Note: Descriptions are shown in the official language in which they were submitted.
CA 02449479 2003-12-02
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MAP3Ks 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/Wafl/Cipl, cyclin G,
Bax, IGF-
BP3, and MDM2 (Levine, Cell (1997) 88:323-331).
Protein kinases (PKs) play a crucial role in regulating cellular processes,
including
growth factor response, cytoskeletal changes, gene expression, and metabolism.
PKs have
CA 02449479 2003-12-02
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very similar sequences and can be grouped based on specificity for the
acceptor amino
acid. Most PKs phosphorylate either serinelthreonine or tyrosine. However,
some PKs,
referred to as mixed-lineage kinases, have features of both serine/threonine
and tyrosine
PKs. All PKs have Src homology (SH) domains and can also be grouped as
receptors or
nonreceptors. Receptor PKs have a transmembrane region, an extracellular
ligand-binding
domain, and an intracellular catalytic domain.
Mitogen activated protein kinase kinase kinase 12 (MAP3K12), is a dual leucine
zipper-bearing kinase, and a member of the mixed lineage protein kinase (MLK)
(Reddy,
U. and Pleasure, D., (1994) Biochem. Biophys. Res. Commun. 202: 613-620).
MAP3Kl2
contains a COOH-terminal and NH2-terminal proline-rich domains suggestive of
src
homology 3 (SH3) domain binding regions, and can be autophosphorylated on
serine and
threonine residues (Holzman, L. et al., (1994) J. Biol. Chem. 269: 30808-
30817). This
kinase activates the SAPK/JNK signaling pathway, and may play a role in
neuronal
differentiation (Hirai, S., (1996) Oncogene 12: 641-650).
MAP3K13 protein, also called LZK (leucine zipper-bearing kinase) contains
double
leucine/isoleucine zippers, has no apparent signal sequence or transmembrane
region but
does contain a kinase catalytic domain, and an acidic domain at its C-terminal
end
Sakuma, H. et al.,( 1997) J. Biol. Chem. 272: 28622-28629). MAP3K13 shares
86.4%
amino acid identity with MAP3K12 and like MAP3K12 it is also a member of the
mixed-
lineage kinase family of proteins which contain similarities to both
serine/threonine and
tyrosine kinases ( Sakuma, H. et al.,( 1997) J. Biol. Chem. 272: 28622-28629).
These
kinases activate the phosphorylation event of c-Jun and turn on JNK-1 (Sakuma,
H. et al.,(
1997) J. Biol. Chem. 272: 28622-28629).
MAP3K12 and MAP3K13 are both highly conserved genes that have been found in
organisms from yeast to man. MAP3K12 has been implicated in neuronal Bell
death (Xu,
Z. et al. (2001) Mol Cell Biol 21:4713-24).
The ability to manipulate the genomes of model organisms such as Drosoplaila
provides a powerful means to analyze biochemical processes that, due to
significant
evolutionary conservation, has 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,
2
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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 random 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 THE INVENTION
We have discovered genes that modify the p53 pathway in Drosophila, and
identified
their human orthologs, hereinafter referred to as MAP3Ks. 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 MAP3K-modulating agents specifically bind to MAP3K
polypeptides and restore p53 function. Other preferred MAP3K-modulating agents
are
nucleic acid modulators such as antisense oligomers and RNAi that repress
MAP3K gene
expression or product activity by, for example, binding to and inhibiting the
respective
nucleic acid (i.e. DNA or mRNA).
MAP3K-specific modulating agents may be evaluated by any convenient in vitro
or if2
viv~ assay for molecular interaction with a MAP3K polypeptide or nucleic acid.
In one
embodiment, candidate p53 modulating agents are tested with an assay system
comprising
a MAP3K 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. MAP3K-modulating
agents
include MAP3K related proteins (e.g. dominant negative mutants, and
biotherapeutics);
MAP3K-specific antibodies; MAP3K-specific antisense oligomers and other
nucleic acid
modulators; and chemical agents that specifically bind MAP3K or compete with
MAP3K
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binding target. In one specific embodiment, a small molecule modulator is
identified
using a kinase assay. In specific embodiments, the screening assay system is
selected
from a binding assay, an apoptosis assay, a cell proliferation assay, an
angiogenesis assay,
and a hypoxic induction assay.
S 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
MAP3K 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.
DETAILED DESCRIPTION OF THE INVENTION
Genetic screens were designed to identify modifiers of the p53 pathway in and
Dr~sophila in which p53 was overexpressed in the wing (Ollmann M, et al., Cell
2000
101: 91-101). The CG8789 gene was identified as a modifier of the p53 pathway.
Accordingly, vertebrate orthologs of these modifiers, and preferably the human
orthologs,
MAP3K 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.
In vitro and in vivo methods of assessing MAP3K function are provided herein.
Modulation of the MAP3K 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.
MAP3K-modulating agents that act by inhibiting or enhancing MAP3K expression,
directly or indirectly, for example, by affecting a MAP3K function such as
enzymatic
(e.g., catalytic) or binding activity, can be identified using methods
provided herein.
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MAP3K modulating agents are useful in diagnosis, therapy and pharmaceutical
development.
Nucleic acids and nolypentides of the invention
Sequences related to MAP3K nucleic acids and polypeptides that can be used in
the
invention are disclosed in Genbank (referenced by Genbank identifier (GI)
number) as
GI#s 5454183 (SEQ ID NO:1), 13645287 (SEQ ID N0:4), and 4758695 (SEQ ID N0:5)
for nucleic acid, and GI#s 5454184 (SEQ ID N0:8) and 4758696 (SEQ ID N0:9) for
polypeptides. Further, sequences of SEQ ID NOs: 2, 3, 6, and 7 can also be
used in the
invention.
MAP3Ks are kinase proteins with kinase domains. The term "MAP3K polypeptide"
refers to a full-length MAP3K protein or a functionally active fragment or
derivative
thereof. A "functionally active" MAP3K fragment or derivative exhibits one or
more
functional activities associated with a full-length, wild-type MAP3K protein,
such as
antigenic or immunogenic activity, enzymatic activity, ability to bind natural
cellular
substrates, etc. The functional activity of MAP3K 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 a
MAP3K,
such as a kinase domain or a binding domain. Protein domains can be identified
using the
PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2;
http:l/pfam.wustl.edul. For example, the kinase domain of MAP3K from GI#
5454184
(SEQ ID N0:8) is located at approximately amino acid residues 125-366 (PFAM
00069).
Likewise, the kinase domain of MAP3K from GI# 4758696 (SEQ ~ N0:9) is located
at
approximately amino acid residues 168-409. Methods for obtaining MAP3K
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:B or 9 (a MAP3K). In further
preferred embodiments, the fragment comprises the entire kinase (functionally
active)
domain.
The term "MAP3K nucleic acid" refers to a DNA or RNA molecule that encodes a
MAP3K polypeptide. Preferably, the MAP3K polypeptide or nucleic acid or
fragment
5
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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 MAP3K.
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 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
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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
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 algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances
in
Applied Mathematics 2:482-489; database: European Bioinformatics Institute
http://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 Gribskov (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, or 7The 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. 1, Chap. 2.10, John Wiley & Sons,
Publishers
(1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). 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, or 7 under stringent hybridization conditions that comprise:
prehybridization of
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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 ,uglml herring sperm
DNA;
hybridization for 18-20 hours at 65° C in a solution containing 6X SSC,
1X Denhardt's
solution, 100 ~.g/ml 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).
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), 5mM 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 ~Cg/ml salmon sperm
DNA, and 10% (wdvol) dextrin 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% formamide, 5 x
SSC, 50 mM
sodium phosphate (pH 7.6), 5X Denhardt's solution, IO% dextrin sulfate, and 20
~Cglml
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 I hour.
Isolation, Production, Expression, and Mis-expression of MAP3K Nucleic Acids
and
Polyneptides
MAP3I~ nucleic acids and polypeptides, useful for identifying and testing
agents that
modulate MAP3K function and for other applications related to the involvement
of
MAP3I~ in the p53 pathway. MAP3K 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
polymerise
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
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
require the addition of specific tags (e.g., generation of fusion proteins).
Overexpression
of a MAP3K protein for assays used to assess MAP3K 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, 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 MAP3K 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 a MAP3K polypeptide can be inserted into any
appropriate expression vector. The necessary transcriptional and translational
signals,
including promoter/enhancer element, can derive from the native MAP3K 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 cosmid DNA. A host cell strain that modulates the
expression
of, modifies, and/or specifically processes the gene product may be used.
To detect expression of the MAP3K gene product, the expression vector can
comprise
a promoter operably linked to a MAP3K 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 MAP3K gene product based on the physical or
functional properties of the MAP3K protein in in vitro assay systems (e.g.
immunoassays).
The MAP3K 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
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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 MAP3K 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, cite purification reference). Alternatively, native MAP3K
proteins can be
purified from natural sources, by standard 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 MAP3K or other genes associated with the pS3
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 MAP3K expression
may
be used in in vivo assays to test for activity of a candidate p53 modulating
agent, or to
further assess the role of MAP3K in a p53 pathway process such as apoptosis or
cell
proliferation. Preferably, the altered MAP3K expression results in a
detectable phenotype,
such as decreased or increased levels of cell proliferation, angiogenesis, or
apoptosis
compared to control animals having normal MAP3K 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 Z~rosophila. 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 DNA (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.
CA 02449479 2003-12-02
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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 arid 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
Zebrafish see Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000);136:375-
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 phosphatelDNA 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 MAP3K
gene
that results in a decrease of MAP3K function, preferably such that MAP3K
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 MAP3K gene is used to
construct a
homologous recombination vector suitable for altering an endogenous MAP3K 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,
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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 Immunol 40:257-264; Declerck PJ
et
al., (1995) J Biol Chem. 270:8397-400).
In another embodiment, the transgenic animal 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 MAP3K gene, e.g., by introduction of
additional
copies of MAP3K, or by operatively inserting a regulatory sequence that
provides for
altered expression of an endogenous copy of the MAP3K gene. Such regulatory
sequences include 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 cre/loxP 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 in 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 MAP3K function and phenotypic
changes are
compared with appropriate control animals such as genetically modified animals
that
receive placebo treatment, and/or animals with unaltered MAP3K expression that
receive
candidate therapeutic agent.
In addition to the above-described genetically modified animals having altered
MAP3K function, animal models having defective p53 function (and otherwise
normal
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MAP3K function), can be used in the methods of the present invention. For
example, a
p53 knockout mouse can be used to assess, in vivo, the activity of a candidate
p53
modulating agent identified in one of the in 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.
ModuIatin~ Agents
The invention provides methods to identify agents that interact with and/or
modulate
the function of MAP3K and/or 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 MAP3K 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 MAP3K activity by administering
a
MAP3K-interacting or -modulating agent.
In a preferred embodiment, MAP3K-modulating agents inhibit or enhance MAP3K
activity or otherwise affect normal MAP3K 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 MAP3K. The phrases "specific modulating agent",
"specifically modulates", etc., are used herein to refer to modulating agents
that directly
bind to the MAP3K polypeptide or nucleic acid, and preferably inhibit,
enhance, or
otherwise alter, the function of the MAP3K. The term also encompasses
modulating
agents that alter the interaction of the MAP3K with a binding partner or
substrate (e.g. by
binding to a binding partner of a MAP3K, or to a protein/binding partner
complex, and
inhibiting function).
Preferred MAP3K-modulating agents include small molecule compounds; MAP3K-
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
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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
libraries. Synthetic compounds may be rationally designed or identified based
on known
or inferred properties of the MAP3K 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 MAP3K-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, and 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 in vitro and an vivo assays to optimize
activity and
minimize toxicity for pharmaceutical development.
Protein Modulators
Specific MAP3K-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 MAP3K-modulating agents. In a preferred
embodiment,
MAP3K-interacting proteins affect normal MAP3K function, including
transcription,
protein expression, protein localization, and cellular or extra-cellular
activity. In another
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embodiment, MAP3K-interacting proteins are useful in detecting and providing
information about the function of MAP3K proteins, as is relevant to p53
related disorders,
such as cancer (e.g., for diagnostic means).
An MAP3K-interacting protein may be endogenous, i.e. one that naturally
interacts
genetically or biochemically with a MAP3K, such as a member of the MAP3K
pathway
that modulates MAP3K expression, localization, and/or activity. MAP3K-
modulators
include dominant negative forms of MAP3K-interacting proteins and of MAP3K
proteins
themselves. Yeast two-hybrid and variant screens offer preferred methods for
identifying
endogenous MAP3K-interacting proteins (Finley, R. L. et al. (1996) in DNA
Cloning-
Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford
University Press, Oxford, 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 MAP3K-interacting protein may be an exogenous protein, such as a MAP3K-
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). MAP3K antibodies are further discussed below.
In preferred embodiments, a MAP3K-interacting protein specifically binds a
MAP3K
protein. In alternative preferred embodiments, a MAP3K-modulating agent binds
a
MAP3K substrate, binding partner, or cofactor.
Antibodies
In another embodiment, the protein modulator is a MAP3K specific antibody
agonist
or antagonist. The antibodies have therapeutic and diagnostic utilities, and
can be used in
screening assays to identify MAP3K modulators. The antibodies can also be used
in
dissecting the portions of the MAP3K pathway responsible for various cellular
responses
and in the general processing and maturation of the MAP3K.
Antibodies that specifically bind MAP3K polypeptides can be generated using
known
methods. Preferably the antibody is specific to a mammalian ortholog of MAP3K
polypeptide, and more preferably, to human MAP3K. Antibodies may be
polyclonal,
monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies,
Fab
CA 02449479 2003-12-02
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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 MAP3K which are particularly antigenic can be selected, for
example, by
routine screening of MAP3K 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. Immunol. 20:483-89;
Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence shown
in any of
SEQ ID NOs:8 or 9. Monoclonal antibodies with affinities of 108 M-1 preferably
10~ 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)
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 MAP3K or
substantially
purified fragments thereof. If MAP3K fragments are used, they preferably
comprise at
least 10, and more preferably, at least 20 contiguous amino acids of a MAP3K
protein. In
a particular embodiment, MAP3K-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
2Q immunized according to conventional protocols.
The presence of MAP3K-specific antibodies is assayed by an appropriate assay
such
as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized
corresponding MAP3K polypeptides. Other assays, such as radioimmunoassays or
fluorescent assays might also be used.
Chimeric antibodies specific to MAP3K 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
(CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse
antibodies
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into a background of human framework regions and constant regions by
recombinant
DNA 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).
MAP3K-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, 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
metals, chemiluminescent moieties, bioluminescent moieties, magnetic
particles, and the
like (U.S. 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.S. Pat.
No.
4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach
their
targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. No.
6,086,900).
When used therapeutically in a patient, the antibodies of the subject
invention are
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
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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 mg/ml to aboutl0 mg/ml.
Immunotherapeutic methods are further described in the Literature (US Pat. No.
5,859,206;
W00073469).
Nucleic Acid Modulators
Other preferred MAP3K-modulating agents comprise nucleic acid molecules, such
as
antisense oligomers or double stranded RNA (dsRNA), which generally inhibit
MAP3K
activity. Preferred nucleic acid modulators interfere with the function of the
MAP3K
nucleic acid such as DNA replication, transcription, translocation of the
MAP3K RNA to
the site of protein translation, translation of protein from the MAP3K RNA,
splicing of the
MAP3K RNA to yield one or more mRNA species, or catalytic activity which may
be
engaged in or facilitated by the MAP3K RNA.
In one embodiment, the antisense oligomer is an oligonucleotide that is
sufficiently
complementary to a MAP3K mRNA to bind to and prevent translation, preferably
by
binding to the 5' untranslated region. MAP3K-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 less 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.
In another embodiment, the antisense oligomer is a phosphothioate morpholino
oligomer (PMO). PMOs are assembled from four different morpholino subunits,
each of
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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 W099/18193; 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 MAP3K 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. elegar2s, l~rosophila, plants, and
humans are known
in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet.
15, 3S8-363
(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); Harnmond, 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); W00129058; WO99326I9; 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. (1993) 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, a MAP3K-specific nucleic acid
modulator is
used in an assay to further elucidate the role of the MAP3K in the p53
pathway, and/or its
relationship to other members of the pathway. In another aspect of the
invention, a
MAP3K-specific antisense oligomer is used as a therapeutic agent for treatment
of p53-
related disease states.
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Assay Systems
The invention provides assay systems and screening methods for identifying
specific
modulators of MAP3K activity. As used herein, an "assay system" encompasses
all the
components required for performing and analyzing results of an assay that
detects and/or
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 MAP3K
nucleic
acid or protein. In general, secondary assays further assess the activity of a
MAP3K
modulating agent identified by a primary assay and may confirm that the
modulating agent
affects MAP3K in a manner relevant to the p53 pathway. In some cases, MAP3K
modulators will be directly tested in a secondary assay.
In a preferred embodiment, the screening method comprises contacting a
suitable
assay system comprising a MAP3K polypeptide with a candidate agent under
conditions
whereby, but for the presence of the agent, the system provides a reference
activity (e.g.
kinase 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 MAP3K
activity, and hence
the p53 pathway.
Primary Assays
The type of modulator tested generally determines the type of primary assay.
Primary assays for sfnall »zoleczsle 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
mitochondrial 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
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
CA 02449479 2003-12-02
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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
MAP3K 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 MAP3K-
interacting
proteins are used in screens to identify small molecule modulators, the
binding specificity
of the interacting protein to the MAP3K protein may be assayed by various
known
methods such as substrate processing (e.g. ability of the candidate MAP3K-
specific
binding agents to function as negative effectors in MAP3K-expressing cells),
binding
equilibrium constants (usually at least about 107 M-1, preferably at least
about 10$1V1-1,
more preferably at least about 109 M-1), and immunogenicity (e.g. ability to
elicit MAP3K
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 MAP3K polypeptide, a fusion protein thereof, or to
cells or
membranes bearing the polypeptide or fusion protein. The MAP3K polypeptide can
be
full length or a fragment thereof that retains functional MAP3K activity. The
MAP3K
polypeptide may be fused to another polypeptide, such as a peptide tag for
detection or
anchoring, or to another tag. The MAP3K polypeptide is preferably human MAP3K,
or is
an ortholog or derivative thereof as described above. In a preferred
embodiment, the
screening assay detects candidate agent-based modulation of MAP3K interaction
with a
binding target, such as an endogenous or exogenous protein or other substrate
that has
MAP3K-specific binding activity, and can be used to assess normal MAP3K gene
function.
Suitable assay formats that may be adapted to screen for MAP3K modulators are
known in the art. Preferred screening assays are high throughput or ultra high
throughput
and thus 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
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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 MAP3K
and p53
pathway modulators (e.g. U.S. Pat. No. 6,165,992 (kinase assays); U.S. Pat.
Nos.
5,550,019 and 6,133,437 (apoptosis assays); and U.S. Pat. No. 6,020,135 (p53
modulation), among others). Specific preferred assays are described in more
detail below.
Kinase assays. In some preferred embodiments the screening assay detects the
ability
of the test agent to modulate the kinase activity of a MAP3K polypeptide. In
further
embodiments, a cell-free kinase assay system is used to identify a candidate
p53
modulating agent, and a secondary, cell-based assay, such as an apoptosis or
hypoxic
induction assay (described below), may be used to further characterize the
candidate p53
modulating agent. Many different assays for kinases have been reported in the
literature
and are well known to those skilled in the art (e.g. U.S. Pat. No. 6,165,992;
Zhu et aL,
Nature Genetics (2000) 26:283-289; and W00073469). Radioassays, which monitor
the
transfer of a gamma phosphate are frequently used. For instance, a
scintillation assay for
p56 (lck) kinase activity monitors the transfer of the gamma phosphate from
gamma -33P
ATP to a biotinylated peptide substrate; the substrate is captured on a
streptavidin coated
bead that transmits the signal (Beveridge M et al., J Biomol Screen (2000)
5:205-212).
This assay uses the scintillation proximity assay (SPA), in which only radio-
ligand bound
to receptors tethered to the surface of an SPA bead are detected by the
scintillant
immobilized within it, allowing binding to be measured without separation of
bound from
free ligand.
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
22
CA 02449479 2003-12-02
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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 a
MAP3K,
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 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 MAP3K function plays a direct role in apoptosis.
For
example, an apoptosis assay may be performed on cells that over- or under-
express
MAP3K relative to wild type cells. Differences in apoptotic response compared
to wild
type cells suggests that the MAP3K 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
MAP3K 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 cytometxy (Gray
JW et
al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55). Cells
transfected with
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a MAP3K 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 a MAP3K, 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
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 kinase assay system. A cell proliferation assay may
also be used
to test whether MAP3K function plays a direct role in cell proliferation or
cell cycle. For
example, a cell proliferation or cell cycle assay may be performed on cells
that over- or
under-express MAP3K relative to wild type cells. Differences in proliferation
or cell
cycle compared to wild type cells suggests that the MAP3K 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 a MAP3K, 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 MAP3K function
plays a
direct role in cell proliferation. For example, an angiogenesis assay may be
performed on
cells that over- or under-express MAP3K relative to wild type cells.
Differences in
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angiogenesis compared to wild type cells suggests that the MAP3K 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
transfected with MAP3K in hypoxic conditions (such as with 0.1% 02, 5% 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 a
MAP3K,
and that optionally has a mutated p53 (e.g. p53 is over-expressed ox 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 MAP3K 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 MAP3K relative to wild type cells. Differences in
hypoxic
response compared to wild type cells suggests that the MAP3K 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
CA 02449479 2003-12-02
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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.
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 cells 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).
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 MAP3K 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 MAP3K-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 MAP3K gene expression, preferably mRNA
expression.
In general, expression analysis comprises comparing MAP3K expression in like
populations of cells (e.g., two pools of cells that endogenously or
reeombinantly express
MAP3K) 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
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TaqMan~, PE Applied Biosystems), or microarray analysis may be used to confirm
that
MAP3K 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 MAP3K
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 MAP3K-
modulating
agent identified by any of the above methods to confirm that the modulating
agent affects
MAP3K in a manner relevant to the p53 pathway. As used herein, MAP3K-
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 MAP3K.
Secondary assays generally compare like populations of cells or animals (e.g.,
two
pools of cells or animals that endogenously or recombinantly express MAP3K) in
the
presence and absence of the candidate modulator. In general, such assays test
whether
treatment of cells or animals with a candidate MAP3K-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
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format. Candidate modulators are typically added to the cell media but may
also be
injected into cells or delivered by any other efficacious means.
Animal Assays
A variety of non-human animal models of normal or defective p53 pathway may be
used to test candidate MAP3K modulators. Models for defective p53 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 MAP3K 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
MAP3K. 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 may be dosed via oral (PO), intraperitoneal (IP), or
intravenous (IV)
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
MAP3K 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 ifi vitro culture. The tumors which express
the MAP3K
endogenously are injected in the flank, 1 x 105 to 1 x 107 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
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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
imrnunohistochemistry 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 MAP3K-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 MAP3K 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 MAP3K is implicated in p53 pathway provides for a variety
of
methods that can be employed for the diagnostic and prognostic evaluation of
diseases and
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 MAP3K
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 a
MAP3K, are identified as amenable to treatment with a MAP3K modulating agent.
In a
preferred application, the p53 defective tissue overexpresses a MAP3K 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 MAP3K cDNA sequences as probes, can determine whether particular
tumors
express or overexpress MAP3K. Alternatively, the TaqMan~ is used for
quantitative RT-
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PCR analysis of MAP3K expression in cell lines, normal tissues and tumor
samples (PE
Applied Biosystems).
Various other diagnostic methods may be performed, fox example, utilizing
reagents
such as the MAP3K oligonucleotides, and antibodies directed against a MAP3K,
as
described above for: (1) the detection of the presence of MAP3K gene
mutations, or the
detection of either over- or under-expression of MAP3K mRNA relative to the
non-
disorder state; (2) the detection of either an over- or an under-abundance of
MAP3K gene
product relative to the non-disorder state; and (3) the detection of
perturbations or
abnormalities in the signal transduction pathway mediated by MAP3K.
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 MAP3K 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.
I. Drosophila 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
CA 02449479 2003-12-02
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members of the p53 pathway. CG8789 was an enhancer of the wing phenotype.
Human
orthologs of the modifiers, are referred to herein as MAP3K.
BLAST analysis (Altschul et al., supra) was employed to identify Targets from
Drosophila modifiers. [For example, representative sequences from MAP3K,
GI#5454184 (SEQ ID N0:8) and GI#4758696 (SEQ ID N0:9) share 52% and 37% amino
acid identity, respectively, with the Drosophila.CG8789.
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
175-182 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 Nov;lO(11):1679-89)
programs.
Using PFAM, the kinase domain of MAP3K from GI# 5454184 (SEQ ID N0:8) is
located
at approximately amino acid residues 125-366 (PFAM 00069). Likewise, the
kinase
domain of MAP3K from GI# 4758696 (SEQ ID N0:9) is located at approximately
amino
acid residues 168-409.
II. High-Throughput In Vitro Fluorescence Polarization Assay
Fluorescently-labeled MAP3K 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 MAP3K activity.
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III. High-Throu.~hput In Vitro BindingAssay.
s3P-labeled MAP3K peptide is added in an assay buffer (100 mM KCI, 20 mM HEPES
pH 7.6, 1 mM MgCl2, 1% glycerol, 0.5% 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. Immunopreci~itations and Immunoblottin~
For coprecipitation of transfected proteins, 3 x 106 appropriate recombinant
cells
containing the MAP3K 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 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 ~,1 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. Kinase assay
A purified or partially purified MAP3K is diluted in a suitable reaction
buffer, e.g., 50
mM Hepes, pH 7.5, containing magnesium chloride or manganese chloride (1-20
mM) and
a peptide or polypeptide substrate, such as myelin basic protein or casein (1-
10 ~,g/ml).
The final concentration of the kinase is 1-20 nM. The enzyme reaction is
conducted in
microtiter plates to facilitate optimization of reaction conditions by
increasing assay
throughput. A 96-well microtiter plate is employed using a final volume 30-100
~,1. The
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reaction is initiated by the addition of 33P-gamma-ATP (0.5 ~,Ci/ml) and
incubated for 0.5
to 3 hours at room temperature. Negative controls are provided by the addition
of EDTA,
which chelates the divalent cation (Mg2+ or Mn2+) required for enzymatic
activity.
Following the incubation, the enzyme reaction is quenched using EDTA. Samples
of the
reaction are transferred to a 96-well glass fiber filter plate (MultiScreen,
Millipore). The
filters are subsequently washed with phosphate-buffered saline, dilute
phosphoric acid
(0.5°Io) or other suitable medium to remove excess radiolabeled ATP.
Scintillation
cocktail is added to the filter plate and the incorporated radioactivity is
quantitated by
scintillation counting (Wallac/Perkin Elmer). Activity is defined by the
amount of
radioactivity detected following subtraction of the negative control reaction
value (EDTA
quench).
VI. 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
50ng1~,l. 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, httn:/lwww.appliedbiosystems.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
~.1 total
volume for 96-well plates and 10 ~1 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
33
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
appreciable amounts is good. The raw data were normalized using 18S rRNA
(universally
expressed in all tissues and cells).
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. Results from various batches of mRNA are
represented
for each batch. 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. . . . . .
colon lung ovary
GI#5454183 (SEQ TaqExp_1005012 11. 30 . 13 . 7
ID NO:1) 4 1 1
GI#13645287 (SEQTaqExp_1005013 11. 30 . 13 . 7
D7 N0:4) 1 2 3
34
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
SEQUENCE LISTING
<110> EXELIXIS,
INC.
<120> MAP3Ks AS p53 PATHWAY
MODIFIERS OF AND METHODS
THE OF USE
<130> EX02-072C-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-O1
<160> 9
<170> Patentln
version 3.1
<210> 1
<211> 3365
<212> DNA
<213> Homo sapiens
<400> 1
agcatccgga gcggagctgcagcagcgccgccttttgtgctgcggccgcggagcccccga60
gggcccagtg ttcaccatcataccaggggccagaggcgatggcttgcctccatgagaccc120
gaacaccctc tccttcctttgggggctttgtgtctaccctaagtgaggcatccatgcgca180
agctggaccc agacacttctgactgcactcccgagaaggacctgacgcctacccatgtcc240
tgcagctaca tgagcaggatgcagggggcccagggggagcagctgggtcacctgagagtc300
gggcatccag agttcgagctgacgaggtgcgactgcagtgccagagtggcagtggcttcc360
ttgagggcct ctttggctgcctgcgccctgtctggaccatgattggcaaagcctactcca420
ctgagcacaa gcagcagcaggaagacctttgggaggtcccctttgaggaaatcctggacc480
tgcagtgggt gggctcaggggcccagggtgctgtcttcctggggcgcttccacggggagg540
aggtggctgt gaagaaggtgcgagacctcaaagaaaccgacatcaagcacttgcgaaagc600
tgaagcaccc caacatcatcactttcaagggtgtgtgcacccaggctccctgctactgca660
tcctcatgga gttctgcgcccagggccagctgtatgaggtactgcgggctggccgccctg720
tcaccccctc cttactggttgactggtccatgggcatcgctggtggcatgaactacctgc780
acctgcacaa gattatccacagggatctcaagtcacccaacatgctaatcacctacgacg840
atgtggtgaa gatctcagattttggcacttccaaggagctgagtgacaagagcaccaaga900
tgtcctttgc agggacagtagcctggatggcccctgaggtgatccgcaatgaacctgtgt960
1
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
ctgagaaggtcgacatctggtcctttggcgtggtgctatgggaactgctgactggtgaga1020
tcccctacaaagacgtagattcctcagccattatctggggtgtgggaagcaacagtctcc1080
atctgcccgtgccctccagttgcccagatggtttcaagatcctgcttcgccagtgctgga1140
atagcaaaccacgaaatcgcccatcattccgacagatcctgctgcatctggacattgcct1200
cagctgatgtactctccacaccccaggagacttactttaagtcccaggcagagtggcggg1260
aagaagtaaaactgcactttgaaaagattaagtcagaagggacctgtctgcaccgcctag1320
aagaggaactggtgatgaggaggagggaggagctcagacacgccctggacatcagggagc1380
actatgaaaggaagctggagagagccaacaacctgtatatggaacttaatgccctcatgt1440
tgcagctggaactcaaggagagggagctgctcaggcgagagcaagctttagagcggaggt1500
gcccaggcctgctgaagccacacccttcccggggcctcctgcatggaaacacaatggaga1560
agcttatcaagaagaggaatgtgccacagaatctgtcaccccatagccaaaggccagata1620
tcctcaaggcggagtctttgctccctaaactagatgcagccctgagtggggtggggcttc1680
ctgggtgtcctaaggcccccccctcaccaggacggagtcgccgtggcaagacccgtcacc1740
gcaaggccagcgccaaggggagctgtggggacctgcctgggcttcgtacagctgtgccac1800
cccatgaacctggaggaccaggaagcccagggggcctaggagggggaccctcagcctggg1860
aggcctgccctcccgccctccgtgggcttcatcatgacctcctgctccgcaaaatgtctt1920
catcgtccccagacctgctgtcagcagcactagggtcccggggccggggggccacaggcg1980
gagctggggatcctggctcaccacctccggcccggggtgacaccccaccaagtgagggct2040
cagcccctggctccaccagcccagattcacctgggggagccaaaggggaaccacctcctc2100
cagtagggcctggtgaaggtgtggggcttctgggaactggaagggaagggacctcaggcc2160
ggggaggaagccgggctgggtcccagcacttgaccccagctgcactgctgtacagggctg2220
ccgtcacccgaagtcagaaacgtggcatctcatcggaagaggaggaaggagaggtagaca2280
gtgaagtagagctgacatcaagccagaggtggcctcagagcctgaacatgcgccagtcac2340
tatctaccttcagctcagagaatccatcagatggggaggaaggcacagctagtgaacctt2400
cccccagtggcacacctgaagttggcagcaccaacactgatgagcggccagatgagcggt2460
ctgatgacatgtgctcccagggctcagaaatcccactggacccacctccttcagaggtca2520
tccctggccctgaacccagctccctgcccattccacaccaggaacttctcagagagcggg2580
gccctcccaattctgaggactcagactgtgacagcactgaattggacaactccaacagcg2640
ttgatgccttgcgccccccagcttccctccctccatgaaagccactcgtattccttgtac2700
atagagaaatatttatatggattatatatatatacatatatatatatatatgcgccacat2760
aatcaacagaaagatggggctgtcccagccgtaagtcaggctcgagggagactgatcccc2820
2
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
tgaccaattcacctgataaactctagggacactggcagctgtggaaatgaatgaggcaca2880
gccgtagagctgtggctaagggcaagccccttcctgccccaccccattccttatattcag2940
caagcaacaaggcaatagaaaagccagggttgtctttatattctttatccccaaataata3000
gggggtggggggaggggcggtgggaggggcaggagagaaaaccacttagactgcactttt3060
ctgttccgtttactctgtttacacattttgcacttgggaggagggaggctaaggctgggt3120
cctcccctctgaggtttctcaggtggcaatgtaactcatttttttgtcccaccatttatc3180
ttctctgcccaagccctgtcttaaggcccagggggaggttaggagactgatagcatgtga3240
tggctcaggctgaagaaccggggttctgtttaagtccctgcttttatcctggtgcctgat3300
tggggtggggactgtcctactgtaacccctgtgaaaaaccttgaaaaataacactccatg3360
cagga 3365
<210> 2
<211> 2830
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<222> (33) .(33)
<223> "n" is A, C, G, or T
<400>
2
cgagggcccagtgttcaccatcataccaggggncagaggcgatggcttgcctccatgaga60
cccgaacaccctctccttcctttgggggctttgtgtctaccctaagtgaggcatccatgc120
gcaagctggacccagacacttctgactgcactcccgagaaggacctgacgcctacccagt180
gtgtacttcgagatgtggtaccccttggtgggcagggtgggggagggcccagcccctccc240
caggtggagagccgccccctgagccttttgccaacagtgtcctgcagctacatgagcagg300
atgcagggggcccagggggagcagctgggtcacctgagagtcgggcatccagagttcgag360
ctgacgaggtgcgactgcagtgccagagtggcagtggcttccttgagggcctctttggct420
gcctgcgccctgtctggaccatgattggcaaagcctactccactgagcacaagcagcagc480
aggaagacctttgggaggtcccctttgaggaaatcctggacctgcagtgggtgggctcag540
gggcccagggtgctgtcttcctggggcgcttccacggggaggaggtggctgtgaagaagg600
tgcgagacctcaaagaaaccgacatcaagcacttgcgaaagctgaagcaccccaacatca660
tcactttcaagggtgtgtgcacccaggctccctgctactgcatcctcatggagttctgcg720
cccagggccagctgtatgaggtactgcgggctggccgccctgtcaccccctccttactgg780
ttgactggtc catgggcatc gctggtggca tgaactacct gcacctgcac aagattatcc 840
acagggatct caagtcaccc aacatgctaa tcacctacga cgatgtggtg aagatctcag 900
3
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
attttggcacttccaaggagctgagtgacaagagcaccaagatgtcctttgcagggacag960
tagcctggatggcccctgaggtgatccgcaatgaacctgtgtctgagaaggtcgacatct1020
ggtcctttggcgtggtgctatgggaactgctgactggtgagatcccctacaaagacgtag1080
attcctcagccattatctggggtgtgggaagcaacagtctccatctgcccgtgccctcca1140
gttgcccagatggtttcaagatcctgcttcgccagtgctggaatagcaaaccacgaaatc1200
gcccatcattccgacagatcctgctgcatctggacattgcctcagctgatgtactctcca1260
caccccaggagacttactttaagtcccaggcagagtggcgggaagaagtaaaactgcact1320
ttgaaaagattaagtcagaagggacctgtctgcaccgcctagaagaggaactggtgatga1380
ggaggagggaggagctcagacacgccctggacatcagggagcactatgaaaggaagctgg1440
agagagccaacaacctgtatatggaacttaatgccctcatgttgcagctggaactcaagg1500
agagggagctgctcaggcgagagcaagctttagagcggaggtgcccaggcctgctgaagc1560
cacacccttcccggggcctcctgcatggaaacacaatggagaagcttatcaagaagagga1620
atgtgccacagaagctgtcaccccatagcaaaaggccagatatcctcaagacggagtctt1680
tgctccctaaactagatgcagccctgagtggggtggggcttcctgggtgtcctaagggcc1740
ccccctcaccaggacggagtcgccgtggcaagacccgtcaccgcaaggccagcgccaagg1800
ggagctgtggggacctgcctgggcttcgtacagctgtgccaccccatgaacctggaggac1860
caggaagcccagggggcctaggagggggaccctcagcctgggaggcctgccctcccgccc1920
tccgtgggcttcatcatgacctcctgctccgcaaaatgtcttcatcgtccccagacctgc1980
tgtcagcagcactagggtcccggggccggggggccacaggcggagctggggatcctggct2040
caccacctccggcccggggtgacaccccaccaagtgagggctcagcccctggctccacca2100
gcccagattcacctgggggagccaaaggggaaccacctcctccagtagggcctggtgaag2160
gtgtggggcttctgggaactggaagggaagggacctcaggccggggaggaagccgggctg2220
ggtcccagcacttgaccccagctgcactgctgtacagggctgccgtcacccgaagtcaga2280
aacgtggcatctcatcggaagaggaggaaggagaggtagacagtgaagtagagctgacat2340
caagccagaggtggcctcagagcctgaacatgcgccagtcactatctaccttcagctcag2400
agaatccatcagatggggaggaaggcacagctagtgaaccttcccccagtggcacacctg2460
aagttggcagcaccaacactgatgagcggccagatgagcggtctgatgacatgtgctccc2520
agggctcagaaatcccactggacccacctccttcagaggtcatccctggccctgaaccca2580
gctccctgcccattccacaccaggaacttctcagagagcggggccctcccaattctgagg2640
actcagactgtgacagcactgaattggacaactccaacagcgttgatgccttgcggcccc2700
cagcttccctccctccatgaaagccactcgtattccttgtacatagagaaatatttatat2760
4
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
aaattatata tatatacata tatatatata tatgcgccac ataatcaaca gaaagatggg 2820
gctgtcccag 2830
<210>
3
<211>
2732
<212>
DNA
<213> sapiens
Homo
<400>
3
cgagggcccagtgttcaccatcataccaggggccagaggcgatggcttgcctccatgaga60
cccgaacaccctctccttcctttgggggctttgtgtctaccctaagtgaggcatccatgc120
gcaagctggacccagacacttctgactgcactcccgagaaggacctgacgcctacccatg180
tcctgcagctacatgagcaggatgcagggggcccagggggagcagctgggtcacctgaga240
gtcgggcatccagagttcgagctgacgaggtgcgactgcagtgccagagtggcagtggct300
tccttgagggcctctttggctgcctgcgccctgtctggaccatgattggcaaagcctact360
ccactgagcacaagcagcagcaggaagacctttgggaggtcccctttgaggaaatcctgg420
acctgcagtgggtgggctcaggggcccagggtgctgtcttcctggggcgcttccacgggg480
aggaggtggctgtgaagaaggtgcgagacctcaaagaaaccgacatcaagcacttgcgaa540
agctgaagcaccccaacatcatcactttcaagggtgtgtgcacccaggctccctgctact600
gcatcctcatggagttctgcgcccagggccagctgtatgaggtactgcgggctggccgcc660
ctgtcaccccctccttactggttgactggtccatgggcatcgctggtggcatgaactacc720
tgcacctgcacaagattatccacagggatctcaagtcacccaacatgctaatcacctacg780
acgatgtggtgaagatctcagattttggcacttccaaggagctgagtgacaagagcacca840
agatgtcctttgcagggacagtagcctggatggcccctgaggtgatccgcaatgaacctg900
tgtctgagaaggtcgacatctggtcctttggcgtggtgctatgggaactgctgactggtg960
agatcccctacaaagacgtagattcctcagccattatctggggtgtgggaagcaacagtc1020
tccatctgcccgtgccctccagttgcccagatggtttcaagatcctgcttcgccagtgct1080
ggaatagcaaaccacgaaatcgcccatcattccgacagatcctgctgcatctggacattg1140
cctcagctgatgtactctccacaccccaggagacttactttaagtcccaggcagagtggc1200
gggaagaagtaaaactgcactttgaaaagattaagtcagaagggacctgtctgcaccgcc1260
tagaagaggaactggtgatgaggaggagggaggagctcagacacgccctggacatcaggg1320
agcactatgaaaggaagctggagagagccaacaacctgtatatggaacttaatgccctca1380
tgttgcagctggaactcaaggagagggagctgctcaggcgagagcaagctttagagcgga1440
ggtgcccaggcctgctgaagccacacccttcccggggcctcctgcatggaaacacaatgg1500
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
agaagcttatcaagaagaggaatgtgccacagaagctgtcaccccatagcaaaaggccag1560
atatcctcaagacggagtctttgctccctaaactagatgcagccctgagtggggtggggc1620
ttcctgggtgtcctaagggccccccctcaccaggacggagtcgccgtggcaagacccgtc1680
accgcaaggccagcgccaaggggagctgtggggacctgcctgggcttcgtacagctgtgc1740
caccccatgaacctggaggaccaggaagcccagggggcctaggagggggaccctcagcct1800
gggaggcctgccctcccgccctccgtgggcttcatcatgacctcctgctccgcaaaatgt1860
cttcatcgtccccagacctgctgtcagcagcactagggtcccggggccggggggccacag1920
gcggagctggggatcctggctcaccacctccggcccggggtgacaccccaccaagtgagg1980
gctcagcccctggctccaccagcccagattcacctgggggagccaaaggggaaccacctc2040
ctccagtagggcctggtgaaggtgtggggcttctgggaactggaagggaagggacctcag2100
gccggggaggaagccgggctgggtcccagcacttgaccccagctgcactgctgtacaggg2160
ctgccgtcacccgaagtcagaaacgtggcatctcatcggaagaggaggaaggagaggtag2220
acagtgaagtagagctgacatcaagccagaggtggcctcagagcctgaacatgcgccagt2280
cactatctaccttcagctcagagaatccatcagatggggaggaaggcacagctagtgaac2340
cttcccccagtggcacacctgaagttggcagcaccaacactgatgagcggccagatgagc2400
ggtctgatgacatgtgctcccagggctcagaaatcccactggacccacctccttcagagg2460
tcatccctggccctgaacccagctccctgcccattccacaccaggaacttctcagagagc2520
ggggccctcccaattctgaggactcagactgtgacagcactgaattggacaactccaaca2580
gcgttgatgccttgcggcccccagcttccctccctccatgaaagccactcgtattccttg2640
tacatagagaaatatttatataaattatatatatatacatatatatatatatatgcgcca2700
cataatcaacagaaagatggggctgtccagcc 2732
<210>
4
<211>
3378
<212>
DNA
<213> sapiens
Homo
<400>
4
gttttggagccctctcttaagtcagaactctgtcccaaaaatcttctgagtgtcatctca60
ggactttggttatactcatggcacgatggccaactttcaggagcacctgagctgctcctc120
ttctccacacttacccttcagtgaaagcaaaaccttcaatggactacaagatgagctcac180
agctatggggaaccacccttctcccaagctgctcgaggaccagcaggaaaaggggatggt240
acgaacagagctaatcgagagcgtgcacagccccgtcaccacaacagtgttgacgagcgt300
aagtgaggattccagggaccagtttgagaacagcgttcttcagctaagggaacacgatga360
atcagagacggcggtgtctcaggggaacagcaacacggtggacggagagagcacaagcgg420
6
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
aactgaagacataaagattcagttcagcaggtcaggcagtggcagtggtgggtttcttga480
aggactatttggatgcttaaggcctgtatggaatatcattgggaaggcatattccactga540
ttacaaattgcagcagcaagatacttgggaagtgccatttgaggagatctcagagctgca600
gtggctgggtagtggagcccaaggagcggtcttcttgggcaagttccgggcggaagaggt660
ggccatcaagaaagtgagagaacagaatgagacggatatcaagcatttgaggaagttgaa720
gcaccctaacatcatcgcattcaagggtgtttgtactcaggccccatgttattgtattat780
catggaatactgtgcccatggacaactctacgaggtcttacgagctggcaggaagatcac840
acctcgattgctagtagactggtccacaggaattgcaagtggaatgaattatttgcacct900
ccataaaattattcatcgtgatctcaaatcacctaatgttttagtgacccacacagatgc960
ggtaaaaatttcagattttggtacatctaaggaactcagtgacaaaagtaccaagatgtc1020
atttgctggcacggtcgcatggatggcgccagaggtgatacggaatgaacctgtctctga1080
aaaagttgatatatggtcttttggagtggtgctttgggagctgctgacaggagagatccc1140
ttacaaagatgtagattcttcagccattatctggggtgttggaagcaacagcctccacct1200
tccagttccttccacttgccctgatggattcaaaatccttatgaaacagacgtggcagag1260
taaacctcgaaaccgaccttcttttcggcagacactcatgcatttagacattgcctctgc1320
agatgtacttgccaccccacaagaaacttacttcaagtctcaggctgaatggagagaaga1380
agtgaaaaaacattttgagaagatcaaaagtgaaggaacttgtatacaccggttagatga1440
agaactgattcgaaggcgcagagaagagctcaggcatgcgctggatattcgtgaacacta1500
tgagcggaagcttgagcgggcgaataatttatacatggaattgagtgccatcatgctgca1560
gctagaaatgcgggagaaggagctcattaagcgtgagcaagcagtggaaaagaagtatcc1620
tgggacctacaaacgacaccctgttcgtcctatcatccatcccaatgccatggagaaact1680
catgaaaaggaaaggagtgcctcacaaatctgggatgcagaccaaacggccagacttgtt1740
gagatcagaagggatccccaccacagaagtggctcccactgcatcccctttgtccggaag1800
tcccaaaatgtccacttctagcagcaagagccgatatcgaagcaaaccacgccaccgccg1860
agggaatagcagaggcagccatagtgactttgccgcaatcttgaaaaaccagccagccca1920
ggaaaattcaccccatcccacttacctgcaccaagctcaatcccaatacccttctcttca1980
tcaccataattctctgcagcagcaataccagcagccccctcctgccatgtcccagagtca2040
ccatcccagactcaatatgcacggacaggacatagcaacctgcgccaacaacctgaggta2100
tttcggcccagcagcagccctgcggagcccactcagcaaccatgCtcagagacagctgcc2160
cggctcgagccctgacctcatctccacagccatggctgcagactgctggagaagttctga2220
gcctgacaagggccaagctggtccctggggctgttgccaggctgacgcttatgacccctg2280
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
ccttcagtgcaggccagaacagtatgggtccttagacataccctctgctgagccagtggg2340
gaggagccctgacctttccaagtcaccagcacataatcctctcttggaaaacgcccagag2400
ttctgagaaaacggaagaaaatgaattcagcggctgtaggtctgagtcatccctcggcac2460
ctctcatctcggcacccctccagcgctacctcgaaaaacaaggcctctgcagaagagtgg2520
agatgactcctcagaagaggaagaaggggaagtagatagtgaagttgaatttccacgaag2580
acagaggccccatcgctgtatcagcagctgccagtcatattcaacctttagctctgagaa2640
tttctctgtgtctgatggagaagagggaaataccagtgaccactcaaacagtcctgatga2700
gttagctgataaacttgaagaccgcttggcagagaagctagacgacctgctgtcccagac2760
gccagagattcccattgacatatcctcacactcggatgggctctctgacaaggagtgtgc2820
cgtgcgccgtgtgaagactcagatgtctctgggcaagctgtgtgtggaggaacgtggcta2880
tgagaaccccatgcagtttgaagaatcggactgtgactcttcagatggggagtgttctga2940
tgccacagttaggaccaataaacactacagctctgctacctggtaatgaaggaatacaca3000
tcctgaagatctcgtgactatactggcatttcagatccaccccacccccagactcatccc3060
actctctcccagcattttgtctgggaagagagactaccccatctttaccaccccctagaa3120
atgagctgcaataacaggaacatgagacttcgcaaatctctggaaaataatatccaaatg3180
aaattaagtctcactgaacatttcaatcaagaatggcagggatctattttattgaatatt3240
ctagctactgtaacattgatatttatttttgtttgacattttaacactttgtactgcaaa3300
gagtgaactatatatgagatagagagacaataatttcttgcaaaaaaaaaaagagataaa3360
agaaagaaca gaaaaaaa 3378
<210> 5
<211> 3569
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<222> (3283)..(3283)
<223> "n" is A, C, G, or T
<400> 5
aatttacatc cattcatgaa tctgtgacgt cagcaagcct ttgggctcct ttgcggtggg 60
ctggaggattgtgtgggtggaatccccctcccctttatttttccaattctgcaaggcttt120
taaaattcaccttacatcttttcaaagcaagaaaatggaacagcatgtgtaggaattctt180
cgttgttgttttggagccctctcttaagtcagaactctgtcccaaaaatcttctgagtgt240
catctcaggactttggttatactcatggcacgatggccaactttcaggagcacctgagct300
g
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
gctcctcttctccacacttacccttcagtgaaagcaaaaccttcaatggactacaagatg360
agctcacagctatggggaaccacccttctcccaagctgctcgaggaccagcaggaaaagg420
ggatggtacgaacagagctaatcgagagcgtgcacagccccgtcaccacaacagtgttga480
cgagcgtaagtgaggattccagggaccagtttgagaacagcgttcttcagctaagggaac540
acgatgaatcagagacggcggtgtctcaggggaacagcaacacggtggacggagagagca600
caagcggaactgaagacataaagattcagttcagcaggtcaggcagtggcagtggtgggt660
ttcttgaaggactatttggatgcttaaggcctgtatggaatatcattgggaaggcatatt720
ccactgattacaaattgcagcagcaagatacttgggaagtgccatttgaggagatctcag780
agctgcagtggctgggtagtggagcccaaggagcggtcttcttgggcaagttccgggcgg840
aagaggtggccatcaagaaagtgagagaacagaatgagacggatatcaagcatttgagga900
agttgaagcaccctaacatcatcgcattcaagggtgtttgtactcaggccccatgttatt960
gtattatcatggaatactgtgcccatggacaactctacgaggtcttacgagctggcagga1020
agatcacacctcgattgctagtagactggtccacaggaattgcaagtggaatgaattatt1080
tgcacctccataaaattattcatcgtgatctcaaatcacctaatgttttagtgacccaca1140
cagatgcggtaaaaatttcagattttggtacatctaaggaactcagtgacaaaagtacca1200
agatgtcatttgctggcacggtcgcatggatggcgccagaggtgatacggaatgaacctg1260
tctctgaaaaagttgatatatggtcttttggagtggtgctttgggagctgctgacaggag1320
agatcccttacaaagatgtagattcttcagccattatctggggtgttggaagcaacagcc1380
tccaccttccagttccttccacttgccctgatggattcaaaatccttatgaaacagacgt1440
ggcagagtaaacctcgaaaccgaccttcttttcggcagacactcatgcatttagacattg1500
cctctgcagatgtacttgccaccccacaagaaacttacttcaagtctcaggctgaatgga1560
gagaagaagtgaaaaaacattttgagaagatcaaaagtgaaggaacttgtatacaccggt1620
tagatgaagaactgattcgaaggcgcagagaagagctcaggcatgcgctggatattcgtg1680
aacactatgagcggaagcttgagcgggcgaataatttatacatggaattgagtgccatca1740
tgctgcagctagaaatgcgggagaaggagctcattaagcgtgagcaagcagtggaaaaga1800
agtatcctgggacctacaaacgacaccctgttcgtcctatcatccatcccaatgccatgg1860
agaaactcatgaaaaggaaaggagtgcctcacaaatctgggatgcagaccaaacggccag1920
acttgttgagatcagaagggatccccaccacagaagtggctcccactgcatcccctttgt1980
ccggaagtcccaaaatgtccacttctagcagcaagagccgatatcgaagcaaaccacgcc2040
accgccgagggaatagcagaggcagccatagtgactttgccgcaatcttgaaaaaccagc2100
cagcccaggaaaattcaccccatcccacttacctgcaccaagctcaatcccaataccctt2160
9
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
ctcttcatca ccataattct ctgcagcagc aataccagca gccccctcct gccatgtccc 2220
agagtcacca tcccagactc aatatgcacg gacaggacat agcaacctgc gccaacaacc 2280
tgaggtattt cggcccagca gcagccctgc ggagcccact cagcaaccat gctcagagac 2340
agctgcccgg ctcgagccct gacctcatct ccacagccat ggctgcagac tgctggagaa 2400
gttctgagcc tgacaagggc caagctggtc cctggggctg ttgccaggct gacgcttatg 2460
acccctgcct tcagtgcagg ccagaacagt atgggtcctt agacataccc tctgctgagc 2520
cagtggggag gagccctgac ctttccaagt caccagcaca taatcctctc ttggaaaacg 2580
cccagagttctgagaaaacggaagaaaatgaattcagcggctgtaggtctgagtcatccc2640
tcggcacctctcatctcggcacccctccagcgctacctcgaaaaacaaggcctctgcaga2700
agagtggagatgactcctcagaagaggaagaaggggaagtagatagtgaagttgaatttc2760
cacgaagacagaggccccatcgctgtatcagcagctgccagtcatattcaacctttagct2820
ctgagaatttctctgtgtctgatggagaagagggaaataccagtgaccactcaaacagtc2880
ctgatgagttagctgataaacttgaagaccgcttggcagagaagctagacgacctgctgt2940
cccagacgccagagattcccattgacatatcctcacactcggatgggctctctgacaagg3000
agtgtgccgtgcgccgtgtgaagactcagatgtctctgggcaagctgtgtgtggaggaac3060
gtggctatgagaaccccatgcagtttgaagaatcggactgtgactcttcagatggggagt3120
gttctgatgccacagttaggaccaataaacactacagctctgctacctggtaatgaagga3180
atacacatcctgaagatctcgtgactatactggcatttcagatccaccccacccccagac3240
tcatcccactctctcccagcattttgtctgggaagagagactnacccatctttacccacc3300
ccctagaaatgagctgcaataacaggaacatgagacttcgcaaatctctggaaaataata3360
tccaaatgaaattaagtctcactgaacatttcaatcaagaatggcagggatctattttat3420
tgaatattctagctactgtaacattgatatttatttttgtttgacattttaacactttgt3480
actgcaaagagtgaactatatatgagatagagagacaataatttcttgcaaaaaaaaaaa3540
gagataaaagaaagaacaaaaaaaaaaaa 3569
<210> 6
<211> 2910
<212> DNA
<213> Homo sapiens
<400> 6
acgatggcca actttcagga gcacctgagc tgctcctctt ctccacactt acccttcagt 60
gaaagcaaaa ccttcaatgg actacaagat gagctcacag ctatggggaa ccacccttct 120
cccaagctgc tcgaggacca gcaggaaaag gggatggtac gaacagagct aatcgagagc 180
gtgcacagcc ccgtcaccac aacagtgttg acgagcgtaa gtgaggattc cagggaccag 240
1~
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
tttgagaacagcgttcttcagctaagggaacacgatgaatcagagacggcggtgtctcag300
gggaacagcaacacggtggacggagagagcacaagtggaactgaagacataaagattcag360
ttcagcaggtcaggcagtggcagtggtgggtttcttgaaggactatttggatgcttaagg420
cctgtatggaatatcattgggaaggcatattccactgattacaaattgcagcagcaagat480
acttgggaagtgccatttgaggagatctcagagctgcagtggctgggtagtggagcccaa540
ggagcggtcttcttgggcaagttccgggcggaagaggtggccatcaagaaagtgagagaa600
cagaatgagacggatatcaagcatttgaggaagttgaagcaccctaacatcatcgcattc660
aagggtgtttgtactcaggccccatgttattgtattatcatggaatactgtgcccatgga720
caactctacgaggtcttacgagctggcaggaagatcacacctcgattgctagtagactgg780
tccacaggaattgcaagtggaatgaattatttgcacctccataaaattattcatcgtgat840
ctcaaatcacctaatgttttagtgacccacacagatgcggtaaaaatttcagattttggt900
acatctaaggaactcagtgacaaaagtaccaagatgtcatttgctggcacggtcgcatgg960
atggcgccagaggtgatacggaatgaacctgtctctgaaaaagttgatatatggtctttt1020
ggagtggtgctttgggagctgctgacaggagagatcccttacaaagatgtagattcttca1080
gccattatctggggtgttggaagcaacagcctccaccttccagttccttccacttgccct1140
gatggattcaaaatccttatgaaacagacgtggcagagtaaacctcgaaaccgaccttct1200
tttcggcagacactcatgcatttagacattgcctctgcagatgtacttgccaccccacaa1260
gaaacttacttcaagtctcaggctgaatggagagaagaagtgaaaaaacattttgagaag1320
atcaaaagtgaaggaacttgtatacaccggttagatgaagaactgattcgaaggcgcaga1380
gaagagctcaggcatgcgctggatattcgtgaacactatgagcggaagcttgagcgggcg1440
aataatttatacatggaattgagtgccatcatgctgcagctagaaatgcgggagaaggag1500
ctcattaagcgtgagcaagcagtggaaaagaagtatcctgggacctacaaacgacaccct1560
gttcgtcctatcatccatcccaatgccatggagaaactcatgaaaaggaaaggagtgcct1620
cacaaatctgggatgcagaccaaacggccagacttgttgagatcagaagggatccccacc1680
acagaagtggctcccactgcatcccctttgtccggaagtcccaaaatgtccacttctagc1740
agcaagagccgatatcgaagcaaaccacgccaccgccgagggaatagcagaggcagccat1800
agtgactttgccgcaatcttgaaaaaccagccagcccaggaaaattcaccccatcccact1860
tacctgcaccaagctcaatcccaatacccttctcttcatcaccataattctctgcagcag1920
caataccagcagccccctcctgccatgtcccagagtcaccatcccagactcaatatgcac1980
ggacaggacatagcaacctgcgccaacaacctgaggtatttcggcccagcagcagccctg2040
cggagcccactcagcaaccatgctcagagacagctgcccggctcgagccctgacctcatc2100
11
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
tccacagccatggctgcagactgctggagaagttctgagcctgacaagggccaagctggt2160
ccctggggctgttgccaggctgacgcttatgacccctgccttcagtgcaggccagaacag2220
tatgggtccttagacataccctctgctgagccagtggggaggagccctgacctttccaag2280
tcaccagcacataatcctctcttggaaaacgcccagagttctgagaaaacggaagaaaat2340
gaattcagcggctgtaggtctgagtcatccctcggcacctctcatctcggcacccctcca2400
gcgctacctcgaaaaacaaggcctctgcagaagagtggagatgactcctcagaagaggaa2460
gaaggggaagtagatagtgaagttgaatttccacgaagacagaggccccatcgctgtatc2520
agcagctgccagtcatattcaacctttagctctgagaatttctctgtgtctgatggagaa2580
gagggaaataccagtgaccactcaaacagtcctgatgagttagctgataaacttgaagac2640
cgcttggcagagaagctagacgacctgctgtcccagacgccagagattcccattgacata2700
tcctcacactcggatgggctctctgacaaggagtgtgccgtgcgccgtgtgaagactcag2760
atgtctctgggcaagctgtgtgtggaggaacgtggctatgagaaccccatgcagtttgaa2820
gaatcggactgtgactcttcagatggggagtgttctgatgccacagttaggaccaataaa2880
cactacagctctgctacctggtaatgaagg 2910
<210> 7
<211> 333
<212> DNA
<213> Homo sapiens
<400> 7
tacctataca tggagtattg tgcccatgga caactctacg aggtcttacg agctggcagg 60
aagatcacac ctcgattgct agtagactgg tccacaggaa ttgcaagtgg aatgaattat 120
ttgcacctcc ataaaattat tcatcgtgat ctcaaatcac ctaatgtttt agtgacccac 180
acagatgcggtaaaaatttcagattttggtacatctaagg aactcagtga caaaagtacc240
aagatgtcatttgctggcacggtcgcatggatggcgccag aggtgatacg gaatgaacct300
gtctctgaaaaagttgatatctggtctatggta 333
<210> 8
<211> 859
<212> PRT
<213> Homo sapiens
<400> 8
Met Ala Cys Leu His Glu Thr Arg Thr Pro Ser Pro Ser Phe Gly Gly
1 5 10 15
Phe Val Ser Thr Leu Ser Glu Ala Ser Met Arg Lys Leu Asp Pro Asp
20 25 30
12
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
Thr Ser Asp Cys Thr Pro Glu Lys Asp Leu Thr Pro Thr His Val Leu
35 40 45
Gln Leu His Glu Gln Asp Ala Gly Gly Pro Gly Gly Ala Ala Gly Ser
50 55 60
Pro Glu Ser Arg Ala Ser Arg Val Arg Ala Asp Glu Val Arg Leu Gln
65 70 75 80
Cys Gln Ser Gly Ser Gly Phe Leu Glu Gly Leu Phe Gly Cys Leu Arg
85 90 95
Pro Val Trp Thr Met Ile Gly Lys Ala Tyr Ser Thr Glu His Lys Gln
100 105 110
Gln Gln Glu Asp Leu Trp Glu Val Pro Phe Glu Glu Ile Leu Asp Leu
115 120 125
Gln Trp Val Gly Ser Gly Ala Gln Gly Ala Val Phe Leu Gly Arg Phe
130 135 140
His Gly Glu Glu Val Ala Val Lys Lys Val Arg Asp Leu Lys Glu Thr
145 150 155 160
Asp Ile Lys His Leu Arg Lys Leu Lys His Pro Asn Ile Ile Thr Phe
165 170 175
Lys Gly Val Cys Thr Gln Ala Pro Cys Tyr Cys Ile Leu Met Glu Phe
180 185 190
Cys Ala Gln Gly Gln Leu Tyr Glu Val Leu Arg Ala Gly Arg Pro Val
195 200 205
Thr Pro Ser Leu Leu Val Asp Trp Ser Met Gly Ile Ala Gly Gly Met
210 215 220
Asn Tyr Leu His Leu His Lys Ile Ile His Arg Asp Leu Lys Ser Pro
225 230 235 240
Asn Met Leu Ile Thr Tyr Asp Asp Val Val Lys Ile Ser Asp Phe Gly
245 250 255
Thr Ser Lys Glu Leu Ser Asp Lys Ser Thr Lys Met Ser Phe Ala Gly
260 265 270
13
Phe Val Ser Thr Leu Ser Glu
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
Thr Val Ala Trp Met Ala Pro Glu Val Ile Arg Asn Glu Pro Val Ser
275 280 285
Glu Lys Val Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Leu Leu
290 295 300
Thr Gly Glu Ile Pro Tyr Lys Asp Val Asp Ser Ser Ala Ile Ile Trp
305 310 315 320
Gly Val Gly Ser Asn Ser Leu His Leu Pro Val Pro Ser Ser Cys Pro
325 330 335
Asp Gly Phe Lys Ile Leu Leu Arg Gln Cys Trp Asn Ser Lys Pro Arg
340 345 350
Asn Arg Pro Ser Phe Arg Gln Ile Leu Leu His Leu Asp Ile Ala Ser
355 360 365
Ala Asp Val Leu Ser Thr Pro Gln Glu Thr Tyr Phe Lys Ser Gln Ala
370 375 380
Glu Trp Arg Glu Glu Val Lys Leu His Phe Glu Lys Ile Lys Ser Glu
385 390 395 400
Gly Thr Cys Leu His Arg Leu Glu Glu Glu Leu Val Met Arg Arg Arg
405 410 415
Glu Glu Leu Arg His Ala Leu Asp Ile Arg Glu His Tyr Glu Arg Lys
420 425 430
Leu Glu Arg Ala Asn Asn Leu Tyr Met Glu Leu Asn Ala Leu Met Leu
435 440 445
Gln Leu Glu Leu Lys Glu Arg Glu Leu Leu Arg Arg Glu Gln Ala Leu
450 455 460
Glu Arg Arg Cys Pro Gly Leu Leu Lys Pro His Pro Ser Arg Gly Leu
465 470 475 480
Leu His Gly Asn Thr Met Glu Lys Leu Ile Lys Lys Arg Asn Val Pro
485 490 495
Gln Asn Leu Ser Pro His Ser Gln Arg Pro Asp Ile Leu Lys Ala Glu
500 505 510
Ser Leu Leu Pro Lys Leu Asp Ala Ala Leu Ser Gly Val Gly Leu Pro
515 520 525
14
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
Gly Cys Pro Lys Ala Pro Pro Ser Pro Gly Arg Ser Arg Arg Gly Lys
530 535 540
Thr Arg His Arg Lys Ala Ser Ala Lys Gly Ser Cys Gly Asp Leu Pro
545 550 555 560
Gly Leu Arg Thr Ala Val Pro Pro His Glu Pro Gly Gly Pro Gly Ser
565 570 575
Pro Gly Gly Leu Gly Gly Gly Pro Ser Ala Trp Glu Ala Cys Pro Pro
580 585 590
Ala Leu Arg Gly Leu His His Asp Leu Leu Leu Arg Lys Met Ser Ser
595 600 605
Ser Ser Pro Asp Leu Leu Ser Ala Ala Leu Gly Ser Arg Gly Arg Gly
610 615 620
Ala Thr Gly Gly Ala Gly Asp Pro Gly Ser Pro Pro Pro Ala Arg Gly
625 630 635 640
Asp Thr Pro Pro Ser Glu Gly Ser Ala Pro Gly Ser Thr Ser Pro Asp
645 650 655
Ser Pro Gly Gly Ala Lys Gly Glu Pro Pro Pro Pro Val Gly Pro Gly
660 665 670
Glu Gly Val Gly Leu Leu Gly Thr Gly Arg Glu Gly Thr Ser Gly Arg
675 680 685
Gly Gly Ser Arg Ala Gly Ser Gln His Leu Thr Pro Ala Ala Leu Leu
690 695 700
Tyr Arg Ala Ala Val Thr Arg Ser Gln Lys Arg Gly Ile Ser Ser Glu
705 710 715 720
Glu Glu Glu Gly Glu Val Asp Ser Glu Val Glu Leu Thr Ser Ser Gln
725 730 735
Arg Trp Pro Gln Ser Leu Asn Met Arg Gln Ser Leu Ser Thr Phe Ser
740 745 750
Ser Glu Asn Pro Ser Asp Gly Glu Glu Gly Thr Ala Ser Glu Pro Ser
755 760 765
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
Pro Ser Gly Thr Pro Glu Val Gly Ser Thr Asn Thr Asp Glu Arg Pro
770 775 780
Asp Glu Arg Ser Asp Asp Met Cys Ser Gln Gly Ser Glu Ile Pro Leu
785 790 795 800
Asp Pro Pro Pro Ser Glu Val Ile Pro Gly Pro Glu Pro Ser Ser Leu
805 810 815
Pro Ile Pro His Gln Glu Leu Leu Arg Glu Arg Gly Pro Pro Asn Ser
820 825 830
Glu Asp Ser Asp Cys Asp Ser Thr Glu Leu Asp Asn Ser Asn Ser Val
835 840 845
Asp Ala Leu Arg Pro Pro Ala Ser Leu Pro Pro
850 855
<210> 9
<211> 966
<212> PRT
<213> Homo Sapiens
<400> 9
Met Ala Asn Phe Gln Glu His Leu Ser Cys Ser Ser Ser Pro His Leu
1 5 10 15
Pro Phe Ser Glu Ser Lys Thr Phe Asn Gly Leu Gln Asp Glu Leu Thr
20 25 30
Ala Met Gly Asn His Pro Ser Pro Lys Leu Leu Glu Asp Gln Gln Glu
35 40 45
Lys Gly Met Val Arg Thr Glu Leu Ile Glu Ser Val His Ser Pro Val
50 55 60
Thr Thr Thr Val Leu Thr Ser Val Ser Glu Asp Ser Arg Asp Gln Phe
65 70 75 80
Glu Asn Ser Val Leu Gln Leu Arg Glu His Asp Glu Ser Glu Thr Ala
85 90 95
Val Ser Gln G1y Asn Ser Asn Thr Val Asp Gly Glu Ser Thr Ser Gly
100 105 110
Thr Glu Asp Ile Lys Ile Gln Phe Ser Arg Ser Gly Ser Gly Ser Gly
115 120 125
16
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
Gly Phe Leu Glu Gly Leu Phe Gly Cys Leu Arg Pro Val Trp Asn Ile
130 135 140
Ile Gly Lys Ala Tyr Ser Thr Asp Tyr Lys Leu Gln Gln Gln Asp Thr
145 150 155 160
Trp Glu Val Pro Phe Glu Glu Ile Ser Glu Leu Gln Trp Leu Gly Ser
165 170 175
Gly Ala Gln Gly Ala Val Phe Leu Gly Lys Phe Arg Ala Glu Glu Val
180 185 190
Ala Ile Lys Lys Val Arg Glu Gln Asn Glu Thr Asp Ile Lys His Leu
195 200 205
Arg Lys Leu Lys His Pro Asn Ile Ile Ala Phe Lys Gly Val Cys Thr
210 215 220
Gln Ala Pro Cys Tyr Cys Ile Ile Met Glu Tyr Cys Ala His Gly Gln
225 230 235 240
Leu Tyr Glu Val Leu Arg Ala Gly Arg Lys Ile Thr Pro Arg Leu Leu
245 250 255
Val Asp Trp Ser Thr Gly Ile Ala Ser Gly Met Asn Tyr Leu His Leu
260 265 270
His Lys Ile Ile His Arg Asp Leu Lys Ser Pro Asn Val Leu Val Thr
275 280 285
His Thr Asp Ala Val Lys Ile Ser Asp Phe Gly Thr Ser Lys Glu Leu
290 295 300
Ser Asp Lys Ser Thr Lys Met Ser Phe Ala Gly Thr Val Ala Trp Met
305 320 315 320
Ala Pro Glu Val Ile Arg Asn Glu Pro Val Ser Glu Lys Val Asp Ile
325 330 335
Trp Ser Phe Gly Val Val Leu Trp Glu Leu Leu Thr Gly Glu Ile Pro
340 345 350
Tyr Lys Asp Val Asp Ser Ser Ala Ile Ile Trp Gly Val Gly Ser Asn
355 360 365
Ser Leu His Leu Pro Val Pro Ser Thr Cys Pro Asp Gly Phe Lys Ile
1~
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
370 375 380
Leu Met Lys Gln Thr Trp Gln Ser Lys Pro Arg Asn Arg Pro Ser Phe
385 390 395 400
Arg Gln Thr Leu Met His Leu Asp Ile Ala Ser Ala Asp Val Leu Ala
405 410 415
Thr Pro Gln Glu Thr Tyr Phe Lys Ser Gln Ala Glu Trp Arg Glu Glu
420 425 430
Val Lys Lys His Phe Glu Lys Ile Lys Ser Glu Gly Thr Cys Ile His
435 440 445
Arg Leu Asp Glu Glu Leu Ile Arg Arg Arg Arg Glu Glu Leu Arg His
450 455 460
Ala Leu Asp Ile Arg Glu His Tyr Glu Arg Lys Leu Glu Arg Ala Asn
465 470 475 480
Asn Leu Tyr Met Glu Leu Ser Ala Ile Met Leu Gln Leu Glu Met Arg
485 490 495
Glu Lys Glu Leu Ile Lys Arg Glu Gln Ala Val Glu Lys Lys Tyr Pro
500 505 510
Gly Thr Tyr Lys Arg His Pro Val Arg Pro Ile Ile His Pro Asn Ala
515 520 525
Met Glu Lys Leu Met Lys Arg Lys Gly Val Pro His Lys Ser Gly Met
530 535 540
Gln Thr Lys Arg Pro Asp Leu Leu Arg Ser Glu Gly Ile Pro Thr Thr
545 550 555 560
Glu Val Ala Pro Thr Ala Ser Pro Leu Ser Gly Ser Pro Lys Met Ser
565 570 575
Thr Ser Ser Ser Lys Ser Arg Tyr Arg Ser Lys Pro Arg His Arg Arg
580 585 590
Gly Asn Ser Arg Gly Ser His Ser Asp Phe Ala Ala Ile Leu Lys Asn
595 600 605
Gln Pro Ala Gln Glu Asn Ser Pro His Pro Thr Tyr Leu His Gln Ala
610 615 620
18
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
Gln Ser Gln Tyr Pro Ser Leu His His His Asn Ser Leu Gln Gln Gln
625 630 635 640
Tyr Gln Gln Pro Pro Pro Ala Met Ser Gln Ser His His Pro Arg Leu
645 650 655
Asn Met His Gly Gln Asp Ile Ala Thr Cys Ala Asn Asn Leu Arg Tyr
660 665 670
Phe Gly Pro Ala Ala Ala Leu Arg Ser Pro Leu Ser Asn His Ala Gln
675 680 685
Arg Gln Leu Pro Gly Ser Ser Pro Asp Leu Ile Ser Thr Ala Met Ala
690 695 700
Ala Asp Cys Trp Arg Ser Ser Glu Pro Asp Lys Gly Gln Ala Gly Pro
705 710 715 720
Trp Gly Cys Cys Gln Ala Asp Ala Tyr Asp Pro Cys Leu Gln Cys Arg
725 730 735
Pro Glu Gln Tyr Gly Ser Leu Asp Ile Pro Ser Ala Glu Pro Val Gly
740 745 750
Arg Ser Pro Asp Leu Ser Lys Ser Pro Ala His Asn Pro Leu Leu Glu
755 760 765
Asn Ala Gln Ser Ser Glu Lys Thr Glu Glu Asn Glu Phe Ser Gly Cys
770 775 780
Arg Ser Glu Ser Ser Leu Gly Thr Ser His Leu Gly Thr Pro Pro Ala
785 790 795 800
Leu Pro Arg Lys Thr Arg Pro Leu Gln Lys Ser Gly Asp Asp Ser Ser
805 810 815
Glu Glu Glu Glu Gly Glu Val Asp Ser Glu Val Glu Phe Pro Arg Arg
820 825 830
Gln Arg Pro His Arg Cys Ile Ser Ser Cys Gln Ser Tyr Ser Thr Phe
835 840 845
Ser Ser Glu Asn Phe Ser Val Ser Asp Gly Glu Glu Gly Asn Thr Ser
850 855 860
Asp His Ser Asn Ser Pro Asp Glu Leu Ala Asp Lys Leu Glu Asp Arg
19
CA 02449479 2003-12-02
WO 02/098889 PCT/US02/17457
865 870 875 880
Leu Ala Glu Lys Leu Asp Asp Leu Leu Ser Gln Thr Pro Glu Ile Pro
885 890 895
Ile Asp Ile Ser Ser His Ser Asp Gly Leu Ser Asp Lys Glu Cys Ala
900 905 910
Val Arg Arg Val Lys Thr Gln Met Ser Leu Gly Lys Leu Cys Val Glu
915 920 925
Glu Arg Gly Tyr Glu Asn Pro Met Gln Phe Glu Glu Ser Asp Cys Asp
930 935 940
Ser Ser Asp Gly Glu Cys Ser Asp Ala Thr Val Arg Thr Asn Lys His
945 950 955 960
Tyr Ser Ser Ala Thr Trp
965
