Note: Descriptions are shown in the official language in which they were submitted.
CA 02309501 2004-04-O1
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NOTE: For additional volumes please contact the Canadian Patent Office.
$OSA/PC,f CA 02453344 2004-O1-21
1
NOVEL NUCLEIC ACIDS AND SECRETED
POLYPEPTIDES
1. CROSS REFERENCE T~ RELATED APPLICATIONS
S This application is a continuation-in-part application of U.S. Application
Serial No.
09/488,725 filed January 21, 2000 entitled "Novel Contigs Obtained from
Various
Libraries", Attorney Docket No. 784; U.S. Application Serial No. 09/491,404
filed January
25, 2000 entitled "Novel Contigs Obtained from Various Libraries"., Attorney
Docket No.
785; U.S. Application Serial No. 09/496,914 filed February 03, 2000 entitled
"Novel Contigs
Obtained from Various Libraries", Attorney Docket No. 787; U.S. Application
Serial No.
09/S 15,126 filed February 28, 2000 entitled "Novel Contigs Obtained from
Various
Libraries", Attorney Docket No. 798; U.S. Application Serial No. 09/S 19;705
filed March
07, 2000 entitled "Novel Contigs Obtained from Various Libraries", Attorney
Docket No.
789; U.S. Application Serial No. 09/540,217 filed March 31, 2000 entitled
"Novel Contigs
1 S Obtained from Various Libraries", Attorney Docket No. 790; U.S.
Application Serial No.
09/5S2,929 filed April 18, 2000 entitled "Novel Contigs Obtained from Various
Libraries",
Attorney Docket No. 791; U.S. Application Serial No. 09/577,408 f led May 18,
2000
entitled "Novel Contigs Obtained from Various Libraries", Attorney Docket No.
792; all of
which are incorporated herein by reference in their entirety.
2. BACKGROUND OF THE INVENTION
2.1 TECHNICAL FIELD
The present invention provides novel polynucleotides and proteins encoded by
such
2S polynucleotides, along with uses for these polynucleotides and proteins,
far example in
therapeutic, diagnostic and research methods.
2.2 BACKGROUND
Technology aimed at the discovery of protein factors (including e.g.,
cytokines, such
as lymphokines, interferons, circulating soluble factors, chemokines, and
interleukins) has
matured rapidly over the past decade. The now routine hybridization cloning
and expression
cloning techniques clone novel polynucleotides "directly'° in the sense
that they rely on
805AIrCT CA 02453344 2004-O1-21
2
information directly related to the discovered protein (i.e., partial
DNA/axnino acid sequence
of the protein in the case of hybridization cloning; activity of the protein
in the case of
expression cloning). More recent "indirect" cloning techniques such as signal
sequence
cloning, which isolates DNA sequences based on the presence of a now well-
recognized
secretory leader sequence motif, as well as various PCR-based or low
stringency
hybridization-based cloning techniques, have advanced the state of the art by
making
available large numbers of DNA/amino acid sequences for proteins that are
known to have
biological activity, for example, by virtue of their secreted nature in the
case of leader
sequence cloning, by virtue of their cell or tissue source in the case of PCR-
based
techniques, or by virtue of structural similarity to other genes of known
biological activity.
Identified polynucleotide and polypeptide sequences have numerous applications
in,
for example, diagnostics, forensics, gene mapping; identification of mutations
responsible
for genetic disorders or other traits, to assess biodiversity, and to produce
many other types
of data and products dependent on DNA and amino acid sequences.
3. SUMMARY OF THE INVENTION
The compositions of the present invention include novel isolated polypeptides,
novel
isolated polynucleotides encoding such polypeptides, including recombinant DNA
molecules,
cloned genes or degenerate variants thereof, especially naturally occurring
variants such as
allelic variants, antisense polynucleotide molecules, and antibodies that
specifically recognize
one or more epitopes present on such polypeptides, as well as hybridomas
producing such
antibodies.
The compositions of the present invention additionally include vectors,
including
expression vectors, containing the polynucleotides of the invention, cells
genetically engineered
to contain such polynucleotides and cells genetically engineered to express
such
polynucleotides.
The present invention relates to a collection or library of at least one novel
nucleic acid
sequence assembled from expressed sequence tags (ESTs) isolated mainly by
sequencing by
hybridization (SBIT), and in some cases, sequences obtained from one or more
public
databases. The invention relates also to the proteins encoded by such
polynucleotides, along
with therapeutic, diagnostic and research utilities for these polynucleotides
and proteins. These
nucleic acid sequences are designated as SEQ ID NO: 1-244, or 489-706 and are
provided in
the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A
is adenine; C is
805A/PCT ~ 02453344 2004-O1-21
3
cytosine; G is guanine; T is thymine; and N is any of the four bases or
unknown. In the amino
acids provided in the Sequence Listing, * corresponds to the stop codon.
The nucleic acid sequences of the present invention also include, nucleic acid
sequences that hybridize to the complement of SEQ ID NO: 1-244, or 489-706
under stringent
S hybridization conditions; nucleic acid sequences which are allelic variants
or species
homologues of any of the nucleic acid sequences recited above, or nucleic acid
sequences that
encode a peptide comprising a specific domain or truncation of the peptides
encoded by SEQ
ID NO: I-244, or 489-706. A polynucleotide comprising a nucleotide sequence
having at least
90% identity to an identifying sequence of SEQ ID NO: 1-244, or 489-706 or a
degenerate
variant or fragment thereof. The identifying sequence can be 100 base pairs in
length.
The nucleic acid sequences of the present invention also include the sequence
information from the nucleic acid sequences of SEQ ID NO: 1-244., or 489-706.
The sequence
information can be a segment of any one of SEQ ID NO: 1-244, or 489-706 that
uniquely
identifies or represents the sequence information of SEQ ID NO: 1-244, or 489-
706.
A collection as used in this application can be a collection of only one
polynueleotide.
The collection of sequence information or identifying information of each
sequence can be
provided on a nucleic acid array. In one embodiment, segments of sequence
information are
provided on a nucleic acid array to detect the polynueleotide that contains
the segment. The
array can be designed to detect foil-match or mismatch to the polynucleotide
that contains the
segment. The collection can also be provided in a computer-readable format.
This invention also includes the reverse or direct complement of any of the
nucleic acid
sequences recited above; cloning or expression vectors containing the nucleic
acid sequences;
and host cells or organisms transformed with these expression vectors. Nucleic
acid sequences
(or their reverse or direct complements) according to the invention have
numerous applications
in a variety of techniques known ~o those skilled in the art of molecular
biology, such as use as
hybridization probes, use as primers for PCR, use in an array, use in computer-
readable media,
use in sequencing full-length genes, use for chromosome and gene mapping, use
in the
recombinant production of protein, and use in the generation of anti-sense DNA
or RNA, their
chemical analogs and the like.
In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: I-244, or
489-
706 or novel segments or parts of the nucleic acids of the invention are used
as primers in
expression assays that are well known in the art. In a particularly preferred
embodiment, the
nucleic acid sequences of SEQ ID NO: 1-244, or 489-706 or novel segments or
parts of the
80St-1IPCT CA 02453344 2004-O1-21
4
nucleic acids provided herein are used in diagnostics for identifying
expressed genes or, as well
known in the art and exemplified by Vollrath et al., Science 258:52-S9 (1992),
as expressed
sequence tags for physical mapping of the human genome.
The isolated polynucleotides of the invention include, but are not limited to,
a
polynucleotide comprising any one of the nucleotide sequences set forth in SEQ
ID NO: I-244,
or 489-706; a polynucleotide comprising any of the full length protein coding
sequences of
SEQ ID NO: 1-244, or 489-706; and a polynucleotide comprising any of the
nucleotide
sequences of the mature protein coding sequences of SEQ ID NO: I-244, or 489-
706. The
polynucleotides of the present invention also include, but are not limited to,
a polynucleotide
I 0 that hybridizes under stringent hybridization conditions to (a) the
complement of any one of the
nucleotide sequences set forth in SEQ ID NO: I-244, or 489-706; (b) a
nucleotide sequence
encoding any one of the amino acid sequences set forth in SEQ ID NO: I-244, or
489-706; (c) a
polynucleotide which is an allelic variant of any polynucleotides recited
above; (d) a
polynucleotide which encodes a species homolog (e.g. orthologs) of ably of the
proteins recited
I 5 above; or (e) a polynucleotide that encodes a polypeptide comprising a
specific domain or
truncation of any of the polypeptides comprising an amino acid sequence set
forth in the
Sequence Listing.
'The isolated polypeptides of the invention include, but are not limited to, a
polypeptide
comprising any of the amino acid sequences set forth in the Sequence Listing;
or the
20 corresponding full length or mature protein. Polypeptides of the invention
also include
polypeptides with biological activity that are encoded by (a) any of the
polynucleotides having
a nucleotide sequence set forth in SEQ ID NO: I-244, or 489-706; or (b)
polynucleotides that
hybridize to the complement of the polynucleotides of (a) under stringent
hybridization
conditions. Biologically active variants of any of the polypeptide sequences
in the Sequence
2S Listing, and "substantial equivalents" thereof (e.g., with at least about
6S%, 70%, 7S%, 80%,
8S%, 90%, 9S%, 98% or 99% amino acid sequence identity) that preferably retain
biological
activity are also contemplated. The polypeptides of the invention may be
wholly or partially
chemically synthesized but are preferably produced by recombinant means using
the genetically
engineered cells (e.g. host cells) of the invention.
30 The invention also provides compositions comprising a polypeptide of the
invention.
Polypeptide compositions of the invention may further comprise an. acceptable
carrier, such
as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
805A/PCT ~ 02453344 2004-O1-21
The invention also provides host cells transformed or transfected with a
polynucleotide of the invention.
The invention also relates to methods for producing a polypeptide of the
invention
comprising growing a culture of the host cells of the invention in a suitable
culture medium
S under conditions permitting expression of the desired polypeptide, and
purifying the
polypeptide from the culture or from the host cells. Preferred embodiments
include those in
which the protein produced by such processes is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a
variety
of techniques known to those skilled in the art of molecular biology. These
techniques
include use as hybridization probes, use as oligomers, or primers, for PCR,
use for
chromosome and gene mapping, use in the recombinant production of protein, and
use in
generation of anti-sense DNA or RNA, their chemical analogs and the like. For
example,
when the expression of an mRNA is largely restricted to a particular cell or
tissue type,
polynucleotides of the invention can be used as hybridization probes to detect
the presence
of the particular cell or tissue mRNA in a sample using, e.g., i~ situ
hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as
expressed sequence tags for identifying expressed genes or, as well known in
the art and
exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed
sequence tags for
physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of
conventional
procedures and methods that are currently applied to other proteins. For
example, a
polypeptide of the invention can be used to generate an antibody that
specifically binds the
polypeptide. Such antibodies, particularly monoclonal antibodies, are useful
for detecting or
quantitating the polypeptide in tissue. The polypeptides of the invention can
also be used as
molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical
condition which comprises the step of administering to a mammalian subject a
therapeutically effective amount of a composition comprising a polypeptide of
the present
invention and a pharmaceutically acceptable carrier.
In particular, the polypeptides and polynucleotides of the invention can be
utilized,
for example, in methods for the prevention and/or treatment of disorders
involving aberrant
protein expression or biological activity.
80SA/PCT ~ 02453344 2004-O1-21
6
The present invention further relates to methods for detecting the presence of
the
polynucleotides or polypeptides of the invention in a sample Such methods can,
for
example, be utilized as part of prognostic and diagnostic evaluation of
disorders as recited
herein and for the identification of subjects exhibiting a predisposition to
such conditions.
S The invention provides a method .for detecting the polynucleotides of the
invention in a
sample, comprising contacting the sample with a compound that binds to and
forms a
complex with the polynucleotide of interest for a period sufficient to form
the complex and
under conditions sufficient to form a complex and detecting the complex such
that if a
complex is detected, the polynucleotide of interest is detected. The invention
also provides a
method for detecting the polypeptides of the invention in a sample comprising
contacting the
sample with a compound that binds to and forms a complex with the polypeptide
under
conditions and for a period sufficient to form the complex and detecting the
formation of the
complex such that if a complex is formed, the polypeptide is detected.
The invention also provides kits comprising polynucleotide probes andlor
1 S monoclonal antibodies, and optionally quantitative standards, for caxrying
out methods of the
invention. Furthermore, the invention provides methods for evaluating the
efficacy of drugs,
and monitoring the progress of patients, involved in clinical trials for the
treatment of
disorders as recited above.
The invention also provides methods for the identification of compounds that
modulate (i.e., increase or decrease) the expression or activity of the
polynucleotides and/or
polypeptides of the invention. Such methods can be utilized, for example, for
the
identification of compounds that can ameliorate symptoms of disorders as
recited herein.
Such methods can include, but are net limited to, assays for identifying
compounds and
other substances that interact with (e.g., bind to) the polypeptides of the
invention. The
2S invention provides a method for identifying a compound that binds to the
polypeptides of the
invention comprising contacting the compound with a polypeptide of the
invention in a cell
for a time sufficient to form a polypeptide/compound complex, wherein the
complex drives
expression of a reporter gene sequence in the cell; and detecting the complex
by detecting
the reporter gene sequence expression such that if expression of the reporter
gene is detected
the compound that binds to a polypeptide of the_invention is identified.
The methods of the invention also provide methods for treatment which involve
the
administration of the polynucleotides or polypeptides of the invention to
individuals
exhibiting symptoms or tendencies. In addition, the invention encompasses
methods for
SOS~p~~ CA 02453344 2004-O1-21
7
treating diseases or disorders as recited herein comprising administering
compounds and
other substances that modulate the overall activity of the target gene
products. Compounds
and other substances can affect such modulation either on the level of target
gene/protein
expression or target protein activity.
The polypeptides of the present invention and the polynucleotides encoding
them are
also useful for the same functions known to one of skill in the art as the
polypeptides and
polynucleotides to which they have homology (set forth in Table 2); for which
they have a
signature region (as set forth in Table 3); or for which they have homology to
a gene family
(as set forth in Table 4). If no homology is set forth for a sequence, then
the polypeptides
and polynucleotides of the present invention are useful for a variety of
applications, as
described herein, including use in arrays for detection.
4. DETAILED DESCT~IPTION OF THE INVENTION
4.1 DEFINITIONS
It must be noted that as used herein and in the appended claims, the singular
forms
"a", "an" and "the" include plural references unless the context clearly
dictates otherwise.
The term "active" refers to those forms of the polypeptide which retain the
biologic
and/or immunologic activities of any naturally occurring polypeptide.
According to the
invention, the terms "biologically active" or "biological activity" refer to a
protein or peptide
having structural, regulatory or biochemical functions of a naturally
occurring molecule.
Likewise "immunologically active" or "immunological activity" refers to the
capability of
the natural, recombinant or synthetic polypeptide to induce a specific immune
response in
appropriate animals or cells and to bind with specific antibodies.
The term "activated cells" as used in this application are those cells which
are
engaged in extracellular or intracellular membrane trafficking, including the
export of
secretory or enzymatic molecules as part of a normal or disease process.
The terms "complementary" or "complementarity" refer to the natural binding of
polynucleotides by base pairing. For example, the sequence 5'-AGT-3' binds to
the
complementary sequence 3'-TCA-5'. Complementarity between two single-stranded
molecules may be "partial" such that only certain portions) of the nucleic
acids bind or it
may be "complete" such that total complementarity exists between the single
stranded
805A/PCT ~ 02453344 2004-O1-21
molecules. The degree of complementarity between the nucleic acid strands has
significant
effects on the efficiency and strength of the hybridization between the
nucleic acid strands.
The term "embryonic stem cells (ES)" refers to a cell that can give rise to
many
differentiated cell types in an embryo or an adult, including the germ cells.
The term "germ
line stem cells (GSCs)" refers to stem cells derived from primordial stem
cells that provide a
steady and continuous source of germ cells for the production of gametes. The
term
"primordial germ cells (PGCs)" refers to a small population of cells set aside
from other cell
lineages particularly from the yolk sac, mesenteries, or gonadal ridges during
embryogenesis
that have the potential to differentiate into germ cells and other cells. PGCs
are the source
from which GSCs and ES cells are derived. The PGCs, the GSCs and the ES cells
are
capable of self renewal. Thus these cells not only populate the germ line and
give rise to a
plurality of terminally differentiated cells that comprise the adult
specialized organs, but are
able to regenerate themselves.
The term "expression modulating fragment," EMF, means a series of nucleotides
which modulates the expression of an operably linked ~RF or another EMF.
As used herein, a sequence is said to "modulate the expression of an operably
linked
sequence" when the expression of the sequence is altered by the presence of
the EMF.
EMFs include, but are not limited to, promoters, and promoter modulating
sequences
(inducible elements). ~ne class of EMFs are nucleic acid fragments which
induce the
expression of an operably linked GRF in response to a specific regulatory
factor or
physiological event.
The terms "nucleotide sequence" or "nucleic acid" or "polynucleotide" or
"oligonucleotide" are used interchangeably and refer to a heteropolymer of
nucleotides or
the sequence of these nucleotides. These phrases also refer to DNA or RNA of
genomic or
synthetic origin which may be single-stranded or double-stranded and may
represent the
sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-
like or RNA-like
material. In the sequences herein A is adenine, C is cytosine, T is thymine, G
is guanine and
N is A, C, G, or T (U) or unknown. It is contemplated that where the
polynucleotide is
RNA, the T (thymine) in the sequences provided herein is substituted with U
(uracil).
Generally, nucleic acid segments provided by this invention rnay be assembled
from
fragments of the genome and short oligonucleotide linkers, or from a series of
oligonucleotides, or from individual nucleotides, to provide a synthetic
nucleic acid which is
~~~~P~-T CA 02453344 2004-O1-21
9
capable of being expressed in a recombinant transcriptional unit comprising
regulatory
elements derived from a microbial or viral operon, or a eukaryotic gene.
The terms "oligonucleotide fragment" or a "polynucleotide fragment",
°'portion,'° or
"segment°' or "probe" or "primer" are used interchangeably and refer to
a sequence of
nucleotide residues which are at least about 5 nucleotides, more preferably at
least about 7
nucleotides, more preferably at least about 9 nucleotides, more preferably at
least about 11
nucleotides and most preferably at least about 17 nucleotides. The fragment is
preferably
less than about 500 nucleotides, preferably less than about 200 nucleotides,
more preferably
less than about 100 nucleotides, more preferably less than about 50
nucleotides and most
preferably less than 30 nucleotides. Preferably the probe is from about 6
nucleotides to
about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more
preferably
from about 17 to 30 nucleotides and most preferably from about 20 to 25
nucleotides.
Preferably the fragments can be used in polymerase chain reaction (PCR),
various
hybridization procedures or microarray procedures to identify or amplify
identical or related
parts of mRNA or DNA molecules. A fragment or segment may uniquely identify
each
polynucleotide sequence of the present invention. Preferably the fragment
comprises a
sequence substantially similar to any one of SEQ ID NO: 1-244, or 489-706.
Probes may, for example, be used to determine whether specific mRNA molecules
are present in a cell or tissue or to isolate similar nucleic acid sequences
from chromosomal
DNA as described by Welsh et al. (Welsh, P.S. et al., 1992, PCR Methods Appl
1:241-250).
They may be labeled by nick translation, Klenow fill-in reaction, PCR, or
other methods
well known in the art. Probes of the present invention, their preparation
and/or labeling are
elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold
Spring Harbor Laboratory, NY; or Ausubel, F.M. et al., 1989, Current Protocols
in
Molecular Biology, John Wiley & Sons, New York NY, both of which are
incorporated
herein by reference in their entirety.
The nucleic acid sequences of the present invention also include the sequence
information from the nucleic acid sequences of SEQ ID NO: 1-244, or 489-706.
The
sequence information can be a segment of any one of SEQ ID NO: 1 ~-244, or 489-
706 that
uniquely identifies or represents the sequence information of that sequence of
SEQ ID NO:
1-244, or 489-706, or those segments identified in Tables 3, 5., 6, and 8. One
such segment
can be a twenty-mer nucleic acid sequence because the probability that a
twenty-mer is fully
matched in the human genome is I in 300. In the human genome, there are three
billion base
CA 02453344 2004-O1-21
pairs in one set of chromosomes. Because 42° possible twenty-mers
exist, there are 300
times more twenty-mers than there are base pairs in a set of human
chromosomes. Using
the same analysis, the probability for a seventeen-mer to be fully matched in
the human
genome is approximately 1 in 5. When these segments are used in arrays for
expression
5 studies, fifteen-mer segments can be used. The probability that the fifteen-
mer is fully
matched in the expressed sequences is also approximately one in five because
expressed
sequences comprise less than approximately 5% of the entire genome sequence.
Similarly, when using sequence information for detecting a single mismatch, a
segment
can be a twenty-five mer. The probability that the twenty-five rrrner would
appear in a human
10 genome with a single mismatch is calculated by multiplying the probability
for a full match
(1-425) times the increased probability for mismatch at each nucleotide
position (3 x 25). The
probability that an eighteen mer with a single mismatch can be detected in an
array for
expression studies is approximately one in five. The probability that a twenty-
mer with a single
mismatch can be detected in a human genome is approximately one in five.
The term "open reading frame," ORF, means a series of nucleotide triplets
coding for
amino acids without any termination colons and is a sequence translatable into
protein.
The terms "operably linked" or "operably associated" refer to functionally
related
nucleic acid sequences. For example, a promoter is operably associated or
operably linked
with a coding sequence if the promoter controls the transcription of the
coding sequence.
While operably linked nucleic acid sequences can be contiguous and in the same
reading
frame, certain genetic elements e.g. repressor genes are not contiguously
linked to the coding
sequence but still control transcription/translation of the coding sequence.
The term "pluripotent" refers to the capability of a cell to differentiate
into a number
of differentiated cell types that are present in an adult organism. A
pluripotent cell is
restricted in its differentiation capability in comparison to a totipotent
cell.
The terms "polypeptide" or "peptide" or "amino acid sequence" refer to an
oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and
to naturally
occurring or synthetic molecules. A polypeptide "fragment," "portion," or
"segment" is a
stretch of amino acid residues of at least about 5 amino acids, preferably at
least about 7
amino acids, more preferably at least about 9 amino acids and most preferably
at least about
17 or more amino acids. The peptide preferably is not greater than about 200
amino acids,
more preferably less than 150 amino acids and most preferably less than 100
amino acids.
CA 02453344 2004-O1-21
11
Preferably the peptide is from about 5 to about 200 amino acids. To be active,
any
polypeptide must have sufficient length to display biological and/or
immunological activity.
The term "naturally occurring polypeptide" refers to polypeptides produced by
cells
that have not been genetically engineered and specifically contemplates
various polypeptides
arising from post-translationa.l modifications of the polypeptide including,
but not limited to,
acetylation, carboxylation, glycosylation, phosphorylation, lipidatioxa and
acylation.
The term "translated protein coding portion" means a sequence which encodes
for the
full-length protein which may include any leader sequence or any processing
sequence.
The term "mature protein coding sequence" means a sequence which encodes a
peptide or protein without a signal or leader sequence. The "mature protein
portion" means
that portion of the protein which does not include a signal or leader
sequence. The peptide
may have been produced by processing in the cell which removes any
leader/signal
sequence. The mature protein portion may or may not include the initial
methionine residue.
The methionine residue may be removed from the protein during processing in
the cell. The
peptide may be produced synthetically or the protein may have been produced
using a
polynucleotide only encoding for the mature protein coding sequence.
The term "derivative" refers to polypeptides chemically modified by such
techniques
as ubiquitination, labeling (e.g., with radionuclides or various enzymes),
covalent polymer
attachment such as pegylation (derivatization with polyethylene glycol) and
insertion or
substitution by chemical synthesis of amino acids such as ornithine, which do
not normally
occur in human proteins.
The term "variant"(or "analog") refers to any polypeptide differing from
naturally
occurring polypeptides by amino acid insertions, deletions, and substitutions,
created using,
a g., recombinant DNA techniques. Guidance in determining which amino acid
residues
may be replaced, added or deleted without abolishing activities of interest,
may be found by
comparing the sequence of the particular polypeptide with that of homologous
peptides and
minimizing the number of amino acid sequence changes made in regions of high
homology
(conserved regions) or by replacing amino acids with consensus sequence.
Alternatively, recombinant variants encoding these same or similar
polypeptides may
be synthesized or selected by making use of the "redundancy" in the genetic
code. Various
codon substitutions, such as the silent changes which produce various
restriction sites, may
be introduced to optimize cloning into a plasmid or viral vector or expression
in a particular
prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may
be
CA 02453344 2004-O1-21
805AIPCT
12
reflected in the polypeptide or domains of other peptides added to the
polypeptide to modify
the properties of any part of the polypeptide, to change characteristics such
as Iigand-
binding afFnities, interchain affinities, or degradation/turnover rate.
Preferably, amino acid '°substitutions" are the result of replacing one
amino acid with
another amino acid having similar structural and/or chemical properties, l,
e., conservative
amino acid replacements. "Conservative" amino acid substitutions may be made
on the
basis of similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the
amphipathic nature of the residues involved. For example, nonpolar
(hydrophobic) amino
acids include alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and
methionine; polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine,
asparagine, and glutamine; positively charged (basic) amino acids include
arginine, lysine,
and histidine; and negatively charged (acidic) amino acids include aspartic
acid and glutamic
acid. "Insertions" or "deletions" are preferably in the range of about 1 to 20
amino acids,
more preferably 1 to I O amino acids. The variation allowed may be
experimentally
determined by systematically making insertions, deletions, or substitutions of
amino acids in
a polypeptide molecule using recombinant DNA techniques and assaying the
resulting
recombinant variants for activity.
Alternatively, where alteration of function is desired, insertions, deletions
or
non-conservative alterations can be engineered to produce altered
polypeptides. Such
alterations can, for example, alter one or more of the biological functions or
biochemical
characteristics of the polypeptides of the invention. For example, such
alterations may
change polypeptide characteristics such as ligand-binding affinities,
interchain affinities, or
degradation/turnover rate. Further, such alterations can be selected so as to
generate
polypeptides that are better suited for expression, scale up and the like in
the host cells
chosen for expression. For example, cysteine residues can be deleted or
substituted with
another amino acid residue in order to eliminate disulfide bridges.
The terms "purified" or "substantially purified" as used herein denotes that
the
indicated nucleic acid or polypeptide is present in the substantial absence of
other biological
macromolecules, e.g., polynucleotides, proteins, and the like. In one
embodiment, the
polynucleotide or polypeptide is purified such that it constitutes at least
95% by weight,
more preferably at least 99% by weight, of the indicated biological
macromolecules present
(but water, buffers, and other small molecules, especially molecules having a
molecular
weight of less than 1000 daltons, can be present).
CA 02453344 2004-O1-21
805A/PCT
13
The term "isolated" as used herein refers to a nucleic acid or polypeptide
separated
from at least one other component (e.g., nucleic acid or polypeptide) present
with the
nucleic acid or polypeptide in its natural source. In one embodiment, the
nucleic acid or
polypeptide is found in the presence of (if anything) only a solvent, buffer,
ion, or other
component normally present in a solution of the same. The terms
'°isolated" and "purified"
do not encompass nucleic acids or polypeptides present in their natural
source.
The term "recombinant," when used herein to refer to a polypeptide or protein,
means
that a polypeptide or protein is derived from recombinant (e.g., microbial,
insect, or
mammalian) expression systems. "Microbial" refers to recombinant polypeptides
or proteins
made in bacterial or fungal (e.g., yeast) expression systems. As a product,
"recombinant
microbial" defines a polypeptide or protein essentially free of native
endogenous substances
and unaccompanied by associated native glycosylation. Polypeptides or proteins
expressed
in most bacterial cultures, e.g., E. coli, will be free of glycosylation
modifications;
polypeptides or proteins expressed in yeast will have a glycosylation pattern
in general
different from those expressed in mammalian cells.
The term "recombinant expression vehicle or vector" refers to a plasmid or
phage or
virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An
expression
vehicle can comprise a transcriptional unit comprising an assembly of (1) a
genetic element
or elements having a regulatory role in gene expression, for example,
promoters or
enhancers, (2) a structural or coding sequence which is transcribed into mRNA
and
translated into protein, and (3) appropriate transcription initiation and
termination sequences.
Structural units intended for use in yeast or eukaryotic expression systems
preferably include
a leader sequence enabling extracellular secretion of translated protein by a
host cell.
Alternatively, where recombinant protein is expressed without a leader or
transport
sequence, it may include an amino terminal methionine residue. This residue
may or may
not be subsequently cleaved from the expressed recombinant protein to provide
a final
product.
The term "recombinant expression system" means host cells which have stably
integrated a recombinant transeriptional unit into chromosomal DNA or carry
the
recombinant transcriptional unit extrachromosomally. Recombinant expression
systems as
defined herein will express heterologous polypeptides or proteins upon
induction of the
regulatory elements linked to the DNA segment or synthetic gene to be
expressed. This term
also means host cells which have stably integrated a recombinant genetic
element or
805L-11TCT CA 02453344 2004-O1-21
14
elements having a regulatory role in gene expression, for example, promoters
or enhancers.
Recombinant expression systems as defined herein will express polypeptides or
proteins
endogenous to the cell upon induction of the regulatory elements linked to the
endogenous
DNA segment or gene to be expressed. The cells can be prokaryotic or
eukaryotic.
The term "secreted" includes a protein that is transported across or through a
membrane, including transport as a result of signal sequences in its amino
acid sequence
when it is expressed in a suitable host cell. "Secreted" proteins include
without limitation
proteins secreted wholly (e.g., soluble proteins) or partially (e.g.,
receptors) from the cell in
which they are expressed. "Secreted" proteins also include without limitation
proteins that
are transported across the membrane of the endoplasmic reti.culum. "Secreted"
proteins are
also intended to include proteins containing non-typical signal sequences
(e.g. Interleukin-1
Beta, see Krasney, P.A. and Young, P.R. (1992) Cytokine 4(2): 134 -143) and
factors
released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see
Arend, W.P. et. al.
(1998) Annu. Rev. Immunol. 16:27-55)
Where desired, an expression vector may be designed to contain a "signal or
leader
sequence" which will direct the polypeptide through the membrane of a cell.
Such a
sequence may be naturally present on the polypeptides of the present invention
or provided
from heterologous protein sources by recombinant DNA techniques.
The term "stringent" is used to refer to conditions that are commonly
understood in
the art as stringent. Stringent conditions can include highly stringent
conditions (i.e.,
hybridization to filter-bound DNA in 0.5 M NaHP04, 7% sodium dodecyl sulfate
(SDS), 1
mM EDTA at 65°C, and washing in O.1X SSCI0.1% SDS at 68°C), and
moderately stringent
conditions (i.e., washing in 0.2X SSC/0.1% SDS at 42°C). Other
exemplary hybridization
conditions are described herein in the examples.
In instances of hybridization of deoxyoligonucleotides, additional exemplary
stringent hybridization conditions include washing in 6X SSC/0.0~% sodium
pyrophosphate
at 37°C (for 14-base oligonucleotides), 48°C (for 17-base
oligonucleotides), 55°C (for 20-
base oligonucleotides), and 60°C (for 23-base oligonucleotides).
As used herein, "substantially equivalent" or "substantially similar" can
refer both to
nucleotide and amino acid sequences, for example a mutant sequence, that
varies from a
reference sequence by one or more substitutions, deletions, or additions, the
net effect of
which does not result in an adverse functional dissimilarity between the
reference and
subject sequences. Typically, such a substantially equivalent sequence varies
from one of
~0~~~,I, CA 02453344 2004-O1-21
1S
those listed herein by no more than about 3S% (i.e., the number of individual
residue
substitutions, additions, and/or deletions in a substantially equivalent
sequence, as
compared to the corresponding reference sequence, divided by the total number
of residues
in the substantially equivalent sequence is about 0.35 or less). Such a
sequence is said to
S have 6S% sequence identity to the listed sequence. In one embodiment, a
substantially
equivalent, e.g., mutant, sequence of the invention varies from a listed
sequence by no more
than 30% (70% sequence identity); in a variation of this embadiment, by no
more than 2S%
(7S% sequence identity); and in a further variation of this embodiment, by no
more than
20% (80% sequence identity) and in a further variation of this embodiment, by
no more than
10% (90% sequence identity) and in a further variation of this embodiment, by
no more that
S% {9S% sequence identity). Substantially equivalent, e.g., mutant, amino acid
sequences
according to the invention preferably have at least 80% sequence identity with
a listed amino
acid sequence, more preferably at least 8S% sequence identity, more preferably
at least 90%
sequence identity, more preferably at least 9S% sequence identity, more
preferably at least
1 S 98% sequence identity, and most preferably at least 99% sequence identity.
Substantially
equivalent nucleotide sequence of the invention can have lower percent
sequence identities,
taking into account, for example, the redundancy or degeneracy of the genetic
code.
Preferably, the nucleotide sequence has at least about 6S% identity, more
preferably at least
about 7S% identity, more preferably at least about 80% sequence identity, more
preferably at
least 8S% sequence identity, more preferably at least 90% sequence identity,
more preferably
at least about 95% sequence identity, more preferably at least 98% sequence
identity, and
most preferably at least 99% sequence identity. For the purposes of the
present invention,
sequences having substantially equivalent biological activity and
substantially equivalent
expression characteristics are considered substantially equivalent. For the
purposes of
2S determining equivalence, truncation of the mature sequence (e.g., via a
mutation which
creates a new stop colon) should be disregarded. Sequence identity may be
determined,
e.g., using the Jotun Heirs method (Heirs, J. (1990) Methods Enzyrnol. 183:626-
64S).
Identity between sequences can also be determined by other methods known in
the art, e.g.
by varying hybridization conditions.
The term "totipotent" refers to the capability of a cell to differentiate into
all of the
cell types of an adult organism.
The term "transformation" means introducing DNA into a suitable host cell so
that
the DNA is replicable, either as an extrachromosomal element, or by
chromosomal
CA 02453344 2004-O1-21
16
integration. The term "transfection" refers to the taking up of an expression
vector by a
suitable host cell, whether or not any coding sequences are in fact expressed.
The term
"infection" refers to the introduction of nucleic acids into a suitable host
cell by use of a
virus or viral vector.
As used herein, an "uptake modulating fragment," UMF, means a series of
nucleotides which mediate the uptake of a linked DNA fragment into a cell.
UMFs can be
readily identified using known UMFs as a target sequence or target motif with
the
computer-based systems described below. The presence and activity of a UMF can
be
confirmed by attaching the suspected UMF to a marker sequence. The resulting
nucleic acid
molecule is then incubated with an appropriate host under appropriate
conditions and the
uptake of the marker sequence is determined. As described above, a UMF will
increase the
frequency of uptake of a linked marker sequence.
Each of the above terms is meant to encompass all that is described for each,
unless
the context dictates otherwise.
4.2 NUCLEIC ACIDS OF THE INVENTION
Nucleotide sequences of the invention are set forth in the Sequence Listing.
The isolated polynucleotides of the invention include a polynucleotide
comprising
the nucleotide sequences of SEQ ID NO: 1-244, or 489-706; a polynucleotide
encoding any
one of the peptide sequences of SEQ ID NO: 1-244, or 489-706; and a
polynucleotide
comprising the nucleotide sequence encoding the mature protein coding sequence
of the
polynucleotides of any one of SEQ ID NO: 1-244, or 489-706. The
polynucleotides of the
present invention also include, but are not limited to, a polynucleotide that
hybridizes under
stringent conditions to (a) the complement of any of the nucleotides sequences
of SEQ ID
NO: 1-244, or 489-706; (b) nucleotide sequences encoding any one of the amino
acid
sequences set forth in the Sequence Listing, or Table 8; (c) a polynucleotide
which is an
allelic variant of any polynucleotide recited above; (d) a polynucleotide
which encodes a
species homolog of any of the proteins recited above; or (e) a polynucleotide
that encodes a
polypeptide comprising a specific domain or truncation of the polypeptides of
SEQ ID NO:
1-244, or 489-706 (for example, as set forth in Tables 3, 5, 6, or 8). Domains
of interest may
depend on the nature of the encoded polypeptide; e.g., domains in receptor-
like polypeptides
include ligand-binding, extracellular, transmembrane, or cytoplasmic domains,
or
combinations thereof; domains in immunoglobulin-like proteins include the
variable
805ti1 PCT CA 02453344 2004-O1-21
17
immunoglobulin-like domains; domains in enzyme-like polypeptides include
catalytic and
substrate binding domains; and domains in ligand polypeptides include receptor-
binding
domains.
The polynucleotides of the invention include naturally occurring or wholly or
partially synthetic DNA, e.g., eDNA and genomic DNA, and RNA, e.g., mRNA. The
polynucleotides may include entire coding region of the cDNA or may represent
a portion of
the coding region of the cDNA.
The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. The corresponding genes can be isolated in accordance with
known methods
using the sequence information disclosed herein. Such methods include the
preparation of
probes or primers from the disclosed sequence information for identification
and/or
amplification of genes in appropriate genomic libraries or other sources of
genomic materials.
Further 5' and 3' sequence can be obtained using methods known in the art. For
example, full
length cDNA or genornic DNA that corresponds to any of the polynucleotides of
SEQ ID NO:
1-244, or 489-706 can be obtained by screening appropriate cDNA or genomic DNA
libraries
under suitable hybridization conditions using any of the polynucleotides of
SEQ ID NO: 1-244,
or 489-706 or a portion thereof as a probe. Alternatively, the polynucleotides
of SEQ ID NO:
1-244, or 489-706 may be used as the basis for suitable primers) that allow
identiEcation
and/or amplification of genes in appropriate genomic DNA or cDNA libraries.
The nucleic acid sequences of the invention can be assembled from ESTs and
sequences
(including cDNA and genomic sequences) obtained from one or more public
databases, such as
dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence
information, representative fragment or segment information, or novel segment
information for
the full-length gene.
The polynucleotides of the invention also provide polynucleotides including
nucleotide sequences that axe substantially equivalent to the polynucleotides
recited above.
Polynucleotides according to tree invention can have, e.g., at least about
65%, at least about
70%, at least about 75%, at least about 80%, 81%, 82%, 83%, 84%, more
typically at least
about 85%, 86%, 87%, 88%, 89%, more typically at least about 90%, 91%, 92%,
93%, 94%,
and even more typically at least about 95%, 96%, 97%, 98%, 99% sequence
identity to a
polynucleotide recited above.
Included within the scope of the nucleic acid sequences of the invention are
nucleic
acid sequence fragments that hybridize under stringent conditions to any of
the nucleotide
805A/PCT ~ 02453344 2004-O1-21
I8
sequences of SEQ ID NO: 1-244, or 489-706, or complements thereof, which
fragment is
greater than about 5 nucleotides, preferably 7 nucleotides, more preferably
greater than 9
nucleotides and most preferably greater than 17 nucleotides. Fragments of,
e.g. 15, 17, or
20 nucleotides or more that are selective for (i.e. specifically hybridize to)
any one of the
polynucleotides of the invention are contemplated. Probes capable of
specifically
hybridizing to a polynucleotide can differentiate polynucleotide sequences of
the invention
from other polynucleotide sequences in the same family of genes ox can
differentiate human
genes from genes of other species, and are preferably based on unique
nucleotide sequences.
The sequences falling within the scope of the present invention are not
limited to these
I O specific sequences, but also include allelic and species variations
thereof. Allelic and species
variations can be routinely determined by comparing the sequence provided in
SEQ ID NO: 1-
244, ox 489-706, a representative fragment thereof, or a nucleotide sequence
at least 90%
identical, preferably 95% identical, to SEQ ID NO: 1-244, or 489-706 with a
sequence from
another isolate of the same species. Furthermore, to accommodate codon
variability, the
I 5 invention includes nucleic acid molecules coding for the same amino acid
sequences as do the
specific ORFs disclosed herein. In other words, in the coding region of an
ORF, substitution of
one codon for another codon that encodes the same amino acid is expressly
contemplated.
The nearest neighbor or homology results for the nucleic acids of the present
invention,
including SEQ ID NO: 1-244, or 489-706 can be obtained by searching a database
using an
20 algorithm or a program. Preferably, a BLAST (Basic Local Alignment Search
Tool) program is
used to search for local sequence alignments (Altshul, S.F. J l~Iol. Evol. 36
290-300 (1993) and
Altschul S.F. et al. J. Mol. Biol. 21:403-410 (1990)). Alternatively a FASTA
version 3 search
against Genpept, using FASTX~' algorithm may be performed.
Species homologs (or orthologs) of the disclosed polynucleotides and proteins
are
25 also provided by the present invention. 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 from the desired species.
The invention also encompasses allelic variants of the disclosed
polynucleotides or
proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide which
30 also encode proteins which are identical, homologous or related to that
encoded by the
polynucleotides.
The nucleic acid sequences of the invention are further directed to sequences
which
encode variants of the described nucleic acids. These amino acid sequence
variants may be
8O5 A mCT CA 02453344 2004-O1-21
19
prepared by methods known in the art by introducing appropriate nucleotide
changes into a
native or variant polynucleotide. There are two variables in the construction
of amino acid
sequence variants: the location of the mutation and the nature of the
mutation. Nucleic
acids encoding the amino acid sequence variants are preferably constructed by
mutating the
polynucleotide to encode an amino acid sequence that does not occur in nature.
These
nucleic acid alterations can be made at sites that differ in the nucleic acids
from different
species (variable positions) or in highly conserved regions (constant
regions). Sites at such
locations will typically be modified in series, e.g., by substituting first
with conservative
choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid)
and then with
more distant choices (e.g., hydrophobic amino acid to a charged amino acid),
and then
deletions or insertions may be made at the target site. Amino acid sequence
deletions
generally range from about 1 to 30 residues, preferably about 1 to 10
residues, and are
typically contiguous. Amino acid insertions include amino- and/or carboxyl-
terminal
fusions ranging in length from one to one hundred or more residues, as well as
intrasequence
insertions of single or multiple amino acid residues. Intrasequence insertions
may range
generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
Examples of
terminal insertions include the heterologous signal sequences necessary for
secretion or for
intracellular targeting in different host cells and sequences such as FLAG or
poly-histidine
sequences useful for purifying the expressed protein.
In a preferred method, polynucleotides encoding the novel amino acid sequences
are
changed via site-directed mutagenesis. This method uses oligonucleotide
sequences to alter
a polynucleotide to encode the desired amino acid variant, as well as
sufficient adjacent
nucleotides on both sides of the changed amino acid to form a stable duplex on
either side of
the site of being changed. In general, the techniques of site-directed
mutagenesis are well
known to those of skill in the art and this technique is exemplified by
publications such as,
Edelman et al., DNA 2:183 (1983). A versatile and efficient method for
producing
site-specific changes in a polynucleotide sequence was published by Zoller and
Smith,
Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino
acid
sequence variants of the novel nucleic acids. When small amounts of template
DNA are
used as starting material, primers) that differs slightly in sequence from the
corresponding
region in the template DNA can generate the desired amino acid variant. PCR
amplification
results in a population of product DNA fragments that differ from the
polynucleotide
template encoding the polypeptide at the position specified by the primer. The
product DNA
g~s~~Z CA 02453344 2004-O1-21
fragments replace the corresponding region in the plasmid and this gives a
polynucleotide
encoding the desired amino acid variant.
A further technique for generating amino acid variants is the cassette
mutagenesis
technique described in Wells et al., Gene 34:31 S (1985); and other
mutagenesis techniques
S well known in the art, such as, for example, the techniques in Sambrook et
al., supra, and
Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent
degeneracy of
the genetic code, other DNA sequences which encode substantially the same or a
functionally equivalent amino acid sequence may be used in the practice of the
invention for
the cloning and expression of these novel nucleic acids. Such DNA sequences
include those
10 which are capable of hybridizing to the appropriate novel nucleic acid
sequence under
stringent conditions.
Polynucleotides encoding preferred polypeptide truncations of the invention
could be
used to generate polynucleotides encoding chimeric or fusion proteins
comprising one or
more domains of the invention and heterologous protein sequences.
1 S The polynucleotides of the invention additionally include the complement
of any of
the polynucleotides recited above. The polynucleotide can be DNA (genomic,
cDNA,
amplified, or synthetic) or RNA. Methods and algorithms for obtaining such
polynucleotides are well known to those of skill in the art and can include,
for example,
methods for determining hybridization conditions that can routinely isolate
polynucleotides
20 of the desired sequence identities.
In accordance with the invention, polynucleotide sequences comprising the
mature
protein coding sequences corresponding to any one of SEQ 1D NO: 1-244, or 489-
706, or
functional equivalents thereof, may be used to generate recombinant DNA
molecules that
direct the expression of that nucleic acid, or a functional equivalent
thereof, in appropriate
2S host cells. Also included are the cDNA inserts of any of the clones
identified herein.
A polynucleotide according to the invention can be joined to any of a variety
of other
nucleotide sequences by well-established recombinant DNA techniques (see
Sambrook J et
al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, NY).
Useful nucleotide sequences for joining to polynucleotides include an
assortment of vectors,
e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like,
that are well
known in the art. Accordingly, the invention also provides a vector including
a
polynucleotide of the invention and a host cell containing the polynucleotide.
In general, the
vector contains an origin of replication functional in at least one organism,
convenient
~~~~~,I, CA 02453344 2004-O1-21
2I
restriction endonuclease sites, and a selectable marker for the host cell.
Vectors according to
the invention include expression vectors, replication vectors, probe
generation vectors, and
sequencing vectors. A host cell according to the invention can be a
prokaryotic or
eukaryotic cell and can be a unicellular organism or part of a multicellular
organism.
The present invention further provides recombinant constructs comprising a
nucleic
acid having any of the nucleotide sequences of SEQ ID NO: 1-244, or 489-706 or
a fragment
thereof or any other polynucleotides of the invention. In one embodiment, the
recombinant
constructs of the present invention comprise a vector, such as a plasmid or
viral vector, into
which a nucleic acid having any of the nucleotide sequences of SEQ ID NO: 1-
244, or 489-
706 or a fragment thereof is inserted, in a forward or reverse orientation. In
the case of a
vector comprising one of the ORFs of the present invention, the vector may
further comprise
regulatory sequences, including for example, a promoter, operably linked to
the ORF. Large
numbers of suitable vectors and promoters are known to those of skill in the
art and are
commercially available for generating the recombinant constructs of the
present invention.
The following vectors are provided by way of example: Bacterial: pBs,
phagescript,
PsiXl74, pBluescript SK, pBs KS, pNHBa, pNHl6a, pNHlBa, pNH46a (Stratagene),
pTrc99A, pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia); Eukaryotic: p~VLneo,
pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
The isolated polynucleotide of the invention may be operably linked to an
expression
control sequence such as the pMT2 or pED expression vectors disclosed in
Kaufman et al.,
Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein
recombinantly.
Many suitable expression control sequences axe known in the art. General
methods of
expressing recombinant proteins are also known and are exemplified in R.
Kaufman,
Methods in Enzymology 185, 537-566 (1990). As defined herein "operably linked"
means
that the isolated polynucleotide of the invention and an expression control
sequence are
situated within a vector or cell in such a way that the protein is expressed
by a host cell
which has been transformed (transfected) with the ligated
polynucleotide/expression control
sequence.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with selectable
markers. Two
appropriate vectors are pKK232-8 and pCM7. Particular named bacterial
promoters include
lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV
immediate
early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and
mouse
~O~~~T CA 02453344 2004-O1-21
22
metallothionein-I. Selection of the appropriate vector and promoter is well
within the level
of ordinary skill in the art. Generally, recombinant expression vectors will
include origins
of replication and selectable markers permitting transformation of the host
cell, e.g., the
ampicillin resistance gene of E. cola and S. cerevisiae TRP1 gene, and a
promoter derived
from a highly expressed gene to direct transcription of a downstream
structural sequence.
Such promoters can be derived from operons encoding glycolytic enzymes such as
3-
phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock
proteins, among
others. The heterologous structural sequence is assembled in appropriate phase
with
translation initiation and termination sequences, and preferably, a leader
sequence capable of
directing secretion of translated protein into the periplasmic space or
extracellular medium.
Optionally, the heterologous sequence can encode a fusion protein including an
amino
terminal identification peptide imparting desired characteristics, e.g.,
stabilization or
simplified purification of expressed recombinant product. Useful expression
vectors for
bacterial use are constructed by inserting a structural DNA sequence encoding
a desired
protein together with suitable translation initiatian and termination signals
in operable
reading phase with a functional promoter. The vector will comprise one or more
phenotypic
selectable markers and an origin of replication to ensure maintenance of the
vector and to, if
desirable, provide amplification within the host. Suitable prokaryotic hosts
for
transformation include E coli, Pacillus subtilis, Salmonella typhimurium and
various species
within the genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may
also be employed as a matter of choice.
As a representative but non-limiting example, useful expression vectors for
bacterial
use can comprise a selectable marker and bacterial origin of replication
derived from
commercially available plasmids comprising genetic elements of the well known
cloning
vector pBR322 (ATCC 37017). Such commercial vectors include, for example,
pKK223-3
(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech,
Madison, WI,
USA). These pBR322 "backbone" sections are combined with an appropriate
promoter and
the structural sequence to be expressed. Following transformation of a
suitable host strain
and growth of the host strain to an appropriate cell density, the selected
promoter is induced
or derepressed by appropriate means (e.g., temperature shift or chemical
induction) and cells
are cultured for an additional period. Cells are typically harvested by
centrifugation,
disrupted by physical or chemical means, and the resulting crude extract
retained for further
purification.
0o« mCT CA 02453344 2004-O1-21
2'~
Polynucleotides of the invention can also be used to induce immune responses.
For
example, as described in Fan et al., Nat. Biotech 17, 870-872 (1999),
incorporated herein
by reference, nucleic acid sequences encoding a polypeptide may be used to
generate
antibodies against the encoded polypeptide following topical administration of
naked
plasmid DNA or following injection, and preferably infra-muscular injection of
the DNA.
The nucleic acid sequences are preferably inserted in a recombinant expression
vector and
may be in the form of naked DNA.
4.3 ANTISENSE
Another aspect of the invention pertains to isolated antisense nucleic acid
molecules
that are hybridizable to or complementary to the nucleic acid molecule
comprising the
nucleotide sequence of SEQ ID NO: 1-244, or 489-706, or fragments, analogs or
derivatives
thereof. An "antisense" nucleic acid comprises a nucleotide sequence that is
complementary
to a "sense'° nucleic acid encoding a protein, e.g., complementary to
the coding strand of a
double-stranded cDNA molecule or complementary to an mRNA sequence. In
specific
aspects, antisense nucleic acid molecules are provided that comprise a
sequence
complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an
entire coding
strand, or to only a portion thereof. Nucleic acid molecules encoding
fragments, homologs,
derivatives and analogs of a protein of any of SEQ ID NO: 1-244, or 489-706 or
antisense
nucleic acids complementary to a nucleic acid sequence of SEQ ID NO: 1-244, or
489-706
are additionally provided.
In one embodiment, an antisense nucleic acid molecule is antisense to a
"coding
region" of the coding strand of a nucleotide sequence of the invention. The
term "coding
region" refers to the region of the nucleotide sequence comprising codons
which are
translated into amino acid residues. In another embodiment, the antisense
nucleic acid
molecule is antisense to a "noncoding region" of the coding strand of a
nucleotide sequence
of the invention. The term "noncoding region" refers to 5' and 3' sequences
that flank the
coding region that are not translated into amino acids (i.e., also referred to
as 5' and 3'
untranslated regions).
Given the coding strand sequences encoding a nucleic acid disclosed herein
(e.g.,
SEQ ID NO: 1-244, or 489-706, antisense nucleic acids of the invention can be
designed
according to the rules of Watson and Crick or Hoogsteen base pairing. The
antisense nucleic
acid molecule can be complementary to the entire coding region of an mRNA, but
more
g05til r CT CA 02453344 2004-O1-21
2~
preferably is an oligonucleotide that is antisense to only a portion of the
coding or noncoding
region of an mRNA. For example, the antisense oligonucleotide can be
complementary to
the region surrounding the translation start site of an mRNA. An antisense
oligonucleotide
can be, for example, about S, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides
in length. An
antisense nucleic acid of the invention can be constructed using chemical
synthesis or
enzymatic ligation reactions using procedures known in the art. For example,
an antisense
nucleic acid (e.g., an antisense oligonucleotide) can be chemically
synthesized using
naturally occurring nucleotides or variously modified nucleotides designed to
increase the
biological stability of the molecules or to increase the physical stability of
the duplex formed
between the antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and
acridine substituted nucleotides can be used.
Examples of modified nucleotides that can be used to generate the antisense
nucleic
acid include: S-fluorouracil, 5-bromouracil, 5-chlorouracil, .5-iodouracil,
hypoxanthine,
xanthine, 4-acetylcytosine, S-(carboxyhydroxylmethyl) uracil, 5-
I S carboxymethylaminomethyl-2-thiouridine, S-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-
methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-
methylcytosine, S-methylcytosine, N6-adenine, 7-methylguanine, 5-
methylaminomethyluracil, S-methoxyaminomethyl-2-thiouracil, beta-D-
mannosylqueosine,
S'-methoxycarboxymethyluracil, S-methoxyuracil, 2-methylthio-N6-
isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-
thiocytosine, S-methyl-
2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic
acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-
carboxypropyl) uracil,
(acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can
be produced
biologically using an expression vector into which a nucleic acid has been
subcloned in an
antisense orientation (i.e., RNA transcribed from the inserted nucleic acid
will be of an
antisense orientation to a target nucleic acid of interest, described further
in the following
subsection).
The antisense nucleic acid molecules of the invention are typically
administered to a
subject or generated i~ situ such that they hybridize with or bind to cellular
mRNA and/or
genomic DNA encoding a protein according to the invention to thereby inhibit
expression of
the protein, e.g., by inhibiting transcription and/or translation. The
hybridization can be by
conventional nucleotide complementarity to form a stable duplex, or, for
example, in the
805A/PCT ~ 02453344 2004-O1-21
case of an antisense nucleic acid molecule that binds to DNA duplexes, through
specific
interactions in the major groove of the double helix. An example of a route of
administration of antisense nucleic acid molecules of the invention includes
direct injection
at a tissue site. Alternatively, antisense nucleic acid molecules can be
modified to target
5 selected cells and then administered systemically. For example, for systemic
administration,
antisense molecules can be modified such that they specifically bind to
receptors or antigens
expressed on a selected cell surface, e.g., by linking the antisense nucleic
acid molecules to
peptides or antibodies that bind to cell surface receptors or antigens. The
antisense nucleic
acid molecules can also be delivered to cells using the vectors described
herein. To achieve
10 sufficient intracellular concentrations of antisense molecules, vector
constructs in which the
antisense nucleic acid molecule is placed under the control of a strong pol II
or pol III
promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is an
a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific
15 double-stranded hybrids with complementary RNA in which, contrary to the
usual a-units,
the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids
Res 15:
6625-6641). The antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (moue et al. (1987) Nucleic Acids Res 15: 6131-6148)
or a
chimeric RNA -DNA analogue (moue et al. ( 1987) FEBS Lett 215: 327-330).
4.4 RIBOZYMES AND PNA MOIETIES
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme.
Ribozymes are catalytic RNA molecules with ribonuclease activity that are
capable of
cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described
in
Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically
cleave
mRNA transcripts to thereby inhibit translation of an mRNA. A ribozyme having
specificity
for a nucleic acid of the invention can be designed based upon the nucleotide
sequence of a
DNA disclosed herein (i.e., SEQ ID NO: 1-244, or 489-706). F'or example, a
derivative of
Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence
of the
active site is complementary to the nucleotide sequence to be cleaved in a
mRNA. See, e.g.,
Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
Alternatively,
mRNA of the invention can be used to select a catalytic RNA having a specific
ribonuclease
80SA/PCT ~ 02453344 2004-O1-21
26
activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993)
Sciehce
261:1411-1418.
Alternatively, gene expression can be inhibited by targeting nucleotide
sequences
complementary to the regulatory region (e.g., promoter and/or enhancers) to
form triple
S helical structures that prevent transcription of the gene in target cells.
See generally, Helene.
{1991) Ahticahcer Drug Des. 6: S69-84; Helene. et al. {1992) Ahn. N. Y. Acad.
Sci.
660:27-36; and Maher (1992) Bioassays 14: 807-1 S.
In various embodiments, the nucleic acids of the invention can be modified at
the
base moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability,
hybridization, or solubility of the molecule. Far example, the deoxyribose
phosphate
backbone of the nucleic acids can be modified to generate peptide nucleic
acids (see Hyrup
et al. (1996) Bioorg Med Chem 4: S-23). As used herein, the terms "peptide
nucleic acids"
or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the
deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only the four
natural
1 S nucleobases are retained. The neutral backbone of PNAs has been shown to
allow for
specific hybridization to DNA and RNA under conditions of low ionic strength.
The
synthesis of PNA oligomers can be performed using standard solid phase peptide
synthesis
protocols as described in Hyrup et al. {1996) above; Perry-O'Keefe et al.
(1996) PATAS 93:
14670-675.
PNAs of the invention can be used in therapeutic and diagnostic applications.
For
example, PNAs can be used as antisense or antigene agents for sequence-
specific modulation
of gene expression by, e.g., inducing transcription or translation arrest or
inhibiting
replication. PNAs of the invention can also be used, e.g., in the analysis of
single base pair
mutations in a gene by, e. g. , PNA directed PCR clamping; as artificial
restriction enzymes
2S when used in combination with other enzymes, e.g., S1 nucleases {Hyrup B.
(1996) above);
or as probes or primers for DNA sequence and hybridization (Hyrup et al.
(1996), above;
Perry-O'Keefe (1996), above).
In another embodiment, PNAs of the invention can be modified, e.g., to enhance
their stability or cellular uptake, by attaching lipophilic or other helper
groups to PNA, by
the formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of drug
delivery known in the art. For example, PNA-DNA chimeras can be generated that
may
combine the advantageous properties of PNA and DNA. Svch chimeras allow DNA
recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the
DNA
g~S~~~T CA 02453344 2004-O1-21
27
portion while the PNA portion would provide high binding affinity and
specificity.
PNA-DNA chimeras can be linked using linkers of appropriate lengths selected
in terms of
base stacking, number of bonds between the nucleobases, and orientation (Hyrup
(1996)
above). The synthesis of PNA-DNA chimeras can be performed as described in
Hyrup
(1996) above and Finn et al. (1996) Nucl Acids Res 24: 3357-63. For example, a
DNA chain
can be synthesized on a solid support using standard phosphoramidite coupling
chemistry,
and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-
thyrnidine
phosphoramidite, can be used between the PNA and the 5' end of DNA (Mag et al.
(1989)
Nucl Acid Res 17: 5973-88). PNA monomers are then coupled in a stepwise manner
to
produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
et al.
( 1996) above). Alternatively, chimerie molecules can be synthesized with a 5'
DNA
segment and a 3' PNA segment. See, Petersen et al. (1975) Bioorg Med Chem Lett
5:
1119-11124.
In other embodiments, the oligonucleotide may include other appended groups
such
as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating transport
across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad.
Sci. U.SA.
86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT
Publication
No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No.
W089/10134).
In addition, oligonucleotides cm be modified with hybridization triggered
cleavage agents
(See, e.g., Krol et al., 1988, BioTechhiques 6:958-976) or intercalating
agents. (See, e.g.,
Zon, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide may be
conjugated to
another molecule, e.g., a peptide, a hybridization triggered cross-linking
agent, a transport
agent, a hybridization-triggered cleavage agent, etc.
4.5 HOSTS
The present invention further provides host cells genetically engineered to
contain
the polynucleotides of the invention. For example, such host cells may contain
nucleic acids
of the invention introduced into the host cell using known transformation,
transfection or
infection methods. The present invention still further provides host cells
genetically
engineered to express the polynucleotides of the invention, wherein such
polynucleotides axe
in operative association with a regulatory sequence heterologous to the host
cell which
drives expression of the polynucleotides in the cell.
805A1i'CT CA 02453344 2004-O1-21
28
Knowledge of nucleic acid sequences allows for modification of cells to
permit, or
increase, expression of endogenous polypeptide. Cells can be modified (e.g.,
by
homologous recombination) to provide increased polypeptide expression by
replacing, in
whole or in part, the naturally occurring promoter with all or part of a
heterologous promoter
so that the cells express the polypeptide at higher levels. The heterologous
promoter is
inserted in such a manner that it is operatively linked to the encoding
sequences. See, for
example, PCT International Publication No. W094/12650, PCT International
Publication
No. W092/20808, and PCT International Publication No. WO91/09955. It is also
contemplated that, in addition to heterologous promoter DNA, amplifiable
marker DNA
IO (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl
phosphate
synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA
may be
inserted along with the heterologous promoter DNA. If linked to the coding
sequence,
amplification of the marker DNA by standard selection methods results in co-
amplification
of the desired protein coding sequences in the cells.
The host cell can be a higher eukaryotic host cell, such as a mammalian cell,
a lower
eukaryotic host cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a
bacterial cell. Introduction of the recombinant canstruct into the host cell
can be effected by
calcium phosphate transfection, DEAE, dextran mediated transfection, or
electroporation
(Davis, L, et al., Basic Methods in Molecular Biology (1986)). The host cells
containing one
of the polynucleotides of the invention, can be used in conventional manners
to produce the
gene product encoded by the isolated fragment (in the case of an ORF) or can
be used to
produce a hetervlogous protein under the control of the EMF.
Any host/vector system can be used to express one or more of the ORFs of the
present invention. These include, but are not limited to, eul~aryotic hosts
such as HeLa cells,
Cv-1 cell, COS cells, 293 cells, and S~3 cells, as well as prokaryotic host
such as E. coli and
B. subtilis. The most preferred cells are those which do not normally express
the particular
polypeptide or protein or which expresses the polypeptide or protein at low
natural level.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other
cells under
the control of appropriate promoters. Cell-free translation systems can also
be employed to
produce such proteins using RNAs derived from the DNA constructs of the
present
invention. Appropriate cloning and expression vectors for use with prokaryotic
and
eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A
Laboratory
805A/L'CT CA 02453344 2004-O1-21
29
Manual, Second Edition, Cold Spring Harbor, New York (1989), the disclosure of
which is
hereby incorporated by reference.
Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7 lines
of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981). Other
cell lines
capable of expressing a compatible vector are, for example, the C127, monkey
COS cells,
Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal
A431 cells,
human Co1o205 cells, 3T3 cells, CV-1 Bells, other transformed primate cell
lines, normal
diploid cells, cell strains derived from in vitro culture of primary tissue,
primary explants,
HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian
expression
vectors will comprise an origin of replication, a suitable promoter and also
any necessary
ribosome binding sites, polyadenylation site, splice donor and acceptor sites,
transcriptional
termination sequences, and 5' flanking nontranscribed sequences. DNA sequences
derived
from the SV40 viral genome, for example, SV40 origin, early promoter,
enhancer, splice,
and polyadenylation sites may be used to provide the required nontranscribed
genetic
elements. Recombinant polypeptides and proteins produced in bacterial culture
are usually
isolated by initial extraction from cell pellets, followed by one or more
salting-out, aqueous
ion exchange or size exclusion chromatography steps. Protein refolding steps
can be used,
as necessary, in completing configuration of the mature protein. Finally, high
performance
liquid chromatography (HPLC) can be employed for final purification steps.
Microbial cells
employed in expression of proteins can be disrupted by any convenient method,
including
freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing
agents.
Alternatively, it may be possible to produce the protein in lower eukaryotes
such as
yeast or insects or in prokaryotes such as bacteria. Potentially suitable
yeast strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Cahdida,
or any yeast strain capable of expressing heterologous proteins. Potentially
suitable bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typlzimurium,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
80SA/PCT ~ 02453344 2004-O1-21
In another embodiment of the present invention, cells and tissues may be
engineered
to express an endogenous gene comprising the polynucleotides of the invention
under the
control of inducible regulatory elements, in which case the regulatory
sequences of the
endogenous gene may be replaced by homologous recombination. As described
herein, gene
5 targeting can be used to replace a gene's existing regulatory region with a
regulatory
sequence isolated from a different gene or a novel regulatory sequence
synthesized by
genetic engineering methods. Such regulatory sequences may be comprised of
promoters,
enhancers, scaffold-attachment regions, negative regulatory elements,
transcriptional
initiation sites, and regulatory protein binding sites or combinations of said
sequences.
10 Alternatively, sequences which affect the structure or stability of the RNA
or protein
produced may be replaced, removed, added, or otherwise modified by targeting.
These
sequence include polyadenylation signals, mRNA stability elements, splice
sites, leader
sequences for enhancing or modifying transport or secretion properties of the
protein, or
other sequences which alter or improve the function or stability of protein or
RNA
15 molecules.
The targeting event may be a simple insertion of the regulatory sequence,
placing the
gene under the control of the new regulatory sequence, e.g., inserting a new
promoter or
enhancer or both upstream of a gene. Alternatively, the targeting event may be
a simple
deletion of a regulatory element, such as the deletion of a tissue-specific
negative regulatory
20 element. Alternatively, the targeting event may replace an existing
element; for example, a
tissue-specific enhancer can be replaced by an enhancer that has broader or
different
cell-type specificity than the naturally occurring elements. Here, the
naturally occurring
sequences are deleted and new sequences are added. In all cases, the
identification of the
targeting event may be facilitated by the use of one or more selectable marker
genes that are
25 contiguous with the targeting DNA, allowing for the selection of cells in
which the
exogenous DNA has integrated into the host cell genome. The identification of
the targeting
event may also be facilitated by the use of one or more marker genes
exhibiting the property
of negative selection, such that the negatively selectable marker is linked to
the exogenous
DNA, but conf gured such that the negatively selectable marker flanks the
targeting
30 sequence, and such that a correct homologous recombination event with
sequences in the
host cell genome does not result in the stable integration of the negatively
selectable marker.
Markers useful for this purpose include the Herpes Simplex Virus thymidine
kinase (TK)
gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.
805A/PCT ~ 02453344 2004-O1-21
31
The gene targeting or gene activation techniques which can be used in
accordance
with this aspect of the invention are more particularly described in U.S.
Patent No.
5,272,071 to Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.;
International
Application No. PCTIUS92/09627 (W093/09222) by Selden et al.; and
International
Application No. PCT/US90/06436 (W091/06667) by Skoultchi et al., each of which
is
incorporated by reference herein in its entirety.
4.6 PoLYPEPTIDES OF TILE IN~~ENTIDN
The isolated polypeptides of the invention include, but are not limited to, a
polypeptide comprising: the amino acid sequences set forth as any one of SEQ
ID NO: 245-
488, or 707-924 or an amino acid sequence encoded by any one of the nucleotide
sequences
SEQ ID NO: 1-244, or 489-706 or the corresponding full length or mature
protein.
Polypeptides of the invention also include polypeptides preferably with
biological or
immunological activity that are encoded by: (a) a polynucleotide having any
one of the
nucleotide sequences set forth in SEQ ID NO: 1-244, or 489-706 or (b)
polynucleotides
encoding any one of the amino acid sequences set forth as SIEQ ID NO: 245-488,
or 707-924
or (c) polynucleotides that hybridize to the complement of the polynucleotides
of either (a)
or (b) under stringent hybridization conditions. The invention also provides
biologically
active or immunologically active variants of any of the amino acid sequences
set forth as
SEQ ID NO: 245-488, or 707-924 or the corresponding full length or mature
protein; and
"substantial equivalents" thereof (e.g., with at least about 65%, at least
about 70%, at least
about 75%, at least about 80%, at Least about 85%, 86%, 87%, 88%, 89%, at
least about
90%, 91%, 92%, 93%, 94%, typically at least about 95%, 96°/~, 97%, more
typically at least
about 98%, or most typically at least about 99% amino acid identity) that
retain biological
activity. Polypeptides encoded by allelic variants may have a similar,
increased, or
decreased activity compared to polypeptides comprising SEQ ID NO: 245-488, or
707-924.
Fragments of the proteins of the present invention which are capable of
exhibiting
biological activity are also encompassed by the present invention. Fragments
of the protein
may be in linear form or they rnay be cyclized using known methods, for
example, as
described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in
R. S.
McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are
incorporated herein by reference. Such fragments may be fused to carrier
molecules such as
805A/PCT ~ 02453344 2004-O1-21
32
immunoglobulins for many purposes, including increasing the valency of protein
binding
sites. Fragments are also identified in Tables 3, 5, 6, and 8.
The present invention also provides both full-length and mature forms (for
example,
without a signal sequence or precursor sequence) of the disclosed proteins.
The protein
coding sequence is identified in the sequence listing by translation of the
disclosed
nucleotide sequences. The predicted signal sequence is set forth in Table 6.
The mature
form of such protein may be obtained and confirmed by expression of a full-
length
polynucleotide in a suitable mammalian cell or other host cell and sequencing
of the cleaved
product. One of skill in the art will recognize that the actual cleavage site
may be different
than that predicted in Table 6. The sequence of the mature form of the protein
is also
determinable from the amino acid sequence of the full-length form. Where
proteins of the
present invention are membrane bound, soluble forms of the proteins are also
provided. In
such forms, part or all of the regions causing the proteins to be membrane
bound are deleted
so that the proteins are fully secreted from the cell in which they are
expressed.
1 S Protein compositions of the present invention may further comprise an
acceptable
carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The present invention further provides isolated polypeptides encoded by the
nucleic
acid fragments of the present invention or by degenerate variants of the
nucleic acid
fragments of the present invention. By "degenerate variant" is intended
nucleotide
fragments which differ from a nucleic acid fragment of the present invention
(e.g., an ORF)
by nucleotide sequence but, due to the degeneracy of the genetic code, encode
an identical
polypeptide sequence. Preferred nucleic acid fragments of the present
invention are the
ORFs that encode proteins.
A variety of methodologies known in the art can be utilized to obtain any one
of the
isolated polypeptides or proteins of the present invention. At the simplest
level, the amino
acid sequence can be synthesized using commercially available peptide
synthesizers. The
synthetically-constructed protein sequences, by virtue of sharing primary,
secondary or
tertiary structural and/or conformational characteristics with proteins may
possess biological
properties in common therewith, including protein activity. This technique is
particularly
useful in producing small peptides and fragments of larger polypeptides.
Fragments are
useful, for example, in generating antibodies against the native polypeptide.
Thus, they may
be employed as biologically active or immunological substitutes for natural,
purified
g05tilPCT CA 02453344 2004-O1-21
33
proteins in screening of therapeutic compounds and in immunological processes
for the
development of antibodies.
The polypeptides and proteins of the present invention can alternatively be
purified
from cells which have been altered to express the desired polypeptide or
protein. As used
herein, a cell is said to be altered to express a desired polypeptide or
protein when the cell,
through genetic manipulation, is made to produce a polypeptide or protein
which it normally
does not produce or which the cell normally produces at a lower level. One
skilled in the art
can readily adapt procedures for introducing and expressing either recombinant
or synthetic
sequences into eukaryotic or prokaryotic cells in order to generate a cell
which produces one
of the polypeptides or proteins of the present invention.
The invention also relates to methods for producing a polypeptide comprising
growing a culture of host cells of the invention in a suitable culture medium,
and purifying
the protein from the cells or the culture in which the cells are grown. Far
example, the
methods of the invention include a process for producing a polypeptide in
which a host cell
I S containing a suitable expression vector that includes a polynucleotide of
the invention is
cultured under conditions that allow expression of the encoded polypeptide.
The
polypeptide can be recovered from the culture, conveniently from the culture
medium, or
from a lysate prepared from the host cells and further purified. Preferred
embodiments
include those in which the protein produced by such process is a full length
or mature form
of the protein.
In an alternative method, the polypeptide or protein is purified from
bacterial cells
which naturally produce the polypeptide or protein. One skilled in the art can
readily follow
known methods for isolating polypeptides and proteins in order to obtain one
of the isolated
polypeptides or proteins of the present invention. These include, but are not
limited to,
immunochromatography, HPLC, size-exclusion chromatography, ion-exchange
chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Proteih
Purification: Principles ahd Practice, Springer-Verlag (1994); Sambrook, et
al., in
Molecular Cloning: A Laboratory Manual; Ausubei et al., Current Protocols iu
Molecular
Biology. Polypeptide fragments that retain biological/immunological activity
include
fragments comprising greater than about 100 amino acids, or greater than about
200 amino
acids, and fragments that encode specific protein domains.
The purified polypeptides can be used in iu vitro binding assays which are
well
known in the art to identify molecules which bind to the polypeptides. These
molecules
~~~A~~.I. CA 02453344 2004-O1-21
34
include but are not limited to, for e.g., small molecules, molecules from
combinatorial
libraries, antibodies or other proteins. The molecules identified in the
binding assay are
then tested for antagonist or agonist activity in in vivo tissue culture or
animal models that
are well known in the art. In brief, the molecules are titrated into a
plurality of cell cultures
or animals and then tested for either cell/animal death or prolonged survival
of the
animal/cells.
In addition, the peptides of the invention or molecules capable of binding to
the
peptides may be complexed with toxins, e.g., ricin or cholera, or with other
compounds that
are toxic to cells. The toxin-binding molecule complex is then targeted to a
tumor or other
cell by the specificity of the binding molecule for SEQ ID NO: 245-488, or 707-
924.
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which are
characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
provided or deliberately engineered. For example, modifications, in the
peptide or DNA
sequence, can be made by those skilled in the art using known techniques.
Modifications of
interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For example,
one or more of the cysteine residues may be deleted or replaced with another
amino acid to
alter the conformation of the molecule. Techniques for such alteration,
substitution,
replacement, insertion or deletion are well known to those skilled in the art
(see, e.g., U.S.
Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement,
insertion or
deletion retains the desired activity of the protein. Regions of the protein
that are important
for the protein function can be determined by various methods known in the art
including the
alanine-scanning method which involved systematic substitution of single or
strings of
amino acids with alanine, followed by testing the resulting alanine-containing
variant for
biological activity. This type of analysis determines the importance of the
substituted amino
acids) in biological activity. Regions of the protein that are important for
protein function
may be determined by the eMATRIX program.
Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and are useful for
screening or other
gOS~~.I. CA 02453344 2004-O1-21
immunological methodologies may also be easily made by those skilled in the
art given the
disclosures herein. Such modifications are encompassed by the present
invention.
The protein may also be produced by operably linking the isolated
polynucleotide of
the invention to suitable control sequences in one or more insect expression
vectors, and
5 employing an insect expression system. Materials and methods for
baculovirus/insect cell
expression systems are commercially available in kit form from, e.g.,
Invitrogen, San Diego,
Calif., U.S.A. (the MaxBatTM kit), and such methods are well known in the art,
as described
in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555
(1987),
incorporated herein by reference. As used herein, an insect cell capable of
expressing a
10 polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i. e., from culture
medium or cell
extracts) using known purification processes, such as gel filtration and ion
exchange
15 chromatography. The purification of the protein may also include an
affinity column
containing agents which will bind to the protein; one or more column steps
over such affinity
resins as concanavalin A-agarose, heparin-toyopearlTM or Cibacrom blue 3GA
SepharoseTM;
one or more steps involving hydrophobic interaction chromatography using such
resins as
phenyl ether, butyl ether, or propyl ether; or immunoaffmity chromatography.
20 Alternatively, the protein of the invention may also be expressed in a form
which will
facilitate purification. For example, it may be expressed as a fusion protein,
such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin
(TRX), or as
a His tag. Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway,
N.J.) and
25 Invitrogen, respectively. The protein can also be tagged with an epitope
and subsequently
purified by using a specific antibody directed to such epitope. One such
epitope ("FLAG~")
is commercially available from Kodak (New Haven, Conn.).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
30 methyl or other aliphatic groups, can be employed to further purify the
protein. Some or all
of the foregoing purification steps, in various combinations, can also be
employed to provide
a substantially homogeneous isolated recombinant protein. The protein thus
purified is
805ti/PCT CA 02453344 2004-O1-21
36
substantially free of other mammalian proteins and is defined in accordance
with the present
invention as an "isolated protein."
The polypeptides of the invention include analogs (variants). This embraces
fragments, as well as peptides in which one or more amino acids has been
deleted, inserted,
or substituted. Also, analogs of the polypeptides of the invention embrace
fusions of the
polypeptides or modifications of the polypeptides of the invention, wherein
the polypeptide
or analog is fused to another moiety or moieties, e.g., targeting moiety or
another therapeutic
agent. Such analogs may exhibit improved properties such as activity and/or
stability.
Examples of moieties which may be fused to the polypeptide or an analog
include, for
example, targeting moieties which provide for the delivery of polypeptide to
pancreatic cells,
e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-
cells, monocytes,
dendritic cells, granulocytes, etc., as well as receptor and ligands expressed
on pancreatic or
immune cells. Other moieties which may be fused to the polypeptide include
therapeutic
agents which are used for treatment, for example, immunosuppressive drugs such
as
cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also,
polypeptides may be
fused to immune modulators, and other cytokines such as alpha or beta
interferon.
4.6.i DETERMINING POLYPEPTIDE AND POLYNUCLEOTIDE
IDENTITY AND SIMILARITY
Preferred identity and/or similarity are designed to give the largest match
between
the sequences tested. Methods to determine identity and similarity are
codified in computer
programs including, but are not limited to, the GCG program package, including
GAP
(Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics
Computer Group,
University of Wisconsin, Madison, WI), BLASTP, BLASTN, BLASTX, FASTA
(Altschul,
S.F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S.F. et
al., Nucleic
Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix
software (Wu
et al., J. Comp. Biol., Vol. 6, pp. 219-235 (1999), herein incorporated by
reference), eMotif
software (Nevill-Manning et al, ISMB-97, Vol. 4, pp. 202-209, herein
incorporated by
reference), Pfam software (Sonnhammer et al.,1\~ucleic Acids Res., Vol. 26(1),
pp. 320-322
(1998), herein incorporated by reference) and the Kyte-Doolittle
hydrophobocity prediction
algorithm (J. Mol Biol, 157, pp. 105-31 (1982), incorporated herein by
reference).
polypeptide sequences were examined by a proprietary algorithm, SeqLoc that
separates the
proteins into three sets of locales: intracellular, membrane, or secreted.
This prediction is
g~SA/PC~I~ CA 02453344 2004-O1-21
37
based upon three characteristics of each polypeptide, including percentage of
cysteine
residues, Kyte-Doolittle scores for the first 20 amino acids of each protein,
and Kyte-
Doolittle scores to calculate the longest hydrophobic stretch of the said
protein. Values of
predicted proteins are compared against the values from a set of 592 proteins
of known
cellular localization from the Swissprot database (http:/lwww.expas .~prot).
Predictions
are based upon the maximum likelihood estimation.
The BLAST programs are publicly available from the National Center for
Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul,
S., et al.
NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., :1. Mol. Biol. 215:403-
410
(1990).
4.7 CHIMERIC AND FUSION PROTEINS
The invention also provides chimeric or fusion proteins. As used herein, a
"chimeric
protein" or "fusion protein" comprises a polypeptide of the invention
operatively linked to
another polypeptide. Within a fusion protein the polypeptide according to the
invention can
correspond to all or a portion of a protein according to the invention. In one
embodiment, a
fusion protein comprises at least one biologically active portion of a protein
according to the
invention. In another embodiment, a fusion protein comprises at least two
biologically
active portions of a protein according to the invention. Within the fusion
protein, the term
"operatively linked" is intended to indicate that the polypeptide according to
the invention
and the other polypeptide are fused in-frame to each other. The polypeptide
can be fused to
the N-terminus or C-terminus, or to the middle.
For example, in one embodiment a fusion protein comprises a polypeptide
according
to the invention operably linked to the extracellular domain of a second
protein.
In another embodiment, the fusion protein is a GST-fusion protein in which the
polypeptide sequences of the invention are fused to the C-terminus of the GST
(i.e.,
glutathione S-transferase) sequences.
In another embodiment, the fusion protein is an immunoglobulin fusion protein
in
which the polypeptide sequences according to the invention comprise one or
more domains
fused to sequences derived from a member of the immunoglobulin protein family.
The
immunoglobulin fusion proteins of the invention can be incorporated into
pharmaceutical
compositions and administered to a subject to inhibit an interaction between a
ligand and a
protein of the invention on the surface of a cell, to thereby suppress signal
transduction in
vivo. The immunoglobulin fusion proteins can be used to affect the
bioavailability of a
805A/PCT ~ 02453344 2004-O1-21
38
cognate ligand. Inhibition of the ligand/protein interaction may be useful
therapeutically for
both the treatment of proliferative and differentiative disorders, e.g.,
cancer as well as
modulating (e.g., promoting or inhibiting) cell survival. Moreover, the
immunoglobulin
fusion proteins of the invention can be used as immunogens to produce
antibodies in a
subject, to purify ligands, and in screening assays to identify molecules that
inhibit the
interaction of a polypeptide of the invention with a ligand.
A chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling-in of cohesive
ends as
appropriate, alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic
ligation. In another embodiment, the fusion gene can be synthesized by
conventional
techniques including automated DNA synthesizers. Alternatively, PCR
amplification of
gene fragments can be carried out using anchor primers that give rise to
complementary
overhangs between two consecutive gene fragments that can subsequently be
annealed and
reamplified to generate a chimeric gene sequence (see, for example, Ausubel et
al. (eds.)
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley ~ Sons, 1992). Moreover,
many expression vectors are commercially available that already encode a
fusion moiety
(e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the
invention can be
cloned into such an expression vector such that the fusion moiety is linked in-
frame to the
protein of the invention.
4.8 GENE THERAPY
Mutations in the polynucleotides of the invention gene may result in loss of
normal
function of the encoded protein. The invention thus provides gene therapy to
restore normal
activity of the polypeptides of the invention; or to treat disease states
involving polypeptides
of the invention. Delivery of a functional gene encoding polypeptides of the
invention to
appropriate cells is effected ex vivo, ih situ, or in viva by use of vectors,
and more
particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a
retrovirus), or ex vivo
by use ofphysical DNA transfer methods (e.g., liposomes or chemical
treatments). See, for
example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25-20 (1998).
For
additional reviews of gene therapy technology see Friedmann, Science, 244:
1275-1281
go5~'- T CA 02453344 2004-O1-21
39
(1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-
460 (1992).
Introduction of any one of the nucleotides of the present invention or a gene
encoding the
polypeptides of the present invention can also be accomplished with
extrachromosomal
substrates (transient expression) or artificial chromosomes (stable
expression). Cells may
also be cultured ex vivo in the presence of proteins of the present invention
in order to
proliferate or to produce a desired effect on or activity in such cells.
Treated cells can then
be introduced in vivo for therapeutic purposes. Alternatively, it is
contemplated that in other
human disease states, preventing the expression of or inhibiting the activity
of polypeptides
of the invention will be useful in treating the disease states. It is
contemplated that antisense
therapy or gene therapy could be applied to negatively regulate the expression
of
polypeptides of the invention.
Other methods inhibiting expression of a protein include the introduction of
antisense
molecules to the nucleic acids of the present invention, their complements, or
their translated
RNA sequences, by methods known in the art. Further, the polypeptides of the
present
invention can be inhibited by using targeted deletion methods, or the
insertion of a negative
regulatory element such as a silencer, which is tissue specific.
The present invention still further provides cells genetically engineered in
vivo to
express the polynucleotides of the invention, wherein such polynucleotides are
in operative
association with a regulatory sequence heterologous to the host cell which
drives expression of
the polynucleotides in the cell. These methods can be used to increase or
decrease the
expression of the polynucleotides of the present invention.
Knowledge of DNA sequences provided by the invention allows for modification
of
cells to permit, increase, or decrease, expression of endogenous polypeptide.
Cells can be
modified (e.g., by homologous recombination) to provide increased polypeptide
expression by
replacing, in whole or in part, the naturally occurring promoter with all or
part of a heterologous
promoter so that the cells express the protein at higher levels. The
heterologous promoter is
inserted in such a manner that it is operatively linked to the desired protein
encoding sequences.
See, for example, PCT International Publication No. WO 94/12650, PCT
International
Publication No. WO 92/20808, and PCT International Publication No. WO
91109955. It is also
contemplated that, in addition to heterologous promoter DNA, amplifiable
marker DNA (e.g.,
ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate
synthase,
aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be
inserted along with
the heterologous promoter DNA. If linked to the desired protein codiilg
sequence,
~~SA~CT CA 02453344 2004-O1-21
amplification of the marker DNA by standard selection methods results in co-
amplification of
the desired protein coding sequences in the cells.
In another embodiment of the present invention, cells and tissues may be
engineered to
express an endogenous gene comprising the polynucleotides of the invention
under the control
5 of inducible regulatory elements, in which case the regulatory sequences of
the endogenous
gene may be replaced by homologous recombination. As described herein, gene
targeting can
be used to replace a gene's existing regulatory region with a regulatory
sequence isolated from
a different gene or a novel regulatory sequence synthesized by genetic
engineering methods.
Such regulatory sequences may be comprised of promoters, enhancers, scaffold-
attachment
10 regions, negative regulatory elements, transcriptional initiation sites,
regulatory protein binding
sites or combinations of said sequences. Alternatively, sequences which affect
the structure or
stability of the RNA or protein produced may be replaced, removed, added, or
otherwise
modified by targeting. These sequences include polyadenylation signals, mRNA
stability
elements, splice sites, leader sequences for enhancing or modifying transport
or secretion
15 properties of the protein, or other sequences which alter or improve the
function or stability of
protein or RNA molecules.
The targeting event may be a simple insertion of the regulatory sequence,
placing the
gene under the control of the new regulatory sequence, e,g., in.serting a new
promoter or
enhancer or both upstream of a gene. Alternatively, the targeting event may be
a simple
20 deletion of a regulatory element, such as the deletion of a tissue-specific
negative regulatory
element. Alternatively, the targeting event may replace an existing element;
for example, a
tissue-specific enhancer can be replaced by an enhancer that has broader or
different cell-type
specificity than the naturally occurring elements. Here, the naturally
occurring sequences are
deleted and new sequences are added. In all cases, the identification of the
targeting event may
25 be facilitated by the use of one or more selectable marker genes that are
contiguous with the
targeting DNA, allowing for the selection of cells in which the exogenous DNA
has integrated
into the cell genome. The identification of the targeting event may also be
facilitated by the use
of one or more marker genes exhibiting the property of negative selection,
such that the
negatively selectable marker is linked to the exogenous DNA, but configured
such that the
30 negatively selectable marker flanks the targeting sequence, and such that a
correct homologous
recombination event with sequences in the host cell genome does not result in
the stable
integration of the negatively selectable marker. Markers useful for this
purpose include the
805A/PCT ~ 02453344 2004-O1-21
41
Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-
guanine
phosphoribosyl-transferase (gpt) gene.
The gene targeting or gene activation techniques which can be used in
accordance with
this aspect of the invention are more particularly described in U.S. Patent
No. 5,272,071 to
Chappel; U.S. Patent No. 5,578,461 to Sherwin et al.; International
Application No.
PCT/LJS92/09627 (W093/09222) by Selden et al.; and International Application
No.
PCT/LTS90/06436 (W091/06667) by Skoultchi et al., each of which is
incorporated by
reference herein in its entirety.
4.9 TIgANSGENIC ANIMALS
In preferred methods to determine biological functions of the polypeptides of
the
invention in vivo, one or more genes provided by the invention are either over
expressed or
inactivated in the germ line of animals using homologous recombination
[Capecchi, Science
244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the
regulatory
1 S control of exogenous or endogenous promoter elements, are known as
transgenic animals.
Animals in which an endogenous gene has been inactivated by homologous
recombination
are referred to as "knockout" animals. Knockout animals, preferably non-human
mammals,
can be prepared as described in IJ.S. Patent No. 5,557,032, incorporated
herein by reference.
Transgenic animals are useful to determine the roles polypeptides of the
invention play in
biological processes, and preferably in disease states. Transgenic animals are
useful as model
systems to identify compounds that modulate lipid metabolism. Transgenic
animals,
preferably non-human mammals, are produced using methods as described in U.S.
Patent No
5,489,743 and PCT Publication No. W094/28122, incorporated herein by
reference.
Transgenic animals can be prepared wherein all or part of a promoter of the
polynucleotides of the invention is either activated or inactivated to alter
the level of
expression of the polypeptides of the invention. Inactivation can be carried
out using
homologous recombination methods described above. Activation can be achieved
by
supplementing or even replacing the homologous promoter to provide for
increased protein
expression. The homologous promoter can be supplemented by insertion of one or
more
heterologous enhancer elements known to confer promoter activation in a
particular tissue.
The polynucleotides of the present invention also make possible the
development,
through, e.g., homologous recombination or knock out strategies, of animals
that fail to
express polypeptides of the invention or that express a variant polypeptide.
Such animals are
805A/PCT ~ 02453344 2004-O1-21
42
useful as models for studying the in vivo activities of polypeptide as well as
for studying
modulators of the polypeptides of the invention.
In preferred methods to determine biological functions of the polypeptides of
the
invention in vivo, one or more genes provided by the invention are either over
expressed or
inactivated in the germ line of animals using homologous recombination
[Capecchi, Science
244:2288-1292 (1989)]. Animals in which the gene is over expressed, under the
regulatory
control of exogenous or endogenous promoter elements, are known as transgenic
animals.
Animals in which an endogenous gene has been inactivated by homologous
recombination
are referred to as "knockout" animals. Knockout animals, preferably non-human
mammals,
can be prepared as described in U.S. Patent No. 5,557,032, incorporated herein
by reference.
Transgenic animals are useful to determine the roles polypeptides of the
invention play in
biological processes, and preferably in disease states. Transgenic animals are
useful as model
systems to identify compounds that modulate lipid metabolism. Transgenic
animals,
preferably non-human mammals, are produced using methods as described in U.S.
Patent No
5,489,743 and PCT Publication No. W094/28122, incorporated herein by
reference.
Transgenic animals can be prepared wherein all or part of the polynucleotides
of the
invention promoter is either activated or inactivated to alter the Ievel of
expression of the
polypeptides of the invention. Inactivation can be carried out using
homologous
recombination methods described above. Activation can be achieved by
supplementing or
even replacing the homologous promoter to provide for increased protein
expression. The
homologous promoter can be supplemented by insertion of one or more
heterologous
enhancer elements known to confer promoter activation in a particular tissue.
4.10 USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit one
or more of the uses or biological activities (including those associated with
assays cited
herein) identified herein. Uses or activities described for proteins of the
present invention
may be provided by administration or use of such proteins or of
polynucleotides encoding
such proteins (such as, for example, in gene therapies or vectors suitable for
introduction of
DNA). The mechanism underlying the particular condition or pathology will
dictate whether
the polypeptides of the invention, the polynucleotides of the invention or
modulators
(activators or inhibitors) thereof would be beneficial to the subject in need
of treatment.
Thus, "therapeutic compositions of the invention" include compositions
comprising isolated
~~SA~CT CA 02453344 2004-O1-21
43
polynucleotides (including recombinant DNA molecules, cloned genes and
degenerate
variants thereof) or polypeptides of the invention (including full length
protein, mature
protein and truncations or domains thereof), or compounds and other substances
that
modulate the overall activity of the target gene products, either at the level
of target
gene/protein expression or target protein activity. Such modulators include
polypeptides,
analogs, (variants), including fragments and fusion proteins, antibodies and
other binding
proteins; chemical compounds that directly or indirectly activate or inhibit
the polypeptides
of the invention (identified, e.g., via drug screening assays as described
herein); antisense
polynucleotides and polynucleotides suitable for triple helix formation; and
in particular
antibodies or other binding partners that specifically recognize one or more
epitopes of the
polypeptides of the invention.
The polypeptides of the present invention may likewise be involved in cellular
activation or in one of the other physiological pathways described herein.
4.10.1 RESEARCH USES AND UTILITIES
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant
protein for analysis, characterization or therapeutic use; as markers for
tissues in which the
corresponding protein is preferentially expressed (either constitutively or at
a particular stage
of tissue differentiation or development or in disease states); as molecular
weight markers on
gels; as chromosome markers or tags (when labeled) to identify chromosomes or
to map
related gene positions; to compare with endogenous DNA sequences in patients
to identify
potential genetic disorders; as probes to hybridize and thus discover novel,
related DNA
sequences; as a source of information to derive PCR primers for genetic
fingerprinting; as a
probe to "subtract-out'° known sequences in the process of discovering
other novel
polynucleotides; for selecting and making oligomers for attachment to a "gene
chip" or other
support, including for examination of expression patterns; to raise anti-
protein antibodies
using DNA immunization techniques; and as an antigen to raise anti-DNA
antibodies or
elicit another immune response. Where the polynucleotide encodes a protein
which binds or
potentially binds to another protein (such as, for example, in a receptor-
ligand interaction),
the polynucleotide can also be ~zsed in interaction trap assays (such as, for
example, that
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding interaction.
805A/PCT ~ 02453344 2004-O1-21
44
The polypeptides provided by the present invention can similarly be used in
assays to
determine biological activity, including in a panel of multiple proteins for
high-throughput
screening; to raise antibodies or to elicit another immune response; as a
reagent (including
the labeled reagent) in assays designed to quantitatively determine levels of
the protein (or
S its receptor) in biological fluids; as markers for tissues in which the
corresponding
polypeptide is preferentially expressed (either constitutively or at a
particular stage of tissue
differentiation or development or in a disease state); and, of course, to
isolate correlative
receptors or ligands. Proteins involved in these binding interactions can also
be used to
screen for peptide or small molecule inhibitors or agonists of the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in the
art. References disclosing such methods include without limitation "Molecular
Cloning: A
Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J.,
E. F.
Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to
Molecular
Cloning Techniques", Academic Press, Bergen S. L. and A. R. Kirnmel eds.,
1987.
4.10.2 NUTRITIONAL USES
Polynucleotides and polypeptides of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein or
amino acid supplement, use as a carbon source, use as a nitrogen source and
use as a source of
carbohydrate. In such cases the polypeptide or polynucleotide of the invention
can be added to
the feed of a particular organism or can be adminstered as a separate solid or
liquid
preparation, such as in the form of powder, pills, solutions, suspensions or
capsules. In the case
of microorganisms, the polypeptide or polynucleotide of the invention can be
added to the
medium in or on which the microorganism is cultured.
4.10. CYTOKINE AND CELL PROLIFERATION/D:IFFERENTIATION
ACTIVITY
A polypeptide of the present invention may exhibit activity relating to
cytokine, cell
proliferation (either inducing or inhibiting) or cell differentiation (either
inducing or
inhibiting) activity or may induce production of other cytokines in certain
cell populations.
A polynucleotide of the invention can encode a polypeptide exhibiting such
attributes.
805A/PCT ~ 02453344 2004-O1-21
Many protein factors discovered to date, including all known cytokines, have
exhibited
activity in one or more factor-dependent cell proliferation assays, and hence
the assays
serve as a convenient confirmation of cytokine activity. The activity of
therapeutic
compositions of the present invention is evidenced by any one of a number of
routine factor
5 dependent cell proliferation assays for cell lines including, without
limitation, 32D, DA2,
DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RBS, DA1, 123, T1165,
HT2,
CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the
invention
can be used in the following:
Assays for T-cell or thymocyte proliferation include without limitation those
10 described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M.
Kruisbeek, D. H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 3, In vitro assays for Mouse Lymphocyte Function
3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-
3500, 1986;
Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
15 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992;
Bowman et al., I.
Immunol. 152:1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node cells
or thymocytes include, without limitation, those described in: Polyclonal T
cell stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E.
e.a. Coligan
20 eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of
mouse and human interleukin-y, Schreiber, R. D. In Current Protocols in
Immunology. J. E.
e.a. Coligan eds. Vol I pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic cells
include, without limitation, those described in: Measurement of° Human
and Murine
25 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P.
E. In Current
Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and
Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173 :1205-1211, 1991; Moreau
et al.,
Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938,
1983; Measurement of mouse and human interleukin 6--Nordan, R. In Current
Protocols in
30 Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto. /991;
Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin I I--Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In
Current Protocols
in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
805A/rCT CA 02453344 2004-O1-21
46
Measurement of mouse and human Interleukin 9--Ciarletta, A., Giannotti, J.,
Clark, S. C.
and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1
pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies,
E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In l~it~o assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and their
cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et
al., Proc.
Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.
11:405-411,
1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512,
1988.
4.10.4 STEM CELL GR~WTH FACT~R ACTIVITY
A polypeptide of the present invention may exhibit stem cell growth factor
activity
and be involved in the proliferation, differentiation and survival of
pluripotent and totipotent
stem cells including primordial germ cells, embryonic stem cells,
hematopoietic stem cells
and/or germ line stem cells. Administration of the polypeptide of the
invention to stem cells
in vivo or ex vivo is expected to maintain and expand cell populations in a
totipotential or
pluripotential state which would be useful for re-engineering damaged or
diseased tissues,
transplantation, manufacture of bio-pharmaceuticals and the development of bio-
sensors.
The ability to produce large quantities of human cells has important working
applications for
the production of human proteins which currently must be obtained from non-
human sources
or donors, implantation of cells to treat diseases such as Parkinson's,
Alzheimer's and other
neurodegenerative diseases; tissues for grafting such as bone marrow, skin,
cartilage,
tendons, bone, muscle (including cardiac muscle), blood vessels, cornea,
neural cells,
gastrointestinal cells and others; and organs for transplantation such as
kidney, liver,
pancreas (including islet cells), heart and Lung.
It is contemplated that multiple different exogenous growth factors and/or
cytokines
may be administered in combination with the polypeptide of the invention to
achieve the
desired effect, including any of the growth factors listed herein, other stem
cell maintenance
factors, and specifically including stem cell factor (SCF), leukemia
inhibitory factor (LIF),
~~S~CT CA 02453344 2004-O1-21
Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6
receptor fused to IL-
6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF,
thrombopoietin (TPO), platelet factor 4 (PF-4), platelet-derived growth factor
(PDGF),
neural growth factors and basic fibroblast growth factor (bFGF).
Since totipotent stem cells can give rise to virtually any mature cell type,
expansion
of these cells in culture will facilitate the production of large quantities
of mature cells.
Techniques for culturing stem cells are known in the art and administration of
polypeptides
of the invention, optionally with other growth factors and/or cytokines, is
expected to
enhance the survival and proliferation of the stem cell populations. This can
be
accomplished by direct administration of the polypeptide of the invention to
the culture
medium. Alternatively, stroma cells transfected with a polynucleotide that
encodes for the
polypeptide of the invention can be used as a feeder layer for the stem cell
populations in
culture or in vivo. Stromal support cells for feeder layers may include
embryonic bone
marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured
embryonic
fibroblasts (see U.S. Patent No. 5,690,926).
Stem cells themselves can be transfected with a polynucleotide of the
invention to
induce autocrine expression of the polypeptide of the invention. This will
allow for
generation of undifferentiated totipotential/pluripotential stem cell lines
that are useful as is
or that can then be differentiated into the desired mature cell types. These
stable cell lines
can also serve as a source of undifferentiated totipotential/pluripotential
mRNA to create
cDNA libraries and templates for polymerase chain reaction experiments. These
studies
would allow for the isolation and identification of differentially expressed
genes in stem cell
populations that regulate stem cell proliferation and/or maintenance.
Expansion and maintenance of totipotent stem cell populations will be useful
in the
treatment of many pathological conditions. For example, polypeptides of the
present
invention may be used to manipulate stern cells in culture to give rise to
neuroepithelial cells
that can be used to augment or replace cells damaged by illness, autoimmune
disease,
accidental damage or genetic disorders. The polypeptide of the invention may
be useful for
inducing the proliferation of neural cells and for the regeneration of nerve
and brain tissue,
i.e. for the treatment of central and peripheral nervous system diseases and
neuropathies, as
well as mechanical and traumatic disorders which involve degeneration, death
or trauma to
neural cells or nerve tissue. In addition, the expanded stem cell populations
can also be
805A/PCT ~ 02453344 2004-O1-21
48
genetically altered for gene therapy purposes and to decrease host rejection
of replacement
tissues after grafting or implantation.
Expression of the polypeptide of the invention and its effect on stem cells
can also
be manipulated to achieve controlled differentiation of the stem cells into
more differentiated
cell types. A broadly applicable method of obtaining pure populations of a
specific
differentiated cell type from undifferentiated stem cell populations involves
the use of a cell-
type specific promoter driving a selectable marker. The selectable marker
allows only cells
of the desired type to survive. For example, stem cells can be induced to
differentiate into
cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et
al., J. Clin.
Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In:
Principles of
Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively,
directed
differentiation of stem cells can be accomplished by culturing the stem cells
in the presence
of a differentiation factor such as retinoic acid aaad an antagonist of the
polypeptide of the
invention which would inhibit the effects of endogenous stem cell factor
activity and allow
differentiation to proceed.
In vitro cultures of stem cells can be used to determine if the polypeptide of
the
invention exhibits stem cell growth factor activity. Stem cells are isolated
from any one of
various cell sources (including hematopoietic stem cells and embryonic stem
cells) and
cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad.
Sci, U.S.A.,
92: 7844-7848 (1995), in the presence of the polypeptide of the invention
alone or in
combination with other growth factors or cytokines. The ability of the
polypeptide of the
invention to induce stem cells proliferation is determined by colony formation
on semi-solid
support e.g. as described by Bernstein et al., Blood, 77: 231-2321 (1991).
4.10.5 HEMATOPOIESIS REGULATING ACTIVITY
A polypeptide of the present invention may be involved in regulation of
hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell
disorders.
Even marginal biological activity in support of colony forming cells or of
factor-dependent
cell lines indicates involvement in regulating hematopoiesis, e.g. in
supporting the growth
and proliferation of erythroid progenitor cells alone or in combination with
other cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in conjunction
with irradiation/chemotherapy to stimulate the production of erythroid
precursors and/or
erythroid cells; in supporting the growth and proliferation of myeloid cells
such as
805tiI PCT CA 02453344 2004-O1-21
49
granulocytes and monocytes/macrophages (i.e., traditional CSF activity)
useful, for example,
in conjunction with chemotherapy to prevent or treat consequent rnyelo-
suppression; in
supporting the growth and proliferation of rnegakaryocytes and consequently of
platelets
thereby allowing prevention or treatment of various platelet disorders such as
thrombocytopenia, and generally for use in place of or complimentary to
platelet
transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells
which are capable of maturing to any and all of the above-mentioned
hematopoietic cells and
therefore find therapeutic utility in various stem cell disorders (such as
those usually treated
with transplantation, including, without limitation, aplastic anemia and
paroxysmal nocturnal
hemoglobinuria), as well as in repopulating the stem cell compartment post
irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in <;onjunction
with bone marrow
transplantation or with peripheral progenitor cell transplantation (homologous
or
heterologous)) as normal cells or genetically manipulated for gene therapy.
Therapeutic compositions of the invention can be used in the following:
Suitable assays for proliferation and differentiation of various hematopoietic
lines are
cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without limitation,
those described in: .Iohansson et al. Cellular Biology 15:141-151, 1995;
Keller et al.,
Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,
1993.
Assays for stem cell survival and differentiation (which will identify, among
others,
proteins that regulate lympho-hematopoiesis) include, without limitation,
those described in:
Methylcellulose colony forming assays, Freshney, M. G. In Culture of
Hematopoietic Cells.
R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.
1994;
Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic
colony forming cells with high proliferative potential, McNiece, I. K. and
Briddell, R. A. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc.,
New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994;
Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of I-
Iematopoietic Cells.
R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y.
1994; Long term
bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter,
M. and Allen,
T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-
179, Wiley-Liss,
goSA/PC,I, CA 02453344 2004-O1-21
Inc., New York, N.Y. 1994; Long term culture initiating cell assay,
Sutherland, H. J. In
Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. 'Vol pp. 139-162,
~Wiley-Liss,
Inc., New York, N.Y. 1994.
4.10.6 'fISSiJE GR~i~VTH ACTIVITY'
A polypeptide of the present invention also may be involved in bone,
cartilage,
tendon, ligament and/or nerve tissue growth or regeneration, as well as in
wound healing and
tissue repair and replacement, and in healing of burns, incisions and ulcers.
A polypeptide of the present invention which induces cartilage and/or bone
growth in
10 circumstances where bone is not normally formed, has application in the
healing of bone
fractures and cartilage damage or defects in humans and other animals.
Compositions of a
polypeptide, antibody, binding partner, or other modulator of the invention
may have
prophylactic use in closed as well as open fracture reduction and also in the
improved
fixation of artificial joints. De novo bone formation induced by an osteogenic
agent
15 contributes to the repair of congenital, trauma induced, or oncologic
resection induced
craniofacial defects, and also is useful in cosmetic plastic surgery.
A polypeptide of this invention may also be involved in attracting bone-
forming
cells, stimulating growth of bone-forming cells, or inducing differentiation
of progenitors of
bone-forming cells. Treatment of osteoporosis, osteoarthritis, bone
degenerative disorders, or
20 periodontal disease, such as through stimulation of bone and/or cartilage
repair or by
blocking inflammation or processes of tissue destruction (collagenase
activity, osteoclast
activity, etc.) mediated by inflammatory processes may also be possible using
the
composition of the invention.
Another category of tissue regeneration activity that may involve the
polypeptide of
25 the present invention is tendon/ligament formation. Induction of
tendon/ligament-like tissue
or other tissue formation in circumstances where such tissue is not normally
formed, has
application in the healing of tendon or ligament tears, deformities and other
tendon or
ligament defects in humans and other animals. Such a preparation employing a
tendon/ligament-like tissue inducing protein may have prophylactic use in
preventing
30 damage to tendon or ligament tissue, as well as use in the improved
fixation of tendon or
ligament to bone or other tissues, and in repairing defects to tendon or
ligament tissue. De
novo tendon/ligament-like tissue formation induced by a composition of the
present
invention contributes to the repair of congenital, trauma induced, or other
tendon or ligament
~~SA/PCT CA 02453344 2004-O1-21
51
defects of other origin, and is also useful in cosmetic plastic surgery for
attachment or repair
of tendons or ligaments. The compositions of the present invention may provide
environment to attract tendon- or ligament-forming cells, stimulate growth of
tendon- or
ligament-forming cells, induce differentiation of progenitors of tendon- or
ligament-forming
cells, or induce growth of tendon/ligament cells or progenitors e~ vivo for
return i~ vivo to
effect tissue repair. The compositions of the invention may also be useful in
the treatment of
tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The
compositions
may also include an appropriate matrix and/or sequestering agent as a carrier
as is well
known in the art.
The compositions of the present invention may also be useful for proliferation
of
neural cells and for regeneration of nerve and brain tissue, i.e. for the
treatment of central
and peripheral nervous system diseases and neuropathies, as well as mechanical
and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a composition may be used in the treatment of
diseases of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present invention
include mechanical and traumatic disorders, such as spinal cord disorders,
head trauma and
cerebrovascular diseases such as stroke. Peripheral neuropathies resulting
from
chemotherapy or other medical therapies may also be treatable using a
composition of the
invention.
Compositions of the invention may also be useful to promote better or faster
closure
of non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
Compositions of the present invention may also be involved in the generation
or
regeneration of other tissues, such as organs (including, for example,
pancreas, liver,
intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac)
and vascular
(including vascular endothelium) tissue, or for promoting the growth of cells
comprising
such tissues. Part of the desired effects may be by inhibition or modulation
of fibrotic
scarring may allow normal tissue to regenerate. A polypeptide of the present
invention may
also exhibit angiogenic activity.
805A/PCT ~ 02453344 2004-O1-21
52
A composition of the present invention may also be useful for gut protection
or
regeneration and treatment of lung or liver fabrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A composition of the present invention may also be useful for promoting or
inhibiting differentiation of tissues described above from precursor tissues
or cells; or for
inhibiting the growth of tissues described above.
Therapeutic compositions of the invention can be used in the following:
Assays for tissue generation activity include, without limitation, those
described in:
International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International
Patent Publication No. W095/05846 (nerve, neuronal); International Patent
Publication No.
W091/07491 (skin, endothelium).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. l, and Rovee, D. T.,
eds.),
Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and
Mertz, J. Invest.
Dermatol 71:382-84 (1978).
4.10.7 IMMUNE STIMULATING OR SUPPRESSING ACTIVITY''
A polypeptide of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays are
described herein. A polynucleotide of the invention can encode a polypeptide
exhibiting
such activities. A protein may be useful in the treatment of various immune
deficiencies and
disorders (including severe combined immunodeficiency (SCID)), e.g., in
regulating (up or
down) growth and proliferation of T and/or B lymphocytes, as well as effecting
the cytolytic
activity of NK cells and other cell populations. These immune deficiencies may
be genetic or
be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or
may result from
autoimmune disorders. More specifically, infectious diseases causes by viral,
bacterial,
fungal or other infection may be treatable using a protein of the present
invention, including
infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania
spp., malaria
spp. and various fungal infections such as candidiasis. Of course, in this
regard, proteins of
the present invention may also be useful where a boost to the immune system
generally may
be desirable, i.e., in the treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention
include, for example, connective tissue disease, multiple sclerosis, systemic
lupus
805A/PCT ~ 02453344 2004-O1-21
53
erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre
syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis,
myasthenia gravis,
graft-versus-host disease and autoimmune inflammatory eye disease. Such a
protein (or
antagonists thereof, including antibodies) of the present invention may also
to be useful in
the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum
sickness, drug
reactions, food allergies, insect venom allergies, mastocytosis, allergic
rhinitis,
hypersensitivity pneumonitis, w-ticaria, angioedema, eczema, atopic
dermatitis, allergic
contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic
conjunctivitis,
atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary
conjunctivitis and
contact allergies), such as asthma (particularly allergic asthma) or other
respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein (or
antagonists
thereof) of the present invention. The therapeutic effects of the polypeptides
or antagonists
thereof on allergic reactions can be evaluated by in vivo animals models such
as the
cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66,
1998), skin
prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin
sensitization test
(Voter et al., Arch. Toxocol. 73: SOl-9), and murine local lymph node assay
{Kimber et al.,
J. Toxicol. Environ. Health 53: 563-79).
Using the proteins of the invention it may also be possible to modulate immune
responses, in a number of ways. Down regulation may be in the form of
inhibiting or
blocking an immune response already in progress or may involve preventing the
induction of
an immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression of T
cell responses is generally an active, non-antigen-specific, process which
requires continuous
exposure of the T cells to the suppressive agent. Tolerance, which involves
inducing
non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that
it is generally antigen-specific and persists after exposure to the tolerizing
agent has ceased.
Operationally, tolerance can be demonstrated by the lack of a T cell response
upon
reexposure to specific antigen in the absence of the tolerizing agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as, for example, B7)), e.g.,
preventing high
level lymphokine synthesis by activated T cells, will be useful in situations
of tissue, skin
and organ transplantation and in graft-versus-host disease (G"VHD). For
example, blockage
BOSL-1/PCT CA 02453344 2004-O1-21
54
of T cell function should result in reduced tissue destruction in tissue
transplantation.
Typically, in tissue transplants, rejection of the transplant is initiated
through its recognition
as foreign by T cells, followed by an immune reaction that destroys the
transplant. The
administration of a therapeutic composition of the invention may prevent
cytokine synthesis
by immune cells, such as T cells, and thus acts as an immunosuppressant.
Moreover, a lack
of costimulation may also be sufficient to anergize the T cells, thereby
inducing tolerance in
a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking
reagents may
avoid the necessity of repeated administration of these blocking reagents. To
achieve
sufficient immunosuppression or tolerance in a subject, it may also be
necessary to block the
function of a combination of B lymphocyte antigens.
The efficacy of particular therapeutic compositions in preventing organ
transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of CaVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of therapeutic compositions of the invention on the
development of that
disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation
of T cells that are reactive against self tissue and which proanote the
production of cytokines
and autoantibodies involved in the pathology of the diseases. Preventing the
activation of
autoreactive T cells may reduce or eliminate disease symptoms. Administration
of reagents
which block stimulation of T cells can be used to inhibit T cell activation
and prevent
production of autoantibodies or T cell-derived cytokines which may be involved
in the
disease process. Additionally, blocking reagents may induce antigen-specific
tolerance of
autoreactive T cells which could lead to long-term relief from the disease.
The efficacy of
blocking reagents in preventing or alleviating autoimmune disorders can be
determined
using a number of well-characterized animal models of human autoimmune
diseases.
Examples include murine experimental autoimmune encephalitis, systemic lupus
erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune
collagen
B~SA~CT CA 02453344 2004-O1-21
arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental
myasthenia
gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp.
840-856).
Upregulation of an antigen function (e.g., a B lymphocyte antigen function),
as a
5 means of up regulating immune responses, may also be useful in therapy.
Upregulation of
immune responses may be in the form of enhancing an existing immune response
or eliciting
an initial immune response. For example, enhancing an immune response may be
useful in
cases of viral infection, including systemic viral diseases such as influenza,
the common
cold, and encephalitis.
10 Alternatively, anti-viral immune responses may be enhanced in an infected
patient by
removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-pulsed
APCs either expressing a peptide of the present invention or together with a
stimulatory
form of a soluble peptide of the present invention and reintroducing the in
vitro activated T
cells into the patient. Another method of enhancing anti-viral immune
responses would be to
15 isolate infected cells from a patient, transfect them with a nucleic acid
encoding a protein of
the present invention as described herein such that the cells express all or a
portion of the
protein on their surface, and reintroduce the transfected cells into the
patient. The infected
cells would now be capable of delivering a costimulatory signal to, and
thereby activate, T
cells in vivo.
20 A polypeptide of the present invention may provide the necessary
stimulation signal
to T cells to induce a T cell mediated immune response against the transfected
tumor cells.
In addition, tumor cells which lack MHC class I or MHC class II molecules, or
which fail to
reexpress sufficient mounts of MHC class I or MHC class II molecules, can be
transfected
with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain
truncated portion)
25 of an MHC class I alpha chain protein and (32 microglobulin protein or an
MHC class II
alpha chain protein and an MHC class II beta chain protein to thereby express
MHC class I
or MHC class II proteins on the cell surface. Expression of the appropriate
class I or class II
MHC in conjunction with a peptide having the activity of a B lymphocyte
antigen (e.g.,
B7-1, B7-2, B7-3) induces a T cell mediated immune response against the
transfected tumor
30 cell. Optionally, a gene encoding an antisense construct which blocks
expression of an MHC
class II associated protein, such as the invariant chain, can also be
cotransfected with a DNA
encoding a peptide having the activity of a B lymphocyte antigen to promote
presentation of
tumor associated antigens and induce tumor specific immunity. Thus, the
induction of a T
80511/ r CT CA 02453344 2004-O1-21
56
cell mediated immune response in a human subject may be sufficient to overcome
tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by
the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek,
D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates
and
Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function
3.1-3.19;
Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad.
Sci. USA
IO 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa
et al., J.
Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986;
Takai et al.,
J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998;
Bertagnolli et
al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092,
1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl/Th2 profiles) include, without limitation, those
described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays fox B cell function:
In vitro
antibody production, Mond, J. J. and Brunswicl~, M. In Current Protocols in
Immunology. J.
E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.
M. Kruisbeek,
D. H. Margulies, E. M. Shevach, W. Strober, Pub. Gxeene Publishing Associates
and
Wiley-Interscience {Chapter 3, In Vitro assays for Mouse Lymphocyte Function
3.1-3.19;
Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-
3500, 1986;
Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783,
1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-
260,
805A/rCT CA 02453344 2004-O1-21
57
1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al.,
Science
264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-
1264,
1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and
Inaba et al.,
Journal of Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz et
al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytornetry 14:891-
897, 1993;
Gorczyca et al., International Journal of ~ncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
development
include, without limitation, those described in: Antica et al., Blood 84:111-
117, 1994; Fine
et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-
2778, 1995;
Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
4.10.8 ACTIVIN/INHIBIN ACTIVITY
A polypeptide of the present invention may also exhibit activin- or inhibin-
related
activities. A polynucleotide of the invention may encode a polypeptide
exhibiting such
characteristics. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to stimulate
the release of follicle stimulating hormone {FSH). Thus, a polypeptide of the
present
invention, alone or in heterodimers with a member of the inhibin family, may
be useful as a
contraceptive based on the ability of inhibins to decrease fextility in female
mammals and
decrease spermatogenesis in male mammals. Administration of sufficient amounts
of other
inhibins can induce infertility in these mammals. Alternatively, the
polypeptide of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin
group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for
advancement
of the onset of fertility in sexually immature mammals, so as to increase the
lifetime
reproductive performance of domestic animals such as, but not limited to,
cows, sheep and
pigs.
805A/PCT ~ 02453344 2004-O1-21
58
The activity of a polypeptide of the invention may, among other means, be
measured
by the following methods.
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1.985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
4.10.9 CHEMOTACTIC/CHEMOKINETIC ACTIVITY
A polypeptide of the present invention may be involved in chemotactic or
chemokinetic activity for mammalian cells, including, for example, monocytes,
fibroblasts,
neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial
cells. A
polynucleotide of the invention can encode a polypeptide exhibiting such
attributes.
Chemotactic and chemokinetic receptor activation can be used to mobilize or
attract a
desired cell population to a desired site of action. Chemotactic or
chemokinetic compositions
(e.g. proteins, antibodies, binding partners, or modulators of the invention)
provide particular
advantages in treatment of wounds and other trauma to tissues, as well as in
treatment of
localized infections. For example, attraction of lymphocytes, rnonocytes or
neutrophils to
tumors or sites of infection may result in improved immune responses against
the tumor or
infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it can
stimulate, directly or indirectly, the directed orientation ox movement of
such cell
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity fox a
population of
cells can be readily determined by employing such protein or peptide in any
known assay for
cell chemotaxis.
Therapeutic compositions of the invention can be used in the following:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration of
cells across a membrane as well as the ability of a protein to induce the
adhesion of one cell
population to another cell population. Suitable assays for movement and
adhesion include,
without limitation, those described in: Current Protocols in Immunology, Ed by
J. E.
Coligan, A. M. Kxuisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub.
Greene
Publishing Associates and Wiley-Tnterscience (Chapter 6.12, Measurement of
alpha and beta
,,;. ,.. :; : .. _ .. .. ~, ,."". ., _. .. ,.,. .. ..
805A/PCT ~ 02453344 2004-O1-21
59
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995;
Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et
al. J. of
Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768,
1994.
4,10.10 HEMOSTATIC AND THROMBOLYTIC ACTIVITY
A polypeptide of the invention may also be involved in hemostatis or
thrombolysis or
thrombosis. A polynucleotide of the invention can encode a polypeptide
exhibiting such
attributes. Compositions may be useful in treatment of various coagulation
disorders
(including hereditary disorders, such as hemophilias) or to enhance
coagulation and other
hemostatic events in treating wounds resulting from trauma, surgery or other
causes. A
composition of the invention may also be useful for dissolving or inhibiting
formation of
thromboses and for treatment and prevention of conditions resulting therefrom
(such as, for
example, infarction of cardiac and central nervous system vessels (e.g.,
stroke).
Therapeutic compositions of the invention can be used in the following:
I S Assay for hemostatic and thrombolytic activity include, without
limitation, those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins 35:467-474, 1988.
4.10.11 CANCER DIAGNOSIS AND THERAPY
Polypeptides of the invention may be involved in cancer cell generation,
proliferation
or metastasis. Detection of the presence or amount of polynucleotides or
polypeptides of the
invention may be useful for the diagnosis and/or prognosis of one or more
types of cancer.
For example, the presence or increased expression of a
polynucleotide/polypeptide of the
invention may indicate a hereditary risk of cancer, a precancerous condition,
or an ongoing
malignancy. Conversely, a defect in the gene or absence of the polypeptide may
be
associated with a cancer condition. Identification of single nucleotide
polymorphisms
associated with cancer or a predisposition to cancer may also be useful for
diagnosis or
prognosis.
Cancer treatments promote tumor regression by inhibiting tumor cell
proliferation,
inhibiting angiogenesis (growth of new blood vessels that is necessary to
support tumor
growth) and/or prohibiting metastasis by reducing tumor cell motility or
invasiveness.
Therapeutic compositions of the invention may be effective in adult and
pediatric oncology
gO~~~T CA 02453344 2004-O1-21
including in solid phase tumors/malignancies, locally advanced tumors, human
soft tissue
sarcomas, metastatic cancer, including lymphatic metastases, blood cell
malignancies
including multiple myeloma, acute and chronic leukemias, and lymphomas, head
and neck
cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers
including
5 small cell carcinoma and non-small cell cancers, breast cancers including
small cell
carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal
cancer,
stomach cancer, colon cancer, colorectal cancer and polyps associated with
colorectal
neoplasia, pancreatic cancers, liver cancer, urologic cancers including
bladder cancer and
prostate cancer, malignancies of the female genital tract including ovarian
carcinoma, uterine
10 (including endometrial) cancers, and solid tumor in the ovarian follicle,
kidney cancers
including renal cell carcinoma, brain cancers including intrinsic brain
tumors,
neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell
invasion in the central
nervous system, bone cancers including osteomas, skin cancers including
malignant
melanoma, tumor progression of human skin keratinocytes, squarnous cell
carcinoma, basal
15 cell carcinoma, hemangiopericytoma and Karposi's sarcoma.
Polypeptides, polynucleotides, or modulators of polypeptides of the invention
(including inhibitors and stimulators of the biological activity of the
polypeptide of the
invention) may be administered to treat cancer. Therapeutic compositions can
be
administered in therapeutically effective dosages alone or in combination with
adjuvant
20 cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy,
and laser
therapy, and may provide a beneficial effect, e.g. reducing tumor size,
slowing rate of tumor
growth, inhibiting metastasis, or otherwise improving overall clinical
condition, without
necessarily eradicating the cancer.
The composition can also be administered in therapeutically effective amounts
as a
25 portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of
the polypeptide or
modulator of the invention with one or more anti-cancer drugs in addition to a
pharmaceutically acceptable carrier for delivery. The use of anti-cancer
cocktails as a cancer
treatment is routine. Anti-cancer drugs that are well known in the art and can
be used as a
treatment in combination with the polypeptide or modulator of the invention
include:
30 Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan,
Carboplatin,
Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine
HCl
(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCI,
Doxorubicin HCI,
Estramustine phosphate sodium, Etoposide (V 16-213), Floxuridine, 5-
Fluorouracil (5-Fu),
S~~~p~T, CA 02453344 2004-O1-21
61
Flutamide, Hydroxyurea (hydroxycarbamide), Lfosfamide, Interferon Alpha-2a,
Interferon
Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine,
Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna,
Methotrexate (MTX), Mitomycin, Mitoxantrone HCI, Octreotide, Plicamycin,
Procarbazine
HCI, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine
sulfate,
Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-
2,
Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.
In addition, therapeutic compositions of the invention may be used for
prophylactic
treatment of cancer. There are hereditary conditions and/or environmental
situations (e.g.
exposure to carcinogens) known in the art that predispose an individual to
developing
cancers. Under these circumstances, it may be beneficial to treat these
individuals with
therapeutically effective doses of the polypeptide of the invention to reduce
the risk of
developing cancers.
Ira vitro models can be used to determine the effective doses of the
polypeptide of the
invention as a potential cancer treatment. These in vitro models include
proliferation assays
of cultured tumor cells, growth of cultured tumor cells in soft agar (see
Freshney, (1987)
Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, NY
Ch 18
and Ch 21), tumor systems in nude mice as described in Giovanella et al., J.
Natl. Can. Inst.,
52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden
Chamber assays
as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and
angiogenesis
assays such as induction of vascularization of the chick chorioallantoic
membrane or
induction of vascular endothelial cell migration as described in Ribatta et
al., Intl. J. Dev.
Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9
(1999), respectively.
Suitable tumor cells lines are available, e.g. from American Type Tissue
Culture Collection
catalogs.
4.10.12 RECEPTOIt/LIGAN1) ACTIVITY
A polypeptide of the present invention may also demonstrate activity as
receptor,
receptor ligand or inhibitor or agonist of receptor/ligand interactions. A
polynucleotide of
the invention can encode a polypeptide exhibiting such characteristics.
Examples of such
receptors and ligands include, without limitation, cytokine receptors and
their ligands,
receptor kinases and their ligands, receptor phosphatases and their ligands,
receptors
involved in cell-cell interactions and their ligands (including without
limitation, cellular
805ti1PCT CA 02453344 2004-O1-21
62
adhesion molecules (such as selectins, integrins and their ligands) and
receptor/ligand pairs
involved in antigen presentation, antigen recognition and development of
cellular and
humoral immune responses. Receptors and Iigands are also useful for screening
of
potential peptide or small molecule inhibitors of the relevant receptorlligand
interaction. A
protein of the present invention (including, without limitation, fragments of
receptors and
ligands) may themselves be useful as inhibitors of receptor/ligand
interactions.
The activity of a polypeptide of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described
in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D.
H.
Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static
conditions
7.28.1- 7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987;
Bierer et al.,
J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160
1989;
Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell
80:661-670, 1995.
By way of example, the polypeptides of the invention may be used as a receptor
for a
ligand(s) thereby transmitting the biological activity of that ligand(s).
Ligands may be
identified through binding assays, affinity chromatography, dihybrid screening
assays,
BIAcore assays, gel overlay assays, or other methods known in the art.
Studies characterizing drugs or proteins as agonist or antagonist or partial
agonists or
a partial antagonist require the use of other proteins as competing ligands.
The polypeptides
of the present invention or ligand(s) thereof may be labeled by being coupled
to
radioisotopes, colorimetric molecules or a toxin molecules by conventional
methods.
("Guide to Protein Purification" Murray P. Deutscher (ed) Methods in
Enzymology Vol. 182
(1990) Academic Press, Inc. San Diego). Examples of radioisotopes include, but
are not
limited to, tritium and carbon-14 . Examples of colorimetric molecules
include, but are not
limited to, fluorescent molecules such as fluorescamine, or rhodamine or other
colorimetric
molecules. Examples of toxins include, but are not limited, to ricin.
4.10.13 DRUG SCREENING
This invention is particularly useful for screening chemical compounds by
using the
novel polypeptides or binding fragments thereof in any of a variety of drug
screening
techniques. The polypeptides or fragments employed in such a test may either
be free in
805A/PCT ~ 02453344 2004-O1-21
63
solution, axed to a solid support, borne on a cell surface or located
intracellularly. One
method of drug screening utilizes eukaryotic or prokaryotic host cells which
are stably
transformed with recombinant nucleic acids expressing the polypeptide or a
fragment
thereof. Drugs are screened against such transformed cells in competitive
binding assays.
Such cells, either in viable or fixed form, can be used for standard binding
assays. One may
measure, for example, the formation of complexes between polypeptides of the
invention or
fragments and the agent being tested or examine the diminution in complex
formation
between the novel polypeptides and an appropriate cell line, which are well
known in the art.
Sources for test compounds that may be screened for ability to bind to or
modulate
(i.e., increase or decrease) the activity of polypeptides of the invention
include ( 1 ) inorganic
and organic chemical libraries, (2) natural product libraries, and (3)
combinatorial libraries
comprised of either random or mimetic peptides, oligonucleotides or organic
molecules.
Chemical Libraries may be readily synthesized or purchased from a number of
commercial sources, and may include structural analogs of known compounds or
compounds
that are identified as "hits" or "Leads" via natural product screening.
The sources of natural product Libraries are microorganisms (including
bacteria and
fungi), animals, plants or other vegetation, or marine organisms, and
libraries of mixtures for
screening may be created by: (1) fermentation and extraction of broths from
soil, plant or
marine microorganisms or (2) extraction of the organisms themselves. Natural
product
libraries include polyketides, non-ribosomal peptides, and (non-naturally
occurring) variants
thereof. For a review, see Science 282:63-68 (1998).
Combinatorial libraries are composed of large numbers of peptides,
oligonucleotides
or organic compounds and can be readily prepared by traditional automated
synthesis
methods, PCR, cloning or proprietary synthetic methods. Of particular interest
are peptide
and oligonucleotide combinatorial libraries. Still other libraries of interest
include peptide,
protein, peptidomimetic, multiparallel synthetic collection, recombinatorial,
and polypeptide
libraries. For a review of combinatorial chemistry and libraries created
therefrom, see
Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples of
peptidomirnetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9{3):205-23
(1998); Hruby
et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Dorner et al., BioorgMed
Chem,
4(5):709-15 (1996) (alkylated dipeptides).
Identification of modulators through use of the various libraries described
herein
permits modification of the candidate "hit" (or "lead") to optimize the
capacity of the "hit"
CA 02453344 2004-O1-21
64
to bind a polypeptide of the invention. The molecules identified in the
binding assay are then
tested for antagonist or agonist activity in in vivo tissue culture or animal
models that are
well known in the art. In brief, the molecules are titrated into a plurality
of cell cultures or
animals and then tested for either cell/animal death or prolonged survival of
the animal/cells.
S The binding molecules thus identified may be complexed with toxins, e.g.,
ricin or
cholera, or with other compounds that are toxic to cells such as
radioisotopes. The
toxin-binding molecule complex is then targeted to a tumor or other cell by
the specificity of
the binding molecule for a polypeptide of the invention. Alternatively, the
binding
molecules may be complexed with imaging agents for targeting and imaging
purposes.
4.10.14 ASSAY FOR RECEPTOR ACTIVITY
The invention also provides methods to detect specific binding of a
polypeptide e.g. a
ligand or a receptor. The art provides numerous assays particularly useful for
identifying
previously unknown binding partners for receptor polypeptides of the
invention. For
1 S example, expression cloning using mammalian or bacterial cells, or
dihybrid screening
assays can be used to identify polynucleotides encoding binding partners. As
another
example, affinity chromatography with the appropriate immobilized polypeptide
of the
invention can be used to isolate polypeptides that recognize and bind
polypeptides of the
invention. There are a number of different libraries used for the
identification of
compounds, and in particular small molecules, that modulate (i. e., increase
or decrease)
biological activity of a polypeptide of the invention. Ligands for receptor
polypeptides of the
invention can also be identified by adding exogenous ligands, or cocktails of
ligands to two
cells populations that are genetically identical except for the expression of
the receptor of the
invention: one cell population expresses the receptor of the invention whereas
the other does
2S not. The responses of the two cell populations to the addition of
ligands(s) are then
compared. Alternatively, an expression library can be co-expressed with the
polypeptide of
the invention in cells and assayed for an autocrine response to identify
potential ligand(s). As
still another example, BIAcore assays, gel overlay assays, or other methods
known in the art
can be used to identify binding partner polypeptides, including, (1) organic
and inorganic
chemical libraries, (2) natural product libraries, and (3) combinatorial
libraries comprised of
random peptides, oligonucleotides or organic molecules.
The role of downstream intracellular signaling molecules in the signaling
cascade of
the polypeptide of the invention can be determined. For example, a chimeric
protein in
~~S~P~Z CA 02453344 2004-O1-21
which the cytoplasmic domain of the polypeptide of the invention is fused to
the
extracellular portion of a protein, whose ligand has been identified, is
produced in a host
cell. The cell is then incubated with the ligand specific for the
extracellular portion of the
chimeric protein, thereby activating the chimeric receptor. Known downstream
proteins
5 involved in intracellular signaling can then be assayed for expected
modifications i.e.
phosphorylation. Other methods known to those in the art can also be used to
identify
signaling molecules involved in receptor activity.
4.10.15 ANTI-INFLAMMATORY ACTIVITY
10 Compositions of the present invention may also exhibit anti-inflammatory
activity.
The anti-inflammatory activity may be achieved by providing a stimulus to
cells involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
15 suppressing production of other factors which more directly inhibit or
promote an
inflammatory response. Compositions with such activities can be used to treat
inflammatory
conditions including chronic or acute conditions), including without
limitation intimation
associated with infection (such as septic shock, sepsis or systemic
inflammatory response
syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis,
20 complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-
induced lung
injury, inflammatory bowel disease, Crohn's disease or resulting from over
production of
cytokines such as TNF or IL-1. Compositions of the invention may also be
useful to treat
anaphylaxis and hypersensitivity to an antigenic substance or material.
Compositions of this
invention may be utilized to prevent or treat conditions such as, but not
limited to, sepsis,
25 acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid
arthritis, chronic
inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1,
graft versus
host disease, inflammatory bowel disease, inflamation associated with
pulmonary disease,
other autoimmune disease or inflammatory disease, an antiproliferative agent
such as for
acute or chronic mylegenous leukemia or in the prevention of premature labor
secondary to
30 intrauterine infections.
4.10.16 LEUKEMIAS
805A/PCT CA 02453344 2004-O1-21
66
Leukemias and related disorders may be treated or prevented by administration
of a
therapeutic that promotes or inhibits function of the polynucleotides and/or
polypeptides of
the invention. Such leukemias and related disorders include but are not
limited to acute
leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,
S promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia,
chronic
myelocytic (granulocytie) leukemia and chronic lymphocytic leukemia (for a
review of such
disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co.,
Philadelphia).
4.10.17 NERVOUS SYSTEM DISORDERS
Nervous system disorders, involving cell types which can be tested for
efficacy of
intervention with compounds that modulate the activity of the polynucleotides
and/or
polypeptides of the invention, and which can be treated upon thLis observing
an indication of
therapeutic utility, include but are not limited to nervous system injuries,
and diseases or
disorders which result in either a disconnection of axons, a diminution or
degeneration of
neurons, or demyelination. Nervous system lesions which may be treated in a
patient
(including human and non-human mammalian patients) according to the invention
include
but are not limited to the following lesions of either the central (including
spinal cord, brain)
or peripheral nervous systems:
(i) 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;
(ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous
system
results in neuronal injury or death, including cerebral infarction or
ischemia, or spinal cord
infarction or ischemia;
(iii)infectious lesions, in which a portion of the n ervous system is
destroyed or
injured as a result of infection, for example, by an abscess or associated
with infection by
human immunodeficiency virus, herpes zoster, or herpes simplex virus or with
Lyme
disease, tuberculosis, syphilis;
(iv) 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, Alzheirner9s disease, Huntington's
chorea, or
amyotrophic lateral sclerosis;
805t11hCT CA 02453344 2004-O1-21
67
(v) lesions associated with nutritional diseases or disorders, in which a
portion of
the nervous system is destroyed or injured by a nutritional disorder or
disorder of
metabolism including but not limited to, vitamin B 12 deficiency, folic acid
deficiency,
Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease
(primary
degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
(vi) neurological lesions associated with systemic diseases including but not
limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus
erythematosus,
carcinoma, or sarcoidosis;
(vii) lesions caused by toxic substances including alcohol, lead, or
particular
neurotoxins; and
(viii) demyelinated lesions in which a portion of the nervous system is
destroyed or
injured by a demyelinating disease including but not limited to multiple
sclerosis, human
immunodeficiency virus-associated myelopathy, transverse myelopathy or various
etiologies, progressive multifocal leukoencephalopathy, and central pontine
myelinolysis.
Therapeutics which are useful according to the invention for treatment of 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,
therapeutics which
elicit any of the following effects may be useful according to the invention:
(i) increased survival time of neurons in culture;
(ii) increased sprouting of neurons in culture or in vivo;
(iii)increas ed production of a neuron-associated molecule in culture or in
vivo,
e.g., choline acetyltransferase or acetylcholinesterase with respect to motor
neurons; or
(iv) 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 be measured by the
method
set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased
sprouting of neurons
may be detected by methods set forth in hestronlc et al. (1980, Exp. Neurol.
70:65-82) or
Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of
neuron-associated molecules may be measured by bioassay, enzymatic assay,
antibody
binding, Northern blot assay, etc., depending on the molecule to be measured;
and motor
neuron dysfunction may be measured by assessing the physical manifestation of
motor
neuron disorder, e.g., weakness, motor neuron conduction velocity, or
functional disability.
CA 02453344 2004-O1-21
68
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
S selectively affect neurons such as arnyotrophic 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 FIereditary
Motorsensory
Neuropathy (Charcot-Marie-Tooth Disease).
4.10.18 OTHER ACTIVITIES
A polypeptide of the invention may also exhibit one or more of the following
additional activities or effects: inhibiting the growth, infection or function
of, or killing,
infectious agents, including, without limitation, bacteria, viruses, fungi and
other parasites;
1 S effecting (suppressing or enhancing) bodily characteristics, including,
without limitation,
height, weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or
organ or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or circadian cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
carbohydrate, vitamins, minerals, co-factors or other nutritional factors or
component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
2S differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies of
the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen in a
vaccine composition to raise an immune response against such protein or
another material or
entity which is cross-reactive with such protein.
4.10.19 IDENTIFICATION OF POLYMORPHISMS
CA 02453344 2004-O1-21
69
The demonstration of polymorphisms makes possible the identification of such
polymorphisms in human subjects and the pharmacogenetic use of this
information for
diagnosis and treatment. Such polymorphisms may be associated with, e.g.,
differential
predisposition or susceptibility to various disease states (such as disorders
involving
inflammation or immune response) or a differential response to drug
administration, and this
genetic information can be used to tailor preventive or therapeutic treatment
appropriately.
For example, the existence of a polymorphism associated with a predisposition
to
inflammation or autoimmune disease makes possible the diagnosis of this
condition in
humans by identifying the presence of the polymorphism.
Polymorphisms can be identified in a variety of ways known in the art which
alI
generally involve obtaining a sample from a patient, analyzing DNA from the
sample,
optionally involving isolation or amplification of the DNA, and identifying
the presence of
the polymorphism in the DNA. For example, PCR may be used to amplify an
appropriate
fragment of genomic DNA which may then be sequenced. Alternatively, the DNA
may be
subjected to allele-specific oligonucleotide hybridization (in which
appropriate
oligonucleotides are hybridized to the DNA under conditions permitting
detection of a single
base mismatch) or to a single nucleotide extension assay (in which an
oligonucleotide that
hybridizes immediately adjacent to the position of the polymorphism is
extended with one or
more labeled nucleotides). In addition, traditional restriction fragment
length polymorphism
analysis (using restriction enzymes that provide differential digestion of the
genomic DNA
depending on the presence or absence of the polymorphism) may be performed.
Arrays with
nucleotide sequences of the present invention can be used to detect
polymorphisms. The
array can comprise modified nucleotide sequences of the present invention in
order to detect
the nucleotide sequences of the present invention. In the alternative, any one
of the
nucleotide sequences of the present invention can be placed on the array to
detect changes
from those sequences.
Alternatively a polymorphism resulting in a change in the amino acid sequence
could
also be detected by detecting a corresponding change in amino acid sequence of
the protein,
e.g., by an antibody specific to the variant sequence.
4.10.20 ARTHRITIS AND INFLAMMATION
The immunosuppressive effects of the compositions of the invention against
rheumatoid arthritis is determined in an experimental animal model system. The
805A/PCT ~ 02453344 2004-O1-21
experimental model system is adjuvant induced arthritis in rats, and the
protocol is described
by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963,
Int. Arch.
Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a
single
injection, generally intradermally, of a suspension of killed Mycobacterium
tuberculosis in
complete Freund's adjuvant (CFA). The route of injection can vary, but rats
may be injected
at the base of the tail with an adjuvant mixture. The polypeptide is
administered in phosphate
buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of
administering
PB S only.
The procedure for testing the effects of the test compound would consist of
IO intradermally injecting killed Mycobacterium tuberculosis in CFA followed
by immediately
administering the test compound and subsequent treatment every other day until
day 24. At
14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an
overall arthritis
score may be obtained as described by J. Holoskitz above. An analysis of the
data would
reveal that the test compound would have a dramatic affect on the swelling of
the joints as
measured by a decrease of the arthritis score.
4.11 THERAPEUTIC METHODS
The compositions (including polypeptide fragments, analogs, variants and
antibodies
or other binding partners or modulators including antisense polynucleotides)
of the invention
have numerous applications in a variety of therapeutic methods. Examples of
therapeutic
applications include, but are not limited to, those exemplified herein.
4.11.1 EXAMPLE
One embodiment of the invention is the administration of an effective amount
of the
polypeptides or other composition of the invention to individuals affected by
a disease or
disorder that can be modulated by regulating the peptides of the invention.
While the mode
of administration is not particularly important, parenteral administration is
preferred. An
exemplary mode of administration is to deliver an intravenous bolus. The
dosage of the
polypeptides or other composition of the invention will normally be determined
by the
prescribing physician. It is to be expected that the dosage will vary
according to the age,
weight, condition and response of the individual patient. Typically, the
amount of
polypeptide administered per dose will be in the range of about 0.01 ~,g/kg to
100 mg/kg of
body weight, with the preferred dose being about 0.1 ~g/kg to I 0 mg/kg of
patient body
g~SA/PCT CA 02453344 2004-O1-21
71
weight. For parenteral administration, polypeptides of the invention will be
formulated in an
injectable form combined with a pharmaceutically acceptable parenteral
vehicle. Such
vehicles are well known in the art and examples include water, saline,
lRnger's solution,
dextrose solution, and solutions consisting of small amounts of the human
serum albumin.
The vehicle may contain minor amounts of additives that maintain the
isotonicity and
stability of the polypeptide or other active ingredient. The preparation of
such solutions is
within the skill of the art.
4.12 PHARMACEUTICAL FORMULATIONS AND ROUTES OF
ADMINISTRATION
A protein or other composition of the present invention (from whatever source
derived, including without limitation from recombinant and non-recombinant
sources and
including antibodies and other binding partners of the polypeptides of the
invention) may be
administered to a patient in need, by itself, or in pharmaceutical
compositions where it is
mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a
variety of
disorders. Such a composition may optionally contain (in addition to protein
or other active
ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers,
solubilizers, and other
materials well known in the art. The term "pharmaceutically acceptable" means
a non-toxic
material that does not interfere with the effectiveness of the biological
activity of the active
ingredient(s). The characteristics of the carrier will depend on the route of
administration.
The pharmaceutical composition of the invention may also contain cytokines,
lymphokines,
or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,
IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO,
TNF1, TNF2,
G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. In
further
compositions, proteins of the invention may be combined with other agents
beneficial to the
treatment of the disease or disorder in question. These agents include various
growth factors
such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF),
transforming
growth factors (TGF-a and TGF-(3), insulin-like growth factor (IGF), as well
as cytokines
described herein.
The pharmaceutical composition may further contain other agents which either
enhance the activity of the protein or other active ingredient or complement
its activity or
use in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein or
other active
005AJrCT CA 02453344 2004-O1-21
72
ingredient of the invention, or to minimize side effects. Conversely, protein
or other active
ingredient of the present invention may be included in formulations of the
particular
clotting factor, cytokine, lymphokine, other hematopoietic factor,
thrombolytic or
anti-thrombotic factor, or anti- inflammatory agent to minimize side effects
of the clotting
factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent (such as IL-lRa, IL-1 HyI, IL-1 Hy2, anti-
TNF,
corticosteroids, immunosuppressive agents). A protein of the present invention
may be
active in multimers (e.g., heterodimers or homodimers) or complexes with
itself or other
proteins. As a result, pharmaceutical compositions of the invention may
comprise a protein
of the invention in such multimeric or complexed form.
As an alternative to being included in a pharmaceutical composition of the
invention
including a first protein, a second protein or a therapeutic agent may be
concurrently
administered with the f rst protein (e.g., at the same time, or at differing
times provided that
therapeutic concentrations of the combination of agents is achieved at the
treatment site).
Techniques for formulation and administration of the compounds of the instant
application
may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co.,
Easton, PA,
latest edition. A therapeutically effective dose further refers to that amount
of the compound
sufficient to result in amelioration of symptoms, e.g., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, a therapeutically effective dose refers to
that ingredient
alone. When applied to a combination, a therapeutically effective dose refers
to combined
amounts of the active ingredients that result in the therapeutic effect,
whether administered
in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein or other active ingredient of the
present invention
is administered to a mammal having a condition to be treated. Protein or other
active
ingredient of the present invention may be administered in accordance with the
method of
the invention either alone or in combination with other therapies such as
treatments
employing cytokines, lymphokines or other hematopoietic factors. When co-
administered
with one or more cytokines, ly~nphokines or other hematopoietic factors,
protein or other
active ingredient of the present invention may be administered either
simultaneously with
the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
~oSA~C,h CA 02453344 2004-O1-21
73
anti-thrombotic factors, or sequentially. If administered sequentially, the
attending physician
will decide on the appropriate sequence of administering protein or other
active ingredient
of the present invention in combination with cytokine(s), lymphokine(s), other
hernatopoietic factor(s), thrombolytic or anti-thrombotic factors.
4.12.1 ROUTES OF ADMINISTRATION
Suitable routes of administration may, for example, include oral, rectal,
transmucosal, or intestinal administration; parenteral delivery, including
intramuscular,
subcutaneous, intramedullary injections, as well as intrathecal, direct
intraventricular,
intravenous, intraperitoneal, intranasal, or intraocular injections.
Administration of protein
or other active ingredient of the present invention used in the pharmaceutical
composition or
to practice the method of the present invention can be carried out in a
variety of conventional
ways, such as oral ingestion, inhalation, topical application or cutaneous,
subcutaneous,
intraperitoneal, parenteral or intravenous injection. Intravenous
administration to the patient
is preferred.
Alternately, one may administer the compound in a local rather than systemic
manner, for example, via injection of the compound directly into a arthritic
joints or in
fibrotic tissue, often in a depot or sustained release formulation. In order
to prevent the
scarring process frequently occurring as complication of glaucoma surgery, the
compounds
may be administered topically, for example, as eye drops. Furthermore, one may
administer
the drug in a targeted drug delivery system, for example, in a liposome coated
with a specific
antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes
will be targeted
to and taken up selectively by the afflicted tissue.
The polypeptides of the invention are administered by any route that delivers
an
effective dosage to the desired site of action. The determination of a
suitable route of
administration and an effective dosage for a particular indication is within
the level of skill
in the art. Preferably for wound treatment, one administers the therapeutic
compound
directly to the site. Suitable dosage ranges for the polypeptides of the
invention can be
extrapolated from these dosages or from similar studies in appropriate animal
models.
Dosages can then be adjusted as necessary by the clinician to provide maximal
therapeutic
benefit.
4.12.2 COMPOSITIONS/FORMULATIONS
~OS~CT CA 02453344 2004-O1-21
Pharmaceutical compositions for use in accordance with the present invention
thus
may be formulated in a conventional manner using one or more physiologically
acceptable
carriers comprising excipients and auxiliaries which facilitate processing of
the active
compounds into preparations which can be used pharmaceutically. These
pharmaceutical
compositions may be manufactured in a manner that is itself known, e.g., by
means of
conventional mixing, dissolving, granulating, dragee-making, levigating,
emulsifying,
encapsulating, entrapping or lyophilizing processes. Proper formulation is
dependent upon
the route of administration chosen. When a therapeutically effective amount of
protein or
other active ingredient of the present invention is administered orally,
protein or other active
ingredient of the present invention will be in the form of a tablet, capsule,
powder, solution
or elixir. When administered in tablet form, the pharmaceutical composition of
the invention
may additionally contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule,
and powder contain from about 5 to 95% protein or other active ingredient of
the present
invention, and preferably from about 25 to 90% protein or other active
ingredient of the
present invention. When administered in liquid form, a liquid carrier such as
water,
petroleum, oils of animal or plant origin such as peanut oil, mineral oil,
soybean oil, or
sesame oil, or synthetic oils may be added. The liquid form of the
pharmaceutical
composition may further contain physiological saline solution, dextrose or
other saccharide
solution, or glycols such as ethylene glycol, propylene glycol or polyethylene
glycol. When
administered in liquid form, the pharmaceutical composition contains from
about 0.5 to 90%
by weight of protein or other active ingredient of the present invention, and
preferably from
about 1 to 50% protein or other active ingredient of the present invention.
When a therapeutically effective amount of protein or other active ingredient
of the
present invention is administered by intravenous, cutaneous or subcutaneous
injection,
protein or other active ingredient of the present invention will be in the
form of a
pyrogen-free, parenterally acceptable aqueous solution. The preparation of
such parenterally
acceptable protein or other active ingredient solutions, having due regard to
pH, isotonicity,
stability, and the like, is within the skill in the art. A preferred
pharmaceutical composition
for intravenous, cutaneous, or subcutaneous injection should contain, in
addition to protein
or other active ingredient of the present invention, an isotonic vehicle such
as Sodium
Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
805AIPCT ~ 02453344 2004-O1-21
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art. For
injection, the agents of the invention may be formulated in aqueous solutions,
preferably in
physiologically compatible buffers such as Hanks's solution, Ringer's
solution, or
physiological saline buffer. For transmucosal administration, penetrants
appropriate to the
5 barrier to be permeated are used in the formulation. Such penetrants are
generally known in
the art.
Fox oral administration, the compounds can be formulated readily by combining
the
active compounds with pharmaceutically acceptable carriers well known in the
art. Such
carriers enable the compounds of the invention to be formulated as tablets,
pills, dragees,
10 capsules, liquids, gels, syrups, slurries, suspensions and the like, for
oral ingestion by a
patient to be treated. Pharmaceutical preparations for oral use can be
obtained from a solid
excipient, optionally grinding a resulting mixture, and processing the mixture
of granules,
after adding suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Suitable
excipients are, in particular, fillers such as sugars, including lactose,
sucrose, mannitol, or
15 sorbitol; cellulose preparations such as, for example, maize starch, wheat
starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-
cellulose,
sodium carboxymethylcellulose, andlor polyvinylpyrrolidone (PVl'). If desired,
disintegrating agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate. Dragee cores are
provided with
20 suitable coatings. For this purpose, concentrated sugar solutions may be
used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol,
andlor titanium dioxide, lacquer solutions, and suitable organic solvents or
solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee coatings for
identification or to
characterize different combinations of active compound doses.
25 Pharmaceutical preparations which can be used orally include push-fit
capsules made
of gelatin, as well as soft, sealed capsules made of gelatin and a
plasticizer, such as glycerol
or sorbitol. The push-fit capsules can contain the active ingredients in
admixture with filler
such as lactose, binders such as starches, and/or lubricants such as talc or
magnesium
stearate and, optionally, stabilizers. In soft capsules, the active compounds
may be dissolved
30 or suspended in suitable liquids, such as fatty oils, liquid paraffin, or
liquid polyethylene
glycols. In addition, stabilizers may be added. All formulations for oral
administration
should be in dosages suitable for such administration. For buccal
administration, the
compositions may take the form of tablets or lozenges forn~ulated in
conventional manner.
~0~~~~T CA 02453344 2004-O1-21
For administration by inhalation, the compounds for use according to the
present
invention are conveniently delivered in the form of an aerosol spray
presentation from
pressurized packs or a nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane,
carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the dosage unit
may be determined
by providing a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin
for use in an inhaler or insufflator may be formulated containing a powder mix
of the
compound and a suitable powder base such as lactose or starch. The compounds
may be
formulated for parenteral administration by injection, e.g., by bolus
injection or continuous
infusion. Formulations for injection may be presented in unit dosage form,
e.g., in ampules
or in mufti-dose containers, with an added preservative. The compositions may
take such
forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and
may contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous
solutions
of the active compounds in water-soluble form. Additionally, suspensions of
the active
compounds may be prepared as appropriate oily injection suspensions. Suitable
lipophilic
solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such
as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions
may contain
substances which increase the viscosity of the suspension, such as sodium
carboxymethyl
cellulose, sorbitol, or dextran. optionally, the suspension rnay also contain
suitable
stabilizers or agents which increase the solubility of the compounds to allow
for the
preparation of highly concentrated solutions. Alternatively, the active
ingredient may be in
powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-
free water, before
use.
The compounds may also be formulated in rectal compositions such as
suppositories
or retention enemas, e.g., containing conventional suppository bases such as
cocoa butter or
other glycerides. In addition to the formulations described previously, the
compounds may
also be formulated as a depot preparation. Such long acting formulations may
be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with suitable
polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
g05A~LT, CA 02453344 2004-O1-21
A pharmaceutical carrier for the hydrophobic compounds of the invention is a
co-
solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-
miscible organic
polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent
system.
VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant
polysorbate
80, and 65% w/v polyethylene glycol 300, made up to volume in absolute
ethanol. The VPD
co-solvent system (VPD:SW) consists of VPD diluted 1:1 with a S% dextrose in
water
solution. This co-solvent system dissolves hydrophobic compounds well, and
itself produces
low toxicity upon systemic administration. Naturally, the proportions of a co-
solvent system
may be varied considerably without destroying its solubility and toxicity
characteristics.
Furthermore, the identity of the co-solvent components may be varied: for
example, other
low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the
fraction size of
polyethylene glycol may be varied; other biocompatible polymers may replace
polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may
substitute for
dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical
compounds
may be employed. Liposomes and emulsions are well known examples of delivery
vehicles
or carriers for hydrophobic drugs. Certain organic solvents such as
dirnethylsulfoxide also
may be employed, although usually at the cost of greater toxicity.
Additionally, the
compounds may be delivered using a sustained-release system, such as
semipermeable
matrices of solid hydrophobic polymers containing the therapeutic agent.
Various types of
sustained-release materials have been established and are well known by those
skilled in the
art. Sustained-release capsules may, depending on their chemical nature,
release the
compounds for a few weeks up to over 100 days. Depending on the chemical
nature and the
biological stability of the therapeutic reagent, additional strategies for
protein or other active
ingredient stabilization may be employed.
The pharmaceutical compositions also may comprise suitable solid or gel phase
carriers or excipients. Examples of such carriers or excipients include but
are not limited to
calcium carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives,
gelatin, and polymers such as polyethylene glycols. Many of the active
ingredients of the
invention may be provided as salts with pharmaceutically compatible counter
ions. Such
pharmaceutically acceptable base addition salts are those salts which retain
the biological
effectiveness and properties of the free acids and which are obtained by
reaction with
inorganic or organic bases such as sodium hydroxide, magnesium hydroxide,
ammonia,
805A/PCT ~ 02453344 2004-O1-21
., 78
trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium
acetate,
potassium benzoate, triethanol amine and the like.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) or other active ingredients) of present invention along with
protein or
peptide antigens. The protein and/or peptide antigen will deliver a
stimulatory signal to both
B and T lymphocytes. B lymphocytes will respond to antigen through their
surface
immunoglobulin receptor. T lymphocytes will respond to antigen through the T
cell receptor
(TCR) following presentation of the antigen by MHC proteins. MHC and
structurally
related proteins including those encoded by class I and class II MHC genes on
host cells will
serve to present the peptide antigens) to T lymphocytes. The antigen
components could
also be supplied as purified MHC-peptide complexes alone or with co-
stimulatory molecules
that can directly signal T cells. Alternatively antibodies able to bind
surface
immunoglobulin and other molecules on B cells as well as antibodies able to
bind the TCR
and other molecules on T cells can be combined with the pharmaceutical
composition of the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome in
which protein of the present invention is combined, in addition to other
pharmaceutically
acceptable carriers, with amphipathic agents such as lipids which exist in
aggregated form as
micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous
solution.
Suitable lipids for liposomal formulation include, without limitation,
monoglycerides,
diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids,
and the like.
Preparation of such liposomal formulations is within the level of skill in the
art, as disclosed,
for example, in U.S. Patent Nos. 4,235,871; 4,501,728; 4,837,028; and
4,737,323, all of
which are incorporated herein by reference.
The amount of protein or other active ingredient of the present invention in
the
pharmaceutical composition of the present invention will depend upon the
nature and
severity of the condition being treated, and on the nature of prior treatments
which the
patient has undergone. Ultimately, the attending physician will decide the
amount of protein
or other active ingredient of the present invention with which to treat each
individual patient.
Initially, the attending physician will administer low doses of protein or
other active
ingredient of the present invention and observe the patient's response. Larger
doses of
protein or other active ingredient of the present invention ~zay be
administered until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
~~~~~.,,I, CA 02453344 2004-O1-21
79
increased further. It is contemplated that the various pharmaceutical
compositions used to
practice the method of the present invention should contain about 0.0I ~.g to
about 100 mg
(preferably about 0.1 ~g to about IO mg, more preferably about 0.1 ~g to about
I mg) of
protein or other active ingredient of the present invention per kg body
weight. For
compositions of the present invention which are useful for bone, cartilage,
tendon or
ligament regeneration, the therapeutic method includes administering the
composition
topically, systematically, or locally as an implant or device. iUhen
administered, the
therapeutic composition for use in this invention is, of course, in a pyrogen-
free,
physiologically acceptable form. Further, the composition may desirably be
encapsulated or
I O injected in a viscous form for delivery to the site of bone, cartilage or
tissue damage.
Topical administration may be suitable for wound healing and tissue repair.
Therapeutically
useful agents other than a protein or other active ingredient of the invention
which may also
optionally be included in the composition as described above, rnay
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
I 5 methods of the invention. Preferably for bone and/or cartilage formation,
the composition
would include a matrix capable of delivering the protein-containing or other
active
ingredient-containing composition to the site of bone and/or cartilage damage,
providing a
structure for the developing bone and cartilage and optimally capable of being
resorbed into
the body. Such matrices may be formed of materials presently in use for other
implanted
20 medical applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential matrices
for the compositions may be biodegradable and chemically defined calcium
sulfate,
25 tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid
and polyanhydrides.
Other potential materials are biodegradable and biologically well-defined,
such as bone or
dermal collagen. Further matrices are comprised of pure proteins or
extracellular matrix
components. Other potential matrices are nonbiodegradable and chemically
defned, such as
sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may
be comprised
30 of combinations of any of the above-mentioned types of material, such as
polylactic acid and
hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be
altered in
composition, such as in calcium-aluminate-phosphate and processing to alter
pore size,
particle size, particle shape, and biodegradability. Presently preferred is a
50:50 (mole
805AIPCT ~ 02453344 2004-O1-21
weight) copolymer of lactic acid and glycolic acid in the form of porous
particles having
diameters ranging from 150 to 800 microns. In some applications, it will be
useful to
utilize a sequestering agent, such as carboxymethyl cellulose or autologous
blood clot, to
prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred
being
cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering
agents
10 include hyaluronic acid, sodium alginate, polyethylene glycol),
polyoxyethylene oxide,
carboxyvinyl polymer and polyvinyl alcohol). The amount of sequestering agent
useful
herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight,
which
represents the amount necessary to prevent desorption of the protein from the
polymer
matrix and to provide appropriate handling of the composition, yet not so much
that the
15 progenitor cells are prevented from infiltrating the matrix, thereby
providing the protein the
opportunity to assist the osteogenic activity of the progenitor cells. In
further compositions,
proteins or other active ingredients of the invention may be combined with
other agents
beneficial to the treatment of the bone and/or cartilage defect, wound, or
tissue in question.
These agents include various growth factors such as epidermal growth factor
(EGF), platelet
20 derived growth factor (PDGF), transforming growth factors (TGF-a and TGF-
(3), and
insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications.
Particularly domestic animals and thoroughbred horses, in addition to humans,
are desired
patients for such treatment with proteins or other active ingredients of the
present invention.
25 The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue
regeneration will be determined by the attending physician considering vaxious
factors which
modify the action of the proteins, e.g., amount of tissue weight desired to be
formed, the site
of damage, the condition of the damaged tissue, the size of a wound, type of
damaged tissue
(e.g., bone), the patient's age, sex, and diet, the severity of any infection,
time of
30 administration and other clinical factors. The dosage may vary with the
type of matrix used
in the reconstitution and with inclusion of other proteins in the
pharmaceutical composition.
For example, the addition of other known growth factors, such as IGF I
(insulin like growth
factor I), to the final composition, may also effect the dosage. Progress can
be monitored by
805t1/i-CT CA 02453344 2004-O1-21
81
periodic assessment of tissue/bone growth and/or repair, for example, X-rays,
histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA). Cells may also be
cultured ex vivo in
the presence of proteins of the present invention in order to proliferate or
to produce a
desired effect on or activity in such cells. Treated cells can then be
introduced in vivo for
therapeutic purposes.
4.12.3 EFFECTIVE DGSAGE
Pharmaceutical compositions suitable for use in the present invention include
compositions wherein the active ingredients are contained in an effective
amount to achieve
its intended purpose. More specifically, a therapeutically effective amount
means an amount
effective to prevent development of or to alleviate the existing symptoms of
the subject
being treated. Determination of the effective amount is well within the
capability of those
skilled in the art, especially in light of the detailed disclosure provided
herein. For any
compound used in the method of the invention, the therapeutically effective
dose can be
estimated initially from appropriate in vitro assays. For example, a dose can
be formulated in
animal models to achieve a circulating concentration range that can be used to
more
accurately determine useful doses in humans. For example, a dose can be
formulated in
animal models to achieve a circulating concentration range that includes the
ICSO as
determined in cell culture (i. e. , the concentration of the test compound
which achieves a
half maximal inhibition of the protein's biological activity). Such
information can be used
to more accurately determine useful doses in humans.
A therapeutically effective dose refers to that amount of the compound that
results in
amelioration of symptoms or a prolongation of survival in a patient. Toxicity
and therapeutic
efficacy of such compounds can be determined by standard pharmaceutical
procedures in
cell cultures or experimental animals, e.g., for determining the LDSO (the
dose lethal to 50%
of the population) and the FDso (the dose therapeutically effective in 50% of
the population).
The dose ratio between toxic and therapeutic effects is the therapeutic index
and it can be
expressed as the ratio between LDSO and EDso. Compounds which exhibit high
therapeutic
goSA/PL-T CA 02453344 2004-O1-21
$2
indices are preferred. The data obtained from these cell culture assays and
animal studies
can be used in formulating a range of dosage for use in human. The dosage of
such
compounds lies preferably within a range of circulating concentrations that
include the
ED50 with little or no toxicity. T'he dosage may vary within this range
depending upon the
dosage form employed and the route of administration utilized. The exact
formulation, route
of administration and dosage can be chosen by the individual physician in view
of the
patient's condition. See, e.g., Fingl et al., 1975, in "The Pharmacological
basis of
Therapeutics", Ch. 1 p.1. Dosage amount and interval may be adjusted
individually to
provide plasma levels of the active moiety which are sufficient to maintain
the desired
effects, or minimal effective concentration (MEC). The MEC will vary for each
compound
but can be estimated from ivc vitro data. Dosages necessary to achieve the MEC
will depend
on individual characteristics and route of administration. However, HPLC
assays or
bioassays can be used to determine plasma concentrations.
Dosage intervals can also be determined using MEC value. Compounds should be
administered using a regimen which maintains plasma levels above the MEC for
10-90% of
the time, preferably between 30-90% and most preferably between 50-90%. In
cases of local
administration or selective uptake, the effective local concentration of the
drug may not be
related to plasma concentration.
An exemplary dosage regimen for polypeptides or other compositions of the
invention will be in the range of about 0.01 pg/kg to 100 mg/kg of body weight
daily, with
the preferred dose being about 0.1 ~g/kg to 25 mg/kg of patient body weight
daily, varying
in adults and children. Dosing may be once daily, or equivalent doses may be
delivered at
longer or shorter intervals.
The amount of composition administered will, of course, be dependent on the
subject
being treated, on the subject's age and weight, the severity of the
affliction, the manner of
administration and the judgment of the prescribing physician.
4.12.4 PACKAGIllTG
The compositions may, if desired, be presented in a pack or dispenser device
which
may contain one or more unit dosage forms containing the active ingredient.
The pack may,
for example, comprise metal or plastic foil, such as a blister pack. The pack
or dispenser
device may be accompanied by instructions for administration. Compositions
comprising a
compound of the invention formulated in a compatible pharmaceutical carrier
may also be
8OStI/PCT CA 02453344 2004-O1-21
83
prepared, placed in an appropriate container, and labeled for treatment of an
indicated
condition.
4.13 ANTIBODIES
Also included in the invention are antibodies to proteins, or fragments of
proteins of
the invention. The term "antibody" as used herein refers to immunoglobulin
molecules and
immunologically active portions of immunoglobulin (Ig) molecules, i.e.,
molecules that
contain an antigen-binding site that specifically binds (immunoreacts with) an
antigen. Such
antibodies include, but are not limited to, polyclonal, monoclonal, chimeric,
single chain,
Fab, Fab° and F~ab~~z fragments, and an Fab expression library. In
general, an antibody molecule
obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD,
which differ
from one another by the nature of the heavy chain present in the molecule.
Certain classes
have subclasses as well, such as IgGr, IgG2, and others. Furthermore, in
humans, the light
chain may be a kappa chain or a lambda chain. I~.eference herein to antibodies
includes a
reference to all such classes, subclasses and types of human antibody species.
An isolated related protein of the invention may be intended to serve as an
antigen, or
a portion or fragment thereof, and additionally can be used as an immunogen to
generate
antibodies that immunospecifically bind the antigen, using standard techniques
for
polyclonal and monoclonal antibody preparation. The full-length protein can be
used or,
alternatively, the invention provides antigenic peptide fragments of the
antigen for use as
immunogens. An antigenic peptide fragment comprises at least 6 amino acid
residues of the
amino acid sequence of the full length protein, such as an amino acid sequence
shown in
SEQ ID NO: 1-244, or 489-706, and encompasses an epitope thereof such that an
antibody
raised against the peptide forms a specific immune complex with the full
length protein or
with any fragment that contains the epitope. Preferably, the antigenic peptide
comprises at
least 10 amino acid residues, or at least 1 S amino acid residues, or at least
20 amino acid
residues, or at least 30 amino acid residues. Preferred epitopes encompassed
by the
antigenic peptide are regions of the protein that are located on its surface;
commonly these
are hydrophilic regions.
In certain embodiments of the invention, at least one epitope encompassed by
the
antigenic peptide is a surface region of the protein, e.g., a hydrophilic
region. A
hydrophobicity analysis of the human related protein sequence will indicate
which regions of
a related protein are particularly hydrophilic and, therefore, are likely to
encode surface
805A/PCT CA 02453344 2004-O1-21
84
residues useful for targeting antibody production. As a means for targeting
antibody
production, hydropathy plots showing regions of hydrophilicity and
hydrophobicity may be
generated by any method well known in the art, including, for example, the
Kyte Doolittle
or the Hopp Woods methods, either with or without Fourier transformation. See,
e.g., Hopp
and Woods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle
1982, J.
Mol. Biol. 157: 105-142, each of which is incorporated herein by reference in
its entirety.
Antibodies that are specific for one or more domains within an antigenic
protein, or
derivatives, fragments, analogs or homologs thereof, are also provided herein.
A protein of the invention, or a derivative, fragment, analog, homolog or
ortholog
thereof, may be utilized as an immunogen in the generation of antibodies that
immunospecifically bind these protein components.
The term "specific for" indicates that the variable regions of the antibodies
of the
invention recognize and bind polypeptides of the invention exclusively (i.e.,
able to
distinguish the polypeptide of the invention from other similar polypeptides
despite sequence
I 5 identity, homology, or similarity found in the family of polypeptides),
but may also interact
with other proteins (for example, S. aureus protein A or other antibodies in
ELISA
techniques) through interactions with sequences outside the variable region of
the antibodies,
and in particular, in the constant region of the molecule. Screening assays to
determine
binding specificity of an antibody of the invention are well known and
routinely practiced in
the art. For a comprehensive discussion of such assays, see Harlow et al.
(Eds), Antibodies
A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY
(1988),
Chapter 6. Antibodies that recognize and bind fragments of the polypeptides of
the
invention are also contemplated, provided that the antibodies are first and
foremost specific
far, as defined above, full-length polypeptides of the invention. As with
antibodies that are
specific for full length polypeptides of the invention, antibodies of the
invention that
recognize fragments are those which can distinguish polypeptides from the same
family of
polypeptides despite inherent sequence identity, homology, or similarity found
in the family
of proteins.
Antibodies of the invention are useful for, for example, therapeutic purposes
(by
modulating activity of a polypeptide of the invention), diagnostic purposes to
detect or
quantitate a polypeptide of the invention, as well as purification of a
polypeptide of the
invention. Kits comprising an antibody of the invention for any of the
purposes described
herein are also comprehended. In general, a kit of the invention also includes
a control
805A/PCT ~ 02453344 2004-O1-21
antigen for which the antibody is immunospecific. The invention further
provides a
hybridoma that produces an antibody according to the invention. Antibodies of
the
invention are useful for detection and/or purification of the polypeptides of
the invention.
Monoclonal antibodies binding to the protein of the invention may be useful
5 diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting and
10 preventing the metastatic spread of the cancerous cells, which may be
mediated by the
protein.
The labeled antibodies of the present invention can be used for i~c vitro, in
vivo, and
in situ assays to identify cells or tissues in which a fragment of the
polypeptide of interest is
expressed. The antibodies may also be used directly in therapies or other
diagnostics. The
15 present invention further provides the above-described antibodies
immobilized on a solid
support. Examples of such solid supports include plastics such as
polycarbonate, complex
carbohydrates such as agarose and Sepharose~, acrylic resins and such as
polyacrylamide
and latex beads. Techniques fox coupling antibodies to such solid supports are
well known
in the art (Weir, D.M. et al., "Handbook of Experimental Immunology" 4th Ed.,
Blackwell
20 Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W.D.
et al., Meth.
Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the
present
invention can be used for in vitro, in vavo, and in situ assays as well as for
immuno-affinity
purification of the proteins of the present invention.
Various procedures known within the art may be used for the production of
25 polyclonal or monoclonal antibodies directed against a protein of the
invention, or against
derivatives, fragments, analogs homologs or orthologs thereof (see, for
example, Antibodies:
A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, NY, incorporated herein by reference). Some of these
antibodies are
discussed below.
4.13.1 POLYCLONAL ANTIBODIES
For the production of polyclonal antibodies, various suitable host animals
(e.g.,
rabbit, goat, mouse or other mammal) may be immunized by one or more
injections with the
805A/PCT ~ 02453344 2004-O1-21
86
native protein, a synthetic variant thereof, or a derivative of the foregoing.
An appropriate
immunogenic preparation can contain, for example, the naturally occurring
immunogenic
protein, a chemically synthesized polypeptide representing the immunogenic
protein, or a
recombinantly expressed immunogenic protein. Furthermore, the protein may be
conjugated
to a second protein known to be immunogenic in the mammal being immunized.
Examples
of such immunogenic proteins include but are not limited to keyhole limpet
hernocyanin,
serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. The
preparation can
further include an adjuvant. Various adjuvants used to increase the
immunological response
include, but are not limited to, Freund's (complete and incomplete), mineral
gels (e.g.,
aluminum hydroxide), surface-active substances (e.g., lysolecithin, pluronic
polyols,
polyanions, peptides, oil emulsions, dinitrophenol, etc.), adjuvants usable in
humans such as
Bacille Calmette-Guerin and Corynebacterium parvum, or similar
immunostimulatory
agents. Additional examples of adjuvants that can be employed include MPL-TDM
adjuvant
(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
The polyclonal antibody molecules directed against the immunogenic protein can
be
isolated from the mammal (e.g., from the blood) and further purified by well
known
techniques, such as affinity chromatography using protein A or protein G,
which provide
primarily the IgG fraction of immune serum. Subsequently, or alternatively,
the specific
antigen which is the target of the immunoglobulin sought, or an epitope
thereof, may be
immobilized on a column to purify the immune specific antibody by
immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for example, by
D.
Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA,
Vol. 14, No. 8
(April 17, 2000), pp. 25-28).
4.13.2 MONOCLONAL ANTIBODIES
The term "monoclonal antibody" (MAb) or "monoclonal antibody composition", as
used herein, refers to a population of antibody molecules that contain only
one molecular
species of antibody molecule consisting of a unique light chain gene product
and a unique
heavy chain gene product. In particular, the complementarity determining
regions (CDRs)
of the monoclonal antibody are identical in all the molecules of the
population. MAbs thus
contain an antigen-binding site capable of immunoreacting with a particular
epitope of the
antigen characterized by a unique binding affinity for it.
805A/PCT ~ 02453344 2004-O1-21
07
Monoclonal antibodies can be prepared using hybridoma methods, such as those
described by Kohler and Milstein, Nature, 256, 495 (1970. In a hybridoma
method, a
mouse, hamster, or other appropriate host animal, is typically immunized with
an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies
that will specifically bind to the immunizing agent. Alternatively, the
lymphocytes can be
immunized in vitro.
The immunizing agent will typically include the protein antigen, a fragment
thereof
or a fusion protein thereof. Generally, either peripheral blood lymphocytes
are used if cells
of human origin are desired, or spleen cells or lymph node cells are used if
non-human
mammalian sources are desired. The lymphocytes are then fused with an
immortalized cell
line using a suitable fusing agent, such as polyethylene glycol, to form a
hybridoma cell
(coding, Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-
103). Immortalized cell lines are usually transformed mammalian cells,
particularly
myeloma cells of rodent, bovine and human origin. Usually, rat or mouse
myeloma cell
lines are employed. The hybridorna cells can be cultured in a suitable culture
medium that
preferably contains one or more substances that inhibit the growth or survival
of the unfused,
immortalized cells. For example, if the parental cells lack the enzyme
hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas
typically will include hypoxanthine, aminopterin, and thymidine ("HAT
medium"), which
substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a
medium such as HAT medium. More preferred immortalized cell lines are marine
myeloma
lines, which can be obtained, for instance, from the Salk Institute Cell
Distribution Center,
San Diego, California and the American Type Culture Collection, Manassas,
Virginia.
Human myelorna and mouse-human heteromyeloma cell lines also have been
described for
the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001
(1984);
Brodeur et al., Monoclonal Antibody Production Techniques and Applications,
Marcel
Dekker, Inc., New York, (1987) pp. 51-63).
The culture medium in which the hybridoma cells are cultured can then be
assayed
for the presence of monoclonal antibodies directed against the antigen.
Preferably, the
binding specificity of monoclonal antibodies produced by the hybridoma cells
is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or
805A/PCT CA 02453344 2004-O1-21
88
enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are
known in
the art. The binding affinity of the monoclonal antibody can, far example, be
determined
by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107, 220
(1980).
Preferably, antibodies having a high degree of specificity and a high binding
affinity for the
target antigen are isolated.
After the desired hybridoma cells are identified, the clones can be subcloned
by
limiting dilution procedures and grown by standard methods. Suitable culture
media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640
medium. Alternatively, the hybridama cells can be grown in vivo as aseites in
a mammal.
The monoclonal antibodies secreted by the subclones can be isolated or
purified from
the culture medium or ascites fluid by conventional immunoglobulin
purification procedures
such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
The monoclonal antibodies can also be made by recombinant DNA methods, such as
those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal
antibodies of
the invention can be readily isolated and sequenced using conventional
procedures (e.g., by
using oligonucleotide probes that are capable of binding specifically to genes
encoding the
heavy and light chains of murine antibodies). The hybridoma cells of the
invention serve as
a preferred source of such DNA. Once isolated, the DNA can be placed into
expression
vectors, which are then transfected into host cells such as simian COS cells,
Chinese hamster
ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant host
cells. The DNA
also can be modified, for example, by substituting the coding sequence for
human heavy and
light chain constant domains in place of the homologous murine sequences (U.S.
Patent No.
4,816,567; Morrison, Nature 368, 812-13 {1994)) or by covalently joining to
the
immunoglobulin coding sequence all or part of the coding sequence for a non-
immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be
substituted
for the constant domains of an antibody of the invention, or can be
substituted for the
variable domains of one antigen-combining site of an antibody of the invention
to create a
ehimeric bivalent antibody.
4.13.3 HUMANIZED ANTIBODIES
805A/PCT ~ 02453344 2004-O1-21
The antibodies directed against the protein antigens of the invention can
further
comprise humanized antibodies or human antibodies. These antibodies are
suitable for
administration to humans without engendering an immune response by the human
against
the administered immunoglobulin. Humanized forms of antibodies are chimeric
S immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,
Fab, Fab',
F(ab')2 or other antigen-binding subsequences of antibodies) that are
principally comprised
of the sequence of a human immunoglobulin, and contain minimal sequence
derived from a
non-human immunoglobulin. Humanization can be performed following the method
of
Winter and co-workers (Jones et al., Nature, 321, 522-525 (1986); Riechmann et
al., Nature,
332, 323-327 (1988); Verhoeyen et al., Science, 239, 1534-1536 (1988)), by
substituting
rodent CDRs or CDR sequences for the corresponding sequences of a human
antibody. (See
also U.S. Patent No. 5,225,539). In some instances, Fv framework residues of
the human
immunoglobulin are replaced by corresponding non-human residues. Humanized
antibodies
can also comprise residues that are found neither in the recipient antibody
nor in the
imported CDR or framework sequences. In general, the humanized antibody will
comprise
substantially all of at least one, and typically two, variable domains, in
which all or
substantially all of the CDR regions correspond to those of a non-human
immunoglobulin
and all or substantially all of the framework regions are those of a human
immunoglobulin
consensus sequence. The humanized antibody optimally also will comprise at
least a portion
of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin
(Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct.
Biol., 2, 593-596
(1992)).
4.13.4 HUMAN ANTII~ODIES
Fully human antibodies relate to antibody molecules in which essentially the
entire
sequences of both the light chain and the heavy chain, including the CDRs,
arise from
human genes. Such antibodies are termed "human antibodies", or "fully human
antibodies"
herein. Human monoclonal antibodies can be prepared by the trioma technique;
the human
B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and
the EBV
hybridoma technique to produce human monoclonal antibodies (see Cole, et al.,
1985 In:
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Human
monoclonal antibodies may be utilized in the practice of the present invention
and may be
produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci
USA 80,
~O~~P~.I, CA 02453344 2004-O1-21
2026-2030) or by transforming human B-cells with Epstein Barr Wirus in vitro
(see Cole, et
al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.,
pp. 77-96).
In addition, human antibodies can also be produced using additional
techniques,
including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227,
381 (1991);
5 Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies
can be made by
introducing human immunoglobulin loci into transgenic animals, e.g., mice in
which the
endogenous immunoglobulin genes have been partially or completely inactivated.
Upon
challenge, human antibody production is observed, which closely resembles that
seen in
humans in all respects, including gene rearrangement, assembly, and antibody
repertoire.
10 This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.
(Bio/Technology 10, 779-
783 (1992)); Lonberg et al. (Nature 368, 856-859 (1994)); Morrison (Nature
368, 812-13
(1994)); Fishwild et al, (Nature Biotechnology 14, 845-51 (1996)); Neuberger
(Nature
Biotechnology 14, 826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol.
13, 65-93
15 (1995)).
Human antibodies may additionally be produced using transgenic nonhuman
animals
that are modified so as to produce fully human antibodies rather than the
animal's
endogenous antibodies in response to challenge by an antigen. (See PCT
publication
W094/02602). The endogenous genes encoding the heavy and light immunoglobulin
chains
20 in the nonhuman host have been incapacitated, and active loci encoding
human heavy and
light chain immunoglobulins are inserted into the host's genome. The human
genes are
incorporated, for example, using yeast artificial chromosomes containing the
requisite
human DNA segments. An animal which provides all the desired modifications is
then
obtained as progeny by crossbreeding intermediate transgenic animals
containing fewer than
25 the full complement of the modifications. The preferred embodiment of such
a nonhuman
animal is a mouse, and is termed the ~enomouseTM as disclosed in PCT
publications WO
96/33735 and WO 96/34096. This animal produces B cells that secrete fully
human
immunoglobulins. The antibodies can be obtained directly from the animal after
immunization with an immunogen of interest, as, for example, a preparation of
a polyclonal
30 antibody, or alternatively from immortalized B cells derived from the
animal, such as
hybridornas producing monoclonal antibodies. Additionally, the genes encoding
the
immunoglobulins with human variable regions can be recovered and expressed to
obtain the
pn'r A IPCT CA 02453344 2004-O1-21
91
antibodies directly, or can be further modified to obtain analogs of
antibodies such as, for
example, single chain Fv molecules.
An example of a method of producing a nonhuman host, exemplified as a mouse,
lacking expression of an endogenous immunoglobulin heavy chain is disclosed in
U.S.
Patent No. 5,939,598. It can be obtained by a method including deleting the S
segment genes
from at least one endogenous heavy chain locus in an embryonic stem cell to
prevent
rearrangement of the locus and to prevent formation of a transcript of a
rearranged
immunoglobulin heavy chain locus, the deletion being effected by a targeting
vector
containing a gene encoding a selectable marker; and producing from the
embryonic stem cell
a transgenic mouse whose somatic and germ cells contain the gene encoding the
selectable
marker.
A method for producing an antibody of interest, such as a human antibody, is
disclosed in U.S. Patent No. 5,916,7X. It includes introducing an expression
vector that
contains a nucleotide sequence encoding a heavy chain into one mammalian host
cell in
culture, introducing an expression vector containing a nucleotide sequence
encoding a light
chain into another mammalian host cell, and fusing the two cells to form a
hybrid cell. The
hybrid cell expresses an antibody containing the heavy chain and the light
chain.
In a further improvement on this procedure, a method for identifying a
clinically
relevant epitope on an immunogen, and a correlative method for selecting an
antibody that
binds immunospecifically to the relevant epitope with high affinity, are
disclosed in PCT
publication W~ 99/53049.
4.13.5 FAB FRAGMENTS AND SINGLE CHAIN ANTIBODIES
According to the invention, techniques can be adapted for the production of
single-chain antibodies specific to an antigenic protein of the invention (see
e.g., U.S. Patent
No. 4,946,778). In addition, methods can be adapted for the construction of
Fab expression
libraries (see e.g., Huse, et al., 1989 Science 246, 1275-1281) to allow rapid
and effective
identification of monoclonal Fab fragments with the desired specificity for a
protein or
derivatives, fragments, analogs or homologs thereof. Antibody fragments that
contain the
idiotypes to a protein antigen may be produced by techniques known in the art
including, but
not limited to: (i) an F~ab')2 fragment produced by pepsin digestion of an
antibody molecule;
(ii) an Fab fragment generated by reducing the disulfide bridges of an F~ab~~2
fragment; (iii) an
805A/PCT ~ 02453344 2004-O1-21
92
Fab fragment generated by the treatment of the antibody molecule with papain
and a reducing
agent and (iv) F~ fragments.
4.13.6 BISPECIFIC ANTIBODIES
Bispecific antibodies a.re monoclonal, preferably human or humanized,
antibodies
that have binding specificities for at least two different antigens. In the
present case, one of
the binding specificities is for an antigenic protein of the invention. The
second binding
target is any other antigen, and advantageously is a cell-surface protein or
receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the
recombinant production of bispecific antibodies is based on the co-expression
of two
immunoglobulin heavy-chainllight-chain pairs, where the two heavy chains have
different
specificities (Milstein and Cuello, Nature, 305, 537-539 (1983)). Because of
the random
assortment of immunoglobulin heavy and light chains, these hybridomas
(quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the
correct bispecific structure. The purification of the correct molecue is
usually accomplished
by affinity chromatography steps. Similar procedures are disclosed in WO
93/08829,
published 13 May 1993, and in Traunecker et al., 1991 EM~O J., 10, 3655-3659.
Antibody variable domains with the desired binding specificities (antibody-
antigen
combining sites) can be fused to immunoglobulin constant domain sequences. The
fusion
preferably is with an immunoglobulin heavy-chain constant domain, comprising
at least part
of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-
chain constant
region (CHl) containing the site necessary for light-chain binding present in
at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if
desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and
are co
transfected into a suitable host organism. For further details of generating
bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology, 121, 210
(1986).
According to another approach described in WO 96/27011, the interface between
a
pair of antibody molecules can be engineered to maximize the percentage of
heterodimers
that are recovered from recombinant cell culture. The preferred interface
comprises at least
a part of the CH3 region of an antibody constant domain. In this method, one
or more small
amino acid side chains from the interface of the first antibody molecule are
replaced with
larger side chains (e.g. tyrosine ar tryptophan). Compensatory "cavities" of
identical or
805A/PCT CA 02453344 2004-O1-21
93
similar size to the large side chains) are created on the interface of the
second antibody
molecule by replacing large amino acid side chains with smaller ones (e.g.
alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over
other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full-length antibodies or antibody
fragments
(e.g. F(ab')2 bispecific antibodies). Techniques for generating bispecific
antibodies from
antibody fragments have been described in the literature. For example,
bispecific antibodies
can be prepared using chemical linkage. Brennan et al., Science 229, 81 (1985)
describe a
procedure wherein intact antibodies are proteolytically cleaved to generate
F(ab')2
fragments. These fragments are reduced in the presence of the dithiol
complexing agent
sodium arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation.
The Fab' fragments generated are then converted to thionitrobenzoate (TNB)
derivatives.
One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by
reduction with
mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced
can be used
as agents for the selective immobilization of enzymes.
Additionally, Fab' fragments can be directly recovered from E, coli and
chemically
coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med, 175, 217-
225 (1992)
describe the production of a fully humanized bispecific antibody F{ab')Z
molecule. Each
Fab' fragment was separately secreted from E. coli and subjected to directed
chemical
coupling in vitro to form the bispecific antibody. The bispecific antibody
thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as
trigger the lytic activity of human cytotoxic lymphocytes against human breast
tumor targets.
Various techniques for making and isolating bispecitic antibody fragments
directly
from recombinant cell culture have also been described. For. example,
bispecific antibodies
have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5),
1547-1 S53
(1992). The leucine zipper peptides from the Fos and Jun proteins were linked
to the Fab'
portions of two different antibodies by gene fusion. The antibody homodimers
were reduced
at the hinge region to form monomers and then re-oxidized to form the antibody
heterodimers. This method can also be utilized for the production of antibody
homodimers.
The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci.
I1SA 90,
6444-6448 (1993) has provided an alternative mechanism for making bispecific
antibody
fragments. The fragments comprise a heavy-chain variable domain (VH) connected
to a
805ti/PCT CA 02453344 2004-O1-21
' 94
light-chain variable domain (V L) by a linker which is too short to allow
pairing between the
two domains on the same chain. Accordingly, the VH and VL domains of one
fragment are
forced to pair with the complementary VL and VH domains of another fragment,
thereby
forming two antigen-binding sites. Another strategy for making bispecific
antibody
fragments by the use of single-chain Fv (sFv) dimers has also been reported.
See, Gruber et
al., J. Immunol. 152, 5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific
antibodies can be prepared. Tutt et al., J. Immunol. 147, 60 (1991).
Exemplary bispecific antibodies can bind to two different epitopes, at least
one of
which originates in the protein antigen of the invention. Alternatively, an
anti-antigenic arm
of an immunoglobulin molecule can be combined with an arm which binds to a
triggering
molecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3,
CD28, or B7),
or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and
FcyIZIII (CD 16)
so as to focus cellular defense mechanisms to the cell expressing the
particular antigen.
Bispecific antibodies can also be used to direct cytotoxic agents to cells
which express a
particular antigen. These antibodies possess an antigen-binding arm and an arm
which binds
a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or
TETA.
Another bispecific antibody of interest binds the protein antigen described
herein and further
binds tissue factor (TF).
4.13.7 HETEROCON.IUGATE ANTIBODIES
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target immune system cells to
unwanted cells
(U.S. Patent No. 4,676,980), and for treatment of HIV infection (WO 91/00360;
WO
92/200373; EP 03089). It is contemplated that the antibodies can be prepared
in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking
agents. For example, immunotoxins can be constructed using a disulfide
exchange reaction
or by forming a thioether bond. Examples of suitable reagents for this purpose
include
iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for
example, in U.S.
Patent No. 4,6?6,980.
4.13.8 EFFECTOR FUNCTION ENGINEERING
805tiIPCT CA 02453344 2004-O1-21
It can be desirable to modify the antibody of the invention with respect to
effector
function, so as to enhance, e.g., the effectiveness of the antibody in
treating cancer. Fox
example, cysteine residues) can be introduced into the Fc region, thereby
allowing
interchain disulfide bond formation in this region. The homodimeric antibody
thus
5 generated can have improved internalization capability and/or increased
complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See
Caron et
al., J. Exp Med., 176, 1191-1 I95 (1992) and Shopes, J. Immunol., 148, 2918-
2922 (1992).
Homodimeric antibodies with enhanced anti-tumor activity can also be prepared
using
heterobifunctional cross-linkers as described in Wolff et al. Cancer Research,
53, 2560-
10 2565 (1993). Alternatively, an antibody can be engineered that has dual Fc
regions and can
thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et
al.,
Anti-Cancer Drug Design, 3, 219-230 (1989).
4.13.9 IMMUNOCONJUGATES
15 The invention also pertains to immunoconjugates comprising an antibody
conjugated
to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an
enzymatically active
toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or
a radioactive
isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
20 been described above. Enzymatically active toxins and fragments thereof
that can be used
include diphtheria A chain, nonbinding active fragments of diphtheria toxin,
exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin .A chain, modeccin A
chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins
{PAPI, PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria
25 officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,
enomycin, and the
tricothecenes. A variety of radionuclides are available for the production of
radioconjugated
antibodies. Examples include 212Bi, 131I, i3'In, 9oY, and is6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling agents such as N-succinimidyl-3-(2-
pyridyldithiol) propionate
30 (SPDP), iminothiolane (IT), bifunetional derivatives of imidoesters (such
as dimethyl
adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes
(such as
glutareldehyde), bis-azido compounds {such as bis (p-azidobenzoyl)
hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates
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96
(such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-
2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as
described in
Vitetta et al., Science, 238: 1098 (1987). Carbon-14-labeled 1-
isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (M%-DTPA) is an exemplary chelating
agent for
conjugation of radionucleotide to the antibody. See W094i11026.
In another embodiment, the antibody can be conjugated to a "receptor" (such
streptavidin) for utilization in tumor pretargeting wherein the antibody-
receptor conjugate is
administered to the patient, followed by removal of unbound conjugate from the
circulation
using a clearing agent and then administration of a "ligand" (e.g., avidin)
that is in turn
conjugated to a cytotoxic agent.
4.14 COMPUTER READABLE SEQUENCES
In one application of this embodiment, a nucleotide sequence of the present
invention
can be recorded on computer readable media. As used herein, "computer readable
media"
refers to any medium which can be read and accessed directly by a computer.
Such media
include, but are not limited to: magnetic storage media, such as floppy discs,
hard disc
storage medium, and magnetic tape; optical storage media such as CD-ROM;
electrical
storage media such as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily appreciate how
any of the
presently known computer readable mediums can be used to create a manufacture
comprising computer readable medium having recorded thereon a nucleotide
sequence of the
present invention. As used herein, "recorded" refers to a process for storing
information on
computer readable medium. A skilled artisan can readily adopt any of the
presently known
methods for recording information on computer readable medium to generate
manufactures
comprising the nucleotide sequence information of the present invention.
A variety of data storage structures are available to a skilled artisan for
creating a
computer readable medium having recorded thereon a nucleotide sequence of the
present
invention. The choice of the data storage structure will generally be based on
the means
chosen to access the stored information. In addition, a variety of data
processor programs
and formats can be used to store the nucleotide sequence information of the
present
invention on computer readable medium. The sequence information can be
represented in a
word processing text file, formatted in commercially-available software such
as V~ordPerfect
805A/PCT ~ 02453344 2004-O1-21
97
and Microsoft Word, or represented in the form of an ASCII file, stored in a
database
application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can
readily adapt
any number of data processor structuring formats (e. g. text file or database)
in order to
obtain computer readable medium having recorded thereon the nucleotide
sequence
information of the present invention.
By providing any of the nucleotide sequences SEQ ID NO: I-244, or 489-706 or a
representative fragment thereof; or a nucleotide sequence at least 95%
identical to any of the
nucleotide sequences of SEQ ID NO: 1-244, or 489-706 in computer readable
form, a skilled
artisan can routinely access the sequence information for a variety of
purposes. Computer
I O software is publicly available which allows a skilled artisan to access
sequence information
provided in a computer readable medium. The examples which follow demonstrate
how
software which implements the BLAST (Altschul et al., J. i~Iol. Biol. 215:403-
410 (1990))
and BLAZE (Bnxtlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on
a Sybase
system is used to identify open reading frames (ORFs) within a nucleic acid
sequence. Such
ORFs may be protein-encoding fragments and rnay be useful in producing
commercially
important proteins such as enzymes used in fermentation reactions and in the
production of
commercially useful metabolites.
As used herein, "a computer-based system" refers to the hardware means,
software
means, and data storage means used to analyze the nucleotide sequence
information of the
present invention. The minimum hardware means of the computer-based systems of
the
present invention comprises a central processing unit (CPU), input means,
output means, and
data storage means. A skilled artisan can readily appreciate that any one of
the currently
available computer-based systems are suitable for use in the present
invention. As stated
above, the computer-based systems of the present invention comprise a data
storage means
having stored therein a nucleotide sequence of the present invention and the
necessary
hardware means and software means for supporting and implementing a search
means. As
used herein, "data storage means" refers to memory which can store nucleotide
sequence
information of the present invention, or a memory access means which can
access
manufactures having recorded thereon the nucleotide sequence information of
the present
invention.
As used herein, '°search means" refers to one or more programs which
are
implemented on the computer-based system to compare a target sequence or
target structural
motif with the sequence information stored within the data storage means.
Search means are
805A/PCT ~ 02453344 2004-O1-21
98
used to identify fragments or regions of a known sequence which match a
particular target
sequence or target motif. A variety of known algorithms are disclosed publicly
and a
variety of commercially available software for conducting search means are and
can be
used in the computer-based systems of the present invention. Examples of such
software
includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and
BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one
of the
available algorithms or implementing software packages far conducting homology
searches
can be adapted for use in the present computer-based systems. As used herein,
a "target
sequence" can be any nucleic acid or amino acid sequence of six or more
nucleotides or two
or more amino acids. A skilled artisan can readily recognize that the longer a
target
sequence is, the less likely a target sequence will be present as a random
occurrence in the
database. The most preferred sequence length of a target sequence is from
about 10 to 300
amino acids, more preferably from about 30 to 100 nucleotide residues.
However, it is well
recognized that searches for commercially important fragments, such as
sequence fragments
involved in gene expression and protein processing, may be of shorter length.
As used herein, "a target structural motif," or "target motif," refers to any
rationally
selected sequence or combination of sequences in which the sequences) are
chosen based on
a three-dimensional configuration which is formed upon the folding of the
target motif.
There are a variety of target motifs known in the art. Protein target motifs
include, but are
not limited to, enzyme active sites and signal sequences. Nucleic acid target
motifs include,
but are not limited to, promoter sequences, hairpin structures and inducible
expression
elements (protein binding sequences).
4.15 TRIPLE HELIX FORMATION
In addition, the fragments of the present invention, as broadly described, can
be used
to control gene expression through triple helix formation or antisense DNA or
RNA, both of
which methods are based on the binding of a polynucleotide sequence to DNA or
RNA.
Polynucleotides suitable for use in these methods are preferably 20 to 40
bases in length and
are designed to be complementary to a region of the gene involved in
transcription (triple
helix-see Lee et al., Nucl. Acids Res. 6, 3073 (1979); Cooney et al., Science
15241, 456
(1988); and Dervan et al., Science 251, 1360 (1991)) or to the mRNA itself
(antisense-
Olmno, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides a.s Antisense
Inhibitors of
Gene Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation
optimally
CA 02453344 2004-O1-21
99
results in a shut-off of RNA transcription from DNA, while antisense RNA
hybridization
blocks translation of an mRNA molecule into polypeptide. Botr~ techniques have
been
demonstrated to be effective in model systems. Information contained in the
sequences of
the present invention is necessary for the design of an antisense or triple
helix
oligonucleotide.
4.16 DIAGNOSTIC ASSAYS AND KITS
The present invention further provides methods to identify the presence or
expression
of one of the ORFs of the present invention, or homolog thereof, in a test
sample, using a
nucleic acid probe or antibodies of the present invention, optionally
conjugated or otherwise
associated with a suitable label.
In general, methods for detecting a polynucleotide of the invention can
comprise
contacting a sample with a compound that binds to and forms a complex with the
polynucleotide for a period sufficient to form the complex, and detecting the
complex, so
that if a complex is detected, a polynucleotide of the invention is detected
in the sample.
Such methods can also comprise contacting a sample under stringent
hybridization
conditions with nucleic acid primers that anneal to a polynucleotide of the
invention under
such conditions, and amplifying annealed polynucleotides, so that if a
polynucleotide is
amplified, a polynucleotide of the invention is detected in the sample.
In general, methods for detecting a polypeptide of the invention can comprise
contacting a sample with a compound that binds to and forms a complex with the
polypeptide for a period sufficient to form the complex, and detecting the
complex, so that if
a complex is detected, a polypeptide of the invention is detected in the
sample.
In detail, such methods comprise incubating a test sample with one or more of
the
antibodies or one or more of the nucleic acid probes of the present invention
and assaying
for binding of the nucleic acid probes or antibodies to components 'within the
test sample.
Conditions for incubating a nucleic acid probe or antibody with a test sample
vary.
Incubation conditions depend on the format employed in the assay, the
detection methods
employed, and the type and nature of the nucleic acid probe or antibody used
in the assay.
~ne skilled in the art will recognize that any one of the commonly available
hybridization,
amplification or immunological assay formats can readily be adapted to employ
the nucleic
acid probes or antibodies of the present invention. Examples of such assays
can be found in
Chard, T., An Introduction to Radioimmunoassay and Related Techniques,
Elsevier Science
805A/PCT ~ 02453344 2004-O1-21
100
Publishers, Amsterdam, The Netherlands (1986); Bullock, G.R. et al.,
Techniques in
Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983),
Vol. 3
(1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory
Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam,
The
Netherlands (1985). The test samples of the present invention include cells,
protein or
membrane extracts of cells, or biological fluids such as sputum, blood, serum,
plasma, or
urine. The test sample used in the above-described method will vary based on
the assay
format, nature of the detection method and the tissues, cells or extracts used
as the sample to
be assayed. Methods for preparing protein extracts or membrane extracts of
cells are well
known in the art and can be readily be adapted in order to obtain a sample
which is
compatible with the system utilized.
In another embodiment of the present invention, kits are provided which
contain the
necessary reagents to carry out the assays of the present invention.
Specifically, the
invention provides a compartment kit to receive, in close confinement, one or
more
containers which comprises: (a) a first container comprising one of the probes
or antibodies
of the present invention; and (b} one or more other containers comprising one
or more of the
following: wash reagents, reagents capable of detecting presence of a bound
probe or
antibody.
In detail, a compartment kit includes any kit in which reagents are contained
in
separate containers. Such containers include small glass containers, plastic
containers or
strips of plastic or paper. Such containers allows one to efficiently transfer
reagents from
one compartment to another compartment such that the samples and reagents are
not
cross-contaminated, and the agents or solutions of each container can be added
in a
quantitative fashion from one compartment to another. Such containers will
include a
container which will accept the test sample, a container which contains the
antibodies used
in the assay, containers which contain wash reagents (such as phosphate
buffered saline,
Tris-buffers, etc.), and containers which contain the reagents used to detect
the bound
antibody or probe. Types of detection reagents include labeled nucleic acid
probes, labeled
secondary antibodies, or in the alternative, if the primary antibody is
labeled, the enzymatic,
or antibody binding reagents which are capable of reacting with the labeled
antibody. One
skilled in the art will readily recognize that the disclosed probes and.
antibodies of the present
invention can be readily incorporated into one of the established kit formats
which axe well
known in the art.
805t-1/PCT CA 02453344 2004-O1-21
101
4.17 MEDICAL IMAGING
The novel polypeptides and binding partners of the invention are useful in
medical
imaging of sites expressing the molecules of the invention (e.g., where the
polypeptide of the
invention is involved in the immune response, for imaging sites of
inflammation or
infection). See, e.g., Kunkel et al., U.S. Pat. NO. 5,413,778. Such methods
involve
chemical attachment of a labeling or imaging agent, administration of the
labeled
polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging
the labeled
polypeptide in vivo at the target site.
4.18 SCREENING ASSAYS
Using the isolated proteins and poiynucleotides of the invention, the present
invention further provides methods of obtaining and identifying agents which
bind to a
polypeptide encoded by an ORF corresponding to any of the nucleotide sequences
set forth
in SEQ ID NO: 1-244, or 489-706, or bind to a specific domain of the
polypeptide encoded
by the nucleic acid. In detail, said method comprises the steps of:
(a) contacting an agent with an isolated protein encoded by an ORF of the
present invention, or nucleic acid of the invention; and
(b) determining whether the agent binds to said protein or said nucleic acid.
In general, therefore, such methods for identifying compounds that bind to a
polynucleotide of the invention can comprise contacting a compound with a
polynucleotide
of the invention for a time sufficient to form a polynucleotide/compound
complex, and
detecting the complex, so that if a polynucleotide/compound complex is
detected, a
compound that binds to a polynucleotide of the invention is identified.
Likewise, in general, therefore, such methods for identifying compounds that
bind to
a polypeptide of the invention can comprise contacting a compound with a
polypeptide of
the invention for a time sufficient to form a polypeptide/compound complex,
and detecting
the complex, so that if a polypeptide/compound complex is detected, a compound
that binds
to a polynucleotide of the invention is identified.
Methods for identifying compounds that bind to a polypeptide of the invention
can
also comprise contacting a compound with a polypeptide of the invention in a
cell for a time
sufficient to form a polypeptidelcompound complex, wherein the complex drives
expression
of a receptor gene sequence in the cell, and detecting the complex by
detecting reporter gene
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102
sequence expression, so that if a polypeptide/compound complex is detected, a
compound
that binds a polypeptide of the invention is identified.
Compounds identified via such methods can include compounds which modulate
the activity of a polypeptide of the invention (that is, increase or decrease
its activity, relative
to activity observed in the absence of the compound). Alternatively, compounds
identified
via such methods can include compounds which modulate the expression of a
polynucleotide
of the invention (that is, increase or decrease expression relative to
expression levels
observed in the absence of the compound). Compounds, such as compounds
identified via
the methods of the invention, can be tested using standard assays well known
to those of
skill in the art for their ability to modulate activity/expression.
The agents screened in the above assay can be, but are not limited to,
peptides,
carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents
can be
selected a.nd screened at random or rationally selected or designed using
protein modeling
techniques.
For random screening, agents such as peptides, carbohydrates, pharmaceutical
agents
and the like are selected at random and are assayed for their ability to bind
to the protein
encoded by the ORF of the present invention. Alternatively, agents may be
rationally
selected or designed. As used herein, an agent is said to be "rationally
selected or designed"
when the agent is chosen based on the configuration of the particular protein.
For example,
one skilled in the art can readily adapt currently available procedures to
generate peptides,
pharmaceutical agents and the like, capable of binding to a specific peptide
sequence, in
order to generate rationally designed antipeptide peptides, for example see
Hurby et al.,
Application of Synthetic Peptides: Antisense Peptides," In Synthetic Peptides,
A User's
Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry
28:9230-8 (1989), or pharmaceutical agents, or the like.
In addition to the foregoing, one class of agents of the present invention, as
broadly
described, can be used to control gene expression through binding to one of
the ORFs or
EMFs of the present invention. As described above, such agents can be randomly
screened
or rationally designed/selected. Targeting the ORF or EMF allows a skilled
artisan to design
sequence specific or element specific agents, modulating the expression of
either a single
ORF or multiple ORFs which rely on the same EMF for expression control. One
class of
DNA binding agents are agents which contain base residues which hybridize or
form a triple
helix formation by binding to DNA or RNA. Such agents can be based on the
classic
805A/PCT ~ 02453344 2004-O1-21
103
phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl
or polymeric
derivatives which have base attachment capacity.
Agents suitable for use in these methods preferably contain 20 to 40 bases and
are
designed to be complementary to a 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, 3.
Neurochem. 56, 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of
Gene
Expression, CRC Press, Boca Raton, FL (1988)). Triple helix-formation
optimally results in
a shut-off of RNA transcription from DNA, while antisense RNA hybridization
blocks
translation of an mRNA molecule into polypeptide. Both techniques have been
demonstrated to be effective in model systems. Information contained in the
sequences of
the present invention is necessary for the design of an antisense or triple
helix
oligonucleotide and other DNA binding agents.
Agents which bind to a protein encoded by one of the ORFs of the present
invention
can be used as a diagnostic agent. Agents which bind to a protein encoded by
one of the
ORFs of the present invention can be formulated using known techniques to
generate a
pharmaceutical composition.
4.19 USE OF NUCLEIC ACIDS AS PROBES
Another aspect of the subject invention is to provide for polypeptide-specific
nucleic
acid hybridization probes capable of hybridizing with naturally occurring
nucleotide
sequences. The hybridization probes of the subject invention may be derived
from any of
the nucleotide sequences SEQ ID NO: 1-244, or 489-706. Because the
corresponding gene
is only expressed in a limited number of tissues, a hybridization probe
derived from any of
the nucleotide sequences SEQ ID NO: 1-244, or 489-706 can be used as an
indicator of the
presence of RNA of cell type of such a tissue in a sample.
Any suitable hybridization technique can be employed, such as, for example, in
situ
hybridization. PCR as described in US Patents Nos. 4,683,195 and 4,965,188
provides
additional uses for oligonucleotides based upon the nucleotide sequences. Such
probes used
in PCR may be of recombinant origin, may be chemically synthesized, or a
mixture of both.
The probe will comprise a discrete nucleotide sequence for the detection of
identical
sequences or a degenerate pool of possible sequences for identification of
closely related
genomic sequences.
805A/PCT ~ 02453344 2004-O1-21
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Other means for producing specific hybridization probes for nucleic acids
include the
cloning of nucleic acid sequences into vectors for the production of mRNA
probes. Such
vectors are known in the art and are commercially available and may be used to
synthesize
RNA probes an vatro by means of the addition of the appropriate RNA polymerase
as T7 or
SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The
nucleotide
sequences may be used to construct hybridization probes for mapping their
respective
genomic sequences. The nucleotide sequence provided herein may be mapped to a
chromosome or specific regions of a chromosome using well-known genetic and/or
chromosomal mapping techniques. These techniques include in situ
hybridization, linkage
analysis against known chromosomal markers, hybridization screening with
libraries or
flow-sorted chromosomal preparations specific to known chromosomes, and the
like. The
technique of fluorescent in situ hybridization of chromosome spreads has been
described,
among other places, in Verma et aI (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York NY.
Fluorescent in situ hybridization of chromosomal preparations and other
physical
chromosome mapping techniques may be correlated with additional genetic map
data.
Examples of genetic map data can be found in the 1994 Genome Issue of Science
(265:1981fj. Correlation between the location of a nucleic acid on a physical
chromosomal
map and a specific disease (or predisposition to a specific disease) may help
delimit the
region of DNA associated with that genetic disease. The nucleotide sequences
of the subject
invention may be used to detect differences in gene sequences between normal,
carrier or
affected individuals.
4.20 PREPARATIOhl OF SUPPORT BOUND OLIGONUCLEOTIDES
Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared
by, for
example, directly synthesizing the oligonucleotide by chemical means, as is
commonly
practiced using an automated oligonucleotide synthesizer.
Support bound oligonucleotides may be prepared by any of the methods known to
those
of skill in the art using any suitable support such as glass, polystyrene or
Teflon. One strategy
is to precisely spot oligonucleotides synthesized by standard synthesizers.
Immobilization can
be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin.
Microbiol. 28(6), 1469-
72); using UV light (Nagata et al., 1985; Dahlen et al , 1987; Morrissey &
Collins, (1989) Mol.
Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA (Keller
et al., 1988;
1989); all references being specifically incorporated herein.
~~~~~T CA 02453344 2004-O1-21
105
Another strategy that may be employed is the use of the strong biotin-
streptavidin
interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad.
Sci. USA 91(8),
3072-6, describe the use of biotinylated probes, although these are duplex
probes, that are
immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads
may be
purchased from Dynal, Oslo. Of course, this same linking chemistry is
applicable to coating
any surface with streptavidin. Biotinylated probes may be purchased from
various sources,
such as, e.g., Operon Technologies (Alameda, CA).
Nunc Laboratories (Naperville, IL) is also selling suitable material that
could be used.
Nunc Laboratories have developed a method by which DNA can be covalently bound
to the
microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface
grafted with
secondary amino groups (>NH) that serve as bridgeheads for further covalent
coupling.
CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be
bound
to CovaLink exclusively at the 5'-end by a phosphoramidate bond, allowing
immobilization of
more than 1 pmol of DNA (Rasmussen et al., (1991) Anal. Biochem. 198(1) 138-
42).
The use of CovaLink NH strips for covalent binding of DNA molecules at the 5'-
end
has been described (Rasmussen et al., (1991). In this technology, a
phosphoramidate bond is
employed (Chu et al., (1983) Nucleic Acids Res. 11(8) 6513-29). This is
beneficial as
immobilization using only a single covalent bond is preferred. The
phosphoramidate bond joins
the DNA to the CovaLink NH secondary amino groups that are positioned at the
end of spacer
arms covalently grafted onto the polystyrene surface through a 2 nm long
spacer arm. To link
an oligonucleotide to CovaLink NH via an phosphoramidate bond, the
oligonucleotide terminus
must have a 5'-end phosphate group. It is, perhaps, even possible for biotin
to be covalently
bound to CovaLink and then streptavidin used to bind the probes.
More specifically, the linkage method includes dissolving DNA in water (7.5
ng/~.1) and
denaturing for 10 min. at 95°C and cooling on ice for 10 min. Ice-cold
0.1 M 1-
methylimidazole, pH 7.0 (1-MeIm~), is then added to a final concentration of
10 mM 1-MeIm~.
A ss DNA solution is then dispensed into CovaLink NH strips (75 ~.1/well)
standing on ice.
Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC),
dissolved in 10 mM 1-MeIrn~, is made fresh and 25 N,l added per well. The
strips are incubated
for 5 hours at SO°C. After incubation the strips are washed using,
e.g., Nunc-Irnmuno Wash;
first the wells are washed 3 times, then they are soaked with washing solution
for 5 min., and
finally they are washed 3 times (where in the washing solution is 0.4 N NaOH,
0.25% SDS
heated to 50°C).
805A/PCT ~ 02453344 2004-O1-21
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It is contemplated that a further suitable method for use with the present
invention is
that described in PCT Patent Application WO 90/03382 (Southern & Maskos),
incorporated
herein by reference. This method of preparing an oligonucleotide bound to a
support involves
attaching a nucleoside 3'-reagent through the phosphate group by a covalent
phosphodiester link
to aliphatic hydroxyl groups earned by the support. The oligonucleotide is
then synthesized on
the supported nucleoside and protecting groups removed from the synthetic
oligonucleotide
chain under standard conditions that do not cleave the oligonucleotide from
the support.
Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen
phosphorate.
An on-chip strategy for the preparation of DNA probe for the preparation of
DNA probe
arrays may be employed. For example, addressable laser-activated
photodeprotection may be
employed in the chemical synthesis of oligonucleotides directly on a glass
surface, as described
by Fodor et al. (1991) Science 251 (4995), 767-73, incorporated herein by
reference. Probes
may also be immobilized on nylon supports as described by Van Ness et al.
(1991) Nucleic
Acids Res., 19(12) 3345-50; or Iinked to Tetlon using the method of Dunean &
Cavalier (1988)
Anal. Biochem. 169(1), 104-8; all references being specifically incorporated
herein.
To link an oligonucleotide to a nylon support, as described by Van Ness et al.
(1991),
requires activation of the nylon surface via alkylation and selective
activation of the 5'-amine of
oligonucleotides with cyanotic chloride.
One particular way to prepare support bound oligonucleotides is to utilize the
light-generated synthesis described by Pease et al., (1994) Proc. Nat'1. Acad.
Sci., USA 91(11),
5022-6, incorporated herein by reference). These authors used current
photolithographic
techniques to generate arrays of immobilized oligonucleotide probes (DNA
chips). These
methods, in which light is used to direct the synthesis of oligonucleotide
probes in high-density,
miniaturized arrays, utilize photolabile 5'-protected N acyl-deoxynucleoside
phosphoramidites,
surface linker chemistry and versatile combinatorial synthesis strategies. A
matrix of 256
spatially defined oligonucleotide probes may be generated in this manner.
4.21 PREPARATION OF hIITCLEIC ACID FRAGMENTS
The nucleic acids may be obtained from any appropriate source, such as cDNAs,
genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC
inserts, and RNA, including mRNA without any amplification steps. For example,
Sambrook
et al. (1989) describes three protocols for the isolation of high molecular
weight DNA from
mammalian cells (p. 9.14-9.23).
805A/PCT ~ 02453344 2004-O1-21
107
DNA fragments may be prepared as clones in M13, plasmid or lambda vectors
and/or
prepared directly from genomic DNA or cDNA by PCR or other amplification
methods.
Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of
DNA
samples may be prepared in 2-500 ml of final volume.
The nucleic acids would then be fragmented by any of the methods known to
those of
skill in the art including, for example, using restriction enzymes as
described at 9.24-9.28 of
Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.
Low pressure shearing is also appropriate, as described by Schriefer et al.
(1990)
Nucleic Acids Res. 18(24), 7455-6, incorporated herein by reference). In this
method, DNA
samples are passed through a small French pressure cell at a variety of low to
intermediate
pressures. A lever device allows controlled application of low to intermediate
pressures to the
cell. The results of these studies indicate that low-pressure shearing is a
useful alternative to
sonic and enzymatic DNA fragmentation methods.
One particularly suitable way for fragmenting DNA is contemplated to be that
using the
two base recognition endonuclease, CviJI, described by Fitzgerald et al.
(1992) Nucleic Acids
Res. 20(14) 3753-62. These authors described an approach far the rapid
fragmentation and
fractionation of DNA into particular sizes that they contemplated to be
suitable for shotgun
cloning and sequencing.
The restriction endonuclease CviJI normally cleaves the recognition sequence
PuGCPy
between the G and C to leave blunt ends. Atypical reaction conditions, which
alter the
specificity of this enzyme (CviJI* *), yield a quasi-random distribution of
DNA fragments form
the small molecule pUCl9 (2688 base pairs). Fitzgerald et al. (1992)
quantitatively evaluated
the randomness of this fragmentation strategy, using a CviJI** digest of pUCl9
that was size
fractionated by a rapid gel filtration method and directly ligated, without
end repair, to a lac Z
minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI**
restricts
pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is
accumulated
at a rate consistent with random fragmentation.
As reported in the literature, advantages of this approach compared to
sonication and
agaxose gel fractionation include: smaller amounts of DNA are required (0.2-
0.5 ~,g instead of
2-5 ~.g); and fewer steps are involved (no preligation, end repair, chemical
extraction, or
agarose gel electrophoresis and elution are needed).
Irrespective of the manner in which the nucleic acid fragments are obtained or
prepared,
it is important to denature the DNA to give single stranded pieces available
for hybridization.
805A/PCT ~ 02453344 2004-O1-21
108
This is achieved by incubating the DNA solution for 2-5 minutes at 80-
90°C. The solution is
then cooled quickly to 2°C to prevent renaturation of the D1~TA
fragments before they are
contacted with the chip. Phosphate groups must also be removed from genomic
DNA by
methods known in the art.
4.22 PREPARATION OF L1NA ARRAYS
Arrays may be prepared by spotting DNA samples on a support such as a nylon
membrane. Spotting may be performed by using arrays of metal pins (the
positions of which
correspond to an array of wells in a microtiter plate) to repeated by transfer
of about 20 n1 of a
DNA solution to a nylon membrane. By offset printing, a density of dots higher
than the density
of the wells is achieved. One to 25 dots may be accommodated in 1 mm2,
depending on the
type of label used. By avoiding spotting in some preselected number of rows
and columns,
separate subsets (subarrays) may be formed. Samples in one suba.rray may be
the same genomic
segment of DNA (or the same gene) from different individuals, or may be
different, overlapped
genomic clones. Each of the subarrays may represent replica spotting of the
same samples. In
one example, a selected gene segment may be amplified from 64 patients. For
each patient, the
amplified gene segment may be in one 96-well plate (all 96 wells containing
the same sample).
A plate for each of the 64 patients is prepared. By using a 96-pin device, all
samples may be
spotted on one 8 x 12 cm membrane. Subarrays may contain 64 samples, one from
each patient.
Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may
be a 1 mm
space between subarrays.
Another approach is to use membranes or plates (available from NUNC,
Naperville,
Illinois) which may be partitioned by physical spacers e.g. a plastic grid
molded over the
membrane, the grid being similar to the sort of membrane applied to the bottom
of multiwell
plates, or hydrophobic strips. A f red physical spacer is not preferred for
imaging by exposure
to flat phosphor-storage screens or x-ray films.
The present invention is illustrated in the following examples Upon
consideration of
the present disclosure, one of skill in the art will appreciate that many
other embodiments and
variations may be made in the scope of the present invention. Accordingly, it
is intended that
the broader aspects of the present invention not be limited to the disclosure
of the following
examples. The present invention is not to be limited in scope by the
exemplified embodiments
which are intended as illustrations of single aspects of the invention, and
compositions and
methods which are functionally equivalent are within the scope of the
invention. Indeed,
numerous modifications and variations in the practice of the invention are
expected to occur to
805AlPCT ~ 02453344 2004-O1-21
109
those skilled in the art upon consideration of the present preferred
embodiments. Consequently,
the only limitations which should be placed upon the scope of the invention
are those which
appear in the appended claims.
All references cited within the body of the instant specification are hereby
incorporated
by reference in their entirety.
5.0 EXAMPLES
5.1 EXAMPLE 1
Novel Nucleic Acid Seauences ~btained From Various Libraries
A plurality of novel nucleic acids were obtained from cDNA libraries prepared
from
various human tissues and in some cases isolated from a genomic library
derived from human
chromosome using standard PCR, SBH sequence signature analysis and Sanger
sequencing
techniques. The inserts of the library were amplified with PCR using primers
specific for the
vector sequences which flank the inserts. Clones from cDNA libraries were
spotted on nylon
membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to
obtain signature
sequences. The clones were clustered into groups of similar or identical
sequences.
Representative clones were selected for sequencing.
In some cases, the 5' sequence of the amplified inserts was then deduced using
a typical
Sanger sequencing protocol. PCR products were purified and subjected to
fluorescent dye
terminator cycle sequencing. Single pass gel sequencing was done using a 377
Applied
Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences.
5.2 EXAMPLE 2
Assemblage of Novel Nucleic Acids
The contigs or nucleic acids of the present invention, designated as SEQ ID
NO: 489-
706 were assembled using an EST sequence as a seed. Then a recursive algorithm
was used to
extend the seed EST into an extended assemblage, by pulling additional
sequences from
different databases (i.e., Hyseq's database containing EST sequences, dbEST,
gb pri, and
UniGene, and exons from public domain genomic sequences predicated by GenScan)
that
belong to this assemblage. The algorithm terminated when there were no
additional sequences
from the above databases that would extend the assemblage. Further, inclusion
of component
sequences into the assemblage was based on a BLASTN hit to the extending
assemblage with
BLAST score greater than 300 and percent identity greater than 95%.
805A/PCT CA 02453344 2004-O1-21
110
Table 8 sets forth the novel predicted polypeptides (including proteins)
encoded by the
novel polynucleotides (SEQ ID NO: 489-706) of the present invention, and their
corresponding translation start and stop nucleotide locations to each of SEQ
ID N~: 489-706.
Table 8 also indicates the method by which the polypeptide was predicted.
Method A refers to
a polypeptide obtained by using a software program called FASTY (available
from
http://fasta.bioch.vir~inia edu) which selects a polypeptide based on a
comparison of the
translated novel polynucleotide to known polynucleotides (W.R. Pearson,
Methods in
Enzymology, 183:63-98 (1990), herein incorporated by reference). Method B
refers to a
polypeptide obtained by using a software program called GenScan for
human/vertebrate
sequences (available from Stanford University, Office of Technology Licensing)
that predicts
the polypeptide based on a probabilistic model of gene structure/compositional
properties (C.
Burge and S. Karlin, J. Mol. Biol., 268:78-94 (1997), incorporated herein by
reference).
Method C refers to a polypeptide obtained by using a Hyseq proprietary
software program that
translates the novel polynucleotide and its complementary strand into six
possible amino acid
sequences (forward and reverse frames) and chooses the polypeptide with the
longest open
reading frame.
5.3 EXAMPLE 3
Novel Nucleic Acids
The novel nucleic acids of the present invention were assembled from sequences
that
were obtained from a cDNA library by methods described in Example 1 above, and
in some
cases sequences obtained from one or more public databases. The nucleic acids
were
assembled using an EST sequence as a seed. Then a recursive algorithm was used
to extend the
seed EST into an extended assemblage, by pulling additional sequences from
different
databases (Hyseq's database containing EST sequences, dbEST, gb pri, and
UniGene) that
belong to this assemblage. The algorithm terminated when there was no
additional sequences
from the above databases that would extend the assemblage. Inclusion of
component sequences
into the assemblage was based on a BLASTN hit to the extending assemblage with
BLAST
score greater than 300 and percent identity greater than 95%.
Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full-length gene cDNA
sequence and its corresponding protein sequence were generated from the
assemblage. Any
frame shifts and incorrect stop codons were corrected by hand editing. During
editing, the
sequences were checked using PASTY and/or BLAST against Genebank (i.e., dbEST,
gb pri,
UniGene, and Genpept) and the Geneseq (Derwent). Other computer programs which
may
805A/PCT ~ 02453344 2004-O1-21
T ~ 111
have been used in the editing process were phredPhrap and Consed (University
of Washington)
and ed-ready, ed-ext and cg-zip-2 (Hyseq, Inc.). The full-length nucleotide
and amino acid
sequences, including splice variants resulting from these procedures are shown
in the
Sequence Listing as SEQ ID NO: 1-488.
S SEQ ID NO: 1-132 were classified as secreted according to their predicted
cellular
localization using the Pfam software program (Sonnhammer et al., Nucleic Acids
Res., Vol.
26(1) pp. 320-322 (1998), and http:/lpfam.wustl.edu/, herein incorporated by
reference).
SEQ ID NO: 133-197 were determined to contain signal peptide sequences and
their
cleavage sites using Neural Network SignalP V 1.1 program (from Center for
Biological
Sequence Analysis, The Technical University of Denmark). The process for
identifying
prokaryotic and eukaryotic signal peptides and their cleavage sites are also
disclosed by
Henrik Nielson, Jacob Engelbrecht, Soren Brunak, and Gunnar von Heijne in the
publication
" Identification of prokaryotic and eukaryotic signal peptides and prediction
of their cleavage
sites" Protein Engineering, Vol. 10, no. l, pp. 1-6 (1997), incorporated
herein by reference.
A maximum S score and a mean S score, as described in the Nielson et al
reference, was
obtained for the polypeptide sequences.
SEQ ID NO: 198-244 were determined to be secreted polypeptides using a
proprietary algorithm, SeqLocTM (Ilyseq Inc.). SeqLocTM classifies the
proteins into three
sets of locales: intracellular, membrane, or secreted. This prediction is
calculated using
maximum likelihood estimation of three characteristics of each polypeptide, 1
) percentage of
cysteine residues, 2) Kyte-Doolittle scores for the first 20 amino acids of
each protein (J.
Mol Biol, 157, pp. 105-31 (1982), incorporated herein by reference), a.nd 3)
Kyte-Doolittle
scores to calculate the longest hydrophobic stretch (LHS) of the said protein
(J. Mol Biol,
157, pp. 105-31 (1982), incorporated herein by reference). The LHS is
calculated by finding
the stretch of 20 amino acid residues in the protein that have the highest sum
of Kyte-
Doolittle hydrophobicity values.
Table 1 shows the various tissue sources of SEQ ID NO: 1-244.
The nearest neighbor results for polypeptides SEQ ID NO: 245-488, that
correspond
to nucleotide sequences SEQ ID NO: 1-244 were obtained by a BLASTP (version
2.0a1
19MP-WashU) searches against Genpept release 124 and the Geneseq release
200112
(Derwent) using BLAST algorithm. The nearest neighbor results showed the
closest
homologue with functional annotation for SEQ ID NO: 245-488 from Genpept 124
and
Geneseq. The translated amino acids sequences for which the nucleic acid
sequence encodes
805tilrCT CA 02453344 2004-O1-21
112
are shown in the Sequence Listing. The homologues with identifiable functions
for SEQ ID
NO: 245-488 are shown in Table 2.
Using eMatrix software package (Stanford University, Stanford, CA) (Wu et al.,
J.
Comp. Biol., Vol. 6, 219-235 (1999), http://motif.stanford.edyernatrix-search/
herein
incorporated by reference), all the polypeptide sequences were examined to
determine
whether they had identifiable signature regions. Scoring matrices of the
eMatrix software
package are derived from the BLOCKS, PRINTS, PFAM, PRODOM, and DOMO
databases. Table 3 shows the accession number of the homologous eMatrix
signature found
in the indicated polypeptide sequence, its description, and the results
obtained which include
accession number subtype; raw score; p-value; and the position of signature in
amino acid
sequence.
Using the Pfam software program (Sonnhammer et al., Nucleic Acids Res., Vol.
26(1) pp. 320-322 (1998) herein incorporated by reference) all the polypeptide
sequences
were examined for domains with homology to certain peptide domains. Table 4
shows the
name of the Pfam model found, the description, the e-value, the Pfam score for
the identified
model within the sequence, number of similar domains found, and the position
of the domain
in the SEQ ID NO: being interrogated. Further description of the Pfam models
can be found
at http://pfam.wustl.eduJ.
The GeneAtlas~ software package (Molecular Simulations Inc. (MSI), San Diego,
CA) was used to predict the three-dimensional structure models for the
polypeptides
encoded by SEQ ID NO 1-244 (i.e. SEQ ID NO: 245-488). Models were generated by
(1)
PSI-BLAST which is a multiple alignment sequence profile-based searching
developed by
Altschul et al, (Nucl. Acids Res. 25, 3389-3408 (1997)), (2) High Throughput
Modeling
(HTM) (Molecular Simulations Inc. (MSI) San Diego, CA,) which is an automated
sequence
and structure searching procedure (http://www.msi.com/), and (3) SeqFoldrM
which is a fold
recognition method described by Fischer and Eisenberg (J. Mol. Biol. 209, 779-
791 (1998)).
This analysis was carried out, in part, by comparing the polypeptides of the
invention with
the known NMR (nuclear magnetic resonance) and x-ray crystal three-dimensional
structures
as templates. Table 5 shows: "PDB ID°°, the Protein DataBase
(PDB) identifier given to
template structure; "Chain ID", identifier of the subcomponent of the PDB
template
structure; "Compound Information", information of the PDB template structure
and/or its
subcomponents; "PDB Function Annotation" gives function of the PDB template as
annotated by the PDB files (http:/www.rcsb.or~lPDB/); start and end amino acid
position of
gO~~~.I. CA 02453344 2004-O1-21
113
the protein sequence aligned; PSI-BLAST score, the verify score, the SeqFold
score, and the
Potentials) of Mean Force (PMF). The verify score is produced by GeneAtlasTM
software
(MSI), is based on Dr. Eisenberg's Profile-3D threading program developed in
Dr. David
Eisenberg's laboratory (US patent no. 5,436,850 and Luthy, Bowie, and
Eisenberg, Nature,
356:83-8S (1992)) and a publication by R. Sanchez and A. Sali, Proc. Natl.
Acad. Sci. USA,
9S:13S97-12502. The verify score produced by GeneAtlas normalizes the verify
score for
proteins with different lengths so that a unified cutoff can be used to select
good models as
follows:
Verify score (normalized) _ (raw score - 1 /2 high score)/( 1 /2 high score)
The PFM score, produced by GeneAtlas~ software (MSI), is a composite scoring
function that depends in part on the compactness of the model, sequence
identity in the
alignment used to build the model, pairwise and surface mean force potentials
(MFP). As
1 S given in table S, a verify score between 0 to 1.0, with 1 being the best,
represents a good
model. Similarly, a PMF score between 0 to 1.0, with 1 being the best,
represents a good
model. A SeqFold~ score of more than SO is considered significant. A good
model may
also be determined by one of skill in the art based all the information in
Table S taken in
totality.
The nucleotide sequence within the sequences that codes for signal peptide
sequences
and their cleavage sites can be determined from using Neural Network SignalP V
1.1
program (from Center for Biological Sequence Analysis, The Technical
University of
Denmark). The process for identifying prokaryotic and eukaryotic signal
peptides and their
cleavage sites are also disclosed by Henrik Nielson, Jacob Engelbrecht, Soren
Brunak, and
Gunnar von Heijne in the publication " Identification of prokaryotic and
eukaryotic signal
peptides and prediction of their cleavage sites" Protein Engineering, Vol. 10,
no. l, pp. 1-6
( 1997), incorporated herein by reference. A maximum S score and a mean S
score, as
described in the Nielson et al reference, was obtained for the polypeptide
sequences. Table 6
shows the position of the last amino acid of the signal peptide in each of the
polypeptides
and the maximum score and mean score associated with that signal peptide.
Table 7 correlates SEQ ID NO: 1-244 to a specific chromosomal location.
Table 8 is a correlation table of the novel polynucleotide sequences SEQ ID
NO: 1-
244, their corresponding polypeptide sequences SEQ ID NO: 24S-488, their
corresponding
~~~~P~.I. CA 02453344 2004-O1-21
114
priority contig nucleotide sequences SEQ ID NO: 489-706, their corresponding
priority
contig polypeptide sequences SEQ ID NO: 707-924, and the US serial number of
the
priority application in which the contig sequence was filed.
805tiIrCT CA 02453344 2004-O1-21
IAS
Tahla 1
Tissue RNA source Librar Name SEQ ID NO:
on in
adrenal Clontech ADR002 4 7 9 13 24-26 31 33 42-43
gland 56 60 71 84-
85 94 98 100-101 118 127
133-134 141-
144 163 182 189 199 201
207 212
adult bladderInvitrogen BLD001 8 12-I3 25 71 122-123
129 134 140 144
178 181 184 241-242
adult brainBioChain ABR013 183
adult brainClontech ABR001 13 22 64 66 102 112 182
186 206 211
238
_
adult brainClontech ABR006 1-5 18 22 24 36-39 47
52 55-56 60-61
93-94 98 106 112 125 128
133 143 145
147 151 157 162 165 179
182 184 189-
191 235 238-239
adult brainClontech ABR008 1-2 4 7-8 I 1 13-14 22
24 27 35-40 42
48-49 56-57 59 61 64 67
70 73 78-82 92
95 98-99 102 104-105 108
112 115 119
121 123 126-130 133 140-143
147 150
153 157 159 162 165 171
176 I 80-182
189-190 195-197 212 222
225-227 231
236-237
241
adult brainGIBCO AB3001 _
1 5 7 13 34 90 99 127
151 207 224 239
adult brainGIBCO ABD003 1 8 11 13 43 55 62 64
75 81 84-85 102
127 129-131 I 33 144 146-147
151- I 52
186 189 197 200-201 207-208
210 225
241
adult brainInvitro ABR014 140 176 189
en
adult brainInvitro ABR015 133 136 208 241
en
adult brainInvitro ABR416 8 13 186
en
_
adult brainInvitro ABT004 11 42 48 60 85 129 133
en 141-142 147 149
adult cervixBioChain CVX001 1 7 12 14 21 24 26-27
35 43 5I 60 62 64
71 84 95 98-99 122-123
127 129-130
140-142 144 146 176 178
186 195 197
200-201 206 211-213 220
222 240
adult colonInvitrogen CLN001 13 71 75 93 122 129 134
141-142 224
241
adult heartGIBCO A~IR001 1 4 6-8 13 21 23-24 42-43
45 53-54 60-
61 67 70 72 81 90-91 98-99
102 115 120
I 30 133 137-140 143-144
146 166-169
178-179 183 186 189 197
204 207 213
224-225
adult kidneyGIBCO AKD001 4 6 8 12-13 20 23-26 34
39-43 53-S4 62
64 66 73 79 81 90 98 102
108 123 127
130 133 135 144-148 163
171-172 179
186 189 197 200 204 206-207
210 213
224-225 227 233 241-242
adult kidneyInvitrogen AKT002 6 12-13 24 39 42-43 51
60 66 86 93 102
I25 132 143 I46-148 156
178 184 186
189 197 200 202 210 219
225 233
adult liverClontech ALV003 36-38 51 73 135-136 165
173-175 224
adult liverInvitrogen ALV002 4-5 12-13 32 39 42 48
51 56 73 81 98
102 120 133 136 140 143-144
163 165
173-175 I78-179 182 189
193 225 23I
241
adult lun GIBCO ALG001 25-26 41 48 60 74 81 146
178 189 197
adult ovaryInvitrogen AOV001 4 7 9 12-14 20-21 23-26
31 34 39-40 42-
43 48 52 60 62 64 73 75
78-79 81 94 96
98 112 1 I 8-119 122-123
127 130 133
805A/PCT
CA 02453344 2004-O1-21
116
Tahla t
Tissue RNA source Library Name SE ID NO:
on in
135 I40 144 146-147 149
153 163 172
178 182 186 193 195 202
206-207 209
213 222 224 231 233 236
239-240 242
adult lacentaClontech APLOO x 43 73 17_8
adult spleenClontech SPLc01 7-8 14 36-38 53-54 59 71
92 104 108
125 129 133 140-1.42 162
237
adult spleenGIBCO ASP001 4 8 13 23-24 27 41 48 64
75 81 102 104
124 141-144 146 189 201
224
adult testisGIBCO ATS001 10 13 34 43 46 60 81 102
123 143-144
189 200 206 224
155 183
bone marrowClontech BMD001 _
1-2 7 12 14 17-19 23 26
46-47 63-64 81
91 102 106 118 122 124
133 144 146
186 I89 198-203 205 207
224-225 236
bone marrowClontech BMD007 11
bone marrowGF BMD002 1-2 11-12 14 x9 28-29 36-38
40-41 43
48-49 59-61 71 73 76 90
93 95 97-98
104 I 15 118 120 122 125
130 134 143
162-163 171 186 189 197
202 206-207
21_2 225 240-241 243
cultured Stratagene ADP001 10 25 42-43 48 75 81 91
122 130 140
readi oc 144 163 178 186 205 241
tes
endothelialStratagene EDT001 1 4 7-8 10-11 13 19-20
23 25 30-31 48
cells 62 64 66 75 78 81 90-91
93 95 98 118
133 137-I39 144-146 150
156 178 I86
197 200 206-207 209 213
215 224 230
241
fetal brainClontech FBR001 35 53-54 129 182
fetal brainClontech FBR004 36-38 70 94 126 171 187
23 x 238
fetal brainClontech FBR006 1-2 5 7 13 15 24 32 35-39
42 56-57 62
67 73 79 83 90 92 98 112
I 14 117 119
123 127 129 135 140-143
150 155 157-
158 162 171-172 176 180-182
190 192
212 2x8 220 222 228 230-231
236-237
241-243
fetal brainGIBCO HFB001 1 4 11-13 15 24 26 30 32
42-44 46 48 64
81 90 94 1 x2 I25 130 133
135 140 143
151 155 162 189 197 200-201
206-208
210 227 241
fetal brainInvitrogen FBT002 9 34 36-38 81 102 127 147
197 207
fetal heartInvitrogen FHR001 4 7-8 10 13-14 21 23 27
34 36-38 43 48
60-61 73 78-79 98-101 104
120 122 126-
127 129 133 143-144 155
160 163 185-
186 197 202 217-218 225
231
fetal kidneClontech FKD001 6 23 66 81 146
fetal kidneyClontech FKD002 19 26 42 60 78-79 92 102
127-130 140
143 182 186 189 202 212
220
fetal kidneInvitro FKD007 122 189
en
fetal liverClontech FLV002 2 11 42 133 173-175 180
224 226
fetal liverClontech FLV004 2 11 3S-38 40 48 98 118
x27 133 136
148 162 173-175 179 182
186 189 x96-
197 202 225
fetal liverInvitrogen FLV001 23 31 42 70 75 122 133
140 172-175
178-179 205
fetal liver-Columbia FLS041 1-13 17 20-21 23 25 30
39 41-43 48 61
spleen ~ University~ 63-64 75-76 79 85 90 95
98-99 102 108
115 120 I27 I 30 133-144
146-147 154
805A/PCT
CA 02453344 2004-O1-21
11'7
TahlP l
Tissue RNA source Librar Name ~ SE ID N~:
on in
173-175 178-179 182 185
187 189 196-
197 201-203 205 210 212
218 224-226
241
fetal liver-Columbia FLS002 1 4-5 8 11 13 17-18 20-25
32 39 41-42
spleen University 48 51 56 63-64 79 90-91
95 98 102 118
130 136 143-144 146 171
173-175 178-
179 182 185-187 193 197
203 214 218
225-226 230 238 243
fetal liver-Columbia _ 1 3 9 13 21 43 50 61 66
FLS003 90 95 98 115
spleen University 122 130 136 173-175 187-188
196 202-
203 218 225 241
fetal lun Clontech FLG001 6 8 32 35 62 122 129 197
211 215
fetal lungInvitrogen FLG003 10 39-40 69 83 98 102
126 135 178 183
189 199 202 224 242
fetal muscleInvitrogen FMS001 4 8 10 2127 33 49 102
122 130 133 164
166-169 192 244
fetal muscleInvitrogen FMS002 7-8 10 13 23 26 33 42
49 61-62 76 86 98
118 122 124 129-130 140-142
164 166-
169 192 204 207 244
fetal skinInvitrogen FSK001 1 4 9-10 12-13 27 36-38
48 50 61-62 64
71 73-75 80-81 87-91 94
99 107 122 130
132-134 140-142 156 163
166-170 172
178-179 181 184 189 196-197
202 212
214 221 227 231 238 241
fetal skinInvitrogen FSK002 10 14 22-23 25 39 48 88-91
98 100-101
104 106 108 115 117-118
120 122 124
130 133 160 176 180 182
186 197 207
225 232 242
fibroblastStratagene LFB001 1 4 8 12-13 30 66 81 117
133 140 144
211 215 238
Genomic Research BAC002 80
DNA-from- Genetics
(CITB
BAC-393I6 BAC libr
)
Genomic Genomic BAC003 80
DNA
DNA-from- from Genetic
BAC-393I6 Research
induced Stratagene NTD001 1 22 30 32 42 84 117 125
144 206 222
neuron-cells
infant Columbia IB2002 4 9-10 15 22-23 33 43
brain 48-49 55 63 67 73
University 75 81 85 90 99 102 120
122 124 135-136
141-142 145-148 151 155
157-158 162
171 182 189 197 200 206-207
224-225
233 238-239
infant Columbia IB2003 7 10 12 22 47 49 53-54
brain 61 75 84-85 90
University 94-95 102 122 133 135
141-i42 147 176
189 204 207 231 239 241
infant Columbia IBM002 12 157-158 224 I
brain
Universi
infant Columbia IBS001 6 10 33 108 135 233
brain
Universi
leukoc Clontech LUC003 14 27 81 146 197 20I 204
to 242
leukocyte GIBCO LUC001 1-2 4 7-8 12-14 17 21-22
25-29 36-38 43
50 61 63-64 71 75 81 88-90
94 98 102
104 118 120 127 130 134
140 146-147
170 186 189 195 197 200-201
205-207
224-225 233 240
OQStiITCT CA 02453344 2004-O1-21
11~
Table 1
Tissue RNAsource Library Name SEQ ID NO:
on in
lung tumorInvitrogen LGT002 1 3-4 12 15 39 42-43 48
50-51 62-63 66
69 75 81 83-84 95 102
116 123 125 137-
139 144 146 178-179 181
185 189 195-
197 204 209-212 216 218
222 224-225
227 231 238 240-242
I m h nodeClontech ALN001 8 13 18 25 35-38
lymphocytesATCC LPC001 4 14 21 25-27 35 46 48
53-54 66 68-69
75 81 88-89 102 104 110
118 122 129-
130 133 145 171 176-177
189 195-196
201 207 212 225-226
macro ha Invitro HMP001 1 12 25 140 144 181 194
a en 197
mammary Invitrogen MMG001 4 6 9 12-13 25 27 31 33-34
36-39 42 48
gland 51 53-56 60-62 70 72 75
81 85 88-89 94-
95 102 119 122 127 129
131-133 140
144 14.6 156 163 172 176
178-179 181
185 189 192 205-206 210
218 224 231
236 238 240-243
melanoma Clontech MEL004 4 52 81 130 133 143 146
186 194 196
from-cell- 200 212 218
line-ATCC-
#CRL-1424
mix of various CTL016 71 122 207
16 vendors
tissues-
mRNAs
mix of various CTL021 189
16 vendors
tissues-
mRNAs
mix B/I/C SUP005 1136173-175181185
mix I B/I/C SUP008 48 55 122 130 173-176
189
mix B/I/C SUP009 1 140 173-175 189
mixed EST clones CGd010 7 20 31 116-I 19 132 150
181 222 236
239
neuronal Stratagene NTU001 3-4 15 60 63 75 120 122
cells 133 140 171
181 196 206 210
pituitary Clontech PIT004 13 20 33 43 66 74 90 123
130 217
land
placenta Clontech PLA003 4-5 7 25 36-39 56 93 100-101
103-104
120 122 134 186-187 189
231
lacenta Invitro APL002 41 75 224
en
prostate Clontech PRT001 20 26 34 62 72 81 143
166-169 178-179
197 202 218 241
rectum Invitrogen REC001 3 25 33 51 74 88-89 122
129 133 155
189 197 224 241 243
retinoic Stratagene NTR001 1 3 34 104 124 129 140
acid- 225 241
induced-
neuronal-cells
saliva Clontech SALs03 179
land
saliva Clontech SAL001 1 18 34 69 71 120 179
land 204 214 235-236
skeletal Clontech SKM001 7 42 49 73 75 102 130
143 223
muscle
small Clontech SIN001 1 4 7-8 10 12 20-22 26
32 36-38 43 48
intestine 51 61 68 71 75 86 91-92
96-99 118 122
124-125 130 133-134 140
143-144 148
160 185 189 196 201 210
212 224 229
234 241
805A/PCT ~ 02453344 2004-O1-21
119
Table 1
Tissue RNA source Librar Name SEA II;i NO:
on in
spinal Clontech SPC001 2 8 13 24 26 35 43 63-64
cord 127-128 130
135 156 178 185 189 196
206 210
stomach Clontech STO001 32 99 143 161 172 189
thalamus Clontech THA002 7 10-11 60 79 98 127 131
136 143 153
183 186 190 206-207 212
227
thymus Clontech THM001 1 14 26 30 46 50 74 79
98 115 118 130
154 196-197 217 222 224
233
thymus Clontech THMc02 I-2 4 10 13 24-25 30 32
36-39 48 61 64-
65 73-74 76-77 79 82 88-89
100-101
105-106 115 118 I20 122
127 130 133
140 146 171 183 185 199
202 206 224-
225 231 237 242
thyroid Clontech THR001 1 4-5 7-8 20-21 24 26
gland 31 43 49 53-54 64
66 70 73 75 81 90 92 98
110 120 124
130 133 140 143-144 162-163
172 189
197 199 201 206-208 212-213
219-220
224 236 238 241-242
trachea Clontech TRC001 4-5 40 48 62 111 144 146
179 200 226
umbilical BioChain FUC001 6 12 18 32 36-38 40 61
70-71 75 84 94-
cord 95 98-99 118 122 127 140
144 156 163
180 184 I 89 205 210 215
241
uterus Clontech UTR001 10 12-13 21 71 130 134
140 144 208 210
213 218
young liverGIBCO ALV001 1 24 27 48 73 85 136 173-175
178-179
189 196-197 201
*The 16 tissue-mRNAs and their vendor source are ass follows: 1) Normal adult
brain
mRNA (Invitrogen), 2) normal kidney mRNA (Invitrogen), 3) normal adult liver
mRNA
(Invitrogen), 4) normal fetal brain mRNA (Invitrogen), 5) normal fetal kidney
mRNA
(Invitrogen), 6) normal fetal liver mRNA (Invitrogen), 7) normal fetal skin
mRIVA
(Invitrogen), 8) human adrenal gland mRNA (Clontech), 9) human bone marrow
mRNA
(Clontech), 10) human leukemia lymphoblastic mRNA (Clontech), 11) human thymus
mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human spinal cord
mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA
(BioChain), 16) human conceptional umbilical cord mRNA (BioChain).
805A/PCT ~ 02453344 2004-O1-21
120
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
245 AAW78199 Homo SapiensHUMA- Human secreted 294899
protein
encoded b Qene 74 clone
HGBAC 11.
245 114915787Mus musculusWAC 291294
245 g111139753Homo SapiensbA48B24.1 (A novel protein280599
containing
a formin binding protein
(FBP28)
domain
246 AAM43622 Homo sapiensHUMA- Human polypeptide 890 99
SEQ ID
NO 300.
246 g118766553Caenorhabditisubiquitin c-terminal 202 25
hydrolase
ele ans
246 AAM71031 Homo SapiensMOLE- Human bone marrow 199 100
expressed
probe encoded protein
SEQ ID NO:
31337.
247 AAM93769 Homo sapiensHELI- Human polypeptide,341299
SEQ ID
NO: 3772.
247 g113559026Homo SapiensbG174L6.2 (MSF: megakaryocyte221 21
stimulatin factor
247 AAB29773 Homo SapiensRHOD- Human megakaryocyte220 21
stimulating factor (MSF),
SEQ ID
NO:1.
248 111967711Homo sa Tsa24 rotein 10136 99
iens
248 1642252 Mus musculusis 24 943792
248 AAB95540 Homo SapiensHELI- Hurnan protein 743999
sequence SEQ ID
N0:18147.
249 115823648Homo sa ALS2CR9 296991
fens
249 118605620Mus museulussimilar to roline-rich 118843
protein 48
249 AAG74705 Homo sapiensHUMA- Human colon cancer929 96
antigen
rotein SEQ ID N0:5469.
250 AAU74823 Homo sa INCY- Human REPTR 6 rotein.378510
iens
250 g113623799Homo sapiensseven-transmembrane receptor378510
Frizzled-
8
250 g110334640Homo sapiensbA425A6.1 (frizzled (Drosophila)378510
homolo 8
251 AAB95372 Homo SapiensHELI- Human protein sequence338999
SEQ ID
N0:17692.
251 g110176983ArabidopsisGTP-binding membrane 183858
protein LepA
thaliana homolog
251 g120515955ThermoanaeroMembrane GTPase LepA 168052
bacter
ten con
ensis
252 AAB58241 Homo SapiensROSE/ Lung cancer associated825 90
of a tide se uence SEQ
ID 579.
252 11780755 Homo sa DJ-1 rotein 825 90
iens
252 116751471Homo sa DJ-1 825 90
iens
253 g121428404DrosophilaLD05365p 227 27
melanoaaster
253 g17190399Chlamydia phospholipase D family 180 31
protein
muridarum
253 g12313422Helicobactermembrane bound endonuclease171 30
(nuc)
Lori 26695
254 11869810 Homo sa SH3-containin Grb-2-like186695
iens 1
254 16120106 Homo sa SH3-containing rotein 186695
iens EEN
254 1 12654853Homo sa SH3-domain GRB2-like 186695
iens 1
255 AAY51529 Homo sa INNO- Human al ha-s nuclein579 86
iens rotein.
255 AAW88131 Homo sa USSH Human al ha s nucleic579 86
iens rotein.
CA 02453344 2004-O1-21
121
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identi
255 AAY07277 Homo Sa UYSA- Human al ha-s nuclein.579 86
iens
256 Qi14587851Homo sa Graft 327899
iens
256 113310137Mus musculusPSGAP-m 293089
256 113310135Mus musculusPSGAP-s 293089
257 AAB58241 Homo SapiensROSE/ Lung cancer associated728 82
oly a tide se uence SEQ
I_D 579.
257 g116751469CercopithecusDJ-1 728 82
aethio s
257 12460318 Homo sa RNA-bindin rotein re 728 82
iens ulator subunit
258 AAM50136 Homo sapiensMILL- Human GTPase activating214510
molecule GAP-5.
258 15020264 Mus musculusCdcA.2 GTPase-activatin 919 48
rotein
258 AAE13842 Homo SapiensCORI- Human lung tumour-specific774 50
rotein 20129.
259 112005724Homo sa mixed linea a kinase 561810
iens MLK1
259 AAE11775 Homo Sa INCY- Human kinase (PKIN)-9530392
iens rotein.
259 g121410177Mus musculusSimilar to mitogen-activated360790
protein
kinase kinase kinase
9
260 AAR85092 Homo SapiensAMGE- EPH-like receptor 489393
protein
tyrosine kinase HEK11.
260 g1551608 Homo sa rece for rotein-tyrosine489393
iens kinase
260 g1755568 Rattus Ehk-3, full length form 482492
norve icus
261 AAQ13290_Homo SapiensUYSF- Leukocyte derived 618 96
growth
aal factor ene.
261 AAB15804 Homo SapiensNEOR- Human chemokine 618 96
PF4 SEQ ID
NO: 46.
261 AAW96716 Homo Sa UNMI A latelet basic 618 96
iens rotein (PBP).
262 AAM00862 Homo SapiensNYSE- Human bone marrow 971 93
protein,
SEQ ID NO: 225.
262 AAM00975 Homo SapiensNYSE- Human bone marrow 827 100
protein,
SEQ ID NO: 451.
262 AAU77835 Homo SapiensMERE Human N-terminal 815 91
acetyl
transferase HUTUDO1).
263 AAG01995 Homo SapiensGEST Human secreted protein,523 100
SEQ ID
NO: 6076.
263 AAU19731 Homo SapiensHUMA- Human novel extracellular439 89
matrix rotein, Se ID
No 381.
263 AAU19744 Homo SapiensHUMA- Human novel extracellular296 95
matrix rotein, Se ID
No 394.
264 AAY44988 Homo sa INCY- Human a idermal 162194
iens rotein-5.
264 114249975Homo sa Similar to Sh3 domain 162194
iens YSC-like 1
264 11944389 Mus musculusSh3 I1 151187
265 g1 19916191Methanosarcinkinesin light chain 508 36
a acetivorans~
str. C2A]
[Methanosarci
na acetivorans
C2A
265 g120907522Methanosarcintetratricopeptide repeat479 36
family protein
a mazei
Goel
265 g12645229Plectonema kinesin light chain 445 37
bo anum
266 AAB94232 Homo SapiensHELI- Human protein sequence307599
SEQ ID
N0:14606.
CA 02453344 2004-O1-21
122
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
266 AAY83079 Homo sa UYNY F-box rotein FBP-11.661 99
iens
266 16164741 Homo sa F-box rotein Fbxl 1 661 99
iens
267 118676718Homo sa FLJ002S8 rotein 393398
iens
267 1 10441465Homo sa actin filament associated152143
iens rotein
2 113129531Gallus actin filament-associated148642
67 anus rotein
_ AAB93991 Homo SapiensHELI- Human protein sequence212099
268 SEQ ID
N0:14091.
268 AAU 17462Homo SapiensHUMA- Novel signal transduction211999
athwa rotein, Se ID 1027.
268 AAU17440 Homo SapiensHUMA- Novel signal transduction204099
athwa rotein, Se ID 1005.
269 ABB50205 Homo sapiensINCY- Human transcription124610
factor
TRFX-56.
269 g117862100DrosophilaLD08718p 723 52
melanoaaster
269 AAM00911 Homo SapiensNYSE- Human bone marrow 472 54
protein,
SEQ ID NO: 387.
270 AAG80184 Homo SapiensWIRT/ Human MEK kinase 698399
MEKK1
rotein fra ent.
270 AAB60291 Homo sa ISIS- Human MEKK1. 698399
iens
270 12815888 Homo sa MEK kinase 1 698399
iens
271 13002590 Homo sa interleukin-1 rece tor-associated350489
iens kinase
271 AAW14306 Homo SapiensT'LTLA- Interleukin-1 349089
receptor-
associated rotein kinase.
271 11220313 Homo sa interleukin-1 rece tor-associated349089
iens kinase
272 AAY97638 Homo sa UNMI A of 1 WD rotein 577788
iens se uence.
272 g15051670Homo Sapiensapoptotic protease activating577788
factor-1
lon isoform APAF-1L
272 AAY97641 Homo SapiensUNMI Apaf 1XL-LlOA protein575587
se uence.
273 AAY97638 Homo sa UNMI A of 1 WD rotein 584788
iens se uence.
273 g15051670Homo Sapiensapoptotic protease activating584788
factor-1
long isoform APAF-1L
_
273 AAY97636 Homo sa UNMI A of 1XL rotein 582588
iens se uence.
274 AAY52186 Homo sapiensCURA- Human enhancer 433 82
of
rudimentary gene (ERH)
amino acid
se uence.
274 AAG03943 Homo SapiensGEST Human secreted protein,433 82
SEQ ID
NO: 8024.
274 g11374695Homo Sapienshuman protein homologous433 82
to DROER
rotein
275 11657835 Mus musculusRho- uanine nucleotide 253180
exchan a factor
275 ABB44551 Homo sapiensSWIT- Human wound healing928 42
related
of a tide SEQ ID NO 8.
275 g15199316Homo Sapiensnon-ocogenic Rho GTPase-specific690 35
GTP exchan a factor
276 g18388704Leishmaniaprobable CG14353 protein678 43
!
ma' or
276 AAG03317 Homo sapiensGEST Human secreted protein,285 98
SEQ ID
_ NO: 7398.
2?6 g115291701DrosophilaLD24014p 115 24
melano
aster
277 AAH78272_Homo SapiensMILL- Coding sequence 252910
of human
aal GTPase activator rotein ~
26651.
CA 02453344 2004-O1-21
123
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number _ identi
277 AAG67550 Homo SapiensMILL- Amino acid sequence252910
of human
GTPase activator rotein
26651.
277 AAH78271_Homo SapiensMILL- Nucleotide sequence252910
of human
gal GTPase activator protein
26651.
278 114488252_ ras-like roteinlVTS58635 108210
Homo sa
iens
278 ABB06136 Homo SapiensCOMP- Human NS protein 681 99
sequence
SEQ ID N0:228.
278 11666073 Homo sa RRP22 rotein 501 51
iens
279 119387193Homo sa StAR-related 1i id transfer111110
iens rotein 4
279 119387189Mus musculusStAR-related 1i id transfer983 86
rotein 4
279 g113542895Mus musculusSimilar to RIKEN cDNA 979 86
2310058622
ene
280 AAB94355 Homo SapiensHELI- Human protein sequence426399
SEQ ID
N0:14877,
280 120372683Homo sa euchromatic histone meth 416399
iens ltransferase 1
280 114211561Homo sa GLP1 237810
iens
281 AAB94355 Homo sapiensHELI- Human protein Sequence393694
SEQ ID
N0:14877.
281 120372683Homo sa euchromatic histone meth 383694
iens Itransferase 1
281 114211561Homo SapiensGLP1 204489
282 AAB94355 Homo SapiensHELI- Human protein sequence410496
SEQ ID
N0:14877.
282 120372683Homo sa euchromatic histone meth 400496
iens Itransferase 1
282 114211561Homo sa GLP1 221994
iens
283 AAB64405 Homo sapiensINCY- Arnino acid sequence204399
of human
intracellular signalling
molecule
INTRA37.
283 g115530218Homo sapiensSimilar to RIKEN cDNA 204399
1300006M19
ene
283 115930031Mus musculusRIKEN cDNA 1300006M19 176785
ene
284 AAW88399 Homo sapiensGEMY Human testis secreted260599
protein
1
dx290 1.
284 g15281051Arabidopsisstress-induced protein 162 28
stil-like protein
thaliana
284 1872116 Gl cine sti (stress inducible 159 26
max rotein)
285 17363368 Mus musculusinhibito ada ter molecule178777
DOK3
285 16492338 Mus musculusada for rotein; DOKL 178777
285 13043919 Homo sa docking rotein 501 37
iens
286 AAM78693 Homo SapiensHYSE- Human protein SEQ 246297
ID NO
1355.
286 i36619Ho rno sa serine/threonine rotein 246297
iens kinase
286 115990456Homo sa PCTAIRE rotein kinase 246297
iens 1
287 AAB64420 Homo SapiensINCY- Amino acid sequence225510
of human
intracellular signalling
molecule
INTRA52.
287 a121411454Mus musculusRIKEN cDNA 4833427E09 19
gene 1685
287 g112484136Rattus SMHS2 _
190384
norve ices
288 AAG67823 Homo SapiensSHAN- Human guanine-nucleotide909 58
releasin factor 52 protein.
288 ABB04984 Homo SapiensMERE Human new ras guanine-908 58
nucleotide-exchange factor
1 SEQ ID
N0:2.
288 g118490322Homo sapiensSimilar to RIKEN cDNA 681 50
6330404M18
ene
ODStI/rCT CA 02453344 2004-O1-21
124
Table 2
SEQ AccessionSpecies Description Score
ID
N~: Number identit
289 ai10801596Mus musculusDoc2 aroma 109878
289 AAW25032 Homo SapiensSHIO Human Doc2-beta colon601 44
cancer
rotein.
289 gi1235722Homo sa Doc2 beta 601 44
iens
290 ABB04984 Homo sapiensMERE Human new ras guanine-170110
nucleotide-exchange factor
1 SEQ ID
N0:2.
290 AAG67823 Homo SapiensSHAN- Human guanine-nucleotide168799
releasin factor 52 rotein.
290 gi18490322Homo SapiensSimilar to RIKEN cDNA 111964
6330404M18
ene
291 AAM40118 Homo SapiensHYSE- Human polypeptide 171070
SEQ ID NO
3263.
291 AAM41904 Homo SapiensNYSE- Human polypeptide 100180
SEQ ID NO
6835.
291 gi4099012Dictyosteliumdrainin 722 39
discoideum
292 AAB34844 Homo SapiensHUMA- Human secreted protein843 84
sequence encoded by gene
44 S:EQ ID
N0:132.
292 1550060 Homo sa GTP-bindin rotein 843 84
iens
292 16969622 Mus musculussmall GTP-bindin rotein 843 84
RAB1A
293 AAH78263~Homo sapiensMILL- Nucleotide sequence310610
of human
aal kinase 14760.
293 1 13194657Homo sa skeletal m osin 1i t chain310610
iens kinase
293 g118073328Homo Sapiensskeletal muscle-specific 310610
myosin light
chain kinase
294 13599940 Mus musculusfacio enital dys lasia 254582
rotein 2
294 g13342246Rattus actin-filament binding 153352
protein Frabin
norve ices
294 115705415Mus musculusactin-bindin rotein frabin-al152654
ha
295 a13851202Homo sa ZO-3 482998
iens
295 g13033501Canis ZO-3 385783
familiaris
295 115214772Mus musculusSimilar to ti ht 'unction368180
rotein 3
296 AAB95184 Homo SapiensHELI- Human protein sequence285499
SEQ ID
N0:17254.
296 ABB03717 Homo SapiensHUMA- Human rnusculoskeletal422 98
system related polypeptide
SEQ ID NO
1664.
296 g13108057Mus musculuschannel interactin PDZ 138 27
domain rotein
297 AAB48307 Homo sa UYYA Human ZF26 rotein. 227479
iens
297 AAY83085 Homo sa UYNY F-box rotein FBP-17.227479
iens
297 110764488Homo sa dactylin 221710
iens
298 AAB48307 Homo sa UYYA Human ZF26 ratein. 211475
iens
298 AAY83085 Homo sa UYNY F-box rotein FBP-17.211475
iens
298 g110764488Homo Sapiensd 200092
actylin
299 AAY97293 Homo Sapiens_ 227578
INCY- Lipid associated
protein
(LIPAP 3335404CD1.
299 15670328 Homo sa co ine III 131749
iens
299 AAM39997 Homo sapiensHYSE- Human polypeptide _
SEQ ID NO 131648
3142.
300 AAE09604 Homo sapiensHUMA- Human gene 12 encoded423 97
novel
rotein HE9RA75, SEQ ID
N0:40.
300 110178646H dra vuI dishevelled 193 48
aris
805A/PCT ~ 02453344 2004-O1-21
125
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number _ identit
300 AAB68347 Homo Sapiens_ 190 41
REGC Amino acid sequence
of
dishevelled (dsh) rotein.
301 AAM41632 Homo sapiensHYSE- Human polypeptide 235610
SEQ ID NO
6563.
301 AAM39846 Homo SapiensHYSE- Human polypeptide 227110
SEQ ID NO
2991.
301 AAW90868 Homo sa GEHO Human d sferlin 113 33
iens roteiii.
302 i51144Musmusculus h2-cal onin 102576
302 117391137Mus musculuscal onin 2 102576
302 a11962 Sus scrofah2-cal onin 102476
303 g1 15082283Homo sapiensSimilar to small glutamine-rich153910
tetratricopeptide repeat
(TPR)-
containin
303 g12909372Homo Sapienssmall glutamine-rich 854 58
tetratricopeptide
(SGT)
303 g14539082Homo Sapienssmall glutamine-rich 854 58
tetratricopeptide
re eat containin rotein
304 111345052Homo sa SM-20 231410
iens
304 114547146Homo sa EGLN1 rotein 231410
iens
304 114547239Mus musculusEGLN1 rotein 156378
305 AAU16939 Homo sapiensHUMA- Human novel secreted161099
protein,
SEQ ID 180.
305 ABB10234 Homo SapiensHUMA- Human cDNA SEQ 161099
ID NO:
542.
305 111527997Homo sa NOTCH2 rotein 1483
iens 99
306 AAM48953 Homo SapiensCHIR Human colon cancer __ 100
related 776
rotein SEQ ID NO: 4.
306 AAG02355 Homo SapiensGEST Human secreted protein,597 96
SEQ ID
NO: 6436.
306 AAU20658 Homo SapiensHUMA- Human secreted 184 43
protein, Seq
ID No 650.
307 AAM78475 Homo sapiensHYSE- Human protein SEQ 589895
ID NO
1137.
307 AAM70260 Homo sapiensMOLE- Human bone marrow 369299
expressed
probe encoded protein
SEQ ID NO:
30566.
307 AAM57843 Homo SapiensMOLE- Human brain expressed369299
single
exon probe encoded protein
SEQ ID
NO: 29948.
308 AAB93852 Homo sapiensHELI- Human protein sequence205399
SEQ ID
NO:1
3705.
308 AAM39549 Homo sapiens_ 205399
HYSE- Human polypeptide
SEQ ID NO
2694.
308 AAG64490 Homo SapiensSHAN- Human lissencephaly204399
protein
43.
309 AAT61456_aHomo SapiensUYJE- C-proteinase clone383699
pCP-1.
al
309 ABB90755 Homo SapiensUYJO Human Tumour Endothelial383699
Marker of a tide SEQ
ID NO 242.
309 AAW13670 Homo SapiensUYJE- C-proteinase encoded383699
by clone
CP-2.
310 AAB93117 Homo SapiensHELI- Human protein sequence197899
SEQ ID
N0:11991.
310 AAB94167 Homo sapiensHELI- Human protein sequence913 100
SEQ ID
N0:14468.
pnG p mCT CA 02453344 2004-O1-21
1z6
Table 2
SEQ Accession Species Description Score
ID
NO: Number identit
310 g16650370 Dictyosteliumrac serine/threonine 113 29
kinase homolog
discoideum
311 AAB65650 Homo SapiensSUGE- Novel protein kinase,201692
SEQ ID
NO: 177.
311 15139689 Homo sa MOK rotein kinase 201692
iens
311 15139691 Mus musculusMOK rotein kinase 166077
312 AAE04546 Homo sapiensINCY- Human G-protein 457599
coupled
rece tor-2 GCREC-2) rotein.
312 g119387136Homo SapiensPYRIN-containing APAFI-like456399
protein
5
312 AAU00023 Homo SapiensBIOJ Human activated 416591
T-lymphocyte
associated se uence 2,
ATLAS-2.
313 AAE18955 Homo sapiensINCY- Human cell cyc3e 155899
' protein and
mitosis-associated molecule
(CCPMAM-3).
313 AAB95737 Homo SapiensHELI- Human protein sequence654 100
SEQ ID
N0:18627.
313 g115722097Homo SapiensbA690P14.1 (novel cyclin463 39
(contains
FLJ10895)
314 AAG67393 Homo SapiensSUGE- Amino acid sequence357599
of human
rotein kinase SGK223.
314 AAG67394 Homo SapiensSUGE- Amino acid sequence104940
of human
rotein kinase SGK269.
314 AAM25743 Homo sapiensHYSE- Human protein sequence100199
SEQ
ID N0:1258.
315 g13327808 Homo Sapienslatent transforming growth804593
factor-beta
bindin rotein 4S
315 12190402 Homo sa latent TGf'-beta bindin 777489
iens rotein-4
315 g13327814 Homo Sapienslatent transforming growth387199
factor-beta
bindin rotein 4
316 16624055 Homo sa similar to rin motif 683 100
iens ; note:
316 11655418 Homo sa an rin motif 551 98
iens
316 AAB92646 Homo SapiensHELI- Human protein sequence497 50
SEQ ID
N0:10979.
317 AAM40636 Homo sapiensHYSE- Human polypeptide 231999
SEQ ID NO
5567.
317 g114307916Mus musculusmyosin phosphatase targeting221782
subunit 3
MYPT3
317 AAM38850 Homo SapiensHYSE- Human polypeptide 215910
SEQ ID NO
1995.
318 1 19343951Homo sa Similar to GTP-rho bindin347099
iens rotein 1
318 119697913 Homo sa rho hilin-1 343796
iens
318 11176422 Mus musculusrho hilin 234171
319 117389232 Homo sa block of roliferation 397410
iens 1
319 113938293 Homo sa Similar to block of roliferation390910
iens 1
319 116416407 Homo sa KM-PA-2 rotein 338910
iens
320 AAU78329 Homo sapiensMILL- Tetratricopeptide 207299
repeat
containin meth ltransferase
TPR
320 AAB56481 Homo sapiensROSE/ Human prostate 769 97
cancer antigen
rotein se uence SEQ ID
N0:1059.
_
320 ABB89399 Homo SapiensHUMA- Human polypeptide 473 95
SEQ ID
NO 1775.
321 g113279080Homo SapiensSimilar to protein interacting206710
with
uanine nucleotide exchan
a factor
~05~CT CA 02453344 2004-O1-21
s ~ 127
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number __ identit
321 g12459833Rattus Maxpl 169186
norve 'cus
321 117386088Homo sa ras effector-like rotein 968 98
iens
322 115529066Homo sa sortin nexin 27 129597
iens
322 AAB45187 Homo sapiensHUMA- Human secreted protein874 100
sequence encoded by gene
15 SEQ ID
N0:128.
322 AAM41948 Homo sapiensHYSE- Human polypeptide 534 94
SEQ ID NO
6879.
323 AAG67293 Homo sapiensLEXI- Amino acid sequence583510
of a human
rotein.
323 g110764778Homo Sapiensphosphoinositol 3-phosphate-binding583510
rotein-2
323 AAB47871 Homo sa INCY- IS1G1'-I. 312210
iens
324 ABB11747 Homo sapiensHYSE- Human IL-1 delta 922 100
homologue,
SEQ ID N0:2117.
324 AAB 19923Horno sapiensHYSE- Human interleukin-1906 100
Hy2
(extended form, artial
se uence).
324 AAB19924 Homo SapiensHYSE- Human interleukin-1868 100
Hy2 (long
version .
325 AAU20396 Homo sapiensHUMA- Human secreted protein,916 100
Seq
ID No 388.
325 AAU20594 Homo sapiensHUMA- Human secreted protein,669 99
Seq
ID No 586.
325 ABB03519 Homo SapiensHUMA- Human musculoskeletal669 99
system related polypeptide
SEQ ID NO
1466.
326 AAB65609 Homo SapiensSUGE- Novel protein kinase,147610
SEQ ID
NO: 135.
326 AAU17266 Horno SapiensHUMA- Novel signal transduction976 98
athwa rotein, Se ID 831.
326 g120901962CaenorhabditisC. elegans CEH-20 protein485 43
ele ans comes ondin se uence F31E3.2d)
327 111596127Homo sa d nein axonemal intermediate318399
iens chain
327 111493148Homo sa intermediate d ein chain 317999
iens
327 g114189848Cynops dynein intermediate chain229069
rrho aster
328 ABB07495 Homo SapiensINCY- Human lipid metabolism901 80
molecule (LMM) polypeptide
(ID:
2970737CD 1 .
' 328 12565396 Mus m_usculusschwannoma-associated 355 39
r
otein
328 AAB90716 Homo sapiens_ 347 42
GEMY Human CI1480 9 protein
se uence SE ID 128.
329 AAB95777 Homo SapiensHELI- Human protein sequence233510
SEQ ID
N0:18722.
329 g11772658Rattus Srgl 207695
norve ices
329 115991284Mus musculuss na tota min XII 205895
330 g115779080Homo Sapiens_ 191110
Similar to RIKEN cDNA
4930513F16
ene
330 AAM95146 Homo SapiensHUMA- Human reproductive 445 83
system
related anti enSE ID NO:
3804..
330 AAG03274 Homo SapiensGEST Human secreted protein,424 100
SEQ ID
NO: 7355.
CA 02453344 2004-O1-21
128
Table 2
SEQ Accession Species Description Score
ID
NO: Number identit
331 gi21309836Trichinellaglutamic acid-rich protein272 22
cNBL1700
s iralis
331 gi9837385 Takifugu retinitis pigmentosa 249 21
GTPase regulator-
rubri es like rotein
331 gi16611639Encephalitozospore wall protein 2 247 21
precursor
on intestinalis
332 AAB65684 Homo sapiensSUGE- Novel protein kinase,113899
SEQ ID
NO: 212.
332 AAI66821 Homo SapiensMILL- Human protein kinase102698
a
al of a tide 13295 codin
se uence.
332 AAG65760 Homo SapiensMILL- Human protein kinase102698
of a tide 13295.
333 AAI66821 Homo SapiensMILL- Human protein kinase133210
a
al of a tide 13295 codin
se uence.
333 AAG65760 Homo sapiensMILL- Human protein kinase133210
of a tide 13295.
333 AAI66820_aHomo sapiensMILL- Human protein kinase133210
al of a tide 13295 encodin
cDNA.
334 AAB65663 Homo SapiensSUGE- Novel protein kinase,711399
SEQ ID
NO: 191.
334 AAG67800 Homo SapiensMILL- Amino acid sequence711399
of human
rotein kinase 14790.
334 AAH78649- Homo sapiensMILL- Nucleotide sequence710399
of human
aal rotein kinase 14790.
335 gi2352277 Homo sa MAP kinase kinase kinase809896
iens
335 gi1504010 Homo SapiensSimilar to Mouse TFIIi-associated779710
transactivator factor
pl7(GB RO:MMU11548):
Containing
ratein kinase motif
335 11932805 Mus musculusMEK kinase 4b 723489
336 118307483 Homo sa hos hoinositide-bindin 232310
iens roteins
336 g118700711Mus musculusdual-specificity Rho- 206590
and Arf GTPase
activatin rotein 1
336 ABB07500 Homo SapiensINCY- Human GTP-binding 847 40
protein
GTPB) (ID: 1299273CD1).
337 AAB65607 Homo SapiensSUGE- Novel protein kinase,390998
SEQ ID
NO: 133.
337 16088096 Homo sa rotein kinase PKNbeta 390998
iens
337 1914100 Homo sa rotein kinase PRK2 200650
iens
338 AAM25715 Homo SapiensNYSE- Human protein sequence358999
SEQ
ID N0:1230.
338 g16448792 Rattus activator of G-protein 151891
signaling 3
norve icus
338 118204662 Mus musculusSimilar to LGN rotein 574 43
339 AAB93093 Homo sapiensHELI- Human protein sequence258510
SEQ ID
N0:11941.
339 118307483 Homo sa hos hoinositide-bindin 258510
iens roteins
339 g118700711Mus musculusdual-specificity Rho- 227088
and Arf=GTPase
activatin rotein 1
340 AAG74346 Homo SapiensHUMA- Human colon cancer712 96
antigen
rotein SEQ ID NO:5110.
340 AAB38475 Homo SapiensHUMA- Fragment of human 504 36
secreted
protein encoded by gene
33 clone
HACBZ59.
340 112005908 Homo sa AD037 499 37
iens
805A/PCT ~ 02453344 2004-O1-21
129
Table 2
SEQ Accession Species Description Score
ID
NO: Number identit
341 g120071809Mus musculusSimilar to expressed 112687
sequence
AI840684
341 g113881165MycobacteriuLAO/AO transport system 783 50
kinase
m tuberculosis
CDC1551
341 g121324296CorynebacteriPutative periplasmic 735 45
protein kinase
um ArgK and related GTPases
of G3E
glutamicumfamily
ATCC 13032
342 120339623 Homo sa KRIT1 isoform 387210
iens
342 19998950 Homo sa an in re eat-containin 387210
iens rotein
342 20 Homo sa krev interaction tra 386499
44278 iens ed 1
11
343 _ Homo sapiensmembrane-associated guanylate471399
_ kinase-
g112003994
related MAGI-3
343 g110945428Homo Sapiensmembrane-associated guanylate465696
kinase
MAGI3
343 g112003992Mus musculusmembrane-associated guanylate445494
kinase-
related MAGI-3
344 AAB93844 Homo SapiensHELI- Human protein sequence102376
SEQ ID
NO;13683.
344 g117980216Drosophilarolling pebbles isoform 916 45
6
melano
aster
344 g117980214Drosophilarolling pebbles isoform 916 45
7
melanogaster
345 g117980216Drosophilarolling pebbles isoform 143247
6
melano
aster
345 g117980214Drosophilarolling pebbles isoform 143247
7
melano
aster
345 g116974692Drosophilarolling pebbles isoform 143247
6
melano
aster
346 g113436428Homo SapiensSimilar to feminization 287285
1 a homolog (C.
ele ans)
346 g13930525 Mus musculussex-determination protein258677
homolog
Fem 1 a
346 114318743 Mus musculusfeminization 1 a homolo 258677
(C. elegans
347 g112274842Homo sa bA157P1.1.1 laminin al 20092 100
iens ha 5)
347 120147503 Homo sa laminin al has chain 20032 99
iens recursor
347 a12599232 Mus musculuslaminin al ha 5 chain 15805 79
348 120269788 Homo sa PH domain containin rotein255310
iens
348 AAU 17064 Homo SapiensHUMA- Novel signal tra.nsduction251098
athwa rotein, Se ID 629.
348 AAU17496 Homo SapiensHUMA- Novel signal transduction897 99
athwa rotein, Se ID 1061.
349 ABA96187- Homo SapiensMERE Human hTSSK3 encoding141510
aal cDNA SE ID NO 1.
349 AAM47999 Homo sa MERE Human hTSSK3 SEQ 141510
iens ID NO 2.
349 AAE19154 Homo sapiensTHOR/ Human kinase polypeptide141510
(PKIN-12 .
350 AAB94849 Homo SapiensHELI- Human protein sequence183099
SEQ ID
N0:16030.
350 AAG01248 Homo SapiensGEST Human secreted protein,288 100
SEQ ID
NO: 5329.
350 AAG68345 Homo sapiensBODA- Human integrin 127 92
85 protein SEQ
ID N0:2.
805A/PCT
CA 02453344 2004-O1-21
130
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
351 gi21309836Trichinellaglutamic acid-rich protein272 22
cNBL1700
s iralis
351 gi9837385Takifugu retinitis pigmentosa 249 21
GTPase regulator-
rubri es like rotein
351 gi21309834Trichineilaglutamic acid-rich protein242 23
cNBL1500
s iralis
352 117940756Homo sa cask-interactin rotein 631110
iens 2
352 117940760Mus musculuscask-interactin rotein 559690
2
352 11794 Homo sa cask-interactin rotein 228342
0758 iens 1
353 _ DrosophilaLD29485p 113254
g116769404
melano
aster
353 19502080 Mus musculustubb su er-famil rotein 364 29
353 19502082 Homo sa tubb su er-famil rotein 363 28
iens
354 AAB65684 Homo SapiensSUGE- Novel protein kinase,151198
SEQ ID
NO: 212.
354 AAI66821 Homo sapiensMILL- Human protein kinase141198
a
al pol peptide 13295 codin
se uence.
354 AAG65760 Homo sapiensMILL- Human protein kinase141198
of a tide 13295.
355 AAB85425 Homo SapiensLEXI- Novel human membrane132110
protein
(NHP).
355 AAW05732 Homo SapiensUSSH Human metastasis 304 28
tumour
su ressor ene KAI1 roduct.
355 1258295 Homo sa C33 antigen 304 28
iens
356 g14689229Rattus b-tomosyn isoform 401963
norveaicus
356 g13790389Rattus m-tomosyn 399163
norve icus
356 g14689231Rattus s-tomosyn isoform 390862
norve icus
357 g119354084Mus musculusSimilar to myosin X 870 43
357 g11755049Bos taurusmyosin X 869 43
357 i6996SS8 Mus musculusm osin X 867 43
358 AAE17499 Homo sapiensINCY- Human secretion 312010
and trafficking
rotein-8 (SAT-8).
358 ABB05693 Homo SapiensGEHU- Human cell 312010
signaling/communication
protein clone
am 2 2013.
358 g114210270Rattus synaptotagmin 3 296595
norve ieus
359 AAM78959 Homo SapiensNYSE- Human protein SEQ 11167 99
ID NO
1621.
3S9 AAM79943 Homo SapiensNYSE- Human protein SEQ 11161 99
ID NO
3589.
359 AAM49177 Homo SapiensHELI- Human MAST205 431862
(hMAST205).
360 121070344Homo sa GAS2-related rotein isoform147598
iens beta
360 121070342Homo sa GAS2-related rotein isoform984 94
iens al ha
360 11666071 Homo sa GAR22 rotein 791 55
iens
361 AAB6S663 Homo sapiensSUGE- Novel protein kinase,854410
SEQ ID
NO: 191.
361 AAG67800 Homo sapiensMILL- Amino acid sequence854410
of human
rotein kinase 14790.
361 AAH78649-Homo SapiensMILL- Nucleotide sequence853499
of human
aal rotein kinase 14790.
805A/PCT ~ 02453344 2004-O1-21
131
Table 2
SEQ AccessionSpecies Description Score /~
ID
NO: Number identit
362 g120379563Homo SapiensSimilar to neural precursor314599
cell
expressed, developmentally
down-
reaulated ene 1
362 1286103 Mus musculusnedd-1 rotein 268984
362 AAG74568 Homo SapiensHUMA- Human colon cancer201798
antigen
rotein SEQ ID N0:5332.
363 117426439Homo sa bA445O16.1 (DXF34 123310
lens
363 114317890Gallus s indlin 117783
anus
363 AAU11882 Homo SapiensMDSP- Human Ndr-interacting110084
protein,
S indlin.
364 AAB56854 Homo SapiensROSEI Human prostate 981 99
cancer antigen
rotein se uence SE ID
N0:1432.
364 ABB90390 Homo SapiensHUMA- Human potypeptide 902 98
SEQ ID
NO 2766.
364 g12330663Schizosaccharcoronin-like protein 593 28
om ces
ombe
365 AAU76874 Homo sa MERE Human E hA full 253283
lens len th kinase.
365 118694546Homo sa Full len th kinase 253283
lens
365 AAE19158 Homo SapiensTHOR/Human kinase polypeptide247x80
PKIN-16).
366 g113603394Homo sapienstype VI collagen alpha 450699
2 chain
recursor
366 i49809Musmusculus al ha-2 colla en 421291
366 i62882Gallus callus a VI colla en subunit 326174
al hat
367 AAE14257 Homo sapiensLEXI- Human calcium-calmodulin243210
de endent rotein kinase.
367 AAEI6267 Homo sa INCY- Human kinase PKIN-13243210
lens rotein.
367 112830367Homo sa serine/threonine kinase 243210
lens 33
368 g117529995Homo Sapiensoxysterol-binding protein-Like491599
protein
OSBPL6
368 112382787Homo sa OSBP-related rotein 6; 491599
lens ORP6
368 AAB95255 Homo sapiensHELI- Human protein sequence379099
SEQ ID
N0:17425.
369 AAB64404 Homo sapiensINCY- Amino acid sequence447410
of human
intracellular signalling
molecule
INTRA36.
369 14539084 Homo sa GRIP1 rotein 436410
lens
369 ABB11493 Homo SapiensHYSE- Human GRIP1 protein427498
homolo ue, SEQ ID NO:1863.
370 g117980216Drosophilarolling pebbles isoform 216942
6
melano
aster
370 g117980214Drosophilarolling pebbles isoform 216942
7
melanogaster
370 g116974692Drosophilarolling pebbles isoform 216942
6
melanoaaster
371 113097174Homo sa Similar to CGI-130 rotein104710
lens
371 14929729 Homo sa CGI-130 rotein 815 82
lens
371 ABB89176 Homo SapiensHUMA- Human polypeptide 471 100
SEQ ID
NO 1552.
372 116945899Homo sa cortactin-bindin rotein 862710
lens 2
372 g117488611Takifugu Brain ankyrin 2 282740
rubri es
372 AAM54754 Homo SapiensMOLE- Human brain expressed282210
single
exon probe encoded protein
SEQ ID
NO: 26859.
805t-llrCT CA 02453344 2004-O1-21
132
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
373 112382789Homo sa OSBP-related rotein 7; 447310
iens ORP7
373 g117529991Homo Sapiensoxysterol-binding protein-like447099
protein
OSBPL7
373 g117529995Homo sapiensoxysterol-binding protein-like216849
protein
OSBPL6
374 117483737Homo sa ZFP106 981899
iens
374 111493840Homo sa zinc fin er rotein 106 981899
iens
374 13372657 Mus musculuszinc fin er rotein 106 732977
375 AAB99783 Homo SapiensBIOW- Human SHC protein 123410
43 protein
SEQ ID N0:2.
375 AAU17199 Homo sapiensHUMA- Novel signal transduction121898
athwa rotein, Se ID 764.
375 ABB16299 Homo SapiensHUMA- Human nervous system506 96
related
of a tide SEQ ID NO 4956.
376 AAE02777 Homo sapiensCURA- Human PRO-C-MG.72 473 49
protein
encoded by DNA-C-MG.72-1776
cDNA clone.
376 AAB90821 Homo SapiensNOJI/ Human shear stress-response467 48
rotein SEQ ID NO: 150.
376 114625021Homo sa MacGAP 467 48
iens
377 AAY14555 Homo SapiensINCY- Human NADH dehydrogenase229894
subunit 1 rotein.
377 g13337443Homo SapiensNADH-ubiquinone oxidoreductase229894
NDUFS2 subunit
377 g114250796Homo SapiensNADH dehydrogenase (ubiGuinone)229894
Fe-
S protein 2 (49kD) (NADH-coenzyme
Q reductase
378 g1550013 Homo sa ribosomal rotein L5 653 72
iens
378 g111640568Homo sa MSTP030 653 72
iens
378 g121483852E uus caballusribosomal protein L5 647 71
379 AAB94219 Homo SapiensHELI- Human protein sequence146510
SEQ ID
N0:14579.
379 AAG67148 Homo sapiensINCY- Amino acid sequence117310
of a
human en me.
379 g12853081ArabidopsisATP binding protein-like778 59
thaliana
380 AAP90342 Homo sa KAGA Human realbumin. 641 87
iens
380 138b999 Homo sa realbumin recursor 641 87
iens
380 Qi189584 Homo sa realbumin 641 87
iens
381 AAG932S6 Homo sa NISC- Human rotein HP10416.105099
iens
381 AAG89214 Homo SapiensGEST Human secreted protein,103498
SEQ ID
NO: 334.
381 AAM40797 Homo sapiensHYSE- Human polypeptide 377 44
SEQ ID NO
5728.
382 AAG93256 Homo sa NISC- Human rotein HP10416.828 83
iens
382 AAG89214 Homo SapiensGEST Human secreted protein,812 82
SEQ ID
NO: 334.
382 AAM40797 Homo SapiensHYSE- Human polypeptide 208 44
SEQ ID NO
5728.
383 AAG93256 Homo sa NISC- Human rotein HP10416.631 99
iens
383 AAG89214 Homo SapiensGEST Human secreted protein,615 98
SEQ ID
NO: 334.
383 115030010Mus musculusRIKEN cDNA 0610033H09 315 59
ene
384 112803105Homo sa nucleobindin 1 196486
iens
384 11144316 Homo sa nucleobindin 194285
iens
CA 02453344 2004-O1-21
805A/PCT
133
Table 2
EQ ID AccessionSpecies Description Score
NO: Number identit
384 AAR49667 Homo sa YOSH- Human nucleobindin.191184
iens
385 120987450Homo sa LOC146433 127499
iens
385 AAB44867 Homo SapiensHUMA- Human secreted 130 96
protein
encoded by gene 38.
385 11517914 Homo sa monocytic leukaemia zinc97 43
iens fm er protein
386 g120987450Homo sa LOC146433 111910
iens
386 AAB44867 Homo SapiensHUMA- Human secreted 130 96
protein
encoded b ene 38.
386 g1643447 Malus x S3-RNase precursor 74 24
domestica
387 g113661132Homo Sapiensnon-biotin containing 292310
~ subunit of 3-
meth Icroton 1 CoA carbo
lace
387 g110934059Homo Sapiensnon-biotin containing 2923100
subunit of 3-
meth lcrotonyl-CoA carbox
lase
387 g113925684Homo sapiens3-methylcrotonyl-CoA 2923100
carboxylase
subunit MCCB
388 AAY48524 Homo SapiensMETA- Human breast tumour-257 100
associated rotein 69.
389 AAG74912 Homo SapiensHUMA- Human colon cancer223 85
antigen
rotein SE ID N0:5676.
390 AAM79919 Homo SapiensHYSE- Human protein S.EQ177510
ID NO
3565.
390 AAM78935 Homo sapiensNYSE- Human protein SEQ 177510
ID NO
1597.
390 AAB50287 Homo SapiensUYNE- Human schizophrenia174910
related
rotein SE ID NO: 21.
391 AAB94276 Homo sapiensHELI- Human protein sequence107010
SEQ ID
N0:14703.
391 g13171934Mus musculusneuronal-STOP rotein 184 34
391 g11370291Rattus STOP protein 183 34
norve icus
392 120073109Mus musculusRIKEN cDNA 1110035L05 350 37
ene
392 AAG74306 Homo sapiensHUMA- Human colon cancer139 35
antigen
rotein SEQ ID N0:5070.
392 g19957242Canis progesterone receptor 120 30
familiaris
393 111386113Homo sa FKSG25 266410
iens
393 113548673Homo sa SCOT-t 265899
iens
393 120988313Homo sa 3-oxoacid CoA transferase264410
iens 2
394 ABB84989 Homo SapiensGETH Human PR09821 protein965 100
se uence SE ID N0:346.
394 AAY27573 Homo SapiensHUMA- Human secreted 331 46
protein
encoded by ene No. 7.
394 AAY31830 Homo SapiensGEMY Ltuman adult brain 328 54
secreted
rotein nh899 8.
395 ABB 14720Homo sapiensHUMA- Human nervous system811 94
related
of a tide SEQ ID NO 3377.
395 AAM64934 Homo sapiensMOLE- Human brain expressed177 100
single
exon probe encoded protein
SEQ ID
NO: 37039.
395 AAU74618 Homo sapiensUYCA- Oestrogen-regulated77 23
LIV-1
famil rotein AX083511
Hs.
396 AAM74834 Homo sapiensMOLE- Human bone marrow 500 93
expressed
probe encoded protein
SEQ ID NO:
35140.
~~S~CT CA 02453344 2004-O1-21
134
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
396 AAM62032 Homo sapiensMOLE- Human brain expressed500 93
single
exon probe encoded protein
SEQ ID
NO: 34137.
396 AAB90685 Homo SapiensI GEMY Human BR595 4 492 95
protein
se uence SEQ ID 51.
397 111094311Homo sa brain link rotein-I 186910
iens
397 AABI2304 Homo sapiensHUMA- Human secreted 185699
protein
encoded b ene 4 clone
HFXHC41.
397 g111094297Rattus brain link protein-1 170591
nerve icus
398 AAB94977 Homo SapiensHELI- Human protein sequence886 100
SEQ ID
N0:16558.
398 118558591Mus musculusvomeronasal rece for 82 22
V1RC19
398 gi~20822105~Mus musculussimilar to vomeronasal 82 22
1 receptor, C3
ref~XP_1447
04.1
399 AAE03765 Homo SapiensHUMA- Human gene 2 encoded408110
secreted protein HCE3C63,
SEQ ID
N0:35.
399 g120988899Mus musculussimilar to deleted in 400997
bladder cancer
chromosome re ion candidate
1
399 AAV83819_Homo sapiensCURI- Tumour suppresser 213953
gene
aal IB3089A (also known as
DBCCRl).
400 AAG03S76 Homo SapiensGEST Human secreted protein,152 81
SEQ ID
NO: 7657.
400 AA006224 Homo SapiensNYSE- Human polypeptide 97 42
SEQ ID NO
20116.
400 AAW76734 Homo SapiensKIRI Human mDia Rho targeting92 51
rotein.
401 AAB74696 Homo SapiensINCY- Human membrane 240510
associated
rotein MEMAP-2.
401 1 15420832Homo sa NOE3-3 240510
iens
401 118490927Homo sa olfactomedin 3 239299
iens
402 AAU12240 Homo sapiensGETH Human PR04399 polypeptide245996
se uence.
402 115420828Homo sa NOE3-1 245996
iens
402 119386930Mus musculuso timedin form B 244396
403 ABB 10359Homo sapiensHUMA- Human cDNA SEQ 122188
I:D NO:
667.
403 AAU18038 Homo SapiensHUMA- Human immunoglobulin116085
of a tide SEQ ID No 183.
403 AAU18085 Homo sapiensHUMA- Human immunogIobulin987 86
of a tide SE ID No 230.
404 119071209Homosa CD109 295196
iens
404 AAB 12127Homo sapiensPROT- Hydrophobic domain294596
protein
isolated from HT-1080
cells.
404 g120070080Mus musculusGPI-anchored alpha-2 209967
macroglobulin-
related rotein
405 AAB94142 Homo sapiensHELI- Human protein sequence328910
SEQ ID
N0:14414.
405 118676648Homo sa FLJ00223 rotein 253999
iens
405 gi590I808DrosophilaBcDNA.GH03694 111641
melano
aster
406 114039834Homo sa elon ation factor G1 136610
iens
406 114285174Homo sa elon ation factor G 136610
iens
805A/PCT
CA 02453344 2004-O1-21
13.5
Tahle 2
SEQ AccessionSpecies Description Score
ID
N~: Number identit
406 gi310102 Rattus elongation factor G 121589
norve ices
407 AAM25864 Homo sapiensHYSE- Human protein sequence146299
SEQ
ID NO:1379.
407 AAB88448 Homo sapiensHELI- Human membrane 142194
or secretory
rotein clone PSEC0233.
407 AAY76143 Homo SapiensHUMA- Human secreted 138098
protein
encoded b ene 20.
408 110716074Mus musculusM83 rotein 309 33
408 Qi13543090Mus musculusSimilar to transmembrane309 33
rotein 6
408 ABB89854 Homo SapiensHUMA- Human polypeptide 290 31
SEQ ID
NO 2230.
409 g116877172Homo sapienscytochrome P450, subfamily281099
IVF,
oly epode 11
409 AAB85779 Homo sapiensINCY- Human drug metabolizing280599
en me (IDNo.6825202CD1).
409 110303605Homo sa CYP4Fl I 280499
iens
410 AAB27230 Homo sapiensINCY- Human EXMAD-8 SEQ 132683
ID NO:
8.
410 g118496364Oncorhynchusotolin-1 581 40
keta
410 118676606Homo sa FLJ0020I rotein 535 41
iens
411 AAB27230 Homo sapiensINCY- Human EXMAD-8 SEQ 149510
ID NO:
8.
411 g118496364Oncorhynchusotolin-1 518 42
keta
41 I g1687606 Lepomis saccular collagen 476 40
macrochirus
412 AAB27230 Homo sapiensdNCY- Human EXMAD-8 SEQ 158890
ID NO:
8.
412 g118496364Oncorhynchusotolin-1 725 46
keta
412 118676606Homo sa FLJ00201 rotein 613 41
iens
413 AAB27230 Homo SapiensINCY- Human EXMAD-8 SEQ 162698
ID NO:
8.
413 g118496364Oncorhynchusotolin-1 583 42
keta
413 118676606Homo sa FLJ00201 rotein 490 37
iens
414 AAU77493 Homo SapiensINCY- Human lipid metabolism213598
enzyme, LMM-1.
414 1505053 Homo sa I sosomal acid 1i ase 115053
iens
414 g1460143 Homo Sapienslysosomal acid lipase/cholesteryl114853
ester
h drolase
415 AAG68347 Homo SapiensBODA- Human zinc finger 283910
protein 59
SEQ ID N0:2.
415 AAB08899 Homo SapiensHUMA- :Human secreted 260299
protein
sequence encoded by gene
9 SEQ ID
N0:56.
415 120146520Homo sa SLTP003 955 100
iens
416 AAB61188 Homo SapiensMILL- Human INTERCEPT 189286
217
rotein.
416 AAB61190 Homo SapiensMILL- Mature human INTERCEPT178885
217 protein.
416 AAB61191 Homo sapiensMILL- Human INTERCEPT 178093
217
extracellular domain.
805A/PCT
CA 02453344 2004-O1-21
136
Table 2
SEQ Accession Species Description ~ Score
ID
NO: Number _ __ identit
417 AAB30550 Homo SapiensPICO- Amino acid sequence183995
of an
al ha-2HS- Tyco rotein
precursor.
417 AAY56991 Homo sa FARB Human fetuin oly 183995
iens epode.
417 AAW61492 Homo SapiensPICO- Human fetuin glycoprotein183995
type
2.
418 AAB30550 Homo SapiensPICO- Amino acid sequence185095
of an
al ha-2HS- 1 co rotein
recursor.
418 AAY56991 Homo sa FARB Human fetuin of 1$5095
iens a tide.
418 AAW61492 Homo sapiensPICO- Human fetuin glycoprotein185095
type
2.
419 AAB30550 Homo sapiensPICO- Amino acid sequence183194
of an
al ha-2HS- I co rotein
recursor.
419 AAY56991 Homo sa FARB Human fetuin of 183194
iens a tide.
419 AAW61492 Homo SapiensPICO- Human fetuin glycoprotein183194
type
2.
420 AAB94839 Homo SapiensHELL- Human protein sequence315310
SEQ ID
N0:16010.
420 AAB73691 Homo SapiensINCY- Human oxidoreductase315310
protein
ORP-24.
420 g118028283Homo Sapiensvery-long-chain acyl-CoA314999
deh dro enase VLCAD
421 g111125672Homo SapiensdJ591C20.1 (novel protein251110
similar to
mouse NG26)
421 AAB94489 Homo sapiensHELI- Human protein sequence135057
SEQ ID
NO:15176.
421 AAY91669 Homo SapiensHUMA- Human secreted 135057
protein
sequence encoded by gene
73 SEQ ID
N0:342.
422 AAD02606- Homo SapiensHYSE- Human angiopoietin,190590
aal CG007a1t1, cDNA.
422 AAB53070 Homo SapiensGETH Human angiogenesis-associated190590
rotein PR0197, SEQ ID
N0:31.
422 AAY72621 Homo SapiensNYSE- Human angiopoietin190590
protein,
CG007a1t1. _
423 1467671 Homo sa ZN-al ha-2- I co rotein 127383
iens
423 i38026Ho rno sa Zn-al hat- 1 co rotein 126483
iens
423 1340442 Homo sa Zn-al ha-2- I co rotein 125983
iens
424 AAE07119 Homo sapiensHUMA- I-3uman gene 12 241198
encoded
secreted protein fragment,
SEQ ID
N0:136.
424 AAE07062 Homo SapiensHUMA- Human gene 12 encoded24I
198
secreted protein HE8FD93,
SEQ ID
N0:79.
424 AAM99932 Homo SapiensHUMA- Human polypeptide 143198
SEQ ID
NO 48.
425 AAI67788 Homo SapiensUYHA- Human lysyl oxidase-like228699
a
al L OXL3) rotein encodin
cDNA.
425 AAD24786- Homo sapiensINCY- Human secreted 228699
protein-3
aal (SECP) cDNA.
425 AAA47799_ Homo SapiensMILL- Human lysyl oxidase228699
related
aal rotein (Lor)-2 cDNA (CDS).
426 AAB88385 Homo SapiensHELI- Human membrane 157099
or secretory
rotein clone PSEC0128.
426 AAD24790- Homo SapiensINCY- Human secreted 154910
protein-7
aal (SECP) cDNA.
805A/PCT
CA 02453344 2004-O1-21
137
Table 2
SEQ AccessionSpecies Description Score /~
ID
NO: Number identit
426 AAA96354 Homo SapiensGETH cDNA encoding a 154910
novel
_ oly a tide desi nated
aal PR06030.
427 AAY71471 Homo SapiensZYMO Human prostaglandin558 95
D2
s nthase (PD2 s nthase).
427 ai189772 Homo sa rosta landin D2 s nthase558 95
iens
427 112963879Homo sa rosta landin D s nthase 558 95
iens
428 AAB24476 Homo SapiensHUMA- Human secreted 129877
protein
sequence encoded by gene
40 SEQ ID
NO:101.
428 g112018147Chlamydomonvegetative cell wall 115 46
protein gpl
as reinhardtii
428 g17715585StreptococcusPspA 114 31
pneumoniae
429 AAB95763 Homo sapiensHELI- Human protein sequence134110
SEQ ID
N0:18691.
429 AAM40777 Homo sapiensNYSE- Human polypeptide 77 34
SEQ ID NO
5708.
429 gi56625Rattus microtubule associated 76 28
protein 2
norve icus
430 AAE04221 Homo SapiensHUMA- Human gene 5 encoded614 97
secreted protein HUVFB80,
SEQ ID
N0:76.
430 AAE04203 Homo sapiensHUMA- Human gene 5 encoded612 97
secreted protein HUVFB80,
SEQ ID
N0:57.
430 AAM90448 Homo SapiensHUMA- Human 256 77
immune/haematopoietic
antigen SEQ
ID N0:18041.
431 gi~20853599~Mus musculussimilar to dJ287G14.1 144781
(exon of a yet
reflXP~1369 unidentified gene, or
part of a
24.1 ~ pseudogene?; similar
to parts of BMP
and Tolloid proteins)
431 gi~4826463~eHomo sapiensdJ287G14.1 (exon of a 620 99
yet unidentified
mb~CAB428 gene, or part of a pseudogene?;
similar
99.1 to arts of BMP and Tolloid
roteins)
431 gi~4557503~reHomo sapienscubilin precursor; cubilin;211 36
intrinsic
fINP 001072 factor-cobalamin receptor;
intrinsic
.l factor B 12-rece for
432 g113774338Homo sapienscytochrome P450 subfamily262499
IIIA
polype tide 43
432 112642642Homo sa cytochrome P450 CYP3A43 262499
iens
432 g111225240Homo sapienscytochrome P450 subfamily261299
IIIA
of a tide 43
433 AAV60292-Homo SapiensVEDA DNA sequence encoding210793
death
aal associated protein (DAP)-7
(cathepsin
D).
433 AAQ89844-Homo sapiensVEDA Human death associated210793
protein
aa1 DAP-7, also called cathe
sin D.
433 AAA46901-Homo SapiensGETH cDNA encoding novel210793
aal of a tide PR0292.
434 AAG01648 Homo SapiensGEST Human secreted protein,281 100
SEQ ID
NO: 5729.
434 AAU81958 Homo SapiensGETH Human PR0346. 254 31
434 AAB80266 Homo sa GETH Human PR0346 rotein.254 31
iens
805A/PCT
CA 02453344 2004-O1-21
138
Tabh 2
SEQ ID AccessionSpecies Description Score
NO: Number identit
435 g13168604Homo Sapiensproline and glutamic 510798
acid rich nuclear
rotein isoform
435 AAW31186 Homo sa DAND Human 160 pol eptide472398
iens 160.2.
435 AAW31185 Homo sa DAND Human 160 0l a tide313980
iens 160.1.
436 AAM25961 Homo SapiensHYSE- Human protein sequence832 100
SEQ
ID N0:1476.
436 ABB84895 Homo sapiensGETH Human PR01190 protein650 99
se uence SEQ ID N0:158.
436 AAY99357 Homo SapiensGETH Human PR01190 (L1NQ604)650 99
amino acid se uence SEQ
ID N0:58.
437 110799172Homo sa uterine-derived 14 kDa 742 100
iens rotein
437 119550451Homo sa o us 2 81 42
iens
437 16957462 Homo sa J159A19.3 novel rotein) 75 35
iens d
438 ABB11737 Homo Sapiens_ 598 100
HYSE- Human secreted
protein
homolo e, SEQ ID N0:2107.
438 AAY02692 Homo SapiensHUMA- Human secreted 459 98
protein
encoded b ene 43 clone
HTADX17.
438 116356681Homo SapiensCD2 family 10 459 98
439 AAY08326 Homo SapiensSTRD Human granulysin 454 96
P522 active
fra went.
439 AAW59874 Homo SapiensHUMA- Amino acid sequence454 96
of the
cDNA clone CAT-1 (HTXET53).
439 AAU84278 Homo sapiensBGHM Human endometrial 269 66
cancer
related rotein, GNLY.
440 119912826Ciona savilarval mesench me s ecifc130449
n 1 rotein
440 16572165 Homo sa d31119A7.5 novel rotein 546 99
iens (isoform 2))
440 AAM68752 Homo SapiensMOLE- Human bone marrow 444 100
expressed
probe encoded protein
SEQ ID NO:
29058.
441 AAB84327 Homo SapiensINCY- Amino acid sequence220299
of a
human lyase and associated
protein
HLYAP-2.
441 AAM93587 Homo SapiensHELI- Human polypeptide,220299
SEQ ID
NO: 3385.
441 AAU83711 Homo SapiensGETH Human PRO protein, 216298
Seq ID No
240.
442 AAU82017 Homo sapiensINCY- Human secreted 101646
protein
SECP43.
442 118676716Homo sa FLJ00257 rotein 100345
iens
442 _ Homo SapiensINCY- CDIFF-15, Incyte 764 44
AAB47134 ID No.
3478571 CD 1.
443 14235144 Homo sa BC39498 1 145265
iens
443 g121265141Homo SapiensSimilar to zinc forger 143365
protein 91 (HPF7,
HTF 10)
443 19802037 Homo sa zinc fm er rotein SBZ:F3140161
iens
444 111493481Homo sa PR02474 433 100
iens
444 ABB06613 Homo sapiensCUBA- G protein-coupled 68 28
receptor
GPCRBa rotein SEQ ID
N0:36.
444 g11171608Plasmodiumrps7 67 38
falci arum
446 112751092Homo sa PNAS-x23 346 100
iens
447 gi65030Xenops ~ transcription factor 94 25
IIF subunit
laevis
447 g121428364DrosophilaGM14375p 94 30
melano
aster
805A/PCT ~ 02453344 2004-O1-21
139
Table 2
SEQ Accession Species Description Score
ID
NO: Number identit
447 g121430981Xenopus RAP74 subunit of transcription93 25
factor
laevis IIF
448 g116265514OdontobutisNADH dehydrogenase subunit?4 36
1
obscura
448 AAW87504 Homo SapiensSIBI- Human N-methyl-D-aspartate72 30
receptor subunit encoded
by clone
NMDA24.
448 AAW87503 Homo SapiensSIBI- Human N-methyl-D-aspartate72 30
receptor subunit encoded
by clone
NMDA22.
449 ABB89065 Homo sapiensHUMA- Human polypeptide 116199
SEQ ID
NO 1441.
449 AAY19743 Homo SapiensHUMA- SEQ ID NO 461 from972 99
W09922243.
449 AAY19541 Homo SapiensHUMA- Amino acid sequence265 100
of a
human secreted rotein.
450 AAB47433 Homo sa BIOD- Human zinc-fm er 223510
iens rotein 43.
450 115929737 Mus musculusSimilar to zinc fm er 182954
rotein 347
450 113752754 Homo sa zinc fin er 1111 178850
iens
451 AAB58357 Homo SapiensROSE/ Lung cancer associated465 98
oly a tide se uence SEQ
ID 695.
451 g113569765Giardia variable surface protein73 53
14f
intestinalis
451 g113569785Giardia variable surface protein71 53
42d
intestinaiis
452 AAG02783 Homo SapiensGEST Human secreted protein,344 100
SEQ ID
NO: 6864.
452 AA013848 Homo SapiensHYSE- Human polypeptide 80 35
SEQ ID NO
27740.
452 1469232 Bos taurusvacuolar H+ATPase subunit70 43
453 1l 1493502Homo sa PR03102 545 100
iens
453 16822268 Mus musculusCIP7 78 36
453 a119070521Homo sa metallothionein 1M 77 29
iens
454 AAG02139 Homo SapiensGEST Human secreted protein,287 98
SEQ ID
NO: 6220.
454 AAM96469 Homo SapiensHUMA- Human reproductive94 95
system
related antigen SEQ m
NO: 5127.
454 g120151347DrosophilaGH06335p 75 26
melano
aster
455 AAU16313 Homo SapiensHUMA- Human novel secreted121598
protein,
Se ID 1266.
455 ABB05662 Homo sapiensGEHU- Human signal transduction980 100
rotein clone am 2 10h17.
455 g121040537Homo SapiensSimilar to RIKEN cDNA 137 40
9130020610
ene
456 1881564 Homo sa ZNF157 104947
iens
456 g1 15787774Homo sapiensbB479F 17.1 (zinc finger104347
protein 157
HZF22 )
456 116797860 Homo sa ZNF317-2 rotein 104150
iens
457 AAG00579 Homo sapiensLEST Human secreted protein,254 94
SEQ ID
NO: 4660.
458 1202219 Mus musculusal ha-tubulin iso a M-a1ha-6405 100
458 g1213862 Oncorhynchusalpha-tubulin 405 100
m kiss
458 g12843123 Homo Sapiensalpha tubulin ~ 405 100
~ ~ ~
$O5 A mC.,T CA 02453344 2004-O1-21
140
Table 2
SEQ Accession Species Description Score
ID
N~: Number identit
459 1532688 Horno sa thrombos ondin- 50 292 97
iens
459 1553801 Homo sa Thrombos ondin 139 100
iens
459 1567240 Mus musculusthrombos ~ondin 1 129 57
460 19885325 Homo sa RAGE-1 796 100
iens
460 AAU85524 Homo sa CORI- L552S lun tumour 663 99
iens rotein.
460 AAB76869 Homo SapiensCORI- Human lung tumour 663 99
protein
related protein sequence
SEQ ID
N0:791.
461 AAU81995 Homo sapierisINCY- Human secreted 171299
protein
SECP21.
461 AAU10030 Homo SapiensUYJO Human elongation 171299
of fatty acids
(ELF) rotein.
461 g114594722Homo Sapienselongation of very long 171299
chain fatty acids
rotein
462 g1159725 Octopus alpha tubulin 274 77
dofleini
462 g12098753 Gecarcinusalpha-2-tubulin 271 75
lateralis
462 g19994 Paracentrotusalpha-tubulin (AA 1-452)271 75
lividus
463 g112314165Homo SapiensbA526D8.4 (novel KRAB 453710
box
containing C2H2 type
zinc finger
rotein
463 AAM79958 Homo SapiensNYSE- Human protein SEQ 235061
ID NO
3604.
463 AAM78974 Homo sapiensNYSE- Human protein SEQ 235061
ID NO
1636.
464 AAB92490 Homo SapiensHELI- Human protein sequence517 60
SEQ ID
N0:10585.
464 AAB92452 Homo sapiensHELI- Human protein sequence129 38
SEQ ID
N0:10484.
464 AAM95329 Homo sapiensHUMA- Human reproductive129 38
system
related anti en SEQ ID
NO: 3987.
465 AAE02058 Homo sapiensHUMA- Human four disulfide536 43
core
domain (FDCD)-containin
rotein.
465 112655452 Homo sa keratin associated rotein495 44
iens 4.7
465 112655456 Homo sa keratin associated rotein471 41
iens 4.9
466 AAM93935 Homo SapiensHELI- Human polypeptide,215610
SEQ ID
NO: 4112.
466 AAG64944 Homo sa SHAN- Human zinc-finger 184
iens protein 44. 010
466 AAM93807 Homo SapiensHELI- Human polypeptide,_
SEQ ID 122755
NO: 3849.
467 112314284 Homo sa dJ353C17.1 (novel rotein)628 99
iens
467 g118892440PyrococcusATP-dependentprotease _ 29
LA (lon) 79
furiosus
DSM
3638
467 1558671 Homo sa re ulato artner for cdk572 33
iens kinase
468 AAM25660 Homo SapiensNYSE- Human protein sequence711 100
SEQ
ID N0:1175.
468 111493560 Homo sa PR02730 711 100
iens
468 ABB89111 Homo sapiensHUMA- Lluman polypeptide634 100
SEQ ID
NO 1487.
469 AAE12784 Homo SapiensINCY- Human delta 1-pyrroline-5-572 100
carboxylate reductase
homologue
(PSCRH).
CA 02453344 2004-O1-21
141
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
469 g115928817Homo SapiensSimilar to pyrroline 572 100
5-carboxylate
reductase isoform
469 AAB74779 Homo sapiensUYFU- Human Py-CR protein543 97
SEQ ID
N0:4.
470 Qi2689443Homo sa 828830 2 307510
iens
470 AAM68615 Homo SapiensMOLE- Human bone marrow 297010
expressed
probe encoded protein
SEQ ID NO:
28921.
470 AAM56237 Homo SapiensMOLE- Human brain expressed297010
single
exon probe encoded protein
SEQ ID
NO: 28342.
471 g11167849Homo sapiensNAD (H)-specific isocitrate199297
dehydrogenase gamma subunit
recursor
471 g1 1673432Homo sapiensNAD(H)-specific isocitrate199297
dehydrogenase gamma-subunit
recursor
471 g14096803Homo sapiensNAD+specific isocitrate 199297
dehydrogenase
amma subunit recursor
472 17459861 Homo sa Zinc fin er rotein ZNF45961 36
iens
472 11160977 Homo sa zinc fm er rotein 957 36
iens
472 AAB21003 Homo SapiensINCY- Human nucleic acid-binding942 35
rotein, NuABP-7.
473 AAB94397 Homo SapiensHELL- Human protein sequence912 100
SEQ ID
N0:14966.
473 AAR13520 Homo SapiensUYSF- Leukocyte derived 72 34
growth
factor analo ue.
473 gi~20823620~Mus musculussimilar to gene 11-1 97 26
protein precursor -
reflXP-1442 malaria parasite (Plasmodium
24.1 falci arum) (fra menu
474 19802037 Homo sa zinc fin er rotein SBZF3257699
iens
474 14235144 Homo sa BC39498 1 146161
iens
474 AAM93961 Homo SapiensHELL- Human polypeptide,142759
SEQ ID
NO: 4169.
475 AAB21040 Homo Sapiens1NCY- Human nucleic acid-binding269792
rotein, NuABP-44.
475 13294544 Homo sa C2H2- a zinc fm er rotein129945
iens
475 15757625 Homo sa C2H2 zinc fm er rotein 129945
iens
476 AAM70492 Homo SapiensMOLE- Human bone marrow 611 56
expressed
probe encoded protein
SEQ ID NO:
30798.
476 AAM58051 Homo SapiensMOLE- Human brain expressed611 56
single
exon probe encoded protein
SEQ ID
NO: 30156.
476 AAE02058 Homo SapiensHUMA- Human four disulfide563 41
core
domain (FDCD)-containin
rotein.
477 ABB04717 Homo SapiensSHAN- Human PP1030 protein127089
SEQ ID
NO:S.
477 AAY86431 Homo SapiensHUMA- Human gene 35-encoded230 94
rotein fra ent, SEQ ID
NO:346.
477 AAY86430 Homo SapiensHUMA- Human gene 35-encoded154 91
rotein fra ent, SEQ ID
NO:345.
_
478 AAY14426 Homo SapiensHUMA-Human secreted protein222 100
encoded b ene 16 clone
HSAVP17.
805AlPCT ~ 02453344 2004-O1-21
142
Table 2
SEQ AccessionSpecies Description Score
ID
NO: Number identit
478 AAY14481 Homo SapiensHUMA- Fragment of human 79 100
secreted
rotein encoded b ene
16.
478 gi14026136MesorhizobiuABC transporter sugar 66 28
permease
m loti
479 112407395Homo sa tri artite motif rotein 116910
iens TRIM7
479 a115150298Homo sa I co enin-interactin 108898
iens rotein 1
479 1 12407397Mus musculustri artite motif rotein 913 84
TRIM7
480 AAB73600 Homo sa SHAN- Zinc fin er rotein290699
iens 57.
480 g16467206Homo Sapiensgonadotropin inducible 146549
transcription
re ressor-4
480 AAY58627 Homo sapiensINCY- Protein regulating141948
gene
ex ression PRGE-20.
481 AAB94433 Homo sapiensHELI- Human protein sequence373899
SEQ ID
N0:15052.
481 a114764499Homo sa zinc fin er rotein 138044
iens
481 1 1504006Homo sa similarto human ZFY rotein.117243
iens
482 AAG03930 Homo sapiensGEST Human secreted protein,90 50
SEQ ID
NO: 8011.
482 g13116064Squalus s-sgkl 89 44
acanthias
482 g13116066Squalus s-sgk2 83 42
acanthus
483 AAB 12318Homo SapiensHUMA- Human secreted 494 96
protein
encoded b ene 18 clone
HE2FL70.
483 gi~2827286~gHomo sapiensnovel antagonist of FGF 68 32
signaling
b~AAC39567
.1
484 AAY08325 Homo SapiensSTRD Human granulysin 327 76
P520 active
fra ent.
484 AAW59874 Homo SapiensHUMA- Amino acid sequence327 76
of the
cDNA clone CAT-1 HTXET53).
484 AAR23732 Homo SapiensMINU Gene 519 cDNA derived327 76
a tide.
485 AAG89277 Homo SapiensGEST Human secreted protein,883 100
SEQ ID
NO: 397.
485 a120147667Homo sa ADP-ribos lation factor-like201 31
iens rotein 1
485 113937801Homo sa ADP-ribos lation factor-like201 31
iens 1
486 118151760Homo sa Offenes Leseraster TB7 867 100
iens
486 AAU16453 Homo SapiensHUMA- Human novel secreted650 97
protein,
Seq ID 1406.
486 AAU16018 Homo sapiensHUMA- Human novel secreted552 94
protein,
Se ID 971.
487 AAE10184 Homo SapiensNYSE- Human bone marrow 332510
derived
rotein, SEQ ID NO: 28.
487 118676608Homo sa FLJ00203 rotein 275 22
iens
487 AAB95523 Homo SapiensHELI- Human protein sequence261 21
SEQ ID
N0:18106.
488 AAB93782 Homo SapiensHELI- Human protein sequence252510
SEQ ID
N0:13516.
488 g120809447Homo sapienssimilar to zinc forger 103543
protein 14 (KOX
6); GIOT-4 for gonadotropin
inducible
transcri tion re ressor-4
488 AAY58627 Homo SapiensINCY- Protein regulating988 44
gene
ex ression PRGE-20.
gOS~PCT CA 02453344 2004-O1-21
143
Table 3
SEQ AccessionDescription Results*
ID
NO: Number
245 BL01159 WW/rs S/WWP domain BL01159 13.85 3.755e-10
roteins. 101-115
245 PR00403 WW DOMAIN SIGNATURE PR00403B 12.19 1.305e-09
101-115
245 DM00215 PROLINE-RICH PROTEIN DM00215 19.43 5.881e-09
3. 403-435
246 PR00450 RECOVERIN FAMILY PR00450C 12.22 1.818e-12
236-257
SIGNATURE
247 PR00659 CHROMOGRANIN SIGNATUREPR00659B 13.09 9.746e-09
539-554
248 BL00115 Eukaryotic RNA polymeriseBLOOI 15Z 3.12 4.176e-09
II 312-360
he to a tide re eat
roteins.
249 BL00904 Protein prenyltransferasesBL00904A 8.30 1.574e-09
alpha 628-677
subunit re eat roteins
roteins.
249 PD02059 CORE POLYPROTEIN PROTEINPD02059B 24.48 7.136e-10
915-949
GAG CONTAINS: P. PD02059B 24.48 5.817e-09
612-646
249 PR00049 WILM'S TUMOUR PROTEIN PR00049D 0.00 9.557e-13
625-639
SIGNATURE PR00049D 0.00 4.000e-12
629-643
PR00049D 0.00 4.000e-12
630-644
PR00049D 0.00 4.000e-12
631-645
PR00049D 0.00 4.000e-12
632-646
PR00049D 0.00 4.000e-12
633-647
PR00049D 0.00 4.000e-12
634-648
PR00049D 0.00 4.000e-12
930-944
PR00049D 0.00 5.125e-12
627-641
PR00049D 0.00 5.125e-12
628-642
PR00049D 0.00 6.899e-11
622-636
PR00049D 0.00 7.126e-11
706-720
PR00049D 0.00 9.168e-11
624-638
PR00049D 0.00 1.214e-10
707-721
PR00049D 0.00 2.071e-10
626-640
PR00049D 0.00 2.071e-10
635-649
PR00049D 0.00 2.071e-10
636-650
PR00049D 0.00 7.857e-10
621-635
PR00049D 0.00 1.153e-09
928-942
PR00049D 0.00 1.458e-09
705-719
PR00049D 0.00 1.610e-09
926-940
PR00049D 0.00 5.576e-09
934-948
PR00049D 0.00 5.729e-09
623-637
PR00049D 0.00 6.797e-09
708-722
PR00049D 0.00 6.949e-09
637-651
PR00049D 0.00 7.102e-09
756-770
249 PR00910 LUTEOVIRUS ORF6 PROTEINPR00910A 2.51 7.429e-09
930-942
SIGNATURE PR00910A 2.51 7.536e-09
938-950
249 PR00211 GLUTELIN SIGNATURE PR00211B 0.86 8.269e-10
632-652
PR00211B 0.86 1.833e-09
625-645
PR00211B 0.86 6.250e-09
623-643
PR00211B 0.86 7.500e-09
628-648
PR00211B 0.86 7.750e-09
629-649
249 PR00806 VINCULIN SIGNATURE PR00806A 6.63 8.767e-09
934-944
249 DM00215 PROL1NE-RICH PROTEIN DM00215 19.43 6.559e-18
3. 615-647
DM00215 19.43 8.875e-15
618-650
DM00215 19.43 2.957e-13
611-643
DM00215 19.43 3.739e-13
616-648
DM00215 19.43 5.891e-13
912-944
DM00215 19.43 9.413e-13
613-645
DM00215 19.43 7.563e-12
614-646
DM00215 19.43 9.250e-12
608-640
I DM00215 19.43 1.353e-11
620-652
DM00215 19.43 2.412e-11
624-656
805A/PCT ~ 02453344 2004-O1-21
144
Table 3
SEQ ID AccessionDescription Results*
NO: Number
DM00215 19.43 4.353e-11
623-655
DM00215 19.43 4.706e-11
605-637
DM00215 19.43 5.235e-11
612-644
DM00215 19.43 5.588e-11
918-950
DM00215 19.43 9.294e-11
609-641
DM00215 19.43 9.647e-1
l 607-639
DM00215 19.43 1.321e-10
619-651
DM00215 19.43 2.929e-10
617-649
DM00215 19.43 5.500e-10
621-653
DM00215 19.43 1.153e-09
914-946
DM00215 19.43 1.915e-09
602-634
DM00215 19.43 2.068e-09
910-942
DM00215 19.43 2.831e-09
622-654
DM00215 19.43 3.288e-09
908-940
DM00215 19.43 3,746e-09
628-660
DM00215 19.43 3.898e-09
909-941
DM00215 19.43 4.051 e-09
785-817
DM00215 19.43 4.203e-09
627-659
DM00215 19.43 5,119e-09
610-642
DM00215 19.43 5.881 e-09
626-658
DM00215 19.43 6.339e-09
629-661
DM0021 S 19.43 6.492e-09
749-781
DM00215 19.43 6.797e-09
917-949
DM00215 19.43 7.254e-09
806-838
DM0021 S 19.43 7.559e-09
687-719
DM00215 19.43 8.017e-09
809-841
DM00215 19.43 9.237e-09
630-662
DM00215 19.43 9.237e-09
916-948
DM00215 19.43 9,695e-09
625-657
250 PR00489 FRIZZLED PROTEIN SIGNATUREPR00489C 9.29 2.250e-28
398-422
PR00489E 9.95 4.808e-25
486-508
PR00489G 8.99 6.478e-25
585-605
PR00489B 13.69 4.273e-24
308-330
PR00489A 11.81 7.353e-24
280-302
PR00489D 15.68 2,703e-22
441-464
PR00489F 14.55 1.675e-21
529-550
250 PR00341 PRION PROTEIN SIGNATUREPR00341D 0.26 5.442e-10
641-656
PR00341C 0.07 8.043e-10_641-656
250 BL00604 Synaptophysin / synaptoporinBL00604F 5.96 5.014e-09625-669
roteins.
250 DM01724 kw ALLERGEN POLLEN CIM1DM01724 8.14 4.553e-09
637-656
HOL-Ll. DM01724 8.14 7.987e-09
640-659
250 PR00527 GASTRIN RECEPTOR PR00527I 5.36 8.412e-09
158-177
SIGNATURE
250 PR00124 ATP SYNTHASE C SUBUNIT PR00124A 8.81 9.069e-09
348-367
SIGNATURE
250 BL00291 Prion protein. BL00291A 4.49 1.867e-10
625-659
BL00291A 4.49 4.931e-09
624-658
BL00291A 4.49 9.379e-09
623-657
250 BL00180 Glutamine synthetase BL00180D 13.26 9.746e-09
roteins. 182-203
251 PR00315 GTP-BINDING ELONGATION PR00315A 11.81 B.OOOe-14
70-83
FACTOR SIGNATURE PR00315C 13.85 3.250e-12
137-147
251 BL00301 GTP-binding elongation BL00301B 20.09 2.080e-24
factors 139-170
roteins. BL00301A 12.41 5.125e-12
70-81
251 BL01176 Initiation factor 2 BL01176B 8.74 7.153e-11
roteins. 136-173
251 PR00449 TRANSFORMING PROTEIN PR00449E 13.50 7.214e-09
P21 221-243
805A/PCT ~ 02453344 2004-O1-21
145
Table 3
SE[ AccessionDescription Itesults*
ID
NO: Number
RAS SIGNATURE
253 PF00614 Phospholipase D. ActivePF00614B 14.45 3.294e-09
site proteins 200-219
motifs.
254 BL50002 Src homology 3 (SH3) BL50002A 14.19 4.7SOe-12
domain 332-350
roteins rofile.
254 PR00452 SH3 DOMAIN SIGNATURE PR004S2B 11.65 5.500e-09
342-3S7
256 BL50002 Src homology 3 (SH3) BL500028 15.18 5.200e-10
domain 693-706
roteins rofile.
256 PF00620 GTPase-activator proteinPF00620B 14.20 6.000e-10
for Rho- 372-388
like GTPases.
256 PD00930 PROTEIN GTPASE DOMAIN PD00930B 33.72 4.000e-18
423-463
ACTIVATION. PD00930A 25.62 6.684e-10
317-342
256 PR00452 SH3 DOMAIN SIGNATURE PR00452D 17.02 2.385e-09
695-707
258 PD00930 PROTEIN GTPASE DOMAIN PD00930B 33.72 2.098e-20
137-177
ACTIVATION.
258 PF00620 GTPase-activator proteinPF00620B 14.20 4.913e-13
for Rho- 86-102
like GTPases.
259 BL00107 Protein kinases ATP-bindingBL00107B 13.31 1.000e-14
region 329-344
roteins.
259 BL00239 Receptor tyrosine kinaseBL002398 25.15 7.286e-18
class II 191-238
proteins. BL00239E 17.14 5.655e-14
301-350
BL00239F 28.15 1.122e-12
355-399
259 BL00790 Receptor tyrosine kinaseBL007900 7.68 5.747e-13
class V 308-340
roteins. BL00790Q 15.61 6.400e-11
366-414
259 BL00240 Receptor tyrosine kinaseBL00240F 17.74 8.953e-18
class III 300-347
roteins. BL00240G 28.45 8.011e-11
347-399
259 PR00452 SH3 DOMAIN SIGNATURE PR00452D 17.02 7.188e-12
102-114
PR00452B 11.65 2.000e-09
69-84
259 PR00109 TYROSINE KINASE CATALYTICPR00109E 14.414.176e-14
374-396
DOMAIN SIGNATURE PR00109D 17.04 9.471e-12
330-352
PR00109C 12.85 3.250e-09
311-321
2S9 PR00761 BINDIN PRECURSOR PR00761E 14.32 1.758e-09
26-44
SIGNATURE PR00761E 1
4.32 4.600e-09 24-42
259 BL50001 Src homology 2 (SH2) _
domain BL50001D 11.00 6.250e-09
329-339
roteins rofile.
259 BL50002 Src homology 3 (SH3) BL50002B 15.18 3.769e-11
domain 100-113
roteins rofile. BL50002A 14.19 7.750e-09
59-77
260 BL00790 Receptor tyrosine kinaseBL00790B 21.59 1.000e-40
class V 62-113
proteins. BL00790C 16.65 1.000e-40
166-219
BL00790E 29.58 1.000e-40
276-323
BL00790G 22.06 1.000e-40
379-422
BL00790J 14.21 1.000e-40
603-642
BL00790K 9.30 1.000e-40
6S5-708
BL007900 7.68 1.000e-40
795-827
BL00790R 16.20 1.000e-40
891-934
BL00790N 13.25 7.618e-33
761-787
BL00790I 20.01 4.094e-28
504-534
BL00790D 12.41 2.125e-27
246-270
BL00790H 13.42 2.957e-27
458-483
BL00790M 8.74 3.483e-27
739-760
BL00790L 11.16 2.350e-25
719-738
BL00790F 15.90 6.143e-25
342-368
BL00790A 19.74 2.688e-18
32-53
BL00790P 12.33 1.261e-16
828-852
260 BL00240 Rece for rosine kinaseBL00240F 17.74 8.640e-18
class III 787-834
CA 02453344 2004-O1-21
146
Table 3
SEQ~ AccessionDescription I~esults*
ID
NO: Number
roteins. BL00240E 11.56 3.778e-16
734-771
260 BL00107 Protein kinases ATP-bindingBL00107A 18.39 6.063e-21
region 748-778
roteins. BL00107B 13.31 1.000e-13
816-83I
260 PR00109 TYROSINE KINASE CATALYTICPR00109D 17.04 7.158e-23
817-839
DOMAIN SIGNATURE PR00109B 12.27 4.706e-20
748-766
PR00109C 12.85 5.765e-12
798-808
PR00109A 15.00 8.269e-11
711-724
260 PR00014 FIBRONECTIN TYPE III PR00014D 12.04 7.545e-13
REPEAT 511-525
SIGNATURE PR00014B 14.77 5.154e-11
470-480
PR00014C 15.44 8.500e-11
493-511
260 BL00239 Receptor tyrosine kinaseBL00239E 17.14 3.813e-26
class II 788-837
proteins. BL00239B 25.15 1.655e-22
682-729
BL00239C 18.75 8.263e-13
735-757
BL00239D 16.81 8.627e-11
760-785
260 BL50001 Src homology 2 (SH2) BL50001B 17.40 8.875e-14
domain 745-765
proteins profile, BL50001C 10.17 2.200e-09
797-807
BL50001D 11.00 6.250e-09
816-826
260 BL00243 Integrins beta chain BL00243I 31.77 6.704e-09
cysteine-rich 273-315
domain proteins.
260 PD02520 RECEPTOR PRECURSOR PD02520C 10.48 7.266e-09
501-517
TRANSMEMBRANE.
261 BL00471 Small cytokines BL00471 23.92 1.000e-40
72-119
(intercrine/chemokine)
C-x-C
subfamil si nat.
261 PR00437 SMALL CXC CYTOKINE PR00437B 14.81 2.421e-22
77-98
FAMILY SIGNATURE PR00437C 14.85 8.579e-19
99-117
PR00437A 9.50 3.813e-11
62-70
261 PR00436 INTERLEUKIN-8 SIGNATUREPR00436C 10.51 6.382e-09
78-101
262 PD00126 PROTEIN REPEAT DOMAIN PD00126A 22.53 3.483e-09
TPR 87-107
NUCLEA.
263 PR00360 C2 DOMAIN SIGNATURE PR00360A 14.59 8.839e-10
57-69
PR00360B 13.61 3.455e-09
82-95
264 PR00499 NEUTROPHIL CYTOSOL PR00499D 10.18 1.875e-12
269-289
FACTOR 2 SIGNATURE
264 BL50002 Src homology 3 (SH3) BL50002A 14.19 3.077e-11
domain 271-289
roteins rofile. BL50002B 15.18 5.800e-10
309-322
264 PR00452 SH3 DOMAIN SIGNATURE PR00452B 11.65 5.645e-10
281-296
PR00452D 17.02 8.773e-10
311-323
265 BL01160 Kinesin light chain BL01160F 9.68 8.161 e-21
repeat proteins. 399-439
BL01160F 9.68 6.243e-17
291-331
BL01160E 8.74 6.938e-17
484-522
BL01160E 8.74 5.140e-16
442-480
BLOT 160E 8.74 7.300e-16
400-438
BL01160E 8.74 3.972e-14
250-288
BL01160E 8.74 5.075e-I4
526-564
BL01160F 9.68 2.017e-13
483-523
BL01160F 9.68 4.913e-13
249-289
BL01160F 9.68 6.009e-13
525-565
BL01160E 8.74 7.300e-13
292-330
BL01160C 2.94 1.354e-12
366-412
BL01160G 13.67 2.948e-12
398-424
BLOI 160F 9.68 6.067e-12
441-481
BL01160F 9.68 6.748e-12
357-397
BL01160G 13.67 1.089e-1
I 248-274
BL01160G 13.67 4.653e-1
1 524-550
g~~~P~T CA 02453344 2004-O1-21
147
Table 3
SEQ AccessionDescription Results*
ID
NO: Number
BL01160C 2.94 7.614e-11
258-304
BL01160E 8.74 9.773e-11
358-396
BL01160G 13.67 4.600e-10
440-466
BL01160C 2.94 4.971e-10
450-496
BL01160I 12.96 7.165e-10
525-572
BL01160I 12.96 9.575e-10
441-488
BL01160C 2.94 1.503e-09
492-538
BL01160G 13.67 4.436e-09
356-382
BL01160G 13.67 5.909e-09
482-508
BL01160I 12.96 8.241e-09
399-446
BL01160I 12.96 9.797e-09
483-530
265 PR00381 KINESIN LIGHT CHAIN PR00381D 13.94 1.318e-10
495-512
SIGNATURE PR00381E 8.75 7.364e-10
413-433
PR00381D 13.94 8.380e-09
537-554
PR00381F 9.13 9.010e-09
406-427
PR00381E 8.75 1.000e-08
305-325
266 PF00646 F-box domain roteins. PF00646A 14.37 3.893e-10
75-88
269 PF00642 Zinc finger C-x8-C-x5-C-x3-HPF00642 11.59 4.673e-10
type 312-322
and similar).
270 PR00109 TYROSINE KINASE CATALYTICPR00109B 12.27 5.059e-12
1198-1216
DOMAIN SIGNATURE
270 BL00107 Protein kinases ATP-bindingBL00107A 18.39 1.818e-15
region 1198-1228
roteins. BL00107B 13.31 1.643e-11
1268-1283
270 BL00239 Receptor tyrosine kinaseBL00239B 25.15 5.792e-13
class II 1133-1180
roteins. BL00239E 17.14 2,528e-09
1240-1289
270 PR00578 LATERAL EYE OPSIN PR00578E 9.62 4.447e-09
779-792
SIGNATURE
270 BL00240 Receptor tyrosine kinaseBL00240E 11.56 5.286e-09
class III 1184-1221
roteins.
271 BL00107 Protein kinases ATP-bindingBL00107A 18.39 8,650e-17
region 356-386
roteins.
271 BL00239 Receptor tyrosine kinaseBL00239B 25.15 7.545e-13
class II 296-343
roteins.
272 BL00678 Trp-Asp (WD) repeat BL00678 9.67 8.615e-1
proteins 1 1038-1048
proteins. BL00678 9.67 9.400e-10
1338-1348
BL00678 9.67 1.474e-09
952-962
BL00678 9.67 3.842e-09
1177-1187
BL00678 9.67 6.684e-09
1380-1390
272 PR00368 FAD-DEPENDENT PYRIDINE PR00368B 12.10 6.760e-09
992-1001
NUCLEOTIDE REDUCTASE
SIGNATURE
272 PR00320 G-PROTEIN BETA WD-40 PR00320A 16.74 7.353e-14
1036-1050
REPEAT SIGNATURE PRU0320C 13.01 3.000e-12
1036-1050
PR00320B 12.19 1.000e-11
1036-1050
PR00320A 16.74 2.862e-11
994-1008
PR00320C 13.01 5.304e-11
994-1008
PR00320A 16.74 6.586e-11
950-964
PR00320B 12.19 4.086e-10
1336-1350
PR003208 12.19 6.657e-10
1175-1189
PR00320A 16.74 6.824e-10
1420-1434
PR00320A 16.74 1.000e-09
1336-1350
PR00320C 13.01 1.000e-09
1336-1350
PR00320C 13.01 3.700e-09
1175-1189
PR00320B 12.19 4.600e-09
950-964
PR00320B 12.19 5.500e-09
994-1008
805A/PCT ~ 02453344 2004-O1-21
148
Table 3
SEQ AccessionDescription Results*
ID
NO: Number
PR00320A 16.74 6.707e-09
1175-1189
PR00320C 13.01 7.300e-09
950-964
PR00320C 13.01 7.300e-09
13?8-1392
273 BL00678 Trp-Asp (WD) repeat BL00678 9.67 8.615e-11
proteins 711-721
proteins. 8L00678 9.67 9.400e-10
1011-1021
BL00678 9.67 1.474e-09
625-635
BL00678 9.67 3.842e-09
850-860
BL00678 9,67 6.684e-09
1053-1063
273 PR00368 FAD-DEPENDENT PYRIDINE PR00368B 12.10 6.760e-09
665-674
NUCLEOTIDE REDUCTASE
SIGNATURE
273 PR00320 G-PROTEIN BETA VJD-40 PR00320A 16.74 7.353e-14
709-723
REPEAT SIGNATURE PR00320C 13.01 3.000e-12
709-723
PR00320B 12.19 1.000e-11
709-723
PR00320A 16.74 2.862e-11
667-681
PR00320C 13.01 5.304e-11
667-681
PR00320A 16.74 6.586e-11
623-637
PR00320B 12.19 4.086e-10
1009-1023
PR00320B 12.19 6.657e-10
848-862
PR00320A 16.74 6.824e-10
1093-1107
PR00320A 16.74 1.000e-09
1009-1023
PR00320C 13.01 1.000e-09
1009-1023
PR00320C 13.01 3.700e-09
848-862
PR00320B 12.19 4.600e-09
623-637
PR00320B 12.19 S.SOOe-09
667-681
PR00320A 16.74 6.707e-09
848-862
PR00320C 13.01 7.300e-09
623-637
PR00320C 13.01 7.300e-09
1051-1065
274 BL01290 Enhancer of rudimentaryBL01290B 17.01 4.231e-39
proteins. 39-78
BL01290A 11.13 6.226e-19
21-49
276 BL00678 Trp-Asp (WD) repeat BL00678 9,67 3.769e-11
proteins 134-144
roteins.
276 PR00320 G-PROTEIN BETA WD-40 PR00320B 12.19 S.SOOe-15
132-146
REPEAT SIGNATURE PR00320A 16.74 4.600e-12
132-146
PR00320C 13.01 8.435e-11
132-146
276 PR00319 BETA G-PROTEIN PR00319B 11.47 8.143e-09
132-146
(TRANSDUCIN) SIGNATURE
278 PR00449 TRANSFORMING PROTEIN PR00449A 13.20 9.206e-14
P21 5-26
RAS SIGNATURE PR00449D 10.79 6.276e-10
119-132
280 PF00791 Domain present in ZO-i PF00791B 28.49 9.053e-12
and UncS- 821-875
like netrin rece tors.
280 PF00023 Ank re eat roteins. PF00023A 16.03 7.750e-10
821-836
280 PD02329 KINASE ACETYLGLUTA~\iIATEPD02329B 16.24 4.838e-09
690-719
NAG DEHYD.
281 PF00791 Domain present in ZO-I PF00791B 28.49 9.053e-12
and UncS- 773-827
like netrin rece tors.
281 PF00023 Ank re eat roteins. PF00023A 16.03 7.750e-10
773-788
281 PD02329 KINASE ACETYLGLUTAMATE PD02329B 16.24 4.838e-09
642-671
NAG DEHYD.
282 PF00791 Domain present in ZO-I PF00791B 28.49 9.053e-12
and UncS- 796-850
like netrin rece torn,
282 PF00023 Ank re eat roteins. PF00023A 16.03 7.750e-10
796-81 I
282 PD02329 KINASE ACETYLGLUTAMATE PD02329B 16.24 4.838e-09
665-694
NAG DEHYD.
286 BL00107 Protein kinases ATP-bindinBL00107A 18.39 I.OOOe-23
re ion 262-292
CA 02309501 2004-04-O1
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