Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED AND TRANSMEM13RANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING T'HE
SAME
FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of
novel DIVA and to the
recombinant production of novel polypeptides.
BACKGROUND OF THE INVENTION
Extracellular proteins play importane roles in, among other things, the
formation; differentiation and
maintenance of multicellular organisms. The fate of many individual cells,
e.g., proliferation, migration,
differentiation, or interaction with other cells, is typically governed by
infbrmation received from other cells
and/or the immediate environment. This information is often transmitted by
secreted polypeptides (for instance,
mitogenic factors, survival factors, cytotoxic factors, differentiation
factors, tieuropeptides, and hormones) which
are, in turn, received and interpreted by diverse cell receptors or membrane-
bound proteins. 7~ese secreted
polypeptides or signaling molecules normally pass through the cellular
secretory pathway to reach their site of
IS action in the extracellular environment.
Secreted proteins have various industrial applications, including as
pharmaceuticals, diagnostics,
biosensors and bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons,
interleukins, erythropoietins, colony stimulating factors, and various other
cytokines, are secretory~proteins.
Their receptors, which are membrane proteins, also have potential as
therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new, native
secreted proteins. Many efforts are
focused on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel
secreted proteins. Examples of screening methods and techniques are described
in the literature [see; for
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Patent
No. 5,536,637)].
Membrane-bound proteins and receptors can play important roles in, among other
things, the formation,
differentiation and maintenance of multicellular organisms. The fate of many
indiviiiual cells, e.g.; proliferation,
migration, differentiation, or interaction with other cells, is typically
governed by information received from
other cells and/or the immediate environment. This information is often
transmitted by secreted polypeptides
(for instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and
hormones) which are, in torn, received and interpreted by diverse cell
receptors or membrane-bound proteins.
Such membrane-bound proteins and cell receptors include, but are not limited
to, cytokine receptors, receptor
kinases, receptor phosphatases, receptors involved in cell-cell interactions;
and cellular. adhesin molecules like
selectiiis and integntis. For iiistans:e, transduction of signals that
regulate cell growth and differentiation is
regulated in part by phosphorylation of various cellular proteins. Protein
tyrosine kinases, enrymes that catalyze
that process, can also act as growth factor receptors. Examples include
fibroblast growth factor receptor and
I
__.. .~_~_ __ ~ _. _... _ __
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nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial
applications, including as
pharmaceutical and diagnostic agents. Receptor immunoadta:sins, for instance,
can be employed as therapeutic
agents to block receptor-ligand interactions. The membrane-bound proteins can
also be employed for screening
of potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. ,
Efforts are being undertaken by both industry and academia to identify new,
native receptor or
membrane-bound proteins. Many efforts are focused on the screening of
mammalian recombinant DNA libraries
to identify the coding sequences for novel receptor or membrane-bound
proteins.
SUMMARY OF THE INVENTION
In one embodiment, the invention provides an isolated nucleic acid molecule
comprising a nucleotide
sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80 % nucleic acid sequence identity, alternatively at least about 81 % nucleic
acid sequence identity, alternatively
at least about 82% nucleic acid sequence identity, alternatively at least
about 83 % nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence identity, alternatively
at least about 85% nucleic acid
sequence identity, alternadveIy at least about 8696 nucleic acid sequence
identity, alternatively at least about 87%
nucleic acid sequence identity, alternatively at least about 88% nucleic acid
sequence identity, alternatively at
.least about 89% nucleic acid sequence identity, alternatively at least about
90% nucleic acid sequence identity,
alternatively at least about 91 % nucleic acid sequence identity,
alternatively at lease about 92 % nucleic acid
sequence identity, alternatively at least about 93 % nucleic acid sequence
identity, alternatively at least about 94 %
nucleic acid sequence identity, alternatively at least about 95 % nucleic acid
sequence identity, alternatively at
least about 96% nucleic acid sequence identity, alternatively at least about
97% nucleic acid sequence identity,
alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99% nucleic acid
sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal peptide, as
disclosed herein~or any other
specifically defined fragment of the full-length amino acid sequenere as
disclosed herein, or (b) the complement
of the DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% nucleic acid sequence identity, alternatively at least about 81 % nucleic
acid sequence identity, alternatively
at least about 82 % nucleic acid sequence identity, alternatively at least
about 83 f6 nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence identity, alternatively
at least about 85% nucleic acid
sequence identity, alternatively at least about 86% nucleic acid sequence
identity, alternatively at least about 87 %
nucleic acid sequence. identity, alle~gativel :at. least about $8.~ nucleic
acid --- uerice :ident alte ~ ivel at
.. . _ _:- . . .: :..~: , _ .: : . . . _. . _. -, : _ . =... :. . ,_ , _ ~.~
rnat y
y ~_
least about 89% nucleic acid sequence identity, altertiatively at least atibut
90% nucleic acid sequence identity,
alternatively at least about 91% nucleic acid sequence identity, alternatively
at Least about 92% nucleic acid
sequence identity, alternatively at least about 93 % nucleic acid sequence
identity, alternatively at least about 94 %
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nucleic acid sequence identity, alternatively at least about 959b ~cleic acid
sequence identity, altet~natively at
Least about 9b '.~ nucleic acid sequence identity, alternatively at least
about 97 °& nucleic acid sequence identity;
alternatively at least about 98 k nucleic acid sequence identity and
alternatively at least about 99 ~ nucleic acid
sequence identity to (a) a DNA molecule comprising the coding sequence of a
full-length PRO polypeptide eDNA
as disclosed herein, the coding sequence of a PRO polypeptide lacking the
signal peptide as disclosed herein,
the coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without the signal
peptide, as disclosed herein or the coding sequence of any other specifically
defined fragment of the full-length
amino acid sequence as disclosed herein, or (ti) the complement of the DNA
molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule
comprising a nucleotide
sequence having at least about 80~ nucleic acid sequence identity,
alternatively.pt least about 81 ~ nucleic acid
sequence identity, alternatively at least about 82 96 nucleic acid sequence
identity, alternatively at least about 83 ~
nucleic acid sequence identity, alternatively at least about 84 ~ nucleic acid
sequence identity, alternatively at
least about 85% nucleic acid sequence identity, alternatively at least about
86~ nucleic acid sequence identity,
alternatively at least about 87~ nucleic acid sequence identity, alternatively
at least about 88'J6 nucleic acid
sequence identity, alternatively at least about 89 ~. nucleic acid sequence
identity, alternatively at least about 90 ~
nucleic acid sequence identity, alternatively at least about 91 % nucleic acid
sequence identity, alternatively at
least about 929~o nucleic acid sequence identity, alternatively at least about
93 °6 nucleic acid sequence identity,
alternatively at least about 94 ~ nucleic acid sequence identity,
alternatively at least about 95 ~ nucleic acid
sequence identity, alternatively at least about 96 ~ nucleic acid sequence
identity, alternatively at least about 97
nucleic acid sequence identity, alternatively at least about 98'~ nucleic acid
sequence identity and alternatively
at least about 99°6 nucleic acid sequence identity to (a) a DNA
molecule that encodes the same mature
polypeptide encoded by any of the human protein cDNAs deposited with the ATCC
as disclosed herein, or (b)
the complement of the DNA molecule of (a).
Another aspect the invention provides an isolated nucleic. acid molecule
comprising a nucleotide
sequence encoding a PRO polypeptide which is either transmembrane domain-
deleted or transmembrane domain-
inactivated,.or is complementary to such encoding nucleotide sequence, wherein
the transmembrane domains)
of such polypeptide are disclos~l herein. Therefore, soluble extracellular
domains of the herein described PRO
polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding
sequence, or-tlte complement
thereof, that may find use as, for example, hybridization probes; for encoding
fragments of a PRO polypeptide
that may optionally encode a polypeptide comprising a binding site for an anti-
PRO antibody or as antisense
oligonucleotide probes. Such nucleic acid fragments are usually at Least about
20 nucleotides in Length,
alternatively at least about 30 nucleotides in length, alternatively at lease
about 40 nucleotides in length,
alternatively at least about 50 nucleotides in length, alternatively at least
about 60 nucleotides in length,
alternatively at least about '70 nucleotides in length, alteriiativel .at
Least about 80 nucleotides ~ in len ~th
Y . . .... . .. . . :-
alternatively at least about 90 nucleotides in length, alternatively at least
about l00 nucleotides in length,
alternatively at least about I 10 nucleotides in length, alternatively at
least about I20 nucleotides in length,
alternatively at ,feast about I30 nucleotides in length, alternatively at
least about 144 nucleotides in length,
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alternatively at least about 150 nucleotides in length, alternatively at /east
about 160 nucleotides in length,
alternatively at least about I70 nucleotides in length, alternatively at least
about I80 nucleotides in length,
alternatively at least about 190 nucleotides in length, alternatively at least
about 200 nucleotides in length,
aitematively at least about 250 nucleotides in length, alternatively at least
about 300 nucleotides in length,
alternatively at least about 350 nucleotides in length, alternatively at least
about 400 nucleotides. in length,
alternatively at least about 450 nucleotides in length, alternatively at least
about 500 nucleotides in length,.
alternatively at least about 600 nucleotides in length, alternatively at least
about 700 nucleotides in length,
aitemadvely at least about 800 nucleotides in length, alternatively at least
about 900 nucleotides in length and
alternatively at least about 1000 nucleotides in length, wherein in this
context the term "about" means the
referenced nucleotide sequence length plus or minus IOYb of that referenced
.length. It is noted that novel
IO fragments of a PRO polypepeide-encoding nucleotide sequence may be
determined in a routine manner by
aligning the PRO polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any
of a number of well known sequence alignritent programs and determining which
PRO polypeptide-encoding
nucleotide sequence fragments) a~-e novel. All of such PRO polypeptide-
encoding nucleotide sequences are
contemplated- herein. Also contemplated are the PRO polypeptide fragments
encoded by these nucleotide
molecule fragments, preferably those PRO polypeptide fragments that comprise a
binding site for an anti-PRO
antibody.
In another embodiment, the invention provides isolated PRO polypeptide encoded
by any of the isolated
nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PRO polypeptide,
comprising an amino acid
sequence having at least about 80 °b amino acid sequence identity,
,alternatively at least about 81 °b amino acid
sequence identity, alternatively at least about 82 9b amino acid sequence
identity, alternatively at least about 83 9'0
amino acid sequence identity, alternatively at least about 84 ~ amino acid
sequence identity, ahernativety at least
about 85'~ amino acid sequence identity, alternatively at least about 8696
amino acid sequence identity,
alternatively at least about 87R& amino acid sequence identity, alternatively
at least about 8896 amino acid
sequence identity, alternatively at least about 89 ~o amino acid sequence
identity, alternatively at least about 90 96
amino acid sequence identity, alternatively at least about 91 k amino acid
sequence identity, altenlatively at least
about 9296 amino acid sequence identity, alternatively at least about 93 96
amino acid sequence identity,
alternatively at least about 9496 amino acid sequence identity, alternatively
at least about 9~~ amino acid
sequence identity, alternatively at least about 96 % amino acid sequence
identity, alternatively at least about 97 ~
amino acid sequence identity, alternatively at least about 98 % amino acid
sequence identity and alternatively at
least about 99'Y amino acid sequence identity to a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as.
disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other
specifically:defined, fragrne~u:of the;full length amino-acid eqaence as
disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
composing 'atiJ aminoacid
sequence having at least about 80'~ amino acid sequence identity,
alternatively at least about 8196 amino acid
sequence identity, alternatively at /east about 82 k amino acid sequence
identity, aEternatively at least about 83 °.b
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amino acid sequence identity, alternatively at least about 84 % amino acid
sequence identity, alternatively at least
about 85~ amino acid sequence identity, alternatively at least about 86Y~
amino acid sequence identity,
alternatively at least about 87~ amino acid sequence identity, alternatively
at least about 88% amino acid
sequence identity, alternatively at least about 89~ amino acid sequence
identity, alternatively at least about 90
amino acid sequence identity, alternatively at least about 91 ~ amino acid
sequence identity, alternatively at least
about 92% amino acid sequence identity, alternatively at least about 93% amino
acid sequence identity,
alternatively at least about 94 % amino acid sequence identity, alternatively
at least about 95 ~ amino acid
sequence identity, alternatively at least about 96 ~ amino acid sequence
identity, alternatively at least about 97 9~
amino acid sequence identity, alternatively at least about 98 ~ amino acid
sequence identity and alternatively at
least about 999b amino acid sequence identify to an amino acid sequence
encoded by any of the human protein
cDNAs deposited with the ATCC as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide
without the N-terminal signal
sequence and/or the initiating methionine and is encoded by a nucleotide
sequence that encodes such an amino
acid sequence as hereinbefore described. Processes for producing the same are
also herein described, wherein
those processes comprise culturing a host cell comprising a vector which
comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of the PRO
polypeptide and recovering the PRO
polypeptide from the cell culture.
Another aspect the invention provides an isolated PRO polypeptide which is
either transmemhrane
domain-deleted or transtnembrane domain-inactivated. Processes for producing
the same are also herein
described, wherein those processes comprise culturing a host cell comprising a
vector which comprises the
appropriate encoding nucleic. acid molecule under conditions suitable for
expression of the PRO polypeptide and
recovering the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists and antagonists of
a native PRO polypeptide
as defined herein. In a particular embodiment, the agonist or antagonist is an
anti-PRO antibody or a small
molecule.
In a further embodiment, the invention concerns a method of identifying
agonists or antagonists.to a
PRO polypeptide which comprise contacting the PRO polypeptide with a candidate
molecule and monitoring a
biological activity mediated by said PRO polypeptide. Preferably, the PRO
polypeptide is a native PRO
polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PRO
polypeptide, or an agonist or antagonist of a PRO potypeptide as herein
described, or an anti-PRO antibody, in
combination wi.h a carrier. Optionally, the carrier is a pharmaceutically
acceptable carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an agonist
or antagonist thereof as hereinbefore described, or an anti-PRO antibody, for
the preparation of a medicament
useful in the treatment of a condition which is responsive to the PRO
polypeptide, an _agonist or antagonist =v
thereof or an anti-PRO antibody.
In other embodiments of the present invention, the invention provides vectors
comprising DNA
encoding any of the herein described polypeptides. Host cell comprising any
such vector are also provided. By
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way of example, the host cells may be CHO cells, E. coli, or yeast. A process
for producing any of the herein
described polypeptides is further provided and. comprises culturing host cells
under conditions suitable for
expression of the desired polypeptide and recovering the desired polypeptide
from the cell culture.
In other embodiments, the invention provides chimeric motecuIes comprising any
of the herein described
polypeptides fused to a heterologous polypeptide or amino xid sequence.
Example of such chimeric molecules
comprise any of the herein described polypeptides fused to an epitope tag
sequence or a Fc region of an
immunoglobulin.
In another embodiment, the invention provides an antibody which binds,
preferably specifically, to any
of the above or belowdescribed polypeptides. Optionally, the antibody is a
monoclonal antibody, humanized
antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleoride probes useful
for isolating genomic
and cDNA nucleotide sequences or as antisense probes, wherein those probes may
be derived from any of the
above or below described nucleotide sequences.
In yet other embodiments, the present invention is directed to methods of
using the PRO polypeptides
of the present invention for a variety of uses based upon the functional
biological assay data. presented in the
Examples below.
BRIEF DESCRIPTION OF THE DRAWINCJS
Figure I shows a nucleotide sequence (SEQ ID NO:1) of a native sequence PR0180
cDNA, wherein
SEQ ID NO:1 is a clone designated herein as "DNA26843-1389".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding
sequence of SEQ ID
NO:l shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID N0:3) of a native sequence PR0218
cDNA, wherein
SEQ ID N0:3 is a clone designated herein as "DNA30867-1335".
Figure 4 shows the amino acid sequence (SEQ ID N0:4) derived from the coding
sequence of SEQ ID
N0:3 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID NO:S) of a native sequence PR0263
cDNA, wherein
SEQ ID NO:S is a clone designated herein as "DNA34431-1177".
Figure 6 shows the amino acid sequence,(SEQ ID N0:6) derived from the coding
sequence of SEA ID
NO:S shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID N0:7) of a native sequence PR0295
cDNA, wherein
SEQ ID N0:7 is a clone designated herein as "DNA38268-1188".
Figure 8 shows the amino acid sequence (SEQ ID N0:8) derived from the coding
sequence of SEQ ID
N0:7 shown in Figure 7. ,
Figure 9 staov~r~,a_-pucleotad_e-se uence S ID NQ:9 of a native W ence
4 . ( ~Q ) . _,_seq . _ .. PRfl874 cDNA, wherein
SEQ ID N0:9 is a clone designated hererri as "DNA40621-1440":
Figure 10 shows the amino acid sequence (SEQ ID NO:IO) derived from the coding
sequence of SEQ
ID N0:9 shown in Figure 9.
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Figure I l shows a nucleotide sequence (SEQ ID NO: I 1) of a native sequence
PR0300 cDNA, wherein
SEQ ID NO:11 is a clone designated herein as "DNA40625-1189"'.
Figure I2 shows the amino acid sequence (SEQ ID NO: I2) derived from the
coding sequence of SEQ
ID NO:11 shown in Figure 11.
Figure 13 shows a nucleotide sequence (SEQ ID N0:13) of a native sequence
PR01864 cDNA, wherein
SEQ -ID N0:13 is a clone designated herein as "DNA454Q9-2511".
Figure 14 shows the amino acid sequence (SEQ ID N0:14) derived from the coding
sequence of SEQ
ID N0:13 shown in Figure 13.
Figure 15 shows a nucleotide sequence (SEQ ID NO: I S) of a native sequence
PR01282 cDNA, wherein
SEQ ID NO:15 is a clone designated herein as "DNA45495-1550".
Figure 16 shows the amino acid sequence (SEQ ID NO:16) derived from the coding
sequence of SEQ
ID NO;15 shown in Figure I5.
Figure 17 shows a nucleotide sequence (SEQ ID N0:17) of a native sequence
PR01063 cDNA, wherein
SEQ ID N0:17 is a clone designated herein as "DNA49$20-1427":
Figure 18 shows the amino acid sequence (SEQ ID N0:18) derived from the coding
sequence of SEQ
ID N0:17 shown in Figure 17.
Figure 19 shows a nucleotide sequence (SEQ ID N0:19) of a native sequence
PR01773 cDNA, wherein
SEQ ID N0:19 is a clone designated herein as "DNA56406-I704".
Figure 20 shows the amino acid sequence. (SEQ ID N0:20) derived from the
coding sequence of SEQ
ID N0:19 shown in Figure 19.
Figure 2i shows a nucleotide sequence (SEQ ID N0:21) of a native sequence
PR01013 cDNA, wherein
SEQ ID N0:21 is a clone designated herein as "DNA56410-1414".
Figure 22 shows the amino acid sequence (SEQ ID N0:22) derived from the coding
sequence of SEQ
ID N0:21 shown in Figure 21.
Figure 23 shows a nucleotide sequence (SEQ-ID N0:23) of a native sequence
PR0937 cDNA, wherein
SEQ ID N0:23 is a clone designated herein as "DNA56436-1448".
Figure 24 shows the amino acid sequence (SEQ ID N0:24) derived from the coding
sequence of SEQ
ID N0:23 shown in Figure 23.
Figure 2S shows a nucleotide sequencx (SEQ ID N0:25) of a native sequence
PR0842 cDNA, wherein
SEQ ID N0:25 is a clone designated herein as "DNA56855-1447'".
Figure 26 shows the amino acid sequence (SEQ ID N0:26) derived from the coding
sequence of SEQ
ID N0:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID N0:27) of a native sequence PROl
I80 cDNA, wherein
SEQ ID N0:27 is a clone designated herein as "DNA56860-1510".
Figure 28 shows the amino acid sequence (SEQ.ID N028) derived from the coding
sequence of SEQ
ID N4.27 shown in Figure 27, .,
Figure 29 shows a nucleotide sequence (SEQ ID N0:29) of a native sequence
PR0831 cDNA, wherein
SEQ ID N0:29 is a clone designated herein as "DNA56862-1343".
7
_ . _......._ ":a"m ~~.~ z. . _ ~ ....., o ...-..,> . . s..~~~::"~~~:.~_.
,~..~. ~ _ _. _ _.. _ ."_, ~... _ ... .._ _ ~, ~ ,.~.-._ _ _. .
_ _ _ ._.._.... ~
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Figure 30 shows the amino acid sequence (SEQ ID N0:30) derived from the coding
sequence of SEQ
ID N0:29 shown in Figure 29.
Figure 31 shows a nucleotide sequence (SEQ ID N0:31) of a native sequence PROl
115 cDNA, wherein
SEQ ID N0:31 is a clone designated herein as "DNA56868-1478".
Figure 32 shows the amino acid sequence (SEQ ID N0:32) derived from the coding
sequence of SEQ
ID N0:31 shown in Figure 31.
Figure 33 shows a nucleotide sequence (SEQ ID N0:33) of a native sequence PRO
1277.cDNA, wherein
SEQ ID N0:33 is a clone designated herein as "DNA56869-1545".
Figure 34 shows the amino acid sequence {SEQ ID N0:34) derived from -the
coding sequence of SfiQ
ID N0:33 shown in Figure 33.
IO Figure 35 shows a nucleotide sequence {SEQ ID N0:35) of a native sequence
PR01074 cDNA, wherein
SEQ ID N0:35 is a clone designated herein as "DNA57704-1452".
Figure 36 shows the amino acid sequence (SEQ ID N0:36) derived from the coding
sequence of SEQ
ID N0:35 shown in Figure 35.
Figure 37 shows a nucleotide sequence (SEQ ID N0:37) of a native sequence PRO
1344 c:DNA, wherein
SEQ ID N0:37 is a clone designated herein as "DNA58723-1588".
Figure 38 shows the amino acid sequence (SEQ ID N0:38) derived from the coding
sequence of SEQ
ID N0:37 shown in Figure 37.
Figure 39 shows a nucleotide sequence (SEQ ID N0:39) of a native sequence PRO
1136 c;DNA, wherein
SEQ ID N0:39 is a clone designated herein as "DNA57827-1493".
Figure 40 shows the amino acid sequence (SEQ ID N0:40) derived from the coding
sequence of SEQ
ID N0:39 shown in Figure 39.
Figure 41 shows a nucleotide sequence {SEQ ID N0:41) of a native sequence PROl
109 cDNA, wherein
SEQ ID N0:41 is a clone designated herein as "DNA58737-1473~.
Figure 42 shows the amino acid sequence (SEQ ID N0:42) derived from the coding
sequence of SEQ
ID N0:41 shown in Figure 41.
Figure 43 shows a nucleotide sequence (SEQ ID N0:43) of a native sequence PRO
1003 cDNA, wherein
SEQ ID N0:43 is a clone designated herein as "DNA58846-1409".
Figure 44 shows the amino acid sequence (SEQ ID N0:44) derived from the coding
sequence of SEQ
ID N0:43 shown in Figure 43.
Figure 45 shows a nucleotide sequence (SEQ ID N0:45) of a native sequence PRO
1138 cDNA, wherein
SEQ ID N0:45 is a clone designated herein as "DNA58850-1495".
Figure 46 shows the amino acid sequence (SEQ ID N0:46) derived from the coding
sequence of SEQ
ID N0:45 shown in Figure 45.
Figure 47 hows ~a nucleotide sequence (SEQ ID N0:47) of a native sequence
.PR0994 c.I~NA, wherein
SEQ ID N0:47 is a clone designated herein as "DNA58855-1422".
Figure 48 shows the amino acid sequence (SEQ ID N0:48) derived from the coding
sequence of SEQ
ID N0:47 shown in Figure 47.
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Figure 49 shows a nucleotide sequence (SEQ ID N0:49) of a native sequence
PR01069 cDNA, wherein
SEQ iD N0:49 is a clone designated herein as "DNA59211-1450p.
Figure 50 shows the amino acid sequence (SEQ ID NO:SO) derived from the coding
sequence of SEQ
ID N0:49 shown in Figure 49.
Figure 51 shows a nucleotide sequence (SEQ ID N0:51) of a native sequence PRO
1411 cDNA, wherein
S SEQ ID NO:S1 is a clone designated herein as "DNAS9212-1627'".
Figure 52 shows the amino acid sequence (SEQ ID NO:S2) derived from the coding
sequence of SEQ
ID NO:S 1 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ ID N0:53) of a: native sequence
PR01129 cDNA, wherein
SEQ ID N0:53 is a clone designated herein as "DNAS9213-1487".
Figure 54 shows the amino acid sequence (SEQ ID N0:54) derived from the coding
sequence of SEQ
ID NO:S3 shown in Figure S3.
Figure SS shows a nucleotide sequence (SEQ ID NO:55) of a native sequence
PR01027 cDNA; wherein
SEQ ID NO:SS is a clone designated herein as "DNAS9605-1418'".
Figure S6 shows the amino acid sequence-(SEQ ID N0:56) derived from the coding
sequence of SEQ
1S ID NO:SS shown in Figure SS.
Figure S7 shows a nucleotide sequence (SEQ ID N0:57) of a native sequence PRO
1106 cDNA, wherein
SEQ ID N0:57 is a cione designated herein as "DNA59609-1470".
Figure 58 shows the amino acid sequence (SEQ ID NO:S8) derived from the coding
sequence of SEQ
ID N0:57 shown in Figure S7:
Figure S9 shows a nucleotide sequence (SEQ ID NO:S9) of a native sequence
PR01291 cDNA, wherein
SEQ ID N0:59 is a clone designated herein as "DNA59610-1556".
Figure 60 shows the amino acid sequence (SEQ ID N0:60) derived from the coding
sequence of SEQ
ID N0:59 shown in Figure S9.
Figure 61 shows a nucleoside sequence (SEQ ID N0:61) of a native sequence
PR03S73 cDNA, wherein
2S SEQ ID N0:61 is a clone designated herein as "DNAS9837-2S4S"'.
Figure 62 shows the amino acid sequence (SEQ ID N0:62) derived from the coding
sequence of SEQ
ID N0:61 shown in Figure 61.
Figure 63 shows a nucleotide sequence (SEQ ID N0:63) of a native sequence
PR03S66 cDNA-, wherein --
SEQ ID N0:63 is a clone designated herein as "DNAS9844-2542".
Figure 64 shows the amino acid sequence (SEQ ID N0:64) derived from the coding
sequence of SEQ
ID N0:63 shown in Figure 63.
Figure 6S shows a nucleotide sequence (SEQ ID N0:65) of a native sequence PRO
1098 cDNA, wherein
SEQ ID NO:bS is a clone designated herein as "DNAS98S4-1459".
Figure 66 shows the amino acid, sequence (SEQ:ID N066) derived from the
coding. sequence bf SEQ
3S ID N0:65 shown in Figure 65.
Figure 67 shows a nucleotide sequence (SEQ ID N0:67) of a native sequence PRO
11 S8 cDNA, wherein
SEQ ID N0:67 is a clone designated herein as "DNA60625-1507".
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Figure 68 shows the amino acid sequence (SEQ ID N0:68) derived from the coding
sequence of SEQ
ID NO:67 shown in Figure 67.
Figure 69 shows a nucleotide sequence (SEQ ID N0:69) of a native sequence
PR01124 cDNA, wherein
SEQ ID NO:69 is a clone designated herein as "DNA60629-1481 ".
Figure 70 shows the amino acid sequence (SEQ ID N0:70) derived from the coding
sequence of SEQ
ID N0:69 shown in Figure 69.
Figure 71 shows a nucleotide sequence (SEQ ID N0:71 ) of a native sequence PRO
1287 cDNA, wherein
SEQ ID N0:71 is a clone designated herein as "DNA61755-1554".
Figure 72 shows the amino acid sequence (SEQ ID N0:72) derived from the coding
sequence of SEQ
ID N0:71 shown in Figure 71.
Figure 73 shows a nucleotide sequence (SEQ ID N0:73) of a native sequence
PR01335 cDNA, wherein
SEQ ID N0:73 is a clone designated herein as "DNA62812-1594".
Figure 74 shows the amino acid sequence (SEQ ID N0:74) derived from the coding
sequence of SEQ
ID N0:73 shown in Figure 73.
Figure 75 shows a nucleotide sequence (SEQ ID N0:75) of a native sequence
PR01315 cDNA, wherein
IS SEQ ID N0:75 is a clone designated herein as "DNA628I5-1576".
Figure 76 shows the amino acid sequence (SEQ ID N0:76) derived from the coding
sequence of SEQ
ID N0:75 shown in Figure 75.
Figure 77 shows a nucleotide sequence (SEQ ID N0:77) of a native sequence PRO
1357 cDNA, wherein
SEQ ID N0:77 is a clone designated herein as "DNA64881-1602".
Figure 78 shows the amino acid sequence (SEQ ID N0:78} derived from the coding
sequence of ;SEQ
ID NO:77 shown in Figure 77.
Figure 79 shows a nucleotide sequence (SEQ ID N0:79) of a native sequence PRO
1356 cDNA, wherein
SEQ ID N0:79 is a clone designated herein as "DNA64886-1601".
Figure 80 shows the amino acid sequence (SEQ ID N0:80) derived from the coding
sequence of SEQ
ID N0:79 shown in Figure 79.
Figure 81 shows a nucleotide sequence (SEQ ID NO:81) of a native sequence
PR01557 cDNA, wherein
SEQ ID N0:81 is a clone designated herein as "DNA64902-1667".
Figure 82 shows the amino acid sequence (SEQ ID N0:82) derived from the coding
sequence of SEQ
ID N0:8I shown in Figure 81.
Figure 83 shows a nucleotide sequence (SEQ ID N0:83) of a native sequence
PR01347 cDNA, wherein
SEQ ID N0:83 is a clone designated herein as "DNA64950-1590".
Figure 84 shows the amino acid sequence (SEQ ID N0:84) derived from the coding
sequence of SEQ
ID N0:83 shown in Figure 83.
Figure $5 shows a nucleotide sequence (SEQ ID NO:85) of a native sequence PRO
1302 cIDNA, wherein
SEQ II? NO:85 is a clone designated herein as "DNA65403=1565".
Figure 86 shows the amino acid sequence (SEQ ID N0:86} derived from the coding
seduence of SEQ
ID N0:85 shown in Figure 85.
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Figure 87 shows a nucleotide sequence (SEQ ID N0:87) of a native sequence PRO
1270 cDNA, wherein
SEQ ID N0:87 is a clone designated herein as "DNA66308-1537".
Figure 88 shows the amino acid sequence (SEQ ID N0:88) derived from the coding
sequence of SEQ
ID N0:87 shown in Figure 87.
Figure 89 shows a nucleotide sequence (SEQ ID. N0:89) of a native sequence PRO
1268 cDNA, wherein
SEQ ID N0:89 is a clone designated herein as "DNA66519-1535".
Figure 90 shows the amino acid sequence (SEQ ID N0:90) derived from the coding
sequence of SEQ
ID N0:89 shown in Figure 89.
Figure 91 shows a nucleotide sequence (SEQ ID N0:91) of a native sequence PRO
1327 cDNA, wherein
SEQ ID N0:91 is a clone designated herein as "DNA66521-1583".
Figure 92 shows the amino acid sequence (SEQ ID N0:92) derived from the coding
sequence of SEQ
ID N0:91 shown in Figure 91.
Figure 93 shows a nucleotide sequence (SEQ ID N0:93) of a native sequence PRO
1328 cDNA, wherein
SEQ ID N0:93 is a clone designated herein as "DNA66658-1584".
Figure 94 shows the amino acid sequence (SEQ ID N0:94) derived from the coding
sequence of SEQ
ID N0:93 shown in Figure 93.
Figure 95 shows a nucleotide sequence (SEQ ID N0:95) of a native sequence PRO
1329 cDNA, wherein
SEQ ID N0:95 is a clone designated herein as "DNA66660-1585".
Figure 96 shows the amino acid sequence (SEQ ID N0:96) derived from the coding
sequence of SEQ
ID N0:95 shown in Figure 95.
Figure 97 shows a nucleoride sequence (SEQ ID N0:97) of a native sequence PRO
1340 c:DNA, wherein
SEQ ID N0:97 is a clone designatP.d herein as "DNA66663-1598".
Figure 98 shows the amino acid sequence (SEQ ID N0:98) derived from the coding
sequence of SEQ
ID N0:97 shown in Figure 97.
Figure 99 shows a nucleotide sequence (SEQ ID N0:99) of a native sequence PRO
1342 eDNA, wherein
SEQ ID N0:99 is a clone designated herein as "DNA66674-1599".
Figure 100 shows the amino acid sequence (SEQ ID NO:100) derived from the
coding sequence of SEQ
ID N0:99 shown in Figure 99.
Figure i01 shows a nucleotide sequence (SEQ ID NO:101) of a native sequence
PR03579 cDNA,
wherein SEQ ID NO: l0I is a clone designated herein as "DNA68862-2546".
Figure 102 shows the amino acid sequence (SEQ ID N0:102) derived from the
coding sequence of SEQ
ID NO:101 shown in Figure 101.
Figure 103 shows a nucleotide sequence (SEQ ID NO:103) of a native sequence
PR01472 cDNA,
wherein SEQ ID N0:103 is a clone designated herein as "DNA68866-1644".
Figure 104 shaves the amino acid sequence (SEQ ID N0:104) derived from the
coding equence of SEQ
ID N0:103 shown in Figure 103.
Figure 105 shows a nucleotide sequence (SEQ ID NO:10_>) of a native sequence
PRU146i cDNA,
wherein SEQ ID N0:105 is a clone designated herein as "DNA6887I-1638".
li
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Figure 106 shows the amino acid sequence {SEQ ID NO: I06) derived from the
coding sequence of SEQ
ID NO:105 shown in Figure 105.
Figure 107 shows a nucleotide sequence (SEQ ID N0:107) of a native sequence
PR01568 cDNA;
wherein SEQ ID N0:107 is a clone designated herein as "DNA68880-1676".
Figure 108 shows the amino acid sequence (SEQ ID NO:108) derived from the
coding sequence of SEQ
ID N0:107 shown in Figure 107.
Figure 109 shows a nucleotide sequence (SEQ ID N0:109) of a native sequence
PRO1753 cDNA;
wherein SEQ ID N0:109 is a clone designated hereiwas "DNA68883-1691 ".
Figure 110 shows the amino acid sequence (SEQ ID NO: I 10) derived from the
coding sequence of SEQ
ID N0:109 shown in Figure 109.
Figure 111 shows a nucleotide sequence (SEQ ID NO:11I) of a native sequence
PRO1570 cDNA,
wherein SEQ ID NO:111 is a clone designated herein as "DNA68885-1678".
Figure 112 shows the amino acid sequence (SEQ iD NO: I I2) derived from the
coding sequence of SEQ
ID NO:111 shown in Figure.ll 1.
Figure 113 shows a nucleotide sequence (SEQ ID NO:1I3) of a native sequence
PRO1446 cDNA,
wherein SEQ ID N0:113 is a clone designated herein as "DNA71277-1636"
Figure I 14 shows the amino acid sequence (SEQ ID N0:114) derived from the
coding sequence of SEQ
ID N0:113 shown in Figure 113.
Figure 115 shows a nucleotide sequence (SEQ ID NO:115) of a native sequence
PRO1565 eDNA,
wherein SEQ ID NO:115 is a clone designated herein as "DNA73727-1673".
Figure 116 shows the amino acid sequence (SEQ ID N0:116) derived from the
coding sequence of SEQ
ID NO:115 shown in Figure 115.
Figure 1I7 shows a nucleotide sequence (SEQ ID N0:117) of a native sequence
PRO1572 cDNA,
wherein SEQ ID N0:117 is a clone designated herein as "DNA73 734-1680".
Figure 1 I8 shows the amino acid sequence {SEQ ID N0:118) derived from the
coding sequence of SEQ
ID NO: I 17 shown in Figure 117.
Figure 119 shows a nucleotide sequence (SEQ ID NO:119) of a native sequence
PR01573 cDNA,
wherein SEQ ID N0:119 is a clone designated herein as "DNA73735-1681".
Figure 120 shows the amino acid sequence (SEQ ID N0:120) derived from the
cbding sequence of SEQ
ID N0:119 shown in Figure 119.
Figure i21 shows a nucleotide sequence (SEQ ID N0:121) of a native sequence
PRO1550 cDIVA,
wherein SEQ ID NO:I2I is a clone designated herein as "DNA76393-1664".
Figure 122 shows the amino acid sequence (SEQ ID N0:122) derived from the
coding sequence of SEQ
ID N0:121 shown in Figure 12I.
Figure 123 shouts a nucleotide sequence (SEQ ID N0:123) of a;native sequence
PROI693 cDNA;'
wherein SEQ ID N0:123 is a clone designated herein as "DNA77301-1708".
Figure 124 shows the amino acid sequence (SEQ ID N0:124) derived from the
coding sequence of SEQ
iD N0:123 shown in Figure 123.
~2
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Figure -125 shows a nucleotide sequence (SEQ ID N0:125) of a native sequence
PR01566 cDNA,
wherein SEQ ID N0:125 is a clone designated herein as "DNA77568-1626".
Figure 126 shows the amino acid sequence (SEQ ID N0:126) derived from the
coding sequence of SEQ
ID NO: I25 shown in Figwe 125.
Figure 127 shows a nucleotide sequence (SEQ ID N0:127) of a native sequence
PR01774 cDNA,
wherein SEQ ID N0:127 is a clone designated herein as "DNA77626-1705".
Figure 128 shows.the amino acid sequence (SEQ ID N0:128) derived from the
coding sequence of SEQ
ID N0:127 shown in Figure 127.
Figure 129 shows a nucleotide sequence (SEQ ID N0:129) of a native sequence
PR01928 cDNA,
wherein SEQ ID N0:129 is a clone designated herein as "DNA81754-2532".
IO Figure I30 shows the amino acid sequence (SEQ 1D N0:130) derived from the
coding sequence of SEQ
ID N0:129 shown in Figure 129.
Figure I31 shows a nucleotide sequence (SEQ ID N0:131) of a native sequence
PR01865 cDNA,
wherein SEQ ID N0:13I is a clone designated herein as "DNA8I757-2512".
Figure 132 shows the amino acid sequence (SEQ ID N0:132) derived from the
coding sequence of SEQ
ID NO: I31 shown in Figure 131.
Figure 133 shows a nucleotide sequence {SEQ ID N0:133) of a native sequence
PR01925 cDNA,
wherein SEQ ID N0:133 is a clone designated herein as "DNA82302-2529".
Figure 134 shows ttae amino acid sequence (SEQ ID N0:134) derived from the
coding sequence of SEQ
ID N0:133 shown in Figure 133.
Figure 135 shows a nucleotide sequence (SEQ ID N0:135) of a native sequence
PR01926 cDNA,
wherein SEQ ID N0:135 is a clone designated herein as "DNA82340-2530".
Figure i36 shows the amino acid sequence {SEQ ID N0:136) derived from the
coding sequence of SEQ
ID I°I0:135 shown in Figure 135.
Figure 137. shows a nucleotide sequence (SEQ ID N0:137) of a native sequence
PR01801 cDNA,
wherein SEQ ID N0:137 is a clone designated herein as "DNA83500-2506" .
Figure 138 shows the amino acid sequence (SEQ ID N0:138) derived from the
coding sequence of SEQ
ID N0:137 shown in Figure 137.
Figure 139 shows a nucleotide sequence (SEQ ID N0:139) of a native sequence
PR04405 cDNA,
wherein SEQ ID N0:139 is a clone designated herein as "DNA84920-2614".
Figure 140 shows the amino acid sequence (SEQ ID N0:140) derived from the
coding sequence of SEQ
ID N0:139 shown in Figure 139
Figure 141 shows a nucleotide sequence (SEQ ID N0:141) of a native sequence
PR03435 cDNA,
wherein SEQ ID N0:141 is a clone designated herein as "DNA85066-2534".
Fagure 142 shows the amino acid sequence (SEQ ID N0:142) derived from the
coding sequence of SEQ
ID' N0:141 shown in Figure 141.
Figure 143 shows a nucleotide sequence (SEQ ID N0:143) of a native sequence
PR03543 cDNA,
wherein SEQ ID N0:143 is a clone designated herein as "DNA86571-2551 ".
I3
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Figure 144 shows the amino acid sequence (SEQ ID N0:144) derived from the
coding sequence of SEQ
ID N0:143 shown in Figure 143.
Figure 145 shows a nucleotide sequence (SEQ ID N0:145) of a native sequence
PR03443 cDNA;
wherein SEQ ID N0:145 is a clone designated herein as "DNA87991-2540".
Figure 146 shows the amino acid sequence (SEQ ID N0:146) derived from the
coding sequence of SEQ
ID NO:145 shown in Figure 145.
Figure 147 shows a nucleotide sequence (SEQ ID N0:147) of a native sequence
PR03442 cDNA,
wherein SEQ ID N0:147 is a clone designated herein as "DNA92238-2539".
Figure 148 shows the amino acid sequence (SEQ ID N0:148) derived from the
coding sequence of SEQ
ID N0:147 shown in Figure 147.
Figure 149 shows a nucleotide sequence (SEQ ID NO:I49) of a native sequence
PR05990 eDNA,
wherein SEQ ID NO:I49 is a clone designated herein as "DNA96042-2682".
Figure 150 shows the amino acid sequence (SEQ ID N0:150) derived from the
coding sequence of SEQ
ID N0:149 shown in Figure 149.
Figure 151 shows a nucleotide sequence (SEQ ID NO:I51) of a native sequence'
PR04342 cDNA,
wherein SEQ ID N0:15I is a clone designated herein as ".DNA96787-2534".
Figure 152 shows the amino acid sequence (SEQ ID N0:152) derived from the
coding sequence of SEQ
ID NO:151 shown in Figure 151.
Figure 153 shows a nucleotide sequence (SEQ ID N0:153) of a native sequence
PR010096 cDNA,
wherein SEQ ID N0:153 is a clone designated herein as "DNA125185-2806".
Figure 154 shows the amino acid sequence (SEQ ID N0:154} derived from the
coding sequence of SEQ
iD N0:153 shown in Figure 153.
Figure 155 shows a nucleotide sequence (SEQ ID N0:155) of a native sequence
PR010272 cDNA,
wherein SEQ ID N0:155 is a clone designated herein as "DNA147531-2821".
Figure 156 shows the amino acid sequence (SEQ ID N0:156) derived from the
coding sequence of SEQ
2$ ID N0:155 shown in Figure 155.
Figure 157 shows a nucleotide sequence (SEQ ID N0:157) of a native sequence
PR05801 cDNA,
wherein SEQ ID N0:157 is a clone designated herein as "DNA115291-2681".
Figure 158 shows the amino acid sequence (SEQ ID N0:158) derived from the
coding sequence of SEQ
ID N0:157 shown in Figure 157.
Figure 159 shows a nucleotide sequence (SEQ ID N0:159) of a native sezluence
PR0201 IO cDNA,
wherein SEQ ID N0:159 is a clone designated herein as "DNA166819".
Figure 160 shows the amino acid sequence (SEQ ID N0:160) derived from the
coding sequence of SEQ
ID NO:I59 shown in Figure 159,
Figure 16i shows a nucleotide sequence (SfiQ ID: N0:161) of a native sequence
PR02004Q:eDNA,
wherein SEQ ID N0:161 is a clone designated herein as "DNA164625-2890".
Figure 162 shows the amino acid sequence (SEQ ID N0:162) derived from the
coding sequence of SEQ
ID N0:161 shown in Figure 161.
14
._.. _,__..._,r-.r,",~.~ ri...~~;,,"~~~~~~",~"~w~ . ..... _ . ~_ ...._. _
____..~.....~.~...__
CA 02481756 2004-10-25
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Figure 163 shows a nucleotide sequence (SEQ ID NO:163) of a native sequence
PRO20233 cDNA,
wherein SEQ ID N0:163 is a clone designated herein as "DNA165608".
Figure 164 shows the amino acid sequence (SEQ ID N0:164) derived from the
coding sequence of SEQ
ID N0:163 shown in Figure 163.
Figure 165 shows a nucleotide sequence (SEQ ID N0:165) of a native sequence
PR019670 cDNA,
wherein SEQ ID N0:165 is a clone designated herein as "DNA131639-2874".
Figure 166 shows the amino acid sequence (SEQ ID NO.-166) derived from the
coding sequence of SEQ
ID N0:165 shown in Figure 165.
Figure 167 shows a nucleotide sequence (SEQ ID N0:167) of a native sequence
PRO1890 cDNA,
wherein SEQ ID N0:167 is a clone designated herein as "DNA79230-2525".
Figure 168 shows the amino acid sequence (SEQ ID N0:168) derived from the
coding sequence of SEQ
ID N0:167 shown in, Figure 167.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
The terms "PRO polypeptide" and "PRO" as used herein and when immediately
followed by a
numerical designation refer to various polypeptides, wherein the complete
designation (i.e., PRO/number) refers
to specific polypeptide sequences as described herein. The terms "PRO/number
pokypeptide" and
"PROlnumber" wherein the term "number" is provided as an actual numerical
designation as used herein
encompass native sequence polypeptides and polypeptide variants (which are
further defined herein). The PRO .
polypeptides described herein may be isolated from a variety of sources, such
as from human tissue types or
from another source, or prepared by recombinant or synthetic methods. The term
"PRO polypeptide" refers to
each individual PRO/number polypeptide disclosed herein. All disclosures im
this specification which refer to
the "PRO polypeptide" refer to each of the polypeptides individually as well
as jointly. For example,
descriptions of the preparation of, purification of, derivation of, formation
of antibodies to or against, .
administration of, compositions containing, treatment of a disease with, etc.,
pertain to.each polypeptide of the
invention individually. The term "PRO polypeptide" also includes variants of
the PROlnumber polypeptides
disclosed herein.
A "native sequence PRO polypeptide" comprises a polypeptide having the same
amino acid sequenrx
as the corresponding PRO polypeptide derived from nature. Such native sequence
PRO polypeptides can be
isolated from nature or can be produced by recombinant or synthetic means. The
term "native sequence PRO
polypeptide" specifically encompasses naturally-occurring truncated or
secreted forms of the specific PRO
polypeptide (e.g., an extracellular domain sequence), naturally-occurring
variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the polypeptide. In
various embodiments of the
invention, the native sequence PRG polypeptides disclosed herein are mature or
full-length native sequence,
polypeptides comprising the full-length amino acids sequences shown in the
accompanying figures. Start and
stop codons are shown in bold font and underlined in the figures., However,
while the PRO polypeptide
disclosed in the accompanying figures are shown to begin with methionine
residues designated herein as amino
CA 02481756 2004-10-25
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acid position 1 in the figures, it is conceivable and possible that other
methionine residues located either upstream
or downstream from the amino acid position 1 in the figures may be employed as
the starting amino acid residue
for the PRO polypeptides.
The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the
PRO polypeptide which
is essentially free of the uansmembrane and cytoplasmic domains. Ordinarily, a
PRO polypeptide ECD will have
less than 1 ~ of such transmembrane andlor cytoplasmic domains and preferably,
will have less than 0.5 ~ of
such domains. It will be understood that any transmembrane domains identified
for the PRO polypeptides of
the present invention are identified pursuant to criteria routinely employed
in the art for identifying that type of
hydrophobic domain. The exact boundaries of a transmembrane domain may vary
but most likely by no more
than about 5 amino acids at eieher end of the domain as initially identified
herein. Optionally,~therefore, an
extracellular domain of a PRO polypeptide may contain from about 5 or fewer
amino acids on either side of the
transmembrane domain/extracellular domain boundary as identified in the
Examples or specification and such
polypeptides, with or without the associated signal peptide, and nucleic acid
encoding them, are comtemplated
by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are
shown in the present specification and/or the accompanying figures. It is
noted, however, that the C-terminal
boundary of a signal peptide may vary, but most likely by no more than about 5
amino acids on either side of
the signal peptide C-ternunal boundary as initially identified herein, wherein
the C-terminal boundary of the
signal peptide may be identified pursuant to criteria routinely employed in
the art for identifying that type of
amino acid sequence element (e:g., NieIsen et al., rot. En . 10:1-6 (1997) and
von Heinje et al., Nucl.~ cids.
Res. 14;4683-4690 (1986)). Moreover, it is also recognized that, in some
cases, cleavage of a signal sequence
from a secreted polypeptide is not entirely uniform, resulting in more than
one secreted species. These mature
polypeptides, where the signal peptide is cleaved within no morenhan about S
amino acids on either side of the
C-terminal boundary of the signal peptide as identified herein, and the
polynucleotides encoding them, are
contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or
below having at least
about 80% amino acid sequence identity with a full-length native sequence PRO
polypeptide sequence as
disclosed herein, a PRO polypeptide sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a PRO polypeptide, with or without the signal peptide, as, disclosed
herein or any other fragment of .
a full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for
instance, PRO poiypeptides wherein one or more amino acid residues are added,
or deleted, at the N- or C-
terminus of the full-length native amino acid sequence. Ordinarily, a PRO
polypeptide variant will have at least
about 80% amino acid sequencx identity, alternatively at least about 81 %
amino ,acid sequence identity,
alternatively at least about 82°6 amino acid sequence identity,
alternatively at least about 83'9b amino acid
sequence identity, alternatively at last about 84 ~ amino acid sequence
identity, alternatively at least about 85 ~
amino acid sequence identity, alternatively at least about 8696 amino acid
sequence identity, alternatively at least
about 87~ amino acid sequence identity, alternatively at least about 88% amino
acid sequence identity,
alternatively at Least about 8996 amino acid sequence identity, alternatively
at least about 90!'~ amino acid
16
. _ _ ... ... .. _ . _.. _. ..,.. .... __..~."w ,_,.,-~, n,.",~,.~ :<- .,"~
.~.~."x.~~. .~,~ ~a..,..~~~.. ~ ., "~.~.....~ _w....._ i
CA 02481756 2004-10-25
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sequcttce identity, alternatively at least about 91 % amino acid sequence
identity, alternatively at least abort 92 '~
amino acid sequencx identity, alternatively at least about 93 ~ amino acid
sequence identity, alternatively at least
about 94~ amino acid sequence identity, alternatively at least about
95°~ amino acid sequence ideatity,
alternatively at least about 96~ amino acid sequence identity, alternatively
at least about 9796 amino acid
sequence identity, alternatively at least about 98 'Y amino acid sequence
identity and alternatively at least about
99 ~ amino acid sequence identity to a full-length native sequence PRO
polypeptide sequence as disclosed lfterein,
a PRO polypeptide sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO
poiypeptide, with or without the signal peptide, as disclosed herein or any
other specifically 'defined fragment
of a full-Length PRO polypeptide sequence as disclosed herein. Oraiinarily,
PRO variant polypeptides are at least
about IO amino acids in length, alternatively at least about 20 amino acids in
length, alternatively at least about
30 amino acids in length, alternatively at least about 40 amino acids in
length, alternatively at least-about 50
amino acids in length, alternatively at least about 60 amino acids in length,
alternatively at lease about 70 amino
acids in length, alternatively at least about 80.amino acids in length,
alternatively at least about 90 amino acids
in length, alternaavely at least about 100 amino acids in length,
alternatively at least about I50 amino aaxds in
length, alternatively at least about 200 amino acids in length, alternatively
aL least about 300 amino adds in
IS length, or more. -
"Percent (96)'amino acid sequence identity" with respect to the PRO
polypeptide sequences ide~ified
herein is defined as the percentage of amino acid residues in a candidate
sequence that are identical with the
amino acid residues in the specific PRO polypeptide sequence, after aligning
the sequences and introducing gaps, . . "
if necessary, to achieve the maximum percent sequence identity, and not
considering any conservative
substitutions.. as part of the .sequence identity. Alignment for purposes of
determining percent amino acid
sequence identity eau be achieved in various ways that are within. the skill
in the art, for instance; using publicly
available computer software such. as BLAST, BLAST-2, ALIGN or Megalign
(DNASTAR) software. Those
skilled in the art can determine appropriate parameters for measuring
alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences bGittg
compared. For purposes herein,
~ however, 96 amino acid sequence identity values are generated using the
sequence comparison computer pragram
ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided
in Table I below. The
ALIGN-2 sequence comparison computer program was authored by Genentech, Inc:
and the source code shown
in Table I below has beg filed with user docximentation in the U.S. Copyright
Office. Washington D. C., 20559;
. where it is registered under U.S. Copyright Registration No. TXUSI0087. The
ALIGN-2 program is publicly
available through Genentech, lnc., South San Francisco, California or may be
compiled from the source; code
provided in Table I below. The ALIGN-2 program should be compiled for use on a
UNIX operating system,
preferably digital UNIX V4.OD. All sequence comparison parameters are set by
the ALIGN-2 program and
do not vary: .
In situations where ALIGN-2:is_employed .for amino acid sequence;
comparisons,ythe 90. atntno-acid
sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which
can alternatively be phrased as a given amino acid sequence A than has or
comprises a certain 96 amino acid
sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows,.
I7
*-trademark
CA 02481756 2004-10-25
w0 o1n631s PGT/USOOIZ3328
100 times the fracxion x%Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the rocs!
number of amino acid residues in
B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of
B to A. As examples of '~ amino acid sequence identity calculations using this
method, Tables 2 and 3
demonstrate how to calculate the % amino acid sequence identity of the amino
acid sequence designated
°Comparison Protein" to the amino acid sequence designated "PRO",
wherein "PRO" represents the amino acid
sequence of a hypothetical PRO polypeptide of interest, "Comparison- Protein"
represents the amino acid
sequence of a polypeptide against which the "PRO" polypeptide of interest is
being compared, and "X, "Y" and
"Z" each represent different_hypotheticai amino acid residues.
Unless spaifically stated otherwise, all % amino acid sequence identity values
used herein are obtained
as described in the immediately preceding paragraph using the ALIGN-2 computer
program. However, % amino
acid sequence identity values may also be obtained as described below by using
the WU-BLAST-2 computer
IS program (Altschul et al., Methods in Enzymoloay 266:460-480 (1996)). Most
of the WU-BY.AST-2 search .
parameters-are set to the default values. Those not set to default values,
i.e., the adjustable parameters, are set
with the foitowing values: overlap span = 1, overlap fraction = 0" 125, word
threshold ~ = 1 I . and scoring
matrix _ BLOSUM62: When WU-BLAST-2 is employed, a % amino acid sequence
.identity value=-is
determined by dividing (a) . the number of matching identical amino acid
residues between the amino acid
sequence of the PRO polypeptide of interest having a sequence derived from the
native PRO polypeptide and
the comparison amino acid sequence of interest (i.e., the sequence against
which the PRO polypepride of interest
is being-,eon~ared which may be a PRO variant polypeptide) as determined by WU-
BLAST 2 by (b) the total
number of amino acid residues of the PRO polypeptide of interest. For example,
in the statement "a polypeptide
comprising an the amino acid sequence A which has or having at least. 80~'o
amino acid sequence identity to the
amino acid sequence B", the amino acid sequence A is the comparison amigo acid
Sequence of interest and the
amino acid sequence B is the amino acid sequence of the PRO polypeptide of
interest.
Percent amino acid sequence identity may also be determined using the sequence
comparison program
NCBI~BLAST2 (Altschul et al., j~ucleic Acids~tes. 25:3389-3402 (1997)). . Ttie
NCBI-BLAST2 sequence
comparison program may be obtained from the
National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search
parameters, wherein all of those
search parameters are set to default vatues including, for example, unmask =
yes, strand = ail, expected
occurrences = 10. minimum low complexity length = 15/5, mufti-pass e-value =
0.01, constant for mufti-pass
= 25, dropoff for final gapped.alignment = 25 and scoring matrix. = BLOSUM62.
In situations where NCBI-BLAST Z is employed for amino acid sequence
comparisons. tile % amino
acid sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence A that has
or comprises a certain 96 amino
acid sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
18
.<_T..
. __ , M"~ . _. ~,.._ ".....w.._ . ... _....__ ~ _ . .,~~ -,. .A-. - -m..~.
~.~ g~,~ x.,~ .,.~.~..-~ ~, ..m _..__ .._. ____... _ .._ .. .
CA 02481756 2004-10-25
wo omns rcr~rsoon33~s
100 times the fraction X/X
where X is the number of amino acid residues scored as identical matches by
the sequence aligrunent program
NCBI-BLAST2 in that program's alignment of A and B, and where V is the total
number of amino acid residues
in B: It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino
acid sequence B, the ~ amino acid sequence identity of A to B will not equal
the %~ amino acid sequence identity
ofBtoA.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a
nucleic acid molecule
which encodes an active PRO golypeptide as defined below and which has at
least about 80~ nucleic acid
sequence identity with a nucleotide acid sequence encoding a full-length
native sequence PRO polypeptide
sequence as disclosed herein, a full-length native sequence PRO polypeptide
sequence tacking the signal peptide
as disclosed herein, an extraceliular domain of a PRO polypeptide, with or
without the signal peptide, as
disclosed herein or any other fragment of a full-length PRO polypeptide
sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic
acid sequence identity,
alternatively at least about 81 °6 nucleic acid sequence identity,
alternatively at least about 82;6 nucleic acid
1 S sequence identity, alternatively at least about 83 ~ nucleic acid sequence
identity, alternatively at least about84 ~
nucleic acid sequence identity, alternatively at least about 85 ~ nucleic acid
sequence identity, alternatively at
least about 86 %. nucleic acid sequence identity, alternatively at least about
87 ~ nucleic acid sequence identity,
alternatively at least about 88~ nucleic acid sequence identity, alternatively
at least about 89~ nucleic acid
sequence identity, alternatively at least about 90 ~ nucleic acid sequence
identity, alternatively at least about 91 ~
nucleic acid sequence identity, alternatively at least about 92~ nucleic acid
sequence identity, alternatively at
least about 93 ~ nucleic acid sequence identity, alternatively at least about
94 ~ nucleic acid sequence identity,
alternatively at least about 95~ nucleic acid sequence identity, alternatively
at least about 96°.li nucleic .acid
sequence identity, alternatively at least about 97 ~ nucleic acid sequence
identity, alternatively at least about 98 %
nucleic acid sequence identity and alternatively at least about 99 ro nucleic
acid sequence identity with a nucleic
acid sequence encoding a full-length native sequence PRO polypeptide sequence
as disclosed herein, a full-length
native sequence PRO polypeptide sequence lacking the signal peptide as
disclosed herein, an extracellular domain
of a PRO polypeptide, with or without the signal sequence, as disclosed herein
or any other fragment of a kull-
length PRO polypeptide sequence as disclosed herein. Variants do not encompass
the native nucleotide
sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in
length, alternatively at least
about 60 nucleotides in length, alternatively at least about 90 nucleotides in
length, alternatively at least about
120 nucleotides in length, alternatively at Least about 150 nucleotides in
length, alternatively at least about 180
nucleotides in length, alternatively at least about 210 nucleotides in length,
alternatively at least about 240
nucleotides in length, alternatively at least about 270 nucleotides in length,
alternatively at least about 300
nucleotides in length, alternatively at least about 450 nucleotides :in
length, alternatively at least about 600
nucleotides in length, alternatively at least about 900 nucleotides in length,
or more.
19
. __.... _ ...___-____-r..-_ __.... _-. .. _ __..__. _. __ __. ... __ . _.~ _.-
~.. .. .__ ~~_ .__. . __:__._
CA 02481756 2004-10-25 '
wo o>'ns3><s
"Percent (9~) nucleic acid sequence identity" with respect to PRO-encoding
nucleic acid sequences
identified herein is defined as the percentage of nucleotides in a candidate
sequence that are identical with the
nucleotides in the PRO nucleic acid sequence of interest, after aligning the
sequences and introducing gaps, if -
necessary, to achieve the maximum percent sequence identity. Alignment for
purposes of determining percent
nucleic acid sequence identity can be achieved in various ways that are within
the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR)
software. For purposes herein, however, fo nucleic. acid sequence identity
values are generated using the
sequence comparison computer pr~gram ALIGN-2, wherein the complete source code
for the ALIGN-2 program
is provided in Table 1 below. The ALIGN-2 sequence comparison computer program
was authored by
Genentech, Inc, and the source code shown in Table 1 below has been filed with
user documentation in the U.S.
IO Copyright Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No.
TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco,
California or may be compiled from the source code provided in Table 1 below.
The ALIGN-2 program should
be compiled for use on a UNIX operating system, preferably digital UNIX V4.OD.
All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
I$ In situations where ALIGN-2 is employed for nucleic acid sequence
comparisons, the % nucleic acid
sequence identity of a given nucleic acid sequence C to, with; or against a
given nucleic acid sequence D (which
can alternatively be phrasal as a given nucleic acid sequence C that has or
comprises a certain % nucleic acid
sequence identity to, with, or against a given nucleic acid sequence D) is
calculated as followsv
20 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-2
in that program's alignment of C and D, and where Z is the total number of
nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence
25 D, the % nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to
C. As examples of 'fo nucleic acid sequence identity calculations, Tables 4
and 5, demonstrate haw to calculate
the % nucleic acid sequence identity of the nucleic acid sequence designated
"Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a
hypothetical PRO-encoding nucleic
acid sequence of interest, "Comparison DNA" represents the nucleotide sequernx
of ~a nucleic acid molecule
30 against which the "PRO-DNA" nucleic acid molecule of interest is being
compared, and "N", "L" and "V" each
represent different hypothetical nucleotides.
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are obtained
as described in the immediately preceding paragraph using the ALIGN-2 computer
program. However, %
nucleic acid sequence identity values may also be obtained as described below
by using the WU-BLAST-2
35 computer program (Altschul et al., ~l~Ietfiods in Enzymology 266:460-480
(I996)). Most of the WU=BLAST-2
search parameters are set to the default values. Thosewot set to default
values, i.e., the adjustable parameters,
are set with the following values: overlap span = 1, overlap fraction = 0.125,
word threshold (T) =-11, and
_........ , K~,."., ,~~.':.x>r",.w-. .;,~,....~,.c,.u~sz..-,~~ ~~..-...",x,~.
_ ".,.,..~......_ ,._w.,.,.,w,",..,...""...EM,. _ .___.._
._..__....._._........ r.,.- "",,.~",9...,.."...-_.-,.---....I._.
CA 02481756 2004-10-25
WO OI/I63I8 PGT/US00~23328
scoring matrix = BLOSL7M62. When WU-BLAST-2 is employed, a :b nucleic acid
sequence identity value
is determined by dividing (a) the number of matching identical nucleotides
between the nucleic acid sequence
of the PRO polypeptide-encoding nucleic acid molecule of interest having a
sequence derived from the native
sequence PRO polypeptide-encoding nucleic acid and the comparison nucleic acid
molecule of interest (i.e., the
sequence against which the PRO polypeptide-encoding nucleic acid molecule of
interest is being compared which
may be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the
total number of nucleotides
of the PRO polypeptide-encoding nucleic acid molecule of interest. For
example, in the statement "an isolated
nucleic acid molecule comprising a nucleic acid sequence A which has or having
at least S0~ nucleic acid
sequence identity to the nucleic acid.sequence B", the nucleic acid sequence A
is the comparison nucleic acid
molecule of interest and the nucleic acid sequence B is the nucleic acid
sequence of the PRO polypeptide-
encoding nucleic acid molecule of interest.
Percent nucleic acid sequence identity may also be determined using the
sequence comparison program
NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). 'Ihe
NCBI-BLAST2 sequence
comparison program may be obtained from the
National Institute of Health, Bethesda, MD. NCBI-BLAST2 uses several search
parameters, wherein all of those
search parameters are set to default values including, for example, unmask =
yes, strand = all, expected
occurrences = I0, minimum low complexity length = 15/5, mufti-pass e-value =
0.01; constant for multi.pass
= 25, dropoff for final gapped alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons, the %
nucleic acid sequence
identity of a given nucleic acid sequence C to, with, or against a given
nucleic acid sequence D (which can
alternatively be phrased as a given nucleic acid sequence C that has or
comprises a certain 9f nucleic acid
sequence identity to, with, or against a given nucleic acid sequence D) is
calculated as follows:
100 times the fraction W!Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program NCBI-
BLAST2 in that program's alignment of C and D, and whore Z is the total number
of nucleotides in D. It will
be appreciated that where the length of nucleic acid sequence C is not equal
to the length of nucleic acid sequence
D, the ~ nucleic acid sequence identity of C to D will not equal the .~
nucleic acid sequence identity of D to
C.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules
that encode an active
PRO polypepeide and.which are; capable of hybridizing, preferably under
stringent hybridization and wash
conditions, to nucleotide sequences encoding a full-length PRO polypeptide as
disclosed herein. PRO variant
polypeptides may be those that are encoded by a PRO variant paiynucleotide.
"Isolated," when used to describe the various polypeptides disclosed herein,
means polypeptide that has
been identified and separaeed andlor recovered from a component of its natural
environment. Contaminant
components of its natural environment are materials that would typically
interfere with diagnostic or therapeutic
uses for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or non-proteinaceous
21
CA 02481756 2004-10-25
wo oinma PCT/USUlin3328
solutes. In preferred embodiments, the polygeptide will be purified (I) to a
degree sufficient to obtain at least ,
1S residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator, or (2) to
homogeneity by SDS-PAGE under non-reducing or reducing conditions using
Coomassie blue or, preferably,
silver stain. Isolated golypeptide includes polypeptide in situ within
recombinant cells, since at least one
component of the PRO. polypeptide natural environment will not be present.
Ordinarily, however, isolated
polypeptide will be prepared by at least one purification step.
An "isolated" PRO polypeptide-encoding nucleic acid or other polypeptide-
encoding nucleic acid is a
nucleic acid molecule that is identified and separated from at least one
contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the polypeptide-
encoding nucleic acid., An isolated
polypeptide-encoding nucleic acid molecule is other than in the form or
setting in which it is fottnd in nature.
Isolated polypeptide-encoding nucleic acid molecules therefore are
distinguishes from the specific polypeptide-
encoding nucleic acid molecule as it exists in natural cells. However, an
isolated polypeptide-enGOding nucleic
acid molecule includes polypeptide-encoding nucleic acid molecules contained
in cells that ordinarily express the
polypeptide where, for example, the nucleic acid molecule is in a chromosomal
location different from that of
natural cells.
1S The term "control sequences" refers to DNA sequences necessary for the
expression of an operably
linked coding sequence in a particular host organism. The control sequences
that are suitable for prokaryotes,
for example, include a promotei, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenyladon signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic
acid sequence. For example, DNA for a presequence or secretory leader is
operabiy linked to DNA for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or
eahancer.is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome
binding.site is operabIy linked to a coding sequence if it is positioned so as
to facilitate uanslation. Generally,
"operably linked" means that the DNA sequences beiztg linked are contiguous,
and, in the case of a secretory
2S leader, contiguous and in reading phase. However, enhancers do not have to
be contiguous. Linking is
accomplished by ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide
adaptors or linkers are used in accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-PRO
monoclonal antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-PRO antibody
compositions with polyepitopic specificity, single chain anti-PRO antibodies,
, and fragments of anti-PRO
antibodies (see below). The term "monoclonal antibody" as used herein refers
to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are
identical except for possible naturally-occurring mutations that may be
present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when
22
CA 02481756 2004-10-25
wo oin631<s
complementary strands are present in an environment below their melting
temperature. The higher the degroe
of desired homology between the probe and hybridizable sequence, the
higher.the relative temperature which
can be used. As a result, it follows that higher relative temperatures would
tend to make 'the reaction conditions
more stringent, while lower temperatures less so. For additional details and
explanation of stringency of .
hybridization reactions, see Ausubel et al., Current Protocols~n Molecular
liiologv, Wiley Interscience
Publishers, (1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may
be identified by those
that: (1) employ , low ionic strength and high temperature for washing, for
example 0..015 M sodium
chloride10.0015 M sodium citrate/0. I °6 sodium dodecyl sulfate at
50°C; (2) employ during hybridization a
denaturing agent, *uch as formamide, for example, 50~ (v/v) formamide with
0.196 bovine scrum
albumin/0.1 ~o Fica11/0. l 96 polyvinylpyrrolidonel50mM sodium phosphate
buffer atpH 6.5 with 750 tnM sodittrn
chloride, 75 mM sodium citrate at 42°C; or (3) employ 5096 formamide, 5
x SSC (0.75 .M NaCI, 0.075 M
sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate,
5 x Detthardt's solution,
sonicated salmon sperm DMA (50 ~cg/ml), O.I96 SDS, and 10% dextran sulfate at
42°C, with washes at 42°C
in 0.2 x SSC (sodium chtorideisodium citrate) and SOX fotTttamide at 55
°C, followed by a high-stringency wash
consisting of O.I x SSC containing EDTA at SS°C.
"Moderately stringent conditions" may be identified as described by Sambrook
et al., of 1 r
Cloning, A Laboratory Manual, New York: Cold Spring Harbor Press; I989, and
include the use of washing
solution and hybridization condieions (e.g., temperature, ionic strength and
~oSDS) less -stringent that .
described above. An example of moderately stringent conditions is overnight
incubation at 37°C in a solution
comprising: 2096 fotYnamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate),
50 mM sodium phosphate (pH .
7.6), 5 x Denhardt's solution, 10~ dextran sulfate, and 20 mglmI denatured
sheared salmon sperm DNA,
followed by washing the filters in 1 x SSC at about 37-50°C. The
skilled artisan will recognize how to adjust
the temperature, ionic saxngth, etc. as necessary to accommodate factors such
as probe length and the like.
The term "epitope tagged" when used herein refers to a chittteric polypeptide
cotnpris'utg a PR0
polypeptide fused to a 'tag polypeptide'. The tag polypeptide has enough
residues to provide an epitope against
which an antibody can be made, yet is shore enough snch~that it does not
interfere with activity of the polypeptide
to which it is fused. The tag polypeptide preferably also is fairly uztique so
that the. arttibody does not
substantially cross-react with othee epitopes. Suitable tag polypeptides
generally have. at least six amino acid
residues and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino
acid residues).
As used herein, the tetxn "immunoadhesin" designates antibody-like molecules
which combine the
.. binding specificity of a heterologous protein (an "adhesin") with the
effector functions of immunogiobulin
constant domains. Structurally, the immunoadhesins comprise a fusion of an
amino acid sequence with the
desired binding specificity which is other than-the antigen recognition and
binding site of an. antibody r:e.~. is.
"heteroiogous"), and an immunoglobulin constant domain sequence. The adhesin
part of an immunoadhesin
molecule typically is a contiguous amino acid sequeace comprising at least the
binding site of a receptor or a
ligand. The . immunoglobuiin constant domain sequence in the immunoadhesin may
be obi from any
x._trademark ~ 23
CA 02481756 2004-10-25
wo oin63zs rc'r~rso
immunogIobuiin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including
IgA-1 and IgA-2), IgE, IgD
or IgM.
"Active" or "activity" for the purposes herein refers to forms) of a PRO
polypeptide which retain a
biological and/or an itnmunological activity of native or naturally-occurring
PRO, wherein "biological ° activity
refers to a biological function (either inhibitory or stimulatory) caused by a
native or naturally-occurring PRO
S other than the ability to induce the production of an antibody against an
antigenic epitope possessed by a native
or naturally-occurring PRO and ati "immunological" activity refers to the
ability to induce the production of an
antibody against an antigenic epitope possessed by a native or naturally-
occurring PRO.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully
blocks, inhibits, or neutralizes a biological activity of a native PRO
polypeptide disclosed herein. In a similar
manner, the term "agonist" is used in the broadest sense and includes any
molecule that mimics a biological
activity of a native PRO polypeptide disclosed herein. Suitable agonist or
antagonist molecules specifically
include agonist or antagonist antibadies or antibody fragments, fragments ar
amino acid sequence variants of
native PRO polypeptides, peptides, antisense oligonucleotides, small organic
molecules, ~ etc.~ Methods for
identifying agonists or antagonists of a PRO polypeptide may comprise
contacting a PRO polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable change in
one or. more biological activities
normally associated with the PRO polypeptide.
"Treatment" refers to both therapeutic treatment and prophylactic or
preventative measures, wherein
the object is to prevent or slow down (lessen) the targeted pathologic
condition or disorder. Those in need of
treatment include those already with the. disorder a~ well as those prone to
have the disorder or those in whom
the disorder is to be prevented.
"Chronic" administration refers to administration of the agents) in a
continuous mode as opposed to
an acute mode, so as to maintain the initial therapeutic effect (activity) for
an extended period of time.
"Intermittent" administration is treatment that is not consecutively done
without interruption, but rather is cyclic
in nature.
"Mammal" for proposes of treatmene refers to any animal classified as a
mammal, including humans,
domestic and farm animals; and zoo, sports, or pet animals, such as dogs,
cats, cattle, horses, sheep, pigs, gaats,
rabbits, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which
are nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often
the physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically
acceptable carriers include buffers such as phosphate, citrate, and other
organic acids; antioxidants including
ascorbic acid; low molecular weight (less than-about 10
residues)_.polypeptide; proteins, such.as serum albumin,. ,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides, disaccliarides,
and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar atcohols
such as mannitol or sorbitot; salt-
24
CA 02481756 2004-10-25
WO Q1/163I8 PCTIUS00~23328
foiming counterions such as sodium; and/or nonionic surfactants such as
TWEEN'~, polyethylene glycol (PEG),
and PLURONICS"'.
"Antibody fragments" comprise a portion of an intact antibody, preferably the
antigen binding or
variable region of the intact antibody. Examples of antibody fragments include
Fab, Fab', F(ab')2, and Fv
fragments; diabodies; linear antibodies (Zapata et al., Protein Eng_. 8(10):
1057-1062 [1995]); single-c>~ain
antibody molecules; arid ~nultispecil'tc antibodies formed from antibody
fragments:
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab"
fragments, each with a single antigen-binding site, and a residual "Fc"
fragment, a designation reflecting the
ability to crystallize readily. Pepsin treatment yields an F(ab')Z fragment
that has two antigen-combining sites
and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding
site; This region consists of a dimer of one heavy- and one fight-chain
variable domain in tight, non-covalent
association. It is in this configuration that the three CDRs of each variable
domain interact to define an antigen-
binding site on the surface of the VN-VL dimer. Collectively, the six CDRs
confer antigen-binding specificity
to the antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific
IS for an antigen) has the ability to recognize and bind antigen, although at
a lower affinity than the entire binding
site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain
(CHl) of the heavy chain. Fab fragments differ from Fab' fragments by the
addition of a few residues at the
carboxy terminus of the heavy chain CHl domain including one ar more cysteines
from the antibody hinge
region. Fab'-SH is the designation herein for Fab' in which the cysteine
residues) of the constant domains bear
a free thiol group. F(ab')= antibody fragments originally were produced as
pairs of Fab' fragments which have
hinge cysteines between them. Other chemical couplings of antibody fragments
are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one
of two clearly distinct types, called kappa and lambda, based on the amino
acid sequences of their constant
domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several of these may be fut~tter divided into subclasses (isotypes),
e.g., IgGl, IgG2, IgG3, IgG4, IgA,
and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and V~ domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide farther comprises a
poIypeptide linker between the V,~ and V~ domains which enables the sFv to
form the desired structure for
antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which
fragments comprise a heavy-chain variable domain (V") connected to a light-
chain variable domain (V,") in the
same polypepdde chain (VH-V J. By using a linker that is too short to allow
pairing between the two domains
CA 02481756 2004-10-25
WO 01!16318 PGT/US00123328.
on the same chain; the domains are forced to pair with the complementary
domains of another chain and create.
two antigen-binding sites. Diabodies are described more fully in, for example,
EP 404,097; WO 93111161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or
recovered from a
component of its natural environment. Contaminant components of its natural
environment are materials which
S would interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and
other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the
antibody will be purified {1)
to greater than 95 q6 by weight of antibody as determined by the Lowry method,
and most preferably more than
99°6 by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or {3) to homogeneity by SDS-
PAGE under reducing or
i0 nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the antibody
in situ within recombinant cells since ai least one component of the
antibody°s natural environment will not be
present. Ordinarily, however, isolated antibody will be prepared by at least
one purification step.
An antibody that "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on
a particular polypeptide is one that binds to that particular polypeptide or
epitope on a particular poIypeptide
15 without substantially binding to any other polypeptide or polypeptide
epitope.
The word "label" when used herein refers to a detectable cornpound or
composition which is conjugated
directly or-indirectly to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself
(e,g. radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical
alteration of a substrate compound or composition which is detectable.
20 By "solid phase" is meant a non-aqueous matrix to which. the antibody of
the present invention can
adhere. Exatrlples of solid phases encompassed herein include those formed
partially or entirely of glass (e.g.,
controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides,
polystyrene, polyvinyl alcohol and
silicones. In certain embodiments, depending on the context, the solid phase
can comprise the well of an assay
plate; in others it is a purification coIuann (e.g., an affinity
chromatography column). This terns also includes
25 a discontinuous solid phase of discrete particles, such as those described
in U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids andlor surfactant
which is useful for delivery of a drug (such as a PRO polypeptide or antibody
thereto) to a mammal. The
components of the Iiposorne are commonly arranged in a bilayer formation,
similar to the.lipid atTartgement of
biological membranes.
30 A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
26
.,-.., .,.v,._ .__,. .aow>~rv~,.s:~a.~--,aenr~.>r:.om
~,....,"~,.:~ce~Wv"'~r"W;-",.-~~~:a~~ea~.r.::,zrmaa.,.~...,m-.gin...
.,y.~u~~~.w.~~~,..a...~-~...W..,.,.,_._.,~..._._ ..
CA 02481756 2004-10-25
WO O11I6318 PC'I°/US00~2332~,
I*
* C-C
increased
from
12
to
1S
* Z
is
average
of
EQ
$ * B
is
average
of
ND
* matchwith stop is M; stop-stop = 0; J (joker) match = 0
_
*/
#defmeM -8 I * value of a match with a stop *I
I~ tnt dayj26](26] _ {
-
Q It
S
T
U
V
'W
X
Y
Z
*/
/*
A
B
C
D
E
F
G
H
I
1
K
L
M
N
O
P
/* { 2, 0,-2. 0, 0,-4, 1; 1: 1. 0: I, 2:_1, 0._M, 1. 0,-2,
A I, 1, 0, 0,-6. 0.-3, 0}.
*!
/* { 0, 3,-4, 3, 2; 5, 0, L2. 0. 0,-3,-2, 2 =M,-1, 1, 0, 0,
B 0, 0,-2,-5, 0,-3, 1},
"/
/* M,-3,-5,-4. 0,-2, 0,_2,-8, 0, ~rS},
C {-2.-4,15,-5: 5,-4,-3; 3,-2, 0,-S.-6.-S,-4,
*/
IS I* _
D M,-1, 2,-1, 0, 0, 0,-2; 7, 0,-4, 2},
*I { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3. 2,
/* _
E { 0, 2,-5, 3, 4,-5, 0, I,-2, 0, 0; 3,-2, 1, M,-1, 2,-1,
*I 0, 0, 0, 2, 7, 0,-4, 3},
l* {-4,-S,-4,-6,-S, 9; 5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-S,-4,-3,-3,
F 0,-1, 0, 0, 7,-S},
*/
/* { 1, 0,-3, I, 0,-5, 5,-2,-3, 0, 2.,-4,-3, 0,_M,-1,-1.-3,
G 1, 0, 0,-1,-7, 0; S. 0},
*/
/* {-1, 1.-3. 1, 1,-2,-2, 6, 2, 0, 0, 2,-2, 2, M, 0. 3, 2;
H I,-1, 0,-2,-3. 0. 0, 2}.
*I
20 I* {-1, 2,-2,-2, 2, i,-3,-2, 5, 0,-2, 2, 2,-2 _M,=2,-2,-2,-I,
I 0, 0; ~4; 5, 0,-1,-2},
*I
I* { 0, 0, 0, 0, 0, 0, 0, 0, 0, U, 0, 0, b, O, M, 0, 0, 0,
1 0, 0, 0, 0, 0, 0, 0, 0},
*I
I* M.-1. 1. 3. 0. 0. 0,-2.-3, 0,-4. 0}.
IC {_I. 0._S. 0, 0,_5,.2, 0.-2. 0, 5; 3. 0. L
*I
!* _
L {-2; 3,-6.-4.-3. 2.-4,-2. 2. 0: 3, 6, 4,-3._M,-3,-2.-3.-3.-1.
*/ 0. 2, 2, 0; 1,-2},
I* {-1,-2.-S,-3,-2, 0.-3,-2, 2. 0. 0. 4, 6,-2. M,-2.-L O: 2,-1,
M 0, 2,-4, 0,-2:1}.
*!
25 /* { 0, 2,-4, 2, I,-4, 0, 2; 2. 0, 1,-3,-2, 2 =M,-1, I, 0,
H 1. 0, 0,-2;-4, 0,-2, 1},
*I
/* M -M,_M,_M,_M,_M ~M,_M, 0,_M,_M,-M,_M =M ~M, M =M, M =M,-
O M},
*I { M,_M =M,_M =M =M,
-
/* _
P { I,-I,-3,-L-1,-5,-I, 0,-2. 0,-I, 3,-2,-1,_M, 6, 0, 0, I,
*I 0. 0.-I,-6, 0,-S. 0},
I* { 0, 1,-5, 2, 2; 5,-1, 3, 2, 0, 1,-2,-1, 1,_M. 0, 4, 1,-I;
Q 1, 0,-2,-5, 0.-4, 3},
*I
l* {-2; 0,-4,-1,-1,-4,-3, 2. 2, 0, 3,-3, 0, 0,
R M, 0, I, 6, 0; 1, 0,-2, 2, 0,-4, 0},
*I
0 /* _
S { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M; I,-1, 0,
*I 2, 1, 0,-1,-2, 0,-3, 0},
!* { I, 0,-2. 0, 0,-3, 0,-I, 0. 0, 0.-1,1, 0. M, 0,-I,-1. I,
T 3. 0, 0,-S. 0.-3. 0},
*!
/* { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
U M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
*/
I* -
V { 0,-2,-2,-2,-2,-1; 1,-2, 4, 0; 2, 2, 2,-2,
*l M,-1; 2,-2,-I. 0, 0, 4,-6, 0,-2, 2},
I* _
VJ {-6,-S; 8,-7, 7. 0. 7; 3; 5. 0,-3; 2,-4,-4, M,-6,-S. 2,-2,-5.
*I 0.-b,I7. 0. 0.-6}.
S /*X*/ {0,0,0,0,0,0,0,0,0,0,0,0,0,0, M,0,0,0,0,0,0,0,0,0,0,0},
I* {-3,-3. 0.-4,-4, 7,-S, 0; 1, ~,-4; 1,-2,-2,
Y M,-5.-4,-4,-3,-3, 0.-2, 0, 0,10,-4},
*!
!* _
Z { 0 1.-S. 2, 3.-S, 0. 2,-Z, 0, 0,-2,I, 1. M. 0; 3. 0, 0.
*! 0. 0; 2.-6, 0,-4. 4}
}~ -
45
SO
27
._. _ .
CA 02481756 2004-10-25
wo o~n63ig PCT/USOOtZ3328
__ Table 1 lcont')
I*
*I
#include
<stdio.h>
#include
<
ctype.h
>
#defmeMA7sJMP16 I * max jumps in a diag *I
.
#defmeMAXGAP24 I * don't continue to penalize gaps larger than
this *I
#defineJMPS 1024 1* max jmps in an path *I
#defineMX 4 I * save if there's at least MX-I bases since Iast
jmp *I
#defmeDMAT 3 /* value of matching bases */ _
#defineDMIS 0 I* penalty for mismatched bases */
#dePmeDINSO 8 l* penalty for a gap *I
#defineDINSI I l* penalty per base */
1$ #definePINSO 8 /* penalty for a gap *I
#defmePINSI 4 /* penalty per residue *l
struct
jmp
{
short n[MAX1MP];
I* size
of jmp
(neg
for defy)
*I
unsignedshort
x(MAXJMPJ;
l* base
no. of
jmp in
seq x
*I
j; /* limits seq to 2"16 -I */
struct
diag
{
int score; I* score at last jmp *I
long offset; l * offset of prev block *I
short ijmp; l* current jmp index *l
struct I * list of jmps *I
jmp
jp;
struct
path
{
int, spc; /* number of leading spaces *I
short n(JMPS]; of jmp (gap) *I
I * size
' int x[JMPS];
l* loc
of jmp
past
elern
before
gap)
*/
j;
char *ofde; l * output file name *I
char *namex(2J;I* seq names: getseqs() *I
char *prog; I* prog name for err msgs *I
char *seqx(2);is seqs: getseqsp *I
int dmax; I * best diag: nwQ *I
int dmax0; I* final ding *I
int dna; I* set if dna: main() *I
int endgaps; I* set if penalizing end gaps *I
int gapx, l * total gaps in seqs *I
gapy;
4S int len0, l~ seq Lens *I
lent;
int ngapx, I* total size of gaps *I
ngapy;
int smax; l* max score: nw() *I
int *xbm; 1* bitmap for matching */
'
long offset; I* current offset in jmp file *I
$0 structdiag *dx; I* hotels diagonals *I
structpath pp[2]; I* holds path for seqs *I
char *callocQ,
*malloc(),
*index(),
*strcpyp;
char *getseqQ,
*g calloc();
28
CA 02481756 2004-10-25
WO O1/1t53I8 pCTIUS00t~3328
Table 1 (cony)
/* Needleman-Wunsch alignment program
* usage: progs file! filet
* where file! and filet are two dna or two protein sequences.
$ * The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ';' ' > ° or ' < ' are igt~red
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1I3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a tmp file in Itmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*/
/!include "nw.h"
1$ ffiuclude "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,Il,O,O,I2,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
}:
static ~bval[26] _ {
1, 2~(1< <('D'-'A'))~(I < <('N'-'A°)), 4, 8, 16, 32, 64,
128, 256, OxFFI~FFFF, 1 < < 10, 1 < < 1 I, 1 < < 12, 1 < < I3, 1 < < 14,
1«15, 1«16, I«I7, I«18, 1«19, 1«20, 1«21. I«22,
2$ 1«23, 1«24, 1«25I(1«('E'-°A'))((I«('Q'-'A'))
}:
main(ac, av) main
int ac;
char *av[ I,
. {
prog = av[0];
if (ac 1= 3) {
fprintf(stderr,"usage: ~s file! file2\n", prog);
3$ fprintf(stderr,"where file! atul filet are two dna or two protein
sequerxes.ln");
fprintf(stderr,"The sequences can be in upper- or lower-.case\n");
fprintf(stderr, "Any lines beginning with ';' or ' < ' are ignored\n"):
fprintf(stderr,"Output is in the file 1"align.outl"\n");
exit(1);
}
namex[0] = av[1];
namex[1] = av[2];
seqx[0] = getseq(namex[0], &IenO);
seqx[1] = getseq(namex[1], &lenl);
4$ xbm = (dna)? dbval : -pbval;
endgaps = 0; I* 1 to penalize endgaps */
ofile = "align.out"; I* output file *I
$0 nwQ; l* fill in the matrix, get the possible jmps *I
readjmpsQ; I* get the actual jmps *I
printQ; I* print scats, alignment *I
cleanup(0); I* unlink any tmp files *I
$$ }
29
i
CA 02481756 2004-10-25
wo oin63is ~ Pc~rrncrsoor~2s
Table 1 (cont'1
I* do the alignment, return best score: main()
* dna: values in Fitch and Smith, PNAS, 80, 1382-138b, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
S * a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y. .
*/
nW
nwQ
I O char *px, *py; I * seqs and ptrs */
int *ndely, *dely; /* keep track of defy *l _
int ndelx, delx; /* keep track of delx *!
int *anp; I* for swapping row0, rowl *I
int mis; I* score for each type */
15 int ins0, insl: I* insertion penalties *l
register id; l* diagonal index *I
register ij; /* jmp index *!
register *col0, *coll; I* score for curr; last row *I
register xx, yy; /*' index into seqs */
dx = (struct diag *)g calloc("to get diags", len0+lenl+I, sizeof(struct
diag));
ndely = (int *)g calloc("to get ndely", lenl+1, siuwf(int));
defy = (int *)g calloc("to get defy", lent+1, sizeof(int)):
col0 = (int *)g'calloc("to get col0". lenl + 1, sizeof(isxt));
coil = (int *)g calloc("to get toll", lenl+1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
ins 1 = (dna)? DINS 1 : P1NS 1;
smax = -10000;
if (endgaps) {
for (col0(O] = dely[Oj = -ins0, yy = 1; yy < = lent; yy++) {
col0[yyj =~ dely[yyj = col0[yy-lj - insl;
ndely[yyj = yy,
col0[Oj = 0; /* Waterman Bull Math Biol 84 *l
else
for (yy = 1; yy < = lenl; yy++)
0 defy[yyj = -ins0;
/* fill in match matrix
*/
for (px = seqx[Oj, zx = 1; xx < = len0; px++, xx++) {
/* initialize first entry in coi
*/
if (endgaps) {
;f(~ _= 1)
c;oll[Oj = deli = -(ins0+insl);
SO else
toll[Oj = delx = col0(Oj - insl;
ndelx = xx;
j
else {
SS toll[Oj = U;
delz = -itas0;
ndelx = 0;
30
CA 02481756 2004-10-25
WO 01/16318 PG"T/US00/23328
~'al~le 1 (cont'1
...nw
for (py = seqx[1]o yy = 1; yy < _ lent; py++, yy+ ~+) {
mis = col0[yy-I];
if (dna)
$ mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
-rnis +_ 'day[*px-'A'j[*pY-°A~]:
I* update penalty for del in x seq;
to * favor new del over oagong del
* ignore MAXGAP if weighting endgaps _
*/
if (endgaps ') cdely[yy] < MAXGAP) {
if (col0[yy] - ins0 > = defy[yy]) {
1$ defy[yy] = col0[yY] - (ins0+insl);
ndely[yy] ° 1:
~ else {
. dely[yy] -= insl;
ndely[yy] + +:
20 :~
~~e{
iif (col0[yy] - (ins0+insl) > = defy[yy]) {
dely(yy] = col0[yy] - (ins0+insl);
ndely[yy] = 1;
25 ~ else
ndely(yy]+ +:
I* update penalty for del in y seq;
30 * favor new del over ongong del
*I
if (endgaps ( ! ndelx c MAXGAP) {
if (coll[yy-I] - ins0 > = deli) {
deix = coll(yy-I] - (ins0+insl);
35 t~delx = 1;
~ eise {
deli -= insl;
ndelx+ +;
40 ~ else {
if (coll(yy-1] - (ins0+insl) > = delx) {
delx = coll[yy-1] - (ins0+insl);
ndeix = l;
. } else
45 ndelx+ +;
I* pick the maximum score; we're favoring
* mis over any deI and delx over defy
50 *I
SS
31
__ ____ __~_ _ __. ___ _ .~~~_ .._~..... _ ____. _ _. ....~~. ~~tt .~4. m._
_w__ . _ ~N..~.,~..4 ~ ~_ _ r _ ____.__
i
CA 02481756 2004-10-25
wo -omng PCTIUSOOI?.3328
Table ~ (cony)
...raw
id=xx-yy+lenl-1;
if (mis > _= deli && mis > = defy[yy])
coll[yy] = anis;
else if (delx > _ dely[yy]) {
col l [yy] = deli;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna 1 ( (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) I ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
1$ offset += sizeaf(struct jmp) + sizeof(offset);
dx(id].jp.n[ij] = ndelx;
~ dx[id].jp.x[ij] = xx;
20 dx[id].score = detx;
else {
collIYY] ° dely[yyl;
ij = dx[id].ijmp;
2$ if (dx[id]:jp.n[0] && (!dna I ~ (ndely[yy] > = MAX1MP
~c& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
3o ij = dx[idj.ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
35 dx[id].jp.n[ij] _ -ndely[yyh
~[id].jp.x[ij] _ ~;
dx[id].score = dely[yy];
if (xx == IenO 8z& yy < lent) {
/* last col
*I
if (endgaps)
coll[yy] -= ins0+insl*(lenl-yy);
if (coll[yy] > smax) {
4S smax = col l [yy];
dmax = id;
j
5o if (endgaps && xx < len0)
coll[yy-1] -= ins0+insl*(IenO-xx);
if (coll[yy-1] > smax) {
smax = toll[yy-1];
dmax = id;
tmp = col0; col0 == toll; toll = tmp;
(vaid) free((char *)ndely);
(void) free((char *)dely);
6~ (void) free((char *xol0);
(void) free((char *koll); j
32
CA 02481756 2004-10-25
WO O1/I6318 ~ PCT/US00123328
Tahle 1 (cont'1
I*
* print() - only routine visible outside this module ,
* static:
* getmatQ -- trace back best path, count matches: printQ.
* pr alignQ -- print alignment of described in array p[ ]: Print()
* dumpblockQ -- dump a block of lines with numbers, stars: pr align()
* numsQ - put out a number line: dutrtpblockQ
* putlineQ - put out a line (name, [num], seq, [num]): dumpblockQ
* stars() - -put a line of stars: dumpblockQ
* striptuune() - strip any path and prefix from a seqname -
*/
1S #inciude "nw.h"
#define SPC 3
#define P LINE 256 I* maximum output line *I
#defme P SPC 3 I* space between name or num and seq */
extern day[26][26]:
int olen; I* set ouq~ut line iengfh *I
FILE *fx; I* output file *I
print() P~~t
int Ix, ly, firstgap, iastgap; I * overlag *I
if ((fx = fopen(ofile, "w")) _ = 0) {
3~ fprintf(stderr,"9~s: can't write ks\n", prog, ofile);
cleanup(i);
fprintf(&, " < first sequence: ~s (length = q6d)\n", namex[0], len0);
fprintf(fx, "<second sequence: 9bs (length = '~d)\n", namex[1], Ienl);
olen = 60;
lx = lenU;
'ly = lent;
firstgap = lastgap = 0;
if (dmax < Ienl - 1) { /* Leading gap in x *I
PP[0] ~~ = f~tgap = lens - dmax - 1:
ly -= pPC~l.sPc:
else if (dmax > leni - 1) { /* leading gap in y *!
pp[I].spc = firstgap = dmax - (lenl - i);
lx -= pp[1].spc;
1
if (dmax0 < len0 - 1) { I* trailing gap in x */
iastgap = IenO - dmax0 -1;
lx -_ lastgap;
}
else if (dmax0 > len0 - i) { I* trailing gap in y *l
lastgap = dmax0 - (IenO - 1);
ly -= Iastgap;
getmat(Ix, ly, firstgap, lastgap);
pr alignQ;
33
___t . mw~.~_rt a _~ . ~.:~n ~ . w . . ~_-.__ _ _ ___ _.~._~ ~:n .~ _:., ~.~, -
~,n..~._ ._- _ _ _ . . _ ~ __~ _ ___
CA 02481756 2004-10-25
wo oW is . PCTIUSf)012332s
Table 1 (cont'~
/*
* trace back the best path. count matches
*/
static
getmat(lx, ly, firstgap, lastgap) getrilat
int lx, ly; I* "core" (mimes endgaps) *!
int firstgap, lastgap; ' !* leading trailing overlap */
{
int nm, i0, ii, siz0, sizl;
1~ char outx[32j;
double pct;
register n0, nl; _
register dear *p0, *pl;
IS /* get total matches, score
*/
i0 = ii = siz0 = seal = 0; '
p0 ~ seqx[0] + pp[lj.spc;
P1 = ~qx[1] + PP[0]~sPc:
zfl n0 = pp[1].spc + 1;
nl = pp[0].spc + 1;
nm - 0;
while ( *p0 && *pl ) {
25 it' (S~) {
pl++;
nl + +;
siz0--;
3o else if (seal) { ,
p0++;
n0++;
seal--;
35 else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm++;
if (n0+-~- _= pp[0].x[i0j)
siz0 = pp[0].n[i0++j;
40 if (nl-~-+ _- pP[1],X[il])
seal = pp[lj.n[i1++];
p0++;
pl++.
45 1
I* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
S~ *I
if (endgaps)
lx = (len0 < lenl)? ien0 : ienl;
else
lx = (lx < ly)? lx : ly;
SS pct = 100.*(double)nm!(double)Ix;
fprintf(fx, "fin");
fprintf(fx, "< ~d match~&s in an overlap of ~d: ~.2f percent similarityln",
~~ (~ _= 1)? "« ~ «es«. ix, pct);
34
CA 02481756 2004-10-25 '
WO 01/16318 PCT/USOOIZ3328 .
Table l~cont')
fprintf(fx, "<gaps in first sequence: ~d", gapx); e.egetIriat
(gapx) {
(void) sprintf(ouGc, '" (96d '~s96s)",
ngapx, (dna)? "base":"residue", (ngapx == I)? "":"s");
fprintf(fx,"96s", outx);
fprintf(fx, ", gaps in second sequence: ~d", gapy);
if (gaPY) 1
(void) sprintf(outx, " (~d ~s~s)",
ngapy, (dna)? "base":"residue", (ngapy == L)? "":"s");
fprintf(fx,"~s", oucx); _
1
if (dna)
fprintf(fx,
"\n<score: 96d (match = Y~d, mismatch = °~d, gap penaity = ~d +
°~d per base)\n°',
smax, DMAT, DMIS, DINSO, DINS1);
else
. fprintf(fx,
0 "\n<score: ?'od (Dayhoff PAM 250 matrix, gap penalty = 96d + ~d per
residue)ln",
smax, PINSO, PINSi);
if (endgaps)
fprintf(fx.
" < endgaps penalized. left endgap: %d 9'os~s, right endgap: ~d ~s~sln",
firstgap, (dna)? "base" : "residue", (firstgap == 1)? "" : "s",
lastgap, (dna)? "base" : "residue", (tastgap =~ 1)? "" : "s");
eL~e
fprintf(fx, "<endgaps not penalized\n");
1
static nm; l* matches in core -- for checking *!
static lmax; ~~* lengths of stripped file names *I
static ij[2]; !* jmp index for a path */
static nc[2]; l* number at start of current line *I
3S static ni[2]; /* current elem number - for gapping *I
static siz[2];
static char *ps[2]; J* ptr to current element *!
static char *po[2]; ,t* ptr to next output char slot *I
static char out[2][P LINE]; I* output line *!
static char star[P LINE]; !* set by stars() */
,*
* print alignment of described in struct.path pp [ ]
*/
static
pr align
pr~alignQ
int nn; I* char count *!
int more;
register i;
for (i = 0, lmax = 0; i < 2; i++)
nn = stripname(namex(i]);
if (nn > Imax)
lmax = nn;
nc[i] - 1~ .
ni(i] = 1:
' siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po(i] = oui(i]; }
CA 02481756 2004-10-25
WO 01!16318 PCT/USOO/Z3328
Table !. (cont'1
for (nn = nm = 0, more = l: more; ) { ...pr allgll
for (i = more = U; i < 2; i++) {
I*
$ * do we have more of this sequence?
*/
if (a*ps[i])
eontinue;
more++;
if (pp[i].spc) { I* leading space *l
*po[i]++ _ , ,~
PP[7.spc__;
else if (siz[i]) { /* in a gap */
. *po[i]++ _ '-':
siz[i]--;
20 else { /* we're putting a seq element
*l
*po[i] _ *ps[i]:
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
25 po[i]++:
ps[i] + +;
l*
* are we at next gap for this seq?
3O *l
if (ni[;] _ = pP[i].x[ij[i]]) {
/*
* we need to merge all gaps
* at this location
35 *l
siz[i] = pp[i].n[ij[i]++];
while (ni[i] _ ~ pp[i].x[ij[i]])
su[i] += PP[i].nlij[i]++];
4~ ni[i] + + ;
if (++nn == olen ~ ~ !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++)
Po[i] = out[i];
nn = o;
SQ
l*
* dump a block of lines, including numbers, stars:
pr align(]
*%
S5 static
dt~mpblo~k~ d~pb~ock
register i;
6Q for (i = 0; i < 2; i++)
*~(i]__ _ 'vo";
36
___ _ __ _ __e ~a,_.~ ~ . ~ ~~ ~ ,~ Mr. __..__ _. r...._. . _ _ _ .__~ .
...____ . .. _ ~ __ _ _ _
i
CA 02481756 2004-10-25
wo oins3is . r~rnlsoor~3zs
~ bla a ~ (cont'1
...dumpblock
(void) putc('1n', fx);
far (i = 0; i < 2; i++)
if (*out[i] && (*out(i] I = ' ' ~ I *(po[i]) . _ ' )) ~
~(i==p)
' nums(i):
if (i == A && *out[I])
starsQ;
lO P~~(i):
if (i == 0 && *out[1])
fprintf(fx, star); _
~(; _= 1)
nums(i);
15 1
1
!*
.
0 * put
out
a
number
line:
dumpblockp
*/
static
nums(ix)nums
int ix; /* index in out[] holding seq line *!
25 1
char mine[P LINE];
register i, j:
register char *pn, *px, *py;
30 for (pn = mine, i = 0; i < lmax+P SPC; i++, pn++)
*pn =
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
~(*PY =_ ~ ' ~ I *pY =_ _')
*pn = ' ,
35 else {
if (i ~ 10 = = 0 ~. I (i = = 1 && nc[ix] ! = 1 )) ~
j = (i < 0)~ _i : i:
for (px = pn; j: j != 10, px-)
*px - JS61U + 'U',
40 if (i < o)
*Px =
*pn = ,
45 i++;
1
1
*Pn = ,\0,:
nc[ix] = i;
for (pn = mine; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
55 /*
* put
out
a
line
.(name,
[num];
seq,
[num]):
dumpblockQ
static
putline(ix)
putlane
60 int ix; {
37
CA 02481756 2004-10-25
WO OI/I6318 PGTIUS00123328
Table I (cony)
o a :putline
int i;
register char *px;
for (px = namex[ixj, i = 0; *px &~c *px !_ :'> px++, i++)
(void) pate(*px, fz);
for (; i < Imax+P SPC: i+~ +)
(void) pule(' ', fx):
I* these count from 1:
* niQ is current element (from 1)
* nc[] is number at start of current line
*/ ,
1 5 for (px = out[ix]; *px;. px++)
(void) putt(*px&Ox7F, fx);
(void) putc('\n', fx);
~*
* put line of stars (seqs always in out[0j, out[lj): dumpblock0
a
*I
static
starsQ
stars
int i;
register char *p0, *p1, cx, *px;
if (!*out[Oj ! ~ (*out[0] __ ' && *(po[0]) _- ' ') ~ ~
!*out[1] ~ I (*out[1] _ _ ' && *(PoLlj) _ - ' '))
px = star;
for (i = lmax+P SPC; i; i--)
*px++ = ' ';
for (p0 = out[Oj, pl = out[1]; *p0 && *pl: p0++, pl++) 1
if (isaipha(*p0) && isalpha(*pl)) {
4d if (xbm[*p0-'A']&xbm[*pl-'A]) {
cx='*';
nm-I- +;
1
else if (!dna && day[*p0-'A'][*pl-'A'] > 0)
cX = . .
else
CX = ,
else
cx =- ,
*px++ = cx:
1
*px++ _ '\n';
*Px = .10,:
SS }
38
f
CA 02481756 2004-10-25
WO 01!16318 PC'1'~S00/23328 .
Table 1 leant')
l*
* snip path or prefix from pn, return len: pr alignQ
*l
static
stripname(pn) str~pname
char *pn; /* file name (may be path) *l
register char *px, *py;
lO py = 0;
for (px = pn; *px; px++)
if (*px =_- ~l~)
PY = px '~" l:
if (PY)
1S (void) sacpy(pn, py);
return(salen(pn));
2S
35
45
SO
SS
39
_ _ .> bfl a .r.~_ a~._~ __.__.....__...._ _..____ _______ ~"~ ~.~.._...
._~___ ,
S
CA 02481756 2004-10-25
PCT/1TSOOA23328
WO OllI6318
Table 1 cont'l
l*
* cleanup() - cleanup any tmp file
* getseq0 -- read in seq, set dna, Ien, maxlen
* g ca11oc0 -- callocQ with error checkin
* readjmpsQ -- get the good jmps, from tmp file if necessary
* writejmps0 -- write a filled array of jmps to a tmp file: nwQ
*l
flinclude "nw.h"
!!include < sys/file. h >
char *jname = "ltmplhomgXXXX3~X"; I* tmp file for jmps *I
FILE *fj;
int cleanupp; I * cleanup tmp file *I
LS tong lseek0;
l*
* remove any tmp file if we blow
*l
cleanup(i) CIeariBIIJ
int i;
if (fj)
(void) unlink(jaame);
exie(i);
I
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with '; , ' <', or ' >' '.
* seq in upper or lower case
*I
char *
getseq(file, len) get'~~
char *file; l* file name *l
int *len; !* seq ten */
char line[1024], *pseq;
register char *px, *py:
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file,~r~)) _= 0) {
fprintf(stderr,~R~s: ran't read ~sln~, prog, file);
exit(I);
Lien = natgc = 0;
while (fgets(line, 1024, fg)) {
if (*Iine =- ' I ~ *line =_ ' <' ~ ~ *line =- ' >')
$0 continue;
for (px = line; *px !_ '\n'; px++)
if (isupper(*px) / J islower(*px))
Lien++;
SS f ((pseq = mailoc{(unsigned)(tlen+6))) _ = 0) I
fprintf{stderr,"Ybs: malloc0 failed to get ~d bytes for '~s\n°', grog.
tlen+ti, file);
exit(1);
P~qfO) = t~q[II = P~qI2l = I~qI31 = '\0';
40
.~____-~.__... ....._._ CA 02481756 2004-10-25
WO 01/I6318 PCTIUSOOl23328
lQl~llG 1 \GVlal. 7
...~BtSP.(j
PY = P~9 + 4:
*Ien = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
If (*line =- ' ~ [ *line =- ' < ° ~ ~ *tine =- ' >')
continue;
for (px = line; *px 1= '1n'; px++) {
IO if (isupper(*px))
*PY++ ~ *Px: _
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU",*(py-1))) '
IS natgc++;
. *py++ _ '\0°;
*PY = °\0°;
20 (void) fclose(fp);
dna = natgc > (tlenJ3);
return(pseq+4);
25 char
g calloc(msg, nx, sz) ~Calloc
char *msg; !* program, catiing routine *!
int nx, sz; !* numtxr and size of elements *I
{
30 char *px, *callocQ;
if ((px = calloc((unsigned)nx. (unsig~ned)sz)) _ = 0) {
if (*msg) {
fprintf(stderr, "~s: g callocQ failed 9&s (n=~d, sz=~d)\n", prog, msg, nx"
sz);
35 exit(1);
}
return(px);
.}
4~
/*
* get final jmps from dx[] or anp file, set ppj], reset dmax: mainQ
*!
readjmps() rP,ad~ Nips
45 {
int fd = -i;
int siz, i0, ii;
register i, j, xx;
SQ if (fj) {
(void) fclose(fj);
if ((fd = open(jname, O_RDUNLY, 0)) < 0) {
fprintf(stderr. "'Yos: can't open() %s\n", prog, jname);
cleanup( i );
55 }
por (i = i0 = il = 0, dmax0 -- dmax, xx = len0; : i++) {
whe7e (1) {
for (j = dx[dmax].ijmp; j > -- 0 && dx[dmax].jp.x[j] > = xx; j--)
41
_.._._ _ __~ .__ ...._~ "~~ . ~ ~~,,,x~~ ~ .~~F=~ _...__.. _. ..__~r~,","...~
~P.m.~..-.~~- _.__.
CA 02481756 2004-10-25
WO O1/I6318 PCT/USOU123328
Table Iscont'1
if (j < o && dx[dmax].offset 8c8c fj) {
...readjmps
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
else
break;
I0 ~
if (i a = JMPS) ~
fprintf(stderr, "9bs: too many gaps in alignmentln", prog); -
cleanup(1);
IS if (j > = o) {
siz = dx[dmax] ;jg.n[j];
xx = dx[dmax].jp.x[j];
dmax + = siz;
~ if (siz < 0) f I * gap in second seq *I
20 PP[i]~nlil] _ _S~;
xx + = siz;
I*id=xz-yy+lenl-1
*%
pp[1].x[il] = xx - dmax + lent - 1;
25 gapy+ +;
ugapy _= siz;
/* ignore MAXGAP when doing endgaps *I
siz = (-siz < MAXGAP ~ E endgaps)? -siz : MAXGAP;
il++;
30 t
else if (siz. > 0) { /* gap in first seq */ ,
PPLQLn[i0] = siz;
pp[0].x[i0] = xx;
gapx++;
3S ngapx + ~ siz;
/* ignore MAXGAP when doing endgaps *!
siz = (siz < MAXGAP ! ~ endgaps)? siz : MAXGAP;
i0++;
40 I
else
break;
j
45 I* reverse the order of jmps
*I
for (j = 0, i0--; j < i0; j + -F' , i0-) {
i = PP[0].nG]: PP[O]~nI1] = PP[Ol~n(i0]; PP[Ol.n[i0] = i;
i = PP[0].xU]: ppfOl.xG] = PFIO].x[i0]; PP[0].x[10] = i;
50 }
for (j = 0, iI--; j < il; j+-t-, il-) {
i = PP[1].n(i]: PP[1].nLt] = PP[1].n[il]; pP[il.n[il] = i;
1 = PP[11.x(1]: PP[1].xtl] = PP[1].x[il]; PP[i]-x[il] = i:
55 if (fd > = 0)
(void) close(fd);
d. (~) {
(void) unlink(jname);
0;
60 offset = 0;
j
42
CA 02481756 2004-10-25
WO 01/16318 PCT/US00/23328
Table 1 tcont')
i*
* write a filled jmp struct offset of the prey one (if any): nwQ
*~
S writejmps(ix) writejmps
int ix; .
char *mktemp0;
if (!fj) {
if (mktemp(jname) <. p) {
fprintf(stderr, "~s: can't mlctemp0 Y6s\n", prog, jname); - _
cleanup(1);
iS if ((fj = fopen(jname, "w")) _ _ ~) {
fprintf(stderr, "96s: can't write Yes\n", prog, jname):
exit(1);
2,0 (void) fwrite((char *)~cdx{ix].jp, sizeof(strtcct jmp), 1, fj);
(void) fwrite((char *)&dx(ix].offset, sizeof(dx[ix].offset), 1, fj);
j
30
40
50
60
43
_ __ . _.. ... _ ~... . .. . . _ _ _ _....~. ~.:L-~a r~. ~~ _ ~, .m. . ~K.~H~
..rc h .v P_. __ _-_ __ _~__~_,. r ~ __ ._..__._ -_
CA 02481756 2004-10-25
WO OIII63I8 ~ PGT/US00123328
Table 2
PRO XXX~XXXX3~X7CX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
°6 amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _ .
5 divided by 15 = 33.3 ~
44
CA 02481756 2004-10-25
wo oins3><8 ~ rcr~soon~2s
Tabls 33
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amine acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the iwo
polypeptide sequences as determined
by ALIGN-2) divided by (the total number of amino acid residues of the PRO
polypeptide) _
5 divided by 10 = 509'v
CA 02481756 2004-10-25
wo .oinm8 PG"T/USOUI23328
bl 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
S k nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) =
6 divided by 14 = 42.9 ~
46
.... .._ __~.... ....~,._ .. ..~. ,x _ . .. __ ._ w __.~ . ~ ~,a
..,x~~e~~".hr.,F,m.~.~~.m~,~...~.____ ___.._~_
CA 02481756 2004-10-25
Wp 01116318 ~ PGT/USOOI23328
PRO-DNA NNI~INNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by. (the total munber of nucleotides of the PRO-DNA nucleic
acid sequence) _
I O 4 divided by 12 = 33.3 9b
47
CA 02481756 2004-10-25
WO OI/I6318 PG"TIUS001~3328
III-. . Cc~,mpositi ns and ethods of the Invent~'on ,
A. Full-Length PRO Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PRO polypeptides. In particular,
cDNAs encoding various PRO
poIypeptides have been identified and isolated, as disclosed in Further detail
in the Examples below. It is noted
that proteins produced in separate expression rounds may be given different
PRO numbers but the UNQ number
is unique for any given DNA and the encoded protein, and will not be changed.
However, for sake of
simplicity, in the present specification the protein encoded by the full
length native nucleic acid molecules
disclosed herein as well as all further native homologues and variants
included in the foregoing definition of
PRO, will be referred to as "PRO/number°, regardless of their origin or
mode of preparation.
As disclosed in the Examples below, various cDNA clones have been deposited
with the ATCC. The
actual nucleotide sequences of those clones can readily be determined by the
skilled artisan by sequencing of the
deposited clone using routine methods in the art. The predicted amino acid
sequence can be determined from
the nucleotide sequence using routine skill. For the PRO polypeptides and
encoding nucleic acids~described
herein, Applicants have identified what is believed to be the reading frame
best identifiable with the sequence
information available at the time.
B. PRO Polypepdde Variants
In addition to the full-length native sequence PRO polypeptides described
herein, it is contemplated that
PRO vaCiants can be prepared. PRO variants can be prepared by introducing
appropriate nucleotide changes into
the PRO DNA; and/or by synthesis of the desired PRO polypeptide. Those skilled
in the art will appreciate that
amino acid changes may alter post-translational processes of the PRO, such as
changing the number or position
of glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PRO: or in various domains of
the PRO described herein,
can be made, for example, using any ~f the techniques and guidelines for
conservative and non-conservative
mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations
may be a substitution, deletion or
insertion of one or more colons encoding the PRO that results in a change in
the amino acid sequence of the
PRO as compared with the native sequence PRO. Optionally the variation is by
substitution of at least one amino
acid with any other amino acid in one or more of the domains of the PRO.
Guidance in determining which
amino acid residue may be inserted, substituted or deleted without adversely
affecting the desired activity may
be found by comparing the sequence of the PRO with that of homologous known
protein molecules and
minimizing the number of amino acid sequence changes made in regions of high
homology. Amino acid
substitutions can be the result of replacing one amino acid with another amino
acid having similar structural
andlor chemical properties, such as the replacement of a leucine with a
serine, i.e., conservative amino acid
replacements. Insertions or_~deletion~may:o ttattall' be tzivthei~an a
of.al~ui h;to 5 aariino aeids~ The variafibn.
P Y g
allowed may fie determined by systematically making insertions, deletions or
substitutions of amino acids in the
sequence and testing the resulting variants,for activity exhibited by the full-
length or mature native sequence.
48
CA 02481756 2004-10-25
wo oins3is rcT~saor~3a~
PRO polypeptide fragments are provided herein. Such fragments may be avncated
at the N-terminus
or C-terminus, or may lack internal residues, for example, when compared with
a full length native protein.
Certain fragments lack amino acid residues that are not essential for a
desired biological activity of the PRO
polypeptide.
PRO fragments may be prepared by any of a number of conventional techniques.
Desired peptide
fragments may be chemically synthesized. An alternative approach involves
generating PRO fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme known to
cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with suitable
restriction enzymes and isolating the
desired fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding
a desired polypeptide fragment, by polymerise chain reaction (PCR).
Oligonucleotides that define the desired
IO termini of the DNA fragment are employed at the 5' and 3' primers in the
PCR. Preferably, PRO polypeptide
fragments share at least one biological andlor immunological activity with the
native PRO polypeptide disclosed
herein.
In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the heading
of preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial
changes, denominated exemplary substitutions in Table 6, or as further
described below in reference to amino
acid classes, are introduced and the products screened.
49
CA 02481756 2004-10-25
WO O1I16318 . ~pCTIUSOU/?.3328
Table 6
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gtn; asn lys
Asn (N) gln; his; lys; arg gln
Asp (D) glu , glu
Cys (C) ser ser
Gtn (Q) asn asn
Glu (E) asp asp _
Gly (G) pro; ala ' ala
His (H) asn; gln; lys; arg. arg
Ite (I) leu; val; met; ala; phe;
norleucine teu
Leu (L) norleucine; ile; val;
, met; ala; phe ile
Lys (I~ arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (T-~ leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
'Tha' (T) sex ser
Trp (W) tyr; phe tyr
Tyr (1~ trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
aia; norleucine teu
Substantial modifications in, function or immunological identity of the PRO
polypepride are accomplished
by selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the potypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or
hydrophobicity of the molecule at the target.site, or (c) the bulk of the side
chain. Naturally occurring residues
are divided into groups based on common side-chain properties:
( 1 ) hydrophobic: norleucine, met, ala, val, teu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
Such substituted residues also may be introduced into the conservative
substitution sites or, more preferably, into
the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucIeotide-mediated .(site-.,
d,recfed). ittitageiiesis; alainne scanning, arid PCR mutagenesis. Site-
directed rriutagenesis [Carter et-a~l:; ttc~_ v
Acids Res., 13:4331 (1986); Zoller et al., ~tucl. Acids Res., 10:64$7 (1987)],
cassette mutagenesis [WeYls et
al., Gene, 34:315 (1985)j, restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA,
.. . ~,~~ , ,.~., rr .". ,r.. .. , ._.. . .._x..~ m ~ .,t A~"., ..... ...
n..w.._.~ «~.". ~.~....m.,._~_ ......
CA 02481756 2004-10-25
CVO 01/16318 . ~ PCT/USOOI?.3328.
X7:415 (1986)] or other known techniques can be performed on the cloned DNA to
produce the PRO variant
DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids along a
contiguous sequence. Among the preferred scanning amino acids are relatively
small, neutral amino acids. Such
amino acids include aIanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid
S among this group because it eliminates the side-chain beyond the beta-carbon
and is Iess likely to alter the main-
chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-
1085 (1989)]. Alanine is also
typically preferred because it is the most common amino acid. Further, it is
frequently found in both buried and
exposed positions [Creighton, .the Pmteins, (W.H. Freeman & Co., N.Y.);.
Chothia, J. Mol. Biol., 15Q:1
(197b)]. If alanine substitution does not yield adequate amounts of variant,
an isoteric amino acid can be used.
C. Modifications of PRO
Covalent modifications of PRO are included within the scope of this invention.
One type of covalent
modification includes reacting targeted amino acid residues of a PRO
polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the N- or C-
terminal residues of the PRO.
Derivatization with bifunctional agents is useful, for instance, for
crosslinking PRO to a water-insoluble support
matrix or surface for use in the method for purifying anti-PRO antibodies, and
vice-versa. Commonly used
crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuceinimide
esters, for example, esters with 4-azidosalieylic acid, homobifunetional
imidoesters, including disuccinimidyl
esters such as 3,3'_-dithiobis(succinimidylpropionate), bifunetional
maleimides such as bis=N-nialeimido-1,8- -
octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioin~adate.
Other modifications include deamidation of glutaminyl and asparag3nyl residues
to the corresponding
glutamyl and aspartyl residues, respectively, hydroxylation of protine and
lysine, phosphorylation of hydmxyl
groups of seryl or threonyi residues, methylation of the a-ami~ groups of
lysine, arginine, and histidine side
chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco,
pp. 79-8b (I983)], acetylation of the N-terminal amine, and amidation of any C-
terminal carboxyl group.
Another type of covalent modification of the PRO polypeptide ~ included within
the scope of this
invention comprises altering the native glycosylation pattern of the
poIypeptide. "Altering the native
glycosylation pattern" is intended for purposes herein to mean deleting one or
more carbohydrate moieties found
in native sequence PRO (either by removing the underlying glycosylation site
or by deleting the glycosylation
by chemical andlor enzymatic means), and/or adding one or more glycosylation
sites that are not present in the
native sequence PRO. In addition, the phrase includes qualieadve changes in
the glycosylation of the native
proteins, involving a change in the nature and proportions of the various
carbohydrate moieties present.
Addition of glyeosylation sites to the PRO polypeptide may be aaeomplished by
altering the amino acid
sequence. The alteration may be made, for example; by the addition of; or
substitution by, one or more serine
or thieonine residues to the native sequence PRO (for O-linked glycosylation
siees). The PRO amino acid
sequence may optionally be altered through changes at the DNA level,
particularly by mutating the DNA
encoding the PRO polypeptide at preselected bases such that colons are
generated that will translate into the
51
...,..,~ ...-.~. _ .______._..-~,~~.~._.._._. .. __ N~.., .~n~,~p..r .....__
._...
CA 02481756 2004-10-25
wo .oW is . . rcrnJSOOr~3z8
desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO
polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide. Such methods
are descn'bed in the art, e.g.,
in WO 87/OS330 published I1 September 1987, and in Aplin and Wriston, CRC
Cr'y,.. Rev. Biochem., pp. 2S9-
306 (1981).
Removal of carbohydrate moieties.present on the PRO polypeptide may be
accomplished chemically
or enzymatically or by mutational substitution of codons encoding for amino
acid residues that serve as targets
for glycosylation. Chemical deglycosyiation techniques are known in the art
and described, for instance, by
Hakimuddin, et al., arch. Bi.ochem. Bic_>phvs., x:52 (1987) and by Edge et
al., anal. Bioche~, x:131
(198I). Enzymatic cleavage of carbohydrate moieties en polypeptides can be
achieved by the use of a variety
of endo- and ~xo-gIycosidases as described by Thotakura et aL, Meth. En~ymol.,
,138:350 (1987).
Another type of covalent modification of PRO comprises linking the PRO
polypeptide to one of a variety
of nonproteinaceous polymers, e:g., polyethylene glycol {PEG), polypropylene
glycol, or polyoxyalkylenes, in
the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689;4,301,144;
4,670,417; 4,791,192 or 4,179,337.
The PRO of the present invention may also be modified in a way to form a
chimeric molecule
comprising PRO fused to another, heterologous polypeptide or amino acid
sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO with
a tag polypeptide
which provides an epitope to which an anti-tag antibody can selectively bind:
The epitope eag is generally placed
at the amino- or carboxyl- terminus of the PRO. The presence of such epitope-
tagged forms of the PRO can be
detected using-an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the PRO to,
be readily purified by 'affinity purification using an anti-tag antibody or
another type of affinity matrix that binds
to the epitope tag, Various tag polypeptides and their respective antibodies
are well known in ehe art: Examples
include poly=histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its
antibody 12CA5-(Field ei al., Mol. Cell. Biol., _8:2159-2165 (1988)]; the c-
myc-tag and the 8F9; 3C7, 6E10,
G4, B7 and 9EI0 antibodies thereto [Evan et at., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the
2S Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky ex
al., Protein Engineering, x(6):547-
553 (1990)]. Other tag polypeptides include. the Flag-peptide [Hopp ee al.,
BioTec~nology, ø:1204-1210
(1988)]; the KT3 epitope peptide [Martin et al., Science, ,.55:192-194
(1992)]; an ~-tubulin epitope peptide
[Skinner et al., 1. Biol. Chem., x:15163-15166 (1991)]; and ehe T7 gene 10
protein peptide tag [Lutz-
Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-b397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the PRO with an
immunoglobulin or a particular region of an immunoglobulin. For a bivalent
form of the chimeric molecule (also
referred to as an "immunoadhesin" ), such a fusion could be to the Fc region
of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane domain deleted
or inacrivated) form of a PRO
;polypeptide tn place of . at least one variable .region within an- I
Briolecute. In- a ai~ticularl . eferred
_: . ::- .:.-: ~ .. ,., .. .. . , g _. p y pr .. .. .,
embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3
regions of an IgG 1 molecule. For the production of immunoglobulin fusions see
also US Patent No. 5,428,130
issued June 27, 1995.
52
CA 02481756 2004-10-25
VifO OI/163I8 PCT/USOOn3328
D. Preparation of PRO
The description below relates primarily to production of PRO by culturing
cells transformed or
transfected with a vector containing PRO nucleic acid. It is, of course,
contemplated that alternative methods,
which are well known in the art, may be employed to prepare PRO. For instance,
the PRO sequence, or
portions thereof, may be produced by direct peptide synthesis using solid-
phase techniques [see, e.g., Stewart
et al., Solid-Phasc Peptide Synthesis, W.H. Freeman Co., San Francisco, CA
(1969); Merrifield, J. Am. Chem.
Soc., 85:2149-2154 (1963)). In vitro protein synthesis may be performed using
manual techniques or by
automation. Automated synthesis may be accomplished, for instance, using an
Applied Biosystems Peptide
Synthesizer (Foster City, CA) using manufacturer's instructions. Various
portions of the PRO may be
chemically synthesized separately and combined using chemical or enrymatic
methods to produce the full-length
PRO.
1. Isolation of DNA Encoding PRO
DNA encoding PR0 may be obtained from a cDNA library prepared from tissue
believed to possess
the PRO mRNA and to express it at a detectable level. Accordingly, human PRO
DNA can be conveniently
IS obtained from a cDNA library prepared from human tissue, such as described
in the Examples. The PRO=
encoding gene may also be obtained from a genotnic library or by known
synthetic procedures (e.g., automated
nucleic acid synthesis).
Libraries can be screened with probes (such as antibodiNS to the PRQ or
oligonuciecitides of ac:least
about 20-80 bases) designed to identify the gene of interest or the protein
encoded by it. Screening the cDNA
or genotnic library with the selected probe may be conducted usutg standard
procedures, such as described in
Sambrook of al., Molecular Cioninsr: A Laboratory Manual (New 'fork: Cold
Spring Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding PRO is to use PCR
methodology [Sambrook et al.,
supra; Dieffenbach et al., ~'CR Primer: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The
oligonucle~tide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon
hybridization to Dl~iA in the library
being screened. Methods of labeling are well known in the art,~and include the
use of radiolabels like'~P-labeled.
ATP, biotinylation or enzyme labeling. Hybridization conditions, including
moderate stringency and high
stringency, are provided in Sambrook ee al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known
sequences deposited and available in public databases such as GettBank or
other private sequence databases.
Sequence ideneity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across
the full-lengtf sequence can be deteryined using methods known in iheart and
as described herein:
Nucleic acid having protein coding sequeiace:niay be atitained by screening
selected. chNAVi: getiomic
libraries using the deducxd amino acid sequetxx disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al., a
ra, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
*-trademark S3
__..w.~ _ . _ . ___,~_ ..~ a.,~.~.~.w .r.~r~~ro~ ...__.~_.. __ .q...~
~...~~~..._,.~ _
i
CA 02481756 2004-10-25
wo .o><ns3>!a rCTlvsoan33Za
___ 2. ~glection and Transforn,~ation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for PRO
production and cultured in conventional nutrient media modified as appropriate
far inducing promoters, selecting
transfotmants, or amplifying the genes encoding the desired sequences. The
culture conditions, such as media,
temperature, pH and the like, can be selected by the skilled artisan without
undue experimentation. In general,
principles, protocols, and practical techniques for maximizing the
productivity of cell cultures can be found in
Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press,
1991 ) and Sambrook et al.,
su ra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCh, CaPO,; liposome-mediated and
electroporation. Depending an the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sunbrook et al., sera, or
electroparation is generally used for
prokaryotes. Infection with Agrobacterium tumefacierrs is used for
transformation of certain plant cells, as
described by Shaw et al. , Gene, 23:315 ( 1983) and WO 89105859 published 29
June 1989. For mammalian cells
without such cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, ViroloQV,
52:456-457 (1978) can be employed. General aspects of mammalian cell host
system transfections have been
described in U.S. Patent No. 4,399,216. . Transformations into yeast are
typically carried out according to the.
method of Van Solingen et al., J. Bact., X0_:946 (1977) and Hsiao et al.,
proc. Natl. Acad. Sci. (USAF, 76:3829
(1979). However, other methods for introducing DNA into cells, Such as by
nuclear.-micwoinjection;> _.,
eleetroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene; polyarnithineday '
also be used. For various techniques for transforming mammalian cells, see
Keown et at., Methods in
Enz~ nologv_, 185:52?-537 (1990) and Mansour et al., Nature, 336:348-352
(1988).
Suitable host cells far cloning or expressing the DNA in the vectors herein
include prokaryote, yeast,
or higher eukaryote cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative
or Gram-positive organisms, for example, Enterobacteriaceae such as E. colt.
Various E, colt strains are
publicly available, such as E. colt K12 strain MM294 (ATCC 31,446); E. colt
X1776 (ATCC 31,537); E. colt
strain W3110 (ATCC 27,325) and KS 772 (ATCC 53,635). Other suitable
prokaryotic host cells include
Enterobacteriaceae such as Escherichia, c.g., E. colt, Enterobacter, Erwinia,
Klebsiella, Proteus, Sadmon~ella,
e.g., Salmonella typhtmurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as.Bucilli such ass B.
subtilis and B, licheruformis (e.g., B. licheniformis 41P disclosed in DD
266,71'0 published 12 April 1989),
Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are
illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host because it is a
common host strain for recombinant
DNA product fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For
example, strain W3110 may be modified to effect a genetic mutation in the
genes encoding proteins endogenous
to the host, with exauiipleS of such-hosts including E: colt W3110 strain 1A2,
which has the csotnplete genotype: _ _:.
3$ tortA ; E, colt W31IO strain 9E4, which has the complete genotype tonA
ptr3; E. colt W3110 strain 27C7
(ATCC 55,244), which has the complete genotype tonA ptr3 phoA El S
(argRlac)169 degP ompT kan'; E, colt
W3110 strain 37D6, which has the complete genotype torul ptr3 phoA EIS fargF-
.lac)169 degP ompT rbs7
54
CA 02481756 2004-10-25
WO OIII63I8 . PCT/US00123328
ilvC;kdn ; E. coli W3110 strain 4084, which is strain 37D6 with a non-
kanamycin resistant degP deletion
mutation; and an E. coli strain having mutant periptasmic protease disclosed
in U. S. Patent No. 4,946,783 issued
7 August 1990. Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase
reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning
or expression hosts for PRO-encoding vectors. Saccharomyces cerevisiae is a
commonly used lower eukaryotic
host microorganism. Others include Schizosaccharomycespombe (Beach and Nurse,
Nature, 290: 140 [1981];
EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No.
4,943,529; Fleer et al.,
Bio/Technoloav, 9:968-975 (1991)) such as, e.g., K, lactis (MW98-8C, CBS683,
CBS4574; Louvencourt et
al., J. Bacteriol., 154(2):737-742 (1983]), K. fragilis (ATCC 12,424), f~
bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24, i78), K. waltii (ATCC 56,500), K drosophilarum (ATCC
36,906; Van den Berg et aL,
Bio/TechnoloQV, 8:135 (1990)), K. thermotolerans, anti K. marxianus; yarrowia
(EP 402,226); Pichiapastoris
(EP 183,070; Sreekrishna et al., ~, Basic MicrobioL, 28:265-278 [1988]);
Candida; Trichoderma reesia (EP
244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-
5263 [1979]); Schwanniorrayces
such as Schwanniomyces occidentalis (EP 394,538 published 3I October 1990);
and filamentous fungi such as;
e.g., Neurospora, Penicidlium, Tolypocladium (WO 91/00357 published 10 January
1991 ), and Aspergillus hosts
such as A. nidulans (Ballance et al., Biochem. Biophvs. Res. Cornmun., 112:284-
289 [1983]; Tilburn et al.,
Gene, 26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 8I : 1470-
1474 [1984]) and A. niger (Kelly
and Hynes, ME BO J., 4:475-479 [1985]): Methylatropic yeasts are suitable
herein and include, but are not
limited to, yeast capable of growth on methanol selected from the genera
consisting of Hanseraula, Culida,
Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotonda. A list of
specific species that are exemplary
of this class of yeasts may be found in C. Anthony, The Biochemistry of
Methylotronhs, 269 (1982).
Suitable host cells for the expression of glycosylated PRO are derived from
multicellular organisms.
Examples of invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf.9, as well as plant
cells. Examples of useful mammalian host cell lines include Chinese hamster
ovary (CHO) and COS cells.
More specific examples include monkey kidney CV1 line transformed by SV40 (COS-
7, ATCC CRL I651);
human embryonic kidney line (293 or 293 cells subcloned for growth in
suspension culture, Graham et al., J.
Gen Virol., 36:59 (1977)); Chinese hamster ovary cellsl-DHFR (CHO, Urlaub and
Chasin, Proc. Natl. Acad.
Sci. SA, 77:4216 ( 1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,
23:243-251 (1980)); human lung
cells.(Wi38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
mammary tumor (MMT
060562, ATCC CCL51). The selection of the appropriate host cell is deemed to
be within the skill in the art.
3. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into
a replicable vector
for cloning (amplifacatiota of he DNA) or for expression. Various vectors are
publicly available. ~e vector
35' may, for example, be in the form of a plasmid, cosmid, viral particle, or
phage. The appropriate nucleic acid
sequence may be inserted into the vector by a variety of procedures. In
general, DNA is inserted into an
appropriate restriction endonuclease sites) using techniques known in the art.
Vector-components generally
CA 02481756 2004-10-25
WO 01/16318 PCT/US00123328
include, but arernot limited to, one or more of a signal sequence, an origin
of replication, one or more marker
genes, an enhancer element, a promoter, and a transcription termination
sequence. Construction of suitable
vectors containing one or more of these components employs standard ligation
techniques which are known to
the skilled artisan.
The PRO may be produced recombinantiy not only directly, but also as a fusion
pol;ypeptide with a
heterologous polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site
at the N-terminus of the mature protein or polypeptide. In general, the signal
sequence may be a component of
'the vector, or it may be a part of the PRO-encoding DNA that is inserted into
the vector. _The signal sequence
may be a prokaryotic signal sequence selected, for example, from the group of
the alkaline phosphatase,
penicillinase, lpp, or heat-stable enterotoxin lI leaders. For yeast secretion
the signal sequence may be, e.g.,
the yeast invertase leader, alpha factor leader (including Saccharamyces and
Kluyveromyces «-factor leaders,
the latter described in U.S. Patent. No. 5,010,182), or acid phosphatase
leader, the C. albicans glucoamylase
leader (EP 362,179 published 4 April 199b), or the signal described in WO
90113646 published 15 November
1990. In mammalian cell expression, mammalian signal sequences may be used to
direct secretion of the
protein, such as signal sequences from secreted polypeptides of the same or
related species, as well as viral
I S secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate
in one or more selected host cells. Such sequences are well known for a
variety of bacteria, yeast, and viruses.
The origin of replication from floe plasmid pBR322 is suitable for most Gram-
negative bacteria; the:2~c plasmid..
origin is suitable for yeast, and various viral origins (SV40, polyoma,
adenovirus, VSV or BPS) are-usefui.for
cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, e.g., the gene encoding D-alanine racemase
for Bacilli.
An example of suitable selectable markers for mammalian cells are those that
enable the identification
of cells competent to take up the PRO-encoding nucleic acid, such as DHFR or
thymidirte kinase. An
appropriate host cell when wild-type DHFR is employed is the CHO cell line
deficient in DHFR activity,
prepared and propagated as described by Urlaub et al., Proc. Nati. Acad. Sci.
USA, 77:4216 (1980). A suitable
selection gene for use in yeast is the trill gene present in the yeast piasmid
YRp7 [Stinchcomb et al., Nature,
282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene,
10:157 (1980)]. The trill gene
provides a selection marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example,
ATCC No. 44076 or PEP4-1 [dories, Genetics, 85:12 (19?7)J.
Expression and cloning vectors usually contain a promoter operably linked to
the PRO-encoding nucleic
acid sequence. to direct mRNA synthesis. Promoters recogntzerl by a variety
_of potential host~cells are .well
known. Promoters suitable for nse with prokaryotic hosts include the (3-
lactamase and lactose promoter systems
[Chang et ai., ature, 275:615 (1978); Goeddel et al., Nature, 281:544
(1979)J,'alkaline phosphatase, a
tryptophan (trp).::.promoter systerra [Goeddel, Nucleic Acids Res., 8:4057
(1980); EP 36,776], and hybrid
56
._.,». _ .,_..
. ____, ,.... ... -.,.. .... r ,T~as.u,-~a~~r~rr,.;~m., , ,~myy.awcz?~ "
..~.~.,r ~,a»...-- _ . ._._..,_,..__.Z........ _ - _..
CA 02481756 2004-10-25
wo .oinms ~ _ PCTmsoor~zs
_ __ promoters such as the tac promoter [deBoer et al., Proc. Natl. Aca~ Sci.
USA; 80:21-25 (1983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding PRO.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-
phosphoglycerate kinase [Hitzeman et aL, J. Biol. Chem., 255:2073 (1980)) or
other glycolytic enzymes [Hess
S et al., J. Adv. Enzyme Reg_, 7:149 (1968); Holland, Biochemistry, 17:4900
(I978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-
6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomer_ase, phosphoglucose
isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription
controlled by growth conditions, are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism,
metallothionein, glyceraldehyde-3-
phosphate dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP 73,657.
PRO transcription from vectors in mammalian host cells is controlled, for
example, by promoters .
1S obtained from the genomes of viruses such as polyoma virus, fowlpox virus
(UK 2;211,504 published 5 July
1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous
mammalian promoters, e.g.,, the
actin promoter or an immunoglobulin promoter, and from heat-shock promoters,
provided such promoters are
compatible with the host cell systems:
Transcription of a DNA encoding the PRO by higher eukaryotes may be increased
by inserting an
enhancer sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 3(?0
bp, that act on a promoter to incc~ease its transcription. Many enhancer
sequences are now laxown from
mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin).
Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on
the late side of the replication
2S origin (bp 100-270), the cytomegalovirus early promoter enhancer, the
polyoma enhancer on the late side of the
replication origin, and adenovirus enhancers. The enhancer may be spliced into
the vector at a position 5' or
3' to the PRO coding sequence, but is preferably located at a site S' from the
promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated
cells from other multicellular organisms) will also contain sequences
necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly
available fronn the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated portion
of the mRNA encoding PRO.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PRO in
recombinant vertebrate cell culture are described in Gething
etaL:ature,_293:620-625,(1981);:~vlanteietal.,:
3S Nature, 281:40-4b (1979); EP i 17,060; and EP 117,058.
S7
CA 02481756 2004-10-25
WO 01!16318 PCT/U'S00/2332>$
4.. Detecting C°~ne A_rnolifieationlEomcession
Gene amplification andlor expression may be measurai in a sample directly, for
example, by
conventional Southern blotting, Northern blotting to quantitatc the
transcription of mRNA [Thomas, Pros. Nati,
cad. Sci. USA, 77:5201-5205 (1980)]; dot blotting (DNA analysis), or in situ
hybridization, usimg an
appropriately labeled probe, based onthe sequences.provided herein.
Alternatively, antibodies may be employed
that can recognize specific duplexes, including DNA duplexes, RNA duplexes,
and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and the assay
may be carried out where the
duplex is bound to a surface, so that upon the formation of duplex on the
surface, the presence of antibody bound
to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as
I O immunohistochemical staining of cells or tissue sections and assay of cell
culture or body fluids, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining andlor assay of
sample fluids may be either monoclonal or polyclonal, and may be prepared in
any mammal. Conveniently, the
antibodies may be prepared against a native sequence PRO polypeptide or
against a synthetic peptide based on
the DNA sequences provided herein or against exogenous sequence fused to PRO
DNA and encoding a specific
IS antibody epitope.
S. Purification of Polypeptide
Forms of PRO may be recovered from culture. medium or from host cell lysates.
If membrane-botind'
it can be released from the membrane using a suitable detergent solution (e.g.
Triton-X 100) or by enzymatic
20 cleavage. Cells employed in expression of PRO can be disrupted by various
physical or chemical means, such
as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing
agents.
It may be desired to purify PRO front recombinant cell proteins or
polypeptides. The following
procedures are exemplary of suitable purification procedures: by fractionation
on an ion-exchange column;
ethanol precipitation; reverse phase. HPLC; chromatography oa silica or an a
ration-exchange resin sus;lt as
2S DEAF; chromatofocusing; SDS-PAGE;, ammonium. sulfate precipitation; gel
filtration using, for example,
Sephadex G-7S; protein A Sepharose columns to remove contaminants such as IgG;
and metal chdlatittg columns
to bind epitope-tagged forms of the PRO. Various methods of protein
purification may be employed and such
methods are known in the art and described for example in Deutscher, ~Mgthods
in Enz~ology, 182 (1990);
Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New
York (1982). The purifteation
30 steps) selected will depend, for example, on the nature of the production
process used and the particular PRO
produced.
E. Uses for~RO
Nucleotide sequences (or thdit ~ lenient) encadmg P-RO. have various
aPPlications in.-the art ~~- - -
...-- : ._ .- ~ _ : . . _ _ .._ . ,
35 molecular biology, including uses as hybridization probes, in cl~rotnosome
and gene mapping and in the
generation of anti-sense RNA and DNA. PRO nucleic acid will also be useful for
the preparation of PRO
polypeptides by the recombinant techniques described herein.
S8
*-trademark
,~..m__ ."~ .<~..M . . .,. .Hw~._~. .~s- .. w M, ..,..w~?,a.»."~..~~.~.,~.-
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...... s._.._... ......... . . ....... . _....... ...... _....... ....._....
_...
CA 02481756 2004-10-25
W0 01/16318 . PCf/US00/23328
The full-length native sequence PRO gene; or portions thereof, may be used as
hybridization probes
for a cDNA library to isolate the full-length PRO cDNA or to isolate still
other cDNAs (for instance, those
encoding naturally-occurring variants of PRO or PRO from other species) which
have a desired sequence identity
to the native PRO sequence disclosed herein. Optionally, the length of the
probes. will be about 20 to about 50
bases. The hybridization probes may be derived from at least partially novel
regions of the full length native
nucleotide sequence wherein those regions may be determined without undue
experimentation or from genomic
sequences including promoters, enhancer elements and introns of native
sequence PRO. By way of example,
a screening method will comprise isolating the coding region of the PRO gene
using the known DNA sequence
to synthesize a selected probe of about 40 bases. Hybridization probes may be
labeled by a variety of labels,
including radionucleotides such as'ZP or'$S, or enzymatic labels such as
alkaline phosphatase coupled to the
probe via avidinibiotin coupling systems. Labeled probes having a sequence
complementary to that of the PRO
gene of the present invention can be used to screen libraries of human cDNA,
genomic DNA or mRNA to
determine which members of such libraries the probe hybridizes to.
Hybridization techniques are described in
further detail in the Examples below.
Any EST sequences disclosed in the present application may similarly be
employed as probes, using
the methods disclosed herein.
Other useful fragments of the PRO nucleic acids include antisense or sense
oligonucleotides comprising
a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding
to target PRO mRNA-(sense)
or PRO DNA (antisense} sequences. Antisense or sense oligonucleotides,
according to the present invention,
comprise a fragment of the coding region of PRO DNA. .Such a fragment
generally comprises at least about' I4
nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive
an antisense or a sense
oligonucleotide, based upon a cDNA sequence encoding a given protein is
described in, for example, Stein and
Cohen (Cancer Res. 48:2659, 1988) and van tier l~rol et al. (BioTechnic~ues
6:958, 1988).
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation
of duplexes that block transcription or aanslation of the target sequence by
one of several means, including
enhanced degradation of the duplexes, premature termination of transcription
or translation, or by other means.
The antisense oligonucleotides thus may be used to block expression of PRO
proteins. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified sugar-
phosphodiester backbones (or other
sugar linkages, such as those described in WO 91/06629) and wherein such sugar
linkages are resistant to
endogenous nucleases. Such oligonucleotides with resistant sugar linkages are
stable in vivo (i.e.; capable of
resisting enzymatic degradation) but retain sequence specificity to be able to
bind to target nucleotide sequences.
Other examples of sense or antisense oligonucleotides include those
oligonucleotides which are
covaiently linked to organic moieties, such as those described in WO 90/
10048, and other moieties that increases
affinity of the oligonucleotide for a target nucleic acid sequence, such as
poly-(L-lysine}. Further still,
intercalating agents, such as ellipticine, and alkylating agents or metal
complexes may be attached to sense or
antisense oligonucleotides to modify binding specificities of the antisense or
sense oligonucleotide for the target
nucleotide sequence.
59
CA 02481756 2004-10-25
WO 01/16318- PCT/US00123328
Antisense or sense oligonucieotides may be introduced into a cell containing
the targ~ nucleic acid
sequence by any gene transfer method, including, for example, CaPO,-mediated
DNA transfection,
electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
In a preferred procedure, an
antisense or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic
acid sequence is contacted with the recombinant retroviral vector, either aet
vivo or ex vivo. Suitable retroviral
vectors include, but are not limited to, those derived from the marine
retrovirus M-MuLV, N2 (a retrovirus
derived from M-MuLV), or the double copy vectors designated DCTSA; DCTSB and
DCTSC (see WO
90113641).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide
sequence by formation of a conjugate with a Iigand binding molecule, as
described in WO 91104753. Suitable
Iigand binding molecules include, but are not limited to, cell surface
receptors, growth factors, other cytokines,
or other ligands that bind to cell surface receptors. Preferably, conjugation
of the ligand binding molecule does
not substantially interfere with the ability of the ligand binding molecule to
bind to its corresponding molecule
or receptor, or block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
Alternatively, a sense or an antisense oIigonucieotide may be introduced into
a cell containing the target
IS nucleic acid sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The
sense or antisense oligonucleotide-lipid complex is preferably dissociated
within the cell by an endogenous lipase.
Antisense or~sense RNA or DNA molecules are generally at lease about 5 bases
in length, about 10
bases in Length, about IS bases in length, about 20 bases in length, about 25
bases in length, about 30 bases in
length, about 35 bases in length; about 40 bases in length, about 4S bases in
length, about SO bases ire length;
about 55 teases in length, about 60 bases in length, about 65 bases in length,
about 70 bases in length, about 75
bases in length, about 80 bases in length, about 8S bases in length, about 90
bases in length, about 95 bases in
length, about 100 bases in length, or more.
The probes may also be employed in PCR techniques to generate a pool of
sequences for' identification
of closely related PRO coding sequences.
Nucleotide sequences encoding a FRO can also be used to construct
hybridization probes for mapping
the gene which encodes that PRO and for the genetic analysis of individuals
with genetic disorders. 'The
nucleotide sequences provided herein may be mapped to a chromosome and
specific regions of a chromosome
using known techniques, such as an situ hybridization, linkage analysis
against known chromosomal markers,
and hybridization screening with libraries.
When the coding sequences for FRO encode a protein which binds to another
protein (example, where
the PRO is a receptor), the PRO can be used in assays to identify the other
proteins or molecules involved in
the binding interaction. By such methods, inhibitors of the receptorlligand
binding interaction can be identified.
Proteins involved in such binding interactions can also be used to screen for
peptide or small molecule inhibitors
of agonists of the binding interaction. Also, the receptor PR0 can be used to
isolate correlative ligand{s):.
Screening assays can be designed to find lead compounds that mimic the
biological activity of a native PRO or
a receptor for PRO. Such screening assays will include assays amenable to high-
throughput screening of
chemical libraries, making them particularly suitable for identifying small
molecule drug candidates. Small
CA 02481756 2004-10-25
wo .omns pc-rn~rsoors3~z$
molecules contemplated include synthetic organic or inorganic compounds. The
assays can be performed in a
variety of formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and
cell based assays, which are well characterized in the art.
Nucleic acids which encode PRO or its modified forms can also be used to
generate either transgenic
animals or "knock out" animals which, in turn, are useful in the development
and screening of therapeutically
useful reagents. A uansgenic animal (e.g., a mouse or rat} is an animal having
cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of the animal at
a prenatal, e.g., an embryonic
stage. A transgene is a DNA which is integrated into the genome of a cell from
which a transgenic animal
develops. In one embodiment, cDNA encoding PRO can be used to clone genomic
DNA encoding PRO in
accordance with established techniques and the genomic sequences used to
generate transgenic animals that
10- contain cells which express DNA encoding PRO. Methods for generating
transgenic animals, particullarly
animals such as mice or rats, have become conventional in the art and are
described, for example, in U.S. Patent
Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted
for PRO transgene incorporation
with tissue-specific enhancers. Transgenic animals that include a copy of a
transgene encoding PRO introduced
into the germ line of the animal' at an embryonic stage can be used to examine
the effect of increased expression
of DNA encoding PRO. Such animals can be used as tester animals for reagents
thought to confer protection
from, for example, pathological conditions associated.with its overexpressian.
In accordance with this facet of
the invention, an animal is treated with the reagent and a reduced incidence
of the pathological condition,
compared to untreated animals bearing the transgene, would indicate a
potential therapeutic intervention for the
pathological condition.
Alternatively, non-human homologues of PRO can be used to construct a PRO
"lanock out" animal
which has a defective or altered gene encoding PRO as a result of homologous
recombination between the
endogenous gene encoding PRO and altered genomic DNA encoding PRO introduced
into an embryonic stem
cell of the animal: For example, cDNA encoding PRO can be used to clone
genomic DNA encoding PRO in
accordance with established techniques. A portion of the genomic DNA encoding
PRO can be deleted or
replaced with another gene, such. as a gene encoding a selectable marker.
which can be used to monitor
integration. Typically, several kilobases of unaltered flanking DNA (both at
the 5' and 3 ° ends) are included
in the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a
description of homologous
recombination vectors]. The vector is introduced into an embryonic stem cell
line (e.g., -by elecunporation) and
cells in which the introduced DNA has homologously recombined with the
endogenous DNA are selected [see
e.g., Li et al., Cgil_, 69:915 (1992)j. The selected cells are then injected
into a blastocyse of an animal (e.g.,
a mouse or rat) to form aggregation chimeras (see e.g., Bradley, in
Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-
152j. A chimeric embryo can
then be implanted into a suitable pseudopregnant: female foster animal and the
embryo brought to term to create
a "knock_out"- animal Piogeny harbo ' ' the:hQmolo oust recombined DNA tn-
_their. erm cells can-be
- - . : : . ,~,;... .-:- ~' , . . . g____. _.__. _ .
- --W
identified by standard techniques and used to breed animals in which all eetIs
of the animal contain the
homologously recombined DNA. Knockout animals can be characterized for
instance, for their ability to defend
against certain pathological conditions and for their development of
pathological conditions due to absence of
61
CA 02481756 2004-10-25
wo -ol~ns~>i8 pcr~,rsoor~32s
the PRO polypeptide.
Nucleic acid encoding the PRO polypeptides may also be used in gene therapy.
In gene therapy
applications, genes are introduced into cells in order to achieve in vivo
synthesis of a therapeutically effective
genetic product, for example for replacement of a defective gene. "Gene
therapy" includes both conventional
gene therapy where a lasting effect is achieved by a single treatment, and the
administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for blocking the
expression of certain genes in
vivo. It has already been shown that short antisense oligonucleotides can be
imported into cells where they act
as inhibitors, despite their low intracellular concentrations caused by their
restricted uptake by the cell
membrane. (Zamecnik et al., Proc. Natl: Acad. Sci. USA 83:4143-4146 [1986]).
The oiigonucleotides can be
modified to enhance their uptake, e.g. by substituting their negatively
charged phosphodiester groups by
uncharged groups.
There are a variety of techniques available for introducing nucleic acids into
viable cells. The
techniques vary depending upon whether the nucleic acid is transferred into
cultured cells in viaro, or in vivo in
the cells of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro
include the use of Iiposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene transfer
techniques include transfection with viral
(typically retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in
Biotechnolosv 11, 205-210 [1993]). In some situations it is desirable to
provide the nucleic acid source with
an agent that targets the target cells, such as an antibody specific for a
cell surface membrane protein or the
target cell, a ligand for a receptor on the target cell, etc. Where liposomes
are employed, proteins which lbind
to a cell surface membrane protein associated with endocytosis may be used for
targeting andlor to facilitate
uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell
type, antibodies for proteins which
undergo internalization in cycling, proteins that target intracellular
localization and enhance intracellular half life.
The technique of receptor-mediated endocytosis is described, for example, by
Wu et al., ,I. 13io1. Chem. 262,
4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene
marking and gene therapy protocols see Anderson et al., Science 256, 808-813
(1992).
The PRO polypeptides described herein may also be employed as molecular weight
markers for protein
electrophoresis purposes and the isolated nucleic acid sequences may be used
for recombinantly expressing those
markers.
The nucleic acid molecules encoding the PRO polypeptides or fragments thereof
described herein are
useful for chromosome identificatian. In this regard, there exists an ongoing
need to identify new chromosome
markers, since relatively few chromosome marking reagents, based upon actual
sequence data are presently
available. Each PRO nucleic acid molecule of the present invention can be used
as a chromosome marker.
The PRO polypeptides and nucleic acid molecules of the present invention may
:.also ~be used.
- dia nosticall for tissue r in I ' fides of the
g y typing, whe a the PRO po ypep present invention may be differentially'
expressed in one tissue as compared to another, preferably in a diseased
tissue as compared to a normal tissue
of the same tissue type. PRO nucleic acid molecules will fmd use for
generating probes for PCR, Northern
62
CA 02481756 2004-10-25
WO Oi/i6318 - PCTIUS00/23328 .
analysis, Southern analysis and Western analysis.
The PRO polypeptides described herein may also be employed as therapeutic
agents. The PRO
polypeptides of the present invention can be formulated according to known
methods to prepare pharmaceutically
useful compositions, whereby the PRO product hereof is combined in admixture
with a pharnnaceutically
acceptable carrier vehicle. Therapeutic formulations are prepared for storage
by mixing the active ingredient
S having the desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers
(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in
the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients or
stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as phosphate,
citrate and other organic acids;
antioxidants including ascorbic acid; low molecular weight (less than about 10
residues) polypeptides; proteins,
such as serum albumin, gelatin or itnmunoglobulins; hydrophilic polymers such
as polyvinylpyrrolidone, amino
acids such as glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides and other
carbohydrates including glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium; andJor nonionic
surfactants such as TWEEN~'~"'',
PLURONICST"' or PEG.
1~ The formulations to be used for in vivo administration must be sterile.
This is readily accomplished by
filtration through sterile filtration membranes, prior to or following
lyophilizaeion and reconstitution.
Therapeutic compositions herein generally are placed into a container having a
sterile access port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
The route of administration is in accord withknown methods, e.g. injection or
infusion by intravenous;
intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or
intralesional z~outes, topical
administration, or by sustained release systems.
Dosages and desired drug concenerations of pharmaceutical compositions of the
present inventiowmay
vary depending on the particular use envisioned. The determination of the
appropriate dosage or route of
administration is well within the skill of an ordinary physician. Animal
experiments provide reliable guidatace
for the determination of effective doses for human therapy. Interspecies
scaling of effecrive doses can be
performed following the principles laid down by Mordenti, J. and Chappell, W.
"The use of interspecies scaling
in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al.,
Eds., Pergamon Press, New
York 1989, pp. 42-96.
When in vivo administration of a PRO polypeptide or agonist or antagonist
thereof is employed, normal
dosage amounts may vary from about 10 nglkg to up to 100 rnglkg of mammal body
weight or more per day,
preferably about 1 ~,glkglday to 10 rngJkglday, depending upon the route of
administration. Guidance as to
particular dosages and methods of delivery is provided in the literature; see,
for example, U.S. Pat. Nos.
4,b57,?60; 5,206,344; or 5,225,212. It is anticipated that different
formulations will be effective for different
. treattrient compounds and different diso~rde~s, that administration
targeting one-organ or tissue; for. example, may; .
necessitate delivery in a manner different from that to another organ or
tissue.
Where sustained-release administration of a PRO polypeptide is desired in a
formulation with release
characteristics suitable for the treatment of any disease or disorder
requiring administrateon of the PRO
63
CA 02481756 2004-10-25
wo .oin631s _.p~y~~~32s
poiypeptide, microencapsulation.of the PRO polypeptide is contemplated.
Microencapsulation of recombinant
proteins for sustained release has been successfully performed with human
growth hormone (rhGI-I), interfeton-
(rhIFN- ), interleukin-2, and MN rgp120: Johnson et al., Nat. ed., 2:795-799
(1996); Yasuda, $io
Ther., 27:1221-1223 (1993); Hora et aL, BioITechnolosv. 8:755-?58 (1990);
Clcland, "Design and Paoduction
of Single Immunization Vaccines Using Polylactide Polygiycoiide Microsphere
Systems," in Vaccine Design:
The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New
York,1995), pp. 439-462;
WO 97103692, WO 96140072, WO 96107399; and U.S. Pat. No. 6,654,010.
The sustained-release formulations of these proteins were developed using poly-
lactic-eoglyoolic acid
(PLGA) polymer due to its biocompatibility and wide range of biodegradable
properties. The degradation
products of PLGA, lactic and glycoiic acids, can be cleared quickly within the
human body. Moreover, the
~10 degradability of this polymer can be adjusted from months to years
depending on its molecular weighs and
composition. Lewis, "Controlled release of bioactive agents from
lactide/glycolide polymer," in: M. Chasin
and R. Larger (F.ds.), )3iodegradable Polymers as Drug 'Delivery S, stems
(Marcel Dekker: New York, 1990),
pp. 1-41.
This invention encompasses methods of screening compounds to identify those
that mimic the PRO
poIypeptide (agonists) or prevent the effect of the PRO polypeptide
(antagonists). Screening assays for
antagonist drug candidates are designed to identify compounds that bind or
complex with the PRO polypeptides
encoded by the genes identified herein, or otherwise interfere with the
interaction of the encoded polypeptides
with other cellular proteins. Such screening assays will include assays
amenable to high-throughput screening
of chemicahlibraries, making there particularly suitable for identifying small
molecule drug candidates
The assays can be performed in a variety of formats, including protein-protein
binding assays,
biochemical screening assays, immunoassays, and cell-based assays, which are
well characterized in the art.
All assays for antagonists are common in that they call for contacting the
drug candidate with a PRO
polypeptide encoded by a nucleic acid identified herein under conditions and
for a time sufficient to allow these
two components to interact.
In binding assays, the -interaction is binding and the ~mplex formed can be
isolated or detected in the
reaction mixture. In a particular embodiment, the PRO polypeptide encoded by
the gene identified herein or the
drug candidate is immobilized on a solid phase, e.g., on a microtiter plate,
by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by coating the
solid surface with a solution of
the PRO polypeptide and drying. Alternatively, an immobilized antibody, e.g.,
a monoclonal antibody, specific
for the PRO polypeptide to be immobilized can be used to anchor it to a solid
surface. The assay is performed
by adding the non-immobilized component, which may be labeled by a detectable
Iabel, to the immobilized
component, e.g., the coated surface containing the anchored component. When
the reaction is complete,~the
non-reacted components are removed, e.g., by washing, and complexes anchored
on the solid surface are
detected. When the originally, non=imrrtobilize~;compprient carries a
detectable-label : the detection=of ~iabel -
_:;; .~ ' ~ . . -: _ -- ° : - -. . ~ _..~:- = '-:-: ,; : , .
immobtltzed on the surface indicates that coinplexing occurred. Wheire the
originally non-immobilized
component does not carry a label, complexing can be detected, for example, by
using a labeled antibody
specifically binding the immobilized complex.
64
CA 02481756 2004-10-25
WO 01116318 PCT/US00/23328 .
if the candidate compound interacts with bui does not bind to a particular PRO
poIypeptide encoded by
a gene identified herein, its interaction with that polypeptide can be assayed
by methods well known for detectizig
protein-protein interactions. Such assays include traditional approaches, such
as, e.g., cross-linking, co-
immunoprecipitation, and co-purification through gradients or chromatographic
columns. In addition, protein-
protein interactions can be monitored by using a yeast-based genetic system
described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et al., Proc.
Natl. Acad. Sci. USA, 88:9578-
9582 (1991)) as disclosed by Chevray and Nathans, roc. Natl. Acad. Sci: USA,
89: 5789-5793 (1991). Many
transcriptional activators, such as yeast GAL49 consist of two physically
discrete modular domains, one acting
as the DNA-binding domain, the other one functioning as the transcription-
activation domain. The yeast
expression system described in the foregoing publications (generally referred
ao as the "two hybrid system")
takes advantage of this property, and employs iwo hybrid proteins, one in
which the target protein is fused to
the DNA-binding domain of GAL4, and another, in which candidate activating
proteins are fused to the
activation domain. The expression of a GALL-IarZ reporter gene under control
of a GAL4-activated prompter
depends on reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting
polypeptides are detected with a chromogenic substrate for (3-galactosidase. A
complete kit
(MATCHMAKER''") for identifying protein-protein interactions between two
specific proteins using. the two-
hybrid technique is commercially available from Clontech. This system can also
be extended to map protein
domains involved in specific protein interactions as well as to pinpoint amino
acid residues that are crucial for
these interactions. _ ~ ,
Compounds that interfere with the interaction of a gene encoding a PRO
polypeptide identif~eil herEin
and other infra- or extracellular components can be tested as follows: usually
a reaction mixture is prepared
containing the product of the gene and the infra- or extracellular component
under conditions and for a time
allowing for the interaction and binding of the two products. To test the
ability of a candidate compound to
inhibit binding, the reaction is run in the absence and in the presence of the
test compound. In addition, a
placebo may be added to a third reaction mixture, to serve as positive conuol.
The binding (complex formation)
between the test compound and the infra- or extracellular component present in
the mixture is monitored as
described hereinabove. The formation of a complex in the control reactions)
but not in the reaction mixture
containing the test compound indicates that the test compound interferes with
the interaction of the test compound
and its reaction partner.
To assay for antagonists, the PRO polypeptide may be added to a cell along
with the compound to be
screened for a particular activity and the ability of the compound to inhibit
the activity of interest in the presence
of the PRO polypeptide indicates that the compound is an antagonist to the PRO
polypeptide. Alternatively,
antagonists may be detected by combining the PRO polypeptide and a potential
antagonist with membrane-bound
PRO polypeptide receptors or recombinant receptors under appropriate
conditions for a competitive.inhibition
assay. The PR0 polypeptide can be labeled; such as-by radioactivity, such that
they umber of PRO~p~lypepti~e : ~.
molecules bound to the receptor can be used to determine the effectiveness of
the potential antagonise: The gene
encoding the receptor can be identified by numerous methods known to those of
skill in the art, for example,
ligand panning and FACS sorting. Coiigan.et ai., ant Protocols in Immun.,
1(2): Chapter 5 (1991).
CA 02481756 2004-10-25
WO O1I16318 .PCT/US001~3328
Preferably, expression cloning is employed wherein polyadenylated RNA is
prepared from a cell responsive to
the PRO polypeptide and a cDNA library created from this RNA is divided into
pools and used to transfect COS
cells or other cells that are not responsive to the PRO polypeptide.
Transfeeted cells that are grown on glass
slides are exposed to labeled PRO polypeptide. The PRO polypeptide can be
labeled by a variety of arteans
including iodination or inclusion of a recognition site for a site=specific
protein kinase. Following fixation and
incubation, the slides are subjected to autoeadiographic analysis. Positive
pools are identified and sub-pools are
prepared and re-transfected using an interactive sub-pooling and re-screening
process, eventually yielding a
single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PRO
polypeptide can be photoaffinity-
Linked with cell membrane or extract preparations that express the receptor
molecule. Cross-linked material is
resolved by PAGE and exposed to X-ray film. The labeled complex containing the
receptor can be excised,
resolved into peptide fragments, and subjected to protein micro-sequencing.
The amino acid sequence obtained
from micro- sequencing would be used to design a set of degenerate
oligonucieotide probes to screen a cDNA
library to identify the gene encoding the putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor
would be incubated with labeled PRO polypeptide in the presence of the
candidate compound. The ability of
the compound to enhance or block this irueracdon could then be measured.
More specific examples of potential antagonists include an oligonucleoride
that binds to the fusions of
immunoglobulin.with PRO polypeptide, and, -in particular, antibodies
including, without>iimitation;.poIy-:and
monoclonal antibodies and antibody fragments, single-chain antibodies, anti-
idiotypic antibodies, and chittteric
or humanized versions of such antibodies or fragments, as well as human
antibodies and antibody fragments.
Alternatively, a potential antagonist may be a closely related protein, for
example, a mutated form of the PRO
polypeptide that recognizes the receptor but imparts no effect, thereby
competitively inhibiting the action of the
PRO -polypeptide.
Another potential PRO polypeptide antagonist is an andsense RNA or DNA
construct prepared using
antisei~se technology, where, e.g., an antisense RNA or DNA molecule acts to
block directly the translation of
mRNA by hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used
to control gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are
based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding
portion of the polynucleotide
sequence, which encodes the mature PRO polypeptides herein, is used to design
an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be
complementary to a region of the gene involved in transcription (triple helix -
see Lee et al., Nucl. Acids Res.,
6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et al., SS
ic'ence, 251:1360 (199I)), thereby
preventing, transcription and the production of the PRO polypeptide. The
antisense RNA oligonucleotide
hybridizes to the mRNA in wivo. and blocks translation .4~ the mRNA molecule
into the PRO polypeptide '. =
(anfisense - Okano, l~Ieurochem., 56:560 (1991); Olieodeoxynucleotides as
Andsense Inhibitors of C~,ene
Expression (CRC Press: l3oca Raton, FL, 1988). The oligonucleotides described
above can also be delivered
to cells such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of the PRO
6G
,.,._s ..,~v.q... _ . .,q,,.,~ . .ms n~.. p~ r m., aM.»_.~... r. ,.v.....
"....-.. .._ . ___..._ "..,. . N .. .~,..~-,~,"..,.~,,b,,ng .w."m.-..~..-
.._..___.....
CA 02481756 2004-10-25
wo .omu8 PCT/USO01Z3328
polypeptide. When antisense DNA is used; oliigodeoxyribottucleotides derived
from the translation-initiation site,
e.g., between about -IO and + IO positions of the target gene nucleotide
sequence, are greferred.
Potential antagonists include small molecules that bind to the active site,
the receptor binding site, or
growth factor or other relevant binding site of the PRO polypeptide, thereby
blocking the normal biological
activity of the PRO polypeptide. Examples of small molecules include, but are
not limited to, small peptides
or peptide-like molecules, preferably soluble peptides, and synthetic non-
peptidyl organic or inorganic
compounds.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA.
Ribozymes act by sequence-specific hybridization to the complementary target
RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential
RNA target can be identified by
known techniques. For furtherdetails see, e.g., Rossi, Current Biology; 4:469-
471 (1994), and PCT publication
No. WO 97133551 (published September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded
and composed of deoxynucIeotides. The base composition of these
oligonucleotides is designed such that it
promotes triple-helix formation via Hoogsteen base-pairing rules, which
generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further details see,
e.g., PCT publication No. WO
97!33551, supra.
These small molecules can be identified by any one or more of the screening
assays discussed
hereinabove andlor by any other screening techniques well known for those
skilled. in the art.
Diagnostic and therapeutic uses of the herein disclosed molecules may also be
based upon the positive
functional assay hits disclosed and described below.
F. Anti-PRO Antibodies
The present invention further provides anti-PRO antibodies. Exemplary
antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate antibodies.
1. Po~lonal Antibodies
The anti-PRO antibodies may comprise polyclonal antibodies. Methods of
preparing polyclonal
antibodies are known to the skilled artisan. Polyclonat antibodies can be
raised in a mammal, for example, by
one or more injections of an immunizing agent and, if desired, an adjuvant.
Typically, the immunizing agent
andlor adjuvant will be injected in the maternal by multiple subcutaneous or
intraperitoneal injections. The
immunizing agent may include the PRO polypeptide or a fusion protein thereof.
It may be useful to conjugate
the immunizing agent to a protein known to be immunogenic in the mammal being
immunized. Examples of
such immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine
th ro lvtiulin, and so tiean sm anhibrtor~.. les of:ad'uvanfs whi ma :be:em 1
~. ~' .g . ____ _~-~ u'~'g, ;~.,~_ ~ ..:::._ ..:J ,..,. , _ci~. :,y...__.
,.PaY~:~~c~u~eFreund;s.,
mr
5 co plete adjuvaiat and MPL-TDM ad~uvant (monophosptaoryl Lipid A, synthetic
trehalose dicorynocriycolate).
The immunization protocol may be selected by one skilled in the art without
undue experimentation.
67
_. .~ , .._ _.__ ~. , .fv...... __ __.. ._.____w~ mKm..."~"~~~w,.*~
be".,~.~~_. _.. .
CA 02481756 2004-10-25
WO O1/I6318 PGTIUS00lZ3328
2. wlonoclona~Antibodies
The anti-PRO antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be
prepared using hybridoma methods, such as those described by Kohler and
Milstein, Nature, 2~C5 :495 (19?5).
In a hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically
bind to the immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
The immunizing agent will typically include the PRO polypeptide or a fusion
protein thereof.
Generally, either.peripheral blood lymphocytes ("PBLs") are used if cells of
human origin are desired, or spleen
cells or lymph node cells are used if non-human mammalian sources are desired.
The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a
hybridoma cell [coding, ~,l~ionoclonal Antibodies: Prineples and Practice,
Academic Press, (I986) pp. 59-103].
Immortalized cell lines are usually transformed mammalian cells, particularly
myeloma cells of rodent, bovine
and human origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridorna cells may be
cultured in a suitable culture medium that preferably contains one or more
substances that inhibit the growth or
survival of the unfused, immortalized cells. For example; if the parental
cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will
include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances prevent the growth of
HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of-:
antibody by the selected antibody-producing cells, and are sensitive to a
medium such as HAT~mediutti~ lNfore.
preferred immortalized cell lines are marine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the
production of human monoclonal antibodies [Kozbor, J. Immunol.,133:3001 (
1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, Marvel Dekker, Inc., New
York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for.the presence of
monoclonal antibodies directed against PRO. Preferably, the binding
specificity of monoclonal antibodies
produced by the hybridoma cells is determined by immunoprecipitation or by an
in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such
techniques.and.assays are
Irnown in the art. The binding affinity of the monoclonal antibody can, for
example, be determined by the
Scatchard analysis of Munson and Pollard, ,~naI. Biochem., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution
procedures and grown by standard methods [coding, su ra . Suitable culture
media for this purpose include,
for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells
may be grown in vivo as ascites in a mammal. -
Tlie monoclonal antibodies secreted by the subclones fray be isolated -or'
purif=ied from theculture
medium or ascites fluid by conventional immunoglobulin purification procedures
such as, for example, protein
A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
68
, , :.....~n. ~. ,~ ~. ,.. ~- ~.._...mr- . ._ .._.~ ,~ . .~-....__ ... ., _
."n.,~.,_... .<,~,.~"-~ _...... . . .__~rc ..-..u,~.. ._.___.__.._._..
_._.._... .. ____
~..
CA 02481756 2004-10-25
WO OI/I6318 PCT/US00l23328 .
The-monoclonal antibodies may also be made by recombinant DNA methods, such as
those described
in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the
invention can be readily isolated
and sequenced using conventional procedures (e.g., by using oligonucleotide
probes that are capable of binding
specifically to genes encoding the heavy and light chains of marine
antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression
vectors, which are then transfected into host cells such as simian COS cells,
Chinese hamster ovary (CHO) cells,
or myeloma cells that do not otherwise produce immunoglobulin protein, to
obtain the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be modified, for
example, by substituting the
coding sequence for human heavy and light chain constant domains in place of
the homologous marine sequences
[U.S. Patent No. 4,816,567; Morrison et al., su ra or by covalently joining to
the immunoglobulin coding
1 O sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a non-immunoglobulin
polypeptide can be substituted for the constant domains of an antibody of the
invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody of the
invention to create a chimeric bivalent
antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well
IS known in the art. For example, one method involves recombinant expression
of immunoglobuiin tight chain and
modified heavy chain. The heavy chain is truncated generally at any point in
the Fc region so as to prevent
heavy chain crosslinking. Alternatively, the relevant cysteine residues are
substituted with another amino acid
residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to
20 produce fragments thereof, particularly, Fab fragments, can be accomplished
using routine techniques known
in the art.
3. Human and Humanized Antibodies
The anti-PRO antibodies of the invention may further comprise humanized
antibodies or human
25 antibodies. Humanized forms of non-human (e.g., marine) antibodies are
chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab°,
Flab°)Z or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human immunoglobulin.
Humanized antibodies include human immunoglobulins (recipient antibody) in
which residues from a
complementary determining region (CDR) of the recipient are replaced by
residues from a CDR of a non-human
30 species (donor antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In
some instances, Fv framework residues of the human immunoglobulin are replaced
by corresponding non-human
residues. Humanized antibodies may also comprise residues which are found
neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the humanized
antibody will comprise
substantially all of at least one, and typically two, variable domains, in
which all or substantially all.of the CDR
35 regions correspond to those of a non-human immunoglobulin and alll or
substantially all of the l~R regions are
those of a human immunoglobulin consensus sequence. The humanized antibody
optimally also will comprise
at least a portion of an immunoglobulin constant region (Fc), typically that
of a human immunogiobulin [Jones
69
CA 02481756 2004-10-25
wo oleW s
et al., aiure, 32:522-525 (1986); Riechmann et al., lure, X3_2:323-329 (1988);
and Presta, err. Qp_.
Struct. Biol., _2:593-596 (1992)].
Methods for humanizing non-human antibodies. are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human. These non-
human amino acid residues are often referred to as "import" residues, which
are typically taken from an "Import"
variable domain. Humanization cam be essentially performed following the
method of Vilinter and co-workers
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., tur , X32,:323-
327 (1988); Verhoeyen et al.;
Science, 239;1534-1536 (1988)], by substituting rodent CDRs or CDR sequences
for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Patent
No. 4,816,567), wherein substantially less than an intact human variable
domain has been substituted by the
corresponding sequence from a non human species. In practice, humanized
antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from
analogous sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage
display libraries [Hoogenboom and 'Winter, Mol. ,yiol., x:381 (1991); Marks et
al., I,~VIoI. Biol.. x.2:581
IS (1991)]. The techniques of Cole et al. and Boerner et al. are also
available for the preparation of human
monoclonal antibodies (Cole et al:, Monoclonal Antibodies and Cancer~herapy,
Alan R. Liss, p. 77 (1985) and
Boerner et al., J. Immunol., 147(1:86-95 (1991)]. Similarly, human antibodies
can be made by introducing
of human immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin
genes have been partially or completely inactivated. Upon challenge, human
antibody production-is observed;
which closely resembles that seen in humans in all respects, including gene
rearrangement, assembly, and
antibody repertoire. This approach is described, for example, in U.S. Patent
Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al.,
BioITechnoloQV I0, 779-783 (1992); Lonbergetal., Nature~68856-859(1994);
Morrison, ature368, 812-13
(1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger,
Nature BiotechnoloQV .~4, 826
(1996);. Lonberg and Huszar, Intern. Rev. immunol. i3 65-93 (1995).
The antibodies may also be affinity matured using known selection and/or
mutagenesis methods as
described above. Preferred affinity matured antibodies have an affinity~which
is five times, more preferably 10
times, even more preferably 20 or 30 times greater than the starting antibody
(generally murine, humanized or
human) from which the matured antibody is prepared.
4. Bispecific Antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding
speciftcities for at least two different antigens. In the present case, one of
the binding specificities is for the
PRO, the other one 'ts .for ariy .other antigen;: and preferably for a cell-
surface protein or receptor or receptor
35, , . subuntt-;~' :.:_:: , - .. . .:_ -: . -
Methods for making bispecific antibodies are.known in the art. Traditionally,
the recombinant
production of bispecific antibodies is based on the co-expressionof two
immunoglobulin heavy-chaiMighi-chain
CA 02481756 2004-10-25
wo oint>3is rcT~saorr~~
pairs, where the two heavy chains have different specificities [Milstein and
Cuello, atu , ~0 :537-539 (I983)].
Because of the random assortment of immunoglobuIin heavy and light chains,
these hybridomas (quadromas)
produce a potential mixture of ten different antibody molecules, of which only
one has the correct bispecific
structure. The purification of the correct molecule is usually accomplished by
affinity chromatography steps.
Similar procedures are disclosed in WO 93108829, published 13. May 1993, and
in Traunecker et al., ,~MBO
1., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
be fused to immunoglobulin constant domain sequences. The fusion preferably is
with an irnmunoglobulin
heavy-chain constant domain, comprising at least part of the hinge, CH2, and
CH3 regions. It is preferred to
have the first heavy-chain constant region (CHI) containing the site necessary
for fight-chain binding present in
at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain
fusions and, if desired, the
immunoglobuIin light chain, are inserted into separate expression vectors, and
are co-transfected into a suitable
host organism. For further details of generating bispecific antibodies see,
for example, Suresh et al., ethods
in Enzymologv, 21:210 (1986).
According to another approach described in WO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maximiie the percentage of heterodimers which
are recovered from recombinant
cell culture. The preferred interface comprises at least a part of the CH3
region of an antibody constant domain.
In this method, one or more small amino acid side chains from the interface of
the first antibody molecule are
repiaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory
"cavities" of identical or similar-
size to the Iarge side chains) are created on the interface of the second
antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or threoriine). This
provides a mechanism for increasing
the yield of the heterodimer over other unwanted end-products such as
homodimers.
Bispeciftc antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')Z
bispeciftc antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared can be prepared using chemical
linkage. Brennan et al. , Science 229:81 ( 1985) describe a procedure wherein
intact antibodies are proteolytically
cleaved to generate F(ab')2 fragments. These fragments are reduced in the
presence of the dithiol complexing
agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab'
fragments generated are then converted to thionitrobenzoate (TNB} derivatives.
One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an
equimolar amount of the other Fab'-TNB derivative to form the bispecific
antibody. The bispecific antibodies
produced can be used as agents for the selective immobilization of enzymes.
Fab' fragments may be dixectly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et cil., J. Ex~. Med. 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')i molecule, . Each Fab' fragment was
~eparatelyaecreted from.E. cola and subjected
to directed chemical cou tin in vitro to form
p g the bispecific antibody.. The bispecific antibody thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic
activity of human cytotoxic lymphocytes against human breast tumor targets.
71
CA 02481756 2004-10-25
wo .omn8 . rcrmsoor~3is
Various technique for making and isolating bispecific antibody fragments
directly from recombinant cell
culture have also been described. For example, bispeciftc antibodies have been
produced using leucine zippers.
Kostelny et al., J.. Immunol. I48(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun
proteins were linked to the Fab' poxtions of two different antibodies by gene
fusion. The antibody homodimers
were reduced at the hinge region to form monomers and then re-oxidized to form
the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)
has provided an alternative
mechanism for malting bispecif c, antibody fragments. The fragments comprise a
heavy-chain variable domain
(V,~ connected to a light-chain variable domain (V J by a linker which is too
short to allow pairing between the
two domains on the same chain. Accbrdingly; the VH and VL domains of one
fragment are forced to pair with
the complementary V, and V" domains of another fragment, thereby forming two
antigen-binding sites. Another
strategy for making bispecafic antibody fragments by the use of single-chain
Fv (sFv) dimers has also peen
reported. See, Gruber et al., J. Immunol. 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies canbe prepared.
Tuts et al., J. Immunol. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given
PRO polypeptide herein.
Alternatively, an anti-PRO polypeptide arm may be combined with an arm which
binds to a triggering molecule
on a.leukocyte such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or
B7), or Fc receptors for IgG
(FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcYRIII (CD 16) so as to focus
cellular defei~se.mechanisiris - =.:~ : ,- _
to the cell expressing the particular PRO polypeptide. Bispecific antibodies
may also be used to locailize
cytotoxic agents to cells which express a particular PRO poiypeptide. These
antibodies possess a PRO-binding
arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such
as EOTUBE, DPTA, DOTA,
or TETA. Another bispecific antibody of interest binds the PRO polypeptide and
further binds tissue factor
(TF).
5. Heteroconju~ate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate
antibodies are composed of two covalently joined antibodies. Such antibodies
have, for example, been proposed
to target immune system cells to unwanted veils [U.S. Patent No. 4,676,980],
and for creattrient of HIV inf~tion
[WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies
may be prepared in vitro
using known methods in synthetic protein chemistry, including those involving
crosslinking agents. For
example, immunotoxins may be constructed using a disulfide exchange reaction
or by forming a thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and
chose disclosed, for example, in U.S. Patent No. 4,676,980.
6. ~f~tor Function En ineerina .
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to
enhance, e.g., the effectiveness of the antibody in treating caper. For
example, cysteine residues) may be
72
,.~ . ~ .,., ,-..... _..._... _ _ .... ....,.~ a ~,.,4F_~ :.~ ~ ~~..w.._ ___.~
.~y.__..__..~.~. .~.H.~.x~...~_____.
.~ CA 02481756 2004-10-25
wo .oin63is . rc~rnrsoorr.~3zs
introduced into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability andJor increased complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See
Caron et al., J. Exp Med., 176:
1191-1195 (1992) and Shopes, J. Immunol.,.148: 2918-2922 (1992). Homodimeric
antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional cross-
linkers as described in Wolff e~t al.
S Cancer Research; 53: 2560-2565 ( 1993). Alternatively, an antibody can be
engineered that has dual Fc regions
and may thereby have enhanced complement lysis and ADCC capabilities. See
Stevenson et al., Anti-Cancer
Drue Design. 3: 219-230 (1989). _
7. Immunoconjugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent
such as a chemotherapeutic agent, toxin (e.g. , an enzymatically active toxin
of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such imrnunoconjugates
have been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aerugirtosa), ricin A chain, abrin A~chain,
modeccin A chain, alpha-sarcin, rlleurites fardii proteins, dianthin proteins,
Phytolaca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin,
sapaonaria o~cinalis inhibitor;
gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes. A variety of radionuclides are
available for the production of radioconjugated antibodies. Examples include
Z'ZBi, "'I, ."'In, 9°Y, and '~Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling
agents such as N.-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT)" bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters
(such as disuccinimidyl suberate),
aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-
diisocyanate), and bis-active fluorine cornpouizds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science, 238: 1098
(1987). Carbon-14-labeled 1-
isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid. (MX-DTPA) is
an exempiary chelating agent
for conjugation of radionucleotide to the antibody. See W094111026.
In another embodiment, the antibody may be conjugated to a "receptor" (such
streptavidin) for
utilization in tumor pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed
by removal of unbound conjugate from the circulation using a clearing agent
and then administration of a
"ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
' Y . ::,' ' _: 8 , Immunoliposonies_
The antibodies disclosed herein may also be formulatal as immunoliposornes.
Liposomes containing
the antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acid.
Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acid. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos.
73
CA 02481756 2004-10-25
wo .ams3ls . Pcr~saon33~s
4,485,045 and 4,544,545. Liposomes with enhanced circulation time, are
disclosed in U.S. Patent No.
5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-
PE). Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired diameter.
Fab' fragments of the antibody of the present invention can be conjugated to
the liposomes as described in Martin
et al ., J. Biol. Chem., ~: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent
(such as Doxorubicin) is optionally contained within the liposome. See Gabizon
etal., J. National Cancer Inst.,
81{19): 1484 (1989).
9. ~harmae:eutical Compositions of Antibodies
Antibodies specifically binding a PRO polypeptide identified herein, as well
as other molecules
identified by the screening assays disclosed hereinbefore, can be administered
for the treaanent of various
disorders in the form of pharmaceutical compositions.
If the PRO polypeptide is intracellular and whole antibodies are used as
inhibitors, internalizing
antibodies are preferred. I~lowever; lipofections or liposomes can also be
used to deliver the antibody, ,ar an
antibody fragment, into cells. ~ where antibody fragments are used, the
smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is preferred.
For example, based upon the variable-
region sequences of an antibody, peptide molecules can be designed that retain
the ability to bind thetarget
protein sequence. Such peptides can be synthesized chemically and/or produced
liy recombinant , DNA
technology. See e:g., Marasco et al., roc. Natl. Acad, Sci,LUSA, ,~0: 7889-
7893 (1993). The foma>>~an
herein may also contain more than one active compound as necessary for the
particular indication being treated,
preferably those with complementary activities that do not adversely affect
each other. Alternatively, or in
addition, the composition may comprise an agent that enhances -its function,
such as, for example, a cytot:oxic
agent, cytolcine, chemotherapeuric agent, or growth-inhibitory agent. Such
molecules are suitably present in
combination in amounts that are effective for the purpose intended.
The active ingredients may also be entrapped in microcapsutes prepared, for
example, by coacervation
techniques or. by interfacial polymerization, for example,
hydmxymethylcellulose or gelatin-microcapsules and
poly-(tnethylmethacylate) microcapsules, respectively, in colloidal drug
delive -ry systetns (for- example,
liposomes, albumin microspheres, microemulsions, nano-particles, and
nanocapsules) or in macroemulsions.
Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.
The formulations to be used for in vivo administration must be sterile. This
is readily accomplished by
filtration through sterile filtration rnembianes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipetmeable matiices of solid .h dro tiobic i" ers coritainiti - the
atitibod whicfi matrices are iii
-,:._. Y: P ;,._: Pq:fm. _. $_ .~: , Y': . ,_:_ .._
the form of shaped articles, e.g.; films, or micz'ocapsules. F.,xamples of
sustained=release inatrices include
polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), potylaetides (U.S.
Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate,
non-degradable ethylene-vinyl
74
CA 02481756 2004-10-25
'WO'O11I6318 , PGT/US00/23328
acetate, degradable lactic acid~glycolic acid copolymers such as the LUPRON
DEPOT "'' (injectable
microspheres composed of lactic acid-glycolic acid copolymer and leuprolide
acetate), and poly-IJ-(-)-3-
hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins for shorter
time periods. When encapsulated
antibodies remain in the body for, a longtime, they may denature or aggregate
as a result of exposure to moisture
at 37°C, resulting in a loss of biological activity and possible
changes in immunogenicity. Rational strategies
can be devised for stabilization .depending on the mechanism involved. For
example, if the aggregarion
mechanism is discovered to be intermodecular S-S bond formation through thio-
disulfide interchange, stabilization
may be achieved by modifying sulthydryl residues, lyophilizing from acidic
solutions, controlling maisture
content, using appropriate additives, and developing specifac polymer matrix
compositions.
G. Uses for anti-PRO Antibodies
The anti-PRO antibodies of the invention have various utilities. For example,
anti-PRO antibodies may
be used in diagnostic assays for PRO, e.g., detecting its expression (and in
some cases, differential expression)
in specific cells, tissues, or serum. Various diagnostic assay techniques
known in the art may be used, such as
competitive binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in
either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A
Manual of Techniques, CRC
Press, Inc. (1987) pp. 147-158J. The antibodies used in the diagnostic assays
can be labeled with a detectable
moiety. The detectable moiety should be capable of producing; either directly
or indirectly, a detectable signal.
For example, the detectable moiety may be a radioisotope, such as'H, "C, 32P'
ass' yr "~I, a fluorescent or
chemiluminescent compound, such as ffuorescein isothiocyanate, rhadamine, or
luciferin, or an enzyme, such
as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any
method known in the art for
conjugating the antibody to the detectable moiety may be employed" including
those methods described by Hunter
et al.; Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974);
Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J, Flistochem. and Cytochem., 30:407 (1982).
BS Anti-PRO antibodies also are useful for the affinity purification of PRO
from recombinant cell culture
or natural sources. In this process, the antibodies against PRO are
immobilized on a suitable support, such a
Sephadex resin or falter paper; using methods well known in the art. The
immobilized antibody then is contacted
with a sample containing the PRO to be purified, and thereafter the support is
washed with a suitable solvent that
will remove substantially all the material in the sample except the PRO, which
is bound to the immobilized
antibody. Finally, the support is washed with another suitable solvent that
will release the PRO from the
antibody.
The following examples are offered for illustrative purposes only, and are not
intended to limit the scope
of the present invention in any way.
Ah patent and literature references cited in the present specification are
hereby incorporated by reference
in their entirety.
CA 02481756 2004-10-25
WO OI1163i8 PCT/ITSOOIZ3328
EXAMPLES
_ _
_ ... ~.._
Commercially available reagents referred to in the examples were used
according to~tiianufacturer's
insuuctions unless otherwise indicated. The source of those cells identified
in the following examples, and
throughout the specification, by ATCC accession numbers is the American Type
Culture Collection, Manassas,
VA.
EXAMPLE 1: Extracellular Domain Homology_Screening to Identify-Novel
Folypentides and cDNA Encoding
Therefor
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public databases (e.g., Dayhoff, GettBank), and proprietary
databases (e.g. LIFESE()'~"'',
Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the
computer program BLAST or
BLAST-2 (Altschul et al., Methods in Enz rmologY 266:460-480(1996)) as a
comparison of the ECD protein
sequences to a 6 frame translation of the EST sequences. Those comparisons
with a BLAST score of 70 (or in
some cases 90) or greater that did not encode known proteins were clustered
and assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, WA).
Using this extracellular domain homology screen, consensus DNA sequences were
assembled relative
to the other identified EST sequences using phrap. In addition, the consensus
DNA sequences obtained were
often (but not always) extended using repeated cycles of BLAST or BLAST-2 and
phrag to extend the consensus
sequence as far as possible using the sources of EST sequences discussed
above.
Based upon the consensus sequences obtained as described above,
oligonucleotides were then
synthesized and used to identify by PCR a cDNA library that contained the
sequence of interest and for use as
probes to isolate a clone of the full-length coding sequence for a PRG
polypeptide. Forward and reverse PCR
primers gezierally range from 20 to 30 nucleotides and are often designed to
give a PCR product of about 100-
1000 by in length. The probe sequences are typically 40-55 by in length. In
some cases, additional
oligonucleotides are synthesized when the consensus sequence is greater than
about I-l.5kbp. In order to screen
several libraries for a full-length clone, DNA from the libraries was screened
by PCR amplification, as per
Ausubei et al., current Pr~ocols in Molecular Biology, with the PCR primer
pair. A positive library was then
used to isolate clones encoding the gene of interest using the probe
oligonucleotide and one of the primer pairs.
The cDNA libraries used to isolate the cDNA clones were constructed by
standard methods using
commercially available reagents such as those from Invitrogen, San Diego, CA.
The cDNA was primed with
oligo dT containing a NotI site, linked with blunt to SalI hemikinased
adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined orientation into
a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the SfiI
site; see, Holmes et al., Science,
25 :1278-1280 (1991)) in tlxe unique Xhol and NotI sites:
_ . _ . :: .;. ..;r a :_ _
76
CA 02481756 2004-10-25
wo oyns~ts . rcr~rsoon33za
._... ~XAMPLR-2: ~sQlation of cDNA clones~,y Amv.[gste iaQ _ . _ . ,~
I. Preparation of oligo dT nricned cDNA tibrary
mRNA was isolated from a'human tissue of interest using reagents and protocols
fmm Invitrogen, San
Diego, CA (Fast Track 2). This RNA was used to generate an oliga dT primed
eDNA library in the vector
pRKSD using reagents and protocols from Life Technologies; Gaithersburg, MD
(Super Script Plasmid System).
In this procedure, the double stranded cDNA was sized to greater than 1000 by
and the SaII/Notl tinkered cDNA
was cloned into XhoIlNotI cleaved vector. pRKSD is a cloning vector that has
an sp6 transcription initiation
site followed by an SfiI restriction enzyme site preceding the XhoIPNotI cDNA
cloning"sites. ,
2. Prevaration of randomprimed cDNA library
A secondary cDNA library was generated in order to preferentially represent
the 5' ends of the primary
cpNA clones. Sp6 RNA was generated from the primary library (described above),
and this RNA was used
to generate a random primed cDNA library in the vector ASST AMY.O using
reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). in this
procedure the double stranded cDNA
was sized to 500-1000 bp, Iinkered with blunt to Noti adapters, cleaved with
$fiI, and cloned into SfiItNotI
cleaved vector. pSST-AMY.O is a cloning vector that has a yeast alcohol
dehydrogenase promoter preceding
the cDNA cloning. sites and the mouse amylase sequence (the mature sequence
without the secretion signal)
followed by the yeast alcohol dehydeagenase terminator, after the cloning
sites. Thus, cDNAs cloned into this
vector that are fused in frame with amylase sequence-will lead to the
secretion of amylase from appmpiiately
transfected yeast colonies. -
3. Transformation and Detection
DNA from the library described in paragraph 2 above was chilled on ice to
which was added
electrocompetent DliIOB bacteria (Life Technologies, ZO mI). The bacteria and
vector mixture .was then
electroporated as recommended by the manufacturer. Subsequently, SOC media
(Life Technologies, I ml) was
added and the mixture was incubated at 37°C for 30 minutes. The
transformattts were then plated onto 20
standard 150 mm LB plates containing ampicillin and incubated for 16 hours
(37°C). Positive colonies were
scraped off the plates and the DNA was isolated from the bacterial-pelIet
using 'standard protocols, e.g. CsCI-
gradient. The purified DNA was then carried ~on to the yeast protocols below.
.
The yeast methods were divided into three categories: (1) Transformation of
yeast with the
plasmidIcDNA combined vector; (2) Detection and isolation of yeast clones
secreting amylase; and (3) PCR
amplification of the insert directly from the yeast colony and purification of
the DNA for sequencing and further
analysis.
The yeast strain used was fiD55-SA (A'fCC-90785). This strain has the
following:; genotype:- FIAT
a ~ ,,. .
aigha, ura3.~5~,.1eu2 3~. teu~i 12; .his3-Il,.his3-I5;-MALr , SUC , GAL .
Preferably; yeast mutant'svcan-be
employed that have deficient post-transtational pathways. Such mutants may
have translocation deficient alleles
in sec7l, sec72, sec62, with truncated sec71 being most poeferred.
Alternatively, antagonists (including
antisense nucleotides andlor ligands) which interfere with the normal
operation of these genes, other proteins r
*-trademark
CA 02481756 2004-10-25
wo o1n631s pc~ricrsoor
_ __., impl~ated in this poi translation pathway (e.g.; SEC6Ip, SEC72p,
~SEC62p, SEC63p, TD7lp or SSAlp~4p~ ° - _ .
or the complex fomiatiowof these proteins may also be preferably employed in
combination with the.amylase-
expressing yeast.
Transformation was performed based on the protocol outlined by Gietz et al.,
Nucl. Acid. Res ;
x:1425 (1992). . Transformed cells were then inoculated from agar into YEPD
complex media broth (I00 ml)
and grown overnight at 30°C. The YEPD broth was prepared as described
in Kaiser et al., Methods in Yeast
Gene ics, Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994): The
overnight culture was then
dilueed to about 2 x 106 cells/anI (approx. ODD=O.I) into fresh YEPD broth
(500 ml) end regrown to 1 x 10'
cellsiml (approx. ODD=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into
GS3 rotor botdes in a
IO Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water,
and centrifuged again in 50 ml falcon tutees at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant
was discarded and the cells were subsequently washed with LiAcITE (10 ml, IO
mM Tris-HC:I,, I mM EDTA
pH 7.5, 100 mM Lii00CCH~, and resuspended into LiAcITE (2.5 m1).
Transformation took place by mixing the prepared cells ( 100 Eel) with freshly
denattued single stranded
salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (i
~cg, vol. < 10 ~1) in
microfuge tubes. The mixture was mixed briefly by vortexiag, then 40 '~
PEGIT'E (600 ~d, 44 Yo polyethylene
glycol-4000, 10 -mM Tris-HCI, 1 mM EDTA, 100 mM Lii00CCHs, pH 7.5) was added.
This mixture was
gently-mixed. and incubated at 30°C white agitating for 30 minutes:
The:cells werenhen heat-- hocluedat'42°C
for,TS rttinutes, and the reaction vessel centrifuged in a microfuge at 12,000
rpm for 5-l0 seconds, decants and
resuspended into TE (S00 N.1,10 mM Tris-HCI, I mM EDTA pH 7.5) followed by
recentrifugation. The cells
were then diluted into TE (i ml) and aliquots (200 ~cl) were spread onto the
selective media previously prepared
in 150 mm growth plates (VWR).
Alternatively, instead of multiple small reactions, the transformation was
performed using a single; large
scale reaction, wherein reagent amounts were scaled up accordingly.
The selective media used was a synthede complete dextrose agar lacking uracil
(SCD-Ura) prepared as
described in Kaiser et al., jMethods in Yeast Genetics, Cold Spring Harbor
Press, Cold Spring Harbor, NY, p.
208-210 (1994). Transformants were grown at 30°C for 2-3 days.
The detection of colonies secreting amylase was perforated by including red
starch in the selective
growth media. Starch was coupled to the red dye (Reactive Red-120, Sigma) as
per the procedure described by
Biely et al., Anal. Biochem., 172: 176-179 (1988), The coupled starch was
incorporaeed into the SCD-Ura agar
plates at a final concentration of 0.15 ~ (w/v), and was buffered with
potassium phosphate to a pH of 7.0 (50-
100 mM final concentration).
The positive .colonies urere picked and streaked .across fresh sele~tive.media
(onto 15~. mm plates) in
r
orde t ob~n wellasolateil.attd idennfisbl site le colonies. aGV.eII isolated
sin le to ' si iv f r Iase
_ _ ... . - _ . . . . g ...8.... ~o ozes.po t a o amy
secretion were detected'by direct inaotporaeion of rod starch into bufferod
SCD-Ura agar. Positive colonies were
determined by their ability to brtak down starch resulting in a clear halo
around the positive colony visualized
directly. . . , .. .
78
*~trademark
--~ .m ~ ..--~--a ~...~ ~,~., ~ ... .._ _..__...__ _..<... ~ ".~~~"~.ri.~~.~.
~- ...._ . _.~,_.-...-".~.w.......__.... _~~.-..».. ..-._._.~_
CA 02481756 2004-10-25
wo .oma3~8 pcTiusoorr~szs
4. Isolate t~~Nl~ øw~R AmP,~ifc~,tion
When a positive colony was isolated, a portion of it was picked by a toothpick
and diluted into sterile
water (30 ~.1) in a.96 well plate. At this time, the positive colonies were
either frozen and stored for subsequent
analysis or immediately amplified. An aliquot of cells (5 ~cl) cuss used as a
template for the PCR reaction in a
25 ~d volume containing: 0.5 ~1 Klentaq.(Clontech, Palo Alto, CA); 4.0 Isl IO
mM dNTP's (Perkin Etmer
* .
Cetus); 2.5 ~1 Kentaq buffer (Clontech); 0.25 ul forward oligo 1; 0.25 p.l
reverse oligo 2; I2.5 ~.1 distilled water.
The sequence of the forward oligonucleotide 1 was:
5'-TGTAAAACGACGGCCAGT~'AAATAGACCTGCAATTATTAATCT-3'_ (SEQ ID N0:169)
The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACC~CCTGCACACCTGCAAATCCATT-3' (SEQ ID NO;170)
IO PCR was then performed as follows:
a. Denature 92°C. 5 minutes
b. 3 cycles of: Denature 92°C, 30 seconds
' Anneal ' S9°C, 30 seconds
IS Extend ' 72°C, 60 Seconds
c. 3 cycles of Denature 92°C, 30 seconds
Anneal 57°C, 30 seconds
Extend 72°C, 60 seconds
d. 25 Cycles Of: DCnature 92°C, 30 Seconds
Area! 55 "C, -30 .seconds
Extend 72°C, 60 seconds
e. Hold 4°C
The underlined regions of the oligonucleotides annealed to the ADH promoter
region and the amylase
region, respectively, and amplified a 307 by region from vector ASST-AMY.O
when no insert was present.
Typically, the first I8 nucleotides of the 5' end of these oligonucieolides
contained annealing sites for the
.sequencing primers. Thus, the total product of the PCR reaction frbm an empty
vector was 343 bp. However,
signal sequence-fused cDNA resulted in considerably longer nucleotide
sequences.
Following the PCR, an aliquot of the reaction (5 ~d) was examined by agarose
gel electrophoresis in
a I % agamse gel using a Ttis-Borate-EDTA (TB~ buffering system as described
by Sambrook et-ai.,yupra:
Clones result'utg in a single strong PCR product larger than 400 by were
further analyzed by DNA sequencing
*.
after purification with a 96 Qiaquick PCR clean-up column (Qiagen lttc.,
Chatsworth, CA).
EXAMPLE 3: isolation of cDNA Clones t,~sing Signal Algorithm ~.nalysis
Various polypeptide-encoding nucleic acid sequences were identified by
applying a piapnetacy signal
ueacc find' a! oritluit.develo b Genentecti; .Inc South San: Francisco, CA a n
F.S'.fs.as=well as ___
~_ _ : m8.- 8 F~ Y . ( , .,_ _ : :-: ;; : . , , . ) P° ::~ -_ .
clustered and assembled EST fragments from public (e.g., GenBazik) andlor
private (L,IFESF.Qm; Idcyte
Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm
computes a secretion signal
score based on the character of the DNA nucleotides surrounding the first and
optionally the second methionine
*-trademark
CA 02481756 2004-10-25
WO 01116318 ~ PCT/fJSa0123328
codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under
consideration. The nucleotides
following the first ATG must code for at least 3S unambiguous amino acids
without any stop colons. If the first
ATG has the required amino acids, the second is not examined. If neither meets
the requirement, the candidate
sequence is not scored. In order to determine whether the EST sequence
contains an authentic signai sequence,
the DNA and corresponding amino acid sequences surrounding the ATG colon are
scored using a.set of seven
sensors (evaluation parameters) known to be associated with secretion signals.
Use of this algorithm resulted
in the identification of numerous polypeptide-encoding nucleic acid sequences.
EXAMPLE 4: Isolation of cDNA Clones Encoding Human PRO Pollr~eptides
Using the techniques described in Examples I to 3 above, numerous full-length
cDNA clones were
identified as encoding PRO polypeptides as disclosed herein. These cDNAs were
then deposited under the terms
of the Budapest Treaty with the American Type Culture Collection, 10801
University Blvd., Manassas, VA
20110-2209, USA (ATCC) as shown in Table 7 below.
Table 7
Material ATCC Dep. No. D~sit Date
DNA26843-1389 203099 August 4, 1998
DNA30867-1335 209807 Apri128, 1998
DNA34431-i 177 209399 October 17, 1997
DNA38268-I 188 209421 October 28, 1997
DNA40621-1440 209922 June 2, 1998
DNA4062S-1189 209788 . April 21, 1998
DNA4S409-2511 203579 January l2, 1999
DNA4S49S-1550 203156 August 25, 1998
DNA49820-1427 209932 June 2, 1998
DNAS6406-1704 203478 November 17, 1998
DNAS6410-1414 209923 June 2, 1998
DNAS6436-1448 209902 May 27, 1998
DNAS68SS-1447 203004 June 23, 1998
DNAS6860-1510 209952 June 9, 1998
DNA56862-1343 203174 September 1, 1998
DNA56868-1478 203024 June 23, 1998
DNAS6869-1545 203161 August 2S, 1998
DNAS7704-1452 209953 June 9, 1998
DNAS8?23-1588 203133 August 18, 1998
DNA57827-1493 203045 July 1, 1998
DNAS8737-1473 203136 August 18, 1998
DNAS8846-1409 209957 June 9, 1998
DNAS88S0-1495 209956 June 9, 1998
DNAS88S5-1422 203018 June 23, I998
DNAS92I I-1450 209960 June 9, 1998
DNAS9212-1627 203245 September 9, 1998
DNAS9213-1487 209959 June 9, 1998
DNA59605-.1418 203005 June 23;,1998
-
DNA59b09-1470 209963 . Junev9, 1998
DNAS96i0-1556 209990 June l6, 1998
DNAS9837-2545 203658 February 9, 1999
DNAS9844-2542 203650 February 9, 1999
DNAS98S4-1459 209974 June I6, 1998
CA 02481756 2004-10-25
WO OI/16318 PGTIUSOOIZ33Z8
Tale 7 lcont')
DNA60625-1507 209975 June I6; 1998
DNA60629-1481 209979 June 16; 1998
DNA61755-1554 203112 August 11, I998
DNA62812-1594 203248 September 9, 1998
DNA62815-1576 203247 September 9, 1998
DNA64881-1602 203240 September 9, 1998
DNA64886-1601 203241 September 9, 1998 .
DNA64902-1667 203317 October 6, 1998
DNA64950-1590 203224 September 15, 1998
DNA65403-1565 203230 September 15, 1998 -
DNA66308-1537 203159 August 25, 1998
.
DNA665I9-1535 203236 September 15, 1998
DNA66521-1583 203225 September 15, 1998
DNA66658-1584 203229 September 15, 1998
DNA66660-1585 203279 September 22, 1998
DNA66663-1598 203268 September 22, 1998
DNA66674-1599 203281 September 22, 1998
T?NA68862-2546 203652 February 9, 1999
DNA68866-1644 203283 September 22, 1998
DNA68871-1638 203280 September 22, 1998
DNA68880-1676 203319 October 6, 1998
DNA68883-1691 203535 December 15, 1998
DNA68885-1678 203311 October 6, 1998
DNA71277-1636 203285 September 22, 1998
DNA73727-1673 203459 November 3, 1998
DNA73734-1680 203363 October 20, 1998
DNA73735-1681 203356 October 20, 1998
-
DNA'76393-1664 203323 October 6, 1998
DNA77301-1708 203407 October 27, 1998
DNA77568-1626 203134 August 18, 1998
DNA77626-1705 203536 December 15, 1998
DNA81754-2532 203542 December 15, 1998
DNA81757-2512 203543 December 15, 1998
DNA82302_2529 203534 December 15, 1998
DNA82340-2530 203547 December 22, 1998
DNA83500-2506 283391 October 29, 1998
DNA84920-2614 203966 Apri127, 1999
DNA85066-2534 203588 January 12, 1999
DNA8657I-2551 203660 February 9, 1999
DNA87991-2540 203656 February 9, 1999
DNA92238-2539 203602 January 20, 1999
DNA96042-2682 PTA-382 July 20, 1999
DNA9678?-2534 203589 January 12, 1999
DNA 125185-2806PTA-1031 December 7, 1999
DNA147531-2821 PTA-1185 January 11, 2000
DNA115291-2681 PTA-202 June 8; 1999
DNA164625-28890PTA-1535 March 2I, 2000
DNA131639-2874 PTA-i784 April 25, 2000
DNA79230-2525 20.3549 December 22, 1998
. ;
v
~
; , ; _ -~ .=
-= - - .. '.;
__ ~; : . -
.The:. a W ..~.
ea
se d posits
.ere: maiiewider
the'psovisions
of the Budapest.Tr
type ttie International
Recognition:
of the Deposit of Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest
Treaty). This assures maintenance of a viable culture of the posit for 30
years from the date of deposit. The
81
CA 02481756 2004-10-25
wo olns3ls p'CT~SOOI~a~
.;d~osits will be made available by ATCC under the ternLS of the Budapest
Treaty,. and subject to ~an agreement
between Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of
the culeure of the deposit to the public upon issuance of the pertinent U.S.
patent or upon laying open to the
public of any U.S. or foreign patent application, whichever comes first, and
assures availability of the progeny
to one determined by the U.S. Commissioner of Patents and Trademarks
The assignee of the present application has agreed that if a culture of the
materials on deposit should
die or be lost or destroyed when cultivated under suitable conditions, the
materials will be promptly replaced on
notification with another of the same. Availability of the deposited material
is not to be construed as a license
to practice the invention in contravention of the rights granted under the
authority of any govenurtent in
accordance with its patens laws.
EXAMPLE 5: Use of PRO as a hybridization probe
The following method describes use of a nucleotide sequence encoding PRO as a
hybridization probe.
~5 . DNA comprising the coding sequence of full-length or mature~PRO as
disclosed herein is employed as
a probe to screen for homologous DNAs (such as those encoding naturally-
occurring variants of PRO) in human
tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the:following
high stringency conditions. Hybridization of radiolabeled PRO-derived probe to
the flters is performed in a.
solution of 50~ fotmamide, Sx SSC, 0.I ~ SDS, 0.1 ~6 sodium pyrophosphate, 50
mM sodium phosphate, pH
6:8, 2x Denhardt's solution, and 109E dezuan sulfate at 42°C for 20
hours. Washing of the filters is performed
in an aqueous solution of 0. Ix SSC and 0. I ~ SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence PRO can
then be identified using standard techniques known in the art.
. _
~XA,MPLE 6: Expression of PRO in E. colt
This example illustrates preparation of an unglycosylated fornt of PR0 by
recombinant expression in
E. colt.
The DNA sequence encoding PRO is initially amplified using selected PCR
pr'nners. The primers
should contain restriction enzyme sites which correspond to the restriction
enzyme sites on the selected
expression vector. A variety of expression vectors may be employed. An.example
of a suitable.vector is
pBR322 (derived from E. cots; see Bolivar et al., Gene, 2:95 (1977)) which
contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction enzyme and:
dephosphorylated. The PGR
amplified se uences areahen ligat~l ~totFtwector 'the vector:vivilt. referabl
include" uenc~s_which.encode
. ~ .. p . Y. ,~9. . . ..: . ,~ ::: t
~. : _ .. _, . ~: ; :: .. . . _ - .. ~:. ~~.:
for an antibiotic resistance gene, a' trp promoter, a polyhis leader
(including the first six STII codons,, pnlyhis
sequence, and enterokinase cleavage site), the PRO coding region, lambda
transcriptional terminator, and an
argU gene. ,
82
,..~ , n~ .>.,,~n~,~~"-~...wr...-~ -- ..__ ~ ... ...v __. .w~_.- ~ .~.~-
....~~n...~.N~....~. -
CA 02481756 2004-10-25
wo omus rc~~soor~3zs
The ligation mixture is then used to transform a selected E. cole strain using
the methods descn'bed in
Sambi'ook et al:, s-,upra. Transformants are identifi~d by their ability to
grow on LB plates and aritibiodc resistant
colonies are then selected. Plasmid DNA can be isolated and aynfitmed by
restriction analysis and DNA
sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with
antibiotics. The overnight culture tray subsequently be used to inoculate a
larger scale culture. The cells are
then grown to a desired optical density, during which the expression promoter
is turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell
pellet obtained by the centrifugation can be solubiIized using various agents
known in the art, and the solubilized
PRO protein can then be purified using a metal cheIating column under
conditions that allow tight binding of the
protein.
PRO may be expressed in E. colt in a poly-His tagged form, using the following
procedure. The DNA
encoding PRO is initially amplified using selected PCR primers. The primers
wilt contain restriction enzyme
sites which correspond to the restriction enzyme sites on the selected
expression vector, and other useful
sequences providing for efficient and reliable translation initiation, rapid
purification on a metal chelation
~ column, and proteolytic removal with enterokinase. The PCR-amplified, poly-
His tagged sequences are then
ligated into an expression vector, which is used to transform an E. colt host
based on swain 52 (W3I10
fuhA(tonA) lon galE rpoHts{htpRts) clpP(IacIq): Transfoctnants are first grown
in LB containing 50 mg/ml'
carbericillin at 30°C with shaking until an O.D.600 of 3-5 is reached.
Cultures are then diluted 50-100 fold into
CRAP media (prepared by mixing x.57 g (NH,~zSO~, 0.7i g sodium citrate~2H20;
1.07 g KCl, 5.36 ~g Difco
yeast extract, 5.3b g Sheffield hycase SF in S00 mL water, as well as 1 i0 mM
MPOS, pH 7.3, 0.55 ~ (wlv)
glucose and 7 mM MgSO,) and grown for approximately 20-30 hours at 30°C
with shaking. Samples are
removed to verify expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell
pellets are frozen until purification and refolding.
E. colt paste.from 0.5 to 1 L fetmentations (6-10 g pellets) is resuspended in
10 volumes (w/v) in'7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium
tetrathionate is added to make final
concentrations of O. iM and 0.02 M, respectively, and the solution is stirred
avernight at 4°C. This step results
in a denatured protein with atI cysteine residues blocked by sulfitolization.
The solution is centrifuged at 40,000
rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-
5 volumes of metal chelate
column buffer (6 M guanidine, 20 nnM Tris; pH 7.4) and filtered through 0.22
micron filters to clarify. 'The
clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal chelate column
equilibrated in the metal chelate
column buffer. The column is washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol
grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractions containing the
desired protein are pooled and stored at 4°C. Protein concentration is
estimated by its absorbance at 280 nm
-using the calculated extinction caefficient based on its amino acid sequence
r fo
The proteins are a Ided by diluting the sample slowly into freshly prepared
refolding buffer consisting
of: 20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycine
and 1 mM EDTA.
Refolding volumes are chosen so that the final protein concentration is
between 50 to 100 micrograms/ml. The
83
CA 02481756 2004-10-25
wo oins3ls pCT/~rs°°n3~zs
refoldutg solucton is stirred gently at 4°C for 12-36 hours. Tlte
refolding reaction is quenched by the addition
of TFA to a final ootxentration of 0.4 ~ (pH of approximately 3). Before
further purification of the protein, the
solution is filtered through a 0.22 micron filter and acetonitrile is added to-
2-10~ final concentration. The
refolded protein is chromatographed on a Poros RI/H reversed phase column
using a mobile buffer of 0.196
TFA with elution with a gradient of acetonitrile from IO to 80~. Aliquots of
fractions with A280 absorbance
S are analyzed on SDS polyacrylamide gels and fractions containing homogeneous
refolded protein are pooled.
Generally, the properly refolded species of most proteins are eluted at the
lowest concentrations of acetonitriIe
since those species are the most compact with their hydrophobic interiors
shielded frbm interaction with the
reversed phase resin.. Aggregated species are usually eluted at higher
acetonitrile concentrations: In addition
to resolving misfolded forms of proteins from the desired form, the reversed
phase step also removes endotoxin
from the samples.
Fractions containing the desired folded PRO polypeptide are pooled and the
acetonitrile removed using
a gentle stream of nitrogen directed at the solution. Proteins are formulated
into 20 mM Hepes, pH 6.8 with
0.14 M sodium chloride and 496 tnannitol by dialysis or by gel filtration
using G25 Superfine (Phatmacia) resins
equilibrated in tine formulation buffer and sterile filtered.
IS Many of the PRO polypeptides disclosed herein were successfully expressed
as described above.
EXAMPLE 7: Expression of PRO in mammalian cells
This example illustrates preparation of a vpotentially glycosylated form of
PRO by recombinant
expression in mammalian cells.
The vector, pRICS (see FP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the PRO DNA is Iigated into pRICS with selected restriction
enzymes to allow insertion of the PRO
DNA using ligation methods such as described in Sambrook et aL, supra. The
resulting vector~is called pRICS-
PRO. _
In one embodiment, the selected host cells tray be 293 cells. Human 293 cells
(ATCC CCL 1573) are
grown co confluence in tissue culture plates in medium such as I)MF..M
supplemented with fetal calf serum and
optionally, nutrient components andlor antibiotics. About 10 kg pRKS-PRO DNA
is mixed with about 1 fcg
DNA encoding the VA RNA gene [Thimmappaya et aL, CeII, x:543 (1982)] and
dissolved in 500 lcl of 1 mM
Tris-HCI, 0.1 mM fiDTA, 0.227 M CaCh. To this mixture is added, drapwise, 500
~d of 50 mM HEPES (pH
7.35), 280 mM NaCI, 1.5 mM NaPO~, and a'precipitate is allowed to form for 10
minutes at 25°C. The
precipitate is suspended and added to the 293 cells and allowed to settle for
about four hours at 37°C. The
culture medium is aspirated off and 2 mI of 20:~ glycerol in PBS is added for
30 seconds. The 293 cells are
then washed with serum free medium, fresh medium is added and the cells are
incubated for about 5 days.
Approximately 24 hours after the tcansfeetiotis, the culuuue medium is.removed
and replaced with culture
tneiiium alone orxtilture~imedium:rontainln 200 CilinI'.sS steine and 200
Cilmt ~sS-u~ethionine. ~lftec
_ ( .,: ) -.: - g (~.,_:.~_ .... ~y. . p. _. _..: _
a I2 hour icxuba6on, the oonditloi~d medium is collected, concentrated on a
spin filter. and loaded onto a 15 96
SDS gel. The processed gel may be dried and exposed to film for a selected
period of time to reveal the
presence of PRO polypeptide. The cultures containing transfected cells may
undergo further incubation (in
*-trademark
. .._......__..~ ~...°.->.~-. . -----.___. .. _.....
_...,.."......~",~e,.~" . . ._..,-,._..~-._r. .____.._........
CA 02481756 2004-10-25
wo oans3><8 PCTnJSOOn~za
serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO may be introduced into 293 cells transiently
using the dextran sulfate
method described by Somparyrac et al., ~roc.~Natl. Acad. Sci., i2:7S75 (198I).
293 cells are grown to
maximal density in a spinner flask and 700 p.g pRICS-PRO DNA is added. The
cells are first concentrated from
the spinner flask by centrifugation and washed with PBS. The DNA-dextran
precipitate is incubated on the cell
S pellet for four hours. The cells .are treated with 2096 glycerol for 90
seconds, washed with tissue culture
medium, and re-introduced into the spinner flask containing tissue culture
medium, 5 ~.g/mI bovine insulin and
0.1 p,glml bovine transferrin. After about four days, the conditioned media is
centrifuged and filtered to remove
cells and debris. The sample containing expressed PRO can then be concentrated
and purified by any selected
method, such as dialysis and/or column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRKS-PRO can be
transfected into
CHO cells using known reagents such as CaPO, or DEAF-dextran. As described
above, the cell cultures can
be incubated, and the medium replaced with culture medium (alone) or medium
containing a radiolabel such as
'SS-methionine. After determining the presence of PRO polypeptide,. the
culture medium may be replaced with
serum free medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium
1S is harvested. The medium containing the expressed PRO can then be
concentrated and purified by any seIr'.cted
method.
Epitope-tagged PRO may also be expressed in hose CHO cells. The PRO may be
subcloned out of the
pRKS vector. The subclone insert can undergo PCR to fuse in frame with a
selected epitope tag such as a poly-
his tag into a Baculovirus expression vector. The poly-his tagged FRO insert
can then be subcloned into a SV40.
driven vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the CHO cells
can be transfected (as described above) with the SV40 driven vector. Labeling
may be performed, as described
above, to verify expression. The culture medium containing the expressed poly-
His Bagged PRO can them be
concentrated and purified by any selected method, such as by Ni2~~-chelate
affinity chromatography.
PRO may also be expressed in CHO andlor COS cells by a transient expression
procedure or in CHO
2S cells by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The
proteins are expressed
as an IgG construct (immunoadhesin), in which the coding sequences for the
soluble forms (e.g. extracellular
domains) of the respective proteins are fused to an IgGl constant region
sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using
standard techniques as described in Ausubel et al., Current Protocols of
Molecular BioIoQV, Urut 3.16, :lohn
Wiley and Sons (1997). CHO expression vectors are constructed to have
compaeible restriction sites S' ar,~d 3'
of the DNA of interest to allow the convenient shuttling of cDNA's. The vector
used expression in CHO r;ells
is as described in Lucas -et aL, Nucl: Acids ices. 24:9 (IT7~1 1779_-~L~996),
~d uses the SV4i) ear~Ty__
3S promoter/enhancer to drive expression of the cDNA of interest and
dilaydrofolate ~ceductase (DIiFR): DHFR
expression permits selection for stable maintenance of the plasmid following
transfection.
8S
CA 02481756 2004-10-25
WO .01/16318 _ PCT7US00/23328
Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO cells
using commercially available iransfeetion reagents Superfect' (Quiagen),
Dosper or Fugene' (Boehringer
Mannheim). The cells are grown as described in Lucas et al., sucrra.
Approximately 3 x 10'' cells are frozen
in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into water bath
and mixed by
vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs
of media and centrifuged at 1000
rpm for 5 minutes. The supernatant is aspirated and the cells are resuspended
in 10 mL of selective media (0.2
~cm filtered PS20 with 5~ 0.2 um diafiltered fetal bovine serum). The~cells
are then aliquoted into a 100 mL
spinner containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner
filled with 150 mL selective growth medium and incubated at 37°C..
After another 2-3 days, 250 mL, 500 mL
and 2000 mL spinners are seeded with 3 x 105 cellslmL. The cell media is
exchanged with fresh media by
centrifugation and resuspension in production medium. Although any suitable
CHO media may be employed,
a production medium described in U.S. Patent No. 5,122,469, issued June 16,
1992 may actually be used. A
3L production spinner is seeded at 1.2 x 106 celIs/mL. On day 0, the cell
number pH ie determined. On day
1, the spinner is sampled and sparging with filtered air is commenced. On day
2, the spinner is sampled, the
1S temperature shifted to 33°C, and 30 mI. of 500 g!L glucose and 0.6
mI. of 10~ antifoam (e.g., 35~
polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken.
Throughout the production,
the pH is adjusted as necessary to keep it at around 7.2. After 10 days, or
until the viability dropped below
70~, the cell culture is harvested by centrifugation and filtering through a
0.22 um filter. The filtrate was either
stored at 4°C or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA
column (Qiagen). Before
purification, imidazole is added to the conditioned media to a concentration
of 5 mM. The conditioned media
is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,
buffer containing 0.3 M NaCI
and 5 mM imidazole at a flow rate of 4-5 mllmin. at 4°C. After loading,
the column is washed with additional
' equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly
purified protein is subsequently desalted into a storage buffer containing 10
mM Hepes, 0.14 M NaCI and 4 °b
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -
80°C.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned
media as follows. The
conditioned medium is pub onto a 5 mI Protein A column (Pharmacia) which had
been equilibrated in 20 _
mM Na phosphate buffer, pH 6.8. After loading, the column is washed
extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1
ml fractions into tubes containing 275 uL of 1 M Tris buffer, pH 9. The highly
purified protein is subsequently
desalted into storage buffer as described above for the poly-His tagged
proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation.
Many of the PRO polypeptides disclosed herein-were successfully,;~expressed as
described. above .. ~ .
86
_ . . _ ~. ~ _ _~ _
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I:X M~: Expression of PRO in Yeast
The following method describes recombinant expressimt of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PRO from
the ADH2/GAPDH promoter. DNA encoding PRO and the promoter is inserted into
suitable restriction enzyme
sites in the selected plasmid to direct intracellular expression of PRO. For
secretion, DNA encoding PRO can
be cloned into the selected plasmid, together with DNA encoding the ADH21GAPDH
promoter, a native PRO
signal peptide or other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory
signallleader sequence, and linker sequences (if needed) for expression of
PRO.
Yeast cells, such as yeast strain AB 110, can then be transformed with the
expression plasmids described
above and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by
1Q precipitation with 10 gb trichloroacuic acid and separation by SDS-PAGE,
followed by staining of the gels with
Coomassie Blue stain.
Recombinant PRO can subsequently be isolated and purified by removing the
yeast cells from the
fermentation medium by centrifugation and then concentrating the medium using
selected cartridge filters. 'The
concentrate containing PRO may further be purified using selected column
chromatography resins.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 9: E~ression of PRO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression of PRO in Baculovirus-
infected insect cells.
The sequence coding for PRO in fused upstream of an epitope tag contained
within a baculovurus
expression vector, Such epitope tags include poly~is tags and immunoglobulin
tags (like Fc regions of IgG).
A variety.of plasnuds may be employed, including plasmids derived from
commercially available plasmids such
as pVLI393 (Novagen). Briefly, the sequence encoding PRO or the desired
portion of the coding sequence of
PRO such as the sequence encoding the extracellular domain of a transtnembrane
protein or the sequence
encoding the mature protein if the protein is extracellular is amplified by
PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking (selected)
restriction enzyme sites. The product
is then digested with those selected restriction enzymes and subcloned into
the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BacuIOGOIdTM Vlrus
DNA (Pharmingen) into Spodopeera frugiperdu ("Sf9") cells (ATCC ~CRL I711 )
using lipofectin.(commerci<~Ily
available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the
released viruses are harvested and used
for further amplifications. Viral infection and protein expression are
performed as described by O'Reilley et
al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
Expressed poly-his tagged FRO can then be purified, for example, by NiZ+-
chelate affinuty
chromatography, as follows. Extracts are prepared from recombinant virus-
infected S~ cells as described by
Rupert et aI:, ature, X62:175-179 (1993). Bnefly; Sf9 cells are washed,
resuspei;ded in sonication buffer-(2 __5.
_ .y ;. . . : . ~ : ~._
inL Hepes; pH 7~9; 12.5 mM MgCiz; 0:1 mIVI EDTA; i0~ glycerol; 0.19 NP-40; 0.4
M KCl), and sorucated
twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and
the supernatant is diluted 50-fold
in loading buffer (50 mM phosphate, 300 mM NaCI, 1096 giycerol,~pH 7.8) and
filtered through a 0.45 ~m
87
CA 02481756 2004-10-25
WO OI/I631$ PCT/USOOIZ33Z8
filter. A Ni2+-NTA agarose column (commercially available from ~iagen) is
prepared with a bed volume of 5
mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer.
The filtered cell extract is
loaded onto the column at 0.5 mL per mia~ute. The column is washed to baseline
A2~ with loading buffer, at
which point fraction collection is started. Next, the column is washed with a
secondary wash buffer (SO mM
phosphate; 300 mM NaCt, 10% glycerol, pH 6.0), which eluees nonspecifically
bound protein. After reaching
A~ baseline again, the column is developed with a 0 to 500 mM Fmidazole
gradient in the secondary wash
buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with
Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen). Fractions containing the
eluted His,Q-tagged PRG are
pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be
performed using known
chromatography techniques, including for instance, Protein A or protein G
column chromatography.
Many of the PRO polygeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 10: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal aneibodies which can
specifically bind PRO.
Techniques for producing -the monoclonal antibodies are known in the art and
are described, for
instance, in Coding, supra. Immunogens that may be employed include purified
PRO, fusion proteins contailung
PR~, and cells expressing recombinant PRO on the cell surface. Selection of
the itrimunogen can be made by
the skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in
complete Freud's
adjuvant and injected subcutaneously or intraperitonealIy in an amount from 1-
100 micrograms. Alternatively,
the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
Hamilton, MT) and
injected into the animal's hind foot pads. The immunized mice are then boosted
10 to I2 days later with
additional immunogen emulsified in the selected adjuvant. Thereafter, for
several weeks, the mice may also be
boosted with additional immunization injections. Serum samples may be
periodically obtained from the nuce
by retro-orbital bleeding for testing in ELISA assays to detect anti-PRO
antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected
with a final intravenous injection of PRO. Three to four days later, the mice
are sacrificed and the spleen cells
are harvested. The spleen cells are then fused (using 35 q6 polyethylene
glycol) to a selected marine myeloma
cell line such as P3X63AgU.l, available from ATCC, No. CRL 1597. The fusions
generate hybridoma cells
which can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, .and
thymidine) medium to inhibit proliferation of non-fused cells, myeloma
hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against PRO.
Determination of
"positive" hybridoma cells secreting the desired monoclonal antibodies against
PRO. is within .the skill in the art.
The positive hybridorna cells can be injected intraperitoneally intoayngeneic
Balb/c mice-to=produce
ascites containing the anti-PRO monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue
culture flasks or roller bottles. Purification of the monoclonal antibodies
produced in the ascites can be
accomplished using ammonium sulfate precipitation, followed by gel exclusion
chromatography. Alternatively,
88
CA 02481756 2004-10-25
w0 o1n63IS PCTIUSOOI23328
affinity chromatography based upon binding of antibody to .protein A or
protein G can be employed.
EXAMPLE 11: Purification of PRO Pol~!peptides Using Specific Antibodies
Native or recombinant PRO polypeptides may be purified by a variety of
standard techniques in the art
of protein purification. For example, pro-PRO polypeptide, mature PRO
polypeptide, or pre-PRO polypepride
is purified by immunoaffinity chromatography using antibodies specific for the
PRO polypeptide of interest. in
general, an imrnunoaffinity colurrin is constructed by covalentIy coupling the
anti-PRO polypeptide antibody to
an activated chromatographic resin.
Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium
sulfate or by purification on immobilized Protein A (Pharmacia LKB
Biotechnology, Pisaataway, N.J.).
Likewise, monoclonal andbodi~ are prepared from mouse ascites fluid by
ammonium sulfate precipitation or
chromatography on immobt~ized Protein A. Partially purified immunoglobulin is
covalently attached. to a
chromatographic resin such as CnBr-activated SEPHAROSETM (Pharmacia LKB
Biotechnology). The antibody
is coupled to the resin, the resin is blocked, and the derivative resin is
washed according to the manufacturer's
instructions.
Such an immunoaffinity column is utilized in the purificauion of PRO
polypeptide by preparing a fraction
from cells containing PRO polypeptide in a soluble form. This preparation is
derived by solubilization of the
whole cell or of a 5ubcellular fraction obtained via differential
centrifugation by the addition of detergent or by
other methods well known in the art. Alteniatively, soluble PRO polypeptide
containing a signal sequence may
be secreted in useful quantity into the medium in which the cells are grown.
A soluble PRO polypeptide-containing preparation is passed over the
immunoaffinity column, and the
column is washed under conditions that allow the preferential absorbance of
PRO polypeptide (e.g., high ionic
strength buffers in. the presence of 'detergent). Then, the column is eluted
under conditions that disrupt
antibodyIPRO polypeptide binding (e.g. , a low pH buffer such as approximately
pH 2-3, or a high concentration
of a chaotrope such as urea or thiocyanate ion), and PRO polypeptide is
collected.
2S
EXAMPLE 12: prug Screening
This invention is particularly useful for screening compounds by using PRO
polypeptides or bisiding
fragment thereof in any of a variety of drug screening techniques. The PRO
polypeptide or fragment employed
in such a test may either be free in solution, affixed to a solid support,
borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably
transformed with r~ombinant nucleic acids expressing the PRO polypeptide or
fragment. Drugs are screened
against such transformed cells in competitive binding assays. Such cells,
either in viable or fixed form, can be
used for standard binding assays. One may measure, for example, the formation
of complexes between PRO
polypeptide; or a fragment and the agent being tested. Alternatively, one can
examine the-dttrl_trtution.tiacomplex
formation between the PRO polypeptfde and its target cell or target receptors
caused by tile agent being tested:
Thus, the present invention provides methods of.screening for drugs or any
other agents which can
affect a PRO polypeptide-associated disease or disorder. These methods
comprise contacting such an agent with
89
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an PRO poiypeptide or fragment thereof and assaying (I) for the presence of a
complex between the agent and
the PRO polypeptide or fragment, or (ii) for the presence of a complex between
the PRO polypeptide or fragment
and the cell, by methods well known in the art. In such competitive binding
assays, the PRO polypeptide or
fragment is typically labeled. After suitable incubation, free PRO polypeptide
or fragment is separated from that
present in bound form, and the amount of free or uncomplexed label is a
measure of the ability of the particular
agent to bind to PRO polypeptide or to interfere with the PRO polypeptidelcell
complex.
Another technique for drug screening provides high throughput screening for
compounds having suitable
binding affinity to a polypeptide and is described in detail in WO 84/03564,
published on September 13, 1984.
Briefly stated, Large numbers of different small peptide test compounds are
synthesized on a solid substrate, such
as plastic pins or some other surface. As applied to a PRO polygeptide, the
peptide test compounds are reacted
with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods
well known in the art.
Purified PRO polypeptide can also be coated directly onto plates for use in
the aforementioned drug screening
techniques. In addition, non-neutralizing antibodies can be used to capture
the peptide and immobilize it on the
solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing
antibodies capable of binding PRO polypeptide specifically compete with a test
compound for binding to 1PR0
polypeptide or fragments thereof: In this manner, _the antibodies can be used
to detect the presence of any
peptide which shares one or more antigenic determinants with PRO polypeptide.
EXAMPLE 13: Rational Drub sign
The goal of rational drug design is to produce structural analogs of
biologically active polypeptide of
interest (i.e., a PRO poIypeptide) or of small molecules with which they
interact, e.g., agonists, antagonists, or
inhibitors. Any of these examples can be used to fashion drugs which are more
active or stable forms of the
PRO polypeptide or which enhance or interfere with the function of the PRO
polypeptide in vivo (cf., Hodgson,
Bio/TechnoIoev, ~: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of
an PRO
polypeptide-inhibitor complex, is detemuned by x-ray crystallography, by
computer modeling or, most typically,
by a combination of the two approaches. Both the shape and charges of the PRO
polypeptide must be ascertaiined
to elucidate the structure and to determine active sites) of the molecule.
Less often, useful information regarding
the structure of the PRO polypeptide may be gained by modeling based on the
structure of homologous proteins.
In both cases, relevant structural information is used to design analogous PRO
polypeptide-like molecules or to
identify efficient inhibitors. Useful examples of rational drug design may
include molecules which have improved
activity or stability as shown by Braxton and Wells, Biochemistyr, X1:7796-
7801 (1992) or which act as
inhibitors, agonists, or antagonists of native peptides as shown by Athauda et
al., J. Biochem., l 13:742-746
(1993)::
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above,
and then to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent
drug design can..be based. It is possible to bypass protein crystallography
altogether by generating anti-idiotypic,
CA 02481756 2004-10-25
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antibodies (anti-ids) to a functional, pharmacologically active antibody. As a-
mirxoc image of a mirror image, _~_
the binding site of the anti-ids would be expected to be an analog of the
original receptor. The anti-id could then
be used to identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated
peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide
may be made available
S to perform such analytical studies as X-ray crystallography. In addition,
knowledge of the PRO polypeptide
amino acid sequence provided herein will provide guidance to those employing
computer modeling techniques
in place of or in addition to x-ray crystallography.
EXAMPLE 14: Pericyte c-Fos Induction ~y 93)
This assay shows that certain polypeptides of the invention act to induce the
expression of c-fos in
pericyte cells and, therefore, are useful not only as diagnostic markers for
particular types of pericyte-associated
tumors but also for giving rise to antagonists which would be expected to be
useful for the therapeutic treatment
of pericyte-associated tumors. Induction of c-fos expression in pericytes is
also indicative of the induction of
angiogenesis and, as such, PRO polypeptides capable of inducing the expression
of e-fos would be expected to
1S be useful for the treatment of conditions where induced angiogenesis would
be beneficial including, far example,
wound healing, and the Like. Specifically, on day I, pericytes are received
from VEC Technologies and all but
5 mI of media is removed from flask. On day 2; the pericytes are trypsinized;
washed, spun and then plated onto
96 well plates. C1n day 7, the media is removed and the pericytes are treated
with 100 ~d. of:PFtO.~polypeptide
test samples and controls (positive control = DME+5 ~ senun +!- PDGF at 500
nglml; negatiue control =
protein 32). Replicates are averaged and SD/CV are determined. Fold increase
over Protein 32 (buffer control)
value indicated by chemiluminescence units (RLU) lurninometer reading verses
frequency zs plotted on a
histogram. Two-fold above Protein 32 value is considered positive for the
assay. ASY Matrix: Growth media
= low glucose DMEM = 20~ FBS + 1X pen strep + IX fungizone. Assay Media = low
glucose DMEM
+5 to FBS.
2S The following polypeptides tested positive in this assay: PR0134~ and
PROI340.
EXAMPLE 15: Abilitlr of PRO Polypeptides to Stimulate the Release of
Proteog~lvcans from Cartila~, a (~ Assav
The ability of various PRO polypeptides to stimulate the release of
proteoglycans from cartilage tissue
was tested as follows.
The metacarphophalangeal joint of 4-6 month old pigs was aseptically
dissected, and articular cartilage
was removed by free hand slicing being careful to avoid the underlying bone.
The cartilage was minced and
cultured in bulk for 24 hours iai a humidified atmosphere of 95 ~ air, S 91;
COZ in sen,~m. free. (SF7..media
(DMEIF12 1:1.) woth 0. I'%. BSA and: 100UIml penicillin 'and I00~,g1m1
streptomyctni_ -~3.fterw~sh~ng t~Fee
3S times, approximately I00 mg of articular cartilage was aliquoted into
mieronics tubes and incubated for an
additional 24 hours in the above SF media. PRO polypeptides were then added at
1 ~ either alone or in
combination with 18 nglml interleukin-la, a known stimulator'of proteoglycan
release from cartilage tissue.
91
CA 02481756 2004-10-25
WO 01116318 PC"T/US00/23328
The supernatant was then harvested and assayed for the amount of proteoglycans
using the 1,9-dimethyl-
methylene blue (DMB) colorimetric assay (Farndale and Buttle, Biochem.
Biaphys. Acta 883:173-177 (1985)).
A positive result in this assay indicates that the test polypeptide will find
use, for example, in the treatment of
sports-related joint problems, articular cartilage defects, osteoarthritis or
rheumatoid arthritis.
When various PRO polypeptides were tested in the above assay, the polypeptides
demonstrated a marked
ability to stimulate release of proteoglycans from cartilage tissue both
basally and after stimulation with
interleukin-1 a and at 24 and 72 hours after treatment, thereby indicating
that these PRO polypeptides are useful
for stimulating proteoglycan release from cartilage tissue. As such, these PRO
polypeptides are useful for the
treatment of sports-related joint problems, articular cartilage defects,
osteoarthritis or rheumatoid arthritis. The
polypeptides testing positive in this assay are: PR01565, PR01693, PRO1801 and
PR010096:
EXAMPLE 16: Detection of PolYpeptides That Affect Glucose or FFA Uptake in
Skeletal Muscle (Assay 106)
This assay is designed to determine whether PRO palypeptides show the ability
to affect glucose or, FFA
uptake by skeletal muscle cells. PRO polypeptides testing positive in this
assay would be expected to be useful
for the therapeutic treatment of disorders where either the stimulation ar
inhibition of glucase uptake by skeletal
muscle would be beneficial including, for example, diabetes or hyper- or hypo-
insulinemia.
In a 96 well format, PRO polypeptides to be assayed are added to primary rat
differentiated skeletal
muscle, and allowed to incubate overnight. Then fresh media with the PRO
polypeptide and +/- insulin are
added to the wells. The sample media is then monitored to determine glucose
and FFA uptake by the skeletal . ,
muscle cells. The insulin will stimulate glucose and FFA uptake by the
skeletal muscle, and insulin in meiiia '
without the PRO polypeptide is used as a positive control, and a limit for
scoring. As the PRO polypeptide being
tested may either stimulate or inhibit glucose and FFA uptake, results are
scored as positive in the assay if
greater than 1.5 times or Less than 0.5 times the insulin control.
The following PRO polypeptides tested positive as either stimulators or
inhibitors of glucose andlor FFA
uptake in this assay: PR44405.
EXAMPLE 17: Identification of PRO Polypeptides That Stimulate TNF-a Release In
Human Blood (Assay 128)
This assay shows that certain PRO polypeptides of the present invention act to
stimulate the release of
TNF-a in human blood. PRO polypeptides testing positive in this assay are
useful-.for, among other things,
research purposes where stimulation of the release of TNF-a would be desired
and for the therapeutic treatment
of conditions wherein enhanced TNF-a release would be beneficial.
Specifically, 200 ~I of human blood .
supplemented with 50mM Hepes buffer (pH 7.2) is aliquotted per well in a 96
well test plate. To each well is
then added 300~c1 of either the test PRO polypeptide in 50 mM Hepes buffer (at
various concentrations) or 50
mM Hepes buffer alone (negative control) and the plates are incubated at
37°C for 6 hours. The samples are
then centrifuged and 50P1 of plasma is collected from each well.and tested for
the presence of TNF-a by ELISA
assay. A positive in the assay is a higher amount of TNF-a in the PRO
polypeptide treated samples as compared
to the negative control samples.
92
.r.. ~. ~.~m.. ~ ~. . M
CA 02481756 2004-10-25
WO OI/I6318 PC"TI~1'SOOi23328
The following PRO polypeptides tweed positive in this assay. PR0263. PR0295;
PR01282, PRO1063,
PR01356, PR03543, and PR05990.
EXAMPLE 18: Tumor Versus Normal Differential Tissue Expression Distribution
Oligonucleotide probes were constructed from some of the PRO polypeptide-
encoding nucleotide
sequences shown in the accompanying figures for use in quantitative PCR
amplification reactions The
oligonucleotide probes were chosen so as to give an approximately 200-600 base
pair amplified fragment from
the 3' end of its associated template in a standard PCR reaction. The
oligonucleotide probes were employed in
standard quantitative PCR amplification reactions with cDNA libraries isolated
from different human tumor and
normal human tissue samples and analyzed by agarose gel electrophoresis so as
to obtain a..quantitative
determination of the level of expression of the PRO polypeptide-encoding
nucleic acid in the various tumor and
normal tissues tested. ~i-actin was used as a control to assure that
equivalent amounts of nucleic acid was used
in eacli reaction. Identification of the differential expression of the PRO
polypeptide-encoding nucleic acid in
one or more tumor tissues as compared to one or more normal tissues of the
same tissue eype renders the
molecule useful diagnostically for the determination of the presence or
absence of tumor in a subject suspected
of possessing a tumor as well as therapeutically as a target for the treatment
of a tumor in a subject possessing
such a tumor. These assays provided the following results.
Molecule is more ~ hey expressed as compared to:
in:
DNA26843-1389 normal lung lung tumor
rectum tumor normal rectum
DNA30867-1335 normal kidney kidney tumor
DNA40621-1440 normal lung lung tumor
DNA40625-1189 normal lung lung tumor
DNA45409-2511 melanoma tumor normal skin
DNA56406-1704 kidney tumor normal kidney
normal skin melanoma tumor
DNA56410-1414 normal stomach stomach tumor
DNA56436-1448 normal skin melanoma tumor
DNA56855-1447 normal esophagus esophageal tumor
rectum tumor normal rectum
DNA56860-1510 normal kidney kidney tumoe
rectum tumor normal rectum
DNA56862-1343 kidney tumor normal kidney
normal lung lung tumor
93
CA 02481756 2004-10-25
wo oms~~s . ~cr~rsoon3328
Molecule is more hiahlv expressed as compared to:
in: _
-.... DNAS6868-1478 normal stomach stomach tumor
normal lung lung tumor
DNA56869-1545 normal esophagus esophageal tumor
$ normal skin melanoma tumor
DNA57704-1452 normal stomach stomach tumor
rectum tumor normal iectum
DNA58723-1588 normal stomach stomach tumor
kidney tumor normal kidney
normal skin melanoma tumor
DNAS7827-1493 normal stomach stomach tumor
normal skin melanoma tumor
DNA~S8737-1473esophageal tumor normal esophagus
noamal stomach stomach tumor
DNA58846-1409 lung tumor normal lung
DNAS8850-1495 esophageal tumor ~ normal esophagus
kidney tumor normal kidney
DNA58855-1422 normal stomach stomach tumor
rectum tumor normal rectum
DNA59211-1450 normal kidney kidney tumor
DNA59212-1627 normal skin melanoma tumor
DNA59213-1487 normal stomach stomach tumor
normal skin melanoma tumor
3$ DNA59605-1418 melanoma tumor normal skin
DNAS9609-1470 esophageal tumor normal esophagus
DNA59610-1556 esophageal tumor normal esophagus
lung tumor normal lung
normal skin melanoma tumor
DNAS9837-2545 normal skin melanoma tumor
4$ DNA59844-2542 normal skin melanoma tumor
esophageal tumor noamal esophagus
DNA59854-1459 normal esophagus esophageal tumor
stomach tumor normal stomach
$0 normal lung lung tumor
DNA60625-1507 normal Ihng lung tumor
DNA60629-1481 normal esophagus esophageal tumor
$$ normal rectum rectum tumor
94
CA 02481756 2004-10-25
PCT/US00/23328
O1II6318 O
VV
olecule ~s more hi~hlv exvressedgs compared to:
in:
DNA617SS-1554 normal stomach -stomach-tumor
kidney tumor normal kidney
DNA62812-1594 normal stomach stomach tumor
normal lung lung tumor
normal rectum rectum tumor
normal skin melanoma tumor
DNA6281S-1576 esophageal tumor normal esophagus
DNA64881-1602 normal stomach stomach tumor -
normal lung lung tumor
DNA64902-1667 esophageal tumor normal esophagus
kidney tumor normal kidney
DNt~65403-1565normal esophagus esophageal tumor
DNA66308-1537 normal lung lung tumor
DNA66519-1535 kidney tumor normal kidney
DNA66521-1583 normal esophagus esophageal tumor
normal stomach stomach tumor
2S normal lung lung tumor
normal rectum rectum tumor
normal skin melanoma tumor
DNA66658-1584 normal lung lung tumor
melanoma tumor normal stdn
DNA666b0-1585 lung tumor normal lung
DNA66674-1599 kidney tumor normal kidney
normal lung lung tumor
DNA68862-2546 melanoma tumor normal skin
DNA68866-1644 normal stomach stomach tumor
DNA68871-1638 lung tumor normal lung
normal skin melanoma tumor
DNA68880-1676 normal lung lung tumor
normal skin melanoma tumor
DNA68883-1691 esophageal tumor normal esophagus
DNA68885-1678 Lung tumor normal lung
SO
DNA71277-1636 normal stomach stomach tumor
DNA73734-1b80 normal Lung Lung tumor
95
t
CA 02481756 2004-10-25
omt~is o rc r~soar~3Zs
w
lecule is more hi h~ Iy expressed~ as compared to:
in:
- ~DNA73735-1681 esophageal tumor normal esophagus
normal kidney kidney tumor
lung tumor normal lung
normal skin melanoma tumor
DNA76393-1664 esophageal tumor normal esophagus
stomach tumor normal stomach
lung tumor normal lung
rectum tumor normal rectum
DNA77568-1626 normal stomach stomach tumor -
lung tumor normal lung
DNA77626-1705 normal rectum rectum tumor
I5
DNA81754-2532. normal skin melanoma tumor
DNA81757-2512 esophageal tumor normal esophagus
normal stomach stomach tumor
melanoma tumor normal skin
DNA82302-2529 normal stomach stomach tumor
normal Iung lung tumor
DNA82340-2530 normal esophagus esophageal tumor
DNA85Q66-2534 lung tumor normal lung
normal skin melanoma tumor
DNA87991-2540 esophageal tumor normal esophagus .
DNA92238-2539 normal skin melanoma tumor
DNA96787-2534 normal kidney kidney tumor
EXAMPLE 19: Identification of Receptor/Li and nteractions
In this assay, various PRO polypeptides are tested for ability to bind to a
panel of potential receptor or
Iigand molecules for the purpose of identifying receptor/ligand interactions.
The identification of a ligand for
a known receptor, a receptor for a known ligand or a novel receptor/Iigand
pair is useful for a variety of
4Q indications including, for example, targeting bioactive molecules (linked
to the ligand or receptor) to ai cell
lrnown to express the receptor or ligand, use of the receptor or Iigand as a
reagent to detect the presence of the
ligand or receptor in a composition suspected of containing the same, wherein
the composition may comprise
cells suspected of expressing the ligand or receptor, modulating the growth of
or another biological or
immunological activity of a cell known to express or respond to the receptor
or Iigand, modulating the immune
response of cells or toward cells that express the receptor or ligand,
allowing the preparaion of agonists,
antagonists and/or. antibodies directed against the receptor or ligand which
will modulate the growth of or a:
biological or immunological activity of a cell expressing the receptor or
Digand, and various other indications
which will be readily apparent to the ordinarily skilled artisan.
96
CA 02481756 2004-10-25
wo om~i$ ~ Pcrnl~2~
The assay is performed as follows. A PRO polypeptide of the present inveneion
suspected of being a
ligand for - a receptor is expressed as a fusion protein containing the Fc
domain of human IgG (an
immunoadhesin). Receptor-ligand binding is detected by allowing interaction of
the immunoadhesin polypeptide
with cells (e.g. Cos cells) expressing candidate PRO polypeptide receptors and
visualization of bound
immunoadhesin with fluorescent reagents directed toward the Fc fusion domain
and examination by microscope.
Cells expressing candidate.receptors are produced by transient txansfection,
in parallel, of defused subsets of a
library of cDNA expression vectors encoding PRO palypepiides that may function
as receptor molecules. Cells
are then incubated for 1 hour in the presence of the PRO polypeptide
immunoadhesin being tested for possible
receptor binding. The cells are then washed and fixed with paraforrnaldehyde.
The cells are then incubated with
fluorescent conjugated antibody directed against the Fc portion of the PRO
polypeptide immunoadhesin (e.g.
FITC conjugated goat anti-human-Fc antibody). The cells are then washed again
and examined by microscope.
A positive interaction is judged by the presence of fluorescent labeling of
cells t=ansfected with cDNA encoding
a particular PRO polypeptide receptor or pool of receptors and an absence of
similar fluorescent labeling of
similarly prepared cells that have been transfected with other cDNA or pools
of cDNA. If a defined pool of
cDNA expression vectors is judged to be positive for interaction with a PRO
polypeptide immunoadhesin, the
individual cDNA species that comprise the pool are tested individually (the
pool is "broken down") to determine
the specific cDNA that encodes a receptor able to interact with the PRO
polypeptide immunoadhesin.
In another embodiment of this assay, an epitope-tagged potential ligand PRO
polypeptide (e.g. 8
histidine "His" tag) is allowed to interact with a panel of potential receptor
PRO palypeptide molecules that have
been expressed as fusions with the Fc domain of human IgG (immunoadhesins}.
Following a 1 hour
co-incubation with the epitoge tagged PRO polypeptide, the candidate receptors
are each immunoprecipitated
with protein A beads and the beads are washed. Potential ligand interaction is
determined by western blot
analysis of the immunoprecipitated complexes with antibody directed towards
the epitope tag. An interaction
is judged to occur if a band of the anticipated molecular weight of the
epitope tagged protein is observed in the
western blot analysis with a candidate receptor, but is not observed t:o occur
with the other members of the panel
of potential receptors.
Using these assays, the following receptor/Iigand interactions have been
herein identified:
(1) PR010272 binds to PR05801.
(2) PR020110 binds to the human IL-17 receptor (Yao et al., G~tokine 9(11):794-
800 (1997); also herein
designated as PRO1) and to PR020040.
(3) PR010096 binds to PR020233.
(4) PR019670 binds to PR01890.
The foregoing written specification is considered to be sufficient to enable
one skilled in the art to
practice the invention. The present invention is not to be limited in scope by
the construct deposited, since.tixe
deposited embodiment is intended'as a single illustration of certain aspects
of the invention-and any~constructs v -
that are functionally equivalent are within the scope of this invention. The
deposit of material herein does not
constitute an admission that the written description herein contained is
inadequate to enable the practice of any
aspect of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the
97
CA 02481756 2004-10-25
iW0 O1/163I8 PCTlUS00~23328
claims to the specific illustrations that it represents. Indeed, various
modifications of the invention in addition
to those shown and described herein will become apparent io those skilled in
the art from the foregoing
description and fall within the scope of the appended claims.
98
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
Sequence Listing
<110> Genentech, Inc.
Eaton,Dan L.
Filvaroff,Ellen
Gerritsen,Mary E.
Goddard,AUdrey
Godowski,Paul 7.
Grimaldi,Christopher ~.
Gurney,AUStin L.
watanabe,Colin K.
wood,william I.
<120> SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC
ACIDS ENCODING THE SAME
<130> P3230R1PCT
<140> PCT/US00/23328
<141> 2000-08-24
<150> PCT/U599/ZO111
<151> 1999-09-O1
<150> PCT/U599/21090
<151> 1999-09-15
<150> US 60/169,495
<151> 1999-12-07
<150> US 60/170,262
<151> 1999-12-09
<150> US 60/175,481
<151> 2000-O1-11
<150> PCT/US00/04341
<151> 2000-02-18
<150> PCT/uS00/04342
<151> 2000-02-18
<150> PCT/U500/04414
<151> 2000-02-22
<150> PCT/US00/05601
<151> 2000-03-O1 .
<150> US 60/187,202
<151> 2000-03-03
<150> US 60/191,007
<151> 2000-03-21
<150> PCT/US00/08439
<151> 2000-03-30
<150> us 60/199,397
<151> 2000-04-25
<150> PCT/US00/14042
<151> 2000-05-22
Page 1
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
<150> US 60/209,832
<151> 200006-05
<160> 170
<210> 1
<211> 1173
<212> DNA
<213> Homo Sapien
<400> 1
ggggcttcgg cgccagcggc cagcgctagt cggtctggta aggatttaca 50
aaaggtgcag gtatgagcag gtctgaagac taacattttg tgaagttgta 100
aaacagaaaa cctgttagaa atgtggtggt ttcagcaagg cctcagtttc 150
cttccttcag cccttgtaat ttggacatct gctgctttca tattttcata 200
cattactgca gtaacactcc accatataga cccggcttta ccttatatca 250
gtgacactgg tacagtagct ccagaaaaat gcttatttgg ggcaatgcta 300
aatattgcgg cagttttatg cattgctacc atttatgttc gttataagca 350
agttcatgct ctgagtcctg aagagaacgt tatcatcaaa ttaaacaagg 400
ctggccttgt acttggaata ctgagttgtt taggactttc tattgtggca 450
aacttccaga aaacaaccct ttttgctgca catgtaagtg gagctgtgct 500
tacctttggt atgggctcat tatatatgtt tgttcagace atcetttcct 550
accaaatgca gcccaaaatc catggcaaac aagtcttctg gatcagactg 600
ttgttggtta tctggtgtgg agtaagtgca cttagcatgc tgacttgctc 650
atcagttttg cacagtggca attttgggac tgatttagaa cagaaactcc 700
attggaaccc cgaggacaaa ggttatgtgc ttcacatgat cactactgca 750
gcagaatggt ctatgtcatt ttccttcttt ggttttttcc tgacttacat 800
tcgtgatttt cagaaaattt ctttacgggt ggaagccaat ttacatggat 850
taaccctcta tgacactgca tcttgcccta ttaacaatga acgaacacgg 900
ctactttcca gagatatttg atgaaaggat aaaatatttc tgtaatgatt 950
atgattctca gggattgggg aaaggttcac agaagttgct tattcttctc 1000
tgaaattttc aaccacttaa tcaaggctga cagtaacact gatgaatgct 1050
gataatcagg aaacatgaaa gaagccattt gatagattat tctaaaggat 1100
atcatcaaga agactattaa aaacacctat gcctatactt ttttatctca 1150
gaaaataaag tcaaaagact atg 1173
<210> 2
<211> 266
<212> PRT
Page 2
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
<213> Homo Sapien
<400> 2
Met Trp Trp Phe Gln Gln Gly Leu Ser Phe Leu Pro Ser Ala Leu
1 5 10 . 15
Val Ile Trp Thr Ser Ala Ala Phe Ile Phe Ser Tyr Ile Thr Ala
20 25 30
Val Thr Leu His His Ile Asp Pro Ala Leu Pro Tyr Ile Ser Asp
35 40 '45
Thr Gly Thr Val Ala Pro Glu Lys Cys Leu Phe Gly Ala Met Leu.
50 55 60
Asn Ile Ala Ala Val Leu Cys Ile Ala Thr Ile Tyr Val Arg Tyr
65 70 75
Lys Gln Val His Ala Leu Ser Pro Glu Glu Asn Val Ile Ile Lys
80 85 90
Leu Asn Lys Ala Gly Leu Val Leu Gly Ile Leu Ser Cys Leu Gly
95 100 105
Leu Ser Ile Val Ala .ASn Phe Gln Lys Thr Thr Leu Phe Ala Ala
110 115 120
His Val Ser Gly Ala Val Leu Thr Phe Gly Met Gly Ser Leu Tyr
125 130 135
Met Phe Val Gln Thr Ile Leu Ser Tyr Gln Met Gln Pro Lys Ile
140 145 150
His Gly Lys Gln Val Phe Trp Ile Arg Leu Leu Leu Val Ile Trp
155 160 165
Cys Gly Val Ser Aha Leu Ser Met Leu Thr Cys Ser Ser Val Leu
170 175 180
His Ser Gly Asn Phe Gly Thr Asp Leu Glu Gln Lys Leu His Trp
185 190 195
Asn Pro Glu Asp Lys Gly Tyr Val Leu His Met I12 Thr Thr Ala
200 205 210
Ala Glu Trp Ser Met Ser Phe Ser Phe Phe Gly Phe Phe Leu Thr
215 220 225
Tyr Ile Arg Asp Phe Gln Lys Ile Ser Leu Arg Val Glu Ala Asn
230 235 240
Leu His Gly Leu Thr ,Leu Tyr Asp Thr Ala Pro Cys Pro Ile Asn
245 250 255
Asn Glu Arg Thr Arg Leu Leu Ser Arg Asp Ile
260 265
<210> 3
<211> 2037
<212> DNA ,
<213> Homo Sapien
<400> 3
Page 3
. , r .... ". _"~,. ,, ",.,. r, .~.,~;_, ~~".. .. .., .,, .-..:~t.~.~a.
.n2...~.r."r.~...:... ",r-...".x, men"aa.,.saw, r.,.., ~".
7..,.z,.a,.....a,.b._._...._.,.-____-._ .t..._. _..___
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
cggacgcgtg ggcggacgcg tgggggagag ccgcagtccc ggctgcagca 50
cctgggagaa ggcagaccgt gtgagggggc ctgtggcccc agcgtgctgt 100
ggcctcgggg agtgggaagt ggaggcagga gccttcctta cacttcgcca 150
tgagtttcct catcgactcc agcatcatga ttacctccca gatactattt 200
tttggatttg ggtggctttt cttcatgcgc caattgttta aaga.ctatga 250
gatacgtcag tatgttgtac aggtgatctt ctccgtgacg tttgcatttt 300
cttgcaccat gtttgagctc atcatctttg aaatcttagg agtattgaat 350
agcagctccc gttattttca ctggaaaatg aacctgtgtg taattctgct 400
gatcctggtt ttcatggtgc ctttttacat tggctatttt attgtgagca 450
atatccgact actgcataaa caacgactgc ttttttcctg tctcttatgg 500
ctgaccttta tgtatttctt ctggaaacta ggagatccct ttcccattct 550
cagcccaaaa catgggatct tatccataga acagctcatc agccgggttg 600
gtgtgattgg agtgactctc atggctcttc tttctggatt tggtgctgtc 650
aactgcccat acacttacat gtcttacttc ctcaggaatg tgactgacac 700
ggatattcta gccctggaac ggcgactgct gcaaaccatg gatatgatca 750
taagcaaaaa gaaaaggatg gcaatggcac ggagaacaat gttccagaag 800
ggggaagtgc ataacaaacc atcaggtttc tggggaatga taaaaagtgt 850
taccacttca gcatcaggaa gtgaaaatct tactcttatt caacaggaag 900
tggatgcttt ggaagaatta agcaggcagc tttttctgga aacagctgat 950
ctatatgcta ccaaggagag aatagaatac tccaaaacct tcaaggggaa 1000
atattttaat tttcttggtt actttttctc tatttactgt gtttggaaaa 1050
ttttcatggc taccatcaat attgtttttg atcgagttgg gaaaacggat 1100
cctgtcacaa gaggcattga gatcactgtg aattatctgg gaatccaatt 1150
tgatgtgaag ttttggtccc aacacatttc cttcattctt gttggaataa 1200
tcatcgtcac atccatcaga ggattgctga tcactcttac caagttcttt 1250
tatgccatct ctagcagtaa gtcctccaat gtcattgtcc tgctattagc 1300
acagataatg ggcatgtact ttgtctcctc tgtgctgctg atccgaatga 1350
gtatgccttt agaataccgc accataatca ctgaagtcct tggagaactg 1400
cagttcaact tctatcaccg ttggtttgat gtgatcttcc tggtcagcgc 1450
tctctctagc atactcttcc tctatttggc tcacaaacag gcaccagaga 1500
agcaaatggc accttgaact taagcctact acagactgtt agaggccagt 1550
Page 4
CA 02481756 2004-10-25
PCT-US00-23328_sec~uence
ggtttcaaaa tttagatata agagggggga aaaatggaac cagggcctga 1600
cattttataa acaaacaaaa tgctatggta gcatttttca ccttcatagc 1650
atactccttc cccgtcaggt gatactatga ccatgagtag catcagccag 1700
aacatgagag ggagaactaa ctcaagacaa tactcagcag agagcatccc 1750
gtgtggatat gaggctggtg tagaggcgga gaggagccaa gaaactaaag 1800
gtgaaaaata cactggaact ctggggcaag acatgtctat ggtagctgag 1850
ccaaacacgt aggatttccg ttttaaggtt cacatggaaa aggttatagc 1900
tttgccttga gattgactca ttaaaatcag agactgtaac aaaaaaaaaa 1950
aaaaaaaaaa agggcggccg cgactctaga gtcgacctgc agaagcttgg 2000
ccgccatggc ccaacttgtt tattgcagct tataatg 2037
<210> 4
<211> 455
<212> PRT
<213> Homo Sapien
<400> 4
Met Ser Phe teu Ile Asp Ser Ser Ile Met Ile Thr Ser Gln Ile
1 5 10 15
Leu Phe Phe Gly Phe Gly Trp Leu Phe Phe Met Arg Gln Leu Phe
20 25 30
Lys Asp Tyr Glu Ile Arg Gln Tyr val val Gln val Ile Phe ser
35 40 45
val Thr Phe Ala Phe ser Cys Thr Met Phe Glu Leu Ile Ile Phe
50 55 60
Glu Ile Leu Gly Val Leu Asn Ser Ser Ser Arg Tyr Phe His Trp
65 70 75
Lys Met Asn Leu Cys Val Ile Leu Leu Ile Leu Val Phe Met Val
80 85 90
Pro Phe Tyr I12 Gly Tyr Phe Ile Val Ser Asn Ile Arg Leu Leu
95 100 105
His Lys Gln Arg Leu Leu Phe Ser Cys Leu Leu Trp Leu Thr Phe
110 115 120
Met Tyr Phe Phe Trp Lys Leu Gly Asp Pro Phe Pro Ile Leu Ser
125 130 135
Pro Lys His Gly Ile Leu Ser Ile Glu Gln Leu Ile Ser Arg Val
140 145 150
Gly val Ile Gly val Thr Leu Met Ala Leu Leu ser Gly Phe Gly
155 160 165
Ala val Asn Cys Pro Tyr Thr Tyr Met Ser Tyr Phe Leu Arg Asn
170 175 180
Val Thr Asp Thr Asp Ile Leu Ala Leu Glu Arg Arg Leu Leu Gln
Page 5
CA 02481756 2004-10-25
PCT-US00-23328_Secp epee
185 190 195
Thr Met Asp Met Ile Ile Ser Lys Lys Lys Arg Met Ala Met Ala
200 205 210
Arg Arg Thr Met Phe Gln Lys Gly Glu Val His Asn Lys Pro Ser
2I5 220 225
Gly Phe Trp Gly Met Ile Lys Ser Val Thr Thr Ser Ala Ser Gly
230 235 240
Ser Glu Asn Leu Thr Leu Ile Gln Gln Glu Val Asp Ala Leu Glu
245 250 255
Glu Leu Ser Arg Gln Leu Phe Leu Glu Thr Ala Asp Leu Tyr Ala
260 265 270
Thr Lys Glu Arg Ile Glu Tyr Ser Lys Thr Phe Lys Gly Lys Tyr
275 280 285
Phe Asn Phe Leu Gly Tyr Phe Phe Ser Ile Tyr Cys Val Trp Lys
290 295 300
Ile Phe Met Ala Thr Ile Asn Ile Val Phe Asp Arg Val Gly Lys
305 310 315
Thr Asp Pro Val Thr Arg Gly I12 Glu I1e Thr Val Asn Tyr Leu
320 325 330
Gly Ile Gln Phe Asp Val Lys Phe Trp Ser Gln His Ile Ser Phe
335 ~ 340 345
Ile Leu val Gly Ile Ile Ile Val Thr Ser Ile Arg Gly Leu Leu
350 355 360
Ile Thr Leu Thr Lys Phe Phe Tyr Ala Ile Ser Ser Ser Lys ser
365 370 375
Ser Asn Val Ile Val Leu Leu Leu Ala Gln Ile Met Gly Met Tyr
380 385 390
Phe val Ser Ser val Leu Leu Ile Arg Met ser Met Pro Leu Glu
395 400 405
Tyr Arg Thr Ile Ile Thr Glu Val Leu Gly Glu Leu Gln Phe Asn
410 415 420
Phe Tyr His Arg Trp Phe Asp Val Ile Phe Leu Val Ser Ala Leu
425 430 435
Ser Ser Ile Leu Phe Leu Tyr Leu Ala His Lys Gln Ala Pro Glu
440 445 450
Lys Gln Met Aia Pro
455
<210> 5
<211> 2372
<212> DNA
<213> Homo Sapien
<400> 5
agcagggaaa tccggatgtc tcggttatga agtggagcag tgagtgtgag 50
Page 6
CA 02481756 2004-10-25
PCT-us00-23328_sequence
cctcaacata gttccagaac tctccatccg gactagttat tgagcatctg 100
cctctcatat caccagtggc catctgaggt gtttccctgg ctctgaaggg 150
gtaggcacga tggccaggtg cttcagcctg gtgttgcttc tcacttccat 200
ctggaccacg aggctcctgg tccaaggctc tttgcgtgca gaagagcttt 250
ccatccaggt gtcatgcaga attatgggga tcacccttgt gagcaaaaag 300
gcgaaccagc agctgaattt cacagaagct aaggaggcct gtaggctgct 350
gggactaagt ttggccggca aggaccaagt tgaaacagcc ttgaaagcta 400
gctttgaaac ttgcagctat ggctgggttg gagatggatt cgtggtcatc 450
tctaggatta gcccaaaccc caagtgtggg aaaaatgggg tgggtgtcct 500
gatttggaag gttccagtga gccgacagtt tgcagcctat tgttacaact 550
catctgatac ttggactaac tcgtgcattc cagaaattat caccaccaaa 600
gatcccatat tcaacactca aactgcaaca caaacaacag aatttattgt 650
cagtgacagt acctactcgg tggcatcccc ttactctaca atacctgccc 700
ctactactac tcctcctgct ccagcttcca cttctattcc acggagaaaa 750
aaattgattt gtgtcacaga agtttttatg gaaactagca ccatgtctac 800
agaaactgaa ccatttgttg aaaataaagc agcattcaag aatgaagctg 850
ctgggtttgg aggtgtcccc acggctctgc tagtgcttgc tctcctcttc 900
tttggtgctg cagctggtct tggattttgc tatgtcaaaa ggtatgtgaa 950
ggccttccct tttacaaaca agaatcagca gaaggaaatg atcgaaacca 1000
aagtagtaaa ggaggagaag gccaatgata gcaaccctaa tgaggaatca 1050
aagaaaactg ataaaaaccc agaagagtcc aagagtccaa gcaaaactac 1100
cgtgcgatgc ctggaagctg aagtttagat gagacagaaa tgaggagaca 1150
cacctgaggc tggtttcttt catgctcctt accctgcccc agctggggaa 1200
atcaaaaggg ccaaagaacc aaagaagaaa gtccaccctt ggttcctaac 1250
tggaatcagc tcaggactgc cattggacta tggagtgcac caaagagaat 1300
gcccttctcc ttattgtaac cctgtctgga tcctatcctc ctacctccaa 1350
agcttcccac ggcctttcta gcctggctat gtcctaataa tatcccactg 1400
ggagaaagga gttttgcaaa gtgcaaggac ctaaaacatc tcatcagtat 1450
ccagtggtaa aaaggcctcc tggctgtctg aggctaggtg ggttgaaagc 1500
caaggagtca ctgagaccaa ggctttctct actgattccg cagctcagac 1550
cctttcttca gctctgaaag agaaacacgt atcccacctg acatgtcctt 1600
Page 7
., ~ . ,.. .:a v a .., _mw "., .:_m' , .v;a. a::. .. u~"aam~w,. .. mvxm* . .
"xna..t . ~:..,~..YP9° ° ~>~,~..m ~.~....,_.., . "."..."",
.,n.,.xmnnvr . "..-.-. ,..._.. ~.-. .........___
CA 02481756 2004-10-25
PCT-uS00-23328_Seguence
ctgagcccgg taagagcaaa agaatggcag aaaagtttag cccctgaaag 1650
ccatggagat tctcataact tgagacctaa tctctgtaaa gctaaaataa 1700
agaaatagaa caaggctgag gatacgacag tacactgtca gcagggactg 1750
taaacacaga cagggtcaaa gtgttttctc tgaacacatt gagttggaat 1800
cactgtttag aacacacaca cttacttttt ctggtctcta ccactgctga 1850
tattttctct aggaaatata cttttacaag taacaaaaat aaaaactctt 1900
ataaatttct atttttatct gagttacaga aatgattact aaggaagatt 1950
actcagtaat ttgtttaaaa agtaataaaa ttcaacaaac atttgctgaa 2000
tagctactat atgtcaagtg ctgtgcaagg tattacactc tgtaattgaa 2050
tattattcct caaaaaattg cacatagtag aacgctatct gggaagctat 2100
ttttttcagt tttgatattt ctagcttatc tacttceaaa ctaattttta 2150
tttttgctga gactaatctt attcattttc tctaatatgg caaccattat 2200
aaccttaatt tattattaac atacctaaga agtacattgt tacctctata 2250
taccaaagca cattttaaaa gtgccattaa caaatgtatc actagccctc 2300
ctttttccaa caagaaggga ctgagagatg cagaaatatt tgtgacaaaa 2350
aattaaagca tttagaaaac tt 2372
<210> 6
<211> 322
<212> PRT
<213> Homo Sapien
<400> '6
Met Ala Arg Cys Phe Ser Leu Val Leu Leu Leu Thr Ser Ile Trp
1 5 20 15
Thr Thr Arg Leu Leu Val Gln Gly Ser Leu Arg Ala Glu Glu Leu
20 25 3a
Ser Ile Gln val Ser Cys Arg Ile Met Gly Ile Thr Leu val Ser
35 40 45
Lys Lys Ala Asn Gln Gln Leu Asn Phe Thr Glu Ala Lys Glu Ala
50 55 60
Cys Arg Leu Leu Gly Leu Ser Leu Ala Gly Lys Asp Gln Val Glu
65 70 75
Thr Ala Leu Lys Ala Ser Phe Glu Thr Cys Ser Tyr Giy Trp Val
80 85 90
Gly Asp G1y Phe Val Val Ile Ser Arg Ile Ser Pro Asn Pro Lys
95 100 105
Cys Gly Lys Asn Gly Val Gly Val Leu I1e Trp Lys Val Pro Val
110 115 120
Page 8
._ s~.. . ..n.-._~__._ .._ .~~ . _ -.-~.--_
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Ser Arg Gln Phe Ala Ala Tyr Cys Tyr Asn Ser Ser Asp Thr Trp
1.25 130 135
Thr Asn Ser Cys Ile Pro Glu Ile Ile Thr Thr Lys Asp Pro Ile
140 145 150
Phe Asn Thr Gln Thr Ala Thr Gln Thr Thr Glu Phe I12 Val Ser
155 160 165
Asp Ser Thr Tyr Ser Val Ala Ser Pro Tyr Ser Thr Ile Pro Ala
170 175 180
Pro Thr Thr Thr Pro Pro Ala Pro Ala Ser Thr Ser Ile Pro Arg
185 190 195
Arg Lys Lys Leu Ile Cys Val Thr Glu Val Phe Met Glu Thr Ser
200 205 210
Thr Met Ser Thr Glu Thr Glu Pro Phe Val Glu Asn Lys Ala Ala
215 220 225
Phe Lys Asn Glu Ala Ala Gly Phe Gly Gly Val Pro Thr Ala Leu
230 235 240
Leu Val Leu Ala Leu Leu Phe Phe Gly Ala Ala Ala Gly Leu Gly
245 250 255
Phe Cys Tyr Val Lys Arg Tyr Val Lys Ala Phe Pro Phe Thr Asn
260 265 270
Lys Asn Gln Gln Lys Glu Met Ile Glu Thr Lys Val Val Lys Glu
275 280 285
Glu Lys Ala Asn Asp Ser Asn Pro Asn Glu Glu Ser Lys Lys Thr
290 295 300
Asp Lys Asn Pro Glu Glu Ser Lys Ser Pro Ser Lys Thr Thr Val
305 310 315
Arg Cys Leu Glu Ala Glu Val
320
<210> 7
<211> 2586
<212> DNA
<213> Homo Sapien
<400> 7
cgccgcgctc ccgcacccgc ggcccgccca ccgcgccgct cccgcatctg 50
cacccgcagc ccggcggcct cccggcggga gcgagcagat ccagtccggc 100
ccgcagcgca actcggtcca gtcggggcgg cggctgcggg cgcagagcgg 150
agatgcagcg gcttggggcc accctgctgt gcctgctgct ggcggcggcg 200
gtccccacgg cccccgcgcc cgctccgacg gcgacctcgg ctccagtcaa 250
gcccggcccg gctctcagct acccgcagga ggaggccacc cteaatgaga 300
tgttccgcga ggttgaggaa ctgatggagg acacgcagca caaattgcgc 350
Page 9
CA 02481756 2004-10-25
PcT-uS00-23328_Sequence
agcgcggtgg aagagatgga ggcagaagaa gctgctgcta aagcatcatc 400
agaagtgaac ctggcaaact tacctcccag ctatcacaat gagaccaaca 450
cagacacgaa ggttggaaat aataccatcc atgtgcaccg agaaattcac 500
aagataacca acaaccaga.c tggacaaatg gtcttttcag agacagttat 550
cacatctgtg ggagacgaag aaggcagaag gagccacgag tgcatcatcg 600
acgaggactg tgggcccagc atgtactgcc agtttgccag cttccagtac 650
acctgccagc catgccgggg ccagaggatg ctctgcaccc gggacagtga 700
gtgctgtgga gaccagctgt gtgtctgggg tcactgcacc aaaatggcca 750
ccaggggcag caatgggacc atctgtgaca accagaggga ctgccagccg 800
gggctgtgct gtgccttcca gagaggcctg ctgttccctg tgtgcacacc 850
cctgcccgtg gagggcgagc tttgccatga ccccgccagc cggcttctgg 900
acctcatcac ctgggagcta gagcctgatg gagccttgga ccgatgccct 950 .
tgtgccagtg gcctcctctg ccagccccac agccacagcc.tggtgtatgt 1000
gtgcaagccg accttcgtgg ggagccgtga ccaagatggg gagatcctgc 1050
tgcccagaga ggtccccgat gagtatgaag ttggcagctt catggaggag 1100
gtgcgccagg agctggagga cctggagagg agcctgactg aagagatggc 1150
gctgggggag cctgcggctg ccgccgctgc actgctggga ggggaagaga 1200
tttagatctg gaccaggctg tgggtagatg tgcaatagaa atagctaatt 1250
tatttcccca ggtgtgtgct ttaggcgtgg gctgaccagg cttcttctta 1300
catcttcttc ccagtaagtt tcccctctgg cttgacagca tgaggtgttg 1350
tgcatttgtt cagctccccc aggctgttct ccaggcttca cagtctggtg 1400
cttgggagag tcaggcaggg ttaaactgca ggagcagttt gccacccctg 1450
tccagattat tggctgcttt gcctctacca gttggcagac agccgtttgt 1500
tctacatggc tttgataatt gtttgagggg aggagatgga aacaatgtgg 1550
agtctccctc tgattggttt tggggaaatg tggagaagag tgccctgctt 1600
tgcaaacatc aacctggcaa aaatgcaaca aatgaatttt ccacgcagtt 1650
ctttccatgg gcataggtaa gctgtgcctt cagctgttgc agatgaaatg 1700
ttctgttcac cctgcattac atgtgtttat tcatccagca gtgttgctca 1750
gctcctacct ctgtgccagg gcagcatttt catatccaag atcaattccc 1800
tctctcagca cagcctgggg agggggtcat tgttctcctc: gtccatcagg 1850
gatctcagag gctcagagac tgcaagctgc ttgcccaagt cacacagcta 1900
Page 10
A~..,.. . . a .:~.,~,u,. . .. , a...., . , _ .. ,M,.. n . r,m w , .~ n ~
~,a"~, -~~, ~~r ~,.~.~a . .r... ,._.~ . ~~.,... ._~..~.~. ... . ... _
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
gtgaagacca gagcagtttc atctggttgt gactctaagc tcagtgctct 1950
ctccactacc ccacaccagc cttggtgcca ccaaaagtgc tccccaaaag 2000
gaaggagaat gggatttttc ttgaggcatg cacatctgga attaaggtca 2050
aactaattct cacatccctc taaaagtaaa ctactgttag gaacagcagt 2100
gttctcacag tgtggggcag ccgtccttct aatgaagaca atgatattga 2150
cactgtccct ctttggcagt tgcattagta actttgaaag gtatatgact 2200
gagcgtagca tacaggttaa cctgcagaaa cagtacttag gtaattgtag 2250
ggcgaggatt ataaatgaaa tttgcaaaat cacttagcag caactgaaga 2300
caattatcaa ccacgtggag aaaatcaaac cgagcagggc tgtgtgaaac 2350
atggttgtaa tatgcgactg cgaacactga actctacgcc actccacaaa 2400
tgatgttttc aggtgtcatg gactgttgcc accatgtatt catccagagt 2450
tcttaaagtt taaagttgca catgattgta taagcatgct ttctttgagt 2500
tttaaattat gtataaacat aagttgcatt tagaaatcaa gcataaatca 2550
cttcaactgc aaaaaaaaaa aaaaaaaaaa aaaaaa 2586
<210> 8
<211> 350
<212> PRT
<213> Homo sapien
<400> 8
Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala
1 5 10 15
Ala Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala
20 25 30
Pro Val Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala
35 40 45
Thr Leu Asn Glu Met Phe Arg Glu val Glu Glu Leu Met Glu Asp
50 55 60
Thr Gln His Lys Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu
65 70 75
Glu Ala Ala Ala Lys Ala Ser Ser Glu Val Asn Leu Ala Asn Leu
80 85 90
Pro Pro Ser Tyr His ~4sn Glu Thr Asn Thr Asp Thr Lys Val Gly
95 100 105
Asn A5n Thr Ile His i/al His Arg Glu Ile His Lys Ile Thr Asn
110 115 120
Asn Gln Thr Gly Gln Met Val Phe Ser Glu Thr Val Ile Thr Ser
125 130 135
Val Gly Asp Glu Glu Gly Arg Arg Ser His Glu Cys Ile Ile Asp
140 145 150
Page 11
a
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln Phe Ala Ser Phe Gln
155 160 165
Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met Leu Cys Thr Arg
170 175 180
Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp Gly His Cys
185 190 195
Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys Asp Asn
200 205 210
Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg Gly
215 220 225
Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
230 235 240
Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu
245 250 255
Leu Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly
260 265 270
Leu Leu Cys Gln Fro His Ser His Ser Leu val Tyr Val Cys Lys
275 280 285
Pro Thr Phe Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu
290 295 300
Pro Arg Glu Val Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu
305 310 315
Glu Val Arg Gln Glu Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu
320 325 330
Glu Met Ala Leu Gly Glu Pro Ala Ala Ala Ala Ala Ala Leu Leu
335 340 345
Gly Gly Glu Glu Ile
350
<210> 9
<211> 1395
<212> DNA
<213> Homo Sapien
<400> 9
cggacgcgtg ggcggacgcg tgggggctgt gagaaagtgc caataaatac 50
atcatgcaac cccacggccc accttgtgaa ctcctcgtgc ccagggctga 100
tgtgcgtctt ccagggctae tcatccaaag gcctaatcca acgttctgtc 150
ttcaatctgc aaatctatgg ggtcctgggg ctcttctgga cccttaactg 200
ggtactggcc ctgggccaat gcgtcctcgc tggagccttt gcctccttct 250
actgggcctt ccacaagccc caggacatcc ctaccttccc cttaatctct 300
gccttcatcc gcacactccg ttaccacact gggtcattgg catttggagc 350
Page 12 .
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
cctcatcctg acccttgtgc agatagcccg ggtcatcttg gagtatattg 400
accacaagct cagaggagtg cagaaccctg tagcccgctg catcatgtgc 450
tgtttcaagt gctgcctctg gtgtctggaa aaatttatca agttcctaaa 500
ccgcaatgca tacatcatga tcgccatcta cgggaagaat ttctgtgtct 550
cagccaaaaa tgcgttcatg ctactcatgc gaaacattgt cagggtggtc 600
gtcctggaca aagtcacaga cctgctgctg ttctttggga agctgctggt 650
ggtcggaggc gtgggggtcc tgtccttctt ttttttctcc ggtcgcatcc 700
cggggctggg taaagacttt aagagccccc acctcaacta ttactggctg 750
cccatcatga cctccatcct gggggcctat gtcatcgcca gcggcttctt 800
cagcgttttc ggcatgtgtg tggacacgct cttcctctgc ttcctggaag 850
acctggagcg gaacaacggc tccctggacc ggccctacta catgtccaag 900
agccttctaa agattctggg caagaagaac gaggcgcccc cggacaacaa 950
gaagaggaag aagtgacagc tccggccctg atccaggact gcaccccacc 1000
cccaccgtcc agccatccaa cctcacttcg ccttacaggt ctccattttg 1050
tggtaaaaaa aggttttagg ccaggcgccg tggctcacgc ctgtaatcca 1100
acactttgag aggctgaggc gggcggatca cctgagtcag gagttcgaga 1150
ccagcctggc caacatggtg aaacctccgt ctctattaaa aatacaaaaa 1200
ttagccgaga gtggtggcat gcacctgtca tcccagctac tcgggaggct 1250
gaggcaggag aatcgcttga acccgggagg cagaggttgc agtgagccga 1300
gatcgcgcca ctgcactcca acctgggtga cagactctgt ctccaaaaca 1350
aaacaaacaa acaaaaagat tttattaaag atattttgtt aactc 1395
<210> 10
<211> 321
<212> PRT
<213> Homo sapien
<400> 10
Arg Thr Arg Gly Arg Thr Arg Gly Gly Cys Glu Lys Val Pro Ile
1 5 10 15
Asn Thr Ser Cys Asn Pro Thr Ala His Leu Val Asn Ser Ser Cys
20 25 30
Pro Gly Leu Met Cys Val Phe Gln Gly Tyr Ser Ser Lys Gly Leu
35 40 45
Ile Gln Arg Ser Val Phe ASn Leu Gln Ile Tyr Gly Val Leu Gly
50 55 60
Leu Phe Trp Thr Leu Asn Trp Val Leu Ala Leu Gly Gln Cys Val
Page 13
.".__ . " .. . .a, ,. ~ .. .._._.,.~ .~n...... , . _. . w.._ r_,w,
,~w,~.~~u~~,w,~,,~.~~,~;"~., ,~"~~,~" "~""" ~~"~,~ .~,.~AY..",. "~.a__.._._
_... _.,.. t
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
65 70 75
Leu Ala Gly Ala Phe Ala Ser Phe Tyr Trp Ala Phe His Lys Pro
80 85 90
Gln Asp Ile Pro Thr Phe Pro Leu Il2 Ser Ala Phe Ile Arg Thr
95 100 105
Leu Arg Tyr His Thr Gly Ser Leu Ala Phe Giy Ala Leu Ile Leu
110 115 120
Thr Leu Val Gln Ile Ala Arg Val Ile Leu Glu Tyr Ile Asp His
125 130 135
Lys Leu Arg Gly Val Gln Asn Pro Val Ala Arg Cys Ile Met Cys
140 145 150
Cys Phe Lys Cys Cys Leu Trp Cys Leu Glu Lys Phe Ile Lys Phe
155 160 165
Leu Asn Arg Asn Ala Tyr Ile Met Ile Ala Ile Tyr Gly Lys Asn
170 175 180
Phe Cys Val Ser Ala Lys Asn Ala Phe Met Leu Leu Met Arg Asn
185 190 195
Ile Val Arg Val Val Val Leu Asp Lys Val Thr Asp Leu Leu Leu
200 205 210
Phe Phe Gly Lys Leu Leu Val Val Gly Gly Val Gly Val Leu Ser
215 220 225
Phe Phe Phe Phe ser Gly Arg Ile Pro Gly Leu Gly Lys Asp Phe
230 235 240
Lys 5er Pro His Leu Asn Tyr Tyr Trp Leu Pro Ile Met Thr Ser
245 250 255
Ile Leu Gly Ala Tyr Val I12 Ala Ser Gly Phe Phe Ser Val Phe
260 265 270
Gly Met Cys Val Asp Thr Leu Phe Leu Cys Phe Leu Glu Asp Leu
275 280 285
Glu Arg Asn Asn Gly Ser Leu ASp Arg Pro Tyr Tyr Met Ser Lys
290 295 300
Ser Leu Leu Lys Ile Leu Gly Lys Lys Asn Glu Ala Pro Pro Asp
305 310 315
Asn Lys ~ys Arg Lys Lys
320
<210> 11
<211> 1901
<212> DNA
<213> Homo Sapien
<400> 11
gccccgcgcc cggcgccggg cgcccgaagc cgggagccac: cgccatgggg 50
gcctgcctgg gagcctgctc cctgctcagc tgcgcgtcct gcctctgcgg 100
Page 14
1
CA 02481756 2004-10-25
PCT~uS00-23328_Sequence
ctctgccccc tgcatcctgt gcagctgctg ccccgccagc cgcaactcca 150
ccgtgagccg cctcatcttc acgttcttcc tcttcctggg ggtgctggtg 200
tccatcatta tgctgagccc gggcgtggag agtcagctct acaagctgcc 250
ctgggtgtgt gaggaggggg ccgggatccc caccgtcctg cagggccaca 300
tcgactgtgg ctccctgctt ggctaccgcg ctgtctaccg catgtgcttc 350
gccacggcgg ccttcttctt cttctttttc accctgctca tgctctgcgt 400
gagcagcagc cgggaccccc gggctgccat ccagaatggg ttttggttct 450
ttaagttcct gatcctggtg ggcctcaccg tgggtgcctt ctacatccct 500
gacggctcct tcaccaacat ctggttctac ttcggcgtcg tgggctcctt 550
cctcttcatc ctcatccagc tggtgctgct catcgacttt gcgcactcct 600
ggaaccagcg gtggctgggc aaggccgagg agtgcgattc ccgtgcctgg 650
tacgcaggcc tcttcttctt cactctcctc ttctacttgc tgtcgatcgc 700
ggccgtggcg ctgatgttca tgtactacac tgagcccagc ggctgccacg 750
agggcaaggt cttcatcagc ctcaacctca ccttctgtgt ctgcgtgtcc 800
atcgctgctg tcctgcccaa ggtccaggac gcccagccca actcgggtct 850
gctgcaggcc tcggtcatca ccctctacac catgtttgtc acctggtcag 900
ccctatccag tatccctgaa cagaaatgca acccccattt gccaacccag 950
ctgggcaacg agacagttgt ggcaggcccc gagggctatg agacccagtg 1000
gtgggatgcc ccgagcattg tgggcctcat catcttcctc ctgtgcaccc 1050
tcttcatcag tctgcgctcc tcagaccacc ggcaggtgaa cagcctgatg 1100
cagaccgagg agtgcccacc tatgctagac gccacacagc agcagcagca 1150
gcaggtggca gcctgtgagg gccgggcctt tgacaacgag caggacggcg 1200
tcacctacag ctactccttc ttccacttct gcctggtgct ggcctcactg 1250
cacgtcatga tgacgctcac caactggtac aagcccggtg agacccggaa 1300
gatgatcagc acgtggaccg ccgtgtgggt gaagatctgt gccagctggg 1350
cagggctgct cctctacctg tggaccctgg tagccccact cctcctgcgc 1400
aaccgcgact tcagctgagg cagcctcaca gcctgccatc tggtgcctcc 1450
tgccacctgg tgcctctcgg ctcggtgaca gccaacctgc cccctcccca 1500 _.
caccaatcag ccaggctgag cccccacccc tgccccagct ccaggacctg 1550
cccctgagcc gggccttcta gtcgtagtgc cttcagggtr_ cgaggagcat 1600
page 15
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
caggctcctg cagagcccca tccccccgcc acacccacac ggtggagctg 1650
cctcttcctt cccctcctcc ctgttgccca tactcagcat ctcggatgaa 1700
agggctccct tgtcctcagg ctccacggga gcggggctgc tggagagagc 1750
ggggaactcc caccacagtg gggcatccgg cactgaagcc ctggtgttcc 1800
tggtcacgtc ccccagggga ccctgccccc ttcctggact tcgtgcctta 1850
ctgagtctct aagacttttt ctaataaaca agccagtgcg tgtaaaaaaa 1900
a 1901
<210> 12
<211> 457
<212> PRT
<213> Homo Sapien
<400> 12
Met Gly Ala Cys Leu Gly Ala Cys Ser Leu Leu Ser Cys Ala Ser
1 5 10 15
Cys Leu Cys Gly Ser Ala Pro Cys Ile Leu Cys Ser Cys Cys Pro
20 25 30
Ala Ser Arg Asn ser Thr val ser Arg Leu Ile Phe Thr Phe Phe
35 40 45
Leu Phe ~eu Gly val Leu val Ser Ile Ile Met Leu Ser Pro Gly
50 55 60
Val Glu Ser Gln Leu Tyr Lys Leu Pro Trp Val Cys Glu Glu Gly
65 70 75.
Ala Gly Ile Pro Thr Val Leu Gln Gly His Ile Asp Cys Gly Ser
80 85 90
Leu Leu Gly Tyr arg Ala val Tyr Arg Met Cys Phe Ala Thr Ala
95 100 105
Ala Phe Phe Phe Phe Phe Phe Thr Leu Leu Met Leu Cys Val Ser
110 115 120
Ser Ser Arg Asp Pro Arg Ala Ala Ile Gln Asn Gly Phe Trp Phe
125 130 135
Phe Lys Phe Leu Ile Leu Val Gly Leu Thr Val Gly Ala Phe~Tyr
140 145 150
Ile Pro Asp Gly Ser Phe Thr Asn Tle Trp Phe Tyr Phe Gly Val
155 160 165
Val Gly Ser Phe Leu Phe Ile Leu Ile Gln Leu Val Leu Leu Ile
170 175 180
Asp Phe Ala His Ser Trp Asn Gln Arg Trp Leu Gly Lys Ala Glu
185 190 195
Glu Cys Asp Ser Arg Ala Trp Tyr Ala Gly Leu Phe Phe Phe Thr
200 205 210
Page 16
__T__. _- _.~ ._ __
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
Leu Leu Phe Tyr Leu Leu Ser Ile Ala Ala Val Ala Leu Met Phe
215 220 225
Met Tyr Tyr Thr Glu Pro Ser Gly Cys His Glu Gly Lys Val Phe
230 235 240
Ile Ser Leu Asn Leu Thr Phe Cys Val Cys Val Ser Ile Ala Ala
245 250 255
Val Leu Pro Lys Val Gln Asp Ala Gln Pro Asn Ser Gly Leu Leu
260 265 270
Gln Ala Ser Val Ile Thr Leu Tyr Thr Met Phe Val Thr Trp Ser
275 280 285
Ala Leu Ser Ser Ile Pro Glu Gln Lys Cys Asn Pro His Leu Pro
290 295 300
Thr Gln Leu Gly Asn Glu Thr Val Val Ala Gly Pro Glu Gly Tyr
305 310 315
Glu Thr Gln Trp Trp Asp Ala Pro Ser Ile Val Gly Leu Ile Ile
320 325 330
Phe Leu Leu Cys Thr Leu Phe Ile Ser Leu Arg Ser Ser Asp His
335 340 345
Arg Gln Val Asn Ser Leu Met Gln Thr Glu Glu Cys Pro Pro Met
350 355 360
Leu Asp Ala Thr Gln Gln Gln Gln Gln Gln Val Ala Ala Cys Glu
365 370 375
Gly Arg Ala Phe Asp Asn Glu Gln Asp Gly Val Thr Tyr Ser Tyr
380 385 390
Ser Phe Phe His Phe Cys Leu Val Leu Ala Ser Leu His Val Met
395 400 405
Met Thr Leu Thr Asn Trp Tyr Lys Pro Gly Glu Thr Arg l.ys Met
410 415 420
Ile Ser Thr Trp Thr Ala Val Trp val Lys Ile Cys Ala Ser Trp
425 430 435
Ala Gly Leu Leu Leu Tyr Leu Trp Thr Leu val Ala Pro Leu Leu
440 445 450
Leu Arg Asn Arg Asp Phe Ser
455
<210> 13
<211> 1572
<212> DNA
<213> Homo Sapien
<400> 13
cgggccagcc tggggcggcc ggccaggaac cacccgttaa ggtgtcttct 50
ctttagggat ggtgaggttg gaaaaagact cctgtaaccc tcctccagga 100
tgaaccacct gccagaagac atggagaacg ctctcaccgg gagccagagc 150
Page 17
CA 02481756 2004-10-25
PC'r-uS00-23328_Sequence
tcccatgctt ctctgcgcaa tatccattcc atcaacccca cacaactcat 200
ggccaggatt gagtcctatg aaggaaggga aaagaaaggc atatctgatg 250
tcaggaggac tttctgtttg tttgtcacct ttgacctctt attcgtaaca 300
ttactgtgga taatagagtt aaatgtgaat ggaggcattg agaacacatt 350
agagaaggag gtgatgcagt atgactacta ttcttcatat tttgatatat 400
ttcttctggc agtttttcga tttaaagtgt taatacttgc atatgctgtg 450
tgcagactgc gccattggtg ggcaatagcg ttgacaacgg cagtgaccag 500
tgccttttta ctagcaaaag tgatcctttc gaagcttttc tctcaagggg 550
cttttggcta tgtgctgccc atcatttcat tcatccttgc ctggattgag 600
acgtggttcc tggatttcaa agtgttacct caagaagcag aagaagaaaa 650
cagactcctg atagttcagg atgcttcaga gagggcagca cttatacctg 700
gtggtctttc tgatggtcag ttttattccc ctcctgaatc cgaagcagga 750
tctgaagaag ctgaagaaaa acaggacagt gagaaaccac ttttagaact 800
atgagtacta cttttgttaa atgtgaaaaa ccctcacaga aagtcatcga 850
ggcaaaaaga ggcaggcagt ggagtctccc tgtcgacagt aaagttgaaa 900
tggtgacgtc cactgctggc tttattgaac agctaataaa gatttattta 950
ttgtaatacc tcacaaacgt tgtaccatat ccatgcacat ttagttgcct 1000
gcctgtggct ggtaaggtaa tgtcatgatt catcctctct tcagtgagac 1050
tgagcctgat gtgttaacaa ataggtgaag aaagtcttgt gctgtattcc 1100
taatcaaaag acttaatata ttgaagtaac acttttttag taagcaagat 1150
acctttttat ttcaattcac agaatggaat ttttttgttt catgtctcag 1200
atttattttg tatttctttt ttaacactct acatttccct tgttttttaa 1250
ctcatgcaca tgtgctcttt gtacagtttt aaaaagtgta ataaaatctg 1300
acatgtcaat gtggctagtt ttatttttct tgttttgcat tatgtgtatg 1350
gcctgaagtg ttggacttgc aaaaggggaa gaaaggaatt gcgaatacat 1400
gtaaaatgtc accagacatt tgtattattt ttatcatgaa atcatgtttt 1450
tctctgattg ttctgaaatg ttctaaatac tcttattttg aatgcacaaa 1500
atgacttaaa ccattcatat catgtttcct ttgcgttcag ccaatttcaa 1550
ttaaaatgaa ctaaattaaa as 1572
<210> 14
<211> 234
<212> PRT
<213> Homo Sapien
Page 18
CA 02481756 2004-10-25
PCT-u500-23328_sequence
<400>
14
Met AsnHisLeu ProGluAsp MetGluAsn AlaLeu ThrGlySer
1 5 10 15
Gln SerSerHis AlaSerLeu ArgAsnIle HisSer IleAsnPro
20 25 30
Thr GlnLeuMet AlaArgIle GiuSerTyr GluGly ArgGluLys
35 40 45
Lys GlyIleSer AspValArg ArgThrPhe CysLeu PheValrthr
50 55 60
Phe AspLeuLeu PheValThr LeuLeuTrp I12Ile GluLeuAsn
65 70 75
Val AsnGlyGly IleGluAsn ThrLeuGlu LysGlu ValMetGln
80 85 90
Tyr AspTyrTyr SerSerTyr PheAspIle PheLeu LeuAlaVal
95 100 105
Phe ArgPheLys ValLeuIle LeuAlaTyr AlaVal CysArgLeu
110 115 120
Arg HisTrpTrp AlaIleAla LeuThrThr AlaVal ThrSerAla
125 130 135.
Phe LeuLeuAla LysValIle LeuSerLys LeuPhe SerGlnGly
140 145 150
Ala PheGlyTyr ValLeuPro IleIleSer PheIle LeuAlaTrp
155 160 165
Ile GluThrTrp PheLeuAsp PheLysVal LeuPro GlnGluAla
170 175 180
Glu GluGluAsn ArgLeuLeu IleValGln AspAla SerGluArg
185 190 195
Ala AlaLeuIle ProGlyGly LeuSerAsp G1yGln PheTyrSer
200 205 210
Pro ProGluSer GluAlaGly SerGluGlu AlaGlu GluLysGln
215 220 225
Asp SerGluLys ProLeuLeu GluLeu
230
<210> 15
<211> 2768
<212> DNA
<213> Homo Sapien
<400> 15
actcgaacgc agttgcttcg ggacccagga ccccctcggg cccgacccgc 50
caggaaagac tgaggccgcg gcctgccccg cccggctccc tgcgccgccg 100
ccgcctcccg ggacagaaga tgtgctccag ggtccctctg etgctgccgc 150
tgctcctgct actggccctg gggcctgggg tgcagggctg cccatccggc 200
Page 19
..., w. . -a.~,:x r.~ ... x. .,..,.."-. .. ,,.,...~... ., " ..xm. .~w ....~..,
a am,xm. ~": x-c...~:.. waa~nr~,m=:.ate., =...m........ _ ......_. ,.
.."..,_.""" , ......."...._.. -..,...
CA 02481756 2004-10-25
PcT-us00-23328_sequence
tgccagtgca gccagccaca gacagtcttc tgcactgccc gccaggggac 250
cacggtgccc cgagacgtgc cacccgacac ggtggggctg tacgtctttg 300
agaacggcat caccatgctc gacgcaggca gctttgccgg cctgccgggc 350
ctgcagctcc tggacctgtc acagaaccag atcgccagcc tgcccagcgg 400
ggtctteeag ccaetegcca acctcagcaa cetggacetg acggceaaca 450
ggctgcatga aatcaccaat gagaccttcc gtggcctgcg gcgcctcgag 500
cgcctctacc tgggcaagaa ccgcatccgc cacatccagc ctggtgcctt 550
cgacacgctc gaccgcctcc tggagctcaa gctgcaggac aacgagctgc 600
gggcactgcc cccgctgcgc ctgccccgcc tgctgctgct ggacctcagc 650
cacaacagcc tcctggccct ggagcccggc atcctggaca ctgccaacgt 700
ggaggcgctg cggctggctg gtctggggct gcagcagctg gacgaggggc 750
tcttcagccg cttgcgcaac ctccacgacc tggatgtgtc cgacaaccag 800
ctggagcgag tgccacctgt gatccgaggc ctccggggcc tgacgcgcct 850
gcggctggcc ggcaacaccc gcattgccca gctgcggccc gaggacctgg 900
ccggcctggc tgccctgcag gagctggatg tgagcaacct aagcctgcag 950
gccctgcctg gcgacctctc gggcctcttc ccccgcctgc ggctgctggc 1000
agctgcccgc aaccccttca actgcgtgtg ccccctgagc tggtttggcc 1050
cctgggtgcg cgagagccac gtcacactgg ccagccctga ggagacgcgc 1100
tgccacttcc cgcccaagaa cgctggccgg ctgctcctgg agcttgacta 1150
cgccgacttt ggctgcccag ccaccaccac cacagccaca gtgcccacca 1200
cgaggcccgt ggtgcgggag cccacagcct tgtcttctag cttggctcct 1250
acctggctta gccccacagc gccggccact gaggccccca gcccgccctc 1300
cactgcccca ccgactgtag ggcctgtccc ccagccccag gactgcccac 1350
cgtccacctg cctcaatggg ggcacatgcc acctggggac acggcaccac 1400
ctggcgtgct tgtgccccga aggcttcacg ggcctgtact gtgagagcca 1450
gatggggcag gggacacggc ccagccctac accagtcacg ccgaggccac 1500
cacggtccct gaccctgggc atcgagccgg tgagccccac ctccctgcgc 1550
gtggggctgc agcgctacct ccaggggagc tccgtgcagc tcaggagcct 1600
ccgtctcacc tatcgcaacc: tatcgggccc tgataagcgg ctggtgacgc 1650
tgcgactgcc tgcctcgctc gctgagtaca cggtcaccca gctgcggccc 1700
aacgccactt actccgtctg tgtcatgcct ttggggcccg ggcgggtgcc 1750
ggagggcgag gaggcctgcg gggaggccca tacaccccca gccgtccact 1800
Page 20
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
ccaaccacgc cccagtcacc caggcccgcg agggcaacct gccgctcctc 1850
attgcgcccg ccctggccgc ggtgctcctg gccgcgctgg ctgcggtggg 1900
ggcagcctac tgtgtgcggc gggggcgggc catggcagca gcggctcagg 1950
acaaagggca ggtggggcca ggggctgggc ccctggaact ggagggagtg 2000
aaggtcccct tggagccagg cccgaaggca acagagggcg gtggagaggc 2050
cctgcccagc gggtctgagt gtgaggtgcc actcatgggc ttcccagggc 2100
ctggcctcca gtcacccctc cacgcaaagc cctacatcta agccagagag 2150
agacagggca gctggggccg ggctctcagc cagtgagatg gccagccccc 2200
tcctgctgcc acaccacgta agttctcagt cccaacctcg gggatgtgtg 2250
cagacagggc tgtgtgacca cagctgggcc ctgttccctc tggacctcgg 2300
tctcctcatc tgtgagatgc tgtggcccag ctgacgagcc ctaacgtccc 2350
cagaaccgag tgcctatgag gacagtgtcc gccctgccct ccgcaacgtg 2400
cagtccctgg gcacggcggg ccctgccatg tgctggtaac gcatgcctgg 2450
gtcctgctgg gctctcccac tccaggcgga ccctgggggc cagtgaagga 2500
agctcccgga aagagcagag ggagagcggg taggcggctg tgtgactcta 2550
gtcttggccc caggaagcga aggaacaaaa gaaactggaa aggaagatgc 2600
tttaggaaca tgttttgctt ttttaaaata tatatattta taagagatcc 2650
tttcccattt attctgggaa gatgtttttc aaactcagag acaaggactt 2700
tggtttttgt aagacaaacg atgatatgaa ggccttttgt aagaaaaaat 2750
aaaagatgaa gtgtgaaa 2768
<210> 16
<211> 673
<212> PRT
<213> Homo Sapien
<400> 16
Met Cys Ser Arg Val Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu
1 S 10 15
Ala Leu Gly Pro Gly Val Gln Gly Cys Pro Ser Gly Cys Gln Cys
20 25 30
Ser Gln Pro Gln Thr Val Phe Cys Thr Ala Arg Gln Gly Thr Thr
35 40 45
val Pro Arg Asp vat Pro Pro Asp Thr val Gly Leu Tyr val Phe
50 55 60
Glu Asn Gly Ile Thr Met Leu Asp Ala Gly Ser Phe Ala Gly Leu
65 70 75
Pro Gly Leu Gln Leu Leu Asp Leu Ser G1n Asn Gln Ile Ala Ser
Page 21
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
80 85 90
Leu Pro Ser Gly Val Phe Gln Pro Leu Ala Asn Leu Ser Asn Leu
95 100 105
Asp Leu Thr Ala Asn Arg Leu His Glu Ile Thr Asn Glu Thr Phe
110 lI5 120
Arg Giy Leu Arg Arg Leu Glu Arg Leu Tyr Leu Gly Lys Asn Arg
125 130 135
Ile Arg His Ile Gln Pro Gly Ala Phe Asp Thr Leu Asp Arg Leu
140 145 150
Leu Glu Leu Lys Leu Gln Asp Asn Glu Leu Arg Ala Leu Pro Pro
155 160 165
Leu Arg Leu Pro Arg Leu Leu Leu Leu Asp Leu Ser His Asn Ser
170 175 180
Leu Leu Ala Leu Glu Pro Gly Ile Leu Asp Thr Ala Asn Val Glu
185 190 195
Ala Leu Arg Leu Ala Gly Leu Gly Leu Gln Gln Leu Asp Glu Gly
200 205 210
Leu Phe Ser Arg Leu Arg Asn Leu His Asp Leu Asp Val Ser Asp
215 220 225
Asn Gln Leu Glu Arg Val Pro Pro Val Ile Arg Gly Leu Arg Gly
230 235 240
Leu Thr Arg Leu Arg Leu Ala Gly Asn Thr Arg Ile Ala Gln Leu
245 250 255
Arg Pro Glu Asp Leu Ala Gly Leu Ala Ala Leu Gln Glu Leu Asp
260 265 270
Val Ser Asn Leu Ser Leu Gln Ala Leu Pro Gly Asp Leu Ser Gly
275 280 285
Leu Phe Pro Arg Leu Arg Leu Leu Ala Ala Ala Arg Asn Pro Phe
290 295 300
Asn Cys Val Cys Pro Leu Ser Trp Phe Gly Pro Trp Val Arg Glu
305 310 315
Ser His Val Thr Leu Ala Ser Pro Glu Glu Thr Arg Cys His Phe
320 325 33'0
Pro Pro Lys Asn Ala Gly Arg Leu Leu Leu Glu Leu Asp Tyr Ala
335 340 345
Asp Phe Gly Cys Pro Ala Thr Thr Thr Thr Ala Thr Val Pro Thr
350 355 360
Thr Arg Pro Val Val Arg Glu Pro Thr Ala Leu Ser Ser Ser Leu
365 370 375
Ala Pro Thr Trp Leu Ser Pro Thr Ala Pro Ala Thr Glu Ala Pro
380 385 390
Ser Pro Pro Ser Thr Ala Pro Pro Thr Val Gly Pro Val Pro Gln
Page 22
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
395 400 405
Pro Gln Asp Cys Pro Pro Ser Thr Cys Leu Asn Gly Gly Thr Cys
410 415 420
His Leu Gly Thr Arg His His Leu Ala Cys Leu Cys Pro Glu Gly
425 430 435
Phe Thr Gly Leu Tyr Cys Glu Ser Gln Met Gly Gln Gly Thr Arg
440 445 450
Pro Ser Pro Thr Pro Val Thr Pro Arg Pro Pro Arg Ser Leu Thr
455 460 465
Leu Gly Ile Glu Pro Val Ser Pro Thr Ser Leu Arg Val Gly Leu
470 475 480
Gln Arg Tyr Leu Gln Gly Ser Ser Val Gln Leu Arg Ser Leu Arg
485 490 495
Leu Thr Tyr Arg Asn Leu Ser Gly Pro Asp Lys Arg Leu Val Thr
500 505 510
Leu Arg Leu Pro Ala Ser Leu Ala Glu Tyr Thr Val Thr Gln Leu
515 520 525
Arg Pro Asn Ala Thr Tyr Ser Val Cys Val Met Pro Leu Gly Pro
530 535 540
Gly Arg Val Pro Glu Gly Glu Glu Ala Cys Gly Glu Ala His Thr
545 550 555
Pro Pro Ala Val His Ser Asn His Ala Pro Val Thr Gln Ala Arg
560 565 570
Glu Gly Asn Leu Pro Leu Leu Ile Ala Pro Ala Leu Ala Ala Val
575 580 585
Leu Leu Ala Ala Leu Ala Ala Val Gly Ala Ala Tyr Cys Val Arg
590 595 600
Arg Gly Arg Ala Met Ala Ala Ala Ala Gln Asp Lys Gly Gln Val
605 610 615
Gly Pro Gly Ala Gly Pro Leu G1u Leu Glu G1y Val Lys Val Pro
620 625 630
Leu Glu Pro Gly Pro !_ys Ala Thr Glu Gly Gly Gly Glu Ala Leu
635 640 645
Pro Ser Gly Ser Glu Cys Glu Val Pro Leu Met Gly Phe Pro Gly
650 655 660
Pro Gly Leu Gln Ser Pro Leu His Ala Lys Pro Tyr Ile
665 670
<210> 17
<211> 1672
<212> DNA
<2i3> Homo Sapien
<400> 17
gcagcggcga ggcggcggtg gtggctgagt ccgtggtggc agaggcgaag 50
Page 23
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
gcgacagctc atgcgggtcc ggatagggct gacgctgctg ctgtgtgcgg 100
tgctgctgag cttggcctcg gcgtcctcgg atgaagaagg cagccaggat 150
gaatccttag attccaagac tactttgaca tcagatgagt cagtaaagga 200
ccatactact gcaggcagag tagttgctgg tcaaatattt cttgattcag 250
aagaatctga attagaatcc tctattcaag aagaggaaga cagcctcaag 300
agccaagagg gggaaagtgt cacagaagat atcagctttc tagagtctcc 350
aaatccagaa aacaaggact atgaagagcc aaagaaagta cggaaaccag 400
ctttgaccgc cattgaaggc acagcacatg gggagccctg ccacttccct 450
tttcttttcc tagataagga gtatgatgaa tgtacatcag atgggaggga 500
agatggcaga ctgtggtgtg ctacaaccta tgactacaaa gcagatgaaa 550
agtggggctt ttgtgaaact gaagaagagg ctgctaagag acggcagatg 600
caggaagcag aaatgatgta tcaaactgga atgaaaatcc ttaatggaag 650
caataagaaa agccaaaaaa gagaagcata tcggtatctc caaaaggcag 700
caagcatgaa ccataccaaa gccctggaga gagtgtcata tgctctttta 750
tttggtgatt acttgccaca gaatatccag gcagcgagag agatgtttga 800
gaagctgact gaggaaggct ctcccaaggg acagactgct cttggctttc 850
tgtatgcctc tggacttggt gttaattcaa gtcaggcaaa ggctcttgta 900
tattatacat ttggagctct tgggggcaat ctaatagccc acatggtttt 950
ggtaagtaga ctttagtgga aggctaataa tattaacatc agaagaattt 1000
gtggtttata gcggccacaa ctttttcagc tttcatgatc cagatttgct 1050
tgtattaaga ccaaatattc agttgaactt ccttcaaatt cttgttaatg 1100
gatataacac atggaatcta catgtaaatg aaagttggtg gagtccacaa 1150
tttttcttta aaatgattag tttggctgat tgcccctaaa aagagagatc 1200
tgataaatgg ctctttttaa attttctctg agttggaatt gtcagaatca 1250
ttttttacat tagattatca taattttaaa aatttttctt tagtttttca 1300
aaattttgta aatggtggct atagaaaaac aacatgaaat attatacaat 1350
attttgcaac aatgccctaa gaattgttaa aattcatgga gttatttgtg 1400
cagaatgact ccagagagct ctactttctg ttttttactt ttcatgattg 1450
gctgtcttcc catttattct ggtcatttat tgctagtgac actgtgcctg 1500
cttccagtag tctcattttc cctattttgc taatttgtta ctttttcttt 1550
gctaatttgg aagattaact catttttaat aaaattatgt ctaagattaa 1600
Page 24
CA 02481756 2004-10-25
PCT-us00-23328_sequence
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1650
aaaaaaaaaa aaaaaaaaaa as 1672
<210> 18
<211> 301
<212> PRT
<213> Homo Sapien
<400> 18
Met Arg Val Arg Ile Gly Leu Thr Leu Leu Leu Cys Ala Val Leu
1 5 10 15
Leu Ser Leu Ala Ser Ala Ser Ser Asp Glu Glu Gly Ser Gln Asp
20 25 30
Glu Ser Leu Asp Ser Lys Thr Thr Leu Thr Ser Asp Glu Ser Val
35 40 45
Lys Asp His Thr Thr Ala Gly Arg Val Val Ala Gly Gln Ile Phe
50 55 60
Leu Asp Ser Glu Glu Ser Glu Leu Glu Ser Ser Ile Gln Glu Glu
65 70 75
Glu Asp Ser Leu Lys Ser Gln Glu Gly Glu Ser Val Thr Glu Asp
80 85 90
Ile Ser Phe Leu Glu 5er Pro Asn Pro Glu Asn Lys Asp Tyr Glu
95 100 105
Glu Pro Lys Lys Val Arg L,ys Pro Ala Leu Thr Ala Ile Glu Gly
110 115 120
Thr Ala His Gly Glu Pro Cys His Phe Pro Phe Leu Phe Leu Asp
125 130 135
Lys Glu Tyr Asp Glu Cys Thr Ser Asp G1y Arg Glu Asp Gly Arg
140 145 150
Leu Trp Cys Ala Thr 'Thr Tyr Asp Tyr Lys Ala Asp Glu Lys Trp
155 160 165
Gly Phe Cys Glu Thr Glu Giu Glu Ala Ala Lys Arg,Arg Gln Met
170 175 180
Gln Glu Ala Glu Met Met Tyr Gln Thr Gly Met Lys Ile Leu Asn
185 190 195
G1y Ser Asn Lys Lys 5er Gln Lys Arg Glu Ala Tyr Arg Tyr Leu
200 2os 210
Gln Lys Ala Ala Ser Met Asn His Thr Lys Ala Leu Glu Arg Val
215 220 225
Ser Tyr,Ala Leu Leu Phe Gly Asp Tyr Leu Pro Gln ASn Ile Gln
230 235 240
A1a A1a Arg Gla Met Phe G7a Lys Leu Thr G1a G1a G1y Ser Pro
245 250 255
Lys Gly Gln Thr Ala Leu Gly Phe Leu Tyr Ala Ser Gly Leu Gly
260 265 270
Page 25
".,..~,N$ ~.~.w,.~..-
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
Val Asn Ser Ser Gln Ala Lys Ala Leu Val Tyr Tyr Thr Phe Gly
275 280 285
Ala Leu Gly Gly Asn Leu Ile Ala His Met Val Leu Val Ser Arg
290 295 300
Leu
<210> 19
<211> 1508
<212> DNA
<213> Homo Sapien
<400> 19
aattcagatt ttaagcccat tctgcagtgg aatttcatga actagcaaga 50
ggacaccatc ttcttgtatt atacaagaaa ggagtgtacc tatcacacac 100
agggggaaaa atgctctttt gggtgctagg cctcctaatc ctctgtggtt 150
ttctgtggac tcgtaaagga aaactaaaga ttgaagacat cactgataag 200
tacattttta tcactggatg tgactcgggc tttggaaact tggcagccag 250
aacttttgat aaaaagggat ttcatgtaat cgctgcctgt ctgactgaat 300
caggatcaac agctttaaag gcagaaacct cagagagact tcgtactgtg 350
cttctggatg tgaccgaccc agagaatgtc aagaggactg cccagtgggt 400
gaagaaccaa gttggggaga aaggtctctg gggtctgatc aataatgctg 450
gtgttcccgg cgtgctggct cccactgact ggctgacact agaggactac 500
agagaaccta ttgaagtgaa cctgtttgga ctcatcagtg tgacactaaa 550
tatgcttcct ttggtcaaga aagctcaagg gagagttatt aatgtctcca 600
gtgttggagg tcgccttgca atcgttggag ggggctatac tccatccaaa 650
tatgcagtgg aaggtttcaa tgacagctta agacgggaca tgaaagcttt 700
tggtgtgcac gtctcatgca ttgaaccagg attgttcaaa acaaacttgg 750
cagatccagt aaaggtaatt gaaaaaaaac tcgccatttg ggagcagctg 800
tctccagaca tcaaacaaca atatggagaa ggttacattg aaaaaagtct 850
agacaaactg aaaggcaata aatcctatgt gaacatggac ctctctccgg 900
tggtagagtg catggaccac gctctaacaa gtctcttccc taagactcat 950
tatgccgctg gaaaagatgc caaaattttc tggatacctc tgtctcacat 1000
gccagcagct ttgcaagact ttttattgtt gaaacagaaa gcagagctgg 1050
ctaatcccaa ggcagtgtga ctcagctaac cacaaatgtc tcctccaggc 1100
tatgaaattg gccgatttca agaacacatc tccttttcaa ccccattcct 1150
tatctgctcc aacctggact catttagatc gtgcttattt ggattgcaaa 1200
Page 26
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
agggagtccc accatcgctg gtggtatccc agggtccctg ctcaagtttt 1250
ctttgaaaag gagggctgga atggtacatc acataggcaa gtcctgccct 1300
gtatttaggc tttgcctgct tggtgtgatg taagggaaat tgaaagactt 1350
gcccattcaa aatgatcttt accgtggcct gccccatgct tatggtcccc 1400
agcatttaca gtaacttgtg aatgttaagt atcatctctt atctaaatat 1450
taaaagataa gtcaacccaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500
aaaaaaaa 1508
<210> 20
<211> 319
<212> PRT
<213> Homo Sapien
<400> 20
Met Leu Phe Trp Val Leu Gly Leu Leu Ile Leu Cys Gly Phe Leu
1 5 10 15
Trp Thr Arg Lys Gly Lys Leu Lys Ile Glu Asp Ile Thr Asp Lys
20 25 30
Tyr Ile Phe Ile Thr Gly Cys Asp Ser Gly Phe Gly Asn Leu Ala
35 40 45
Ala Arg Thr Phe Asp Lys Lys Gly Phe His Val Ile Ala Ala Cys
50 55 60
Leu Thr Glu Ser Gly Ser Thr Ala Leu Lys Ala Glu Thr Ser Glu
65 70 75
Arg Leu Arg Thr Val Leu Leu Asp Val Thr Asp Pro Glu Asn Val
80 85 90
Lys Arg Thr Ala Gln Trp Val Lys Asn Gln Val Gly Glu Lys Gly
95 100 105
Leu Trp Gly Leu Ile Asn Asn Ala Gly Val Pro Gly Val Leu Ala
110 115 120
Pro Thr Asp Trp Leu Thr Leu Glu Asp Tyr Arg Glu Pro Ile Glu
125 130 135
Val Asn Leu Phe Gly Leu Ile Ser Val Thr Leu Asn Met Leu Pro
140 145 150
Leu val Lys Lys Ala Gln Gly Arg Val Ile Asn val Ser Ser val
155 160 165
Gly Gly Arg Leu Aia Ile Val Gly Gly Gly Tyr Thr Pro Ser Lys
170 175 180
Tyr Ala Val Glu Gly Phe Asn Asp Ser Leu Arg Arg Asp Met Lys
I85 190 195
Ala Phe Gly Val His Val Ser Cys Ile Glu Pro Gly Leu Phe Lys
200 205 210
Page 27
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Thr Asn Leu Ala Asp Pro Val Lys Val Ile Glu Lys Lys Leu Ala
215 220 225
Ile Trp Glu Gln Leu ser Pro Asp Ile Lys Gln Gln Tyr Gly Glu
230 235 240
Gly Tyr Ile Glu Lys 5er Leu asp Lys Leu Lys Gly Asn Lys Ser
245 250 255
Tyr Val Asn Met Asp Leu Ser Pro Val Val Glu Cys Met Asp His
260 265 X70
Ala Leu Thr Ser Leu Phe Pro Lys Thr His Tyr Ala Ala Gly Lys
275 280 285
Asp Ala Lys Ile Phe Trp Ile Pro Leu Ser His Met Pro Ala Ala
290 295 300
Leu Gln Asp Phe Leu Leu Leu Lys Gln Lys Ala Glu Leu Ala Asn
305 310 315
Pro Lys Ala Val
<210> 21
<211> 1849
<212> DNA
<213> Homo Sapien
<400> 21
ctgaggcggc ggtagcatgg agggggagag tacgtcggcg gtgctctcgg 50
gctttgtgct cggcgcact:c gctttccagc acctcaacac ggactcggac 100
acggaaggtt ttcttcttgg ggaagtaaaa ggtgaagcca agaacagcat 150
tactgattcc caaatggatg atgttgaagt tgtttataca attgacattc 200
agaaatatat tccatgctat cagcttttta gcttttataa ttcttcaggc 250
gaagtaaatg agcaagcact gaagaaaata ttatcaaatg tcaaaaagaa 300
tgtggtaggt tggtacaaat tccgtcgtca ttcagatcag atcatgacgt 350
ttagagagag gctgcttcac aaaaacttgc aggagcattt ttcaaaccaa 400
gaccttgttt ttctgctatt aacaccaagt ataataacag aaagctgctc 450
tactcatcga ctggaacatt ccttatataa acctcaaaaa ggactttttc 500
acagggtacc tttagtggtt gccaatctgg gcatgtctga acaactgggt 550
tataaaactg tatcaggttc ctgtatgtcc actggtttta gccgagcagt 600
acaaacacac agctctaaat tttttgaaga agatggatcc ttaaaggagg 650
tacataagat aaatgaaatg tatgcttcat tacaagagga attaaagagt 700
atatgcaaaa aagtggaaga cagtgaacaa gcagtagata aactagtaaa: 750
ggatgtaaac agattaaaac gagaaattga gaaaaggaga ggagcacaga 800
ttcaggcagc aagagagaag aacatccaaa aagaccctca ggagaacatt 850
Page 28
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
tttctttgtc aggcattacg gacctttttt ccaaattctg aatttcttca 900
ttcatgtgtt atgtctttaa aaaatagaca tgtttctaaa agtagctgta 950
actacaacca ccatctcgat gtagtagaca atctgacctt aatggtagaa 1000
cacactgaca ttcctgaagc tagtccagct agtacaccac aaa~tcattaa 1050
gcataaagcc ttagacttag atgacagatg gcaattcaag agatctcggt 1100
tgttagatac acaagacaaa cgatctaaag caaatactgg tagtagtaac 1150
caagataaag catccaaaat gagcagccca gaaacagatg aagaaattga 1200
aaagatgaag ggttttggtg aatattcacg gtctcctaca ttttgatcct 1250
tttaacctta caaggagatt tttttatttg gctgatgggt aaagccaaac 1300
atttctattg tttttactat gttgagctac ttgcagtaag ttcatttgtt 1350
tttactatgt tcacctgttt gcagtaatac acagataact cttagtgcat 1400
ttacttcaca aagtactttt tcaaacatca gatgctttta tttccaaacc 1450
tttttttcac ctttcactaa gttgttgagg ggaaggctta cacagacaca 1500
ttctttagaa ttggaaaagt gagaccaggc acagtggctc acacctgtaa 1550
tcccagcact tagggaagac aagtcaggag gattgattga agctaggagt 1600
tagagaccag cctgggcaac gtattgagac catgtctatt aaaaaataaa 1650
atggaaaagc aagaatagcc ttattttcaa aatatggaaa gaaatttata 1700
tgaaaattta tctgagtcat taaaattctc cttaagtgat acttttttag 1750
aagtacatta tggctagagt tgccagataa aatgctggat atcatgcaat 1800
aaatttgcaa aacatcatct aaaatttaaa aaaaaaaaaa aaaaaaaaa 1849
<210> 22
<211> 409
<212> PRT
<213> Homo Sapien
<400> 22
Met Glu Gly Glu Ser Thr Ser Ala Val Leu Ser Gly Phe Val Leu
1 5 10 15 ~ _
Gly Ala Leu Ala Phe Gln His Leu Asn Thr Asp Ser Asp Thr Glu
20 25 30
Gly Phe Leu Leu Gly Glu Val Lys Gly Glu Ala Lys Asn Ser Ile
35 40 45
Thr Asp Ser Gln Met Asp Asp Val Glu Val Val Tyr Thr Ile Asp
50 55 60
Ile Gln Lys Tyr Ile Pro Cys Tyr Gln Leu Phe Ser Phe Tyr Asn
65 70 75
Ser Ser Gly Glu Val Asn Glu Gln Ala Leu Lys Lys Ile Leu Ser
Page 29
CA 02481756 2004-10-25
PCT-us00-23328_Sequence
80 85 90
Asn Val Lys Lys Asn Val Val Gly Trp Tyr Lys Phe Arg Arg His
95 100 105
Ser Asp Gln Ile Met Thr Phe Arg Glu Arg Leu Leu His Lys Asn
110 115 120
Leu Gln Glu His Phe Ser Asn Gln Asp Leu Val Phe Leu Leu Leu
125 130 135
Thr Pro Ser Ile Ile Thr Glu Ser Cys Ser Thr His Arg Leu Glu
140 145 150
His Ser Leu Tyr Lys Pro Gln Lys Gly Leu Phe His Arg Val Pro
155 160 165
Leu Val Val Ala Asn Leu G1y Met Ser Glu Gln Leu Gly Tyr Lys
170 175 180
Thr Val ser Gly ser cys Met ser Thr Gly Phe ser Arg Ala val
185 190 195
Gln Thr His Ser Ser Lys Phe Phe Glu Glu Asp Gly Ser Leu Lys
200 205 210
Glu Val His Lys Ile Asn Glu Met Tyr Ala Ser Leu Gln Glu Glu
215 220 225
Leu Lys Ser Ile Cys Lys Lys val Glu Asp Ser Glu Gln Ala Val
230 235 240
Asp Lys Leu val Lys Asp Val Asn Arg Leu Lys Arg Glu Ile Glu
245 250 255
Lys Arg Arg Gly Ala Gln Ile Gln Ala Ala Arg Glu Lys Asn Ile
260 265 270
Gln Lys Asp Pro Gln Glu Asn Ile Phe Leu Cys Gln Ala Leu Arg
275 280 285
Thr Phe Phe Pro Asn Ser Glu Phe Leu His Ser Cys Val Met Ser
290 295 300
Leu Lys Asn Arg His Val Ser Lys Ser Ser Cys Asn Tyr Asn His
305 310 315
His Leu Asp Val Val Asp Asn Leu Thr Leu Met val Glu His Thr
320 325 330
Asp Ile Pro Giu Ala Ser Pro Ala Ser Thr Pro Gln Ile Ile Lys
335 340 345
His Lys Ala Leu Asp Leu Asp Asp Arg Trp G1n Phe Lys Arg Ser
350 355 360
Arg Leu Leu Asp Thr Gln Asp Lys Arg Ser Lys Ala Asn Thr Gly
365 370 375
Ser Ser Asn Gln ASp Lys Ala Ser Lys Met Ser Ser Pro G1u Thr
380 385 390
Asp Glu Glu Ile Glu Lys Met Lys Gly Phe Gly Glu Tyr Ser Arg
Page 30
CA 02481756 2004-10-25
PCT-u500-23328_Seguence
395 400 405
Ser Pro Thr Phe
<210> 23
<211> 2651
<212> DNA
<213> Homo Sapien
<400> 23
ggcacagccg cgcggcggag ggcagagtca gccgagccga gtccagccgg 50
acgagcggac cagcgcaggg cagcccaagc agcgcgcagc gaacgcccgc 100
cgccgcccac accctctgcg gtccccgcgg cgcctgccac ccttccctcc 150
ttccccgcgt ccccgcctcg ccggccagtc agcttgccgg gttcgctgcc 200
ccgcgaaacc ccgaggtcac cagcccgcgc ctctgcttcc ctgggccgcg 250
cgccgcctcc acgccctcct tctcccctgg cccggcgcct ggcaccgggg 300
accgttgcct gacgcgaggc ccagctctac ttttcgcccc gcgtctcctc 350
cgcctgctcg cctcttccac caactccaac tccttctccc tccagctcca 400
ctcgctagtc cccgactccg ccagccctcg gcccgctgcc gtagcgccgc 450
ttcccgtccg gtcccaaagg tgggaacgcg tccgccccgg cccgcaccat 500
ggcacggttc ggcttgcccg cgCttctctg caccctggca gtgctcagcg 550
ccgcgctgct ggctgccgag ctcaagtcga aaagttgctc ggaagtgcga 600
cgtctttacg tgtccaaagg cttcaacaag aacgatgccc ccctccacga 650
gatcaacggt gatcatttga agatctgtcc ccagggttct acctgctgct 700
ctcaagagat ggaggagaag tacagcctgc aaagtaaaga tgatttcaaa 750
agtgtggtca gcgaacagtg caatcatttg caagctgtct ttgcttcacg 800
ttacaagaag tttgatgaat tcttcaaaga actacttgaa aatgcagaga 850
aatccctgaa tgatatgttt gtgaagacat atggccattt atacatgcaa 900
aattctgagc tatttaaaga tctcttcgta gagttgaaac gttactacgt 950
ggtgggaaat gtgaacctgg aagaaatgct aaatgacttc tgggctcgcc 1000
tcctggagcg gatgttccgc ctggtgaact cccagtacca ctttacagat 1050
gagtatctgg aatgtgtgag caagtatacg gagcagctga agcccttcgg 1100
agatgtccct cgcaaattga agctccaggt tactcgtgct tttgtagcag 1150
cccgtacttt cgctcaaggc ttagcggttg cgggagatgt cgtgagcaag 1200
gtctccgtgg taaaccccac agcccagtgt acccatgccc tgttgaagat 1250 -
gatctactgc tcccactgcc ggggtctcgt gactgtgaag ccatgttaca 1300
Page 31
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
actactgctc aaacatcatg agaggctgtt tggccaacca aggggatctc 1350
gattttgaat ggaacaattt catagatgct atgctgatgg tggcagagag 1400
gctagagggt cctttcaaea ttgaatcggt catggatccc atcgatgtga 1450
agatttctga tgctattatg aacatgcagg ataatagtgt tcaagtgtct 1500
cagaaggttt tccagggatg tggacccccc aagcccctcc cagctggacg 1550
aatttctcgt tccatctctg aaagtgcctt cagtgctcgc ttcagaccac 1600
atcaccccga ggaacgccca accacagcag ctggcactag tttggaccga 1650
ctggttactg atgtcaagga gaaactgaaa caggccaaga aattctggtc 1700
ctcccttccg agcaacgttt gcaacgatga gaggatggct gcaggaaacg 1750
gcaatgagga tgactgttgg aatgggaaag gcaaaagcag gtacctgttt 1800
gcagtgacag gaaatggatt agecaaccag ggcaacaacc cagaggtcca 1850
ggttgacacc agcaaaccag acatactgat ccttegtcaa atcatggctc 1900
ttcgagtgat gaccagcaag atgaagaatg catacaatgg gaacgacgtg 1950
gacttctttg atatcagtga tgaaagtagt ggagaaggaa gtggaagtgg 2000
ctgtgagtat cagcagtgcc cttcagagtt tgactacaat gccactgacc 2050
atgctgggaa gagtgccaat gagaaagccg acagtgctgg tgtccgtcct 2100
ggggcacagg cctacctcct cactgtcttc tgcatcttgt tcctggttat 2150
gcagagagag tggagataat tctcaaactc tgagaaaaag tgttcatcaa 2200
aaagttaaaa ggcaccagtt atcacttttc taccatccta gtgactttgc 2250
tttttaaatg aatggacaac aatgtacagt ttttactatg tggccactgg 2300
tttaagaagt gctgactttg ttttctcatt cagttttggg aggaaaaggg 2350
actgtgcatt gagttggttc ctgctccccc aaaccatgtt aaacgtggct 2400
aacagtgtag gtacagaact atagttagtt gtgcatttgt gattttatca 2450
ctctattatt tgtttgtatg tttttttctc atttcgtttg tgggtttttt 2500
tttccaactg tgatctcgcc ttgtttctta caagcaaacc agggtccctt 2550
cttggcacgt aacatgtacg tatttctgaa atattaaata gctgtacaga 2600
agcaggtttt atttatcatg ttatcttatt aaaagaaaaa gcccaaaaag 2650
c 2651
<210> 24
<211> 556
<212> PRT
<213> Homo sapien
<400> 24
Met Ala Arg Phe Gly Leu Pro Ala Leu Leu Cys Thr Leu Ala Val
Page 32
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
1 5 10 15
Leu Ser Ala Ala Leu Leu Ala Ala Glu Leu Lys Ser Lys Ser Cys
20 25 30
Ser Glu Val Arg Arg Leu Tyr Val Ser Lys Gly Phe Asn Lys Asn
35 40 45
Asp Ala Pro Leu His Glu Ile Asn Gly Asp His Leu Lys Ile Cys
50 55 60
Pro Gln Gly Ser Thr Cys Cys Ser Gln Glu Met Glu Glu Lys Tyr
65 70 75
Ser Leu Gln Ser Lys Asp Asp Phe Lys Ser Val Val Ser Glu Gln
80 85 90
Cys Asn His Leu Gln Ala Val Phe Ala Ser Arg Tyr Lys Lys Phe
95 100 105
Asp Glu Phe Phe Lys Glu Leu Leu Glu Asn Ala Glu Lys Ser Leu
110 115 120
Asn Asp Met Phe Val Lys Thr Tyr Gly His Leu Tyr Met Gln Asn
125 130 135
Ser Glu Leu Phe Lys Asp Leu Phe Val Glu Leu Lys Arg Tyr Tyr
140 145 150
Val Val Gly Asn Val Asn Leu Glu Glu Met Leu Asn Asp Phe Trp
155 160 165
Ala Arg Leu Leu Glu Arg Met Phe Arg Leu Val Asn ser Gln Tyr
170 175 180
His Phe Thr Asp Glu Tyr Leu Glu Cys Val Ser Lys Tyr Thr Glu
185 190 195
Gln Leu Lys Pro Phe Gly Asp Val Pro Arg Lys Leu Lys Leu Gln
zoo 205 210
Val Thr Arg Ala Phe Val Ala Ala Arg Thr Phe Ala Gln Gly Leu
215 220 225
Ala Val Ala Gly Asp Val Val Ser Lys Val Ser Val Val Asn Pro
230 235 Z40
Thr Ala Gln Cys Thr His Ala Leu Leu Lys Met Ile Tyr Cys Ser
245 250 255
His Cys Arg Gly Leu Val Thr Val Lys Pro Cys Tyr Asn Tyr Cys
260 265 270
Ser Asn Ile Met Arg Gly Cys Leu Ala Asn Gln Gly Asp Leu Asp
275 280 285
Phe Glu Trp Asn Asn Phe Ile Asp Ala Met Leu Met Val Ala Glu
290 295 300
Arg Leu Glu Gly Pro Phe ASn Ile Glu Ser Val Met Asp Pro Ile
305 310 315
Asp Val Lys Ile Ser Asp Ala Ile Met Asn Met Gln Asp Asn Ser
Page 33
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
320 325 330
Val Gln Val Ser Gln Lys Val Phe Gln Gly Cys Gly Pro Pro Lys
335 340 345
Pro Leu Pro Ala Gly Arg Ile Ser Arg Ser Ile Ser Glu Ser Ala
350 355 360
Phe Ser Ala Arg Phe Arg Pro His His Pro Glu Glu Arg Pro Thr
365 370 375
Thr Ala Ala Gly Thr Ser Leu Asp Arg Leu Val Thr Asp Val Lys
380 385 390
Glu Lys Leu Lys Gln Ala Lys Lys Phe Trp Ser Ser Leu Pro Ser
395 400 405
Asn Val Cys Asn Asp Glu Arg Met Ala Ala Gly Asn Gly Asn Glu
410 415 420
Asp Asp Cys Trp Asn Gly Lys Gly Lys Ser Arg Tyr Leu Phe Ala
425 430 435
Val Thr Gly Asn Gly Leu Ala Asn Gln Gly Asn Asn Pro Glu Val
440 445 450
Gln Val Asp Thr Ser Lys Pro Asp Ile Leu Ile Leu Arg Gln Ile
455 460 465
Met Ala Leu Arg Val Met Thr Ser Lys Met Lys Asn Ala Tyr Asn
470 475 480
Gly Asn Asp Val Asp Phe Phe Asp Ile Ser Asp Glu Ser Ser Gly
485 490 495
Glu Gly Ser Gly Ser Gly Cys Glu Tyr Gln Gln Cys Pro Ser Glu
500 505 510
Phe Asp Tyr Asn Ala Thr Asp His Ala Gly Lys Ser Ala Asn Glu
515 520 525
Lys Ala Asp Ser Ala Gly Val Arg Pro Gly Ala Gln Ala Tyr Leu
530 535 540
Leu Thr Val Phe Cys Ile Leu Phe Leu Val Met Gln Arg Glu Trp
545 550 - 555
Arg
<210> 25
<211> 870
<212> DNA
<213> Homo Sapien
<400> 25
ctcgccctca aatgggaacg ctggcctggg actaaagcat agaccaccag 50
gctgagtatc ctgacctgag tcatccccag ggatcaggag cctccagcag 100
ggaaccttcc attatattct tcaagcaact tacagctgca ccgacagttg 150
cgatgaaagt tctaatctct tccctcctcc tgttgctgcc actaatgctg 200
Page 34
CA 02481756 2004-10-25
PCT-u500-23328_seguence
atgtccatgg tctctagcag cctgaatcca ggggtcgcca gaggccacag 250
ggaccgaggc caggcttcta ggagatggct ccaggaaggc ggccaagaat 300
gtgagtgcaa agattggttc ctgagagccc cgagaagaaa attcatgaca 350
gtgtctgggc tgccaaagaa gcagtgcccc tgtgatcatt tcaagggcaa 400
tgtgaagaaa acaagacacc aaaggcacca cagaaagcca aacaagcatt 450
ccagagcctg ccagcaattt ctcaaacaat gtcagctaag aagctttgct 500
ctgcctttgt aggagctctg agcgcccact cttccaatta aacattctca 550
gccaagaaga cagtgagcac acctaccaga cactcttctt ctcccacctc 600
actctcccac tgtacccacc cctaaatcat tccagtgctc tcaaaaagca 650
tgtttttcaa gatcattttg tttgttgctc tctctagtgt cttcttctct 700
cgtcagtctt agcctgtgcc ctccccttac ccaggcttag gcttaattac 750
ctgaaagatt ccaggaaact gtagcttcct agctagtgtc atttaacctt 800
aaatgcaatc aggaaagtag caaacagaag tcaataaata tttttaaatg 850
tcaaaaaaaa aaaaaaaaaa g70
<210> 26
<211> 119
<212> PRT
<213> Homo Sapien
<400> 26
Met Lys Val Leu Ile Ser Ser Leu Leu Leu Leu Leu Pro Leu Met
1 5 10 15
Leu Met Ser Met Val Ser Ser Ser Leu Asn Pro Gly Val Ala Arg
20 25 30
Gly His Arg Asp Arg Gly Gln Ala Ser Arg Arg Trp Leu Gln Glu
35 40 45
Gly Gly Gln Glu Cys Glu Cys Lys Asp Trp Phe Leu Arg Ala Pro
50 55 60
Arg Arg Lys Phe Met Thr Val Ser Gly Leu Pro Lys Lys:Gln Cys
65 70 75
Pro Cys Asp His Phe Lys Gly Asn Val Lys Lys Thr Arg His Gln
80 85 90
Arg His His Arg Lys Pro Asn Lys His Ser Arg Ala Cys Gln Gln
95 100 105
Phe Leu Lys Gln Cys Gln Leu Arg Ser Phe Ala Leu Pro Leu
110 115
<210> 27
<211> 1371
<212> DNA
<213> Homo Sapien
Page 35 ~ .
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
<400> 27
ggacgccagc gcctgcagag gctgagcagg gaaaaagcca gtgccccagc SO
ggaagcacag ctcagagctg gtctgccatg gacatcctgg tcccactcct 100
gcagctgctg gtgctgcttc ttaccctgcc cctgcacctc atggctctgc 150
tgggctgctg gcagcccctg tgcaaaagct acttccccta cctgatggcc 200
gtgctgactc ccaagagcaa ccgcaagatg gagagcaaga aacgggagct 250
cttcagccag ataaaggggc ttacaggagc ctccgggaaa gtggccctac 300
tggagctggg ctgcggaacc ggagccaact ttcagttcta cccaccgggc 350
tgcagggtca cctgcctaga cccaaatccc cactttgaga agttcctgac 400
aaagagcatg gctgagaaca ggcacctcca atatgagcgg tttgtggtgg 450
ctcctggaga ggacatgaga cagctggctg atggctccat ggatgtggtg 500
gtctgcactc tggtgctgtg ctctgtgcag agcccaagga aggtcctgca 550
ggaggtccgg agagtactga gaccgggagg tgtgctcttt ttctgggagc 600
atgtggcaga accatatgga agctgggcct tcatgtggca gcaagttttc 650
gagcccacct ggaaacacat tggggatggc tgctgcctca ccagagagac 700
ctggaaggat cttgagaacg cccagttctc cgaaatccaa atggaacgac 750
agccccctcc cttgaagtgg ctacctgttg ggccccacat catgggaaag 800
gctgtcaaac aatctttccc aagctccaag gcactcattt gctccttccc 850
cagcctccaa ttagaacaag ccacccacca gcctatctat cttccactga 900
gagggaccta gcagaatgag agaagacatt catgtaccac ctactagtcc 950
ctctctcccc aacctctgcc agggcaatct ctaacttcaa tcccgccttc 1000
gacagtgaaa aagctctact tctacgctga cccagggagg aaacactagg 1050
accctgttgt atcctcaact gcaagtttct ggactagtct cccaacgttt 1100
gcctcccaat gttgtccctt tccttcgttc ccatggtaaa gctcctctcg 1150
ctttcctcct gaggctacac ccatgcgtct ctaggaactg gtcacaaaag 1200
tcatggtgcc tgcatccctg ccaagccccc ctgaccctct ctccccacta 1250
ccaccttctt cctgagctgg gggcaccagg gagaatcaga gatgctgggg 1300
atgccagagc aagactcaaa gaggcagagg ttttgttctc aaatattttt 1350
taataaatag acgaaaccac g 1371
<210> 28 ,,
<211> 277
<212> PRT
<213> Homo Sapien
Page 36
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
<400> 28
Met Asp Ile Leu Val Pro Leu Leu Gln Leu Leu Val Leu Leu Leu
1 5 10 15
Thr Leu Pro Leu His Leu Met Ala Leu Leu Gly Cys Trp Gln Pro
20 25 30
Leu Cys Lys Ser Tyr Phe Pro Tyr Leu Met Ala Val Leu Thr Pro
35 40 45
Lys Ser Asn Arg Lys Met Glu Ser Lys Lys Arg Glu Leu Phe Ser
50 55 60
Gln Ile Lys Gly Leu Thr Gly Ala Ser Gly Lys Val Ala Leu Leu
65 70 75
Glu Leu Gly Cys Gly Thr Gly Ala Asn Phe Gln Phe Tyr Pro Pro
80 85 90
Gly Cys Arg Val Thr Cys Leu Asp Pro Asn Pro His Phe Glu Lys
95 100 105
Phe Leu Thr Lys Ser Met Ala Glu Asn Arg His Leu Gln Tyr Glu
110 115 120
Arg Phe Val Val Ala Pro Gly Glu Asp Met Arg Gln Leu Ala Asp
125 130 135
Gly Ser Met Asp Val Val Val Cys Thr Leu Val Leu Cys Ser Val
140 145 150
Gln Ser Pro Arg Lys Val Leu Gln Glu Val Arg Arg Val Leu Arg
155 160 165
Pro Gly Gly Val Leu Phe Phe Trp Glu His Val Ala Glu Pr0 Tyr
170 175 180
Gly Ser Trp Ala Phe Met Trp Gln Gln Val Phe Glu Pro Thr Trp
185 190 195
Lys His Ile Gly Asp Gly Cys Cys Leu Thr Arg Glu Thr Trp Lys
200 20.5 210
Asp Leu Glu Asn Ala Gln Phe Ser Glu Ile Gln Met Glu Arg Gln
215 220 225
Pro Pro Pro Leu Lys Trp Leu Pro Val Gly Pro His Ile Met Gly
230 235 240
Lys Ala Val Lys Gln Ser Phe Pro Ser Ser Lys Ala Leu Ile Cys
245 250 255
Ser Phe Pro Ser Leu Gln Leu Glu Gln Ala Thr His Gln Pro Ile
260 265 270
Tyr Leu Pro Leu Arg Gly Thr
275
<210> 29
<211> 494
<212> DNA
<213> Homo Sapien
Page 37
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
<400> 29
caatgtttgc ctatccacct cccccaagcc cctttaccta tgctgctgct SO
aacgctgctg ctgctgctgc tgctgcttaa aggctcatgc ttggagtggg 100
gactggtcgg tgcccagaaa gtctcttctg ccactgacgc ccccatcagg 150
gattgggcct tctttccccc ttcctttctg tgtctcctgc ctcatcggcc 200
tgccatgacc tgcagccaag cccagccccg tggggaaggg gagaaagtgg 250
gggatggcta agaaagctgg gagataggga acagaagagg gtagtgggtg 300
ggctaggggg gctgccttat ttaaagtggt tgtttatgat tcttatacta 350
atttatacaa agatattaag gccctgttca ttaagaaatt gttcccttcc 400
cctgtgttca atgtttgtaa agattgttct gtgtaaatat gtctttataa 450
taaacagtta aaagctgaaa aaaaaaaaaa aaaaaaaaaa aaaa 494
<210> 30
<211> 73
<212> PRT
<213> Homo Sapien
<400> 30
Met Leu Leu Leu Thr Leu Leu Leu Leu Leu Leu Leu Leu Lys Gly
1 5 10 15
Ser Cys Leu Glu Trp Gly Leu Val Gly Ala Gln Lys Val Ser Ser
20 25 30
Ala Thr Asp Ala Pro I12 Arg Asp Trp Ala Phe Phe Pro Pro Ser
35 40 45
Phe Leu Cys Leu Leu Pro t-~is Arg Pro Ala Met Thr Cys Ser Gln
50 55 60
Ala Gln Pro Arg Gly Glu Gly Glu Lys val Gly Asp Gly
65 70
<210> 31
<211> 1660
<212> DNA
<213> Homo Sapien
<400> 31
gtttgaattc cttcaactat acccacagtc caaaagcaga etcactgtgt 50
cccaggctac cagttcctcc aagcaagtca tttcccttat ttaaccgatg 100
tgtccctcaa acacctgagt gctactccct atttgcatct gttttgataa 150
atgatgttga caccctccac cgaattctaa gtggaatcat gtcgggaaga Z00
gatacaatee ttggectgtg tatcetegca ttageettgt etttggccat 250
gatgtttacc ttcagattca tcaccaccct tctggttcae attttcattt 300
cattggttat tttgggattg ttgtttgtct gcggtgtttt atggtggctg 350
tattatgact ataccaacga cctcagcata gaattggaca cagaaaggga 400
Page 38
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
aaatatgaag tgcgtgctgg ggtttgctat cgtatccaca ggcatcacgg 450
cagtgctgct cgtcttgatt tttgttctca gaaagagaat aaaattgaca 500
gttgagcttt tccaaatcac aaataaagcc atcagcagtg ctcccttcct 550
gctgttccag ccactgtgga catttgccat cctcattttc ttctgggtcc 600
tctgggtggc tgtgctgctg agcctgggaa ctgcaggagc tgcccaggtt 650
atggaaggcg gccaagtgga atataagccc ctttcgggca ttcggtacat 700
gtggtcgtac catttaattg gcctcatctg gactagtgaa ttcatccttg 750
cgtgccagca aatgactata gctggggcag tggttacttg ttatttcaac 800
agaagtaaaa atgatcctcc tgatcatccc atcctttcgt ctctctccat 850
tctcttcttc taccatcaag gaaccgttgt gaaagggtca tttttaatct 900
ctgtggtgag gattccgaga atcattgtca tgtacatgca aaacgcactg 950
aaagaacagc agcatggtgc attgtccagg tacctgttcc gatgctgcta 1000
ctgctgtttc tggtgtcttg acaaatacct gctccatctc aaccagaatg 1050
catatactac aactgctatt aatgggacag atttctgtac atcagcaaaa 1100
gatgcattca aaatcttgtc caagaactca agtcacttta catctattaa 1150
ctgctttgga gacttcataa tttttctagg aaaggtgtta gtggtgtgtt 1200
tcactgtttt tggaggactc atggctttta actacaatcg ggcattccag 1250
gtgtgggcag tccctctgtt attggtagct ttttttgcct acttagtagc 1300
ccatagtttt ttatctgtgt ttgaaactgt gctggatgca cttttcctgt 1350
gttttgctgt tgatetggaa acaaatgatg gatcgtcaga aaagccctac 1400
tttatggatc aagaatttct gagtttcgta aaaaggagca acaaattaaa 1450
caatgcaagg gcacagcagg acaagcactc attaaggaat gaggagggaa 1500
cagaactcca ggccattgtg agatagatac ccatttaggt atctgtacct 1550
ggaaaacatt tccttctaag agccatttac agaatagaag atgagaccac 1600
tagagaaaag ttagtgaatt tttttttaaa agacctaata aaccctattc 1650
ttcctcaaaa 1660
<210> 32
<211> 445
<212> PRT .
<213> Homo Sapien
<400> 32
Met Ser Gly Arg Asp Thr Ile Leu Gly Leu Cys Ile Leu Ala Leu
l 5 10 15
Ala Leu Ser Leu Ala Met Met Phe Thr Phe Arg Phe Ile Thr Thr
Page 39
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
20 25 30
Leu Leu Yal His I12 Phe Ile Ser Leu Yal Ile Leu Gly Leu Leu
35 40 45
Phe Val Cys Gly Val Leu Trp Trp Leu Tyr Tyr Asp Tyr Thr Asn
50 55 60
Asp Leu Ser Ile Glu Leu Asp Thr Glu Arg Glu Asn Met Lys Cys
65 70 75
Val Leu Gly Phe Ala Ile Yal Ser Thr Gly Ile Thr Ala Val Leu
80 85 90
Leu val Leu Ile Phe val Leu Arg Lys Arg Ile Lys Leu Thr val
95 100 105
Glu Leu Phe Gln Ile Thr Asn Lys Ala Ile Ser Ser Ala Pro Phe
110 115 120
Leu Leu Phe Gln Pro Leu Trp Thr Phe Ala Ile Leu Ile Phe Phe
125 130 135
Trp Val Leu Trp Val Ala Val Leu Leu Ser Leu Gly Thr Ala Gly
140 145 150
Ala Ala Gln Val Met Glu Gly Gly Gln Val Glu Tyr Lys Pro Leu
155 160 165
Ser Gly Ile Arg Tyr Met Trp Ser Tyr His Leu Ile Gly Leu Ile
170 175 180
Trp Thr Ser Glu Phe Ile Leu Ala Cys Gln Gln Met Thr Ile.Ala
185 190 195
Gly Ala val Yal Thr Cys Tyr Phe Asn Arg Ser Lys ASn Asp Pro
200 205 210
Pro Asp His Pro Ile Leu Ser Ser Leu Ser Ile Leu Phe Phe Tyr
215 220 225
His Gln Gly Thr Val ~Val Lys Gly Ser Phe Leu Ile Ser Yal Val
230 235 240
Arg Ile Pro Arg Ile Ile Yal Met Tyr Met Gln Asn Ala Leu Lys
245 250 255
Glu Gln Gln His Gly Ala Leu Ser Arg Tyr Leu Phe Arg Cys Cys
260 265 270
Tyr Cys Cys Phe Trp Cys Leu Asp Lys Tyr Leu Leu His Leu Asn
275 280 285
Gln Asn Ala Tyr Thr Thr Thr Ala Ile Asn Gly Thr Asp Phe Cys
290 295 300
Thr Ser Ala Lys Asp Ala Phe Lys Ile Leu Ser Lys Asn Ser Ser
305 310 315
His Phe Thr Ser Ile Asn Cys Phe Gly Asp Phe Ile IIe Phe Leu
320 325 330
Gly Lys Val Leu Yal Yal Cys Phe Thr Val Phe Gly Gly Leu Met
Page 40
.< .. ,.:., ..x . ..~ ., .. . ,.~ _....< ,«ou -, ....., , ... w.,_ .. .ar. ,
~. ~ > , ~5,~~,z~ .,gym ,..,,~:"A., fR,~.,. ."~.,~",... w ~~.-.... .._ ..r
..._._ ......
CA 02481756 2004-10-25
PCT-US00-23328_Seguence
335 340 345
Ala Phe Asn Tyr Asn Arg Ala Phe Gln Val Trp Ala Val Pro Leu
350 355 360
Leu Leu Val Ala Phe Phe Ala Tyr Leu Val Ala His Ser Phe Leu
365 370 375
Ser Val Phe Glu Thr Val Leu Asp Ala Leu Phe Leu Cys Phe Ala
380 385 390
Val Asp Leu Glu Thr Asn Asp Gly Ser Ser Glu Lys Pro Tyr Phe
395 400 405
Met Asp Gln Glu Phe Leu Ser Phe val Lys Arg 5er Asn Lys Leu
410 415 420
Asn Asn Ala Arg Ala Gln Gln Asp Lys His Ser Leu Arg Asn Glu
425 430 435
Glu Gly Thr Glu Leu Gln Ala zle val Arg
440 445
<210> 33
<211> 2773
<212> DNA
<213> Homo Sapien
<400> 33
gttcgattag ctcctctgag aagaagagaa aaggttcttg gacctctccc 50
tgtttcttcc ttagaataat ttgtatggga tttgtgatgc aggaaagcct 100
aagggaaaaa gaatattcat tctgtgtggt gaaaattttt tgaaaaaaaa 150
attgccttct tcaaacaagg gtgtcattct gatatttatg aggactgttg 200
ttctcactat gaaggcatct gttattgaaa tgttccttgt tttgctggtg 250
actggagtac attcaaacaa agaaacggca aagaagatta aaaggcccaa 300
gttcactgtg cctcagatca actgcgatgt caaagccgga aagatcatcg 350
atcctgagtt cattgtgaaa tgtccagcag gatgccaaga ccccaaatac 400
catgtttatg gcactgacgt gtatgcatcc tactccagtg tgtgtggcgc 450
tgccgtacac agtggtgtgc ttgataattc aggagggaaa atacttgttc 500
ggaaggttgc tggacagtct ggttacaaag ggagttattc caacggtgtc 550
caatcgttat ccctaccacg atggagagaa tcctttatcg tcttagaaag 600
taaacccaaa aagggtgtaa cctacccatc agctcttaca tactcatcat 650
cgaaaagtcc agctgccca.a gcaggtgaga ccacaaaagc ctatcagagg 700
ccacctattc cagggacaac tgcacagccg gtcactctga tgcagcttct 750
ggctgtcact gtagctgtgg ccacccccac caccttgcca aggccatccc 800
cttctgctgc ttctaccacc agcatcccca gaccacaatc agtgggccac 850
Page 41
CA 02481756 2004-10-25
PCT-U500-23328_Sequence
aggagccagg agatggatct ctggtccact gccacctaca caagcagcca 900
aaacaggccc agagctgatc caggtatcca aaggcaagat ccttcaggag 950
ctgccttcca gaaacctgtt ggagcggatg tcagcctggg acttgttcca 1000
aaagaagaat tgagcacaca gtCtttggag ccagtatccc tgggagatcc 1050
aaactgcaaa attgacttgt cgtttttaat tgatgggagc accageattg 1100
gcaaacggcg attccgaatc cagaagcagc tcctggctga tgttgcccaa 1150
gctcttgaca ttggccctgc cggtccactg atgggtgttg tccagtatgg 1200
agacaaccct gctactcact ttaacctcaa gacacacacg aattctcgag 1250
atctgaagac agccatagag aaaattactc agagaggagg actttctaat 1300
gtaggtcggg ccatctcctt tgtgaccaag aacttctttt ccaaagccaa 1350
tggaaacaga agcggggctc ccaatgtggt ggtggtgatg gtggatggct 1400
ggcccacgga caaagtggag gaggcttcaa gacttgcgag agagtcagga 1450
atcaacattt tcttcatca.c cattgaaggt gctgctgaaa atgagaagca 1500
gtatgtggtg gagcccaact ttgcaaacaa ggccgtgtgc agaacaaacg 1550
gcttctactc gctccacgtg cagagctggt ttggcctcca caagaccctg 1600
cagcctctgg tgaagcgggt ctgcgacact gaccgcctgg cctgcagcaa 1650
gacctgcttg aactcggctg acattggctt cgtcatcgac ggctccagca 1700
gtgtggggac gggcaacttc cgcaccgtcc tccagtttgt gaccaacctc 1750
accaaagagt ttgagatttc cgacacggac acgcgcatcg gggccgtgca 1800
gtacacctac gaacagcggc tggagtttgg gttcgacaag tacagcagca 1850
agcctgacat cctcaacgcc atcaagaggg tgggctactg gagtggtggc 1900
accagcacgg gggctgccat caacttcgcc ctggagcagc tcttcaagaa 1950
gtccaagccc aacaagagga agttaatgat cctcatcacc gacgggaggt 2000
cctacgacga cgtccggatc ccagccatgg ctgcccatct gaagggagtg 2050
atcacctatg cgataggcgt tgcctgggct gcccaagagg agctagaagt 2100
cattgccact caccccgcca gagaccactc cttctttgtg gacgagtttg 2150
acaacctcca tcagtatgtc cccaggatca tccagaacat ttgtacagag 2200
ttcaactcac agcctcggaa ctgaattcag agcaggcaga gcaccagcaa 2250
gtgctgcttt actaactgac gtgttggacc accccaccgc ttaatggggc 2300
acgcacggtg catcaagtct tgggcagggc atggagaaac aaatgtcttg 2350
ttattattct ttgccatcat gctttttcat attccaaaac ttggagttac 2400
aaagatgatc acaaacgtat agaatgagcc aaaaggctac atcatgttga 2450
Page 42
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
gggtgctgga gattttacat tttgacaatt gttttcaaaa taaatgttcg 2500
gaatacagtg cagcccttac gacaggctta cgtagagctt ttgtgagatt 2550
tttaagttgt tatttctgat ttgaactctg taaccctcag caagtttcat 2600
ttttgtcatg acaatgtagg aattgctgaa ttaaatgttt agaaggatga 2650
aaaataaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2700
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2750
aaaaaaaaaa aaaaaaaaaa aag 2773
<210> 34
<211> 678
<21Z> PRT
<213> Homo Sapien
<400> 34
Met Arg Thr Val Val Leu Thr Met Lys Ala Ser Val Ile Glu Met
1 5 10 15
Phe Leu Val Leu Leu Val Thr Gly Va1 His Ser Asn Lys Glu Thr
20 25 30
Ala Lys Lys Ile Lys Arg Pro Lys Phe Thr Val Pro Gln Ile Asn
35 40 45
Cys Asp vat Lys Ala Gly Lys Ile Ile Asp Pro Glu Phe Ile val
SO 55 60
Lys Cys Pro Ala Gly Cys Gln Asp Pro Lys Tyr His Val Tyr Gly
65 70 75
Thr Asp Val Tyr Ala Ser Tyr Ser Ser Val Cys Gly Ala Ala Val
80 85 90
His Ser Gly Val Leu Asp Asn Ser Gly Gly Lys Ile Leu Val Arg
95 100 105
Lys Val Ala Gly Gln Ser Gly Tyr Lys Gly Ser Tyr Ser Asn Gly
110 115 120
val Gln Ser Leu Ser Leu Pro Arg Trp Arg Glu Ser Phe Ile val
125 130 135
Leu Glu Ser Lys Pro Lys Lys Gly Val Thr Tyr Pro Ser Ala Leu
140 145 150
Thr Tyr Ser Ser Ser Lys Ser Pro Ala Ala Gln Ala Gly Glu Thr
155 160 165
Thr Lys Ala Tyr Gln Arg Pro Pro Ile Pro Gly Thr Thr Ala Gln
170 175 180
Pro Val Thr Leu Met Gln Leu Leu Ala Val Thr Val Ala Val Ala
185 190 195
Thr Pro Thr Thr Leu Pro Arg Pro Ser Pro Ser Ala Ala Ser Thr
200 205 210
Page 43
CA 02481756 2004-10-25
PCT-u500-23328_ Sequence
ThrSerIlePro ArgProGln SerValGly HisArg SerGlnGlu
215 220 225
MetAspLeuTrp SerThrAla ThrTyrThr SerSer GlnAsnArg
230 235 240
ProArgAlaASp ProGlyIle GlnArgGln AspPro SerGlyAla
245 250 255
AlaPheGlnLys ProValGly AlaAspVal SerLeu GlyLeuVal
260 265 270
ProLysGluGlu LeuSerThr GlnSer~eu GluPro ValSerLeu
275 280 285
GlyAspProAsn CysLysIle AspLeuSer PheLeu IleAspGly
290 295 300
SerThrSerIle GlyLysArg ArgPheArg IleGln LysGlnLeu
305 310 315
LeuAlaAspVal AlaGlnAla LeuAspIle GlyPro AlaGlyPro
320 325 330
LeuMetGlyVal ValGlnTyr GlyAspAsn ProAla ThrHisPhe
335 340 345
AsnLeuLysThr HisThrAsn SerArgAsp LeuLys ThrAlaIle
350 355 360
GluLysIleThr GlnArgGly GlyLeuSer AsnVal GlyArgAla
365 370 375
IleSerPheVal ThrLysAsn PhePheSer LysAla AsnGlyAsn
380 385 390
ArgSerGlyAla ProAsnVal ValValVal MetVal AspGlyTrp
395 400 405
ProThrAspLys ValGluGlu AlaSerArg LeuAla ArgGluSer
410 415 420
GlyIleAsnIle PhePheIle ThrIleGlu GlyAla AlaGluAsn
425 430 435
GluLysGlnTyr ValValGlu ProAsnPhe AlaAsn LysAlaVal
440 445 450
CysArgThrAsn GlyPheTyr SerLeuHis ValGln SerTrpPhe
455 460 465
GlyLeuHisLys ThrLeuGln ProLeuVal LysArg ValCysAsp
470 475 48 0
ThrAspArgLeu AlaCysSer LysThrCys LeuAsn SerAlaAsp
485 490 495
IleGlyPheVal IleAspGly SerSerSer ValGly ThrGlyAsn
500 505 5I0
PheArgThrVal LeuGlnPhe ValThrAsn LeuThr LysGluPhe
515 520 525
page 44
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
GluIle SerAspThr AspThr ArgIleGly AlaValGln TyrThr
530 535 540
TyrGlu GlnArgLeu GluPhe GlyPheAsp LysTyrSer SerLys
545 550 555
ProAsp IleLeuAsn AlaIle LysArgVal GlyTyrTrp SerGly
560 565 570
GlyThr SerThrGly AlaAla IleAsnPhe AlaLeuGlu GlnLeu
575 580 585
PheLys LysSerLys ProAsn LysArgLys LeuMetIle LeuIle
590 595 600
ThrAsp GlyArgSer TyrAsp AspValArg IleProAla MetAla
605 610 615
AlaHis LeuLysGly ValIle ThrTyrAla Il~eGlyVal AlaTrp
620 625 630
AlaAla Gln.GluGlu LeuGlu ValIleAla ThrHisPro AlaArg
635 640 645
AspHis SerPhePhe ValAsp GluPheAsp AsnLeuHis GlnTyr
650 655 660
ValPro ArgIleIle GlnAsn IieCysThr GluPheAsn SerGln
665 670 675
ProArg Asn
<210> 35
<211> 2095
<212> DNA
<213> Homo Sapien
<400> 35
ccgagcacag gagattgcct gcgtttagga ggtggctgcg ttgtgggaaa 50
agctatcaag gaagaaattg ccaaaccatg tctttttttc tgttttcaga 100
gtagttcaca acagatctga gtgttttaat taagcatgga atacagaaaa 150
caacaaaaaa cttaagcttt aatttcatct ggaattccac agttttctta 200
gctccctgga cccggttgac ctgttggctc ttcccgctgg ctgctctatc 250
acgtggtgct ctccgactac tcaccccgag tgtaaagaac cttcggctcg 300
cgtgcttctg agctgctgtg gatggcctcg gctctctgga ctgtccttcc 350
gagtaggatg tcactgagat ccctcaaatg gagcctcctg ctgctgtcac 400
tcctgagttt ctttgtgatg tggtacctca gccttcccc.a ctacaatgtg 450
atagaacgcg tgaactggat gtacttctat gagtatgagc cgatttacag 500
acaagacttt cacttcacac ttCgagagca ttcaaactgc tctcatcaaa 550
atccatttct ggtcattctg gtgacctccc acccttcaga tgtgaaagcc 600
Page 45
...,._. ~ , .,. .. ,., . . r.. ...,o-. ~, , x . _rn". ....,:~ e~w. v_.. ,
s~..",. ~yp~d, qyY~-.,.~. ,... ~. , ~.. ~ ,-., .p.a,.. ..... , -.__.. - _ _
- ,..... -::, ",. , -,.~ .. ,w. . .;... ~ e- - -. ~. .....
t
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
aggcaggcca ttagagttac ttggggtgaa aaaaagtctt ggtggggata 650
tgaggttctt acatttttct tattaggcca agaggctgaa aaggaagaca 700
aaatgttggc attgtcctta gaggatgaac accttcttta tggtgacata 750
atccgacaag attttttaga cacatataat aacctgacct tgaaaaccat 800
tatggcattc aggtgggtaa ctgagttttg ccccaatgcc aagtacgtaa 850
tgaagacaga cactgatgtt ttcatcaata ctggcaattt agtgaagtat 900
cttttaaacc taaaccactc agagaagttt ttcacaggtt atcctctaat 950
tgataattat tcctatagag gattttacca aaaaacccat atttcttacc 1000
aggagtatcc tttcaaggtg ttccctccat actgcagtgg gttgggttat 1050
ataatgtcca gagatttggt gccaaggatc tatgaaatga tgggtcacgt 1100
aaaacccatc aagtttgaag atgtttatgt cgggatctgt ttgaatttat 1150
taaaagtgaa cattcatatt ccagaagaca caaatctttt ctttctatat 1200
agaatccatt tggatgtctg tcaactgaga cgtgtgattg cagcccatgg 1250
cttttcttcc aaggagatca tcactttttg gcaggtcatg ctaaggaaca 1300
ccacatgcca ttattaactt cacattctac aaaaagccta gaaggacagg 1350
ataccttgtg gaaagtgtta aataaagtag gtactgtgga aaattcatgg 1400
ggaggtcagt gtgctggctt acactgaact gaaactcatg aaaaacccag 1450
actggagact ggagggttac acttgtgatt tattagtcag gcccttcaaa 1500
gatgatatgt ggaggaatta aatataaagg aattggaggt ttttgctaaa 1550
gaaattaata ggaccaaaca atttggacat gtcattctgt agactagaat 1600
ttcttaaaag ggtgttactg agttataagc tcactaggct gtaaaaacaa 1650
aacaatgtag agttttattt attgaacaat gtagtcactt gaaggttttg 1700
tgtatatctt atgtggatta ccaatttaaa aatatatgta gttctgtgtc 1750
aaaaaacttc ttcactgaag ttatactgaa caaaatttta cctgtttttg 1800
gtcatttata aagtacttca agatgttgca gtatttcaca gttattatta 1850
tttaaaatta cttcaaettt gtgtttttaa atgttttgac gatttcaata 1900
caagataaaa aggatagtga atcattcttt acatgcaaac attttccagt 1950
tacttaactg atcagtttat tattgataca tcactccatt aatgtaaagt 2000
cataggtcat tattgcatat cagtaatctc ttggactttg ttaaatattt 2050
tactgtggta atatagagaa gaattaaagc aagaaaatct gaaaa 2095
<210> 36
<211> 331
<212> PRT
Page 46
CA 02481756 2004-10-25
PCT-u500-23328_Sepuence
<213> Homo Sapien
<400> 36
Met Ala Ser Ala Leu Trp Thr Val Leu Pro Ser Arg Met Ser Leu
1 5 10 15
Arg Ser Leu Lys Trp Ser Leu Leu Leu Leu Ser Leu Leu Ser Phe
zo z5 30
Phe Val Met Trp Tyr Leu Ser Leu Pro His Tyr Asn Val Ile Glu
35 40 -45
Arg Val Asn Trp Met Tyr Phe Tyr Glu Tyr Glu Pro Ile Tyr Arg
50 55 60
Gln Asp Phe His Phe Thr Leu Arg Glu His Ser Asn Cys Ser His
65 70 75
Gln Asn Pro Phe Leu Val Ile Leu Val Thr Ser His Pro Ser Asp
80 85 90
Val Lys Ala Arg Gln Ala Ile Arg Val Thr Trp Gly Glu Lys Lys
95 100 105
Ser Trp Trp Gly Tyr Glu Val Leu Thr Phe Phe Leu Leu Gly Gln
110 115 120
Glu Ala Glu Lys Glu Asp Lys Met Leu Ala Leu Ser Leu GIu Asp
125 130 135
Glu His Leu Leu Tyr Gly Asp Ile Ile Arg Gln Asp Phe Leu Asp
140 145 150
Thr Tyr Asn Asn Leu Thr Leu Lys Thr Ile Met Ala Phe Arg Trp
155 160 165
val Thr Glu Phe Cys Pro Asn Ala Lys Tyr val Met Lys Thr Asp
170 175 180
Thr Asp Val Phe Ile Asn Thr Gly Asn Leu Val Lys Tyr Leu Leu
185 190 195
Asn Leu Asn His Ser Glu Lys Phe Phe Thr Gly Tyr Pro Leu Ile
200 205 210
Asp Asn Tyr Ser Tyr Arg Gly Phe Tyr Gln Lys Thr His Ile Ser
215 220 225
Tyr Gln Glu Tyr Pro Phe Lys Val Phe Pro Pro Tyr Cys Ser Gly
230 235 240
Leu Gly Tyr Ile Met 5er Arg Asp Leu Val Pro Arg Ile Tyr Glu
245 250 255
Met Met Gly His Val Lys Pro Ile Lys Phe Glu Asp Val Tyr Val
260 265 270
Gly Ile Cys Leu Asn Leu Leu Lys Val Asn Ile His Ile Pro Glu
275. 280 285
Asp Thr Asn Leu Phe Phe Leu Tyr Arg Ile His Leu Asp Val Cys
290 295 300
Page 47
,r.. , . .. ,.,m,. .... _. _. _...._ _...,r .,_ .....mM .. ... _.._.. .._..__
~.._..
CA 02481756 2004-10-25
PCT-u500-23328_sequence
Gln Leu Arg Arg Val Ile Ala Ala His Gly Phe Ser Ser Lys Glu
305 310 315
Ile Ile Thr Phe Trp Gln Val Met Leu Arg Asn Thr Thr Cys His
320 325 330
Tyr
<210> 37
<211> 2846
<212> DNA.
<213> Homo Sapien
<400> 37
cgctcgggca ccagccgcgg caaggatgga gctgggttgc tggacgcagt 50
tggggctcac ttttcttcag ctccttctca tctcgtcctt gccaagagag 100
tacacagtca ttaatgaagc ctgccctgga gcagagtgga atatcatgtg 150
tcgggagtgc tgtgaatatg atcagattga gtgcgtctgc cccggaaaga 200
gggaagtcgt gggttatacc atcccttgct gcaggaatga ggagaatgag 250
tgtgactcct gcctgatcca cccaggttgt accatctttg aaaactgcaa 300
gagctgccga aatggctcat gggggggtac cttggatgac ttctatgtga 350
aggggttcta ctgtgcagag tgccgagcag gctggtacgg aggagactgc 400
atgcgatgtg gccaggttct gcgagcccca aagggtcaga ttttgttgga 450
aagctatccc ctaaatgctc actgtgaatg gaccattcat gctaaacctg 500
ggtttgtcat ccaactaaga tttgtcatgt tgagtctgga gtttgactac 550
atgtgccagt atgactatgt tgaggttcgt gatggagaca accgcgatgg 600
ccagatcatc aagcgtgtct gtggcaacga gcggccagct cctatccaga 650
gcataggatc ctcactccac gtcctcttcc actccgatgg ctccaagaat 700
tttgacggtt tccatgccat ttatgaggag atcacagcat gctcctcatc 750
cccttgtttc catgacggca cgtgcgtcct tgacaaggct ggatcttaca 800
agtgtgcctg cttggcaggc tatactgggc agcgctgtga aaatctcctt 850
gaagaaagaa actgctcaga ccctgggggc ccagtcaatg ggtaccagaa 900
aataacaggg ggccctgggc ttatcaacgg acgccatgct aaaattggca 950
ccgtggtgtc tttcttttgt aacaactcct atgttcttag tggcaatgag 1000
aaaagaactt gccagcagaa tggagagtgg tcagggaaac agcccatctg 1050
cataaaagcc tgccgagaac caaagatttc agacctggtg agaaggagag 1100
ttcttccgat gcaggttcag tcaagggaga caccattaca ccagctatac 1150
tcagcggcct tcagcaagca gaaactgcag agtgccccta ccaagaagcc 1200
Page 48
CA 02481756 2004-10-25
PC'r-uS00-23328_5equence
agcccttccc tttggagatc tgcccatggg ataccaacat ctgcataccc 1250
agctccagta tgagtgcatc tcacccttct accgccgcct gggcagcagc 1300
aggaggacat gtctgaggac tgggaagtgg agtgggcggg caccatcctg 1350
catccctatc tgcgggaaaa ttgagaacat cactgctcca aagacccaag 1400
ggttgcgctg gccgtggcag gcagccatct acaggaggac cagcggggtg 1450
catgacggca gcctacacaa gggagcgtgg ttcctagtct gcagcggtgc 1500
cctggtgaat gagcgcactg tggtggtggc tgcccactgt gttactgacc 1550
tggggaaggt caccatgatc aagacagcag acctgaaagt tgttttgggg 1600
aaattctacc gggatgatga ccgggatgag aagaccatcc agagcctaca 1650
gatttctgct atcattctgc atcccaacta tgaccccatc ctgcttgatg 1700
ctgacatcgc catcctgaag ctcctagaca aggcccgtat cagcacccga 1750
gtccagccca tctgcctcgc tgccagtcgg gatctcagca cttccttcca 1800
ggagtcccac atcactgtgg ctggctggaa tgtcctggca gacgtgagga 1850
gccctggctt caagaacgac acactgcgct ctggggtggt cagtgtggtg 1900
gactcgctgc tgtgtgagga gcagcatgag gaccatggca tcccagtgag 1950
tgtcactgat aacatgttct gtgccagctg ggaacccact gccccttctg 2000
atatctgcac tgcagagaca ggaggcatcg cggctgtgtc cttcccggga 2050
cgagcatctc ctgagccacg ctggcatctg atgggactgg tcagctggag 2100
ctatgataaa acatgcagcc acaggctctc cactgccttc accaaggtgc 2150
tgccttttaa agactggatt gaaagaaata tgaaatgaac catgctcatg 2200
cactccttga gaagtgtttc tgtatatccg tctgtacgtg tgtcattgcg 2250
tgaagcagtg tgggcctgaa gtgtgatttg gcctgtgaac ttggctgtgc 2300
cagggcttct gacttcaggg acaaaactca gtgaagggtg agtagacctc 2350
cattgctggt aggctgatgc cgcgtccact actaggacag ccaattggaa 2400
gatgccaggg cttgcaagaa gtaagtttct tcaaagaaga ccatatacaa 2450
aacctctcca ctccactgac ctggtggtct tccccaactt tcagttatac 2500
gaatgccatc agcttgacca gggaagatct gggcttcatg aggccccttt 2550
tgaggctctc aagttctaga gagctgcctg tgggacagcc cagggcagca 2600
gagctgggat gtggtgcatg cctttgtgta catggccaca gtacagtctg 2650
gtccttttcc ttccccatct cttgtacaca ttttaataaa ataagggttg 2700
gcttctgaac tacaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2750
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2800
Page 49
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2846
<210> 38
<211> 720
<212> PRT
<213> Homo Sapien
<400> 38
Met Glu Leu Gly Cys Trp Thr Gln Leu Gly Leu Thr Phe Leu Gln
1 5 10 -15
Leu Leu Leu Ile Ser Ser Leu Pro Arg Glu Tyr Thr Val Ile Asn
20 25 30
Glu A1a Cys Pro Gly Ala Glu Trp Asn Ile Met Cys Arg Glu Cys
35 40 45
Cys Glu Tyr Asp Gln Ile Glu Cys Val Cys Pro Gly Lys Arg Glu
50 55 60
Val Val Gly Tyr Thr Ile Pro Cys Cys Arg Asn Glu Glu Asn Glu
65 70 75
Cys Asp Ser Cys Leu Ile His Pro Gly Cys Thr Ile Phe Glu Asn
80 85 90
Cys Lys Ser Cys Arg Asn Gly Ser Trp Gly Gly Thr Leu Asp Asp
95 100 105
Phe Tyr Val Lys Gly Phe Tyr Cys Ala Glu Cys Arg Ala Gly Trp
110 115 120
Tyr Gly Gly Asp Cys Met Arg Cys Gly Gln Val Leu Arg Ala Pro
125 130 135
Lys Gly Gln Ile Leu Leu Glu ser Tyr Pro Leu Asn Ala His Cys
140 145 150
Glu Trp Thr Ile His Ala Lys Pro Gly Phe Val Ile Gln Leu Arg
155 160 165
Phe val Met Leu Ser Leu Glu Phe Asp Tyr Met Cys Gln Tyr Asp
170 175 180
Tyr Val Glu Val Arg Asp Gly Asp Asn Arg Asp Gly Gln Ile Ile
185 190 195
Lys Arg val Cys Gly Asn Glu Arg Pro Ala Pro Ile Gln Servile
200 205 210
Gly Ser Ser Leu His Val Leu Phe His ser Asp Gly Ser Lys Asn
215 220 225
Phe Asp Gly Phe His Ala Ile Tyr Giu Glu Ile Thr Ala Cys Ser
230 235 240
Ser ser Pro Cys Phe His Asp Gly Thr Cys val Leu Asp Lys Ala
245 250 255
Gly Ser Tyr ~ys Cys A1a Cys Leu Ala Gly Tyr Thr Gly Gln Arg
260 265 270
Page 50
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Cys Glu Asn Leu Leu Glu Glu Arg Asn Cys Ser Asp Pro Gly Gly
275 280 285
Pro val Asn Gly Tyr G1n Lys I1e Thr Gly Gly Pro Gly Leu Ile
290 295 300
Asn Gly Arg His Ala Lys Ile Gly Thr Val Val 5er Phe Phe Cys
305 310 315
Asn Asn Ser Tyr Val Leu Ser Gly Asn Glu Lys Arg Thr Cys Gln
320 325 330
Gln Asn Gly Glu Trp Ser Gly Lys Gln Pro Ile Cys I1e Lys Ala
335 340 345
Cys Arg Glu Pro Lys Ile Ser Asp Leu val Arg Arg Arg val Leu
350 355 360
Pro Met Gln Val Gln Ser Arg Glu Thr Pro Leu His Gln Leu Tyr
365 370 375
Ser Ala Ala Phe Ser Lys Gln Lys Leu Gln Ser Ala Pro Thr Lys
380 385 390
Lys Pro Ala Leu Pro Phe Gly Asp Leu Pro Met Gly Tyr Gln His
395 400 405
Leu His Thr Gln Leu Gln Tyr Glu Cys Ile Ser Pro Phe Tyr Arg
410 415 420
Arg Leu Gly Ser Ser Arg Arg Thr Cys Leu Arg Thr Gly Lys Trp
425 430 435
Ser Gly Arg Ala Pro Ser Cys Ile Pro Ile Cys Gly Lys Ile Glu
440 445 450
Asn Ile Thr Ala Pro Lys Thr Gln Gly Leu Arg Trp Pro Trp Gln
455 460 465
Ala Ala Ile Tyr Arg Arg Thr Ser Gly Val His Asp Gly Ser Leu
470 475 480
His Lys Gly Ala Trp Phe Leu Val Cys Ser Gly Ala Leu Val Asn
485 490 495
G1u Arg Thr Val Val Val Ala Ala His Cys val Thr Asp Leu Gly
500 505 510
Lys Val Thr Met Ile Lys Thr Ala Asp Leu Lys Val Val Leu Gly
515 520 525
Lys Phe Tyr Arg Asp Asp Asp Arg Asp Glu Lys Thr Ile Gln Ser
530 . 535 540
Leu Gln Ile Ser Ala Ile Ile Leu His Pro Asn Tyr Asp Pro Ile
545 550 555
Leu Leu Asp Ala Asp Ile Ala Ile Leu Lys Leu Leu Asp Lys Ala
560 565 570
Arg Ile Ser Thr Arg Val Gln Pro Ile Cys Leu Ala AIa Ser Arg
575 580 585
Page 51
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
Asp Leu Ser Thr Ser Phe Gln Glu Ser His I12 Thr Val Ala Gly
590 595 600
Trp Asn Val Leu Aia Asp Val Arg Ser Pro Gly Phe Lys Asn Asp
605 610 615
Thr Leu Arg Ser Gly val val Ser val val Asp Ser Leu Leu Cys
620 625 630
Glu Glu Gln His Glu Asp His Gly Ile Pro Val Ser Val Thr Asp
63 5 640 f 4 5
Asn Met Phe Cys Ala Ser Trp Glu Pro Thr Ala Pro Ser Asp Ile
650 655 660
Cys Thr Ala Glu Thr Gly Giy Ile Ala Ala val Ser Phe Pro Gly
665 670 675
Arg Ala Ser Pro Glu Pro Arg Trp His Leu Met Giy Leu val Ser
680 685 690
Trp Ser Tyr Asp Lys Thr Cys Ser His Arg Leu Ser Thr Ala Phe
695 700 705
Thr Lys Vai Leu Pro Phe Lys Asp Trp Ile Glu Arg Asn Met Lys
710 715 720
<210> 39
<211> 2571
<212> DNA
<213> Homo Sapien ,'
<400> 39
ggttcctaca tcctctcatc tgagaatcag agagcataat cttcttacgg SO
gcccgtgatt tattaacgtg gcttaatctg aaggttctca gtcaaattct 100
ttgtgatcta ctgattgtgg gggcatggca aggtttgctt aaaggagctt 150
ggctggtttg ggcccttgta gctgacagaa ggtggccagg gagaatgcag 200
cacactgctc ggagaatgaa ggcgcttctg ttgctggtct tgccttggct 250
cagtcctgct aactacattg acaatgtggg caacctgcac ttcctgtatt 300
cagaactctg taaaggtgcc tcccactacg gcctgaccaa agataggaag 350
aggegctcae aagatggetg tecagacggc tgtgegagce tcacageeac.400
ggctccctcc ccagaggttt ctgcagctgc caccatctcc ttaatgacag 450
acgagcctgg cctagacaac cctgcctacg tgtcctcggc agaggacggg 500
cagccagcaa tcagcccagt ggactctggc cggagcaacc gaactagggc 550
acggcccttt gagagatcca ctattagaag cagatcattt aaaaaaataa 600
atcgagcttt gagtgttctt cgaaggacaa agagcgggag tgcagttgcc 650
aaccatgccg accagggcag ggaaaattct gaaaacacca ctgcccctga 700
agtctttcca aggttgtacc acctgattcc agatggtgaa attaccagca 750
Page 52
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
tcaagatcaa tcgagtagat cccagtgaaa gcctctctat taggctggtg 800
ggaggtagcg aaaccccact ggtccatatc attatccaac acatttatcg 850
tgatggggtg atcgccagag acggccggct actgccagga gacatcattc 900
taaaggtcaa cgggatggac atcagcaatg tccctcacaa ctacgctgtg 950
cgtctcctgc ggcagccctg ccaggtgctg tggctgactg tgatgcgtga 1000
acagaagttc cgcagcagga acaatggaca ggccccggat gcctacagac 1050
cccgagatga cagctttcat gtgattctca acaaaagtag ccccgaggag 1100
cagcttggaa taaaactggt gcgcaaggtg gatgagcctg gggttttcat 1150
cttcaatgtg ctggatggcg gtgtggcata tcgacatggt cagcttgagg 1200
agaatgaccg tgtgttagcc atcaatggac atgatcttcg atatggcagc 1250
ccagaaagtg cggctcatct gattcaggcc agtgaaagac gtgttcacct 1300
cgtcgtgtcc cgccaggttc ggcagcggag ccctgacatc tttcaggaag 1350
ccggctggaa cagcaatggc agctggtccc cagggccagg ggagaggagc 1400
aacactccca agcccctcca tcctacaatt acttgtcatg agaaggtggt 1450
aaatatccaa aaagaccccg gtgaatctct cggcatgacc gtcgcagggg 1500
gagcatcaca tagagaatgg gatttgccta tctatgtcat cagtgttgag 1550
cccggaggag tcataagcag agatggaaga ataaaaacag gtgacatttt 1600
gttgaatgtg gatggggtcg aactgacaga ggtcagccgg agtgaggcag 1650
tggcattatt gaaaagaaca tcatcctcga tagtactcaa agctttggaa 1700
gtcaaagagt atgagcccca ggaagactgc agcagcccag cagccctgga 1750
ctccaaccac aacatggccc cacccagtga ctggtcccca tcctgggtca 1800
tgtggctgga attaccacgg tgcttgtata actgtaaaga tattgtatta 1850
cgaagaaaca cagctggaag tctgggcttc tgcattgtag gaggttatga 1900
agaatacaat ggaaacaaac cttttttcat caaatccatt gttgaaggaa 1950
caccagcata caatgatgga agaattagat gtggtgatat tcttcttgct 2000 -
gtcaatggta gaagtacatc aggaatgata catgcttgct tggcaagact 2050
gctgaaagaa cttaaaggaa gaattactct aactattgtt tcttggcctg 2100
gcactttttt atagaatcaa tgatgggtca gaggaaaaca gaaaaatcac 2150
aaataggcta agaagttgaa acactatatt tatcttgtca gtttttatat 2200
ttaaagaaag aatacattgt aaaaatgtca ggaaaagtat gatcatctaa 2250
tgaaagccag ttacacctca gaaaatatga ttccaaaaaa attaaaacta 2300
ctagtttttt ttcagtgtgg aggatttctc attactctac aacattgttt 2350
Page 53 .
_.___ . ,~.... ,~ e..~__.~ __.. . _ . ?.. .. __......
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
atattttttc tattcaataa aaagccctaa aacaactaaa atgattgatt 2400
tgtatacccc actgaattca agctgattta aatttaaaat ttggtatatg 2450
ctgaagtctg ccaagggtac attatggcca tttttaattt acagctaaaa 2500
tattttttaa aatgca.ttgc tgagaaacgt tgctttcatc aaacaagaat 2550
aaatattttt cagaagttaa a 2571
<210> 40
<211> 632
<z12> PRT
<213> Homo Sapien
<400> 40
Met Lys Ala Leu Leu Leu Leu Val Leu Pro Trp Leu Ser Pro Ala
1 S 10 15
Asn Tyr Ile Asp Asn Val Gly Asn Leu His Phe Leu Tyr Ser Glu
20 25 30
Leu Cys Lys Gly Ala Ser His Tyr Gly Leu Thr Lys Asp Arg Lys
35 40 45
Arg Arg Ser Gln Asp Gly Cys Pro asp Gly Cys Ala Ser Leu Thr
50 55 60
Ala Thr Ala Pro Ser Pro Glu Val Ser Ala A7a Ala Thr Ile Ser
65 70 75
Leu Met Thr Asp Glu Pro Gly Leu Asp Asn Pro Ala Tyr. Val Ser
80 85 90
Ser Ala Glu ASp Gly Gln Pro Ala Ile Ser Pro val Asp Ser Gly
95 100 105
Arg Ser Asn Arg Thr Arg Ala Arg Pro Phe Glu Arg Ser Thr Ile
110 115 120
Arg Ser Arg Ser Phe Lys Lys Ile Asn Arg Ala Leu Ser Val Leu
125 130 135
Arg Arg Thr Lys Ser Gly Ser Ala Val Ala Asn His Ala Asp Gln
140 145 150
Gly Arg Glu Asn Ser Glu Asn Thr Thr Ala Pro Glu Val Phe Pro
155 160 165
Arg Leu Tyr His Leu Ile Pro Asp Gly Glu Ile Thr Ser Ile Lys
170 175 180
Ile Asn Arg val Asp Pro Ser Glu Ser Leu 5er Ile Arg Leu Val
18S 190 195
Gly G1y Ser Glu Thr Pro Leu val His Ile Ile Ile Gln His Ile
200 205 210
Tyr Arg ASp Gly Val Ile Ala Arg Asp Gly Arg Leu Leu Pro Gly
215 220 225
Asp Ile Ile Leu Lys wa't ASn Gly Met Asp I1e Ser Asn val Pro
Page 54
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
230 235 240
His Asn Tyr Ala Val Arg Leu Leu Arg Gln Pro cys Gln val Leu
245 250 255
Trp Leu Thr Val Met Arg Glu Gln Lys Phe Arg Ser Arg Asn Asn
260 265 270
Gly Gln Ala Pro Asp Ala Tyr Arg Pro Arg Asp Asp Ser Phe His
275 280 285
val Ile Leu Asn Lys Ser Ser Pro Glu Glu Gln Leu Gly Ile Lys
290 295 300
Leu Val Arg Lys Val Asp Glu Pro Gly Val Phe Ile Phe Asn Val
305 310 315
Leu Asp Gly Gly Val Ala Tyr Arg His Gly Gln Leu Glu Glu Asn
320 325 330
Asp Arg val Leu Ala Ile Asn Gly His asp Leu Arg Tyr Gly Ser
335 340 345
Pro Glu Ser Ala Ala His Leu Ile Gln Ala Ser Glu Arg Arg Val
350 355 360
His Leu Val Val Ser Arg Gln Val Arg Gln Arg Ser Pro Asp Ile
365 370 375
Phe Gln Glu Ala Gly Trp Asn Ser Asn Gly Ser Trp Ser Pro Gly
380 385 390
Pro Gly Glu Arg Ser Asn Thr Pro Lys Pro Leu His Pro Thr Ile
395 400 405
Thr cys His Glu Lys val val Asn Ile Gln Lys asp Pro Gly Glu
410 415 420
Ser Leu Gly Met Thr val Ala Gly Gly Ala Ser His Arg Glu Trp
425 430 435
Asp Leu Pro Ile Tyr val Ile ser val Glu Pro Gly Gly val Ile
440 445 450
ser Arg Asp G1y Arg Ile Lys Thr Gly asp Ile Leu Leu Asn val
455 460 465
Asp Gly Val Glu Leu Thr G1a Val Ser Arg Ser Glu Ala Va1 Ala
470 475 480
Leu Leu Lys Arg Thr ser Ser Ser Ile val Leu Lys Ala Leu Glu
485 490 495
val Lys Glu Tyr Glu Pro Gln Glu Asp Cys Ser Ser Pro Ala Ala
500 505 510
Leu Asp Ser Asn His Asn Met Ala Pro Pro Ser Asp Trp Ser Pro
515 520 525
Ser Trp Val Met Trp Leu Gia Leu Pro Arg Cys Leu Tyr Asn Cys
530 535 540
Lys asp Ile val Leu Arg Arg Asn Thr Ala Gly Ser Leu Gly Phe
Page 55
CA 02481756 2004-10-25
PCT-uS00-23328_seguence
545 550 555
Cys Ile Val Gly Gly Tyr Glu Glu Tyr Asn Gly Asn Lys Pro Phe
560 565 570
Phe Ile Lys Ser Ile Val Glu Gly Thr Pro Ala Tyr Asn Asp Gly
575 580 585
Arg Ile Arg Cys Gly Asp Ile Leu Leu Ala Val Asn Gly Arg Ser
590 595 600
Thr Ser Gly Met Ile His Ala Cys Leu Ala Arg Leu Leu Lys Glu
605 610 615
Leu Lys Gly Arg Ile Thr Leu Thr Ile Val Ser Trp Pro Gly Thr
620 625 630
Phe Leu
<210> 41
<211> 1964
<212> DNA
<213> Homo Sapien
<400> 41
accaggcatt gtatcttcag ttgtcatcaa gttcgcaatc agattggaaa 50
agctcaactt gaagctttct tgcctgcagt gaagcagaga gatagatatt 100
attcacgtaa taaaaaacat gggcttcaac ctgactttcc acctttccta 150
caaattccga ttactgttgc tgttgacttt gtgcctgaca gtggttgggt 200
gggccaccag taactacttc gtgggtgcca ttcaagagat tcctaaagca 250
aaggagttca tggctaattt ccataagacc ctcattttgg ggaagggaaa 300
aactctgact aatgaagcat ccacgaagaa ggtagaactt gacaactgtc 350
cttctgtgtc tccttacctc agaggccaga gcaagctcat tttcaaacca 400
gatctcactt tggaagaggt aeaggcagaa aatcccaaag tgtccagagg 450
ccggtatcgc cctcaggaat gtaaagcttt acagagggtc gccatcctcg 500
ttccccaccg gaacagagag aaacacctga tgtacctgct ggaacatctg 550
catcccttcc tgcagaggca gcagctggat tatggcatct acgtcatcca 600
ccaggctgaa ggtaaaaagt ttaatcgagc caaactcttg aatgtgggct 650
atctagaagc cctcaaggaa gaaaattggg actgctttat attccacgat 700
gtggacctgg tacccgagaa tgactttaac ctttacaagt gtgaggagca 750
tcccaagcat ctggtggttg gcaggaacag cactgggtac aggttacgtt 800
acagtggata ttttgggggt gttactgccc taagcagaga gcagtttttc 850
aaggtgaatg gattctctaa caactactgg ggatggggag gcgaagacga 900
tgacctcaga ctcagggttg agctccaaag aatgaaaatt tcccggcccc 950
Page 56
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
tgcctgaagt gggtaaatat acaatggtct tccacactag agacaaaggc 1000
aatgaggtga acgcagaacg gatgaagctc ttacaccaag tgtcacgagt 1050
ctggagaaca gatgggttga gtagttgttc ttataaatta gtatctgtgg 1100
aacacaatcc tttatatatc aacatcacag tggatttctg gtttggtgca 1150
tgaccctgga tcttttggtg atgtttggaa gaactgattc tttgtttgca 1200
ataattttgg cctagagact tcaaatagta gcacacatta agaacctgtt 1250
acagctcatt gttgagctga atttttcctt tttgtatttt cttagcagag 1300
ctcctggtga tgtagagtat aaaacagttg taacaagaca gctttcttag 1350
tcattttgat catgagggtt aaatattgta atatggatac ttgaaggact 1400
ttatataaaa ggatgactca aaggataaaa tgaacgctat ttgaggactc 1450
tggttgaagg agatttattt aaatttgaag taatatatta tgggataaaa 1500
ggccacagga aataagactg ctgaatgtct gagagaacca gagttgttct 1550
cgtccaaggt agaaaggtac gaagatacaa tactgttatt catttatcct 1600
gtacaatcat ctgtgaagtg gtggtgtcag gtgagaaggc gtccacaaaa 1650
gaggggagaa aaggcgacga atcaggacac agtgaacttg ggaatgaaga 1700
ggtagcagga gggtggagtg tcggctgcaa aggcagcagt agctgagctg 1750
gttgcaggtg ctgatagcct tcaggggagg acctgcccag gtatgccttc 1800
cagtgatgcc caccagagaa tacattctct attagttttt aaagagtttt 1850
tgtaaaatga ttttgtacaa gtaggatatg aattagcagt ttacaagttt 1900
acatattaac taataataaa tatgtctatc aaatacctct gtagtaaaat 1950
gtgaaaaagc aaaa 1964
<210> 42
<211> 344
<212> PRT
<213> Homo Sapien
<400> 42 .
Met Gly Phe Asn Leu Thr Phe His Leu Ser Tyr Lys Phe Arg Leu
1 5 10 15
Leu Leu Leu Leu Thr Leu Cys Leu Thr Val Val Gly Trp Ala Thr
20 25 30
ser Asn Tyr Phe val Gly Ala Ile G1n Glu Ile Pro Lys Ala Lys
35 40 45
Glu Phe Met ATa ASn Phe His Lys Thr Leu Ile Leu Gly Lys Gly
S0 55 60
Lys Thr Leu Thr Asn Glu Ala Ser Thr Lys Lys Val Glu Leu Asp
65 70 75
Page 57
CA 02481756 2004-10-25
PCT-US00-23328_5equence
Asn Cys Pro Ser Val Ser Pro Tyr Leu Arg Gly Gln ser Lys Leu
80 85 90
Ile Phe Lys Pro Asp Leu Thr Leu Glu Glu Val Gln Ala Glu Asn
95 100 105
Pro Lys Val Ser Arg Gly Arg Tyr Arg Pro Gln Glu Cys Lys Ala
1l0 115 120
Leu Gln Arg Val Ala Ile Leu Val Pro His Arg Asn Arg Glu tys
125 130 135
His Leu Met Tyr Leu Leu Glu His Leu His Pro Phe Leu Gln Arg
140 145 150
Gln Gln Leu Asp Tyr Gly Ile Tyr Val Ile His Gln Ala Glu Gly
155 160 165
Lys Lys Phe Asn Arg Ala Lys Leu Leu Asn Val Gly Tyr Leu Glu
170 175 180
Ala Leu Lys Glu Glu ,asn Trp Asp Cys Phe Ile Phe His asp Val
185 190 195
Asp Leu Val Pro Glu Asn Asp Phe Asn Leu Tyr Lys Cys Glu Glu
200 205 210
His Pro Lys His Leu Val Val Gly Arg Asn Ser Thr Gly Tyr Arg
215 220 225
Leu Arg Tyr Ser Gly Tyr Phe Gly Gly Val Thr Ala Leu Ser Arg
230 235 240
Glu Gln Phe Phe Lys Val Asn Gly Phe Ser Asn Asn Tyr Trp Gly
245 250 255
Trp Gly Gly Glu Asp Asp Asp Leu Arg Leu Arg Val Glu Leu Gln
260 265 270
Arg Met Lys Ile Ser Arg Pro Leu Pro Glu Val Gly Lys Tyr Thr
275 280 285
Met Val Phe His Thr Arg Asp Lys Gly ASn Glu Val Asn Ala Glu
290 295 300
Arg Met Lys Leu Leu His Gln Val Ser Arg Val Trp Arg Thr Asp
305 3I0 315
Gly Leu Ser Ser cys Ser Tyr Lys Leu Val Ser Val Glu His Asn
320 325 330
Pro Leu Tyr Ile Asn Ile Thr Val Asp Phe Trp Phe Gly Ala
335 340
<210> 43~
<211> 485
<212> DNA
<213> Homo sapien
<400> 43
gctcaagacc cagcagtggg acagccagac agacggcacg atggcactga 50
Page 58
,rv., .. ~.. , n. .. __.._.__ ~... ~~.r ~ ...~. _ .....w. __~r,~.Y . M_r,_._ .
. _ _ _._.__._..~.m.~. .",..~..~~",~ ..~.~"~~.~._.~.~ ~~n .~~...~~.~.
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
gctcccagat ctgggccgct tgcctcctgc tcctcctcct cctcgccagc 100
ctgaccagtg gctctgtttt cccacaacag acgggacaac ttgcagagct 150
gcaaccccag gacagagctg gagccagggc cagctggatg cccatgttcc 200
agaggcgaag gaggcgagac acccacttcc ccatctgcat tttctgctgc 250
ggctgctgtc atcgatcaaa gtgtgggatg tgctgcaaga ~cgtagaacct 300
acctgccctg cccccgtccc ctcccttcct tatttattcc tgctgcccca 350
gaacataggt cttggaataa aatggctggt tcttttgttt tccaaaaaaa 400
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 450
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 485
<210> 44
<211> 84
<212> PRT
<213> Homo Sapien
<400> 44
Met Ala Leu Ser 5er Gln Ile Trp Ala Ala Cys Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Ala Ser Leu Thr Ser Gly Ser val Phe Pro Gln Gln
20 25 30
Thr Gly Gln Leu Ala Glu Leu Gln Pro Gln Asp Arg Ala Gly Ala
35 40 45
Arg Ala Ser Trp Met Pro Met Phe Gln Arg Arg Arg Arg Arg ASp
SO 55 60
Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg
65 70 75
Ser Lys Cys Gly Met Cys Cys Lys Thr
80
<210> 45
<211> 1076
<212> DNA
<213> Homo Sapien
<400> 45
gtggcttcat ttcagtggct gacttccaga gagcaatatg gctggttccc 50
caacatgcct caccctcatc tatatccttt ggcagctcac agggtcagca 100
gcctctggac ccgtgaaaga gctggtcggt tccgttggtg gggccgtgac 150
tttccccctg aagtccaaag taaagcaagt tgactctatt gtctggacct 200
tcaacacaac ccctcttgtc accatacagc cagaaggggg cactatcata 250
gtgacccaaa atcgtaatag ggagagagta gacttcccag atggaggcta 300
ctccctgaag ctcagcaaac tgaagaagaa tgactcaggg atctactatg 350
tggggatata cagctcatea ctccagcagc cctccaccca ggagtacgtg 400
Page 59
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
ctgcatgtct acgagcacct gtcaaagcct aaagtcacca tgggtctgca 450
gagcaataag aatggcacct gtgtgaccaa tctgacatgc tgcatggaac S00
atggggaaga ggatgtgatt tatacctgga aggccctggg gcaagcagcc 550
aatgagtccc ataatgggtc catcctcccc atctcctgga gatggggaga 600
aagtgatatg accttcatct gcgttgccag gaaccctgtc agcagaaact 650
tctcaagccc catccttgcc aggaagctct gtgaaggtgc tgctgatgac 700
ccagattcct ccatggtcct cctgtgtctc ctgttggtgc ccctcctgct 750
cagtctcttt gtactggggc tatttctttg gtttctgaag agagagagac 800
aagaagagta cattgaagag aagaagagag tggacatttg tcgggaaact 850
cctaacatat gcccccattc tggagagaac acagagtacg acacaatccc 900
tcacactaat agaacaatcc taaaggaaga tccagcaaat acggtttact 950
ccactgtgga aataccgaaa aagatggaaa atccccactc actgctcacg 1000
atgccagaca caccaaggct atttgcctat gagaatgtta tctagacagc 1050
agtgcactcc cctaagtctc tgctca 1076
<210> 46
<211> 335
<212> PRT
<213> Homo Sapien
<400> 46
Met Ala Gly Ser Pro Thr Cys Leu Thr Leu Ile Tyr Ile Leu Trp
1 - 5 10 15
Gln Leu Thr Gly Ser Ala Ala Ser Gly Pro Val Lys Glu Leu Val
20 25 30
Gly Ser Val Gly Gly Ala Val Thr Phe Pro Leu Lys Ser Lys Val
35 40 45
Lys Gln Val Asp Ser Ile Val Trp Thr Phe Asn Thr Thr Pro Leu
50 55 60
vai Thr Iie Gln Pro Glu Gly Gly Thr Ile zie val Thr Gln Asn
65 70 75
Arg Asn Arg Glu Arg Val Asp Phe Pro Asp Gly Gly Tyr Ser Leu
80 85 90
Lys Leu Ser Lys Leu Lys Lys Asn asp Ser Giy Ile Tyr Tyr val
95 100 105
Gly Ile Tyr Ser Ser Ser Leu Gln Gln Pro Ser Thr Gln Glu Tyr
110 115 120
Val Leu His Val Tyr G1a His Leu Ser Lys Pro Lys Val Thr Met
125 130 135
Gly Leu Gln Ser Asn Lys Asn Gly Thr Cys Val Thr Asn Leu Thr
Page 60
.... ,... a .._ yn.,,hwn v.,.,x.x . , wme_ .:~~,a~roy~Mr d ~« a . .....,." ..
.".... ",. .,_a. w,,awa,..,.V m. ..n~,.. m. "_-,~..~,-,..... _. .. __.
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
140 145 150
Cys Cys Met Glu His Gly Glu Glu Asp Val Ile Tyr Thr Trp Lys
I55 160 165
Ala Leu Gly Gln Ala Ala Asn Glu Ser His Asn Gly Ser Ile Leu
170 175 180
Pro Ile Ser Trp Arg Trp Gly Glu Ser asp Met Thr Phe Ile Cys
185 190 195
Val Ala Arg Asn Pro Val Ser Arg Asn Phe Ser Ser Pro Ile Leu
200 205 210
Ala Arg Lys Leu Cys Glu Gly Ala Ala Asp asp Pro Asp Ser Ser
21S 220 225
Met Val Leu Leu Cys Leu Leu Leu Val Pro Leu Leu Leu Ser Leu
230 235 240
Phe Val Leu Gly Leu Phe Leu Trp Phe Leu Lys Arg Glu Arg Gln
245 250 255
Glu Glu Tyr Ile Glu Glu Lys Lys Arg Val Asp Ile Cys Arg Glu
260 265 270
Thr Pro Asn Ile Cys Pro His Ser Gly Glu Asn Thr Glu Tyr Asp
275 280 285
Thr Ile Pro His Thr Asn Arg Thr Ile Leu Lys Glu Asp Pro Ala
290 295 300
Asn Thr Val Tyr Ser Thr Val Glu Ile Pro Lys Lys Met Glu Asn
305 310 315
Pro His Ser Leu Leu Thr Met Pro Asp Thr Pro Arg Leu Phe Ala
320 325 330
Tyr Glu Asn val Ile
335
<210> 47
<211> 766
<212> ANA
<213> Homo Sapien
<400> 47
ggctcgagcg tttctgagcc aggggtgacc atgacctgct gcgaaggatg 50
gacatcctgc aatggattca gcctgctggt tctactgctg ttaggagtag 100
ttctcaatgc gatacctcta attgtcagct tagttgagga agaccaattt 150
tctcaaaacc ccatctcttg ctttgagtgg tggttcccag gaattatagg 200
agcaggtctg atggccattc cagcaacaac aatgtccttg acagcaagaa 250
aaagagcgtg,ctgcaacaac agaactggaa tgtttc~tttc atcatttttc 300
agtgtgatea eagtcattgg tgctetgtat tgeatgctga tatctatcca 350
ggctctctta aaaggtcctc tcatgtgtaa ttctccaagc aacagtaatg 400
Page 6I
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
ccaattgtga attttcattg aaaaacatca gtgacattca tccagaatcc 450
ttcaacttgc agtggttttt eaatgactct tgtgcacctc ctactggttt 500
caataaaccc accagtaacg acaccatggc gagtggctgg agagcatcta 550
gtttccactt cgattctgaa gaaaacaaac ataggcttat ccacttctca 600
gtatttttag gtctattgct tgttggaatt ctggaggtcc tgtttgggct 650
cagtcagata gtcatcggtt tccttggctg tctgtgtgga gtctctaagc 700
gaagaagtca aattgtgtag tttaatggga ataaaatgta agtatcagta 750
gtttgaaaaa aaaaaa 766
<210> 48
<211> 229
<212> PRT
<213> Homo Sapien
<400> 48
Met Thr Cys Cys Glu Gly Trp Thr Ser Cys Asn Gly Phe Ser Leu
1 5 10 15
Leu Val Leu Leu Leu ~eu Gly Val Val Leu Asn Ala Ile Pro Leu
20 25 30
Ile val 5er Leu val Glu Glu Asp Gln Phe Ser Gln Asn Pro Ile
35 40 45
Ser Cys Phe Glu Trp Trp Phe Pro Gly Ile Ile Gly Ala Gly Leu
50 55 60
Met Ala Ile Pro Ala Thr Thr Met Ser Leu Thr Ala Arg Lys Arg
65 70 75
Ala Cys Cys Asn Asn Arg Thr Gly Met Phe Leu Ser Ser Phe Phe
80 85 90
Ser val Ile Thr val Ile Gly Ala Leu Tyr Cys Met Leu Ile Ser
95 100 105
Ile Gln Ala Leu Leu Lys Gly Pro Leu Met Cys Asn Ser Pro Ser
110 115 120
Asn Ser Asn Ala Asn Cys Glu Phe Ser Leu Lys Asn Ile Ser Asp
125 130 135
Ile His Pro Glu Ser Phe Asn Leu Gln Trp Phe Phe Asn Asp Ser
140 145 150
Cys Ala Pro Pro Thr Gly Phe Asn Lys Pro Thr Ser Asn Asp Thr
155 160 165
Met Ala Ser Gly Trp Arg Ala Ser Ser Phe His Phe Asp Ser Glu
170 175 180
Glu Asn Lys His Arg Leu Ile His Phe Ser Val Phe Leu G1y Leu
185 190 195
Leu Leu Val Gly Ile Leu Giu Val Leu Phe Gly Leu Ser Gln Ile
200 205 210
Page 62
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
val Ile Gly Phe Leu Gly Cys Leu cys Gly val Ser Lys Arg Arg
215 220 225
Ser Gln Ile Val
<210> 49
<211> 636
<212> DNA
<213> Homo Sapien
<400> 49
atccgttctc tgcgctgcca gctcaggtga gccctcgcca aggtgacctc 50
gcaggacact ggtgaaggag cagtgaggaa cctgcagagt cacacagttg 100
ctgaccaatt gagctgtgag cctggagcag atccgtgggc tgcagacccc 150
cgccccagtg cctctccccc tgcagccctg cccctcgaac tgtgacatgg 200
agagagtgac cctggccctt ctcctactgg caggcctgac tgccttggaa 250
gccaatgacc catttgccaa taaagacgat cccttctact atgactggaa 300
aaacctgcag ctgagcggac tgatctgcgg agggctcctg gccattgctg 350
ggatcgcggc agttctgagt ggcaaatgca aatacaagag cagccagaag 400
cagcacagtc ctgtacctga gaaggccatc ccactcatca ctccaggctc 450
tgccactact tgctgagcac aggactggcc tccagggatg gcctgaagcc 500
taacactggc ccccagcacc tcctcccctg ggaggcctta tcctcaagga 550
aggacttctc tccaagggca ggctgttagg cccctttctg atcaggaggc 600
ttctttatga attaaactcg ccccaccacc ccctca 636
<210> 50
<211> 89
<212> PRT
<2I3> Homo Sapien
<400> 50
Met Glu Arg Val Thr Leu Ala Leu Leu Leu Leu Ala Gly Leu Thr
1 5 10 15
Ala Leu Glu Ala Asn Asp Pro Phe Ala Asn Lys Asp Asp Pro Phe
20 25 30
Tyr Tyr Asp Trp Lys Asn Leu Gln Leu Ser Gly Leu Ile Cys Gly
35 40 45
Gly Leu Leu Aia I1e Ala Giy Ile Ala Ala Val Leu Ser Gly Lys
50 55 60
Cys Lys Tyr Lys Ser. Ser Gln Lys Gln His Ser Pro val Pro Glu
65 70 75
Lys Ala Ile Pro Leu Ile Thr Pro Gly Ser Ala Thr Thr Cys
80 85
Page 63
~.,~.~, " ~. ,.... ....... ". ~a,~~,~~,a,;~.~ ~.,~~,n,..s~:. ._~.... -- .__
_....
CA 02481756 2004-10-25
PCT-US00-23328_seq~ence
<210> 51
<211> 1734
<212> DNA
<213> Homo 5apien
<400> 51
gtggactctg agaagcccag gcagttgagg acaggagaga gaaggctgca 50
gacccagagg gagggaggac agggagtcgg aaggaggagg acagaggagg 100
gcacagagac gcagagcaag ggcggcaagg aggagaccct ggtgggagga 150
agacactctg gagagagagg gggctgggca gagatgaagt tccaggggcc 200
cctggcctgc ctcctgctgg ccctctgcct gggcagtggg gaggctggcc 250
ccctgcagag cggagaggaa agcactggga caaatattgg ggaggccctt 300
ggacatggcc tgggagacgc cctgagcgaa ggggtgggaa aggccattgg 350
caaagaggcc ggaggggcag ctggctctaa agtcagtgag gcccttggcc 400
aagggaccag agaagcagtt ggcactggag tcaggcaggt tccaggcttt 450
ggcgcagcag atgctttggg caacagggtc ggggaagcag cccatgctct 500
gggaaacact gggcacgaga ttggcagaca ggcagaagat gtcattcgac 550
acggagcaga tgctgtccgc ggctcctggc agggggtgcc tggccacagt 600
ggtgcttggg aaacttctgg aggccatggc atctttggct ctcaaggtgg 650
ccttggaggc cagggccagg gcaatcctgg aggtctgggg actccgtggg 700
tccacggata ccccggaaac tcagcaggca gctttggaat gaatcctcag 750
ggagctccct ggggtcaagg aggcaatgga gggccaccaa actttgggac 800
caacactcag ggagctgtgg cccagcctgg ctatggttca gtgagagcca 850
gcaaccagaa tgaagggtgc acgaatcccc caccatctgg ctcaggtgga 900
ggctccagca actctggggg aggcagcggc tcacagtcgg gcagcagtgg 950
cagtggcagc aatggtgaca acaacaatgg cagcagcagt ggtggcagca 1000
gcagtggcag cagcagtggc agcagcagtg gcggcagcag tggcggcagc 1050
agtggtggca gcagtggcaa cagtggtggc agcagaggtg acagcggcag 1100
tgagtcctcc tggggatcca gcaccggctc ctcctccggc aaccacggtg 1150
ggagcggcgg aggaaatgga cataaacccg ggtgtgaaaa gccagggaat 1200
gaagcccgcg ggagcgggga atctgggatt cagggcttca gaggacaggg 1250
agtttccagc aacatgaggg aaataagcaa agagggcaat cgcctccttg 1300
gaggctctgg agaeaa~Ctat cgggggcaag ggtcgagctg gggcagtgga 1350
ggaggtgacg ctgttggtgg agtcaatact gtgaactctg a.gacgtctcc 1400
tgggatgttt aactttgaca ctttctggaa gaattttaaa tccaagctgg 1450
Page 64
.. w M ..... ._~,.~_._ ..~..... _M. .~e. .. w. ~,~ H.~w~~,"~~~,-~"~
~...m~._~..._ _... _
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
gtttcatcaa ctgggatgcc ataaacaagg accagagaag ctctcgcatc 1500
ccgtgacctc cagacaagga gccaccagat tggatgggag cccccacact 1550
ccctccttaa aacaccaccc tctcatcact aatctcagcc cttgcccttg 1600
aaataaacct tagctgcccc acaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1650
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1700
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1734
<210> S2
<211> 440
<212> PRT
<213> Homo Sapien
<400> 52
Met Lys Phe Gln Gly Pro Leu Ala Cys Leu Leu Leu Ala Leu Gys
1 5 10 15
Leu Gly Ser Gly Glu Ala Gly Pro Leu Gln Ser Giy Glu Glu Ser
20 25 30
Thr Gly Thr Asn Ile Gly Glu Ala Leu Gly His Gly Leu Gly Asp
35 40 45
Ala Leu Ser Glu G50y Val Gly Lys Ala I55 Gly Lys Glu Ala G
Gly Ala Ala Gly Ser Lys Val Ser Glu Ala Leu Gly Gln Gly Thr
65 70 75
Arg Glu Ala Val Gly Thr Gly Val Arg Gln Val Pro Gly Phe Gly
80 85 90
Aia Ala Asp Ala Leu Gly Asn Arg Vai G1y Giu Ala Aia His Ala
95 100 105
Leu Gly Asn Thr Gly His Glu Ile Gly Arg Gln Ala Glu Asp val
110 115 120
Ile Arg His Gly Ala Asp Ala Val Arg Gly Ser Trp Gln Gly Val
125 130 135
Pro Gly His Ser Gly Ala Trp Glu Thr Ser Gly Gly His Gly Ile
140 145 150
Phe Gly Ser Gln Gly Gly Leu Gly Gly Gln Gly Gln Gly Asn Pro
155 160 165
Gly Gly Leu Gly Thr Pro Trp Val His G1y Tyr Pro Gly Asn Ser
170 175 180
Ala Gly Ser Phe Gly Met Asn Pro Gln Gly Ala Pro Trp Giy Gln
185 190 195
Giy Gly Asn Gly Gly Pro Pro Asn Phe GIy Thr Asn Thr G7n Gly
200 205 210
Ala Val Ala Gln Pro Gly Tyr Gly Ser Val Arg Aia Ser Asn Gln
215 220 225
Page 65
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
Asn Glu Gly Cys Thr Asn Pro Pro Pro Ser Gly Ser Gly Gly Gly
230 235 240
Ser Ser Asn Ser Gly Gly Gly Ser Gly Ser Gln Ser Gly Ser Ser
245 250 255
Gly Ser Gly Ser Asn Gly Asp Asn Asn Asn Gly Ser Ser Ser Gly
260 265 270
Gly Ser Ser Ser Gly Ser Ser Ser Gly Ser Ser Ser Gly Gly Ser
275 280 285
Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Asn Ser Gly Gly Ser
290 295 300
Arg Gly Asp Ser Gly Ser Glu Ser Ser Trp Gly Ser Ser Thr Gly
305 310 315
Ser Ser Ser Gly Asn His Gly Gly Ser Gly Gly Gly Asn Gly His
320 325 330
Lys Pro Gly Cys Glu Lys Pro Gly Asn Glu Ala Arg Gly Ser Gly
335 340 345
Glu Ser Gly Ile Gln Gly Phe Arg Gly Gln Gly val Ser Ser Asn
350 355 360
Met Arg Glu Ile Ser Lys Glu Gly Asn Arg Leu Leu Gly Gly Ser
365 370 375
Gly Asp Asn Tyr Arg Gly Gln Gly Ser Ser Trp Gly Ser Gly Gly
380 385 390
Gly Asp Ala Val Gly Gly Val Asn Thr Val Asn Ser Glu Thr Ser
395 400 405
Pro Gly Met Phe Asn Phe Asp Thr Phe Trp Lys Asn Phe Lys Ser
410 415 420
Lys Leu Gly Phe Ile Asn Trp Asp Ala Ile Asn Lys Asp Gln Arg
425 430 435
Ser Ser Arg Ile Pro
440
<210> 53
<211> 1676
<212> DNA
<213> Homo Sapien
<400> 53
ggagaagagg ttgtgtggga caagctgctc ccgacagaag gatgtcgctg 50
ctgagcctgc cctggctggg cctcagaccg gtggcaatgt ccccatggct 100
actcctgctg ctggttgtgg gctcctggct actcgcccgc atcctggctt 150
ggacctatgc cttctataa.c aactgccgcc ggctccagtg ttteccacag 200
cccccaaaac ggaactggtt ttggggtcac ctgggcctga tcactcctac 250
agaggagggc ttgaaggact cgacccagat gtcggccacc tattcccagg 300
Page 66
CA 02481756 2004-10-25
Pct-US00-23328_5equence
gctttacggt atggctgggt cccatcatcc ccttcatcgt tttatgccac 350
cctgacacca tccggtctat caccaatgcc tcagctgcca ttgcacccaa 400
ggataatctc ttcatcaggt tcctgaagcc ctggctggga gaagggatac 450
tgctgagtgg cggtgacaag tggagccgcc accgtcggat gctgacgccc 500
gccttccatt tcaacatcct gaagtcctat ataacgatct tcaacaagag 550
tgcaaacatc atgcttgaca agtggcagca cctggcctca gagggcagca 600
gtcgtctgga catgtttgag cacatcagcc tcatgacctt ggacagtcta 650
cagaaatgca tcttcagctt tgacagccat tgtcaggaga ggcceagtga 700
.atatattgcc accatcttgg agctcagtgc ccttgtagag aaaagaagcc 750
agcatatcct ccagcacatg gactttctgt attacctctc ccatgacggg 800
cggcgcttcc acagggcctg ccgcctggtg catgacttca cagacgctgt 850
catccgggag cggcgtcgca ccctccccac tcagggtatt gatgattttt 900
tcaaagacaa agccaagtcc aagactttgg atttcattga tgtgcttctg 950
ctgagcaagg atgaagatgg gaaggcattg tcagatgagg atataagagc 1000
agaggctgac accttcatgt ttggaggcca tgacaccacg gccagtggcc 1050
tctcctgggt cctgtacaac cttgcgaggc acccagaata ccaggagcgc 1100
tgccgacagg aggtgcaaga gcttctgaag gaccgcgatc ctaaagagat 1150
tgaatgggac gacctggccc agctgccctt cctgaccatg tgcgtgaagg 1200
agagcctgag gttacatccc ccagctccct tcatctcccg atgctgcacc 1250
caggacattg ttctcccaga tggccgagtc atccccaaag gcattacctg 1300
cctcatcgat attatagggg tccatcacaa cccaactgtg tggccggatc 1350
ctgaggtcta cgaccccttc cgctttgacc cagagaacag caaggggagg 1400
tcacctctgg cttttattcc tttctccgca gggcccagga actgcatcgg 1450
gcaggcgttc gccatggcgg agatgaaagt ggtcctggcg ttgatgctgc 1500
tgcacttccg gttcctgcca gaccacactg agccccgcag gaagctggaa 1550
ttgatcatgc gcgccgaggg cgggctttgg ctgcgggtgg agcccctgaa 1600
tgtaggcttg cagtgacttt ctgacccatc cacctgtttt tttgcagatt 1650
gtcatgaata aaacggtgct gtcaaa 1676
<210> 54
<211> 524
<212> PRT
<213> Homo Sapien
<400> 54
Page 67
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Met Ser Leu Leu Ser ~.eu Pro Trp Leu Gly Leu Arg Pro Val Ala
1 5 IO 15
Met Ser Pro Trp Leu Leu Leu Leu Leu Val Val Gly Ser Trp Leu
20 25 30
Leu Ala Arg Ile Leu Ala Trp Thr Tyr Ala Phe Tyr Asn Asn Cys
35 40 45
Arg Arg Leu Gln Cys Phe Pro Gln Pro Pro Lys Arg Asn Trp Phe
50 S 5 -60
Trp Gly His Leu Gly Leu Ile Thr Pro Thr Glu Glu Gly Leu Lys
65 70 75
Asp Ser Thr Gln Met Ser Ala Thr Tyr Ser Gln Gly Phe Thr Val
80 85 90
Trp Leu Gly Pro Lle Ile Pro Phe Ile Val Leu Cys His Pro Asp
95 100 105
Thr Ile Arg Ser Ile Thr Asn Ala Ser Ala Ala Ile Ala Pro Lys
110 115 120
Asp Asn Leu Phe Ile Arg Phe Leu Lys Pro Trp Leu Gly Glu Gly
125 130 135
Ile Leu Leu Ser Gly Gly Asp Lys Trp Ser Arg His Arg Arg Met
140 I45 150
Leu Thr Pro Aia Phe His Phe Asn Ile Leu Lys Ser Tyr Iie Thr
155 160 ' 165
Ile Phe Asn Lys Ser Ala Asn Ile Met Leu Asp Lys Trp Gln His
170 175 180
Leu Ala Ser Glu Gly Ser Ser Arg Leu Asp Met Phe Glu His Ile
185 190 195
Ser Leu Met Thr Leu Asp Ser Leu Gln Lys Cys Ile Phe Ser Phe
20o zo5 zlo
Asp Ser His Cys Gln Glu Arg Pro Ser Glu Tyr Ile Ala Thr Iie
215 220 225
Leu Glu Leu Ser Ala Leu Val Glu Lys Arg Ser Gln His Ile Leu
230 235 240
Gln His Met Asp Phe Leu Tyr Tyr Leu Ser His Asp Gly Arg Arg
24S 250 255
Phe His Arg Ala Cys Arg Leu Vai His Asp Phe Thr Asp Ala Val
260 265 270
Ile Arg Glu Arg Arg Arg Thr Leu Pro Thr Gln Gly Ile Asp Asp
z7s zso z85
Phe Phe Lys Asp Lys Ala Lys Ser Lys Thr Leu Asp Phe Ile Asp
290 295 300
Val Leu Leu Leu Ser Lys ASp Glu Asp GIy Lys Ala Leu Ser Asp
305 310 315
Page 68
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
Glu Asp Ile Arg Ala Glu Ala Asp Thr Phe Met Phe Gly Gly His
320 325 330
Asp Thr Thr Ala Ser Gly Leu Ser Trp Val Leu Tyr Asn Leu Ala
335 340 345
Arg His Pro Glu Tyr Gln Glu Arg Cys Arg Gln Glu Val Gln Glu
350 355 360
Leu Leu Lys Asp Arg As'p Pro Lys Glu Ile Glu Trp Asp Asp Leu
365 370 375
Aia Gln Leu Pro Phe Leu Thr Met Cys Val Lys Glu Ser Leu Arg
380 385 390
Leu His Pro Pro Aia Pro Phe Ile Ser Arg Cys Cys Thr Gln Asp
395 400 405
Ile Val Leu. Pro Asp Gly Arg Val Ile Pro Lys Gly Ile Thr Cys
410 415 420
Leu Ile asp Ile Ile Gly Val His His Asn Pro Thr Val Trp Pro
425 430 435
Asp Pro Glu Val Tyr Asp Pro Phe Arg Phe Asp Pro Glu Asn Ser
440 445 450
Lys Gly Arg Ser Pro Leu Ala Phe Ile Pro Phe Ser Ala Gly Pro
455 460 465
Arg Asn Cys Ile Gly Gln Ala Phe Ala Met Ala Glu Met Lys Val
470 475 480
Val Leu Ala Leu Met Leu Leu His Phe Arg Phe Leu Pro Asp His
485 490 495
Thr Glu Pro Arg Arg Lys Leu Glu Leu Ile Met Arg Ala Glu Gly
500 505 510
Gly Leu Trp Leu Arg Val Glu Pro Leu Asn Val Gly Leu Gln
515 520
<210> 55
<211> 644
<212> DNA
<213> Homo Sapien
<400> 55
atcgcatcaa ttgggagtac catcttcctc atgggaccag tgaaacagct 50
gaagcgaatg tttgagccta ctcgtttgat tgcaactatc atggtgctgt 100
tgtgttttgc acttaccctg tgttctgcct tttggtggca taacaaggga 150
cttgcactta tcttctgcat tttgcagtct ttggcattga cgtggtacag 200
cctttccttc ataccatttg caagggatgc tgtgaagaag tgttttgccg 250
tgtgtcttgc ataattcatg gceagtttta tgaagctttg gaaggcacta 300
tggacagaag ctggtggaca gttttgtaac tatcttcgaa acctctgtct 350
tacagacatg tgccttttat cttgcagcaa tgtgttgctt gtgattcgaa 400
Page 69
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
catttgaggg ttacttttgg aagcaacaat acattctcga acctgaatgt 450
cagtagcaca ggatgagaag tgggttctgt atcttgtgga gtggaatctt 500
cctcatgtac ctgtttcctc tctggatgtt gtcccactga attcccatga 550
atacaaacct attcagcaac agcaaaaaaa aaaaaaaaaa aaaaaaaaaa 600
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 644
<210>
56
<211>
77
<212>
PRT
<213>
Homo
Sapien
<400>
56
Met Pro LysGln LeuLysArg MetPheGlu ProThr Arg
Gly Val
1 5 10 15
Leu Ala IieMet ValLeuLeu CysPheAla LeuThr Leu
Iie Thr
20 25 , 30
Cys Ala TrpTrp HisAsnLys GlyLeuAla LeuIle Phe
Ser Phe
35 40 45
Cys Leu SerLeu AlaLeuThr TrpTyrSer LeuSer Phe
Ile Gln
SO 55 60
Ile Phe ArgAsp AlaValLys LysCysPhe AlaVal Cys
Pro Ala
65 70 75
Leu
Ala
<210> 57
<211> 3334
<212> DNA
<213> Homo Sapien
<400> 57
cggctcgagc tcgagccgaa tcggctcgag gggcagtgga gcacccagca SO
ggccgccaac atgctctgtc tgtgcctgta cgtgccggtc atcggggaag 100
cccagaccga gttccagtac tttgagtcga aggggctccc tgccgagctg 150
aagtccattt tcaagctcag tgtcttcatc ccctcccagg aattctccac 200
ctaccgccag tggaagcaga aaattgtaca agctggagat aaggaccttg 250
atgggcagct agactttgaa gaatttgtcc attatctcca agatcatgag 300
aagaagctga ggctggtgtt taagattttg gacaaaaaga atgatggacg 350
cattgacgcg caggagatca tgcagtccct gcgggacttg ggagtcaaga 400
tatctgaaca gcaggcagaa aaaattctca agagcatgga taaaaacggc 450
acgatgacca tcgactggaa cgagtggaga gactaccacc tcctccaccc 500
cgtggaaaac atccccgaga tcatcctcta ctggaagcat tccacgatct 550
Page 70
_. _. .. . __.. _ _ ___ _,. . . ..~..~ _ . ...,Y. ~ tt z. H .~7..
~~u;~,,~.~~,, ~ wn ,. ~ ~ . a. _a~. ~ d4 ~.~ ~ ~. _ M..~~. . a ,~.__m~_
~____._~.
~a.~
CA 02481756 2004-10-25
PCT-US00-23328_Sec~uence
ttgatgtggg tgagaatcta acggtcccgg atgagttcac agtggaggag 600
aggcagacgg ggatgtggtg gagacacctg gtggcaggag gtggggcagg 650
ggccgtatcc agaacctgca cggcccccct ggacaggctc aaggtgctca 700
tgcaggtcca tgcctcccgc agcaacaaca tgggcatcgt tggtggcttc 750
actcagatga ttcgagaagg aggggccagg tcactctggc ggggcaatgg 800
catcaacgtc ctcaaaattg cccccgaatc agccatcaaa ttcatggcct 850
atgagcagat caagcgccta gttggtagtg accaggagac tctgaggatt 900
cacgagaggc ttgtggcagg gtccttggca ggggccatcg cccagagcag 950
catctaccca atggaggtcc tgaagacccg gatggcgctg cggaagacag 1000
gccagtactc aggaatgctg gactgcgcca ggaggatcct ggccagagag 1050
ggggtggccg ccttctacaa aggctatgtc cccaacatgc tgggcatcat 1100
cccctatgcc ggcatcgacc ttgcagtcta cgagacgctc aagaatgcct 1150
ggctgcagca ctatgcagtg aacagcgcgg accccggcgt gtttgtgctc 1200
ctggcctgtg gcaccatgtc cagtacctgt ggccagctgg ccagctaccc 1250
cctggcccta gtcaggaccc ggatgcaggc gcaagcctct attgagggcg 1300
ctccggaggt gaccatgagc agcctcttca aacatatcct gcggaccgag 1350
ggggccttcg ggctgtacag ggggctggcc cceaacttca tgaaggtcat 1400
cccagctgtg agcatcagct acgtggtcta cgagaacctg aagatcaccc 1450
tgggcgtgca gtcgcggtga cggggggagg gccgcccgge agtggactcg 1500
ctgatcctgg gccgcagcct ggggtgtgca gccatctcat tctgtgaatg 1550
tgccaacact aagctgtctc gagccaagct gtgaaaaccc tagacgcacc 1600
cgcagggagg gtggggagag ctggcaggec cagggcttgt cctgctgacc 1650
ccagcagacc ctcctgttgg ttccagcgaa gaccacaggc attccttagg 1700
gtccagggtc agcaggctcc gggctcacat gtgtaaggac aggacatttt 1750
ctgcagtgcc tgccaatagt gagcttggag cctggaggcc ggcttagttc 1800
ttccatttca cccttgcagc cagctgttgg ccacggcccc tgccctctgg 1850
tctgccgtgc atctccctgt gccctcttgc tgcctgcctg tctgctgagg 1900
taaggtggga ggagggctac agcccacatc ccaccccctc gtccaatccc 1950
ataatccatg atgaaaggtg aggtcacgtg gcctcccagg cctgacttcc 2000
caacctacag cattgacgcc aacttggctg tgaaggaaga ggaaaggatc 2050
tggccttgtg gtcactggca tctgagccct gctgatggct ggggctctcg 2100
ggcatgcttg ggagtgcagg gggctcgggc tgcctggcct ggctgcacag 2150
Page 71
.. ro x ,r , ..~..._..... ._......,._.wn~.F4
~~,.a~~irv".c>s~".~..".x0.~~~.e.,..e~.mr.m.m- s. ".o-m .m~a..~-.xm,~
t
CA 02481756 2004-10-25
PCT-US00-23328_Sepuence
aaggcaagtg ctggggctca tggtgctctg agctggcctg gaccctgtca 2200
ggatgggccc cacctcagaa ccaaactcac tgtccccact gtggcatgag 2250
ggcagtggag caccatgttt gagggcgaag ggcagagcgt ttgtgtgttc 2300
tggggaggga aggaaaaggt gttggaggcc ttaattatgg actgttggga 2350
aaagggtttt gtccagaagg acaagccgga caaatgagcg acttctgtgc 2400
ttccagagga agacgaggga gcaggagctt ggctgactgc tcagagtctg 2450
ttctgacgcc ctgggggttc ctgtccaacc ccagcagggg cgcagcggga 2500
ccagccccac attccacttg tgtcactgct tggaacctat ttattttgta 2550
tttatttgaa cagagttatg tcctaactat ttttatagat ttgtttaatt 2600
aatagcttgt cattttcaag ttcatttttt attcatattt atgttcatgg 2650
ttgattgtac cttcccaac~c ccgcccagtg ggatgggagg aggaggagaa 2700
ggggggcctt gggccgctgc agtcacatct gtccagagaa attccttttg 2750
ggactggagg cagaaaagcg gccagaaggc agcagccctg gctcctttcc 2800
tttggcaggt tggggaaggg cttgccccca gccttaggat ttcagggttt 2850
gactgggggc gtggagagag agggaggaac ctcaataacc ttgaaggtgg 2900
aatccagtta tttcctgcgc tgcgagggtt tctttatttc actcttttct 2950
gaatgtcaag gcagtgaggt gcctctcact gtgaatttgt ggtgggcggg 3000
ggctggagga gagggtgggg ggctggctcc gtccctccca gccttctgct 3050
gcccttgctt aacaatgccg gccaactggc gacctcacgg ttgcacttcc 3100
attccaccag aatgacctga tgaggaaatc ttcaatagga tgcaaagatc 3150
aatgcaaaaa ttgttatata tgaacatata actggagtcg tcaaaaagca 3200
aattaagaaa gaattggacg ttagaagttg tcatttaaag cagccttcta 3250
ataaagttgt ttcaaagctg aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3300
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 3334
<210> 58
<211> 469
<212> PRT
<213> Homo Sapien
<400> 58
Met Leu Cys Leu Cys Leu Tyr Val Pro Val I12 Gly Glu Ala Gln
1 5 10 15
Thr Glu Phe Gln Tyr Phe Glu Ser Lys Gly Leu Pro Ala Glu Leu
20 25 30
Lys Ser Ile Phe Lys Leu Ser Val Phe Ile Pro Ser Gln Glu Phe
35 40 45
Page 72
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
SerThrTyrArg GlnTrp Ly5GlnLys IleValGln AlaGlyAsp
50 55 60
LysAspLeuAsp GlyGln LeuAspPhe GluGluPhe ValHisTyr
65 70 75
LeuGlnAspHis GluLys LysLeuArg LeuValPhe LysIleLeu
80 85 90
AspLysLysAsn AspGly ArgIleAsp AlaGlnGlu IleMetGln
g5 100 105
SerLeuArgAsp LeuGly ValLysIle SerGluGln GlnAlaGlu
110 115 120
LysIleLeuLys SerMet AspLysAsn GlyThrMet ThrIleAsp
125 130 135
TrpAsnGluTrp ArgAsp TyrHisLeu LeuHisPro ValGluAsn
140 145 150
IleProGluIle IleLeu TyrTrpLys HisSerThr IlePheAsp
155 160 165
ValGlyGluAsn LeuThr valProAsp GluPheThr ValGluGlu
170 175 180
ArgGlnThrGly MetTrp TrpArgHis LeuValAla GlyGlyGly
185 190 195
AlaGlyAlaVal SerArg ThrCysThr AlaProLeu AspArgLeu
200 205 210
LysValLeuMet GlnVal HisAlaSer ArgSerAsn AsnMetGly
215 220 225
IleValGlyGly PheThr GlnMetIle ArgGluGly GlyAlaArg
230 235 240
SerLeuTrpArg GlyAsn GlyIleAsn ValLeuLys IleAlaPro
245 250 255
GluSerAIaIle LysPhe MetAlaTyr GluGlnIle LysArgLeu
260 265 270
ValGlySerAsp GlnGlu ThrLeuArg IleHisGlu ArgLeuVal
z75 2so zs5
AlaGlySerLeu AlaGly AlaI1eAla GlnSerSer IleTyrPro
290 295 300
MetGluValLeu LysThr ArgMetAla LeuArgLys ThrGlyGln
305 310 315
TyrSerGlyMet LeuAsp CysAlaArg ArgIleLeu AlaArgGlu
320 325 330
GlyValAlaAla PheTyr LysGlyTyr ValProAsn MetLeuGly
335 340 345
IleIleProTyr AlaGly IleAspLeu AlaValTyr GluThrLeu
350 355 360
Page 73
CA 02481756 2004-10-25
PCT-u500-23328_Sec~uence
LysAsnAla TrpLeuGln HisTyrAla Asn SerAlaAsp Pro
Val
365 370 375
GlyValPhe ValLeuLeu AlaCysGly Met SerSerThr Cys
Thr
380 385 390
GlyGlnLeu AlaSerTyr ProLeuAla Val ArgThrArg Met
Leu
395 400 405
GlnAlaGln AlaSerIle GluGlyAla Glu ValThrMet Ser
Pro
410 41s 420
SerLeuPhe LysHisIle LeuArgThr Gly AlaPheGly Leu
Glu
425 430 435
TyrArgGly LeuAlaPro AsnPheMet Val IleProAla Val
Lys
440 445 450
SerIleSer TyrValVal TyrGluAsn Lys LleThrLeu Gly
Leu
455 460 465
Val Gln Ser Arg
<210> 59
<211> 1658
<212> DNA '
<213> Homo Sapien
<400> 59
ggaaggcagc ggcagctcca ctcagccagt acccagatac gctgggaacc 50
ttccccagcc atggcttccc tggggcagat cctcttctgg agcataatta 100
gcatcatcat tattctggct ggagcaattg cactcatcat tggctttggt 150
atttcaggga gacactccat cacagtcact actgtcgcct cagctgggaa 200
cattggggag gatggaatcc tgagctgcac ttttgaacct gacatcaaac 250
tttctgatat cgtgatacaa tggctgaagg aaggtgtttt aggcttggtc 300
catgagttca aagaaggcaa agatgagctg tcggagcagg atgaaatgtt 350
cagaggccgg acagcagtgt ttgctgatca agtgatagtt ggcaatgcct 400
etttgegget gaaaaaegtg caaeteaeag atgctggeae etacaaatgt 450
tatatcatca cttctaaagg caaggggaat gctaaccttg agtataaaac 500
tggagccttc ageatgccgg aagtgaatgt ggactataat gccagctcag 550
~agaccttgcg gtgtgaggct ccccgatggt tcccccagcc cacagtggtc 600
tgggcatccc aagttgacca gggagccaac ttctcggaag tctccaatac 650
cagctttgag ctgaactctg agaatgtgac catgaaggtt gtgtctgtgc 700
tctacaatgt tacgatcaac aacacatact cctgtatgat tgaaaatgac 750
attgccaaag caacagggga tatcaaagtg acagaatcgg agatcaaaag 800
gcggagtcac ctacagctgc taaactcaaa ggcttctctg tgtgtctctt 850
Page 74
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
ctttctttgc catcagctgg gcacttctgc ctctcagccc ttacctgatg 900
ctaaaataat gtgccttggc cacaaaaaag catgcaaagt cattgttaca 950
acagggatct acagaactat ttcaccacca gatatgacct agttttatat 1000
ttctgggagg aaatgaattc atatctagaa gtctggagtg agcaaacaag 1050
agcaagaaac aaaaagaagc caaaagcaga aggctccaat atgaacaaga 1100
taaatctatc ttcaaagaca tattagaagt tgggaaaata attcatgtga 1150
actagacaag tgtgttaaga gtgataagta aaatgcacgt ggagacaagt 1200
gcatccccag atctcaggga cctccccctg cctgtcacct ggggagtgag 1250
aggacaggat agtgcatgtt ctttgtctct gaatttttag ttatatgtgc 1300
tgtaatgttg ctctgaggaa gcccctggaa agtctatccc aacatatcca 1350
catcttatat tccacaaatt aagctgtagt atgtacccta agacgctgct 1400
aattgactgc cacttcgcaa ctcaggggcg gctgcatttt agtaatgggt 1450
caaatgattc actttttatg atgcttccaa aggtgccttg gcttctcttc 1500
ccaactgaca aatgccaaag ttgagaaaaa tgatcataat tttagcataa 1550
acagagcagt cggggacacc gattttataa ataaactgag caccttcttt 1600
ttaaacaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1650
aaaaaaaa 1658
<210> 60
<211> 282
<212> PRT
<213> Homo Sapien
<400> 60
Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser Ile Ile Ser Ile
1 5 10 15
Ile Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile Ile Gly Phe Gly
20 25 30
Ile Ser Gly Arg His Ser Ile Thr Val Thr Thr Va1 Ala Ser Ala.
35 40 45~
Gly Asn Ile Gly Glu Asp Gly Ile Leu Ser Cys Thr Phe Glu Pro
50 55 60
Asp Ile Lys Leu Ser Asp Ile Val Ile Gln Trp Leu Lys Glu Gly
65 70 75
val Leu Gly Leu Val His Glu Phe Lys Glu Gly Lys Asp Glu Leu
80 85 90
Ser Glu Gln Asp Glu Met Phe Arg Gly Arg Thr Ala Val Phe Ala
95 100 105
Asp Gln Val Ile Val Gly Asn Ala Ser Leu Arg Leu Lys Asn Val
Page 75
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
110 115 120
Gln Leu Thr Asp Ala Gly Thr Tyr Lys Cys Tyr Ile Ile Thr Ser
125 130 135
Lys Gly Lys Gly Asn Ala Asn Leu Glu Tyr Lys Thr Gly Ala Phe
140 145 150
Ser Met Pro Glu Val Asn Val Asp Tyr Asn Ala Ser Ser Glu Thr
155 160 165
Leu Arg Cys Glu Ala Pro Arg Trp Phe Pro Gln Pro Thr Val Val
170 175 180
Trp Ala Ser Gln Val Asp Gln Gly Ala Asn Phe Ser Glu Val Ser
185 190 195
Asn Thr Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met Lys Val
200 205 210
Val Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser Cys
215 220 225
Met Ile Glu Asn Asp I12 Ala Lys Ala Thr Gly Asp Ile Lys Val
230 235 240
Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu ASn
245 250 255
Ser Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp
260 265 270
Ala Leu Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys
275 280
<210> 61
<211> 1617
<212> DNA
<213> Homo Sapien
<400> 61
tgacgtcaga atcaccatgg ccagctatcc ttaccggcag ggctgcccag 50
gagctgcagg acaagcacca ggagcccctc cgggtagcta ctaccctgga 100
ccccccaata gtggagggca gtatggtagt gggctaccec ctggtggtgg 150
ttatgggggt cctgcccctg gagggcctta tggaccacca gctggtggag 200
ggccctatgg acaccccaat cctgggatgt tcccctctgg aact:ccagga 250
ggaccatatg gcggtgcagc tcccgggggc ccctatggtc agccacctcc 300
aagttcctac ggtgcccagc agcctgggct ttatggacag ggtggcgccc 350
ctcccaatgt ggatcctgag gcctactcct ggttccagtc ggtggactca 400
gatcacagtg gctatatctc catgaaggag ctaaagcagg ccctggtcaa 450
ctgcaattgg tcttcattca atgatgagac ctgcctcatg atgataaaca 500
tgtttgacaa gaccaagtca ggccgcatcg atgtctacgg cttctcagcc 550
Page 76
CA 02481756 2004-10-25
PCT-u500-23328_Seguence
ctgtggaaat tcatccagca gtggaagaac ctcttccagc agtatgaccg 600
ggaccgctcg ggctccatta gctacacaga gctgcagcaa gctctgtccc 650
aaatgggcta caacctgagc ccccagttca cccagcttct ggtctcccgc 700
tactgcccac gctctgccaa tcctgccatg cagcttgacc gcttcatcca 750
ggtgtgcacc cagctgcagg tgctgacaga ggccttccgg gagaaggaca 800
cagctgtaca aggcaacatc cggctcagct tcgaggactt cgtcaccatg 850
acagcttctc ggatgctatg acccaaccat ctgtggagag tggagtgcac 900
cagggacctt tcctggcttc ttagagtgag agaagtatgt ggacatctct 950
tcttttcctg tccctctaga agaacattct cccttgcttg atgcaacact 1000
gttccaaaag agggtggaga gtcctgcatc atagccacca aatagtgagg 1050
accggggctg aggccacaca gataggggcc tgatggagga gaggatagaa 1100
gttgaatgtc ctgatggcca tgagcagttg agtggcacag cctggcacca 1150
ggagcaggtc cttgtaatgg agttagtgtc.cagtcagctg agctccaccc 1200 '
tgatgccagt ggtgagtgtt catcggcctg ttaccgttag tacctgtgtt 1250
ccctcaccag gccatcctgt caaacgagcc cattttctcc aaagtggaat 1300
ctgaccaagc atgagagaga tctgtctatg ggaccagtgg cttggattct 1350
gccacaccca taaatccttg tgtgttaact tctagctgcc tggggctggc 1400
cctgctcaga caaatctgct ccctgggcat ctttggccag gcttctgccc 1450
cctgcagctg ggacccctca cttgcctgcc atgctctgct cggcttcagt 1500
ctccaggaga cagtggtcac ctctccctgc caatactttt tttaatttgc 1550
attttttttc atttggggcc aaaagtccag tgaaattgta agcttcaata 1600
aaaggatgaa actctga 1617
<210> 62
<211> 284
<212> PRT
<213> Homo Sapien
<400> 6Z
Met Ala Ser Tyr Pro Tyr Arg Gln Gly cys Pro Gly Ala Ala Gly
1 S 10 15
Gln Ala Pro Gly Ala Pro Pro Gly Ser Tyr Tyr Pro Gly Pro Pro
20 25 30
Asn Ser Gly Gly Gln Tyr Gly Ser Gly ~eu Pra Pro Gly Gly Gly
35 40 45
Tyr Gly Gly Pro Ala pro Gly Gly'Pro Tyr~Gly Pro Pro Ala Gly
50 55 60
Gly Gly Pro Tyr Gly His Pro Asn Pro Gly Met Phe Pro Ser Gly
Page 77
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
65 70 75
Thr Pro Gly Gly Pro Tyr Gly Gly Ala Ala Pro Gly Gly Pro Tyr
80 85 90
Gly Gln Pro Pro Pro Ser Ser Tyr Gly Ala Gln Gln Pro Gly Leu
95 100 105
Tyr Gly Gln Gly G1y Ala Pro Pro Asn Val Asp Pro Glu Ala Tyr
110 115 120
Ser Trp Phe Gln Ser val Asp Ser Asp His Ser Gly Tyr Ile Ser
125 130 135
Met Lys Glu Leu Lys Gln Ala Leu Val Asn Cys Asn Trp Ser Ser
140 145 150
Phe Asn Asp Glu Thr Cys Leu Met Met Ile Asn Met Phe Asp Lys
1S5 160 165
Thr Lys Ser Gly Arg Ile Asp Val Tyr Gly Phe Ser Ala Leu Trp
170 175 180
Lys Phe Ile Gln Gln Trp Lys Asn Leu Phe Gln Gln Tyr Asp Arg
185 190 195
Asp Arg Ser Gly Ser Ile Ser Tyr Thr Glu Leu Gln Gln Ala Leu
200 205 210
Ser Gln Met Gly Tyr Asn Leu Ser Pro Gln Phe Thr Gln Leu Leu
215 220 . 225
val ser Arg Tyr Cys Pro Arg Ser Ala Asn Pro Ala Met Gln Leu
230 235 240
Asp Arg Phe Ile Gln Val Cys Thr Gln Leu Gln Val Leu Thr Glu
245 250 255
Ala Phe Arg Glu Lys Asp Thr Ala Val Gln Gly Asn Ile Arg Leu
260 265 270
Ser Phe Glu Asp Phe Val Thr Met Thr Ala Ser Arg Met Leu
275 280
<Z10> 63
<211> 1234
<212> DNA
<213> Homo Sapien
<400> 63
caggatgcag ggccgcgtgg cagggagctg cgctcctctg ggcctgctcc 50
tggtctgtct tcatctccca ggcctctttg cccggagcat cggtgttgtg 100
gaggagaaag tttcccaaaa cttcgggacc aacttgcctc agctcggaca 150
accttcctcc actggcccct ctaactctga acatccgcag cccgctctgg 200
accctaggtc taatgacttg gcaagggttc ctctgaagct cagcgtgcct 250 _
ccatcagatg gcttcccacc tgcaggaggt tctgcagtgc agaggtggcc 300
tccatcgtgg gggctgcctg ccatggattc ctggccccct gaggatcctt 350
Page 78
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
ggcagatgat ggctgctgcg gctgaggacc gcctggggga agcgctgcct 400
gaagaactct cttacctctc cagtgctgcg gccctcgctc cgggcagtgg 450
ccctttgcct ggggagtctt ctcccgatgc cacaggcctc tcacctgagg 500
cttcactcct ccaccaggac tcggagtcca gacgactgcc ccgttctaat 550
tcactgggag ccgggggaaa aatcctttcc caacgccctc cctggtctct 600
catccacagg gttctgcctg atcacccctg gggtaccctg aatcccagtg 650
tgtcctgggg aggtggaggc cctgggactg gttggggaac.gaggcccatg 700
ccacaccctg agggaatctg gggtatcaat aatcaacccc caggtaccag 750
ctggggaaat attaatcggt atccaggagg cagctgggga aatattaatc 800
ggtatccagg aggcagctgg gggaatatta atcggtatcc aggaggcagc 850
tgggggaata ttcatctata cccaggtatc aataacccat ttcctcctgg 900
agttctccgc cctcctggct cttcttggaa catcccagct ggcttcccta 950
atcctccaag ccctaggttg cagtggggct agagcacgat agagggaaac 1000
ccaacattgg gagttagagt cctgctcccg ccccttgctg tgtgggctca 1050
atccaggccc tgttaacatg tttccagcac tatccccact tttcagtgcc 1100
tcccctgctc atctccaata aaataaaagc acttatgaaa aaaaaaaaaa 1150
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1234
<210> 64
<211> 325
<212> PRT
<213> Homo Sapien
<400> 64
Met Gln Gly Arg Val Ala Gly Ser Cys Ala Pro Leu Gly Leu Leu
1 S 10 15
Leu Val Cys Leu His Leu Pro Gly Leu Phe Ala Arg Ser Ile Gly
20 25 - 30
Val Val Glu Glu Lys Val Ser Gln Asn Phe Gly Thr Asn Leu Pro
35 40 45
Gln Leu Gly Gln Pro Ser Ser Thr Gly Pro Ser Asn Ser Glu His
50 55 60
Pro Gln Pro Ala Leu Asp Pro Arg Ser ASn Asp Leu Ala Arg Val
65 70 75
Pro Leu Lys Leu Ser Val Pro Pro Ser Asp Gly Phe Pro Pro Ala
g0 85 90
Gly Gly Ser Ala Val Gln Arg Trp Pro Pro Ser Trp Gly Leu Pro
95 100 105
Page 79
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
Ala Met Asp Ser Trp Pro Pro Glu Asp Pro Trp Gln Met Met Ala
110 115 120
Ala Ala Ala Glu Asp Arg Leu Gly Glu Ala Leu Pro Glu Glu Leu
125 130 135
Ser Tyr Leu Ser Ser Ala Ala Ala Leu Ala Pro GIy Ser Gly Pro
140 145 150
Leu Pro Gly Glu Ser Ser Pro Asp Ala Thr Gly Leu Ser Pro Glu
155 160 165
Ala Ser Leu Leu His Gln Asp Ser Glu Ser Arg Arg Leu Pro Arg
170 175 180
Ser Asn Ser Leu Gly Ala Gly Gly Lys Ile Leu Ser Gln Arg Pro
185 190 195
Pro Trp Ser Leu Ile His Arg Val Leu Pro Asp His Pro Trp Gly
200 205 210
Thr Leu Asn Pro Ser Val Ser Trp Gly Gly Gly Gly Pro Gly Thr
215 220 225
Gly Trp Gly Thr Arg Pro Met Pro His Pro Glu Gly 21e Trp Gly
230 235 240
Ile Asn Asn Gln Pro Pro Gly Thr ser Trp Gly Asn Ile Asn Arg
245 250 255
Tyr Pro Gly Gly Ser Trp Gly Asn Ile Asn Arg Tyr Pro Gly Gly
260 265 270
Ser Trp Gly Asn Ile Asn Arg Tyr Pro Gly Gly Ser Trp Gly Asn
275 280 ' 285
Ile His Leu Tyr Pro Gly Ile Asn Asn Pro Phe Pro Pro Gly val
290 295 300
Leu Arg Pro Pro Gly Ser Ser Trp Asn Ile Pro Ala Gly Phe Pro
305 310 315
Asn Pro Pro Ser Pro Arg Leu Gln Trp Gly
320 325
<210> 65
<211> 422
<212> DNA
<213> Homo Sapien
<400> 65
aaggagaggc caccgggact tcagtgtctc ctccatccca ggagcgcagt 50
ggccactatg gggtctgggc tgccccttgt cctcctcttg accctccttg 100
gcagctcaca tggaacaggg ccgggtatga ctttgcaact gaagctgaag 150
gagtcttttc tgacaaattc ctcctatgag tccagcttcc tgg~attgct 200
tgaaaagctc tgcctcctcc tccatctccc ttcagggacc agcgtcaccc 250
tccaccatgc aagatctcaa caccatgttg tctgcaacac atgacagtca 300
Page 80
..a. ,.,, _ ,..~".m . »m . ,._ert_.._._._ ._ ,~ ~., ~ _~..____ ...~~_. .
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
ttgaagcctg tgtccttct~t ggcccgggct tttgggccgg ggatgcagga 350
ggcaggcccc gaccctgtct ttcagcaggc ccccaccctc ctgagtggca 400
ataaataaaa ttcggtatgc tg 422
<210> 66
<211> 78
<212> PRT
<213> Homo Sapien -
<400> 66
Met Gly Ser Gly Leu Pro Leu Val Leu Leu Leu Thr Leu Leu Gly
1 5 10 15
Ser Ser His Gly Thr Gly Pro Gly Met Thr Leu Gln Leu Lys Leu
20 25 30
Lys Glu Ser Phe Leu Thr Asn Ser Ser Tyr Glu Ser Ser Phe Leu
35 40 45
Glu Leu Leu Glu Lys Leu cys Leu Leu Leu His Leu Pro Ser Gly
50 55 60
Thr Ser Val Thr Leu His His Ala Arg Ser Gln His His Val Val
65 70 75
cys Asn Thr
<210> 67
<211> 744
<212> DNA
<213> HOmo Sapien
<400> 67
acggaccgag ggttcgaggg agggacacgg accaggaacc tgagctaggt 50
caaagacgcc cgggccaggt gccccgtcgc aggtgcccct ggccggagat 100
gcggtaggag gggcgagcgc gagaagcccc ttcctcggcg ctgccaaccc 150
gccacccagc ccatggcgaa ccccgggctg gggctgcttc tggcgctggg 200
cctgccgttc ctgctggccc gctggggccg agcctggggg caaatacaga 250
ccacttctgc aaatgagaat agcactgttt tgccttcatc caccagctcc 300
agctccgatg gcaacctgcg tccggaagcc atcactgcta tcatcgtggt 350
cttctccctc ttggctgcct tgctcctggc tgtggggctg gcactgttgg 400
tgcggaagct tcgggagaag cggcagacgg agggcaccta ccggcccagt 450
agcgaggagc agttctccca tgcagccgag gcccgggccc ctcaggactc 500
caaggagacg gtgcagggct gcctgcccat ct~ggtcccc tctcctgcat 550
ctgtctccct tcattgctgt gtgaccttgg ggaaaggcag tgccctctct 600
gggcagtcag atccacccag tgcttaatag cagggaagaa ggtacttcaa 650
Page 81
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
agactctgcc cctgaggtca agagaggatg gggctattca cttttatata 700
tttatataaa attagtagtg agatgtaaaa aaaaaaaaaa aaaa 744
<210> 68
<211> 123
<21z> PRT
<213> Homo Sapien
<400> 68
Met Ala Asn Pro Gly Leu Gly Leu Leu Leu Ala Leu Gly Leu Pro
1 5 10 15
Phe Leu Leu Ala Arg Trp Gly Arg Ala Trp Gly Gln Ile Gln Thr
20 25 30
Thr Ser Ala Asn Glu Asn Ser Thr Val Leu Pro Ser Ser Thr Ser
35 40 45
Ser Ser Ser Asp Gly Asn Leu Arg Pro Glu Ala Tle Thr Ala Ile
50 55 60
Ile Val Val Phe Ser Leu Leu Ala Ala Leu Leu Leu Ala Val Gly
65 70 75
Leu Ala Leu Leu Val Arg Lys Leu Arg Glu Lys Arg Gln Thr Glu
80 85 90
Gly Thr Tyr Arg Pro Ser Ser Glu Glu Gln Phe Ser His Ala Ala
95 100 105
Glu Ala Arg Ala Pro Gln Asp Ser Lys Glu Thr Val Gln Gly Cys
110 115 120
Leu Pro Ile
<210> 69
<211> 3265
<212> oNA
<213> Homo Sapien
<400> 69
gccaggaata actagagagg aacaatgggg ttattcagag gttttgtttt 50
cctcttagtt ctgtgcctgc tgeaccagtc aaatacttcc ttcattaagc 100
tgaataataa tggctttgaa gatattgtca ttgttataga tcctagtgtg 150
ccagaagatg aaaaaataat tgaacaaata gaggatatgg tgactacagc 200
ttctacgtac ctgtttgaag ccacagaaaa aagatttttt ttcaaaaatg 250
tatctatatt aattcctgag aattggaagg aaaatcctca gtacaaaagg 300
ccaaaacatg aaaaccataa acatgctgat gttatagttg caccacctac 350
actcccaggt agagatgaac catacaccaa gcagttcaca gaatgtggag 400
agaaaggcga atacattcac ttcacccctg accttctact tggaaaaaaa 450
caaaatgaat atggaccacc aggcaaactg tttgtccatg agtgggctca 500
Page 82
CA 02481756 2004-10-25
PCT-uS00-23328-Sequence
cctccggtgg ggagtgtttg atgagtacaa tgaagatcag cctttctacc 550
gtgctaagtc aaaaaaaatc gaagcaacaa ggtgttccgc aggtatctct 600
ggtagaaata gagtttataa gtgtcaagga ggcagctgtc ttagtagagc 650
atgcagaatt gattctacaa caaaactgta tggaaaagat tgtcaattct 700
ttcctgataa agtacaaaca gaaaaagcat ccataatgtt tatgcaaagt 750
attgattctg ttgttgaatt ttgtaacgaa aaaacccata atcaagaagc 800
tccaagccta caaaacataa agtgcaattt tagaagtaca tgggaggtga 850
ttagcaattc tgaggatttt aaaaacacca tacccatggt gacaccacct 900
cctccacctg tcttctcatt gctgaagatc agtcaaagaa ttgtgtgctt 950
agttcttgat aagtctggaa gcatgggggg taaggaccgc ctaaatcgaa 1000
tgaatcaagc agcaaaacat ttcctgctgc agactgttga aaatggatcc 1050
tgggtgggga tggttcactt tgatagtact gccactattg taaataagct 1100
aatccaaata aaaagcagtg atgaaagaaa cacactcatg gcaggattac 1150
ctacatatcc tctgggagga acttccatct gctctggaat taaatatgca 1200
tttcaggtga ttggagagct acattcccaa ctcgatggat ccgaagtact 1250
gctgctgact gatggggagg ataacactgc aagttcttgt attgatgaag 1300
tgaaacaaag tggggccatt gttcatttta ttgctttggg aagagctgct 1350
gatgaagcag taatagagat gagcaagata acaggaggaa gtcattttta 1400
tgtttcagat gaagctcaga acaatggcct cattgatgct tttggggctc 1450
ttacatcagg aaatactgat ctctcccaga agtcccttca gctcgaaagt 1500
aagggattaa cactgaatag taatgcctgg atgaacgaca ctgtcataat 1550
tgatagtaca gtgggaaagg acacgttctt tctcatcaca tggaacagtc 1600
tgcctcccag tatttctctc tgggatccca gtggaacaat aatggaaaat 1650
ttcacagtgg atgcaacttc caaaatggcc tatctcagta ttccaggaac 1700
tgcaaaggtg ggcacttggg catacaatct tcaagccaaa gcgaacccag 1750
aaacattaac tattacagta acttctcgag cagcaaattc ttctgtgcct 1800
ccaatcacag tgaatgctaa aatgaataag gacgtaaaca gtttccccag 1850
cccaatgatt gtttacgcag aaattctaca aggatatgta cctgttcttg 1900
gagccaatgt gactgctttc attgaatcac agaatggaca tacagaagtt 1950
ttggaacttt tggataatgg tgcaggcgct gattctttca agaatgatgg 2000
agtctactcc aggtatttta cagcatatac agaaaatggc agatatagct 2050
taaaagttcg ggctcatgga ggagcaaaca ctgccaggct aaaattacgg 2100
Page 83
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
cctccactga atagagccgc gtacatacca ggctgggtag tgaacgggga 2150
aattgaagca aacccgccaa gacctgaaat tgatgaggat actcagacca 2200
ccttggagga tttcagccga acagcatccg gaggtgcatt tgtggtatca 2250
caagtcccaa gccttccctt gcctgaccaa tacccaccaa gtcaaatcac 2300
agaccttgat gccacagttc atgaggataa gattattctt acatggacag 2350
caccaggaga taattttgat gttggaaaag ttcaacgtta tatcataaga 2400
ataagtgcaa gtattcttga tctaagagac agttttgatg atgctcttca 2450
agtaaatact actgatctgt caccaaagga ggccaactcc aaggaaagct 2500
ttgcatttaa accagaaaat atctcagaag aaaatgcaac ccacatattt 2550
attgccatta aaagtataga taaaagcaat ttgacatcaa aagtatccaa 2600
cattgcacaa gtaactttgt ttatccctca agcaaatcct gatgacattg 2650
atcctacacc tactcctact cctactccta ctcctgataa aagtcataat 2700
tctggagtta atatttctac getggtattg tctgtgattg ggtctgttgt 2750
aattgttaac tttattttaa gtaccaccat ttgaacctta acgaagaaaa 2800
aaatcttcaa gtagacctag aagagagttt taaaaaacaa aacaatgtaa 2850
gtaaaggata tttctgaatc ttaaaattca tcccatgtgt gatcataaac 2900
tcataaaaat aattttaaga tgtcggaaaa ggatactttg attaaataaa 2950
aacactcatg gatatgtaaa aactgtcaag attaaaattt aatagtttca 3000
tttatttgtt attttatttg taagaaatag tgatgaacaa agatcctttt 3050
tcatactgat acctggttgt atattatttg atgcaacagt tttctgaaat 3100
gatatttcaa attgcatcaa gaaattaaaa tcatctatct gagtagtcaa 3150
aatacaagta aaggagagca aataaacaac atttggaaaa aaaaaaaaaa 3200
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3250
aaaaaaaaaa aaaaa 3265
<210> 70
<211> 919
<212> PRT
<213> Homo Sapien
<400> 70
Met Gly Leu Phe Arg Gly Phe val Phe Leu Leu val Leu cys Leu
1 5 10 15
Leu His Gln Ser Asn Thr Ser Phe Ile Lys Leu Asn Asn Asn Gly
20 25 30
Phe Glu Asp Ile val Ile val Ile Asp Pro Ser val Pro Glu Asp
35 40 45
Page 84
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
Glu Lys Ile Ile Glu Gln Ile Glu Asp Met Val Thr Thr Ala Ser
50 55 60
Thr Tyr Leu Phe Glu Ala Thr Glu Lys Arg Phe Phe Phe Lys Asn
65 70 75
Val Ser Ile Leu Ile Pro Glu Asn Trp Lys Glu Asn Pro Gln Tyr
80 85 90
Lys Arg Pro Lys His Glu Asn His Lys His Ala Asp Val Ile Val
95 100 105
Ala Pro Pro Thr Leu Pro Gly Arg Asp Glu Pro Tyr Thr Lys Gln
110 115 120
Phe Thr Glu Cys Gly Glu Lys Gly Glu Tyr Ile His Phe Thr Pro
125 130 135
Asp Leu Leu Leu Gly Lys Lys Gln Asn Glu Tyr Gly Pro Pro Gly
140 145 150
Lys Leu Phe Val His Glu Trp Ala His Leu Arg Trp Gly Val Phe
155 160 165
Asp Glu Tyr Asn Glu Asp Gln Pro Phe Tyr Arg Ala Lys Ser Lys
170 175 180
Lys Ile Glu Ala Thr Arg Cys Ser Ala Gly Ile ser Gly Arg Asn
185 190 19S
Arg val Tyr Lys Cys Gln Gly Gly Ser Cys Leu Ser Arg Ala Cys
200 205 210
Arg Ile Asp Ser Thr ~Thr Lys Leu Tyr Gly Lys Asp Cys Gln Phe
215 220 225
Phe Pro Asp Lys Val Gln Thr Glu Lys Ala Ser Ile Met Phe Met
230 235 240
Gln Ser Ile Asp Ser Val val Glu Phe Cys Asn Glu Lys Thr His
245 250 255
Asn Gln Glu Ala Pro Ser Leu Gln Asn Ile Lys Cys Asn Phe Arg
260 265 270
Ser Thr Trp Glu Val Ile Ser Asn Ser Giu Asp Phe Lys Asn Thr
275 280 285
Ile Pro Met val Thr Pro Pro Pro Pro Pro Va) Phe Ser Leu Leu
290 295 300
Lys Ile Ser Gln Arg :Ile Val Cys Leu Val Leu Asp Lys Ser Gly
305 310 315
Ser Met Gly Gly Lys Asp Arg Leu Asn Arg Met Asn Gln Ala Ala
320 325 330
Lys His Phe Leu Leu G1n Thr Val G1a Asn Gly Ser Trp Val Gly
335 340 345
Met val His Phe Asp 5er Thr Aia Thr Ile val Asn Lys Leu Ile
350 355 360
Page 85
r ~, a _ . my ~_ ~.. .. ..~ v . .~_,~a.~~~_.. T ~~ ~ ~~. ~ .. ..w _ _ ~~... ,
~~.~ ~ . .~, .r, ~~ ~~~ ~~,~ ... .r~.~~.m~.~ ~._ .Rn~.~,~ Mmti, .~,. ...A~
t
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Gln Ile Lys ser Ser Asp Glu Arg Asn Thr Leu Met Ala Gly Leu
365 370 37S
Pro Thr Tyr Pro Leu Gly Gly Thr 5er Ile Cys Ser Gly I12 Lys
380 385 390
Tyr Ala Phe Gln Val Ile Gly Glu Leu His Ser Gln Leu Asp Gly
395 400 40S
Ser Glu Val Leu Leu Leu Thr Asp Gly Glu Asp Asn Thr Ala Ser
410 415 420
Ser Cys Ile Asp Glu Val Lys Gln Ser Gly Ala Ile Val His Phe
425 430 435
Ile Ala Leu Gly Arg EAIa Ala Asp Glu Ala Val Ile Glu Met Ser
440 445 450
Lys Ile Thr Gly Gly ser His Phe Tyr Val Ser Asp Glu Ala Gln
455 460 465
Asn Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu Thr 5er Gly Asn
470 475 480
Thr Asp Leu Ser Gln Lys ser Leu Gln Leu Glu Ser Lys Gly Leu
485 490 495
Thr Leu Asn Ser Asn Ala Trp Met Asn Asp Thr Val Ile Ile Asp
500 505 510
ser Thr val Gly Lys .a,sp Thr Phe Phe Leu Ile Thr Trp Asn 5er
515 520 525
Leu Pro Pro Ser Ile 5er Leu Trp Asp Pro Ser Gly Thr Ile Met
530 535 540
Glu Asn Phe Thr Val Asp Ala Thr Ser Lys Met Ala Tyr Leu Ser
545 550 555
Ile Pro Gly Thr Ala Lys Val Gly Thr Trp Ala Tyr Asn Leu Gln
560 565 570
Ala Lys Ala Asn Pro Glu Thr Leu Thr Ile Thr Val Thr ser Arg
575 580 585
Ala Ala Asn Ser Ser Val Pro Pro Ile Thr Val Asn Ala Lys Met
590 595 600
Asn Lys Asp Val Asn Ser Phe Pro Ser Pro Met Ile Val Tyr Ala
605 610 615
Glu Ile Leu Gln Gly Tyr Val Pro val Leu Gly Ala Asn Val Thr
620 625 630
Ala Phe Ile Glu Ser Gln Asn Gly His Thr Glu Val Leu Glu Leu
635 640 645
~eu Asp Asn Gly Ala Gly Ala Asp Ser Phe Lys Asn Asp Gly val
650 655 660
Tyr Ser Arg Tyr Phe Thr Ala Tyr Thr Glu Asn Gly Arg Tyr Ser
665 670 675
Page 86
.w. .. h T ~ ~ ~... . ~..~~~~e~w~ d ;.~z ~ . ..re , m~..~~ .v~.:.~~~., . ~
a.uz., a s ~.._ _«~m .~n _...,_. ,..- _ __.__ ___
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Leu Lys Val Arg Ala His Gly Gly Ala Asn Thr Ala Arg Leu Lys
680 685 690
Leu Arg Pro Pro Leu Asn Arg Ala Ala Tyr Tle Pro Gly Trp val
695 700 705
val Asn Gly Glu Ile Glu Ala Asn Pro Pro Arg Pro Glu Ile Asp
710 715 720
Glu Asp Thr Gln Thr Thr Leu Glu Asp Phe ser Arg Thr Ala her
725 730 735
Gly Gly Ala Phe Val Val Ser Gln Val Pro Ser Leu Pro Leu Pro
740 745 750
Asp Gln Tyr Pro Pro Ser Gln Ile Thr Asp Leu Asp Ala Thr val
755 760 765
His Glu Asp Lys Ile Iie Leu Thr Trp Thr Ala Pro Gly Asp Asn
770 775 780
Phe Asp val Gly Lys val Gln Arg Tyr Ile Ile Arg Ile Ser Ala
785 790 795
Ser Ile Leu Asp Leu Arg Asp Ser Phe Asp Asp Ala Leu Gln val
800 805 810
Asn Thr Thr Asp Leu Ser Pro Lys Glu Ala Asn Ser Lys Glu Ser
815 820 825
Phe Ala Phe Lys Pro Glu Asn Ile Ser Glu Glu Asn Ala Thr His
830 835 840
Ile Phe Ile Ala Ile Lys Ser Ile Asp Lys Ser Asn Leu Thr Ser
845 850 855
Lys Val Ser Asn Ile Ala Gln val Thr Leu Phe Ile Pro Gln Ala
860 865 870
Asn Pro Asp Asp Ile Asp Pro Thr Pro Thr Pro Thr Pro Thr Pro
875 880 885
Thr Pro Asp Lys Ser His Asn Ser Gly val Asn Ile Ser Thr Leu
890 895 900
val Leu Ser val Ile Gly Ser vai val Ile val Asn Phe Tle Leu
905 910 915
Ser Thr Thr Ile
<210> 71
<211> 3877
<212> DNA
<213> Homo Sapien
<400> 71
ctccttaggt ggaaaccctg ggagtagagt actgacagca aagaccggga 50
aagaccatac gtccccgggc aggggtgaca acaggtgtca tctttttgat 100
ctcgtgtgtg gctgccttcc tatttcaagg aaagacgcca aggtaatttt 150
Page 87
CA 02481756 2004-10-25
PCT-us00-23328_sequence
gacccagagg agcaatgatg tagccacctc ctaaccttcc cttcttgaac 200
ccccagttat gccaggattt actagagagt gtcaactcaa ccagcaagcg 250
gctccttcgg cttaacttgt ggttggagga gagaaccttt gtggggctgc 300
gttctcttag cagtgctcag aagtgacttg cctgagggtg gaccagaaga 350
aaggaaaggt cccctcttgc tgttggctgc acatcaggaa ggctgtgatg 400
ggaatgaagg tgaaaacttg gagatttcac ttcagtcatt gcttctgcct 450
gcaagatcat cctttaaaag tagagaagct gctctgtgtg gtggttaact 500
ccaagaggca gaactcgttc tagaaggaaa tggatgcaag cagctccggg 550
ggccccaaac gcatgcttcc tgtggtctag cccagggaag cccttccgtg 600
ggggccccgg ctttgaggga tgccaccggt tctggacgca tggctgattc 650
ctgaatgatg atggttcgcc gggggctgct tgcgtggatt tcccgggtgg 700
tggttttgct ggtgctcctc tgctgtgcta tctctgtcct gtacatgttg 750
gcctgcaccc caaaaggtga cgaggagcag ctggcactgc ccagggccaa 800
cagccccacg gggaaggagg ggtaccaggc cgtccttcag gagtgggagg 850
agcagcaccg caactacgtg agcagcctga agcggcagat cgcacagctc 900
aaggaggagc tgcaggagag gagtgagcag ctcaggaatg ggcagtacca 950
agccagcgat gctgctggcc tgggtctgga caggagcccc ccagagaaaa 1000
cccaggccga cctcctggcc ttcctgcact cgcaggtgga caaggcagag 1050
gtgaatgctg gcgtcaagct ggccacagag tatgcagcag tgcctttcga 1100
tagctttact ctacagaagg tgtaccagct ggagactggc cttacccgcc 1150
accccgagga gaagcctgtg aggaaggaca agcgggatga gttggtggaa 1200
gccattgaat cagccttgga gaccctgaac aatcctgcag agaacagccc 1250
caatcaccgt ccttacacgg cctctgattt catagaaggg atctaccgaa 1300
cagaaaggga caaagggaca ttgtatgagc tcaccttcaa aggggaccac 1350
aaacacgaat tcaaacggct catcttattt cgaccattca gccccatcat 1400
gaaagtgaaa aatgaaaagc tcaacatggc caacacgctt atcaatgtta 1450
tcgtgcctct agcaaaaagg gtggacaagt tccggcagtt catgcagaat 1500
ttcagggaga tgtgcattga gcaggatggg agagtccatc tcactgttgt 1550
ttactttggg aaagaagaaa taaatgaagt caaaggaata cttgaaaaca 1600
cttccaaagc tgccaacttc aggaacttta ccttcatcca gctgaatgga 1650
gaattttctc ggggaaaggg acttgatgtt ggagcccgct tctggaaggg 1700
Page 88
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
aagcaacgtc cttctctttt tctgtgatgt ggacatctac ttcacatctg 1750
aattcctcaa tacgtgtagg ctgaatacac agccagggaa gaaggtattt 1800
tatccagttc ttttcagtca gtacaatcct ggcataatat acggccacca 1850
tgatgcagtc cctcccttgg aacagcagct ggtcataaag aaggaaactg 1900
gattttggag agactttgga tttgggatga cgtgtcagta tcggtcagac 1950
ttcatcaata taggtgggtt tgatctggac atcaaaggct ggggcggaga 2600
ggatgtgcac ctttatcgca agtatctcca cagcaacctc atagtggtac 2050
ggacgcctgt gcgaggactc ttccacctct ggcatgagaa gcgctgcatg 2100
gacgagctga cccccgagca gtacaagatg tgcatgcagt ccaaggccat 2150
gaacgaggca tcccacggcc agctgggcat gctggtgttc aggcacgaga 2200
tagaggetea cettegcaaa cagaaaeaga agacaagtag caaaaaaaca 2250
tgaactccca gagaaggatt gtgggagaca ctttttcttt ccttttgcaa 2300
ttactgaaag tggctgcaae agagaaaaga cttccataaa ggacgacaaa 2350
agaattggac tgatgggtca gagatgagaa agcctccgat ttctctctgt 2400
tgggcttttt acaacagaaa tcaaaatctc cgctttgcct gcaaaagtaa 2450
cccagttgca ccctgtgaag tgtctgacaa aggcagaatg cttgtgagat 2500
tataagccta atggtgtgga ggttttgatg gtgtttacaa tacactgaga 2550
cctgttgttt tgtgtgctca ttgaaatatt catgatttaa gagcagtttt 2600
gtaaaaaatt cattagcatg aaaggcaagc atatttctcc tcatatgaat 2650
gagcctatca gcagggctct agtttctagg aatgctaaaa tatcagaagg 2700
caggagagga gataggctta ttatgatact agtgagtaca ttaagtaaaa 2750
taaaatggac cagaaaagaa aagaaaccat aaatatcgtg tcatattttc 2800
cccaagatta accaaaaata atctgcttat ctttttggtt gtccttttaa 2850
ctgtctccgt ttttttcttt tatttaaaaa tgcacttttt ttcccttgtg 2900
agttatagtc tgcttattta attaccactt tgcaagcctt acaagagagc 2950
acaagttggc ctacattttt atatttttta agaagatact ttgagatgca 3000
ttatgagaac tttcagttca aagcatcaaa ttgatgccat atccaaggac 3050
atgccaaatg ctgattctgt eaggeaetga atgteaggea ttgagacata 3100
gggaaggaat ggtttgtact aatacagacg tacagatact ttctctgaag 3150
agtattttcg aagaggagca actgaacact ggaggaaaag aaaatgacac 3200
tttctgcttt acagaaaagg aaactcattc agactggtga tatcgtgatg 3250
tacctaaaag tcagaaacca cattttctcc tcagaagtag ggaccgcttt 3300
Page 89
CA 02481756 2004-10-25
PCT-us00-23328_sequence
cttacctgtt taaataaacc aaagtatacc gtgtgaacca aacaatctct 3350
tttcaaaaca gggtgctcct cctggcttct ggcttccata agaagaaatg 3400
gagaaaaata tatatatata tatatatatt gtgaaagatc aatccatctg 3450
ccagaatcta gtgggatgga agtttttgct acatgttatc caccccaggc 3500
caggtggaag taactgaatt attttttaaa ttaagcagtt ctactcaatc 3550
accaagatgc ttctgaaaat tgcattttat taccatttca aactattttt 3600
taaaaataaa tacagttaac atagagtggt ttcttcattc atgtgaaaat 3650
tattagccag caccagatgc atgagctaat tatctctttg agtccttgct 3700
tctgtttgct cacagtaaac tcattgttta aaagcttcaa gaacattcaa 3750
gctgttggtg tgttaaaaaa tgcattgtat tgatttgtac tggtagttta 3800
tgaaatttaa ttaaaacaca ggccatgaat ggaaggtggt attgcacagc 3850
taataaaata tgatttgtgg atatgaa 3877
<210> 72
<211> 532
<212> PRT
<213> Homo Sapien
<400> 72
Met Met Met Val Arg Arg Gly Leu Leu Ala Trp Ile Ser Arg Val
1 5 10 15
Val Val Leu Leu Val Leu Leu Cys Cys Ala Ile Ser Val Leu Tyr
20 25 30
Met Leu Ala Cys Thr Pro Lys Gly Asp Glu Glu Gln Leu Ala Leu
35 40 45
Pro Arg Ala Asn Ser Faro Thr Gly Lys Glu Gly Tyr Gln Ala Val
50 55 60
Leu Gln Glu Trp Glu Glu Gln His Arg Asn Tyr Val Ser Ser Leu
65 70 75
Lys Arg Gln Ile Ala C7ln Leu Lys Glu Glu Leu Gln Glu Arg ser
80 85 90
Glu Gln Leu Arg Asn Gly Gln Tyr Gln Ala Ser Asp Ala Ala Gly
95 100 105
Leu Gly Leu Asp Arg Ser Pro Pro Glu Lys Thr Gln Ala Asp Leu
lI0 115 120
Leu Ala Phe Leu His ser Gln Val Asp Lys Ala Glu Val Asn Ala
125 130 135
Gly val Lys Leu Ala T'hr Glu Tyr Ala Ala Val Pro Phe Asp Ser
140 145 150
Phe Thr Leu Gln Lys Val Tyr Gln Leu Glu Thr Gly Leu Thr Arg
155 160 165
Page 90
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
His Pro Glu Glu Lys Pro Val Arg Lys Asp Lys Arg Asp Glu Leu
170 175 180
val Glu Ala Ile Glu Ser Ala Leu Glu Thr Leu Asn Asn Pro Ala
185 190 195
Glu Asn Ser Pro Asn His Arg Pro Tyr Thr Ala Ser Asp Phe Ile
200 205 210
Glu Gly Ile Tyr Arg Thr Glu Arg Asp Lys Gly Thr Leu Tyr Glu
215 220 225
Leu Thr Phe Lys Gly Asp His Lys His Glu Phe Lys Arg Leu Ile
230 235 240
Leu Phe Arg Pro Phe Ser Pro Ile Met Lys Val Lys Asn Glu Lys
245 250 255
Leu Asn Met Ala Asn Thr Leu Ile Asn Val Ile Val Pro Leu Ala
260 265 270
Lys Arg Val Asp 2~5 Phe Arg Gln Phe z8t0 Gln Asn Phe Arg 2
Met Cys Ile Glu Gln Asp Gly Arg Val His Leu Thr Val Val Tyr
290 295 300
Phe Gly Lys Glu.Glu Ile Asn Glu Val Lys Gly Ile Leu Glu Asn
305 310 315
Thr Ser Lys Ala Ala Asn Phe Arg Asn Phe Thr Phe Ile Gln Leu
320 325 330
Asn Gly Glu Phe Ser Arg Gly Lys Gly Leu Asp Val Gly Ala Arg
335 340 345
Phe Trp Lys Gly Ser Asn Val Leu Leu Phe Phe Cys Asp Val Asp
350 355 360
Ile Tyr Phe Thr Ser Glu the Leu Asn Thr Cys Arg Leu Asn Thr
365 370 375
Gln Pro Gly Lys Lys Val Phe Tyr Pro Val Leu Phe Ser Gln Tyr
380 385 390
Asn Pro Gly Ile Ile Tyr Gly His His Asp Ala Val Pro Pro Leu
395 400 405
Glu Gln Gln Leu Val Ile Lys Lys Glu Thr Gly Phe Trp Arg Asp
410 415 420
Phe Gly Phe Gly Met Thr Cys Gln Tyr Arg Ser Asp Phe I12 Asn
425 430 435
Ile Gly Gly Phe Asp Leu Asp Ile Lys Gly Trp Gly Gly Glu Asp
440 445 450
Val His Leu Tyr Arg Lys Tyr Leu His Ser ASn Leu Ile Val Val
455 460 465
Arg Thr Pro Val Arg Gly Leu Phe His Leu Trp His Glu Lys Arg
470 475 480
Page 91
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
cys Met Asp Glu Leu Thr Pro Glu Gln Tyr Lys Met cys Met Gln
485 490 495
Ser Lys Ala Met Asn Glu Ala Ser His Gly Gln Leu Gly Met Leu
500 505 510
Val Phe Arg His Glu Ile Glu Ala His Leu Arg Lys Gln Lys Gln
515 520 525
Lys Thr Ser Ser Lys Lys Thr -
530
<210> 73
<211> 1701
<212> DNA
<213> Homo Sapien
<220>
<221> unsure
<222> 1528
<223> unknown base
<400> 73
gagactgcag agggagataa agagagaggg caaagaggca gcaagagatt 50
tgtcctgggg atccagaaac ccatgatacc ctactgaaca ccgaatcccc 100
tggaagccca cagagacaga gacagcaaga gaagcagaga taaatacact 150
cacgccagga gctcgctcgc tctctctctc tctctctcac tcctccctcc 200
ctctctctct gcctgtccta gtcctctagt cctcaaattc ccagtcccct 250
gcaccccttc ctgggacact atgttgttct ccgccctcct gctggaggtg 300
atttggatcc tggctgcaga tgggggtcaa cactggacgt atgagggccc 350
acatggtcag gaccattggc cagcctctta ccctgagtgt ggaaacaatg 400
cccagtcgcc catcgatatt cagacagaca gtgtgacatt tgaccctgat 450
ttgcctgctc tgcagcccca cggatatgac cagcctggca ccgagccttt 500
ggacctgcac aacaatggcc acacagtgca actctctctg ccctctaccc 550
tgtatctggg tggacttccc cgaaaatatg tagctgccca gctccacctg 600
cactggggtc agaaaggats~ cccagggggg tcagaacacc agatcaacag 650
tgaagccaca tttgcagagc tccacattgt acattatgac tctgattcct 700
atgacagctt gagtgaggct. gctgagaggc ctcagggcct ggctgtcctg 750
ggcatcctaa ttgaggtggg tgagactaag aatatagctt atgaacacat 800
tctgagtcac ttgcatgaag tcaggcataa agatcagaag acctcagtgc 850
ctcccttcaa cctaagagag ctgctcccca aacagctggg,gcagtacttc 900
cgctacaatg gctcgctcac aactccccct tgctaccaga gtgtgctctg 950
gacagttttt tatagaaggt= cccagatttc aatggaacag ctggaaaagc 1000
Page 92
CA 02481756 2004-10-25
PCT-u500-23328_sequence
ttcaggggac attgttctcc acagaagagg agccctctaa gcttctggta 1050
cagaactacc gagcccttca gcctctcaat cagcgcatgg tctttgcttc 1100
tttcatccaa gcaggatcct cgtataccac aggtgaaatg ctgagtctag 1150
gtgtaggaat cttggttggc tgtctctgcc ttctcctggc tgtttatttc 1200
attgctagaa agattcggaa gaagaggctg gaaaaccgaa agagtgtggt 1250
cttcacctca gcacaagcca cgactgaggc ataaattcct tctcagatac 1300
catggatgtg gatgacttcc cttcatgcct atcaggaagc ctctaaaatg 1350
gggtgtagga tctggccaga aacactgtag gagtagtaag cagatgtcct 1400
ccttcccctg gacatctctt agagaggaat ggacccaggc tgtcattcca 1450
ggaagaactg cagagccttc agcctctcca aacatgtagg aggaaatgag 1500
gaaatcgctg tgttgttaat gcagaganca aactctgttt agttgcaggg 1550
gaagtttggg atatacccca aagtcctcta ccccctcact tttatggccc 1600
tttccctaga tatactgcgg gatctctcct taggataaag agttgctgtt 1650
gaagttgtat atttttgatc aatatatttg gaaattaaag tttctgactt 1700
t 1701 .
<210> 74
<211> 337
<212> PRT
<213> Homo Sapien
<400> .74
Met Leu Phe Ser Ala Leu Leu Leu Glu Val Ile Trp Ile Leu Ala
1 5 10 15
Ala Asp Gly Gly Gln His Trp Thr Tyr Glu Gly Pro His Gly Gln
20 25 30
Asp His Trp Pro Ala Ser Tyr Pro Glu Cys Gly Asn Asn Ala Gln
35 40 45
ser Pro Ile Asp Ile Gln Thr Asp Ser val Thr Phe Asp Pro Asp
50 55 60
Leu Pro Ala Leu Gln Pro His G1y Tyr Asp Gln Pro Gly Thr Glu
65 70 75
Pro Leu Asp Leu His Asn Asn Gly His Thr Val Gln Leu Ser Leu
80 85 90
Pro Ser Thr Leu Tyr Leu Gly Gly Leu Pro Arg Lys Tyr Val Ala
95 100 105
Ala Gln Leu His Leu His Trp G1y Gln Lys Gly Ser Pro Gly Gly
110 115 120
Ser Glu His Gln Ile Asn Ser Glu Ala Thr Phe Ala Glu Leu His
125 130 135
Page 93
. ~w.., ~ . s ~,." . : .. ....~... .~ . . , . .. _ . ~.. .,.. . .. . . _ . .
...... . a .,a,~ . .... "... .. .. _. __ _.__... ~ _._ ~ .~. _. ..,. _ .. . _
_ __._._ ~ _ ._ . _ ..
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Ile Val His Tyr Asp Ser Asp Ser Tyr Asp Ser Leu Ser Glu Ala
140 145 150
Ala Glu Arg Pro Gln Gly Leu Ala Val Leu Gly Ile Leu Ile Glu
155 160 165
Val Gly Glu Thr Lys Asn Ile Ala Tyr Glu His Ile Leu Ser His
170 175 180
Leu His Glu Val Arg His Lys Asp Gln Lys Thr Ser Val Pro Pro
185 190 195
Phe Asn Leu Arg Glu Leu Leu Pro Lys Gln Leu Gly Gln Tyr Phe
200 205 210
Arg Tyr Asn Gly Ser Leu Thr Thr Pro Pro Cys Tyr Gln Ser val
215 220 225
Leu Trp Thr Val Phe Tyr Arg Arg Ser Gln Ile Ser Met Glu Gln
230 235 240
Leu Glu Lys Leu Gln Gly Thr Leu Phe Ser Thr Glu Glu Glu Pro
245 250 255
Ser Lys Leu Leu Val Gln Asn Tyr Arg Ala Leu Gln Pro Leu Asn
260 265 270
Gln Arg Met Val Phe Ala Ser Phe I1e Gln Ala Gly 5er Ser Tyr
275 280 ~ 285
Thr Thr Gly Glu Met Leu Ser Leu Gly Val Gly Ile Leu Val Gly
290 295 300
Cys Leu Cys Leu Leu Leu Ala Val Tyr Phe Ile Ala Arg Lys Ile
305 310 315
Arg Lys Lys Arg Leu Glu Asn Arg Lys Ser val val Phe Thr Ser
320 325 330
Ala Gln Ala Thr Thr Glu Ala
335
<210> 75
<211> 1743
<212> DNA
<213> Homo Sapien
<400> 75
tgccgctgcc gccgctgctg ctgttgctcc tggcggcgcc ttggggacgg 50
gcagttccct gtgtctctgg tggtttgcct aaacctgcaa acatcacctt 100
cttatccatc aacatgaaga atgtcctaca atggactcca ccagagggtc 150
ttcaaggagt taaagttact tacactgtgc agtatttcat cacaaattgg 200
cccaccagag gtggcactga ctacagatga gaagtccatt tctgttgtcc 250
tgacagctcc agagaagtgg aagagaaatc cagaagacct tcctgtttcc 300
atgcaacaaa tatactccaa tctgaagtat aacgtgtctg tgttgaatac 350
Page 94
~~.~, __.. . .. . r~ ,~, 1. >rna~ri ,r~~ ...~m~,~.w. ~~"~~~xa~,.__~. _......
_~._._._.~_.~, ., ..__ ._ _. . ___..___~____..~.___.w..__mr__.~._..~~~s.
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
taaatcaaac agaacgtggt cccagtgtgt gaccaaccac acgctggtgc 400
tcacctggct ggagccgaac actctttact gcgtacacgt ggagtccttc 450
gtcccagggc cccctcgccg tgctcagcct tctgagaagc agtgtgccag 500
gactttgaaa gatcaatcat cagagttcaa ggctaaaatc atcttctggt 550
atgttttgcc catatctatt accgtgtttc ttttttctgt gatgggctat 600
tccatctacc gatatatcca cgttggcaaa gagaaacacc cagcaaattt 6~0
gattttgatt tatggaaatg aatttgacaa aagattcttt gtgcctgctg 700
aaaaaatcgt gattaacttt atcaccctca atatctcgga tgattctaaa 750
atttctcatc aggatatgag tttactggga aaaagcagtg atgtatccag 800
ccttaatgat cctcagccca gcgggaacct gaggccccct caggaggaag 850
aggaggtgaa acatttaggg tatgcttcgc atttgatgga aattttttgt 900
gactctgaag aaaacacgga aggtacttct ctcacccagc aagagtccct 950
cagcagaaca atacccccgg ataaaacagt cattgaatat gaatatgatg 1000
tcagaaccac tgacatttgt gcggggcctg aagagcagga gctcagtttg 1050
caggaggagg tgtccacaca aggaacatta ttggagtcgc aggcagcgtt 1100
ggcagtcttg ggcccgcaaa cgttacagta ctcatacacc cctcagctcc 1150
aagacttaga ccccctggcg caggagcaca cagactcgga ggaggggccg 1200
gaggaagagc catcgacgac cctggtcgac tgggatcccc aaactggcag 1250
gctgtgtatt ccttcgctgt ccagcttcga ccaggattca gagggctgcg 1300
agccttctga gggggatggg ctcggagagg agggtcttct atctagactc 1350
tatgaggagc cggctccaga caggccacca ggagaaaatg aaacctatct 1400
catgcaattc atggaggaa-t gggggttata tgtgcagatg gaaaactgat 150
gccaacactt ccttttgcc-t tttgtttcct gtgcaaacaa gtgagtcacc 1500
cctttgatcc cagccataaa gtacctggga tgaaagaagt tttttccagt 1550
ttgtcagtgt ctgtgagaat tacttatttc ttttctctat tctcatagca 1600
cgtgtgtgat tggttcatgc atgtaggtct cttaacaatg atggtgggcc 1650
tctggagtcc aggggctggc cggttgttct atgcagagaa agcagtcaat 1700
aaatgtttgc cagactgggt gcagaattta ttcaggtggg tgt 1743
<210> 76
<211> 442
<212> PRT
<213> Homo Sapien
<400> 76
Met Ser Tyr Asn Gly t_eu His Gln Arg Val Phe Lys Glu Leu Lys
Page 95
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
1 5 10 15
LeuLeuThr LeuCys SerIleSer SerGlnIle GlyPro ProGlu
20 25 30
ValAlaLeu ThrThr AspGluLys SerIleSer ValVal LeuThr
35 40 45
AlaProGlu LysTrp LysArgAsn ProGluAsp LeuPro ValSer
50 55 60
MetGlnGln IleTyr SerAsnLeu LysTyrAsn ValSer ValLeu
65 70 75
AsnThrLys SerASn ArgThrTrp SerGlnCys ValThr AsnHis
80 85 90
ThrLeuVal LeuThr ~TrpLeuGlu ProAsnThr LeuTyr CysVal
95 100 105
HisValGlu SerPhe ValProGly ProProArg ArgAla GlnPro
110 115 120
SerGluLys GlnCys AlaArgThr LeuLysAsp GlnSer SerGlu
125 130 135
PheLysAla LysIle I12PheTrp TyrValLeu ProI12 SerIle
140 145 150
Thr val Phe Leu Phe Ser Val Met Gly Tyr Ser Ile Tyr Arg Tyr
155 160 165
Ile His Val Gly Lys Glu Lys His Pro Ala Asn Leu Ile Leu Ile
170 175 180
Tyr Gly Asn Glu Phe Asp Lys Arg Phe Phe Val Pro Ala Glu Lys
185 190 195
Ile Val Ile Asn Phe Ile Thr Leu Asn Ile ser Asp Asp Ser Lys
200 205 210
Ile Ser His Gln Asp Met Ser Leu Leu Gly Lys Ser Ser asp Val
215 220 225
Ser Ser Leu Asn Asp Pro Gln Pro Ser Gly Asn Leu Arg Pro Pro
230 235 240
Gln Glu Glu Glu Glu Val Lys His Leu Gly Tyr Ala Ser His Leu
24S 250 255
Met Glu Ile Phe Cys Asp Ser Glu Glu Asn Thr G7u Gly Thr Ser
260 265 270
Leu Thr Gln Gln Glu 5er Leu Ser Arg Thr Ile Pro Pro Asp Lys
275 280 285
Thr Val Ile Glu Tyr Glu Tyr Asp Val Arg Thr Thr Asp Ile Cys
290 295 300
Ala Gly Pro Glu Glu Gln Glu Leu Ser Leu Gln Glu Glu Val Ser
305 310 315
Thr Gln Gly Thr Leu L.eu Glu Ser Gln Ala Ala Leu Ala Val Leu
Page 96
. _ . _;,~ ,~_ .~~_..._ . ~;~zx.r _.uF... ..~,... _ ___s ~ , ~,~ ~~: ",j .~
...~ ,,4 ~~.~,u.a , ...~._~_ ~.}..._a. ~__..___.._.. ~~..._. _._....__ ___ .
~ _ ~._.~W__~
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
320 325 330
Gly Pro Gln Thr Leu Gln Tyr Ser Tyr Thr Pro Gln Leu Gln Asp
335 340 345
Leu Asp Pro Leu Ala Gln Glu His Thr Asp Ser Glu Glu Gly Pro
350 355 360
Glu Glu Glu Pro Ser Thr Thr Leu Val Asp Trp Asp Pro Gln Thr
365 370 375
Gly Arg Leu Cys Ile Pro Ser Leu Ser Ser Phe Asp Gln Asp Ser
380 385 390
Glu Gly Cys Glu Pro Ser Glu Gly Asp Gly Leu Gly Glu Glu Gly
395 400 405
Leu Leu Ser Arg Leu Tyr Glu Glu Pro Ala Pro Asp Arg Pro Pro
410 415 420
Gly Glu Asn Glu Thr Tyr Leu Met Gln Phe Met Glu Glu Trp Gly
425 430 435
Leu Tyr Val Gln Met Glu Asn
440
<210> 77
<211> 1636
<212> DNA
<213> Homo Sapien
<400> 77
gaggagcggg ccgaggactc cagcgtgccc aggtctggca tcctgcactt 50
gctgccctct gacacctggg aagatggccg gcccgtggac cttcaccctt 100
ctctgtggtt tgctggcagc caccttgatc caagccaccc tcagtcccac 150
tgcagttctc atcctcggcc caaaagtcat caaagaaaag ctgacacagg 200
agctgaagga ccacaacgcc accagcatcc tgcagcagct gccgctgctc 250
agtgccatgc gggaaaagcc agccggaggc atccctgtgc tgggcagcct 300
ggtgaacacc gtcctgaagc acatcatctg gctgaaggtc atcacagcta 350
acatcctcca gctgcaggtg aagccctcgg ccaatgacca ggagctgcta 400
gtcaagatcc ccctggacat ggtggctgga ttcaacacgc ccctggtcaa 450
gaccatcgtg gagttccaca tgacgactga ggcccaagcc accatccgca 500
tggacaccag tgcaagtggc cccacccgcc tggtcctcag tgactgtgcc 550
accagccatg ggagcctgcg catccaactg ctgtataagc tctccttcct 600
ggtgaacgcc ttagctaagr_ aggtcatgaa cctcctagtg ccatccctgc 650
ccaatctagt gaaaaaccag ctgtgtcccg tgatcgaggc ttccttcaat 700
ggcatgtatg cagacctcct gcagctggtg aaggtgccca tttccctcag 750
cattgaccgt ctggagtttg accttctgta tcctgccatc aagggtgaca 800
Page 97
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
ccattcagct ctacctgggg gccaagttgt tggactcaca gggaaaggtg 850
accaagtggt tcaataactc tgcagcttcc ctgacaatgc ccaccctgga 900
caacatcccg ttcagcctca tcgtgagtea ggacgtggtg aaagctgcag 950
tggctgctgt gctctctcca gaagaattca tggtcctgtt ggactctgtg 1000
cttcctgaga gtgcccatcg gctgaagtca ageatcgggc tgatcaatga 1050
aaaggctgca gataagctgg gatctaccca gatcgtgaag atcctaactc 1100
aggacactcc cgagtttttt atagaccaag gccatgccaa ggtggcccaa 1150
ctgatcgtgc tggaagtgtt tccctccagt gaagccctcc gccctttgtt 1200
caccctgggc atcgaagcca gctcggaagc tcagttttac accaaaggtg 1250
accaacttat actcaacttg aataacatca gctctgatcg gatccagctg 1300
atgaactctg ggattggctg gttccaacct gatgttctga aaaacatcat 1350
cactgagatc atccactcca tcctgctgcc gaaccagaat ggcaaattaa 1400
gatctggggt cccagtgtca ttggtgaagg ccttgggatt cgaggcagct 1450
gagtcctcac tgaccaagga tgcccttgtg cttactccag cctccttgtg 1500
gaaacccagc tctcctgtct cccagtgaag acttggatgg cagccatcag 1550
ggaaggctgg gtcceagctg ggagtatggg tgtgagctct atagaccatc 1600
cctctctgca atcaataaac acttgcctgt gaaaaa 1636
<210> 78
<211> 484
<212> PRT
<213> Homo Sapien
<400> 78
Met Ala Gly Pro Trp Thr Phe Thr Leu Leu Cys Gly Leu Leu Ala
1 5 10 15
Ala Thr Leu Ile Gln Ala Thr Leu Ser Pro Thr Ala val Leu Ile
20 25 30
Leu Gly Pro Lys val Ile Lys Glu Lys Leu Thr Gln Glu Leu Lys
35 40 4S
Asp His Asn Ala Thr Ser Ile Leu Gln Gln Leu Pro Leu Leu Ser
50 5S 60
Ala Met Arg Glu Lys Pro Ala Gly Gly Ile Pro Val Leu Gly Ser
65 a0 75
Leu val Asn Thr val I_eu Lys His Ile Ile Trp Leu Lys val Ile
80 8S 90
Thr Ala Asn Ile Leu Gln Leu Gln val Lys Pro Ser Ala Asn Asp
95 100 105
Gln Glu ~eu Leu val Lys Ile Pro Leu Asp Met val Ala Gly Phe
Page 98
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
110 115 120
Asn Thr Pro Leu Val Lys Thr Ile Val Glu Phe His Met Thr Thr
125 130 135
Glu Ala Gln Ala Thr Ile Arg Met Asp Thr Ser Ala Ser Gly Pro
140 145 150
Thr Arg Leu Val Leu Ser Asp Cys Ala Thr Ser His Gly Ser Leu
155 160 165
Arg Ile Gln Leu Leu Tyr Lys Leu Ser Phe Leu Val Asn Ala Leu
170 175 180
Ala Lys Gln Val Met Asn Leu Leu Val Pro Ser Leu Pro Asn Leu
185 190 195
Val Lys Asn Gln Leu Cys Pro Val Ile Glu Ala Ser Phe Asn Gly
200 205 210
Met Tyr Ala Asp Leu Leu Gln Leu Val Lys Val Pro Ile Ser Leu
215 220 225
Ser Ile Asp Arg Leu Glu Phe Asp Leu Leu Tyr Pro Ala Ile Lys
230 235 240
Gly Asp Thr Ile Gln Leu Tyr Leu Gly Ala Lys Leu Leu Asp Ser
245 250 255
Gln G1y Lys Val Thr Lys Trp Phe Asn Asn Ser Ala Ala Ser Leu
260 265 270
Thr Met Pro Thr Leu Asp Asn Ile Pro Phe Ser Leu Ile Val Ser
275 280 285
Gln Asp Val Val Lys Ala Ala Val Ala Ala Val Leu Ser Pro Glu
290 295 300
Glu Phe Met Val Leu Leu Asp Ser Val Leu Pro Glu Ser Ala His
305 310 315
Arg leu Lys Ser Ser Ile Gly Leu Ile Asn Glu Lys Ala Ala Asp
320 325 330
Lys Leu Gly Ser Thr Gln Ile Val Lys Ile Leu Thr Gln Asp Thr
335 340 345
Pro Glu Phe Phe Ile Asp Gln Gly His Ala Lys Val Ala Gln Leu
350 355 360
Ile Val Leu Glu Val Phe Pro Ser Ser Glu Ala Leu Arg Pro Leu
365 370 375
Phe Thr Leu Gly Ile Glu Ala Ser Ser Glu Ala Gln Phe Tyr Thr
380 385 390
Lys Gly Asp Gln Leu Ile Leu Asn Leu Asn Asn Ile Ser Ser Asp
395 400 405
Arg Ile Gln Leu Met Asn ser Gly Ile Gly Trp Phe Gln Pro Asp
410 415 420
Val Leu Lys Asn Ile :Cle Thr Glu Ile Ile His Ser Ile Leu Leu
Page 99
."... . ~~ ~,w, a ~. a . .N... .... ~.,. . _ . _ ._.._.. _. . . . . .. .
I
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
425 430 435
Pro Asn Gln Asn Gly Lys Leu Arg Ser Gly Val Pro Val Ser Leu
440 445 450
Val Lys Ala Leu Gly Phe Glu Ala Ala Glu Ser Ser Leu Thr Lys
455 460 465
Asp Ala Leu Val Leu Thr Pro Ala Ser Leu Trp Lys Pro Ser Ser
470 475 480
Pro val Ser Gln
<210> 79
<211> 1475
<212> DNA
<213> Homo Sapien
<400> 79
gagagaagtc agcctggcag agagactctg aaatgaggga ttagaggtgt 50
tcaaggagca agagcttcag cctgaagaca agggagcagt ccctgaagac 100
gcttctactg agaggtctgc catggcctct cttggcctcc aacttgtggg 150
ctacatccta ggccttctgg ggcttttggg cacactggtt gccatgctgc 200
tccccagctg gaaaacaagt tcttatgteg gtgccagcat tgtgacagca 250
gttggcttct ccaagggcct ctggatggaa tgtgccacac acagcacagg 300
catcacccag tgtgacatct atagcaccct tctgggcctg cccgctgaca 350
tccaggctgc ccaggccatg atggtgacat ccagtgcaat ctcctccctg 400
gcctgcatta tctctgtggt gggcatgaga tgcacagtct tctgccagga 450
atcccgagcc aaagacagag tggcggtagc aggtggagtc tttttcatcc 500
ttggaggcct cctgggattc attcctgttg cctggaatct tcatgggatc 550
ctacgggact tctactcacr_ actggtgcct gacagcatga aatttgagat 600
tggagaggct ctttacttgg gcattatttc ttccctgttr_ tccctgatag 650
ctggaatcat cctctgcttt tcctgctcat cccagagaaa tcgctccaac 700
tactacgatg cctaccaagc ccaacctctt gccacaagga gctctccaag 750
gcctggtcaa cctcccaaag tcaagagtga gttcaattcc tacagcctga 800
cagggtatgt gtgaagaacc aggggccaga gctggggggt ggctgggtct 850
gtgaaaaaca gtggacagca ccccgagggc cacaggtgag ggacactacc 900
actggatcgt gtcagaaggt gctgctgagg atagactgac tttggccatt 950
ggattgagca aaggcagaaa tgggggctag tgtaacagca tgcaggttga 1000
attgccaagg atgctcgcca tgccagcctt tctgttttcc tcaccttgct 1050
gctcccctgc cctaagtccc caaccctcaa cttgaaaccc cattccctta 1100
Page 100
CA 02481756 2004-10-25
ACT-uS00-23328_Sequence
agccaggact cagaggatc:c ctttgccctc tggtttacct gggactccat 1150
ccccaaaccc actaatcaca tcccactgac tgaccctctg tgatcaaaga 1200
ccctctctct ggctgaggta ggctcttagc tcattgctgg ggatgggaag 1250
gagaagcagt ggcttttgt.g ggcattgctc taacctactt ctcaagcttc 1300
cctccaaaga aactgattgg ccctggaacc tccatcccac tcttgttatg 1350
actccacagt gtccagacta atttgtgcat gaactgaaat aaaaccatcc 1400
tacggtatcc agggaacaga aagcaggatg caggatggga ggacaggaag 1450
gcagcctggg acatttaaaa aaata 1475
<210> 80
<211> 230
<212> PRT
<213> Homo Sapien
<400> 80
Met Ala Ser Leu Gly Leu Gln Leu Val Gly Tyr Ile Leu Gly Leu
1 5 10 15
Leu Gly Leu Leu Gly Thr Leu Val Ala Met Leu Leu Pro Ser Trp
20 25 30
Lys Thr Ser Ser Tyr Val Gly Ala Ser Ile Val Thr Ala Val Gly
35 40 45
Phe Ser Lys Gly Leu Trp Met Glu Cys Ala Thr His Ser Thr Gly
50 55 60
Ile Thr Gln Cys Asp Ile Tyr Ser Thr Leu Leu Gly Leu Pro Ala
65 70 75
Asp Ile Gln Ala Ala Gln Ala Met Met val Thr Ser Ser Ala Ile
80 85 90
Ser Ser Leu A1a Cys Ile Ile Ser Val Val Gly Met Arg Cys Thr
95 100 105
val Phe Cys Gln Glu 5er Arg Ala Lys Asp Arg val Ala val Ala
110 115 120
Gly Gly Val Phe Phe Iie Leu Gly Gly Leu Leu Gly Phe Ile Pro
125 130 135
val Ala Trp Asn Leu His Giy Ile Leu Arg Asp Phe Tyr Ser Pro
140 145 150
Leu Val Pro Asp Ser Met Lys Phe Glu Ile Gly Glu Ala Leu Tyr
155 I60 165
Leu Gly Ile Ile Ser Ser Leu Phe Ser Leu Ile Ala Gly Ile Ile
170 175 180
Leu Cys Phe Ser Cys Ser Ser Gln Arg ASn Arg 5er Asn Tyr Tyr
185 190 195
Asp Ala Tyr Gln Ala Gln Pro Leu Ala Thr Arg Ser Ser Pro Arg
Page 101
CA 02481756 2004-10-25
PCT-u500-23328_sequence
200 205 210
Pro Gly Gln Pro Pro Lys Val Lys 5er Glu Phe Asn Ser Tyr Ser
215 220 225
Leu Thr Gly Tyr Val
230
<210> 81
<211> 1732 _
<212> DNA -
<213> Homo Sapien
<400> 81
cccacgcgtc cgcgcctctc ccttctgctg gaccttcctt cgtCtctcca 50
tctctccctc ctttccccgc gttctctttc cacctttctc ttcttcccac 100
cttagacctc ccttcctgcc ctcctttcct gcccaccgct gcttcctggc 150
ccttctcega ccccgctcta gcagcagacc tcctggggtc tgtgggttga 200
tctgtggccc ctgtgcctcc gtgtcctttt cgtctccctt cctcccgact 250
ccgctcccgg accagcggcc tgaccctggg gaaaggatgg ttcccgaggt 300
gagggtcctc tcctccttgc tgggactcgc gctgctctgg ttccccctgg 350
actcccacgc tcgagcccgc ccagacatgt tctgcctttt ccatgggaag 400
agatactccc ccggcgagag ctggcacccc tacttggagc cacaaggcct 450
gatgtactgc ctgcgctgta cctgctcaga gggcgcccat gtgagttgtt 500
accgcctcca ctgtccgcct gtccactgcc cccagcctgt gacggagcca 550
cagcaatgct gtcccaagtg tgtggaacct cacactccct ctggactccg 600
ggccccacca aagtcctgcc agcacaacgg gaccatgtac caacacggag 650
agatcttcag tgcccatgag ctgttcccct cccgcctgcc caaccagtgt 700
gtcctctgca gctgcacaga gggccagatc tactgcggcc tcacaacctg 750
cccegaaeca ggetgeeeag eacceeteee aetgeeagac tcetgetgee 800
aagcctgcaa agatgaggca agtgagcaat cggatgaaga ggacagtgtg 850
cagtcgctcc atggggtgag acatcctcag gatccatgtt ccagtgatgc 900
tgggagaaag agaggcccgg gcaccccagc ccccactggc ctcagcgccc 950
ctctgagctt catccctcgc cacttcagac ccaagggagc aggcagcaca 1000
actgtcaaga tcgtcctgaa ggagaaacat aagaaagcct gtgtgcatgg 1050
cgggaagacg tactcccacg gggaggtgtg gcacccggcc ttccgtgcct 1100
tcggcccctt gccctgcatc ctatgcacct gtgaggatgg ccgecaggac 1150
tgccagcgtg tgacctgtcc caccgagtac ccctgccgtc accccgagaa 1200
agtggctggg aagtgctgca agatttgccc agaggacaaa gcagaccctg 1250
Page 102
CA 02481756 2004-10-25
PCT-uS00-23328_sequence
gccacagtga gatcagttct accaggtgtc ccaaggcacc gggccgggtc 1300
ctcgtccaca catcggtatc cccaagccca gacaacctgc gtcgctttgc 1350
cctggaacac gaggcctcgg acttggtgga gatctacctc tggaagctgg 1400
taaaagatga ggaaactgag gctcagagag gtgaagtacc tggcccaagg 1450
ccacacagcc agaatcttcc acttgactca gatcaagaaa gtcaggaagc 1500
aagacttcca gaaagaggca cageacttcc gactgctcgc tggcccccac 1550
gaaggtcact ggaacgtctt cctagcccag accctggagc tgaaggtcac 1600
ggccagtcca gacaaagtga ccaagacata acaaagacct aacagttgca 1650
gatatgagct gtataattgt tgttattata tattaataaa taagaagttg 1700
cattaccctc aaaaaaaaaa aaaaaaaaaa as 1732
<210> 82
<211> 451
<212> PRT
<213> Homo Sapien
<400> 82
Met Val Pro Glu Val Arg Val Leu 5er Ser Leu Leu Gly Leu Ala
1 5 10 15
Leu Leu Trp Phe Pro Leu Asp Ser His Ala Arg Ala Arg Pro ASp
20 25 30
Met Phe Cys Leu Phe His Gly Lys Arg Tyr Ser Pro Gly Glu Ser
35 40 45
Trp His Pro Tyr Leu Glu Pro Gln Gly Leu Met Tyr Cys Leu Arg
50 55 60
Cys Thr Cys Ser Glu Gly Ala His val Ser Cys Tyr Arg Leu His
65 70 75
Cys Pro Pro Val His Cys Pro Gln Pro Val Thr Glu Pro Gln Gln
80 85 90
Cys Cys Pro Lys Cys val Glu Pro His Thr Pro Ser Gly Leu Arg
95 100 105
Ala Pro Pro Lys Ser Cys Gln His Asn Gly Thr Met Tyr Gln His
110 115 120
Gly Glu zie Phe Ser Ala His Glu Leu Phe Pro Ser Arg Leu Pro
125 130 135
Asn Gln Cys Val Leu Cys Ser Cys Thr Glu Gly Gln zle Tyr Cys
140 145 150
Gly Leu Thr Thr Cys Pro Glu Pro Gly Cys Pro Ala Pro Leu Pro
I5S 160 165
Leu Pro Asp Ser Cys Cys Gln Ala Cys Lys Asp Glu Ala Ser Glu
170 175 180
Page 103
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Gln Ser Asp Glu Glu Asp Ser Val Gln Ser Leu His Gly Val Arg
185 190 195
His Pro Gln Asp Pro Cys Ser Ser Asp Ala Gly Arg Lys Arg Gly
200 205 210
Pro Gly Thr Pro Ala Pro Thr Gly Leu Ser Ala Pro Leu Ser Phe
215 220 225
Ile Pro Arg His Phe Arg Pro Lys Gly Ala Gly Ser Thr Thr Val
230 235 240
Lys Ile Val Leu Lys Glu Lys His Lys Lys Ala Cys Val His Gly
245 250 255
Gly Lys Thr Tyr Ser His Gly Glu Val Trp His Pro Ala Phe Arg
260 265 270
Ala Phe Gly Pro Leu Pro Cys Ile Leu Cys Thr Cys Glu Asp Gly
275 280 285
Arg Gln Asp Cys Gln Arg Val Thr Cys Pro Thr Glu Tyr Pro Cys
290 295 300
Arg His Pro Glu Lys Val Ala Gly Lys Cys Cys Lys Ile Cys Pro
305 310 315
Glu Asp Lys Ala Asp Pro Gly His Ser Glu Ile Ser Ser Thr Arg
320 325 330
Cys Pro Lys Ala Pro Gly Arg Vai Leu Val His Thr Ser Val Ser
335 340 345
Pro Ser Pro Asp Asn Leu Arg Arg Phe Aia Leu Glu His Glu Ala
350 355 360
Ser Asp Leu Val Glu Ile Tyr Leu Trp Lys Leu Val Lys Asp Glu
365 370 375
Glu Thr Glu Ala Gln Arg Gly Glu Val Pro Gly Pro Arg Pro His
380 385 390
Ser Gln Asn Leu Pro Leu Asp Ser Asp Gln Glu Ser Gin Glu Ala
395 400 405
Arg Leu Pro Glu Arg Gly Thr Ala Leu Pro Thr Ala Arg Trp Pro
410 415 420
Pro Arg Arg Ser Leu Glu Arg Leu Pro Ser Pro Asp Pro Gly Ala
425 430 435
Glu Gly His Gly Gln Ser Arg Gln Ser Asp Gln Asp Ile Thr Lys
440 445 450
Thr
<210> 83
<211> 2052
<212> DNA
<213> Homo Sapien
<400> 83
Page 104
CA 02481756 2004-10-25
RCT-uS00-23328_Sequence
gacagctgtg tctcgatgga gtagactctc agaacagcgc agtttgccct 50
ccgctcacgc agagcctctc cgtggcttcc gcaccttgag cattaggcca 100
gttctcctct tctctctaat ccatccgtca cctctcctgt catccgtttc 150
catgccgtga ggtccattca cagaacacat ccatggctct catgcteagt 200
ttggttctga gtctcctcaa gctgggatca gggcagtggc aggtgtttgg 250
gccagacaag cctgtccagg ccttggtggg ggaggacgca gcattctcct 300
gtttcctgtc tcctaagacc aatgcagagg ccatggaagt gcggttcttc 350
aggggccagt tctctagcgt ggtccacctc tacagggacg ggaaggacca 400
gccatttatg cagatgccac agtatcaagg caggacaaaa ctggtgaagg 450
attctattgc ggaggggcgc atctctctga ggctggaaaa cattactgtg 500
ttggatgctg gcctctatgg gtgcaggatt agttcccagt cttactacca 550
gaaggccatc tgggagctac aggtgtcagc actgggctca gttcctctca 600
tttccatcac gggatatgtt gatagagaca tccagctact ctgtcagtcc 650
tcgggctggt tcccccggcc cacagcgaag tggaaaggtc cacaaggaca 700
ggatttgtcc acagactcca ggacaaacag agacatgcat ggcctgtttg 750
atgtggagat ctctctgacc gtccaagaga acgccgggag catatcctgt 800
tccatgcggc atgctcatct gagccgagag gtggaatcca gggtacagat 850
aggagatacc tttttcgagc ctatatcgtg gcacctggct accaaagtac 900
tgggaatact ctgctgtggc ctattttttg gcattgttgg actgaagatt 950
ttcttctcca aattccagtg gaaaatccag gcggaactgg actggagaag 1000
aaagcacgga caggcagaat tgagagacgc ccggaaacac gcagtggagg 1050
tgactctgga tccagagacg gctcacccga agctctgcgt ttctgatctg 1100
aaaactgtaa cccatagaaa agctccccag gaggtgcctc actctgagaa 1150
gagatttaca aggaagagtg tggtggcttc tcagagtttc caagcaggga 1200
aacattactg ggaggtggac ggaggacaca ataaaaggtg gcgcgtggga 1250
gtgtgccggg atgatgtgga caggaggaag gagtacgtga ctttgtctcc 1300
cgatcatggg tactgggtcc tcagactgaa tggagaacat ttgtatttca 1350
cattaaatcc ccgttttatc agcgtcttcc ccaggacccc acctacaaaa 1400
ataggggtct tcctggacta tgagtgtggg accatctcct tcttcaacat 1450
aaatgaccag tcccttattt ataccctgac atgtcggttt gaaggcttat 1500
tgaggcccta cattgagtat ccgtcctata atgagcaaaa tggaactccc 1550
atagtcatct gcccagtcac ccaggaatca gagaaagagg cctcttggca 1600
Page 105
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
aagggcctct gcaatcccag agacaagcaa cagtgagtcc tcctcacagg 1650
caaccacgcc cttcctcccc aggggtgaaa tgtaggatga atcacatccc 1700
acattcttct ttagggatat taaggtctct ctcccagatc caaagtcccg 1750
cagcagccgg ccaaggtggc ttccagatga agggggactg gcctgtccac 1800
atgggagtca ggtgtcatgg ctgccctgag ctgggaggga agaaggctga 1850
cattacattt agtttgctca cactccatct ggctaagtga tcttgaaata 1900
ccacctctca ggtgaagaac cgtcaggaat tcccatctca caggctgtgg 1950
tgtagattaa gtagacaagg aatgtgaata atgcttagat cttattgatg 2000
acagagtgta tcctaatggt ttgttcatta tattacactt tcagtaaaaa 2050
as 2052
<210> 84
<211> 500
<212> PRT
<213> Homo Sapien
<400> 84
Met Ala Leu Met Leu Ser Leu vat Leu Ser Leu Leu Lys Leu Gly
1 5 10 15 .
Ser Gly Gln Trp Gln val Phe Gly Pro Asp Lys Pro val Gln Ala
20 25 30
Leu val Gly Glu Asp Ala Ala Phe Ser Cys Phe Leu Ser Pro Lys
35 40 45
Thr ASn Ala GlU Ala Met G1U Vai Arg Phe Phe Arg Gly Gln Phe
50 55 60
Ser Ser val val His Leu Tyr Arg Asp Gly Lys Asp Gln Pro Phe
65 70 75
Met Gln Met Pro Gin Tyr Gln Gly Arg Thr Lys Leu val Lys Asp
80 85 90
Ser Ile Ala Glu Gly Arg Ile Ser Leu Arg Leu Glu Asn Ile Thr
95 100 105
val Leu Asp Ala Gly Leu Tyr Gly Cys Arg Ile Ser~Ser Gin Ser
110 115 120
Tyr, Tyr Gln Lys 125 Iie Trp Glu Leu ~3~ val Ser Aia Leu i35
Ser val Pro Leu Ile Ser Ile Thr Gly Tyr val Asp Arg Asp Ile
140 145 150
Gln Leu Leu Cys Gln Ser Ser G1y Trp Phe Pro Arg Pro Thr A1a
155 160 165
Lys Trp Lys Gly Pro Gln Giy Gln Asp Leu Ser Thr Asp Ser Arg
170 175 180
Page 106
CA 02481756 2004-10-25
PCT-u500-23328_Se~ Luence
Thr Asn Arg Asp Met His Gly Leu Phe Asp Vai Glu Ile Ser Leu
185 190 195
Thr Val Gln Glu Asn Ala Gly Ser Ile Ser Cys Ser Met Arg His
200 205 210
Ala His Leu Ser Arg Glu i/al Glu Ser Arg Val Gln Ile Gly Asp
2I5 , 220 225
Thr Phe Phe Glu Pro Ile Ser Trp His Leu Ala Thr Lys Val Leu
230 235 2-40
Gly Ile Leu Cys Cys Gly Leu Phe Phe Gly Ile Val Gly Leu Lys
245 250 255
Ile Phe Phe Ser Lys Phe Gln Trp Lys Ile Gln Ala Glu Leu Asp
260 265 270
Trp Arg Arg Lys His Gly Gln Ala Glu Leu Arg Asp Ala Arg Lys
275 280 285
His Ala Val Glu Val Thr Leu Asp Pro Glu Thr Ala His Pro Lys
290 295 300
Leu Cys Val Ser Asp Leu Lys Thr Val Thr His Arg Lys Ala Pro
305 310 315
Gln Glu Val Pro His Ser Glu Lys Arg Phe Thr Arg Lys Ser Val
320 325 330
Val Ala Ser G1n Ser Phe Gln Ala Gly Lys His Tyr Trp Glu Val
335 340 345
Asp Gly Gly His 35~ Lys Arg Trp Arg 355 Gly Val Cys Arg 36d
Asp Val Asp Arg Arg Lys Glu Tyr Val Thr Leu Ser Pro Asp His
365 370 375
Gly Tyr Trp Val Leu Arg Leu Asn Gly Glu His Leu Tyr Phe Thr
380 385 390
Leu Asn Pro Arg Phe Ile Ser Val Phe Pro Arg Thr Pro Pro Thr
395 400 405
Lys Ile Gly Va1 Phe Leu Asp Tyr Glu Cys Gly Thr Ile Ser Phe
410 415 420
Phe Asn Ile Asn Asp Gln Ser Leu Ile Tyr Thr Leu Thr Cys Arg
425 430 435 .
Phe Glu Gly Leu Leu Arg Pro Tyr Ile Glu Tyr Pro Ser Tyr Asn
440 445 450
Glu Gln Asn Gly Thr Pro Ile Val Ile Cys Pro Val Thr Gln Glu
455 460 465
Ser Glu Lys Glu Ala Ser Trp Gln Arg Ala Ser Ala Ile Pro Glu
470 475 480
Thr Ser ASn Ser Glu Ser Ser Ser Gln Ala Thr Thr Pro Phe Leu
485 490 495
Page 107
.. ,.., .._ u. .,.;.M.. ...". s.,aa ,~,~ ...~~~.~ , ~~~, ..
.~"~~.x.~m.a._~.za,~.,-ew...~. .__.~w...~.._.._. ___....__--"
~m~_~rt~~~T~..~.~~A,~."w~ ,m,.~~.,~~~~
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
Pro Arg Gly Glu Met
500
<210> 85
<211> 1665
<212> DNA
<213> Homo Sapien
<400> 85
aacagacgtt ccctcgcggc cctggcacct ctaaccccag acatgctgct 50
gctgctgctg cccctgctct gggggaggga gagggcggaa ggacagacaa 100
gtaaactgct gacgatgcag agttccgtga cggtgcagga aggcctgtgt 150
gtccatgtgc cctgctcctt ctcctacccc tcgcatggct ggatttaccc 200
tggcccagta gttcatggct actggttccg ggaaggggcc aatacagacc 250
aggatgctcc agtggccaca aacaacccag ctcgggcagt gtgggaggag 300
actcgggacc gattccacct ccttggggac ccacatacca agaattgcac 350
cctgagcatc agagatgcc:a gaagaagtga tgcggggaga tacttctttc 400
gtatggagaa aggaagtata aaatggaatt ataaacatca ccggctctct 450
gtgaatgtga cagccttgac ccacaggccc aacatcctca tcccaggcac 500
cctggagtcc ggctgecccc agaatctgac ctgctctgtg ccctgggcct 550
gtgagcaggg gacaccccct atgatctcct ggatagggac ctccgtgtcc 600
cccctggacc cctccaccac ccgctccteg gtgctcaccc tcatcccaca 650
gccccaggac catggcacca gcctcacctg tcaggtgacc ttccctgggg 700
ccagcgtgac cacgaacaag accgtccatc tcaacgtgtc ctacccgcct 750
cagaacttga ccatgactgt cttccaagga gacggcacag tatccacagt 800
cttgggaaat ggcteatctc tgtcactccc agagggccag tctctgcgcc 850
tggtctgtgc agttgatgca gttgacagca atccccctgc caggctgagc 900
ctgagctgga gaggcctgac cctgtgcccc tcacagccct caaacccggg 950
ggtgctggag ctgcettggg tgcacctgag ggatgcagct gaattcacct 1000
gcagagctca gaaccctctc ggctctcagc aggtctacct gaacgtctcc 1050
ctgcagagca aagccacatc aggagtgact cagggggtgg tcgggggagc 1100
tggagccaca gccctggtct tcctgtcctt ctgcgtcatc ttcgttgtag 1150
tgaggtcctg caggaagaaa tcggcaaggc cagcagcggg cgtgggagat 1200
acgggcatag aggatgcaaa cgctgtcagg ggttcagcct ctcaggggcc 1250
cctgactgaa ccttgggcag aagacagtcc cccagaccag cctcccecag 1300-
cttctgcccg ctcctcagtg ggggaaggag agctccagta tgcatccctc 1350
Page 108
~..W ,.._. . . p~r tz. rv._.,. _ .~.~z. ,~., r..n__. .~_._._ a, . #n_~ .m, .-
_, ~. _ ......~~. . _._~m..~ _. ~,..«._.~..x.._.. _...~_... .._..~~__..~._.
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
agcttccaga tggtgaagcc ttgggactcg cggggacagg aggccactga 1400
caccgagtac tcggagatca agatccacag atgagaaact gcagagactc 1450
accctgattg agggatcaca gcccctccag gcaagggaga agtcagaggc 1500
tgattcttgt agaattaaca gccctcaacg tgatgagcta tgataacact 1550
atgaattatg tgcagagtga aaagcacaca ggctttagag tcaaagtatc 1600
tcaaacctga atccacactg tgccctccct tttatttttt taactaaaag 1650
acagacaaat tccta 1665
<210> 86
<211> 463
<212> PRT
<213> Homo Sapien
<400> 86
Met Leu Leu Leu Leu Leu Pro Leu Leu Trp Gly Arg Glu Arg Ala
1 5 10 15
Glu Gly Gln Thr Ser Lys Leu Leu Thr Met Gln Ser Ser Val Thr
20 z5 30
val Gln Glu Gly Leu Cys Val His Val Pro Cys Ser Phe Ser Tyr
35 40 45
Pro Ser His Gly Trp Ile Tyr Pro Gly Pro Val Val His Gly Tyr
50 55 60
Trp Phe Arg Glu Gly Ala Asn Thr Asp Gln Asp Ala Pro Val Ala
65 70 75
Thr Asn Asn Pro Ala .Arg Ala Val Trp Glu Glu Thr Arg Asp Arg
80 85 90
Phe His Leu Leu Gly ,Asp Pro His Thr Lys Asn Cys Thr Leu Ser
95 100 105
Ile Arg Asp Ala Arg Arg Ser asp Ala Gly Arg Tyr Phe Phe Arg
110 115 120
Met Glu Lys Gly Ser Ile Lys Trp Asn Tyr Lys His His Arg Leu
125 130 135
Ser Val Asn Val Thr Ala Leu Thr His Arg Pro Asn Ile Leu Ile
140 145 150
Pro Gly Thr Leu Glu Ser Gly Cys Pro Gln Asn Leu Thr Cys Ser
155 160 165
Val Pro Trp Ala Cys Glu Gln Gly Thr Pro Pro Met Ile Ser Trp
170 175 180
Ile Gly Thr Ser Val Ser Pro Leu Asp Pro Ser Thr Thr Arg Ser
185 190 195
Ser Val Leu Thr Leu Ile Pro Gln Pro Gln Asp His Gly Thr 5er
2oa 205 210
Leu Thr Cys Gln Val Thr Phe Pro Gly Ala Ser Val Thr Thr Asn
Page 109 w
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
215 220 225
Lys Thr val His Leu Asn val Ser Tyr Pro Pro Gln Asn Leu Thr
230 235 240
Met Thr val Phe Gln Gly Asp Gly Thr val Ser Thr val Leu Gly
245 250 255
Asn Gly Ser Ser Leu Ser Leu Pro Glu Gly Gln Ser Leu Arg Leu
260 265 270
val Cys Ala val Asp Ala val Asp ser Asn Pro Pro Ala Arg Leu
275 280 285
Ser Leu Ser Trp Arg Giy Leu Thr Leu Cys Pro Ser Gln Pro Ser
290 295 300
Asn Pro Gly val Leu Glu Leu Pro Trp Val His Leu Arg Asp Ala
305 310 315
Ala Glu Phe Thr Cys Arg Ala Gln Asn Pro Leu Gly Ser Gln Gln
320 325 330
val Tyr Leu Asn val Ser Leu Gln Ser Lys Ala Thr Ser Gly val
335 340 345
Thr Gln Gly val val Gly Gly Ala Gly Ala Thr Ala Leu val Phe
3S0 355 360
Leu ser Phe Cys val Ile Phe val val val Arg Ser Cys Arg Lys
365 370 375
Lys Ser Ala Arg Pro Ala Ala Gly val Gly Asp Thr Gly I1e Glu
380 385 390
Asp Ala Asn Ala val Arg Gly Ser Ala Ser Gln Gly Pro Leu Thr
395 400 405
Glu Pro Trp Ala Glu Asp Ser Pro Pro Asp Gln Pro Pro Pro Ala
410 415 420
Ser Ala Arg Ser Ser Val Gly Glu Gly Glu Leu Gln Tyr Ala Ser
425 430 435
Leu Ser Phe Gln Met val Lys Pro Trp Asp Ser Arg Gly Gln Glu
440 445 450
Ala Thr ASp Thr G1a 'ryr Ser Glu Ile Lys Ile His Arg
455 460
<210> 87
<211> 1176
<212> DNA
<213> Homo Sapien
<400> 87
agaaagctgc actctgttga gctccagggc gcagtggagg gagggagtga 50
aggagctctc tgtacccaag gaaagtgcag ctgagactca gacaagatta 100
caatgaacca actcagcttc ctgctgtttc tcatagcgac caccagagga 150
tggagtacag atgaggctaa tacttacttc aaggaatgga cctgttcttc 200
Page 110
.,. ._ ». R_ ~ ,~~ ..".~ ..m. ~ ~ ~ ..,,,. , ."t,..__ n~ ~ ,~ ~_.~« ~~~". ~, M
.n.,..~ ae..,~,rp .~",o r _.._ __ ...__ __ ~ ..-._~ r,~~~.,u~~.~..T.~ _~,~ ~
,.~ ~,x~",A~P.~,~
i
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
gtctccatct ctgcccagaa gctgcaagga aatcaaagac gaatgtccta 250
gtgcatttga tggcctgtat tttctccgca ctgagaatgg tgttatctac 300
cagaccttct gtgacatgac ctctgggggt ggcggctgga ccctggtggc 350
cagcgtgcat gagaatgaca tgcgtgggaa gtgcacggtg ggcgatcgct 400
ggtccagtca gcagggcagc aaagcagact acccagaggg ggacggcaac 450
tgggccaact acaacacctt tggatctgca gaggcggcca cgagcgatga 500
ctacaagaac cctggctact acgacatcca ggccaaggac ctgggcatct 550
ggcacgtgcc caataagtcc cccatgcagc actggagaaa cagctccctg 600
ctgaggtacc gcacggacac tggcttcctc cagacactgg gacataatct 650
gtttggcatc taccagaaat atccagtgaa atatggagaa ggaaagtgtt 700
ggactgacaa cggcccggtg atccctgtgg tctatgattt tggcgacgcc 750
cagaaaacag catcttatta ctcaccctat ggccagcggg aattcactgc 800
gggatttgtt cagttcaggg tatttaataa cgagagagca gccaacgcct 850
tgtgtgctgg aatgagggtc accggatgta acactgagca tcactgcatt 900
ggtggaggag gatactttcc agaggccagt ccccagcagt gtggagattt 950
ttctggtttt gattggagtg gatatggaac tcatgttggt tacagcagca 1000
gccgtgagat aactgaggca gctgtgcttc tattctatcg ttgagagttt 1050
tgtgggaggg aacccagacc tctcctccca accatgagat cccaaggatg 1100
gagaacaact tacccagtag ctagaatgtt aatggcagaa gagaaaacaa 1150
taaatcatat tgactcaaga aaaaaa 1176
<210> 88
<211> 313
<212> PRT
<213> Homo Sapien
<400> 88
Met Asn Gln Leu Ser Phe Leu Leu Phe Leu I1e Ala Thr Thr Arg
1 5 10 15
Gly Trp Ser Thr Asp Glu Ala Asn Thr Tyr Phe Lys Glu Trp Thr
20 25 30
Cys Ser Ser Ser Pro Ser Leu Pro Arg Ser Cys Lys Glu Ile Lys
35 40 45
asp Glu Cys Pro Ser Ala Phe Asp Gly Leu Tyr Phe Leu Arg Thr
50 55 60
Glu Asn Gly Val Ile Tyr Gln Thr Phe Cys Asp Met Thr Ser Gly:
65 70 75
Gly G1y Gly Trp Thr L_eu Val Ala Ser Val His Glu Asn Asp Met
Page 11I
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
80 85 90
Arg Gly Lys Cys Thr Val Gly Asp Arg Trp Ser Ser Gln Gln Gly
95 100 105
Ser Lys Ala Asp Tyr Pro Glu Gly Asp Gly Asn Trp Ala Asn Tyr
110 115 120
Asn Thr Phe Gly Ser Ala Glu Ala Ala Thr Ser Asp Asp Tyr Lys
125 130 135
Asn Pro Gly Tyr Tyr Asp Ile Gln Ala Lys Asp Leu G1y Ile Trp
140 145 150
His Val Pro Asn Lys Ser Pro Met Gln His Trp Arg Asn Ser Ser
155 160 165
Leu Leu Arg Tyr Arg Thr Asp Thr Gly Phe Leu Gln Thr ~eu Gly
170 175 180
His Asn Leu Phe Gly Ile Tyr Gln Lys Tyr Pro Val Lys Tyr Gly
185 190 195
Glu Giy Lys Cys Trp Thr Asp Asn Gly Pro Val Ile Pro Val Val
200 205 210
Tyr Asp Phe Gly Asp Ala Gln Lys Thr Ala Ser Tyr Tyr Ser Pro
215 220 225
Tyr Giy Gln Arg Glu Phe Thr Ala Gly Phe Val Gln Phe Arg Val
230 235 240
Phe Asn Asn Glu Arg Ala Ala Asn Ala Leu Cys Ala Gly Met Arg
245 250 255
Val Thr Gly Cys Asn Thr Glu His His Cys Ile Gly Gly Gly Gly
260 265 270
Tyr Phe Pro Glu Ala Ser Pro Gln Gln Cys Gly Asp Phe Ser Gly
275 280 285
Phe Asp Trp Ser Gly Tyr Gly Thr His Val Gly Tyr Ser Ser Ser
290 295 300
Arg Glu Ile Thr Glu Vila Ala Val Leu Leu Phe Tyr Arg
305 310
<210> 89
<211> 759
<212> DNA
<213> Homo Sapien
<400> 89
ctagatttgt cggcttgcgg ggagacttca ggagtcgctg tctctgaact 50
tccagcctca gagaccgccg cccttgtccc cgagggccat gggccgggtc 100
tcagggcttg tgccctctcg cttcctgacg ctcctggcgc atctggtggt 150
cgtcatcacc ttattctggt cccgggacag caacatacag gcctgcctgc 200
ctctcacgtt cacccccgag gagtatgaca agcaggacat tcagctggtg-250
Page 112
,,~ _~:~4,.~..-~~-_: -~.~~--R-
~~:...~~~.."_~..,ae:...~~~~~,~,..E.~...~.e.~..~...~__._.___..____._________.___
__ ___~ ..
;n._ _ _ _ -
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
gccgcgctct ctgtcaccct gggcctcttt gcagtggagc tggccggttt 300
cctctcagga gtctccatgt tcaacagcac ccagagcctc atctccattg 350
gggctcactg tagtgcatcc gtggccctgt ccttcttcat attcgagcgt 400
tgggagtgca ctacgtattg gtacattttt gtcttctgca gtgcccttcc 450
agctgtcact gaaatggctt tattcgtcac cgtctttggg ctgaaaaaga 500
aacccttctg attaccttca tgacgggaac ctaaggacga agcctacagg 5~0
ggcaagggcc gcttcgtatt cctggaagaa ggaaggcata ggcttcggtt 600
ttcccctcgg aaactgcttc tgctggagga tatgtgttgg aataattacg 650
tcttgagtct gggattatcc gcattgtatt tagtgctttg taataaaata 700
tgttttgtag taacattaag acttatatac agttttaggg gacaattaaa 750
aaaaaaaaa 759
<210> 90
<211> 140
<212> PRT
<213> Homo Sapien
<400> 90
Met Gly Arg Val Ser Gly Leu Val Pro Ser Arg Phe Leu Thr Leu
1 5 10 15
Leu Ala His Leu Val Val Val Ile Thr Leu Phe Trp Ser Arg Asp
20 25 30
Ser Asn Ile Gln Ala Cys Leu Pro Leu Thr Phe Thr Pro Glu Glu
35 40 45
Tyr Asp Ly.s Gln Asp Ile Gln Leu Val Ala Ala Leu Ser Val Thr
50 55 60
Leu Gly Leu Phe Ala Val Glu Leu Ala Gly Phe Leu Ser Gly Val
65 70 75
Ser Met Phe Asn Ser Thr Gln Ser Leu Ile Ser Ile Gly Ala His
80 85 90
Cys Ser Ala Ser Val Ala Leu ser Phe Phe Ile Phe Glu Arg Trp
95 100 105
Glu Cys Thr Thr Tyr Trp Tyr Ile Phe Val Phe Cys Ser Ala Leu
110 115 120
Pro Ala Val Thr Glu Met Ala Leu Phe Val Thr Val Phe Gly Leu
125 130 135
Lys Lys Lys Pro Phe
140
<210> 91
<211> 1871
<212> DNA
<2I3> Homo Sapien
Page 113
~~ . ,.. r~, r . ~,h~ . »~~m_ ~~.~.,,~ _ . ~.~" ,n.,~~,w~ ~. u.. ~",. .n__
....
S
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
<400> 91
ctgggacccc gaaaagagaa ggggagagcg aggggacgag agcggaggag 50
gaagatgcaa ctgactcgct gctgcttcgt gttcctggtg cagggtagcc 100
tctatctggt catctgtggc caggatgatg gtcctcccgg ctcagaggac 150
cctgagcgtg atgaccacga gggccagccc cggccccggg tgcctcggaa 200
gcggggccac atctcaccta agtcccgccc catggccaat tccactctcc 250
tagggctgct ggccccgcct ggggaggctt ggggcattct tgggcagccc 300
cccaaccgcc cgaaccacag ccccccaccc tcagccaagg tgaagaaaat 350
ctttggctgg ggcgacttct actccaacat caagacggtg gccctgaacc 400
tgctcgtcac agggaagatt gtggaccatg gcaatgggac cttcagcgtc 450
cacttccaac acaatgccac aggccaggga aacatctcca tcagcctcgt S00
gccccccagt aaagctgtag agttccacca ggaacagcag atcttcatcg 550
aagccaaggc ctccaaaatc ttcaactgcc ggatggagtg ggagaaggta 600
gaacggggcc gccggacctc gctttgcacc cacgacccag ccaagatctg 650
ctcccgagac cacgctcaga gctcagccac ctggagctgc tcccagccct 700
tcaaagtcgt ctgtgtctac atcgccttct acagcacgga ctatcggctg 750
gtccagaagg tgtgcccaga ttacaactac catagtgata ccccctacta 800
cccatctggg tgacccgggg caggccacag aggccaggcc agggctggaa 850
ggacaggcct gcccatgcag gagaccatct ggacaccggg cagggaaggg 900
gttgggcctc aggcagggag gggggtggag acgaggagat gccaagtggg 9S0
gccagggcca agtctcaagt ggcagagaaa gggtcccaag tgctggtccc 1000
aacctgaagc tgtggagtga ctagatcaca ggagcactgg aggaggagtg 1050
ggctctctgt gcagcctcac agggctttgc cacggagcca cagagagatg 1100
ctgggtcccc gaggcctgtg ggcaggccga tcagtgtggc cccagatcaa 1150
gtcatgggag gaagctaagc ccttggttct tgccatcctg aggaaagata 1200
gcaacaggga gggggagatt tcatcagtgt ggacagcctg tcaacttagg 1250
atggatggct gagagggctt cctaggagcc agtcagcagg gtggggtggg 1300
gccagaggag ctctccagcc ctgcctagtg ggcgccctga gccccttgtc 1350
gtgtgctgag catggcatga ggctgaagtg gcaaccctgg ggtctttgat 1400
gtcttgaeag attgaeeatc tgtctecage eaggecaccc cttteeaaaa 1450
ttccctcttc tgccagtac~ ccccctgtac cacccattgc tgatggcaca 1500
cccatcctta agctaagaca ggacgattgt ggtcctccca cactaaggcc 1550
Page 114
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
acagcccatc cgcgtgctgt gtgtccctct tccaccccaa cccctgctgg 1600
ctcctctggg agcatccatg tcccggagag gggtccctca acagtcagcc 1650
tcacctgtca gaccggggtt ctcccggatc tggatggcgc cgccctctca 1700
gcagcgggca cgggtggggc ggggccgggc cgcagagcat gtgctggatc 1750
tgttctgtgt gtctgtctgt gggtgggggg aggggaggga agtcttgtga 1800
aaccgctgat tgctgacttt tgtgtgaaga atcgtgttct tggagcagga 1850
aataaagctt gccccggggc a 1871
<210> 92
<211> 252
<212> PRT
<213> Homo Sapien
<400> 92
Met Gln Leu Thr Arg Cys Cys Phe val Phe Leu val Gln Gly ser
1 S 10 15
Leu Tyr Leu val Ile Cys Gly Gln Asp Asp Gly Pro Pro Gly Ser
20 25 30
Glu Asp Pro Glu Arg Asp Asp His Glu Gly Gln Pro Arg Pro Arg
35 40 45
val Pro Arg Lys Arg Gly His Ile Ser Pro Lys Ser Arg Pro Met
50 55 60
Ala Asn Ser Thr Leu Leu Gly Leu Leu Ala Pro Pro G1y Glu Ala
65 70 75
Trp Gly Ile Leu Gly Gln Pro Pro Asn Arg Pro Asn His Ser Pro
80 85 90
Pro Pro Ser Ala Lys val Lys Lys Ile Phe Gly Trp Gly Asp Phe
95 100 105
Tyr Ser Asn Ile Lys Thr val Ala Leu Asn Leu Leu val Thr Gly
110 115 120
Lys Ile val Asp His Gly Asn Gly Thr Phe Ser val His Phe Gln
125 130 135
His Asn Ala Thr Gly Gln Gly Asn Ile Ser Ile Ser Leu val Pro.
140 145 ~ 150
Pro Ser Lys Ala val Glu Phe His Gln Glu Gln Gln Ile Phe Ile
155 160 165
Glu Ala Lys Ala Ser Lys Ile Phe Asn Cys Arg Met Glu Trp Glu
170 175 180
Lys val Glu Arg Gly Arg Arg Thr Ser Leu Cys Thr His Asp Pro
185 190 195
Ala Lys Ile Cys Ser Arg asp His Ala Gln Ser Ser Ala Thr Trp
200 205 2I0
Ser Cys Ser Gln Pro Phe Lys val val Cys Val Tyr Ile Ala Phe
Page 115
CA 02481756 2004-10-25
PC'f-uS00-23328_Sequence
215 220 225
Tyr Ser Thr Asp Tyr Arg Leu val Gln Lys val Cys Pro Asp Tyr
230 235 240
Asn Tyr His Ser Asp Thr Pro Tyr Tyr Pro Ser Gly
245 250
<210> 93
<211> 902
<212> DNA -
<213> Homo Sapien
<400> 93
cggtggccat gactgcggcc gtgttcttcg gctgcgcctt cattgccttc 50
gggcctgcgc tcgcccttta tgtcttcacc atcgccatcg agccgttgcg 100
tatcatcttc ctcatcgccg gagctttctt ctggttggtg tctctactga 150
tttcgtccct tgtttggttc atggcaagag tcattattga caacaaagat 200
ggaccaacac agaaatatct gctgatcttt ggagcgtttg tctctgtcta 250
tatccaagaa atgttccgat ttgcatatta taaactctta aaaaaagcca 300
gtgaaggttt gaagagtata aacccaggtg agacagcacc ctctatgcga 350
ctgctggcct atgtttctgg cttgggcttt ggaatcatga gtggagtatt 400
ttcctttgtg aataccctat ctgactcctt ggggccaggc acagtgggca 450
ttcatggaga ttctcctcaa ttcttccttt attcagcttt catgacgctg 500
gtcattatct tgctgcatgt attctggggc attgtatttt ttgatggctg 550
tgagaagaaa aagtggggca tcctccttat cgttctcctg acccacctgc 600
tggtgtcagc ccagaccttc ataagttctt attatggaat aaacctggcg 650
tcagcattta taatcctggt gctcatgggc acctgggcat tcttagctgc 700
gggaggcagc tgccgaagcc tgaaactctg cctgctctgc caagacaaga 750
actttcttct ttacaaccag cgctccagat aacctcaggg aaccagcact 800
tcccaaaccg cagactacat ctttagagga agcacaactg tgcctttttc 8S0
tgaaaatccc tttttctggt ggaattgaga aagaaataaa actatgcaga 900
to 902
<210> 94 _
<211> 257
<212> PRT
<213> Homo Sapien
<400> 94 ,
Met Thr Ala Ala Val Phe Phe Gly Cys Ala Phe Ile Ala Phe Gly
1 5 10 15
Pro Ala Leu Ala Leu Tyr Val Phe Thr Ile Ala Ile Glu Pro Leu
20 25 30
Page 116
..- w . . ..~~__.y.
CA 02481756 2004-10-25
PCT-uS00-23328_sequence
Arg Ile Ile Phe Leu Ile Ala Gly Ala Phe Phe Trp Leu Val Ser
35 40 45
Leu Leu Ile Ser Ser Leu val Trp Phe Met Ala Arg Val Ile Ile
50 55 60
Asp Asn Lys Asp Gly Pro Thr Gln Lys Tyr Leu Leu Ile Phe Gly
65 70 75
Ala Phe Val Ser Val Tyr Ile Gln Glu Met Phe Arg Phe Ala '~yr
80 85 90
Tyr Lys Leu Leu Lys Lys Ala Ser Glu Gly Leu Lys Ser Ile Asn
95 100 105
Pro Gly Glu Thr Aia Pro Ser Met Arg Leu Leu Ala Tyr Val Ser
lI0 115 120
Gly Leu Gly Phe Gly Ile Met Ser Gly Val Phe Ser Phe Val Asn
125 130 135
Thr Leu Ser Asp Ser Leu Gly Pro Gly Thr Val Gly Ile His Gly
140 145 150
Asp Ser Pro Gln Phe Phe Leu Tyr Ser Ala Phe Met Thr Leu Val
155 160 165
Ile Ile Leu Leu His Val Phe Trp Gly Iie Val Phe Phe Asp Gly
170 175 180
Cys Glu Ly5 Lys Lys Trp Gly Ile Leu Leu Ile Val Leu Leu Thr
185 190 195
His Leu Leu Val Ser Ala Gln Thr Phe Ile Ser Ser Tyr Tyr Gly
200 205 210
Ile Asn Leu Ala Ser Ala Phe Ile Ile Leu Val Leu Met Gly Thr
215 220 225
Trp Ala Phe Leu Ala Ala Gly Gly Ser Cys Arg Ser Leu Lys Leu
230 235 240
Cys Leu Leu Cys Gln Asp Lys Asn Phe Leu Leu Tyr Asn Gln Arg
245 250 255
Ser Arg
<210> 95
<211> 1073
<212> DNA
<213> Homo Sapien
<400> 95
aatttttcac cagagtaaac ttgagaaacc aactggacct tgagtattgt 50
acattttgcc tcgtggaccc aaaggtagca atctgaaaca tgaggagtac 100
gattctactg ttttgtcttc taggateaac fcggtcatta ccacagctca 150
aacctgcttt gggactccct cccacaaaac tggctccgga tcagggaaca 200
Page 117
:~.,~~.~. :~-..., _
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
ctaccaaacc aacagcagtc aaatcaggtc tttccttctt taagtctgat 250
accattaaca cagatgctca cactggggcc agatctgcat ctgttaaatc 300
ctgctgcagg aatgacacct ggtacccaga cccacccatt gaccetggga 350
gggttgaatg tacaacagca actgcaccca catgtgttac caatttttgt 400
cacacaactt ggagcccagg gcactatcct aagctcagag gaattgccac 450
aaatcttcac gagcctcatc atccattcct tgttcccggg aggcatcctg 500
cccaccagtc aggcaggggc taatccagat gtccaggatg gaagccttcc 550
agcaggagga gcaggtgtaa atcctgccac ccagggaacc ccagcaggcc 600
gcctcccaac tcccagtggc acagatgacg actttgcagt gaccacccct 650
gcaggcatcc aaaggagcac acatgccatc gaggaagcca ccacagaatc 700
agcaaatgga attcagtaag ctgtttcaaa ttttttcaac taagctgcct 750
cgaatttggt gatacatgtg aatctttatc attgattata ttatggaata 800
gattgagaca cattggatag tcttagaaga aattaattct taatttacct 850
gaaaatattc ttgaaatttc agaaaatatg ttctatgtag agaatcccaa 900
cttttaaaaa caataattca atggataaat ctgtctttga aatataacat 950
tatgctgcct ggatgatatg catattaaaa catatttgga aaactggaaa 1000
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1050
aaaaaaaaaa aaaaaaaaaa aaa 1073
<210> 96
<211> 209
<212> PRT
<213> Homo 5apien .
<400> 96
Met Arg Ser Thr Ile Leu Leu Phe Cys Leu Leu Gly Ser Thr Arg
1 S 10 15
Ser Leu Pro Gln Leu t_ys Pro Ala Leu Gly Leu Pro Pro Thr Lys
20 25 30
Leu Ala Pro Asp Gln Gly Thr Leu Pro Asn Gln Gln Gln Ser Asn
35 40 45
Gln Val Phe Pro Ser Leu Ser Leu Ile Pro Leu Thr Gln Met Leu
50 55 60
Thr Leu Gly Pro Asp Leu His Leu Leu Asn Pro Ala Ala Gly Met
65 70 75
Thr Pro Gly Thr Gln Thr His Pro Leu Thr Leu Gly Gly Leu Asn
80 85 gp
Val Gln G1n G1n Leu t-lis Pro His Val Leu Pro Ile Phe Val Thr
95 100 105
Page 118
~~~. ,. .r.
CA 02481756 2004-10-25
PcT-US00-23328_Sequence
Gln Leu Gly Ala Gln Giy Thr Ile Leu Ser Ser Glu Glu Leu Pro
110 115 120
Gln Ile Phe Thr Ser Leu Ile Ile His Ser Leu Phe Pro Gly Giy
125 130 135
Ile Leu Pro Thr Ser Gln Ala Gly Ala Asn Pro Asp Val Gln Asp
140 145 150
Gly Ser Leu Pro Ala Gly Gly Ala Gly Val Asn Pro Ala Thr Gln
155 160 165
Gly Thr Pro Ala Gly Arg Leu Pro Thr Pro Ser Gly Thr Asp Asp
170 175 180
Asp Phe Ala Val Thr Thr Pro Ala Gly Ile Gln Arg Ser Thr His
185 190 195
Ala Ile Glu Glu Ala Thr Thr Glu Ser Ala Asn Gly Ile Gln
200 205
<210> 97
<211> 2848
<212> DNA
<213> Homo sapien
<400> 97
gctcaagtgc cctgccttgc cccacccagc ccagcctggc cagagccccc 50
tggagaagga gctctcttct tgcttggcag ctggaccaag ggagccagtc 100
ttgggcgctg gagggcctgt cctgaccatg gtccctgcct ggctgtggct 150
gctttgtgtc tccgtccccc aggctctccc caaggcccag cctgcagagc 200
tgtctgtgga agttccagaa aactatggtg gaaatttccc tttatacctg 250
accaagttgc cgctgccccg tgagggggct gaaggccaga tcgtgctgtc 300
aggggactca ggcaaggcaa ctgagggccc atttgctatg gatccagatt 350
ctggcttcct gctggtgacc agggccctgg accgagagga gcaggcagag 400
taccagctac aggtcaccct ggagatgcag gatggacatg tcttgtgggg 450
tccacagcct gtgcttgtgc acgtgaagga tgagaatgac caggtgcccc 500
atttctctca agccatctac agagctcggc tgagccgggg taccaggcct 550
ggcatcccct tcctcttcct tgaggcttca gaccgggatg agccaggcac 600
agccaactcg gatcttcgat tccacatcct gagccaggct ccagcccagc 650
cttccccaga catgttccag ctggagcctc ggctgggggc tctggccctc 700
agccccaagg ggagcaccag ccttgaccac gccctggaga ggacctacca 750
gctgttggta caggtcaagg acatgggtga ccaggcctca ggccaccagg 800
ccactgccac cgtggaagtc tccatcatag agagcacctg ggtgtcccta 850
gagcctatcc acctggcaga gaatctcaaa gtcctatacc cgcaccacat 900
Page 119
CA 02481756 2004-10-25
PCT-uS00-23328_5equence
ggcccaggta cactggagtg ggggtgatgt gcactatcac ctggagagcc 950
atcccccggg accctttgaa gtgaatgcag agggaaacct ctacgtgacc 1000
agagagctgg acagagaagc ccaggctgag tacctgctcc aggtgcgggc 1050
tcagaattcc catggcgagg actatgcggc ccctctggag ctgcacgtgc 1100
tggtgatgga tgagaatgac aacgtgccta tctgccctcc ccgtgacccc 1150
acagtcagca tccctgagct cagtccacca ggtactgaag tgactagact 1200
gtcagcagag gatgcagatg cccccggctc ccccaattcc cacgttgtgt 1250
atcagctcct gagccctgag cctgaggatg gggtagaggg gagagccttc 1300
caggtggacc ccacttcagg cagtgtgacg ctgggggtgc tcccactccg 1350
agcaggccag aacatcctgc ttctggtgct ggccatggac ctggcaggcg 1400
cagagggtgg cttcagcagc acgtgtgaag tcgaagtcgc agtcacagat 1450
atcaatgatc acgcccctga gttcatcact tcccagattg ggcctataag 1500
cctccctgag gatgtggagc ccgggactct ggtggccatg ctaacagcca 1550
ttgatgctga cctcgagccc gccttccgcc tcatggattt tgccattgag 1600
aggggagaca cagaagggac ttttggcctg gattgggagc cagactctgg 1650
gcatgttaga ctcagactct gcaagaacct cagttatgag gcagctccaa 1700
gtcatgaggt ggtggtggtg gtgcagagtg tggcgaagct ggtggggcca 1750
ggcccaggcc ctggagccac cgctacggtg actgtgctag tggagagagt 1800
gatgccaccc cccaagttgg accaggagag ctacgaggcc agtgtcccca 1850
tcagtgcccc agccggctct ttcctgctga ccatccagcc ctccgacccc 1900
atcagccgaa ccctcaggtt ctccctagtc aatgactcag agggctggct 1950
ctgcattgag aaattctccg gggaggtgca caccgcccag tccctgcagg 2000
gcgcccagcc tggggacacc tacacggtgc ttgtggaggc ccaggataca 2050
gccctgactc ttgcccctgt gccctcccaa tacctctgca caccccgcca 2100
agaccatggc ttgatcgtga gtggacccag caaggacccc gatctggcca 2150
gtgggcacgg tccctacag<: ttcacccttg gtcccaaccc cacggtgcaa 2200
cgggattggc gcctccagac tctcaatggt tcccatgcct acctcacctt 2250
ggccctgcat tgggtggagc cacgtgaaca cataatcccc gtggtggtca 2300
gccacaatgc ccagatgtgg cagctcctgg ttcgagtgat cgtgtgtcgc 2350
tgcaacgtgg aggggcagtg eatgegcaag gtgggccgca tgaagggcat 2400
gcccacgaag ctgtcggcag tgggcatcet tgtaggcacc ctggtagcaa 2450
taggaatctt cctcatcctc attttcaccc actggaccat gtcaaggaag 2500
Page 120
a_ . ,.,-~"
~r' ., ., " .~ ..,: s~.x. ., x.. .,...r ~., r ,.,.. r _ .n.... r. n ,n"m r.._
r.,-. n.nx.,r3 Kt,N ._(YA.kv:.'u~f~,cz....7H4Faa~srrv*xsa.....~M..,~....s wy
s:...,'F5~,°"'-..".y,FN'F3,h;:".a.~Padd W3:ld~A ...~u .a..,..w
....~r~a.a..m.wn..,e.msm ,v.nvm~, ,vr....:aammn~>a.,.,~...r.........,._-..,
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
aaggacccgg atcaaccagc agacagcgtg cccctgaagg cgactgtctg 2550
aatggcccag gcagctctag ctgggagctt ggcctctggc tccatctgag 2600
tcccctggga gagagcccag cacccaagat ccagcagggg acaggacaga 2650
gtagaagccc ctccatctgc cctggggtgg aggcaccatc accatcacca 2700
ggcatgtctg cagagcctgg acaccaactt tatggactgc ccatgggagt 2750
gctccaaatg tcagggtgtt tgcccaataa taaagcccca gagaactggg 2800
ctgggcccta tgggaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaag 2848
<210> 98
<211> 807
<212> PRT
<213> Homo sapien
<400> 98
Met Va1 Pro Ala Trp Leu Trp Leu Leu Cys Val Ser Val Pro Gln
1 5 10 15
Ala Leu Pro Lys Ala Gln Pro Ala Glu Leu Ser Val Glu Val Pro
20 25 30
Glu Asn Tyr Gly Gly Asn Phe Pro Leu Tyr Leu Thr Lys Leu Pro
35 40 45
Leu Pro Arg Glu Gly Ala Glu Gly Gln Ile Val Leu Ser Gly Asp
50 55 60
Ser Gly Lys Ala Thr Glu Gly Pro Phe Ala Met Asp Pro Asp Ser
65 70 75
Gly Phe Leu Leu Val Thr Arg Ala Leu Asp Arg Glu Glu Gln Ala
80 85 90
Glu Tyr Gln Leu Gln "val Thr Leu Glu Met Gln Asp Gly His Val
95 100 105
Leu Trp Gly Pro Gln Pro Val Leu Val His val Lys Asp Glu Asn
110 115 120
Asp Gln Val Pro His Phe Ser Gln Ala Ile Tyr Arg Ala Arg Leu
125 130 135
Ser Arg Gly Thr Arg Pro Gly Ile Pro Phe Leu Phe Leu Glu Ala
140 145 150
Ser Asp Arg Asp Glu Pro Giy Thr Ala Asn Ser Asp Leu Arg Phe
155 160 - 165
His Ile Leu Ser Gln Ala Pro Ala Gln Pro Ser Pro Asp Met Phe
170 175 180
Gln Leu Glu Pro Arg Leu Gly Ala Leu Ala Leu Ser Pro Lys Gly
185 190 195
Ser Thr Ser Leu Asp His Ala Leu Glu Arg Thr Tyr Gln Leu Leu
200 205 210
Page 121
CA 02481756 2004-10-25
PCT-u500-23328_Se~uence
Val Gln Val Lys Asp Met Gly Asp Gln Ala Ser Gly His Gln Ala
215 220 225
Thr Ala Thr Val Glu Val Ser Ile Ile Glu Ser Thr Trp Val Ser
230 235 ~ 240
Leu Glu Pro Ile His Leu Ala Glu Asn Leu Lys Val Leu Tyr Pro
245 250 255
His His Met Ala Gln Val His Trp Ser Gly Gly Asp Val His Tyr
260 265 X70
His Leu Glu Ser His Pro Pro Gly Pro Phe Glu Val Asn Ala Glu
275 280 285
Gly Asn Leu Tyr Val Thr Arg Giu Leu Asp Arg Glu Ala Gln Ala
290 295 300
Glu Tyr Leu Leu Gln Val Arg Ala Gln Asn Ser Hi5 Gly Glu Asp
305 310 315
Tyr Ala Ala Pro Leu Glu Leu His Val Leu Val Met Asp Glu Asn
320 325 330
Asp Asn Val Pro Ile Cys Pro Pro Arg Asp Pro Thr.Val Ser Ile
335 340 345
Pro Glu Leu Ser Pro Pro Gly Thr Glu Val Thr Arg Leu Ser Ala
350 355 360
Glu Asp Ala Asp Ala Pro Gly Ser Pro Asn Ser His Val Val Tyr
' 365 370 375
Gln Leu Leu Ser Pro Glu Pro Glu Asp Gly Val Glu Gly Arg Ala
380 385 390
Phe Gln Val Asp Pro 'Thr Ser Gly Ser Val Thr Leu Gly Val Leu
395 400 405
Pro Leu Arg Ala Gly Gln Asn Ile Leu Leu Leu Val Leu Ala Met
410 415 420
Asp Leu Ala Gly A1a G1u Gly Gly Phe Ser Ser Thr Cys Glu Val
425 430 435
Glu Val Ala Val Thr Asp Ile Asn Asp His Ala Pro Glu Phe Ile
440 445 450
Thr Ser Gln Ile Gly Pro I1e Ser Leu Pro Glu Asp Val Glu Pro
455 460 465
Gly Thr Leu Val Ala Met Leu Thr Ala Ile Asp Ala Asp Leu Glu
470 475 480
Pro Ala Phe Arg Leu Met Asp Phe Ala Ile Glu Arg Gly Asp Thr
485 490 495
Glu Gly Thr Phe Gly Leu Asp Trp,Glu Pro Asp Ser Gly His Val
500 505 510
Arg Leu Arg Leu cys Lys Asn Leu Ser Tyr Glu Ala Ala Pro Ser
515 520 525
Page 122
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
His Glu val val val val val Gln ser val Ala Lys Leu val Gly
530 535 540
Pro Giy Pro Gly Pro Gly Ala Thr Ala Thr val Thr val Leu val
545 550 ~ 555
Glu Arg Val Met Pro Pro Pro Lys Leu Asp Gln Glu Ser Tyr Glu
560 565 570
Ala Ser val Pro Ile Ser Ala Pro Ala Gly Ser Phe Leu Leu Thr
575 580 585
Ile Gln Pro Ser Asp Pro Ile Ser Arg Thr Leu Arg Phe Ser Leu
590 595 600
val Asn Asp Ser Glu Gly Trp Leu Cys Ile G1a Lys Phe Ser Gly
605 610 615
Glu Val His Thr Ala Gln Ser Leu Gln Gly Ala Gln Pro Gly Asp
620 625 630
Thr Tyr Thr Val Leu Val Glu Ala Gln Asp Thr Ala Leu Thr Leu
635 640 645
Ala Pro Val Pro Ser Gln Tyr Leu Cys Thr Pro Arg Gln Asp His
650 655 660
Gly Leu Ile Val Ser Gly Pro Ser Lys Asp Pro Asp Leu Ala Ser
665 670 675
Gly His Gly Pro Tyr Ser Phe Thr Leu Gly Pro Asn Pro Thr Val
680 685 690
Gln Arg Asp Trp Arg Leu Gln Thr Leu Asn Gly Ser HiS Ala Tyr
695 700 705
Leu Thr Leu Ala Leu His Trp val Glu Pro Arg Glu His Ile I1e
710 715 720
Pro Val Val val Ser His Asn Ala Gln Met Trp Gln Leu Leu Val
725 730 735
Arg val Ile val Cys Arg Cys Asn val Glu Gly Gln Cys Met Arg
740 745 750
Lys Val Gly Arg Met Lys Gly Met Pro Thr Lys Leu Ser Ala Vai
755 760 765
Gly Ile Leu val Gly Thr Leu val Ala Ile G1y Ile Phe Leu Ile
770 775 780
Leu Ile Phe Thr His Trp Thr Met Ser Arg Lys Lys Asp Pro Asp
785 790 795
Gln Pro Ala Asp Ser Val Pro Leu Lys Ala Thr Val
800 805
<210> 99
<211> 2436
<212> DNA
<213> Homo Sapien
<400> 99
Page 123
CA 02481756 2004-10-25
ggctgace t PCT-US00-23328_5equence
g gctacattgc ctggaggaag cctaaggaac ccaggcatcc 50
agctgcccac gcctgagtcc aagattcttc ccaggaacac aaacgtagga 100
gacccacgct cctggaagca ccagccttta tctcttcacc ttcaagtccc 150
ctttetcaag aatcctctgt tctttgcect ctaaagtctt ggtacatcta 200
ggacccaggc atcttgcttt ccagccacaa agagacagat gaagatgcag 250
aaaggaaatg ttctccttat gtttggtcta ctattgcatt tagaagctgc 3Q0
aacaaattcc aatgagacta gcacctctgc caacactgga tccagtgtga 350
tctccagtgg agccagcaca gccaccaact ctgggtccag tgtgacctcc 400
agtggggtca gcacagecac catctcaggg tccagcgtga cctccaatgg 450
ggtcageata gtcaccaact ctgagttcca tacaacctcc agtgggatca 500
gcacagccac caactctgag ttcagcacag cgtccagtgg gatcagcata 550
gccaccaact ctgagtcca.g caeaacctcc agtggggcca gcacagccac 600
caactctgag tccagcacac cctccagtgg ggccagcaca gtcaccaact 650
ctgggtccag tgtgacctcc agtggagcca gcactgccac caactctgag 700
tccagcacag tgtccagtag ggecagcact gccaccaact ctgagtctag 750
cacactctcc agtggggcca gcacagccac caactctgac tccagcacaa 800
ectccagtgg ggctagcaca gccaccaact ctgagtccag cacaacctcc 850
agtggggcca gcacagccac caactetgag tccagcacag tgtccagtag 900
ggccagcact gccaccaact ctgagtecag cacaacctcc agtggggcca 950
gcacagccac caactctgag tccagaacga cctccaatgg ggctggcaca 1000
gccaccaact ctgagtccag cacgacctcc agtggggcca gcacagccac 1050
caactctgac tccagcacag tgtccagtgg ggecagcact gccaccaact 1100
ctgagtccag cacgacetcc agtggggcca gcacagccac caactctgag 1150
tccagcacga cctccagtgg ggctagcaca gccaccaact ctgactccag 1200
cacaacctcc agtggggccg gcacagccac caactctgag tccagcacag 1250
tgtccagtgg gatcagcaca gtcaccaatt etgagtccag cacaccctcc 1300
agtggggcca acacagccac caactctgag tccagtacga cetccagtgg 1350
ggccaacaca gccaccaact ctgagtccag cacagtgtcc agtggggcca 1400
gcactgccac caactctgag tccagcacaa cctecagtgg ggtcagcaca 1450
gccaccaact ctgagtccag cacaacctcc agtggggcta gcacagccac 1500
caactctgac tccagcacaa cctccagtga ggccagcaca gccaecaact 1550
ctgagtctag cacagtgtec agtgggatca gcacagtcac caattctgag 1600
Page 124
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
tccagcacaa cctccagtgg ggccaacaca gccaccaact ctgggtccag 1650
tgtgacctct gcaggctctg gaacagcagc tctgactgga atgcacacaa 1700
cttcccatag tgcatctact gcagtgagtg aggcaaagcc tggtgggtcc 1750
ctggtgccgt gggaaatc~rt cctcatcacc ctggtctcgg ttgtggcggc 1800
cgtggggctc tttgctgggc tcttcttctg tgtgagaaac agcctgtccc 1850
tgagaaacac ctttaacaca gctgtctacc accctcatgg cctcaaccat 1900
ggccttggtc caggccctgg agggaatcat ggagcccccc acaggcccag 1950
gtggagtcct aactggttct ggaggagacc agtatcatcg atagccatgg 2000
agatgagcgg gaggaacagc gggccctgag cagccccgga agcaagtgcc 2050
gcattcttca ggaaggaaga gacctgggca cccaagacct ggtttccttt 2100
cattcatccc aggagacccc tcccagcttt gtttgagatc ctgaaaatct 2150
tgaagaaggt attcctcacc tttcttgcct ttaccagaca ctggaaagag 2200
aatactatat tgctcattta gctaagaaat aaatacatct catctaacac 2250
acacgacaaa gagaagctgt gcttgccccg gggtgggtat ctagctctga 2300
gatgaactca gttataggag aaaacctcca tgctggactc catctggcat 2350
tcaaaatctc cacagtaaaa tccaaagacc tcaaaaaaaa aaaaaaaaaa 2400
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 2436
<210> 100
<211> 596
<212> PRT
<213> Homo Sapien
<400> 100
Met Lys Met Gln Lys Gly Asn Val Leu Leu Met Phe Gly Leu Leu
1 5 10 15
Leu His Leu Glu Ala Ala Thr Asn Ser Asn Glu Thr Ser Thr Ser
20 25 30
Ala Asn Thr Gly Ser Ser val Ile Ser Ser Gly Ala Ser Thr Ala
35 40 45
Thr Asn Ser Gly Ser 5er val Thr Ser Ser Gly val Ser Thr Ala
50 55 60
Thr Ile ser Gly ser Ser val Thr ser Asn Gly val ser zle val
65 70 75
Thr Asn Ser Glu Phe His Thr Thr Ser Ser Gly Ile Ser Thr Ala
80 85 90
Thr Asn Ser-Glu Phe Ser Thr Ala Ser Ser Gly Ile Ser Ile Ala
95 100 105
Thr Asn Ser Glu Ser Ser Thr Thr Ser Ser Gly Ala Ser Thr Ala
Page 125
CA 02481756 2004-10-25
PCT-u500-23328_sequence
110 115 120
ThrAsn SerGluSer SerThrPro SerSerGly Ser ThrVal
Ala
125 130 135
ThrAsn SerGlySer SerValThr SerSerGly Ser ThrAla
Ala
240 145 150
ThrAsn SerGluSer SerThrVal SerSerArg Ser ThrAla
Ala
155 160 165
ThrAsn SerGluSer SerThrLeu SerSerGly Ser ThrAla
Ala
170 175 180
ThrAsn SerAspSer SerThrThr SerSerGly Ser ThrAla
Ala
185 190 195
ThrAsn SerGluSer SerThrThr SerSerGly Ser ThrAla
Ala
200 205 210
ThrAsn SerGluSer SerThrVal SerSerArg Ser ThrAla
Ala
215 220 225
ThrAsn SerGluSer SerThrThr SerSerGly Ser ThrAla
Ala
230 235 240
ThrAsn SerGluSer ArgThrThr SerAsnGly Gly ThrAla
Ala
245 250 255
ThrAsn SerGluSer SerThrThr SerSerGly Ser ThrAla
Ala
260 265 270
ThrAsn SerAspSer SerThrVa1 SerSerGly Ser ThrAla
Ala
275 280 285
ThrAsn SerGluSer SerThrThr SerSerGly Ser ThrAla
Ala
290 295 300
ThrAsn SerGluSer SerThrThr SerSerGly Ser ThrAla
Ala
305 310 315
ThrAsn SerAspSer SerThrThr SerSerGly Gly ThrAla
Ala
320 325 330
ThrAsn SerGluSer SerThrVal SerSerGly Ser ThrVal
Ile
335 340 345
ThrAsn SerGluSer SerThrPro SerSerGly Asn ThrAla
Ala
350 355 360
ThrAsn SerGluSer SerThrThr SerSerGly Asn ThrAla
Ala
365 370 375
ThrAsn SerGluSer SerThrVal SerSerGly Ser ThrAla
Ala
380 385 390
ThrAsn serGluSer SerThrThr serSerGly Ser ThrAla
val
395 400 405
ThrAsn SerGluSer SerThrThr SerSerGly Ser ThrAla
Ala
410 415 420
ThrAsn SerAspSer SerThrThr SerSerGlu Ser ThrAla
Ala
Page
126
CA 02481756 2004-10-25
PC'r-US00-23328_Sequence
425 430 435
Thr Asn Ser Glu Ser Ser Thr val Ser Ser Gly Ile Ser Thr val
440 445 450
Thr Asn Ser Glu Ser Ser Thr Thr Ser Ser Gly Ala Asn Thr Ala
455 460 465
Thr Asn Ser Gly Ser ser val Thr ser Ala Gly ser Gly Thr Ala
470 475 480
Ala Leu Thr Gly Met His Thr Thr Ser His Ser Ala ser Thr Ala
485 490 495
Val Ser Glu Ala Lys Pro Gly Gly Ser Leu val Pro Trp Glu Ile
500 505 510
Phe Leu Ile Thr Leu val ser val val Ala Ala val Gly Leu Phe
515 520 525
Ala Gly Leu Phe Phe cys Va7 Arg Asn Ser Leu Ser Leu Arg Asn
530 535 540
Thr Phe Asn Thr Ala vai Tyr His Pro His Gly Leu Asn His Gly
545 550 555
Leu Gly Pro Gly Pro Gly Gly Asn~His Gly Ala Pro His Arg Pro
560 565 570
Arg Trp Ser Pro Asn Trp Phe Trp Arg Arg Pro val ser Ser Ile
575 580 585
Ala Met Glu Met Ser Gly Arg Asn Ser Gly Pro
590 595
<210> 101
<211> 1728
<212> DNA
<213> Homo Sapien
<400> 101
ggccggacgc ctccgcgtta cgggatgaat taacggcggg ttccgcacgg 50
aggttgtgac ccctacggag ccccagcttg cccacgcacc ccactcggcg 100
tcgcgcggcg tgccctgctt gtcacaggtg ggaggctgga actatcaggc 150
tgaaaaacag agtgggtact ctcttctggg aagctggcaa caaatggatg 200
atgtgatata tgcattccag gggaagggaa attgtggtgc ttctgaaccc 250
atggtcaatt aacgaggcag tttctagcta ctgcacgtac ttcataaagc 300
aggactctaa aagctttgga atcatggtgt catggaaagg gatttacttt 350
atactgactc tgttttgggg aagctttttt ggaagcattt tcatgctgag 400
tcccttttta cctttgatgt ttgtaaaccc atcttggtat cgctggatca 450
acaaccgcct tgtggcaaca tggcteaccc tacctgtggc attattggag 500 -
accatgtttg gtgtaaaagt gattataact ggggatgcat ttgttcctgg 550
Page 127
.., .. ~r .r",., u.,ax a . ,._ <.., . , w , _. ,, ~,:, . ,. , ...:..ran,.
,~,~sa~~,.:.,.~ xirrr... ...:<.,~, ," " .~ .........,.._
i
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
agaaagaagt gtcattatca tgaaccatcg gacaagaatg gactggatgt 600
tcctgtggaa ttgcctgatg cgatatagct acctcagatt ggagaaaatt 650
tgcctcaaag cgagtctcaa aggtgttcct ggatttggtt gggccatgca 700
ggctgctgcc tatatcttca ttcataggaa atggaaggat gacaagagcc 750
atttcgaaga catgattgat tacttttgtg atattcacga accacttcaa 800
ctcctcatat tcccagaagg gactgatctc acagaaaaca gcaagtctcg 850
aagtaatgca tttgctgaaa aaaatggact tcagaaatat gaatatgttt 900
tacatccaag aactacaggc tttacttttg tggtagaccg tctaagagaa 950
ggtaagaacc ttgatgctgt ccatgatatc actgtggcgt atcctcacaa 1000
cattcctcaa tcagagaagc acctcctcca aggagacttt cccagggaaa 1050
tccactttca cgtccaccgg tatccaatag acaccctccc cacatccaag 1100
gaggaccttc aactctggtg ccacaaacgg tgggaagaga aagaagagag 1150
gctgcgttcc ttctatcaag gggagaagaa tttttatttt accggacaga 1200
gtgtcattcc accttgcaag tctgaactca gggtccttgt ggtcaaattg 1250
ctctctatac tgtattggac ectgttcagc cctgcaatgt gcctactcat 1300
atatttgtac agtcttgtta agtggtattt tataatcacc attgtaatct 1350
ttgtgctgca agagagaata tttggtggac tggagatcat agaacttgca 1400
tgttaccgac ttttacacaa acagccacat ttaaattcaa agaaaaatga 1450
gtaagattat aaggtttgcc atgtgaaaac ctagagcata ttttggaaat 1500
gttctaaacc tttctaagct cagatgcatt tttgcatgac tatgtcgaat 1550
atttcttact gccatcatta tttgttaaag atattttgca ettaattttg 1600
tgggaaaaat attgctacaa ttttttttaa tctctgaatg taatttcgat 1650
actgtgtaca tagcagggag tgatcggggt gaaataactt gggccagaat 1700
attattaaac aatcatcagg cttttaaa 1728
<210> 102
<211> 414
<212> PRT
<213> Homo Sapien
<400> 102
Met His Ser Arg Gly Arg Glu Ile Val Val Leu Leu Asn Pro Trp
1 5 10 15
Ser Ile Asn Glu Ala Val Ser Ser Tyr Cys Thr Tyr Phe Ile Lys
20 25 30
Gln Asp Ser Lys Ser Phe Gly Ile Met Val Ser Trp Lys Gly Ile
35 40 45
Page 128
.~, j :~-,w.
_,:xc , ,ry~wnu: a. r..f.~.ra~~~ M.w.:...v .__w..._._._,..._. ..
~.~,rt..SNN,,. ,..,."Hrr
.>.w;~,.,~>mr~,~.,.c~:.t<~~:,a,~:ASttt~~.~~~x....";~,.~.zmsrr~ ~-
.~.n~~.ea,um.,~ ~...,..~",.,a",."....~."".,...~,"~.m,~ .
.........,.~...,~...".",~,.....-A,v..".......,..
CA 02481756 2004-10-25
PCT-u500-23328_seguence
Tyr Phe Ile Leu Thr Leu Phe Trp Gly Ser Phe Phe Gly Ser Iie
50 55 60
Phe Met Leu Ser Pro Phe Leu Pro Leu Met Phe Val Asn Pro Ser
65 70 75
Trp Tyr Arg Trp Ile Asn ASn Arg Leu Val Ala Thr Trp Leu Thr
80 85 90
Leu Pro Val Ala Leu Leu Glu Thr Met Phe Gly Val Lys Val Ile
95 100 IO5
Ile Thr Gly Asp Aia Phe Val Pro G1y Glu Arg Ser Val Ile Ile
110 115 120
Met Asn His Arg Thr Arg Met Asp Trp Met Phe Leu Trp Asn Cys
125 130 135
Leu Met Arg Tyr Ser Tyr Leu Arg Leu Glu Lys Ile Cys Leu Lys
140 145 150
Ala Ser Leu Lys Gly Val Pro Gly Phe Gly Trp Ala Met Gln Ala
155 160 165
Ala Ala Tyr ile Phe Ile His Arg Lys Trp Lys Asp Asp Lys Ser
170 175 180
His Phe Glu Asp Met Ile Asp Tyr Phe Cys Asp Ile His G1u Pro
185 190 195
Leu Gln Leu Leu Ile Phe Pro Glu Gly Thr Asp Leu Thr Glu Asn
200 205 210
Ser Lys Ser Arg Ser Asn Ala Phe Ala Glu Lys Asn Gly Leu Gln
215 220 225
Lys Tyr Glu Tyr Val Leu His Pro Arg Thr Thr Gly Phe Thr Phe
230 235 240
Va1 Va1 Asp Arg Leu Arg Glu Gly Lys Asn Leu Asp A1a Val His
245 250 255
Asp Ile Thr Val Ala Tyr Pro His Asn Ile Pro Gln Ser Glu Lys
260 265 270
His Leu Leu Gln Gly Asp Phe Pro Arg Glu Ile His Phe His Val
275 280 285
His Arg Tyr Pro Ile Asp Thr Leu Pro Thr Ser Lys Glu Asp Leu
290 295 300
Gln Leu Trp Cys His Lys Arg Trp Glu Glu Lys Glu Glu Arg Leu
305 310 315
Arg Ser Phe Tyr Gln Gly Glu Lys Asn Phe Tyr Phe Thr Gly Gln
320 325 330
Ser Vai Ile Pro pro Cys Lys Ser Glu Leu Arg Val Leu Val Val
335 340 345
Lys Leu Leu Ser Ile Leu Tyr Trp Thr Leu Phe Ser Pro Ala Met
350 355 360
Page 129
CA 02481756 2004-10-25
PCT-0500-23328_5equence
Cys Leu Leu Ile Tyr Leu Tyr Ser Leu Val Lys Trp Tyr Phe Ile
365 370 375
Ile Thr Ile val Ile Phe vai Leu Gln Glu Arg Ile Phe Gly Gly
380 385 390
Leu Glu Ile Iie Glu Leu Ala Cys Tyr Arg Leu Leu His Lys Gln
395 400 405
Pro His Leu Asn Ser Lys Lys Asn Glu
410 -
<210> 103
<211> 2403
<212> DNA
<213> Homo sapien
<400> 103
cggctcgagc ggctcgagtg aagagcctct ccacggctcc tgcgcctgag 50
acagctggcc tgacctccaa atcatccatc cacccctgct gtcatctgtt 100
ttcatagtgt gagatcaacc cacaggaata tccatggctt ttgtgctcat 150
tttggttctc agtttctacg agctggtgtc aggacagtgg caagtcactg 200
gaccgggcaa gtttgtccag gccttggtgg gggaggacgc cgtgttctcc 250
tgctccctct ttcctgagac cagtgcagag gctatggaag tgcggttctt 300
caggaatcag ttccatgctg tggtccacct ctacagagat ggggaagact 350
gggaatctaa gcagatgcca cagtatcgag ggagaactga gtttgtgaag 400
gactccattg caggggggcg tgtctctcta aggctaaaaa acatcactcc 450
ctcggacatc ggcctgtatg ggtgctggtt cagttcccag atttacgatg 500
aggaggccac ctgggagctg cgggtggcag cactgggctc acttcctctc 550
atttccatcg tgggatatgt tgacggaggt atccagttac tctgcctgtc 600
ctcaggctgg ttcccccagc ccacagccaa gtggaaaggt ceacaaggac 650
aggatttgtc ttcagactcc agagcaaatg cagatgggta cagcctgtat 700
gatgtggaga tctccattat agtccaggaa aatgctggga gcatattgtg 750
ttccatccac cttgctgagc agagtcatga ggtggaatcc aaggtattga 800
taggagagac gtttttccag ccctcacctt ggcgcctggc ttctatttta 850
ctcgggttac tctgtggtgc cctgtgtggt gttgtcatgg ggatgataat 900
tgttttcttc aaatccaaag ggaaaatcca ggcggaactg gactggagaa 950
gaaagcacgg acaggcagaa ttgagagacg cccggaaaca cgcagtggag 1000
gtgactctgg atccagagac ggctcacccg aagctctgcg tttctgatct 1050
gaaaactgta acccatagaa aagctcccca ggaggtgcct cactctgaga 1100
agagatttac aaggaagagt gtggtggctt ctcagggttt ccaagcaggg 1150
Page 130
n. ".. . _. z...,.,.. > .. .,x... <.or~,.r. r ... ,scw.,5.
r~m..,.>..SA'"''~»'~'~~=Y'~,..,.°rrv..w,.m«ro,~.:~ ~.n.EZ~.~gy~a..Tr~
~.,a~.a..,...~s .~-.-~....,~.,m,=.,. r.a"..":a....a~~s- ......o.~"~,.---~--,---
-__,~_--_-
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
agacattact gggaggtgga cgtgggacaa aatgtagggt ggtatgtggg 1200
agtgtgtcgg gatgacgtag acagggggaa gaacaatgtg actttgtctc 1250
ccaacaatgg gtattgggtc ctcagactga caacagaaca tttgtatttc 1300
acattcaatc cccattttat cagcctcccc cccagcaccc ctcctacacg 1350
agtaggggtc ttcctggact atgagggtgg gaccatctcc ttcttcaata 1400
caaatgacca gtcccttatt tataccctgc tgacatgtca gtttgaaggc 1450
ttgttgagac cctatatcca gcatgcgatg tatgacgagg aaaaggggac 1500
tcccatattc atatgtccag tgtcctgggg atgagacaga gaagaccctg 1550
cttaaagggc cccacaccac agacccagac acagccaagg gagagtgctc 1600
ccgacaggtg gccccagctt cctctccgga gcctgcgcac agagagtcac 1650
gccccccact ctcctttagg gagctgaggt tcttctgccc tgagccctgc 1700
agcagcggca gtcacagctt ccagatgagg ggggattggc ctgaccctgt 1750
gggagtcaga agccatggct gccctgaagt ggggacggaa tagactcaca 1800
ttaggtttag tttgtgaaaa ctccatccag ctaagcgatc ttgaacaagt 1850
cacaacctcc caggctcctc atttgctagt cacggacagt gattcctgcc 1900
tcacaggtga agattaaaga gacaacgaat gtgaatcatg cttgcaggtt 1950
tgagggcaca gtgtttgcta atgatgtgtt tttatattat acattttccc 2000
accataaact ctgtttgctt attccacatt aatttacttt tctctatacc 2050
aaatcaccca tggaatagtt attgaacacc tgctttgtga ggctcaaaga 2100
ataaagagga ggtaggattt ttcactgatt ctataagccc agcattacct 2150
gataccaaaa ccaggcaaag aaaacagaag aagaggaagg aaaactacag 2200
gtccatatcc ctcattaaca cagacacaaa aattctaaat aaaattttaa 2250
caaattaaac taaacaatat atttaaagat gatatataac tactcagtgt 2300
ggtttgtccc acaaatgcag agttggttta atatttaaat atcaaccagt 2350
gtaattcagc acattaataa agtaaaaaag aaaaccataa aaaaaaaaaa 2400
aaa 2403
<210> 104
<211> 466
<212> PRT
<213> Homo Sapien
<400> 104
Met Ala Phe Val Leu I1a Leu Val Leu Ser Phe Tyr G1a Leu Val
1 5 10 15
Ser Gly Gln Trp Gln Val Thr Gly Pro Gly Lys Phe Val Gln Ala
Page 131
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
20 25 30
Leu Val Gly Glu Asp Ala Val Phe Ser Cys Ser Leu Phe Pro Glu
35 40 45
Thr ser Ala Glu Ala Met Glu val Arg Phe Phe Arg ASn Gln Phe
50 55 60
His Ala val val His Leu Tyr Arg Asp Gly Glu Asp Trp Glu Ser
65 70 75
Lys Gln Met Pro Gln Tyr Arg Gly Arg Thr Glu Phe Val Lys Asp
80 85 90
Ser Ile Ala Gly Gly ,a,rg val Ser Leu Arg Leu Lys Asn Ile Thr
95 100 105
Pro Ser Asp Ile Gly Leu Tyr Gly Cys Trp Phe Ser Ser Gln Ile
ll0 115 120
Tyr Asp Glu Glu Ala Thr Trp Glu Leu Arg Val Ala Ala Leu Gly
125 130 135
Ser Leu Pro Leu Ile Ser Ile Val Gly Tyr Val Asp Gly Gly Ile
140 145 150
Gln Leu Leu Cys Leu Ser Ser Gly Trp Phe Pro Gln Pro Thr Ala
155 160 165
Lys Trp Lys Gly Pro Gln Gly Gln Asp Leu Ser Ser Asp Ser Arg
170 175 180
Ala Asn Ala Asp Gly Tyr Ser Leu Tyr Asp val Glu Ile Ser Ile
185 190 195
Ile Val Gln Glu Asn Ala Gly Ser Ile Leu Cys Ser Ile His Leu
200 205 210
Ala Glu Gln Ser His Glu val Glu Ser Lys val Leu Ile Gly Glu
215 220 225
Thr Phe Phe Gln Pro Ser Pro Trp Arg Leu Ala Ser Ile Leu Leu
230 235 240
Gly Leu Leu Cys Gly Ala Leu Cys Gly Val Val Met Gly Met Ile
245 250 255
Ile Val Phe Phe Lys Ser Lys Gly Lys Ile Gln Ala Glu Leu Asp
260 265 270
Trp Arg Arg Lys His ~Gly Gln Ala Glu Leu Arg Asp Ala Arg Lys
275 280 285
His Ala val Glu val Thr Leu ASp Pro Glu Thr Ala His Pro Lys
290 295 300
Leu Cys Val Ser Asp Leu Lys Thr Val Thr His Arg Lys Ala Pro
305 310 315
Gln. Glu Vai Pro His Ser Glu Lys Arg Phe Thr Arg Lys Ser Val
320 325 330
Val Ala Ser Gln Gly Phe Gln Ala Gly Arg His Tyr Trp Glu Val
Page 132
_ _ _ ,.. _ .w . . .. ... v, .,_ ~ _..., r _._ _ . . .. _~
.,~"~~~""~~,..~.s.~s=. . -.,~~~..- -.P._._... , ___. _. ...... __ .. _~~ ..
~.._,._."."".~".,r..~~~..h"-a,~,~ :a~. ~,,~~.
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
335 340 345
Asp Val Gly Gln Asn Val Gly Trp Tyr Val Gly Val Cys Arg Asp
350 355 360
Asp Val Asp Arg Gly Lys Asn Asn Val Thr Leu Ser Pro Asn Asn
365 370 375
Gly Tyr Trp Val Leu Arg Leu Thr Thr Glu His Leu Tyr Phe Thr
380 385 390
Phe Asn Pro His Phe Ile Ser Leu Pro Pro Ser Thr Pro Pro Thr
395 400 405
Arg Val Gly Val Phe Leu Asp Tyr Glu Gly Gly Thr Ile Ser Phe
410 4I5 420
Phe Asn Thr Asn Asp Gln Ser Leu Ile Tyr Thr Leu Leu Thr Cys
425 430 435
Gln Phe Glu Gly Leu Leu Arg Pro Tyr Ile Gln His Ala Met Tyr
440 445 450
Asp Glu Glu Lys Gly Thr Pro Ile Phe Ile Cys Pro Val Ser Trp
455 460 465
Gly
<210> 105
<211> 2103
<212> DNA
<213> Homo Sapien
<400> 105
ccttcacagg actcttcatt gctggttggc aatgatgtat cggccagatg 50
tggtgagggc taggaaaaga gtttgttggg aaccctgggt tatcggcctc 100
gtcatcttca tatccctgat tgtcctggca gtgtgcattg gactcactgt 150
tcattatgtg agatataatc aaaagaagac ctacaattac tatagcacat 200
tgtcatttac aactgacaaa ctatatgctg agtttggcag agaggcttct 250
aacaatttta cagaaatgag ccagagactt gaatcaatgg tgaaaaatgc 300
attttataaa tctccattaa gggaagaatt tgtcaagtct caggttatca 350
agttcagtca acagaagcat ggagtgttgg ctcatatgct gttgatttgt 400
agatttcact ctactgagga tcctgaaact gtagataaaa ttgttcaact 450
tgttttacat gaaaagctgc aagatgctgt aggaccccct aaagtagatc 500
ctcactcagt taaaattaaa aaaatcaaca agacagaaac agacagctat 550
ctaaaccatt gctgcggaac acgaagaagt aaaactctag gtcagagtct 600
caggatcgtt ggtgggacag aagtagaaga gggtgaatgg ccctggcagg 65'0
ctagcctgca gtgggatggg agtcatcgct gtggagcaac cttaattaat 700
Page 133
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
gceacatggc ttgtgagtgc tgeteactgt tttacaaeat ataagaacec 750
tgecagatgg actgcttect ttggagtaae aataaaaect tcgaaaatga 800
aacggggtct eeggagaata attgteeatg aaaaataeaa aeacccatca 850
catgaetatg atatttetct tgeagagett tetagccetg ttecetacac 900
aaatgcagta eatagagttt gtetcectga tgeatcctat gagtttcaac 950
caggtgatgt gatgtttgtg acaggatttg gagcactgaa aaatgatggt 1000
tacagtcaaa atcatctteg acaagcacag gtgactctca tagacgctac 1050
aaettgcaat gaaecteaag ettaeaatga egeeataaet cetagaatgt 1100
tatgtgctgg etcettagaa ggaaaaacag atgeatgcca gggtgactct 1150
ggaggaccac tggttagttc agatgctaga gatatctggt accttgctgg 1200
aatagtgagc tggggagatg aatgtgcgaa acccaacaag cctggtgttt 1250
ataetagagt taeggecttg egggaetgga ttactteaaa aaetggtate 1300
taagagacaa aagcctcatg gaacagataa catttttttt tgttttttgg 1350
gtgtggaggc catttttaga gatacagaat tggagaagac ttgcaaaaca 1400
gctagatttg actgatctea ataaactgtt tgcttgatgc atgtattttc 1450
tteccagctc tgttccgcac gtaagcatec tgcttctgce agatcaacte 1500 -
tgteatctgt gagcaatagt tgaaacttta tgtacataga gaaatagata 1550
ataeaatatt aeattaeage etgtattcat ttgtteteta gaagttttgt 1600
cagaattttg acttgttgac ataaatttgt aatgcatata tacaatttga 1650
agcaetcctt ttetteagtt eetcagetcc tetcatttca gcaaatatec 1700
attttcaagg tgcagaacaa ggagtgaaag aaaatataag aagaaaaaaa 1750
tcccctacat tttattggca cagaaaagta ttaggtgttt ttcttagtgg 1800
aatattagaa atgatcatat tcattatgaa aggtcaagca aagacagcag 1850
aataecaate aetteatcat ttaggaagta tgggaaetaa gttaaggaag 1900
tccagaaaga agceaagata tatcettatt ttcatttcca aacaactact 1950
atgataaatg tgaagaagat tctgtttttt tgtgacctat aataattata 2000
eaaaetteat geaatgtaet tgttetaage aaattaaage aaatatttat 2050
ttaacattgt taetgaggat gteaacatat aacaataaa~ tataaatcae 2100
eea 2103
<210> 106
<211> 423
<212> PRT
<213> Homo Sapien
Page 134
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
<400> 106
Met Met Tyr Arg Pro asp Val Val Arg Ala Arg Lys Arg Val Cys
1 5 10 15
Trp Glu Pro Trp V210 Ile Gly Leu Val I25 Phe Ile Ser Leu I30
Val Leu Ala val Cys Ile Gly Leu Thr Val His Tyr Val Arg Tyr
35 40 45
Asn Gln Lys Lys Thr Tyr Asn Tyr Tyr Ser Thr Leu Ser Phe Thr
50 55 60
Thr Asp Lys Leu Tyr Ala Glu Phe Gly Arg Glu Ala Ser Asn Asn
65 70 75
Phe Thr Glu Met Ser Gln Arg Leu Glu Ser Met Val Lys Asn Ala
80 85 90
Phe Tyr Lys Ser Pro Leu Arg Glu Glu Phe Val Lys Ser Gln Val
95 100 105
Ile Lys Phe Ser Gln Gln Lys His Gly Val Leu Ala His Met Leu
110 115 120
Leu Ile Cys Arg Phe His Ser Thr Glu Asp Pro Glu Thr Val Asp
125 130 135
Lys Ile Val Gln Leu val Leu His Glu Lys Leu Gln Asp Ala Val
140 145 150
Gly Pro Pro Lys Val Asp Pro His Ser Val Lys Ile Lys Lys Ile
155 160 165
Asn Lys Thr Glu Thr Asp Ser Tyr Leu Asn His Cys Cys Gly Thr
170 175 180
arg Arg Ser Lys.Thr Leu Gly Gin Ser Leu Arg Ile val Gly Gly
185 190 195
Thr Glu~val Glu Glu Gly Glu Trp Pro Trp Gln Ala Ser Leu Gln
200 205 210
Trp Asp Gly Ser His Arg Cys Gly Ala Thr Leu Ile Asn Ala Thr
215 220 225
Trp Leu Val Ser Ala Ala His Cys Phe Thr Thr Tyr Lys Asn Pro
230 235 240
Ala Arg Trp Thr Ala Ser Phe Gly Val Thr Ile Lys Pro Ser Lys
245 250 255
Met Lys Arg Gly Leu Arg Arg Ile Ile Val His Glu Lys Tyr Lys
260 265 270
His Pro Ser His Asp Tyr Asp Ile Ser Leu Ala Glu Leu Ser Ser
275 280 285
Pro Val Pro Tyr Thr Asn Ala Val His Arg Val Cys Leu Pro Asp
2g~ 295 300
Ala Ser Tyr Glu Phe Gln Pro Gly Asp Val Met Phe Val Thr Gly
305 310 315
Page 135
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
Phe Gly Ala Leu Lys Asn Asp Gly Tyr Ser Gln Asn His Leu Arg
320 325 330
Gln Ala Gln Val Thr Leu Ile Asp Ala Thr Thr Cys Asn Glu Pro
335 340 345
Gln Ala Tyr Asn Asp Ala Ile Thr Pro Arg Met Leu Cys Ala Gly
350 355 360
Ser Leu Glu Gly Lys Thr Asp Ala Cys Gln Gly Asp Ser Gly Gly
365 370 375
Pro Leu Vai Ser Ser Asp Ala Arg Asp Ile Trp Tyr Leu Ala Gly
380 385 390
Ile val Ser Trp Gly Asp Glu Cys Ala Lys Pro Asn Lys Pro Gly
395 400 405
Val Tyr Thr Arg Val Thr Ala Leu Arg Asp Trp Ile Thr Ser Lys
410 415 420
Thr Gly Ile
<210> 107
<211> 2397
<212> DNA
<213> Homo Sapien
<400> 107
agagaaagaa gcgtctccag ctgaagccaa tgcagccctc cggctctccg 50
cgaagaagtt ccctgccccg atgagccccc gccgtgcgtc cccgactatc 100
cccaggcggg cgtggggcac cgggcccagc gccgacgatc gctgccgttt 150
tgcccttggg agtaggatgt ggtgaaagga tggggcttct cccttacggg 200
gctcacaatg gccagagaag attccgtgaa gtgtctgcgc tgcctgctct 250
acgccctcaa tctgctcttt tggttaatgt ccatcagtgt gttggcagtt 300
tctgcttgga tgagggacta cctaaataat gttctcactt taactgcaga 350
aacgagggta gaggaagcag tcattttgac ttactttcct gtggttcatc 400
cggtcatgat tgctgtttgc tgtttcctta tcattgtggg gatgttagga 450
tattgtggaa cggtgaaaag aaatctgttg cttcttgcat ggtactttgg 500
aagtttgctt gtcattttct gtgtagaact ggcttgtggc gtttggacat 550
atgaacagga acttatggtt ccagtacaat ggtcagatat ggtcactttg 600
aaagccagga tgacaaatta tggattacct agatatcggt ggcttactca 650
tgcttggaat ttttttcaga gagagtttaa gtgctgtgga gtagtatatt 700
tcactgactg gttggaaatg acagagatgg actggccccc agattcctgc 750
tgtgttagag aattcccagg atgttccaaa caggcccacc aggaagatct 800
Page 136
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
cagtgacctt tatcaagagg gttgtgggaa gaaaatgtat tcctttttga 850
gaggaaccaa acaactgcag gtgctgaggt ttctgggaat ctccattggg 900
gtgacacaaa tcctggccat gattctcacc attactctgc tctgggctct 950
gtattatgat agaagggagc ctgggacaga ccaaatgatg tccttgaaga 1000
atgacaactc tcagcacctg tcatgtccct cagtagaact gttgaaacca 1050
agcctgtcaa gaatctttga acacacatcc atggcaaaca gctttaatac 1200
acactttgag atggaggagt tataaaaaga aatgtcacag aagaaaacca 1150
caaacttgtt ttattggact tgtgaatttt tgagtacata ctatgtgttt 1200
cagaaatatg tagaaataaa aatgttgcca taaaataaca cctaagcata 1250
tactattcta tgctttaaaa tgaggatgga aaagtttcat gtcataagtc 1300
accacctgga caataattga tgcccttaaa atgctgaaga cagatgtcat 1350
acccactgtg tagcctgtgt atgactttta ctgaacacag ttatgttttg 1400
aggcagcatg gtttgattag catttccgca tccatgcaaa cgagtcacat 1450
atggtgggac tggagccata gtaaaggttg atttacttct accaactagt 1500
atataaagta ctaattaaat gctaacatag gaagttagaa aatactaata 1550
acttttatta ctcagcgatc tattcttctg atgctaaata aattatatat 1600
cagaaaactt tcaatattgg tgactaccta aatgtgattt ttgctggtta 1650
ctaaaatatt cttaccactt aaaagagcaa gctaacacat tgtcttaagc 1700
tgatcaggga ttttttgtat ataagtctgt gttaaatctg tataattcag 1750
tcgatttcag ttctgataat gttaagaata accattatga aaaggaaaat 1800
ttgtcctgta tagcatcatt atttttagcc tttcctgtta ataaagcttt 1850
actattctgt cctgggctta tattacacat ataactgtta tttaaatact 1900
taaccactaa ttttgaaaat taccagtgtg atacatagga atcattattc 1950
agaatgtagt ctggtcttta ggaagtatta ataagaaaat ttgcacataa 2000
cttagttgat tcagaaagga cttgtatgct gtttttctcc caaatgaaga 2050
ctctttttga cactaaacac tttttaaaaa gcttatcttt gccttctcca 2100
aacaagaagc aatagtctcc aagtcaatat aaattctaca gaaaatagtg 2150
ttctttttct ccagaaaaat gcttgtgaga atcattaaaa catgtgacaa 2200
tttagagatt ctttgtttta tttcactgat taatatactg tggc~aat'ta 2250
cacagattat taaatttttt tacaagagta tagtatattt atttgaaatg 2300
ggaaaagtgc attttactgt attttgtgta ttttgtttat ttctcagaat 2350
atggaaagaa aattaaaatg tgtcaataaa tattttctag agagtaa 2397
Page I37
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
<210> 108
<211> 305
<212> PRT
<213> Homo sapien
<400> 108
Met Ala Arg Glu Asp Ser val Lys Cys Leu Arg Cys Leu Leu Tyr
1 5 10 15
Ala Leu Asn Leu Leu Phe Trp Leu Met Ser Iie Ser Val Leu Ala
20 25 30
val Ser Ala Trp Met Arg Asp Tyr Leu Asn Asn Val Leu Thr Leu
35 40 ~ 4S
Thr Ala Glu Thr Arg Val Glu Glu Ala val Ile Leu Thr Tyr Phe
50 55 60
Pro val val His Pro val Met Ile Ala val cys Cys Phe Leu Ile
65 70 75
Ile Val Gly Met Leu Gly Tyr Cys Gly Thr Val Lys Arg Asn Leu
80 85 90
Leu Leu Leu Ala Trp Tyr Phe Gly Ser Leu Leu val Ile Phe Cys
95 100 105
val Glu Leu Ala Cys Gly Val Trp Thr Tyr Glu Gln Glu Leu Met
110 115 120
Val Pro Val Gln Trp Ser Asp Met val Thr Leu Lys Ala Arg Met
125 130 135
Thr Asn Tyr Gly Leu Pro Arg Tyr Arg Trp Leu Thr His Ala Trp
140 145 150
Asn Phe Phe Gln Arg Glu Phe Lys Cys Cys Gly Val Val Tyr Phe
155 160 165
Thr Asp Trp Leu Glu Met Thr Giu Met Asp Trp Pro Pro Asp Ser
170 175 180
Cys Cys Val Arg Glu Phe Pro Gly Cys Ser Lys Gln Ala His Gln
185 190 195
Glu Asp Leu Ser Asp Leu Tyr Gln Glu Gly Cys Gly Lys Lys Met
200 205 210
Tyr Ser Phe Leu Arg Gly Thr Lys Gln Leu Gln val Leu Arg Phe
215 220 225
Leu Gly Ile Ser Ile Gly val Thr Gln Ile Leu Ala Met Ile Leu
230 235 240
Thr Ile Thr Leu Leu 'Trp Ala Leu Tyr Tyr Asp Arg Arg Glu Pro
245 250 255
Gly Thr Asp Gln Met Met Ser Leu Lys Asn Asp Asn Ser G1n His
260 265 270
Leu Ser Cys Pro Ser Val Glu Leu Leu Lys Pro Ser Leu Ser Arg
275 280 285
Page 138
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
Ile Phe Glu His Thr ser Met Ala Asn Ser Phe Asn Thr His Phe
290 295 300
Glu Met Glu Glu Leu
305
<210> 109
<211> 2339
<212> DHA
<213> Homo Sapien
<400> 109
ccaaggccag agctgtggac accttatccc actcatcctc atcctcttcc 50
tctgataaag cccctaccag tgctgataaa gtctttctcg tgagagccta 100
gaggccttaa aaaaaaaagt gcttgaaaga gaaggggaca aaggaacacc 150
agtattaaga ggattttcca gtgtttctgg cagttggtcc agaaggatgc 200
ctccattcct gcttctcacc tgcctcttca tcacaggcac ctccgtgtca 250
cccgtggccc tagatccttg ttctgcttac atcagcctga atgagccctg 300
gaggaacact gaccaccagt tggatgagtc tcaaggtcct cctctatgtg 350
acaaccatgt gaatgggga.g tggtaccact tcacgggcat ggcgggagat 400
gccatgccta ccttctgca.t accagaaaac cactgtggaa cccacgcacc 450
tgtctggctc aatggcagcc accccctaga aggcgacggc attgtgcaac 500
gccaggcttg tgccagcttc aatgggaact gctgtctctg gaacaccacg 550
gtggaagtca aggcttgccc tggaggctac tatgtgtatc gtctgaccaa 600
gcccagcgtc tgcttccacg tctactgtgg tcatttttat gacatctgcg 650
acgaggactg ccatggcagc tgctcagata ccagcgagtg cacatgcgct 700
ccaggaactg tgctaggccc tgacaggcag acatgctttg atgaaaatga 750
atgtgagcaa aacaacggtg gctgcagtga gatctgtgtg aacctcaaaa 800
actcctaccg ctgtgagtgt ggggttggcc gtgtgctaag aagtgatggc 850
aagacttgtg aagacgttga aggatgccac aataacaatg gtggctgcag 900
ccactcttgc cttggatctg agaaaggcta ccagtgtgaa tgtccccggg 950
gcctggtgct gtctgaggat aaccacactt gccaagtccc tgtgttgtgc 1000
aaatcaaatg ccattgaagt gaacatcccc agggagctgg ttggtggcct 1050
ggagctcttc ctgaccaaca cctcctgccg aggagtgtcc aacggcaccc 1100
atgtcaacat cctcttctct ctcaagacat gtggtacagt ggtcgatgtg 1150
gtgaatgaca agattgtggc cagcaacctc gtgacaggtc tacccaagca 1200
gaccccgggg agcagcgggg acttcatcat ccgaaccagc aagctgctga 1250
Page 139
.,..,:;sz z ~. .
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
tcccggtgac ctgcgagttt ccacgcctgt acaccatttc tgaaggatac 1300
gttcccaacc ttcgaaactc cccactggaa atcatgagcc gaaatcatgg 1350
gatcttccca ttcactctgg agatcttcaa ggacaatgag tttgaagagc 1400
cttaccggga agctctgccc accctcaagc ttcgtgactc cctctacttt 1450
ggcattgagc ccgtggtgc,a cgtgagcggc ttggaaagct tggtggagag 1500
ctgctttgcc -acccccacct ccaagatcga cgaggtcctg aaatactacc 15-50
tcatccggga tggctgtgtt tcagatgact cggtaaagca gtacacatcc 1600
cgggatcacc tagcaaagca cttccaggtc cctgtcttca agtttgtggg 1650
caaagaccac aaggaagtgt ttctgcactg ccgggttctt gtctgtggag 1700
tgttggacga gcgttcccgt tgtgcccagg gttgccaccg gcgaatgcgt 1750
cgtggggcag gaggagagga ctcagccggt ctacagggcc agacgctaac 1800
aggcggcccg atccgcatcg actgggagga ctagttcgta gccatacctc 1850
gagtccctgc attggacggc tctgctcttt ggagcttctc cccccaccgc 1900
cctctaagaa catctgccaa cagctgggtt cagacttcac actgtgagtt 1950
cagactccca gcaccaactc actctgattc tggtccattc agtgggcaca 2000
ggtcacagca ctgctgaaca atgtggcctg ggtggggttt catctttcta 2050
gggttgaaaa ctaaactgtc cacccagaaa gacactcacc ccatttccct 2100
catttctttc ctacacttaa atacctcgtg-tatggtgcaa tcagaccaca 2150
aaatcagaag ctgggtataa tatttcaagt tacaaaccct agaaaaatta 2200
aacagttact gaaattatga cttaaatacc caatgactcc ttaaatatgt 2250
aaattatagt tataccttga aatttcaatt caaatgcaga ctaattatag 2300
ggaatttgga agtgtatcaa taaaacagta tataatttt 2339
<210> 110
<211> 545
<212> PRT
<213> Homo sapien
<400> 110
Met Pro Pro Phe Leu Leu Leu Thr Cys Leu Phe Ile Thr Gly Thr
1 5 10 15
Ser Val Ser Pro Val Ala Leu Asp Pro Cys Ser Ala Tyr Ile Ser
20 25 30
Leu Asn Glu Pro Trp Arg Asn Thr Asp His Gln Leu Asp Glu Ser
35 40 45
Gln Gly Pro Pro Leu Cys Asp Asn His Val Asn Gly Glu Trp Tyr
50 SS 60
His Phe Thr Gly Met Ala Gly Asp Ala Met P-ro Thr Phe Cys Ile
Page 140
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
65 70 75
Pro Glu Asn His Cys Gly Thr His Ala Pro Val Trp Leu Asn Gly
80 85 90
Ser His Pro Leu Glu Gly Asp Gly Ile val Gln Arg Gln Ala Cys
95 100 105
Ala Ser Phe Asn Gly Asn Cys Cys Leu Trp Asn Thr Thr Val Glu
120 115 120
Val Lys Ala Cys Pro Gly Gly Tyr Tyr Val Tyr Arg Leu Thr Lys
125 130 135
Pro Ser val Cys Phe His val Tyr Cys Gly His Phe Tyr Asp Ile
140 145 150
Cys Asp Glu Asp Cys His Gly Ser Cys Ser Asp Thr Ser Glu Cys
155 160 165
Thr Cys Ala Pro Gly Thr Val Leu Gly Pro Asp Arg Gln Thr Cys
170 175 180
Phe Asp Glu Asn Glu Cys Glu Gln Asn Asn Gly Gly Cys Ser Glu
185 190 195
Ile Cys val Asn Leu Lys Asn Ser Tyr Arg Cys Glu Cys Gly val
200 205 210
Gly Arg Val Leu Arg Ser ASp Gly Lys Thr Cys Glu Asp Val Glu
215 220 225
Gly Cys His Asn Asn Asn Gly Gly Cys Ser His Ser Cys Leu Gly
230 235 240
Ser Glu Lys Gly Tyr Gln Cys Glu Cys Pro Arg Gly Leu Val Leu
245 250 255
ser Glu Asp Asn His Thr Cys Gln val Pro val Leu Cys Lys Ser
260 265 270
Asn Ala Ile Glu Val Asn Ile Pro Arg Glu Leu Val Gly Gly Leu
275 280 285
Glu Leu Phe Leu Thr Asn Thr Ser Cys Arg Gly val ser Asn Gly
29~ 295 300
Thr His Val Asn Ile Leu Phe Ser Leu Lys Thr Cys Gly Thr Val
305 320 315
Val Asp Val Val Asn Asp Lys Ile Val Ala Ser Asn Leu Val Thr
320 325 330
Gly Leu Pro Lys Gln Thr Pro Gly Ser Ser Gly Asp Phe Ile Ile
335 340 345
Arg Thr Ser Lys Leu Leu Ile Pro val Thr Cys Glu Phe Pro Arg
350 355 360
Leu Tyr Thr Ile ser Glu Gly Tyr Val Pro Asn Leu Arg Asn Ser
365 370 375
Pro Leu Glu Ile Met Ser Arg Asn His Gly Ile Phe Pro Phe Thr
Page 141
. __ ,~~_ .a".~.~a~_...~ . .__. _. ..~.~v.~ .. smvnr~ ~..... __~. ~. _~~_e~ .
~~~,...~.... ~.. ..u .rt_.~_.~.~~ ~ ~~~~ ~w~n-~~w, .,~ ~~~~ m.__.N..~,v
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
380 385 390
Leu Glu Ile Phe Lys Asp Asn Glu Phe Glu Glu Pro Tyr Arg Glu
395 400 405
Ala Leu Pro Thr Leu Lys Leu Arg Asp Ser Leu Tyr Phe Gly Ile
410 415 420
Glu Pro Val Val His Val Ser Gly Leu Glu Ser Leu Val Glu Ser
425 430 435
Cys Phe Ala Thr Pro Thr Ser Lys Ile Asp Glu Val Leu Lys Tyr
440 445 450
Tyr Leu Ile Arg Asp Gly Cys Val Ser Asp Asp Ser val Lys Gln
455 460 465
Tyr Thr Ser Arg Asp His Leu Ala Lys His Phe Gln Val Pro Val
470 475 480
Phe Lys Phe val Gly Lys Asp His Lys Glu val Phe Leu His Cys
485 490 495
Arg val Leu val Cys Gly val Leu Asp Glu Arg Ser Arg Cys Ala
500 505 510
Gln Gly Cys His Arg Arg Ntet Arg Arg Gly Ala Gly Gly Glu Asp
515 520 525
Ser Ala Gly Leu Gln Gly Gln Thr Leu Thr Gly Gly Pro Ile Arg
530 535 540
Ile Asp Trp Glu Asp
545
<210> 111
<211> 2063
<212> DNA
<213> Homo Sapien
<400> 111
gagagaggca gcagcttgct cagcggacaa ggatgctggg cgtgagggac 50
caaggcctgc cctgcactcg ggcctcctcc agccagtgct gaccagggac 100
ttctgacctg ctggccagcc aggacctgtg tggggaggcc ctcctgctgc 150
cttggggtga caatctcagc tccaggctac agggagaccg ggaggatcac 200
agagccagca tgttacagga tcctgacagt gatcaacctc tgaacagcct 250
cgatgtcaaa cccctgcgca aaccccgtat ccccatggag accttcagaa 300
aggtggggat ccccatcatc atageactac tgagcctggc gagtatcatc 350
attgtggttg tcctcatcaa ggtgattctg gataaatact acttcctctg 400
cgggcagcct ctccacttca tcccgaggaa gcagctgtgt gacggagagc 450
tggactgtcc cttgggggag gacgaggagc actgtgtcaa gagcttcccc 500
gaagggcctg cagtggcagt ccgcctctcc aaggaccgat ccacactgca 550
Page 142
-, , 4 , .. . K w <... . ~ .~,..._y .r .. , t. x~,_ rt a~.~a.. .. ._ ..__ _
~__ .. _.~ . ._~.~~ ..~ _ _ ..~ ; ~. ~~.K ~~~ . , ~. ~_x~~~..~~»,~~ .. r. ,_~
.. .m..~._
CA 02481756 2004-10-25
PCT-U500-23328_Sequence
ggtgctggac tcggccacag ggaactggtt ctctgcctgt ttcgacaact 600
tcacagaagc tctcgctgag acagcctgta ggcagatggg ctacagcaga 650
gctgtggaga ttggcccaga ccaggatctg gatgttgttg aaatcacaga 700
aaacagccag gagcttcgca tgcggaactc aagtgggccc tgtctctcag 750
gctccctggt ctccctgcac tgtcttgcct gtgggaagag cctgaagacc 800
ccccgtgtgg tgggtgggga ggaggcctct gtggattctt ggccttggca 850
ggtcagcatc cagtacgaca aacagcacgt ctgtggaggg agcatcctgg 900
acccccactg ggtcctcacg gcagcccact gcttcaggaa acataccgat 950
gtgttcaact ggaaggtgcg ggcaggctca gacaaactgg gcagcttccc 1000
atccctggct gtggecaaga tcatcatcat tgaattcaac cccatgtacc 1050
ccaaagacaa tgacatcgcc ctcatgaagc tgcagttccc actcactttc 1100
tcaggcacag tcaggcccat ctgtctgccc ttctttgatg aggagctcac 1150
tccagccacc ccactctgga tcattggatg gggctttacg aagcagaatg 1200
gagggaagat gtctgacata ctgctgcagg cgtcagtcca ggtcattgac 1250
agcacacggt gcaatgcaga cgatgcgtac cagggggaag tcaccgagaa 1300
gatgatgtgt gcaggcatcc cggaaggggg tgtggacacc tgccagggtg 1350
acagtggtgg gcccctgatg taccaatctg accagtggca tgtggtgggc 1400
atcgttagct ggggctatgg ctgcgggggc ccgageaccc caggagtata 1450
caccaaggtc tcagcctatc tcaactggat ctacaatgtc tggaaggctg 1500
agctgtaatg ctgctgcccc tttgcagtgc tgggagccgc ttccttcctg 1550
ccctgcccac ctggggatcc cccaaagtca gacacagagc aagagtcccc 1600
ttgggtacac ccctctgccc acagcctcag catttcttgg agcagcaaag 1650
ggcctcaatt cctgtaagag accctcgcag eccagaggcg cecagaggaa 1700
gtcagcagcc ctagctcggc cacacttggt gctcccagca tcccagggag 1750
agacacagcc cactgaacaa ggtctcaggg gtattgctaa gccaagaagg 1800
aactttccca cactactgaa tggaagcagg ctgtcttgta aaagcccaga 1850
tcactgtggg ctggagagga gaaggaaagg gtctgcgcca gccctgtccg 1900
tcttcaccca tccccaagcc tactagagca agaaaccagt tgtaatataa 1950
aatgcactgc cctactgttg gtatgactac cgttacctac tgttgtcatt 2000
gttattacag ctatggccac tattattaaa gagctgtgta acatctctgg 2050
caaaaaaaaa aaa 2063
<210> 112
Page 143
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
<211> 432
<212> PRT
<213> Homo Sapien
<400> 112
Met Leu Gln Asp Pro Asp Ser Asp Gln Pro Leu Asn Ser Leu Asp
1 5 10 15
Val Lys Pro Leu Arg Lys Pro Arg Ile Pro Met Glu Thr Phe Arg
20 25 30
Lys Val Gly Ile Pro Ile Ile Ile Ala Leu Leu Ser Leu Ala Ser
35 40 45
Ile Ile Ile Val Val Val Leu Ile Lys Val Ile Leu Asp Lys Tyr
50 55 60
Tyr Phe Leu Cys Gly Gln Pro Leu His Phe Ile Pro Arg Lys Gln
65 70 75
Leu Cys Asp Gly Glu Leu Asp Cys Pro Leu Gly Glu Asp Glu Glu
80 85 90
His Cys val Lys Ser Phe Pro Glu Gly Pro Aia Val Ala Val Arg
95 100 105
Leu Ser Lys Asp Arg Ser Thr Leu Gln Val Leu Asp Ser Ala Thr
110 115 120
Gly Asn Trp Phe Ser Ala Cys Phe Asp Asn Phe Thr Glu Ala Leu
125 130 135
Ala Glu Thr Ala Cys Arg Gln Met Gly Tyr Ser Arg Ala Val Glu
140 145 150
Ile Gly Pro Asp Gln Asp Leu Asp Val Val Glu Ile Thr Glu Asn
155 160 165
Ser Gln Glu Leu Arg Met Arg Asn Ser Ser Gly Pro Cys Leu Ser
170 175 180
Gly Ser Leu Val Ser Leu His Cys Leu Ala Cys Gly Lys Ser Leu
185 190 . 195
Lys Thr Pro Arg Val Val Gly Gly Glu Glu Ala Ser Val Asp Ser
200 205 210
Trp Pro Trp Gln Val Ser Ile Gln Tyr Asp Lys Gln His Val Cys
215 220 225
Gly Gly Ser Ile Leu Asp Pro His Trp Val Leu Thr Ala Ala His
230 235 240
Cys Phe Arg Lys His 'Thr Asp Val Phe Asn Trp Lys Val Arg Ala
245 250 255
Gly Ser Asp Lys Leu Gly Ser Phe Pro Ser Leu Ala Val Ala Lys
260 265 270
I1a I1a I1a I1a Gla Phe Asn Pro Met Tyr Pro Lys ASp Asn Asp
275 280 285
Ile Ala Leu Met Lys Leu Gln Phe Pro Leu Thr Phe Ser Gly Thr
Page 144
,. ~ ,
.~..
~.~....~Ta. ~,~ .,.~~..,~ ,.. ~F~u~ ~_ .m~_..~ ~«~..~~.~,.n..r~~~,
r.R.~:n~.~n. .: ~~.3.~~~~~~~~~~~~,~a~~n .~w~~.,~~~,~A.~~_
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
290 295 300
Val Arg Pro Ile Cys Leu Pro Phe Phe Asp Glu Glu Leu Thr Pro
305 310 315
Ala Thr Pro Leu Trp Ile Ile Gly Trp Gly Phe Thr Lys Gln Asn
320 325 330 '
Gly Gly Lys Met Ser Asp Ile Leu Leu Gln Ala Ser Val Gln Val
335 340 345
Ile Asp Ser Thr Arg Cys Asn Ala Asp Asp Ala Tyr Gln Gly Glu
350 355 360
val Thr Glu Lys Met Met Cys Ala Gly Ile Pro Glu Gly Gly Val
365 370 375
Asp Thr Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Tyr Gln Ser
380 385 390
Asp Gin Trp His Val Val Gly Ile Val Ser Trp Gly Tyr Gly Cys
395 400 405
Giy Gly Pro Ser Thr Pro Gly Val Tyr Thr Lys Val Ser Ala Tyr
410 415 420
Leu Asn Trp Ile Tyr Asn Val Trp Lys Ala Glu Leu
425 430
<210> 113
<211> 1768
<212> DNA
<213> Homo Sapien
<400> 113
ggctggactg gaactcctgg tcccaagtga tccacccgcc tcagcctccc SO
aaggtgctgt gattataggt gtaagccacc gtgtctggcc tctgaacaac 100
tttttcagca actaaaaaag ccacaggagt tgaactgcta ggattctgac 150
tatgctgtgg tggctagtgc tcctactcct acctacatta aaatctgttt 200
tttgttctct tgtaaetagc etttaectte etaaeaeaga ggatctgtca 250
ctgtggctct ggcccaaacc tgaccttcac tctggaacga gaacagaggt 300
ttctacccac accgtcccc~t cgaagccggg gacagcctca ccttgctggc 350
ctctcgctgg agcagtgccc tcaccaactg tctcacgtct ggaggcactg 400
actcgggcag tgcaggtagc tgagcctctt ggtagctgcg gctttcaagg 450
tgggccttgc cctggccgta gaagggattg acaagcccga agatttcata 500
ggcgatggct cccactgccc aggcatcagc cttgctgtag tcaatcactg 550
ccctggggcc aggacgggcc gtggacacct gctcagaagc agtgggtgag 600
acatcacgct gcccgcccat ctaacctttt catgtcctgc acatcacctg 650
atccatgggc taatctgaac tctgtcccaa ggaacccaga gcttgagtga 700
Page 145
_ . ~-a-,.~.~.-_~...--v-
CA 02481756 2004-10-25
PCT-0500-23328_Sequence
gctgtggctc agacccagaa ggggtctgct tagaccacct ggtttatgtg 750
acaggacttg cattctcctg gaacatgagg gaacgccgga ggaaagcaaa 800
gtggcaggga aggaacttgt gccaaattat gggtcagaaa agatggaggt 850
gttgggttat cacaaggcat cgagtctcct gcattcagtg gacatgtggg 900
ggaagggctg ccgatggcgc atgacacact cgggactcac ctctggggcc 950
atcagacagc cgtttccgcc ccgatecacg taccagctgc tgaagggcaa 1000
ctgcaggccg atgctctcat cagccaggca gcagccaaaa tctgcgatca 1050
ccagccaggg gcagccgtct gggaaggagc aagcaaagtg accatttctc 1100
ctcccctcct tccctctgag aggccctcct atgtccctac taaagccacc 1150
agcaagacat agctgacagg ggctaatggc tcagtgttgg cccaggaggt 1200
cagcaaggcc tgagagctga tcagaagggc ctgctgtgcg aacacggaaa 1250
tgcctccagt aagcacaggc tgcaaaatcc ccaggcaaag gactgtgtgg 1300
ctcaatttaa atcatgttct agtaattgga gctgtcccca agaccaaagg 1350
agctagagct tggttcaaat gatctccaag ggcccttata ccccaggaga 1400
ctttgatttg aatttgaaae cccaaatcca aacctaagaa ccaggtgcat 1450
taagaatcag ttattgccgg gtgtggtggc etgtaatgcc aacattttgg 1500
gaggccgagg cgggtagatc acctgaggtc aggag.ttcaa gaccagcctg 1550
gccaacatgg tgaaacccct gtctctacta aaaatacaaa aaaactagcc 1600
aggcatggtg gtgtgtgcct gtatcccagc tactcgggag gctgagacag 1650
gagaattact tgaacctggg aggtgaagga ggctgagaca ggagaatcac 1700
ttcagcctga gcaacacagc gagactctgt ctcagaaaaa ataaaaaaag 1750
aattatggtt atttgtaa 1768
<210> 114
<211> 109
<212> PRT
<213> Homo sapien
<400> 114
Met Leu Trp Trp Leu Val Leu Leu Leu Leu Pro Thr Leu Lys Ser
1 5 10 15
Val Phe cys Ser Leu Val Thr Ser Leu Tyr Leu Pro Asn Thr Glu
20 25 30
Asp Leu Ser Leu Trp Leu Trp Pro Lys Pro Asp Leu His Ser Gly ,
35 40 45
Thr Arg Thr Glu Val Ser Thr His Thr Va7 Pro Ser Lys Pro Gly
50 55 60
Thr Ala Ser Pro cys Trp Pro Leu Ala Gly Ala VaT Pro Ser Pro
Page 146
,.. " .,. ,~,.. "#F x ..,u,~,~ .m~,~.T . ~,~".r~.H~ ~"~,,~~. ~,.»,. .x f ~~.~~
..~.~.ri.~o~~ ..~~...m.,~~n.~ ~ ..~mm~w..wm...~~.~. ~. .~~.~.~
~~.~.._.~....~..R_ _ ___.._.
°"T
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
65 70 75
Thr val Ser Arg Leu Glu Ala Leu Thr Arg Ala Val Gln Val Ala
80 85 90
Glu Pro Leu Gly Ser cys Gly Phe Gin Gly Gly Pro cys Pro Gly
95 100 105
Arg Arg Arg Asp
<210> 115
<211> 1197
<212> DNA
<213> Homo Sapien
<400> 115
cagcagtggt ctctcagtc~c tctcaaagca aggaaagagt actgtgtgct 50
gagagaccat ggcaaagaat cctccagaga attgtgaaga ctgtcacatt 100
ctaaatgcag aagcttttaa atccaagaaa atatgtaaat cacttaagat 150
ttgtggactg gtgtttggta tcctggccct aactctaatt gtcctgtttt 200
gggggagcaa gcacttctgg ccggaggtac ccaaaaaagc ctatgacatg 250
gagcacactt tctacagcaa tggagagaag aagaagattt acatggaaat 300
tgatcctgtg accagaactg aaatattcag aagcggaaat ggcactgatg 350
aaacattgga agtgcacgac tttaaaaacg gatacactgg catctacttc 400
gtgggtcttc aaaaatgttt tatcaaaact cagattaaag tgattcctga 450
attttctgaa ccagaagagg aaatagatga gaatgaagaa attaccacaa 500
ctttctttga acagtcagtg atttgggtcc cagcagaaaa gcctattgaa 550
aaccgagatt ttcttaaaaa ttccaaaatt ctggagattt gtgataacgt 600
gaccatgtat tggatcaatc ccactctaat atcagtttct gagttacaag 650
actttgagga ggagggagaa gatcttcact ttcctgccaa cgaaaaaaaa 700
gggattgaac aaaatgaaca gtgggtggtc cctcaagtga aagtagagaa 750
gacccgtcac gccagacaag caagtgagga agaacttcca ataaatgact 800
atactgaaaa tggaatagaa tttgatccca tgctggatga gagaggttat 850
tgttgtattt actgccgtcg aggcaaccgc tattgccgcc gcgtctgtga 900
acctttacta ggctactacc catatccata ctgctaccaa ggaggacgag 950
tcatctgtcg tgtcatcatg ccttgtaact ggtgggtggc ccgcatgctg 1000
gggagggtct aataggagg~ ttgagctcaa atgcttaaac tgctggcaac 1050
atataataaa tgcatgctat~tcaatgaatt tctgcctatg aggcatctgg 1100
cccctggtag ccagctctcc agaattactt gtaggtaatt cctctcttca 1150
Page 147
,~ _a,,
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
tgttctaata aacttctaca ttatcaccaa aaaaaaaaaa aaaaaaa 1197
<210> 116
<211> 317
<212> PRT
<213> Homo Sapien
<400> 116
Met Ala Lys Asn Pro Pro Glu Asn Cys Glu Asp Cys His Ile Leu
1 5 10 15
Asn Ala Glu Ala Phe Lys Ser Lys Lys I12 Cys Lys Ser Leu Lys
20 25 30
Ile Cys Gly Leu Val Phe Gly Ile Leu Ala Leu Thr Leu Ile Val
35 40 45
Leu Phe Trp Gly Ser Lys His Phe Trp Pro Glu Val Pro Lys Lys
50 55 60
Ala Tyr Asp Met Glu His Thr Phe Tyr Ser Asn Gly Glu Lys Lys
65 70 75
Lys Ile Tyr Met Glu Ile Asp Pro Val Thr Arg Thr Glu Ile Phe
80 85 90
Arg Ser Gly Asn Gly Thr Asp Glu Thr Leu Glu Val His Asp Phe
95 100 105
Lys Asn Gly Tyr Thr Gly Ile Tyr Phe Val Gly Leu Gln Lys Cys
110 115 120
Phe Ile Lys Thr Gln Ile Lys Val Ile Pro Glu Phe Ser Glu Pro
125 130 135
Glu Glu Glu Ile Asp Glu Asn Glu Glu Ile Thr Thr Thr Phe Phe
140 145 150
Glu Gln Ser Val Ile Trp Val Pro Ala Glu Lys Pro Ile Glu Asn
155 160 165
Arg Asp Phe Leu Lys Asn Ser Lys Ile Leu Glu Ile Cys Asp Asn
170 175 180
val Thr Met Tyr Trp Ile Asn Pro Thr Leu Ile Ser val Ser Glu
185 190 195
Leu Gln Asp Phe Glu Glu Glu Gly Glu Asp Leu His Phe Pro Ala
200 205 210
Asn Glu Lys Lys Gly Ile Glu Gln Asn Glu Glr1 Trp Val Val Pro
215 220 225
Gln Val Lys Vai Glu Lys Thr Arg His Ala Arg Gln Ala Ser Glu
230 235 240
Glu Glu Leu Pro I7e Asn ASp Tyr Thr Glu Asn Gly I12 Glu Phe
24S 250 255
Asp Pro Met Leu ASp Glu Arg Gly Tyr Cys Cys Ile Tyr Cys Arg
260 265 270
Arg Gly Asn Arg Tyr Cys Arg Arg Val Cys Glu Pro Leu Leu Gly
Page 148
,_.. _.,,_ ...r.~.. ~ . .4 . ~ ,.~~~~ W,.~.~ ;~~,~~~,~a~~,~ R dn~ H ~.~ ~ ~,an
~~.~~~ ,_.,~..u~w~,. ~Rw~.n, ~.~ ~ .ati.
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
275 280 285
Tyr Tyr Pro Tyr Pro Tyr Cys Tyr Gln Gly Gly Arg Val Ile Cys
290 295 300
Arg Val Ile Met Pro Cys Asn Trp Trp Val Ala Arg Met Leu Gly
305 310 315
Arg val
<210> 117
<211> 2121
<212> DNA
<213> Homo Sapien
<400> 117
gagctcccct caggagcgcg ttagcttcac accttcggca gcaggagggc 50
ggcagcttct cgcaggcggc agggcgggcg gccaggatca tgtccaccac 100
cacatgccaa gtggtggcgt tcctcctgtc catcctgggg ctggccggct 150
gcatcgcggc caccgggatg gacatgtgga gcacccagga cctgtacgac 200
aaccccgtca cctccgtgtt ccagtacgaa gggctctgga ggagctgcgt 250
gaggcagagt tcaggcttca ccgaatgcag gccctatttc accatcctgg 300
gacttccagc catgctgcag gcagtgcgag ccctgatgat cgtaggcatc 350
gtcctgggtg ccattggcct cctggtatcc atctttgccc tgaaatgcat 400
ccgcattggc agcatggagg actctgccaa agccaacatg acactgacct 450
ccgggatcat gttcattgtc tcaggtcttt gtgcaattgc tggagtgtct 500
gtgtttgcca acatgctggt gactaacttc tggatgtcca cagctaacat 550
gtacaeegge atgggtggga tggtgcagae tgtteagaee aggtaeaeat 600
ttggtgcggc tctgttcgtg ggctgggtcg ctggaggcct cacactaatt 650
gggggtgtga tgatgtgcat egeetgccgg ggcetggeac eagaagaaac 700
caactacaaa gccgtttctt atcatgcctc aggccacagt gttgcctaca 750
agcctggagg cttcaaggcc agcactggct ttgggtccaa caceaaaaac 800
aagaagatat acgatggagg tgcccgcaca gaggacgagg tacaatctta 850
tccttccaag cacgactatg tgtaatgctc taagacctct cagcacgggc 900
ggaagaaact cccggagagc tcacccaaaa aacaaggaga tcccatctag 950
atttcttctt gcttttgact cacagctgga agttagaaa.a gcctcgattt 1000
catctttgga gaggccaaat ggtcttagcc tcagtctctg tctctaaata 1050
ttccaccata aaacagctga gttatttatg aattagaggc tatagctcac 1100
attttcaatc ctctatttct ttttttaaat ataactttct actctgatga 1150
Page 149
n . _.. . _ , ., r ,~ , ..,. .4 K,. .._~ " . <,_.. ,n ,.a.r~. ....ri.p,~.,~
~~. .n.. "._ ,.,. . ~,~,~"G .~,"~, x~d:~"~~.~ ~~~~.~"~, ~"~-».~ ~-_
.,r.:~."".n,~ ,.~~~rov..~,oN ,~~..~..." r
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
gagaatgtgg ttttaatctc tctctcacat tttgatgatt tagacagact 1200
ccccctcttc ctcctagtca ataaacccat tgatgatcta tttcccagct 1250
tatccccaag aaaacttttg aaaggaaaga gtagacccaa agatgttatt 1300
ttctgctgtt tgaattttgt ctccccaccc ccaacttggc tagtaataaa 1350
cacttactga agaagaagca ataagagaaa gatatttgta atctctccag 1400
cccatgatct cggttttctt acactgtgat cttaaaagtt accaaaccaa 1450
agtcattttc agtttgaggc aaccaaacct ttctactgct gttgacatct 1500
tcttattaca gcaacaccat tctaggagtt tcctgagctc tccactggag 1550
tcctctttct gtcgcgggtc agaaattgtc cctagatgaa tgagaaaatt 1600
atttttttta atttaagtcc taaatatagt taaaataaat aatgttttag 1650
taaaatgata cactatctct gtgaaatagc ctcaccccta catgtggata 1700
gaaggaaatg aaaaaataat tgctttgaca ttgtctatat ggtactttgt 1750
aaagtcatgc ttaagtacaa attccatgaa aagctcacac ctgtaatcct 1800
agcactttgg gaggctgagg aggaaggatc acttgagecc agaagttcga 1850
gactagcctg ggcaacatgg agaagccctg tctctacaaa atacagagag 1900
aaaaaatcag ccagtcatgg tggcatacac ctgtagtccc agcattccgg 1950
gaggctgagg tgggaggatc acttgagccc agggaggttg gggctgcagt 2000
gageeatgat caeaceaetg caetecagec aggtgaeata gegagatcet 2050
gtctaaaaaa ataaaaaata aataatggaa cacagcaagt cctaggaagt 2100
aggttaaaac taattcttta a 2121
<210> 118
<211> 261
<212> PRT
<213> Homo Sapien
<400> 118
Met Ser Thr Thr Thr Cys Gln~Va1 Val Ala Phe Leu Leu Ser Ile
1 5 10 15
Leu Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp
20 25 30
Ser Thr Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln
35 40 45
Tyr Glu Gly Leu Trp Arg Ser Cys val Arg Gln Ser Ser Gly Phe
50 55 60
Thr Glu Cys Arg Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala:Met
65 70 75
Leu Gln Ala Val Arg Ala Leu Met Ile val Gly Ile Val Leu Gly
80 85 90
Page 150
x.. . ", . ....~,. ..... ,.w.,., .g. _,......,..~ r*.-"..~.s-a , ..bv..,"..u
ro.,~ s h~ z..~ ,< .~..,..~.~:a. -... Rp2z.. .- ....-r.a",n.. am.
..~.._,..._~_v... . .,d.~.._........~...____,"._.A..-_y.,.._._ ____."..
."..._~.._..__,.,_
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
Ala Ile Gly Leu Leu Val Ser Ile Phe Ala Leu Lys Cys Ile Arg
95 100 105
I12 Gly Ser Met Glu Asp Ser Ala Lys Ala Asn Met Thr Leu Thr
110 115 120
Ser Gly Ile Met Phe Ile Val Ser Gly Leu Cys Ala Ile Ala Gly
125 130 135
Val Ser Val Phe Ala Asn Met Leu Val Thr Asn Phe Trp Met Ser
140 145 150
Thr Ala Asn Met Tyr Thr Gly Met Gly Gly Met Val Gln Thr Val
155 160 165
Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe Val Gly Trp Val
170 175 180
Ala Gly Gly Leu Thr Leu Iie Gly Gly Val Met Met Cys Ile Aia
1ss 190 19s
Cys Arg Gly Leu Ala Pro Giu Glu Thr Asn Tyr Lys Ala Val Ser
200 205 210
Tyr His Ala Ser Giy His Ser Val Ala Tyr Lys Pro Gly Gly Phe
215 220 225
Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
230 235 240
Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro
245 250 255
Ser Lys His Asp Tyr Val
260
<210> 119
<211> 2010
<212> DNA
<213> Homo Sapien
<400> 119
ggaaaaactg ttctcttctg tggcacagag aaccctgctt caaagcagaa 50
gtagcagttc cggagtccag ctggctaaaa ctcatcccag aggataatgg 100
caacccatgc cttagaaatc gctgggctgt ttcttggtgg tgttggaatg 150
gtgggcacag tggctgtcac tgtcatgcct cagtggagag tgtcggcctt 200
cattgaaaac aacatcgtgg tttttgaaaa cttctgggaa ggactgtgga 250
tgaattgcgt gaggcaggct aacatcagga tgcagtgcaa aatctatgat 300
tccctgctgg ctctttctcc ggacctacag gcagccagag gactgatgtg 350
tgctgcttcc gtgatgtcct tcttggcttt catgatggcc atccttggca 400
tgaaatgcac caggtgcacg ggggacaatg agaaggtgaa ggctcacatt 450
ctgctgacgg ctggaatcat cttcatcatc acgggcatgg tggtgctcat 500
Page 151
CA 02481756 2004-10-25
PCT-u500-23328_Sequence
ccctgtgagc tgggttgcca atgccatcat cagagatttc tataactcaa 550
tagtgaatgt tgcccaaaaa cgtgagcttg gagaagctct ctacttagga 600
tggaccacgg cactggtgct gattgttgga ggagctctgt tctgctgcgt 650
tttttgttgc aacgaaaaga gcagtagcta cagatactcg ataccttccc 700
atcgcacaac ccaaaaaagt tatcacaccg gaaagaagtc accgagcgtc 750
tactccagaa gtcagtatgt gtagttgtgt atgttttttt aactttacta 800
taaagccatg caaatgacaa aaatctatat tactttctca aaatggaccc 850
caaagaaact ttgatttact gttcttaact gcctaatctt aattacagga 900
actgtgcatc agctatttat gattctataa gctatttcag cagaatgaga 950
tattaaaccc aatgctttga ttgttctaga aagtatagta atttgttttc 1000
taaggtggtt caagcatcta ctctttttat catttacttc aaaatgacat 1050
tgctaaagac tgcattattt tactactgta atttctccac gacatagcat 1100
tatgtacata gatgagtgta acatttatat ctcacataga gacatgctta 1150
tatggtttta tttaaaatga aatgccagtc cattacactg aataaataga 1200
actcaactat tgcttttcag ggaaatcatg gatagggttg aagaaggtta 1250
ctattaattg tttaaaaaca gcttagggat taatgtcctc catttataat 1300
gaagattaaa atgaaggctt taatcagcat tgtaaaggaa attgaatggc 1350
tttctgatat gctgtttttt agcctaggag ttagaaatcc taacttcttt 1400
atcctcttct cccagaggct ttttttttct tgtgtattaa attaacattt 1450
ttaaaacgca gatattttgt caaggggctt tgcattcaaa ctgcttttcc 1500
agggctatac tcagaagaaa gataaaagtg tgatctaaga aaaagtgatg 1550
gttttaggaa agtgaaaata tttttgtttt tgtatttgaa gaagaatgat 1600
gcattttgac aagaaatcat atatgtatgg atatatttta ataagtattt 1650
gagtacagac tttgaggttt catcaatata aataaaagag cagaaaaata 1700
tgtcttggtt ttcatttgct taccaaaaaa acaacaacaa aaaaagttgt 1750
cctttgagaa cttcacctgc tcctatgtgg gtacctgagt caaaattgtc 1800
atttttgttc tgtgaaaaat aaatttcctt cttgtaccat ttctgtttag 1850
ttttactaaa atctgtaaat actgtatttt tctgtttatt ccaaatttga 1900
tgaaactgac aatccaattt gaaagtttgt gtcgacgtct gtctagctta 1950
aatgaatgtg ttctatttgc tttatacatt tatattaata aattgtacat 2000
ttttctaatt 2010
<210> 120
Page 152
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
<211> 225
<212> PRT
<213> Homo Sapien
<400> 120
Met Ala Thr His Ala Leu Glu Ile Ala Gly Leu Phe Leu Gly Gly
1 5 10 15
Val Gly Met Val Gly Thr Val Ala Val Thr Val Met Pro Gln Trp
20 25 30
Arg Val Ser Ala Phe Ile Glu Asn Asn Ile Val Val Phe Glu Asn
35 40 45
Phe Trp Glu Gly Leu Trp Met Asn Cys Val Arg Gln Ala Asn Ile
50 55 60
Arg Met Gln Cys Lys Ile.Tyr Asp Ser Leu Leu Ala Leu Ser Pro
65 70 75
Asp Leu Gl.n Ala Ala Arg Gly Leu Met Cys Ala Ala Ser Val Met
80 85 90
Ser Phe Leu Ala Phe Met Met Ala Ile Leu Gly Met Lys Cys Thr
95 100 105
Arg Cys Thr Gly, Asp Asn Glu Lys Val Lys Ala His Ile Leu Leu
110 115 120
Thr Ala Gly Ile Ile Phe Ile Ile Thr Gly Met Val Val Leu Ile
125 130 135
Pro Val Ser Trp Val Ala Asn A1a Ile Ile Arg Asp Phe Tyr Asn
140 145 150
Ser Iie Val Asn Val Ala Gln Lys Arg Glu Leu G1y Glu Ala Leu
155 160 165
Tyr Leu Gly Trp Thr Thr Ala Leu val Leu Ile Val Gly Gly Ala
170 175 180
Leu Phe Cys Cys Val Phe Cys Cys Asn Giu Lys Ser Ser Ser Tyr
185 190 195
Arg Tyr Ser Ile Pro Ser His Arg Thr Thr Gln Lys Ser Tyr His
200 205 210
Thr Gly Lys Lys Ser Pro Ser Val Tyr Ser Arg Ser Gln Tyr Val
215 220 225
<210> 121
<211>.1257
<212> DNA
<213> Homo Sapien
<400> 121
ggagagaggc gcgcgggtga aaggcgcatt gatgcagcct gcggcggcct 50
cggagcgcgg cggagccaga cgctgaccac gttcctctcc tcggtctcct 100
ccgcctccag ctccgcgctg cccggcagcc gggagccatg cgaccccagg 150
gccccgccgc ctccccgcag cggctccgcg gcctcctgct gctcctgctg 200
Page 253
,_.ISYnY.. ,. L . , .- mnw ..~a. ~..... . , .__.»_., ...... _ _,.,... . »
.,...,v..w,nvnrar.. ".7wKww,. »-..-.......,... . .. .... .w... H mswdt2w.~w vw-
m~...mw.M.»..~... -m.N.nmnwvo-.-
zr~;M'MIY2 azi:~pyw~FffPz&WPTRAk~ a~au~.n,..,.,»»_.....,.»___..»,. .
CA 02481756 2004-10-25
PCT-uS00-23328_sequence
ctgcagctgc ccgcgccgtc gagcgcctct gagatcccca aggggaagca 250
aaaggcgcag ctccggcaga gggaggtggt ggacctgtat aatggaatgt 300
gcttacaagg gccagcagga gtgcctggtc gagacgggag ccctggggcc 350
aatgttattc cgggtacacc tgggatccca ggtcgggatg gattcaaagg 400
agaaaagggg gaatgtctga gggaaagctt tgaggagtcc tggacaccca 450
actacaagca gtgttcatgg agttcattga attatggcat agatcttggg 500
aaaattgcgg agtgtacatt tacaaagatg cgttcaaata gtgctctaag 550
agttttgttc agtggctcac ttcggctaaa atgcagaaat gcatgctgtc 600
agcgttggta tttcacattc aatggagctg aatgttcagg acctcttccc 650
attgaagcta taatttattt ggaccaagga agccctgaaa tgaattcaac 700
aattaatatt catcgcactt cttctgtgga aggactttgt gaaggaattg 750
gtgctggatt agtggatgtt gctatctggg ttggcacttg ttcagattac 800
ccaaaaggag atgcttctac tggatggaat tcagtttctc gcatcattat 850
tgaagaacta ccaaaataaa tgctttaatt ttcatttgct acctcttttt 900
ttattatgcc ttggaatggt tcacttaaat gacattttaa ataagtttat 950
gtatacatct gaatgaaaag caaagctaaa tatgtttaca gaccaaagtg 1000
tgatttcaca ctgtttttaa atctagcatt attcattttg cttcaatcaa 1050
aagtggtttc aatatttttt ttagttggtt agaatacttt cttcatagtc 1100
acattctctc aacctataat ttggaatatt gttgtggtct tttgtttttt 1150
ctcttagtat agcattttta aaaaaatata aaagctacca atctttgtac 1200
aatttgtaaa tgttaagaat tttttttata tctgttaaat aaaaattatt 1250
tccaaca 1257
<210> 122
<211> 243
<212> PRT
<213> HOmo Sapien
<400> 12 2
Met Arg Pro Gln Gly Pro Ala Ala Ser Pro Gln Arg Leu Arg Gly
1 5 10 15
Leu Leu Leu Leu Leu Leu Leu Gln Leu Pro Ala Pro Ser Ser Ala
20 25 30
Ser Glu Ile Pro L35 Gly Lys Gln Lys A~Oa Gln Leu Arg Gln A45
Glu Val Val Asp Leu Tyr Asn Gly Met Cys Leu Gln Gly Pro Ala
50 55 60
Page 154
CA 02481756 2004-10-25
PCT-us00-23328_sequence
Gly val Pro Gly Arg Asp Gly Ser Pro Gly Ala Asn Val Ile Pro
65 70 75
Giy Thr Pro Gly Iie Pro Gly Arg Asp Gly Phe Lys Gly Glu Lys
80 85 90
Gly Glu Cys Leu Arg Glu Ser Phe Glu Glu Ser Trp Thr Pro Asn
95 100 105
Tyr Lys Gln Cys Ser Trp Ser Ser Leu A5n Tyr Gly Ile Asp Leu
110 11s z2o
Gly Lys Ile Ala Glu Cys Thr Phe Thr Lys Met Arg Ser Asn Ser
125 130 135
Ala Leu Arg Val Leu Phe Ser Gly Ser Leu Arg Leu Lys Cys Arg
140 145 150
Asn Ala Cys Cys Gln Arg Trp Tyr Phe Thr Phe Asn Giy Ala Glu
155 160 165
Cys Ser Gly Pro Leu Pro Ile Glu Ala Ile Ile Tyr Leu Asp Gln
170 175 180
G1y Ser Pro Glu Met Asn Ser Thr Ile Asn Ile His Arg Thr Ser
185 190 195
Ser Val Glu Gly Leu Cys Glu Gly Tle Gly Ala Gly Leu Val Asp
200 205 210
val Ala Ile Trp val Gly Thr Cys ser Asp Tyr Pro Lys Gly Asp
215 220 225
Ala Ser Thr Gly Trp Asn Ser Val Ser Arg Ile Ile Ile G1a Glu
230 235 240
Leu Pro Lys
<210> 123
<211> 2379
<212> oNA
<213> Homo Sapien
<400> 123
gctgagcgtg tgcgcggtac ggggctctcc tgccttctgg gctccaacgc 50
agctctgtgg ctgaactggg tgctcatcac gggaactgct gggctatgga 100
atacagatgt ggcagctcag gtagccccaa attgcctgga agaatacatc 150
atgtttttcg ataagaagaa attgtaggat ccagtttttt ttttaaccgc 200
cccctcccca ccccccaaaa aaactgtaaa gatgcaaaaa cgtaatatcc 250
atgaagatcc tattacctag gaagattttg atgttttgct gcgaatgcgg 300
tgttgggatt tatttgttct tggagtgttc tgcgtggctg gcaaagaata 350
atgttcCaaaatcggtccat ctcccaaggg gtccaatttt tcttcctggg 400
tgtcagcgag ccctgactca ctacagtgca gctgacaggg gctgtcatgc 450
Page 155
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
aactggcccc taagccaaag caaaagacct aaggacgacc tttgaacaat 500
acaaaggatg ggtttcaatg taattaggct actgagcgga tcagctgtag 550
cactggttat agcccccact gtcttactga caatgctttc ttctgccgaa 600
cgaggatgcc ctaagggctg taggtgtgaa ggcaaaatgg tatattgtga 650
atctcagaaa ttacaggaga taccctcaag tatatctgct ggttgcttag 700
gtttgtccct tcgctataac agccttcaaa aacttaagta taatcaattt 750
aaagggctca accagctcac ctggctatac cttgaccata accatatcag 800
caatattgac gaaaatgctt ttaatggaat acgcagactc aaagagctga 850
ttcttagttc caatagaatc tcctattttc ttaacaatac cttcagacct 900
gtgacaaatt tacggaactt ggatctgtcc tataatcagc tgcattctct 950
gggatctgaa cagtttcggg gcttgcggaa gctgctgagt ttacatttac 1000
ggtctaactc cctgagaacc atecctgtgc gaatattcca agactgccgc 1050
aacctggaac ttttggacct gggatataac cggatccgaa gtttagccag 1100
gaatgtcttt gctggcatga tcagactcaa agaacttcac ctggagcaca 1150
atcaattttc caagctcaac ctggcccttt ttccaaggtt ggtcagcctt 1200 .
cagaaccttt acttgcagtg gaataaaatc agtgtcatag gacagaccat 1250
gtcctggacc tggagctcc.t tacaaaggct tgatttatca ggcaatgaga 1300
tcgaagcttt cagtggaccc agtgttttcc agtgtgtccc gaatctgcag 1350
cgcctcaacc tggattccaa caagctcaca tttattggtc aagagatttt 1400
ggattcttgg atatccctca atgacatcag tcttgctggg aatatatggg 1450
aatgcagcag aaatatttgc tcccttgtaa actggctgaa aagttttaaa 1500 .
ggtctaaggg agaatacaat tatctgtgcc agtcccaaag agctgcaagg 1550
agtaaatgtg atcgatgcag tgaagaacta cagcatctgt ggcaaaagta 1600
ctacagagag gtttgatctg gccagggctc tcccaaagcc gacgtttaag 1650
cccaagctcc ccaggccgaa gcatgagagc aaaccccctt tgcccccgac 1700
ggtgggagcc acagagcccg gcccagagac cgatgctgac gccgagcaca 1750
tctctttcca taaaatcatc gcgggcagcg tggcgctttt cctgtccgtg 1800
ctcgtcatcc tgctggttat ctacgtgtca tggaagcggt accctgcgag 1850
catgaagcag ctgcagcagc gctccctcat gcgaaggcac aggaaaaaga 1900
aaagacagtc cctaaagcaa atgactccca gcacccagga attttatgta 1950
gattataaac ccaccaacac ggagaccagc gagatgctgc tgaatgggac 2000
gggaccctgc acctataaca aatcgggctc cagggagtgt gaggtatgaa 2050
Page I56 a
:r" . ~ .. _
CA 02481756 2004-10-25
PCT-uS00-23328_Sequence
ccattgtgat aaaaagagct cttaaaagct gggaaataag tggtgcttta 2100
ttgaactctg gtgactatca agggaacgcg atgccccccc tccccttccc 2150
tctccctctc actttggtgg caagatcctt ccttgtccgt tttagtgcat 2200
tcataatact ggtcattttc ctctcataca taatcaaccc attgaaattt 2250
aaataccaca atcaatgtga agcttgaact ccggtttaat ataataccta 2300
ttgtataaga ccctttactg attccattaa tgtcgcattt gttttaagat 2350
aaaacttctt tcataggtaa aaaaaaaaa 2379
<210> 124
<211> 513
<212> PRT
<213> Homo Sapien
<400> 124
Met Gly Phe Asn val Ile Arg Leu Leu ser Gly Ser Ala val Ala
1 S 10 15
Leu Val Ile Ala Pro Thr Val Leu Leu Thr Met Leu Ser Ser Ala
20 25 30
Glu Arg G1y Cys Pro Lys Gly Cys Arg Cys Glu Gly Lys Met Val
35 40 45
Tyr Cys Glu Ser Gln Lys Leu Gln Glu Ile Pro Ser Ser Ile Ser
50 55 60
Ala Gly Cys Leu Gly Leu Ser Leu Arg Tyr Asn Ser Leu Gln Lys
65 70 75
Leu Lys Tyr Asn Gln Phe Lys Gly Leu Asn Gln Leu Thr Trp Leu
80 85 90
Tyr Leu Asp His Asn His Ile Ser Asn Ile Asp Glu Asn Ala Phe
95 100 105
Asn Gly Ile Arg Arg Leu Lys Glu Leu Ile Leu Ser Ser Asn Arg
110 115 120
Ile Ser Tyr Phe Leu Asn Asn Thr Phe Arg Pro Val Thr Asn Leu
125 130 13S
Arg Asn Leu Asp Leu Ser Tyr Asn Gln Leu His Ser Leu Gly Ser
140 145 150
Glu Gln Phe Arg Gly Leu Arg Lys Leu Leu Ser Leu His Leu Arg
155 160 165
Ser Asn Ser Leu Arg Thr Ile Pro Val Arg Ile Phe Gln Asp Cys
170 175 180
Arg Asn Leu Glu Leu Leu Asp Leu Gly Tyr Asn Arg Ile Arg Ser
185 190 195
Leu Ala Arg Asn Val Phe Ala Gly Met Ile Arg Leu Lys Glu Leu
200 205 210
Page 157
CA 02481756 2004-10-25
PCT-US00-23328_Sequence
His Leu Glu His Asn Gln Phe Ser Lys Leu Asn Leu Ala Leu Phe
215 220 225
Pro Arg Leu Val Ser Leu Gln Asn Leu Tyr Leu Gln Trp Asn Lys
230 235 240
Ile Ser Val Ile Gly Gln Thr Met Ser Trp Thr Trp Ser Ser Leu
245 250 255
Gln Arg Leu Asp Leu Ser Gly Asn Glu Ile Glu Ala Phe Ser Gly
260 265 270
Pro Ser Val Phe Gln Cys Val Pro Asn Leu Gln Arg Leu Asn Leu
275 280 285
Asp Ser Asn Lys Leu Thr Phe Ile Gly Gln Glu Ile Leu Asp Ser
290 295 300
Trp Ile Ser Leu Asn Asp Ile Ser Leu Ala Gly Asn Ile Trp Glu
305 310 315
Cys Ser Arg Asn Ile Cys Ser Leu Val Asn Trp Leu Lys Ser Phe
320 325 330
Lys Gly Leu Arg Glu Asn Thr Ile Ile Cys Ala Ser Pro Lys Glu
335 340 345
Leu Gln Gly Val Asn Val Tle Asp Ala Val Lys Asn Tyr Ser Ile
350 355 360
Cys Gly Lys Ser Thr Thr Glu Arg Phe Asp Leu Ala Arg Ala Leu
365 370 375
Pro Lys Pro Thr Phe Lys Pro Lys Leu Pro Arg Pro Lys His Glu
380 385 390
Ser Lys Pro Pro Leu Pro Pro Thr Val Gly Ala Thr Glu Pro Gly
395 400 405
Pro Glu Thr Asp Ala Asp Ala Glu His Iie Ser Phe His Lys Tle
410 415 420
Ile Ala Gly ser Val Ala Leu Phe Leu Ser Val Leu val Ile Leu
425 430 435
Leu Val Ile Tyr Val Ser Trp Lys Arg Tyr Pro Ala Ser Met Lys
440 445 450
Gln Leu Gln Gln Arg Ser Leu Met Arg Arg His Arg Lys Lys Lys
455 460 465
Arg Gln Ser Leu Lys Gln Met Thr Pro Ser Thr Gln Glu Phe Tyr
470 475 480
Val Asp Tyr Lys Pro Thr Asn Thr Glu Thr Ser Glu Met Leu Leu
485 490 495
Asn Gly Thr Gly Pro Cys Thr Tyr Asn Lys Ser Giy Ser Arg Glu
500 505 510
Cys Glu val
Page 158
