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CA 02395811 2002-06-21
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Nucleic Acids, Proteins, and Antibodies
[1] This application refers to a "Sequence Listing" that is provided only on
electronic
media in computer readable form pursuant to Administrative Instructions
Section 801(a)(i).
The Sequence Listing forms a part of this description pursuant to Rule 5.2 and
Administrative Instructions Sections 801 to 806, and is hereby incorporated in
its entirety.
[2] The Sequence Listing is provided as an electronic file
(PTZ11PCT_seqList.txt,
17,511 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
methods useful for diagnosing, treating, preventing and/or prognosing
disorders related to
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these novel polypeptides. The invention further relates to screening methods
for identifying
agonists and antagonists of polynucleotides and polypeptides of the invention.
The present
invention further relates to methods and/or compositions for inhibiting or
enhancing the
production and function of the polypeptides of the present invention.
Background of the Invention
[4] Innate immunity, or natural resistance to infection, arises through a
combination
of anatomical barriers (e.g., skin and mucous membranes), and cellular and
humoral
immune effectors. As opposed to acquired immure responses, which are antigen-
specific
and develop over the course of days to weeks, innate immune mechanisms are,non-
specific
with respect to antigens and are usually the first line of defense against
infection. The major
components of the innate immune system have traditionally been thought to
comprise a
number of immune cell types, including neutrophils, eosinophils, basophils,
macrophages,
natural killer cells and mast cells, as well as serum proteins such as those
involved in the
complement cascade.
[5] More recently, a class of peptides with broad and potent antimicrobial
activity,
known as defensins, have been proposed as key components of the innate immune
response
(see, e.g., Hancock and Diamond, Trends in Microbiology 8(9): 402-410,
(2000)). These
proteins; also known as human neutrophil peptides, are generally small
cationic peptides of
about 30 amino acids, with a conserved cysteine motif. Defensiris have been
found to be
expressed in cytoplasmic granules of phagocytic cells (such as eosinophils,
macrophages,
and in particular, neutrophils), epithelial and mucosal tissues (e.g.,
bronchial, pulmonary,
gastrointestinal, gingival, and reproductive epithelia); and keratinocytes.
[6] A growing body of experimental and clinical evidence supports the role of
defensiris in immune system function. Among the most compelling observations
are that
plasma levels of defensins are elevated in patients with infections, such as
bacterial
pneumonia, meningitis, and septicemia; and patients with abnormally low
defensin levels
are more susceptible to infection (Hancock and Diamond, Trends in Microbiology
8(9):
402-410, (2000)). Furthermore, overexpressian of defensins in cell culture
confers
resistance to bacterial and viral infections, while defensin-deficient mice
are highly prone to
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bacterial infection (see Gropp et al., Hurn. Gene Ther. 10:957-964 (1999);
Wilson et al.,
Science 286:113-117 (1999)).
(7] It is thought that these cytotoxic proteins contribute to innate immunity
primarily
by disrupting the cytoplasmic membrane of microorganisms (or protein envelope
of some
viruses), possibly by forming transmembrane channels (Lehrer, et al., J. Clin.
Invest.
84:553-561 (1989); Kagan, et al, P~oc. Natl. Acad. Sci. (USA) 87: 210-214
(1990)).
However, several recent findings suggest that the role of defensins in immune
responses
may be more. complex. For example, there is evidence that defensins may exert
chemotactic effects over immune cells, possibly by interacting with chemokine
receptors
(Yang et al., Science 286:525-528 (1999)). Furthermore, defensins have also
been found to-
induce histamine release from mast cells, influence complement activation,
enhance serum
IgG antibody responses, and enhance T cell proliferation and function
(reviewed in Halder
et al., Blood 95(9),: 2890-2896 (2000)).
[8J , The discovery of new human defensin-Iike polynucleotides, the
polypeptides
encoded by them, and antibodies that immunospecifically bind these
polypeptides, satisfies
a need in the art by providing new compositions which are useful in the
diagnosis,
treatment, prevention and/or prognosis of disorders and/or conditions,
including, but not
limited to, infectious diseases (e.g., bacterial --including antibiotic-
resistant bacterial
strains, fungal, parasitic, and viral infections); immunodeficiencies (e.g.,
specific granule
deficiency, neonatal innate immune deficiency, and AIDS); and inflammatory
disorders ,
(e.g., gastrointestinal inflammation and endocarditis).
Summary of the Invention
[9] ~ 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
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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
[10] Table 1A summarizes some of the polynucleotides encompassed by the
invention
(including cDNA clones related to the sequences (Clone ID NO:Z), contig
sequences
(contig identifier (Contig ID:) and contig nucleotide sequence identifier (SEQ
ID NO:X))
and further summarizes certain characteristics of these polynucleotides and
the polypeptides'
encoded thereby. The first column provides the gene number in the application
for each
clone identifier. The second column provides a unique clone identifier, "Clone
ID NO:Z",
for a cDNA clone related to each contig sequence disclosed in Table 1A. The
third column
provides a unique contig identifier, "Contig ID:" for each of the contig
sequences disclosed
in Table 1A. The fourth column provides the sequence identifier, "SEQ ID
NO:X", for
each of the contig sequences disclosed in Table 1A. The fifth column, "ORF
(From-To)",
provides the location (i.e., nucleotide position numbers) within the
polynucleotide
sequence of SEQ ID NO:X that delineate the preferred open reading frame (ORF)
that
encodes the amino acid sequence shown in the sequence listing and referenced
in Table 1A
as SEQ ID NO:Y (column 6). Column 7 lists residues comprising predicted
epitopes
contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID
NO:Y).
Identification of potential 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
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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
s3P .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 ignal, emanating from each gene target, was measured
using a
Phosphorimager. Gene expression was reported as Phosphor Stimulating
Luminescence
(PSL) which reflects the level of phosphor signal generated from the probe
hybridized to
each of the gene targets represented on the array. A local background signal
subtraction was
performed before the total signal generated from each array was used to
normalize gene
expression between the different hybridizations. The value presented after
"[array code]:"
represents the mean of the duplicate values, following background subtraction
and probe
normalization. One of skill in the art could routinely use this information to
identify normal
and/or diseased tissues) which show a predominant expression pattern of the
corresponding
polynucleotide of the invention or to identify polynucleotides which show
predominant
and/or specific tissue and/or cell expression. Column 9 provides the
chromosomal location
of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was
determined
by finding exact matches to EST and cDNA sequences contained in the NCBI
(National
Center fox Biotechnology Information) UniGene database. Given a presumptive
chromosomal location, disease locus association was determined by comparison
with the
Morbid Map, derived from Online Mendelian Inheritance in Man (Online Mendelian
Inheritance in Man, OMIMTM. McKusick-Nathans Institute for Genetic Medicine,
Johns
CA 02395811 2002-06-21
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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.
[1l] 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,
r
"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).
[12] 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.
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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 iri 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.
[13] Table 3 provides polynucleotide sequences that may be disclaimed
according to
certain embodiments of the invention. The first column provides a unique clone
identifier,
"Clone ID", for a cDNA clone related to contig sequences disclosed in Table
1A. The
second column provides the sequence identifier, "SEQ ID NO:X", for contig
sequences
disclosed in Table 1A. The third column provides the unique contig identifier,
"Contig
ID:", for contigs disclosed in Table 1A. 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 colunrin provides a unique integer 'b' where 'b' is any integer
between 15 and the
final nucleotide of SEQ ID NO:X, where both a and b correspond to the
positions of
nucleotide residues shown in SEQ ID NO:X, and where b is greater.than or equal
to a + 14.
For each of the polynucleotides shown as SEQ ID NO:X, the uniquely defined
integers can
be substituted into the general~formula of a-b, and used to describe
polynucleotides which
may be preferably excluded from the invention..In certain embodiments,
preferably
excluded from the' invention are at least one, two, three, four, five, ten, or
more of the
polynucleotide sequence(s).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).
[14] Table 4 provides a key to the tissue/cell source identifier code
disclosed in Table
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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.
[15] Table 5 provides a key to the OMIM reference identification numbers
disclosed
in Table 1A, column 10. OMIM reference identification numbers (Column 1) were
derived
from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man,
OMIM.
McKusick-Nathans Institute for Genetic Medicine, Johns. Hopkins University
(Baltimore,
MD) and National Center for Biotechnology Inforrt~ation, 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. '
[16j Table 6 summarizes ATCC Deposits, Deposit dates, and ATCC designation
numbers of deposits made with the ATCC in connection with the present
application.
[17] Table 7 shows the cDNA libraries sequenced, and ATCC designation numbers
and vector information relating to these cDNA libraries.
[18] ~ Table 8 provides a physical characterization of clones encompassed by
the
invention. The first column provides the unique clone identifier, "Clone ID
NO:Z", for
certain cDNA clones of the invention, as described in Table 1A. The second
column
provides the size of the cDNA insert contained in the corresponding cDNA
clone.
Definitions
[19] The following definitions are provided to facilitate understanding of
certain terms
used throughout this specification.
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[20] 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, genoniic 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.
[21] As used herein, a "polynucleotide" refers to a molecule having a wucleic
acid
sequence encoding SEQ ID NO:Y or a fragment or variant thereof; a nucleic acid
sequence
contained in SEQ ID NO:X (as described in column 3 of Table 1A) or the
complement
thereof; a cDNA sequence contained in Clone ID NO:Z (as described in column 2
of Table
1A and contained within a library deposited with the ATCC); a nucleotide
sequence
encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as
defined in
column 6 of Table 1B or a fragment or variant thereof; or a nucleotide coding
sequence in
SEQ ID NO:B as defined in column 6 of Table 1B or the complement thereof.. For
example, the polynucleotide can contain the nucleotide sequence of the hill
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).
[22] In the present invention, "SEQ ID NO:X" was often generated by
overlapping
sequences contained in multiple clones (contig analysis). A representative
clone containing
all or most of the sequence for SEQ ID NO:X is deposited at Human Genome
Sciences, Inc.
(HGS) in a catalogued and archived library. As shown, for example, in column 2
of Table
1A, each clone is identified by a cDNA Clone ID (identifier generally referred
to herein as '
Clone ID NO:Z). Each Clone ID is unique to an individual clone and the Clone
ID is all the
information needed to retrieve a given clone from the HGS. library.
Furthermore, certain
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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.
[23] In specific embodiments, the polyrlucleotides of the invention are at
least 15, at
. ' least 30, at least 50; at least 100, at least 125, at least 500, or at
least 1000 continuous
nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15
kb, 10 kb, 7.Skb,
kb, 2.5 kb; 2.0 kb, or 1 kb., in length. In a further embodiment,
polynucleotides of the
invention comprise ~. 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).
[24] 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
CA 02395811 2002-06-21
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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 1B or the complement thereof. "Stringent
hybridization
conditions" refers to an overnight incubation at 42 degree C in a solution
comprising 50%
formamide, Sx SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium
phosphate
(pH 7.6), Sx Denhardt's solution, 10% dextran sulfate, and 20 ~.g/ml
denatured, sheared
salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65.
degree C.
(25] Also contemplated axe nucleic acid molecules that hybridize to the
polynucleotides of the present invention at lower stringency hybridization
conditions.
Changes in the stringency of hybridization and signal detection are primarily
accomplished
through the manipulation of formamide concentration (lower percentages of
formamide
result in lowered stringency); salt conditions, ~or temperature. For example,
lower
stringency conditions include an overnight incubation at 37 degree C in a
solution
comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH2P04; 0.02M EDTA, pH 7.4),
0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA; followed by
washes
at 50 degree C with 1XSSPE, 0.1% SDS. In addition, to achieve even lower
stringency,
washes performed following stringent hybridization can be done at higher salt
concentrations (e.g. 5X SSC).
[26] 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.
[27] Of course, a polynucleotide which hybridizes only to polyA+ sequences
(such as~
any 3' . terminal polyA+ tract of a cDNA shown in the sequence listing), or
to. a
complementary stretch of T (or U) residues, would not be included in the
definition of
"polynucleotide," since such a polynucleotide would hybridize to any nucleic
acid molecule
containing a poly (A) stretch or the complement thereof (e.g., practically any
double-
stranded cDNA clone generated using oligo dT as a primer)..
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[28] 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.
[29] . The polypeptide of the present invention can be composed of amino acids
joined
to each other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres, and may
contain amino acids other than the 20 gene-encoded amino acids. The
polypeptides may be
modified by either natural processes, such as posttranslational processing, or
by chemical
modification techniques which are well known in the art. Such modifications
are well
described in basic texts and in more detailed monographs, as well as in a
voluminous
research literature. Modifications can occur anywhere in a polypeptide,
including the
peptide backbone, the amino acid side-chains and the amino or carboxyl
termini. It will be
appreciated that the same type of modification may be present in the same or
varying
degrees at several sites in a given polypeptide. Also; a given polypeptide may
contain
many types of modifications. Polypeptides may be branched, for example, as a
result of
ubiquitination, and they may be cyclic, with or without branching. Cyclic,
branched, and
branched cyclic polypeptides may result from posttranslation natural processes
or may be
made by synthetic methods. Modifications include acetylation, acylation, ADP-
ribosylation, amidation, covalent attachment of flavin, covalent attachment of
a heme
moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent
attachment of
a lipid or lipid derivative, covalent attachment of phosphotidylinositol,
cross-linking,
cyclization, disulfide bond formation, demethylation, formation of covalent
cross-links,
formation of cysteine, formation of pyroglutamate, formylation, gamma-
carboxylation,
12
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glycosylation, GPI anchor formation, hydroxylation, iodination, m~ethylation,
myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated addition of
amino acids to
proteins such as arginylation, and ubiquitination. (See, for instance,
PROTEINS -
STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H.
Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT.
MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs.
1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al.,
Ann. N.Y.
Acad. Sci. 663:48-62 (1992)).
[30] "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.
[31] "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,
antigerlicity [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.~
[32] The polypeptides of the invention can be assayed for fiirlctional
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
defensin-like
polypeptides (including fragments and variants) of the invention for activity
using assays as
described in the examples 22, 23, 32, 33, or 35 below. '
[33], "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
13.
CA 02395811 2002-06-21
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in a given activity as compared to the polypeptide of the present invention
(i.e., the
candidate polypeptide will exhibit greater activity or not more than about 25-
fold less and,
preferably, not more than about tenfold less activity, and most preferably,
not more than
about three-fold less activity relative to the polypeptide of the present
invention).
[34] 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. .
14
CA 02395811 2002-06-21
WO 01/55306 PCT/USO1/01304
w
OA
w
w
w
.
,
.
bD
~~
o
e~
'~''
Pa
U
+' N
~ ~ a1
.u
'*"
'
~ o f~
U
o d
~'
~~
w ~ ~
.G
~
~H
~
eC
w
0
w
w
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~,
~aA ~
z
0
w
Ax
ao
~z
.
~
O M
U o
U
Z x
w
~
z
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CA 02395811 2002-06-21
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[35] The first column in Table 1A provides the gene number in the application
corresponding to the clone identifier. The second column in Table 1A provides
a unique
"Clone ID NO:Z" for a cDNA clone related to each contig sequence disclosed ir~
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.
[36] ~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.
[37] 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.
[38] Column 7 in Table 1A lists residues comprising epitopes contained in the
polypeptides encoded by the preferred ORF (SEQ .ID NO:Y), as predicted using
the
algorithm of 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.
16
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It will be appreciated that depending on the analytical criteria used to
predict 'antigenic
determinants, the exact address of the determinant may vary slightly.
(39] Column 8 in Table 1A provides an expression profile and library code:
count for
each of the contig sequences (SEQ ID NO:X) disclosed in Table 1A, which can
routinely be
combined with the information provided in Table 4 and used to determine the
tissues, cells,
and/or cell 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 skillYin the art could
routinely use
this information to identify normal and/or diseased tissues) which show a
predominant
z
expression pattern of the corresponding polynucleotide of the invention or' to
identify
polynucleotides which show predominant and/or specific tissue~andlor cell
expression.
. . [40] , Column 9 in Table 1A provides a chromosomal map location for
certain
polynucleotides of the invention. Chromosomal location was determined by
fording exact
matches to EST and cDNA 'sequences contained in the NCBI (National Center for
17
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Biotechnology Information) UniGene database. Each sequence in the UniGene
database is
assigned to a "cluster"; all of the ESTs, cDNAs, and STSs in a cluster are
believed to be
derived from a single gene. Chromosomal mapping data is often available for
one or more
sequences) in a UniGene cluster; this data (if consistent) is then applied to
the cluster as a
whole. Thus, it is possible to infer the chromosomal location of a new
polynucleotide
sequence by determining its identity with a mapped UniGene cluster. .
[41] ~ 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.
[42] , Once a presumptive chromosomal location was determined for a
polynucleotide
of the invention, an associated disease locus was identified by comparison
with a database
of diseases which have been experimentally associated with genetic loci. The
database used
was the Morbid Map, derived from OMIMTM (supra). If the putative chromosomal
location
of a polynucleotide of the invention (Query sequence) was associated with a
disease in the
Morbid Map database, an OMIM reference identification number was noted in
column 10,
Table 1A, labelled "OMIM Disease References)". Table 5 is a key to the OMIM
reference
identification numbers (column 1), and provides a description of the
associated disease in
Column 2.
18
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TABLE 1B
Clone ID SEQ ID CONTIG BAC ID: SEQ ID EXON
A
NO:Z NO:X ID: N From-To
O
:B
H6EAQ38 11 1032576 AC004840 _ 1-2233
_
_
13
19
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(43] 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).
CA 02395811 2002-06-21
WO 01/55306 PCT/USO1/01304
O ~ N ~ t~
oo ~O
' ~ l~ ~ vwf'
d' N
~O CO M ~
O V1
z
~
~
-
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t
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O
n
t
V1I~NOOd'
zw
0 o ao o
a
~n N ~n v1
N d'
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y y
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wz W
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21
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WO 01/55306 PCT/USO1/01304
[44] Table 2 further characterizes certain encoded polypeptides of the
invention, by
providing the results of comparisons to protein and protein family databases.
The first
column provides a unique clone identifier, "Clone ID NO:", corresponding to a
cDNA
clone disclosed in Table 1A. The second column provides the unique contig
identifier,
"Contig ID:" which allows correlation with the information in Table 1A. The
third column
provides the sequence identifier, "SEQ ID NO:", for the contig polynucleotide
sequences.
The fourth column provides the analysis method by which the 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.
[45] 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
22
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and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode ~ the
polypeptide sequence that generates an HSP are delineated by columns 8 and 9
of Table 2.
[46] 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 bf 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 PFAlVI hit is provided in
column 6.
Column 7 provides the score returned by SIMMER 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.
[47] 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.'
[48] The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are
sufficiently accurate and otherwise suitable for a variety Qf uses well known
in the art and
described fi.~rther 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
23
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probes will also hybridize to nucleic acid molecules in biological samples,
thereby enabling
immediate applications 'in chromosome mapping, linkage analysis, tissue
identification
and/or typing, and a variety of forensic and diagnostic methods of the
invention. Similarly,
polypeptides identified from SEQ ID NO:Y may be used to generate antibodies
which bind
specifically to these polypeptides, or fragments thereof, and/or to the
polypeptides encoded
by the cDNA clones identified in, for example, Table 1A.
[49] 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).
[50] ~ Accordingly, for those applications requiring precision in the
nucleotide
sequence or the amino acid sequence, the present invention provides not only
the generated
nucleotide sequence identified as SEQ ID NO:X, and a predicted translated
amino acid
sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA
containing cDNA
Clone ID NO:Z ' (deposited with the ATCC on October 5, 2000, and receiving
ATCC
designation numbers PTA 2574 and PTA 2575; deposited with the ATCC on January
5,
2001,. and having depositor reference numbers TS-1, TS-2, AC-1, and AC-2;
and/or as set
forth, for example, in Table 1A, 6 and 7). The nucleotide sequence of each
deposited clone
can readily be determined by sequencing the deposited clone in accordance with
known
methods. Further, techniques known in the art can be used to verify the
nucleotide
sequences of SEQ ID NO:X.
[51] 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-Lengtla .Genes
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[52] 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'I.
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,
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 hen tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL),
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.
[53] Several quality-controlled kits are commercially available for purchase.
Similar
reagents and methods to those above are supplied in kit form from GibcoBRL for
both 5'
and 3' RACE for recovery of full length genes. A second kit is available from
Clontech
which is a modif cation 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
CA 02395811 2002-06-21
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the first-strand cDNA. This obviates the necessity for the dA-tailing reaction
which results
in a polyT stretch that is difficult to sequence past.
[54] An alternative to generating 5' or 3' cDNA from RNA is to use cDNA
library
double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is
synthesized with an antisense cDNA-specific primer and a_plasmid-anchored
primer. These
primers are removed and a symmetric PCR reaction is performed with a nested
cDNA-
specific antisense primer and the plasmid-anchored primer.
RNA Ligase Pf-otocol For Generating The 5' or 3' End Sequences To Obtain Full
Leugth
Genes
[55] 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 cam 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):163-164
(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
26'
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strand cDNA synthesis using a gene specific oligonucleotide. The first strand
synthesis -
reaction can then be used as a template for PCR amplification of the desired
5' 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
conErm that the 5' end sequence belongs to the relevant gene.
[56] 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 PTA 2574 and PTA 2575; deposited with the ATCC on January 5, 2001, and
receiving ATCC designation numbers TS-l, 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
lAor 2 by procedures hereinafter further described, and others apparent to
those skilled in
the art.
[57] 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.
[58] Vectors Lambda Zap (U.S. Patent Nos. 5,128,256 and 5,286,636), Uni-Zap XR
(IJ.S. Patent Nos. 5,128, 256 and 5,286,636), Zap Express (LT.S. Patent Nos.
5,128,256 and
5,286;636), pBluescript (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:
7583-7600 (1988);
Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17: 9494 (1989)) and
pBK (Alting-
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Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from
Stratagene
r
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.
[59] Vectors pSportl, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were
obtained from Life Technologies, Ine., P. O. Box 6009, Gaithersburg, MD 20897.
All Sport
vectors contain an ampicillin resistance gene and may be transformed into E.
coli strain
DHlOB, also available from Life Technologies. See, for instance, Gruber, C.
E., et al.,
Focus 1 S: 59- (1993). Vector lafmid. BA (Bento Soares, Columbia University,
New Yorlc,
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 DH10B; 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).
[60] 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.
[61] ~ ' Also provided in the present invention are allelic variants,
orthologs, and/or
species homologs. Procedures known in the art can be used to obtain full-
length genes,
allelic variants, splice variants, full-length coding portions, orthologs,
and/or species
homologs of genes corresponding to SEQ ID NO:X or the complement thereof,
polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement
thereof,
and/or the cDNA contained in Clone ID NO:Z, using information from the
sequences
disclosed herein or the clones deposited with the ATCC. For example, allelic
variants
and/or species homologs may be isolated and identified by making suitable
probes or
primers from the sequences provided herein and screening a suitable nucleic
acid source for
allelic variants and/or the desired homologue.
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[62] 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.
[63] 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.
[64] The polypeptides of the present invention are preferably provided in an
isolated
foam, 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.
[65] The present invention provides a polynucleotide comprising, or
alternatively
consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA
sequence
contained in Clone ID NO:Z. The present invention also provides a polypeptide
comprising, or alternatively, consisting of, the polypeptide sequence of SEQ
ID NO:Y, a
polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide
encoded by
the cDNA contained in Clone ID NO:Z, and/or the polypeptide sequence encoded
by a
nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1B.
Polynucleotides
encoding a polypeptide comprising, or alternatively consisting of the
polypeptide sequence
of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by
the
cDNA contained in Clone ID NO:Z, and/or a polypeptide sequence encoded by a
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B are also
encompassed by the
invention. The present invention further encompasses a polynucleotide
comprising, or
alternatively consisting of, the complement of the nucleic acid sequence of
SEQ ID NO:X,
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a nucleic acid sequence encoding a polypeptide encoded by the complement of
the nucleic
acid sequence of SEQ ID NO:~, and/or the cDNA contained in Clone ID NO:Z.
[66] Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the sequences delineated in Table 1B column 6, or any combination thereof.
Additional,
representative examples of polynucleotides of the invention comprise, or
alternatively
consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more
of the
complementary strands) of the sequences delineated in Table 1B column 6, or
any
combination thereof. In further embodiments, the above-described
polynucleotides of the
invention comprise, or alternatively consist of, sequences delineated in Table
1B, column 6,
and have a nucleic acid sequence which is different from that of the BAC
fragment having
the sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In 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.
[67] Further, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. Additional,
representative
examples of polynucleotides of the invention comprise, or alternatively
consist of, one, two,
three, four, five, six, seven, eight, nine, ten, or more of the complementary
strands) of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID
NO:Z (see Table 1B, column 1), or any combination thereof. In further
embodiments, the
above-described. polynucleotides of the invention comprise, or alternatively
consist of,
CA 02395811 2002-06-21
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sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID
NO:Z (see Table 1B, column 1) and have a nucleic acid sequence which is
different from
that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see
Table 1B,
column 5). In additional embodiments, the above-described polynucleotides of
the
invention comprise, or alternatively consist of, sequences delineated in
column 6 of Table
1B which correspond to the same Clone ID NO:Z (see Table 1B, column 1) and
have a
nucleic acid sequence which is different from that published for the BAC clone
identified as
BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated
in column 6 of Table 1B which correspond to the same Clone ID NO:Z (see Table
1B,
column 1) and have a nucleic acid sequence which is different from that
contained in the
BAC.clone identified as BAC ID NO:A (see Table 1B, column 4). Polypeptides
encoded
by these polynucleotides, other polynucleot'ides 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.
[68] Further, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
contig
sequence identifer SEQ ID NO:X (see Table 1B, column 2), or any combination
thereof.
Additional, representative examples of polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
complementary strands) of the, sequences delineated in column 6 of Table 1B
which
correspond to the same contig sequence identifer SEQ ID NO:X (see Table 1B,
column 2),
or any combination thereof. In further embodiments, the above-described
polynucleotides
of the invention comprise, or alternatively consist of, sequences delineated
in column 6 of
Table 1B which correspond to the same contig sequence identifer SEQ ID NO:X
(see Table
1B, column 2) and have a nucleic acid sequence which is different from that of
the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column
5). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist of, sequences delineated in column 6 of Table 1B which
correspond to
the same contig sequence identifer SEQ ID NO:X (see Table 1B, column 2) and
have a
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nucleic acid sequence which is different from that published for the BAC clone
identified as
BAC ID NO:A (see Table 1B, column 4). In additional embodiments, the above-
described
polynucleotides of the invention comprise, or alternatively consist of,
sequences delineated
in column 6 of Table 1B which correspond to the same contig sequence identifer
SEQ ID
NO:X (see Table 1B, column 2) and have a nucleic acid sequence which is
different from
that contained in the BAC clone identified as BAC ID NO:A (See Table 1B,
column 4).
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention. Additionally, fragments and variants of the above-described
polynucleotides and
polypeptides are also encompassed by the invention.
[69J Moreover, representative examples of polynucleotides of the invention
comprise,
or alternatively consist of, one, two, three,, four, five, six, seven, eight,
nine, ten, or more of
the sequences delineated in the same row of Table 1B column 6, or any
combination
thereof. Additional, representative examples of polynucleotides of the
invention comprise,
or alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of
the complementary strands) of the sequences delineated in the same row of
Table 1B
column 6, or any combination thereof. In preferred embodiments, the
polynucleotides of
the invention comprise, or alternatively consist of, one, two, three, four,
five, six, seven,
eight, nine, ten, or more of the complementary strands) of the sequences
delineated in the
same row of Table 1B column 6, wherein sequentially delineated sequences in
the table (i.e.
corresponding to those exons located closest to each other) are directly
contiguous in a 5' to
3' orientation. In further embodiments, above-described polynucleotides of the
invention
comprise, or alternatively consist of, sequences delineated in the same row of
Table 1B,
column 6, 'and have a nucleic acid sequence which is different from that of
the BAC
fragment having the sequence disclosed in SEQ ID NO:B (see Table 1B, column
5). In
additional embodiments, the above-described polynucleotides of the invention
comprise, or
alternatively consist ot~, sequences delineated in the same row of Table 1B,
column 6, and
have a nucleic acid sequence which is different'from that.published for the
BAC clone
identified as BAC ID NO:A (see Table 1B, column 4). In additional embodiments,
the
above-described polynucleotides of the invention comprise, or alternatively
consist of,
sequences delineated in the same row of Table 1B, column 6, and have a nucleic
acid
sequence which is different from that contained in the BAC clone identified as
BAC ID
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NO:A (see Table 1B, column 4). Polypeptides encoded by these polynucleotides,
other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
are also encompassed by the invention.
[70] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, foux, 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.
[71] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, one, two, three, four, five, six, seven, eight,
nine, ten, or more of the
sequences delineated in column 6 of Table 1B which correspond to the same
Clone ID
NO:Z (see Table 1B, column 1), and the polynucleotide sequence of SEQ ID NO:X
(e.g., as
defined in Table 1A or 1B) or fragments or variants thereof. In preferred
embodiments, the
delineated sequences) and polynucleotide sequence of SEQ ID NO:X correspond to
the
same Clone ID NO:Z. Polypeptides encoded by these polynucleotides, other
polynucleotides that encode these polypeptides, and antibodies that bind these
polypeptides
are also encompassed by the invention.
[72] 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.
[73] In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of the
sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize
to the
complement of these 20 contiguous polynucleotides . under stringent
hybridization
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conditions or alternatively, under lower stringency conditions, are also
encompassed by the
invention. Polypeptides encoded by these polynucleotides and/or nucleic
acids,'other
polynucleotides and/or nucleic acids that encode these polypeptides, and
antibodies that
bind these polypeptides are also encompassed by the invention. Additionally,
fragments
and variants of the above-described polynucleotides, nucleic acids, and
polypeptides are
also encompassed by the invention.
[74] 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 lov~rer 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 specific embodiments, polynucleotides o.f 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 pblynucleotides
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.
[76] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X and the 5' 10
polynucleotides of the
sequence of one of the sequences delineated in column 6 of Table 1B are
directly
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contiguous. Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by
these polynucleotides and/or nucleic acids, other polynucleotides and/or
nucleic acids
encoding these polypeptides, and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-described
polynucleotides, nucleic acids, and polypeptides, are also encompassed by the
invention.
[77] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of
one of the sequences delineated in column 6 of Table IB and the 5' 10
polynucleotides of
another sequence in column 6 are directly contiguous. Nucleic acids which
hybridize to the
complement of these 20 contiguous polynucleotides under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed by the
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids,
other
polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies that bind
these polypeptides are also encompassed by the invention. Additionally,
fragments and
variants of the above-described polynucleotides, nucleic. acids, and
polypeptides are also
encompassed by the invention.
[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
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.
[79]~ In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of, a polynucleotide sequence in which the 3' 10
polynucleotides of
' one sequence in column 6 corresponding to the same contig sequence identifer
SEQ ID
NO:X (see Table 1B, column 2) are directly. contiguous. Nucleic acids which
hybridize to
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the complement of these 20 contiguous polynucleotides under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed by the
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids,
other
polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies that bind
these polypeptides are also encompassed by the invention. Additionally,
fragments and
variants of the above-described polynucleotides, nucleic acids, and
polypeptides are also
encompassed by the invention.
[80] In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of
another sequence in column 6 corresponding to the same row are directly
contiguous. In
preferred embodiments, the 3' 10 polynucleotides of one of the sequences
delineated~in
column 6 of Table 1B is directly contiguous with the 5' 10 polynucleotides of
the next
sequential exon delineated in Table 1B, column 6. Nucleic acids which
hybridize to the
complement of these 20 contiguous polynucleotides under stringent
hybridization
conditions or alternatively, under lower stringency conditions, are also
encompassed by the
invention. Polypeptides encoded by these polynucleotides and/or nucleic acids,
~ other
polynucleotides and/or nucleic acids encoding these polypeptides, and
antibodies that bind
these polypeptides are also encompassed by the invention. Additionally,
fragments and
variants of the above-described polynucleotides, nucleic acids, and
polypeptides are also
encompassed by the invention.
[81] Many polynucleotide sequences, such as EST sequences, are publicly
available
and accessible through sequence databases and may have been publicly available
prior to
conception of the present invention. Preferably, such related polynucleotides
are
specifically excluded from the scope of the present invention. Accordingly,
for each contig
sequence (SEQ ID NO:X) listed in the fourth column of Table 1A, preferably
excluded are
one or more polynucleotides comprising a nucleotide sequence described by the
general
formula of a-b, where a is any integer between 1 and the final nucleotide
minus' 15 of SEQ
ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where
both a and b
correspond to the positions of nucleotide residues shown in SEQ ID NO:x, and
where b is
greater than or equal to a + 14. More specifically, preferably excluded are
one or more
polynucleotides comprising a nucleotide sequence described by the general
formula of a-b,
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where a and b are integers as defined in columns 4 and 5, respectively, of
Table 3. In
specific embodiments, the polynucleotides of the invention do not consist of
at least one,
two, three, four, five, ten, or more of the specific polynucleotide sequences
referenced by
the Genbank Accession No. as disclosed in column 6 of Table 3 (including for
example,
published sequence in connection with a particular BAC clone). In further
embodiments,
preferably excluded from the invention are the specific polynucleotide
sequences)
contained in the clones corresponding to at least one, two, three, four, five,
ten, or more of
the available material having the accession numbers identified in the sixth
column of this
Table (including for example, the actual sequence contained in an identified
BAC clone).
In no way is this listing meant to encompass all of the sequences which may be
excluded by
the general formula, it is just a representative example. All references
available through
these accessions are hereby incorporated by reference in their entirety.
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TABLE 3
SEQ
ID
lone NO: ontig ST Disclaimer
ID X ID: Range ccession #'s
NO: Z of a
Range
of b
H6EAQ38 11 10325761 - 153815 - 1552AI961321, AI001128, AI343334,
AW135558, AA777237, AA478021,
AI623324, AI825954, AA232184,
AA478177, AA182540, AI085995,
AI864561, AA308520, AI872887,
H44644,
H43602, AA182585, AA852700,
AA243086, AA233362, 836915,
AI687192, AA852699, AI648561,
AA776508, and AC004840.
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TABLE 4
Code Description Tissue Organ Cell DiseaseVector
Line
AR022a_Heart a_Heart
AR023a_Liver a_Liver
AR024a_mammary glanda_mammar
gland
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 activated
AR030Blood B cells Blood B cells
resting resting
AR031Blood T cells Blood T cells
activated activated
AR032Blood T cells Blood T cells
resting resting
AR033brain brain
AR034breast breast
AR035breast cancer breast cancer
AR036Cell Line CAOV3Cell Line
CAOV3
AR037cell line PA-1 cell line
PA-1
AR038celllinetransformedcell line
transformed
AR039colon colon
AR040colon(9808co65R)colon (9$08co65R)
AR041colon (9809co15)colon (9809co15)
AR042colon cancer colon cancer'
AR043colon cancer colon cancer
(9808co64R) (9808co64R)
AR044colon cancer colon cancer
9809co14 9809co14
AR045com clone 5 corn clone
- 5
AR046com clone 6 corn clone
6
AR047com clone2 cornclone2
AR048com clone3 corn 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
AR055Heart Heart
AR056Human Lung (clonetech)Human Lung
(clonetech)
AR057Human Mammary Human Mammary
(clontech) (clontech)
AR058Human Thymus Human Thymus
, (clonetech)
(clonetech)
AR059Jurkat (unstimulated)Jurkat
(unstimulated)
AR060Kidne Kidne
AR061Liver Liver
AR062Liver (Clontech)Liver (Clontech)
AR063L m hoc tes Lym hoc tes
chronic
39
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lymphocytic chronic lymphocytic
leukaemia ,
leukaemia
AR064Lymphocytes Lymphocytes
diffuse large
B cell lymphomadiffuse large
B cell
1 m homa
AR06SLymphocytes Lymphocytes
follicular
lym homa follicular
lymphoma
AR066normal breast normal breast
AR067Normal Ovarian Normal Ovarian
(4004901) (4004901)
AR068Normal Ovary Normal Ovary
95086045
95086045
AR069Normal Ovary Normal Ovary
97016208
97016208
AR070Normal Ovary Normal Ovary
98066005
98066005
AR071Ovarian Cancer Ovarian Cancer
AR072Ovarian Cancer Ovarian Cancer
(97026001) (97026001)
AR073Ovarian Cancer Ovarian Cancer
(97076029) (97076029)
AR074Ovarian Cancer Ovarian Cancer
(98046011) (98046011)
AR07SOvarian Cancer Ovarian Cancer -
(9806G019) (98066019)
AR076Ovarian Cancer Ovarian Cancer
(98076017) (98076017)
AR077Ovarian Cancer Ovarian Cancer
(98096001) (98096001)
AR078ovarian cancer ovarian cancer
15799
15799
AR079Ovarian Cancer Ovarian Cancer
,
17717AID 17717AID
AR080Ovarian Cancer Ovarian Cancer
400466481 400466481
AR081Ovarian Cancer Ovarian Cancer
4005315A1 4005315A1
AR082ovarian cancer ovarian cancer
94127303
94127303
AR083Ovarian Cancer Ovarian Cancer
96069304
96069304
AR084Ovarian Cancer Ovarian Cancer
97076029
97076029 .
ARO8SOvarian Cancer Ovarian Cancer
98076045
98076045
AR086ovarian cancer ovarian cancer
98096001
98096001
AR087Ovarian Cancer Ovarian Cancer
990SC032RC 990SC032RC
AR088Ovarian cancer Ovarian cancer
9907 C00 9907
3rd C00 3rd
AR089Prostate Prostate
AR090Prostate (clonetech)Prostate
(clonetech)
AR091rostate cancer rostate cancer
AR092prostate cancerprostate
#15176 cancer
#15176
AR093prostate cancerprostate
#15509 cancer
#15509
AR094prostate cancerprostate
#15673 cancer
#15673
AR09SSmall IntestineSmall Intestine
(Clontech)
(Clontech)
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AR096S teen S leen
AR097Thymus T cells Thymus T
activated cells
activated
AR098Thymus T cells Thymus T
resting cells
restin
AR099Tonsil Tonsil
AR100Tonsil geminal Tonsil geminal
center center centroblast
centroblast
AR101Tonsil germinalTonsil germinal
center B center B
cell cell
AR102Tonsil 1 m h Tonsil lym
node h node
AR103Tonsil memory Tonsil memory
B cell B
cell
AR104Whole Brain Whole Brain
AR105Xeno~raft ES-2 Xeno~raft
ES-2
AR106Xenograft SW626Xenograft
SW626
41
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TABLE 5
OMIM ~ Description
Reference
No entry.
Polynucleotide and Polypeptide Variants
[82] 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
polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9
of Table 2,
the nucleotide sequence as defined in column 6 of Table 1B, nucleotide
sequences encoding
the polypeptide encoded by the nucleotide sequence as defined in column 6 of
Table 1B, the
cDNA sequence contained in Clone ID NO:Z, and/or nucleotide sequences encoding
the
polypeptide encoded by the cDNA sequence contained in Clone ID NO:Z.
[83] The present invention also encompasses variants of the polypeptide
sequence
- disclosed in SEQ ID NO:Y, the polypeptide sequence as defined in column 7 of
Table 1A, a
polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a
polypeptide sequence encoded by the nucleotide sequence as defined in columns
8 and 9 of
Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined
in column 6
of Table 1B, a polypeptide sequence encoded by the complement of the
polynucleotide
sequence in SEQ ID NO:X, and/or a polypeptide sequence encoded by the cDNA
sequence
contained in Clone ID NO:Z.
[84] "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. ~'
' . 42
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[85] Thus, one aspect of the invention provides an isolated nucleic acid
molecule
comprising, ox alternatively consisting of, a polynucleotide having a
nucleotide sequence
selected from the group consisting of: (a) a nucleotide sequence described in
SEQ ID NO:X
or contained in the cDNA sequence of Clone ID NO:Z; (b) a nucleotide sequence
in SEQ
ID NO:X or the cDNA in Clone ID NO:Z which encodes the complete amino acid
sequence
of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in
Clone ID
NO:Z;, (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in Clone ID NO:Z
which
encodes a mature polypeptide; (d) a nucleotide sequence in SEQ ID NO:X or the
cDNA
sequence of Clone ID NO:Z, which encodes a biologically active fragment of a
polypeptide;
(e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of Clone ID
NO:Z,
which encodes an antigenic fragment of a polypeptide; (f) a nucleotide
sequence encoding
a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or
the
complete amino acid sequence encoded by the cDNA in Clone ID NO:Z; (g) a
nucleotide
sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID
NO:Y or
the amino acid sequence encoded by. the cDNA in Clone ID NO:Z; (h) a
nucleotide
sequence encoding a biologically active fragment of a polypeptide having the
complete
amino acid sequence of SEQ ID NO:Y or the complete' amino acid sequence
encoded by the
cDNA in Clone ID NO:Z; (i) a nucleotide sequence encoding an antigenic
fragment of a
polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the
complete
amino acid sequence encoded by .the cDNA in Clone ID NO:Z; and (j) a
nucleotide
sequence complementary to any of the nucleotide sequences in (a), (b), (c),
(d), (e), (f), (g),
(h), or (i) above.
[86] 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
43
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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.
[87] In a preferred embodiment, the invention encompasses nucleic acid
molecules
which comprise, or alternatively, consist of a polynucleotide which hybridizes
under
stringent hybridization conditions, or alternatively, under lower stringency
conditions, to a
polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as
are polypeptides encoded
by these polynucleotides. In another preferred embodiment, polynucleotides
which
hybridize to the complement of these nucleic acid molecules under stringent
hybridization
conditions, or alternatively, under lower stringency conditions, are also
encompassed by the
invention, as are polypeptides encoded by these polynucleotides.
[88] ~ 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
4~1.
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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.
[89] The present invention is also directed to proteins which comprise, or
alternatively
consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%,
97%, 98%,
99% or 100%, identical to, for example, any of the amino acid sequences in
(a), (b), (c), or
(d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid
sequence
encoded by the cDNA contained in Clone ID NO:Z, the amino acid sequence of the
polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in
columns 8
and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the
nucleotide
sequence in SEQ ID NO:B as defined in column 6 of Table 1B, the amino acid
sequence as
defined in column 7 of Table 1A, an amino acid sequence encoded by the
nucleotide
sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement
of the
polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are
also
provided (e.g., those fragments described herein). Further proteins encoded by
polynucleotides which hybridize to the complement of the nucleic acid
molecules encoding
these amino acid sequences under stringent hybridization conditions or
alternatively, under
lower stringency conditions, are also encompassed by the invention, as are the
polynucleotides encoding these proteins.
[90] By a nucleic acid having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence of the present invention, it is
intended that the
nucleotide sequence of the nucleic acid is identical to the reference sequence
except that the
nucleotide sequence may include up to five point mutations per each 100
nucleotides of the
reference nucleotide sequence encoding the polypeptide. In other words, to
obtain a nucleic
acid having a nucleotide sequence at least 95% identical to a reference
nucleotide sequence,
up to 5% of the nucleotides in the reference sequence mayv 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.
[91] 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
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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.
[92] 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 S' and 3' of the subject
sequence, which
are not matched/aligned, as a percent of the total bases of the query
sequence. Whethex a
nucleotide is matchedlaligned is determined by results of the FASTDB sequence
alignment.
This percentage is then subtracted from the percent identity, calculated by
the above
FASTDB program using the specified parameters, to arrive at a final percent
identity score.
This corrected score is what is used for the purposes of the present
invention. Only bases
outside the 5' and 3' bases of the subject sequence, as displayed by the
FASTDB alignment,
which are not matched/aligned with the query sequence, are calculated for the
purposes of
manually adjusting the percent identity score.
[93] 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,
46
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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.
[94] 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 amine 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.
[95] As a practical matter, whether any particular polypeptide is at least
80%, 85%,
90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid
sequence of a
polypeptide referred to in Table 1A (e.g., the amino acid sequence identified
in column 6)
or Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the
polynucleotide
sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the
amino acid
sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID
NO:B~ as
defined in column 6 of Table 1B or a fragment thereof, the amino acid sequence
of the
polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment
thereof, or
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
47
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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.
[96] If the subject sequence is shorter than the query sequence due to N- or C-
terminal
deletions, not because of internal deletions, a manual correction must be made
to the results.
This is because the FASTDB program does not account for N- and C-terminal
truncations
of the subject sequence when calculating global percent identity. For subject
sequences
truncated at. the N- and C-termini, relative to the query sequence, the
percent identity is
corrected by calculating the number of residues of the query sequence that are
N- and C-
terminal of the subject sequence, which are not matched/aligned with a
corresponding
subject residue, as a percent of the total bases of the query sequence.
Whether a residue is
matched/aligned is determined by results of the FASTDB sequence alignment.
This
percentage is then subtracted from the percent identity, calculated by the
above FASTDB
program using the specified parameters, to arnve 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.
[97] For example, a 90 amino acid residue subject sequence is aligned with a
100
residue query sequence to determine percent identity. The deletion occurs at
the N-
terminus of the subject sequence and therefore, the FASTDB alignment does not
show a
matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired
residues
represent 10% of the sequence (number of residues at the N- and C- termini not
matched/total number of residues in the query sequence) so 10% is
subtracted'from the
percent identity score calculated by the FASTDB~ program. If the remaining 90
residues
were perfectly matched the final percent identity would be 90%. In another
example, a 90
residue subject sequence is compared with a 100 residue query sequence. This
time the
48
CA 02395811 2002-06-21
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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.
[98] The polynucleotide variants of the invention may contain alterations in
the
coding regions, non-coding regions, or both. Especially preferred are
polynucleotide
variants containing alterations which produce silent substitutions, additions,
or deletions,
but do not alter the properties or activities of the encoded polypeptide.
Nucleotide variants
produced by silent substitutions due to the degeneracy of the genetic code are
preferred.
Moreover, polypeptide variants in which less than 50; less than 40, less than
30, less than
20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are
substituted, deleted, or
added in any combination are also preferred. Polynucleotide variants can be
produced for a
variety of reasons, e.g., to optimize codon expression for a particular host
(change codons in
the human mRNA to those preferred by a bacterial host such as E. coli).
[99] Naturally occurring variants are called "allelic variants," and refer to
one of
several alternate forms of a gene occupying a given locus on a chromosome of
an organism.
(Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic
variants
can vary at either the polynucleotide and/or polypeptide level and are
included in the
present invention. Alternatively, non-naturally occurring variants may be
produced by
mutagenesis techniques or by direct synthesis.
[100] Using known methods of protein engineering and recombinant DNA
technology,
variants may be generated to improve or alter the characteristics of the
polypeptides of the
present invention. For instance, one or more amino acids can be deleted from
the N-
terminus or C-terminus of the polypeptide of the present invention without
substantial loss
of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-
2988 (1993))
reported variant KGF proteins having heparin binding activity even after
deleting 3, 8, or 27
amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up
to ten times
higher activity after deleting 8-10 amino acid residues from the carboxy
terminus of this
protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)
49
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[101] Moreover, ample evidence demonstrates that variants often retain a
biological
activity similar to that of the naturally occurring protein. For example,
Gayle and
coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive
mutational
analysis of human cytokine IL-1 a. They used random mutagenesis to generate
over 3,500
individual IL-1 a mutants that averaged 2.5 amino acid changes per variant
over the entire
length of the molecule. Multiple mutations were examined at every possible
amino acid
position. The investigators found that "[m]ost of the molecule could be
altered with little
effect on either [binding or biological activityj." 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.
(102] 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 and7or 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.
[103] 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.
[104] 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 nucleic acid molecule, for instance, as a hybridization probe or a
polymerase chain
reaction (PCR) primer. Uses of the nucleic acid molecules of the present
invention that do
not encode a polypeptide having functional activity include, inter alia, (1)
isolating a gene
or allelic or splice variants thereof in a cDNA library; (2) in situ
hybridization (e.g.,
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"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).
[105] 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. _
[106] The functional activity of the polypeptides, and fragments, variants and
derivatives of the invention, can be assayed by various methods. .
[107] 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 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
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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.
[108] 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.
[109] In addition,_ assays described herein (see Examples) and otherwise known
in the
art may routinely be applied to measure the ability of polypeptides of the
present invention
and fragments, variants and derivatives thereof to elicit polypeptide related
biological
activity (either i~z vitro or in vivo). Other methods will be known to the
skilled artisan and
are within the scope of the invention.
[110] 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. '
[111] For example, guidance concerning how to make phenotypically silent amino
acid
substitutions is provided in Bowie et al., "Deciphering the Message in Protein
Sequences:
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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.
[112) 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.
[113] 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:I08I-1085 (1989). The resulting mutant molecules can then
be tested
for biological activity.
[114] 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 compound, such as a compound to increase the stability and/or
solubility of
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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.
[115] For example, polypeptide variants containing amino acid substitutions of
charged
amino acids with other charged or neutral amino acids may produce proteins
with improved
characteristics, such as less aggregation. Aggregation of pharmaceutical
formulations both
reduces activity and increases clearance due to the aggregate's immunogenic
activity. See
Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al.,
Diabetes 36: 838-845
(1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377
(1993).
[116] 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.
[117] 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
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SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the
complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence
encoded
by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or
fragments
thereof; and (e) the amino acid sequence encoded by cDNA contained in Clone ID
NO:Z or
fragments thereof; wherein the fragments or variants have 1-5, 5-10, 5-25, S-
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
[118] 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 TD NO:Z or the complementary strand
thereto; is
a portion of the polynucleotide sequence encoding the polypeptide encoded by
the cDNA
contained in Clone ID NO:Z or the complementary strand thereto; . is a portion
of a
polynucleotide sequence encoding the amino acid sequence encoded by the region
of SEQ
ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand
thereto;, is
a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns
8 and 9 of
Table 2 or the complementary strand thereto; is a portion of the
polynucleotide sequence in
SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence
encoding
a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence
encoding a
portion of a polypeptide encoded by SEQ ID NO:X; is. a polynucleotide sequence
encoding
a portion of a polypeptide encoded by the complement of the polynucleotide
sequence in
SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid
sequence
encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1B or the
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.
[119] 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
CA 02395811 2002-06-21
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about 150 nt in length. A fragment "at least 20 nt in length," fox example, is
intended to
include 20 or more contiguous bases from the cDNA sequence contained in Clone
ID
NO:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary
stand
thereto. In this context "about f 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.
[120) 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, 3141-3150, 3151-3200, 3241-
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, 595'1-6000, 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)
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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.
[121] 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-
1?50,
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, 30$1-3100, 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, 4'701-
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, 6101-6.150, 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, 5851-6900, 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
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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.
[122] Moreover, representative examples of polynucleotide fragments of the
invention
comprise, or alternatively consist of, a nucleic acid sequence comprising one,
two, three,
four, five, six, seven, eight, nine, ten, or more of the above described
polynucleotide
fragments of the invention in combination with a polynucleotide sequence
delineated in
Table 1B column 6. Additional, representative examples of polynucleotide
fragments of the
invention comprise, or alternatively consist of, a nucleic acid sequence
comprising one,
two, three, four, five, six, seven, eight, nine, ten, or more of the above
described
polynucleotide fragments of the invention in combination with a polynucleotide
sequence
that is the complementary strand of a sequence delineated in column 6 of Table
1B. In
further embodiments,. the above-described polynucleotide fragments of the
invention
comprise, or alternatively consist of, sequences delineated in Table 1B,
column 6, and have
a nucleic acid sequence which is different from that of the BAC fragment
having the
sequence disclosed in SEQ ID NO:B (see Table 1B, column 5). In additional
embodiments,
the above-described polynucleotide fragments of the invention comprise, or
alternatively
consist of, sequences delineated in Table 1B, column 6, and have a nucleic
acid sequence
which is different from that published for the BAC clone identified as BAC ID
NO:A (see
Table 1B, column 4). In additional embodiments, the above-described
polynucleotides of
the invention comprise, or alternatively consist of, sequences delineated
Table 1B, column
6, arid 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.
[123] 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
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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, one, two, three, four, five, six, seven, eight,
nine, ten, or more
fragments of the sequences delineated in column 6 of Table 1B which correspond
to the
same Clone ID NO:Z (see Table 1B, column 1), and the polynucleotide sequence
of SEQ
ID NO:X (e.g., as defined in Table 1A or 1B) or fragments or variants thereof.
Polypeptides encoded by these polynucleotides, other polynucleotides that
encode these
polypeptides, and antibodies that bind these polypeptides are also encompassed
by the
invention.
[125] 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.
[126] . 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 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.
[127] ~ In additional specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein)
are directly
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contiguous Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower
stringency conditions, are also encompassed by the invention. Polypeptides
encoded by
these polynucleotides and/or nucleic acids, other polynucleotides and/or
nucleic acids
encoding these polypeptides; and antibodies that bind these polypeptides are
also
encompassed by the invention. Additionally, fragments and variants of the
above-described
polynucleotides, nucleic acids, and polypeptides are also encompassed by the
invention.
[128] In further specific embodiments, polynucleotides of the invention
comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of a
fragment or variant of the sequence of SEQ ID NO:X and the 5' 10
polynucleotides of the
sequence of one of the. sequences delineated in column 6 of Table 1B are
directly
contiguous. Nucleic acids which hybridize to the complement of these 20
contiguous
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.
[129] . In specific embodiments, polynucleotides of the invention comprise, or
alternatively consist of a polynucleotide sequence in which the 3' 10
polynucleotides of one
of the sequences delineated in column 6 of Table 1B and the 5' 10
polynucleotides of
another sequence in column 6 are directly contiguous.. In preferred
embodiments, the 3' 10
polynucleotides of one of the sequences delineated in column 6 of Table 1B is
directly
contiguous with the 5' 10 polynucleotides of the next sequential exon
delineated in Table
1B, column 6. Nucleic acids which hybridize to the complement of these 20
contiguous
polynucleotides under stringent hybridization conditions or alternatively,
under lower
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.
so
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[130] , In the present invention, a "polypeptide fragment" refers to an amino
acid
sequence which is a portion of that contained in SEQ ID NO:Y, a portion of an
amino acid
sequence encoded by the portion of SEQ ID NO:X as defined in columnns 8 and 9
of Table
2, a portion of an amino acid sequence encoded by the polynucleotide sequence
of SEQ ID
NO:X, a portion of an amino acid sequence encoded by the complement of the
polynucleotide sequence in SEQ ID NO:X, and/or a portion of an amino acid
sequence
encoded by the cDNA contained in Clone ID NO:Z. Protein (polypeptide)
fragments may
be "free-standing," or comprised within a larger polypeptide of which the
fragment forms a
part or region, most preferably as a single continuous region. Representative
examples of
polypeptide fragments of the invention, include, for example, fragments
comprising, or
alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60,
61-80, 81-
100, 101-120; 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-
260,261-280,
281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-
460, 461-
480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640,
641-660,
661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-
840, 841-
860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-
1020, 1021-
1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-
1180,
1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-Y300, 1301-1320,
1321-
1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the
end of the
coding region of cDNA and SEQ ID NO: Y. In a preferred embodiment, polypeptide
fragments of the invention include, for example, fragments comprising, or
alternatively
consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100,
101-120,
121-140, 141-160, 161-180, 181-200, 201-220., 221-240,,241-260, 261-280, 281-
300, 301-
320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480,
481-500,
501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-
680, 681-
700, 701-720,. 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860,
861-880,
881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040,
1041--
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,
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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.
[131] Even if deletion of one or more amino acids from the N-terminus of a
protein
results in modification of loss of one or more biological functions of the
protein, other
functional activities (e.g., biological activities, ability to multimerize,
ability to bind a-
ligand) may still be retained. For example, the ability of shortened muteins
to induce and/or
bind to antibodies which recognize the complete or mature forms of the
polypeptides
generally will be retained when less than the majority of the residues of the
complete or
mature polypeptide are removed from the N-terminus. Whether a particular
polypeptide
lacking N-terminal residues of a complete polypeptide retains such immunologic
-activities
can readily be determined by routine methods described herein and otherwise
known in the
art. It is not unlikely that a mutein with a large number of deleted N-
terminal amino acid
residues may retain some biological or immunogenic activities. In fact,
peptides composed
of as few as six amino acid residues may often evoke an immune response.
[132] Accordingly, polypeptide fragments include the secreted protein as well
as the
mature form. Further preferred polypeptide fragments include the secreted
protein or the
mature form having a continuous series of deleted residues from the amino or
the carboxy
terminus, or both. For example, any number of amino acids, ranging from 1-60,
can be
deleted from the amino terminus of either the secreted polypeptide or the
mature form.
Similarly, any number of amino acids, ranging from 1-30, can be deleted from
the carboxy
terminus of the secreted protein or mature form. Furthermore, any combination
of the
above amino and carboxy terminus deletions are preferred. Similarly,
polynucleotides
encoding these polypeptide fragments are also preferred.
[133] 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/or a
polypeptide encoded by the cDNA contained in Clone~ID NO:Z). In particular, N-
terminal
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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.
[134] ~ The present invention further provides polypeptides having one or more
residues
from the carboxy terminus of the amino acid sequence of a polypeptide
disclosed herein
(e.g., a polypeptide of SEQ ID NO:Y, a polypeptide encoded by the
polynucleotide
sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ
ID
NO:X as defined in columns 8 and 9 of Table 2, and/or a polypeptide encoded by
the cDNA
contained in Clone ID NO:Z). In particular, C-terminal deletions may be
described by the
general formula 1-n, where n is any whole integer ranging from 6 to q-1, and
where n
corresponds to the position of amino acid residue in a polypeptide of the
invention.
Polynucleotides encoding these polypeptides are also encompassed by the
invention.
(135] 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.
~1~i6~' Al~n a:~ mentinnPrl ahnVP PVPn 1f rlPlatlnn of nnP nr mnra amino
arirle from tha
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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.
[137] 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.
[138] Any polypeptide , sequence encoded by, for example, the polynucleotide
sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for
example, in
Tables 1A and 2), the cDNA contained in Clone ID NO:Z, or the polynucleotide
sequence
as defined in column 6 of Table 1B, may be analyzed to determine certain
preferred regions
of the polypeptide. For example, the amino acid~sequence of a polypeptide
encoded by a
polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide~of SEQ ID NO:Y
and the
polypeptide encoded by the portion of SEQ ID NO:X as defined in columnns 8 and
9 of
Table 2) or the cDNA contained in Clone ID NO:Z may be analyzed using the
default
parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Parlc
St.,
Madison, WI 53715 USA; http://www.dnastar.com/).
[139] 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
tum-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.
[140] Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic
regions, Emini
surface-forming regions, .and Jameson-Wolf regions of high antigenic index
(i.e., containing
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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)
cari 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
lileely 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.
[141] 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.
[142] Other preferred polypeptide fragments are biologically active fragments.
Biologically active fragments are those exhibiting activity similar, but not
necessarily
identical, to an activity of the polypeptide of the present invention. The
biological activity
of the fragments may include an improved desired activity, or a decreased
undesirable
activity.
[143]' 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.
[144] 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 1B or the complement thereto; the polypeptide sequence
encoded by
the cDNA contained in Clone ID NO:Z; or the polypeptide sequence encoded by a
polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement
of the
sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined
in
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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
deEned 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.
[145] The term "epitopes," as used herein, refers to portions of a polypeptide
having
antigenic or immunogenic activity in an animal, preferably a mammal, and most
preferably
in a human. In a preferred embodiment, the present invention encompasses a
polypeptide
comprising an epitope, as well as the polynucleotide encoding this
polypeptide. An
"immunogenic epitope," as used herein, is defined as a portion of a protein
that elicits an
antibody response in an animal, as determined by any method known in the art,
for
example, by the methods for generating antibodies described infra. (See, for
example,
Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 (1983)). The term
"antigenic
epitope," as used herein, is defined as a portion of a protein to which an
antibody can
immunospecifically bind its antigen as determined by any method well known in
the art,
for example, by the immunoassays described herein. Immunospecific binding
excludes
non-specific binding but does not necessarily exclude cross-;reactivity with
other antigens.
Antigenic epitopes need not necessarily be immunogenic.
[146] 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.)
[147] 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 1 l, at least 12, at least 13, at least 14, at least 1S, 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
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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)).
[148] Non-limiting examples of epitopes of polypeptides that can be used to
generate
antibodies of the invention include a polypeptide comprising, or alternatively
consisting of,
at least one, two, three, four, five, six or more of the portions) of SEQ ID
NO:Y specified
in column 7 of Table 1A. These polypeptide fragments have been determined to
bear
antigenic epitopes of the proteins of the invention by the analysis of the
Jameson-Wolf
antigenic index which is included in the DNAStar suite of computer programs.
By
"comprise" it is intended that a polypeptide contains at least one, two,
three, four, five, six .
or more of the portions) of SEQ ID NO:Y shown in column 7 of Table 1A, but'it
may
contain additional flanking residues on either the amino or carboxyl termini
of the recited
portion. Such additional flanking sequences are preferably sequences naturally
found
adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y: The
flanking
sequence may, however, be sequences from .a heterolgous polypeptide, such as
from
another protein described herein or-from a heterologous polypeptide not
described herein.
In particular embodiments, epitope portions of a polypeptide of the invention
comprise one,
two, three, or more of the portions of SEQ ID NO:Y shown in column 7 of Table
1A.
(149] 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
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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).
[150] 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 ih vivo
immunization is used, animals may be immunized with free peptide; however,
anti-peptide
antibody titer may be boosted by coupling the peptide to a maciomolecular
carrier, such as
keyhole limpet hemacyanin (I~LH) or tetanus toxoid. For instance, peptides
containing
cysteine residues may be coupled to a carrier using a linker such as
maleimidobenzoyl- N-
hydroxysuccinimide ester (MBS), while other peptides may be coupled to
carriers using a
more general linking agent such as glutaraldehyde. Animals such as rabbits,
rats and mice
are immunized with either free or carrier- coupled peptides, for instance, by
intraperitoneal
and/or intradermal injection of emulsions containing about 100 ~.g of peptide
or carrier
protein and Freund's adjuvant or any other adjuvant known for stimulating an
immune
response. Several booster injections may be needed, for instance, at intervals
of about two
weeks, to provide a useful titer of anti-peptide antibody which can be
detected, for example,
by ELISA assay using free peptide adsorbed to a solid surface. The titer of
anti-peptide
antibodies in serum from an immunized animal may be increased by selection of
anti-
peptide antibodies, for instance, by adsorption to the peptide on a solid
support and elution
of the selected antibodies according to methods well known in the art.
[151] As one of skill in the art will appreciate, and as discussed- above,,
the polypeptides
of the. present invention (e.g., those comprising an immunogenic or antigenic
epitope) can
be fused to heterologous polypeptide sequences. For example, polypeptides of
the present
invention (including fragments or variants thereof), may be fused with the
constant domain
of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHl, 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
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preferred embodiment, polypeptides and/or antibodies of the present invention
(including
fragments or variants thereof) are fused with the mature form of human serum
albumin (i.e.,
amino acids 1 - 585 of human serum albumin as shown in Figures 1 and 2 of EP
Patent 0
322 094) which is herein incorporated by reference in its entirety. In another
preferred
embodiment, polypeptides and/or antibodies of the present invention (including
fragments
or variants thereof) are fused with polypeptide fragments comprising, or
alternatively
consisting of, amino acid residues 1-z of human serum albumin, where z is an
integer from
369 to 419, as described in U.S. Patent 5,766,883 herein incorporated by
reference in its
entirety. Polypeptides and/or antibodies of the present invention (including
fragments or
variants thereof) may be fused to either the N- or C-terminal end of the
heterologous protein
(e.g., .immunoglobulin Fc polypeptide or human serum albumin polypeptide).
Polynucleotides encoding fusion proteins of the invention are also encompassed
by the
invention.
[152] 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
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 fra-me of the gene is translationally fused
to an amino-
terminal tag consisting of six histidine residues. The tag serves as a matrix
binding domain
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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
[153] 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.
[154] 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.
[155] 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.
[156] Moreover, fusion proteins may also be engineered to improve
characteristics of
the polypeptide of the present invention. For instance, a region of additional
amino acids,
particularly charged amino acids, may be added to the N-terminus of the
polypeptide to
improve stability and persistence during purification from the host cell or
subsequent
handling and storage. Also, peptide moieties may be added to the polypeptide
to facilitate
purification. Such regions may be removed prior to final preparation of the
polypeptide.
The addition of peptide moieties to facilitate handling of polypeptides are
familiar and
routine techniques in the art.
[157] 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
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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).
[158] Moreover, the polypeptides of the present invention can be fused to
marlcer
sequences, such as a polypeptide which facilitates purification of the fused
polypeptide. In
preferred embodiments, the marker amino acid sequence is a hexa-histidine
peptidc, such as
the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
CA;
91311), among others, many of which are commercially available. As described
in Gentz et
al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-
histidine provides for
' convenient purification of the fusion protein. Another peptide tag useful
for purification,
the "HA" tag, corresponds to an epitope derived from the influenza
hemagglutinin protein
(Wilson et al., Cell 37:767 (1984)).
[159] 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
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modulate the activities of polypeptides of the invention, such methods can be
used to
generate polypeptides with altered activity, as well as agonists and
antagonists of the
polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238;
5,830,721; 5,834,252;
and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997);
Harayama,
Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-
76 (1999);
and Lorenzo and Blasco, Biotechniques 24(2):308- 13 (1998) (each of these
patents and
publications are hereby incorporated by reference in its entirety). In one
embodiment,
alteration of polynucleotides corresponding to SEQ ID NO:X and the
polypeptides encoded
by these polynucleotides may be achieved by DNA shuffling. DNA shuffling
involves the
assembly of two or more DNA segments by homologous or site-specific
recombination to
generate variation in the polynucleotide sequence. In another embodiment,
polynucleotides
of the invention, or the encoded poIypeptides, 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.
(160] 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
[161] 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.
[162] 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 vinzs, it may be packaged in vitro using an appropriate
packaging cell line
and then transduced into host cells. -
. . 72
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[163] 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 contaim sites for transcription initiation,
termination, and, in the
transcribed region, a ribosome binding site for translation. The coding
portion of the
transcripts expressed by the constructs will preferably include a translation
initiating codon
at the beginning and a termination codon (UAA, UGA or UAG) appropriately
positioned at
the end of the polypeptide to be translated.
[164] 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. 20I 178));
insect cells
such as Drosophila S2 and Spodoptera Sf~ cells; animal cells such as CHO, COS,
293, and
Bowes melanoma cells; and plant cells. Appropriate culture mediums and
conditions for
the above-described host cells are known in the art.
[165] Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-
9,
available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNHBA,
pNHl6a,
pNHl8A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a,
pKK223-3, pKK233-3, pDR540, pRITS available from Pharmacia Biotech, Inc. Among
preferred eukaryotic~vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG
available
from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
Preferred
expression vectors for use in yeast systems include, but are not limited to
pYES2, pYD 1,
pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZaIph, pPIC9, pPIC3.5, pHIL-D2, pHIL-
S1, pPIC3.5K, pPIC9K, and PA08f5 (all available from Invitrogen, Carlbad, CA).
Other
suitable vectors will be readily apparent to the skilled artisan.
[166] 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 synthasebased vectors are the
availabilty of cell
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lines (e.g., the marine 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 iri
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 marine 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.
[167] 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.
[168]~ 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 the polypeptides of the present invention may in fact be expressed by a
host cell lacking
a recombinant vector.
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[169] 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 94112650; Roller et al., Proc. Nat!. Acad.
Sri. 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).
[170] Polypeptides of the invention cari be recovered and purified from
recombinant
cell cultures by well-known methods including ammonium sulfate or ethanol
precipitation,
acid extraction, anion or ration 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.
[171] ' 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-terminal
methionine on
most proteins also is efficiently removed in most prokaryotes, for some
proteins, this
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prokaryotic removal process is inefficient, depending on the nature of the
amino acid to
which the N-terminal methionine is covalently linked.
[172] In one embodiment, the yeast Piehia pastor-is 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 pasto~is must generate high levels. of alcohol oxidase due, in part, to
the relatively
low affinity of alcohol oxidase for 02. Consequently, in a growth medium
depending on
methanol as a main carbon source, the promoter region of one of the two
alcohol oxidase
genes (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 Piehia
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.
[173] 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 AOXl
promoter linked to the Pichia pasto~is alkaline phosphatase (PHO) secretory
signal peptide
(i.e., leader) located upstream of a multiple cloning site.
[174] Many other yeast vectors could be used in place of pPIC9K, such as,
pYES2,
pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, PHIL-D2,
pHIL-S1, pPIC3.5K, and PA0815, as one skilled in the art would readily
appreciate, as
long as the proposed expression construct provides appropriately located
signals for
transcription, translation,. secretion (if desired), and the like, including
an in-frame AUG as
required.
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[175] In another embodiment, high-level expression of a heterologous coding
sequence,
such as, for example, a polynucleotide of the present invention, may be
achieved by cloning
the heterologous polynucleotide of the invention into an expression vector
such as, for
example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of
methanol.
[176] ~ 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 o'r
replace endogenous genetic material (e.g., coding sequence), and/or to include
genetic
material (e.g., heterologous polynucleotide sequences) that is operably
associated with
polynucleotides of the invention, and which activates, alters, and/or
amplifies endogenous
polynucleotides. For example, techniques known in the art may be used to
operably
associate heterologous control regions (e.g., promoter and/or enhancer) and
endogenous
polynucleotide sequences via homologous recombination (see, e.g., U.S. Patent
No.
5,641,670, issued June 24, 1997; International Publication No. WO 96/2941 l,
published
September 26, 1996; International Publication No. WO 94/12650, published
August 4,,
1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and
Zijlstra et al.,
Nature 342:435-438 (1989), the disclosures of each of which are incorporated
by reference
in their entireties).
[177] 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, sarcosine; citrulline, homocitrulline, cysteic acid, t-
butylglycine, t-
butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids,
designer
amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl
amino acids,
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and amino acid analogs in general. Furthermore, the amino acid can be D
(dextrorotary) or
L (levorotary).
(178] The invention encompasses polypeptides of the present invention which
are
differentially modified during or after 'translation, e.g., by glycosylation,
acetylation,
phosphorylation, amidation, derivatization by known protecting/blocking
groups,
proteolytic cleavage, linkage to an antibody molecule or other cellular
ligand, etc. Any of
numerous chemical modifications may be carried out by known techniques,
including but
not limited, to specific chemical cleavage by cyanogen bromide, trypsin,
chymotrypsin,
papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction;
metabolic
synthesis in the presence of tunicamycin; etc.
[179] 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.
[180] 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, dichlorotriazinylamirie 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 (lzll, 1231, lasl, lsll), carbon (14C), sulfur (3sS), tritium
(3H), indium (111In,
llaln, 113mIn, llsmln), technetium (g9Tc,99mTc), thallium (2olTi), gallium
(68Ga, 67Ga),
palladium (lo3Pd), molybdenum (99Mo), xenon (lssxe), fluorine (18F), 153sm~
177Lu, ls9Gd,
149Pm, 140La, 175, 166H~, 90Y, 475, 186Re, 188Re, 142Pr, 105, and 97Ru.
[181]~ In specific embodiments, a polypeptide of the present invention or
fragment or
variant thereof is attached to macrocyclic chelators that associate with
radiometal ions,
includin but not limited to 177Lu Soy ls6Ho and ls3sm to ' of a tides. Iri a
referred
g > > > > > p Yp p p
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embodiment, the radiometal ion associated with the macrocyclic chelators is 1
j lIn. In
another preferred embodiment, the radiometal ion associated with the
macrocyclic chelator
is 9°Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-
tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA). In other specific
embodiments, DOTA is attached to an antibody of the invention or fragment
thereof via a
linker molecule. Examples of linker molecules useful for conjugating DOTA to a
polypeptide are commonly known in the art - see, for example, DeNardo et al.,
Clin Cancer
Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7
(1999); and
Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby
incorporated by
reference in their entirety.
[182] As mentioned, the proteins of the invention may be modified by either
natural
processes, such as posttranslational processing, or by chemical modification
techniques
which are well known in the art. It will be appreciated that the same type of
modification
may be present in the same or varying degrees at several sites in a given
polypeptide:
Polypeptides of the invention may be branched, for example, as a result of
ubiquitination,
and they may be cyclic, with or without branching. Cyclic, branched, and
branched cyclic
polypeptides may result from posttranslation natural processes-or may be made
by synthetic
methods. Modifications include acetylation, acylation, ADP-ribosylation,
amidation,
covalent attachment of flavin, covalent attachment of a heme moiety, covalent
attachment
of a nucleotide or nucleotide derivative, covalent attachment of a lipid or
lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond
formation, demethylation, formation of covalent cross-links, formation of
cysteine,
formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation,
GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation, oxidation,
pegylation,
proteolytic processing, phosphorylation, prenylation, racemization,
selenoylation, sulfation,
transfer-RNA mediated addition of amino acids to proteins such as
arginylation, and
ubiquitination. (See, for instance, PROTEINS -, STRUCTURE AND MOLECULAR
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)).
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[183] 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.
(184] 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.
[185] 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.,Biotech~col. 56:59-72 (1996); Vorobjev et al.,
Nucleosides
Nucleotides 18:2745-2750 (1999); and Caliceti et al., Biocohjug. Chem. 10:638-
646 (1999),
the disclosures of each of which are incorporated herein by reference
[186] 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),
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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. Sulthydryl groups may also be used as a
reactive group for
attaching the polyethylene glycol molecules. Preferred for therapeutic
purposes is
attachment at an amino group, such as attachment at the N-terminus or lysine
group.
[187] 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 maybe 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.
[188] One may specifically desire proteins chemically modified at the N-
terminus.
Using polyethylene glycol as an illustration of the present composition, one
may select from
a variety of polyethylene glycol molecules (by molecular weight, branching,
etc.), the
proportion of polyethylene glycol molecules to protein (polypeptide) molecules
in the
reaction mix, the type of pegylation reaction to be performed, and the method
of obtaining
the selected N-terminally pegylated protein. The method of obtaining the N-
terminally
pegylated preparation (i.e., separating this moiety from other monopegylated
moieties if
necessary) may be by purification of the N-terminally pegylated material from
a population
of pegylated protein molecules. Selective proteins chemically modified at the
N-terminus
modification may be accomplished by reductive alkylation which exploits
differential
reactivity of different types of primary amino groups (lysine versus the N-
terminal)
available for derivatization in a particular protein. Under the appropriate
reaction
conditions, substantially selective derivatization of the protein at the N-
terminus with a
carbonyl group containing polymer is achieved. .
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[189J 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.
[190] 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
(C1SO2CHZCF3). Upon reaction of protein with tresylated MPEG, polyethylene
glycol is
directly attached to amine groups of the protein. Thus, the invention includes
protein-
polyethylene glycol conjugates produced by reacting proteins of the invention
with a
polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
[191] 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.
[192] The number of polyethylene glycol moieties attached to each protein of
the
invention (i.e., the degree of substitution) may also vary. For example, the
pegylated
proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 12, 15, 17,
20, or more polyethylene glycol molecules. Similarly, the average degree of
substitution
within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-
13, 12-14, 13-
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15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per
protein molecule.
Methods for determining the degree of substitution are discussed, for example,
in Delgado
et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
[193] The polypeptides of the invention can be recovered and purified from
chemical
synthesis and recombinant cell cultures by standard methods which include, but
are not
limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion
or cation
exchange chromatography, phosphocellulose chromatography, hydrophobic
interaction
chromatography, affinity chromatography, hydroxylapatite chromatography and
lectin
chromatography. Most preferably, high performance~liquid chromatography
("HPLC") is
employed for purification. Well known techniques for refolding protein may be
employed
to regenerate active conformation when the polypeptide is denatured during
isolation and/or
purification.
[194] The polypeptides of the invention may be in monomers or multimers (i.e.,
dimers, trimers, tetramers and higher multimers). Accordingly, the present
invention relates
to monomers and multimers of the polypeptides of the invention, their
preparation, and
compositions (preferably, Therapeutics) containing them. In specific
embodiments, the
polypeptides of ,the invention are monomers, dimers, trimers or tetramers. In
additional
embodiments, the multimers of the invention are at least dimers, at least
trimers, or at least
tetramers.
[195] ~ Multimers encompassed by the invention may be homorriers 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
arid 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
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amino acid sequences) or a homotrimer (e.g., containing three polypeptides
having identical
andlor different amino acid sequences). In.additional embodiments, the
homomeric
multimer of the invention. is at least a homodimer, at least a homotrimer, or
at least a.
homotetramer.
(196J A,s 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 multirner of the invention is at least a heterodimer, at least a
heterotrimer, or at
least a heterotetramer.
[197] Multimers of the invention may be the result of hydrophobic,
hydrophilic, ionic
and/or covalent associations and/or may be indirectly linked by, for example,
liposome
formation. Thus, in one embodiment, multimers of the invention, such as, for
example,
homodimers or homotrimers, are formed when polypeptides of the invention
contact one
another in solution. In another embodiment, heteromultimers of the invention,
such as, for
example, heterotrimers or heterotetramers, are formed when polypeptides of the
invention .
contact antibodies to the polypeptides of the invention (including antibodies
to the
heterologous polypeptide sequence in a fusion protein of the invention) in
solution. In other
embodiments, multimers of the invention are formed by covalent associations
with andlor
between the polypeptides of the invention. Such covalent associations may
involve one or
more amino acid residues contained in the polypeptide sequence (e.g., that
recited in SEQ
ID NO:Y, encoded' by the portion of SEQ ID NO:X as defined in columns 8 and 9
of Table
2, and/or encoded by the cDNA contained in Clone ID NO:Z). In one instance,
the covalent
associations are cross-linking between cysteine residues located within the
polypeptide
sequences which interact in the native (i.e., naturally occurring)
polypeptide. In another
instance, the covalent associations are the consequence of chemical or
z'ecombinant
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
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associations of fusion .proteins of the invention are between heterologous
polypeptide
sequence from another protein that is capable of forming covalently associated
multiiners,
such as for example, osteoprotegerin (see, e.g., International- Publication
NO: WO
98/49305, the contents of which are herein incozporated 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.
(198] 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,
(198$)), and have since been found in a variety of different proteins.' Among
the known
leucine zippers are naturally occurring peptides and derivatives thereof that
dimerize or
trimerize. Examples of leucine zipper domains suitable for producing soluble
multimeric~
proteins of the invention are those described in PCT application WO 94/10308,
hereby
incorporated by reference. Recombinant fusion proteins comprising a
polypeptide of the
invention fused to a polypeptide sequence that dimerizes or trimerizes in
solution are
expressed in suitable host cells, and the resulting soluble multimeric fusion
protein is
recovered from the culture supernatant using techniques known in the art.
(199] 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 I3oppe 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
palypeptides of the invention.
[200] 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
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are associated by interactions between heterologous polypeptide sequence
contained in
Flag~ fusion proteins of the invention and anti-Flag~ antibody.
[201] 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-limes 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).
[202] 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
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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
[203] 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 fox assaying specific antibody-antigen binding. Antibodies,
of the
invention include, but are not limited to, polyclonal, monoclonal,
multispecific, human,
humanized or chimeric antibodies, single chain antibodies, Fab fragments,
F(ab')
fragments, fragments produced by a Fab expression library, anti-idiotypic
(anti-Id)
antibodies (including, e.g., anti-Id antibodies to antibodies of the
invention), intracellularly-
made antibodies (i.e., intrabodies), and epitope-binding fragments of any of
the above. The
term "antibody," as used herein, refers to immunoglobulin molecules and
immunologically
active portions of immunoglobulin molecules, i.e., molecules that contain an
antigen
binding site that immunospecifically bi-nds an antigen. The immunoglobulin
molecules of
the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),
class (e.g., IgGl,
IgG2, IgG3,,IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. In
preferred
embodiments, the iminunoglobulin molecules of the invention are IgGl . In
other preferred
embodiments, the immunoglobulin molecules of the invention are IgG4.
[204] 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
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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.
[205]' The antibodies of the present invention may be monospecific,
bispecific,
trispecific or of greater multispecificity. Multispecific antibodies may be
specific for
different epitopes of a polypeptide of the present invention or may be
specific for both a
polypeptide of the present invention as well as for a heterologous epitope,
such as a
heterologous polypeptide or solid support material. See, e.g., PCT
publications WO
93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Irnmunol.
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).
[206] Antibodies of the present invention may be described or specified in
terms of the
epitope(s) or portions) of a polypeptide of the present invention which they
recognize or
specifically bind. The epitope(s) or polypeptide portions) may be specified as
described
herein, e.g., by N-terminal and C-terminal positions; or by size in contiguous
amino acid
residues, or listed in the Tables and Figures. Preferred epitopes of the
invention include the
predicted epitopes shown in column 7 of Table 1A, as well as polynucleotides
that encode
these epitopes. Antibodies which specifically bind any epitope or polypeptide
of the present
invention may also be excluded. Therefore, the present invention includes
antibodies that
specifically bind polypeptides of the present invention, and allows for the
exclusion of the
same.
[207] 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. Im specific embodiments, antibodies of the
present
invention cross-react with murine, rat and/or rabbit homologs .of human
proteins and,the
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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-Z M, 5 X 10-3 M,
10-3 M, 5 X 10'
4 M, 10'~ M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M, 10-6M, 5 X 10-' M, 10' M, 5 X 10-
8 M, 10-8 M,
X 10-9 M, 10-9 M, 5 X 10-1° M, 10-'° M, 5 X 10-11 M, 10-'1 M, 5
X 10-12 M, 10-12 M, 5 X
10-13 M, 10-is M, 5 X 10-14 M, 10-14 M, 5 X 10-15 M, or 10-15 M.
[208] 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%.
[209] 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) may be determined by
techniques described
herein or otherwise known in the art. For example, receptor activation can be
determined
by detecting the phosphorylation (e.g., tyrosine or serine/threonirle) of the
receptor or its
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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.
[210] 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.
1'60(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998);
Pitard et al., J.
Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241
(1997);
Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al.,
Neuron
14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek
et al.,
Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in
their
entireties).
(211] Antibodies of the present invention maybe 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 present invention in biological samples. See, e.g., Harlow et al.,
Antibodies: A
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Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);
incorporated by
reference herein in its entirety.
[212] 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.
[213] 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.
[214] The antibodies of the present invention may be generated by any suitable
method
known in the art. Polyclonal antibodies to an antigen-of interest can be
produced by
various procedures well known in the art. For example, a polypeptide of the
invention can
be administered to various host animals including, but not limited to,
rabbits, mice, rats, etc.
to induce the production of sera containing polyclonal antibodies specific for
the antigen.
Various adjuvants may be used to increase the immunological response,
depending~on the
host species, and include but are not limited to, Freund's (complete and
incomplete),
mineral gels such as aluminum hydroxide, surface active substances such as
lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG (bacille
Calrnette-
Guerin) and corynebacterium parvum. Such adjuvants are also well known in the
art.
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[215] 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.
[216] 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.
[217] Accordingly, the present invention provides methods of generating
monoclonal
antibodies as well as antibodies produced by the method comprising culturing a
hybridoma
cell secreting an antibody of the invention wherein, preferably, the hybridoma
is generated
by fusing splenocytes isolated from a mouse immunized with an antigen of the
invention
with myeloma cells and then screening the hybridomas resulting from the fusion
for
hybridoma clones that secrete an antibody_ able to bind a polypeptide of the
invention.
[218] Another well known method for producing both polyclonal and monoclonal
human B cell lines is transformation using Epstein Burr Virus (EBV). Protocols
for.
generating EBV=transformed B cell lines are commonly known in the art, such
as, for
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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.
[219] In general,'the sample containing human B cells is~ iniloci~lated with
EBV, and
cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of
the B95-8 cell
line (ATCC #VR-1492). Physical signs of EBV transformation can generally be
seen
towards the end of the 3-4 week culture period. By phase-contrast microscopy,
transformed
cells may appear large, clear, hairy and tend to aggregate in tight clusters
of cells. Initially,
EBV lines are generally polyclonal. However, over prolonged periods of cell
cultures, EBV
lines may become monoclonal or polyclonal as a result of the selective
outgrowth of
particular B cell clones. Alternatively, polyclonal EBV transformed lines may
be subcloned~
(e.g., by limiting dilution culture) or fused with a suitable fusion partner
and plated at
limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners
for EBV
transformed cell lines include mouse myeloma cell lines (e.g., SP210, 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.
[220] Antibody fragments which recognize specific epitopes may be generated by
known techniques.. For example, Fab and F(ab')2 fragments of the invention may
be
produced by proteolytic cleavage of immunoglobulin molecules, using enzymes
such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2
fragments contain the variable region, the light chain constant region and the
CHl domain
of the heavy chain.
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[221] 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 M13 binding domains
expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused
to either the phage gene III or gene VIII protein. Examples of phage display
methods that
can be used to make the antibodies of the present invention include those
disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.
Immunol. Methods
184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958
(1994);~Persic et
al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280
(1994);
PCT application No. ~PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737;
WO 92/01047; WO 92/18619; WO 93111236; WO 95/15982; WO 95/20401; and U.S.
Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and
5,969,108; each of
which is incorporated herein by reference in its entirety.
[222] 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
(I992); 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).
[223] Examples of techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Patents 4,946,778 and 5,258,498;
Huston et al.,
Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and
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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. 5,585089; 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 (LJ.S.
Patent No. 5,565,332).
[224] 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 iminunoglobulin '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
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96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein
by
reference in its entirety.
[225] 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 imrnunoglobulin
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.
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[226] - Completely human antibodies which recognize a selected epitope can be
generated using a technique referred.to as "guided selection." In this
approach a selected
non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the
selection of a
completely human antibody recognizing the same epitope. (Jespers et al.,
Biotechnology
12:899-903 (1988)).
(227] 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 multimeri~ation
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.
[228] 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.
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-,
Pol~nucleotides Encoding Antibodies
[229] The invention further provides polynucleotides comprising a nucleotide
sequence
encoding an antibody of the invention and fragments thereof. The invention
also
encompasses polynucleotides that hybridize under stringent or alternatively,
under lower
stringency hybridization conditions, e.g., as defined 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:~ 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.
[230] 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.
[231] 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.
[232] 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.,
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recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for
example, the
techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory
Manual,
2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et
al., eds.,
1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are
both
incorporated by reference herein in their entireties ), to generate antibodies
having a
different amino acid sequence, for example to create amino acid substitutions,
deletions,
and/or insertions.
[233] In a specific embodiment, the amino acid. sequence of the heavy and/or
light
chain variable domains rnay be inspected to identify the sequences of the
complementarity
determining regions (CDRs) by methods that are well know in the art, e.g., by
comparison
to known amino acid sequences of other heavy and light chain variable regions
to determine
the regions of sequence hypervariability. Using routine recombinant DNA
techniques, one
or more of the CDRs may be inserted within framework regions, e.g., into human
framework regions to humanize a non-human antibody, as described supra. The
framework
regions may be naturally occurring or consensus framework regions, and
preferably human
framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479
(1998) for a listing
of human framework regions). Preferably, the polynucleotide generated by the
combination
of the framework regions and CDRs encodes an antibody that specifically binds
a
polypeptide of the invention. Preferably, as discussed supra, one or more
amino acid
substitutions may be made within the framework regions, and, preferably, the
amino acid
substitutions improve binding of the antibody to its antigen. Additionally,
such methods
may be used to make amino acid substitutions or deletions of one or more
variable region
cysteine residues participating in an intrachain disulfide bond to generate
antibody
molecules lacking one or more intrachain disulfide, bonds. Other alterations
to the
polynucleotide are encompassed by the present invention and within the skill
of the art.
[234] 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
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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.
[235] ~ Alternatively, techniques described for the production ~of single
chain antibodies
(U.S. Patent No. 4,946,778; Bird, Science 242:423- 42 (1988); Huston et al.,
Proc. Natl.
Acad. Sci. USA 85:5879-5883 (1988); and Ward et al.; Nature 334:544-54 (1989))
can be
adapted to produce single chain antibodies. Single chain antibodies are formed
by linking
the heavy and light chain fragments of the Fv region via an amino acid bridge,
resulting in a
single chain polypeptide. Techniques for the assembly of functional Fv
fragments in E. coli
may also be used (Skerra et al., Science 242:1038- 1041 (1988)).
Methods of Producing Antibodies
[236] 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.
[237] 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
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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.
[238] . The expression vector is transferred to a host cellby conventional
techniques and
the transfected cells are then cultured by conventional techniques to produce
an antibody of
the invention. Thus, the invention includes host cells containing a
polynucleotide encoding
an antibody of~the invention, or a heavy or light chain thereof, or a single
chain antibody of
the invention, operably linked to a heterologous promoter. In preferred
embodiments for
the expression of double-chained antibodies, vectors encoding both the heavy
and light
chains may be co-expressed in the host cell for expression of the entire
irnmunoglobulin
molecule, as detailed below.
[239] A variety of host-expression vector systems may be utilized to express
the
antibody molecules of the invention. Such host-expression systems represent
vehicles by
which the coding sequences of interest may be produced and subsequently
purified, but also
represent cells which may, when transformed or transfected with the
appropriate nucleotide
coding sequences, express an antibody molecule of the invention in situ. These
include but
are not limited to'microorganisms such as bacteria (e.g., E. coli, B.
subtilis) transformed
with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors
containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia)
transformed
with recombinant yeast expression vectors containing antibody coding
sequences; insect
cell ,systems infected with recombinant virus expression vectors (e.g.,
baculovirus)
containing antibody coding sequences; plant cell systems infected with
recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing
antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK,
293, 3T3
cells) harboring recombinant expression constructs containing promoters
derived from the
genome of mammalian cells (e.g., metallothionein promoter) or from mammalian
viruses
(e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial
cells such as Escherichia coli, and more preferably, eukaryotic cells,
especially for the
expression of whole recombinant antibody molecule, are used for the expression
of a
recombinant antibody molecule. For example, mammalian cells such as Chinese
hamster
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ovary cells (CHO), in conjunction with a vector uch 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)). -
[240] In bacterial systems, a number of expression vectors may be
advantageously
selected depending upon the use intended for the .antibody molecule being
expressed. For
example, when a large quantity of such a protein is to be produced, for the
generation of
pharmaceutical compositions of an antibody molecule, vectors which direct the
expression
of high levels of fusion protein products that are readily purified may be
desirable. Such
vectors include, but are not limited, to the E. coli expression vector pUR278
(Ruther et al.,
EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated~
individually into the vector in frame with the lac Z coding region so that a
fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109
(1985); Van
Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX
vectors may
also be used to express foreign polypeptides as fusion proteins with
glutathione S-
transferase (GST). In general, such fusion proteins are soluble and can easily
be purified
from lysed cells by adsorption and binding to matrix glutathione-agarose beads
followed
by elution in the presence of free glutathione. The pGEX vectors are designed
to include
thrombin or factor Xa protease cleavage sites so that the cloned target gene
product can be
released from the GST moiety. , a
[241] In an insect system, Autographa californica nuclear polyhedrosis virus
(AcNPV)
is used as a vector to express foreign genes. The virus grows in Spodopte~a
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).
[242] 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 wivo
recombination. Insertion in
a non- essential region of the 'viral gerlome (e.g., region E1 or E3)
will.result in a
recombinant virus that is viable and capable of expressing the antibody
molecule . in
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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
f, 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)).
[243] In addition, a host cell strain may be chosen which modulates the
expression of
the inserted sequences, or modifies and processes the gene product in the
specific fashion
desired. Such modifications (e.g., glycosylation)~ and processing (e.g.,
cleavage) of protein
products may be important for the function of the protein. Different host
cells have
characteristic and specific mechanisms for the post-translational processing
and
modification of proteins and gene products. Appropriate cell lines or host
systems can be
chosen to ensure the correct modification and processing of the foreign
protein expressed.
To this end, eukaryotic host cells which possess the cellular machinery for
proper
processing of the primary transcript, glycosylation, and phosphorylation of
the gene
product may be used. Such mammalian host cells include but are not limited to
CHO,
VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and.in particular, breast cancer
cell
lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal
mammary
gland cell line such as, for example, CRL7030 and Hs578Bst.
[244] 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
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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.
[245] 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 (Szybalslca & Szybalslci, Proc. Natl. Acad.
Sci. USA
48:202 (1992)), arid adenine phosphoribosyltransferase (Lowy et al., Cell
22:817 (1980))
genes can be employed in tk-, hgprt- or aprt- cells, respectively. Also,
antimetabolite
resistance can be used as the basis of selection for the following genes:
dhfr, which confers
resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980);
O'Hare et
al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance
to
mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072
(1981)); neo,
which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-
505; Wu
and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol.
32:573-
596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,
Ann. Rev.
Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and
hygro,
which confers resistance to hygromycin. (Santerre et al., Gene 30:147 (1984)).
Methods
commonly known in the art of recombinant DNA technology may be routinely
applied to
select the desired recombinant clone, and such methods are described, for
example, in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, NY
(1993); I~riegler, Gene Transfer and Expression, A Laboratory Manual, Stockton
Press, NY
(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols
in Human
Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1
(1981), which are incorporated by reference herein in their entireties.
[246] The expression levels of an antibody molecule can be increased by vector
amplification (for a review, see~Bebbington and Hentschel, The use of vectors
based on
gene amplification for the expression of cloned genes in mammalian cells in
DNA cloning,
Vol.3. (Academic Press, New York, 1987)). When a marker in the vector system
expressing antibody is amplifiable, increase in the level of inhibitor present
in culture of
host cell will increase the number of copies of the marker gene. Since the
amplified region
is associated with the antibody gene, production of the antibody will also
increase (Grouse
et al., Mol. Cell. Biol. 3:257 (1983)). ~ '
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[247] 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., Bioltechrzoloby 10:169(1992) and in Biblia and Robinson
Biotechnol.
Prog. 11:1 (1995) which are incorporated in their entirities by reference
herein.
[248] 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.
[249] 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
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polypeptide sequences described herein or, otherwise known ~in the art, to
facilitate
purification. '
[250] 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 ifa 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;
Gillie's et al.,~
PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which
are
incorporated by reference in their entireties.
[251] 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 diners 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.
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Immunol. 1S4:SS90-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA
89:11337-
11341 (1992) (said references incorporated by reference in their entireties).
[252] 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, human
proteins, such as
hIL-S, have been fused with Fc portions for the purpose of high-throughput
screening
assays to identify antagonists of hIL-S. (See, Bennett et al., J.
Molecular.Recognition 8:52-
58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).
[253] Moreover, the antibodies or fragments thereof of the present invention
can be
fused to marker sequences, such as a peptide to facilitate purification. In
preferred
embodiments, the marker amino acid sequence is a hexa-histidine peptide, such
as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA,
91311),
among others, many of which are commercially available. As described in Gentz
et al.,
Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine
provides for
convenient purification of the fusion protein. Other peptide tags useful for
purification
include, but are not limited to, the "HA" tag, which corresponds to an epitope
derived from
the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and
the "flag" tag.
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[254] The present invention further encompasses antibodies or fragments
thereof
conjugated to a diagnostic or therapeutic agent. The antibodies can be used
diagnostically
to, for example, monitor the development or progression of a tumor as part of
a clinical
testing procedure to, e.g., determine the efficacy of a given treatment
regimen. Detection
can be facilitated by coupling the antibody to a detectable substance.
Examples of
detectable substances include various enzymes, prosthetic groups, fluorescent
materials,
luminescent materials, bioluminescent materials, radioactive materials,
positron emitting
metals _using various positron emission tomographies, and nonradioactive
paramagnetic
metal ions. The detectable substance may be coupled or conjugated either
directly to the
antibody (or fragment thereof) or indirectly, through an intermediate (such
as, for example,
a linker known in the 'art) using techniques known in the art. See, for
example, U.S. Patent
No. 4,741,900 for metal ions which can be conjugated to antibodies for use as
diagnostics
according .to the present invention. Examples of suitable enzymes include
horseradish
peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
examples of
suitable prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples
of suitable fluorescent materials include umbelliferone, fluorescein,
fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin; and
examples of
suitable radioactive material include 125I, 131I, 111In or 99Tc.
[255] 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, . rilelphalan,
carmustine
(BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, .
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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).
[256] 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 W mor
necrosis factor, a-interferon, 13-interferon, nerve growth factor, platelet
derived growth
factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-
beta, AIM I
(See, International Publication No. WO 97133899), AIM II (See, International
Publication
No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immur2ol., 6:1567-1574
(1994)),
VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or
an anti-
angiogenic agent, e.g., angiostatin .or endostatin; or, biological response
modifiers such as,
for example, lymphokines; interleukin-1 ("IL=1 "), interleukin-2 ("IL-2"),
interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF",),
granulocyte
colony stimulating factor ("G-CSF"), or other growth factors.
[257] Antibodies may also be attached to solid supports, which are
particularly useful
for immunoassays or purification of the target antigen. Such solid supports
include, but are
not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene,
polyvinyl chloride or
polypropylene'.
[258] Techniques . for conjugating such therapeutic moiety to antibodies are
well
known. See, for example, Arnon et al., "Monoclonal Antibodies For
Immunotargeting Of
Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy,
Reisfeld et al.
(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug
Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp.
623-53
(Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In
Cancer
Therapy:. A .Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications,
Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future
Prospective Of
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
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Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of
Antibody-
Toxin Conjugates", Immunol. Rev. 62:119-58~ (1982).
[259] 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.
[260] 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
therap eutic. _
Immunophenotyping
[261] 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)).
[262] 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-sel-f' 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
[263] 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"
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immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion
precipitin
reactions, immunodiffusion assays, agglutination assays, complement-fixation
assays,
immunoradiometric assays, fluorescent immunoassays, and protein A
immunoassays, to
name but a few. Such assays are routine and well known in the art (see, e.g.,
Ausubel et al,
eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New
York, which is incorporated by reference herein in its entirety). Exemplary
immunoassays
are described briefly below (but are not intended by way of limitation).
[264] ~ I 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° G, 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. l, John Wiley & Sons, Inc., New York, section 10.16.1.
[265] Western blot analysis generally comprises preparing protein samples,
electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20%
SDS-PAGE
depending on the molecular weight of the antigen), transferring the protein
sample from the
polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon,
blocking the
membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing
the
membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with
primary
antibody (the antibody of interest) diluted in blocking buffer, washing the
membrane in
washing buffer, blocking the membrane with a secondary antibody (which
recognizes .the
primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic
substrate (e.g.,
horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32P or 125I)
diluted in blocking buffer, . washing the membrane in wash buffer, and
detecting the
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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.
[266] 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.
[267] The binding affinity of an antibody to an antigen and the off rate of an
antibody-
antigen interaction can be determined by competitive binding assays. One
example of a
competitive binding assay is a radioimmunoassay comprising the incubation of
labeled
antigen (e.g., 3H or 125I) with the antibody of interest in the presence of
increasing
amounts of unlabeled antigen, and the .detection of the antibody bound to the
labeled
antigen. The affinity of the antibody of interest for a particular antigen and
the binding off
rates can be determined from the data by scatchard plot analysis. Competition
with a
second antibody can also be determined using radioimmunoassays. In this case,
the antigen
is incubated with antibody of interest conjugated to a labeled compound (e.g.,
3H or 125I)
in the presence of increasing amounts of an unlabeled second antibody.
[268] 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
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known in the art. Antibodies that bind antigen transfected cells, but not
vector-only
transfected cells, are antigen specific.
Therapeutic Uses
[269) 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.
[270] In a specific and preferred. embodiment, the present invention is
directed to
antibody-based therapies which involve administering antibodies of the
invention to an
animal, preferably a mammal, and most preferably a human, patient for treating
one or more
diseases, disorders, or conditions, including but not limited to: neural
disorders, immune
system disorders, muscular disorders, reproductive disorders, gastrointestinal
disorders,
pulmonary disorders, cardiovascular disorders, renal disorders, proliferative
disorders,
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 1A; or
a
conformational epitope, including fragments, analogs and derivatives thereof
as described
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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.
[271] 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.
[272] 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., IZJ-2, IL-3 and IL-7), for example, which serve
to increase the
number or activity of effector cells which interact with the antibodies.
[273] The .antibodies of the invention may be administered alone, or in
combination
with other types of treatments (e.g., radiation therapy, chemotherapy,
hormonal therapy,
immunotherapy and anti-tumor agents). Generally, administration of products of
a species
origin or species reactivity (in the case of antibodies) that is the same
species as that of the
patient is preferred. Thus, in a preferred ~ embodiment, human antibodies,
fragments
derivatives, analogs, or nucleic ~ acids,. are administered to a human patient
for therapy or
prophylaxis.
[274] It is preferred to use high affinity and/or potent ih vivo inhibiting
and/or
neutralizing antibodies against polypeptides or polynucleotides of the present
invention,
fragments or regions thereof, for both immunoassays directed to and therapy of
disorders
related to polynucleotides or polypeptides, including fragments thereof, of
the present
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invention. Such antibodies, fragments, or regions, will preferably have an
affinity for
polynucleotides or polypeptides of the invention, including fragments thereof.
Preferred
binding affinities include those with a dissociation constant or Kd less than
5 X 10-2 M, 10-2
M, 5 X 10-3 M, 10-3 1VI, 5 X 104 M, 10-4 M, 5 X 10-5 M, 10-5 M, 5 X 10-6 M, 10-
6 M, 5 X
10-~ M, 10-~ M, 5 X 10-8 M, 10-$ M, 5 X 10-9 M, 10-9 M, 5 X 10-1° M, 10-
1° M, 5 X 10-11 M,
10-11 M, 5 X 10-12 M, 10-12 M, 5 X 10-13 M, 10-13 M, 5 X 10-14 M, 10-14 M, 5 X
10-15 M, and
10-1s M. .
Gene Therapy
[275] 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 andlor 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.
[276) Any of the methods for gene therapy available in the art can be used
according to
the present invention. Exemplary methods are described below.
[277] For general reviews of the methods of gene therapy, .see Goldspiel et
al., Clinical
Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev,
Ann.
Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932
(1993); and '
Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH
11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al. (eds.), Current
Protocols.in
Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer
and
Expression, A Laboratory Manual, Stockton Press, NY (1990).
[278] In a preferred embodiment, the compound comprises nucleic acid sequences
encoding an antibody, said nucleic acid sequences being part of expression
vectors that
express the antibody or fragments or chimeric proteins or heavy or light
chains thereof in a
suitable host. In particular, such nucleic acid sequences have promoters
operably linked to
the antibody coding region, said promoter being inducible or constitutive,
and, optionally,
tissue-specif c. In another particular embodiment; nucleic acid molecules are
used in which
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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 86:89.32-8935 (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. .
[279] 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.
[280] In a specific embodiment, the nucleic acid sequences are directly
administered ih
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
rriicrocapsules, 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; W093/14I88, WO 93/20221). Alternatively,
the nucleic acid can be introduced intracellularly and incorporated within
host cell DNA for
expression, by homologous recombination (Koller and Smithies, Proc.. Natl.
Acad. Sci..
USA 86:8932-8935 (.1989); Zijlstra et al., Nature 342:435-438 (1989)).
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[281] In a specific embodiment, viral vectors that contains nucleic acid
sequences
encoding an antibody of the invention are used. For example, a retroviral
vector can be
used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral
vectors
contain the components necessary for the correct packaging of the viral genome
and
integration into the host cell DNA. The nucleic acid sequences encoding the
antibody to be
used in gene therapy are cloned into one or more vectors, which facilitates
delivery of the
gene into a patient. More detail about retroviral vectors can be found in
Boesen et al.,
Biotherapy 6:291-302 (1994), which describes,the use of a retroviral vector to
deliver the
mdrl gene to hematopoietic stem cells in order to make the~stem cells more
resistant to
chemotherapy. Other references illustrating the use of retroviral vectors in
gene therapy
are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood
83:1467-1473
(1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and
Grossman
and Wilson, Curr: Opin. in Genetics and Devel. 3:110-114 (1993).
[282] 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 cari 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 W094/12649and Wang, et al., Gene Therapy 2:775-783
(1995).
In a preferred embodiment, adenovirus vectors are used.
[283] Aderio-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).
[284] Another approach to gene therapy involves transferring a gene to cells
in tissue
culture by such methods as electroporation, lipofection, calcium phosphate
mediated
transfection, or viral infection. Usually, the method of transfer includes the
transfer of a
selectable marker to the cells. The cells are then placed under selection to
isolate those
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cells that have taken up and are expressing the transferred gene. Those cells
are then
delivered to a patient. .
[285] In this embodiment, the nucleic acid is introduced into a cell prior to
administration in vivo of the resulting recombinant cell. Such introduction
can be carried
out by any method known in the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or bacteriophage
vector containing
the, nucleic acid sequences, cell fusion, chromosome-mediated gene transfer,
microcell-
mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known
in the art
for the introduction of foreign genes into cells (see, e.g., Loeffler and
Behr, Meth. Enzymol.
217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline,
Pharmac.
Ther. 29:69-92m.(1985) and may be 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 hat the nucleic acid is expressible by the cell and preferably
heritable and
expressible by its cell progeny.
(286] 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.
(287] 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.
[288] In a preferred embodiment, the cell used for gene therapy is autologous
to the
patient.
[289] In an embodiment in which recombinant cells are used in gene therapy,
nucleic
acid sequences encoding an antibody are introduced into the cells such that
they are
expressible by the cells or their progeny, and the recombinant cells are then
administered in
vivo for therapeutic effect. In a specific embodiment, stem or progenitor
cells are used.
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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)).
[290] 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
[291] 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
[292] 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 not limited to animals such as cows, pigs, horses,
chickens, cats,
dogs, etc., and is preferably a mammal, and most preferably human.
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[293] Formulations and methods of administration that can be employed when the
compound comprises a nucleic acid or an immunoglobulin are described above;
additional
appropriate formulations and routes of administration can be selected from
among those
described herein below.
[294] 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 intradermah 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.
(295] 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, fox example, and not by way of limitation, local infusion
during
surgery, topical application, e.g., in conjunction with a wound dressing after
surgery, by
injection, by means of a catheter, by means of a suppository, or by means of
an implant,
said implant being of a porous, non-porous, or gelatinous material, including
membranes,
such as sialastic membranes, or fibers. Preferably, when administering a
protein, including
an antibody, of the invention, care must be taken to use materials to which
the protein does
not absorb.
[296] 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
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(eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-
327; see
generally ibid.)
[297] In yet another embodiment, the compound or composition can be delivered
in a
controlled release system. In one embodiment, a pump may be used (see Langer,
supra;
Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery
88:507 -
(1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another
embodiment,
polymeric materials can be used (see Medical Applications of Controlled
Release, Langer
and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug
Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York
(1984);
Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see
also Levy
et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989);
Howard et al.,
J.Neurosurg. 71:105 (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)).
[298] Other controlled release systems are discussed in the review by Langer
(Science
249:1527-1533 (1990)).
[299] 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 o.f 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.
[300] 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
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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
Garners can be sterile liquids, such as water and oils, including those of
petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and
the like. Water is a preferred carrier when the pharmaceutical composition is
administered
intravenously. Saline solutions and aqueous dextrose and glycerol solutions
can also be
employed as liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,.
flour, challe, 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.
[301] In, a preferred embodiment, the composition is formulated in accordance
with
routine procedufes 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
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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.
[302] The compounds of the invention can be formulated as neutral or salt
forms.
Pharmaceutically acceptable salts include those formed with anions such as,
those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those
formed with
cations such as those derived from sodium, potassium, ammonium, calcium,
ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[303] 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
~cliriical 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 o~ 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.
[304] ~ For antibodies, the dosage administered to a patient is typically 0.1
mg/kg to 100
nig/kg of the patient's body weight. Preferably, the dosage administered to a
patient is
between 0.1 mglkg and 20 mg/kg of the patient's body weight, more preferably 1
mg/kg to
mg/kg of the patient's body weight. Generally, human antibodies have a longer
half life
within the human body than antibodies from other species due to the immune
response to
the foreign polypeptides. Thus, lower dosages of human antibodies and less
frequent
administration is often possible. Further, the dosage and frequency of
administration of
antibodies of the invention may be reduced by enhancing uptake and tissue
penetration
(e.g., into the brain) of the antibodies by modifications such as, for
example, lipidation.
[305] 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. .
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Diagnosis and Imaging
[306] Labeled antibodies, and derivatives and analogs thereof, which
specifically bind
to a polypeptide of interest can be used for diagnostic purposes to detect,
diagnose, or
monitor diseases, disorders, and/or conditions associated with the aberrant
expression
and/or activity of a polypeptide of the invention. The invention provides for
the detection
of aberrant expression of a polypeptide of interest, comprising (a) assaying
the expression
of the polypeptide of interest in cells or body fluid of an individual using
one or more.
antibodies specific to the polypeptide interest and (b) comparing the level of
gene
expression with a standard gene expression level, whereby an increase or
decrease in the
assayed polypeptide gene expression level compared to the standard expression
level is
indicative of aberrant expression.
[307] 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.
[308] 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 (1.987)). 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 (lI2In), and
technetium
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(99Tc); luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and biotin.
[309] One facet of the invention is the detection and diagnosis of a disease
or disorder
associated with aberrant expression of a polypeptide of interest in an animal,
preferably a
mammal and most preferably a human. In one embodiment, diagnosis comprises: a)
administering (for example, parenterally, subcutaneously, or
intraperitoneally) to a subject '
an effective amount of a labeled molecule which specifically binds to the
polypeptide of
interest; b) waiting for a time interval following the administering for
permitting the labeled
molecule to~ preferentially concentrate at sites in the subject where the
polypeptide is
expressed (and for unbound labeled molecule to be cleared to background
level); c)
determining background level; and d) detecting the labeled molecule in the
subject, such
that detection of labeled molecule above the background level indicates that
the subject has
a particular disease or disorder associated with aberrant expression of the
polypeptide of
interest. Background level can be determined by various methods including,
comparing the
amount of labeled molecule detected to a standard value previously determined
for a
particular system.
[310] 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)).
[311] 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 they 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
to 10 days.
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[3~2] In an. embodiment, monitoring of, the disease or disorder is carried out
by
repeating the method for .diagnosing the disease or disease, for example, one
month after
initial diagnosis, six months after initial diagnosis, one year. after initial
diagnosis, etc.
[313] Presence of the labeled molecule can be detected in the patient using
methods
known in the art for i~z 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.
[314] In a specific embodiment, the molecule is labeled with a radioisotope
and is
detected in the patient using a radiation responsive surgical instrument
(Thurston et al., U.S.
Patent No. 5,441,050). In another embodiment, the molecule is labeled with a
fluorescent
' compound and is -detected in the patient using a fluorescence responsive
scanning
instrument. In another embodiment, the molecule is labeled with a positron
emitting metal
and is detected in the patent using positron emission-tomography. In yet
another
embodiment, the molecule is labeled with a paramagnetic label and is detected
in a patient
using magnetic resonance imaging (MRI).
Kits .
[315] 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).
[316] In another specific embodiment of the present ineention, the kit is a
diagnostic kit
.for use in screening serum containing antibodies specific against
proliferative and/or
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cancerous polynucleotides and polypeptides. Such a kit may include a control
antibody that
does not react with the polypeptide of interest. Such a kit may include a
substantially
isolated polypeptide antigen comprising an epitope which is specifically
immunoreactive
with at least one anti-polypeptide antigen antibody. Further, such a kit
includes means for
detecting the binding of said antibody to the antigen (e.g., the antibody may
be conjugated
to a fluorescent compound such as fluorescein or rhodamine which can be
detected by flow
cytometry). In specific embodiments, the kit may include a recombinantly
produced or
chemically synthesized polypeptide antigen. The polypeptide antigen of the kit
may also be
attached to a solid support.
[317] 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.
[318] 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.
[319] 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).
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[320] 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).
[321] 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
[322] 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.
[323] The polynucleotides of the present invention are useful for chromosome
identification. There exists an ongoing need to identify new chromosome
markers, since
few chromosome marking reagents, based on actual sequence data (repeat
polymorphisms),
are presently available. Each sequence is specifically targeted to and can
hybridize with a
particular location on an individual human chromosome, thus each
polynucleotide of the
present invention can routinely be used as a chromosome marker using
techniques known in
the art. Table 1A, column 9 provides the chromosome location of some of the
polynucleotides of the invention.
[324] 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.
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[325] Similarly, somatic hybrids provide a rapid method of PCR mapping the
polynucleotides to particular chromosomes. Three or more clones can be
assigned per day
using a single thermal cycler. Moreover, sublocalization of the
polynucleotides can be
achieved with panels of specific chromosome fragments. Other gene mapping
strategies
that can be used include in situ hybridization, prescreening with labeled flow-
sorted'
chromosomes, preselection by hybridization to construct chromosome specific-
cDNA
libraries, and computer mapping techniques (See, e.g., Shuler, Trends
Biotechnol 16:456-
459 (1998) which is hereby incorporated by reference in its entirety).
[326] Precise chromosomal location of the polynucleotides can also be achieved
using
fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread.
This
technique uses polynucleotides as short as 500 or 600 bases; however,
polynucleotides
2,000-4,000 by are preferred. Fox a review of this technique, see Verma et
al., "Human
Chromosomes: a Manual of Basic Techniques," Pergamon Press, New York (1988).
[327] ~ 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).
[328] Thus, the present invention also provides a method for chromosomal.
localization
which involves (a) preparing PCR primers from the polynucleotide sequences in
Table 1A
and/or Table 2 and SEQ ID NO:X and (b) screening somatic cell hybrids
containing
individual chromosomes.
[329] 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); lHamilton 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.
[330] 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
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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 1A,
as
determined using techniques described herein and by reference to Table 5.
Assuming 1
megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized
to a
chromosomal region associated with the disease could be one of 50-500
potential causative
genes.
[331] 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 linleage analysis.
[332] Furthermore, increased or decreased expression of the gene in affected
individuals as compared to unaffected individuals can be assessed using the
polynucleotides
of the invention. Any of,these alterations (altered expression, chromosomal
rearrangement,
or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and
prognostic
methods, kits and reagents encompassed by the present invention are briefly
described
below and more thoroughly elsewhere herein (see e.g., the sections labeled
"Antibodies",
"Diagnostic Assays", and "Methods for Detecting Diseases"). '
[333] 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).
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[334] . 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 orie
polynucleotide probe
containing a nucleotide sequence that will specifically hybridize with a
polynucleotide of
the invention and a suitable container. In a specific embodiment, the kit
includes two
polynucleotide probes defining an internal region of the polynucleotide of the
invention,
where each.probe has one strand containing a 31'mer-end internal to the
region. In a further
embodiment, the probes may be useful as primers for polymerase chain reaction
amplification.
[335] 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 standardlevel.
[336] By "measuring the expression level of polynucleotides of the invention"
is
intended qualitatively or quantitatively measuring or estimating the level of
the polypeptide
of the invention or the level of the mRNA encoding the polypeptide of the
invention in a
first biological sample either directly (e.g., by determining or estimating
absolute protein
level or mRNA level) or relatively (e.g., by comparing to the polypeptide
level or mRNA
level in a second biological sample). Preferably, the polypeptide level or
mRNA level in
the first biological sample is measured or estimated and compared to a
standard polypeptide
level or mRNA level, the standard being taken from a second biological sample
obtained
from an individual not having the related disorder or being determined by
averaging levels
from a population of individuals not having a related disorder. As will be
appreciated in the
art, once a standard polypeptide level or mRNA level is known, it can be used
repeatedly as
a standard for comparison.
[337] 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
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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 prefeiTed source.
[338] The methods) provided above may preferably be applied in a diagnostic
method
and/or kits in which polynucleotides andlor 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.
[339] The present invention encompasses polynucleotides of the present
invention that
are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or
according to
other methods known in the art. The use of PNAS would serve as the preferred
form if the
polynucleotides of the invention are incorporated onto a solid support, or
gene chip. For the
purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide
type of DNA
analog and the monomeric units for adenine, guanine, thymine and cytosine are
available
commercially (Perceptive Biosystems). Certain components of DNA, such as
phosphorus,
phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As
disclosed by
Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666
(1993), PNAs
bind specifically and tightly to complementary DNA strands and are not
degraded by
nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This
is probably
because there is no electrostatic repulsion between the two strands; and also
the polyamide
backbone is more flexible. Because of this, PNA/DNA duplexes bind under a
wider range
of stringency conditions than DNA/DNA duplexes, making it easier to perform
multiplex
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
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hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting
point
(Tm) by 8°-20° C, vs. 4°-16° C for the
DNA/DNA 15-mer duplex. Also, the absence of
charge groups in PNA means that hybridization can be done at low ionic
strengths and
reduce possible interference by salt during the analysis.
[340] 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
erythroleulcemia, acute megalearyocytic leukemia, and acute undifferentiated
leukemia, etc.;
and chronic myelogenous leukemias including chronic myelomonocytic leukemia,
chronic
granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats,
dogs, cows,
pigs, horses, rabbits and humans. Particularly preferred are humans.
[341] 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)
[342] 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
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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.
[343] 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 Olcano, 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
D.ervan~ et al., Science 251: 1360 (1991). Both methods rely on binding of the
polynucleotide to a complementary DNA or RNA. For these techniques, preferred
polynucleotides are usually oligonucleotides 20 to 40 bases in length and
complementary to'
either the region of the gene involved in transcription (triple helix - see
Lee et al., Nucl.
Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et
al.,
Science 251:1360 (1991)) or to~the mRNA itself (antisense - Okano, J.
Neurochem. 56:560
(1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC
Press,
Boca Raton, FL (1988)). Triple helix formation optimally results in a shut-off
of RNA
transcription from DNA, while antisense RNA hybridization blocks translation
of an
mRNA molecule 'into polypeptide. 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 described elsewhere
herein (see,
e.g., the section labeled "Antisense and Ribozyme (Antagonists)").
[344] 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
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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).
(345] 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.
[346] 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. ° '
[347] 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
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
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the DQa class II HLA gene. Similarly, polynucleotides of the present invention
can be used
as polymorphic markers for forensic purposes.
[348] 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 ~. similar fashion, these reagents can be used to screen tissue
cultures for
contamination. Additional non-limiting examples of such uses are further
described herein.
[349] 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, polypeptidesr 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.
[350] 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.
[351] In the very least, the polynitcleotides 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 Polxpeptides
[352] Each of the polypeptides identified herein can be used in numerous ways.
The
following description should be considered exemplary and utilizes known
techniques.
[353] 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).
[354] Antibodies can be used to assay levels of polypeptides encoded by
polynucleotides of the invention in a biological sample using classical
immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et al., ~J.
Cell. Biol. 101:976-
985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other
antibody-based
methods useful for detecting protein gene expression include immunoassays,
such as the
enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable
antibody assay labels are known in the art and include enzyme labels, such as,
glucose
oxidase; radioisotopes, such as iodine (1311, l2sh lzsh lzll)~ carbon (14C),
sulfur (35S), tritium
(3H), indium (llsmln, 113mIn, llaln, 111In), and technetium (99Tc, ~9'~Tc),
thallium (2olTi),
gallium (68Ga, 6~Ga), palladium (losPd), molybdenum (99Mo), xenon (ls3Xe),
fluorine (18F),
ls3Sm l~~Lu ls9Gd la9Pm laoLa l~s~,b 166Ho 9oY aaSc ls6Re lsaRe lazPr lost
9~Ru,
> > > > > > > > > > > > > >
luminescent labels, such as luminol; and fluorescent labels, such as
fluorescein and
rhodamine, and' biotin.
[355] 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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[356] A protein-specific antibody or antibody fragment which has been labeled
with an
appropriate detectable imaging moiety, such as a radioisotope (for example,
i3lh n2In,
99m~.~' (131I' 125I' 1231, lall), carbon (14C), sulfur (35S), tritium (3H),
indium (115mIn' 113mIn'
llzln, mIn), and technetium (99Tc, 99mTc), thallium (2oiTi), gallium (68Ga,
6~Ga), palladium
t
203Pd , m01 bdenum g9M0 xenon (133Xe) fluorine (18F 153sm l~~Lu IS~Gd 149Pm
140La
( ) y ( )> > > > > > > >
os~,b 166H~ 90Y 47s~ 186Re 138Re 142Pr 105 9~Ru) a radio o a ue substance, or
a
> > > > > > > > > - p q
material detectable by nuclear magnetic resonance, is introduced (for example,
parenterally,
subcutaneously or intraperitoneally) into the mammal to be examined for immune
system
disorder. It will be understood in the art that the size of the subject and
the imaging system
used will determine the quantity of imaging moiety needed to produce
diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity of
radioactivity
injected will normally range from about 5 to 20 millicuries of 99mTc. The
labeled antibody
or antibody fragment will then preferentially accumulate at the location of
cells which
express the polypeptide encoded by a polynucleotide of the invention. In vivo
tumor
imaging is described in S.W. $urchiel et al., "Immunopharmacokinetics of
Radiolabeled
Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical
Detection of Cance~~, .S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing
Inc.
(1982)).
[357] 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.
[358] 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.
[359] 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
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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. "Toxin" also includes a cytostatic or cytocidal agent, a
therapeutic agent or a
radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or
other radioisotopes
such aS for exam 1e lo3Pd 133xe 1311 sate S~Co 65Zn 85Sr 32P 35S 90Y 153Sm
153Gd
p > > > > > > > > > > > > >
169~,b~ slCr~ s4Mn~ ~sSe, 113Sn, 9°Yttrium, ll~Tin, 186Rhenium,
ls6Holmium, and ls$Rhenium;
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 111In: In a further specific embodiment, the invention provides a
method for
the specific destruction of cells (e.g., the destruction of tumor cells) by
administering
polypeptides of the invention or antibodies of the invention in association
with the
radioisotope 1311. '
[360] . 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). ~ ~ '
[361] 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
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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. .
[362] Moreover, polypeptides of the. present invention can be used to treat or
prevent
diseases .or conditions such as, for example, neural disorders, immune system
disorders,
muscular disorders, reproductive disorders, gastrointestinal disorders,
pulmonary disorders,
cardiovascular disorders, renal disorders, proliferative disorders, and/or
cancerous diseases
and conditions. For example, patients can be administered a polypeptide of the
present
invention in an effort to replace absent or decreased levels of the
polypeptide {e.g., insulin),
to supplement absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S for
hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of
a polypeptide
(e.g., an oncogene or tumor supressor), to activate the activity of a
polypeptide (e.g., by
binding to a receptor), to reduce the activity of a membrane bound receptor by
competing
with it for free ligand (e.g., soluble TNF receptors used in reducing
inflammation), or to
bring about a desired response (e.g., blood vessel growth inhibition,
enhancement of the
immune response to proliferative cells or tissues).
[363] 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).
[364] 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.
a
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Diagnostic Assays
[365] The compounds of the present invention are useful for diagnosis,
treatment,
prevention and/or prognosis of various disorders in mammals, preferably
humans.. Such
disorders include, but are not limited to, those described herein under the
section heading
"Biological Activities".
[366] 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 polypept'ide 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 dia-gnostic assays may be
performed in
vivo or in vitro, such as, for example, on blood samples, biopsy tissue or
autopsy tissue.
[367] 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.
[368] In certain embodiments, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
diagnose and/or prognose diseases and/or disorders associated with the
tissues) in which
the polypeptide of the invention is expressed, including one, two, three,
four, five, or more
tissues disclosed in Table 1A, column 8 (Tissue Distribution Library Code).
[369] 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 rriRNA level) or relatively (e.g., by comparing to the polypeptide
level or mRNA
level in a second biological sample). Preferably, the polypeptide expression
Ievel 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
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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.
[370] By "biological sample" is intended any biological sample obtained from
an
individual, cell line, tissue culture, or other source containing polypeptides
of the invention
(including portions thereof) or mRNA. As indicated, biological samples include
body fluids
(such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue
sources found to
express the full length or fragments thereof of a polypeptide or mRNA. Methods
for
obtaining tissue biopsies and body fluids from mammals are well known in the
art. Where
the biological sample is to include mRNA, a tissue biopsy is the preferred
source.
[371] 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, Sl 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).
[372] 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.
[373]. Assaying polypeptide levels in a biological sample can occur using
antibody-based techniques. For example, polypeptide expression in tissues can
be studied
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with classical immunohistological methods (Jalkanen et al., J. Cell. Biol.
101:976-985
(1985); Jalleanen, 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 oxidase, and radioisotopes, such as iodine (1251, lzll), carbon (14C),
sulfur (35S),
tritium (3H), iridium (lzIn), and technetium (99mTc), and fluorescent labels,
such as
fluorescein and rhodamine, and biotin. -
[374] 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., 1'988, "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.
[375] 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.
[376] 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.
[377] 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
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techniques employing a fluorescently labeled antibody coupled with light
microscopic, flow
cytometric, or fluorimetric detection. '
[378] 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
thereot~ or polypeptide is preferably applied by overlaying the labeled
antibody (or
fragment) onto a biological sample. Through the use of such a procedure, it is
possible to
determine not only the presence of the gene product, or conserved variants or
peptide
fragments, or polypeptide binding, but also its distribution in the examined
tissue. Using
the present invention, those of ordinary skill will readily perceive that any
of a wide variety
of histological methods (such as staining procedures) can be modified in order
to achieve
such in situ detection.
[379] 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.
[380] 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.
[381] 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,
14.4.
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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. '
[382] The binding activity of a given lot of antibody or antigen polypeptide
may be
determined according to well known methods. Those skilled in the art will be
able to
determine operative and optimal assay conditions for each determination by
employing
routine experimentation.
[383] 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) andlor 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.
[384] 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
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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); N'euberger et al., NatuYe
314:268
(1985).
[385] 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.
[386] A polypeptide-specific antibody or antibody fragment which has been
labeled
with an appropriate detectable imaging moiety, such as a radioisotope (for
example, 1311,
llaln~ 99m.Lc)~ a radio-opaque substance, or a material detectable by nuclear
magnetic
resonance, is introduced (for example, parenterally, subcutaneously or
intraperitoneally)
into the mammal to be examined for a disorder. It will be understood in the
art that the size
of the subject and the imaging system used will determine the quantity of
imaging moiety
needed to produce diagnostic images. In the case of a radioisotope moiety, for
a human
subject, the quantity of radioactivity injected will normally range from about
5 to 20
millicuries of 99mTC. The labeled antibody or antibody fragment will then
preferentially
accumulate at the location of cells which contain the antigenic protein. In
vivo tumor
imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of
Radiolabeled
Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The
Radiochemical
Detectiora of CanceY, S.W. Burchiel and B. A. Rhodes, eds.,.Masson Publishing
Inc.
(1982)).
[387] 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) (Volley, A., "The Enzyme
Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological
Associates Quarterly Publication, Walkersville, MD); Volley et al., J. ClirZ.
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,
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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.
(388] 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 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.
[389] 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 fluoresceirl isothiocyanate, rhodamine, phycoerythrin,
phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
(390] The antibody can also be detectably labeled using fluorescence emitting
metals
such as 152Eu, 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).
[391J The antibody also can be detectably labeled by coupling it to a
chemiluminescent
compound. The presence of the chemiluminescent-tagged antibody is then
determined by
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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.
[392] 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
lucifer'in, luciferase and aequorin.
Methods for Detecting Diseases
(393] In general, a disease may be detected in a patient based on the presence
of one or
more proteins of the invention and/or polynucleotides encoding such proteins
in a biological
sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from
the patient. In
other words, such proteins may be used as markers to indicate the presence or
absence of a
disease or disorder, including cancer and/or as described elsewhere herein. In
addition, such
proteins may. be useful for the detection of other diseases and cancers. The
binding agents
provided herein generally permit detection of the level of antigen that binds
to the agent in
the biological sample. Polynucleotide primers and probes may be used to detect
the level of
mRNA encoding polypeptides of the invention,, which is also indicative of the
presence or
absence of a disease or disorder, including cancer. In general, polypeptides
of the invention
should be present at a level that is at least three fold higher in diseased
tissue than in normal
tissue.
[394] 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.
[395] 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
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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.
[396] The solid support may be any material known to those of skill in the art
to which
polypeptides of the invention may be attached. For example, the solid support
may be a test
well in a microtiter plate or a nitrocellulose or other suitable membrane.
Alternatively, the
support may be a bead or disc, such as glass fiberglass, latex or a plastic
material such as
polystyrene or polyvinylchloride. The support may also be a magnetic particle
or a fiber
optic sensor, such as those disclosed, for example, in U.S. Patent No.
5,359,681. The
binding agent may be immobilized on the solid support using a variety of
techniques known
to those of skill in the art, which are amply described in the patent and
scientific literature.
In the context of the present invention, the term "immobilization" refers to
both
noncovalent association, such as adsorption, and covalent attachment (which
may be a
direct linkage between the agent and functional groups on the support or may
be a linkage
by way of a cross-linking agent). Immobilization by adsorption to a well in a
microtiter
plate or to a membrane is preferred. In such cases, adsorption may be achieved
by
contacting the binding agent, in a suitable buffer, with the solid support for
the suitable
amount of time. The contact time varies with temperature, but is typically
between about 1
hour and about 1 day. In general, contacting a well of plastic microtiter
plate (such as
polystyrene or polyvinylchloride) with an amount of binding agent ranging from
about 10
ng to about 10 ug, and preferably about 100 ng to about 1 ug, is sufficient to
immobilize an
adequate amount of binding agent.
[397] 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
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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
[398] 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. .
[399] Thus, for example, cells from a patient may be engineered with a
polynucleotide
(DNA or RNA) comprising a promoter operably linked to a polynucleotide of the
present
invention ex vivo, with the engineered cells then being provided to a patient
to be treated
with the polypeptide of the present invention. Such methods are well-known in
the art. For
example, see Belldegrun, A., et aL, J. Natl. Cancer Inst. 85: 207-216 (I993);
Ferrantini, M.
et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J.
Immunology 153:
4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura,
H., et al.,
Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene
Therapy 7:1-
(1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang,
J.-F. et
al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by
reference. In
one embodiment, the cells which are engineered are arterial cells. The
arterial cells may be
reintroduced into the patient through direct injection to the artery, the
tissues surrounding
the artery, or through catheter injection.
[400] As discussed in more detail below, the polynucleotide constructs can be
delivered by any method that delivers injectable materials to the cells o-f an
animal, such as,
injection into the interstitial space of tissues (heart, muscle, skin, lung,
liver, and the like).
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The polynucleotide constructs may be delivered'in a pharmaceutically
acceptable liquid or
aqueous carrier..
[401] 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.
[402] 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, pXTl and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL
available from Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from
Invitrogen. Other suitable vectors will be readily apparent to the skilled
artisan. ,
[403] Any strong promoter known to those skilled in the art can be used for
driving the
expression of the polynucleotide sequence. Suitable promoters include
adenoviral
promoters; such as the adenoviral major late promoter; or heterologous
promoters, such as
the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)
promoter;
inducible promoters, such as the MMT promoter, the metallothionein promoter;
heat shock
promoters; the albumin promoter; the ApoAI promoter; human globin promoters;
viral
thymidine kinase promoters, such as the Herpes Simplex thymidine kinase
promoter;
retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The
promoter also may be the native promoter for the polynucleotide of the present
invention.
[404] 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 calls. 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.
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[405] The polynucleotide construct can be delivered to the interstitial space
of tissues
within the an animal, including of muscle, skin, brain, lung, liver, spleen,
bone marrow,
thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder,
stomach,
intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and
connective tissue.
Interstitial space of the tissues comprises the intercellular, fluid,
mucopolysaccharide matrix
among the reticular fibers of organ tissues, elastic fibers in the walls of
vessels or chambers,
collagen fibers of fibrous tissues, or that same matrix within connective
tissue ensheathing
muscle cells or in the lacunae of bone. It is similarly the space occupied by
the plasma of the
circulation and the lymph fluid of the lymphatic channels. Delivery to the
interstitial space of
muscle tissue is preferred for the reasons discussed below. They may be
conveniently
delivered by injection into the tissues comprising these cells. They are
preferably delivered
to and expressed in persistent, non-dividing cells which are differentiated,
although delivery
and expression may be achieved in non-differentiated or less completely
differentiated cells,
such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle
cells are
particularly competent in their ability to take up and express
polynucleotides.
(406] 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 mglkg body weight to about
50 mg/leg
body weight. Preferably the dosage will be from about 0:005 mg/lcg 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. -
[407] 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.
[408] 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:
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[409] 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.
[410] 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. . .
[411] Cationic liposomes . are readily available. Fox example,
N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylamrrionium (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). .
[412] 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
Iiposomes is explained in the literature, see, e.g., P. Felgner et al., Proc.
Natl. Acad. Sci.
USA 84:7413-7417, which, is herein incorporated by reference. Similar methods
can be
used to prepare liposomes from other cationic lipid materials. ~ .
[413] 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
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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.
[414] For example, commercially dioleoylphosphatidyl choline ' (DOPC),
dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine
(DOPE)
can be used in various combinations to make conventional liposomes, with or
without the
addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared
by
drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a
sonication
vial. The sample is placed under~a vacuum pump overnight and is hydrated the
following
day with deionized water. The sample is then sonicated for 2 hours in a capped
vial, using a
Heat Systems model 350 sonicator equipped with an inverted cup (bath type)
probe at the
maximum setting while the bath is circulated at 15EC. Alternatively,
negatively charged
vesicles can be prepared without sonication to produce multilamellar vesicles
.or by
extrusion through nucleopore membranes to produce unilamellar vesicles of
discrete size.
Other methods are known and available to those of skill in the art.
[4I5] . ~ The liposomes can comprise multilamellar vesicles (MLVs), small
unilamellar
vesicles (SUVs), or large unilarnellar 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 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.
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Res. Common. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348
(1979));
detergent dialysis (Epoch, 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.
[416] 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.
[417] U.S. Patent No. 5,676,954 (which is herein incorporated by reference)
reports on
the injection of genetic material, complexed with cationic Iiposomes carriers,
into mice.
U.S. Patent Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466,
5,693,622,
5,580,859, 5,703,055, and international publication rio. 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.
[418] In certain embodiments, cells are engineered, ex vivo or ih vivo, using
a retroviral
particle containing RNA which comprises a sequence encoding a polypeptide of
the present
invention. Retroviruses.from which the fetroviral 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.
[419] The retroviral. plasmid vector is employed to transduce packaging cell
lines to
form producer cell .lines. Examples of packaging cells which may be
transfected include,
but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-
H2,
RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller,
Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in
its
entirety. The vector may transduce the packaging cells through any means known
in the
art. Such means include, but are not limited to, electroporation, the use of
liposomes, and
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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.
[420] 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 i~
vivo. The transduced eukaryotic cells will express a polypeptide of the
present invention.
[421] 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 e1 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).
[422] Suitable adenoviral vectors useful in the present invention are
described, for
example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993);
Rosenfeld
et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-
769 (1993);
Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692
(1993); and
U.S. Patent No. 5,652,224, which, are herein incorporated by reference. For
example, the
adenovirus vector Ad2 is useful and can be grown in human 293 cells. These
cells contain
the E1 region of adenovirus and constitutively express Ela and Elb, which
complement the
defective adenoviruses by providing the products of the genes deleted from the
vector. In
addition to Ad2, other varieties of adenovirus (e.g., Ad3, AdS, and Ad7) are
also useful in
the present invention.
[423] 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
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cells and can express a polynucleotide of interest which is operably linked to
a promoter,
but cannot replicate in most cells. Replication deficient adenoviruses may be
deleted in one
or more of all or a portion of the following genes: Ela, Elb, E3, E4, E2a, or
Ll through L5.
[424] 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.
[425] 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 Sainbrook 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.
[426] 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;
Roller et
al., Proc. Nat). 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
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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.
[427] 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 am
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.
[428] 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.
[429] 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.
[430] 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.
[431] The polynucleotide encoding a polypeptide of the present invention may
contaim
a secretory signal sequence that facilitates secretion of the protein.
Typically, the signal
sequence is positioned in the coding region of the polynucleotide to be~
expressed towards or
at the 5' end of the coding region. The signal sequence may be homologous or
heterologous
to the polynucleotide of interest and may be homologous or heterologous to the
cells to be
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transfected. Additionally, the signal sequence may be chemically synthesized
using
methods known in the art.
[432] Any mode of administration of any of the above-described
polyriucleotides
constructs can be used so long as the mode results in the expression of one or
more
molecules in an amount sufficient to provide a therapeutic effect: This
includes direct
needle injection, systemic injection, catheter infusion, biolistic injectors,
particle
accelerators (i.e., "gene guns"), gelfoam sponge depots, other commercially
available depot
materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid
(tablet or pill)
pharmaceutical formulations, and decanting or topical applications during
surgery. For
example, direct injection of naked calcium phosphate-precipitated plasmid into
rat liver and
rat spleen or a protein-coated plasmid into the portal vein has resulted in
gene expression of
the foreign gene in the rat livers (I~aneda et al., Science 243:375 (1989)).
[433] 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.
[434] Another method of local administration is to contact a polynucleotide
construct
of the present invention in or around a surgical wound. For example, a patient
can undergo
surgery and the polynucleotide construct can be coated on the surface of
tissue inside the
wound or the construct'can be injected into areas of tissue inside the wound.
[435] 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.
[436] 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
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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.
[437] 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.
[438] 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
[439] Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, can be used in assays to test for one or more biological
activities. If these
polynucleotides or polypeptides, or agonists or antagonists of the present,
invention, do
exhibit activity in a particular assay, it is likely that these molecules may
be involved in the
diseases associated with the biological activity. Thus, the polynucleotides
and
polypeptides, and agonists or antagonists could be used to diagnose, prognose,
prevent
and/or treat the associated disease.
j440] Defensin-like proteins are believed to be involved in biological
activities
associated with microbial cytotoxicity, anti-viral activity, chemotaxis;
immunostimulation,
inflammation, and lymphocyte proliferation. Accordingly, compositions of the
invention
(including polynucleotides, polypeptides and antibodies of the invention, and
fragments and
variants thereof) maybe used in the diagnosis; prognosis, prevention, and/or
treatment of
diseases and/or disorders associated with immunological disorders and/or
conditions.
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[441] In preferred embodiments, compositions of the invention (including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) are used in the diagnosis, prognosis, prevention, and/or treatment of
infectious
diseases (e.g., bacterial, fungal, parasitic, viral infections, and/or those
described in the
sections entitled "Immune Activity" and "Infectious Diseases");
immunodeficiencies (e.g.,
specific granule deficiency, neonatal innate immune deficiency, AIDS, and/or
as described
in the section entitled "Immune Activity"), autoimmune disorders (e.g.,
multiple sclerosis,
and/or as described in the section entitled "Immune Activity"), and
inflammatory disorders
(e.g., gastrointestinal inflammation, endocarditis, and/or as described in the
section entitled
"Immune Activity").
[442] In another preferred embodiment, compositions of the invention
(including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) are used in the diagnosis, prognosis, prevention, and/or treatment of
antibiotic-
resistant infections.
[443] In another preferred embodiment, compositions of the invention
(including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) are used, as inimunostimulants, for example, in the prevention of
infection in
neonates and/or as vaccine adjuvants.
[444] In another preferred embodiment, compositions of the invention
(including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) are used in the diagnosis, prognosis, prevention, and/or treatment of
cystic fibrosis.
[445] In another preferred embodiment, compositions of the invention
(including
polynucleotides, polypeptides and antibodies of the invention, and fragments
and variants
thereof) are used in the diagnosis, prognosis, prevention, and/or treatment of
neoplastic
disorders, as described. in the section entitled "Hyperproliferative
Disorders".
[446] 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 progriose 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).
[447] Thus, polynucleotides, translation products and antibodies of the
invention are
useful as immunostimulants, as well as in the diagnosis, prognosis, prevention
and/or
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treatment of infectious diseases, immunodeficiencies, inflammatory disorders,
and
neoplastic disorders.
[448] 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
[449] 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.
[450] In another embodiment, a polypeptide of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to treat
diseases and disorders of the immune system and/or to inhibit or enhance an
immune
response generated by cells associated with the tissues) in, which the
polypeptide of the
invention is expressed, including one, two, three, four, five, or more tissues
disclosed in
Table 1A, column 8 (Tissue Distribution Library Code).
[451] Polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention may be useful in treating; preventing, diagnosing, and/or.
prognosing
immunodeficiencies, including both congenital and acquired immunodeficiencies.
Examples of B cell immunodeficiencies in which immunoglobulin levels B cell
function
and/or B cell numbers are decreased include: X-linked agammaglobulinemia
(Breton's
disease), X-linked infantile agammaglobulinemia, X-linked imrnunodeficiency
with hyper
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IgM, non X-linked immunodeficiency with hyper IgM, X-linked
lymphoproliferative
syndrome (XLP), agammaglobulinemia including congenital and acquired
agammaglobulinemia, adult onset agammaglobulinemia, late-onset
agammaglobulinemia,
dysgammaglobulinemia, hypogammaglobulinemia, unspecified
hypogammaglobulinemia,
recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective
IgA'
deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with
or without
IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with
increased
IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain
deletions, kappa chain
deficiency, B cell lymphoproliferative .disorder (BLPD), common variable.
immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired),
and
transient hypogammaglobulinemia of infancy.
[452] 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.
[453] Examples of congenital immunodeficiencies in which . T cell and/or B
cell
function and/or number is decreased include; but are not limited to: DiGeorge
anomaly,
severe combined immunodeficiencies (SCID) (including, but not limited to, X-
linked SLID,
autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside
phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte
syndrome),
Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third
and fourth
pharyngeal pouch syndrome, 22q11.2 deletion, chronic 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.
[454] 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.
[455] 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- ,
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Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate
dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP),
leukocyte
adhesion deficiency, complement component. deficiencies (including C1, C2, C3,
C4, C5,
C6, C7, C8 ~andlor C9 deficiencies), reticular dysgenesis, thymic
alymphoplasia-aplasia,
immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with
immunodeficiency, neonatal neutropenia, short limbed dwarfisrri, and Nezelof
syndrome-
combined immunodeficiency with Igs.
[456] 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. .
[457] In a preferred, embodiment polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention could be used as an agent to
boost
immunoresponsiveness among immunodeficient individuals. In specific
embodiments,
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention could be used as an agent to boost immunoresponsiveness among B cell
and/or T
cell immunodeficient individuals.
[458] 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.
[459] Autoimmilne. diseases or disorders that may be treated, prevented,
diagnosed
and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists
or
antagonists of the present invention include, but are not limited to, one or
more of the
following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing
spondylitis,
multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis,
autoimmune hemolytic
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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.
[460] 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.
[461] Additional disorders that are likely to have an autoimmune component
that may
be treated, prevented, diagnosed and/or prognased with the compositions of the
invention
include, .but are not limited to, scleroderma with anti-collagen antibodies
(often
characterized, e.g., by nucleolar and other nuclear antibodies), mixed
connective tissue
disease (often characterized, e.g., by antibodies to extractable nuclear
antigens (e.g.,
ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone
ANA),
pernicious anemia (often characterized, e.g., by antiparietal cell,
microsomes, and intrinsic
factor, antibodies), idiopathic Addison's disease (often characterized, e.g.,
by humoral and
cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by
antispermatozoal
antibodies), glomerulonephritis (often, characterized, e.g., by glomerular
basement
membrane antibodies or immune complexes), bullous pemphigoid (often
characterized, e.g.,
by IgG and complement in basement membrane), Sjogren's syndrome (often
characterized,
e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)),
diabetes
mellitus (often characterized, e.g., by cell-mediated and humoral islet cell
antibodies), and
adrenergic drug resistance (including adrenergic drug resistance with asthma
or cystic
fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).
[462] Additional disorders that may have an autoimmune component that may be
treated, prevented, diagnosed and/or prognosed with the compositions of the
invention.
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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 derrriatitis
(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. .
[463] 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 specif c
preferred embodiment,
rheumatoid arthritis .is treated, prevented, and/or , diagnosed using
polynucleotides,
polypeptides, antibodies, and/or agonists or antagonists of the present
invention.
[464] 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.
[465] 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.
[466] In a preferred embodiment, the autoimmune diseases and disorders andlor
conditions associated with the diseases and disorders recited above are
treated, prevented,
diagnosed and/or prognosed using polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention
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(467] In preferred embodiments, polypeptides, antibodies, polynucleotides
and/or
agonists or antagonists of the present invention are used as a
immunosuppressive agent(s).
[468] 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.
[469] Allergic reactions andnconditions, 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.
[470] Additionally, polypeptides or polynucleotides of the invention, and/or
agonists or
antagonists thereof, may be used to treat, prevent, diagnose and/or prognose
IgE-mediated
allergic reactions. Such allergic reactions include, but .are not limited to,
asthma, rhinitis,
and eczema. In specific embodiments, polynucleotides, polypeptides,
antibodies, and/or
agonists or antagonists of the present invention may be used to modulate IgE
concentrations
in vitro or in vivo.
[471]. 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,
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or polynucleotides of the invention, and/or agonists or antagonists of the
invention may
inhibit the activation, proliferation and/or differentiation of cells involved
in an
inflammatory response, these molecules can be used to prevent and/or treat
chronic and
acute inflammatory conditions. Such inflammatory conditions include, but are
not limited
to, for example, inflammation associated with infection (e.g., septic shock,
sepsis, or
systemic inflammatory response syndrome), ischemia-reperfusion injury,
endotoxin
lethality, complement-mediated hyperacute rejection, nephritis, cytokine or
chemokine
induced lung injury, inflammatory bowel disease, Crohn's disease, over
production of
cytokines (e.g., TNF or IL-I.), respiratory disorders (e.g., asthma arid
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 andJor 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).
[472] 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,
balariitis, 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, orchids,
osteochondritis, otitis, pericarditis, peritendonitis, peritonitis,
pharyngitis, phlebitis,
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poliomyelitis, prostatitis, pulpitis, retinitis, rhiriitis, salpingitis,
scleritis, sclerochoroiditis,
scrotitis, sinusitis, spondylitis, steatitis~ stomatitis, synovitis, ~
syringitis, tendonitis,
tonsillitis, urethritis, and vaginitis.
[473] 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.
Ire 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.
[474] 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.
[475J 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 newimmune 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.
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[476] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are used as a vaccine adjuvant that
enhances immune
responsiveness to an antigen. In a specific embodiment, polypeptides,
antibodies,
polynucleotides and/or agonists or antagonists of the present invention are
used as an
adjuvant to enhance tumor-specific immune responses.
[477] 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 ofAIDS,
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.
[478] 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:
[479] 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
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typhi, Salmonella paratyphi, Meisser~ia meningitidis, Streptococcus
pneumoniae, Group B
streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,
Enterohemorrhagic E, coli,
and Borrelia buYgdorferi.
(480] 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.
(48I] 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. .
(482] 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.
[483J In one embodiment, polypeptides, antibodies, polynucleotides andlor
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.
[484] 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.
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[485] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an activator of T
cells.
[486] 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.
[487] 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.
[488] In another specific embodiment, pblypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as an agent to
increase serum
immunoglobulin concentrations.
[489] 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.
[490] 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:'
[491] 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.
[492] 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
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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).
[493] 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.
[494] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of tfie 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.
[495] 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.
[496] 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.
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[497J In another specific embodiment, polypeptides, antibodies,
polynucleotides andlor
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.
[498] 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
S
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. "
[499] 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.
[500] 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.
[501] 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.
[502] 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.
[503] 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
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allergies, inflammation, bowel disease, injury and diseases/disorders
associated with
pathogens.
[504] 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.
[505] 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.
[506] In another specific embodirizent, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention are used as a therapy for
chronic
hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of
undetermined significance (MGUS), Waldenstrom's disease, related idiopathic
monoclonal
gammopathies, and plasmacytomas.
[507] 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
neutrQphils, 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.
[508] 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.
[S09] In another specific embodiment, polypeptides, antibodies,
polynucleotides andlor
agonists or antagonists of the present invention are used to enhance or
inhibit complement
mediated cell lysis.
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[510] 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.
[511] 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.
[512] In another specific embodiment, polypeptides, antibodies,
polynucleotides and/or
agonists or antagonists of the present invention rnay be employed to treat
adult respiratory
distress syndrome CARDS).
[5I3] 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 ofmucosal surfaces.
[514] 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, ~andlor treated with
polynucleotides or
polypeptides, and/or agonists of the present invention include, but are not
limited to, HIV
infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction
anemia,
thrombocytopenia, and hemoglobinuria. -
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(515] 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.
[516] 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; pblypeptides, antibodies, and/or agonists or
antagonists of the
present invention include, but are not limited to, acute myelogenous leukemia,
chronic
myelogenous leukemia, Hodgkin's disease, non-Hodglcin'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.
[517] 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.
[518] 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. .
[519] 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.
[520] Antagonists of the invention include, for example, binding and/or
inhibitory
antibodies, antisense nucleic acids, ribozymes or soluble forms of the
polypeptides of the
present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of
the invention
include, for example, binding or stimulatory antibodies, and soluble forms of
the
polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides,
antibodies,
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polynucleotides 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.
[521] In another embodiment, polypeptides, antibodies, polynucleotides and/or
agonists
or antagonists of the present invention are administered to an animal
(including, but not
limited to, those listed above; and also including transgenic animals)
incapable of producing
functional endogenous antibody molecules or having an otherwise compromised
endogenous immune system, but which is capable of producing human
immunoglobulin
molecules by means of a. reconstituted or partially reconstituted immune
system from
another animal (see, e.g., published PCT Application Nos. W098/24893,
WO/9634096,
WO/9633735, and WO/9110741). Administration of polypeptides, antibodies,
polynucleotides and/or agonists or antagonists of the present invention to
such animals is
useful for the generation of monoclonal antibodies against the polypeptides,
antibodies,
polynucleotides and/or agonists or, antagonists of the present invention.
Blood-Related Disorders
[522] The polynucleotides, polypeptides, antibodies, a.nd/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.
[523] 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,
thromboembolisril,-
pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic
attack,
unstable angina. In specific embodiments, the polynucleotides, polypeptides,
antibodies,
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and/or agonists or antagonists of the present invention may be used for the
prevention of
occulsion of saphenous grafts, for reducing the risk of periprocedural
thrombosis as might
accompany angioplasty procedures, for reducing the risk of stroke in patients
with atrial
fibrillation including nonrheumatic atrial fibrillation, for reducing the risk
of embolism
associated.with mechanical heart valves and or mural valves disease. Other
uses for the
polynucleotides, polypeptides, antibodies, andlor 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).
[524] In another embodiment, a polypeptide _ of the invention, or
polynucleotides,
antibodies, agonists, or antagonists corresponding to that polypeptide, may be
used to
prevent, diagnose, p~ognose, and/or treat diseases and disorders of the blood
and/or blood
forming organs associated with the tissues) in which the polypeptide of the
invention is
expressed, including one, two, three, four, five, ~or more tissues disclosed
in Table 1A,
column 8 (Tissue Distribution Library Code).
[525] The polynucleotides, polypeptides,' antibodies, and/or agonists or
antagonists of
the present invention may be used to modulate hematopoietic activity (the
formation of
blood cells). For example, the polynucleotides, polypeptides, antibodies,
andlor agonists or
antagonists of the present invention may be used to increase the quantity of
all or subsets of
blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells),
myeloid cells
(e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and
platelets. The
ability to decrease the quantity of blood cells or subsets of blood cells may
be useful in the
prevention, detection, diagnosis and/or treatment of anemias and leukopenias
described
below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention may be used to decrease the quantity of
all or subsets of
blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells),
myeloid cells
(e.g., basophils,~ eosinophils, neutrophils, mast cells, macrophages)' and
platelets.. The
ability to decrease the quantity of blood cells or subsets of blood cells may
be useful in the
prevention, detection, diagnosis and/or treatment of leukocytoses, such as,
for example
eosinophilia.
[526] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be used to prevent, treat, or diagnose blood
dyscrasia.
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[527] 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 1.2
deficiency) and folic
acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune
helolytic
anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal
hemoglobinuria).
The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists
of the present
invention may be useful in treating, preventing, and/or diagnosing anemias
associated with
diseases including but not limited to, anemias associated with systemic lupus
erythematosus, cancers, lymphomas, chronic renal disease, and enlarged
spleens. The
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention may be useful in treating, preventing, 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.
[528] ~ 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. '
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[529] 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 thrombotie
thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet
disorders (e.g.,
storage pool disease such as Chediak-Higashi and Hermanslcy-Pudlak syndromes,
thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome),
hemolytic-
uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and
Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic
Telangiectsia, also
known as Rendu-Osler-Weber syndrome, allergic purpura (Herioch Schonlein
purpura) and
disseminated intravascular coagulation.
[530] The effect bf the polynucleotides, polypeptides, antibodies, and/or
agonists or
antagonists of the present invention on the clotting time of blood may be
monitored using
L
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.
[531] 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. .
[532] In another embodiment, the .polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating diseases and disorders characterized by or
associated with
increased or decreased numbers of white blood cells. Leukopenia occurs when
the number
of white blood cells decreases below normal. Leukopenias include, but are not
limited to,
neutropenia and lymphocytopenia. An increase- in the number of white blood
cells
compared to normal is known as leukocytosis. The body generates increased
numbers of
white blood cells during infection. Thus, leukocytosis may simply be a normal
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physiological parameter that reflects infection. Alternatively, leukocytosis
may be an
indicator of injury or other disease such as cancer. Leokocytoses, include but
are not
limited to, eosinophilia, and accumulations of macrophages. In specific
embodiments, the
polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of
the present
invention may be useful in diagnosing, prognosing, preventing, and/or treating
leukopenia.
In other specific embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosixig,
preventing, and/or treating leukocytosis.
[533] Leukopenia may be a generalized decreased in all types of white blood
cells, or
may be a specific depletion of particular types of white blood cells. Thus, in
specific
embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists
of the present invention may be useful in diagnosing, prognosing, preventing,
and/or
treating decreases in neutrophil numbers, known as neutropenia. Neutropenias
that may be
diagnosed, prognosed, prevented, and/or treated by the polynucleotides,
polypeptides,
antibodies, and/or agonists or antagonists of the present invention include,
but are not
limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic
neutropenia,
neutropenias resulting from or associated with dietary deficiencies (e.g.,
vitamin B 12
deficiency or folic acid deficiency), neutropenias resulting from or
associated with drug
treatments (e.g., antibiotic 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 ari enlarged spleen (e.g.,
Felty syndrome,
malaria and sarcoidosis), and some drug treatment regimens.
[534] The polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of
the present invention may be useful in diagnosing, prognosing, preventing,
and/or treating
lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but
not
limited lymphocytopenias resulting from or associated with stress, drug
treatments (e.g.,
drug treatment with corticosteroids, cancer chemotherapies, and%or radiation
therapies),
AIDS infection and/or other diseases such as, for example, cancer, rheumatoid
arthritis,
systemic lupus erythematosus, chronic infections, some viral infections and/or
hereditary
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disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined
irnmunodeficiency, ataxia teiangiectsia). _
[535] 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 andlor macrophage
function
including, but not limited to, Gaucher's disease, Niemann-Pick disease,
Letterer-Siwe
disease and Hand-Schuller-Christian disease.
[536] 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.
[537] In yet another embodiment, the polynucleotides, polypeptides,
antibodies, and/or
agonists or antagonists of the present invention may be useful in diagnosing,
prognosing,
preventing, and/or treating leukemias and lymphomas including, but not limited
to, acute
lymphocytic (lymphpblastic). leukemia (ALL), acute myeloid (myelocytic,
myelogenous,
myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g.,
B cell
leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia),
chronic myelocytic
(myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-
hodgkin's
lymphoma, Burkitt's lymphoma, and mycosis fungoides.
[538] 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.
[539] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in treating,
preventing, and/or-
diagnosing myeloproliferative disorders, including but not limited to,
polycythemia vera,
relative polycythemia, secondary polycythemia, myelofibrosis, acute
myelofibrosis,
agnogenic myelod metaplasia, thrombocythemia, (including both primary and
seconday ,
thrombocythemia) and chronic myelocytic leukemia.
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[540] 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.
[541] 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.
[542] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonises 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 agonises 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.
[543] 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.
[544] In other embodiments, the polynucleotides, polypeptides, antibodies,
and/or
agonists or antagonists of the present invention may be useful in preventing,
diagnosing,
and/or treating primary hematopoietic disorders.
Hyperproliferative Disorders
[545] 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.
[546] For example, by increasing an immune response, particularly increasing
antigenic
qualities of the hyperproliferative disorder or by proliferating,
differentiating, or mobilizing
T-cells, hyperproliferative disorders can be treated. This immune response may
be
increased by either enhancing an existing immune response, or by initiating a
new immune
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response. Alternatively, decreasing an immune response may also be a method of
treating
hyperproliferative disorders, such as a chemotherapeutic agent.
[547] Examples of hyperproli~erative 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.
[548j 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:
Aeute
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 Leulcemia, Adult Hodgkin's Disease, Adult Hodgkin's
Lymphoma,
Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver
Cancer, Adult Soft Tissue Sarcoma, . AIDS-Related Lymphoma, AIDS-Related
Malignancies, Anal Cancer, Astrocytoma, Bile.Duct Cancer, Bladder Cancer, Bone
Cancer,
Brain Stem Glioma, Brain Tumors; Breast Cancer, Cancer of the Renal Pelvis and
Ureter,
Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma,
Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood
(Primary)
Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute
Lymphoblastic
Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma,
Childhood
Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial
Germ
Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma,
Childhood
Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia,
Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal
and
Supratentorial Primitive 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-CeII Lymphoma, Endocrine
Pancreas
Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer,
Esophageal
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Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer,
Extracranial
Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer,
Eye
Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric
Cancer,
Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors,
Gestational
Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neclc Cancer,
Hepatocellular Cancer,
Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal
Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet
Cell
Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and
Oral
Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,
Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant
Thymoma,
Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous
Neck
Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck
Cancer,
Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic
Syndrome,
Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal
Cavity
and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-
Hodgkin's
Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung
Cancer,
Occult Primary Metastatic ,Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-
/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,
Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial
Cancer,
Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer,
Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer,
Pheochromocytoma,
Pituitary Tumor, Plasma Cell Neoplasni/Multiple Myeloma, Primary Central
Nervous
System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal
Cell
Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma,
Salivary
Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell
Lung
Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer,
Stomach
Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell
Lymphoma,
Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the
Renal Pelvis
and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,
Ureter and
Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma,
Vaginal
Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's
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Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease,
besides
neoplasia, located in an organ system listed above.
[549] 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.)
[550] 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.
Hyperpla tic disorders which can be diagnosed, prognosed, prevented, and/or
treated with
compositions of the invention (including polynucleotides, polypeptides,
agonists or
antagonists) include, but are not limited to', angiofollicular mediastinal
lymph node
hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic
hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia,
cementum
hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia,
cystic
hyperplasia, cystic hyperplasia of the breast, denture hyperplasia,, ductal
hyperplasia,
endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial
hyperplasia~ gingival
hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary
hyperplasia,
intravascular papillary endothelial hyperplasia, nodular hyperplasia of
prostate, nodular
regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous
hyperplasia, and verrucous hyperplasia.
[551] Metaplasia is a form of controlled cell growth in which one type of
adult or fully
differentiated cell substitutes for another type of adult cell. Metaplastic
disorders which can
be diagnosed, prognosed, prevented, and/or treated with compositions of the
invention
(including polynucleotides, polypeptides, agonists , or antagonists) include,
but are not
limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical
metaplasia,
autoparenchymatous metaplasia, connective tissue metaplasia, epithelial
metaplasia,
intestinal metaplasia, metaplastic anemia, metaplastic
ossification,_metaplastic polyps,
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myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia,
squamous
metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.
.
[552] Dysplasia is frequently a forerunner of cancer, and is found mainly in
the
epithelia; it is the most disorderly form of non-neoplastic cell growth,
involving a loss in
individual cell uniformity and in the architectural orientation of cells.
Dysplastic cells often
have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
Dysplasia
characteristically occurs where there exists chronic irritation or
inflammation. Dysplastic
disorders which can be diagnosed, prognosed, prevented, and/or treated with
compositions
of the invention (including polynucleotides, polypeptides, agonists or
antagonists) include,
but are not limited to, anhidrotic ectodermal dysplasia, anterofacial
dysplasia, asphyxiating
thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,
cerebral dysplasia,
cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia,
congenital
ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia,
craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,
ectodermal dysplasia,
enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis
hemimelia,
dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial
dysplasia,
faciodigitogenital dysplasia, familial fibrous dysplasia , of j aws, familial
white folded
dysplasia, fibromuscular dysplasia, fbrous 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, bculoauriculovertebral dysplasia,
oculodentodigital
dysplasia, oculovertebral dysplasia, odontogenic dysplasia,
ophthalmomandibulomelic
dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia,
pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-
optic
dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.
[553] Additional pre-neoplastic disorders which can be diagnosed, prognosed,
prevented, and/or treated with compositions of the invention (including
polynucleotides,
polypeptides, agonists or antagonists) include, but are not limited to, benign
dysproliferative
disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy,
intestinal polyps,
colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's
disease, Farmer's
Skin, solar cheilitis, and solar keratosis.
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[554] 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).
[555] 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, andlor 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.
[556] ' 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, intestinalY 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.
[557] 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.
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(558] 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 inyeloma, 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, mesothe.lioma, Ewing's tumor,
Ieiomyosarcoma, 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.
[559] ~ 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
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
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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.
[S60] 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.
[S61] 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.
[S62] 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.
[563] 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.
[S64] 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 retxovirus, or more preferably an adenoviral vector (See G
J. Nabel, et.
al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a
most
preferred embodiment, the viral vector is defective and will not transform non-
proliferating
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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.
[565] Polynucleotides of the present. invention may be useful in repressing
expression
of oncogenic genes or antigens. By "repressing expression of the orlcogenic
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.
[566] 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); Hoclce, Nature 320:275 (1986); Wilson, et
al., Proc.
Natl. Acad. Sci. U.~S.A. 85:3014), vaccinia virus system (Chakrabarty et al.,
Mol. Cell Biol.
5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature
313:812
(1985)) known to those skilled in the art. These references are exemplary only
and are
hereby incorporated .by reference. In order to specifically deliver or
transfect cells which
are abnormally proliferating and spare non-dividing cells, it is preferable to
utilize a
retrovirus, or adenoviral (as described in the art and elsewhere herein)
delivery system
known to, those of skill in the art. Since host DNA replication is required
for retroviral
DNA to integrate and the retrovirus will be unable to self replicate due to
the lack of the
retrovirus genes needed for its life cycle. Utilizing such a retroviral
delivery system for
polynucleotides of the present invention will target said gene and constructs
to abnormally
proliferating cells and will spare the non-dividing normal cells.
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[567] 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 adiniriistered to disease
sites at the
time of surgical intervention.
[568] 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.
[589] 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 rrialignant 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.
[570] 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.
[571] A summary of the ways in which the antibodies of the present invehtion
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
axe 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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[572] 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
[573] 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. .
[574J 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
disordexs
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,
5X10-'M, 10-
'M., SX10-$M, 10-$M, SX10-9M, 10-9M, SX10-1°M, 10-1°M, 5X10-11M,
10-11M, SX10-12M,
10-12M, 5X10-13M, 10-13M, 5X10-14M, 10-iaM, 5X10-15M, and 10-15M.
[575] Moreover, polypeptides of the present invention are useful in inhibiting
the
angiogenesis of proliferative cells or tissues, either alone, ~ as a protein
fusion, or in
combination with other polypeptides directly or indirectly, as described
elsewhere herein. In
a most preferred embodiment, .said anti-angiogenesis effect may be achieved
indirectly, for
example, through the inhibition of hematopoietic, tumor-specific cells, such
as tumor-
associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-
53 (1998),
which is hereby incorporated by reference). Antibodies directed to
polypeptides or
polynucleotides of the .present invention may also result in inhibition of
angiogenesis
directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-
61 (1998),
Which is hereby incorporated by reference)). '
[576] 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 (FaslAPO-1), TNF-receptor-
related
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apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand
(TRAIL;)
receptor-1 and -2 (See Schulze-Osthoff I~, et.al., Eur J Biochem 254(3):439-59
(1998),
which is hereby incorporated by reference). Moreover, in another preferred
embodiment of
the present invention, said polypeptides may induce apoptosis through other
mechanisms,
such as in the activation of other proteins which will activate apoptosis, or
through
stimulating the expression of said proteins; either alone or in combination
with small
molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, anti-
inflammatory
proteins (See for example, Mutat Res 400(1-2):447-S5 (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).
[577] 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.
[578] 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.
[579] 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
[580] 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. . -
[581] 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.)
[582] In addition, compositions of the invention can be used to diagnose,
prognose,
prevent, andlor 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, Bartter'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
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nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney
stones, and
membranous nephropathy), and autoimmune disorders of the kidney (e.g.,
systemic lupus
erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial
proliferative glomerulonephritis).
[583] Compositions of the invention can also be used to diagnose, prognose,
prevent,
and/or treat sclerotic or necrotic disorders of the kidney (e.g.,
glomerulosclerosis, diabetic
nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing
glomerulonephritis,
and renal papillary necrosis), cancers of the kidney (e.g., nephroma,
hypernephroma,
nephroblastoma, renal cell cancer, transitional cell cancer, renal
adenocarcinoma, squamous
cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g.,
nephrocalcinosis, pyuria,
edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia,
hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and
hyperphosphatemia).
[S84] 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
polynucleoti.des are
described in more detail herein.
Cardiovascular Disorders
[585] 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.
[586] 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, Eisenmerlger complex, hypoplastic left heart syndrome, levocardia,
tetralogy of
fallot, transposition of great vessels, double outlet right ventricle,
tricuspid atresia,
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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. .
[587] 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.
[588] 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.
[589] 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.
[590] Myocardial diseases include, but are not limited to; alcoholic
cardiomyopathy,
congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular
~stenosis,
pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas
cardiomyopathy,
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endocardial fibroelastosis, endomyocardial fibrosis, Learns Syndrome,
myocardial
reperfusion injury, and myocarditis.
[591] 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.
[592] Cardiovascular diseases also include vascular diseases such as
aneurysms,
angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
Llippel-
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 verso-occlusive disease, Raynaud's disease, CREST
syndrome, retinal
vein occlusion, Scimitar syndrome, superior versa cava syndrome,
telangiectasia, atacia
telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose
veins, varicose
ulcer, vasculitis, and venous insufficiency.
(593] 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.
[594] 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.
[595] 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 hematorna, subdural hematoma, subaraxhnoid hemorrhage,
cerebral
infarction, cerebral ischemia (including transient), subclavian steal
syndrome,
periventricular leukomalacia, vascular headache, cluster headache, migraine,
and
vertebrobasilar insufficiency.
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[596] 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.
(597] Ischemic disorders include, but are not limited to, cerebral ischemia,
ischemic
colitis, compartment syndromes, anterior compartment syndrome, myocardial
ischemia,
reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but
is not limited to,
aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous
lymph node
syndrome, thromboangiitis obliterans, hypersensitivity vasculitis,. Schoenlein-
Henoch
purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
[598] 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.
Respiratory Disorders
[599] 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.
[640] 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 rhiriitis), 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
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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 pueumoniae (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. (Legionnaires' disease), and
Chlamydia
psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox
(varicella).
[601] 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 neoforma~cs; aspergillosis, caused by Aspergillus spp.;
candidiasis, caused by
Candida; and mucormycosis)), Pheumocystis carihii (pneumocystis pneumonia),
atypical
pneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunistic infection
pneumonia,
nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural
disorders
(e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous
pneumothorax,
complicated spontaneous pneumothorax, tension pneumothorax)), obstructive
airway
diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD),
emphysema, chronic
or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung
(coal workers'
pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis,
and benign
pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g.,
fibrosing
alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis,
desquamative
interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X
(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
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embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis,
lung abscess (caused
by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic
fibrosis.
Anti-An~iogenesis Activity
[602] The naturally occurring balance between endogenous stimulators and
inhibitors
of angiogenesis is one in which inhibitory influences predominate. Rastinejad
et al., Cell
56:345-355 (1989). In those rare instances in which neovascularization occurs
under normal
physiological conditions, such as wound healing, organ regeneration, embryonic
development, and female reproductive processes, angiogenesis is stringently
regulated and
spatially and temporally delimited. Under conditions of pathological
angiogenesis such as
that characterizing solid tumor growth, these regulatory controls fail.
Unregulated
angiogenesis becomes pathologic and sustains progression of many neoplastic
and non-
neoplastic diseases. 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. Ehgl. 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. Folkrrian and Klagsbrun, Scie~zce 235:442-447
(1987).
[603] The present invention provides for treatment of diseases or disorders
associated
with neovascularization by administration of the polynucleotides and/or
polypeptides of the
invention, as well as agonists or antagonists of the present invention.
Malignant and
metastatic conditions which can be treated with the polynucleotides and
polypeptides, or
agonists or antagonists of the invention include, but are not limited to,
malignancies, solid
tumors, and cancers described herein and otherwise known in the art (for a
review of such
disorders, see Fishman et al., Medicine, Zd Ed., J. B. Lippincott Co.,
Philadelphia T
(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 .
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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 iri order to
therapeutically treat a cancer
or tumor. Cancers which may be treated with polynucleotides, polypeptides,
antagonists
and/or agonists include, but are not limited to solid tumors, including
prostate, lung, breast,
ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,.
biliary tract, colon,
rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary
tumors and
metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-
small cell
lung cancer; colorectal cancer; advanced malignancies; and blood born tumors
such as
leukemias. For example, polynucleotides, polypeptides, antagonists and/or
agonists may be
delivered topically, in order to treat cancers such as skin cancer, head and
neck tumors,
breast tumors, and ICaposi'.s sarcoma.
(604] 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.
[605] 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,
retrolerital fibroplasia,
rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth)
of the eye;
rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis;
wasculogenesis;
granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma;
trachoma;
vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral
collaterals;
arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber
Syndrome; plaque
neovascularization; telangiectasia; hemophiliac joints; angiofibroma;
fibromuscular
dysplasia; wound granulation; Crohn's disease; and atherosclerosis.
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[606] 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. .
[607] 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 rioted above,
the present
invention also provides methods for treating neovascular diseases of the eye,
including for
example, corneal neovascularization, neovascular glaucoma, proliferative
diabetic
retinopathy, retrolental fibroplasia and macular degeneration.
[608] 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.,
Su~v. Ophthal. 22:291-312 (1978).
[609] 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 neovascixlarization, including for
example, corneal
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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.
[610] 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 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.
[611] 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.
[612] Within another aspect of the present invention, methods are provided.
for treating
neovascular glaucoma, comprising the step of administering to a patient a
therapeutically
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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.
[613] 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
inorease 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.
[614] 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.
[615] 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.
[616] 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,
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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,
Osier-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. ,
[617] 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.
[618] . Polynucleotides, polypeptides, agonists and/or agonists of the present
invention
may be incorporated into surgical sutures in order to prevent stitch
granulomas.
[619] Polynucleotides, polypeptides, agonists and/or agonists may be utilized
in a wide
variety of surgical procedures. For example, within one aspect of the present
invention a
compositions (in the form of, for example, a spray or film) may be utilized to
coat or spray
an area prior to removal of a tumor, in order to isolate normal surrounding
tissues from
malignant tissue, and/or to prevent the spread of disease to surrounding
tissues: Within
other aspects of the present invention, compositions (e.g., in the form of a
spray) may be
delivered via endbscopic procedures in order to coat tumors, or inhibit
angiogenesis in a
desired locale. Within yet other aspects of the present invention, surgical
meshes which
have been coated with anti- angiogenic compositions of the present invention
may be
utilized in any procedure wherein a surgical mesh might be utilized. For
example, within
one embodiment of the invention a surgical mesh laden with an anti-angiogenic
composition may be utilized during abdominal cancer resection surgery (e.g.,
subsequent to
colon resection) in order to provide support to the structure, and to release
an amount of the
anti-angiogenic factor. ' .
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[620] Within further aspects of the present invention, methods are provided
for treating
tumor excision sites, comprising administering a polynucleotide, polypeptide,
agonist
andlor 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
neurosi~rgical operations.
[621] 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. '
[622] 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.
[623] 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.
[624] ~ Representative examples of vanadium complexes include oxo vanadium
complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes
include
metavanadate and orthovanadate complexes such as, for example, -ammonium
metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl
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complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate
including
vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
[625] Representative examples of tungsten and molybdenum complexes also
include
oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten
oxide
complexes. Suitable tungstate complexes include ammonium tungstate, calcium
tungstate,
sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides
include tungsten
(IV) oxide and ~ tungsten (VI) oxide. , Suitable oxo molybdenum complexes
include
molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate
complexes include ~ ammonium molybdate and its hydrates, sodium molybdate and
its
hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides
include
molybdenum (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.
[626] ~ A wide variety of other anti-angiogenic factors may also be utilized
Within the
context of the present invention. Representative examples include platelet
factor 4;
protamine sulphate; sulphated chitin derivatives (prepared from queen crab
shells), (Murata
et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan
Complex (SP-
PG) (the function of this compound may be enhanced by the presence of steroids
such as
estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix
metabolism,
including for example, proline analogs, cishydroxyproline, d,L-3,4-
dehydroproline,
Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile famerete; 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.
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Diseases at the Cellular Level
[627] 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, glioblastorna, lung cancer, intestinal cancer,
testicular cancer,
stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoina; 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.
[628] 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.
[629] Additional diseases or conditions associated with increased cell
survival that
could be treated or detected by polynucleotides or polypeptides, or agonists
or 'antagonists
of the present invention include, but are not limited to, progression, and/or
metastases of
malignancies and related disorders such as leukemia (including acute leukemias
(e.g., acute
lymphocytic leukemia, acute myelocytic leukemia (including. myeloblastic,
promyelocytic,
myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g.~
chronic
myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)),
polycythemia
vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple
myeloma,
Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors
including, but not
limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma,
liposarcoma,
chondros~arcoma, osteogenic sarcoma, chordorna, angiosarcoma,
endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma,, synovioma, mesothelioma,
Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,
breast
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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, meriangioma, melanoma,
neuroblastoma, and retinoblastoma.
[630] 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.
Wound Healing and Epithelial Cell Proliferation
[631] 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 .
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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
[632] 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, Bla.ir-Brown grafts, bone graft,
brephoplastic
grafts, cutis graft, delayed graft, dermic graft, epidermis graft, fascia
graft, full thickness
graft, heterologous graft, xenograft, homologous graft, hyperplastic graft,
lamellar graft,
mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft,
pedicle graft,
penetrating graft, split skin graft, thick split graft. Polynucleotides or
polypeptides, as well
as agonists or antagonists of the present invention, can be used to promote
skin strength and
to improve the~appearance of aged skin.
[633) It is believed that polynucleotides or polypeptides, as well as agonists
or
a
antagonists of the present invention, will also produce changes in
hepatocyte,proliferation;
and epithelial cell proliferation in the lung, breast, pancreas, stomach,
small intestine, and
large intestine. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could promote proliferation of epithelial cells such as
sebocytes, hair.
follicles, hepatocytes, type II pneumocytes, 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.
[634] 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
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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.
[635] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention, could further be used in full regeneration of skin in full and
partial thickness skin
defects, including burns, (i.e., repopulation of hair follicles, sweat glands,
and sebaceous
glands), treatment of other skin defects such as psoriasis. Polynucleotides or
polypeptides,.
as well as agonists or antagonists of the present invention, could be used to
treat
epidermolysis bullosa, a defect in adherence of the epidermis to the
underlying dermis
which results in frequent, open and painful blisters by accelerating
reepithelialization of
these lesions. Polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention, could also be used to treat gastric and doudenal ulcers and
help heal by
scar formation of the mucosal lining and regeneration of glandular mucosa and
duodenal
mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's
disease and
ulcerative colitis, are diseases which result in destruction of the mucosal
surface of the
small or large intestine, respectively. Thus, polynucleotides or polypeptides,
as well as
agonists or antagonists of the present invention, could be used to promote the
resurfacing of
the mucosal surface to aid more rapid healing and to prevent progression of
inflammatory
bowel disease. Treatment with polynucleotides or polypeptides, agonists or
antagonists of
the present invention, is expected to have a significant effect on the
production of mucus
throughout the gastrointestinal tract and could be used to protect the
intestinal mucosa from
injurious-substances that are ingested or following surgery. Polynucleotides
or polypeptides,
as well as agonists or antagonists of the present invention, could be used to
treat diseases
associate with the under expression. .
[636] Moreover, polynucleotides or ~polypeptides, as well as agonists or
antagonists of
the present invention, could be used to prevent and heal damage to the lungs
due to various
pathological states. Polynucleotides or polypeptides, as well as agonists or
antagonists. of
the present invention, which could stimulate proliferation and differentiation
and promote
the repair of alveoli and brochiolar epithelium .to prevent or treat acute or
chronic lung
damage. For example, emphysema, which results in the progressive loss of
aveoli, and
inhalation injuries, i.e., resulting from smoke inhalation and burns, that
cause necrosis of
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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.
[637] 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 az't).
[638] 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 Activity and Neurological Diseases
[639] The polynucleotides, polypeptides and agonists or antagonists of the
invention
may be used for the diagnosis andlor treatment of diseases, disorders, damage
or~injury of
the brain and/or nervous system. Nervous system disorders that can be treated
with the
compositions of the invention (e.g., polypeptides, polynucleotides, and/or
agonists or
antagonists), include, but are not limited to, nervous system injuries, and,
diseases or
disorders which result in either a disconnection of axons, a diminution or
degeneration of
neurons, or demyelination. Nervous system lesions which may be treated in a
patient
(including human and non-human mammalian patients) according to the methods of
the
invention, include but are not limited to, the' following lesions of either
the central
(including spinal cord, brain) or peripheral nervous systems: (1) ischemic
lesions, in which
a lack of oxygen in a portion of the nervous system results in neuronal injury
or death,
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including cerebral infarction or ischemia, or spinal cord infarction or
ischemia; (2)
traumatic lesions, including lesions caused by physical injury or associated
with surgery, for
example, lesions which sever a portion of the nervous system, or compression
injuries; (3)
malignant lesions, in which a portion of the nervous system is destroyed or
injured by
malignant tissue which is either a nervous system associated malignancy or a
malignancy
derived from non-nervous system tissue; (4) infectious lesions, in which a
portion of the
nervous system is destroyed or injured as a result of infection, for example,
by an abscess or
associated with infection by human immunodeficiency virus, herpes zoster, or
herpes
simplex virus or with Lyme disease, tuberculosis, or syphilis; (5)
degenerative lesions, in
which a portion of the nervous system is destroyed or injured as a result of a
degenerative
process including but not limited to, degeneration associated with Parkinson's
disease,
Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis
(ALS); (6)
lesions associated with nutritional diseases or disorders, in which a portion
of the nervous
system is destroyed or injured by a nutritional disorder or disorder of
metabolism including,
but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke
disease, tobacco-
alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the
corpus
callosum), and alcoholic cerebellar degeneration; (7) neurological lesions
associated with
systemic diseases including, but not limited to, diabetes (diabetic
neuropathy, Bell's palsy),
systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by
toxic
substances including alcohol, lead, or particular neurotoxins; and (9)
demyelinated lesions
in ,which a portion of the nervous system is destroyed or injured by a
demyelinating disease
including, but not limited to, multiple sclerosis, human immunodeficiency
virus-associated
myelopathy, transverse inyelopathy or various etiologies, progressive
inultifocal
leukoencephalopathy, and central pontine ~riyelinolysis.
[640] In one embodiment, the polypeptides, polynucleotides, or agonists or
antagonists
of the invention are used to protect neural cells from the damaging effects of
hypoxia. In ~a
further preferred embodiment, the polypeptides, polynucleotides, or agonists
or antagonists
of the invention are used to protect neural cells from the damaging effects of
cerebral
hypoxia. According to this embodiment, the compositions of the invention are
used to treat
or prevent neural cell injury associated with cerebral hypoxia. In one non-
exclusive aspect
of this embodiment, the polypeptides, polynucleotides, or agonists or
antagonists of the
invention,' are used to treat or prevent neural cell injury associated with
cerebral ischemia.
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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.
[641] 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 axe used to treat or prevent cerebral neural cell
injury associated
with a stroke.
[642) In another preferred embodixxient, 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.
[643) 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., P~oc Natl Acad Sci USA 97:3637-
42 (2000) or
in Arakawa et~al., J. Neurosci., 10:3507-15 (1990); increased sprouting of
neurons may be
detected by methods known in the art, such as, for example, the methods set
forth in
Pestronk et al., Exp. Neurol., 70:65-82 (1980); or Brown et al., Ann. Rev.
Neu~osci.,
4:17-42 (1981); increased production of neuron-associated molecules may be
measured by
bioassay, enzymatic ,assay, antibody binding, Northern blot assay, etc., using
techniques
known in the art and depending on the molecule to be measured; and motor
neuron
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dysfunction may be measured by assessing the physical manifestation of motor
neuron
disorder, e.g., weakness, motor neuron conduction velocity, or functional
disability. -
[644] 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).
[645] 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.
[646] Additionally, polypeptides, polynucleotides and/or agonists or
antagonists of the
invention, may be useful in protecting neural cells from diseases, damage,
disorders, or
injury, associated with cerebrovascular disorders including, but not limited
to, carotid artery
diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya
Disease), cerebral
amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral
arteriosclerosis, cerebral
arteriovenous malformations, cerebral artery, diseases, cerebral embolism and
thrombosis -
(e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome),
cerebral
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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).
[647] 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 andlor 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.
[648] 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.
[649] Additional neuroiogic 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
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infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian
Steal
Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-
infarct
dementia, periventricular leukomalacia, vascular headache such as cluster
headache and
migraine..
[650] 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 and temporal lobe epilepsy,
post-traumatic
epilepsy, status epilepticus such as Epilepsia Partialis Continua, and
Hallervorden-Spatz
Syndrome.
[651] 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 Hemori-hagic
Encephalomyelitis,
Visna, and cerebral malaria.
[652] 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
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includes lymphocytic choriomeningitis, Bacterial meningtitis which includes
Haemophilus
Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as
Waterhouse-
Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis,
fungal
meningitis such as Cryptococcal Meningtitis, subdural effusion,
meningoencephalitis such
as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis,
neurosyphilis
such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and
postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob
Syndrome,. Bovine
Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and
cerebral
toxoplasmosis. , _
[653] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
central nervous system neoplasms such as brain neoplasms that include
cerebellar
neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such
as choroid
plexus .neoplasms, hypothalamic neoplasms and supratentorial neoplasms,
meningeal
neoplasms, spinal cord neoplasms which include epidural neoplasms,
demyelinating
diseases such as Canavan Diseases, . diffuse cerebral sceloris which includes
adrenoleulcodystrophy, encephalitis periaxialis, globoid cell leukodystrophy,
diffuse
cerebral sclerosis such as metachromatic leukodystrophy, allergic
encephalomyelitis, .
necrotizing hemorrhagic encephalomyelitis, progressive multifocal
leukoencephalopathy,
multiple sclerosis, central pontine myelinolysis, transverse myelitis,
neuromyelitis optica,
Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous
Syndrome,
Meningism, spinal cord dise~.ses 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(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease,
homocystinuria, Laurence-Moon- Biedl~Syndrome, Lesch-Nyhan Syndrome, Maple
Syrup
Urine Disease, mucolipidosis such as . fucosidosis, neuronal ceroid-
lipofuscinosis,
oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria,
Prader-
Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis,
WAGR
Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube
defects
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such as anencephaly which includes hydrangencephaly, Arnold-Cbairi .
Deformity,
encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina
bifida
cystica and spina bifida occulta.
[654] Additional neurologic diseases which can be treated or detected with
polynucleotides, polypeptides, agonists, and/or antagonists of the present
invention include
hereditary motor and sensory neuropathies which include Gharcot-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, anomia, 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 angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia,
muscle
hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as
muscle rigidity
such as stiff man syndrome, muscle spasticity, paralysis such as facial
paralysis which
includes Herpes Zoster Oticus, Gastxoparesis, 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
andwocal
cord paralysis, paresis, phantom limb, taste.disorders such as ageusia and
dysgeusia, vision
disorders such as amblyopia, blindness, color vision defects, diplopia,
hemianopsia,
scotorria and subnormal vision, sleep disorders such as hypersomnia which
includes Kleine-
Leviri 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, arnyotrophic lateral sclerosis, Lambent-Eaton
Myasthenic
Syndrome, motor neuron disease, muscular atrophy such as spinal muscular
atrophy,
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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 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.
[655] ~ 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
[656j ~ Polynucleotides or polypeptides, or agonists or antagonists of the
present
invention, may be used to treat, prevent, diagnose, and/or pro.gnose disorders
and/or
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diseases related to hormone imbalance, and/or disorders or diseases of the
endocrine
system.
[657] Hormones secreted by the glands of the endocrine system control physical
growth, sexual function, metabolism, and other functions. Disorders may be
classified in
two ways: disturbances in the production of hormones, and the inability of
tissues to
respond to hormones. The etiology of these hormone imbalance or endocrine
system
diseases, disorders or conditions may be genetic, somatic, such as cancer and
some
autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or
infectious.
Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or
antagonists of the
present invention can be used as a marker or detector of a particular disease
or disorder
related to the endocrine system and/or hormone imbalance.
[658] 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).
[659] Endocrine, system andlor hormone imbalance disorders and/or diseases
include
disorders and/or diseases. of the pancreas, such as, for example, diabetes
mellitus, diabetes
insipidus, congenital pancreatic agenesis, pheochromocytoma--islet cell tumor
syndrome;
disorders and/or diseases of the adrenal glands such as, for example,
Addison's Disease,
corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome,
hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the
pituitary gland,
such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism,
pituitary
adenoma, panhypopituitarisW , 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 andlor
diseases of the
hypothalamus.
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[660] 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 or ovaries further
include, for example,
ovarian cancer, polycystic ovary syndrome, I~linefelter'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.
[661] 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.
[662] 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
[663] The polynucleotides or polypeptides, or agonists or antagonists of the
invention
may be used for the diagnosis, treatment, or prevention of diseases andlor
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.
[664] Reproductive system disorders and/or diseases include diseases and/or
disorders
of the testes, including testicular atrophy, testicular feminization,
cryptorchism (unilateral
and bilateral), anorchia,, ectopic testis, epididymitis and orchitis
(typically resulting from
infections such as, for example, gonorrhea, mumps, tuberculosis, and
syphilis), testicular
torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell
carcinomas,
teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal
tumors (e.g.,
Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal
hernia, and
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disorders of sperm production (e..g., immotile cilia syndrome, aspermia,
asthenozoospermia,
azoospermia, oligospermia, and teratozoospermia).
[665] 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, gr. anulomatous
prostatitis,
malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate
neoplastic
disorders, including adenocarcinomas, transitional cell carcinomas, ductal
carcinomas, and
squamous cell carcinomas.
[666] Additionally, the compositions of the invention may be useful in the
diagnosis,
treatment, and/or prevention of disorders or diseases of the penis and
urethra, including
_ .inflammatory disorders, such as balanoposthitis, balanitis xerotica
obliterans, phimosis,
paraphimosis, syphilis, herpes' simplex virus, gonorrhea, non-gonococcal
urethritis,
chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma
acuminatum, condyloma latum, and pearly penile papules; urethral
abnormalities, such as
hypospadias, epispadias, and phimosis; premalignant lesions, including
Erythroplasia of '
Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-
Lowenstein,
and varrucous carcinoma; penile cancers, including squamous cell carcinomas,
carcinoma
in situ,. verrucous carcinoma, and disseminated penile carcinoma; urethral
neoplastic
disorders, including penile urethral carcinoma, brlbomembranous urethral
carcinoma, and
prostatic urethral carcinoma; and erectile disorders, such as priapism,
Peyronie's disease,
erectile dysfunction, and impotence.
[667] 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.
[668] 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.
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[669] 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.
[670] Disorders and/or diseases of the uterus include dysmenorrhea,
retroverted uterus,
endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea,
Cushing's
' syndrome, hydatidiform moles, Asherman's syndrome, premature menopause,
precocious
puberty; uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant
hormonal'
signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas,
and
sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or
antagonists of the
invention may be useful as a marker or detector of, as well as in the
diagnosis, treatment,
and/or prevention ~of congenital uterine abnormalities, such as bicornuate
uterus, septate
uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary
rudimentary horn,
unicornuate uterus with a non-communicating cavitary rudimentary horn,
unicornuate
uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus,
and T-shaped
uterus.
[671] 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 mucirious
adenocarcinoma, and
Ovarian Krukenberg tumors).
[672] 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
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carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell
neoplasia,
and columnar cell neoplasia).
[673] 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, pblypeptides, and agonists or antagonists
of the present
invention may be used in the diagnosis, treatment, and/or prevention of
diseases that can
complicate pregnancy, including heart disease, heart failure, rheumatic heart
disease,
congenital heart disease, mitral valve prolapse, high blood pressure, anemia,
kidney disease,
infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious
hepatitis,
chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease,
thyroiditis,
hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis
of the liver,
primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid
arthritis,
myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian
cysts,
gallbladder disorders,and obstruction of the intestine.
[674] 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
ahnnrmal fetal nncitinnl chnnlrler dvctn~i~ nrnlancPrl nmhiliral rnrrl
arnnintir flnirl
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pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease,
pelvic
congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia,
ruptured fallopian
tube, and Mittelschmerz.
Infectious Disease ' ~ . '
[677] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention can be used to treat or detect infectious agents. For example, by
increasing the
immune response, particularly increasing the proliferation and differentiation
of B and/or T
cells, infectious diseases may be treated. The immune response may be
increased by either
enhancing an existing immune response, or by initiating a new immune response.
Alternatively, polynucleotides or polypeptides, as well as agonists or
antagonists of the
present invention may also directly inhibit the infectious agent, without
necessarily eliciting
an immune response.
[678] Viruses are one example of an infectious agent that can cause disease or
symptoms that can be treated or detected by a polynucleotide or polypeptide
and/or agonist
or antagonist of the present invention. Examples of viruses, include, but are
not limited to
Examples of viruses, include, but are not limited to the following DNA and RNA
viruses
and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus,
Birnaviridae,
Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, 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, hernorrhagic fever, Measles, Mumps,
Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually
transmitted
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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.
[679] 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., Clostridium botulinum, Clostridium dificile, Clostridium perfringens,
Clostridium
tetani), Coccidioides, Corynebacterium (e.g., Cor~nebacterium diptheriae),
Cryptococcus,
Dermatocycoses, E. coli (e.g., Enterotoxigenic~E. coli and Enterohemorrhagic
E. coli),
Enterobacter (e.g. Erzterobacter 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), Leptospira, Listeria (e.g.,
Listeria
monocytogerzes), 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.,
Pseudomorzas aervcgirzosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp.,
Leptospira
spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus
aureus),
Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus
przeumoniae 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,
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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. In specific embodiments,
polynucleotides, polypeptides, agonists or antagonists of the invention are
used to treat:
tetanus, diptheria, botulism, and/or meningitis type B.
[680] 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,
Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g.,
Plasmodiurn
virax, Plasmodium falciparium, Plasmodium mala~iae and Plasmodium ovate).
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.
[681] 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
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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
[682] Polynucleotides or polypeptides, as well as agonists'or antagonists of
the present
invention can be used to differentiate, proliferate, and attract cells,
leading to the
regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of
tissues could
be used to repair, replace, or protect tissue damaged by congenital defects,
trauma (wounds,
burns, incisions, or ulcers), age, disease (e.g. osteoporosis,
osteocarthritis, periodontal
disease, liver failure), surgery, including cosmetic plastic surgery,
fibrosis, reperfusion
injury, or systemic cytokine damage.
[683] 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.
[684] 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
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.
[685] 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
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neuropathy (e.g., resulting from chemotherapy or other medical therapies),
localized
neuropathies, and central nervous system diseases (e.g., Alzheimer's disease,
Parkinson's
disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager
syndrome),
could all be treated using the polynucleotides or polypeptides, as well as
agonists or
antagonists of the present invention.
Gastrointestinal Disorders .
[686] 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 (careinoid tumor of the small intestine, non-Hodgkin's
lymphoma of
the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.
[687] Gastrointestinal disorders include dysphagia, odynophagia, inflammation
of the
esophagus, peptic esophagitis, gastric reflex, submucosal fibrosis and
stricturing, Mallory-
Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas,
gastric retention
disorders, gastroenteritis, gastric atrophy, gastriclstomach cancers, polyps
of the stomach,
autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis
(bacterial, viral,
eosinophilic, stress-induced, chronic .erosive, atrophic, plasma cell, and
Menetrier's), and
peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst,
mesenteric
lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms,
peritonitis,
pneumoperitoneum, bubphrenic abscess,).
[688j Gastrointestinal disorders also include disorders associated with the.
small
intestine, such as malabsorption syndromes, distension, irritable bowel
syndrome, sugar
intolerance, celiac disease, duodenal ulcers, duodenitis, tropical spree,
Whipple's disease,
intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of
the ileum,
Meckel's diverticulum, multiple diverticula, failure of complete rotation of
the small and
large intestine, lymphoma, and bacterial and parasitic diseases (such as
Traveler's diarrhea,
typhoid and paratyphoid, cholera, infection 'by Roundworms (Ascariasis
lumbricoides),
Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis),
Tapeworms (Taenia saginata, Echinococcus gra~culosus, Diphyllobothrium spp.,
and T.
solium).
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[689] 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, 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).
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[690] 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)).
[691] Gallbladder diseases include gallstones (cholelithiasis and
choledocholithiasis),
postcholecystectomy syndrome, diverticulosis of the gallbladder, acute
cholecystitis,
chronic cholecystitis, bile duct tumors, and mucocele.
[692] 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, coloriic
neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma),
colon
carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis,
megacolon
[Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis,
sigmoin
neoplasms]), constipation, Crohn's disease, diairhea (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, ileal neoplasms, intestinal polyps, jejunal neoplasms,
rectal
neoplasms), intestinal obstruction (afferent loop syndrome, duodenal
obstruction, impacted
feces, intestinal pseudo-obstruction [cecal volvulus], intussusception),
intestinal
perforation, intestinal polyps (colonic polyps, gardner syndrome, peutz-
jeghers syndrome),
jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop
syndrome, celiac
disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's
disease),
mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-
losing
enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases,
fecal incontinence,
hemorrhoids, proctitis, rectal fistula, rectal prolapse, recfocele), peptic
ulcer (duodenal
ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-
Ellison
syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases
(e.g.,
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achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular
ectasia, gastric
fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic),
gastroparesis, stomach
dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric
polyps, gastric
adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer,
stomach
volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis,
hyperemesis
gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis. '
[693] 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., GREST 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
[694] Polynucleotides or polypeptides, as well as agonists or antagonists of
the present
invention may have chemotaxis activity. A chemotaxic molecule attracts or
mobilizes cells
(e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils,
epithelial and/or
endothelial cells) to a particular site in the body, such as inflammation,
infection, or site of
hyperproliferation. The mobilized cells can then fight off and/or heal the
particular trauma
or abnormality.
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[695] 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 filiroblasts, which can be
used to treat
wounds.
[696] 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
[697] 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.
[698] 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 _
9
known techniques.
[699] 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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CA 02395811 2002-06-21
WO 01/55306 PCT/USO1/01304
containing the molecule to observe binding, stimulation, or inhibition of
activity of either
the polypeptide or the molecule.
[700] 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.
[701] 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.
[702] 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.
[703] 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.
[704] , 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.
[705] As an alternative approach for receptor identification, the labeled
polypeptides
can be photoaffinity linked with cell membrane or extract preparations that
express the
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WO 01/55306 PCT/USO1/01304
receptor molecule: Cross-linked material is resolved by PAGE analysis. and
exposed to X-
ray film. The labeled complex containing the receptors of the polypeptides can
be excised,
resolved into peptide fragments, and subjected to protein microsequencing. The
amino acid
sequence obtained from microsequencing would be used to design a set of
degenerate
oligonucleotide probes to screen a cDNA library to identify the genes encoding
the putative
receptors.
[706] 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. Biotechfaiques 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 PCR, random nucleotide insertion or other
methods
prior to recombination. ,In another embodiment, one or more components,
motifs, sections,
parts, domains, fragments, etc., of the polypeptide of the present invention
may be
recombined with one or more components, motifs, sections, parts, domains,
fragments, etc.
of one or more heterologous molecules. In preferred embodiments, the
heterologous
molecules are family members. In further preferred embodiments, the
heterologous
molecule is a growth factor. such as, for example, platelet-derived growth
factor (PDGF),
insulin-like growth factor (IGF-I), transforming growth factor (fiGF)-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).
238
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