Note: Descriptions are shown in the official language in which they were submitted.
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
LCEs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. provisional patent applications
60/296,076
filed 6/5/2001, 60/328,605 filed 10/10/2001, 60/357,253 filed 2/15/2002, and
60/361,196
filed 3/1/2002. The contents of the prior applications are hereby incorporated
in their
entirety.
BACKGROUND OF THE INVENTION
The p53 gene is mutated in over 50 different types of human cancers, including
familial and spontaneous cancers, and is believed to be the most commonly
mutated gene
in human cancer (Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et
al.,
Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of mutations in the
p53 gene
are missense mutations that alter a single amino acid that inactivates p53
function.
Aberrant forms of human p53 are associated with poor prognosis, more
aggressive tumors,
metastasis, and short survival rates (Mitsudomi et al., Clin Cancer Res 2000
Oct;
6(10):4055-63; Koshland, Science (1993) 262:1953).
The human p53 protein normally functions as a central integrator of signals
including
DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives,
Cell
(1998) 95:5-8). In response to these signals, p53 protein levels are greatly
increased with
the result that the accumulated p53 activates cell cycle arrest or apoptosis
depending on
the nature and strength of these signals. Indeed, multiple lines of
experimental evidence
have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997)
88:323-331).
For example, homozygous p53 "knockout" mice are developmentally normal but
exhibit
nearly 100% incidence of neoplasia in the first year of life (Donehower et
al., Nature
(1992) 356:215-221).
The biochemical mechanisms and pathways through which p53 functions in normal
and cancerous cells are not fully understood, but one clearly important aspect
of p53
function is its activity as a gene-specific transcriptional activator. Among
the genes with
known p53-response elements are several with well-characterized roles in
either regulation
of the cell cycle or apoptosis, including GADD45, p21/Waf1/Cipl, cyclin G,
Bax, IGF-
BP3, and MDM2 (Levine, Cell (1997) 88:323-331).
Several essential organs (e.g., lungs, kidney, lymphatic system and
vasculature) are
made up of complex networks of tube-like structures that serve to transport
and exchange
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
fluids, gases, nutrients and waste. The formation of these complex branched
networks
occurs by the evolutionarily conserved process of branching morphogenesis, in
which
successive ramification occurs by sprouting, pruning and remodeling of the
network.
During human embryogenesis, blood vessels develop via two processes:
vasculogenesis,
whereby endothelial cells are born from progenitor cell types; and
angiogenesis, in which
new capillaries sprout from existing vessels.
Branching morphogenesis encompasses many cellular processes, including
proliferation, survival/apoptosis, migration, invasion, adhesion, aggregation
and matrix
remodeling. Numerous cell types contribute to branching morphogenesis,
including
endothelial, epithelial and smooth muscle cells, and monocytes. Gene pathways
that
modulate the branching process function both within the branching tissues as
well as in
other cells, e.g., certain monocytes can promote an angiogenic response even
though they
may not directly participate in the formation of the branch structures.
An increased level of angiogenesis is central to several human disease
pathologies,
including rheumatoid arthritis and diabetic retinopathy, and, significantly,
to the growth,
maintenance and metastasis of solid tumors (for detailed reviews see Liotta LA
et al, 1991,
Cell 64:327-336; Folkman J., 1995 Nature Medicine 1:27-31; Hanahan D and
Folkman
J, 1996 Cell 86:353-364). Impaired angiogenesis figures prominently in other
human
diseases, including heart disease, stroke, infertility, ulcers and
scleroderma.
The transition from dormant to active blood vessel formation involves
modulating the
balance between angiogenic stimulators and inhibitors. Under certain
pathological
circumstances an imbalance arises between local inhibitory controls and
angiogenic
inducers resulting in excessive angiogenesis, while under other pathological
conditions an
imbalance leads to insufficient angiogenesis. This delicate equilibrium of pro-
and anti
angiogenic factors is regulated by a complex interaction between the
extracellular matrix,
endothelial cells, smooth muscle cells, and various other cell types, as well
as
environmental factors such as oxygen demand within tissues.
Most known angiogenesis genes, their biochemical activities, and their
organization
into signaling pathways are employed in a similar fashion during angiogenesis
in human,
mouse and Zebrafish, as well as during branching morphogenesis of the
Drosoplvila
trachea. Accordingly, Drosophila tracheal development and zebrafish vascular
development provide useful models for studying mammalian angiogenesis (Metzger
RJ,
Krasnow MA. Science. 1999. 284:1635-9; Roman BL, and Weinstein BM. Bioessays
2000, 22:882-93).
2
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
In mammals, most of the fatty acids that are synthesized de novo possess chain
lengths
of 16-18 carbons. These long chain fatty acids constitute more than 90% of all
fatty acids
present in cells. They are important components of membranes, and they
represent the
largest energy storage reservoir in animals. The highest rate of de novo fatty
acid
synthesis occurs in liver, which converts excess glucose into fatty acids for
storage and
transport. Glycolysis converts glucose to pyruvate, which is converted to
ciliate in the
mitochondria and transported to the cytosol. Cytosolic ATP citrate lyase
generates acetyl-
CoA, the precursor of fatty acids and cholesterol. Acetyl-CoA is carboxylated
by acetyl-
CoA carboxylase (ACC) to form malonyl-CoA. The multifunction enzyme fatty acid
synthase (FAS) uses malonyl-CoA, acetyl-CoA, and NADPH to elongate fatty acids
in 2-
carbon increments. The principal end product of FAS in rodents is palmitic
acid, which
contains 16 carbons and is designated 16:0. A high proportion of this palmitic
acid is then
converted to stearate.
At a molecular Ievel, fatty acid elongases have been characterized most
extensively by
genetic studies in yeast. Yeast EL01 elongates C14 to C16 fatty acids and is
designated a
long chain fatty acid elongase (Toke DA et al, 1996, J Biol Chem 271:18413-
18422). The
EL02 and EL03 genes encode very long chain elongases that produce fatty acids
of 24 to
26 carbons (Oh CS et al, 1997, J Biol Chem 272:17376-17384). The mouse gene,
Cig30,
encodes the mouse version of EL02, and Sscl encodes the ortholog of EL03, both
of
which are very long chain elongases (Tvrdik P et al., 2000, J Cell Biol
149:707-718).
CIG30 and other Fatty acid elongases have been implicated in sphingolipid
biosynthesis.
In certain cells, sphingosine kinase is exported to extracellular space, where
it
phosphorylates sphingosine to make phospho-sphingosine (S1P) (Hla et al.,
Science 2001,
294:1875-8). S1P is a potent second messenger that binds to the EDGl GPCR to
mediate
' migration, survival, morphogenesis and proliferation. S1P and EDGl have been
shown to
be involved in endothelial cell migration and vascular maturation in vivo
(Ancellin et al., J
Biol Chem 2002, 277:6667-75).
A murine long chain fatty acyl elongase (LCE) that shares sequence identity
with
previously identified very long chain fatty acid elongases has been
identified. LCE
mRNA is highly expressed in liver and adipose tissue and is thought to
catalyze the rate
limiting condensing step in addition of 2-carbon units to C12-C16 fatty acids
(Moon YA
et al., 2001, J Biol Chem 276:45358-66). One of the human LCE genes, ELOVIA~,
has
been identified as a disease gene -Autosomal Dominant Stargardt-like Macular
Dystrophy
(STGD3) - associated with inherited forms of macular degeneration
characterized by
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
decreased visual acuity, macular atrophy and extensive fundus flecks (Zhang et
al., Nat
Genet 2001, 27:89-93). AlI afflicted members in five related families carry a
5 by deletion
(the so-called 797-801 del AACTT) of the gene.
The ability to manipulate the genomes of model organisms such as Drosophila
provides a powerful means to analyze biochemical processes that, due to
significant
evolutionary conservation, have direct relevance to more complex vertebrate
organisms.
Due to a high level of gene and pathway conservation, the strong similarity of
cellular
processes, and the functional conservation of genes between these model
organisms and
mammals, identification of the involvement of novel genes in particular
pathways and
their functions in such model organisms can directly contribute to the
understanding of the
correlative pathways and methods of modulating them in mammals (see, for
example,
Mechler BM et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res.
37: 33-
74; Watson KL., et al., 1994 J Cell Sci. 18: 19-33; Miklos GL, and Rubin GM.
1996 Cell
86:521-529; Wassarman DA, et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth
DR.
1999 Cancer Metastasis Rev. 18: 261-284). For example, a genetic screen can be
carried
out in an invertebrate model organism having underexpression (e.g. knockout)
or
overexpression of a gene (referred to as a "genetic entry point") that yields
a visible
phenotype, Additional genes are mutated in a xandom or targeted manner. When a
gene
mutation changes the original phenotype caused by the mutation in the genetic
entry point,
the gene is identified as a "modifier" involved in the same or overlapping
pathway as the
genetic entry point. When the genetic entry point is an ortholog of a human
gene
implicated in a disease pathway, such as p53, modifier genes can be identified
that may be
attractive candidate targets for novel therapeutics.
All references cited herein, including sequence information in referenced
Genbank
identifier numbers and website references, are incorporated herein in their
entireties.
SUMMARY OF TI3E INVENTION
We have discovered genes that modify the p53 pathway in Drosophila, and
identified
their human orthologs, hereinafter referred to as LCEs, The invention provides
methods
for utilizing these p53 modifier genes and polypeptides to identify candidate
therapeutic
agents that can be used in the treatment of disorders associated with
defective p53
function. Preferred LCE-modulating agents specifically bind to LCE
polypeptides and
restore p53 function. Other preferred LCE-modulating agents are nucleic acid
modulators
such as antisense oligomers and RNAi that repress LCE gene expression or
product
4
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
activity by, for example, binding to and inhibiting the respective nucleic
acid (i.e. DNA or
mRNA).
LCE-specific modulating agents may be evaluated by any convenient in vitro or
in
vivo assay for molecular interaction with an LCE polypeptide or nucleic acid.
In one
embodiment, candidate p53 modulating agents are tested with an assay system
comprising
a LCE polypeptide or nucleic acid. Candidate agents that produce a change in
the activity
of the assay system relative to controls are identified as candidate p53
modulating agents.
The assay system may be cell-based or cell-free. LCE-modulating agents include
LCE
related proteins (e.g. dominant negative mutants, and biotherapeutics); LCE-
specific
antibodies; LCE-specific antisense oligomers and other nucleic acid
modulators; and
chemical agents that specifically bind LCE or compete with LCE binding target.
In one
specific embodiment, a small molecule modulator is identified using a binding
assay. In
specific embodiments, the screening assay system is selected from an apoptosis
assay, a
cell proliferation assay, an angiogenesis assay, and a hypoxic induction
assay.
In another embodiment, candidate p53 pathway modulating agents are further
tested
using a second assay system that detects changes in the p53 pathway, such as
angiogenic,
apoptotic, or cell proliferation changes produced by the originally identified
candidate
agent or an agent derived from the original agent. The second assay system may
use
cultured cells or non-human animals. In specific embodiments, the secondary
assay
system uses non-human animals, including animals predetermined to have a
disease or
disorder implicating the p53 pathway, such as an angiogenic, apoptotic, or
cell
proliferation disorder (e.g. cancer).
The invention further provides methods for modulating the p53 pathway in a
mammalian cell by contacting the mammalian cell with an agent that
specifically binds a
LCE polypeptide or nucleic acid. The agent may be a small molecule modulator,
a nucleic
acid modulator, or an antibody and may be administered to a mammalian animal
predetermined to have a pathology associated the p53 pathway.
The invention further provides methods of identifying candidate branching
morphogenesis modulating agents and methods of modulating branching
morphogenesis
in mammalian cells using an LCE.
DETAILED DESCRIPTION OF THE INVENTION
Genetic screens were designed to identify modifiers of the p53 pathway in
Drosoplaila
in which p53 was overexpressed in the wing (Ollmann M, et al., Cell 2000 101:
91-101).
5
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
The baldspot gene was identified as a modifier of the p53 pathway.
Accordingly,
vertebrate orthologs of these modifiers, and preferably the human orthologs,
LCE genes
(i.e., nucleic acids and polypeptides) are attractive drug targets for the
treatment of
pathologies associated with a defective p53 signaling pathway, such as cancer.
We have further identified an LCE (ELOVL4) that is involved in branching
morphogenesis, specifically angiogenesis and vasculogenesis, as further
described in the
Examples. Accordingly, ELOVL4 is an attractive drug target for the treatment
of
pathologies related to branching morphogenesis, including the treatment of
tumors whose
growth is associated with increased angiogenesis.
In vitro and in vivo methods of assessing LCE function are provided herein.
Modulation of the LCE or their respective binding partners is useful for
understanding the
association of the p53 pathway and its members in normal and disease
conditions and for
developing diagnostics and therapeutic modalities for p53 related pathologies.
Modulation
of the ELOVL4 or its binding partners is further useful for elucidating the
process of
branching morphogenesis and the association of branching morphogenesis with
the p53
pathway, and for developing diagnostic and therapeutic modalities for
pathologies
associated with the branching morphogenesis. As used herein, branching
morphogenesis
encompasses the numerous cellular process involved in the formation of
branched
networks, including proliferation, survival/apoptosis, migration, invasion,
adhesion,
aggregation and matrix remodeling. As used herein, pathologies associated with
branching morphogenesis encompass pathologies where branching morphogenesis
contributes to maintaining the healthy state, as well as pathologies whose
course may be
altered by modulation of the branching morphogenesis.
LCE-modulating agents that act by inhibiting or enhancing LCE expression,
directly or
indirectly, for example, by affecting an LCE function such as enzymatic (e.g.,
catalytic) or
binding activity, can be identified using methods provided herein. LCE
modulating agents
are useful in diagnosis, therapy and pharmaceutical development.
Nucleic acids and nolypentides of the invention
Sequences related to LCE nucleic acids and polypeptides that can be used in
the
invention are disclosed in Genbank (referenced by Genbank identifier (GI)
number) as
GI#s 10444344 (SEQ ID NO:1), 13129087 (SEQ ID N0:3), 10440044 (SEQ ID N0:4),
12044042 (SEQ )D N0:5), 12232378 (SEQ m N0:6), and 18576451 (SEQ 1D N0:8) for
nucleic acid, and GI#s 10444345 (SEQ m N0:9), 13129088 (SEQ ~ N0:11), 10440045
6
WO 02/099068 PCT/US02/17739
ac
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
(SEQ ID N0:12), 12044043 (SEQ 1D N0:13), 12232379 (SEQ ID N0:14), and 17454617
(SEQ B7 N0:16) for polypeptides. Additionally, nucleic acid sequences of SEQ
ID NOs:
2 and 7, and polypeptide sequences of SEQ ID NOs: 10 and 15 can also be used
in the
invention.
LCEs are fatty acid elongase proteins with GNS1/SUR4 domains. The term "LCE
polypeptide" refers to a full-length LCE protein or a functionally active
fragment or
derivative thereof. A "functionally active" LCE fragment or derivative
exhibits one or
more functional activities associated with a full-length, wild-type LCE
protein, such as
antigenic or immunogenic activity, enzymatic activity, ability to bind natural
cellular
substrates, etc. The functional activity of LCE proteins, derivatives and
fragments can be
assayed by various methods known to one skilled in the art (Current Protocols
in Protein
Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, New
Jersey) and
as further discussed below. For purposes herein, functionally active fragments
also
include those fragments that comprise one or more structural domains of an
LCE, such as
a binding domain. Protein domains can be identified using the PFAM program
(Bateman
A., et al., Nucleic Acids Res, 1999, 27:260-2; http://pfam.wustl.edu). For
example, the
GNS1/SUR4 domain (PFAM 01151) of LCE from GI#s 10444345, 13129088, 12232379,
and 17454617 (SEQ ID NOs:9, I I, I4, and 16, respectively) are located
respectively at
approximately amino acid residues 1-235, 10 to 265, 9 to 289, and 55 to 270.
Methods for
obtaining LCE polypeptides are also further described below. In some
embodiments,
preferred fragments are functionally active, domain-containing fragments
comprising at
least 25 contiguous amino acids, preferably at least 50, more preferably 75,
and most
preferably at least 100 contiguous amino acids of any one of SEQ ID NOs:9, 10,
11, 12,
13, 14, 15, or 16 (an LCE). In further preferred embodiments, the fragment
comprises the
entire GNS 1/SUR4 (functionally active) domain.
The term "LCE nucleic acid" refers to a I~NA or RNA molecule that encodes a
LCE
polypeptide. Preferably, the LCE polypeptide or nucleic acid or fragment
thereof is from
a human, but can also be an ortholog, or derivative thereof with at least 70%
sequence
identity, preferably at least 80%, more preferably 85%, still more preferably
90%, and
most preferably at least 95% sequence identity with LCE. Normally, orthologs
in different
species retain the same function, due to presence of one or more protein
motifs and/or 3-
dimensional structures. Orthologs are generally identified by sequence
homology
analysis, such as BLAST analysis, usually using protein bait sequences.
Sequences are
assigned as a potential ortholog if the best hit sequence from the forward
BLAST result
7
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
retrieves the original query sequence in the reverse BLAST (Huynen MA and Bork
P,
Proc Natl Acad Sci (1998) 95:5849-5856; Huynen MA et al., Genome Research
(2000)
10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL
(Thompson JD et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to
highlight
conserved regions and/or residues of orthologous proteins and to generate
phylogenetic
trees. In a phylogenetic tree representing multiple homologous sequences from
diverse
species (e.g., retrieved through BLAST analysis), orthologous sequences from
two species
generally appear closest on the tree with respect to all other sequences from
these two
species. Structural threading or other analysis of protein folding (e.g.,
using software by
ProCeryon, Biosciences, Salzburg, Austria) may also identify potential
orthologs. In
evolution, when a gene duplication event follows speciation, a single gene in
one species,
such as Drosophila, may correspond to multiple genes (paralogs) in another,
such as
human. As used herein, the term "orthologs" encompasses paralogs. As used
herein,
"percent (%) sequence identity" with respect to a subject sequence, or a
specified portion
of a subject sequence, is defined as the percentage of nucleotides or amino
acids in the
candidate derivative sequence identical with the nucleotides or amino acids in
the subject
sequence (or specified portion thereof), after aligning the sequences and
introducing gaps,
if necessary to achieve the maximum percent sequence identity, as generated by
the
program WU-BLAST-2.Oa19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410;
http://blast.wustl.edu/blast/README.html) with all the search parameters set
to default
values. The HSP S and HSP S2 parameters are dynamic values and are established
by the
program itself depending upon the composition of the particular sequence and
composition
of the particular database against which the sequence of interest is being
searched. A %
identity value is determined by the number of matching identical nucleotides
or amino
acids divided by the sequence length for which the percent identity is being
reported.
"Percent (%) amino acid sequence similarity" is determined by doing the same
calculation
as for determining % amino acid sequence identity, but including conservative
amino acid
substitutions in addition to identical amino acids in the computation.
A conservative amino acid substitution is one in which an amino acid is
substituted for
another amino acid having similar properties such that the folding or activity
of the protein
is not significantly affected. Aromatic amino acids that can be substituted
for each other
are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino
acids are
leucine, isoleucine, methionine, and valine; interchangeable polar amino acids
are
glutamine and asparagine; interchangeable basic amino acids are arginine,
lysine and
8
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
histidine; interchangeable acidic amino acids are aspartic acid and glutamic
acid; and
interchangeable small amino acids are alanine, serine, threonine, cysteine and
glycine.
Alternatively, an alignment for nucleic acid sequences is provided by the
local
homology algoritlun of Smith and Waterman (Smith and Waterman, 1981, Advances
in
Applied Mathematics 2:482-489; database: European Bioinformatics Institute
http:l/www.ebi.ac.uk/MPsrch/; Smith and Waterman, 1981, J. of Molec.Biol.,
147:195-
197; Nicholas et al., 1998, "A Tutorial on Searching Sequence Databases and
Sequence
Scoring Methods" (www.psc.edu) and references cited therein.; W.R. Pearson,
1991,
Genomics 11:635-650). This algorithm can be applied to amino acid sequences by
using
the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences
and
Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research
Foundation, Washington, D.C., USA), and normalized by Crribskov (Gribskov 1986
Nucl. Acids Res. 14(6):6745-6763). The Smith-Waterman algorithm may be
employed
where default parameters are used for scoring (for example, gap open penalty
of 12, gap
extension penalty of two). From the data generated, the "Match" value reflects
"sequence
identity."
Derivative nucleic acid molecules of the subject nucleic acid molecules
include
sequences that hybridize to the nucleic acid sequence of any of SEQ ID NOs: l,
2, 3, 4, 5
,6 7, or 8. The stringency of hybridization can be controlled by temperature,
ionic
strength, pH, and the presence of denaturing agents such as formamide during
hybridization and washing. Conditions routinely used are set out in readily
available
procedure texts (e.g., Current Protocol in Molecular Biology, Vol. l, Chap.
2.10, John
Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold
Spring
Harbor (19$9)). In some embodiments, a nucleic acid molecule of the invention
is capable
of hybridizing to a nucleic acid molecule containing the nucleotide sequence
of any one of
SEQ ID NOs:l, 2, 3, 4, 5, 6, 7, or 8 under stringent hybridization conditions
that comprise:
prehybridization of filters containing nucleic acid for 8 hours to overnight
at 65° C in a
solution comprising 6X single strength citrate (SSC) (1X SSC is 0.15 M NaCI,
0.015 M
Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and
100 ~,g/ml
herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution
containing 6X
SSC, 1X Denhardt's solution, 100 ,uglml yeast tRNA and 0.05% sodium
pyrophosphate;
and washing of filters at 65° C for 1h in a solution containing 0.2X
SSC and 0.1% SDS
(sodium dodecyl sulfate).
9
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
In other embodiments, moderately stringent hybridization conditions are used
that
comprise: pretreatment of filters containing nucleic acid for 6 h at
40° C in a solution
containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH7.5), SmM EDTA, 0.1 % PVP,
0.1 % Ficoll, 1 % BSA, and 500 ~,g/ml denatured salmon sperm DNA;
hybridization for
18-20h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM
Tris-HCl
(pH7.5), 5mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ~.g/ml salmon sperm
DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at
55° C in
a solution containing 2X SSC and 0.1% SDS.
Alternatively, low stringency conditions can be used that comprise: incubation
for 8
hours to overnight at 37° C in a solution comprising 20% forrnamide, 5
x SSC, 50 mM
sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20
p,g/ml
denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to
20
hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.
Isolation, Production, Expression, and Mis-expression of LCE Nucleic Acids and
Polypeptides
LCE nucleic acids and polypeptides, useful for identifying and testing agents
that
modulate LCE function and for other applications related to the involvement of
LCE in the
p53 pathway. LCE nucleic acids and derivatives and orthologs thereof may be
obtained
using any available method. For instance, techniques for isolating cDNA or
genomic
DNA sequences of interest by screening DNA libraries or by using polymerase
chain
reaction (PCR) are well known in the art. In general, the particular use for
the protein will
dictate the particulars of expression, production, and purification methods.
For instance,
production of proteins for use in screening for modulating agents may require
methods
that preserve specific biological activities of these proteins, whereas
production of proteins
for antibody generation may require structural integrity of particular
epitopes. Expression
of proteins to be purified for screening or antibody production may require
the addition of
specific tags (e.g., generation of fusion proteins). Overexpression of an LCE
protein for
assays used to assess LCE function, such as involvement in cell cycle
regulation or
hypoxic response, may require expression in eukaryotic cell lines capable of
these cellular
activities. Techniques for the expression, production, and purification of
proteins are well
known in the art; any suitable means therefore may be used (e.g., Higgins SJ
and Hames
BD (eds.) Protein Expression: A Practical Approach, Oxford University Press
Inc., New
York 1999; Stanbury PF et al., Principles of Fermentation Technology, 2nd
edition,
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification
Protocols,
Humana Press, New Jersey, 1996; Coligan JE et al, Current Protocols in Protein
Science
(eds.), 1999, John Wiley & Sons, New York). In particular embodiments,
recombinant
LCE is expressed in a cell line known to have defective p53 function (e.g.
SAOS-2
osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-
29 and
DLD-1 colon cancer cells, among others, available from American Type Culture
Collection (ATCC), Manassas, VA). The recombinant cells are used in cell-based
screening assay systems of the invention, as described further below.
The nucleotide sequence encoding an LCE polypeptide can be inserted into any
appropriate expression vector. The necessary transcriptional and translational
signals,
including promoter/enhancer element, can derive from the native LCE gene
and/or its
flanking regions or can be heterologous. A variety of host-vector expression
systems may
be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia
virus,
adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus);
microorganisms such as yeast containing yeast vectors, or bacteria transformed
with
bacteriophage, plasmid, or cosnnid DNA. A host cell strain that modulates the
expression
of, modifies, andJor specifically processes the gene product may be used.
To detect expression of the LCE gene product, the expression vector can
comprise a
promoter operably linked to an LCE gene nucleic acid, one or more origins of
replication,
and, one or more selectable markers (e.g. thymidine kinase activity,
resistance to
antibiotics, etc.). Alternatively, recombinant expression vectors can be
identified by
assaying for the expression of the LCE gene product based on the physical or
functional
properties of the LCE protein in in vitro assay systems (e.g. immunoassays).
The LCE protein, fragment, or derivative may be optionally expressed as a
fusion, or
chimeric protein product (i.e. it is joined via a peptide bond to a
heterologous protein
sequence of a different protein), for example to facilitate purification or
detection. A
chimeric product can be made by ligating the appropriate nucleic acid
sequences encoding
the desired amino acid sequences to each other using standard methods and
expressing the
chimeric product. A chimeric product may also be made by protein synthetic
techniques,
e.g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984)
310:105-111).
Once a recombinant cell that expresses the LCE gene sequence is identified,
the gene
product can be isolated and purified using standard methods (e.g. ion
exchange, affinity,
and gel exclusion chromatography; centrifugation; differential solubility;
electrophoresis).
Alternatively, native LCE proteins can be purified from natural sources, by
standard
11
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
methods (e.g. immunoaffinity purification). Once a protein is obtained, it may
be
quantified and its activity measured by appropriate methods, such as
immunoassay,
bioassay, or other measurements of physical properties, such as
crystallography.
The methods of this invention may also use cells that have been engineered for
altered
expression (mis-expression) of LCE or other genes associated with the p53
pathway. As
used herein, mis-expression encompasses ectopic expression, over-expression,
under-
expression, and non-expression (e.g. by gene knock-out or blocking expression
that would
otherwise normally occur).
Genetically modified animals
Animal models that have been genetically modified to alter LCE expression may
be
used in iya vivo assays to test for activity of a candidate p53 modulating
agent, or t~ further
assess the role of LCE in a p53 pathway process such as apoptosis or cell
proliferation.
Preferably, the altered LCE expression results in a detectable phenotype, such
as
decreased or increased levels of cell proliferation, angiogenesis, or
apoptosis compared to
control animals having normal LCE expression. The genetically modified animal
may
additionally have altered p53 expression (e.g. p53 knockout). Preferred
genetically
modified animals are mammals such as primates, rodents (preferably mice),
cows, horses,
goats, sheep, pigs, dogs and cats. Preferred non-mammalian species include
zebrafish, C.
elegans, and Drosophila. Preferred genetically modified animals are transgenic
animals
having a heterologous nucleic acid sequence present as an extrachromosomal
element in a
portion of its cells, i.e. mosaic animals (see, for example, techniques
described by
Jakobovits, 1994, Curr. Biol. 4:761-763.) or stably integrated into its germ
line I~NA (i.e.,
in the genomic sequence of most or all of its cells). Heterologous nucleic
acid is
introduced into the germ line of such transgenic animals by genetic
manipulation of, for
example, embryos or embryonic stem cells of the host animal.
Methods of making transgenic animals are well-known in the art (for transgenic
mice
see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U.S. Pat.
Nos.
4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by
Wagner et al.,
and Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No.,
4,945,050,
by Sandford et al.; for transgenic Drosophila see Rubin and Spradling, Science
(1982)
218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see Berghammer
A.J. et
al., A Universal Marker for Transgenic Insects (1999) Nature 402:370-371; for
transgenic
12
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Zebrafish see Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000);136:3.75-
3830); for
microinjection procedures for fish, amphibian eggs and birds see Houdebine and
Chourrout, Experientia (1991) 47:897-905; for transgenic rats see Hammer et
al., Cell
(1990) 63:1099-1112; and for culturing of embryonic stem (ES) cells and the
subsequent
production of transgenic animals by the introduction of DNA into ES cells
using methods
such as electroporation, calcium phosphate/DNA precipitation and direct
injection see,
e.g., Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J.
Robertson,
ed., IRL Press (1987)). Clones of the nonhuman transgenic animals can be
produced
according to available methods (see Wilmut, I. et al. (1997) Nature 385:810-
813; and PCT
International Publication Nos. WO 97/07668 and WO 97/07669).
In one embodiment, the transgenic animal is a "knock-out" animal having a
heterozygous or homozygous alteration in the sequence of an endogenous LCE
gene that
results in a decrease of LCE function, preferably such that LCE expression is
undetectable
or insignificant. Knock-out animals are typically generated by homologous
recombination
with a vector comprising a transgene having at least a portion of the gene to
be knocked
out. Typically a deletion, addition or substitution has been introduced into
the transgene
to functionally disrupt it. The transgene can be a human gene (e.g., from a
human
genomic clone) but more preferably is an ortholog of the human gene derived
from the
transgenic host species. For example, a mouse LCE gene is used to construct a
homologous recombination vector suitable for altering an endogenous LCE gene
in the
mouse genome. Detailed methodologies for homologous recombination in mice are
available (see Capecchi, Science (1989) 244:1288-1292; Joyner et al., Nature
(1989)
338:153-156). Procedures for the production of non-rodent transgenic mammals
and other
animals are also available (Houdebine and Chourrout, supra; Pursel et al.,
Science (1989)
244:1281-1288; Simms et al., Bio/Technology (1988) 6:179-183). In a preferred
embodiment, knock-out animals, such as mice harboring a knockout of a specific
gene,
may be used to produce antibodies against the human counterpart of the gene
that has been
knocked out (Claesson MH et al., (1994) Scan J Imrnunol 40:257-264; Declerck
PJ et
al., (1995) J Biol Chem. 270:8397-400).
In another embodiment, the txansgenic aniriial is a "knock-in" animal having
an
alteration in its genome that results in altered expression (e.g., increased
(including
ectopic) or decreased expression) of the LCE gene, e.g., by introduction of
additional
copies of LCE, or by operatively inserting a regulatory sequence that provides
for altered
expression of an endogenous copy of the LCE gene. Such regulatory sequences
include
13
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
inducible, tissue-specific, and constitutive promoters and enhancer elements.
The knock-
in can be homozygous or heterozygous.
Transgenic nonhuman animals can also be produced that contain selected systems
allowing for regulated expression of the transgene. One example of such a
system that
may be produced is the crelloxP recombinase system of bacteriophage P1 (Lakso
et al.,
PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase
system
is used to regulate expression of the transgene, animals containing transgenes
encoding
both the Cre recombinase and a selected protein are required. Such animals can
be
provided through the construction of "double" transgenic animals, e.g., by
mating two
transgenic animals, one containing a transgene encoding a selected protein and
the other
containing a transgene encoding a recombinase. Another example of a
recombinase
system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et
al.
(1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a preferred
embodiment,
both Cre-LoxP and Flp-Frt are used in the same system to regulate expression
of the
transgene, and for sequential deletion of vector sequences in the same cell
(Sun X et al
(2000) Nat Genet 25:83-6).
The genetically modified animals can be used in genetic studies to further
elucidate the
p53 pathway, as animal models of disease and disorders implicating defective
p53
function, and for ifi vivo testing of candidate therapeutic agents, such as
those identified in
screens described below. The candidate therapeutic agents are administered to
a
genetically modified animal having altered LCE function and phenotypic changes
are
compared with appropriate control animals such as genetically modified animals
that
receive placebo treatment, and/or animals with unaltered LCE expression that
receive
candidate therapeutic agent.
In addition to the above-described genetically modified animals having altered
LCE
function, animal models having defective p53 function (and otherwise normal
LCE
function), can be used in the methods of the present invention. For example, a
p53
knockout mouse can be used to assess, irZ vivo, the activity of a candidate
p53 modulating
agent identified in one of the ifa vitro assays described below. p53 knockout
mice are
described in the literature (Jacks et al., Nature 2001;410:1111-1116, 1043-
1044;
Donehower et al., supra). Preferably, the candidate p53 modulating agent when
administered to a model system with cells defective in p53 function, produces
a detectable
phenotypic change in the model system indicating that the p53 function is
restored, i.e.,
the cells exhibit normal cell cycle progression.
14
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Modulating Agents
The invention provides methods to identify agents that interact with and/or
modulate
the function of LCE andlor the p53 pathway. Such agents are useful in a
variety of
diagnostic and therapeutic applications associated with the p53 pathway, as
well as in
further analysis of the LCE protein and its contribution to the p53 pathway.
Accordingly,
the invention also provides methods for modulating the p53 pathway comprising
the step
of specifically modulating LCE activity by administering a LCE-interacting or -
modulating agent.
In a preferred embodiment, LCE-modulating agents inhibit or enhance LCE
activity or
otherwise affect normal LCE function, including transcription, protein
expression, protein
localization, and cellular or extra-cellular activity. In a further preferred
embodiment, the
candidate p53 pathway- modulating agent specifically modulates the function of
the LCE.
The phrases "specific modulating agent", "specifically modulates", etc., are
used herein to
refer to modulating agents that directly bind to the LCE polypeptide or
nucleic acid, and
preferably inhibit, enhance, or otherwise alter, the function of the LCE. The
term also
encompasses modulating agents that alter the interaction of the LCE with a
binding partner
or substrate (e.g. by binding to a binding partner of an LCE, or to a
proteinlbinding partner
complex, and inhibiting function).
Preferred LCE-modulating agents include small molecule compounds; LCE-
interacting proteins, including antibodies and other biotherapeutics; and
nucleic acid
modulators such as antisense and RNA inhibitors. The modulating agents may be
formulated in pharmaceutical compositions, for example, as compositions that
may
comprise other active ingredients, as in combination therapy, and/or suitable
carriers or
excipients. Techniques for formulation and administration of the compounds may
be
found in "Remington's Pharmaceutical Sciences" Mack Publishing Co., Easton,
PA, 19~
edition.
Small molecule modulators
Small molecules, are often preferred to modulate function of proteins with
enzymatic
function, and/or containing protein interaction domains. Chemical agents,
referred to in
the art as "small molecule" compounds are typically organic, non-peptide
molecules,
having a molecular weight less than 10,000, preferably less than 5,000, more
preferably
less than 1,000, and most preferably less than 500. This class of modulators
includes
chemically synthesized molecules, for instance, compounds from combinatorial
chemical
15~
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
libraries. Synthetic compounds may be rationally designed or identified based
on known
or inferred properties of the LCE protein or may be identified by screening
compound
libraries. Alternative appropriate modulators of this class are natural
products, particularly
secondary metabolites from organisms such as plants or fungi, which can also
be
identified by screening compound libraries for LCE-modulating activity.
Methods for
generating and obtaining compounds are well known in the art (Schreiber SL,
Science
(2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).
Small molecule modulators identified from screening assays, as described
below, can
be used as lead compounds from which candidate clinical compounds may be
designed,
optimized, and synthesized. Such clinical compounds may have utility in
treating
pathologies associated with the p53 pathway. The activity of candidate small
molecule
modulating agents may be improved several-fold through iterative secondary
functional
validation, as further described below, structure determination, arid
candidate modulator
modification and testing. Additionally, candidate clinical compounds are
generated with
specific regard to clinical and pharmacological properties. For example, the
reagents may
be derivatized and re-screened using i~ vitro and ifZ vivo assays to optimize
activity and
minimize toxicity for pharmaceutical development.
Protein Modulators
Specific LCE-interacting proteins are useful in a variety of diagnostic and
therapeutic
applications related to the p53 pathway and related disorders, as well as in
validation
assays for other LCE-modulating agents. In a preferred embodiment, LCE-
interacting
proteins affect normal LCE function, including transcription, protein
expression, protein
localization, and cellular or extra-cellular activity. In another embodiment,
LCE-
interacting proteins are useful in detecting and providing information about
the function of
LCE proteins, as is relevant to p53 related disorders, such as cancer (e.g.,
for diagnostic
means).
An LCE-interacting protein may be endogenous, i.e. one that naturally
interacts
genetically or biochemically with an LCE, such as a member of the LCE pathway
that
modulates LCE expression, localization, and/or activity. LCE-modulators
include
dominant negative forms of LCE-interacting proteins and of LCE proteins
themselves.
Yeast two-hybrid and variant screens offer preferred methods for identifying
endogenous
LCE-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning-
Expression Systems:
A Practical Approach, eds. Glover D. ~z. Hames B. D (Oxford University Press,
Oxford,
I6
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
England), pp. 169-203; Fashema SF et al., Gene (2000) 250:1-14; Drees BL Curr
Opin
Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999)
27:919-29;
and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative preferred
method for
the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M,
Nature
(2000) 405:837-846; Yates JR 3rd, Trends Genet (2000) 16:5-8).
An LCE-interacting protein may be an exogenous protein, such as an LCE-
specific
antibody or a T-cell antigen receptor (see, e.g., Harlow and Lane (1988)
Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using
antibodies: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory
Press). LCE antibodies are further discussed below.
In preferred embodiments, an LCE-interacting protein specifically binds an LCE
protein. In alternative preferred embodiments, an LCE-modulating agent binds
an LCE
substrate, binding partner, or cofactor.
Antibodies
In another embodiment, the protein modulator is an LCE specific antibody
agonist or
antagonist. The antibodies have therapeutic and diagnostic utilities, and can
be used in
screening assays to identify LCE modulators. The antibodies can also be used
in
dissecting the portions of the LCE pathway responsible for various cellular
responses and
in the general processing and maturation of the LCE.
Antibodies that specifically bind LCE polypeptides can be generated using
known
methods. Preferably the antibody is specific to a mammalian ortholog of LCE
polypeptide, and more preferably, to human LCE. Antibodies may be polyclonal,
monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies,
Fab
fragments, F(ab')2 fragments, fragments produced by a FAb expression
library, anti-
idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the
above.
Epitopes of LCE which are particularly antigenic can be selected, for example,
by routine
screening of LCE polypeptides for antigenicity or by applying a theoretical
method for
selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati.
Acad. Sci.
U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Irnmunol. 20:483-89; Sutcliffe
et al.,
(1983) Science 219:660-66) to the amino acid sequence shown in any of SEQ ID
NOs:9,
10, 11, 12, 13, 14, 15, or 16. Monoclonal antibodies with affinities of 1081V1-
1 preferably
109 M-1 to 101° M-1, or stronger can be made by standard procedures as
described (Harlow
and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d ed)
17
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and
4,618,577).
Antibodies may be generated against crude cell extracts of LCE or
substantially purified
fragments thereof. If LCE fragments are used, they preferably comprise at
least 10, and
more preferably, at least 20 contiguous amino acids of an LCE protein. In a
particular
embodiment, LCE-specific antigens and/or immunogens are coupled to carrier
proteins
that stimulate the immune response. For example, the subject polypeptides are
covalently
coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is
emulsified
in Freund's complete adjuvant, which enhances the immune response. An
appropriate
immune system such as a laboratory rabbit or mouse is immunized according to
conventional protocols.
The presence of LCE-specific antibodies is assayed by an appropriate assay
such as a
solid phase enzyme-linked imrnunosorbant assay (ELISA) using immobilized
corresponding LCE polypeptides. Other assays, such as radioimmunoassays or
fluorescent assays might also be used.
Chimeric antibodies specific to LCE polypeptides can be made that contain
different
portions from different animal species. For instance, a human immunoglobulin
constant
region may be linked to a variable region of a murine mAb, such that the
antibody derives
its biological activity from the human antibody, and its binding specificity
from the
murine fragment. Chimeric antibodies are produced by splicing together genes
that
encode the appropriate regions from each species (Morrison et al., Proc. Natl.
Acad. Sci.
(1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et
al., Nature
(1985) 31:452-454). Humanized antibodies, which are a form of chimeric
antibodies, can
be generated by grafting complementary-determining regions (CI?Rs) (Carlos, T.
M., J. M.
Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of
human
framework regions and constant regions by recombinant I~NA technology
(Riechmann
LM, et al., 1988 Nature 323: 323-327). Humanized antibodies contain ~10%
murine
sequences and ~90% human sequences, and thus further reduce or eliminate
immunogenicity, while retaining the antibody specificities (Co MS, and Queen
C. 1991
Nature 351: 501-501; Morrison SL. 1992 Ann. Rev. Immun. 10:239-265). Humanized
antibodies and methods of their production are well-known in the art (U.S.
Pat. Nos.
5,530,101, 5,585,089, 5,693,762, and 6,180,370).
LCE-specific single chain antibodies which are recombinant, single chain
polypeptides
formed by linking the heavy and light chain fragments of the Fv regions via an
amino acid
bridge, can be produced by methods known in the art (U.S. Pat. No. 4,946,778;
Bird,
18
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988)
85:5879-
5883; and Ward et al, Nature (1989) 334:544-546).
Other suitable techniques for antibody production involve in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively to selection of
libraries of
antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-
1281). As
used herein, T-cell antigen receptors are included within the scope of
antibody modulators
(Harlow and Lane, 1988, supra).
The polypeptides and antibodies of the present invention may be used with or
without
modification. Frequently, antibodies will be labeled by joining, either
covalently or non
covalently, a substance that provides for a detectable signal, or that is
toxic to cells that
express the targeted protein (Menard S, et al., Int J. Biol Markers (1989)
4:131-134). A
wide variety of labels and conjugation techniques are known and are reported
extensively
in both the scientific and patent literature. Suitable labels include
radionuclides, enzymes,
substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting
Lanthanide
1S metals, chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the
Like (U.5. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149;
and 4,366,241). Also, recombinant immunoglobulins may be produced (U.5. Pat.
No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach
their
targets by conjugation with membrane-penetrating toxin proteins (U.5. Pat. No.
6,086,900).
When used therapeutically in a patient, the antibodies of the subject
invention axe
typically administered parenterally, when possible at the target site, or
intravenously. The
therapeutically effective dose and dosage regimen is determined by clinical
studies.
Typically, the amount of antibody administered is in the range of about 0.1
mg/kg -to
about 10 mg/kg of patient weight. For parenteral administration, the
antibodies are
formulated in a unit dosage injectable form (e.g., solution, suspension,
emulsion) in
association with a pharmaceutically acceptable vehicle. Such vehicles are
inherently
nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution,
dextrose
solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils,
ethyl
oleate, or liposome carriers may also be used. The vehicle may contain minor
amounts of
additives, such as buffers and preservatives, which enhance isotonicity and
chemical
stability or otherwise enhance therapeutic potential. The antibodies'
concentrations in
such vehicles are typically in the range of about 1 mglml to aboutl0 mglml.
19
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Immunotherapeutic methods are further described in the literature (US Pat. No.
5,859,206;
W00073469).
Specific biotherapeutics
In a preferred embodiment, an LCE-interacting protein may have biotherapeutic
applications. Biotherapeutic agents formulated in pharmaceutically acceptable
carriers
and dosages may be used to activate or inhibit signal ixansduction pathways.
This
modulation may be accomplished by binding a ligand, thus inhibiting the
activity of the
pathway; or by binding a receptor, either to inhibit activation of, or to
activate, the
receptor. Alternatively, the biotherapeutic may itself be a ligand capable of
activating or
inhibiting a receptor. Biotherapeutic agents and methods of producing them are
described
in detail in U.S. Pat. No. 6,146,628.
LCE ligand(s), antibodies to the ligand(s) or the LCE itself may be used as
biotherapeutics to modulate the activity of LCE in the p53 pathway.
Nucleic Acid Modulators
Other preferred LCE-modulating agents comprise nucleic acid molecules, such as
antisense oligomers or double stranded RNA (dsRNA), which generally inhibit
LCE
activity. Preferred nucleic acid modulators interfere with the function of the
LCE nucleic
acid such as DNA replication, transcription, translocation of the LCE RNA to
the site of
protein translation, translation of protein from the LCE RNA, splicing of the
LCE RNA to
yield one or more mRNA species, or catalytic activity which may be engaged in
or
facilitated by the LCE RNA.
In one embodiment, the antisense oligomer is an oligonucleotide that is
sufficiently
complementary to an LCE mRNA to bind to and prevent translation, preferably by
binding
to the 5' untranslated region. LCE-specific antisense oligonucleotides,
preferably range
from at least 6 to about 200 nucleotides. In some embodiments the
oligonucleotide is
preferably at least 10, 15, or 20 nucleotides in length. In other embodiments,
the
oligonucleotide is preferably Iess than 50, 40, or 30 nucleotides in length.
The
oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or
modified
versions thereof, single-stranded or double-stranded. The oligonucleotide can
be modified
at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide
may
include other appending groups such as peptides, agents that facilitate
transport across the
cell membrane, hybridization-triggered cleavage agents, and intercalating
agents.
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
In another embodiment, the antisense oligomer is a phosphothioate morpholino .
oligomer (PMO). PMOs are assembled from four different morpholino subunits,
each of
which contain one of four genetic bases (A, C, G, or T) linked to a six-
membered
morpholine ring. Polymers of these subunits are joined by non-ionic
phosphodiamidate
intersubunit linkages. Details of how to make and use PMOs and other antisense
oligomers are well known in the art (e.g. see W099118193; Probst JC, Antisense
Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281;
Summerton
J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev. :7:187-95; US Pat. No.
5,235,033; and US Pat No. 5,378,841).
Alternative preferred LCE nucleic acid modulators are double-stranded RNA
species
mediating RNA interference (RNAi). RNAi is the process of sequence-specific,
post-
transcriptional gene silencing in animals and plants, initiated by double-
stranded RNA
(dsRNA) that is homologous in sequence to the silenced gene. Methods relating
to the use
of RNAi to silence genes in C. elegans, Dr-osoplaila, plants, and humans are
known in the
I5 art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15,
358-363 (1999);
Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S.
M.,
et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2,
239-245
(2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et
al.,
Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000);
Bernstein, E.,
et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15,
188-200
(2001); WO0129058; W09932619; Elbashir SM, et al., 2001 Nature 411:494-498).
Nucleic acid modulators are commonly used as research reagents, diagnostics,
and
therapeutics. For example, antisense oligonucleotides, which are able to
inhibit gene
expression with exquisite specificity, are often used to elucidate the
function of particular
genes (see, for example, U.S. Pat. No. 6,165,790). Nucleic acid modulators are
also used,
for example, to distinguish between functions of various members of a
biological pathway.
For example, antisense oligomers have been employed as therapeutic moieties in
the
treatment of disease states in animals and man and have been demonstrated in
numerous
clinical trials to be safe and effective (Milligan JF, et al, Current Concepts
in Antisense
Drug Design, J Med Chem. (19'93) 36:1923-1937; Tonkinson JL et al., Antisense
Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996)
14:54-65).
Accordingly, in one aspect of the invention, an LCE-specific nucleic acid
modulator is
used in an assay to further elucidate the role of the LCE in the p53 pathway,
and/or its
relationship to other members of the pathway. In another aspect of the
invention, an LCE-
21
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
specific antisense oligomer is used as a therapeutic agent for treatment of
p53-related
disease states.
Assay Systems
The invention provides assay systems and screening methods for identifying
specific
modulators of LCE activity. As used herein, an "assay system" encompasses all
the
components required for performing and analyzing results of an assay that
detects andlor
measures a particular event. In general, primary assays are used to identify
or confirm a
modulator's specific biochemical or molecular effect with respect to the LCE
nucleic acid
or protein. In general, secondary assays further assess the activity of a LCE
modulating
agent identified by a primary assay and may confirm that the modulating agent
affects
LCE in a manner relevant to the p53 pathway. In some cases, LCE modulators
will be
directly tested in a secondary assay.
In a preferred embodiment, the screening method comprises contacting a
suitable
assay system comprising an LCE polypeptide with a candidate agent under
conditions
whereby, but for the presence of the agent, the system provides a reference
activity (e.g.
binding activity), which is based on the particular molecular event the
screening method
detects. A statistically significant difference between the agent-biased
activity and the
reference activity indicates that the candidate agent modulates LCE activity,
and hence the
p53 pathway.
Primary Assays
The type of modulator tested generally determines the type of primary assay.
Primary assays for small molecule modulators
For small molecule modulators, screening assays are used to identify candidate
modulators. Screening assays may be cell-based or may use a cell-free system
that
recreates or retains the relevant biochemical reaction of the target protein
(reviewed in
Sittampalam GS et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying
references). As used herein the term "cell-based" refers to assays using live
cells, dead
cells, or a particular cellular fraction, such as a membrane, endoplasmic
reticulum, or
mitochondria) fraction. The term "cell free" encompasses assays using
substantially
purified protein (either endogenous or recombinantly produced), partially
purified or crude
cellular extracts. Screening assays may detect a variety of molecular events,
including
22
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand
binding),
transcriptional activity (e.g., using a reporter gene), enzymatic activity
(e.g., via a property
of the substrate), activity of second messengers, immunogenicty and changes in
cellular
morphology or other cellular characteristics. Appropriate screening assays may
use a wide
range of detection methods including fluorescent, radioactive, colorimetric,
spectrophotometric, and amperometric methods, to provide a read-out for the
particular
molecular event detected.
Cell-based screening assays usually require systems for recombinant expression
of
LCE and any auxiliary proteins demanded by the particular assay. Appropriate
methods
for generating recombinant proteins produce sufficient quantities of proteins
that retain
their relevant biological activities and are of sufficient purity to optimize
activity and
assure assay reproducibility. Yeast two-hybrid and variant screens, and mass
spectrometry
provide preferred methods for determining protein-protein interactions and
elucidation of
protein complexes. In certain applications, when LCE-interacting proteins are
used in
screens to identify small molecule modulators, the binding specificity of the
interacting
protein to the LCE protein may be assayed by various known methods such as
substrate
processing (e.g. ability of the candidate LCE-specific binding agents to
function as
negative effectors in LCE-expressing cells), binding equilibrium constants
(usually at least
about 107 M-1, preferably at least about 108 M-1, more preferably at least
about 109 M-1),
and immunogenicity (e.g. ability to elicit LCE specific antibody in a
heterologous host
such as a mouse, rat, goat or rabbit). For enzymes and receptors, binding may
be assayed
by, respectively, substrate and ligand processing.
The screening assay may measure a candidate agent's ability to specifically
bind to or
modulate activity of a LCE polypeptide, a fusion protein thereof, or to cells
or membranes
bearing the polypeptide or fusion protein. The LCE polypeptide can be full
length or a
fragment thereof that retains functional LCE activity. The LCE polypeptide may
be fused
to another polypeptide, such as a peptide tag for detection or anchoring, or
to another tag.
The LCE polypeptide is preferably human LCE, or is an ortholog or derivative
thereof as
described above. In a preferred embodiment, the screening assay detects
candidate agent-
based modulation of LCE interaction with a binding target, such as an
endogenous or
exogenous protein or other substrate that has LCE -specific binding activity,
and can be
used to assess normal LCE gene function.
Suitable assay formats that may be adapted to screen for LCE modulators are
known in
the art. Preferred screening assays are high throughput or ultra high
throughput and thus
23
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
provide automated, cost-effective means of screening compound libraries for
lead
compounds (Fernandes PB, Curr Opin Chem Biol (1998) 2:597-603; Sundberg SA,
Curr
Opin Biotechnol 2000, 11:47-53). In one preferred embodiment, screening assays
uses
fluorescence technologies, including fluorescence polarization, time-resolved
fluorescence, and fluorescence resonance energy transfer. These systems offer
means to
monitor protein-protein or DNA-protein interactions in which the intensity of
the signal
emitted from dye-labeled molecules depends upon their interactions with
partner
molecules (e.g., Selvin PR, Nat Struct Biol (2000) 7:730-4; Fernandes PB,
supra;
Hertzberg RP and Pope AJ, Curr Opin Chem Biol (2000) 4:445-451).
A variety of suitable assay systems may be used to identify candidate LCE and
p53
pathway modulators (e.g. U.S. Pat. No. 6,020,135 (p53 modulation)). Specific
preferred
assays are described in more detail below.
Elongase assays. Assays for elongases are well-known in the art. In one
example, an
elongation assay uses chromatographic (e.g., HPLC) analysis of labeled
elongation
products to assess LCE activity. Long chain fatty acids may be labeled with 14-
C (Moon
YA et al. (2001) J Biol Chem 276:45358-45366). Briefly, fatty acid elongation
activity is
measured in microsomes prepared from transfected cells. Fatty acyl-CoAs or BSA
bound
fatty acids may be used as substrates for the reactions. Substrate mixtures
also include [2-
14C]malonyl-CoA. Elongation reactions are initiated when microsomal proteins
and
substrates are mixed. After termination of the reactions, fatty acids are
collected, washed,
and counted. Elongase activity is measured and expressed as the amount of
radioactivity
incorporated into the fatty acids.
Apoptosis assays. Assays for apoptosis may be performed by terminal
deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling
(TUNEL)
assay. The TUNEL assay is used to measure nuclear DNA fragmentation
characteristic of
apoptosis ( Lazebnik et al., 1994, Nature 371, 346), by following the
incorporation of
fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747). Apoptosis
may further
be assayed by acridine orange staining of tissue culture cells (Lucas, R., et
al., 1998, Blood
15:4730-41). An apoptosis assay system may comprise a cell that expresses an
LCE, and
that optionally has defective p53 function (e.g. p53 is over-expressed or
under-expressed
relative to wild-type cells). A test agent can be added to the apoptosis assay
system and
changes in induction of apoptosis relative to controls where no test agent is
added, identify
24
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
candidate p53 modulating agents. In some embodiments of the invention, an
apoptosis
assay may be used as a secondary assay to test a candidate p53 modulating
agents that is
initially identified using a cell-free assay system. An apoptosis assay may
also be used to
test whether LCE function plays a direct role in apoptosis. For example, an
apoptosis
assay may be performed on cells that over- or under-express LCE relative to
wild type
cells. Differences in apoptotic response compared to wild type cells suggests
that the LCE
plays a direct role in the apoptotic response. Apoptosis assays are described
further in US
Pat. No. 6,133,437.
Cell proliferation and cell cycle assays. Cell proliferation may be assayed
via
bromodeoxyuridine (BRDU) incorporation. This assay identifies a cell
population
undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA.
Newly-synthesized DNA may then be detected using an anti-BRDU antibody
(Hoshino et
al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth.
107, 79), or by
other means.
Cell Proliferation may also be examined using [3H]-thymidine incorporation
(Chen; J.,
1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73).
This
assay allows for quantitative characterization of S-phase DNA syntheses. In
this assay,
cells synthesizing DNA will incorporate [3H]-thymidine into newly synthesized
DNA.
Incorporation can then be measured by standard techniques such as by counting
of
radioisotope in a scintillation counter (e.g., Beckman LS 3800 Liquid
Scintillation
Counter).
Cell proliferation may also be assayed by colony formation in soft agar
(Sambrook et
al., Molecular Cloning, Cold Spring Harbor (1989)). For example, cells
transformed with
LCE are seeded in soft agar plates, and colonies are measured and counted
after two
weeks incubation.
Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray
JW et
al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55). Cells
transfected with
an LCE may be stained with propidium iodide and evaluated in a flow cytometer
(available from Becton Dickinson).
Accordingly, a cell proliferation or cell cycle assay system may comprise a
cell that
expresses an LCE, and that optionally has defective p53 function (e.g. p53 is
over-
expressed or under-expressed relative to wild-type cells). A test agent can be
added to the
assay system and changes in cell proliferation or cell cycle relative to
controls where no
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
test agent is added, identify candidate p53 modulating agents. In some
embodiments of
the invention, the cell proliferation or cell cycle assay may be used as a
secondary assay to
test a candidate p53 modulating agents that is initially identified using
another assay
system such as a cell-free assay system. A cell proliferation assay may also
be used to test
whether LCE function plays a direct role in cell proliferation or cell cycle.
For example, a
cell proliferation or cell cycle assay rnay be performed on cells that over-
or under-express
LCE relative to wild type cells. Differences in proliferation or cell cycle
compared to wild
type cells suggests that the LCE plays a direct role in cell proliferation or
cell cycle.
Angiogenesis. Angiogenesis may be assayed using various human endothelial cell
systems, such as umbilical vein, coronary artery, or dermal cells. Suitable
assays include
Alamar Blue based assays (available from Biosource International) to measure
proliferation; migration assays using fluorescent molecules, such as the use
of Becton
Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of
cells
through membranes in presence or absence of angiogenesis enhancer or
suppressors; and
tubule formation assays based on the formation of tubular structures by
endothelial cells
on Matrigel~ (Becton Dickinson). Accordingly, an angiogenesis assay system may
comprise a cell that expresses an LCE, and that optionally has defective p53
function (e.g.
p53 is over-expressed or under-expressed relative to wild-type cells). A test
agent can be
added to the angiogenesis assay system and changes in angiogenesis relative to
controls
where no test agent is added, identify candidate p53 modulating agents. In
some
embodiments of the invention, the angiogenesis assay may be used as a
secondary assay to
test a candidate p53 modulating agents that is initially identified using
another assay
system. An angiogenesis assay may also be used to test whether LCE function
plays a
direct role in cell proliferation. For example, an angiogenesis assay may be
performed on
cells that over- or under-express LCE relative to wild type cells. Differences
in
angiogenesis compared to wild type cells suggests that the LCE plays a direct
role in
angiogenesis.
Hypoxic induction. The alpha subunit of the transcription factor, hypoxia
inducible
factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia
in vitro.
Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be
important in tumour cell survival, such as those encoding glyolytic enzymes
and VEGF.
Induction of such genes by hypoxic conditions may be assayed by growing cells
26
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
transfected with LCE in hypoxic conditions (such as with 0.1°Io 02,
5°lo C02, and balance
N2, generated in a Napco 7001 incubator (Precision Scientific)) and normoxic
conditions,
followed by assessment of gene activity or expression by Taqman~. For example,
a
hypoxic induction assay system may comprise a cell that expresses an LCE, and
that
optionally has a mutated p53 (e.g. p53 is over-expressed or under-expressed
relative to
wild-type cells). A test agent can be added to the hypoxic induction assay
system and
changes in hypoxic response relative to controls where no test agent is added,
identify
candidate p53 modulating agents. In some embodiments of the invention, the
hypoxic
induction assay may be used as a secondary assay to test a candidate p53
modulating
agents that is initially identified using another assay system. A hypoxic
induction assay
may also be used to test whether LCE function plays a direct role in the
hypoxic response.
For example, a hypoxic induction assay may be performed on cells that over- or
under-
express LCE relative to wild type cells. Differences in hypoxic response
compared to wild
type cells suggests that the LCE plays a direct role in hypoxic induction.
Cell adhesion. Cell adhesion assays measure adhesion of cells to purified
adhesion
proteins, or adhesion of cells to each other, in presence or absence of
candidate
modulating agents. Cell-protein adhesion assays measure the ability of agents
to modulate
the adhesion of cells to purified proteins. For example, recombinant proteins
are
produced, diluted to 2.Sg/mL in PBS, and used to coat the wells of a
microtiter plate. The
wells used for negative control are not coated. Coated wells are then washed,
blocked
with 1% BSA, and washed again. Compounds are diluted to 2x final test
concentration
and added to the blocked, coated wells. Cells are then added to the wells, and
the unbound
cells are washed off. Retained cells are labeled directly on the plate by
adding a
membrane-permeable fluorescent dye, such as calcein-AM, and the signal is
quantified in
a fluorescent microplate reader.
Cell-cell adhesion assays measure the ability of agents to modulate binding of
cell
adhesion proteins with their native ligands. These assays use cells that
naturally or
recombinantly express the adhesion protein of choice. In an exemplary assay,
cells
expressing the cell adhesion protein are plated in wells of a multiwell plate.
Cells
expressing the ligand are labeled with a membrane-permeable fluorescent dye,
such as
BCECF , and allowed to adhere to the monolayers in the presence of candidate
agents.
Unbound cells are washed off, and bound cells are detected using a
fluorescence plate
reader.
27
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
High-throughput cell adhesion assays have also been described. In one such
assay,
small molecule ligands and peptides are bound to the surface of microscope
slides using a
microarray spotter, intact cells are then contacted with the slides, and
unbound Bells are
washed off. In this assay, not only the binding specificity of the peptides
and modulators
against cell lines are determined, but also the functional cell signaling of
attached cells
using immunofluorescence techniques in situ on the microchip is measured
(Falsey JR et
al., Bioconjug Chem. 2001 May-Jun;l2(3):346-53).
Tubulogenesis. Tubulogenesis assays monitor the ability of cultured cells,
generally
endothelial cells, to form tubular structures on a matrix substrate, which
generally
simulates the environment of the extracellular matrix. Exemplary substrates
include
Matrigel~ (Becton Dickinson), an extract of basement membrane proteins
containing
laminin, collagen IV, and heparin sulfate proteoglycan, which is liquid at
4° C and forms a
solid gel at 37° C. Other suitable matrices comprise extracellular
components such as
collagen, fibronectin, and/or fibrin. Cells are stimulated with a pro-
angiogenic stimulant,
and their ability to form tubules is detected by imaging. Tubules can
generally be detected
after an overnight incubation with stimuli, but longer or shorter time frames
may also be
used. Tube formation assays are well known in the art (e.g., Jones MK et al.,
1999, Nature
Medicine 5:1418-1423). These assays have traditionally involved stimulation
with serum
or with the growth factors FGF or VEGF. Serum represents an undefined source
of
growth factors. In a preferred embodiment, the assay is performed with cells
cultured in
serum free medium, in order to control which process or pathway a candidate
agent
modulates. Moreover, we have found that different target genes respond
differently to
stimulation with different pro-angiogenic agents, including inflammatory
angiogenic
factors such as TNF-alpa. Thus, in a further preferred embodiment, a
tubulogenesis assay
system comprises testing an LCE's response to a variety of factors, such as
FGF, VEGF,
phorbol myristate acetate (PMA), TNF-alpha, ephrin, etc.
Cell Migration. An invasion/migration assay (also called a migration assay)
tests the
ability of cells to overcome a physical barrier and to migrate towards pro-
angiogenic
signals. Migration assays are known in the art (e.g., Paik JH et al., 2001, J
Biol Chem
276:11830-11837). In a typical experimental set-up, cultured endothelial cells
are seeded
onto a matrix-coated porous lamina, with pore sizes generally smaller than
typical cell
size. The matrix generally simulates the environment of the extracellular
matrix, as
28
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
described above. The lamina is typically a membrane, such as the transwell
polycarbonate
membrane (Corning Costar Corporation, Cambridge, MA), and is generally part of
an
upper chamber that is in fluid contact with a lower chamber containing pro-
angiogenic
stimuli. Migration is generally assayed after an overnight incubation with
stimuli, but
longer or shorter time frames may also be used. Migration is assessed as the
number of
cells that crossed the lamina, and may be detected by staining cells with
hemotoxylin
solution (VWR Scientific, South San Francisco, CA), or by any other method fox
determining cell number. In another exemplary set up, cells are fluorescently
labeled and
migration is detected using fluorescent readings, for instance using the
Falcon HTS
FluoroBlok (Becton Dickinson). While some migration is observed in the absence
of
stimulus, migration is greatly increased in response to pro-angiogenic
factors. As
described above, a preferred assay system for migration/invasion assays
comprises testing
an LCE's response to a variety of pro-angiogenic factors, including tumor
angiogenic and
inflammatory angiogenic agents, and culturing the cells in serum free medium.
Sprouting assay. A sprouting assay is a three-dimensional in vitro
angiogenesis
assay that uses a cell-number defined spheroid aggregation of endothelial
cells
("spheroid"), embedded in a collagen gel-based matrix. The spheroid can serve
as a
starting point for the sprouting of capillary-like structures by invasion into
the
extracellular matrix (termed "cell sprouting") and the subsequent formation of
complex
anastomosing networks (Korff and Augustin, 1999, J Cell Sci 112:3249-58). In
an
exemplary experimental set-up, spheroids are prepared by pipetting 400 human
umbilical
vein endothelial cells into individual wells of a nonadhesive 96-well plates
to allow
overnight spheroidal aggregation (Korff and Augustin: J Cell Biol 143: 1341-
52, 1998).
Spheroids are harvested and seeded in 900p,1 of methocel-collagen solution and
pipetted
into individual wells of a 24 well plate to allow collagen gel polymerization.
Test agents
are added after 30 min by pipetting 100 ~,1 of 10-fold concentrated working
dilution of the
test substances on top of the gel. Plates are incubated at 37°C for
24h. Dishes are fixed at
the end of the experimental incubation period by addition of paraformaldehyde.
Sprouting
intensity of endothelial cells can be quantitated by an automated image
analysis system to
determine the cumulative sprout length per spheroid.
29
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Primary assays for antibody modulators
For antibody modulators, appropriate primary assays test is a binding assay
that tests
the antibody's affinity to and specificity for the LCE protein. Methods for
testing antibody
affinity and specificity are well known in the art (Harlow and Lane, 1988,
1999, supra).
The enzyme-linked immunosorbant assay (ELISA) is a preferred method for
detecting
LCE-specific antibodies; others include FACS assays, radioimmunoassays, and
fluorescent assays.
Primary assays for nucleic acid modulators
For nucleic acid modulators, primary assays may test the ability of the
nucleic acid
modulator to inhibit or enhance LCE gene expression, preferably mRNA
expression. In
general, expression analysis comprises comparing LCE expression in like
populations of
cells (e.g., two pools of cells that endogenously or recombinantly express
LCE) in the
presence and absence of the nucleic acid modulator. Methods for analyzing mRNA
and
protein expression are well known in the art. For instance, Northern blotting,
slot blotting,
ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan~, PE
Applied
Biosystems), or microarray analysis may be used to confirm that LCE mRNA
expression
is reduced in cells treated with the nucleic acid modulator (e.g., Current
Protocols in
Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc.,
chapter 4;
Freeman WM et al., Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med
2001,
33:142-147; Blohm DH and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-
47).
Protein expression may also be monitored. Proteins are most commonly detected
with
specific antibodies or antisera directed against either the LCE protein or
specific peptides.
A variety of means including Western blotting, ELISA, or in situ detection,
are available
(Harlow E and Lane D, 1988 and 1999, supra).
Secondary Assays
Secondary assays may be used to further assess the activity of LCE-modulating
agent
identified by any of the above methods to confirm that the modulating agent
affects LCE
in a manner relevant to the p53 pathway. As used herein, LCE-modulating agents
encompass candidate clinical compounds or other agents derived from previously
identified modulating agent. Secondary assays can also be used to test the
activity of a
modulating agent on a particular genetic or biochemical pathway or to test the
specificity
of the modulating agent's interaction with LCE.
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Secondary assays generally compare like populations of cells or animals (e.g.,
two
pools of cells or animals that endogenously or recombinantly express LCE) in
the presence
and absence of the candidate modulator. In general, such assays test whether
treatment of
cells or animals with a candidate LCE-modulating agent results in changes in
the p53
pathway in comparison to untreated (or mock- or placebo-treated) cells or
animals.
Certain assays use "sensitized genetic backgrounds", which, as used herein,
describe cells
or animals engineered for altered expression of genes in the p53 or
interacting pathways.
Cell-based assays
Cell based assays may use a variety of mammalian cell lines known to have
defective
p53 function (e.g. SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3
cervical
cancer cells, HT-29 and DLD-1 colon cancer cells, among others, available from
American Type Culture Collection (ATCC), Manassas, VA). Cell based assays may
detect endogenous p53 pathway activity or may rely on recombinant expression
of p53
pathway components. Any of the aforementioned assays may be used in this cell-
based
format. Candidate modulators are typically added to the cell media but may
also be
injected into cells or delivered by any other efficacious means.
Aninzal Assays
A variety of non-human animal models of normal or defective p53 pathway may be
used to test candidate LCE modulators. Models for defective pS3 pathway
typically use
genetically modified animals that have been engineered to mis-express (e.g.,
over-express
or lack expression in) genes involved in the p53 pathway. Assays generally
require
systemic delivery of the candidate modulators, such as by oral administration,
injection,
etc.
In a preferred embodiment, p53 pathway activity is assessed by monitoring
neovascularization and angiogenesis. Animal models with defective and normal
p53 are
used to test the candidate modulator's affect on LCE in Matrigel~ assays.
Matrigel~ is an
extract of basement membrane proteins, and is composed primarily of laminin,
collagen
IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at
4° C, but rapidly
forms a solid gel at 37° C. Liquid Matrigel~ is mixed with various
angiogenic agents,
such as bFGF and VEGF, or with human tumor cells which over-express the LCE.
The
mixture is then injected subcutaneously(SC) into female athymic nude mice
(Taconic,
Germantown, NY) to support an intense vascular response. Mice with Matrigel~
pellets
31
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
may be dosed via oral (PO), intraperitoneal (IP), or intravenous (1V) routes
with the
candidate modulator. Mice are euthanized 5 - 12 days post-injection, and the
Matrigel~
pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit).
Hemoglobin
content of the gel is found to correlate the degree of neovascularization in
the gel.
In another preferred embodiment, the effect of the candidate modulator on LCE
is
assessed via tumorigenicity assays. In one example, xenograft human tumors are
implanted SC into female athymic mice, 6-7 week old, as single cell
suspensions either
from a pre-existing tumor or from in vitro culture. The tumors which express
the LCE
endogenously are injected in the flank, 1 x 105 to 1 x 10~ cells per mouse in
a volume of
100 p,L using a 27gauge needle. Mice are then ear tagged and tumors are
measured twice
weekly. Candidate modulator treatment is initiated on the day the mean tumor
weight
reaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus
administration. Depending upon the pharmacokinetics of each unique candidate
modulator, dosing can be performed multiple times per day. The tumor weight is
assessed.
by measuring perpendicular diameters with a caliper and calculated by
multiplying the
measurements,of diameters in two dimensions. At the end of the experiment, the
excised
tumors maybe utilized for biomarker identification or further analyses. For
immunohistochemistry staining, xenograft tumors are fixed in 4%
paraformaldehyde,
O.1M phosphate, pH 7.2, for 6 hours at 4°C, immersed in 30% sucrose in
PBS, and rapidly
frozen in isopentane cooled with liquid nitrogen.
Diagnostic and therapeutic uses
Specific LCE-modulating agents are useful in a variety of diagnostic and
therapeutic
applications where disease or disease prognosis is related to defects in the
p53 pathway,
such as angiogenic, apoptotic, or cell proliferation disorders. Accordingly,
the invention
also provides methods for modulating the p53 pathway in a cell, preferably a
cell pre-
determined to have defective p53 function, comprising the step of
administering an agent
to the cell that specifically modulates LCE activity. Preferably, the
modulating agent
produces a detectable phenotypic change in the cell indicating that the p53
function is
restored, i.e., for example, the cell undergoes normal proliferation or
progression through
the cell cycle.
The discovery that LCE is implicated in p53 pathway provides for a variety of
methods that can be employed for the diagnostic and prognostic evaluation of
diseases and
32
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
disorders involving defects in the p53 pathway and for the identification of
subjects having
a predisposition to such diseases and disorders.
Various expression analysis methods can be used to diagnose whether LCE
expression
occurs in a particular sample, including Northern blotting, slot blotting,
ribonuclease
protection, quantitative RT-PCR, and microarray analysis. (e.g., Current
Protocols in
Molecular Biology (1994) Ausubel FM et al., eds., John Wiley & Sons, Inc.,
chapter 4;
Freeman WM et al., Biotechniques (1999) 26:112-125; Kallioniemi OP, Ann Med
2001,
33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001, 12:41-47).
Tissues
having a disease or disorder implicating defective p53 signaling that express
an LCE, are
identified as amenable to treatment with an LCE modulating agent. In a
preferred
application, the p53 defective tissue overexpresses an LCE relative to normal
tissue. For
example, a Northern blot analysis of mRNA from tumor and normal cell lines, or
from
tumor and matching normal tissue samples from the same patient, using full or
partial LCE
cDNA sequences as probes, can determine whether particular tumors express or
overexpress LCE. Alternatively,~the TaqMan~ is used for quantitative RT-PCR
analysis
of LCE expression in cell lines, normal tissues and tumor samples (PE Applied
Biosystems).
Various other diagnostic methods may be performed, for example, utilizing
reagents
such as the LCE oligonucleotides, and antibodies directed against an LCE, as
described
above for: (1) the detection of the presence of LCE gene mutations, or the
detection of
either over- or under-expression of LCE mRNA relative to the non-disorder
state; (2) the
detection of either an over- or an under-abundance of LCE gene product
relative to the
non-disorder state; and (3) the detection of perturbations or abnormalities in
the signal
transduction pathway mediated by LCE.
Thus, in a specific embodiment, the invention is drawn to a method for
diagnosing a
disease in a patient, the method comprising: a) obtaining a biological sample
from the
patient; b) contacting the sample with a probe for LCE expression; c)
comparing results
from step (b) with a control; and d) determining whether step (c) indicates a
likelihood of
disease. Preferably, the disease is cancer, most preferably a cancer as shown
in TABLE 1.
The probe may be either DNA or protein, including an antibody.
EXAMPLES
The following experimental section and examples are offered by way of
illustration
and not by way of limitation.
33
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
I. Droso~hila p53 screen
The Drosophila p53 gene was overexpressed specifically in the wing using the
vestigial margin quadrant enhancer. Increasing quantities of Drosophila p53
(titrated
using different strength transgenic inserts in 1 or 2 copies) caused
deterioration of normal
wing morphology from mild to strong, with phenotypes including disruption of
pattern and
polarity of wing hairs, shortening and thickening of wing veins, progressive
crumpling of
the wing and appearance of dark "death" inclusions in wing blade. In a screen
designed to
identify enhancers and suppressors of Drosophila p53, homozygous females
carrying two
copies of p53 were crossed to 5663 males carrying random insertions of a
piggyBac
transposon (Eraser M et al., Virology (1985) 145:356-361). Progeny containing
insertions
were compared to non-insertion-bearing sibling progeny for enhancement or
suppression
of the p53 phenotypes. Sequence information surrounding the piggyBac insertion
site was
used to identify the modifier genes. Modifiers of the wing phenotype were
identified as
members of the p53 pathway. baldspot was an enhancer of the wing phenotype.
Human
orthologs of the modifiers are referred to herein as LCE.
BLAST analysis (Altschul et al., supra) was employed to identify Targets from
Drosophila modifiers. [For example, representative sequences from LCE, GI#s
10444345,
13129088, 12232379, and 17454617 (SEQ ID NOs:9, 11, 14, and 16, respectivley)
share
45%, 48%, 25%, and 41% amino acid identity, respectively, with the
l~rosophila.baldspot.
Various domains, signals, and functional subunits in proteins were analyzed
using the
PSORT (Nakai K., and Horton P., Trends Biochem Sci, 1999, 24:34-6; Kenta
Nakai,
Protein sorting signals and prediction of subcellular localization, Adv.
Protein Chem. 54,
277-344 (2000)), PFAM (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2;
htt~://pfam.wustl.edu), SMART (Ponting CP, et al., SMART: identification and
annotation
of domains from signaling and extracellular protein sequences. Nucleic Acids
Res. 1999
Jan 1;27(1):229-32), TM-HMM (Erik L.L. Sonnhammer, Gunnar von Heijne, and
Anders
Krogh: A hidden Markov model for predicting transmembrane helices in protein
sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular
Biology, p
175-I82 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen
Menlo Park, CA: AAAI Press, 1998), and clust (Remm M, and Sonnhammer E.
Classification of transmembrane protein families in the Caenorhabditis elegans
genome
and identification of human orthologs. Genome Res. 2000 Noe;lO(11):1679-89)
programs.
Using PFAM, GNS1/SUR4 domain (PFAM 01151) of LCE from GI#s 10444345,
13129088, 12232379, and 17454617 (SEQ ID NOs:9, 11, 14, and 16, respectively)
are
34
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
located respectively at approximately amino acid residues I-235, 10 to 265, 9
to 289, and
55 to 270. Further, using TM-HMM, GI# 10444345 (SEQ ID N0:9) has 7
transmembrane
domains with start and end amino acids of (4,21) (26,48) (52,74) (81,103)
(131,153)
(166,188) (203,225); GI# 13129088 (SEQ ll~ NO:11) has 6 transmembrane domains
with
start and end coordinates of (34,51) (66,88) (137,156) (161,183) (195,217)
(232,254);
GI#12232379 (SEQ ID N0:14) has 7 transmembrane domains with start and end
coordinates of (47,64) (77,99) (125,147) (154,173) (183,205) (217,239)
(249,267); and
GI#17454617 (SEQ m N0:16) has 7 transmembrane domains with start and end
coordinates of (33,55) (60,82) (86,108) (115,137) (165,187) (200,222)
(237,259).
II. High-Throughput In Vitro Fluorescence Polarization Assay
Fluorescently-labeled LCE peptide/substrate are added to each well of a 96-
well
microtiter plate, along with a test agent in a test buffer (10 mM HEPES, 10 mM
NaCI, 6
mM magnesium chloride, pH 7.6). Changes in fluorescence polarization,
determined by
using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech
Laboratories, Inc), relative to control values indicates the test compound is
a candidate
modifier of LCE activity.
III. Fligh-Throughput In Vitro Binding Assay_
33P-labeled LCE peptide is added in an assay buffer (100 mM ICI, 20 mM HEPES
pH
7.6, I mM MgCl2, I% glycerol, O.S% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml
BSA, cocktail of protease inhibitors) along with a test agent to the wells of
a Neutralite-
avidin coated assay plate and incubated at 25°C for 1 hour.
Biotinylated substrate is then
added to each well and incubated for 1 hour. Reactions are stopped by washing
with PBS,
and counted in a scintillation counter. Test agents that cause a difference in
activity
relative to control without test agent are identified as candidate p53
modulating agents.
IV. Immunoprecipitations and Immunoblotting
For coprecipitation of transfected proteins, 3 x 106 appropriate recombinant
cells
containing the LCE proteins are plated on 10-cm dishes and transfected on the
following
day with expression constructs. The total amount of DNA is kept constant in
each
transfection by adding empty vector. After 24 h, cells are collected, washed
once with
phosphate-buffered saline and lysed for 20 min on ice in 1 ml of lysis buffer
containing 50
mM Hepes, pH 7.9, 250 mM NaCI, 20 mM -glycerophosphate, 1 mM sodium
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
orthovanadate, 5 mM p-nitrophenyl phosphate, 2 mM dithiothreitol, protease
inhibitors
(complete, Roche Molecular Biochemicals), and 1% Nonidet P-40. Cellular debris
is
removed by centrifugation twice at 15,000 x g for 15 min. The cell lysate is
incubated
with 25 ~,l of M2 beads (Sigma) for 2 h at 4 °C with gentle rocking.
After extensive washing with lysis buffer, proteins bound to the beads are
solubilized
by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel
electrophoresis,
transferred to polyvinylidene difluoride membrane and blotted with the
indicated
antibodies. The reactive bands are visualized with horseradish peroxidase
coupled to the
appropriate secondary antibodies and the enhanced chemiluminescence (ECL)
Western
blotting detection system (Amersham Pharmacia Biotech).
V. Expression analysis
All cell lines used in the following experiments are NCI (National Cancer
Institute)
lines, and are available from ATCC (American Type Culture Collection,
Manassas, VA
20110-2209). Normal and tumor tissues were obtained from Impath, UC Davis,
Clontech,
Stratagene, and Ambion.
TaqMan analysis was used to assess expression levels of the disclosed genes in
various
samples.
RNA was extracted from each tissue sample using Qiagen (Valencia, CA) RNeasy
kits, following manufacturer's protocols, to a final concentration of
50ng/N,1. Single
stranded cDNA was then synthesized by reverse transcribing the RNA samples
using
random hexamers and 500ng of total RNA per reaction, following protocol
4304965 of
Applied Biosystems (Foster City, CA, http://www.a~~liedbiosystems.com/ ).
Primers for expression analysis using TaqMan assay (Applied Biosystems, Foster
City,
CA) were prepared according to the TaqMan protocols, and the following
criteria: a)
primer pairs were designed to span introns to eliminate genomic contamination,
and b)
each primer pair produced only one product.
Taqman reactions were carried out following manufacturer's protocols, in 25
N.l total
volume for 96-well plates and 10 ~,l total volume for 384-well plates, using
300nM primer
and 250 nM probe, and approximately 25ng of cDNA. The standard curve for
result
analysis was prepared using a universal pool of human cDNA samples, which is a
mixture
of cDNAs from a wide variety of tissues so that the chance that a target will
be present in
appreciable amounts is good. The raw data were normalized using 18S rRNA
(universally
expressed in all tissues and cells).
36
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
For each expression analysis, tumor tissue samples were compared with matched
normal tissues from the same patient. A gene was considered overexpressed in a
tumor
when the level of expression of the gene was 2 fold or higher in the tumor
compared with
its matched normal sample. In cases where normal tissue was not available, a
universal
pool of cDNA samples was used instead. In these cases, a gene was considered
overexpressed in a tumor sample when the difference of expression levels
between a
tumor sample and the average of all normal samples from the same tissue type
was greater
than 2 times the standard deviation of all normal samples (i.e., Tumor -
average(all normal
samples) > 2 x STDEV(all normal samples) ).
Results are shown in Table 1. Data presented in bold indicate that greater
than 50% of
tested tumor samples of the tissue type indicated in row 1 exhibited over
expression of the
gene listed in column 1, relative to normal samples. Underlined data indicates
that
between 25% to 49% of tested tumor samples exhibited over expression. A
modulator
identified by an assay described herein can be further validated for
therapeutic effect by
administration to a tumor in which the gene is overexpressed. A decrease in
tumor growth
confirms therapeutic utility of the modulator. Prior to treating a patient
with the
modulator, the likelihood that the patient will respond to treatment can be
diagnosed by
obtaining a tumor sample from the patient, and assaying for expression of the
gene
targeted by the modulator. The expression data for the genes) can also be used
as a
diagnostic marker for disease progression. The assay can be performed by
expression
analysis as described above, by antibody directed to the gene target, or by
any other
available detection method.
Table 1
breast.. . lung . _
colon . o~
GI# 10444344 (SEQ 1 12. 29.3 14. 7
ID NO:1) 8 2
GI#10440044 (SEQ 3 12. 30.9 14. 7
ID N0:4) 4 3
VI. Loss of function of the Drosophila CIG30/LCE gene is associated with
tracheal
defects
Genetic screens were designed to identify modifiers of branching morphogenesis
in
Drosoplaila. Briefly, Drosophila embryos (approximately stage 16) that were
homozygous
for lethal insertions of a piggyBac (Eraser M et al., Virology (1985) 145:356-
361) or P-
element transposon were screened for tracheal defects using monoclonal
antibody 2A12
37
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
(Samakovlis C, et al., Development (1996) 122:1395-1407; Patel NH. (1994)
Practical
Uses in Cell and Molecular Biology. Eds LSB Goldstein and EA Fryberg. Vol 44
pp446-
488. San Diego Academic Press). Sequence information surrounding the
transposon
insertion site was used to identify the gene mutated by the insertion. The
homozygous
disruption of the Drosoplaila CIG30 (baldspot) gene was identified as
associated with
tracheal defects.
VII. Loss of function of a zebrafish LCEs is associated with
branching_morpho~enesis
defects
Using antisense technologies, we identified vasculature defects associated
with loss-
of-function of the zebrafish (Danio rerio) LCE that we have designated
DrCIG30L4,
whose nucleic acid and polypeptide sequences are presented, respectively, in
SEQ ID
N0:7and SEQ ID N0:15. We have identified ten candidate zebrafish LCE genes. Of
these, four were tested for involvement in angiogenesis using essentially the
following
methods. Wild type, one-cell stage embryos from the Tiibingen strain were
treated with
antisense morpholino oligonucleotide (PMOs) that targeted predicted zebrafish
genes.
PMOs were dissolved in injection buffer (0.4 mM MgSO4, 0.6 mM CaCl2, 0,7 mM
KCI,
58 mM NaCI, 25 mM Hepes [pH 7,6]), and 2-8 ng was injected into zebrafish
embryos at
the 1-cell stage.
Larvae were fixed at 4 days post fertilization (dpf) in 4% para-formaldehyde
in
phosphate-buffered saline (PBS) for 30 minutes. Fixed larvae were dehydrated
in
methanol and stored over night at -20°C. After permeabilization in
acetone (30 minutes at
-20°C), embryos were washed in PBS and incubated in the staining buffer
(100 mM Tris-
HCl [pH 9.5], 50mM MgCl2, 100mM NaCI, 0.1 % Tween-20) for 45 minutes. Staining
reaction was started by adding 2.25 ~1 nitro blue tetrazolium (NBT, Sigma) and
1.75 p,1 5-
bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) per m1 of staining buffer
(stock
solutions: 75 mg/ml NBT in 70% N,N-dimethylformamide, 50 mg/ml BLIP in N,N-
dimethylformamide).
The fixed specimens were scanned for changes in blood vessel formation.
Treatment
of embryos with a PMO corresponding to the complement of nucleotides 692-712
of SEQ
~ N0:7 (nucleotide sequence), produced a dose-dependant block of
vasculogenesis and
angiogenesis. Following injection of 2ng of the PMO, the intersegmental
vessels (ISV)
were disrupted; at 4ng of the PMO, the loss of ISV was much more severe.
Following
38
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
injection of ~ng of PMO, the loss of blood vessels is more severe with few ISV
and the
subintestinal vein (SIV) was almost complete disrupted.
VIII. ELOVL4 is an an~o eg~nesis ,gene
Based on analysis of the zebrafish DrCIG30L4, we have identified ELOVL4 as an
angiogenesis gene. We used computation analysis, specifically BLAST, Smith-
Waterrnan, and CLUSTALW analysis, to identify human ELOVL4 ("HsELOVL4,"GI#s
12044043 and 12232379, SEQ ID NOs:l3 and 14) as the ortholog of DrCIG30L4. The
Drosophila CIG30 protein sequence was BLASTed against a Homo sapierzs amino
acid
sequence database to identify the family of CIG30 and ELOV (fatty acid
elongases). The
Homo Sapiens sequences were similarly used to BLAST analyze the available
zebrafish
amino acid sequences both from public databases as well as our intexnal
sequence
databases. Initially, four zebrafish homologs to the CIG30/ELOV family were
identified.
To reassess the orthology of the DrCIG30L4 and other zebrafish orthologs,
these were
BLAST analyzed individually against the Homo Sapiens amino acid sequence
database. In
the case of DrCIG30L4, the top "hit" was Ho»ao Sapiens ELOVL4. Ho~rao sapiens
ELOVL4 amino acid sequence was then used to BLAST against the zebxafish amino
acid
sequence database; the DrCIG30L4 sequence was the mutual best "hit". ClustalW
alignment and phylogenetic analysis of the zebrafish CIG30/ELOV family also
showed
that HsELOVL4 and DrCIG30L4 are most closely related.
Without intent to be limiting, we have contemplated links between ELOVLA~ and
branching moiphogenesis. As described above, ELOVL4 is associated with
Autosomal
Dominant Stargardt-like Maculax Dystrophy (STDG3). While a direct link between
STGD3, ELOVL4 and angiogenesis has not been made, the PMO results in zebrafish
2,5 suggest a possible causal link between a defect in blood vessel formation
and the'
phenotypes associated with STGD3.
Thus, we have identified ELOVE4 as an attractive drug target for the treatment
of
pathologies associated with branching morphogenesis, such as angiogenesis,
particularly
pathologies that are also associated with defective p53 signaling.
39
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
SEQUENCE LISTING
<110> EXELIXIS, INC.
<120> LCEs AS MODIFIERS OF THE p53 PATHWAY AND METHODS OF USE
<130> EX02-0850-PC
<150> US 60/296,076
<151> 2001-06-05
<150> US 60/328,605
<151> 2001-10-10
<150> US 60/357,253
<151> 2002-02-15
<150> US 60/361,196
<151> 2002-03-01
<160> 16
<170> PatentIn version 3.1
<210> 1
<211> 7094
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (7004)..(7004)
<223> "n" is A, C, G. or T
<400>
1
ttccggcagtggctcatgtctgtaatctcagcactttgggaagctgaagtgggcagatca60
cttgaggtcaggagttcaagtactagcctggccaacatggcgaaaactgtctctactaaa120
agcacaaaaattagcaaggcgtggtggcacacatctgtagtcccagcactcaggggactg180
aggtacgagaatcgcttgaactcaggaggcagaggttgcggtgagccaagatcaggccac240
tgcactccagcctgggcgacagagcaagactctgtctcataataaaaaattttaaaaaat300
agacatagtccctgcattcatggagctcaagaactagtaggaaaaacaggcattgatcaa360
gaaatcacaaaaacaactaattataaactctcactgaagaaaagaaataccatgctctga420
gaacagtttatataaaataagggtaccagaccaagtccaagggtcaaggaatgtttcctc480
tagaatataatttctgaagatttttcctcttcttttttcttttttgagacagagtctcgc540
tctgttgcccaggctggagtgcagtgacacaatctctgctcagtgcaacccacgcctgcc600
agattcaagcaattctecctgcctcagcctgccaagtagctggaattacaggtatccgcc660
actacacctagctaatttttgtatttttagtggagatggggtttcaccatgtgggccagg720
ctggtcttgaactcctgacctcaggtgatctgcccacctcagectcccaaagtgctggga780
1
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
taacaggcgtgagccactgcacctagcttttttttttttttttgagacagagtctcactc840
tgtcacccaggctggagtgcagtggtgtgatcacagctcagtggcacgatcacagctcac900
tgcagccttgacctccctggctcaagtgatCCtCCCtCCtcagtccctgagtagctggga960
ccacaggtgggcgctaccatgcccagctaatttttgtactttttgtagagatggattttc1020
accatgttgcccaggctggtctcaaattcctgggctcaagcaatccccccacctcagctt1080
cctacagtactgggattacaggcgtgagccaccgtgcccagccccgagtgttcttctttc1140
cctcttccacatacacctcctccaagcctcaccattaaacctcacagggaaagacaatgt1200
taaatatcttcacagagaaatccaggactgagtatatattctccatatgcacttgtaaac1260
ctcccataatgattcaccttgccatttccatgtgtctagtccacaatatcattttatcta1320
cattatctggcttgcatactaatcacaaccttgtgacataagcaggcgagtatgactata1380
tgcatttttttctgaagtagaatgtgacaaaggctaggtaacttgcctaaaatcacatgg1440
ctcattaatgggggtgctgggacttgaacttgggtcttgtaagacctagatggcattatt1500
cttgtaatattcattcttttatttattcattcatccatagacatgtattgatcacctttt1560
gattagctgtcaggctatatatggagccatcaggaaccactgaaggtttttttttttttt1620
tttttttttgagacggagtctcactctgtcacccaggctggagtgcagtggcacgatctc1680
tgctcactgcaagctctgcctcccaggttcacgccattctcctgcctcagcctcccgagt1740
agctgggactacaggcgcctgccaccacgcccggctaattttttgtattttttagtagag1800
acggggtttgacggtgttagccaggatggtctcgatctcctgacctcatgatctgcccgc1860
ctcggcctcccaaggtgctgggattacaggcgtgaaccaccgtgcccggccgaaccactg1920
aaggtttttaagcaggaaagcagagctgttttctggatgagcaaacagaaagtagtggtt1980
ttccaagtacagtctgagacaacctataggaccagaatctctgcagttgaggctcaggaa2040
tctggtaatcagccaggtataggaactcttttctgattgcaatgcagtgaagagcagaag2100
cactgtattagagaaagaggcagtgcaaccaggtaacgtgaccaggtgagaagtgatgag2160
gtacagagacaaagagatgcacttttgagtcacttagatggcactgataggacttccact2220
acaccctcgcatagacagtggctgaggttcaggaaatagagctggggttcctacttggat2280
cctctggctctagagctttactgcacatagccatttatacccacatcttgattttaatta2340
ttttatatctatgtttcttagcactttttgcaaatttccaccttatctcaaactgccctc2400
aagccttgtatttctccttcgctttcataaaacctaggaaagaaataagggacagccaag2460
taaaacttttaaaagttttagaacatttatttctttggggctggttacacaggcgagaaa2520
gaagtagatttggttagggagagaaaacaacaggccttggggagatacactggctctccc2580
cctccctaaaccctaagaggcctccaggaaacctgaagacaataattccagaagcccaga2640
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
gggtgacccc atttcctctc tccatggtta ttactgtcag tctg~agcag ttcaggaatt 2700
caggaaacta taaagaaacc acaacagcct caacaaccca aacatcaaca tcaacaacct 2760
caacaataaaactccttaaaattcatctccttccacccactcacaaccgcagactcgaag2820
ctaggaggtggaagggactacagaagctctgcgttgcccaggttagtatttgctcatcac2880
aggcctgggtttcccaggatctcagggagcctggaaactgacgcctccatttctgggtgg2940
gagcaccaaagcctaaggacacctttcctctctcttcactgctaagcaggtcaagattaa3000
agcaaaccgaggcaaaggccacggttgacagttccaagggaacccgcaaggccgcacagg3060
atggggtggacgttttacgggagaaaaggctggggaagtgggcgggcgatggcctacgac3120
gggacttggggcggggtgtgcgaaacgcctggcaggcgggcccttgagtatgaccaatca3180
gaatgcggactgcgtcccaggggcggagcagaggcgtatcttggtcgagattggatagcg3240
gcggggcgcaggaaagaggtcgcgccagcccgggcaggcagctttgcaagtccgcgttat3300
atatcgcagtggctgcgcccgggatagctggctgcgccgccgcgcacatgcctaggttcg3360
acgccctcctccctttgcccaggagttccttctgtcccggctctgttccgtctcgecccg3420
aggttcacgccatcctcggagccccagcctttcacccagcgcctccaagctttggacctt3480
gacttctgcaaaactagatggtcacagccatgaatgtctcacatgaagtaaatcagctgt3540
tccagccctataacttcgagctgtccaaggacatgaggccctttttcgaggagtattggt3600
gagactttgggagagggaaaggccatgccagggccccggggccggggcgcgagggggtgg3660
cattgaatgcccacaatgattttcttacagagcagagttatgggactcccttgtactggc3720
ttcacactacctttgtccgaggtgcaggtagtatgtggcacctcaaagagattagagctg3780
aaagcaagcaagcagaaataagaggatgagggagaacgtggaaatatacagaagtcaaag3840
agagcgtgtagtgagaagggtcttggaggcgtgaggttgatctgaagccttactagatga3900
cttccccgtcctttgtggctgtgtgtgcgcgcgtgtgtgcacagtgcagggcccggggga3960
cagcctgccctttacaattatcccatcgttccccaggtgctccaccgttcctgctgtgga4020
gaaggaggcgaagtcagagagctcttccaagctttccccaggaagagctctctggctttg4080
ccttaaagctccccagaggttttggaggctgactttatgctccaaccaatttgccatacc4140
ccagccagggagaagctattgccacattcttcttacgccacagccctggtatgcttctag4200
gagccccaaccagggagaagctattgccacacttcttacaccacagccctgggctgcttc4260
taggcgcgggccagacagccgtcacccacctctaaccccactgagagagcaataatcaca4320
gaaaccttggacatagctcctgccctgtgctagatactttgtatacgttaatacccccag4380
gagagatgtagtattcgcgctctacaaatgaggaagccaaggctcagagaattaagttgg4440
tttttccaatgtcacatagctagtaagtggcagaactgggactccaacccagagcactca4500
3
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
cctggagagatgagtgggcatttctctaatCagcacccacccatgagcccatccctctgc4560
cttctgcttgccagggcaacctcattccccatagccctgatctacctggttctcatcgct4620
gtggggcagaactacatgaaggaacgcaagggcttcaacctgcaagggcctctcatcctc4680
tggtccttctgccttgcaatcttcaggtaagaccccatcccactccctgcctcttctcta4740
gatcttagaccaccattctatcccttgaagctttcccgattgcatcaacctccatcctgt4800
ccccaagttgccttgtaacactctccccatCtCattCtCggCtttagttCccctcccagc4860
cttcaccctcttctgttatattatcctgttttccttctcttttagacccctacctggtgg4920
tctctgctttgtccctcttgccttcgtagagtctgtactcgcaactaatttctccagcct4980
ctaatagtgtcacctcctaacaccctctctcaagaccctccaataacttccccatatttt5040
ctcccccaaactccttcagtcttcccytatccctctacacatctcctcccaggctcctaa5100
cccctcccaagaaccccattccttaactcaactttcagtcccatccccctgcattccctg5160
atctttctccagccctcrtctcctctagactactgattggatctaccagattggcttcag5220
gatctcaggagctttgacccacccsctgtggacaggtggggaggtggtagagcttggaac5280
acagtaacctggccaagccgggggaagggtggattattggtgcctggggatgacacttac5340
accatcttcctttgtcccatttcagtatcctgggggcagtgaggatgtggggcattatgg5400
ggactgtgctacttaccgggggcctaaagcaaaccgtgtgcttcatcaacttcatcgata5460
attccacagtcaaattctggtcctgggtctttcttctcagcaaggtcatagaactcggtg5520
agtggcaaagctttgtctttctggtgccttgtgaactgcatccttcctcagggccctccc5580
ttcacccatcccatggagggtctctttcctacccttgggtcccaataatacctctcaccc5640
aagcccctctacagattctctgccacaaagacccecttccctcccctgagaatttctccc5700
gtgtccctacatccagtgcagagggtggtcccagcactgggtggtatgccaactatgact5760
ctccatctcccaggagacacagccttcatcatcctgcgtaagcggccactcatctttatt5820
cactggtaccaccacagcacagtgctcgtgtacacaagctttggatacaagaacaaagtg5880
cctgcaggaggctggttcgtcaccatgaactttggtgttcatgccatcatgtacacctac5940
tacactctgaaggctgccaacgtgaagccccccaagatgctgcccatgctcatcaccagc6000
ctgcagatcttgcagatgtttgtaggagccatcgtcagcatcctcacgtacatctggagg6060
caggatcagggatgccacaccacgatggaacacttattctggtccttcatcttgtatatg6120
acctatttcatcctctttgcccacttcttctgccagacctacatcaggcccaaggtcaaa6180
gccaagaccaagagccagtgaaggtttggagagaacaatgaagctccaggctctctcttc6240
tccagggcaccaagaggctgggcttagttttgggagaatgattaggttgccttacctgca6300
tggtttccccagaggatgtgtgccccaaggtggctggaatttttgacagacaagaagggt6360
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
gaccttgggatgggggtgtggtctgttactttaatgtttctgtttttaatgtgaaggcca6420
agcaggccctgggatgggagtggggcggaggagggtcctaagagctgattatttaatttc6480
tatccagaaatctttcttcttcttgctctgtttttttaaattaaagatttcaacaaaatt6540
ttgagagttgggggatttggggggaaagagggctgctgtgatggcaggaggctgctacca6600
aggggatgatctgcaggtgggacgcctgagggtgtgtggaagggtgagaggcacacacac6660
agacactgaaagaatcctaggcctggtaggcacttaacaaatgtctgttacagaccagaa6720
ttttattgctgttagagacccaagcccctcataggaacagtgagaaacaggtgcagaaag6780
gcggagtaactttatctaaagtcataggctccctgaatagcagagctgacacctacaagg6840
aagcgttggagaccagatctaccagctagcctccctgagaccacgaggtggcgccgcagc6900
accggctgtggccgatgccagccaggtagccggtttcccacgtcccccgcacgcacgcac6960
ctctttgctgcaggaatcccgggctgccccgacctggagtaggnggggtggtgagtggga7020
ctgagtccctagaagcctggaccctcasttcgttccctgtacatccagctcgcctgtaga7080
cagtggggga ggat 7094
<210> 2
<211> 828
<212> DNA
<213> Homo Sapiens
<400>
2
ctagatggtcacagccatgaatgtctcacatgaagtaaatcagctgttccagccctataa60
cttcgagctgtccaaggacatgaggccctttttcgaggagtattgggcaacctcattccc120
catagccctgatctacctggttctcatcgctgtggggcagaactacatgaaggaacgcaa180
gggcttcaacctgcaagggcctctcatcctctggtccttctgccttgcaatcttcagtat240
cctgggggcagtgaggatgtggggcattatggggactgtgctacttaccgggggcctaaa300
gcaaaccgtgtgcttcatcaacttcatcgataattccacagtcaaattctggtcctgggt360
ctttcttctcagcaaggtcatagaactcggagacacagccttcatcatcctgcgtaagcg420
gccactcatctttattcactggtaccaccacagcacagtgctcgtgtacacaagctttgg480
atacaagaacaaagtgcctgcaggaggctggttcgtcaccatgaactttggtgttcatgc540
catcatgtacacctactacactctgaaggctgccaacgtgaagccccccaagatgctgcc600
catgctcatcaccagcctgcagatcttgcagatgtttgta ggagccatcg tcagcatcct660
cacgtacatctggaggcaggatcagggatgccacaccacg atggaacact tattctggtc720
cttcatcttgtatatgacctatttcatcctctttgcccac ttcttctgcc agacctacat780
caggcccaaggtcaaagccaagaccaagagccagtgaa 818
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
<210> 3
<211> 3045
<212> DNA
<213> Homo Sapiens
<400>
3
actaagaccgcaaggcattcatttcctcctacggtggatgcggacgccgggaggaggaga60
gccccagagagaggagctgggagcggaggcgcaggcaatgctcagccctggatgtagctg120
agaggctgggagaagagacgaccgctggagaccgagcggcgtggggaagacctagggggg180
tgggtgggggaagcagacaggagaacactcgaaatcaagcgctttacagattattttatt240
ttgtatagagaacacgtagcgactccgaagatcagccccaatgaacatgtcagtgttgac300
tttacaagaatatgaattcgaaaagcagttcaacgagaatgaagccatccaatggatgca360
ggaaaactggaagaaatctttcctgttttctgctctgtatgctgcctttatattcggtgg420
tcggcacctaatgaataaacgagcaaagtttgaactgaggaagccattagtgctctggtc480
tctgacccttgcagtcttcagtatattcggtgctcttcgaactggtgcttatatggtgta540
cattttgatgaccaaaggcctgaagcagtcagtttgtgaccagggtttttacaatggacc600
tgtcagcaaattctgggcttatgcatttgtgctaagcaaagcacccgaactaggagatac660
aatattcattattctgaggaagcagaagctgatcttcctgcactggtatcaccacatcac720
tgtgctcctgtactcttggtactcctacaaagacatggttgccgggggaggttggttcat780
gactatgaactatggcgtgcacgccgtgatgtactcttactatgccttgcgggcggcagg840
tttccgagtctcccggaagtttgccatgttcatcaccttgtcccagatcactcagatgct900
gatgggctgtgtggttaactacctggtcttctgctggatgcagcatgaccagtgtcactc960
tcactttcagaacatcttctggtcctcactCatgtaCCtCdgCtaCCttgtgCtCttCtg1O2O
ccatttcttctttgaggcctacatcggcaaaatgaggaaaacaacgaaagctgaatagtg1080
ttggaactgaggaggaagccatagctcagggtcatcaagaaaaataatagacaaaagaaa1140
atggcacaaggaatcacacgtggtgcagctaaaacaaaacaaaacatgagcaaacacaaa1200
acccaaggcagcttagggataattaggttgatttaacccagtaagtttatgatcctttta1260
gggtgaggactcactgagtgcacctccatctccaagcactgctgctggaagaccccattc1320
cctctttatctatcaactctaggacaagggagaacaaaagcaagccagaagcagaggaga1380
ctaatcaaaggcaaacaaaggctattaacacataggaaaatatgtatttactaagtgtca1440
catttctctaagatgaaagatttttactctagaaactgtgcgagcacaacacacacaatc1500
ctttctaactttatggacactaaactggagccaatagaaaagacaaaaatgaaagagaca1560
cagggtgtatatctagaacgataatgcttttgcagaaactaaagcctttttaagaaatgc1620
cagctgctgtagaccccatgagaaaagatgtcttaatcatccttatgaaaacagatgtaa1680
6
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
acaactatatttcaactaacttcatcttcactgcatagcctcaggctagtgagtttgcca1740
aaaccaaagggggtgaatacttccccaagattcttcctgggaggatggaaacagtgcagc1800
ccaggtcccatgggggcagctccatcccagagcatttctgatagttgaactgtaatttct1860
actcttaagtgagatatgaagtattatccttttgttcagttgccccgggcttttgaacag1920
aagagtaaatacagaattgaaaaagataaacactcaaccaaacaatgtgaaaacgggttc1980
tgtagtatttgtaaaaaggcccggcccaggaccactgtgagctggaaaagggagaaaggc2040
agtgggaaaagaggtgagccgaagatcaattcgacagacagacggtgtgtatgcccctcc2100
ctgtttgacttcacacacactcataactttccaaatgaaaccccacagtatagcgcatat2160
tttcgatatttttgtgaattccaaaaggaaatcacagggctgttcgaaatattgggggaa2220
cactgtgtttctgcatcatctgcatttgctccccaagcaatgtagaggtgtttaaagggc2280
cctctgctggctgagtggcaatactacaacaaacttcaaggcaagtttggctgaaaacag2340
ttgacaacaaagggcccccatacacttatccctcaaattttaagtgatatgaaatacttg2400
tcatgtctttggccaaatcagaagatattcatcctgcttcaagtcagcttcagaaatgtt2460
ttaaaagggactttagctctggaactcaaaatcaatttattaagagccatattctttaaa2520
aaaaaaagctggataatattatctgtaatatttcagtcctttacaagccaaatacatgtg2580
tcaatgtttctagtatttcaaagaagcaattatgtaaagttgttcaatgtgacataatag2640
tattataattggttaagtagcttaatgattaggcaaactagatgaaaagattaggggctt2700
ccacactgcatagatcacacgcacatagccacgcatacacacacagacacacagatgtgg2760
ggtacactgaatttcaaagcccaaatgaatagaaacacattttctggctagcagaaaaaa2820
acaaaacaaaactgttgtttctctttcttgctttgagagtgtacagtaaaagggattttt2880
tcgaattatttttatattattttagctttaattgtgctgtcgttcatgaaacagagctgc2940
tctgcttttctgtcagagatggcaagggctttttcagcatctcgtttatgtgtggaattt3000
aaaaagaataaagttttattccattctgaaaaaaaaaaaaaaaaa 3045
<210> 4
<211> 3045
<212> DNA
<213> Homo sapiens
<400> 4
actaagaccg caaggcattc atttcctcct acggtggatg cggacgccgg gaggaggaga 60
gccccagaga gaggagctgg gagcggaggc gcaggcaatg ctcagccctg gatgtagctg 120
agaggctggg agaagagacg accgctggag accgagcggc gtggggaaga cctagggggg 180
tgggtggggg aagcagacag gagaacactc gaaatcaagc gctttacaga ttattttatt 240
ttgtatagag aacacgtagc gactccgaag atcagcccca atgaacatgt cagtgttgac 300
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
tttacaagaatatgaattcgaaaagcagttcaacgagaatgaagccatccaatggatgca360
ggaaaactggaagaaatctttcctgttttctgctctgtatgctgcctttatattcggtgg420
tcggcacctaatgaataaacgagcaaagtttgaactgaggaagccattagtgctctggtc480
tctgacccttgcagtcttcagtatattcggtgctcttcgaactggtgcttatatggtgta540
cattttgatgaccaaaggcctgaagcagtcagtttgtgaccagggtttttacaatggacc600
tgtcagcaaattctgggcttatgcatttgtgctaagcaaagcacccgaactaggagatac660
aatattcattattctgaggaagcagaagctgatcttcctgcactggtatcaccacatcac720
tgtgctcctgtactcttggtactcctacaaagacatggttgccgggggaggttggttcat780
gactatgaactatggcgtgcacgccgtgatgtactcttactatgccttgcgggcggcagg840
tttccgagtctcccggaagtttgccatgttcatcaccttgtcccagatcactcagatgct900
gatgggctgtgtggttaactacctggtcttctgctggatgcagcatgaccagtgtcactc960
tcactttcagaacatcttctggtcctcactcatgtacctcagctaccttgtgctcttctg1020
ccatttcttctttgaggcctacatcggcaaaatgaggaaaacaacgaaagctgaatagtg1080
ttggaactgaggaggaagccatagctcagggtcatcaagaaaaataatagacaaaagaaa1140
atggcacaaggaatcacacgtggtgcagctaaaacaaaacaaaacatgagcaaacacaaa1200
acccaaggcagcttagggataattaggttgatttaacccagtaagtttatgatcctttta1260
gggtgaggactcactgagtgcacctccatctccaagcactgctgctggaagaccccattc1320
cctctttatctatcaactctaggacaagggagaacaaaagcaagccagaagcagaggaga1380
ctaatcaaaggcaaacaaaggctattaacacataggaaaatatgtatttactaagtgtca1440
catttctctaagatgaaagatttttactctagaaactgtgcgagcacaacacacacaatc1500
ctttctaactttatggacactaaactggagccaatagaaaagacaaaaatgaaagagaca1560
cagggtgtatatctagaacgataatgcttttgcagaaactaaagcctttttaagaaatgc1620
cagctgctgtagaccccatgagaaaagatgtcttaatcatccttatgaaaacagatgtaa1680
acaactatatttcaactaacttcatcttcactgcatagcctcaggctagtgagtttgcca1740
aaaccaaagggggtgaatacttccccaagattcttcctgggaggatggaaacagtgcagc1800
ccaggtcccatgggggcagctccatcccagagcatttctgatagttgaactgtaatttct1860
actcttaagtgagatatgaagtattatccttttgttcagttgccccgggcttttgaacag1920
aagagtaaatacagaattgaaaaagataaacactcaaccaaacaatgtgaaaacgggttc1980
tgtagtatttgtaaaaaggcccggcccaggaccactgtgagctggaaaagggagaaaggc2040
agtgggaaaagaggtgagccgaagatcaattcgacagacagacggtgtgtatgcccctcc2100
ctgtttgacttcacacacactcataactttccaaatgaaaccccacagtatagcgcatat2160
8
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
tttcgatatttttgtgaattccaaaaggaaatcacagggctgttcgaaatattgggggaa2220
cactgtgtttctgcatcatctgcatttgctccccaagcaatgtagaggtgtttaaagggc2280
cctctgctggctgagtggcaatactacaacaaacttcaaggcaagtttggctgaaaacag2340
ttgacaacaaagggcccccatacacttatccctcaaattttaagtgatatgaaatacttg2400
tcatgtctttggccaaatcagaagatattcatcctgcttcaagtcagcttcagaaatgtt2460
ttaaaagggactttagctctggaactcaaaatcaatttattaagagccatattctttaaa2520
aaaaaaagctggataatattatctgtaatatttcagtcctttacaagccaaataCatgtg2580
tcaatgtttctagtatttcaaagaagcaattatgtaaagttgttcaatgtgacataatag2640
tattataattggttaagtagcttaatgattaggcaaactagatgaaaagattaggggctt2700
ccacactgcatagatcacacgcacatagccacgcatacacacacagacacacagatgtgg2760
ggtacactgaatttcaaagcccaaatgaatagaaacacattttctggctagcagaaaaaa2820
acaaaacaaaactgttgtttctctttcttgctttgagagtgtacagtaaaagggattttt2880
tcgaattatttttatattattttagctttaattgtgctgtcgttcatgaaacagagctgc2940
tctgcttttetgtcagagatggcaagggctttttcagcatctcgtttatgtgtggaattt3000
aaaaagaataaagttttattccattctgaaaaaaaaaaaaaaaaa 3045
<210> 5
<211> 2900
<212> DNA
<213> Homo Sapiens
<400>
tgaggagcaggagaagacgcagccgggccgccgccgttagaggggttcccggccgccgct60
cgccccgtcggccgccaccgcctccggggtcagccctctctctgggtctccgctttctcc120
tgccgccagcgcccgctcatcgccgcgatggggctcctggactcggagccgggtagtgtc180
ctaaacgtagtgtccacggcactcaacgacacggtagagttctaccgctggacctggtcc240
atcgcagataagcgtgtggaaaattggcctctgatgcagtctccttggcctacactaagt300
ataagcactctttatctcctgtttgtgtggctgggtccaaaatggatgaaggaccgagaa360
ccttttcagatgcgtctagtgctcattatctataattttgggatggttttgcttaacctc420
tttatcttcagagagttattcatgggatcatataatgcgggatatagctatatttgccag480
agtgtggattattctaataatgttcatgaagtcaggatagctgctgctctgtggtggtac540
tttgtatctaaaggagttgagtatttggacacagtgttttttattctgagaaagaaaaac600
aaccaagtttctttccttcatgtgtatcatcactgtacgatgtttaccttgtggtggatt660
ggaattaagtgggttgcaggaggacaagcattttttggagcccagttgaattcctttatc720
9
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
catgtgatta tgtactcata ctatgggtta actgcatttg gcccatggat tcagaaatat 780
ctttggtgga aacgatacct gactatgttg caactgattc aattccatgt gaccattggg 840
cacacggcac tgtctcttta cactgactgc cccttcccca aatggatgca ctgggctcta 900
attgcctatg caatcagctt catatttctc tttcttaact tctacattcg gacatacaaa 960
gagcctaagaaaccaaaagctggaaaaacagccatgaatggtatttcagcaaatggtgtg1020
agcaaatcagaaaaacaactcatgatagaaaatggaaaaaagcagaaaaatggaaaagca1080
aaaggagattaaattgaactgggccttaactgttgttgacagtgaggaaaaactcccata1140
tcatataaaatttcagggaaaacagaagcaaaggagagcttgggggtggggagaaaagac1200
aaatgtgctctatgtcctagtaactcttagactgagtaaagtgttaataccatacccaga1260
tgttttatttatgaagtttttattttaaacattttttttaaaaattagccttgatattct1320
ccagaccaaagcaatcattaagtgactttggggattctccccctgttcacatccagttgt1380
ctaaaggatgagatttttcatgtatcttatagtcactcattcttcggtctgaattttaga1440
cgatcacagaaacggtctttatgaattattttgataaattactaattatcttatctactg1500
actgaaatcagtggtgttacattttcttgtccaaagctgaaaatgtgtatacacttaaac1560
ttgcacatttgaattcatttgctgaccggaatggtcaaatctctccacctctagtcagag1620
tataattttggttgtaattaaatttttaaaatctgctgatctctgtagaatcttagaggc1680
ttgatgatgatggtgttggtgaaaataagaaagaattgcagtaaagtcttgtctggtgac1740
ccagagatcaccatgacttgaggcacaaatcactgtggggaaacaattttttgtgatgaa1800
aaggcagcatttgaatactcctgttagtagcagaaatatattatgaaattaagattattg1860
tctgattgaaacatgaaacaactcatgtctttattagtaacatcataagatagttacatt1920
tatgtgctgttagaatatgttaatttttatcaggctttccttgttttgatttatggctgt1980
tcctgatttttcatatgtggaaatatacctacctcttccgttggaaagaacatttaaaat2040
taaataaattttaattaaaaaatcaaggagtcttctaatgtaaattttaatgttaacttt2100
caaatccactagtattttttttgcttttatgacaaatagcatacaccaaacatttctgtg2160
aaactatccttctctttcaatgtgtttaattttggagtaacgttttccttgtgactaagt2220
tgcaagatcttatttattaactaggtatgaagtataaacccattttggtgcaatattctt2280
gactccttggtgctaaagattgttaaattcaatgcttgatgttacaaggtgttgttaaaa2340
cacaaaatgaataaaagtgagagtagtcagaactataacattcaatttgctatttacaaa2400
tgaagtatttcatgtaatataagtgaacaactggaaataaagtaggaaagaatttgtatc2460
atgttttactacataggttaattttttaagggatgttgcaaagggattactagagaaaga2520
caaaatgtgaccaaaaaaaagcatgaatatttcttaagtatctcaacaacatgtcaaagc2580
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
tgcatgtgtaggatgtatgctgtttgtacaaactatttcagaatattttgtaagctataa2640
catatttattgtgcattaaaattaaatactttttccccaaaggcatgcagtcatgagaat2700
tacagaaaatttgcaacatataaagtagtttgatctaagaggattcaacacctttgtttt2760
gttgctcagtgtgtaatgactgagatttgtaaatctttgtgaacattctgtactggttcc2820
caagagctattcattccctgctacctgatttcagcacaataaatatacttctgctgtggg2880
aaaaaaaaaa aaaaaaaaaa 2900
<210> 6
<211> 2900
<212> DNA
<213> Homo sapiens
<400>
6
tgaggagcaggagaagacgcagccgggccgccgccgttagaggggttcccggccgccgct60
cgccccgtcggccgccaccgcctccggggtcagccctctctCtgggtCtCCgCtttctCC120
tgccgccagcgcccgctcatcgccgcgatggggctcctggactcggagccgggtagtgtc180
ctaaacgtagtgtccacggcactcaacgacacggtagagttctaccgctggacctggtcc240
atcgcagataagcgtgtggaaaattggcctctgatgcagtctccttggcctacactaagt300
ataagcactctttatctcctgtttgtgtggctgggtccaaaatggatgaaggaccgagaa360
ccttttcagatgcgtctagtgctcattatctataattttgggatggttttgcttaacctc420
tttatcttcagagagttattcatgggatcatataatgcgggatatagctatatttgccag480
agtgtggattattctaataatgttcatgaagtcaggatagctgctgctctgtggtggtac540
tttgtatctaaaggagttgagtatttggacacagtgttttttattctgagaaagaaaaac&00
aaccaagtttctttccttcatgtgtatcatcactgtacgatgtttaccttgtggtggatt660
ggaattaagtgggttgcaggaggacaagcattttttggagcccagttgaattcctttatc720
catgtgattatgtactcatactatgggttaactgcatttggcccatggattcagaaatat780
ctttggtggaaacgatacctgactatgttgcaactgattcaattccatgtgaccattggg840
cacacggcactgtctctttacactgactgccccttccccaaatggatgcactgggctcta900
attgcctatgcaatcagcttcatatttctctttcttaacttctacattcggacatacaaa960
gagcctaagaaaccaaaagctggaaaaacagccatgaatggtatttcagcaaatggtgtg1020
agcaaatcagaaaaacaactcatgatagaaaatggaaaaaagcagaaaaatggaaaagca1080
aaaggagattaaattgaactgggccttaactgttgttgacagtgaggaaaaactcccata1140
tcatataaaatttcagggaaaacagaagcaaaggagagcttgggggtggggagaaaagac1200
aaatgtgctctatgtcctagtaactcttagactgagtaaagtgttaataccatacccaga1260
tgttttatttatgaagtttttattttaaacattttttttaaaaattagccttgatattct1320
11
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
ccagaccaaagcaatcattaagtgactttggggattctccccctgttcacatccagttgt1380
ctaaaggatgagatttttcatgtatcttatagtcactcattcttcggtctgaattttaga1440
cgatcacagaaacggtctttatgaattattttgataaattactaattatcttatctactg1500
actgaaatcagtggtgttacattttcttgtccaaagctgaaaatgtgtatacacttaaac1560
ttgcacatttgaattcatttgctgaccggaatggtcaaatctctccacctctagtcagag1620
tataattttggttgtaattaaatttttaaaatctgctgatctctgtagaatcttagaggc1680
ttgatgatgatggtgttggtgaaaataagaaagaattgcagtaaagtcttgtctggtgac1740
ccagagatcaccatgacttgaggcacaaatcactgtggggaaacaattttttgtgatgaa1800
aaggcagcatttgaatactcctgttagtagcagaaatatattatgaaattaagattattg1860
tctgattgaaacatgaaacaactcatgtctttattagtaacatcataagatagttacatt1920
tatgtgctgttagaatatgttaatttttatcaggctttccttgttttgatttatggctgt1980
tcctgatttttcatatgtggaaatatacctacctcttccgttggaaagaacatttaaaat2040
taaataaattttaattaaaaaatcaaggagtcttctaatgtaaattttaatgttaacttt2100
caaatccactagtattttttttgcttttatgacaaatagcatacaccaaacatttctgtg2160
aaactatccttctctttcaatgtgtttaattttggagtaacgttttccttgtgactaagt2220
tgcaagatcttatttattaactaggtatgaagtataaacccattttggtgcaatattctt2280
gactccttggtgctaaagattgttaaattcaatgcttgatgttacaaggtgttgttaaaa2340
cacaaaatgaataaaagtgagagtagtcagaactataacattcaatttgctatttacaaa2400
tgaagtatttcatgtaatataagtgaacaactggaaataaagtaggaaagaatttgtatc2460
atgttttactacataggttaattttttaagggatgttgcaaagggattactagagaaaga2520
caaaatgtgaccaaaaaaaagcatgaatatttcttaagtatctcaacaacatgtcaaagc2580
tgcatgtgtaggatgtatgctgtttgtacaaactatttcagaatattttgtaagctataa2640
catatttattgtgcattaaaattaaatactttttccccaaaggcatgcagtcatgagaat2700
tacagaaaatttgcaacatataaagtagtttgatctaagaggattcaacacctttgtttt2760
gttgctcagtgtgtaatgactgagatttgtaaatctttgtgaacattctgtactggttcc2820
caagagctattcattccctgctacctgatttcagcacaataaatatacttctgctgtggg2880
aaaaaaaaaa aaaaaaaaaa 2900
<210> 7
<211> 1307
<212> DNA
<213> Danio rerio
<400> 7
12
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
cgagcgcaccggcatgagccccacatggcgcgactgcgcatgctcagttgcggacagacc60
ggtgtccagtcaccgtcagacgcaccgctcagaagcgtttgcctcatatgctccggtccc120
gttagtgacactccgtttgccttcgcgttattattgccaaaaaactgcggggttgatgcg180
tttaaacgtacgctgtagatttaaatatagacaattattcactcgcatattttacctggg240
ctgcagcacacctgatgtcgacataggcacgcgctcgtaaggataatcatcttagaacta300
gcgccatggagacggtcgttcacctgatgaatgactctgtagagttttacaaatggagcc360
ttaccatagcagacaagcgtgtggagaaatggccgatgatgtcatctcctctgcccactc420
tggggatcagtgttttgtacctgctcttcctttgggccggccctctttacatgcagaacc480
gcgagcctttccagctcaggaaaacactcattgtgtacaacttcagcatggtgctgctta540
acttctacatctgcaaagagctgctcctgggctccagagcagccggatacagctacctct600
gccagcctgtcaactactccaatgatgttaatgaagtcaggatagcatctgctctgtggt660
ggtattacatctccaagggagtggagtttctggacacggtcttcttcatcatgaggaaga720
agtttaatcaggtcagcttcctgcacgtctatcaccactgcacaatgttcatcctgtggt780
ggatcggcatcaagtgggttcctggtggacagtctttctttggcgcaacgattaactcag840
gcattcatgtgctgatgtacggctactacggcctggcagcgtttggcccgaagatccaga900
agtacctgtggtggaagaaatacctcactattattcagatgatccagttccacgtcacca960
ttgtccatgctgcttactctctctacacgggctgtccattcccagcatggatgcaaagtg1020
ctttgattggctatgccgatacattcatcatcctgttggccaatttttactaccagacct1080
accgtcgccagccacttcctaagacagccaaattcgcagttaacggcgtctccatgtcaa1140
ccaacggcaccagcaagacagccgaggtcacggaaaatggaaagaaacag acgaaaggaa1200
aaggaaagcacgattaaaacgaatcttggatggagataaaccattacaga ctgtttggta1260
gttgtaaaaacaaaacaaacatgctgattgtatttctgggacaataa 1307
<210> 8
<211> 1338
<212> DNA
<213> Homo sapiens
<400>
8
gcagtggctgcgcccgggatagctggctgcgccgccgcgcacatgcctaggttcgacgcc60
ctcctccctttgcccaggagttccttctgtcccggctctgttccgtctcgccccgaggtt120
cacgccatcctcggagccccagcctttcacccagcgcctccaagctttggaccttgactt180
ctgcaaaactagatggtcacagccatgaatgtctcacatgaagtaaatcagctgttccag240
ccctataacttcgagctgtccaaggacatgaggccctttttcgaggagtattgggcaacc300
tcattccccatagccctgatctacctggttctcatcgctgtggggcagaactacatgaag360
13
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
gaacgcaagggcttcaacctgcaagggcctctcatcctctggtccttctgccttgcaatc420
ttcagtatcctgggggcagtgaggatgtggggcattatggggactgtgctacttaccggg480
ggcctaaagcaaaccgtgtgcttcatcaacttcatcgataattccacagtcaaattctgg540
tcctgggtctttcttctcagcaaggtcatagaactcggagacacagccttcatcatcctg600
cgtaagcggccactcatctttattcactggtaccaccacagcacagtgctcgtgtacaca660
agctttggatacaagaacaaagtgcctgcaggaggctggttcgtcaccatgaactttggt720
gttcatgccatcatgtacacctactacactctgaaggctgccaacgtgaagccccccaag780
atgctgcccatgctcatcaccagcctgcagatcttgcagatgtttgtaggagccatcgtc840
agcatcctcacgtacatctggaggcaggatcagggatgccacaccacgatggaacactta900
ttctggtccttcatcttgtatatgacctatttcatcctctttgcccacttcttctgccag960
acctacatcaggcccaaggtcaaagccaagaccaagagccagtgaaggtttggagagaac1020
aatgaagctccaggctctctcttctccagggcaccaagaggctgggcttagttttgggag1080
aatgattaggttgccttacctgcatggtttccccagaggatgtgtgccccaaggtggctg1140
gaatttttgacagacaagaagggtgaccttgggatgggggtgtggtctgttactttaatg1200
tttctgtttttaatgtgaaggccaagcaggccctgggatgggagtggggcggaggagggt1260
cctaagagctgattatttaatttctatccagaaatctttcttcttcttgctctgtttttt1320
taaattaaagatttcaac 1338
<210> 9
<211> 236
<212> PRT
<213> Homo sapiens
<400> 9
Ala Thr Ser Phe Pro Ile Ala Leu Ile Tyr Leu Val Leu Ile Ala Val
1 5 10 15
Gly Gln Asn Tyr Met Lys Glu Arg Lys Gly Phe Asn Leu Gln Gly Pro
20 25 30
Leu Ile Leu Trp Ser Phe Cys Leu Ala Ile Phe Ser I1e Leu Gly Ala
35 40 45
Va1 Arg Met Trp Gly Ile Met Gly Thr Val Leu Leu Thr Gly Gly Leu
50 55 60
Lys Gln Thr Val Cys Phe Ile Asn Phe Ile Asp Asn Ser Thr Val Lys
65 70 75 80
14
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Phe Trp Ser Trp Val Phe Leu Leu Ser Lys Val Ile Glu Leu Gly Asp
85 90 95
Thr Ala Phe Ile Ile Leu Arg Lys Arg Pro Leu Ile Phe Ile His Trp
100 105 110
Tyr His His Ser Thr Val Leu Val Tyr Thr Ser Phe Gly Tyr Lys Asn
115 120 125
Lys Val Pro Ala Gly Gly Trp Phe Val Thr Met Asn Phe Gly Val His
130 135 140
Ala Ile Met Tyr Thr Tyr Tyr Thr Leu Lys Ala Ala Asn Val Lys Pro
145 150 155 160
Pro Lys Met Leu Pro Met Leu Ile Thr Ser Leu Gln Ile Leu Gln Met
165 170 175
Phe Val Gly Ala Ile Val Ser Ile Leu Thr Tyr Ile Trp Arg Gln Asp
180 185 190
Gln Gly Cys His Thr Thr Met Glu His Leu Phe Trp Ser Phe Ile Leu
195 200 205
Tyr Met Thr Tyr Phe Ile Leu Phe Ala His Phe Phe Cys Gln Thr Tyr
210 215 220
Ile Arg Pro Lys Val Lys Ala Lys Thr Lys Ser Gln
225 230 235
<210> 10
<211> 270
<212> PRT
<213> Homo sapiens
<400> 20
Met Val Thr Ala Met Asn Val Ser His Glu Val Asn Gln Leu Phe Gln
l 5 10 15
Pro Tyr Asn Phe Glu Leu Ser Lys Asp Met Arg Pro Phe Phe Glu Glu
20 25 30
Tyr Trp Ala Thr Ser Phe Pro Ile Ala Leu Ile Tyr Leu Val Leu Ile
35 40 45
Ala Val Gly Gln Asn Tyr Met Lys Glu Arg Lys Gly Phe Asn Leu Gln
50 55 60
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Gly Pro Leu Ile Leu Trp Ser Phe Cys Leu Ala Ile Phe Ser Ile Leu
65 70 75 80
G1y Ala Val Arg Met Trp Gly Ile Met Gly Thr Val Leu Leu Thr Gly
85 90 95
Gly Leu Lys Gln Thr Val Cys Phe Ile Asn Phe Ile Asp Asn Ser Thr
100 105 110
Val Lys Phe Trp Ser Trp Val Phe Leu Leu Ser Lys Val Ile Glu Leu
115 120 125
Gly Asp Thr Ala Phe Ile Ile Leu Arg Lys Arg Pro Leu Ile Phe Ile
130 135 140
His Trp Tyr His His Ser Thr Val Leu Val Tyr Thr Ser Phe Gly Tyr
145 150 155 160
Lys Asn Lys Val Pro Ala Gly Gly Trp Phe Val Thr Met Asn Phe Gly
165 170 175
Val His Ala Ile Met Tyr Thr Tyr Tyr Thr Leu Lys Ala Ala Asn Val
180 185 190
Lys Pro Pro Lys Met Leu Pro Met Leu Ile Thr Ser Leu Gln Ile Leu
195 200 205
Gln Met Phe Val Gly Ala Ile Val Ser I1e Leu Thr Tyr Ile Trp Arg
210 215 220
Gln Asp Gln Gly Cys His Thr Thr Met Glu His Leu Phe Trp Ser Phe
225 230 235 240
Ile Leu Tyr Met Thr Tyr Phe Ile Leu Phe Ala His Phe Phe Cys Gln
245 250 255
Thr Tyr Ile Arg Pro Lys Val Lys Ala Lys Thr Lys Ser Gln
260 265 270
<210> 11
<211> 265
<212> PRT
<213> Homo sapiens
<400> 11
Met Asn Met Ser Val Leu Thr Leu Gln Glu Tyr Glu Phe Glu Lys Gln
16
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
1 5 10 15
Phe Asn Glu Asn Glu Ala Ile Gln Trp Met Gln Glu Asn Trp Lys Lys
20 25 30
Ser Phe Leu Phe Ser Ala Leu Tyr Ala Ala Phe Ile Phe Gly Gly Arg
35 40 45
His Leu Met Asn Lys Arg Ala Lys Phe Glu Leu Arg Lys Pro Leu Val
50 55 60
Leu Trp Ser Leu Thr Leu Ala Val Phe Sex Ile Phe Gly Ala Leu Arg
65 70 75 80
Thr Gly Ala Tyr Met Val Tyr Ile Leu Met Thr Lys Gly Leu Lys Gln
85 90 95
Ser Val Cys Asp Gln Gly Phe Tyr Asn Gly Pro Val Ser Lys Phe Trp
100 105 110
Ala Tyr Ala Phe Val Leu Ser Lys Ala Pro Glu Leu Gly Asp Thr Ile
115 120 125
Phe Ile Ile Leu Arg Lys Gln Lys Leu Ile Phe Leu His Trp Tyr His
130 135 140
His Ile Thr Val Leu Leu Tyr Ser Trp Tyr Ser Tyr Lys Asp Met Val
145 150 155 160
Ala Gly Gly Gly Trp Phe Met Thr Met Asn Tyr Gly Val His Ala Val
165 170 175
Met Tyr Ser Tyr Tyr Ala Leu Arg Ala Ala Gly Phe Arg Val Ser Arg
180 185 190
Lys Phe Ala Met Phe Ile Thr Leu Ser Gln Ile Thr Gln Met Leu Met
195 200 205
Gly Cys Val Val Asn Tyr Leu Val Phe Cys Trp Met Gln His Asp Gln
210 215 220
Cys His Ser His Phe Gln Asn Ile Phe Trp Ser Ser Leu Met Tyr Leu
225 230 235 240
Ser Tyr Leu Val Leu Phe Cys His Phe Phe Phe Glu A1a Tyr Ile Gly
245 250 255
17
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Lys Met Arg Lys Thr Thr Lys Ala Glu
260 265
<210> 12
<211> 265
<212> PRT
<213> Homo Sapiens
<400> 12
Met Asn Met Ser Val Leu Thr Leu Gln Glu Tyr Glu Phe Glu Lys Gln
1 5 10 15
Phe Asn Glu Asn Glu A1a Ile Gln Trp Met Gln Glu Asn Trp Lys Lys
20 25 30
Ser Phe Leu Phe Ser Ala Leu Tyr Ala Ala Phe Ile Phe Gly Gly Arg
35 40 45
His Leu Met Asn Lys Arg Ala Lys Phe Glu Leu Arg Lys Pro Leu Val
50 55 60
Leu Trp Ser Leu Thr Leu Ala Val Phe Ser Ile Phe Gly Ala Leu Arg
65 70 75 80
Thr Gly Ala Tyr Met Val Tyr Ile Leu Met Thr Lys Gly Leu Lys Gln
85 90 95
Ser Val Cys Asp Gln Gly Phe Tyr Asn Gly Pro Val Ser Lys Phe Trp
100 105 110
Ala Tyr Ala Phe Val Leu Ser Lys Ala Pro Glu Leu Gly Asp Thr Ile
115 120 125
Phe Ile Ile Leu Arg Lys Gln Lys Leu Ile Phe Leu His Trp Tyr His
130 135 140
His Ile Thr Val Leu Leu Tyr Ser Trp Tyr Ser Tyr Lys Asp Met Val
145 150 155 160
Ala Gly Gly Gly Trp Phe Met Thr Met Asn Tyr Gly Val His Ala Val
165 170 175
Met Tyr Ser Tyr Tyr Ala Leu Arg Ala Ala Gly Phe Arg Val Ser Arg
180 185 190
Lys Phe Ala Met Phe Ile Thr Leu Ser Gln Ile Thr Gln Met Leu Met
195 200 205
18
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Gly Cys Val Val Asn Tyr Leu Val Phe Cys Trp Met Gln His Asp Gln
210 215 220
Cys His Ser His Phe Gln Asn Ile Phe Trp Ser Ser Leu Met Tyr Leu
225 230 235 240
Ser Tyr Leu Val Leu Phe Cys His Phe Phe Phe Glu Ala Tyr Ile Gly
245 250 255
Lys Met Arg Lys Thr Thr Lys Ala Glu
260 265
<210> 13
<211> 314
<212> PRT
<213> Homo Sapiens
<400> 13
Met Gly Leu Leu Asp Ser Glu Pro Gly Ser Val Leu Asn Val Val Ser
1 5 10 15
Thr Ala Leu Asn Asp Thr Val Glu Phe Tyr Arg Trp Thr Trp Ser Ile
20 25 30
Ala Asp Lys Arg Val Glu Asn Trp Pro Leu Met Gln Ser Pro Trp Pro
35 40 45
Thr Leu Ser Ile Ser Thr Leu Tyr Leu Leu Phe Val Trp Leu Gly Pro
50 55 60
Lys Trp Met Lys Asp Arg Glu Pro Phe Gln Met Arg Leu Val Leu Ile
65 70 75 80
Ile Tyr Asn Phe Gly Met Val Leu Leu Asn Leu Phe Ile Phe Arg Glu
85 90 95
Leu Phe Met Gly Ser Tyr Asn Ala Gly Tyr Ser Tyr Ile Cys Gln Ser
100 105 110
Val Asp Tyr Ser Asn Asn Val His Glu Val Arg Ile Ala Ala Ala Leu
115 120 125
Trp Trp Tyr Phe Val Ser Lys Gly Val Glu Tyr Leu Asp Thr Val Phe
130 135 140
Phe Ile Leu Arg Lys Lys Asn Asn Gln Val Ser Phe Leu His Val Tyr
19
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
145 150 155 160
His His Cys Thr Met Phe Thr Leu Trp Trp Ile Gly Ile Lys Trp Val
165 170 175
Ala Gly Gly Gln Ala Phe Phe Gly Ala Gln Leu Asn Ser Phe Ile His
180 185 190
Val Ile Met Tyr Ser Tyr Tyr Gly Leu Thr Ala Phe Gly Pro Trp Ile
195 200 205
Gln Lys Tyr Leu Trp Trp Lys Arg Tyr Leu Thr Met Leu Gln Leu Ile
210 215 220
Gln Phe His Val Thr Ile Gly His Thr Ala Leu Ser Leu Tyr Thr Asp
225 230 235 240
Cys Pro Phe Pro Lys Trp Met His Trp Ala Leu Ile Ala Tyr Ala Ile
245 250 255
Ser Phe Ile Phe Leu Phe Leu Asn Phe Tyr Ile Arg Thr Tyr Lys Glu
260 265 270
Pro Lys Lys Pro Lys Ala Gly Lys Thr Ala Met Asn Gly Ile Ser Ala
275 280 285
Asn Gly Val Ser Lys Ser Glu Lys Gln Leu Met Ile Glu Asn Gly Lys
290 295 300
Lys Gln Lys Asn Gly Lys Ala Lys Gly Asp
305 310
<210> 14
<211> 314
<212> PRT
<213> Homo sapiens
<400> 14
Met Gly Leu Leu Asp Ser Glu Pro Gly Ser Val Leu Asn Val Val Ser
1 5 10 15
Thr Ala Leu Asn Asp Thr Val Glu Phe Tyr Arg Trp Thr Trp Ser Ile
20 25 30
Ala Asp Lys Arg Val Glu Asn Trp Pro Leu Met Gln Ser Pro Trp Pro
35 40 45
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Thr Leu Ser Ile Ser Thr Leu Tyr Leu Leu Phe Val Trp Leu Gly Pro
50 55 60
Lys Trp Met Lys Asp Arg Glu Pro Phe Gln Met Arg Leu Val Leu Ile
65 70 75 80
Ile Tyr Asn Phe Gly Met Val Leu Leu Asn Leu Phe Tle Phe Arg Glu
85 90 95
Leu Phe Met Gly Ser Tyr Asn Ala Gly Tyr Ser Tyr Ile Cys Gln Ser
100 105 ' 110
Val Asp Tyr Ser Asn Asn Val His Glu Val Arg Ile Ala Ala Ala Leu
115 120 125
Trp Trp Tyr Phe Val Ser Lys Gly Val Glu Tyr Leu Asp Thr Val Phe
130 135 140
Phe Ile Leu Arg Lys Lys Asn Asn Gln Val Ser Phe Leu His Val Tyr
145 150 155 160
His His Cys Thr Met Phe Thr Leu Trp Trp Ile Gly Ile Lys Trp Val
165 170 175
Ala Gly Gly Gln Ala Phe Phe Gly Ala Gln Leu Asn Ser Phe Ile His
180 185 190
Val Ile Met Tyr Ser Tyr Tyr Gly Leu Thr Ala Phe Gly Pro Trp Ile
195 200 205
Gln Lys Tyr Leu Trp Trp Lys Arg Tyr Leu Thr Met Leu Gln Leu Ile
210 215 220
Gln Phe His Val Thr Ile Gly His Thr Ala Leu Ser Leu Tyr Thr Asp
225 230 235 240
Cys Pro Phe Pro Lys Trp Met His Trp Ala Leu Ile Ala Tyr Ala Ile
245 250 255
Ser Phe Ile Phe Leu Phe Leu Asn Phe Tyr Ile Arg Thr Tyr Lys Glu
260 265 270
Pro Lys Lys Pro Lys Ala Gly Lys Thr Ala Met Asn Gly Ile Ser Ala
275 280 285
Asn Gly Val Ser Lys Ser Glu Lys Gln Leu Met Ile Glu Asn Gly Lys
290 ~ 295 300
21
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Lys Gln Lys Asn Gly Lys Ala Lys Gly Asp
305 310
<210> 15
<211> 303
<212> PRT
<213> Danio rerio
<400> 15
Met Glu Thr Val Val His Leu Met Asn Asp Ser Val Glu Phe Tyr Lys
1 5 10 15
Trp Ser Leu Thr Ile Ala Asp Lys Arg Val Glu Lys Trp Pro Met Met
20 25 30
Ser Ser Pro Leu Pro Thr Leu GIy Ile Ser Val Leu Tyr Leu Leu Phe
35 40 45
Leu Trp Ala Gly Pro Leu Tyr Met Gln Asn Arg Glu Pro Phe Gln Leu
50 55 60
Arg Lys Thr Leu Ile Val Tyr Asn Phe Ser Met Val Leu Leu Asn Phe
65 70 75 80
Tyr Ile Cys Lys Glu Leu Leu Leu Gly Ser Arg Ala Ala Gly Tyr Ser
85 90 95
Tyr Leu Cys Gln Pro Val Asn Tyr Ser Asn Asp Vat Asn Glu Val Arg
100 105 110
Ile Ala Ser Ala Leu Trp Trp Tyr Tyr Ile Ser Lys Gly Val Glu Phe
115 120 125
Leu Asp Thr Val Phe Phe Ile Met Arg Lys Lys Phe Asn Gln Val Ser
130 135 140
Phe Leu His Val Tyr His His Cys Thr Met Phe Ile Leu Trp Trp Ile
145 150 155 160
Gly Ile Lys Trp Val Pro Gly Gly Gln Ser Phe Phe Gly Ala Thr Ile
165 170 175
Asn Ser Gly Ile His Val Leu Met Tyr Gly Tyr Tyr Gly Leu Ala Ala
180 185 190
Phe Gly Pro Lys Ile Gln Lys Tyr Leu Trp Trp Lys Lys Tyr Leu Thr
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
195 200 205
Ile I1e Gln Met Ile Gln Phe His Val Thr Ile Val His Ala Ala Tyr
210 215 220
Ser Leu Tyr Thr Gly Cys Pro Phe Pro Ala Trp Met Gln Ser Ala Leu
225 230 235 240
Ile Gly Tyr Ala Asp Thr Phe Ile Ile Leu Leu Ala Asn Phe Tyr Tyr
245 250 255
Gln Thr Tyr Arg Arg Gln Pro Leu Pro Lys Thr Ala Lys Phe Ala Val
260 265 270
Asn Gly Val Ser Met Ser Thr Asn Gly Thr Ser Lys Thr Ala Glu Val
275 280 285
Thr Glu Asn Gly Lys Lys Gln Thr Lys Gly Lys Gly Lys His Asp
290 295 300
<210> 16
<211> 270
<212> PRT
<213> Homo sapiens
<400> 16
Met Val Thr Ala Met Asn Val Ser His Glu Val Asn Gln Leu Phe Gln
1 5 10 15
Pro Tyr Asn Phe Glu Leu Ser Lys Asp Met Arg Pro Phe Phe G1u Glu
20 25 30
'Tyr Trp Ala Thr Ser Phe Pro Ile Ala Leu Ile Tyr Leu Val Leu Ile
35 40 45
Ala Val Gly Gln Asn Tyr Met Lys Glu Arg Lys Gly Phe Asn Leu Gln
50 55 60
Gly Pro Leu Ile Leu Trp Ser Phe Cys Leu Ala Ile Phe Ser Ile Leu
65 70 75 80
Gly Ala Val Arg Met Trp Gly Ile Met Gly Thr Val Leu Leu Thr Gly
85 90 95
Gly Leu Lys Gln Thr Val Cys Phe Ile Asn Phe Ile Asp Asn Ser Thr
100 105 110
23
CA 02448112 2003-11-20
WO 02/099068 PCT/US02/17739
Val Lys Phe Trp Ser Trp Val Phe Leu Leu Ser Lys Val Ile Glu Leu
115 120 125
Gly Asp Thr Ala Phe Ile Ile Leu Arg Lys Arg Pro Leu I1e Phe Ile
130 135 140
His Trp Tyr His His Ser Thr Val Leu Val Tyr Thr Ser Phe Gly Tyr
145 150 155 160
Lys Asn Lys Val Pro Ala Gly Gly Trp Phe Val Thr Met Asn Phe Gly
165 170 175
Val His Ala Ile Met Tyr Thr Tyr Tyr Thr Leu Lys Ala Ala Asn Val
180 185 190
Lys Pro Pro Lys Met Leu Pro Met Leu Ile Thr Ser Leu Gln Ile Leu
195 200 205
Gln Met Phe Val Gly Ala Ile Val Ser Ile Leu Thr Tyr Ile Trp Arg
210 215 220
Gln Asp Gln Gly Cys His Thr Thr Met Glu His Leu Phe Trp Ser Phe
225 230 235 240
Ile Leu Tyr Met Thr Tyr Phe Ile Leu Phe Ala His Phe Phe Cys Gln
245 250 255
Thr Tyr Ile Arg Pro Lys Val Lys Ala Lys Thr Lys Ser Gln
260 265 270
24
