceutical composition include, the effect of a
compound on a
cell line or a patient tissue sample. The effect of the compound or
composition on the cell
line and/or tissue sample can be determined utilizing techniques known to
those of skill in the
art including, but not limited to, rosette formation assays and cell lysis
assays. In accordance
with the invention, in vitro assays which can be used to determine whether
administration of
a specific compound is indicated, include in vitro cell culture assays in
which a patient tissue
sample is grown in culture, and exposed to or otherwise administered a
compound, and the
effect of such compound upon the tissue sample is observed.
TherapeuticlProphylactic Administration and Composition
[290] The invention provides methods of treatment, inhibition and prophylaxis
by
administration to a subject of an effective amount of a compound or
pharmaceutical
composition of the invention; preferably a polypeptide or antibody of the
invention. In a
preferred embodiment, the compound is substantially purified (e.g.,
substantially free from
substances that limit its effect or produce undesired side-effects). The
subject is preferably
an animal, including but riot limited to animals such as cows, pigs, horses,
chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
[291] Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional
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appropriate formulations and routes of administration can be selected from
among those
described herein below.
[292] Various delivery systems are known and can be used to administer a
compound of
the invention, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant
cells capable of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and
Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as
part of a
retroviral or other vector, etc. Methods of introduction include ~ but are not
limited to
intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural,
and oral routes. The compounds or compositions may be administered by any
convenient
route, for example by infusion or bolus injection; by absorption through
epithelial or
mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.)
and may be
administered together with other biologically active agents. Administration
can be systemic
or local. In addition, it may be desirable to introduce the pharmaceutical
compounds or
compositions of the invention into the central nervous system by any suitable
route,
including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such as an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
[293] 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 vVound 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.
[294] 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.)
[295] 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;
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Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 (1980);
Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment,
polymeric
materials can be used (see Medical Applications of Controlled Release, Langer
and Wise
(eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug
Bioavailability, Drug
Product Design and Performance, Smolen and Ball (eds.), Wiley, New York
(1984); Ranger
and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also
Levy et al.,
Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et
al.,
J.Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release
system can be
placed in proximity of the therapeutic target, e.g., the brain, thus requiring
only a fraction of
the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled
Release, supra,
vol. 2, pp. 115-138 (1984)).
[296] Other controlled release systems are discussed. in the review by Langer
(Science
249:1527-1533 (1990)).
[297] In a specific embodiment where the compound of the invention is a
nucleic acid
encoding a protein, the nucleic acid can be administered in vivo to promote
expression of its
encoded protein, by constructing it as part of an appropriate nucleic acid
expression vector
and administering it so that it becomes intracellular, e.g., by use of a
retroviral vector (see
U.S. Patent No. 4,980,286), or by direct injection, or by use of microparticle
bombardment
(e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface'
receptors or
transfecting agents, or by administering it in linkage to a homeobox-like
peptide which is
known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.
USA 88:1864-1868
(1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly
and incorporated
within host cell DNA for expression, by homologous recombination. ,
[298] 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
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intravenously. Saline solutions and aqueous dextrose and glycerol solutions
can also be
employed as liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if desired,
can also contain
minor amounts of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules,
powders, sustained-release formulations and the like. The composition can be
formulated as
a suppository, with traditional binders and carriers such as triglycerides.
Oral formulation
can include standard carriers such as pharmaceutical grades of mannitol,
lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of
suitable pharmaceutical carriers are described in "Remington's Pharmaceutical
Sciences" by
E.W. Martin. Such compositions will contain a therapeutically effective amount
of the
compound, preferably in purified form, together with a suitable amount of
carrier so as to
provide the form for proper administration to the patient. The formulation
should suit the
mode of administration.
[299] 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:
[300] 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.
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[301] The amount of the compound of the invention which will be effective in
the
treatment, inhibition and prevention of a disease or disorder associated with
aberrant
expression and/or activity of a polypeptide of the invention can be determined
by standard
clinical techniques. In addition, in vitro assays may optionally be employed
to help identify
optimal dosage ranges. The precise dose to be employed in the formulation will
also depend
on the route of administration, and the seriousness of the disease or
disorder, and should be
decided according to the judgment of the practitioner and each patient's
circumstances.
Effective doses may be extrapolated from dose-response curves derived from in
vitro or
animal model test systems.
[302] 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 mglkg of the patient's body weight, more preferably 1
mg/kg to
mg/kg of the patient's body weight. Generally, human antibodies have a longer
half life
within the human body than antibodies from other species due to the immune
response to the
foreign polypeptides. Thus, lower dosages of human antibodies and less
frequent
administration is often possible. Further, the dosage and frequency of
administration of
antibodies of the invention may be reduced by enhancing uptake and tissue
penetration (e.g.,
into the brain) of the antibodies by modifications such as, for example,
lipidation.
[303] 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
[304] Labeled antibodies, and derivatives and analogs thereof, which
specifically bind to.
a polypeptide of interest can be used for diagnostic purposes to detect,
diagnose, or monitor
diseases, disorders, and/or conditions associated with the aberrant expression
and/or activity __
of a polypeptide of the invention. The invention provides for the detection of
aberrant
expression of a polypeptide of interest, comprising (a) assaying the
expression of the
polypeptide of interest in cells or body fluid of an individual using one or
more antibodies
specific to the polypeptide interest and (b) comparing the level of gene
expression with a
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standard gene expression level, whereby an increase or decrease in the assayed
polypeptide
gene expression level compared to the standard expression level is indicative
of aberrant
expression.
[305] 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.
(306] Antibodies of the invention can be used to assay protein levels in a
biological
sample using classical immunohistological methods known to those of skill in
the art (e.g.,
see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J.
Cell . Biol. 105:3087-
3096 (1987)). Other antibody-based methods useful for detecting protein gene
expression
include immunoassays, such as the enzyme linked immunosorbent assay (ELISA)
and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in the art
and include
enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I,
121I), carbon
(14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);
luminescent labels,
such as luminol; and fluorescent labels, such as fluorescein and rhodamine,
and biotin.
[307J One facet of the invention is the detection and diagnosis of a disease
or disorder
associated with aberrant expression of a polypeptide of interest in an animal,
preferably a
mammal and most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject
an effective amount of a labeled molecule which specifically binds to the
polypeptide of
interest; b) waiting for a time interval following the administering for
permitting the labeled
molecule to preferentially concentrate at sites in the subject where the
polypeptide is
expressed (and for unbound labeled molecule to be cleared to background
level); c)
determining background level; and d) detecting the labeled molecule in the
subject, such that
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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.
[308] . 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)).
[309] 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.
[310] In an embodiment, monitoring of the disease or disorder is carned 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.
[311] Presence of the labeled molecule can be detected in the patient using
methods
known in the art for in vivo scanning. These methods depend upon the type of.
label used.
Skilled artisans will be able to determine the appropriate method for
detecting a particular
label. Methods and devices that may be used in the diagnostic methods of the
invention
include, but are not limited to, computed tomography (CT), whole body scan
such as position
emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
(312] In a specif c 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.
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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
[313] 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).
[314] In another specific embodiment of the present invention, the kit is a
diagnostic kit
for use in screening serum containing antibodies specific against
proliferative and/or
cancerous polynucleotides and polypeptides. Such a kit may include a control
antibody that
does not react with. the polypeptide of interest. Such a kit may include a
substantially
isolated polypeptide antigen comprising an epitope which is specifically
immunoreactive
with at least one anti-polypeptide antigen antibody. Further, such a kit
includes means for
detecting the binding of said antibody to the antigen (e.g., the antibody may
be conjugated to
a fluorescent compound such as fluorescein or rhodamine which can be detected
by flow
cytonietry). In specific embodiments, the kit may include a recombinantly
produced or
chemically synthesized polypeptide antigen. The polypeptide antigen of the kit
may also be
attached to a solid support.
[315] 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.
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[316] 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.
[317] 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).
[318] The solid surface reagent in the above assay is prepared by known
techniques for
attaching protein material to solid support material, such as polymeric beads,
dip sticks, 96-
well plate or filter material. These attachment methods generally include non-
specific
adsorption of the protein to the support or covalent attachment of the
protein, typically
through a free amine group, to a chemically reactive group on the solid
support, such as an
activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin
coated plates can
be used in conjunction with biotinylated antigen(s).
(319] Thus, the invention provides an assay system or kit for carrying out
this diagnostic
method. The kit generally includes a support with surface- bound recombinant
antigens, and
a reporter-labeled anti-human antibody for detecting surface-bound anti-
antigen antibody.
Uses of the Polynucleotides
[320] 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. - .
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[321] The ponynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers, since few
chromosome marking reagents, based on actual sequence data (repeat
polymorphisms), are
presently available. Each sequence is specifically targeted to and can
hybridize with a
particular location on an individual human chromosome, thus each
polynucleotide of the
present invention can routinely be used as a chromosome marker using
techniques known in
the art. Table 1A, column 9 provides the chromosome location of some of the
polynucleotides of the invention.
[322] Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably at least 15 by (e.g., 15-25 bp) from the sequences shown in SEQ ID
NO:X.
Primers can optionally be selected using computer analysis so that primers do
not span more
than one predicted exon in the genomic DNA. These primers are then used for
PCR
screening of somatic cell hybrids containing individual human chromosomes.
Only those
hybrids containing the human gene corresponding to SEQ ID NO:X will yield an
amplified
fragment.
[323] Similarly, somatic hybrids provide a rapid method of PCR mapping the
ponynucleotides to particular chromosomes. Three or more clones can be
assigned per day
using a single thermal cycler. Moreover, sublocalization of the
polynucleotides can .be
achieved with panels of specific chromosome fragments. Other gene mapping
strategies that
can be used include in situ hybridization, prescreening with labeled flow-
sorted
chromosomes, preselection by hybridization to construct chromosome specific-
cDNA
libraries, and computer mapping techniques (See, e.g., Shiner, Trends
Biotechnol 16:456-459
(1998) which is hereby incorporated by reference in its entirety).
[324] Precise chromosomal location of the polynucleotides can. also be
achieved using
fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
This
technique uses polynucneotides as short as 500 or 600 bases; however,
polynucleotides 2,000-
4,000 by are preferred., For a review of this technique, see Verma et al.,
"Human
Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).
[325] 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).
[326] . Thus; the present invention also provides a method for chromosomal
localization
which involves (a) preparing PCR primers from the ponynucneotide sequences in
Table 1A
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and/or Table 2 and SEQ ID NO:X and (b). screening somatic cell hybrids
containing
individual chromosomes.
(327] The polynucleotides of the present invention would likewise be useful
for radiation
hybrid mapping, HAPPY mapping, and long range restriction mapping. For a
review of these
techniques and others known in the art, see, e.g. Dear, "Genome Mapping: A
Practical
Approach," IRL Press at Oxford University Press, London (1997); Aydin, J. Mol.
Med.
77:691-694 (1999); Hacia et al., Mol. Psychiatry 3:483-492 (1998); Herrick et
al.,
Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-
280 (2000);
and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by
reference in its
entirety.
[328] Once a polynucleotide has been mapped to a precise chromosomal location,
the
physical position of the polynucleotide can be used in linkage analysis.
Linkage analysis
establishes coinheritance between a chromosomal location and presentation of a
particular
disease. (Disease mapping data are found, for example, in V. McKusick,
Mendelian
Inheritance in Man (available on line through Johns Hopkins University Welch
Medical
Library)). Column 10 of Table 1A provides an OMIM reference identification
number of
diseases associated with the cytologic band disclosed in column 9 of Table 1
A, as determined
using techniques described herein and by reference to Table 5. Assuming 1
megabase
mapping resolution and one gene per 20 kb, a cDNA precisely localized to a
chromosomal
region associated with the disease could be one of 50-500 potential causative
genes.
(329] Thus, once coinheritance is established, differences in a polynucleotide
of the
invention and the corresponding gene between affected and unaffected
individuals can be
examined. First, visible structural alterations in the chromosomes, such as
deletions or
translocations, are examined in chromosome spreads or by PCR. If no structural
alterations
exist, the presence .of point mutations are ascertained. Mutations observed in
some or all
affected individuals, but not in normal individuals, indicates that the
mutation may cause the
disease. However, complete sequencing of the polypeptide and the corresponding
gene from.
several normal individuals is required to distinguish the mutation from a
polymorphism. If a
new polymorphism is identified, this polymorphic polypeptide can be used for
further linkage
analysis.
[330] Furthermore, increased or decreased expression of the gene in affected
individuals
as compared to unaffected individual's can be assessed using the
polynucleotides of the
invention. Any of these alterations '(altered expression, chromosomal
rearrangement, or
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mutation) can be used as a diagnostic or prognostic marker. Diagnostic and
,prognostic
methods, kits and reagents encompassed by the present invention are briefly
described below
and more thoroughly elsewhere herein (see e.g., the sections labeled
"Antibodies",
"Diagnostic Assays", and "Methods for Detecting Diseases").
[331] Thus, the invention also provides a diagnostic method useful during
diagnosis of a
disorder, involving measuring the expression level of polynucleotides of the
present
invention in cells or body fluid from an individual and comparing the measured
gene
expression level with a standard level of polynucleotide expression level,
whereby an
increase or decrease in the gene expression level compared to the standard is
indicative of a
disorder. Additional non-limiting examples of diagnostic methods encompassed
by the
present invention are more thoroughly described elsewhere herein (see, e.g.,
Example 12).
[332] In still another embodiment, the invention includes a kit for analyzing
samples for
the presence of proliferative and/or cancerous polynucleotides derived from a
test subject. In
a general embodiment, the kit includes at least one ~polynucleotide probe
containing a
nucleotide sequence that will specifically hybridize with a polynucleotide of
the invention
and a suitable container. In a specific embodiment, the kit includes two
polynucleotide
probes. defining an internal region of the polynucleotide of the invention,
where each probe
has one strand containing a 31'mer-end internal to the region. In a further
embodiment, the
probes may be useful as primers for polymerise chain reaction,amplification.
[333] Where a diagnosis of a related disorder, including, for example,
diagnosis of a
tumor, has already been made according to conventional methods, the present
invention is
useful as a prognostic indicator, whereby . patients exhibiting enhanced or
depressed
polynucleotide of the invention expression will experience a worse clinical
outcome relative
to patients expressing the gene at a level nearer the standard level.
[334] By "measuring the expression level of polynucleotides of the invention"
is
intended qualitatively or quantitatively measuring or estimating the level of
the polypeptide
of the invention or the level of the mRNA encoding the polypeptide of the
invention in a first
biological sample either directly (e.g., by determining or estimating absolute
protein level or
mRNA level) or relatively (e.g., by comparing- to the polypeptide level or
mRNA level in a
second biological sample). Preferably, the polypeptide level or mRNA level in
the first
biological sample is measured or estimated and compared to a standard
polypeptide level or
mRNA level, the standard being taken from a second biological sample obtained
from an
individual not having the related disorder or being determined by averaging
levels from a
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population of individuals not having a related disorder. As will be
appreciated in the art,
once a standard polypeptide level or mRNA level is known, it can be used
repeatedly as a
standard for comparison. ,
[335] By "biological sample" is intended any biological sample obtained from
an
individual, body fluid; cell line, tissue culture, or other source which
contains polypeptide of
the present invention or the corresponding mRNA. As indicated, biological
samples include
body fluids (such as semen, lymph, vaginal pool, sera, .plasma, -urine,
synovial fluid and
spinal fluid) which contain the polypeptide of the present invention, and
tissue sources found
to express the polypeptide of the present invention. Methods for obtaining
tissue biopsies
and body fluids from mammals are well known in the art. Where the biological
sample is to .
include mRNA, a tissue biopsy'is the preferred source.
[336] The methods) provided above may preferably be applied in a diagnostic
method
and/or kits in Which polynucleotides and/or polypeptides of the invention are
attached to a
solid support. In one exemplary method, the support may be a "gene chip" or a
"biological
chip" as described in US Patents 5,837,832, 5,874,219, and 5,856,174. Further,
such a gene
chip with polynucleotides of the invention attached may be used to identify
polymorphisms
between the isolated polynucleotide sequences of the invention, with
polynucleotides isolated
from a test subject. The knowledge of such polymorphisms (i.e. their location,
as well as,
their existence) would be beneficial in identifying disease loci for many
disorders, such as for
example, in neural disorders, immune system disorders, muscular disorders,
reproductive
disorders, gastrointestinal disorders, pulmonary disorders, digestive
disorders, metabolic
disorders, cardiovascular disorders, renal disorders, proliferative disorders,
and/or 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.
[337] The present invention encompasses polynucleotides of the present
invention that
are chemically .synthesized, or reproduced as peptide nucleic acids (PNA), or
according to
other methods known in the art. The use of PNAs would serve as the preferred
form if the
polynucleotides of the invention are incorporated onto a solid support, or
gene chip. For the
purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide
type of DNA
analog and the monomeric units for adenine, guanine, thymine and cytosine are
available
commercially (Perceptive Biosystems). Certain components of DNA, such as
phosphorus,
phosphorus 'oxides, or deoxyribose derivatives, are not present in PNAs. As
disclosed by
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Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365; 666
(1993), PNAs
bind specifically and tightly to complementary DNA strands and are not
degraded by
nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This
is probably
because there is no electrostatic repulsion between the two strands, and also
the polyamide
backbone is more flexible. Because of this, PNA/DNA duplexes bind under a
wider range of
stringency conditions than DNA/DNA duplexes, making it easier to perform
multiplex
hybridization. Smaller probes can be used than with DNA due to the strong
binding. In
addition, it is more likely that single base mismatches can be determined with
PNA/DNA
hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting
point
(Tm) by 8°-20° C, vs. 4°-16° C for the
DNA/DNA 1 S-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.
S
[338] The compounds of the present invention have uses which include, but are
not
limited to, detecting cancer in mammals. In particular the invention is useful
during
diagnosis of pathological cell proliferative neoplasias which include, but are
not limited to:
acute myelogenous leukemias including acute monocytic leukemia, acute
myeloblastic
leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute
erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated
leukemia, etc.;
and chronic myelogenou's leukemias including chronic myelomonocytic Teukemia,
chronic
granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats,
dogs, cows,
pigs, horses, rabbits and humans. Particularly preferred are humans.
[339] Pathological cell proliferative disorders are often associated with
inappropriate
activation of proto-oncogenes. (Gelmann, E. P. et al., "The Etiology of Acute
Leukemia:
Molecular Genetics and Viral Oncology," in Neoplastic Diseases of the Blood,
Vol 1.,
Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to
result from the
qualitative alteration of a normal cellular gene product, or from the
quantitative modification
of gene expression by insertion into the chromosome of a viral sequence, by
chromosomal
translocation of a gene to a more actively transcribed region, or by some
other mechanism.
(Gelmann et al., supra) It is likely that mutated or altered expression of
specific genes is
involved in the pathogenesis of some leukemias, among other tissues and cell
types.
(Gelmann et al., supra) Indeed, the human counterparts of the oncogenes
involved in some
animal neoplasias have been amplified or translocated in some cases of human
leukemia and
carcinoma. (Gelmann et al., supra)
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[340] For example; c-myc expression is highly amplified in. the non-
lymphocytic
leukemia cell line HL-60. When HL-60 cells are chemically induced to stop
proliferation,
the level of c-myc is found to be downregulated. (International Publication
Number WO
91/15580). However, it has been shown that exposure of HL-60 cells to a DNA
construct that
is complementary to the 5' end of c-myc or c-myb blocks translation of the
corresponding
mRNAs which downregulates expression of the c-myc or c-myb proteins and causes
arrest of
cell proliferation and differentiation of the treated cells. (International
Publication Number
WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi
et al., Proc.
Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would
appreciate the present
invention's usefulness is not be limited to treatment, prevention, and/or
prognosis of
proliferative disorders of cells and tissues of hematopoietic origin, in light
of the numerous
cells and cell types of varying origins which are known to exhibit
proliferative phenotypes.
[341] In addition to the foregoing, a polynucleotide of the present invention
can be used
to control gene expression through triple helix formation or through antisense
DNA or RNA.
Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:
560 (1991);
"Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC-Press,
Boca Raton,
FL (1988). Triple helix formation is discussed in, for instance Lee et al.,
Nucleic Acids
Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et
al.,-Science
251: 1360 (1991). Both methods rely on binding of the polynucleotide to a
complementary
DNA or RNA. For these techniques, preferred polynucleotides are usually
oligonucleotides
20 to 40 bases in length and complementary to either the region of the gene
involved in
transcription (triple helix - see Lee et al., Nucl~. Acids Res. 6:3073 (1979);
Cooney et al.,
Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the
mRNA itself
(antisense - Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix
formation
optimally results in a shut-off of RNA transcription from DNA, while antisense
RNA
hybridizatiomblocks translation of an mRNA molecule into polypeptide. The
oligonucleotide
described above can also be delivered to cells such that the antisense RNA or
DNA may be
expressed in vivo to inhibit production of polypeptide of the present
invention antigens. Both
techniques are effective in model systems, and the information disclosed
herein can be used
to design antisense or triple helix polynucleotides in an effort to treat
disease, and in
particular, for the treatment of proliferative diseases and/or conditions. Non-
limiting
antisense and triple helix methods encompassed by the present invention are
more thoroughly
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described elsewhere herein (see, e.g., the section labeled "Antisense and
Ribozyme
(Antagonists)").
[342] Polynucleotides of the present invention are also useful in gene
therapy. One goal -
of gene therapy is to insert a normal gene into an organism having a defective
gene, in an
effort to correct the genetic defect. The polynucleotides disclosed in the
present invention
offer a means of targeting such genetic defects in a highly accurate manner.
Another goal is
to insert a new gene that was not present in the host genome, thereby
producing a new trait in
the host cell. Additional non-limiting examples of gene therapy methods
encompassed by the
present invention are more thoroughly described elsewhere herein (see, e.g.,
the sections
labeled "Gene Therapy Methods", and Examples 16, 17 and 18).
[343] 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.
[344] 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.
[345] Forensic biology also benefits from using DNA-based identification
techniques as
disclosed herein. DNA sequences taken from very small biological samples such
as tissues,
e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial
fluid, amniotic fluid,
breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc.,
can. be
amplified using PCR. In one prior art technique, gene sequences amplified from
polymorphic loci, such as DQa class II HLA gene, are used in forensic biology
to identify
individuals. (Erlich, H., PCR Technology, Freeman and Co. ( 1992)). Once these
specific
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polymorphic loci are amplified, they are digested with one or more restriction
enzymes,
yielding an identifying set of bands on a Southern blot probed with DNA
corresponding to
the DQa class II HLA gene. Similarly, polynucleotides of the present invention
can be used
as polymorphic markers for forensic purposes.
[346] There is also a need for reagents capable of identifying the source of a
particular
tissue. Such need arises, for example, in forensics when presented with tissue
of unknown
origin. Appropriate reagents can'comprise, for example, DNA probes or primers
prepared
from the sequences of the present invention, specific to tissues, including
but not limited to
those shown in Table 1A. Panels of such reagents can identify tissue by
species and/or by
organ type. In a similar fashion, these reagents can be used to screen tissue
cultures for
contamination. Additional non-limiting examples of such uses are further
described herein.
[347] The polynucleotides of the present invention are also useful as
hybridization
probes for differential identification of the tissues) or cell types) present
in a biological
sample. Similarly, polypeptides and antibodies directed to polypeptides of the
present
invention are useful to provide immunological probes for differential
identification of the
tissues) (e.g., immunohistochemistry assays) or cell types) (e.g.,
immunocytochemistry
assays). In addition, for a number of disorders of the above tissues or cells,
significantly
higher or lower levels of gene expression of the polynucleotides/polypeptides
of the present
invention may be detected in certain tissues (e.g., tissues expressing
polypeptides and/or
polynucleotides of the present invention, for example, those disclosed in
column 8 of Table
1A, and/or cancerous and/or wounded tissues) or bodily fluids (e.g., semen,
lymph, vaginal
pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an
individual having
such a disorder, relative to a "standard" gene expression level, i.e., the
expression level in
healthy tissue from an individual not having the disorder.
[348] Thus, the invention provides a diagnostic method of a disorder, which
involves: (a)
assaying gene expression level in cells or body fluid of an individual; (b)
comparing the gene
expression level with a standard gene expression level, whereby an increase or
decrease in
the assayed gene expression level compared to the standard expression level is
indicative of a
disorder.
[349] 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
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attachment to a "gene chip" or other support, to raise anti-DNA antibodies
using DNA
immunization techniques, and as an antigen to elicit an immune response.
Uses of the Polypeptides
[350] Each of the polypeptides identified herein can be used in numerous ways.
The
following description should be considered exemplary and utilizes known
techniques.
[351] Polypeptides and antibodies directed to polypeptides of the present
invention are
useful to provide immunological probes for differential identification of the
tissues) (e.g.,
immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et
al., J.
Histochem. Cytochem. 29:577-580 (1981)) or cell types) (e.g.,
immunocytochemistry
assays).
[352] Antibodies can be used to assay levels of polypeptides encoded by
polynucleotides
of the invention in a biological sample using classical immunohistological
methods known to
those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-
985 (1985); Jalkanen,
et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods
useful for
detecting protein gene expression include immunoassays, such as the enzyme
linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay
labels are known in the art and include enzyme labels, such as, glucose
oxidase;
radioisotopes, such as iodine ('31I, lzsh lz3h lzll)~ carbon (14C), sulfur
(35S), tritium (3H),
indium (115mIn', 113mIn' ilzln' 111In), and technetium (99TC, 99mTC), thallium
(z°1T1), gallium
(68Ga, 67Ga), palladium (lo3Pd), molybdenum (99Mo), xenon (133Xe), fluorine
('8F), 1's3Sm,
177Lu 159Gd 149Pm 140La 175Yb 166H~ 90Y 47SC 186Re 188Re 142Pr lOSRh 97Ru~
> > > > > > > > > > > > >
luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
[353] In addition to assaying levels of polypeptide of the present invention
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. '
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[354] A protein-specific antibody or antibody fragment which has been labeled
with an
appropriate detectable imaging moiety, such as a radioisotope (for example,
'3'I, "ZIn, 99mTc,
(i3~h 125h lz3h i2'I), carbon ('4C), sulfur (35S), tritium (3H), indium
("5"'In, "3"'In, "2In, "'In),
and technetium (99Tc, 99"'Tc)_, thallium (2°'Ti), gallium (68Ga, 6~Ga),
palladium ('°3Pd),
mol bdenum 99Mo , xenon '33Xe , fluorine '8F 'S3Sm 177Lu 'S9Gd '49Pm
'4°La "SYb
Y ( ) ( ) ( > > > > > > >
166H~' 9oY~ 4~sc~ ~s6Re, 'BgRe, ~42Pr, losRh, 97Ru), a radio-opaque substance,
or a material
detectable by nuclear magnetic resonance, is introduced (for example,
parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined for immune
system
disorder. It will be understood in the art that the size of the subject and
the imaging system
used will determine the quantity of imaging moiety needed to produce
diagnostic images. In
the case of a radioisotope moiety, for a human subject, the quantity of
radioactivity injected
will normally range from about 5 to 20 millicuries of 99mTc. The labeled
antibody or
antibody fragment will then preferentially accumulate at the location of cells
which express
the polypeptide encoded by a polynucleotide of the invention. In vivo tumor
imaging is
described in S.W. Burchiel et al.; "Immunopharmacokinetics of Radiolabeled
Antibodies and
Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of
Cancer,
S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
[355] In one embodiment, the invention provides a method for the specific
delivery of
compositions of the invention to cells by administering polypeptides of the
invention (e.g.,
polypeptides encoded by. polynucleotides of the invention and/or antibodies)
that are
associated with heterologous polypeptides or nucleic acids. In one example,
the invention
provides a method for delivering a therapeutic protein into the 'targeted
cell. In another
example, the invention provides a method for delivering a single stranded
nucleic acid (e.g.;
antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can
integrate into the
cell's genome or replicate episomally and that can be transcribed) into the
targeted cell.
[356] In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides of the
invention in association with toxins or cytotoxic prodrugs.
[357] By "toxin" is meant one or more compounds that bind ,and activate
endogenous
cytotoxic effector systems, radioisotopes, holotoxins, modified toxins,
catalytic subunits of
toxins, or any molecules or enzymes not normally present in or on the surface
of a cell that
under defined conditions cause the cell's death. Toxins, that may be used
according to the
methods of the invention include, but are not limited to, radioisotopes known
in the art,
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compounds such as, for example, antibodies (or complement fixing containing
portions
thereof) that bind an inherent or induced endogenous cytotoxic effector
system, thymidine
kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin
A, diphtheria
toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin
and cholera
toxin. "Toxin" also includes a cytostatic or cytocidal agent, a therapeutic
agent or a
radioactive metal ion, e.g., alpha-emitters such as, for example, z'3Bi, or
other radioisotopes
such as, for example, lo3Pd, 133Xe, 1311, 6gGe, s7Co, 6sZn, BsSr, 3zP, 3sS,
901, ls3Sm, ls3Gd,
169Yb, slCr, saMn, ~sse, "3Sn, 9°Yttrium, "'Tin, 'g6Rhenium,
'66Holmium, and '$BRhenium;
luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and.biotin. In a specific embodiment, the invention provides a
method for the
specific destruction of cells (e.g., the destruction of tumor cells) by
administering
polypeptides of the invention or antibodies of the invention in association
with the
radioisotope 9°Y. In another specific embodiment, the invention
provides a method for the
specific destruction of cells (e.g., the destruction of tumor cells) by
administering
polypeptides of the invention or antibodies of the invention in' association
with the
radioisotope "'In. In a further specific embodiment, the invention provides a
method for the
specific destruction of cells (e.g., the destruction of tumor cells) by
administering
polypeptides of the invention or antibodies of the invention in association
with the
radioisotope '3' I.
[358] Techniques known in the art may be applied to label polypeptides of the
invention
(including antibodies). Such techniques include, but are not limited to, the
use of
bifunctional conjugating agents (see e.g., U.S. Patent Nos. 5,756,065;
5,714,631; 5,696,239;
5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119;
4,994,560;
and 5,808,003; the contents of each of which are hereby incorporated by
reference in its
entirety).
[359] Thus, the invention provides a diagnostic method of a disorder, which
involves (a)
assaying the expression level of a polypeptide of the present invention in
cells or body fluid
of an individual; and (b) comparing the assayed polypeptide expression level
with a standard
polypeptide expression level, whereby an increase or decrease in the assayed
polypeptide
expression level compared to the standard expression level is indicative of a
disorder. With
respect to cancer, the presence of a relatively high amount of transcript in
biopsied tissue
from an individual may indicate a predisposition for the development of the
disease, or may
provide a means for detecting the disease prior to the appearance of actual
clinical symptoms.
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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.
[360] Moreover, polypeptides of the present invention can be used to treat or
prevent
diseases or conditions such as, for example, neural disorders, immune system
disorders,
muscular disorders, reproductive disorders, gastrointestinal disorders,
pulmonary disorders,
cardiovascular disorders, renal disorders, prolifeTative disorders, and/or
cancerous diseases
and conditions. For example, patients can be administered a polypeptide of the
present
invention in an effort to replace absent or decreased levels of the
polypeptide (e.g., insulin),
to supplement absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S for
hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of
a polypeptide
(e.g., an oncogene or tumor supressor), to activate the activity of a
polypeptide (e.g., by
binding to a receptor), to reduce the activity of a membrane bound receptor by
competing
with it for free ligand (e.g., soluble TNF receptors used in reducing
inflammation), or to
bring about a desired response (e.g., blood vessel growth inhibition,
enhancement of the
immune response to proliferative cells or tissues).
[361] Similarly, antibodies directed to a polypeptide of the present invention
can also be
used to treat disease (as described supra, and elsewhere herein). For example,
administration
of an antibody directed to a polypeptide of the present invention can bind,
and/or neutralize
the polypeptide, and/or reduce overproduction of the polypeptide. Similarly,
administration
of an antibody can activate the polypeptide, such as by binding to a
polypeptide bound to a
membrane (receptor).
[362] At the very least, the polypeptides of the present invention can be used
as
molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration
columns
using methods well known to those of skill in the art. Polypeptides can also
be used to raise
antibodies, which. in turn are used to measure protein expression from a
recombinant cell, as
a way of assessing transformation of the host cell. Moreover, the polypeptides
of the present
invention can be used to test the biological activities described herein.
Diagnostic Assays
[363] The compounds of the present invention are useful for diagnosis,
treatment,
prevention and/or prognosis of various disorders in mammals, preferably
humans. Such
disorders include, but are not limited to, those described herein under the
section heading
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"Biological Activities".
[364] For a number of disorders, substantially altered (increased or
decreased) levels of
gene expression can be detected in tissues, cells or bodily fluids (e.g.,
sera, plasma, urine,
semen, synovial fluid or spinal fluid) taken from an individual having such a
disorder,
relative to a "standard" gene expression level, that is, the expression level
in tissues or bodily.
fluids from an individual not having the disorder. Thus, the invention
provides a diagnostic ,
method useful during diagnosis of a disorder, which involves measuring the
expression level
of the gene encoding the polypeptide in tissues, cells or body fluid from an
individual and
comparing the measured gene expression level with a standard gene expression
level,
whereby an increase or decrease in the gene expression levels) compared to the
standard is
indicative of a disorder. These diagnostic assays may be performed in vivo or
in vitro, such
as, for example, on blood samples, biopsy tissue or autopsy tissue:
[365] The present invention is also useful as. a prognostic indicator, whereby
patients
exhibiting enhanced or depressed gene expression will experience a worse
clinical outcome
relative to patients expressing the gene at a level nearer the standard level.
[366] In certain embodiments, a polypeptide of the i-nvention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose diseases and/or disorders associated with the
tissues) in which the
polypeptide of the invention is expressed, including one, two, three, four,
five, or more
tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[367] By "assaying the expression level of the gene encoding the polypeptide"
is
intended qualitatively or quantitatively measuring or estimating the level of
the polypeptide
of the invention or the level of the mRNA encoding the polypeptide of the
invention in a first
biological sample either directly (e.g., by determining or estimating absolute
protein level or
mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA
level in a
second biological sample). Preferably, the polypeptide expression level or
mRNA level in
the first biological sample is measured or estimated and compared to a
standard polypeptide
level or mRNA level, the standard being taken from a second biological sample
obtained
from an individual not having the disorder or being determined by averaging
levels from a
population of individuals not having the disorder. As will be appreciated in
the art, once a
standard polypeptide level or mRNA level is known, it can be used repeatedly
as a standard
for comparison.
(368] By "biological sample" is intended any biological sample obtained from
an
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individual, cell line, tissue culture, or other source containing polypeptides
of the invention
(including portions thereof] or mRNA. As indicated, biological samples include
body fluids
(such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue
sources found to
express the full length or fragments thereof of a polypeptide or mRNA. Methods
for
obtaining tissue biopsies and body fluids from mammals are well known in the
art. Where
the biological sample is to include mRNA, a tissue biopsy is the preferred
source.
[369] Total cellular RNA can be isolated from a biological sample using any
suitable
technique such as the single-step guanidinium-thiocyanate-phenol-chloroform
method
described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987-).
Levels of
mRNA encoding the polypeptides of the invention are then assayed using any
appropriate
method. These include Northern blot analysis, S 1 nuclease mapping, the
polymerise chain
reaction (PCR), reverse transcription in combination with -the polymerise
chain reaction
(RT-PCR), and reverse transcription in combination with the ligase chain
reaction (RT-LCR).
[370] The present invention also relates to diagnostic assays such as
quantitative and
diagnostic assays for detecting levels of polypeptides of the invention, in a
biological sample
(e.g., cells and tissues), including determination of normal and abnormal
levels of
polypeptides. Thus, for instance, a diagnostic assay in accordance with the
invention for
detecting over-expression of polypeptides of the invention compared to normal
control tissue
samples may be used to detect the presence of tumors. Assay techniques that
can be used to
determine levels of a polypeptide, such as a polypeptide of the present
invention in a sample
derived from a host are well-known to those of skill in the art. Such assay
methods include
radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA
assays.
Assaying polypeptide levels in a biological sample can occur using any art-
known method.
[371] Assaying polypeptide levels in a biological sample can occur using
antibody-based
techniques. For example, polypeptide expression in tissues can be studied with
classical
immunohistological methods (Jalkanen et al., J. Cell: Biol. 101:976-985
(1985); Jalkanen,
M., et al., J. Cell . Biol. 105:3087-3096 (1987)). Other antibody-based
methods useful for
detecting polypeptide gene expression include immunoassays, such as the enzyme
linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody
assay
labels are known in the art and include enzyme labels, such as, glucose
oxidise, and
radioisotopes, such as iodine ('zsh lzll), carbon ('4C), sulfur (3sS), tritium
(3H), indium ("zIn),
and technetium (99mTc), and fluorescent labels, such as fluorescein and
rhodamine, and
biotin.
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[372] The tissue or cell type to be analyzed will generally include those
which are
known, or. suspected, to express the gene of inteest (such as, for example,
cancer). The
protein isolation methods employed herein may, for example, be such as those
described in
Harlow and Lane (Harlow, E. and Lane, D., 1988, "Antibodies: A Laboratory
Manual", Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, New York), which is
incorporated
herein by reference in its entirety. The isolated cells can be derived from
cell culture or from
a patient. The analysis,of cells taken from culture may be a necessary step in
the assessment
of cells, that could be used as part of a cell-based gene therapy technique
or, alternatively, to
test the effect of compounds on the expression of the gene.
[373] For example, antibodies, or fragments of antibodies, such as those
described
herein, may be used to quantitatively or qualitatively detect the presence of
gene products or
conserved variants or peptide fragments thereof. This can be accomplished, for
example, by
immunofluorescence techniques employing a fluorescently labeled antibody
coupled with
light microscopic, flow cytometric, or fluorimetric detection.
[374] In a preferred embodiment, antibodies, or fragments of antibodies
directed to any
one or all of the predicted epitope domains of the polypeptides of the
invention (shown in
column 7 of Table 1A) may be used to quantitatively or qualitatively detect
the presence of
gene products or conserved variants or peptide fragments thereof. This can be
accomplished,
for example, by immunofluorescence techniques employing a fluorescently
labeled antibody
coupled with light microscopic, flow cytometric, or fluorimetric detection.
[375] In an additional preferred embodiment, antibodies, or fragments of
antibodies
directed to a conformational epitope of a polypeptide of the invention may be
used to
quantitatively or qualitatively detect the presence of gene products or
conserved variants or
peptide fragments thereof. This can be accomplished, for example, by
immunofluorescence
techniques employing a fluorescently labeled antibody coupled with light
microscopic, flow
cytometric, or fluorimetric detection.
[376] The antibodies (or fragments thereof), and/or polypeptides of the
present invention
may, additionally, be employed histologically, as in immunofluorescence,
immunoelectron
microscopy or non-immunological assays, for in situ detection of gene products
or conserved
variants or peptide fragments thereof. In situ detection may be accomplished
by removing a
histological specimen from, a patient, and applying thereto a labeled antibody
or polypeptide
of the present invention. The antibody (or fragment thereof) or polypeptide is
preferably
applied by overlaying the labeled antibody (or fragment) onto a biological
sample. Through
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the use of such a procedure, it is possible to determine not only the presence
of the gene
product, or conserved variants or peptide fragments, or polypeptide binding,
but also its
distribution in the examined tissue. Using the present invention, those of
ordinary skill will
readily perceive that any of a wide variety of histological methods (such as
staining
procedures) can be modified in order to achieve such in situ detection.
[377] Immunoassays and non-immunoassays for gene products or conserved
variants or
peptide fragments thereof will typically comprise incubating a sample, such as
a biological
fluid, a tissue extract, freshly harvested cells, or lysates of cells which
have been incubated in
cell culture, in the presence of a detectably labeled antibody capable of
binding gene products
or conserved variants or peptide fragments thereof, and detecting the bound
antibody by any
of a number of techniques well-known in the art. ,
[378] The biological sample may be brought in contact with and immobilized
onto a
solid phase support or carrier such as nitrocellulose, or other solid.support
which is capable
of immobilizing cells, cell particles or soluble proteins. The support may
then be washed
with suitable buffers followed by treatment with the detectably labeled
antibody or detectable
polypeptide of the invention. The solid phase support may then be washed with
the buffer a
second time to remove unbound antibody or polypeptide. Optionally the
,antibody is
subsequently labeled. The amount of bound label on solid support may then be
detected by
conventional means.
[379] By "solid phase support or carrier" is intended any support capable of
binding an
antigen or an antibody. Well-known supports or carriers include glass,
polystyrene,
polypropylene, polyethylene, dextran, nylon, amylases, natural and modified
celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier can be
either soluble to
some extent or insoluble for the purposes of the present invention. The
support material may
have virtually any possible structural configuration so long as the coupled
molecule is
capable of binding to an antigen or antibody. Thus, the support configuration
may be
spherical, as in a bead, or cylindrical, as in the inside surface of a test
tube, or the external
surface of a rod. Alternatively, the surface may be flat such as a sheet, test
strip, etc.
Preferred supports include polystyrene beads. Those skilled in the art will
know many other
suitable carriers for binding antibody or antigen, or will be able to
ascertain the same by use
of routine experimentation.
[380] The binding activity of a given lot of antibody or antigen polypeptide
may be
determined according to well known methods. Those skilled in the art will be
able to
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determine operative and optimal assay conditions for each determination by
employing
routine experimentation.
[381] In addition to assaying polypeptide levels or polynucleotide levels in a
biological
sample obtained from an individual, polypeptide or polynucleotide can also be
detected in
vivo by imaging. For example, in one embodiment of the invention, polypeptides
and/or
antibodies of the invention are used to image diseased cells, such as
neoplasms. In another
embodiment, polynucleotides of the invention (e.g., polynucleotides
complementary to all or
a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one
or a
combination of the epitopes of a polypeptide of the invention, antibodies
directed to a
conformational epitope of a polypeptide of the invention, or antibodies
directed to the full
length polypeptide expressed on the cell surface of a mammalian cell) are used
to image
diseased or neoplastic cells.
[382] Antibody labels or markers for in vivo imaging of polypeptides of the
invention
include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For
X-radiography; suitable labels include radioisotopes such as barium or cesium,
which emit
detectable radiation but are not overtly harmful to the subject. Suitable
markers for NMR
and ESR include those with a detectable characteristic spin, such as
deuterium, which may be,
incorporated into the antibody by labeling of nutrients for the relevant
hybridoma. Where in
vivo imaging is used to detect enhanced levels of polypeptides for diagnosis
in humans, it
may be preferable to use human antibodies or ''humanized" chimeric monoclonal
antibodies.
Such antibodies can be produced using techniques described herein or otherwise
known in
the art. For example methods for producing chimeric antibodies are known in
the art. See,
for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214
(1986);
Cabilly et al., U.S. Patent No. 4,816,567; Taniguchi et al., EP 171496;
Morrison et al., EP
173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne
et al.,
Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).
[383] Additionally, any polypeptides of the invention whose presence can be
detected,
can be administered. For example, polypeptides of the invention labeled with a
radio-opaque
or other appropriate compound can be administered and visualized in vivo, as
discussed,
above for labeled antibodies. Further, such polypeptides can be utilized for
in vitro.
diagnostic procedures.
[384] ~ A polypeptide-specific antibody or antibody fragment which has been
labeled with
an appropriate detectable imaging moiety, such as a radioisotope (for example,
'31I, "ZIn,
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99mTC), a radio-opaque substance, or a material detectable by nuclear magnetic
resonance, is
introduced (for example, parenterally, subcutaneously or intraperitoneally)
into the mammal
to be examined for a disorder. It will be understood in the art that the size
of the subject and
the imaging system used will determine the quantity of imaging moiety needed
to produce
diagnostic images. In the case of a radioisotope moiety, for a human subject,
the quantity of
radioactivity injected will normally range from about 5 to 20 millicuries of
99mTc. The
labeled antibody or antibody fragment will then preferentially accumulate at
the location of
cells which contain the antigenic protein. In vivo tumor imaging is described
in S.W.
Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments"
(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W.
Burchiel and
B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
[385] With respect to antibodies, one of the ways in which an antibody of the
present
'invention can be detectably labeled is by linking the same to a reporter
enzyme and using the
linked product in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological
Associates Quarterly Publication, Walkersville, MD); Voller et al., J. Clin.
Pathol. 31:507-
520 (1978); Butler, J.E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.),
1980, Enzyme
Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa, E. et al., (eds.), 1981,
Enzyme
Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the
antibody
will react with an appropriate substrate, preferably a chromogenic substrate,
in such a~manner
as to produce a chemical moiety which can be detected, for example, by
spectrophotometric,
fluorimetric or by visual means. Reporter enzymes which can be used to
detectably label the
antibody include, but are not limited to, malate dehydrogenase, staphylococcal
nuclease,
delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-
glycerophosphate,
dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline
phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease,
catalase, glucose-6-
phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally,
the
detection can be accomplished by colorimetric methods which employ a
chromogenic
substrate for the reporter enzyme. Detection may also be accomplished by
visual comparison
of the extent of enzymatic reaction of a substrate in comparison with
similarly prepared
standards.
[386] Detection may also-be accomplished using any of a variety of other
immunoassays.
For example, by radioactively labeling the antibodies or antibody fragments,
it is possible to
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detect polypeptides through the use of a radioimmunoassay (RIA) (see, for
example,
Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on
Radioligand
Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by
reference
herein). The radioactive isotope can be detected by means including, but not
limited to, a
gamma counter, a scintillation counter, or autoradiography.
[387] It is also possible to label the antibody with a fluorescent compound.
When the
fluorescently labeled antibody is exposed to light of the proper wave length,
its presence can
then be detected due to fluorescence. Among the most commonly used fluorescent
labeling
compounds are fluorescein isothiocyanate; rhodamine, phycoerythrin,
phycocyanin,
allophycocyanin, ophthaldehyde and fluorescamine.
[388] The antibody can also be detectably labeled using fluorescence emitting
metals
such as lszEu, or others of the lanthanide series. These metals can be
attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA)
or
ethylenediaminetetraacetic acid (EDTA).
[389] The antibody also can be detectably labeled by coupling it to a
chemiluminescent
compound. The presence of the cherriiluminescent-tagged antibody is then
determined by
detecting the presence of luminescence that arises during the course of a
chemical reaction.
Examples of particularly useful chemiluminescent labeling compounds are
luminol,
isoluminol, theromatic acridinium ester, imidazole, acridinium salt and
oxalate ester.
[390] Likewise, a bioluminescent compound may be used to label the antibody of
the
present invention. Bioluminescence is a type of chemiluminescence found in
biological
systems in, which a catalytic protein increases the efficiency of the
chemiluminescent
reaction. The presence of a bioluminescent protein is determined by detecting
the presence
of luminescence. Important bioluminescent compounds for purposes of labeling
are
luciferin, luciferase and aequorin.
Methods for Detective Diseases
[391] In general, a disease may be detected in a patient based on the presence
of one or
more proteins of the invention and/or polynucleotides encoding such proteins
in a biological
sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from
the patient. In
other words, such proteins may be used as markers to indicate the presence or
absence of a
disease or disorder, including cancer and/or as described elsewhere herein. In
addition, such
proteins may be useful for the detection of other diseases and cancers. The
binding agents
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provided herein generally permit detection of the level of antigen that binds
to the agent in
the biological sample. Polynucleotide primers and probes may be used to detect
the level of
mRNA encoding polypeptides of the invention, which is also indicative of the
presence or
absence of a disease or disorder, including cancer. In general, polypeptides
of the invention
should be present at a level that is at least three fold higher in diseased
tissue than in normal
tissue.
[392] There are a variety of assay formats known to those of ordinary skill in
the art for
using a binding agent to detect polypeptide markers in a sample. See, e.g.,
Harlow and Lane,
supra. In general, the presence or absence of a disease in a patient may be
determined by (a)
contacting a biological sample obtained from a patient with a binding agent;
(b) detecting in
the sample a level of polypeptide that binds to the binding agent; and (c)
comparing the level
of polypeptide with a predetermined cut-off value.
[393] In a preferred embodiment, the assay involves the use of a binding
agents)
immobilized on a solid support to bind to and remove the polypeptide of the
invention from
the remainder of the sample. The bound polypeptide may then be detected using
a detection
reagent that contains a reporter group and specifically binds to the binding
agent/polypeptide
complex. Such detection reagents may comprise, for example, a binding agent
that
specifically binds to the polypeptide or an antibody or other agent that
specifically binds to
the binding agent, such as an anti-immunoglobulin, protein G, protein A or a
~lectin.
Alternatively, a competitive assay may be utilized, in which a polypeptide'is
labeled with a
reporter group and allowed to bind to the immobilized binding agent after
incubation of the
binding agent with the sample. The extent to which components of the sample
inhibit the
binding of the labeled polypeptide to the binding agent is indicative of the
reactivity of the
sample with the immobilized binding agent. Suitable polypeptides for use
within such assays
include polypeptides of the invention and portions thereof, or antibodies, to
which the
binding agent, binds, as described above.
[394] The solid support may be any material known to those of skill. in the
art to which
polypeptides of the invention may be attached. For example, the solid support
may be a test
well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the
support may be a bead or disc, such as glass fiberglass, latex or a plastic
material such as
polystyrene or polyvinylchloride. The support may also be a magnetic particle
or a fiber optic
sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
The binding agent
may be immobilized on the solid support using a variety of techniques known to
those of skill
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in the art, which are amply described in the patent and scientific literature.
In the context of
the present invention, the teim. "immobilization" refers to both noncovalent
association, such
as adsorption, and covalent attachment (which may be a direct linkage between
the agent and
functional groups on the support or may be a linkage by way of a cross-linking
agent).
Immobilization by adsorption to a well in a microtiter plate or to a membrane
is preferred. Im
such cases, adsorption may be achieved by contacting the binding agent, in a
suitable buffer,
with the solid support for the suitable amount of time. The contact time
varies with .
temperature, but is typically between about 1 hour and about 1 day. In
general, contacting a
well of plastic microtiter plate (such as polystyrene or polyvinylchloride)
with an amount of
binding agent ranging from about 10 ng to about 10 ug, and preferably about
100 ng to about
1 ug, is sufficient to immobilize an adequate amount of binding agent.
[395] Covalent attachment of binding agent to a solid support may generally be
achieved
by first reacting the support with a bifunctional reagent that will react with
both the support
and a functional group, such as a hydroxyl or amino group, on the binding
agent. For
example, the binding agent may be covalently attached to supports having an
appropriate
polymer coating using benzoquinone or by condensation of an aldehyde group on
the support
with an amine and an active hydrogen on the binding partner (see, e.g., Pierce
Immunotechnology Catalog and Handbook, 1991, at A12-A13).
Gene Therapy Methods
[396] Also encompassed by the invention are gene therapy methods for treating
or
preventing disorders, diseases and conditions. The gene therapy methods relate
to the
introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences
into an
animal to achieve expression of the polypeptide of the present invention. This
method
requires a polynucleotide which codes for a polypeptide of the present
invention operatively
linked to a promoter and any other genetic elements necessary for the
expression of the
polypeptide by the target tissue. Such gene therapy and delivery techniques
are known in the
art, see, for example, W090/11092, which.is herein incorporated by reference.
(397] Thus, for example, cells from a patient may be engineered with a
polynucleotide
(DNA or RNA) comprising a promoter operably linked to a polynucleotide of the
present
invention ex vivo, with the engineered cells then being provided to a patient
to be treated
with the polypeptide of the present invention. Such methods are well-known in
the art. For
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example, see Belldegrun; A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993);
Ferrantini, M. et
al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.
Immunology 153: 4604-
4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H.,
et al., Cancer
Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-
10 (1996);
Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et
al., Cancer
Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In
one
embodiment, the cells which are engineered are arterial cells. The arterial
cells may be
reintroduced into the patient through direct injection to the artery, the
tissues surrounding the
artery, or through catheter injection.
[398] 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 Garner.
[399] In one embodiment, the polynucleotide of the present invention is
delivered as a
naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to
sequences
that are free from any delivery vehicle that acts to assist, promote or
facilitate entry into the
cell, including viral sequences, viral particles, liposome formulations,
lipofectin or
precipitating agents and the like. However, the polynucleotide of the present
invention can
also be delivered in liposome formulations and lipofectin formulations and the
like can be
prepared by methods well known to those skilled in the art. Such methods are
described, for
example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are
herein
incorporated by reference.
[400] The polynucleotide vector constructs used in the gene therapy method are
preferably constructs that will not integrate into the host genome nor will
they contain
sequences that allow for replication. Appropriate vectors include pWLNEO,
pSV2CAT,
pOG44, pXTI and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEFl/V5, pcDNA3.1, and pRc/CMV2 available from
Invitrogen. Other suitable vectors will be readily apparent to the skilled
artisan.
[401] Any strong promoter known to those skilled in the art can be used for
driving the
expression of the polynucleotide sequence. Suitable promoters include
adenoviral promoters,
such as the adenoviral major late promoter; or heterologous promoters, such as
the
cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)
promoter; inducible
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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
acon promoter; and human growth hormone promoters. The promoter also may be
the native
' promoter for the polynucleotide of the present invention.
[402] 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.
[403] The polynucleotide construct can be delivered to the interstitial space
of tissues
within the an animal, including of muscle, skin, brain, lung, liver, spleen,
bone marrow;
thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder,
stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and
connective tissue.
Interstitial space of the tissues comprises the intercellular, fluid,
mucopolysaccharide matrix
among the reticular fibers of organ tissues, elastic fibers in the,walls of
vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within connective
tissue ensheathing
r
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 maybe 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.
[404] For the naked nucleic acid sequence injection, an effective dosage
amount of DNA
or RNA will be in the range of from about 0.05 mg/kg body weight to about 50
mg/kg body
weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg
and more
preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan
of ordinary
skill will appreciate, this dosage will vary according to the tissue site of
injection. The
appropriate and effective dosage of nucleic acid sequence can readily~be
determined by those
of ordinary skill in the art and may depend on the condition being treated and
the route of
administration.
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[405] 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.
[406] 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.
[407] 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.
[408] In certain embodiments, the polynucleotide constructs are complexed in a
liposome preparation. Liposomal preparations for use in the instant invention
include
cationic (positively charged), anionic (negatively charged) and neutral
preparations.
However, cationic liposomes are particularly preferred because a tight charge
complex can be
formed between the cationic liposome and the polyanionic nucleic acid.
Cationic liposomes
have been shown to mediate intracellular delivery of plasmid DNA (Felgner et
al., Proc. Natl.
Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by
reference); mRNA
(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is
herein
incorporated by reference); and purified transcription factors (Debs et al.,
J. Biol. Chem.
(1990) 265:10189-10192, which is herein incorporated by reference), in
functional form.
[409] Cationic liposomes are readily available. For example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are
particularly
useful and are available under the trademark Lipofectin, from GIBCO BRL, Grand
Island,
N.Y. (See, also, Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84:7413-
7416, which is
herein incorporated by reference). Other commercially available liposomes
include
transfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).
[410] Other cationic liposomes can be prepared from readily available
materials using
techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092
(which is
herein incorporated by reference) for a description of the synthesis of DOTAP
(1,2-
bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA
liposomes
is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl.
Acad. Sci. USA
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84:7413-7417, which is herein incorporated by reference. Similar methods can
be used to
prepare liposomes from other cationic lipid materials.
[411] Similarly, anionic and neutral liposomes are readily available, such as
from Avanti
Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily
available materials.
Such materials include phosphatidyl, choline, cholesterol, phosphatidyl
ethanolamine,
dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG),
dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can
also be mixed
with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for
making
liposomes using these materials are well known in the art.
[412] 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, DOPGlDOPC 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.
[413] The liposomes can comprise multilamellar vesicles (MLVs), small
unilamellar
vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being
preferred. The
various liposome-nucleic acid complexes are prepared using methods well known
in the art.
See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527,
which is herein ._
incorporated by reference. For example, MLVs containing nucleic acid can be
prepared by
depositing a thin film of phospholipid on the walls of a glass tube and
subsequently hydrating
with a solution of the material to be encapsulated. SUVs are prepared by
extended sonication
of MLVs to produce a homogeneous population of unilamellar liposomes. The
material to be
entrapped is added to a suspension of preformed MLVs and then sonicated. When
using
liposomes containing cationic lipids, the dried lipid film is resuspended in
an appropriate
solution such as sterile water or an isotonic buffer solution such as 10 mM
Tris/NaCI,
sonicated, and then the preformed liposomes are mixed directly with the DNA.
The liposome
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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 Caz+-
EDTA
chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483;
Wilson et al.,
Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim.
Biophys. Acta
443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977);
Fraley et al.,
Proc. Natl. Acad. Sci. USA 76:3348 ( 1979)); detergent dialysis (Enoch, H. and
Strittmatter,
P., Proc. Natl. Acad. Sci. USA 76:145 ( 1979)); and reverse-phase evaporation
(REV) (Fraley
et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D.,
Proc. Natl.
Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)),
which are
herein incorporated by reference.
[414] Generally, the ratio of DNA to liposomes will be from about 10:1 to
about 1:10.
Preferably, the ration will be from about 5:1 to about 1:5. More preferably,
the ration will be
about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.
[415J U.S. Patent No. 5,676,954 (which is herein incorporated by reference)
reports on
the injection of genetic material, complexed with cationic liposomes Garners,
into mice. U.S.
Patent Nos. 4,897,355; 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622,
5,580,859,
5,703,055, and international publication no. WO 94/9469 (which are herein
incorporated by
reference) provide cationic lipids for use in transfecting DNA into cells and
mammals. U.S.
Patent Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international
publication no.
WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to
mammals.
[416] In certain embodiments, cells are engineered, ex vivo or in vivo, using
a retroviral
particle containing RNA which comprises a sequence encoding a polypeptide of
the present
invention. Retroviruses from which the retroviral plasmid vectors may be
derived include,
but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus,
Rous sarcoma
Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus,
human
immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor
virus.
[417] 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+envAm 12, and DAN cell lines as described in Miller, Human
Gene
Therapy 1:5-14 (1990), which is incorporated herein by reference in its
entirety. The vector
may transduce the packaging cells through any means known in the art. Such
means include,
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but are not limited to, electroporation, the use of liposomes, and CaP04
precipitation. In one
alternative, the retroviral plasmid vector may be encapsulated into a
liposome, or coupled to a
lipid, and then administered to a host.
[418] The producer cell line generates infectious retroviral vector particles
which include
polynucleotide encoding a polypeptide of the present invention. Such
retroviral vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or in vivo. The
transduced eukaryotic cells will express a polypeptide of the present
invention.
[419] ' In certain other embodiments, cells are engineered, ex vivo or in
vivo, with
polynucleotide contained in an adenovirus vector. Adenovirus can be
manipulated such that
it encodes and expresses a polypeptide of the present invention, and at the
same time is
inactivated in terms of its ability to replicate in a normal lytic viral life
cycle. Adenovirus
expression is achieved without integration of the viral DNA into the host cell
chromosome,
thereby alleviating concerns about insertional mutagenesis. Furthermore,
adenoviruses have
been used as live enteric vaccines for many years with an excellent safety
profile (Schwartz
et al. Am. Rev. Respir. Dis.109:233-238-(1974)). Finally, adenovirus mediated
gene transfer
has been demonstrated in a number of instances including transfer of alpha-1-
antitrypsin and
CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science
252:431-434;
Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to
attempt to
establish adenovirus as a causative agent in human cancer were uniformly
negative (Green,
M. et al. ( 1979) Proc. Natl. Acad. Sci. USA 76:6606).
(420] Suitable adenoviral vectors useful in the present invention are
described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993);
Rosenfeld
et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-
769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692
(1993); and
U.S. Patent No. 5,652,224, which are herein incorporated by reference. For
example, the
adenovirus vector Ad2 is useful and can be grown in human 293 cells. These
cells contain the.
El region of adenovirus and constitutively express Ela and Elb, which
complement the
defective adenoviruses by providing the products of the genes deleted from the
vector. In
addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are
also useful in the
present invention.
[421] 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
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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: E 1 a, E 1 b, E3, E4, E2a, or L 1 through L5.
[422] 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, N., Curr. Topics in
Microbiol.
Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate
its DNA into
non-dividing cells. Vectors containing as little as 300 base pairs of AAV can
be packaged
and can integrate, but space . for exogenous DNA is limited to about 4.5 kb.
Methods 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.
[423] For example, an appropriate AAV vector for use in the present invention
will
include all the sequences necessary for DNA replication, encapsidation, and
host-cell
integration. The polynucleotide construct is inserted into the AAV vector
using standard
cloning methods, such as those found in Sambrook et al., Molecular Cloning: A
Laboratory
Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then
transfected
into packaging cells which are infected with a helper virus, using any
standard technique,
including lipofection, electroporation, calcium phosphate precipitation, etc.
Appropriate
helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or
herpes viruses.
Once the packaging cells.are transfected and infected, they will produce
infectious AAV viral
particles which contain the polynucleotide construct. These viral particles
are then used to
transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells
will contain the
polynucleotide construct integrated into its genome, and will express a
polypeptide of the
invention.
[424] Another method of gene therapy involves operably associating
heterologous
control regions and endogenous polynucleotide sequences (e.g. encoding a
polypeptide of the
present invention) via homologous recombination (see, e.g., U.S. Patent No.
5,641,670,
issued June 24, 1997; International Publication No. WO 96/29411, published
September 26,
1996; International Publication No. WO 94/12650, published August 4, 1994;
Koller et al.,
Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature
342:435-438
(1989), which are herein encorporated by reference. This method involves the
activation of a
gene which is present in the target cells, but which is not normally expressed
in the cells, or is
expressed at a lower level than desired.
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[425) 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.
[426] 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.
[427] 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.
[428] 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.
[429] The polynucleotide encoding a polypeptide of the present invention may
contain a
secretory signal sequence that facilitates secretion of the protein.
Typically, the signal
sequence is positioned in the coding region of the polynucleotide to be
expressed towards or
at the S' 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.
[430] 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 ari amount sufficient to provide a therapeutic effect. This includes direct
needle injection,
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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)).
[431] A preferred method of local administration is by direct injection.
Preferably, a
recombinant molecule of the present invention complexed with a delivery
vehicle is
administered by direct injection into or locally within the area of arteries.
Administration of a
composition locally within the area of arteries refers to injecting the
composition centimeters
and preferably, millimeters within arteries.
[432] Another method of local administration is to contact a polynucleotide
construct of
the present invention in or around a, surgical woumd. 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.
[433] Therapeutic compositions useful in systemic administration, include
recombinant
molecules of the present invention complexed to a targeted delivery vehicle of
the present
invention. Suitable delivery vehicles for use with systemic administration
comprise
liposomes comprising ligands for targeting the vehicle to a particular site.
In specific
embodiments, suitable delivery vehicles for use with systemic administration
comprise
liposomes comprising polypeptides of the invention for targeting the vehicle
to a particular
site.
[434] 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.
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[435] 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.
[436] Therapeutic compositions of the present invention can be administered to
any
animal, preferably to mammals and birds. Preferred mammals include humans,
dogs, cats,
mice, rats, rabbits sheep, cattle, horses and pigs, with humans being
particularly preferred.
Biological Activities
[437] Polynucleotides or polypeptides, or agonists or antagonists of the ~
present
invention, cam be used in assays to test for one or more biological
activities. . If these
polynucleotides or polypeptides, or agonists or antagonists of the present
invention, do
exhibit activity in a particular assay, it is likely that these molecules may
be involved in the
diseases associated with the biological activity. Thus, the polynucleotides
and polypeptides,
and agonists or antagonists could be used to diagnose, prognose, prevent
and/or treat the
associated disease.
(438] Many members of the ABC Transport Receptor family of proteins are
believed' to
be involved in- the active transport of small hydrophilic molecules across the
cytoplasmic
membrane. ABC Transport family members have been implicated in confernng upon
cells
resistance to a wide range of drugs, such as chemotherapeutic drugs (Gottesman
et al., JBiol.
Chem., 263:12163-6 (1988). Furthermore, dysfunction of ABC Transport Receptor
polypeptides have been implicated in such diseases and/or disorders as cystic
fibrosis and
unregulated insulin secretion in patients with Familial persistent
hyperinsulinemic
hypoglycemia of infancy. Accordingly, compositions of the invention (including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) may be used in the diagnosis, prognosis, prevention and/or treatment
of diseases
and/or disorders associated with aberrant ABC Transport.Receptor activity.
(439] In preferred embodiments, compositions of the invention (including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) may be used in the diagnosis, prognosis, detection and/or treatment
of diseases
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and/or disorders relating to blood disorders (e.g., insulin secretion
disorders, and/or as
described under "Immune activity" and "Cardiovascular Disorders" below) and
neoplastic
disorders (e.g., as described under "Hyperproliferative Disorders" below).
[440] In certain embodiments, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose diseases and/or disorders associated with the
tissues) in which the
polypeptide of the invention is expressed, including one, two, three, four,
five, or more of the
tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[441J Thus, polynucleotides, translation products and antibodies of the
invention are
useful in the diagnosis, prognosis, prevention and/or treatment of diseases
and/or disorders
associated with activities that include, but are not limited to, cellular drug
resistance, cystic
fibrosis, and unregulated insulin secretion.
[442] More generally, polynucleotides, translation products and antibodies
corresponding to this gene may be useful for the diagnosis, prognosis,
prevention and/or
treatment of diseases and/or disorders associated with the following systems.
Immune Activity
[443] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, diagnosing and/or
prognosing
diseases, disorders, and/or conditions of the immune system, by, for example,
activating or
inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of
immune cells.
Immune cells develop through a process called' hematopoiesis, producing
myeloid (platelets,
red blood cells, neutrophils, and macrophages) and lymphoid (B and T
lymphocytes) cells
from pluripotent stem cells. The etiology of these immune diseases, disorders,
and/or
conditions may be genetic, somatic, such as cancer and some autoimmune
diseases, acquired
(e.g., by chemotherapy or toxins), or infectious. Moreover, polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention can be
used as a marker or
detector of a particular immune system disease or disorder.
[444] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to treat
diseases and disorders of the immune system and/or to inhibit or enhance an
immune
response generated by cells associated with the tissues) in which the
polypeptide of the
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invention is expressed, including one, two, three, four, five, or more tissues
disclosed in
Table 1A, column 8 (Tissue Distribution Library Code).
[445] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, diagnosing, and/or
prognosing
immunodeficiencies, including both congenital and acquired immunodeficiencies.
Examples
of B cell immunodeficiencies in which immunoglobulin levels B cell function
and/or B cell
numbers are decreased include: X-linked agammaglobulinemia (Biuton's disease),
X-linked
infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-
linked
immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP),
agammaglobulinemia including congenital and acquired agammaglobulinemia, adult
onset
agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,
hypogainmaglobulinemia, unspecified hypogammaglobulinemia, recessive
agatiimaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA
deficiency,
selective IgG subclass deficiencies, IgG subclass deficiency (with or without
IgA deficiency),
Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM,
antibody
deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain
deficiency, B
cell lymphoproliferative disorder (BLPD), common variable immunodeficiency
(CVID),
common variable immunodeficiency (CVI) (acquired), and transient
hypogammaglobulinemia of infancy.
[446] In specific embodiments, ataxia-telangiectasia or conditions associated
with ataxia-
telangiectasia are treated, prevented, diagnosed, and/or prognosing using the
polypeptides or
polynucleotides of the invention, and/or agonists or antagonists thereof.
[447] Examples of congenital immunodeficiencies in which T cell and/or B cell
function
and/or number is decreased include, but are not limited to: DiGeorge anomaly,
severe
combined immunodeficiencies (SCID) (including, but riot limited to, X-linked
SCID,
autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside
phosphorylase
(PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-
Aldrich
syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth
pharyngeal pouch
syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer
cell
deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with
predominant
T cell defect (unspecified), and unspecified immunodeficiency of cell mediated
immunity.
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[448] In specific embodiments, DiGeorge anomaly or conditions associated with
DiGeorge anomaly are treated, prevented, diagnosed, and/or prognosed using
polypeptides or
polynucleotides of the invention, or antagonists or agonists thereof.
[449] Other immunodeficiencies that may be treated, prevented, diagnosed,
and/or
prognosed using polypeptides or polynucleotides of the invention, and/or
agonists or
antagonists thereof, include, but are not limited to, chronic granulomatous
disease, Chediak-
Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate
dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP),
leukocyte
adhesion deficiency, complement component deficiencies (including CI, C2, C3,
C4, C5, C6,
C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-
aplasia,
immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with
immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof
syndrome-
combined immuriodeficiency with Igs.
[450] In a preferred embodiment, the immunodeficiencies and/or conditions
associated
with the immunodeficiencies recited above are treated, prevented, diagnosed
and/or
prognosed using polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention.
[451] In a preferred embodiment polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention could be used as an agent to
boost
immurioresponsiveness among immunodeficient individuals. In specific
embodiments,
polynucleotides, polypeptides, antibodies, and/or agonists~ or antagonists of
the present
invention could be used as an agent to boost immunoresponsiveness among B cell
and/or T
cell immunodeficient individuals.
[452] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, diagnosing and/or
prognosing
autoimmune disorders. Many autoimmune disorders result from inappropriate
recognition of
self as foreign material by immune cells. This inappropriate recognition
results in an
immune response leading to the destruction of the host tissue. Therefore, the
administration
of polynucleotides and polypeptides of the invention that can inhibit an
immune response,
particularly the proliferation, differentiation, or. chemotaxis of T-cells,
may be an effective
therapy in preventing autoimmune disorders.
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[453] Autoimmune diseases or disorders that may be treated, prevented,
diagnosed
and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists
or antagonists
of the present invention include, but are not limited to, one or more of the
following:
systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis,
multiple
sclerosis, autoimmune thyroiditis, Hashirrioto's thyroiditis, autoimmune
hemolytic anemia,
hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura,
autoimmune
neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g.,
Henloch-
Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus
vulgaris,
myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant
diabetes mellitus.
[454] Additional disorders that are likely to have an autoimmune component
that may be
treated, prevented, and/or diagnosed with the compositions of the invention
include; but are
not limited to, type II collagen-induced arthritis, antiphospholipid syndrome,
dermatitis,
allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic
heart disease,
neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff
Man Syndrome,
autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin
dependent
diabetes mellitus, and autoimmune inflammatory eye disorders.
[455] Additional disorders that are likely to have an autoimmune component
that may be
treated, prevented, diagnosed and/or prognosed with the compositions of the
invention
include, but are not limited to, scleroderma with anti-collagen antibodies
(often characterized,
e.g., by nucleolar and other nuclear antibodies), mixed connective tissue
disease (often
characterized, e.g., by antibodies to extractable nuclear antigens (e.g.,
ribonucleoprotein)),
polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia
(often
characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor
antibodies),
idiopathic Addison's disease (often characterized, e.g., by humoral and cell-
mediated adrenal
cytotoxicity, infertility (often characterized, e.g., by antispermatozoal
antibodies),
glomerulonephritis (often characterized, e.g., by glomerular basement membrane
antibodies
or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG
and
complement in basement membrane), Sjogren's syndrome (often characterized,
e.g., by
multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes
mellitus
(often characterized, e.g., by cell-mediated and humoral islet cell
antibodies), and adrenergic
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drug resistance (including adrenergic drug resistance with asthma or cystic
fibrosis) (often
characterized, e.g., by beta-adrenergic receptor antibodies).
(456] Additional disorders that may have an autoimmune component that may be
treated,
prevented, diagnosed and/or prognosed with the compositions of the invention
include, but
are not limited to, chronic active hepatitis (often characterized, e.g., by
smooth muscle
antibodies), primary biliary cirrhosis (often characterized, e.g., by
mitochondria antibodies),
other endocrine gland failure (often characterized, e.g., by specific tissue
antibodies in some
cases), vitiligo (often characterized, e.g., by melanocyte antibodies),
vasculitis (often
characterized, e.g., by Ig and complement in vessel walls and/or low serum
complement),
post-MI (often characterized,~e.g., by myocardial antibodies), cardiotomy
syndrome (often
characterized, e.g., by myocardial antibodies), urticaria (often
characterized, e.g., by IgG and
IgM antibodies to IgE), atopic dermatitis (often characterized, e.g.-, by IgG
and IgM
antibodies to IgE), asthma ('often characterized, e.g., by IgG and IgM
antibodies to IgE), and
many other inflammatory, granulomatous, degenerative, and atrophic disorders. -
[457] In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated, prevented,
diagnosed and/or prognosed using for example, antagonists or agonists,
polypeptides or
polynucleotides, or antibodies of the present invention. In a specific
preferred embodiment,
rheumatoid arthritis is treated, prevented, and/or diagnosed using
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention.
[458] In another specific preferred embodiment, systemic lupus erythematosus
is treated,
prevented, and/or diagnosed using polynucleotides, polypeptides, antibodies,
and/or agonists
or antagonists of the present invention. In another specific preferred
embodiment, idiopathic
thrombocytopenia purpura is treated, prevented, and/or diagnosed using
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention.
[459] In another specific preferred embodiment IgA nephropathy is treated,
prevented,
and/or diagnosed using polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention.
[460] In a preferred embodiment, the autoimmune diseases and disorders and/or
conditions associated with the diseases and disorders recited above are
treated, prevented,
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diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies,
and/or agonists
or antagonists of the present invention
[461] In preferred embodiments, polypeptides, antibodies, polynucleotides
and/or
agonists or antagonists of the present invention are used as a
immunosuppressive agent(s).
[462] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, prognosing, and/or
diagnosing
diseases, disorders, and/or conditions of hematopoietic cells.
Polynucleotides, polypeptides,
antibodies, and/or agonists or antagonists of the present invention could be
used to increase
differentiation and proliferation of hematopoietic cells, including the
pluripotent stem cells,
in an effort to treat or prevent those diseases, disorders, and/or conditions
associated with a
decrease in certain (or many) types hematopoietic cells, including but not
limited to,
leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively,
Polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention could be
used to increase differentiation and proliferation of hematopoietic cells,
including the
pluripotent stem cells, in an effort to treat or prevent those diseases,
disorders, and/or
conditions associated with an increase in certain (or many) types of
hematopoietic cells,
including but not limited to, histiocytosis.
[463] Allergic reactions and conditions, such as asthma (particularly allergic
asthma) or
other respiratory problems, may also be treated, prevented, diagnosed and/or
prognosed using
polypeptides, antibodies, or polynucleotides of the invention, and/or agonists
or antagonists
thereof. Moreover, these molecules can be used to treat, prevent, prognose,
and/or diagnose
anaphylaxis, hypersensitivity to an antigenic molecule, or blood group
incompatibility.
[464] Additionally, polypeptides or polynucleotides of the invention, and/or
agonists or
antagonists thereof, may be used to treat, prevent, diagnose and/or prognose
IgE-mediated
allergic reactions. Such allergic reactions include, but are not limited to,
asthma, rhinitis, and
eczema. In specific embodiments, polynucleotides, polypeptides, antibodies,
and/or agonists
or antagonists of the present.invention may be used to modulate IgE
concentrations in vitro
or m mvo.
[465] Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the present invention have uses in the diagnosis, prognosis,
prevention, and/or
treatment of inflammatory conditions. For example, since polypeptides,
antibodies, or
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polynucleotides of the invention, and/or agonists or antagonists of the
invention may inhibit
the activation, proliferation and/or differentiation of cells involved in an
inflammatory
response, these molecules can be used to prevent and/or treat chronic and
acute inflammatory
conditions. Such inflammatory conditions include, but are not limited to, for
example,
inflammation associated with infection (e.g., septic shock, sepsis, or
systemic inflammatory
response syndrome), ischemia-reperfusion injury, endotoxin lethality,
complement-mediated
hyperacute rejection, nephritis, cytokine or chemokine induced lung injury,
inflammatory
bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-
1.), respiratory
disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g.,
inflammatory bowel
disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast);
CNS disorders (e.g.,
multiple sclerosis; ischemic brain injury andlor stroke, traumatic brain
injury,
neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's
disease); AIDS-
related dementia; and prion disease); cardiovascular disorders (e.g.,
atherosclerosis,
myocarditis, cardiovascular disease, and cardiopulmonary bypass
complications); as well as
many additional diseases, conditions, and disorders that are characterized by
inflammation
(e.g., hepatitis, rheumatoid arthritis, gout, trauma, pancreatitis,
sarcoidosis, dermatitis, renal
ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus,
diabetes
mellitus, and allogenic transplant rejection).
(466] Because inflammation is a fundamental defense mechanism, inflammatory
disorders can effect virtually any tissue of the body. Accordingly,
polynucleotides,
polypeptides, and antibodies of the invention, as well as agonists. or
antagonists thereof, have
uses in the treatment of tissue-specific inflammatory disorders, including,
but not limited to,
adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis,
blepharitis, bronchitis,
bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis,
cochlitis, colitis, conjunctivitis,
cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis,
eustachitis,
fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis,
hepatosplenitis, keratitis,
labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis,
metritis, mucitis,
myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis,
osteochondritis, otitis,
pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis,
poliomyelitis, prostatitis,
pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis,
scrotitis, sinusitis,
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spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis,
tonsillitis, urethritis, and
vaginitis.
[467] In specific embodiments, polypeptides, antibodies, or polynucleotides of
the
invention, and/or agonists or antagonists thereof, are useful to diagnose,
prognose, prevent,
and/or treat organ transplant rejections and graft-versus-host disease. Organ
rejection occurs
by host immune cell destruction of the transplanted tissue through an immune
response.
Similarly, an immune response is also involved in GVHD, but, in this case, the
foreign
transplanted immune cells destroy the host tissues. Polypeptides, antibodies,
or
polynucleotides of the invention, and/or agonists or antagonists thereof, that
inhibit an
immune response, particularly the activation, proliferation, differentiation,
or chemotaxis of
T-cells, may be an effective therapy in preventing organ rejection or GVHD. In
specific
embodiments, polypeptides, antibodies, or polynucleotides of the invention,
and/or agonists
or antagonists thereof, that inhibit an immune response, particularly the
activation,
proliferation, differentiation, or chemotaxis of T-cells, may be an effective
therapy in
preventing experimental allergic and hyperacute xenograft rejection.
[468] In other embodiments, polypeptides, antibodies, or polynucleotides of
the
invention, and/or agonists or antagonists thereof, are useful to diagnose,
prognose, prevent,
and/or treat immune complex diseases, including, but not limited to, serum
sickness, post
streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-
induced
vasculitis.
[469] Polypeptides, antibodies, polynucleotides and/or agonists or antagonists
of the
invention can be used to treat, detect, and/or prevent infectious agents. For
example, by
increasing the immune response, particularly increasing the proliferation
activation and/or
differentiation of B and/or T cells, infectious diseases may be treated,
detected, and/or
prevented. The immune response may be increased by either enhancing an
existing immune
response, or by initiating a new immune response. Alternatively,
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may also
directly inhibit the infectious agent (refer to section of application listing
infectious agents,
etc), without necessarily eliciting an immune response.
[470] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are used as a vaccine adjuvant that
enhances immune
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responsiveness to an antigen. In a specific embodiment, polypeptides,
antibodies,
polynucleotides and/or agonists or antagonists of the present invention are
used as an
adjuvant to enhance tumor-specific immune responses.
[471] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an adjuvant to
enhance anti-viral .
immune responses. Anti-viral immune responses that may be enhanced using the,
compositions of the invention as an adjuvant, include virus and virus
associated diseases or
symptoms described herein or otherwise known in the art. In specific
embodiments, the
compositions of the invention are used as an adjuvant to enhance an immune
response to a
virus, disease, or symptom selected from the group consisting of: AIDS,
meningitis, Dengue,
EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the
compositions of
the invention are used aS an adjuvant to enhance an immune response to a
virus, disease, or
symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial
virus,
Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza,
measles,
cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes
simplex, and yellow
fever.
[472] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an adjuvant to
enhance anti-
bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal
immune responses
that may be enhanced using the compositions of the invention as an adjuvant,
include
bacteria or fungus and bacteria or fungus associated diseases or symptoms
described herein-
or otherwise known in the art. In specific embodiments, the compositions of
the invention
are used as an adjuvant to enhance an immune response to a bacteria or fungus,
disease, or
symptom selected from the group consisting of: tetanus, Diphtheria, botulism,
and meningitis
type B.
[473] In another specific embodiment, the compositions of the invention are
used as an
adjuvant to enhance an immune response to a bacteria or fungus, disease, or
symptom
selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae,
Salmonella
typhi, Salmonella paratyphi, Meisseria meningitides, Streptococcus pneumoniae,
Group B
streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E. coli,
and Borrelia burgdorferi.
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[474] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an adjuvant to
enhance anti-
parasitic immune responses. Anti-parasitic immune responses that may be
enhanced using the
compositions of the invention as an adjuvant, include parasite and parasite
associated
diseases or symptoms described herein or, otherwise known in the art. In
specific
embodiments, the compositions of the invention are used as an adjuvant to
enhance an
immune response to a parasite. In another specific embodiment, the
compositions of the
invention are used as an adjuvant to enhance an immune response to Plasmodium
(malaria)
or Leishmania.
[475] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may also be employed to treat
infectious
diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis;
for example, by
preventing the recruitment and activation of mononuclear phagocytes.
[476] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an antigen for
the generation of
antibodies to inhibit or enhance immune mediated responses against
polypeptides of the
invention.
[477] In one embodiment, polypeptides, antibodies, polynucleotides and/or
agonists or
antagonists of the present invention are administered to an animal (e.g.,
mouse, rat, rabbit,
hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep,
dog, cat, non-
human primate, and human, most preferably human) to boost the immune system to
produce
increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE),
to induce
higher affinity antibody production and immunoglobulin class switching (e.g.,
IgG, IgA,
IgM, and IgE), and/or to increase an immune response.
[478] In another specific embodiment, polypeptides; antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a stimulator of B
cell
responsiveness to pathogens.
[479] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an activator of T
cells.
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[480] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent that
elevates the immune
status of an individual prior to their receipt of immunosuppressive therapies.
[481] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
induce higher affinity
antibodies.
[482] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
increase serum
immunoglobulin concentrations.
[483] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
accelerate recovery of
immunocompromised individuals.
[484] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists. or' antagonists of the present invention are used as an agent to
boost
immunoresponsiveness among aged populations and/or neonates.
[485] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an immune system
enhancer prior
to, during, or after bone marrow transplant and/or other transplants (e.g.,
allogeneic or
xenogeneic organ transplantation). With respect to transplantation,
compositions of the
invention may be administered prior to, concomitant with, and/or after
transplantation. In a
specific embodiment, compositions of the invention are administered after
transplantation,
prior to the beginning,of recovery of T-cell populations. In another specific
embodiment,
compositions of the invention are first administered after transplantation
after the beginning
of recovery of T cell populations, but prior to full recovery of B cell
populations.
[486] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to boost
immunoresponsiveness among individuals having an acquired loss of B cell
function.
Conditions resulting in an acquired loss of B cell function that may be
ameliorated or treated
by administering the polypeptides, antibodies, polynucleotides and/or agonists
or antagonists
thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow
transplant, and B
cell chronic lymphocytic leukemia (CLL).
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[487] In another specific embodiment; polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to boost
immunoresponsiveness among individuals having a temporary immune deficiency.
Conditions resulting in a temporary immune deficiency that may be ameliorated
or treated by
administering the polypeptides, antibodies, polynucleotides and/or agonists or
antagonists
thereof, include, but are not limited to, recovery from viral infections
(e.g., influenza),
conditions associated with malnutrition, recovery from infectious
mononucleosis, or
conditions associated with stress, recovery from measles, recovery from blood
transfusion,
and recovery from surgery.
[488] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a regulator of
antigen presentation
by monocytes, dendritic cells, and/or B-cells. In one embodiment,
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention enhance
antigen presentation or antagonizes antigen presentation in vitro or in vivo.
Moreover, in
related embodiments, said enhancement or antagonism of antigen presentation
may be useful
as an anti-tumor treatment or to modulate the immune system.
[489] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
direct an individual's
immune system towards development of a humoral response (i.e. TH2) as opposed
to a TH1
cellular response:
[490] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means to induce
tumor
proliferation and thus make it more susceptible to anti-neoplastic agents. For
example,
multiple myeloma is a slowly dividing disease and is thus refractory to
virtually all anti-
neoplastic regimens. If these cells were forced to proliferate more rapidly
their susceptibility
profile would likely change.
[491] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a stimulator of B
cell production
in pathologies such as AIDS, chronic lymphocyte disorder and/or Common
Variable
Immunodificiency.
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[492] ~ . In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
generation and/or
regeneration of lymphoid tissues following surgery, trauma or genetic defect.
In another
specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists
or
antagonists of the present invention are used in the pretreatment of bone
marrow samples
prior to transplant.
[493] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a gene-based
therapy for
genetically inherited disorders resulting in immuno-
incompetence/immunodeficiency such as
observed among SCID patients.
[494] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
activating
monocytes/macrophages to defend against parasitic diseases that effect
monocytes such as
Leishmania.
(495J In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
regulating secreted
cytokines that are elicited by polypeptides of the invention.
[496] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists ~of the present invention are used in one or more of the
applications decribed
herein, as they may apply to veterinary medicine.
[497] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
blocking various
aspects of immune responses to foreign agents or self. Examples of.diseases or
conditions in
which blocking of certain aspects of immune responses may be desired include
autoimmune
disorders such as lupus,. and arthritis, as well as immunoresponsiveness to
skin allergies,,
inflammation, bowel disease, injury and diseases/disorders associated with
pathogens.
[498] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
preventing the B cell
proliferation and Ig secretion associated with autoimmune diseases such as
idiopathic
thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.
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[499] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a inhibitor of B
and/or T cell
migration in endothelial cells. This activity disrupts tissue architecture or
cognate responses
and is useful, for example in disrupting immune responses, and blocking
sepsis.
[500] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a theiapy for
chronic
hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of
undetermined significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonal
gammopathies, and plasmacytomas.
[501] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may be employed for instance
to inhibit
polypeptide chemotaxis and activation of macrophages and their precursors, and
of
neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated
and CD8
cytotoxic T cells and natural killer cells, in certain autoimmune and chronic
inflammatory
and infective diseases. Examples of autoimmune diseases are described herein
and include
multiple sclerosis, and insulin-dependent diabetes.
[502] The polypeptides, antibodies, polynucleotides and/or agonists or
antagonists of the
present invention may also be employed to treat idiopathic hyper-eosinophilic
syndrome by,
for example, preventing eosinophil production and migration.
[503] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used to enhance or
inhibit complement
mediated cell lysis.
[504] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used to enhance or
inhibit antibody
dependent cellular cytotoxicity.
[505] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may also be employed for
treating
atherosclerosis, for example, by.preventing monocyte infiltration in the
artery wall.
[506] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may be employed to treat
adult respiratory
distress syndrome CARDS).
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(507] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention may be useful for stimulating
wound and
tissue repair, stimulating angiogenesis, and/or stimulating the repair of
vascular or lymphatic
diseases or disorders. Additionally, agonists and antagonists of the invention
may be used to
stimulate the regeneration of mucosal surfaces.
[508] In a specific embodiment, polynucleotides or polypeptides, and/or
agonists thereof
are used to diagnose, prognose, treat, and/or prevent a disorder characterized
by primary or
acquired immunodeficiency, deficient serum immunoglobulin production,
recurrent
infections, and/or immune system dysfunction. Moreover, polynucleotides or
polypeptides,
and/or agonists thereof may be used to treat or prevent infections of the
joints, bones, skin,
and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis,
septic arthritis, and/or
osteomyelitis), autoimmune diseases (e.g., those disclosed herein),
inflammatory 'disorders,
and malignancies, and/or any disease or disorder or condition associated with
these
infections, diseases, disorders and/or malignancies) including, but not
limited to, CVID,
other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis,
sinusitis, otitis
media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g.,
severe herpes
zoster), and/or pneumocystis carnii. Other diseases and disorders that may be
prevented,
diagnosed, prognosed, and/or treated with polynucleotides or polypeptides,
and/or agonists
of the present invention include, but are not limited to, HIV infection, HTLV-
BLV infection,
lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and
hemoglobinuria.
[509J In another embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists
or antagonists of the present invention are used to treat, and/or diagnose an
individual having
common variable immunodeficiency disease ("CVID"; also known as "acquired
agammaglobulinemia" and "acquired hypogammaglobulinemia") or a subset of this
disease.
[510J In a specific embodiment, polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be used to diagnose,
prognose, prevent,
and/or treat cancers or neoplasms including immune cell or immune tissue-
related cancers or
neoplasms. Examples of cancers or neoplasms that may be prevented, diagnosed,
or treated
by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present
invention include, but are not limited to, acute myelogenous leukemia, chronic
myelogenous
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leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia
(ALL)
Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's
lymphoma,
EBV-transformed diseases, and/or diseases and disorders described in the
.section entitled
"Hyperproliferative Disorders" elsewhere herein.
[511] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
decreasing cellular
proliferation of Large B-cell Lymphomas.
[512] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a means of
decreasing the
involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.
[513] In specific embodiments, the compositions of the invention are used as
an agent to
boost immunoresponsiveness among B cell immunodeficient individuals, such as,
for
example, an individual who has undergone a partial or complete splenectomy.
[514] Antagonists of the invention include, for example, binding and/or
inhibitory
antibodies, antisense nucleic acids, ribozymes or soluble forms of the
polypeptides of the
present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of
the invention
include, for example, binding or stimulatory antibodies, and soluble forms of
the
polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides,
antibodies,
polynucleotides and/or agonists or antagonists of the present invention may be
employed in a
composition with a pharmaceutically acceptable carrier, e.g., as described
herein.
[515] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are administered to an animal
(including, but not
limited to, those listed above, and also including transgenic animals)
incapable of producing
functional endogenous antibody molecules or having an otherwise compromised
endogenous
immune system, but which is capable of producing human immunoglobulin
molecules by
means of a reconstituted or partially reconstituted immune system from another
animal (see,
e.g., published PCT Application Nos. W098/24893, WO/9634096, WO/9633735, and
WO/9110741). Administration of polypeptides, antibodies, polynucleotides
and/or agonists
or antagonists of the present invention to such animals is useful for the
generation of
monoclonal antibodies against. the polypeptides, antibodies, polynucleotides
and/or agonists
or antagonists of the present invention.
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Blood-Related Disorders
[516] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be used to modulate hemostatic (the stopping of
bleeding) or
thrombolytic (clot dissolving) activity. For example, by increasing hemostatic
or
thrombolytic activity, polynucleotides or polypeptides, and/or agonists or
antagonists of the
present invention could be used to treat or prevent blood coagulation
diseases; disorders,
and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia),
blood platelet
diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds
resulting from
trauma, surgery, or other causes. Alternatively, polynucleotides,
polypeptides, antibodies,
and/or agonists or antagonists of the present invention that can decrease
hemostatic or
thrombolytic activity could be used to inhibit or dissolve clotting. These
molecules could be
important in the treatment or prevention of heart attacks (infarction),
strokes, or scarring.
[517] In specific embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may .be used to prevent,
diagnose, prognose,
and/or treat thrombosis, arterial thrombosis, venous thrombosis,
thromboembolism,
pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic
attack,
unstable angina. In specific embodiments, the polynucleotides, polypeptides,
antibodies,
and/or agonists or antagonists of the present invention may be used for the
prevention of
occulsion of saphenous grafts, for reducing the risk of periprocedural
thrombosis as might
accompany angioplasty procedures, for reducing the risk of stroke in patients
with atrial
fibrillation including nonrheumatic atrial fibrillation, for reducing the risk
of embolism
associated with mechanical heart valves and or mural valves disease. Other
uses for the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention, include, but are not limited to, the prevention of occlusions in
extrcorporeal
devices (e.g., intravascular canulas, vascular access shunts in hemodialysis
patients,
hemodialysis machines, and cardiopulmonary bypass machines).
[518] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists; or antagonists corresponding to that polypeptide, may be
used to
prevent, diagnose, prognose, and/or treat diseases and disorders of the blood
andJor blood
forming organs associated with the tissues) in which the polypeptide of the
invention is
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expressed, including one, two, three, four, five, or more tissues disclosed in
Table 1A,
column 8 (Tissue Distribution Library Code).
[519] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be used to modulate hematopoietic activity (the
formation of blood
cells). For example, the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention may be used to increase the quantity of
all or subsets of
blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells),
myeloid cells
(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets. The ability
to decrease the quantity of blood cells or subsets of blood cells may be
useful in the
prevention, detection, diagnosis and/or treatment of anemias and leukopenias
described
below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention may be used to decrease the. quantity of
all or subsets of
blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells),
myeloid cells
(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets.. The ability
to decrease the quantity of blood cells or subsets of blood cells may be
useful in the
prevention, detection, diagnosis and/or treatment of leukocytoses, such as,
for example
eosinophilia.
[520] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be used to prevent, treat, or diagnose blood
dyscrasia.
[521] Anemias are conditions in which the number of red blood cells or amount
of
hemoglobin (the protein that carries oxygen) in them is below normal. Anemia
may be
caused by excessive bleeding, decreased red blood cell production, or
increased red blood
cell destruction (hemolysis). The polynucleotides, polypeptides, antibodies,
and/or agonists
or antagonists of the present invention may be useful in treating, preventing,
and/or
diagnosing anemias. Anemias that may be treated prevented or diagnosed by the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention include iron deficiency anemia, hypochromic anemia, microcytic
anemia,
chlorosis, hereditary siderob;astic anemia, idiopathic acquired sideroblastic
anemia, red cell
aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B 12
deficiency) and folic
acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune
helolytic
anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal
hemoglobinuria).
The polynucleotides, polypeptides, antibodies, and/or. agonists or antagonists
of the present
invention may be useful in treating, preventing, and/or diagnosing anemias
associated with
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diseases including but not limited to, anemias associated with systemic lupus
erythematosus,
cancers, lymphomas, chronic renal disease, and enlarged spleens. The
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may be
useful in treating, preventing, and/or diagnosing anemias arising from drug
treatments such
as anemias associated with methyldopa, dapsone, and/or sulfadrugs.
Additionally, the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention may be useful in treating, preventing, and/or diagnosing anemias
associated with
abnormal red blood cell architecture including but not limited to, hereditary
spherocytosis,
hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency; and
sickle cell
anemia.
[522] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating, preventing, and/or diagnosing
hemoglobin
abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C
disease,
hemoglobin S-C disease, and hemoglobin E disease). Additionally, the
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention may be
useful in diagnosing, prognosing, preventing, and/or treating thalassemias,
including, but not
limited to major and minor forms of alpha-thalassemia and beta-thalassemia.
[523] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing; and/or treating bleeding disorders including, but not limited to,
thrombocytopenia
(e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic
purpura), Von
Willebrand's disease, hereditary platelet disorders (e.g., storage pool
disease such as
Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction,
thromboasthenia, arid Bernard-Soulier syndrome), hemolytic-uremic syndrome,
hemophelias
such as hemophelia A or Factor VII deficiency and Christmas disease or Factor
IX
deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-
Weber
syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated
intravascular
coagulation.
[524] The effect of the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention on the clotting time of blood may be
monitored using any
of the clotting tests known in the art including, but not limited to, whole
blood partial
thromboplastin time (PTT), the activated partial thromboplastin time (aPTT),
the activated
clotting time (ACT), the recalcified activated clotting time, or the Lee-White
Clotting time.
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[525] Several diseases and a variety of drugs can cause platelet dysfunction.
Thus, in a
specific embodiment, the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention may be useful in diagnosing, prognosing,
preventing,
and/or treating acquired platelet dysfunction such as platelet dysfunction
accompanying
kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and
systemic lupus
erythematosus as well as platelet dysfunction associated with drug treatments,
including
treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs
(used for arthritis,
pain, and sprains), and penicillin in high doses.
[526] In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders characterized by or
associated with
increased or decreased numbers of white blood cells. Leukopenia occurs when
the number of
white blood cells decreases below normal. Leukopenias include, but are not
limited to,
neutropenia and lymphocytopenia. An increase in the number of white blood
cells compared
to normal is known as leukocytosis. The body generates increased numbers of
white blood
cells during infection. Thus, leukocytosis may simply be a normal
physiological parameter
that reflects infection. Alternatively, leukocytosis may be an indicator of
injury or other
disease such as cancer. Leokocytoses, include but are not limited to,
eosinophilia, and
accumulations of macrophages. In specific embodiments, the polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention may be
useful in
diagnosing, prognosing, preventing, and/or treating leukopenia. In other
specific
embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may 'be useful in diagnosing, prognosing, preventing,
and/or treating
leukocytosis.
[527] Leukopenia may be a generalized decreased in all types of white blood
cells, or
may be a specific depletion of particular types of white blood cells. Thus, in
specific
embodiments, the polynucleotides, polypeptides, antibodies; and/or agonists or
antagonists of
the present invention may be useful in diagnosing, prognosing, preventing,
and/or treating
decreases in neutrophil numbers, known as neutropenia. Neutropenias that may
be
diagnosed, prognosed, prevented, and/or treated by the polynucleotides,
polypeptides,
antibodies, and/or agoriists or antagonists of the present invention include,
but are not limited
to, infantile genetic agranulocytosis, familial neutropenia, cyclic
neutropenia, neutropenias
resulting from or associated with dietary deficiencies (e.g., vitamin B 12
deficiency or folic
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acid deficiency), neutropenias resulting from or associated with drug
treatments (e.g.,
antibiotic regimens such as penicillin treatment, sulfonamide treatment,
anticoagulant
treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer.
chemotherapy), and
neutropenias resulting from increased neutrophil destruction that may occur in
association
with some bacterial or viral infections, allergic disorders, autoimmune
diseases, conditions in
which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and
sarcoidosis),
and some drug treatment regimens.
[528] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in diagnosing, prognosing, preventing, and/or
treating
lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but
not
limited lymphocytopenias resulting from or associated with stress, drug
treatments (e.g., drug
treatment with corticosteroids, cancer chemotherapies, and/or radiation
therapies), AIDS
infection and/or other diseases such as; for example, cancer, rheumatoid
arthritis, systemic
lupus erythematosus, chronic infections, some viral infections and/or
hereditary disorders
(e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined
immunodeficiency,
ataxia telangiectsia).
[529] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in diagnosing, prognosing, preventing, and/or
treating
diseases and disorders associated with macrophage numbers and/or macrophage
function
including, but not limited to, Gaucher's disease, Niemann-Pick disease,
Letterer-Siwe disease
and Hand-Schuller-Christian disease.
[530J In another embodiment, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders associated with eosinophil
numbers and/or
eosinophil function including, but not limited to, idiopathic
hypereosinophilic syndrome,
eosinophilia-myalgia syndrome; and Hand-Schuller-Christian disease.
[531] In yet another embodiment, the polynucleotides, polypeptides,
antibodies, and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating leukemias and lymphomas including, but not limited
to, acute
lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic,
myelogenous,
myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g.,
B cell
leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia),
chronic myelocytic
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(myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-
hodgkin's
lymphoma, Burkitt's lymphoma, and mycosis fungoides.
[532J ~ In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders of plasma cells including,
but not limited
to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies
of
undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's
macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon. '
[533] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in treating,
preventing, and/or
diagnosing myeloproliferative disorders, including but not limited to,
polycythemia vera,
relative polycythemia, secondary polycythemia, myelofibrosis, acute
myelofibrosis,
agnogenic myelod metaplasia, thrombocythemia, (including both primary and
seconday
thrombocythemia) and chronic myelocytic leukemia.
[534] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as a treatment
prior to surgery,
to increase blood cell production.
[535] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
enhance the
migration, phagocytosis, superoxide production, antibody dependent cellular
cytotoxicity of
neutrophils, eosionophils and macrophages.
[536] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
increase the
number of stem cells in circulation prior to stem cells pheresis. In another
specific
embodiment, the polynucleotides, polypeptides, antibodies, and/or agoriists or
antagonists of
the present invention may be useful as an agent to increase the number of stem
cells in
circulation prior to platelet pheresis.
[537] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful as an agent to
increase
cytokine production.
[538] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in preventing,
diagnosing,
and/or treating primary hematopoietic disorders.
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Hy_perproliferative Disorders
[539] In certain embodiments, polynucleotides or polypeptides, or agonists or
antagonists of the present invention can be used to treat or detect
hyperproliferative disorders,
including neoplasms.~ Polynucleotides or polypeptides, or agonists or
antagonists of the
present invention may inhibit the proliferation of the disorder through direct
or indirect
interactions. Alternatively, Polynucleotides or polypeptides, or.agonists or
antagonists of the
present invention may proliferate other cells which can inhibit the
hyperproliferative
disorder.
[540] 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.
[541] Examples of hyperproliferative disorders that can be treated or detected
by
polynucleotides or polypeptides, or agonists or antagonists of the present
invention include,
but are not limited to neoplasms located in the: colon, abdomen, bone, breast,
digestive
system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,
pituitary,
testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and
peripheral),
lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital
tract.
[542] Similarly, other hyperproliferative disorders can also be treated or
detected by
polynucleotides or polypeptides, or agonists or antagonists of the present
invention.
Examples of such hyperproliferative disorders include, but are not limited to:
Acute
Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute
Lymphocytic
Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary)
Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic
Leukemia,
Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's
Lymphoma,
Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver
Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related
Malignancies,
Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain
Stem
Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter,
Central
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Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar
Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)
Hepatocellular
Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic
Leukemia,
Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood
Cerebellar
Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell
Tumors,
Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood
Hypothalamic
and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood
Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and
Supratentorial Primitive -Neuroectodermal Tumors, Childhood Primary Liver
Cancer,
Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual
Pathway
and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous
Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet
Cell
Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal
Cancer,
Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial
Germ Cell
Tumor, Extragonadal-Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye
Cancer, Female
Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer,
'Gastrointestinal
Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational
Trophoblastic
Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer,
Hodgkin's
Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer,
Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell
Pancreatic Cancer,
Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer,
Liver
Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male
Breast
Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,
Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic
Primary
Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma,
Multiple
Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia,
Myeloid Leukemia, Myeloproliferatiye Disorders, Nasal Cavity and Paranasal
Sinus Cancer,
Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,
Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic
Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma,
Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous
Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor,
Ovarian Low
Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura,
Parathyroid
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Cancer, Penile Cancer,- Pheochromocytoma, Pituitary Tumor, Plasma Cell
Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary
Liver
Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and
Ureter Cancer,
Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas,
Sezary
Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft
Tissue
Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive
Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer,
Thymoma,
Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,
Transitional Renal
Pelvis and Ureter Cancer, Trophoblasfic Tumors, Ureter and Renal Pelvis Cell
Cancer,
Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual
Pathway and
Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms'
Tumor,
and any other hyperproliferative disease, besides neoplasia, located in an
organ system listed
above.
[543] In another preferred embodiment, polynucleotides or polypeptides, or
agonists or
antagonists of the present invention are used to diagnose, prognose, prevent,
and/or treat
premalignant conditions and to prevent progression to a neoplastic or
malignant state,
including but not limited to those disorders described above. Such uses are
indicated in
conditions known or suspected of preceding progression to neoplasia or cancer,
in particular,
where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or
most particularly,
dysplasia has occurred (for review of such abnormal growth conditions, see
Robbins and
Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp.
68-79.)
[544] Hyperplasia is a form of controlled cell proliferation, 'involving an
increase in cell
number in a tissue or organ, without significant alteration' in structure or
function.
Hyperplastic disorders which can be diagnosed, prognosed, prevented, and/or
treated with
compositions of the invention (including polynucleotides, polypeptides,
agonists or
antagonists) include, but are not limited to, angiofollicular mediastinal
lymph node
hyperplasia; angiolymphoid hyperplasia with eosinophilia, atypical melanocytic
hyperplasia,
basal cell hyperplasia, benign giant lymph node hyperplasia, cementum
hyperplasia,
congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic
hyperplasia, cystic
hyperplasia of the breast, denture hyperplasia, ductal hyperplasia,
endometrial hyperplasia,
fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia,
inflammatory
fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular
papillary endothelial
hyperplasia, nodular hyperplasia of prostate, nodular regenerative
hyperplasia,
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pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous
hyperplasia.
[545] Metaplasia is a form of controlled cell growth in which one type of
adult or fully
differentiated cell substitutes for another type of adult cell. Metaplastic
disorders which can
be diagnosed, prognosed, prevented, ~ and/or treated with compositions of the
invention
(including polynucleotides, polypeptides, agonists or antagonists) include,
but are not limited
to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia,
autoparenchymatous metaplasia, connective tissue metaplasia, epithelial
metaplasia,
intestinal metaplasia, metaplastic anemia, metaplastic ossification,
metaplastic polyps,
myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia,
squamous
metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
[546J Dysplasia is frequently a forerunner of cancer, and is found mainly in
the epithelia;
it is the most disorderly form of non-neoplastic cell growth, involving a loss
in individual cell
uniformity and in the architectural orientation of cells. Dysplastic cells
often have abnormally
large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia
characteristically occurs
where there exists chronic irritation or inflammation. Dysplastic disorders
which can be
diagnosed, prognosed, prevented, . and/or treated with compositions of the
invention
(including polynucleotides, polypeptides, agonists or antagonists) include,
but are not limited
to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating
thoracic dysplasia,
atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia,
cervical dysplasia,
chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal
dysplasia,
crariiodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial
dysplasia, dentin
dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia,
encephalo-
ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis
multiplex,
dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital
dysplasia, familial
fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular
dysplasia, fibrous
dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal
dysplasia, hidrotic
ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic
dysplasia,
mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini
dysplasia,
monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial
dysplasia,
oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral
dysplasia,
odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental
dysplasia,
polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial
dysplasia, retinal
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dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and
ventriculoradial
dysplasia.
[547] Additional pre-neoplastic disorders which can be diagnosed, prognosed,
prevented,
and/or treated with compositions of the invention (including polynucleotides,
polypeptides,
agonists or antagonists) include, but are not limited to, benign
dysproliferative disorders (e.g.,
benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps,
colon polyps, and
esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin,
solar
cheilitis, and solar keratosis.
[548] In another embodiment, a polypeptide . of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose disorders associated with the tissues) in which the
polypeptide of
the invention is expressed, including one, two, three, four, five, or more
tissues disclosed in
Table 1A, column 8 (Tissue Distribution Library Code).
[549] In another embodiment, polynucleotides, polypeptides, antibodies, and/or
agonists
or antagonists of the present invention conjugated to a toxin or a radioactive
isotope, as
described herein, may be used to treat cancers and neoplasms, including, but
not limited to
those described herein. In a further preferred embodiment, polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention conjugated
to a toxin or a
radioactive isotope, as described herein, may be used to treat acute
myelogenous leukemia.
[550] Additionally, polynucleotides, polypeptides, and/or agonists or
antagonists of the
invention may affect apoptosis, and therefore, would be useful in treating a
number of
diseases associated with increased cell survival or the inhibition of
apoptosis. For example,
diseases associated with increased cell survival or the inhibition of
apoptosis that could be
diagnosed, prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or
agonists or antagonists of the invention, include cancers (such as follicular
lymphomas,
carcinomas with p53 mutations, and hormone-dependent tumors, including, but
not limited to
colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,
glioblastoma, ._
lung cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma,
myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,
chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and
ovarian
cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome,
Hashimoto's
thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid arthritis)
and viral
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infections (such as herpes viruses, pox viruses and adenoviruses),
inflammation, graft v. host
disease, acute graft rejection, and chronic graft rejection.
[551] In preferred embodiments, polynucleotides, polypeptides, and/or agonists
or
antagonists of the invention are used to inhibit growth, progression, and/or
metastasis of
cancers, in particular those listed above.
[552] Additional diseases or conditions associated with increased cell
survival that could
be diagnosed, prognosed, prevented, and/or treated by polynucleotides,
polypeptides, and/or
agonists or antagonists of the invention, include, but are not limited to,
progression, and/or
metastases of malignancies and related disorders such as leukemia (including
acute
leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia
(including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia))
and
chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and
chronic lymphocytic
leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-
Hodgkin's
disease),. multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, and
solid tumors including, but not limited to, sarcomas and carcinomas such as
fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma; chordoma,
angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous
cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland
carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer,
testicular
tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,
epithelial carcinoma,
glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,
pinealoma,
emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma,
neuroblastoma, and retinoblastoma.
[553] Diseases associated with increased apoptosis that could be diagnosed,
prognosed,
prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or
antagonists of
the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's
disease,
Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa,
cerebellar
degeneration and brain tumor or prior associated disease); autoimmune
disorders (such as,
multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary
cirrhosis, Behcet's
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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.
[554] ' Hyperproliferative diseases and/or disorders that could be diagnosed,
prognosed,
prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or
antagonists of
the invention, include, but are not limited to, neoplasms located in the
liver, abdomen, bone,
breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal,
parathyroid,
pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous
system (central and
peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and
urogenital tract.
[555] Similarly, other hyperproliferative disorders can also be diagnosed,
prognosed,
prevented, and/or treated by polynucleotides, polypeptides, and/or agonists or
antagonists of
the invention.. Examples of such hyperproliferative disorders include, but are
not limited to:
hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,
purpura,
sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's
Disease,
histiocytosis, and any other hyperproliferative disease, besides neoplasia,
located in an organ
system listed above.
[556] Another preferred embodiment utilizes polynucleotides of the present
invention to
inhibit aberrant cellular division, by gene therapy using the present
invention, and/or protein
fusions or fragments thereof.
[557] 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.
[558] Another embodiment of the present invention provides. a method of
treating cell-
proliferative disorders in individuals comprising administration of one or
more active gene
copies of the present invention to an abnormally proliferating cell or cells.
In a preferred
embodiment, polynucleotides of the.present invention is a DNA construct
comprising a
recombinant expression vector effective in expressing a DNA sequence encoding
said
polynucleotides. In another preferred embodiment of the present invention, the
DNA
construct encoding the poynucleotides of the present invention is inserted
into cells to be
treated utilizing a retrovirus, or more preferably an adenoviral vector (See G
J. Nabel, et. al.,
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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.
[559] 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.
[560] For local administration to abnormally proliferating cells,
polynucleotides of the
present invention may be administered by any method known to those of skill in
the art
including, but not limited to transfection, electroporation, microinjection of
cells, or in
vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any
other method
described throughout the specification. The polynucleotide of the present
invention may be
delivered by known gene delivery systems such as, but not limited to,
retroviral vectors
(Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et
al., Proc. Natl.
Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.
Cell Biol. 5:3403
(1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812
(1985)) known
to those skilled in the art. These references are exemplary only and are
hereby incorporated
by reference. In order to specifically deliver or transfect cells which are
abnormally
proliferating and spare non-dividing cells,-it is preferable to utilize a
retrovirus, or adenoviral
(as described in the art and elsewhere herein) delivery system known to those
of skill in the
art. Since host DNA replication is required for retroviral DNA to integrate
and the retrovirus
will be unable to self replicate due to the lack of the retrovirus genes
needed for its life cycle.
Utilizing such a retroviral delivery system for polynucleotides of the present
invention will
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target said gene and constructs to abnormally proliferating cells and_will
spare the non-
dividing normal cells.
[561] 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.
[562] 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.
[563] 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.
[564] 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.
[565] 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.
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[566] 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.
[567] 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.
[568] 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 SX10'6M, 10'6M,
SX10~~M, 10'7M,
SX10'8M, 10'8M, SX10~9M, 10'9M, SX10-1°M, 10''°M, SX10''1M,
10''1M, SX10-12M, 10-iZM,
SX10'13M, 10'13M, SX10-14M, 10'14M, SX10'15M, and 10'~SM.
(569] 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-angiogeriesis 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)).
[570] 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 ligarid
(TRAIL)
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receptor-1 and -2 (See Schulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59
(I998), which
is hereby incorporated by reference). Moreover, in another preferred
embodiment of the
present invention, said polypeptides may induce apoptosis through other
mechanisms, such
as in the activation of other proteins which will activate apoptosis, or
through stimulating the
expression of said proteins, either alone or in combination with small
molecule drugs or
adjuviants, such as apoptonin, galectins, thioredoxins, anti-inflammatory
proteins (See for
example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998),
Chem
Biol Interact. Apr 24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int
J Tissue
React;20(1):3-15 (1998), which are all hereby incorporated by reference).
[571J Polypeptides, including protein fusions to, or fragments thereof, of the
present
invention are useful in inhibiting the metastasis of proliferative cells or
tissues. Inhibition
may occur as a direct result of administering polypeptides, or .antibodies
directed to said
polypeptides as described elsewere herein, or indirectly, such as activating
the expression of
proteins known to inhibit metastasis, for example alpha 4 integrins, (See,
e.g., Curr Top
Microbiol Immunol 1998;231:125-41, which is hereby incorporated by reference).
Such
thereapeutic affects of the present invention may be achieved either alone, or
in combination
with small molecule drugs or adjuvants.
[572] 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.
[573] 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.
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Renal Disorders
[574] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention, may be used to treat, prevent, diagnose, and/or prognose
disorders of the
renal system. Renal disorders which can be diagnosed, prognosed, prevented,
and/or treated
with compositions of the invention include, but are not limited to, kidney
failure, nephritis,
blood vessel disorders of kidney, metabolic and congenital kidney disorders,
urinary
disorders of the kidney, autoimmune disorders, sclerosis and necrosis,
electrolyte imbalance,
and kidney cancers.
[575] Kidney diseases which can be diagnosed, prognosed, prevented, and/or
treated
'with compositions of the invention include, but are not limited to, acute
kidney failure,
chronic kidney failure, atheroembolic renal failure, end-stage renal disease,
inflammatory
diseases of the kidney (e.g., acute glomerulonephritis, postinfectious
glomerulonephritis,
rapidly progressive glomerulonephritis, nephrotic syndrome, membranous
glomerulonephritis, familial nephrotic syndrome, membranoproliferative
glomerulonephritis
I and II, mesangial proliferative glomerulonephritis, chronic .
glomerulonephritis, acute
tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-
streptococcal
glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis,
interstitial
nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders
of the kidneys
(e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis,
malignant
nephrosclerosis, renal vein thrombosis, renal underperfusion, renal
retinopathy, renal
ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and
kidney disorders
resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis,
urolithiasis (renal
lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections,
urinary retention, and
acute or chronic unilateral obstructive uropathy.)
[576] In addition, compositions of the invention can be used to diagnose,
prognose,
prevent, and/or treat metabolic and congenital disorders of the kidney (e.g.,
uremia, renal
amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria,
nephrogenic
diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis
(renal rickets),
Hartnup disease, Banter's syndrome, Liddle's syndrome, polycystic kidney
disease,
medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-
patella
syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney,
diabetic
nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney
stones, and
membranous nephropathy), and autoimmune disorders of the kidney (e.g.,
systemic lupus '
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erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial
proliferative glomerulonephritis).
(577] Compositions of the invention can also be used to diagnose, prognose,
prevent,
and/or treat sclerotic or necrotic disorders of the kidney (e.g.,
glomerulosclerosis, diabetic
nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing
glomerulonephritis,
and renal papillary necrosis), cancers of the kidney (e.g., nephroma,
hypernephroma,
nephroblastoma, renal cell cancer, transitional cell cancer, renal
adenocarcinoma, squamous
cell cancer, and WiIW 's tumor), and electrolyte imbalances (e.g.,
nephrocalcinosis, pyuria,
edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia,
hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphosphatemia).
[578] 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 may be administered
as part of a
Therapeutic, described in more detail below. Methods of delivering
polynucleotides are
described in more detail herein.
Cardiovascular Disorders
[579] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or prognose
cardiovascular disorders,
including, but not limited to, peripheral artery disease, such as limb
ischemia.
[580] Cardiovascular disorders include, but are not limited to, cardiovascular
abnormalities, such as arterio-arterial fistula, arteriovenous fistula,
cerebral arteriovenous
malformations, congenital heart defects, pulmonary atresia, and Scimitar
Syndrome.
Congenital heart defects include, but are not limited to, aortic coarctation,
cor triatriatum,
coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus
arteriosus, Ebstein's
anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of
fallot, transposition of great vessels, double outlet right ventricle,
tricuspid atresia, persistent
truncus arteriosus, and heart septal defects, such as aortopulmonary septal
defect, endocardial
cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart
septal defects.
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[581] Cardiovascular disorders also include, but are not limited to, heart
disease, such as
arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output,
cardiac
tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest,
congestive
heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema,
heart
hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right
ventricular
hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart
valve diseases,
myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis
(including
constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome,
pulmonary
heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia,
cardiovascular
pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and
cardiovascular
tuberculosis.
[582] Arrhythmias include, but are not limited to, 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.
[583] Heart valve diseases include, but are not limited to, aortic valve
insufficiency,
aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve
prolapse, tricuspid
valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary
atresia,
pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia,
tricuspid valve
insufficiency, and tricuspid.valve stenosis.
[584] Myocardial diseases include, but are not limited to, alcoholic
cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular
stenosis,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy,
endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome,
myocardial
reperfusion injury, and myocarditis.
[585] Myocardial ischemias include, but are not limited to, coronary disease,
such as
angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary
thrombosis, coronary
vasospasm, myocardial infarction and myocardial stunning.
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[586] 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.
[587] Aneurysms include, but are not limited to, dissecting aneurysms, false
aneurysms,
infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,
coronary
aneurysms, heart aneurysms, and iliac aneurysms.
[588] Arterial occlusive diseases include, but are not limited to,
arteriosclerosis,
intermittent claudication, carotid stenosis, fibromuscular dysplasias,
mesenteric vascular
occlusion, Moyamoya disease, renal artery obstruction, retinal artery
occlusion, and
thromboangiitis obliterans.
[589] Cerebrovascular disorders include, but are not limited to, carotid
artery diseases,
cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral
arteriosclerosis,
cerebral arteriovenous malformation, cerebral artery diseases, cerebral
embolism and
thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's
syndrome, cerebral
hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage,
cerebral
infarction, cerebral ischemia (including transient), subclavian steal
syndrome, periventricular .
leukomalacia, vascular headache, cluster headache, migraine, and
vertebrobasilar
insufficiency.
[590] Embolisms include, but are not limited to, air embolisms, amniotic fluid
embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary
embolisms,
and thromoboembolisms. Thrombosis include, but are not limited to, coronary
thrombosis,
hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis,
sinus thrombosis,
Wallenberg's syndrome, and thrombophlebitis.
[591] Ischemic disorders include, but are not limited to, cerebral ischemia,
ischemic
colitis, compartment syndromes, anterior compartment syndrome, myocardial
ischemia,
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reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but
is not limited to,
aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous
lymph node
syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-
Henoch
purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
[592] 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 may be administered
as part of a
Therapeutic, described in more detail below. Methods of delivering
polynucleotides are
described in more detail herein.
Respirator~Disorders
[593] Polynucleotides or polypeptides, or agonists or antagonists of the
present invention
may be used to treat, prevent, diagnose, and/or prognose diseases and/or
disorders of the
respiratory system.
[594] Diseases and disorders of the respiratory system include, but are not
limited to,
nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic
rhinitis; atrophic rhinitis,
vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas,
cancer of the nose
and juvenile papillomas, vocal cord polyps, nodules (singer's nodules),
contact ulcers, vocal
cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial),
tonsillitis, tonsillar
cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat
cancers (e.g., cancer of
the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g.; squamous
cell carcinoma,
small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma),
allergic disorders
(eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic
allergic alveolitis,
allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic
bronchopulmonary
aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis),
Goodpasture's
syndrome)), pneumonia (e.g.; bacterial pneumonia (e.g., Streptococcus
pneumoniae
(pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia),
Gram-
negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.),
Mycoplasma
pneumoniae pneumonia; Hemophilus influenzae pneumonia, Legionella pneumophila
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(Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral
pneumonia (e.g.,
influenza, chickenpox (varicella).
[595] Additional diseases and disorders of the respiratory system include, but
are not
limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial
viral infection,
mumps, erythema infectiosum (fifth disease), roseola infantum, progressive
rubella
panencephalitis, german measles, and subacute sclerosing panencephalitis),
fungal
pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal
infections in
people with severely suppressed immune systems (e.g., cryptococcosis, caused
by
Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.;
candidiasis, caused by
Candida; and mucormycosis)), Pneumocystis carinii (prieumocystis pneumonia),
atypical
pneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunistic infection
pneumonia,
nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural
disorders
(e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous
pneumothorax,
complicated spontaneous pneumothorax, tension pneumothorax)), obstructive
airway
diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD),
emphysema, chronic
or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung
(coal workers'
pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis,
and benign
pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g.,
fibrosing
alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis,
desauamative
interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X
(e.g., Letterer-Siwe
disease, Hand-Schiiller-Christian disease, eosinophilic granuloma), idiopathic
pulmonary
hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute
respiratory distress
syndrome (also called, e.g., adult respiratory distress syndrome), edema,
pulmonary
embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis,
lung abscess (caused
by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic
fibrosis.
Anti-Angio~enesis Activity
[596] 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
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spatially and temporally delimited. Under conditions of pathological
angiogenesis such as
that characterizing solid tumor growth, these regulatory controls fail.
Unregulated
angiogenesis becomes pathologic and sustains progression of many neoplastic
and non-
neoplastic diseases. A number of serious diseases are dominated by abnormal
neovascularization including solid tumor growth and metastases, arthritis,
some types of eye
disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-
634 (1991);
Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.
Microvasc. Res.
29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and
Weinhouse,
Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-
743
(1982); and Folkman et al., Science 221:719-725 (1983). In a number of
pathological
conditions, the process of angiogenesis contributes to the disease state. For
example,
significant data have accumulated which suggest that the growth of solid
tumors is dependent
on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).
[597] The present invention provides for treatment of diseases or disorders
associated
with neovascularization by administration of the polynucleotides and/or
polypeptides of the
i1.., ention, as well as agonists or antagonists of the present invention.
Malignant and
metastatic conditions which can be treated with the polynucleotides and
polypeptides, or
agonists or antagonists of the invention include, but are not limited to,
malignancies, solid
tumors, and cancers described herein and otherwise known in the art (for a
review of such
disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co.,
Philadelphia
(1985)).Thus, the present invention provides a method of treating an
angiogenesis-related
disease and/or disorder, comprising administering to an individual in need
thereof a
therapeutically effective amount of a polynucleotide, polypeptide, antagonist
and/or agonist
of the invention. For example, polynucleotides, polypeptides, antagonists
and/or agonists
may be utilized in a variety of additional methods in order to therapeutically
treat a cancer or
tumor. Cancers which may be treated with polynucleotides, polypeptides,
antagonists and/or
agonists include, but are not limited to solid tumors, including prostate,
lung, breast, ovarian,
stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract,
colon, rectum,
cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors
and metastases;
melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell
lung cancer;
colorectal cancer; advanced malignancies; and blood born tumors such as
leukemias. For
example, polynucleotides, polypeptides, antagonists and/or agonists may be
delivered
topically, in order to treat cancers such as skin cancer, head and neck
tumors, breast tumors,
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and Kaposi's sarcoma.
[598) 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.
[599] . 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
~eovascularization; telangiectasia; hemophiliac joints; angiofibroma;
fibromuscular
dysplasia; wound.granulation; Crohn's disease; and atherosclerosis.
[600] 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.
[601] Within one embodiment of the present invention polynucleotides,
polypeptides,
antagonists and/or agonists of the invention are directly injected into a
hypertrophic scar or
keloid, in order to prevent the progression of these lesions. This therapy is
of particular value
in the prophylactic treatment of conditions which are known to result in the
development of
hypertrophic scars and keloids (e.g., burns), and is preferably initiated
after the proliferative
phase has had time to progress (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
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fibroplasia and macular degeneration.
[602] Moreover, Ocular disorders associated with neovascularization which can
be
treated with the polynucleotides and polypeptides of the present invention
(including agonists
and/or antagonists) include, but are not limited to: neovascular glaucoma,
diabetic
retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of
prematurity
macular degeneration, corneal graft neovascularization, as well as other eye
inflammatory
diseases, ocular tumors and diseases associated with choroidal or iris
neovascularization. See,
e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and~Gartner
et al., Surv.
Ophthal. 22:291-312 (1978).
[603] Thus, within one aspect of the present invention methods are provided
for treating
neovascular diseases of the eye such as corneal neovascularization (including
corneal graft
neovascularization), comprising the step of administering to a patient a
therapeutically .
effective amount of a compound (as described above) to the cornea, such that
the formation
of blood vessels is inhibited. Briefly, the cornea is a tissue which normally
lacks blood
vessels. In certain pathological conditions however, capillaries may extend
into the cornea
from the pericorneal vascular plexus of the limbus. When the cornea becomes
vascularized,
it also becomes clouded; resulting in a decline in the patient's visual
acuity. Visual loss may
become complete if the cornea completely opacitates. A wide variety of
disorders can result
in corneal neovascularization, including for example, corneal infections
(e.g., trachoma,
herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological
processes (e.g.,
graft 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.
[604] 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 administered
several times daily.
Alternatively, anti-angiogenic compositions, prepared as described above, may
also be
administered directly to the cornea., Within preferred embodiments, the anti-
angiogenic
composition is prepared with a muco-adhesive polymer which binds to cornea.
Within
further embodiments, the anti-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 .
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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.
[605] 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.
[606J 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 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.
(607J 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.
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[608] 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.
[609] 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.
[610] Moreover, disorders and/or states, which can be treated, prevented,
diagnosed,
and/or prognosed with the the polynucleotides, polypeptides,, agonists and/or
agonists of the
invention include, but are not limited to, solid tumors, blood born tumors
such as leukemias,
tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas,
acoustic
neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid
arthritis,
psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy,
retinopathy of
prematurity, macular degeneration, corneal graft rejection, neovascular
glaucoma, retrolental
fibroplasia, ~rubeosis, retinoblastoma, and uvietis, delayed wound healing,
endometriosis;
vascluogenesis, granulations, hypertrophic scars (keloids), nonunion
fractures, scleroderma,
trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals,
cerebral
collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-
Webber
Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints,
angiofibroma
fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis,
birth control
agent by preventing vascularization required for embryo implantation
'controlling
menstruation, diseases that have angiogenesis as a pathologic consequence such
as cat
scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori),
Bartonellosis and
bacillary angiomatosis.
[611] 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.
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[612] Polynucleotides, polypeptides, agonists and/or agonists of the present
invention
may be incorporated into surgical sutures in order to prevent stitch
granulomas.
[613] 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 fiom
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.
[614] Within further aspects of the present invention, methods are provided
for treating
tumor excision sites, comprising administering a polynucleotide, polypeptide,
agonist and/or
agonist to the resection margins of a tumor subsequent to excision, such that
the local
recurrence of cancer and the formation of new blood vessels at the site is
inhibited. Within
one embodiment of the invention, the anti-angiogenic compound is administered
directly to
the tumor excision site (e.g., applied by swabbing, brushing or otherwise
coating the.
resection margins of the tumor with the anti-angiogenic compound).
Alternatively, the anti-
angiogenic compounds may be incorporated into known surgical pastes prior to
administration. Within particularly preferred embodiments of the invention,
the anti-
angiogenic compounds are applied after hepatic resections for malignancy, and
after
neurosurgical operations.
[615] 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.
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[616] The polynucleotides, polypeptides, agonists and/or agonists of the
present
invention may also be administered along with other anti-angiogenic factors.
Representative
examples of other anti-angiogenic factors include: Anti-Invasive Factor,
retinoic acid and
derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of
Metalloproteinase-l, Tissue
Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1,
Plasminogen Activator
Inhibitor-2, and various forms of the lighter "d group" transition metals.
[617] 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.
[618] 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 complexes
include, for
example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate
hydrates
such as vanadyl sulfate mono- and trihydrates.
[619] Representative examples of tungsten and molybdenum complexes also
include oxo
complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide
complexes. Suitable tungstate complexes include ammonium tungstate, calcium
tungstate,
sodium tungstate dehydrate, and tungstic acid. Suitable tungsten oxides
include tungsten (IV)
oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include
molybdate,
molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes
include
ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and
potassium
molybdate and its hydrates. Suitable .molybdenum oxides include molybdenum
(VI) oxide,
molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes
include, for
example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum
complexes
include hydroxo. derivatives derived from, for example, glycerol, tartaric
acid, and sugars.
[620] 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
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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
36:312-316,.
1992); Thalidomide; Angostatic steroid; AGM-1470; carboxynaminolmidazole; and
metalloproteinase inhibitors such as BB94.
Diseases at the Cellular Level
[621] Diseases associated with increased cell survival or the inhibition of
apoptosis that
could be treated, prevented, diagnosed, and/or prognosed using polynucleotides
or
polypeptides, as well as antagonists or agonists of the present invention,.
include cancers
(such as follicular lymphomas, carcinomas with p53 mutations, and hormone-
dependent
tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic
cancer,
melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,
testicular cancer,
stomach cancer, neuroblastoma, myxoma; myoma, lymphoma, endothelioma,
osteoblastoma,
osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate
cancer,
Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple
sclerosis,
Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's
disease, Crohn's
disease, polymyositis, systemic lupus erythematosus and immune-related
glomerulonephritis
and rheumatoid arthritis) and viral infections (such as herpes viruses, pox
viruses and
adenoviruses), inflammation, graft v. host disease, acute graft rejection, and
chronic graft
rejection.
[622] ~ In preferred embodiments, polynucleotides, polypeptides, and/or
antagonists of the
invention are used to inhibit growth, progression, and/or metasis of cancers,
in particular
those listed above.
[623] Additional diseases or conditions associated with increased cell
survival that could
be treated or detected by polynucleotides or polypeptides, or agonists or
antagonists of the
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present invention include, but are not limited to, progression, and/or
metastases of
malignancies and related disorders such as leukemia (including acute leukemias
(e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,
promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g.,
chronic
myelocytic (granulocytic) leukemia,and chronic lymphocytic leukemia)),
polycythemia vera,
lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple
myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors
including, but not
limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma,
chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,
Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,
breast
cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell
carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma,
seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular
tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma,
glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma,
neuroblastoma, and retinoblastoma.
[624] Diseases associated with increased apoptosis that could be treated,
prevented,
diagnosed, and/or prognesed using polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, include, but are not limited to, AIDS;
neurodegenerative
disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic
lateral sclerosis,
Retinitis pigmentosa,~ Cerebellar degeneration and brain tumor or prior
associated disease);
autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,
Hashimoto's
thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,
polymyositis, systemic lupus
erythematosus and immune-related glomerulonephritis and rheumatoid arthritis)
myelodysplastic syndromes (such as aplastic anemia), graft v. host disease,
ischemic injury
(such as that caused by myocardial infarction, stroke and reperfusion injury),
liver injury
(e.g., hepatitis related liver injury, ischemia/reperfusion injury,
cholestosis (bile duct injury)
and liver cancer); toxin-induced liver disease (such as that caused by
alcohol), septic shock,
cachexia and anorexia.
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Wound Healin and Epithelial Cell Proliferation
[625] In accordance with yet a further aspect of the present invention, there
is provided a
process for utilizing polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, for therapeutic purposes, for example, to stimulate
epithelial cell
proliferation and basal keratinocytes for the purpose of wound healing, and to
stimulate hair
follicle production and healing of dermal wounds. Polynucleotides or
polypeptides, as well as
agonists or antagonists of the present invention, may be clinically useful in
stimulating
wound healing including surgical wounds, excisional wounds, deep wounds
involving
damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds,
oral cavity
wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,
venous stasis ulcers,
burns resulting from heat exposure or chemicals, and other abnormal wound
healing
conditions such as uremia, malnutrition, vitamin deficiencies and
complications associated
with systemic treatment with steroids, radiation therapy. and antineoplastic
drugs and
antimetabolites. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could be used to promote dermal reestablishment subsequent
to dermal
loss
[626] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention, could be.used to increase the adherence of skin grafts to a wound
bed and to
stimulate re-epithelialization from the wound bed. The following are types of
grafts that
polynucleotides or polypeptides, agonists or antagonists of the present
invention, could be
used to increase adherence to a wound bed: autografts, artificial skin,
allografts, autodermic
graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft,
brephoplastic
grafts, cubs 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, Oilier-Thiersch graft,.omenpal graft, patch graft,
pedicle graft,
penetrating graft, split skin graft, thick split graft. Polynucleotides or
polypeptides, as well as
agonists or antagonists of the present invention, can be used to promote skin
strength and to
improve the appearance of aged skin.
[627] It is believed that polynucleotides or polypeptides, as well as agonists
or
antagonists of the present invention, will also produce changes in hepatocyte
proliferation,
and epithelial cell proliferation in the lung, breast, pancreas, stomach,
small intestine, and
large intestine. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
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present 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.
Polynucleotides or polypeptides, agonists or antagonists of the present
invention, may
promote proliferation of endothelial cells, keratinocytes, and basal
keratinocytes.
[628] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention, could also be used to reduce the side effects of gut toxicity that
result from
radiation, chemotherapy treatments or viral infections. Polynucleotides or
polypeptides, as
well as agonists or antagonists of the present invention, may have a
cytoprotective effect on
the small intestine mucosa. Polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention, may also stimulate healing of mucositis
(mouth ulcers)
that result from chemotherapy and viral infections.
[629] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention, could further be used in full regeneration of skin in full and
partial thickness skin
defects, including burns, (i.e., repopulation of hair follicles, sweat glands,
and sebaceous
glands), treatment of other skin defects such as psoriasis. Polynucleotides or
polypeptides, as
well as agonists or antagonists of the present invention, could be used to
treat epidermolysis
bullosa, a defect in adherence of the epidermis to the underlying dermis which
results in
frequent, open and painful blisters by accelerating reepithelialization of
these lesions.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention,
could also be used to treat gastric and doudenal ulcers and help heal by scar
formation of the
rriucosal lining and regeneration of glandular mucosa and duodenal mucosal
lining more
rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative
colitis, are
diseases which result in destruction of the mucosal surface of the small or
large intestine,
respectively. Thus, polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could be used to promote the resurfacing of the mucosal
surface to aid
more rapid healing and to prevent progression of inflammatory bowel disease.
Treatment
with polynucleotides or polypeptides, agonists or antagonists of the present
invention, is
expected to have a significant effect on the production of mucus throughout
the
gastrointestinal tract and could be used to protect the intestinal mucosa from
injurious
substances that are ingested or following surgery. Polynucleotides or
polypeptides, as well as
agonists or antagonists of the present invention, could be used to treat
diseases associate with
the under expression.
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[630] Moreover, polynucleotides or. polypeptides, as well as agonists or
antagonists of
the present invention, could be used to prevent and heal damage to the lungs
due to various
pathological states. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, which could stimulate proliferation and differentiation and
promote the
repair of alveoli and brochiolar epithelium fo prevent or treat acute or
chronic lung damage.
For example, emphysema, which results in the progressive loss of aveoli, and
inhalation
injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis
of the
bronchiolar epithelium and alveoli could be effectively treated using
polynucleotides or
polypeptides, agonists or antagonists of the present invention. Also,
polynucleotides or
polypeptides, as well as agonists or .antagonists of the present invention,
could be used to
stimulate the proliferation of and differentiation of type II pneumocytes,
which may help treat
or prevent disease such as hyaline membrane diseases, such as infant
respiratory distress
syndrome and bronchopulmonary displasia, in premature infants.
[631] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention, could stimulate the proliferation and differentiation of
hepatocytes and, thus, could
be used to alleviate or treat liver diseases and pathologies such as fulminant
liver failure
caused by cirrhosis, liver damage caused by viral hepatitis and toxic
substances (i.e.,
acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
[632] In addition, polynucleotides or polypeptides, as well as agonists or
antagonists of
the present invention, could be used treat or prevent the onset of diabetes
mellitus. In
patients with newly diagnosed Types I and II diabetes, where some islet cell
function
remains, polynucleotides or polypeptides, as well as agonists or antagonists
of the present
invention, could be used to maintain the islet function so as to alleviate,
delay or prevent
permanent manifestation of the disease. Also, polynucleotides or polypeptides,
as well as
agonists, or antagonists of the present invention, could be used as an
auxiliary in islet cell
transplantation to improve or promote islet cell function.
Neural Activitrr and Neurological Diseases
[633] The polynucleotides, polypeptides and agonists or antagonists of the
invention may
be used for the diagnosis and/or treatment of diseases, disorders, damage or
injury of the
brain ' and/or nervous system. Nervous system disorders that can be treated
with the
compositions of the invention (e.g., polypeptides, polynucleotides, and/or
agonists or
antagonists), include, but are not limited to, nervous system injuries, and
diseases or
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disorders which result in either a disconnection of axons, a diminution or
degeneration of
neurons, or demyelination. Nervous system lesions which may be treated in a
patient
(including human and non-human mammalian patients) according to the methods of
the
invention, include but are not limited to, the following lesions of either the
central (including
spinal cord, brain) or peripheral nervous systems: ( 1 ) ischemic lesions, in
which a lack of
oxygen in a portion of the nervous system results in neuronal injury or death,
including
cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2)
traumatic lesions,
including lesions caused by physical injury or associated with surgery, for
example, lesions
which sever a portion of the nervous system, or compression injuries; (3)
malignant lesions,
in which a portion of the nervous system is destroyed or injured by malignant
tissue which is
either a nervous system associated malignancy or a malignancy derived from non-
nervous
system tissue; (4) infectious lesions, in which a portion of the nervous
system is destroyed or
injured as a result of infection, for example, by an abscess or associated
with infection by
human immunodeficiency virus, herpes zoster, or herpes simplex virus or with
Lyme disease,
tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the
nervous system
is destroyed or injured as a result of a degenerative process including but
not limited to,
degeneration associated with Parkinson's disease, Alzheimer's disease,
Huntington's chorea,
or amyotrophic lateral sclerosis (ALS); (6) lesions associated with
nutritional diseases or
disorders, in which a portion of the nervous system is destroyed or injured by
a nutritional
disorder or disorder of metabolism including, but not limited to, vitamin 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, multiple sclerosis,
human
immunodeficiency virus-associated myelopatfiy, transverse myelopathy or
various etiologies,
progressive multifocal leukoencephalopathy, and central pontine myelinolysis.
[634] In one embodiment, the polypeptides, polynucleotides, or agonists or
antagonists
of the invention are used to protect neural cells from the damaging effects of
hypoxia. In a
further ,preferred embodiment; the polypeptides, polynucleotides, or agonists
~or antagonists
of the invention are used to protect neural cells from the damaging effects of
cerebral
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hypoxia. According to this embodiment, the compositions of the invention are
used to treat
or prevent neural cell injury associated with cerebral hypoxia. In one non-
exclusive aspect of
this embodiment, the polypeptides, polynucleotides, or agonists or antagonists
of the
invention, are used to treat or prevent neural cell injury associated with
cerebral ischemia. In
another non-exclusive aspect of this embodiment, the polypeptides,
polynucleotides, or
agonists or antagonists of the invention are used to treat or prevent neural
cell injury
associated with cerebral infarction.
[635J In another preferred embodiment, the polypeptides, polynucleotides, or
agonists or
antagonists of the invention are used to treat or prevent neural cell injury
associated with a
stroke. In a specific embodiment, the polypeptides, polynucleotides, or
agonists or
antagonists of the invention are used to treat or prevent cerebral neural cell
injury associated
with a stroke.
[636] In another preferred embodiment, the polypeptides, polynucleotides, or
agonists or
antagonists of_the invention are used to treat or prevent neural cell injury
associated with a
heart attack. In a specific embodiment, the polypeptides, polynucleotides, or
agonists or
antagonists of the invention are used to treat or prevent cerebral neural cell
injury associated
with a heart attack.
[637] 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
either in the
presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting
of neurons in
culture or in vivo; (3) increased production of a neuron-associated molecule
in culture or in
vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to
motor neurons; or
(4) decreased symptoms of neuron dysfunction in vivo. Such effects may be
measured by
any method known in the art. In preferred, non-limiting embodiments, increased
survival of
neurons may routinely be measured using a method set forth herein or otherwise
known in
the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA 97:3637-
42 (2000) or
in Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased sprouting of
neurons may be
detected by methods known in the art, such as, for example, the methods set
forth in Pestronk
et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci.,
4:17-42 (1981);
increased production of neuron-associated molecules may be measured by
bioassay,
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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.
[638] 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).
[639] Further, polypeptides or polynucleotides of the invention may play a
role in
neuronal survival; synapse formation; conductance; neural differentiation,
etc. Thus,
compositions of the .invention (including polynucleotides, polypeptides, and
agonists or
antagonists) may be used to diagnose and/or treat or prevent diseases or
disorders associated
with these roles, including, but not limited to, learning and/or cognition
disorders. The
compositions of the invention may also be useful in the treatment or
prevention of
neurodegenerative disease states and/or behavioural disorders. Such
neurodegenerative
disease states and/or behavioral disorders include, but are ,not limited to,
Alzheimer's
Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome,
schizophrenia,
mania, dementia, paranoia, obsessive compulsive disorder, panic disorder,
learning
disabilities, ALS, psychoses, autism, and altered behaviors, including
disorders in feeding,
sleep patterns, balance, and perception. In addition, compositions of the
invention may also
play a role in the treatment, prevention and/or detection of developmental
disorders
associated with the developing embryo, or sexually-linked. disorders.
[640] Additionally, polypeptides, polynucleotides and/or agonists or
antagonists of the
invention, may be useful in protecting neural cells from diseases, damage,
disorders, or
injury, associated with cerebrovascular disorders including, but not 'limited
to, carotid artery
diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya
Disease), cerebral
amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral
arteriosclerosis, cerebral
arteriovenous malformations, cerebral artery diseases, cerebral embolism and
thrombosis
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(e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome),
cerebral
hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage),
cerebral
infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian
Steal Syndrome, or
vertebrobasilar insufficiency), vascular dementia (e.g., mufti-infarct),
leukomalacia,
periventricular, and vascular headache (e.g., cluster headache or migraines).
[641] In accordance with yet a further aspect of the present invention, there
is provided a
process for utilizing polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, for therapeutic purposes, for example, to stimulate
neurological cell
proliferation and/or differentiation. Therefore, polynucleotides,
polypeptides, agonists and/or
antagonists of the invention may be used to treat and/or detect neurologic
diseases.
Moreover, polynucleotides or polypeptides, or agonists or antagonists of the
invention, can
be used as a marker or detector of a particular nervous system disease or
disorder.
[642] Examples of neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
brain diseases, such as metabolic brain diseases which includes
phenylketonuria such as
maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate
dehydrogenase
complex deficiency, . Wernicke's Encephalopathy, brain edema, brain neoplasms
such as
cerebellar neoplasms which include infratentorial neoplasms, cerebral
ventricle neoplasms
such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial
neoplasms,
canavan disease, cerebellar diseases such as cerebellar ataxia which include
spinocerebellar
degeneration such as ataxia telangiectasia, cerebellar dyssynergia,
Friederich's Ataxia,
Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms
such as
infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis
periaxialis, globoid
cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing
panencephalitis.
[643] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
cerebrovascular disorders (such as carotid artery diseases which include
carotid artery
thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid
angiopathy, cerebral
aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous
malformations,
cerebral artery diseases, cerebral embolism and thrombosis such as carotid
artery thrombosis,
sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as
epidural
hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction,
cerebral
ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and
vertebrobasilar
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insufficiency, vascular dementia such as multi-infarct dementia,
periventricular
leukomalacia, vascular headache such as cluster headache and migraine.
[644] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's
Disease
and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease
and
progressive supranuclear palsy, vascular dementia such as multi-infarct
dementia,
encephalitis which include encephalitis periaxialis, viral encephalitis such
as epidemic
encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne
encephalitis and West
Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as
uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and
subacute
sclerosing panencephalitis, encephalomalacia such as periventricular
leukomalacia, epilepsy
such as generalized epilepsy which includes infantile spasms, absence
epilepsy, myoclonic
epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial
epilepsy such as
complex partial epilepsy, frontal lobe epilepsy arid temporal lobe epilepsy,
post-traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua, and
Hallervorden-Spatz
Syndrome.
[645] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus,
hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria,
narcolepsy which
includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome,
Reye's
Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma
and
Zellweger Syndrome, central nervous system infections such as AIDS Dementia
Complex,
Brain Abscess, subdural empyema, encephalomyelitis such as Equine
Encephalomyelitis,
Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic
Encephalomyelitis, Visna,
and cerebral malaria.
[646] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
meningitis such as arachnoiditis, aseptic meningtitis such as viral
meningtitis which includes
lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus
Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as
Waterhouse-
Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis,
fungal
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meningitis such as Cryptococcal Meningtitis, subdural effusion,
meningoencephalitis such as
uvemeningoencephalitic syndrome, myelitis such as transverse myelitis,
neurosyphilis such
as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and
postpoliomyelitis
syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine
Spongiform
Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral
toxoplasmosis.
[647] Additional neurologic diseases 'which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
central nervous system neoplasms such.as brain neoplasms that include
cerebellar neoplasms
such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid
plexus
neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal
neoplasms,
spinal cord neoplasms which include epidural neoplasms, demyelinating diseases
such as
Cariavan Diseases, diffuse cerebral sceloris which includes
adrenoleukodystrophy,
encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral
sclerosis such as
metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing
hemorrhagic
encephalomyelitis, progressive multifocal leukoencephalopathy, multiple
sclerosis, central
pontine W yelinolysis, transverse myelitis, neuromyelitis optica, Scrapie,
Swayback, Chronic
Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal
cord
diseases such as amyotonia congenita, amyotrophic lateral sclerosis, spinal
muscular atrophy
such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord
neoplasms such as
epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff Man Syndrome, mental
retardation
such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down
Syndrome, Gangliosidoses such as gangliosidoses G(Ml), Sandhoff Disease, Tay-
Sachs
Disease, Hartnup Disease, homocystinuria, Laurence-Moon- Biedl Syndrome, Lesch-
Nyhan
Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis,
neuronal ceroid-
lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as, maternal
phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi
Syndrome,
Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as
holoprosencephaly; neural tube defects such as anencephaly which includes
hydrangencephaly, Arnold=Chairi Deformity, encephalocele, meningocele,
meningomyelocele, spinal dysraphism such as spina bifida cystica and spina
bifida occulta:
[648] Additional neurologic diseases. which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
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hereditary motor and sensory neuropathies which include Charcot-Marie Disease,
Hereditary
optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-
Hoffmann Disease,
Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and
Familial
Dysautonomia, Neurologic manifestations (such as agnosia that include
Gerstmann's
Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder,
cataplexy,
communicative disorders such as hearing disorders that includes deafness,
partial hearing
loss, loudness recruitment and tinnitus, language disorders such as aphasia
which include
agraphia, ariomia, broca aphasia, and Wemicke Aphasia, Dyslexia such as
Acquired
Dyslexia, language development disorders, speech disorders such as aphasia
which includes
anomia, broca aphasia and Wernicke Aphasia, articulation disorders,
communicative
disorders such as speech disorders which include dysarthria, echolalia, mutism
and stuttering,
voice disorders such as aphonia and hoarseness, decerebrate state, delirium,
fasciculation,
hallucinations, meningism, movement disorders such as angelmari syndrome,
ataxia,
athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic,
torticollis and-
tremor, muscle hypertonia such as muscle rigidity such as stiff man syndrome,
muscle
spasticity, paralysis such as facial paralysis which includes Herpes Zoster
Oticus,
Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's Syndrome,
Homer's
Syndrome, Chronic progressive external ophthalmoplegia such as Kearns
Syndrome, Bulbar
Paralysis, Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard
Syndrome,
quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom
limb, taste
disorders such as ageusia and dysgeusia, vision disorders such as amblyopia,
blindness, color
vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep
disorders such as
hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism,
spasm
such as trismus, unconsciousness such as coma, persistent vegetative state and
syncope and
vertigo, neuromuscular diseases such as amyotonia congenita, amyotrophic
lateral sclerosis,
Lambert-Eaton Myastheriic~ Syndrome, motor neuron disease, muscular atrophy
such as
spinal muscular ,atrophy, Charcot-Marie Disease and Werdnig-Hoffmann Disease,
Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia
Atrophica,
Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis; Multiplex
Paramyloclonus, Tropical Spastic Paraparesis and Stiff Man Syndrome,
peripheral nervous
system diseases such as acrodynia, amyloid neuropathies, autonomic nervous
system diseases
such as Adie's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Homer's
Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve
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Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which
includes
Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-
Rosenthal
Syndrome, ocular motility disorders which includes amblyopia, nystagmus,
oculomotor nerve
paralysis, ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome,
Chronic
Progressive External Ophthalmoplegia which includes Kearns Syndrome,
Strabismus such as
Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such
as Optic
Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic
Neuritis such as
Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis,
Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic
neuropathies such as diabetic foot.
[649] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel
syndrome,
thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve
compression syndrome,
neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and
trigeminal
neuralgia, neuritis such as experimental allergic neuritis, optic neuritis,
polyneuritis,
polyradiculoneuritis and radiculities such as polyradiculitis, hereditary
motor and sensory
neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's
Disease,
Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory
and
Autonomic .Neuropathies which include Congenital Analgesia and Familial
Dysautonomia,
POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).
Endocrine Disorders
[650] Polynucleotides or. polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or prognose disorders
and/or diseases
related to hormone imbalance, and/or disorders or diseases of the endocrine
system.
[651] Hormones secreted by the glands of the endocrine system control physical
growth,
sexual function, metabolism, and other functions. Disorders may be classified
in two ways:
disturbances in the production of hormones, and the inability of tissues to
respond to
hormones. The etiology of these hormone imbalance or endocrine system
diseases, disorders
or conditions may be genetic, somatic, such as cancer and some autoimmune
diseases,
acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover,
polynucleotides,
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polypeptides, antibodies, and/or agonists or antagonists of the present,
invention can be used
as a marker or detector of a particular disease or disorder related to the
endocrine system
and/or hormone imbalance.
[652] Endocrine system and/or hormone imbalance and/or diseases encompass
disorders
of uterine motility including, but not limited to: complications with
pregnancy and labor
(e.g., pre-term labor; post-term pregnancy, spontaneous abortion, and slow or
stopped labor);
and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and
endometriosis).
[653] Endocrine system and/or hormone imbalance disorders and/or diseases
include
disorders and/or diseases of the pancreas, such as, for example, diabetes
mellitus, diabetes
insipidus, congenital pancreatic agenesis, pheochromocytonia--islet cell tumor
syndrome;
disorders and/or diseases of the adrenal glands such as, for example,
Addison's Disease,
corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome,
hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the
pituitary gland,
such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism,
pituitary
adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases
of the
thyroid, including but not limited to, hyperthyroidism, hypothyroidism,
Plummer's disease,
Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis
(Hashimoto's
thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic
thyroiditis), Pendred's
syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling
defect, thymic
aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid
carcinoma, Medullary
thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for
example,
hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the
hypothalamus.
[654] In addition, endocrine system and/or hormone imbalance disorders and/or
diseases
may also include disorders and/or diseases of the testes or ovaries, including
cancer. Other
disorders and/or diseases of the testes of ovaries further include, for
example, ovarian cancer,
polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome
(bilateral
anorchia), congenital . absence of Leydig's cells, cryptorchidism, Noonan's
syndrome,
myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias
of the testis
and neo-testis.
[655] Moreover, endocrine system and/or hormone imbalance disorders and/or
diseases
may also include disorders and/or diseases such as, for example, polyglandular
deficiency
syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and
disorders
and/or cancers of endocrine tissues.
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[656] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose, prognose, prevent, and/or treat endocrine diseases and/or disorders
associated with
the tissues) in which the polypeptide of the invention is expressed, including
one, two, three,
four, five, or more tissues disclosed in Table 1A, column 8 (Tissue
Distribution Library
Code). _
Reproductive System Disorders
[657] The polynucleotides or polypeptides, or agonists or antagonists of the
invention
may be used for the diagnosis, treatment, or prevention of diseases and/or
disorders of the
reproductive system. Reproductive system disorders that can be treated by the
compositions
of the invention, include, but are not limited to, reproductive system
injuries, infections, .
neoplastic disorders, congenital defects, and diseases or disorders which
result in infertility,
complications with pregnancy, labor, or parturition, and postpartum
difficulties.
[658] Reproductive system disorders and/or diseases include diseases and/or
disorders of
the testes, including testicular atrophy, testicular feminization,
cryptorchism (unilateral and
bilateral), anorchia, ectopic testis, epididymitis and orchids (typically
resulting from
infections such as, foi example, gonorrhea, mumps, tuberculosis, and
syphilis), testicular
torsion, vasitis nodosa, germ cell tumors. (e.g., seminomas, embryonal cell
carcinomas,
teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal
tumors (e.g.,
Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal
hernia, and
disorders of sperm production (e.g., immotile cilia syndrome, aspermia,
asthenozoospermia,
azoospermia, oligospermia, and teratozoospermia).
[659] Reproductive system disorders also include disorders of the prostate
gland, such as
acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute
bacterial prostatitis,
chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous
prostatitis,
malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate
neoplastic disorders,
including adenocarcinomas, transitional cell carcinomas, ductal carcinomas,
and squamous
cell carcinomas.
(660] Additionally, the compositions of the invention may be useful in the
diagnosis, r
treatment, and/or prevention of disorders or diseases of the penis and
urethra, including
inflammatory disorders, such as balanoposthitis, balanitis xerotica
obliterans, phimosis,
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paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal
urethritis,
chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma
acuminatum, condyloma latum, and pearly penile papules; urethral
abnormalities, such as
hypospadias, epispadias, and phimosis; premalignant lesions, including
Erythroplasia of
Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-
Lowenstein, and
varrucous carcinoma; penile cancers., including squamous cell carcinomas,
carcinoma in situ,
verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic
disorders,
including penile urethral carcinoma, bulbomembranous urethral carcinoma, and
prostatic
urethral carcinoma; and erectile disorders, such as priapism, Peyronie's
disease, erectile
dysfunction, and impotence.
[661] Moreover, diseases and/or disorders of the vas deferens include
vasculititis and
CBAVD (congenital bilateral absence ~of the vas deferens); additionally, the
polynucleotides,
polypeptides, and agonists or antagonists of the present invention may be used
in the
diagnosis, treatment, and/or prevention of diseases and/or disorders of the
seminal vesicles,
including hydatid disease, congenital chloride diarrhea, and polycystic kidney
disease.
[662] Other disorders and/or diseases of the male reproductive system include,
for
example, Klinefelter's syndrome, Young's syndrome, premature ejaculation,
diabetes
mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple
sclerosis, and
gynecomastia.
(663] Further, the polynucleotides, polypeptides, and agonists or antagonists
of the
present invention may be used in the diagnosis, treatment, and/or prevention
of diseases
and/or disorders of the vagina and vulva, including bacterial vaginosis,
candida vaginitis,
herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma
venereum, scabies,
human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia
vaginitis,
gonorrhea, trichomonas vaginitis, condyloma acuminatum; syphilis, molluscum
contagiosum,
atrophic vaginitis, Paget's disease, lichen-sclerosus, lichen planus,
vulvodynia, toxic shock
syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic
disorders, such as
squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma,
melanomas, cancer of
Bartholin's gland, and vulvar intraepithelial neoplasia.
[664] Disorders and/or diseases of the uterus include dysmenorrhea,
retroverted uterus,
endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea,
Cushing's
syndrome, hydatidiform moles, Asherman's syndrome, premature menopause,
precocious
puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant
hormonal
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signals), and neoplastic disorders, . such as adenocarcinomas,
keiomyosarcomas, and
sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or
antagonists of the
invention may be useful as a marker or detector of, as well as in the
diagnosis, treatment,
and/or prevention of congenital uterine abnormalities, such as bicornuate
uterus, septate
uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary
rudimentary horn,
unicornuate uterus with a non-communicating cavitary rudimentary horn,
unicornuate uterus
with a'communicating cavitary horn, arcuate uterus, uterine didelfus, and T-
shaped uterus.
[665] Ovarian diseases and/or disorders include anovulation, polycystic ovary
syndrome
(Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian
insensitivity to
gonadotropins, ovarian overproduction of androgens, right ovarian vein
syndrome,
amenorrhea, hirutism, and ovarian cancer (including, but not limited to,
primary and
secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of
the ovary,
ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and
Ovarian
Krukenberg tumors).
[666] Cervical diseases and/or disorders include cervicitis, chronic
cervicitis,
' mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian
cysts, cervical
erosion, cervical incompetence; and cervical neoplasms (including, for
example, cervical
carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell
neoplasia,
and columnar cell neoplasia).
[667] Additionally, diseases and/or disorders of the reproductive system
include
disorders and/or diseases of pregnancy, including miscarriage and stillbirth,
such as early
abortion, late abortion, spontaneous abortion, induced abortion, therapeutic
abortion,
threatened abortion, missed abortion, incomplete abortion, complete abortion,
habitual
abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh
incompatibility, vaginal bleeding during pregnancy, gestational diabetes,
intrauterine growth
retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta
previa,
hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of
pregnancy.
Additionally, the polynucleotides, polypeptides, and agonists or antagonists
of the present
invention may be used in the diagnosis, treatment, and/or prevention of
diseases that can
complicate pregnancy, including heart disease, heart failure, rheumatic heart
disease,
congenital heart disease, mitral valve prolapse, high blood pressure, anemia,
kidney disease,
infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious
hepatitis,
chlamydia, HIV, AIDS,.and genital herpes), diabetes mellitus, Graves' disease,
thyroiditis,
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hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis
of the liver,
primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid
arthritis,
myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian
cysts,
gallbladder disorders,and obstruction of the intestine.
[668] Complications associated with labor and parturition include premature
rupture of
the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that
progresses too
slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal),
breathing problems, and
abnormal fetal position), shoulder dystocia, prolapsed umbilical cord,
amniotic fluid
embolism, and aberrant uterine bleeding.
[669] Further, diseases and/or disorders of the postdelivery period, including
endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis,
pulmonary
embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, .mastitis,
cystitis,
postpartum hemorrhage, and inverted uterus.
[670] Other disorders and/or diseases of the female reproductive system that
may be
diagnosed, treated, and/or prevented by the polynucleotides, polypeptides, and
agonists or
antagonists of the present invention include, for example, Turner's syndrome,
pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease,
pelvic .
congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia,
ruptured fallopian
tube, and Mittelschmerz.
Infectious Disease
[671] Polytiucleotides or polypeptides, as well as agonists or antagonists of
the present
invention can be used to treat or detect infectious agents. For example, by
increasing the
immune response, particularly increasing the proliferation and differentiation
of B and/or T
cells, infectious diseases may be treated. The immune response may be
increased by either
enhancing an , existing immune response, or by initiating a new immune
response.
Alternatively, polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention may also directly inhibit the infectious agent, without
necessarily eliciting
an immune response.
[672] 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
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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, Dengue, EBV, HIV, Flaviviridae,
Hepadnaviridae
(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes
Zoster),
Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),
Orthomyxoviridae
(e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, .
Papovaviridae,
Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),
Reoviridae (e.g.,
Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g.,
Rubivirus).
Viruses falling within these families can cause a variety of diseases or
symptoms, including,
but not limited to: arthritis, bronchiollitis, respiratory syncytial 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.
[673] Similarly, bacterial and fungal agents that can cause disease or
symptoms and that
can be treated or detected by a polynucleotide or polypeptide and/or agonist
or antagonist of
the present invention include, but not limited to, the following Gram-Negative
and Gram-
positive bacteria, bacterial families, and fungi: Actinomyces (e.g.,
Norcardia),
Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g.,
Bacillus anthrasis),
Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia
(e.g., Borrelia
burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia; Clostridium (e.g.,
Clo'stridium
botulinum, Clostridium dificile, Clostridium perfringens, Clostridium tetani),
Coccidioides,
Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus,
Dermatocycoses, E. coli
(e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacter
(e.g.
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Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g.,,
Salmonella typhi,
Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella),
Erysipelothrix,
Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella
(e.g.,
Legionella pneumophila), Le.ptospira, Listeria (e.g., Listeria monocytogenes),
Mycoplasma,
Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis),
Vibrio (e.g.,
Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria
meningitidis),
Pasteurellacea, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa),
Rickettsiaceae,
Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella
sp,p.,
Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and
Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C
Streptococci), and
Ureaplasmas. These bacterial, parasitic, and fungal families can cause
diseases or symptoms,
including, but not limited to: antibiotic-resistant infections, bacteremia,
endocarditis,
septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis,
gingivitis, bacterial
diarrhea, opportunistic infections (e.g., AIDS related infections),
paronychia, prosthesis-
related infections, dental caries, Reiter's Disease, respiratory tract
infections, such as
Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease,
dysentery,
paratyphoid fever, food poisoning, Legionella disease, chronic and acute
inflammation,
erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g.,
mengitis types A
and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers,
anthrax,
spontaneous abortions, birth defects, pneumonia, lung infections, ear
infections, deafness,
blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's.disease;
colitis, vaginosis,
sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis,
tuberculosis, lupus,
botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever,
sexually transmitted
diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary
tract infections,
wound infections, noscomial infections. Polynucleotides or polypeptides,
agonists or
antagonists of the invention, can be used to treat or detect any of these
symptoms or diseases.
Iri specific embodiments, polynucleotides, polypeptides, agonists or
antagonists of the
invention are used to treat: tetanus, diptheria, botulism, and/or meningitis
type B.
[674] Moreover, parasitic agents causing disease or symptoms that can be
treated,
prevented, and/or diagnosed by a polynucleotide or polypeptide and/or agonist
or antagonist
of the present invention include, but not, limited to, the following families
or class:
Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,
Dourine,
Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma,
Theileriasis,
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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), 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, prevent, and/or diagnose any of these
symptoms or diseases. .
In specific embodiments, polynucleotides, polypeptides, or agonists or
antagonists of the
invention are used to treat, prevent, and/or diagnose malaria.
[675] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention of the present invention could either be by administering an
effective amount of a
polypeptide to the patient, or by removing cells from the patient, supplying
the cells with a
polynucleotide of the present invention, and returning the engineered cells to
the patient (ex
vivo therapy). Moreover, the polypeptide or polynucleotide of the present
invention can be
used as an antigen in a vaccine-to raise an immune response against infectious
disease.
Regeneration
[676] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention can be used to differentiate, proliferate, and attract cells,
leading to the regeneration
of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could
be used to
repair, replace, or protect tissue damaged by congenital defects, trauma
(wounds, burns,
incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,
periodontal disease, liver
y failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion
injury, or systemic
cytokine damage.
[677] 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.
[678] Moreover, polynucleotides or polypeptides, as well as agonists or
antagonists of
the present invention, may increase regeneration of tissues difficult to heal.
For example,
increased tendon/ligament regeneration would quicken recovery time after
damage.
Polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention
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could also be used prophylactically in an effort to avoid damage. Specific
diseases that could
be treated include of tendinitis, carpal tunnel syndrome, and other tendon or
ligament defects.
A further example of tissue regeneration of non-healing wounds includes
pressure ulcers,
ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
[679] Similarly, nerve and brain tissue could also be regenerated by using
polynucleotides or polypeptides, as well as agonists or antagonists of the
present invention, to
proliferate and differentiate nerve cells. Diseases that could be treated
using this method
include central and peripheral nervous system diseases, neuropathies, or
mechanical and
traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular
disease, and
stoke). Specifically, diseases associated with peripheral nerve injuries,
peripheral neuropafhy
(e.g., resulting from chemotherapy or other medical therapies), localized
neuropathies, and
central nervous system diseases (e.g., Alzheimer's disease, Parkinson's
disease, Huntington's
disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be
treated using
the polynucleotides or polypeptides, as well as agonists or antagonists of the
present
invention.
Gastrointestinal Disorders
[680] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose,.and/or prognose
gastrointestinal disorders,
including inflammatory diseases and/or conditions, infections, cancers (e.g.,
intestinal
neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of
the small
intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
[681] Gastrointestinal disorders include dysphagia, odynophagia, inflammation
of the
esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and
stricturing, Mallory-
Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas,
gastric retention
disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps
of the stomach,
autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis
(bacterial, viral,
eosinophilic, stress-induced,~chronic erosive, atrophic, plasma cell, and
Menetrier's), and
peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst,
mesenteric
lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms,
peritonitis,
pneumoperitoneum, bubphrenic abscess,).
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[682] Gastrointestinal disorders also include disorders associated with the
small
intestine, such as malabsorption syndromes, distension, irritable bowel
syndrome, sugar
intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue,
Whipple's disease,
intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of
the ileum,
Meckel's diverticulum, multiple diverticula, failure of complete rotation of
the small and
large intestine, lymphoma, and bacterial and parasitic diseases (such as
Traveler's diarrhea,
typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis
lumbricoides),
Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis),
Tapeworms
(.Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T.
solium).
[683] Liver diseases and/or disorders include intrahepatic cholestasis
(alagille syndrome,
biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome),
hepatic vein
thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary
syndrome,
hepatorenal syndrome, portal hypertension (esophageal and gastric varices),
liver abscess
(amebic liver abscess), liver.cirrhosis (alcoholic, biliary and experimental),
alcoholic liver
diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic
echinococcosis, fascioliasis,
amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic),
cholestasis,
portal hypertension, liver enlargement, ascites, hepatitis (alcoholic
hepatitis, animal hepatitis,
chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug
induced), toxic
hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C,
hepatitis D, hepatitis E),
Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis,
hepatic
encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary
biliary
cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma,
hemangiomas, bile stones,
liver failure (hepatic encephalopathy, acute liver failure), and liver
neoplasms
(angiomyolipoma, calcified liver metastases, cystic liver metastases,
epithelial tumors,
fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma,
hepatobiliary
cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer,
liver
hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver,
nodular
regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts,
Polycystic liver
disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors
[Mesenchymal
hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis,
Lipomas,
Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct
epithelium (Bile
duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular
hyperplasia,
Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular,
hepatoblastoma,
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hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma,
cystadenocarcinoma,
tumors of blood vessels, angiosarcoma, Karposi's sarcoma,
hemangioendothelioma, other
tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma,
carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]),
peliosis
hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent
porphyria, porphyria
cutanea tarda), Zellweger syndrome).
[684] Pancreatic diseases and/or disorders include acute pancreatitis, chronic
pancreatitis
(acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms
(adenocarcinoma of.the
pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic
neoplasms,
islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g.,
cystic fibrosis, cyst
(pancreatic pseudocyst, pancreatic fistula, insufficiency)).
[685] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis),
postcholecystectomy'syndrome, diverticulosis of the gallbladder, acute
cholecystitis, 'chronic
cholecystitis, bile duct tumors, and mucocele.
[686] . Diseases and/or disorders of the large intestine include antibiotic-
associated colitis,
diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and
bacterial
infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic
diseases (colitis, colonic
neoplasms [colon cancer, adenomatous colon polyps (e.g., vinous adenoma),
colon
carcinoma, colorectal cancer]; colonic diverticulitis, colonic diverticulosis,
megacolon
[Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis,
sigmoin
neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea,
dysentery), duodenal
diseases (duodenal neoplasms, duodenal. obstruction, duodenal ulcer,
duodenitis), enteritis
(enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis),
immunoproliferative
small intestinal disease, inflammatory bowel disease (ulcerative colitis,
Crohn's disease),
intestinal atresia, parasitic diseases (anisakiasis, balantidiasis,
blastocystis infections,
cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal
fistula (rectal
fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal
neoplasms, deal
neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal
obstruction
(afferent loop syndrome, duodenal obstruction, impacted feces, intestinal
pseudo-obstruction
[cecal volvulus], intussusception), intestinal perforation, intestinal polyps
(colonic polyps,
gardner syndrome, peutz-jeghers syndrome), jejunal diseases (jejunal
neoplasms),
malabsorption syndromes (blind loop syndrome, celiac disease, lactose
intolerance, short
bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular
occlusion, .
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pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal
lymphagiectasis),
rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis,
rectal fistula, rectal
prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis,
hemorrhage,
perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy
syndromes
(dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric
reflux (bile
reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet
obstruction, gastritis
(atrophic or hypertrophic), gastroparesis; stomach dilatation, stomach
diverticulum, stomach
neoplasms- (gastric cancer, gastric polyps, gastric adenocarcitloma,
hyperplastic gastric
polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis,
visceroptosis,
vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and
vomiting)
and hemorrhagic colitis.
[687] Further diseases and/or disorders of the gastrointestinal system include
biliary tract
diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal
fistula, gastric fistula,
intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract
neoplasms, esophageal
neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell
carcinoma,
gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of
the pancreas,
mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms,
pancreatoblastoma,
and peritoneal neoplasms), esophageal disease (e.g., bullous diseases,
candidiasis, glycogenic
acanthosis, ulceration, barrett -esophagus varices, atresia, cyst,
diverticulum (e.g., Zenker's
diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders
(e.g., CREST
syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux),
neoplasms,
perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis,
esophagitis,
diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such
as, gastroenteritis
(e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g.,
hematemesis, melena,
peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps,
gastric
adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic
hernia, femoral
hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia),
and intestinal
diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).
Chemotaxis
[688] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes-cells
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(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.
[689] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention may increase chemotaxic activity of particular cells. These
chemotactic molecules
can then be used to treat inflammation, infection, hyperproliferative
disorders, or any
immune system disorder by increasing the number of cells targeted to a
particular location in
the body. For example, chemotaxic molecules can be used to treat wounds and
other trauma
to tissues by attracting immune cells to the injured location. Chemotactic
molecules of the
present invention can also attract fibroblasts, which can be used to treat
wounds:
[690] It is also contemplated that polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention may inhibit chemotactic activity. These
molecules could
also be used to treat disorders. Thus, polynucleotides or polypeptides, as
well as agonists or
antagonists of the present invention could be used as an inhibitor of
chemotaxis.
Binding_Activity
[691] A polypeptide of the present invention may be used to screen for
molecules that
bind to the polypeptide or for molecules to which the polypeptide binds. The
binding of the
polypeptide and the molecule may activate (agonist), increase, inhibit
(antagonist), or
decrease activity of the polypeptide or the molecule bound. Examples of such
molecules
include antibodies, oligonucleotides, proteins (e.g., receptors),or small
molecules.
[692] 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.
[693] Preferably, the screening for these molecules involves producing
appropriate cells
which express the polypeptide. Preferred cells include cells from mammals,
yeast,
Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane
containing the
expressed polypeptide) are then preferably contacted with a test compound
potentially
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containing the molecule to observe binding, stimulation, or inhibition of
activity of either the
polypeptide or the molecule.
[694] 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.
[695] Alternatively, the assay can be carried out using cell-free
preparations,
polypeptide/molecule affixed to a solid support, chemical libraries, or
natural product
mixtures. The assay may also simply comprise the steps of mixing a candidate
compound
with a solution containing a polypeptide, measuring polypeptide/molecule
activity or
binding, and comparing the polypeptide/molecule_ activity or binding to a
standard.
[696] 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.
[697] Additionally, the receptor to which the polypeptide of the present
invention binds
can be identified by numerous methods known to those of skill in the art, for
example, ligand
panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1 (2),
Chapter 5,
(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 grown on
glass slides are exposed to the polypeptide of the present invention, after
they have been
labeled. The polypeptides can be labeled by a variety of means including
iodination or
inclusion of a recognition site for a site-specific protein_kinase.
[698] 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.
[699] 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.
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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.
[700] Moreover, the techniques of gene-shuffling, motif shuffling, exon-
shuffling,
and/or codon-shuffling (collectively referred to as "DNA shuffling") may be
employed to
modulate the activities of the polypeptide of the present invention thereby
effectively
generating agonists and antagonists of the polypeptide of the present
invention. See
generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and
5,837,458, and
Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.
Trends
Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-
76 (1999); and
Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these
patents
and publications are hereby incorporated by reference). In one embodiment,
alteration of
polynucleotides and corresponding polypeptides may be achieved by DNA
shuffling. DNA
shuffling involves the assembly of two or more DNA segments into a desired
molecule by
homologous, or site-specific, recombination. In another embodiment,
polynucleotides and
corresponding polypeptides may be altered by being subjected to random
mutagenesis by
error-prone PGR, random nucleotide insertion or other methods prior to
recombination. In
another embodiment, one or more components, motifs, sections, parts, domains,
fragments,
etc., of the polypeptide of the present invention may be recombined with one
or more
components, motifs, sections, parts, domains, fragments, etc. of one or more
heterologous
molecules. In preferred embodiments, the heterologous molecules are family
members. In
further preferred embodiments, the heterologous molecule is a growth factor
such as, for
example, platelet-derived growth factor (PDGF), insulin-like growth factor
(IGF-I),
transforming growth factor (TGF)-alpha, epidermal growth factor (EGF),
fibroblast growth
factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-
6, .
BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth
differentiation
factors (GDFs), nodal, MIS, inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3,
TGF-betas,
and glial-derived neurotrophic factor (GDNF)..
[701] Other preferred fragments are biologically active fragments of the
polypeptide of
the present invention. Biologically active fragments are those exhibiting
activity similar, but
not necessarily identical, to an activity of the polypeptide of the present
invention. The
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biological activity of the fragments may include an improved desired activity,
or a decreased
undesirable activity.
[702] Additionally, this invention provides a method of screening compounds to
identify
those which modulate the action of the polypeptide of the present invention.
An example of
such an assay comprises combining a mammalian fibroblast cell, a the
polypeptide of the
present invention, the compound to be screened and 3 [H] thymidine under cell
culture
conditions where the fibroblast cell would normally proliferate. A control
assay may be
performed in the absence of the compound to be screened and compared to the
amount of
fibroblast proliferation in the presence of the compound to determine if the
compound
stimulates proliferation by determining the uptake of 3 [H] thymidine in each
case. The
amount of fibroblast cell proliferation is measured by liquid scintillation
chromatography
which measures the incorporation of 3 [H] thymidine. Both agonist and
antagonist
compounds may be identified by this procedure.
[703] . In another method, a mammalian cell or membrane preparation expressing
a
receptor for a polypeptide of the present invention is incubated with a
labeled polypeptide of
the present invention in the presence of the compound. The ability of the
compound to
enhance or block this interaction could then be measured. Alternatively, the
response of a
known second messenger system following interaction of a compound to be
screened and the
receptor is measured and the ability of the compound to bind to the receptor
and elicit a
second messenger: response is measured to determine if the compound is a
potential agonist
or antagonist. Such second messenger systems include but are riot limited to,
cAMP
guanylate cyclase, ion channels or phosphoinositide hydrolysis.
[704] All of these above assays can be used as diagnostic or prognostic
markers. The
molecules discovered using these assays can be used to treat disease or to
bring about a
particular result in a patient (e.g., blood vessel growth) by activating or
inhibiting the
polypeptide/molecule. Moreover, the assays can discover agents which may
inhibit or
enhance the production of the polypeptides of the invention from suitably
manipulated cells
or tissues.
[705] Therefore, the invention includes a method of identifying compounds
which bind
to a polypeptide of the invention comprising the steps of: (a) incubating a
candidate binding
compound with ~a polypeptide of the present invention; and (b) determining if
binding has
occurred. Moreover, the invention includes a method of identifying
agonists/antagonists
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comprising the steps of: (a) incubating a candidate compound with a
polypeptide of the
present invention, (b) assaying a biological activity, and (b) determining if
a biological
activity of the polypeptide has been altered. ;
Targeted Delivery
[706] In another embodiment, the invention provides a method of delivering
compositions to targeted cells expressing a receptor for a polypeptide of the
invention, or
cells expressing a cell bound form of a polypeptide of the invention.
[707] As discussed herein, polypeptides or antibodies of the invention may be
associated
with heterologous polypeptides, heterologous nucleic acids, toxins, or
prodrugs via
hydrophobic, hydrophilic, ionic and/or covalent interactions. In one
embodiment, the
invention provides a method for the specific delivery of compositions of the
invention to cells
by administering polypeptides of the invention (including antibodies) that are
associated with
heterologous polypeptides or nucleic acids. In one example, the invention
provides a method
for delivering a therapeutic protein into the targeted cell. In another
example, the invention
provides a method for delivering a single stranded nucleic acid (e.g.,
antisense or ribozymes)
or double stranded nucleic acid (e.g., DNA that can integrate into the cell's
genome or
replicate episomally and that can be transcribed) into the targeted cell.
[708] In another embodiment, the invention provides a method for the specific
destruction of cells (e.g., the destruction of tumor cells) by administering
polypeptides of the
invention (e.g., polypeptides of the invention or antibodies of the invention)
in association
with toxins or cytotoxic prodrugs.
[709] By "toxin" is meant compounds that bind, and activate endogenous
cytotoxic
effector systems, radioisotopes, holotoxins, modified toxins, catalytic
subunits of toxins, or
any molecules or enzymes not normally present in or on the surface- of a cell
that under
defined conditions cause the cell's death. Toxins that may be used according
to the methods
of the invention include, but are not limited to, radioisotopes known in the
art, compounds
such as, for example, antibodies (or complement fixing containing portions
thereof) that bind
an inherent or induced endogenous cytotoxic effector system, thymidine kinase,
endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A,
diphtheria toxin,
saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and
cholera toxin. By
"cytotoxic prodrug" is meant a non-toxic compound that is converted by an
enzyme,
\ normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs
that may be
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used according to the methods of the invention include, but are not limited
to, glutamyl
derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of
etoposide or
mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide
derivatives of
doxorubicin.
Drug Screening
[710] Further contemplated is the use of the polypeptides of the present
invention, or the
polynucleotides encoding these polypeptides, to screen for molecules which
modify the
activities of the polypeptides of the present invention. Such a method would
include
contacting the polypeptide of the present invention~with a selected compounds)
suspected of
having antagonist or agonist activity, and assaying the activity of these
polypeptides
following binding.
[711] This invention is particularly useful for screening therapeutic
compounds by using
the polypeptides of the present invention, or binding fragments thereof, in
any of a variety of
drug screening techniques. The polypeptide or fragment employed in such a test
may be
affixed to a solid support, expressed on a cell surface, free in solution, or
located
intracellularly. One method of drug screening utilizes eukaryotic or
prokaryotic host cells
which are stably transformed with recombinant nucleic acids expressing the
polypeptide or
fragment. Drugs are screened against such transformed cells in competitive
binding assays.
One may measure, for example, the formulation of complexes between the agent
being tested
and a polypeptide of the present invention.
[712] Thus, the present invention provides methods of screening for drugs or
any othei
agents which affect activities mediated by the polypeptides of the present
invention. These
methods comprise' contacting such an agent with a polypeptide of the present
invention or a
fragment thereof and assaying for the presence of a complex between the agent
and the.
polypeptide or a fragment thereof, by methods well known in the art. In such a
competitive
binding assay, the agents to screen are typically labeled. Following
incubation, free agent is.
separated from that present in bound form, and the amount of free or
uncomplexed label is a
measure of the ability of a particular agent to bind to the polypeptides of
the present
invention.
(713] Another technique for drug screening provides high throughput screening
for
compounds having suitable binding affinity to the polypeptides of the present
invention, and
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is described in great detail in European Patent Application 84/03564,
published on September
13, 1984, which is incorporated herein by reference herein. Briefly stated,
large numbers of
different small peptide test compounds are synthesized on a solid substrate,
such as plastic
pins or some other surface. The peptide test compounds are reacted with
polypeptides of the
present invention and washed. Bound polypeptides are then detected by methods
well known
in the art. Purified polypeptides are coated directly onto plates for use in
the aforementioned
drug screening techniques. In addition, non-neutralizing antibodies may be
used to capture
the peptide and immobilize it on the solid support.
[714] This invention also contemplates the use of competitive drug screening
assays in
which neutralizing antibodies capable of binding polypeptides of the present
invention
specifically compete with a test compound for binding to the polypeptides or
fragments
thereof. In this manner, the antibodies are used to detect the presence of any
peptide which
shares one or more antigenic epitopes with a polypeptide of the invention.
Antisense And Ribozyme (Antagonists)
[715] In specific embodiments, antagonists according to the present invention
are nucleic
acids corresponding to the sequences contained in SEQ ID NO:X, or the
complementary
strand thereof, and/or to cDNA sequences contained in cDNA Clone ID NO:Z
identified for
example, in Table )A. In one embodiment, antisense sequence is generated
internally,~by the
organism, in another embodiment, the antisense sequence is separately
administered (see, for
example, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as
Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Antisense
technology
can be used to control gene expression through antisense DNA or RNA, or
through triple-
helix formation. Antisense techniques are discussed for example, in Okano, J.,
Neurochem.
56:560 (1'991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC
Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for
instance; Lee et al.,
Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988);
and Dervan
et al., Science 251:1300 (1991). The methods are based on binding of a
polynucleotide to a
complementary DNA or RNA.
[716] For-example, the use of c-myc and c-myb antisense 'RNA constructs to
inhibit the
growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines
was previously
described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments
were
performed in vitro by incubating cells with the oligoribonucleotide. A similar
procedure for
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in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides
for a given
antisense RNA is, produced as follows: A sequence complimentary to the first
15 bases of the
open reading frame is flanked by an EcoRl site on the 5 end and a HindIII site
on the 3 end.
Next, the pair of oligonucleotides is heated at 90°C for one minute and
then annealed in 2X
ligation buffer (20mM TRIS HCl pH 7.5, lOmM MgCl2, l OMM dithiothreitol (DTT)
and 0.2
mM ATP) and then ligated to the EcoRl/Hind III site of the retroviral vector
PMV7 (WO
91/15580).
[717] For example, the 5' coding portion of a polynucleotide that encodes the
polypeptide of the present invention may be used to design an antisense RNA
oligonucleotide
of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed
to be
complementary to a region of the gene involved in transcription thereby
preventing
transcription and the production of the receptor. The antisense RNA
oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into receptor
polypeptide.
[718] In one embodiment, the antisense nucleic acid of the invention is
produced
intracellularly by transcription from an exogenous sequence. For example, a
vector or a
portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of
the invention.
Such a vector would contain a sequence encoding the antisense nucleic acid.
Such a vector
can remain episomal or become chromosomally integrated, as long as it can be
transcribed to
produce the desired antisense RNA. Such vectors can be constructed by
recombinant DNA
technology methods standard in the art. Vectors can be plasmid, viral, or
others known in the
art, used for replication and expression in vertebrate cells. Expression of
the sequence
encoding the polypeptide of the present invention or fragments thereof, can be
by any
promoter known in the art to act in vertebrate, preferably human cells. Such
promoters can
be inducible or constitutive. Such promoters include, but are not limited to,
the SV40 early
promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter
contained
in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell
22:787-797
(1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci.
U.S.A. 78:1441-
1445 (1981), the regulatory sequences of the metallothionein gene (Brinster,
et al., Nature
296:39-42 (1982)), etc.
[719] The antisense nucleic acids of the invention comprise a sequence
complementary
to at least a portion of an RNA transcript of a gene of the present invention.
However,
absolute complementarity, although preferred, is not required. A sequence
"complementary
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to at least a portion of an RNA," referred to herein, means a sequence having
sufficient
complementarity to be able to hybridize with the RNA, forming a stable duplex;
in the case
of double stranded antisense nucleic acids, a single strand of the duplex DNA
may thus be
tested, or triplex formation may be assayed. The ability to hybridize will
depend on both the
degree of complementarity and the length of the antisense nucleic acid.
Generally, the larger
the hybridizing nucleic acid, the more base mismatches with a RNA it may
contain and still
form a stable duplex (or triplex as the case may be). One skilled in the art
can ascertain a
tolerable degree of mismatch by use of standard procedures to determine the
melting point of
the hybridized complex.
[720] Oligonucleotides that are complementary to the 5' end of the message,
e.g., the 5'
untranslated sequence up to and including the AUG initiation codon, should
work most
efficiently at inhibiting translation. However; sequences complementary to the
3'
untranslated sequences of mRNAs have been shown to be effective at inhibiting
translation
of mRNAs as .well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus,
oligonucleotides complementary to either the 5'- or 3'- non- translated, non-
coding regions of
polynucleotide sequences described herein could be used in an antisense
approach to inhibit
translation .of endogenous mRNA. Oligonucleotides complementary to the 5'
untranslated
region of the mRNA should include the complement of the AUG start codon.
Antisense
oligonucleotides complementary to mRNA- coding regions are less efficient
inhibitors of
translation but could be used in accordance with the invention. Whether
designed to
hybridize to the 5'-, 3'- or coding region of mRNA of the present invention,
antisense nucleic
acids should be at least six nucleotides in length, and are preferably
oligonucleotides ranging
from 6 to about 50 nucleotides in length. In specific aspects the
oligonucleotide is at least 10 \
nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50
nucleotides.
[721] The polynucleotides of the invention can be DNA or RNA or chimeric
mixtures or
derivatives or modified versions thereof, single-stranded or double-stranded.
The
oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate
backbone, for
example, to improve stability of the molecule, hybridization, etc. The
oligonucleotide may
include other appended groups such as peptides (e.g., for targeting host cell
receptors in
vivo), or agents facilitating transport across the cell membrane (see, e.g.,
Letsinger et al.,
1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc.
Natl. Acad.
Sci. 84:648-652; PCT Publication No. W088/09810, published December 15, 1988)
or the
blood-brain barrier .(see, e.g., PCT Publication No. W089/10134, published
April 25, 1988),
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hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988,
BioTechniques 6:958-
976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549).
To this end, the
oligonucleotide may be conjugated to another molecule, e.g., a peptide,
hybridization
triggered cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[722] The antisense oligonucleotide may comprise at least one modified base
,moiety
which is selected from the group including, but not limited to, 5-
fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine,
inosine,
N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-
adenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-
D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-
methylthio-N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-
5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil, 3-(3-amino-
3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
[723] The antisense oligonucleotide may also comprise at least one modified
sugar
moiety selected from the group including, but not limited to, arabinose, 2-
fluoroarabinose,
xylulose, and hexose.
[724] In yet another embodiment, the antisense oligonucleotide comprises at
least one
modified phosphate backbone selected from the group including, but not limited
to, a
phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a
phosphoramidate, a
phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a
formacetal or
analog thereof.
[725] In yet another embodiment, the antisense oligonucleotide is an a-
anomeric
oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded
hybrids with
complementary RNA in which, contrary to the usual b-units, the strands run
parallel to each
other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The
oligonucleotide is a 2'-0-
methylribonucleotide (moue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a
chimeric
RNA-DNA.analogue (moue et al., 1987, FEBS Lett. 215:327-330).
[726] Polynucleotides of the invention may be synthesized by standard methods
known
in the art, e.g. by use of an automated DNA synthesizer (such as are
commercially available
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from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate
oligonucleotides
may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res.
16:3209),
methylphosphonate oligonucleotides can be prepared by use of controlled pore
glass polymer
supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451),
etc.
[727] While antisense nucleotides complementary to the coding region sequence
could
be used, those complementary to the transcribed untranslated region are most
preferred.
[728] Potential antagonists according to the invention also include catalytic
RNA, or a
ribozyme (See, e.g., PCT International Publication WO 90/11364, published
October 4,
1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave
mRNA at
site specific recognition sequences can be used to destroy mRNAs, the use of
hammerhead
ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations
dictated by
flanking regions that form complementary base pairs with the target mRNA. The
sole
requirement is that the target mRNA have the following sequence of two bases:
5'-UG-3'.
The construction and production of hammerhead ribozymes is well known in the
art and is
described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There
are
numerous potential hammerhead ribozyme cleavage sites within the nucleotide
sequence of
SEQ ID NO:X. Preferably, the ribozyme is engineered so that the cleavage
recognition site
is located near the 5' end of the mRNA; i.e., to increase efficiency and
minimize the
intracellular accumulation of non-functional mRNA transcripts.
[729] As in the antisense approach, the ribozymes of the invention can be
composed of
modified oligonucleotides (e.g., for improved stability, targeting, etc.) and
should be
delivered to cells which express in vivo. DNA constructs encoding the-ribozyme
may be
introduced into the cell in the same manner as described above for the
introduction of
antisense encoding DNA. A preferred method of delivery involves using a DNA
construct
"encoding" the ribozyme under the control of a strong constitutive promoter,
such as, for
example, pol III or pol II promoter, so that transfected cells will produce
sufficient quantities
of the ribozyme to destroy endogenous messages and inhibit translation. Since
ribozymes
unlike antisense molecules, are catalytic, a lower intracellular concentration
is required for
efficiency.
[730] Antagonist/agonist compounds may be employed to inhibit the cell growth
and
proliferation effects of the polypeptides of the present invention on
neoplastic cells and
tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or
prevent abnormal
cellular growth and proliferation, for example, in tumor formation or growth.
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[731] The antagonist/agonist may also be employed to prevent hyper-vascular
diseases,
and prevent the. proliferation of epithelial lens cells after extracapsular
cataract surgery.
Prevention of the mitogenic activity of the polypeptides of the present
invention may also be
desirous in cases such as restenosis after balloon angioplasty.
[732] The antagonist/agonist may also be employed to prevent the growth of
scar tissue
during wound healing.
[733] The antagonist/agonist may also be employed to treat the diseases
described
herein.
[734] Thus, the invention provides a method of treating disorders or diseases,
including
but not limited to the disorders or diseases listed throughout this
application, associated with
overexpression of a polynucleotide of the present invention by administering
to a patient (a)
an antisense molecule directed to the polynucleotide of the present invention,
and/or (b) a
ribozyme directed to the polynucleotide of the present invention.
Binding Peptides and Other Molecules .
[735] The invention also encompasses screening methods for identifying
polypeptides
and nonpolypeptides that bind polypeptides of the invention, and the binding
molecules
identified thereby. These binding molecules are useful, for example, as
agonists and
antagonists of the polypeptides of the invention. Such agonists and
antagonists can be used,
in accordance with the invention, in the therapeutic embodiments described in
detail, below.
This method comprises the steps of:
a. contacting polypeptides of the invention with a plurality of molecules; and
b. identifying a molecule that binds the polypeptides of the invention.
[736] The step of contacting the polypeptides of the -invention with the
plurality of
molecules may be effected in a number of ways. For example, one may
contemplate
immobilizing the polypeptides on a solid support and bringing a solution of
the plurality of
molecules in contact with the immobilized polypeptides. Such a procedure would
be akin to
an affinity chromatographic process, with the affinity matrix being comprised
of the
immobilized polypeptides of the invention. The molecules having a selective
affinity for the
polypeptides can then be purified by affinity selection. The nature of the
solid support,
process for attachment of the polypeptides to the solid support, solvent, and
conditions of the
affinity isolation or selection are largely conventional and well known to
those of ordinary
skill in the art.
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[737] Alternatively, one may also separate a plurality of polypeptides into
substantially
separate fractions comprising a subset of or individual polypeptides. For
instance, one can
separate the plurality of polypeptides by gel electrophoresis, column
chromatography, or like
method known to those of ordinary skill for the separation of polypeptides.
The individual
polypeptides can also be produced by a transformed host cell in such a way as
to be
expressed on or about its outer surface (e.g., a recombinant phage).
Individual isolates can
then be "probed" by the polypeptides of the invention, optionally in the
presence of an
induces should one be required for expression; to determine if any selective
affinity
interaction takes place between the polypeptides and the individual clone.
Prior to contacting
the polypeptides with each fraction comprising individual polypeptides, the
polypeptides
could first be transferred to a solid support for additional convenience. Such
a solid support
may simply be a piece of filter membrane, such as one made of nitrocellulose
or nylon. In
this manner, positive clones could be identified from a collection of
transformed host cells of
an expression library, which harbor a DNA construct encoding a polypeptide
having a
selective affinity for polypeptides of the invention. Furthermore, the amino
acid sequence of
the polypeptide having a selective affinity for the polypeptides of the
invention can be
determined directly by conventional means or the coding sequence of the DNA
encoding the
polypeptide can frequently be determined more conveniently. The primary
sequence can then
be deduced from the corresponding DNA sequence. If the amino acid sequence is
to be
determined from the polypeptide itself, one may use microsequencing
techniques. The
sequencing technique may include mass spectroscopy.
[738J In certain situations, it may be desirable to wash away any unbound
polypeptides
from a mixture of the polypeptides of the invention and the plurality of
polypeptides prior to
attempting to determine or to detect the presence of a selective affinity
interaction. Such a
wash step may be particularly desirable when the polypeptides of the invention
or the
plurality of polypeptides are bound to a solid support.
[739] ~ The plurality of molecules provided according to this method may be
provided by
way of diversity libraries, such as random or combinatorial peptide or
nonpeptide libraries
which can be screened for molecules that specifically bind polypeptides of the
invention.
Many libraries are known in the art that can be used, e.g., chemically
synthesized libraries,
recombinant (e.g., phage display libraries), and in vitro translation-based
libraries. Examples
of chemically synthesized libraries are described in Fodor et al., 1991,
Science 251:767-773;
Houghten et.al., 1991, Nature 354:84-86; Lam et al., 1991, Nature-354:82-84;
Medynski,
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1994, Bio/Technology 12:709-710;Gallop et al., 1994, J. Medicinal Chemistry
37(9):1233-
1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et
al., 1994,
Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992,
Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. 5c1. USA 91:1614-1618; Salmon et
al., 1993,
Proc. Natl. Acad: Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242;
and
Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.
[740] Examples of phage display libraries are described in Scott and Smith,
1990,
Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R.
B., et al.,
1992, J: Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-
157; Kay et al.,
1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.
[741] In vitro translation-based libraries include but are not limited to
those described in
PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al.,
1994, Proc.
Natl. Acad. Sci. USA 91:9022-9026.
[742] By way of examples of nonpeptide libraries, a benzodiazepine library
(see e.g.,
Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted
for use. Peptoid
libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can
also be used.
Another example of a library that can be used, in which the amide
functionalities in peptides
have been permethylated to generate a chemically transformed combinatorial
library, is
described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).
[743] The variety of non-peptide libraries that are useful in the present
invention is great.
For example, Ecker arid Crooke, 1995, Bio%Technology 13:351-360 list
benzodiazepines,
hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones,
arylacetic
acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and
oxazolones as
among the chemical species that form the basis of various libraries.
[744] Non-peptide libraries can be classified broadly into two types:
decorated
monomers and oligomers. Decorated monomer libraries employ a relatively simple
scaffold
structure upon which a variety functional groups is added. Often the scaffold
will be a
molecule with a known useful pharmacological activity. For example, the
scaffold might be
the benzodiazepine structure.
[745] Non-peptide oligomer libraries utilize a large number of monomers that
are
assembled together in' ways that create new shapes that depend omthe order of
the monomers.
Among the monomer units that have been used are carbamates, pyrrolinones, and
morpholinos. Peptoids, peptide-like oligomers in which the side chain is
attached to the alpha
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amino group rather than the alpha carbon, form the basis of another version of
non-peptide
oligomer libraries. The first non-peptide oligomer libraries utilized a single
type of monomer
and thus contained a repeating backbone. Recent libraries have utilized more
than one
monomer, giving the libraries added flexibility.
[746] Screening the libraries can be accomplished by any of a variety of
commonly
known methods. See, e.g., the following references, which disclose screening
of peptide
libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott
and Smith,
1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427;
Oldenburg et
al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell
76:933-945; Staudt
et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566;
Tuerk et al., 1992,
Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature
355:850-852; U.S.
Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all
to Ladner et al.;
Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.
[747] In a specific embodiment, screening to identify a molecule that binds
polypeptides
of the invention can be carried out by contacting the library members with
polypeptides of
the invention immobilized on a solid phase and harvesting those library
members that bind to
the polypeptides of the invention. Examples of such screening methods, termed
"panning"
techniques are described by way of example in Parmley and Smith, 1988, Gene
73:305-318;
Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO
94/18318; and in
references cited herein.
[748] In another embodiment; the two-hybrid system for selecting interacting
proteins in
yeast (Fields and Song, 1989; Nature 340:245-246; Chien et al., 1991, Proc.
Natl. Acad. Sci.
USA 88:9578-9582) can be used to identify molecules that specifically bind to
polypeptides
of the invention.
[749] Where the binding molecule is a polypeptide, the polypeptide can be
conveniently
selected from any peptide library, including random peptide libraries,
combinatorial peptide
libraries, or biased peptide libraries. The term "biased" is used herein to
mean that the method
of generating the library is manipulated so as to restrict one or more
parameters that govern
the diversity of the resulting collection of molecules, in this case peptides.
[750J Thus, a truly random peptide library would generate a collection of
peptides iri
which the probability.of finding a particular amino acid at a given position
of the peptide is
the same for all 20 amino acids. A bias can be introduced into the library,
however, by
specifying, for example, that a lysine occur every fifth amino acid or that
positions 4, 8, and 9
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of a decapeptide library be fixed to include only arginine. Clearly, many
types of biases can
be contemplated, and the present invention is not restricted to any particular
bias.
Furthermore, the present invention contemplates specific types of peptide
libraries, such as
phage displayed peptide libraries and those that utilize a. DNA construct
comprising a lambda
phage vector with a DNA insert.
[751] As mentioned above, in the case of a binding molecule that is a
polypeptide, the
polypeptide may have about 6 to less than about 60 amino acid residues,
preferably about 6
to about 10 amino acid residues, and most preferably, about 6 to about 22
amino acids. In
another embodiment, a binding polypeptide has in the range of 15-100 amino
acids, or 20-50
amino acids.
[752] The selected binding polypeptide can be obtained by chemical synthesis
or
recombinant expression.
Other Activities
[753] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention, as a
result of the ability to stimulate vascular endothelial cell growth, may be
employed in
treatment for stimulating re-vascularization of ischemic tissues due to
various disease
conditions such as thrombosis, arteriosclerosis, and other cardiovascular
conditions. The
polypeptide, polynucleotide, agonist, or antagonist of the present invention
may also be
employed to stimulate angiogenesis and limb regeneration, as discussed above.
[754] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
also be employed for treating wounds due to injuries, burns, post-operative
tissue repair, and
ulcers since they are mitogenic to various cells of different origins, such as
fibroblast cells
and skeletal muscle cells, and therefore, facilitate the repair or replacement
of damaged or
diseased tissue.
[755] A polypeptide, polynucleotide,, agonist, or antagonist of the present
invention may
also be employed stimulate neuronal growth and to treat and prevent neuronal
damage which
occurs in certain neuronal disorders or neuro-degenerative conditions such as
Alzheimer's
disease, Parkinson's disease, and AIDS-related complex. A polypeptide,
polynucleotide,
agonist, or antagonist of the present invention may have the ability to
stimulate chondrocyte
growth, therefore, they may be employed to enhance bone and periodontal
regeneration and -
aid in tissue transplants or bone grafts.
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[756] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
be also be employed to prevent skin aging due to sunburn by stimulating
keratinocyte
growth. .
[757] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
also be employed for preventing hair loss, since FGF family members activate
hair-forming
cells and promotes melanocyte growth. Along the same lines, a polypeptide,
polynucleotide,
agonist, or antagonist of the present invention may be employed to stimulate
growth and
differentiation of hematopoietic cells and bone marrow cells when used in
combination with
other cytokines.
[758] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
also be employed to maintain organs before transplantation or for supporting
cell culture of
primary tissues. A polypeptide, polynucleotide, agonist, or antagonist of the
present
invention may also be employed for inducing tissue of mesodermal origin to
differentiate in
early embryos.
[759] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
also increase or decrease the differentiation or proliferation of embryonic
stem cells, besides,
as discussed above, hematopoietic lineage.
[760] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
also be used to modulate mammalian characteristics, such as body height,
weight, hair color,
eye color, skin, percentage of adipose tissue, pigmentation, size, and shape
(e.g., cosmetic
surgery). Similarly, a polypeptide, polynucleotide, agonist, or antagonist of
the present
invention may be used to modulate mammalian metabolism affecting catabolism,
anabolism,
processing, utilization, and storage of energy.
[761] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
be used to change a mammal's mental state or physical state by influencing
biorhythms,
caricadic rhythms, depression (including depressive disorders), tendency for
violence,
tolerance for pain, reproductive capabilities (preferably by Activin or
Inhibin-like activity),
hormonal or endocrine levels, appetite, libido, memory, stress, or other
cognitive qualities.
(762] A polypeptide, polynucleotide, agonist, or antagonist of the present
invention may
also be used as a food additive or preservative, such as to increase or
decrease storage
capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals,
cofactors or other
nutritional components.
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[763] The above-recited applications have uses in a wide variety of hosts.
Such hosts
include, but are not limited to, human, murine, rabbit, goat, guinea pig,
camel, horse, mouse,
rat, hamster, pig, micro-pig, chicken, goat; cow, sheep, dog, cat, non-human
primate, and
human. In specific embodiments, the host is a mouse, rabbit, goat,. guinea
pig, chicken, rat,
hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a
mammal. In most
preferred embodiments, the host is a human.
Other Preferred Embodiments
[764] Other preferred embodiments of the claimed invention include an isolated
nucleic
acid molecule comprising a nucleotide sequence which is at least 95% identical
fo a sequence
of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ
ID NO:X or
the complementary strand thereto, the nucleotide sequence as defined in column
5 of Table
1A or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or
cDNA
contained in Clone ID NO:Z.
[765] Also preferred is a nucleic acid molecule wherein said sequence of
contiguous
nucleotides is included in the nucleotide sequence of the portion of SEQ ID
NO:X as defined
in column S, "ORF (From-To)", in Table 1A.
[766] Also preferred is a nucleic acid molecule wherein said sequence of
contiguous
nucleotides is included in the nucleotide sequence of the portion of SEQ ID
NO:X as defined
in columns 8 and 9, "NT From" and "NT To" respectively, in Table 2.
[767] Also preferred is an isolated nucleic acid molecule comprising a
nucleotide
sequence which is at least 95% identical to a sequence of at least about 150
contiguous
nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary
strand
thereto, the nucleotide sequence as defined in column 5 of Table 1 A or
columns 8 and 9 of
. Table 2 or the complementary strand thereto, and/or cDNA contained in Clone
ID NO:Z.
[768] Further preferred is an isolated nucleic acid molecule comprising a
nucleotide
sequence which is at least 95% identical to a sequence of at least about 500
contiguous
nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary
strand
thereto, the nucleotide sequence as defined in column 5 of Table 1A or columns
8 and 9 of
Table 2 or the complementary strand thereto, and/or cDNA contained in Clone ID
NO:Z.
[769] A further preferred embodiment is a nucleic acid molecule comprising a
nucleotide
sequence which is at least 95% identical to the nucleotide sequence of the
portion of SEQ ID
NO:X defined in column 5, "ORF (From-To)", in Table 1A.
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[770] A further preferred embodiment is a nucleic acid molecule comprising a
nucleotide
sequence which is at least 95% identical to the nucleotide sequence of the
portion of SEQ ID
NO:X defined in columns 8 and 9, "NT From" and "NT To", respectively, in Table
2.
[771] A further preferred embodiment is an isolated nucleic acid molecule
comprising a
nucleotide sequence which is at least 95% identical to the complete nucleotide
sequence of
SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as
defined in
column 5 of Table 1A or columns 8 and 9 of Table 2 or the complementary strand
thereto,
and/or cDNA contained in Clone ID NO:Z.
[772] Also preferred is an isolated nucleic acid molecule which hybridizes
under
stringent hybridization conditions to a nucleic acid molecule comprising a
nucleotide
sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide
sequence as
defined in column 5 of Table 1A or columns 8 and 9 of Table 2 or the
complementary strand
thereto, and/or cDNA contained in Clone ID NO:Z, wherein said nucleic acid
molecule
which hybridizes does not hybridize under stringent hybridization conditions
to a nucleic acid
molecule having a nucleotide sequence consisting of only A residues or of only
T residues.
[773] Also preferred is a composition of matter comprising a DNA molecule
which
comprises the cDNA contained in Clone ID NO:Z.
[774] Also preferred is an isolated nucleic acid molecule comprising a
nucleotide
sequence which is at least 95% identical to a sequence of at least 50
contiguous nucleotides
of the cDNA sequence contained in Clone ID NO:Z.
[775] Also preferred is an isolated nucleic acid molecule, wherein said
sequence of at
least 50 contiguous nucleotides is included in the nucleotide sequence of an
open reading
frame sequence encoded by cDNA contained in Clone ID NO:Z.
[776] Also preferred is an isolated nucleic acid molecule comprising a
nucleotide
sequence which is at least 95% identical to sequence of at least 150
contiguous nucleotides in
the nucleotide sequence encoded by cDNA contained in Clone ID NO:Z.
[777] A further preferred embodiment is an isolated nucleic acid molecule
comprising a
nucleotide sequence which is at least 95% identical to sequence of at least
500 contiguous
nucleotides in the nucleotide sequence encoded by cDNA contained in Clone ID
NO:Z.
[778] A further preferred embodiment is- an isolated nucleic acid molecule
comprising a
nucleotide sequence which is at least 95% identical to the complete nucleotide
sequence
encoded by cDNA contained in Clone ID NO:Z.
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[779] A further preferred embodiment is a method for detecting in a biological
sample a
nucleic acid molecule comprising a nucleotide sequence which is at least 95%
identical to a
sequence of at least 50 contiguous nucleotides in a sequence selected from the
group
consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary
strand thereto;
the nucleotide sequence as defined in column 5 of Table 1 A or columns 8 and 9
of Table 2 or
the complementary strand thereto; and a nucleotide sequence encoded by cDNA
contained in
Clone ID NO:Z; which method comprises a 'step of comparing a nucleotide
sequence of at
least one nucleic acid molecule in said sample with a sequence selected from
said group and
determining whether the sequence of said nucleic acid molecule in said sample
is at least
95% identical to said selected sequence.
[780] Also preferred is the above method wherein said step of comparing
sequences
comprises determining the extent of nucleic acid hybridization between nucleic
acid
molecules in said sample and a .nucleic acid molecule comprising said sequence
selected
from said group. Similarly, also preferred is the above method, wherein said
step of
comparing sequences is performed by comparing the nucleotide sequence
determined from a
nucleic acid molecule in said sample with said sequence selected from said
group. The
nucleic acid molecules can comprise DNA molecules or RNA molecules.
[781] A further preferred embodiment is a method for identifying the species,
tissue or
cell type of a biological sample which method comprises a step of detecting
nucleic acid
molecules in said sample, if any, comprising a nucleotide sequence that is at
least 95%
identical to a sequence of at least 50 contiguous nucleotides in a sequence
selected from the
group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary
strand
thereto; the nucleotide sequence as defined in column 5 of Table 1A or columns
8 and 9 of
Table 2 or the complementary strand thereto; and a nucleotide sequence of the
cDNA
contained in Clone ID NO:Z.
[782] . The method for identifying the species, tissue or cell type of a
biological sample
can comprise a step of detecting nucleic acid molecules comprising a
nucleotide sequence in
a panel of at least two nucleotide sequences, wherein at least one sequence in
said panel is at
least 95%.identical to a sequence of at least 50 contiguous nucleotides in a
sequence selected
from said group.
[783] Also preferred is a method for diagnosing in a subject a pathological
condition
associated with abnormal structure or expression of a nucleotide sequence of
SEQ ID NO:X
or the complementary strand thereto; the nucleotide sequence as defined in
column 5 of Table
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1A or columns 8 and 9 of Table 2 or the complementary strand thereto; or the
cDNA
contained in Clone ID NO:Z which encodes a protein, wherein the method
comprises a step
of detecting in a biological sample obtained from said subject nucleic acid
molecules, if any,
comprising a nucleotide sequence that is at least 95% identical to a sequence
of at least 50
contiguous nucleotides in a sequence selected from the group consisting of: a
nucleotide
sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide
sequence as
defined in column 5 of Table 1 A or columns 8 and 9 of Table 2 or the
complementary strand
thereto; and a nucleotide sequence of cDNA contained in Clone ID NO:Z.
[784] The method for diagnosing a pathological condition can comprise a step
of
detecting nucleic acid molecules comprising a nucleotide sequence in a panel
of at least two
nucleotide sequences, wherein at least one sequence in said panel is at least
95% identical to
a sequence of at least 50 contiguous nucleotides in a sequence selected from
said group.
[785] Also preferred is a composition of matter comprising isolated nucleic
acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise a panel
of at least two nucleotide sequences, wherein at least one sequence in said
panel is at least
95% identical to a sequence of at least 50 contiguous nucleotides in a
sequence selected from
the group consisting of: a nucleotide sequence of SEQ ID NO:X or the
complementary strand -.
thereto; the nucleotide sequence as defined in column 5 of Table 1A or columns
8 and 9 of
Table 2 or the complementary strand thereto; and a nucleotide sequence encoded
by cDNA
contained in Clone ID NO:Z. The nucleic acid molecules can comprise DNA
molecules or
RNA molecules.
[786] Also preferred is a composition of matter comprising isolated nucleic
acid
molecules wherein the nucleotide sequences of said nucleic acid molecules
comprise a DNA
microarray or "chip" of at least l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,
30, 40, 50, 100, 150,
200, 250, 300, 500, 1000, 2000, 3000, or 4000 nucleotide sequences, wherein at
least one
sequence in said DNA microarray or "chip" is at least 95% identical to a
sequence of at least
50 contiguous nucleotides in a sequence selected from the group consisting
of:, a nucleotide
sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1A; and a
nucleotide
sequence encoded by a human cDNA clone identified by a cDNA "Clone ID" in
Table 1A.
[787] Also preferred is an isolated polypeptide comprising an amino acid
sequence at
least 90% identical to a sequence of at least about 10 contiguous amino .
acids in the
polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or
the
complementary strand thereto; the polypeptide encoded by the nucleotide
sequence as
263
CA 02395178 2002-06-19
WO 01/55208 PCT/USO1/01357
defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA
contained in
Clone ID NO:Z.
[788] Also preferred is an isolated polypeptide comprising an amino acid
sequence at
least 95% identical to a sequence of at least about 30 contiguous amino acids
in the amino
acid 'sequence of SEQ. ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the
complementary strand thereto; the polypeptide encoded by the nucleotide
sequence as
defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA
contained in
Clone ID NO:Z.
[789] Further preferred is an isolated polypeptide comprising an amino acid
sequence at
least 95% identical to a sequence of at least about 100 contiguous amino acids
in the amino
acid sequence of SEQ ID NO:Y; a polypeptide. encoded by SEQ ID NO:X or the
complementary strand thereto; the polypeptide encoded by the nucleotide
sequence as
defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA
contained in
Clone ID NO:Z.
[790] Further preferred is an isolated polypeptide comprising an amino acid
sequence at
least 95% identical to the complete amino acid sequence of SEQ ID NO:Y; a
polypeptide
encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide
encoded by
the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a
polypeptide
encoded by cDNA contained in Clone ID NO:Z.
[791] Further preferred is an isolated polypeptide comprising an amino acid
sequence at
least 90% identical to a sequence of at least about 10 contiguous amino acids
in the complete
amino acid sequence of a polypeptide encoded by contained in Clone ID NO:Z
[792] Also preferred is a polypeptide wherein said sequence of contiguous
amino acids is
included in the amino acid sequence of a portion of said polypeptide encoded
by cDNA
contained in Clone ID NO:Z; a polypeptide encoded by SEQ ID NO:X or the
complementary
strand thereto; the polypeptide encoded by the nucleotide sequence as defined
in columns 8
and 9 of Table 2; and/or the polypeptide sequence of SEQ ID NO:Y.
[793] Also preferred is an isolated polypeptide comprising an amino acid
sequence at
least 95% identical to a sequence of at least about 30 contiguous amino acids
in the amino
acid sequence of a polypeptide encoded by the cDNA contained in Clone ID NO:Z.
[794] Also preferred is an isolated polypeptide comprising an amino acid
sequence at
least 95% identical to a sequence of at least about 100 contiguous amino acids
in the amino
acid sequence of a polypeptide encoded by cDNA contained in Clone ID NO:Z.
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CA 02395178 2002-06-19
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[795] Also preferred is an isolated polypeptide comprising an amino acid
sequence at
least 95% identical to the amino acid sequence of a polypeptide encoded by the
cDNA
contained in Clone ID NO:Z.
[796] Further preferred is an isolated antibody which binds specifically to a
polypeptide
comprising an amino acid sequence that is at least 90% identical to a sequence
of at least 10
contiguous amino acids in a sequence selected from the group consisting of: a
polypeptide
sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the
complementary
strand thereto; the polypeptide encoded by the nucleotide sequence as defined
in columns 8
and 9 of Table 2; and a polypeptide encoded by the cDNA contained in Clone ID
NO:Z.
[797] Further preferred is a method for detecting in a biological sample a
polypeptide
comprising an amino acid sequence which is at least 90% identical to a
sequence of at least
contiguous amino acids in a sequence selected from the group consisting of: a
polypeptide
sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the
complementary
strand thereto; the polypeptide encoded by the nucleotide sequence as defined
in columns 8
and 9 of Table 2; and a polypeptide encoded by the cDNA contained in Clone ID
NO:Z;
which method comprises a step of comparing an amino acid sequence of at least
one
polypeptide molecule in said sample with a sequence selected from said group
and
determining whether the sequence of said polypeptide molecule in said sample
is at least 90%
identical to said sequence of at least 10 contiguous amino acids.
[798] Also preferred is the above method wherein said step of comparing an
amino acid
sequence of at least one polypeptide molecule in said sample with a sequence
selected from
said group comprises determining the extent of specific binding of
polypeptides in said
sample to an antibody which binds specifically to a polypeptide comprising an
amino acid
sequence that is at. least 90% identical to a sequence of at least 10
contiguous amino acids in
a sequence selected from the group consisting of: a polypeptide sequence of
SEQ ID NO:Y; a
polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the
polypeptide
encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2;
and a
polypeptide encoded by the cDNA contained in Clone ID NO:Z.
[799] Also preferred is the above method wherein said step of comparing
sequences is
performed by comparing the amino acid sequence determined from a polypeptide
molecule in
said sample with said sequence selected from said group.
[800] Also preferred is a method for identifying the species, tissue or cell
type of a
biological sample which method comprises a step of detecting polypeptide
molecules in said
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