SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE SAME

POLYPEPTIDES SECRETES ET TRANSMEMBRANAIRES ET ACIDES NUCLEIQUES CODANT POUR CES POLYPEPTIDES

English Abstract

The present invention is directed to novel polypeptides and to nucleic acid
molecules
encoding those polypeptides. Also provided herein are vectors and host cells
comprising those nucleic acid sequences, chimeric polypeptide molecules
comprising
the polypeptides of the present invention fused to heterologous polypeptide
sequences, antibodies which bind to the polypeptides of the present invention
and to
methods for producing the polypeptides of the present invention.

Claims

WHAT IS CLAIMED IS:

1. Isolated nucleic acid having at least 80% nucleic acid sequence identity to
a
nucleotide sequence that encodes an amino acid sequence shown in SEQ ID
NO:456.
2. Isolated nucleic acid having at least 80% nucleic acid sequence identity to
a
nucleotide sequence shown in SEQ ID NO:455.
3. Isolated nucleic acid having at least 80% nucleic acid sequence identity to
the full-
length coding sequence of the nucleotide sequence shown in SEQ ID NO:455.
4. Isolated nucleic acid having at least 80% nucleic acid sequence identity to
the full-
length coding sequence of the DNA deposited under ATCC accession number PTA-
1632.
5. A vector comprising the nucleic acid of any one of Claims 1 to 4.
6. A host cell transformed with the vector of Claim 5.
7. The host cell of Claim 6, wherein said cell is a CHO cell.
8. The host cell of Claim 6, wherein said cell is an E. coli.
9. The host cell of Claim 6, wherein said cell is a yeast cell.
10. A process for producing a PRO19645 (SEQ ID NO:456) polypeptide comprising
culturing the host cell of Claim 6 under conditions suitable for expression of
said
PRO19645 polypeptide and recovering said PRO19645 polypeptide from the cell
culture.
11. An isolated polypeptide having at least 80 % amino acid sequence identity
to the
amino acid sequence shown in SEQ ID NO:456.
12. An isolated polypeptide having at least 80% amino acid sequence identity
to an
amino acid sequence encoded by the full-length coding sequence of the DNA
deposited under ATCC accession number PTA-1632.



13. A chimeric molecule comprising a polypeptide according to Claim 11 fused
to a
heterologous amino acid sequence.
14. The chimeric molecule of Claim 13, wherein said heterologous amino acid
sequence is an epitope tag sequence.
15. The chimeric molecule of Claim 13, wherein said heterologous amino acid
sequence is a Fc region of an immunoglobulin.
16. An antibody which specifically binds to a polypeptide according to Claim
11.
17. The antibody of Claim 16, wherein said antibody is a monoclonal antibody,
a
humanized antibody or a single-chain antibody.
18. Isolated nucleic acid having at least 80% nucleic acid sequence identity
to:
(a) a nucleotide sequence encoding the polypeptide shown in SEQ ID NO:456,
lacking its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown in SEQ ID NO:456, with its associated signal peptide; or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown
in SEQ ID NO:456, lacking its associated signal peptide.
19. An isolated polypeptide having at least 80% amino acid sequence identity
to:
(a) an amino acid sequence of the polypeptide shown in SEQ ID NO:456, lacking
its
associated signal peptide;
(b) an amino acid sequence of an extracellular domain of the polypeptide shown
in
SEQ ID NO:456, with its associated signal peptide; or
(c) an amino acid sequence of an extracellular domain of the polypeptide shown
in
SEQ ID NO:456, lacking its associated signal peptide.
20. A method for detecting the presence of tumor in an mammal, said method
comprising comparing the level of expression of PRO19645 polypeptide (SEQ ID
NO:456) in (a) a test sample of cells taken from said mammal and (b) a control
sample of normal cells of the same cell type, wherein a higher level of
expression of
said PRO19645 polypeptide in the test sample as compared to the control sample
is
indicative of the presence of tumor in said mammal.


21. The method of Claim 20, wherein said tumor is adrenal tumor, lung tumor,
colon
tumor, breast tumor, prostate tumor, rectal tumor, cervical tumor or liver
tumor.
22. An oligonucleotide probe derived from the nucleotide sequence shown in SEQ
ID
NO:455.

Description

CA 02534018 2001-02-28
LA PI2ESENTE P:~RTIE DE CETTE DEiYL~NDF OLr CE DRE~%~TS
COyIPREND PLUS D'U~I' TOLYIE.
CECI EST LE TOiYIE ~ DE
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadian des
Brevets.
~JIVI~~ ~:'~'L~~C~.TI~I~IS / PATENTS
THIS SECTION OF THE APPLICATION I PATEN T COr~f'I'AiNS l~iO~E
THAN ONE VOLUME.
THIS IS VOLUyIE OF
NOTE: For additional volumes please contact the Canadian Patent Office.


CA 02534018 2001-02-28
WO 01/fi8848 PCT1US01J06520
SECRETED AND TRANSMEMBRANE POLYPEPTIDES AND NUCLEIC ACIDS ENCODING THE
SAME
FIELD OF THE INVENTION
The present invention relates generally to the identification and isolation of
novel DNA and to the
recombinant production of novel polypeptides.
BACKGROUND OF THE INVENTION
Extracellular proteins play important 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
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 turn, received and interpreted by diverse cell receptors or membrane-
bound proteins. These secreted
polypeptides or signaling molecules normally pass through the cellular
secretory pathway to reach their site of
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
cytoldnes, 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
secretedproteins.. Examplesof screenin$methods and techniquesrye described in
the lieratuzve~se~, 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
individual 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 turn, 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
selectins and integrins. For instance, transduction of signals that regulate
cell growth and differentiation is
regulated in part by phosphorylation of various cellular proteins. Protein
tyrosine kinases, enzymes that catalyze
that process, can also act as growth factor receptors. Examples include
fibroblast growth factor receptor and
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CA 02534018 2001-02-28
WO 01Ifi8848 PCT/US01/Ofi520
nerve growth factor receptor.
Membrane-bound proteins and receptor molecules have various industrial
applications, including as
pharmaceutical and diagnostic agents. Receptor immunoadhesins, 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
receptorlligand 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, 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 % 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 sequence 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 % 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 % 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
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CA 02534018 2001-02-28
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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 comprising the coding sequence of a
full-length PRO polypeptide cDNA
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 (b) the complement of the DNA
molecule of (a).
Tn 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 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, 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 % nucleic acid sequence
identity, alternatively at least about 97 %
nucleic acid sequence identity, akternatively at least about 98 % nucleic acid
sequence identity and alternatively
at least about 99 % 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 iransmembrane domain-deleted or
transmembrane domain-inactivated,
or is complementary to such encoding nucleotide sequence, wherein the
transmembrane domains) of such
polypeptide are disclosed 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 the 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 length,
usually at least about 10 nucleotides in


alternatively at least about 15 nucleotides in length, alternativelylength,
at least about 20 nucleotides in


alternatively at least about 30 nucleotides in length, alternativelylength,
at least about 40 nucleotides in


alternatively at least about 50 nucleotides in length, alternativelylength,
at least about 60 nucleotides in


alternatively at least about 70 nucleotides in length, length,
alternatively at least about 80 nucleotides in


alternatively at least about 90 nucleotides in length, alternativelylength,
at least about 100 nucleotides in


alternatively at least about 110 nucleotides in length, length,
alternatively at least about 120 nucleotides in


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alternatively at least about 130 nucleotides in length, alternatively at least
about 140 nucleotides in length,
alternatively at least about 150 nucleotides in length, alternatively at least
about 160 nucleotides in length,
alternatively at least about 170 nucleotides in length, alternatively at least
about 180 nucleotides in length,
alternatively at least about 190 nucleotides in length, alternatively at least
about 200 nucleotides in length,
alternatively 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,
alternatively 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 10% of that referenced
length. It is noted that novel
fragments of a PRO polypeptide-encoding nucleotide sequence may be determined
in a routine manner by aligning
the PRO pokypeptide-encoding nucleotide sequence with other laiown nucleotide
sequences using any of a number
of well Irnown sequence alignment programs and determining which PRO
polypeptide-encoding nucleotide
sequence fragments) are novel. All of such PRO polypeptide-encoding nucleotide
sequences are contemplated
herein. Also contemplated are the PRO pokypeptide 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% amino acid sequence identity, alternatively
at least about 81% amino acid
sequence identity, alternatively at least about 82% amino acid sequence
identity, alternatively at least about 83
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 86 %
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%
amino acid sequence identity, alternatively at least about 98% amino acid
sequence identity and alternatively at
least about 99% 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 fragment of the full-length amino acid sequence as disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid
sequence having at least about 80% amino acid sequence identity, alternatively
at least about 81% amino acid
sequence identity, alternatively at least about 82 % amino acid sequence
identity, alternatively at least about 83 %
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CA 02534018 2001-02-28
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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 86% 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%
amino acid sequence identity, alternatively at least about 98 % amino acid
sequence identity and alternatively at
least about 99 % amino acid sequence identity to an amino acid sequence
encoded by any of the human protein
IO 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 transmembrane
domain-deleted or transmembrane 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 polypeptide as herein
described, or an anti-PRO antibody, in
combination with 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
polypepdde, an agonist or antagonist 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 way of
example, the host cells may be CHO cells, E. coli, or yeast. A process for
producing any of the herein described
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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 molecules comprising any
of the herein described
polypeptides fused to a heterologous polypeptide or amino acid 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 below descn'bed polypeptides. Optionally, the antibody is a
monoclonal antibody, humanized
antibody, antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligomicleotide probes which
may be useful for
isolating genomic and cDNA nucleotide sequences, measuring or detecting
expression of an associated gene or
as antisense probes, wherein those probes may be derived from any of the above
or below described nucleotide
sequences. Preferred probe lengths are described above.
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 DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequence PR0276
cDNA, wherein
SEQ ID NO:1 is a clone designated herein as "DNA16435-1208".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding
sequence of SEQ ID
NO:1 shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID N0:3) of a native sequence PR0284
cDNA, wherein
SEQ ID N0:3 is a clone designated herein as "DNA23318-1211".
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 PR0193
cDNA, wherein
SEQ ID NO:S is a clone designated herein as "DNA23322-1393".
Figure 6 shows the amino acid sequence (SEQ 1D N0:6) derived from the coding
sequence of SEQ ID
NO:S shown in Figure 5.
Figure 7 shows a nucleotide sequence (SEQ ID N0:7) of a native sequence PR0190
cDNA, wherein
SEQ ID N0:7 is a clone designated herein as "DNA23334-1392".
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 shows a nucleotide sequence (SEQ ID N0:9) of a native sequence PR0180
cDNA, wherein
SEQ ID N0:9 is a clone designated herein as "DNA26843-1389".
Figure 10 shows the amino acid sequence (SEQ ID NO:10) derived from the coding
sequence of SEQ
ID N0:9 shown in Figure 9.
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Figure 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequence
PR0194 cDNA, wherein
SEQ ID NO:11 is a clone designated herein as "DNA26844-1394".
Figure 12 shows the amino acid sequence (SEQ ID N0:12) derived from the coding
sequence of SEQ
ID NO:l l shown in Pigure 11.
Figure 13 shows a nucleotide sequence (SEQ ID N0:13) of a native sequence
PR0218 cDNA, wherein
SEQ ID N0:13 is a clone designated herein as "DNA30867-1335".
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 N0:15) of a native sequence
PR0260 cDNA, wherein
SEQ ID N0:15 is a clone designated herein as "DNA33470-1175".
Figure 16 shows the amino acid sequence (SEQ ID N0:16) derived from the coding
sequence of SEQ
ID N0:15 shown in Figure 15.
Figure 17 shows a nucleotide sequence (SEQ ID N0:17) of a native sequence
PR0233 cDNA, wherein
SEQ ID N0:17 is a clone designated herein as "DNA34436-1238".
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 )D N0:19) of a native sequence
PR0234 cDNA, wherein
SEQ ID N0:19 is a clone designated herein as "DNA35557-1137".
Figure 20 shows the amino acid sequence (SEQ ID N0:20) dexived from the coding
sequence of SEQ
ID N0:19 shown in Figure 19.
Figure 21 shows a nucleotide sequence (SEQ ID N0:21) of a native sequence
PR0236 cDNA, wherein
SEQ ID N0:21 is a clone designated herein as "DNA35599-1168".
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
PR0244 cDNA, wherein
SEQ ID N0:23 is a clone designated herein as "DNA35668-1171".
Figure 24 shows the amino acid sequence (SEQ ID N0:24) derived from the coding
sequence of SEQ
ID N0:23 shown in Pigure 23.
Figure 25 shows a nucleotide sequence (SEQ ID N0:25) of a native sequence
PR0262 cDNA, wherein
SEQ ID N0:25 is a clone designated herein as "DNA36992-1168".
Figure 26 shows the amino acid sequence (SEQ ID N0:26) derived from the coding
sequence of SEQ
1D N0:25 shown in Figure 25.
Figure 27 shows a nucleotide sequence (SEQ ID N0:27) of a native sequence
PR0271 cDNA, wherein
SEQ ID N0:27 is a clone designated herein as "DNA39423-1182" .
Figure 28 shows the amino acid sequence (SEQ ID N0:28) derived from the coding
sequence of SEQ
ID N0:27 shown in Figure 27.
Figure 29 shows a nucleotide sequence (SEQ ID N0:29) of a native sequence
PR0268 cDNA, wherein
SEQ ID N0:29 is a clone designated herein as "DNA39427-1179".


CA 02534018 2001-02-28
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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 1D N0:31) of a native sequence
PR0270 cDNA, wherein
SEQ ID N0:31 is a clone designated herein as "DNA39510-1181".
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
PR0355 cDNA, wherein
SEQ ID N0:33 is a clone designated herein as "DNA39518-1247".
Figure 34 shows the amino acid sequence (SEQ ID N0:34) derived from the coding
sequence of SEQ
ID N0:33 shown in Figute 33.
Figure 35 shows a nucleotide sequence (SEQ ID N0:35) of a native sequence
PR0298 cDNA, wherein
SEQ m N0:35 is a clone designated herein as "DNA39975-1210".
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
PR0299 cDNA, wherein
SEQ ID N0:37 is a clone designated herein as "DNA39976-1215".
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
PR0296 cDNA, wherein
SEQ ID N0:39 is a clone designated herein as "DNA39979-1213".
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.
Figute 41 shows a nucleotide sequence (SEQ ID N0;41) of a native sequence
PR0329 cDNA, wherein
SEQ ID N0:41 is a clone designated herein as "DNA40594-1233".
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
PR0330 cDNA, wherein
SEQ ID N0:43 is a clone designated herein as "DNA40603-1232".
Figure 44 shows the amino acid sequence (SEQ >D 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
PR0294 cDNA, wherein
SEQ ID N0:45 is a clone designated herein as "DNA40604-1187".
Figure 46 shows the amino acid sequence (SEQ 117 N0:46) derived from the
coding sequence of SEQ
ID N0:45 shown in Figure 45.
Figure 47 shows a nucleotide sequence (SEQ ID N0:47) of a native sequence
PR0300 cDNA, wherein
SEQ ID N0:47 is a clone designated herein as "DNA40625-1189".
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 1D N0:49) of a native sequence
PR0307 cDNA, wherein
SEQ ID N0:49 is a clone designated herein as "DNA41225-1217".
Figure 50 shows the amino acid sequence (SEQ ID N0:50) derived from the coding
sequence of SEQ
ll~ N0:49 shown in Figure 49.
Figure 51 shows a nucleotide sequence (SEQ ID N0:51) of a native sequence
PR0334 cDNA, wherein
SEQ ID N0:51 is a clone designated herein as "DNA41379-1236".
Figure 52 shows the amino acid sequence (SEQ ID N0:52) derived from the coding
sequence of SEQ
1D N0:51 shown in Figure 51.
Figure 53 shows a nucleotide sequence (SEQ ID N0:53) of a native sequence
PR0352 cDNA, wherein
SEQ ID N0:53 is a clone designated herein as "DNA41386-1316".
Figure 54 shows the amino acid sequence (SEQ 1D N0:54) derived from the coding
sequence of SEQ
ID N0:53 shown in Figure 53.
Figure 55 shows a nucleotide sequence (SEQ ID N0:55) of a native sequence
PR0710 cDNA, wherein
SEQ 1D N0:55 is a clone designated herein as "DNA44161-1434".
Figure 56 shows the amino acid sequence (SEQ ID N0:56) derived from the coding
sequence of SEQ
ID N0:55 shown in Figure 55.
Figure 57 shows a nucleotide sequence (SEQ ID N0:57) of a native sequence
PR0873 cDNA, wherein
SEQ ID N0:57 is a clone designated herein as "DNA44179-1362".
Figure 58 shows the amino acid sequence (SEQ ID N0:58) derived from the coding
sequence of SEQ
ID N0:5'7 shown in Figure 57.
Figure 59 shows a nucleotide sequence (SEQ ID N0:59) of a native sequence
PR0354 cDNA, wherein
SEQ ID N0:59 is a clone designated herein as "DNA44192-1246".
Figure 60 shows the amino acid sequence (SEQ ID N0:60) derived from the coding
sequence of SEQ
ID N0:59 shown in Figure 59.
Figure 61 shows a nucleotide sequence (SEQ ID N0:61) of a native sequence
PR01151 cDNA, wherein
SEQ ID N0:61 is a clone designated herein as "DNA44694-1500".
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
PR0382 cDNA, wherein
SEQ ID N0:63 is a clone designated herein as "DNA45234-1277".
Figure 64 shows the amino acid sequence (SEQ )D N0;64) derived from the coding
sequence of SEQ
ID N0:63 shown in Figure 63.
Figure 65 shows a nucleotide sequence (SEQ ID N0:65) of a native sequence
PR01864 cDNA, wherein
SEQ D7 N0:65 is a clone designated herein as "DNA45409-2511".
Figure 66 shows the amino acid sequence (SEQ ID N0:66) derived from the coding
sequence of SEQ
ID N0:65 shown in Figure 65.
Figure 67 shows a nucleotide sequence (SEQ ID N0:67) of a native sequence
PR0386 cDNA, wherein
SEQ ID N0:67 is a clone designated herein as "DNA45415-1318".
9


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Figure 68 shows the amino acid sequence (SEQ ID N0:68) derived from the coding
sequence of SEQ
ID N0:67 shown in Figure 67.
Figure 69 shows a nucleotide sequence (SEQ 1D N0:69) of a native sequence
PROS41 cDNA, wherein
SEQ ID N0:69 is a clone designated herein as "DNA45417-1432".
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
PR08S2 cDNA, wherein
SEQ ID N0:71 is a clone designated herein as "DNA4S493-1349".
Figure 72 shows the amino acid sequence (SEQ ID N0:72) derived from the coding
sequence of SEQ
1D N0:71 shown in Figure 71,
Figure 73 shows a nucleotide sequence (SEQ ID N0:73) of a native sequence
PR0700 cDNA, wherein
SEQ ID N0:73 is a clone designated herein as "DNA46776-1284".
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.
Figures 75A-75B show a nucleotide sequence (SEQ ID N0:75) of a native sequence
PR0708 cDNA,
wherein SEQ ID N0:75 is a clone designated herein as "DNA48296-1292".
Figure 76 shows the amino acid sequence (SEQ ID N0:76) derived from the coding
sequence of SEQ
ID N0:75 shown in Figures 75A-75B.
Figure 77 shows a nucleotide sequence (SEQ 1D N0:77) of a native sequence
PR0707 cDNA, wherein
SEQ ID N0:77 is a clone designated herein as "DNA48306-1291".
Figure 78 shows the amino acid sequence (SEQ ID N0:78) derived from the coding
sequence of SEQ
ID N0:77 shown in Figure 77.
Figure 79 shows a nucleotide sequence (SEQ ID N0:79) of a native sequence
PR0864 cDNA, wherein
SEQ ID N0:79 is a clone designated herein as "DNA48328-1355".
Figure 80 shows the amino acid sequence (SEQ ID N0:80) derived from the coding
sequence of SEQ
2$ ID N0:79 shown in Figure 79.
Figure 81 shows a nucleotide sequence (SEQ ID N0:81) of a native sequence
PR0706 cDNA, wherein
SEQ ID N0:81 is a clone designated herein as "DNA48329-1290".
Figure 82 shows the amino acid sequence (SEQ ID N0:82) derived from the coding
sequence of SEQ
ID N0:81 shown in Figure 81.
Figure 83 shows a nucleotide sequence (SfiQ ID N0:83) of a native sequence
PR0732 cDNA, wherein
SEQ ID N0:83 is a clone designated herein as "DNA48334-1435".
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 85 shows a nucleotide sequence (SEQ ID N0:85) of a native sequence
PR0537 cDNA, wherein
SEQ II7 N0:85 is a clone designated herein as "DNA49141-1431".
Figure 86 shows the amino acid sequence (SEQ ID N0:86) derived from the coding
sequence of SEQ
ID N0:85 shown in Figure 8S.


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Figure 87 shows a nucleotide sequence (SEQ ID N0:87) of a native sequence
PR0545 cDNA, wherein
SEQ ID N0:87 is a clone designated herein as "DNA49624-1279".
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
PR0718 cDNA, wherein
SEQ ID N0:89 is a clone designated herein as "DNA49647-1398".
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
PR0872 cDNA, wherein
SEQ ID N0:91 is a clone designated herein as "DNA49819-1439".
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
PR0704 cDNA, wherein
SEQ ID N0:93 is a clone designated herein as "DNA50911-1288".
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
PR0705 cDNA, wherein
SEQ ID N0:95 is a clone designated herein as "DNA50914-1289".
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 nucleotide sequence (SEQ ID N0:97) of a native sequence
PR0871 cDNA, wherein
SEQ ID N0:97 is a clone designated herein as "DNA50919-1361".
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
PR0702 cDNA, wherein
SEQ ID N0:99 is a clone designated herein as "DNA50980-1286".
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 101 shows a nucleotide sequence (SEQ ID NO:101) of a native sequence
PR0944 cDNA, wherein
SEQ ID NO:101 is a clone designated herein as "DNA52185-1370".
Figure 102 shows the amino acid sequence (SEQ ID N0:102) derived from the
coding sequence of SEQ
ID NO:101 shown in Pigure 101.
Figure 103 shows a nucleotide sequence (SEQ ID N0:103) of a native sequence
PR0739 cDNA, wherein
SEQ ID N0:103 is a clone designated herein as "DNA52756".
Figure 104 shows the amino acid sequence (SEQ ID N0:104) derived from the
coding sequence of SEQ
ID N0:103 shown in Figure 103.
Figure 105 shows a nucleotide sequence (SEQ ID N0:105) of a native sequence
PR0941 cDNA, wherein
SEQ ID N0:105 is a clone designated herein as "DNA53906-1368".
11


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Figure 106 shows the amino acid sequence (SEQ ID N0:106) 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
PR01082 cDNA,
wherein SEQ )D N0:107 is a clone designated herein as "DNA53912-1457".
Figure 108 shows the amino acid sequence (SEQ ID N0:108) 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
PR01133 cDNA,
wherein SEQ 117 N0:109 is a clone designated herein as "DNA53913-1490".
Figure 110 shows the amino acid sequence (SEQ ID NO:110) derived from the
coding sequence of SEQ
ID N0:109 shown in Figure 109.
Figure 111 shows anucleotide sequence (SEQ TD NO:111) of a native sequence
PR0983 cDNA, wherein
SEQ ID NO:111 is a clone designated herein as "DNA53977-1371".
Figure 112 shows the amino acid sequence (SEQ ID N0:112) derived from the
coding sequence of SEQ
ID NO:111 shown in Figure 111.
Figure 113 shows a nucleotide sequence (SEQ ID N0:113) of a native sequence
PR0784 cDNA, wherein
SEQ ID N0:113 is a clone designated herein as "DNA53978-1443".
Figure 114 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
PR0783 cDNA, wherein
SEQ ID NO:115 is a clone designated herein as "DNA53996-1442".
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 117 shows a nucleotide sequence (SEQ ID N0:117) of a native sequence
PR0940 cDNA, wherein
SEQ ID N0:117 is a clone designated herein as "DNA54002-1367".
Figure 118 shows the amino acid sequence (SEQ ID N0:118) derived from the
coding sequence of SEQ
ID N0:117 shown in Figure 117.
Figure 119 shows a nucleotide sequence (SEQ ID N0:119) of a native sequence
PR0768 cDNA, wherein
SEQ ID N0:119 is a clone designated herein as "DNA55737-1345".
Figure 120 shows the amino acid sequence (SEQ ID N0:120) derived from the
coding sequence of SEQ
ID N0:119 shown in Figure 119.
Figure 121 shows a nucleotide sequence (SEQ ID N0:121) of a native sequence
PR01079 cDNA,
wherein SEQ ID N0:121 is a clone designated herein as "DNA56050-1455".
Figure 122 shows the amino acid sequence (SEQ ID N0:122) derived from the
coding sequence of SEQ
ID N0:121 shown in Figure 121.
Figure 123 shows a nucleotide sequence (SEQ ID N0:123) of a native sequence
PR01078 cDNA,
wherein SEQ ID N0:123 is a clone designated herein as "DNA56052-1454".
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.
12


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Figure 125 shows a nucleotide sequence (SEQ ID N0:125) of a native sequence
PR01018 cDNA,
wherein SEQ ID N0:125 is a clone designated herein as "DNA56107-1415".
Figure 126 shows the amino acid sequence (SEQ ID N0:126) derived from the
coding sequence of SEQ
ID N0:125 shown in Figure 125.
Figure 127 shows a nucleotide sequence (SEQ ID N0:127) of a native sequence
PR0793 cDNA, wherein
SEQ ID N0:127 is a clone designated herein as "DNA56110-1437".
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
PR01773 cDNA,
wherein SEQ ID N0:129 is a clone designated herein as "DNA56406-1704".
Figure 130 shows the amino acid sequence (SEQ ID N0:130) derived from the
coding sequence of SEQ
ID N0:129 shown in Figure 129.
Figure 131 shows a nucleotide sequence (SEQ ID N0:131) of a native sequence
PR01014 cDNA,
wherein SEQ ID N0:131 is a clone designated herein as "DNA56409-1377".
Figure 132 shows the amino acid sequence (SEQ ID N0:132) derived from the
coding sequence of SEQ
ID N0:131 shown in Figure 131.
Figure 133 shows a nucleotide sequence (SEQ ID N0:133) of a native sequence
PR01013 cDNA,
wherein SEQ ID N0:133 is a clone designated herein as "DNA56410-1414".
Figure 134 shows the 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
PR0937 cDNA, wherein
SEQ ID N0:135 is a clone designated herein as "DNA56436-1448".
Figure 136 shows the amino acid sequence (SEQ ID N0:136) derived from the
coding sequence of SEQ
ID N0:135 shown in Figure 135.
Figure 137 shows a nucleotide sequence (SEQ ID N0:137) of a native sequence
PR01477 cDNA,
wherein SEQ ID NO:I37 is a clone designated herein as "DNA56529-1647".
Figure 138 shows the amino acid sequence (SEQ ID N0:138) derived fmm 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
PR0842 cDNA, wherein
SEQ 1D N0:139 is a clone designated herein as "DNA56855-1447".
Figure 140 shows the amino acid sequence (SEQ ID N0:140) derived from the.
coding sequence of SEQ
ID NO:I39 shown in Figure 139.
Figure 141 shows a nucleotide sequence (SEQ ID N0:141) of a native sequence
PR0839 cDNA, wherein
SEQ ID N0:141 is a clone designated herein as "DNA56859-1445".
Figure 142 shows the amino acid sequence (SfiQ 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
PR01180 cDNA,
wherein SEQ 1D N0:143 is a clone designated herein as "DNA56860-1510".
13


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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
PR01134 cDNA,
wherein SEQ ID N0:145 is a clone designated herein as "DNA5686S-1491".
Figure 146 shows the amino acid sequence (SEQ ID N0:146) derived from the
coding sequence of SEQ
ID N0:145 shown in Figure 145.
Figure 147 shows a nucleotide sequence (SEQ ID N0:147) of a native sequence
PR01115 cDNA,
wherein SEQ ID N0:147 is a clone designated herein as "DNA56868-1478".
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 N0:149) of a native sequence
PR01277 cDNA,
wherein SEQ ID N0:149 is a clone designated herein as "DNA56869-1545".
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 N0:151) of a native sequence
PR01135 cDNA,
1S wherein SEQ ID N0:151 is a clone designated herein as "DNA56870-1492".
Figure 152 shows the amino acid sequence (SEQ ID N0:152) derived from the
coding sequence of SEQ
ID NO:1S1 shown in Figure 151.
Figure 153 shows a nucleotide sequence (SEQ ID N0:153) of a native sequence
PR0827 cDNA, wherein
SEQ ID N0:153 is a clone designated herein as "DNA57039-1402".
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 1D N0:155) of a native sequence
PR01057 cDNA,
wherein SEQ ID N0:155 is a clone designated herein as "DNA57253-1382".
Figure 156 shows the amino acid sequence (SEQ ID N0:156) derived from the
coding sequence of SEQ
1D NO:I55 shown in Figure I55.
Figure 157 shows a nucleotide sequence (SEQ ID N0:157) of a native sequence
PR01113 cDNA,
wherein SEQ ID N0:157 is a clone designated hereia as "DNA57254-1477".
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 sequence
PR01006 cDNA,
wherein SEQ 1D N0:159 is a clone designated herein as "DNA57699-1412" .
Figure 160 shows the amino acid sequence (SEQ ID N0:160) derived from the
coding sequence of SEQ
ID N0:159 shown in Figure 159.
Figure 161 shows a nucleotide sequence (SEQ ID N0:161) of a native sequence
PR01074 cDNA,
wherein SEQ ID N0:161 is a clone designated herein as "DNA57704-1452".
Figure 162 shows the amino acid sequence (SEQ ID N0:162) derived from the
coding sequence of SEQ
1D N0:161 shown in Figure 161
14


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Figure 163 shows a nucleotide sequence (SEQ ID N0:163) of a native sequence
PR01073 cDNA,
wherein SEQ ID N0:163 is a clone designated hexein as "DNA57710-1451".
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
PR01136 cDNA,
wherein SEQ ID N0:165 is a clone designated herein as "DNA57827-1493".
Figure 166 shows the amino acid sequence (SEQ ID N0:166) 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
PR01004 cDNA,
wherein SEQ ID N0:167 is a clone designated herein as "DNA57844-1410" .
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.
Figure 169 shows a nucleotide sequence (SEQ ID N0:169) of a native sequence
PR01344 cDNA,
wherein SEQ ID N0:169 is a clone designated herein as "DNA58723-1588".
Figure 170 shows the amino acid sequence (SEQ ID N0:170) derived from the
coding sequence of SEQ
ID N0:169 shown in Figure 169.
Figure x71 shows a nucleotide sequence (SEQ ID N0:171) of a native sequence
PRO1110 cDNA,
wherein SEQ ID N0:171 is a clone designated herein as "DNA58727-1474".
Figure 172 shows the amino acid sequence (SEQ ID N0:172) derived from the
coding sequence of SEQ
ID N0:171 shown in Figure 171.
Figure 173 shows a nucleotide sequence (SEQ ID N0:173) of a native sequence
PR01378 cDNA,
wherein SEQ ID N0:173 is a clone designated herein as "DNA58730-1607".
Figure 174 shows the amino acid sequence (SEQ ID N0:174) derived from the
coding sequence of SEQ
ID N0:173 shown in Figure 173.
Figure 175 shows a nucleotide sequence (SEQ ID N0:175) of a native sequence
PR01481 cDNA,
wherein SEQ ID N0:175 is a clone designated herein as "DNA58732-1650".
Figure 176 shows the amino acid sequence (SEQ ID N0:176) derived from the
coding sequence of SEQ
ID N0:175 shown in Figure 175.
Figure 177 shows a nucleotide sequence (SEQ ID N0:177) of a native sequence
PR01109 cDNA,
wherein SEQ ID N0:177 is a clone designated herein as "DNA58737-1473".
Figure 178 shows the amino acid sequence (SEQ ID N0:178) derived from the
coding sequence of SEQ
ID N0:177 shown in Figure 177.
Figure 179 shows a nucleotide sequence (SEQ ID N0:179) of a native sequence
PR01383 cDNA,
wherein SEQ ID N0:179 is a clone designated herein as "DNA58743-1609".
Figure 180 shows the amino acid sequence (SEQ ID N0:180) derived from the
coding sequence of SEQ
ID N0:179 shown in Figure 179.
Figure 181 shows a nucleotide sequence (SEQ ID N0:181) of a native sequence
PR01072 cDNA,
wherein SEQ ID N0:181 is a clone designated herein as "DNA58747-1384".


CA 02534018 2001-02-28
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Figure 182 shows the amino acid sequence (SEQ ID N0:182) derived from the
coding sequence of SEQ
ID N0:181 shown in Figure 181.
Figure 183 shows a nucleotide sequence (SEQ ID N0:183) of a native sequence
PR01189 cDNA,
wherein SEQ ID N0:183 is a clone designated herein as "DNAS8828-1519".
Figure 184 shows the amino acid sequence (SEQ ID N0:184) derived from the
coding sequence of SEQ
1D N0:183 shown in Figure 183.
Figure 185 shows a nucleotide sequence (SEQ ID N0:185) of a native sequence
PR01003 cDNA,
wherein SEQ ID N0:185 is a clone designated herein as "DNA58846-1409".
Figure 186 shows the amino acid sequence (SEQ ID N0:186) derived from the
coding sequence of SEQ
ID N0:185 shown in Figure 185.
Figure 187 shows a nucleotide sequence (SEQ ID N0:187) of a native sequence
PR01108 cDNA,
wherein SEQ ID N0:187 is a clone designated herein as "DNA58848-1472".
Figure 188 shows the amino acid sequence (SEQ ID N0:188) derived from the
coding sequence of SEQ
ID N0:187 shown in Figure 187.
Figure 189 shows a nucleotide sequence (SEQ ID N0:189) of a native sequence
PR01137 eDNA,
wherein SEQ ID N0:189 is a clone designated herein as "DNA58849-1494".
Figure 190 shows the amino acid sequence (SEQ ID N0:190) derived from the
coding sequence of SEQ
ID N0:189 shown in Figure 189.
Figure 191 shows a nucleotide sequence (SEQ ID N0:191) of a native sequence
PR01138 cDNA,
wherein SEQ ID N0:191 is a clone designated herein as "DNA58850-1495".
Figure 192 shows the amino acid sequence (SEQ ID N0:192) derived from the
coding sequence of SEQ
ID N0:191 shown in Figure 191.
Figure 193 shows a nucleotide sequence (SEQ 1D N0:193) of a native sequence
PR01415 cDNA,
wherein SEQ ID N0:193 is a clone designated herein as "DNAS8852-1637".
Figure 194 shows the amino acid sequence (SEQ ID N0:194) derived from the
coding sequence of SEQ
ID N0:193 shown in Figure 193.
Figure 195 shows a nucleotide sequence (SEQ ID N0:195) of a native sequence
PR01054 cDNA,
wherein SEQ ID N0:195 is a clone designated herein as "DNA58853-1423".
Figure 196 shows the amino acid sequence (SEQ lD N0:196) derived from the
coding sequence of SEQ
ID N0:195 shown in Figure 195.
Figure 197 shows a nucleotide sequence (SEQ 1D NO:197) of a native sequence
PR0994 cDNA, wherein
SEQ ID N0:197 is a clone designated herein as "DNA58855-1422".
Figure 198 shows the amino acid sequence (SEQ ID N0:198) derived from the
coding sequence of SEQ
ID N0:197 shown in Figure 197.
Figure 199 shows a nucleotide sequence (SEQ ID N0:199) of a native sequence
PR01069 cDNA,
wherein SEQ ID N0:199 is a clone designated herein as "DNAS9211-1450".
Figure 200 shows the amino acid sequence (SEQ ID N0:200) derived from the
coding sequence of SEQ
ID N0:199 shown in Figure 199.
16


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Figure 201 shows a nucleotide sequence (SEQ ID N0:201) of a native sequence
PR01411 cDNA,
wherein SEQ 1D N0:201 is a clone designated herein as "DNA59212-1627".
Figure 202 shows the amino acid sequence (SEQ ID N0:202) derived from the
coding sequence of SEQ
ID N0:201 shown in Figure 201.
Figure 203 shows a nucleotide sequence (SEQ ID N0:203) of a native sequence
PR01129 cDNA,
wherein SEQ ID N0:203 is a clone designated herein as "DNA59213-1487".
Figure 204 shows the amino acid sequence (SEQ ID N0:204) derived from the
coding sequence of SEQ
)D N0:203 shown in Figure 203.
Figure 20S shows a nucleotide sequence (SEQ ID N0:205) of a native sequence
PR01359 cDNA,
wherein SEQ ID N0:205 is a clone designated herein as "DNA59219-1613".
Figure 206 shows the amino acid sequence (SEQ ID N0:206) derived from the
coding sequence of SEQ
ID N0:205 shown in Figure 205.
Figure 207 shows a nucleotide sequence (SEQ ID N0:207) of a native sequence
PR01139 cDNA,
wherein SEQ ID N0:207 is a clone designated herein as "DNA59497-1496".
Figure 208 shows the amino acid sequence (SEQ ID N0:208) derived from the
coding sequence of SEQ
ID N0:207 shown in Figure 207.
Figure 209 shows a nucleotide sequence (SEQ ID N0:209) of a native sequence
PR01065 cDNA,
wherein SEQ ID N0:209 is a clone designated herein as "DNA59602-1436".
Figure 210 shows the amino acid sequence (SEQ ID N0:210) derived from the
coding sequence of SEQ
ID N0:209 shown in Figure 209.
Figure 211 shows a nucleotide sequence (SEQ ID N0:211) of a native sequence
PR01028 cDNA,
wherein SEQ ID N0:211 is a clone designated herein as "DNA59603-1419".
Figure 212 shows the amino acid sequence (SEQ ID N0:212) derived from the
coding sequence of SEQ
ID N0:211 shown in Figure 211.
Figure 213 shows a nucleotide sequence (SEQ ID N0:2I3) of a native sequence
PROI027 cDNA,
wherein SEQ ID N0:213 is a clone designated herein as "DNA59605-1418".
Figure 214 shows the amino acid sequence (SEQ m N0:214) derived from the
coding sequence of S8Q
ID N0:213 shown in Figure 213.
Pigure 215 shows a nucleotide sequence (SEQ ID N0:215) of a native sequence
PR01140 cDNA,
wherein SEQ ID N0:215 is a clone designated herein as "DNA59607-1497".
Figure 216 shows the amino acid sequence (SEQ ID N0:216) derived from the
coding sequence of SEQ
ID N0:215 shown in Figure 215.
Figure 217 shows a nucleotide sequence (SEQ ID N0:217) of a native sequence
PR01291 cDNA,
wherein SEQ ID N0:217 is a clone designated herein as "DNA59610-1556".
Figure 218 shows the amino acid sequence (SEQ ID N0:218) derived from the
coding sequence of SEQ
ID N0:217 shown in Figure 217.
Figure 219 shows a nucleotide sequence (SEQ ID N0:219) of a native sequence
PR01105 cDNA,
wherein SEQ ID N0:219 is a clone designated herein as "DNA59612-1466".
17


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Figure 220 shows the amino acid sequence (SEQ ID N0:220) derived from the
coding sequence of SEQ
ID N0:219 shown in Figure 219.
Figure 221 shows a nucleotide sequence (SEQ ID N0:221) of a native sequence
PR01026 cDNA,
wherein SEQ ID N0:221 is a clone designated herein as "DNA59613-1417".
Figure 222 shows the amino acid sequence (SEQ ID N0:222) derived from the
coding sequence of SEQ
ID N0:221 shown in Figure 221.
Figure 223 shows a nucleotide sequence (SEQ ID N0:223) of a native sequence
PR01104 cDNA,
wherein SEQ ID N0:223 is a clone designated herein as "DNA59616-1465".
Figure 224 shows the amino acid sequence (SEQ ID N0:224) derived from the
coding sequence of SEQ
ID N0:223 shown in Figure 223.
Figure 225 shows a nucleotide sequence (SEQ ID N0:225) of a native sequence
PRO1100 cDNA,
wherein SEQ ID N0:225 is a clone designated herein as "DNA59619-1464".
Figure 226 shows the amino acid sequence (SEQ ID N0:226) derived from the
coding sequence of SEQ
ID N0:225 shown in Figure 225.
Figure 227 shows a nucleotide sequence (SEQ ID N0:227) of a native sequence
PR01141 cDNA,
wherein SEQ TD N0:227 is a clone designated herein as "DNA59625-1498".
Figure 228 shows the amino acid sequence (SEQ ID N0:228) derived from the
coding sequence of SEQ
ID N0:227 shown in Figure 227.
Figure 229 shows a nucleotide sequence (SEQ ID N0:229) of a native sequence
PR01772 cDNA,
wherein SEQ ID N0:229 is a clone designated herein as "DNA59817-1703".
Figure 230 shows the amino acid sequence (SEQ ID N0:230) derived from the
coding sequence of SEQ
ID N0:229 shown in Pigure 229.
Figure 231 shows a nucleotide sequence (SEQ ID N0:231) of a native sequence
PR01064 cDNA,
wherein SBQ ID N0:231 is a clone designated herein as "DNA59827-1426".
Figure 232 shows the amino acid sequence (SEQ ID N0:232) derived from the
coding sequence of SEQ
ID N0:231 shown in Figure 231.
Figure 233 shows a nucleotide sequence (SEQ ID N0:233) of a native sequence
PR01379 cDNA,
wherein SEQ II7 N0:233 is a clone designated herein as "DNA59828-1608".
Figure 234 shows the amino acid sequence (SEQ ID N0:234) derived from the
coding sequence of SEQ
ID N0:233 shown in Figure 233.
Figure 235 shows a nucleotide sequence (SEQ ID N0:235) of a native sequence
PR03573 cDNA,
wherein SEQ ID N0:235 is a clone designated herein as "DNA59837-2545".
Figure 236 shows the amino acid sequence (SEQ ID N0:236) derived from the
coding sequence of SEQ
ID N0:235 shown in Figure 235.
Figure 237 shows a nucleotide sequence (SEQ ID N0:237) of a native sequence
PR03566 cDNA,
wherein SEQ ID N0:237 is a clone designated herein as "DNA59844-2542".
Figure 238 shows the amino acid sequence (SEQ ID N0:238) derived from the
coding sequence of SEQ
ID N0:237 shown in Figure 237.
18


CA 02534018 2001-02-28
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Figure 239 shows a nucleotide sequence (SEQ ID N0:239) of a native sequence
PR01156 cDNA,
wherein SEQ ID N0:239 is a clone designated herein as "DNA59853-1505".
Figure 240 shows the amino acid sequence (SEQ ID N0:240) derived from the
coding sequence of SEQ
ID N0:239 shown in Figure 239.
Figure 241 shows a nucleotide sequence (SEQ ID N0:241) of a native sequence
PR01098 cDNA,
wherein SEQ ID N0:241 is a clone designated herein as "DNA59854-1459".
Figure 242 shows the amino acid sequence (SEQ ID N0:242) derived from the
coding sequence of SEQ
ID N0:241 shown in Figure 241.
Figure 243 shows a nucleotide sequence (SEQ ID N0:243) of a native sequence
PR01128 cDNA,
wherein SEQ ID N0:243 is a clone designated herein as "DNA59855-1485".
Figure 244 shows the amino acid sequence (SEQ ID N0:244) derived from the
coding sequence of SEQ
ID N0:243 shown in Figure 243.
Figure 245 shows a nucleotide sequence (SEQ ID N0:245) of a native sequence
PR01248 cDNA,
wherein SEQ ID N0:245 is a clone designated herein as "DNA60278-1530".
Figure 246 shows the amino acid sequence (SEQ ID N0:246) derived from the
coding sequence of SEQ
ID N0:245 shown in Figure 245.
Figure 247 shows a nucleotide sequence (SEQ ID N0:247) of a native sequence
PR01127 cDNA,
wherein SEQ ID N0:247 is a clone designated herein as "DNA60283-1484".
Figure 248 shows the amino acid sequence (SEQ ID N0:248) derived from the
coding sequence of SEQ
ID N0:247 shown in Figure 247.
Figure 249 shows a nucleotide sequence (SEQ ID N0:249) of a native sequence
PR01316 cDNA,
wherein SEQ ID N0:249 is a clone designated herein as "DNA60608-1577".
Figure 250 shows the amino acid sequence (SEQ ID N0:250) derived from the
coding sequence of SEQ
ID N0:249 shown in Figure 249.
Figure 251 shows a nucleotide sequence (SEQ ID N0:251) of a native sequence
PR01197 cDNA,
wherein SEQ ID N0:251 is a clone designated herein as "DNA60611-1524".
Figure 252 shows the amino acid sequence (SEQ ID N0:252) derived from the
coding sequence of SEQ
ID N0:251 shown in Figure 251.
Figure 253 shows a nucleotide sequence (SEQ ID N0:253) of a native sequence
PR01125 cDNA,
wherein SEQ ID N0:253 is a clone designated herein as "DNA60619-1482".
Figure 254 shows the amino acid sequence (SEQ ID N0:254) derived from the
coding sequence of SEQ
ID N0:253 shown in Figure 253.
Figure 255 shows a nucleotide sequence (SEQ ID N0:255) of a native sequence
PR01158 cDNA,
wherein SEQ ID N0:255 is a clone designated herein as "DNA60625-1507".
Figure 256 shows the amino acid sequence (SEQ ID N0:256) derived from the
coding sequence of SEQ
ID N0:255 shown in Figure 255.
Figure 257 shows a nucleotide sequence (SEQ ID N0:257) of a native sequence
PR01124 cDNA,
wherein SEQ ID N0:257 is a clone designated herein as "DNA60629-1481".
19


CA 02534018 2001-02-28
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Figure 258 shows the amino acid sequence (SEQ ID N0:258) derived from the
coding sequence of SEQ
ID N0:257 shown in Figure 257.
Figure 259 shows a nucleotide sequence (SEQ ID N0:259) of a native sequence
PR01380 cDNA,
wherein SEQ ID N0:259 is a clone designated herein as "DNA60740-1615".
Figure 260 shows the amino acid sequence (SEQ ID N0:260) derived from the
coding sequence of SEQ
ID N0:259 shown in Figure 259.
Figure 261 shows a nucleotide sequence (SEQ ID N0:261) of a native sequence
PR01377 cDNA,
wherein SEQ ID N0:261 is a clone designated herein as "DNA61608-1606".
Figure 262 shows the amino acid sequence (SEQ ID N0:262) derived from the
coding sequence of SEQ
ID N0:261 shown in Figure 261.
Figure 263 shows a nucleotide sequence (SEQ ID N0:263) of a native sequence
PR01287 cDNA,
wherein SEQ ID N0:263 is a clone designated herein as "DNA61755-1554" .
Figure 264 shows the amino acid sequence (SEQ ID N0:264) derived from the
coding sequence of SEQ
ID N0:263 shown in Figure 263.
Figure 265 shows a nucleotide sequence (SEQ ID N0:265) of a native sequence
PR01249 cDNA,
1S wherein SEQ ID N0:265 is a clone designated herein as "DNA62809-1531".
Figure 266 shows the amino acid sequence (SEQ ID N0:266) derived from the
coding sequence of SEQ
ID N0:265 shown in Figure 265.
Figure 267 shows a nucleotide sequence (SEQ ID N0:267) of a native sequence
PR01335 cDNA,
wherein SEQ ID N0:267 is a clone designated herein as "DNA62812-1594".
Figure 268 shows the amino acid sequence (SEQ ID N0:268) derived from the
coding sequence of SEQ
ID N0:267 shown in Figure 267.
Figure 269 shows a nucleotide sequence (SEQ ID N0:269) of a native sequence
PR03572 cDNA,
wherein SEQ ID N0:269 is a clone designated herein as "DNA62813-2544".
Figure 270 shows the amino acid sequence (SEQ ID N0:270) derived from the
coding sequence of SEQ
2S ID N0:269 shown in Figure 269.
Figure 271 shows a nucleotide sequence (SEQ ID N0:271) of a native sequence
PR01599 cDNA,
wherein SEQ ID N0:271 is a clone designated herein as "DNA62845-1684",
Figure 272 shows the amino acid sequence (SEQ ID N0:272) derived from the
coding sequence of SEQ
ID N0:271 shown in Figure 271.
Figure 273 shows a nucleotide sequence (SEQ ID N0:273) of a native sequence
PR01374 cDNA,
wherein SEQ ID N0:273 is a clone designated herein as "DNA64849-1604".
Figure 274 shows the amino acid sequence (SEQ ID N0:274) derived from the
coding sequence of SEQ
ID N0:273 shown in Figure 273.
Figure 275 shows a nucleotide sequence (SEQ ID N0:275) of a native sequence
PR01345 cDNA,
wherein SEQ ID N0:275 is a clone designated herein as "DNA64852-1589".
Figure 276 shows the amino acid sequence (SEQ ID N0:276) derived from the
coding sequence of SEQ
ID N0:275 shown in Figure 275.


CA 02534018 2001-02-28
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Figure 277 shows a nucleotide sequence (SEQ ID N0:277) of a native sequence
PR01311 cDNA,
wherein SEQ ID N0:277 is a clone designated herein as "DNA64863-1573".
Figure 278 shows the amino acid sequence (SEQ ID N0:278) derived from the
coding sequence of SEQ
ID N0:277 shown in Figure 277.
Figure 279 shows a nucleotide sequence (SEQ ID N0:279) of a native sequence
PR01357 cDNA,
wherein SEQ ID N0:279 is a clone designated herein as "DNA64881-1602".
Figure 280 shows the amino acid sequence (SEQ ID N0:280) derived from the
coding sequence of SEQ
ID N0:279 shown in Figure 279.
Figure 281 shows a nucleotide sequence (SEQ ID N0:281) of a native sequence
PR01557 cDNA,
wherein SEQ ID N0:281 is a clone designated herein as "DNA64902-1667".
Figure 282 shows the amino acid sequence (SEQ ID N0:282) derived from the
coding sequence of SEQ
ID N0:281 shown in Figure 281.
Figure 283 shows a nucleotide sequence (SEQ ID N0:283) of a native sequence
PR01305 cDNA,
wherein SEQ ID N0:283 is a clone designated herein as "DNA64952-1568".
Figure 284 shows the amino acid sequence (SEQ ID N0:284) derived from the
coding sequence of SEQ
ID N0:283 shown in Figure 283.
Figure 28S shows a nucleotide sequence (SEQ ID N0:285) of a native sequence
PR01302 cDNA,
wherein SEQ ID N0:285 is a clone designated herein as "DNA65403-1565".
Figure 286 shows the amino acid sequence (SEQ ID N0:286) derived from the
coding sequence of SEQ
ID N0:285 shown in Figure 285.
Figure 287 shows a nucleotide sequence (SEQ ID N0:287) of a native sequence
PR01266 cDNA,
wherein SEQ ID N0:287 is a clone designated herein as "DNA65413-1534".
Figure 288 shows the amino acid sequence (SEQ ID N0:288) derived from the
coding sequence of SEQ
ID N0:287 shown in Figure 287.
Figures 289A-289B show a nucleotide sequence (SEQ ID N0:289) of a native
sequence PR01336
cDNA, wherein SEQ ID N0:289 is a clone designated herein as "DNA65423-1595".
Figure 290 shows the amino acid sequence (SEQ ID N0:290) derived from the
coding sequence of SEQ
ID N0:289 shown in Figures 289A-289B.
Figure 291 shows a nucleotide sequence (SEQ ID N0:291) of a native sequence
PR01278 cDNA,
wherein SEQ ID N0:291 is a clone designated herein as "DNA66304-1546".
Figure 292 shows the amino acid sequence (SEQ ID N0:292) derived from the
coding sequence of SEQ
ID N0:291 shown in Figure 291.
Figure 293 shows a nucleotide sequence (SEQ ID N0:293) of a native sequence
PR01270 cDNA,
wherein SEQ ID N0:293 is a clone designated herein as "DNA66308-1537".
Figure 294 shows the amino acid sequence (SEQ ID N0:294) derived from the
coding sequence of SEQ
ID N0:293 shown in Figure 293.
Figure 295 shows a nucleotide sequence (SEQ ID N0:295) of a native sequence
PR01298 cDNA,
wherein SEQ ID N0:295 is a clone designated herein as "DNA66511-1563".
21


CA 02534018 2001-02-28
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Figure 296 shows the amino acid sequence (SEQ LD N0:296) derived from the
coding sequence of SEQ
ID N0:295 shown in Figure 295.
Figure 297 shows a nucleotide sequence (SEQ ID N0:297) of a native sequence
PR01301 cDNA,
wherein SEQ ID N0:297 is a clone designated herein as "DNA66512-1564".
Figure 298 shows the amino acid sequence (SEQ ID N0:298) derived from the
coding sequence of SEQ
ID N0:297 shown in Figure 297.
Figure 299 shows a nucleotide sequence (SEQ ID N0:299) of a native sequence
PR01268 cDNA,
wherein SEQ ID N0:299 is a clone designated herein as "DNA66519-1535".
Figure 300 shows the amino acid sequence (SEQ ID N0:300) derived from the
coding sequence of SEQ
ID N0:299 shown in Figure 299.
Figure 301 shows a nucleotide sequence (SEQ ID N0:301) of a native sequence
PR01327 cDNA,
wherein SEQ m N0:301 is a clone designated herein as "DNA66521-1583".
Figure 302 shows the amino acid sequence (SEQ ID N0;302) derived from the
coding sequence of SEQ
ID N0:301 shown in Figure 301.
Figure 303 shows a nucleotide sequence (SEQ ID N0:303) of a native sequence
PR01328 cDNA,
ZS wherein SEQ ID N0:303 is a clone designated herein as "DNA66658-1584".
Figure 304 shows the amino acid sequence (SEQ ID N0:304) derived from the
coding sequence of SEQ
ID N0:303 shown in Figure 303.
Figure 305 shows a nucleotide sequence (SEQ ID N0:305) of a native sequence
PR01329 cDNA,
wherein SEQ ID N0:305 is a clone designated herein as "DNA66660-1585".
Figure 306 shows the amino acid sequence (SEQ ID N0:306) derived from the
coding sequence of SEQ
ID N0:305 shown in Figure 305.
Figure 307 shows a nucleotide sequence (SEQ ID N0:307) of a native sequence
PR01339 cDNA,
wherein SEQ ID N0:307 is a clone designated herein as "DNA66669-1597".
Figure 308 shows the amino acid sequence (SEQ ID N0:308) derived from the
coding sequence of SEQ
ID N0:307 shown in Figure 307.
Figure 309 shows a nucleotide sequence (SEQ ID N0:309) of a native sequence
PR01342 cDNA,
wherein SEQ 1D N0:309 is a clone designated herein as "DNA66674-1599".
Figure 310 shows the amino acid sequence (SEQ ID N0:310) derived from the
coding sequence of SEQ
ID N0:309 shown in Figure 309.
Figures 311A-311B show a nucleotide sequence (SEQ ID N0:311) of a native
sequence PR01487
cDNA, wherein SEQ ID N0:311 is a clone designated herein as "DNA68836-1656".
Figure 312 shows the amino acid sequence (SEQ ID N0:312) derived from the
coding sequence of SEQ
ID N0:311 shown in Figures 311A-311B.
Figure 313 shows a nucleotide sequence (SEQ ID N0:313) of a native sequence
PR03579 cDNA,
wherein SEQ ID N0:313 is a clone designated herein as "DNA68862-2546".
Figure 314 shows the amino acid sequence (SEQ ID N0:314) derived from the
coding sequence of SEQ
ID N0:313 shown in Figure 313.
22


CA 02534018 2001-02-28
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Figure 315 shows a nucleotide sequence (SEQ ID N0:315) of a native sequence
PR01472 cDNA,
wherein SEQ ID N0:315 is a clone designated herein as "DNA68866-1644",
Figure 316 shows the amino acid sequence (SEQ ID N0:316) derived from the
coding sequence of SEQ
ID N0:315 shown in Figure 315.
Figure 317 shows a nucleotide sequence (SEQ ID N0:317) of a native sequence
PR01385 cDNA,
wherein SEQ ID N0:317 is a clone designated herein as "DNA68869-1610".
Pigure 318 shows the amino acid sequence (SEQ ID N0:318) derived from the
coding sequence of SEQ
ID N0:317 shown in Figure 317.
Figure 319 shows a nucleotide sequence (SEQ ID N0:319) of a native sequence
PR01461 cDNA,
wherein SEQ ID N0:319 is a clone designated herein as "DNA68871-1638".
Figure 320 shows the amino acid sequence (SEQ ID N0:320) derived from the
coding sequence of SEQ
ID N0:319 shown in Figure 319.
Figure 321 shows a nucleotide sequence (SEQ ID N0:321) of a native sequence
PR01429 cDNA,
wherein SEQ ID N0:321 is a clone designated herein as "DNA68879-1631".
Figure 322 shows the amino acid sequence (SEQ ID N0:322) derived from the
coding sequence of SEQ
)D N0:321 shown in Figure 321.
Figure 323 shows a nucleotide sequence (SEQ ID N0:323) of a native sequence
PR01568 cDNA,
wherein SEQ ID N0:323 is a clone designated herein as "DNA68880-1676".
Figure 324 shows the amino acid sequence (SEQ ID N0:324) derived from the
coding sequence of SEQ
ID N0:323 shown in Figure 323.
Figure 325 shows a nucleotide sequence (SEQ ID N0:325) of a native sequence
PR01569 cDNA,
wherein SEQ ID N0:325 is a clone designated herein as "DNA68882-1677".
Figure 326 shows the amino acid sequence (SEQ ID N0:326) derived from the
coding sequence of SEQ
ID N0:325 shown in Figure 325.
Figure 327 shows a nucleotide sequence (SEQ ID N0:327) of a native sequence
PR01753 cDNA,
wherein SEQ ID N0:327 is a clone designated herein as "DNA68883-1691".
Figure 328 shows the amino acid sequence (SEQ ID N0:328) derived from the
coding sequence of SEQ
ID N0:327 shown in Figure 327.
Figure 329 shows a nucleotide sequence (SEQ ID N0:329) of a native sequence
PR01570 cDNA,
wherein SEQ ID N0:329 is a clone designated herein as "DNA68885-1678".
Figure 330 shows the amino acid sequence (SEQ ID N0:330) derived from the
coding sequence of SEQ
ID N0:329 shown in Figure 329.
Figure 331 shows a nucleotide sequence (SEQ ID N0:331) of a native sequence
PR01559 cDNA,
wherein SEQ ID N0:331 is a clone designated herein as "DNA68886".
Figure 332 shows the amino acid sequence (SEQ ID N0:332) derived from the
coding sequence of SEQ
ID N0:331 shown in Figure 331.
Figure 333 shows a nucleotide sequence (SEQ ID N0:333) of a native sequence
PR01486 cDNA,
wherein SEQ ID N0:333 is a clone designated herein as "DNA71180-1655".
23


CA 02534018 2001-02-28
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Figure 334 shows the amino acid sequence (SEQ TD N0:334) derived from the
coding sequence of SEQ
ID N0:333 shown in Figure 333.
Figure 335 shows a nucleotide sequence (SEQ 1D N0:335) of a native sequence
PR01433 cDNA,
wherein SEQ ID N0:335 is a clone designated herein as "DNA71184-1634".
Figure 336 shows the amino acid sequence (SEQ ID N0:336) derived from the
coding sequence of SEQ
ID N0:335 shown in Figure 335.
Figure 337 shows a nucleotide sequence (SEQ ID N0:337) of a native sequence
PR01490 cDNA,
wherein SEQ 1D N0:337 is a clone designated herein as "DNA71213-1659".
Figure 338 shows the amino acid sequence (SEQ ID N0:338) derived from the
coding sequence of SEQ
ID N0:337 shown in Figure 337.
Figure 339 shows a nucleotide sequence (SEQ ID N0:339) of a native sequence
PR01482 cDNA,
wherein SEQ ID N0:339 is a clone designated herein as "DNA71234-1651".
Figure 340 shows the amino acid sequence (SEQ )D N0:340) derived from the
coding sequence of SEQ
ID N0:339 shown in Figure 339.
Figure 341 shows a nucleotide sequence (SEQ ID N0:341) of a native sequence
PR01449 cDNA,
wherein SEQ ID N0:341 is a clone designated herein as "DNA71269-1621".
Figure 342 shows the amino acid sequence (SEQ ID N0:342) derived from the
coding sequence of SEQ
ID N0:341 shown in Figure 341.
Figure 343 shows a nucleotide sequence (SEQ ID N0:343) of a native sequence
PR01446 cDNA,
wherein SEQ ID N0:343 is a clone designated herein as "DNA71277-1636".
Figure 344 shows the amino acid sequence (SEQ ID N0:344) derived from the
coding sequence of SEQ
ID N0:343 shown in Figure 343.
Figure 345 shows a nucleotide sequence (SEQ ID N0:345) of a native sequence
PR01604 cDNA,
wherein SEQ ID N0:345 is a clone designated herein as "DNA71286-1687".
Figure 346 shows the amino acid sequence (SEQ ID N0:346) derived from the
coding sequence of SEQ
ID N0:345 shown in Figure 345.
Figure 347 shows a nucleotide sequence (SEQ ID N0:347) of a native sequence
PR01491 cDNA,
wherein SEQ ID N0:347 is a clone designated herein as "DNA71883-1660".
Figure 348 shows the amino acid sequence (SEQ )D N0:348) derived from the
coding sequence of SEQ
ID N0:347 shown in Figure 347.
Figure 349 shows a nucleotide sequence (SEQ ID N0:349) of a native sequence
PR01431 cDNA,
wherein SEQ )D N0:349 is a clone designated herein as "DNA73401-1633".
Figure 350 shows the amino acid sequence (SEQ ID N0:350) derived from the
coding sequence of SEQ
ID N0:349 shown in Figure 349.
Figures 351A-351B show a nucleotide sequence (SEQ ID N0:351) of a native
sequence PR01563
cDNA, wherein SEQ ID N0:351 is a clone designated herein as "DNA73492-1671".
Figure 3S2 shows the amino acid sequence (SEQ ID N0:352) derived from the
coding sequence of SEQ
ID N0:351 shown in Figures 351A-351B.
24


CA 02534018 2001-02-28
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Figure 353 shows a nucleotide sequence (SEQ ID N0:353) of a native sequence
PR01571 eDNA,
wherein SEQ ID N0:353 is a clone designated herein as "DNA73730-1679".
Figure 354 shows the amino acid sequence (SEQ ID N0:354) derived from the
coding sequence of SEQ
ID N0:353 shown in Figure 353.
Figure 355 shows a nucleotide sequence (SEQ ID N0:355) of a native sequence
PR01572 eDNA,
wherein SEQ )D N0:35S is a clone designated herein as "DNA73734-1680".
Figure 356 shows the amino acid sequence (SEQ ID N0:356) derived from the
coding sequence of SEQ
ID N0:355 shown in Figure 355.
Figure 357 shows a nucleotide sequence (SEQ ID N0:357) of a native sequence
PR01573 eDNA,
wherein SEQ ID N0:357 is a clone designated herein as "DNA73735-1681".
Figure 358 shows the amino acid sequence (SEQ ID N0:358) derived from
the~coding sequence of SEQ
ID N0:357 shown in Figure 357.
Figure 359 shows a nucleotide sequence (SEQ ID N0:359) of a native sequence
PR01508 eDNA,
wherein SEQ ID N0:359 is a clone designated herein as "DNA73742-1662".
Figure 360 shows the amino acid sequence (SEQ ID N0:360) derived from the
coding sequence of SEQ
ID N0:359 shown in Figure 359.
Figure 361 shows a nucleotide sequence (SEQ ID N0:361) of a native sequence
PR0148S cDNA,
wherein SEQ U~ N0:361 is a clone designated herein as "DNA73746-1654".
Figure 362 shows the amino acid sequence (SEQ ID N0:362) derived from the
coding sequence of SEQ
ID N0:361 shown in Figure 361.
Figure 363 shows a nucleotide sequence (SEQ ID N0:363) of a native sequence
PR01564 cDNA,
wherein SEQ ID N0:363 is a clone designated herein as "DNA73760-1672".
Figure 364 shows the amino acid sequence (SEQ ID N0:364) derived from the
coding sequence of SEQ
ID N0:363 shown in Figure 363.
Figure 365 shows a nucleotide sequence (SEQ ID N0:365) of a native sequence
PR01550 cDNA,
wherein SEQ ID N0:365 is a clone designated herein as "DNA76393-1664".
Figure 366 shows the amino acid sequence (SEQ ID N0:366) derived from the
coding sequence of SEQ
ID N0:365 shown in Figure 365.
Figure 367 shows a nucleotide sequence (SEQ ID N0:367) of a native sequence
PR01757 eDNA,
wherein SEQ ID N0:367 is a clone designated herein as "DNA76398-1699".
Figure 368 shows the amino acid sequence (SEQ ID N0:368) derived from the
coding sequence of SEQ
ID N0:367 shown in Figure 367.
Figure 369 shows a nucleotide sequence (SEQ ID N0:369) of a native sequence
PR01758 cDNA,
wherein SEQ ID N0:369 is a clone designated herein as "DNA76399-1700".
Figure 370 shows the amino acid sequence (SEQ ID N0:370) derived from the
coding sequence of SEQ
ID N0:369 shown in Figure 369.
Figure 371 shows a nucleotide sequence (SEQ ID N0:371) of a native sequence
PR01781 cDNA,
wherein SEQ ID N0:371 is a clone designated herein as "DNA76522-2500".


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Figure 372 shows the amino acid sequence (SEQ ID N0:372) derived from the
coding sequence of SEQ
ID N0:371 shown in Figure 371.
Figure 373 shows a nucleotide sequence (SEQ ID N0:373) of a native sequence
PR01606 cDNA,
wherein SEQ ID N0:373 is a clone designated herein as "DNA76533-1689".
Figure 374 shows the amino acid sequence (SEQ ID N0:374) derived from the
coding sequence of SEQ
ID N0:373 shown in Figure 373.
Figure 375 shows a nucleotide sequence (SEQ ID N0:375) of a native sequence
PR01784 cDNA,
wherein SEQ ID N0:375 is a clone designated herein as "DNA77303-2502".
Figure 376 shows the amino acid sequence (SEQ ID N0:376) derived from the
coding sequence of SEQ
ID N0:375 shown in Figure 375.
Figure 377 shows a nucleotide sequence (SEQ ID N0:377) of a native sequence
PR01774 cDNA,
wherein SEQ )D N0:377 is a clone designated herein as "DNA77626-1705".
Figure 378 shows the amino acid sequence (SEQ ID N0:378) derived from the
coding sequence of SEQ
ID N0:377 shown in Figure 377.
Figure 379 shows a nucleotide sequence (SEQ ID N0:379) of a native sequence
PR01605 cDNA,
wherein SEQ ID N0:379 is a clone designated herein as "DNA77648-1688".
Figure 380 shows the amino acid sequence (SEQ ID N0:380) derived from the
coding sequence of SEQ
ID N0:379 shown in Figure 379.
Figure 381 shows a nucleotide sequence (SEQ ID N0:381) of a native sequence
PR01928 cDNA,
wherein SEQ ID N0:381 is a clone designated herein as "DNA81754-2532".
Figure 382 shows the amino acid sequence (SEQ )D N0:382) derived from the
coding sequence of SEQ
ID N0:381 shown in Figure 381.
Figure 383 shows a nucleotide sequence (SEQ ID N0:383) of a native sequence
PR01865 cDNA,
wherein SEQ ID N0:383 is a clone designated herein as "DNA81757-2512".
Figure 384 shows the amino acid sequence (SEQ ID N0:384) derived from the
coding sequence of SEQ
ID N0:383 shown in Figure 383.
Figure 385 shows a nucleotide sequence (SEQ ID N0:385) of a native sequence
PR01925 cDNA,
wherein SEQ ID N0:385 is a clone designated herein as "DNA82302-2529".
Figure 386 shows the amino acid sequence (SEQ ID N0:386) derived from the
coding sequence of SEQ
ID N0:385 shown in Figure 385.
Figure 387 shows a nucleotide sequence (SEQ ID N0:387) of a native sequence
PR01926 cDNA,
wherein SEQ ID N0:387 is a clone designated herein as "DNA82340-2530" .
Figure 388 shows the amino acid sequence (SEQ ID N0:388) derived from the
coding sequence of SEQ
ID N0:387 shown in Figure 387.
Figure 389 shows a nucleotide sequence (SEQ ID N0:389) of a native sequence
PR02630 cDNA,
wherein SEQ ID N0:389 is a clone designated herein as "DNA83551".
Figure 390 shows the amino acid sequence (SEQ ID N0:390) derived from the
coding sequence of SEQ
ID N0:389 shown in Figure 389.
26


CA 02534018 2001-02-28
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Figure 391 shows a nucleotide sequence (SEQ ID N0:391) of a native sequence
PR03443 cDNA,
wherein SEQ ID N0:391 is a clone designated herein as "DNA87991-2540".
Figure 392 shows the amino acid sequence (SEQ ID N0:392) derived from the
coding sequence of SEQ
)D N0:391 shown in Figure 391.
Figure 393 shows a nucleotide sequence (SEQ ID N0:393) of a native sequence
PR03301 cDNA,
wherein SEQ ID N0:393 is a clone designated herein as "DNA88002".
Figure 394 shows the amino acid sequence (SEQ ID N0:394) derived from the
coding sequence of SEQ
ID N0:393 shown in Figure 393.
Figure 395 shows a nucleotide sequence (SEQ ID N0:395) of a native sequence
PR03442 cDNA,
wherein SEQ ID N0:395 is a clone designated herein as "DNA92238-2539".
Figure 396 shows the amino acid sequence (SEQ ID N0:396) derived from the
coding sequence of SEQ
ID N0:395 shown in Figure 395.
Figure 397 shows a nucleotide sequence (SEQ ID N0:397) of a native sequence
PR04978 cDNA,
wherein SEQ ID N0:397 is a clone designated herein as "DNA95930".
Figure 398 shows the amino acid sequence (SEQ ID N0:398) derived from the
coding sequence of SEQ
ID N0:397 shown in Figure 397.
Figure 399 shows a mxcleotide sequence (SEQ ID N0:399) of a native sequence
PR05801 cDNA,
wherein SEQ ID N0:399 is a clone designated herein as "DNA115291-2681".
Figure 400 shows the amino acid sequence (SEQ ID N0:400) derived from the
coding sequence of SEQ
ID N0:399 shown in Figure 399.
Figure 401 shows a nucleotide sequence (SEQ ID N0:401) of a native sequence
PR019630 cDNA,
wherein SEQ ID N0:401 is a clone designated herein as "DNA23336-2861".
Figure 402 shows the amino acid sequence (SEQ ID N0:402) derived from Lhe
coding sequence of SEQ
ID N0:401 shown in Figure 401.
Figure 403 shows a nucleotide sequence (SEQ ID N0:403) of a native sequence
PR0203 cDNA, wherein
SEQ )D N0:403 is a clone designated herein as "DNA30862-1396".
Figure 404 shows the amino acid sequence (SEQ ID N0:404) derived from the
coding sequence of SEQ
ID N0:403 shown in Figure 403.
Figure 405 shows a nucleotide sequence (SEQ ID N0:405) of a native sequence
PR0204 cDNA, wherein
SEQ ID N0:405 is a clone designated herein as "DNA30871-1157".
Figure 406 shows the amino acid sequence (SEQ ID N0:406) derived from the
coding sequence of SEQ
ID N0:405 shown in Pigure 405.
Figure 407 shows a nucleotide sequence (SEQ ID N0:407) of a native sequence
PR0210 cDNA, wherein
SEQ ID N0:407 is a clone designated herein as "DNA32279-1131".
Figure 408 shows the amino acid sequence (SEQ ID N0:408) derived from the
coding sequence of SEQ
ID N0:407 shown in Figure 407.
Figure 409 shows a nucleotide sequence (SEQ ID N0:409) of a native sequence
PR0223 cDNA, wherein
SEQ ID N0:409 is a clone designated herein as "DNA33206-1165".
27


CA 02534018 2001-02-28
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Figure 410 shows the amino acid sequence (SEQ 1D N0:410) derived from the
coding sequence of SEQ
ID N0:409 shown in Figure 409.
Figure 411 shows a nucleotide sequence (SEQ ID N0:411) of a native sequence
PR0247 cDNA, wherein
SEQ ID N0:411 is a clone designated herein as "DNA35673-1201".
Figure 412 shows the amino acid sequence (SEQ ID N0:412) derived from the
coding sequence of SEQ
S ID N0:411 shown in Figure 411.
Figure 413 shows a nucleotide sequence (SEQ ID N0:413) of a native sequence
PR0358 cDNA, wherein
SEQ ID N0:413 is a clone designated herein as "DNA47361-1154-2".
Figure 414 shows the amino acid sequence (SEQ ID N0:414) derived from the
coding sequence of SEQ
ID N0:413 shown in Figure 413.
Figure 415 shows a nucleotide sequence (SEQ ID N0:415) of a native sequence
PR0724 cDNA, wherein
SEQ ID N0:415 is a clone designated herein as "DNA49631-1328".
Figure 416 shows the amino acid sequence (SEQ ID N0:416) derived from the
coding sequence of SEQ
ID N0:415 shown in Figure 415.
Figure 417 shows a nucleotide sequence (SEQ ID N0:417) of a native sequence
PR0868 cDNA, wherein
SEQ ID N0:417 is a clone designated herein as "DNA52594-1270".
Figure 418 shows the amino acid sequence (SEQ ID N0:418) derived from the
coding sequence of SEQ
ID N0:417 shown in Figure 417.
Figure 419 shows a nucleotide sequence (SEQ ID N0:419) of a native sequence
PR0740 cDNA, wherein
SEQ ID N0:419 is a clone designated herein as "DNA55800-1263".
Figure 420 shows the amino acid sequence (SEQ ID N0:420) derived from the
coding sequence of SEQ
ID N0:419 shown in Figure 419.
Figure 421 shows a nucleotide sequence (SEQ ID N0:421) of a native sequence
PR01478 cDNA,
wherein SEQ ID N0:421 is a clone designated herein as "DNA56531-1648".
Figure 422 shows the amino acid sequence (SEQ ID N0:422) derived from the
coding sequence of SEQ
B7 N0:421 shown in Figure 421.
Figvre 423 shows a nucleotide sequence (SEQ ID N0:423) of a native sequence
PR0162 cDNA, wherein
SEQ ID N0:423 is a clone designated herein as "DNA56965-1356".
Figure 424 shows the amino acid sequence (SEQ ID N0:424) derived from the
coding sequence of SEQ
ID N0:423 shown in Figure 423.
Figure 42S shows a nucleotide sequence (SEQ ID N0:425) of a native sequence
PR0828 cDNA, wherein
SEQ ID N0:425 is a clone designated herein as "DNA57037-1444".
Figure 426 shows the amino acid sequence (SEQ ID N0:426) derived from the
coding sequence of SEQ
ID N0:425 shown in Figure 425.
Figure 427 shows a nucleotide sequence (SEQ ID N0:427) of a native sequence
PR0819 cDNA, wherein
SEQ ID N0:427 is a clone designated herein as "DNA57695-1340".
Figure 428 shows the amino acid sequence (SEQ ID N0:428) derived from the
coding sequence of SEQ
ID N0:427 shown in Figure 427.
28


CA 02534018 2001-02-28
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Figure 429 shows a nucleotide sequence (SEQ ID N0:429) of a native sequence
PR0813 cDNA, wherein
SEQ ID N0:429 is a clone designated herein as "DNA57834-1339".
Figure 430 shows the amino acid sequence (SEQ ID N0:430) derived from the
coding sequence of SEQ
ID N0:429 shown in Figure 429.
Figure 431 shows a nucleotide sequence (SEQ ID N0:431) of a native sequence
PR01194 cDNA,
wherein SEQ ID N0:431 is a clone designated herein as "DNA57841-1522".
Figure 432 shows the amino acid sequence (SEQ ID N0:432) derived from the
coding sequence of SEQ
ID N0:431 shown in Figure 431.
Figure 433 shows a nucleotide sequence (SEQ ID N0:433) of a native sequence
PR0887 cDNA, wherein
SEQ ID N0:433 is a clone designated herein as "DNA58130".
Figure 434 shows the amino acid sequence (SEQ ID N0:434) derived from the
coding sequence of SEQ
ID N0:433 shown in Figure 433.
Figure 435 shows a nucleotide sequence (SEQ ID N0:435) of a native sequence
PR01071 cDNA,
wherein SEQ ID N0:435 is a clone designated herein as "DNA58847-1383".
Figure 436 shows the amino acid sequence (SEQ ID N0:436) derived from the
coding sequence of SEQ
ID N0:435 shown in Figure 435.
Figure 437 shows a nucleotide sequence (SEQ ID N0:437) of a native sequence
PR01029 cDNA,
wherein SEQ ID N0:437 is a clone designated herein as "DNA59493-1420".
Figure 438 shows the amino acid sequence (SEQ ID N0:438) derived from the
coding sequence of SEQ
ID N0:437 shown in Figure 437.
Figure 439 shows a nucleotide sequence (SEQ ID N0:439) of a native sequence
PR01190 cDNA,
wherein SEQ ID N0:439 is a clone designated herein as "DNA59586-1520".
Figure 440 shows the amino acid sequence (SEQ ID N0:440) derived from the
coding sequence of SEQ
ID N0:439 shown in Figure 439.
Figure 441 shows a nucleotide sequence (SEQ ID N0:441) of a native sequence
PR04334 cDNA,
wherein SEQ ID N0:441 is a clone designated herein as "DNA59608-2577".
Figure 442 shows the amino acid sequence (SEQ )I7 N0:442) derived from the
coding sequence of SEQ
ID N0:441 shown in Figure 441.
Figure 443 shows a nucleotide sequence (SEQ ID N0:443) of a native sequence
PR01155 cDNA,
wherein SEQ iD N0:443 is a clone designated herein as "DNA59849-1504".
Figure 444 shows the amino acid sequence (SEQ ID N0:444) derived from the
coding sequence of SEQ
ID N0:443 shown in Figure 443.
Figure 445 shows a nucleotide sequence (SEQ ID N0:445) of a native sequence
PR01157 cDNA,
wherein SEQ ID N0:445 is a clone designated herein as "DNA60292-1506".
Figure 446 shows the amino acid sequence (SEQ ID N0:446) derived from the
coding sequence of SEQ
ID N0:445 shown in Figure 445.
Figure 447 shows a nucleotide sequence (SEQ 1D N0:447) of a native sequence
PROI122 cDNA,
wherein SEQ ID N0:447 is a clone designated herein as "DNA62377-1381-1".
29


CA 02534018 2001-02-28
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Figure 448 shows the amino acid sequence (SEQ ID N0:448) derived from the
coding sequence of SEQ
ID N0:447 shown in Figure 447.
Figure 449 shows a nucleotide sequence (SEQ ID N0:449) of a native sequence
PR01183 cDNA,
wherein SEQ ID N0:449 is a clone designated herein as "DNA62880-1513".
Figure 450 shows the amino acid sequence (SEQ ID N0:450) derived from the
coding sequence of SEQ
ID N0:449 shown in Figure 449.
Figure 451 shows a nucleotide sequence (SEQ ID NO_:451) of a native sequence
PR01337 cDNA,
wherein SEQ 1D N0:451 is a clone designated herein as "DNA66672-1586".
Figure 452 shows the amino acid sequence (SEQ ID N0:452) derived from the
coding sequence of SEQ
ID N0:451 shown in Figure 451.
Figure 453 shows a nucleotide sequence (SEQ ID N0:453) of a native sequence
PR01480 cDNA,
wherein SEQ m N0:453 is a clone designated herein as "DNA67962-1649".
Figure 454 shows the amino acid sequence (SEQ ID N0:454) derived from the
coding sequence of SEQ
ID N0:453 shown in Figure 453.
Figure 45S shows a nucleotide sequence (SEQ ID N0:455) of a native sequence
PR019645 cDNA,
wherein SEQ ID N0:455 is a clone designated herein as "DNA69555-2867".
Figure 456 shows the amino acid sequence (SEQ 1D N0:456) derived from the
coding sequence of SEQ
ID N0:455 shown in Figure 455.
Figure 457 shows a nucleotide sequence (SEQ ID N0:457) of a native sequence
PR09782 cDNA,
wherein SEQ ID N0:457 is a clone designated herein as "DNA71162-2764" .
Figure 458 shows the amino acid sequence (SEQ ID N0:458) derived from the
coding sequence of SEQ
ID N0:457 shown in Figure 457.
Figure 459 shows a nucleotide sequence (SEQ ID N0:459) of a native sequence
PR01419 cDNA,
wherein SEQ ID N0:459 is a clone designated herein as "DNA71290-1630".
Figure 460 shows the amino acid sequence (SEQ ID N0:460) derived from the
coding sequence of SEQ
ID N0:459 shown in Figure 459.
Figure 4b1 shows a nucleotide sequence (SEQ ID N0:461) of a native sequence
PR01575 cDNA,
wherein SEQ ID N0:461 is a clone designated herein as "DNA76401-1683".
Figure 462 shows the amino acid sequence (SEQ ID N0:462) derived from the
coding sequence of SEQ
ID N0:461 shown in Figure 461.
Figure 463 shows a nucleotide sequence (SEQ ID N0:463) of a native sequence
PR01567 cDNA,
wherein SEQ ID N0:463 is a clone designated herein as "DNA76541-1675".
Figure 464 shows the amino acid sequence (SEQ ID N0:464) derived from the
coding sequence of SEQ
ID N0:463 shown in Figure 463.
Figure 465 shows a nucleotide sequence (SEQ ID N0:465) of a native sequence
PR01891 cDNA,
wherein SEQ 1D N0:465 is a clone designated herein as "DNA76788-2526".
Figure 466 shows the amino acid sequence (SEQ ID N0:466) derived from the
coding sequence of SEQ
)D N0:465 shown in Figure 465,


CA 02534018 2001-02-28
WO 01/68848 PCT/US01J06i20
Figure 467 shows a nucleotide sequence (SEQ ID N0:467) of a native sequence
PR01889 cDNA,
wherein SEQ ID N0:467 is a clone designated herein as "DNA77623-2524".
Figure 468 shows the amino acid sequence (SEQ ID N0:468) derived from the
coding sequence of SEQ
ID N0:467 shown in Figure 467.
Figure 469 shows a nucleotide sequence (SEQ ID N0:469) of a native sequence
PR01785 cDNA,
wherein SEQ TD N0:469 is a clone designated herein as "DNA80136-2503".
Figure 470 shows the amino acid sequence (SEQ ID N0:470) derived from the
coding sequence of SEQ
ID N0:469 shown in Figure 469.
Figure 471 shows a nucleotide sequence (SEQ ID N0:471) of a native sequence
PR06003 cDNA,
wherein SEQ 1D N0:471 is a clone designated herein as ~DNA83568-2692".
Figure 472 shows the amino acid sequence (SEQ ID N0:472) derived from the
coding sequence of SEQ
ID N0:471 shown in Figure 471.
Figure 473 shows a nucleotide sequence (SEQ ID N0:473) of a native sequence
PR04333 cDNA,
wherein SEQ ID N0:473 is a clone designated herein as "DNA84210-2576".
Figure 474 shows the amino acid sequence (SEQ ID N0:474) derived from the
coding sequence of SEQ
ID N0:473 shown in Figure 473.
Figure 475 shows a nucleotide sequence (SEQ ID N0:475) of a native sequence
PR04356 cDNA,
wherein SEQ 1D N0:475 is a clone designated herein as "DNA86576-2595".
Figure 476 shows the amino acid sequence (SEQ ID N0:476) derived from the
coding sequence of SEQ
ID N0:475 shown in Figure 475.
Figure 477 shows a nucleotide sequence (SEQ ID N0:477) of a native sequence
PR04352 cDNA,
wherein SEQ ID N0:477 is a clone designated herein as ~DNA87976-2593".
Figure 478 shows the amino acid sequence (SEQ ID N0:478) derived from the
coding sequence of SEQ
ID N0:477 shown in Figure 477.
Figure 479 shows a nucleotide sequence (SEQ ID N0:479) of a native sequence
PR04354 cDNA,
wherein SEQ ID N0:479 is a clone designated herein as "DNA92256-2596".
Figure 480 shows the amino acid sequence (SEQ ID N0:480) derived from the
coding sequence of SEQ
ID N0:479 shown in Figure 479.
Figure 48I shows a nucleotide sequence (SEQ ID N0:481) of a native sequence
PR04369 cDNA,
wherein SEQ ID N0:481 is a clone designated herein as "DNA92289-2598".
Figure 482 shows the amino acid sequence (SEQ ID N0:482) derived from the
coding sequence of SEQ
ID N0:481 shown in Figure 481.
Figure 483 shows a nucleotide sequence (SEQ ID N0:483) of a native sequence
PR06030 cDNA,
wherein SEQ ID N0:483 is a clone designated herein as "DNA96850-2705".
Figure 484 shows the amino acid sequence (SEQ ID N0:484) derived from the
coding sequence of SEQ
ID N0:483 shown in Figure 483.
Figure 485 shows a nucleotide sequence (SEQ ID N0:485) of a native sequence
PR04433 cDNA,
wherein SEQ ID N0:485 is a clone designated herein as "DNA96855-2629".
31


CA 02534018 2001-02-28
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Figure 486 shows the amino acid sequence (SEQ ID N0:486) derived from the
coding sequence of SEQ
ID N0:485 shown in Figure 485.
Figure 487 shows a nucleotide sequence (SEQ ID N0:487) of a native sequence
PR04424 cDNA,
wherein SEQ ID N0:487 is a clone designated herein as "DNA96857-2636".
Figure 488 shows the amino acid sequence (SEQ >D N0:488) derived from the
coding sequence of SEQ
ID N0:487 shown in Figure 487.
Figure 489 shows a nucleotide sequence (SEQ ID N0:489) of a native sequence
PR06017 cDNA,
wherein SEQ ID N0:489 is a clone designated herein as "DNA96860-2700".
Figure 490 shows the amino acid sequence (SEQ ID N0:490) derived from the
coding sequence of SEQ
ID N0:489 shown in Figure 489.
Figure 491 shows a nucleotide sequence (SEQ ID N0:491) of a native sequence
PR019563 cDNA,
wherein SEQ ID N0:491 is a clone designated herein as "DNA96861-2844".
Figure 492 shows the amino acid sequence (SEQ ID N0:492) derived from the
coding sequence of SEQ
ID N0:491 shown in Figure 491.
Figure 493 shows a nucleotide sequence (SEQ ID N0:493) of a native sequence
PR06015 cDNA,
wherein SEQ ID N0:493 is a clone designated herein as "DNA96866-2698".
Figure 494 shows the amino acid sequence (SEQ ID N0:494) derived from the
coding sequence of SEQ
ID N0:493 shown in Figure 493.
Figure 495 shows a nucleotide sequence (SEQ ID N0:495) of a native sequence
PR05779 cDNA,
wherein SEQ ID N0:495 is a clone designated herein as "DNA96870-2676".
Figure 496 shows the amino acid sequence (SEQ ID N0:496) derived from the
coding sequence of SEQ
ID N0:495 shown in Figure 495.
Figure 497 shows a nucleotide sequence (SEQ ID N0:497) of a native sequence
PR05776 cDNA,
wherein SEQ ID N0:497 is a clone designated herein as "DNA96872-2674".
Figure 498 shows the amino acid sequence (SEQ ID N0:498) derived from the
coding sequence of SEQ
ID N0:497 shown in Figure 497.
Figure 499 shows a nucleotide sequence (SEQ ID N0:499) of a native sequence
PR04430 cDNA,
wherein SEQ ID N0:499 is a clone designated herein as "DNA96878-2626".
Figure 500 shows the amino acid sequence (SEQ ID N0:500) derived from the
coding sequence of SEQ
ID N0:499 shown in Figure 499.
Figure 501 shows a nucleotide sequence (SEQ ID N0:501) of a native sequence
PR04421 cDNA,
wherein SEQ ID N0:501 is a clone designated herein as "DNA96879-2619".
Figure 502 shows the amino acid sequence (SEQ ID N0:502) derived from the
coding sequence of SEQ
ID N0:501 shown in Figure 501.
Figure 503 shows a nucleotide sequence (SEQ ID N0:503) of a native sequence
PR04499 cDNA,
wherein SEQ ID N0:503 is a clone designated herein as "DNA96889-2641".
Figure 504 shows the amino acid sequence (SEQ ID N0:504) derived from the
coding sequence of SEQ
ID N0:503 shown in Figure 503.
32


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/OG520
Figure SOS shows a nucleotide sequence (SEQ ID N0:505) of a native sequence
PR04423 cDNA,
wherein SEQ ID N0:505 is a clone designated herein as "DNA96893-2621".
Figure 506 shows the amino acid sequence (SEQ ID N0:506) derived from the
coding sequence of SEQ
ID NO:SOS shown in Figure 505.
Figure 507 shows a nucleotide sequence (SEQ TD N0:507) of a native sequence
PR05998 cDNA,
wherein SEQ ID N0:507 is a clone designated herein as "DNA96897-2688".
Figure 508 shows the amino acid sequence (SEQ ID N0:508) derived from the
coding sequence of SEQ
ID N0:507 shown in Figure 507.
Figure S09 shows a nucleotide sequence (SEQ ID N0:509) of a native sequence
PR04501 cDNA,
wherein SEQ 1D N0:509 is a clone designated herein as "DNA98564-2643".
Figure 510 shows the amino acid sequence (SEQ ID NO:S10) derived from the
coding sequence of SEQ
ID N0:509 shown in Figure 509.
Figure 511 shows a nucleotide sequence (SEQ ID N0:511) of a native sequence
PR06240 cDNA,
wherein SEQ ID NO:S11 is a clone designated herein as "DNA107443-2718".
Figure 512 shows the amino acid sequence (SEQ ID N0:512) derived from the
coding sequence of SEQ
ID N0:511 shown in Figure 511.
Figure SI3 shows a nucleotide sequence (SEQ ID N0:513) of a native sequence
PR06245 cDNA,
wherein SEQ 1D N0:513 is a clone designated herein as "DNA107786-2723".
Fignre S14 shows the amino acid sequence (SEQ ID N0:514) derived from the
coding sequence of SEQ
ID N0:513 shown in Figure 513.
Figure S15 shows a nucleotide sequence (SEQ 1D N0:515) of a native sequence
PR06175 cDNA,
wherein SEQ ID N0:515 is a clone designated herein as "DNA108682-2712".
Figure 516 shows the amino acid sequence (SEQ ID NO:S16) derived from the
coding sequence of SEQ
ID N0:51S shown in Figure 515.
Figure 517 shows a nucleotide sequence (SEQ 1D N0:517) of a native sequence
PR09742 cDNA,
wherein SEQ ID N0:517 is a clone designated herein as "DNA108684-2761".
Figure 518 shows the amino acid sequence (SEQ ID N0:518) derived from the
coding sequence of SEQ
ID NO:SI7 shown in Figure 517.
Figure 519 shows a nucleotide sequence (SEQ 1D N0:519) of a native sequence
PR07179 cDNA,
wherein SEQ ID N0:519 is a clone designated herein as ~DNA108701-2749".
Figure 520 shows the amino acid sequence (SEQ ID N0:520) derived from the
coding sequence of SEQ
ID N0:519 shown in Figure 519.
Figure 521 shows a nucleotide sequence (SEQ ID N0:521) of a native sequence
PR06239 cDNA,
wherein SEQ ID N0:521 is a clone designated herein as "DNA108720-2717".
Figure 522 shows the amino acid sequence (SEQ ID N0:522) derived from the
coding sequence of SEQ
ID N0:521 shown in Figure 521.
Figure 523 shows a nucleotide sequence (SEQ ID N0:523) of a native sequence
PR06493 cDNA,
wherein SEQ 1D N0:523 is a clone designated herein as "DNA108726-2729".
33


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Figure 524 shows the amino acid sequence (SEQ ID N0:524) derived from the
coding sequence of SEQ
ID N0:523 shown in Figure 523.
Figures 525A-525B show a nucleotide sequence (SEQ 1D N0:525) of a native
sequence PR09741
cDNA, wherein SEQ 1D N0:525 is a clone designated herein as "DNA108728-2760".
Figure 526 shows the amino acid sequence (SEQ ID N0:526) derived from the
coding sequence of SEQ
ID N0:525 shown in Figures 525A-525B.
Figure 527 shows a nucleotide sequence (SEQ ID N0:527) of a native sequence
PR09822 cDNA,
wherein SEQ ID N0:527 is a clone designated herein as "DNA108738-2767".
Figure 528 shows the amino acid sequence (SEQ 1D N0:528) derived from the
coding sequence of SEQ
ID N0:527 shown in Figure 527.
Figure 529 shows a nucleotide sequence (SEQ ID N0:529) of a native sequence
PR06244 cDNA,
wherein SEQ ID N0:529 is a clone designated herein as "DNA108743-2722".
Figure 530 shows the amino acid sequence (SEQ ID N0:530) derived from the
coding sequence of SEQ
ID N0:529 shown in Figure 529.
Figure 531 shows a nucleotide sequence (SEQ 1D N0:531) of a native sequence
PR09740 cDNA,
wherein SEQ ID N0:531 is a clone designated herein as "DNA108758-2759".
Figure 532 shows the amino acid sequence (SEQ ID N0:532) derived from the
coding sequence of SEQ
ID N0:531 shown in Figure 53I.
Figure 533 shows a nucleotide sequence (SEQ ID N0:533) of a native sequence
PR09739 eDNA,
wherein SEQ ID N0:533 is a clone designated herein as "DNA108765-2758".
Figure 534 shows the amino acid sequence (SEQ ID N0:534) derived from the
coding sequence of SEQ
ID N0:533 shown in Figure 533.
Figure 535 shows a nucleotide sequence (SEQ ID N0:535) of a native sequence
PR07177 cDNA,
wherein SEQ ID N0:535 is a clone designated herein as "DNA108783-2747".
Figure 536 shows the amino acid sequence (SEQ ID N0:536) derived from the
coding sequence of SEQ
ID N0:535 shown in Figure 535.
Figure 537 shows a nucleotide sequence (SEQ ID N0:537) of a native sequence
PR07178 cDNA,
wherein SEQ ID N0:537 is a clone designated herein as "DNA108789-2748".
Figure 538 shows the amino acid sequence (SEQ ID N0:538) derived from the
coding sequence of SEQ
m N0:537 shown in Figure 537.
Figure 539 shows a nucleotide sequence (SEQ ID N0:539) of a native sequence
PR06246 eDNA,
wherein SEQ ID N0:539 is a clone designated herein as "DNA108806-2724".
Figure 540 shows the amino acid sequence (SEQ ID N0:540) derived from the
coding sequence of SEQ
ID N0:539 shown in Figure 539.
Figure 541 shows a mzcleotide sequence (SEQ ID N0:541) of a native sequence
PR06241 eDNA,
wherein SEQ ID N0:541 is a clone designated herein as "DNA108936-2719".
Figure 542 shows the amino acid sequence (SEQ ID N0:542) derived from the
coding sequence of SEQ
1D N0:541 shown in Figure 541.
34


CA 02534018 2001-02-28
WO O1/G8848 PCT/US01/06520
Figure 543 shows a nucleotide sequence (SEQ ID N0:543) of a native sequence
PR09835 cDNA,
wherein SEQ >D N0:543 is a clone designated herein as "DNA119510-2771".
Figure 544 shows the amino acid sequence (SEQ ID N0:544) derived from the
coding sequence of SEQ
ID N0:543 shown in Figure 543.
Figure 545 shows a nucleotide sequence (SEQ ID N0:545) of a native sequence
PR09857 cDNA,
wherein SEQ ID N0:545 is a clone designated herein as "DNA119517-2778".
Figure 546 shows the amino acid sequence (SEQ ID N0:546) derived from the
coding sequence of 5EQ
1D N0:545 shown in Figure 545.
Figure 547 shows a nucleotide sequence (SEQ ID N0:547) of a native sequence
PR07436 cDNA,
wherein SEQ ID N0:547 is a clone designated herein as "DNA119535-2756".
Figure 548 shows the amino acid sequence (SEQ ID N0:548) derived from the
coding sequence of SEQ
ID NO:S47 shown in Figure 547.
Figure 549 shows a nucleotide sequence (SEQ ID N0:549) of a native sequence
PR09856 cDNA,
wherein SEQ ID N0:549 is a clone designated herein as "DNA119537-2777".
Figure 550 shows the amino acid sequence (SEQ ID N0:550) derived from the
coding sequence of SEQ
ID N0:549 shown in Figure 549.
Figure 551 shows a nucleotide sequence (SEQ ID N0:551) of a native sequence
PR019605 cDNA,
wherein SEQ ID N0:551 is a clone designated herein as "DNA119714-2851".
Figure 552 shows the amino acid sequence (SEQ ID N0:552) derived from the
coding sequence of SEQ
ID N0:551 shown in Figure 551.
Figure 553 shows a nucleotide sequence (SEQ ID N0:553) of a native sequence
PR09859 cDNA,
wherein SEQ ID N0:553 is a clone designated herein as "DNA125170-2780".
Figure 554 shows the amino acid sequence (SEQ ID N0:554) derived from the
coding sequence of SEQ
ID NO:S53 shown in Figure 553.
Figure 555 shows a nucleotide sequence (SEQ ID N0:555) of a native sequence
PR012970 cDNA,
wherein SEQ ID N0:555 is a clone designated herein as "DNA129594-2841".
Figure S56 shows the amino acid sequence (SEQ ID N0:556) derived from the
coding sequence of SEQ
ID N0:555 shown in Figure 555.
Figure 557 shows a nucleotide sequence (SEQ ID N0:557) of a native sequence
PROI9626 cDNA,
wherein SEQ ID N0:557 is a clone designated herein as "DNA129793-2857".
Figure 558 shows the amino acid sequence (SEQ ID N0:558) derived from the
coding sequence of SEQ
ID N0:557 shown in Figure 557.
Figure 559 shows a nucleotide sequence (SEQ ID N0:559) of a native sequence
PR09833 cDNA,
wherein SEQ ID NO:S59 is a clone designated herein as "DNA130809-2769".
Figure 560 shows the amino acid sequence (SEQ ID NO:S60) derived from the
coding sequence of SEQ
ID N0:559 shown in Figure 559.
Figure 561 shows a nucleotide sequence (SEQ ID N0:561) of a native sequence
PR019670 cDNA,
wherein SEQ 1D N0:561 is a clone designated herein as "DNA131639-2874".


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/0652U
Figure 562 shows the amino acid sequence (SEQ ID N0:562) derived from the
coding sequence of SEQ
ID N0:561 shown in Figure 561.
Figure 563 shows a nucleotide sequence (SEQ ID N0:563) of a native sequence
PR019624 cDNA,
wherein SEQ ID N0:563 is a clone designated herein as "DNA131649-2855".
Figure 564 shows the amino acid sequence (SEQ ID N0:564) derived from the
coding sequence of SEQ
ID N0:563 shown in Figure 563.
Figure 565 shows a nucleotide sequence (SEQ ID N0:565) of a native sequence
PR019680 cDNA,
wherein SEQ 117 N0:565 is a clone designated herein as "DNA131652-2876".
Figure 566 shows the amino acid sequence (SEQ ID N0:566) derived from the
coding sequence of SEQ
ID N0:565 shown in Figure 565.
Figure 567 shows a nucleotide sequence (SEQ ID N0:567) of a native sequence
PR019675 cDNA,
wherein SEQ ID N0:567 is a clone designated herein as "DNA131658-2875".
Figure 568 shows the amino acid sequence (SEQ ID N0:568) derived from the
coding sequence of SEQ
ID N0:567 shown in Figure 567.
Figure 569 shows a nucleotide sequence (SEQ ID N0:569) of a native sequence
PR09834 cDNA,
wherein SEQ B7 N0:569 is a clone designated herein as "DNA132162-2770".
Figure 570 shows the amino acid sequence (SEQ ID N0:570) derived from the
coding sequence of SEQ
ID N0:569 shown in Figure 569.
Figure 571 shows a nucleotide sequence (SEQ ID N0:571) of a native sequence
PR09744 cDNA,
wherein SEQ ID N0:571 is a clone designated herein as "DNA136110-2763".
Figure 572 shows the amino acid sequence (SEQ ID N0:572) derived from the
coding sequence of SEQ
TD N0:571 shown in Figure 571.
Figure 573 shows a nucleotide sequence (SEQ ID N0:573) of a native sequence
PR019644 cDNA,
wherein SEQ )D N0:573 is a clone designated herein as "DNA139592-2866".
Figure 574 shows the amino acid sequence (SEQ ID N0:574) derived from the
coding sequence of SEQ
ID N0:573 shown in Figure 573.
Figure 575 shows a nucleotide sequence (SEQ ID N0:575) of a native sequence
PR019625 cDNA,
wherein SEQ ID N0:575 is a clone designated herein as "DNA139608-2856".
Figure 576 shows the amino acid sequence (SEQ ID N0:576) derived from the
coding sequence of SEQ
ID N0:575 shown in Figure 575.
Figure 577 shows a nucleotide sequence (SEQ ID N0:577) of a native sequence
PR019597 cDNA,
wherein SEQ ID N0:577 is a clone designated herein as "DNA143292-2848".
Figure 578 shows the amino acid sequence (SEQ ID N0:578) derived from the
coding sequence of SEQ
ID N0:577 shown in Figure 577.
Figure 579 shows a nucleotide sequence (SEQ ID N0:579) of a native sequence
PR016090 cDNA,
wherein SEQ ID N0:579 is a clone designated herein as "DNA144844-2843".
Figure 580 shows the amino acid sequence (SEQ ID N0:580) derived from the
coding sequence of SEQ
ID N0:579 shown in Figure 579.
36


CA 02534018 2001-02-28
WO 01/68848 rc:~rmwnvb,iv
Figure 581 shows a nucleotide sequence (SEQ ID N0:581) of a native sequence
PR019576 cDNA,
wherein SEQ ID N0:581 is a clone designated herein as "DNA144857-2845".
Figure 582 shows the amino acid sequence (SEQ ID N0:582) derived from the
coding sequence of SEQ
1D NO:S81 shown in Figure 581.
Figure S83 shows a nucleotide sequence (SEQ ID N0:583) of a native sequence
PR019646 cDNA,
wherein SEQ U~ N0:583 is a clone designated herein as "DNA145841-2868".
Figure 584 shows the amino acid sequence (SEQ ID N0:584) derived from the
coding sequence of SEQ
ID N0:583 shown in Figure 583.
Figure 585 shows a nucleotide sequence (SEQ ID N0:585) of a native sequence
PR019814 cDNA,
wherein SEQ ID NO:S85 is a clone designated herein as "DNA148004-2882".
Figure 586 shows the amino acid sequence (SEQ ID N0:586) derived from the
coding sequence of SEQ
ID N0:585 shown in Figure 585.
Figure 587 shows a nucleotide sequence (SEQ ID N0:587) of a native sequence
PR019669 cDNA,
wherein SEQ ID N0:587 is a clone designated herein as "DNA149893-2873".
Figure 588 shows the amino acid sequence (SEQ ID N0:588) derived from the
coding sequence of SEQ
ID N0:587 shown in Figure 587.
Figure 589 shows a nucleotide sequence (SEQ ID N0:589) of a native sequence
PR019818 cDNA,
wherein SEQ ID N0:589 is a clone designated herein as "DNA149930-2884".
Figure 590 shows the amino acid sequence (SEQ ID N0:590) derived from the
coding sequence of SEQ
ID N0:589 shown in Figure 589.
Figure 591 shows a nucleotide sequence (SEQ iD N0:591) of a native sequence
PR020088 cDNA,
wherein SEQ ID N0:591 is a clone designated herein as "DNA150157-2898".
Figure 592 shows the amino acid sequence (SEQ ID N0:592) derived from the
coding sequence of SEQ
ID N0:591 shown in Figure 591.
Figure 593 shows a nucleotide sequence (SEQ ID N0:593) of a native sequence
PR016089 cDNA,
wherein SEQ ID N0:593 is a clone designated herein as "DNA150163-2842".
Figure 594 shows the amino acid sequence (SEQ ID N0:594) derived from the
coding sequence of SEQ
ID N0:593 shown in Figure 593.
Figure 595 shows a nucleotide sequence (SEQ ID N0:59S) of a native sequence
PR020025 cDNA,
wherein SEQ ID N0:595 is a clone designated herein as "DNA153579-2894".
Figure 596 shows the amino acid sequence (SEQ ID N0:596) derived from the
coding sequence of SEQ
ID N0:595 shown in Figure 595.
Figure 597 shows a nucleotide sequence (SEQ ID N0:597) of a native sequence
PR020040 cDNA,
wherein SEQ ID N0:597 is a clone designated herein as "DNA164625-2890".
Figure 598 shows the amino acid sequence (SEQ ID N0:598) derived from the
coding sequence of SEQ
ID N0:597 shown in Figure 597.
Figure 599 shows a nucleotide sequence (SEQ ID N0:599) of a native sequence
PR0791 cDNA, wherein
SEQ ID N0:599 is a clone designated herein as "DNA57838-1337".
37


CA 02534018 2001-02-28
WO 01/G8848 PCT/USO1/OG520
Figure 600 shows the amino acid sequence (SEQ ID N0:600) derived from the
coding sequence of SEQ
ID N0:599 shown in Figure 599.
Figure 601 shows a nucleotide sequence (SEQ ID N0:601) of a native sequence
PR01131 cDNA,
wherein SEQ ID N0:601 is a clone designated herein as "DNA59777-1480".
Figure 602 shows the amino acid sequence (SEQ ID N0:602) derived from the
coding sequence of SEQ
ID N0:601 shown in Figure 601.
Figure 603 shows a nucleotide sequence (SEQ ID N0:603) of a native sequence
PR01343 cDNA,
wherein SEQ ID N0:603 is a clone designated herein as "DNA66675-1587".
Figure 604 shows the amino acid sequence (SEQ )D N0:604) derived from the
coding sequence of SEQ
ID N0:603 shown in Figure 603.
Figure 605 shows a nucleotide sequence (SEQ ID N0:605) of a native sequence
PR01760 cDNA,
wherein SEQ ID N0:605 is a clone designated herein as "DNA76532-1702".
Figure 606 shows the amino acid sequence (SEQ ID N0:6(~ derived from the
coding sequence of SEQ
ID N0:605 shown in Figure 605.
Figure 607 shows a nucleotide sequence (SEQ ID N0:607) of a native sequence
PR06029 eDNA,
wherein SEQ ID N0:607 is a clone designated herein as "DNA105849-2704".
Figure 608 shows the amino acid sequence (SEQ ID N0:608) derived from the
coding sequence of SEQ
ID N0:607 shown in Figure 607.
Figure 609 shows a nucleotide sequence (SEQ ID N0:609) of a native sequence
PR01801 cDNA,
wherein SEQ ID N0:609 is a clone designated herein as "DNA83500-2506".
Figure 610 shows the amino acid sequence (SEQ DJ N0:610) derived from the
coding sequence of SEQ
ID N0:609 shown in Figure 609.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. 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 polypeptide"
and "PRO/number" 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 in 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 PRO/number polypeptides disclosed
herein.
A "native sequence PRO polypeptide" comprises a polypeptide having the same
amino acid sequence as
38


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
the corresponding PRO polypeptide derived from nature. Sucli 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 PRO 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
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 transmembrane and cytoplasmic domains. Ordinarily,
a PRO polypeptide ECD will have
less than 1 % of such transmembrane and/or 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 either 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-terminal 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., Nielsen et al., Prot. En~. 10:1-6 (1997) and von
Heinje et al., Nucl. Acids. 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 more than about 5
amino acids on either side of the
C-terminal boundary of the signal pepride as identified herein, and the
polynucleotides encoding them, are
contemplated by the present invention.
"PRO polypeptide variant" means an active PRO polygeptide 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
39


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
polypeptides 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 sequence identity, alternatively at least about 81 % amino acid sequence
idemity, alternatively at least about
82% amino acid sequence identity, alternatively at least about 83% amino acid
sequence identity, alternatively
at least about 84 % amino acid sequence identity, alternatively at least about
85 % amino acid sequence identity,
S alternatively at least about 86% 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 9196 amino acid sequence identity, alternatively at least about 9296
amino acid sequence identity,
alternatively at least about 93 96 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 abort 97% amino acid
sequence identity, alternatively at least
about 98 % 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 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 speciFcally defined fragment of a
full-length PRO polypeptide sequence
as disclosed herein. Ordinarily, PRO variant polypeptides are at least about
10 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 least
about 70 amino acids in length,
alternatively at least about 80 amino acids in length, alternatively at least
about 90 amino acids in length,
alternatively at least about 100 amino acids in length, alternatively at least
about 150 amino acids in length,
alternatively at least about 200 amino acids in length, alternatively at least
about 300 amino acids in length, or
more.
"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide
sequences identified
herein is defined as the percentage of amino acid residues is 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 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. 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 being compared. For purposes
herein, however, % amino acid
sequence identity values are generated using the sequence comparison computer
program 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. 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


CA 02534018 2001-02-28
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.
In situations where ALIGN-2 is employed for amino acid sequence comparisons,
the % 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 % amino acid
sequence identity to, with, or against a given amino acid sequence B) is
calculated as.follows:
100 times the fraction 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 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 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 hypothetical amino acid residues.
Unless specifically 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, q6 amino
acid sequence identity values may also be obtained as described below by using
the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzvmolo;~y 266:460-480 (1996)). Most of
the WU-BLAST-2 search
parameters are set to the default values. Those not 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 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 polypeptide of
interest is being compared 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% amino acid sequence
identity to the amino acid sequence
B", the amino acid sequence A is the comparison amino acid sequence of
interest and the amina 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., Nucleic Acids Res. 25:3389-3402 (1997)).
41


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
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 = 10, minimum low complexity length = 15/5, multi-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 amino acid sequence
comparisons, the % 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 % amino acid
sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
NCBI-BLAST2 in that program's alignment of A and B, and where Y 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 96 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 polypeptide 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 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. Ordinarily, a PRO
variant polynucleotide will have 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, 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 % nucleic acid
sequence identity, alternatively at least
about 97 To nucleic acid sequence identity, alternatively at least about 98 Yb
nucleic acid sequence identity and
alternatively at least about 99 % nucleic acid sequence identity with a
nucleic acid sequence encoding a full-length
native sequence PRO polypepdde 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
full-length PRO polypeptide sequence
as disclosed herein. Variants do not encompass the native nucleotide sequence.
42


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/06520
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.
"Percent (%) 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, % nucleic acid sequence identity
values are generated using the
sequence comparison computer program 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, Ine. and the source code shown in Table 1 below has been tiled with
user documentation in the U.S.
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 UNTX 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 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 phrased 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 follows:
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
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 % nucleic acid sequence identity calculations, Tables 4 and 5,
demonstrate how 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 sequence of a
nucleic acid molecule against
which the "PRO-DNA" nucleic acid molecule of interest is being compared, and
"N", "L" and "V" each represent
43


CA 02534018 2001-02-28
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 computer
program (Altschul et al., Methods in Enzymology 266:460-480 (1996)). Most of
the WU-BLAST-2 search
parameters are set to the default values. Those not 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 scoring
matrix = BLOSUM62. When WU-BLAST-2 is employed, a % 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 80
% 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)).
25 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 % 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 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
D, the % nucleic acid sequence identity of C to D will not equal the 9'o
nucleic acid sequence identity of D to C.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules
that encode an active PRO
polypeptide and which are capable of hybridizing, preferably under stringent
hybridization and wash conditions,
44


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
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 polynucleatide.
"Isolated, " when used to describe the various polypeptides disclosed herein,
means polypeptide that has
been identified and separated and/or 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
solutes. In preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least
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 polypeptide includes polypeptide in situ within
recombinant cells, since at least one
10 component of the PRO polypeptide natural environment will not be pzesent.
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 polypepdde-
encoding nucleic acid. An isolated
15 polypeptide-encoding nucleic acid molecule is other than in the form or
setting in which it is found in nature.
Isolated polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-
encoding nucleic acid molecule as it exists in natural cells. However, an
isolated polypeptide-encoding 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. '
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 promoter, optionally an operator sequence, and a
ribosome binding site. Eukaryotic cells
are lmown to utilize promoters, polyadenylation 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 operably
linked to DNA for a polypeptide
if it is expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the transcription of the
sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the case of
a secretory leader, contiguous and
in reading phase. However, enhancers do not have to be contiguous. Linking is
accomplished by ligalion 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


CA 02534018 2001-02-28
WO O1/188~8 PCT/USOI/0(s20
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 complementary
strands are present in an environment below their melting temperature. The
higher the degree 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 in Molecular Biolo~y, 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 chloride/0.0015
M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C; (2) employ
during hybridization a denaturing agent, such
as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum
albumin/0.1 % Ficoll/0.1 %
polyvinylpyrrolidone/SOmM sodium phosphate buffer at pH 6.5 with 750 mM sodium
chloride, 75 mM sodium
citrate at 42°C; or (3) employ 50% 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 Denhardt's solution,
sonicated salmon sperm DNA (50
/cg/ml), 0.1 % SDS, and 10 % dextran sulfate at 42 °C, with washes at
42 °C in 0.2 x SSC (sodium chloride/sodium
citrate) and SO % formamide at 55 ° C, followed by a high-stringency
wash consisting of 0.1 x SSC containing
EDTA at 55 ° C.
"Moderately stringent conditions" may be identified as described by Sambrook
et al., Molecular Cloning:
A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and
hybridization conditions (e.g., temperature, ionic strength and %SDS) less
stringent that those described above.
An example of moderately stringent conditions is overnight incubation at
37°C in a solution comprising: 20%
formamide, 5 x SSC (I50 mM NaCI, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.~, 5 x
Denhardt's solution, 10% dextran sulfate, and 20 mg/ml 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 strength, etc. as necessary to accommodate factors such as probe length
and the like.
The term "epitope tagged" when used herein refers to a chimeric polypeptide
comprising a PRO
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 short enough such that it does not
interfere with activity of the polypeptide
to which it is fused. The tag polypeptide preferably also is fairly unique so
that the antibody does not substantially
cross-react with other 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 term "immunoadhesin" designates antibody-like molecules
which combine the binding
specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of an amino acid
sequence with the desired binding
*-trademark 46


CA 02534018 2001-02-28
WO 01/G8848 PCT/USO1/06520
specificity which is other than the antigen recognition and binding site of an
antibody (i.e., is "heterologous"),
and an immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is
a contiguous amino acid sequence comprising at least the binding site of a
receptor or a ligand. The
immunoglobulin constant domain sequence in the immunoadhesin may be obtained
from any immunoglobulin,
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 ~rafers to forms) of a PRO
polypeptide which retain a
biological and/or an immunological 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
other than the ability to induce the production of an antibody against an
antigenic epitope possessed by a native
or naturally-occurring PRO and an "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 antibodies or antibody fragments, fragments or
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.
"Treatrnent" 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 as 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 purposes of treatment 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, goats,
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;
Iow molecular weight (less than about 10 residues) polypeptide; proteins, such
as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as poIyvinylpyrrolidone; amino
acids such as glycine, glutamine,
47


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
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; and/or nonionic surfactants such as TWEENT''~,
polyethylene glycol (PEG), and
PLURONICST'"
"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')Z, and Fv fragments;
diabodies; linear antibodies (Zapata et al., Protein Ena. 8(10): 1057-1062
[1995]); single-chain antibody
molecules; and multispecific 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')2 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 light-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 VH-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 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
(CH1) 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 or 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 lmown.
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 further divided into subclasses (isotypes),
e.g., IgGl, IgG2, IgG3, IgG4, IgA,
and IgA2.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a
polypeptide linker between the VH and V~ domains which enables the sFv to form
the desired structure for antigen
binding. For a review of sFv, see Pluckthun in The Pharmacolog~of Monoclonal
Antibodies, vol. II3,
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 (VH) connected to a light-chain
variable domain (V,,) in the same
48


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO110G520
polypeptide chain (VH V~. By using a linker that is too short to allow pairing
between the two domains 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 93/11161; 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
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 ~ by weight of antibody as determined by the Lowry method, and
most preferably more than 99
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 nonreducing
conditions using Coomassie blue or, preferably, silver stain. Isolated
antibody includes the antibody in situ within
recombinant cells since at 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 polypeptide without
substantially binding to any other polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound 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.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the
present invention can
adhere. Examples 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 column (e.g., an affinity chromatography
column). This term also includes 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 and/or surfactant
which is useful for delivery of a drug (such as a PRO polypeptide or antibody
thereto) to a mammal. The
components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of
biological membranes.
A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
An "effective amount" of a polypeptide disclosed herein or an agonist or
antagonist thereof is an amount
sufficient to carry out a specifically stated purpose. An "effective amount"
may be determined empirically and
in a routine manner, in relation to the stated purpose.
49


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
Table 1
/*
* C-C
increased
from
12
to
15


* Z
is
average
of
EQ


* B
is
average
of
ND


* match
with
stop
is
M;
stop-stop
=
0;
J
(joker)
match
=
0


*/


#defineM -8 /* value of a match with a stop */


int day[26][26] _ {


/* _
A B C D E F G H I J K L M N O P Q R S T U V W X Y Z
*/


7* { 2, 0,-2, 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0,_M, 1,
A 0,-2, 1, 1, 0, 0,-6, 0,-3, 0},
*l


I* { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2 _M,-1,
B 1, 0, 0, 0, 0,-2,-5, 0,-3, 1},
*1


/* {-2,-4,15,-5, 5,-4,-3,-3, 2, 0,-5,-6,-5,~, M,-3,-5,
C 4, 0,-2, 0,-2,-8, 0, 0,-5},
*/


/* { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1,
D 2,-1, 0, 0, 0,-2,-7, 0,-4, 2},
*I


I* { 0, 2,-5, 3, 4; 5, 0, 1,-2, 0, 0,-3,-2, 1 =M,-1,
E 2,-1, 0, 0, 0,-2, 7, 0,-4, 3},
*/


/* {-4,-5,-4,-6,-5, 9; 5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3,
F 0,-1, 0, 0, 7,-5},
*!


/* { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M; 1,-1,-3,
G 1, 0, 0,-1,-7, 0,-5, 0},
*/


I* {-1, 1,-3, 1, 1, 2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0,
H 3, 2,-1,-1, 0,-2,-3, 0, 0, 2},
*l


/* {-1, 2,-2,-2,-2, 1,-3,-2, 5, 0, 2, 2, 2,-2 -M,-2,-2,
I 2,-1, 0, 0, 4,-5, 0,-1; 2},
*/


/* { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, O, M, 0,
J 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
*/


/* {-1, 0,-5, 0, 0,-5, 2, 0,-2, 0, 5,-3, 0, 1 =M,-1,
K 1, 3, 0, 0, 0, 2,-3, 0,-4, 0},
*/


/* {-2,-3; 6, 4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3 =M,-3,-2,-3,-3,-1,
L 0, 2,-2, 0,-1, 2},
*l


/* {-1,-2,-5,3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1,
M 0,-2,-1, 0, 2,-4, 0,-2,-1},
*/


/* { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1,
N 1, 0, 1, 0, 0,-2,-4, 0,-2, 1},
*/


/* M =M =M, M =M, M =M _M, 0 =M, M, M,_M =M =M,_M,_M,_M,
O M -M},
*/ ~M =M, M =M =M =M,


/* _
P { 1,-1,-3,-1; 1,-5,-1, 0,-2, 0,-1,-3,-2,-1 -M, 6,
*! 0, 0, 1, 0, 0,-1,-6, 0,-5, 0},


!* { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1; 2,-1, 1 =M, 0,
Q 4, 1,-1,-1, 0,-2,-5, 0,-4, 3},
*1


/* {-2, 0,-4,-1; 1,-4,-3, 2,-2, 0, 3,-3, 0, 0, M, 0,
R 1, 6, 0,-1, 0,-2, 2, 0,-4, 0},
*l


/* { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M, 1,-1,
S 0, 2, 1, 0,-1,-2, 0,-3, 0},
*/


/* { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, O, M, 0,-1,-1,
T 1, 3, 0, 0,-5, 0; 3, 0},
*/


/* { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 =M, 0,
U 0, 0, 0, 0, 0, 0, 0, 0, 0, 0},
*1


/* { 0,-2,-2, 2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2, M,-1,-2,-2,-1,
V 0, 0, 4,-6, 0,-2,-2},
*/


/* {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4 =M,-6,-5,
W 2,-2,-5, 0,-6,17, 0, 0,-6},
*/


/* { o, o, o, o, o, o, o, o, o, o, o, o, o, o =M, o,
x o, o, o, o, o, o, o, o, o, o},
*l


/* {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2, M,-5,-4,-4;
Y 3,-3, 0, 2, 0, 0,10,-4},
*I


/* { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,-M, 0,
Z 3, 0, 0, 0, 0,-2,-6, 0,-4, 4}
*/


};


45
55


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 1 cont'Z
/*


*/


#include<stdio.h>


#include<
ctype.
h
>



#defmeMAX1MP /* max jumps in a diag *!
16


#defmeMA?CGAP /* don't continue to penalize
24 gaps larger than this */


#defmeJMPS 1024 I * max jmps in an path
*I


#defineMX 4 /* save if there's at least
MX-1 bases since last jmp
*/



#defineDMAT 3 /* value of matching bases
*/


#defmeDMIS 0 /* penalty for mismatched
bases */


#defmeDINSO8 /* penalty for a gap */


#defmeDINSI1 /* penalty per base */


1S #defmePINSO8 /* penalty for a gap */


#defmePINSl4 /* penalty per residue */


struct
jmp
{


shortn[MAXJMP];
/* size
of jmp
(neg
for
defy)
*/


unsigned
short
x[MA3CJMP];
/*
base
no.
of
jmp
in
seq
x
*!


}; /* limits seq to 2" 16 -1
*/


strnct
diag
{


int score; /* score at last jmp */


2S long offset; l* offset of pxev block
*/


shortijmp; l* current jmp index */


struct /* list of jmps */
jmp
jp;



struct
path
{


int spc; l* number of leading spaces
*!


shortn[JMPS];/*of jmp (gap) *!
size


int x[JMPS];
/* loc
of jmp
(last
elem
before
gap)
*/



3S


char *ofile; !* output file name *!


char *namex[2J;/* seq names: getseqs0 */


char *prog; /* prog name for err msgs
*/


char *seqx[2];/* seqs: getseqsQ */


int dmax; /* best diag: nw0 */


int dmax0; /* final diag */


int dna; ~ /* set if dna: main()
*/


int endgaps;/* set if penalizing end
gaps */


int gapx, /* total gaps in seqs */
gapy;


int len0, /* seq lens */
lenl;


int ngapx, /* total size of gaps */
ngapy;


int smax; /* max score: nwp */


int *xbm; /* bitmap for matching */


long offset; /* current offset in jmp
file */


SO structdiag *dx; /* holds diagonals */


structpath pp[2]; /* holds path for seqs */


char *callocQ,
*mallocQ,
*index0,
*strcpyp;


char *getseqQ,
*g calloc0;


5 5


51


CA 02534018 2001-02-28
WO 01/68848 PCTlUS01/06520
Table 1 (cont'1
/* Needleman-Wunsch alignment program
* usage: progs filel filet
* where filel and filet are two dna or two protein sequences.
S * The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with ';', ' >' or ' <' are ignored
* Max file length is 65535 (limited by unsigned short x in the jmp struct)
* A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA
* Output is in the file "align.out"
* The program may create a mtp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*!
#include "nw.h"
1S #include "day.h"
static _dbval[26] _ {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
j;
static -pbval[26] _ {
1, 2~(1< <('D'-'A'))~(1< <('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14,
1«15, 1«16, 1«17, 1«18, 1«19, 1«20, 1«21, 1«22,
1«23, 1«24, 1«25(1«('E'-'A'))I(1«('Q'-'A'))
};
main(ac, aV) lrialn
int ac;
char *avp;
{
grog = av[O];
~(~!=3){
fprintf(stderr,"usage: %s filet filet\n", prog);
fprintf(stderr, "where filet and filet are two dna or two protein
sequences.\n");
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.out\"\n");
exit(1);
namex[0] = av[1];
namex[1] = av[2];
seqx[0] = getseq(namex[0], &len0);
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : ~bval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; - /* output file */
S0 nwQ; /* fill in the matrix, get the possible jmps */
readjmps0; /* get the actual jmps */
print(); !* print state, alignment */
cleanup(0); /* unlink any tmp files */
1
52


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/06520
Table l~cont')
/* do the alignment, return best score: maim
* dna: values in Fitch and Smith, PNAS, 80, 1382-1386, 1983
* pro: PAM 250 values
* When scores are equal, we prefer mismatches to any gap, prefer
* a new gap to extending an ongoing gap, and prefer a gap in seqx
* to a gap in seq y.
*!
nw() nW
{
char *px, *py; /* seqs and ptrs *I
int *ndely, *dely; /* keep track of dely */
int ndelx, deli; /* keep track of deli */
int *tmp; l* for swapping row0, cowl *!
int mis; /* score for each type */
int ins0, insl; I* insertion penalties *I
register id; /* diagonal index */
register ij; /* jmp index */
register *col0, *coll; /* score for curr, last row */
register xx, yy; /* index into seqs */
dx = (struct diag *)g_calloc("to get diags", len0+lenl+ 1, sizeof(struct
diag));
ndely = (int *)g calloc("to get ndely", lenl+1, sizeof(int));
dely = (int *)g calloc("to get dely", lenl+1, sizeof(int));
col0 = (int *)g caltoc("to get col0", lenl+1, sizeof(int));
coll = (int *)g calloc("to get coil", lenl+1, sizeof(int));
ins0 -_ (dna)? DINSO : PINSO;
insl = (dna)? DINSl : PINSl;
smax = -10000;
if (endgaps) {
for (col0[0] = defy[0] _ -ins0, yy = 1; yy < = lenl; yy-i-+) {
col0[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yy] = yy;
col0[0] = 0; /* Waterman Bull Math Biol 84 */
1
else
for (yy = 1; yy < = lent; yy++)
dely[yy] _ -~;
/* fill in match matrix
*/
for (px = seqx[0], xx = 1; xx < = len0; px++, xx++) {
/* initialize first entry in col
*/
if (endgaps) {
~(~ _= 1)
toll[0] = deli = -(ins0+insl);
else
col l [0] = deli = col0[0] - ins 1;
ndelx = xx;
else {
coil[o] = o;
delx = -ins0;
ndelx = 0;
1
53


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 1 (cony)
for (py = seqx[l], yy = 1; yy <= lenl; py++,
yy++) {


mis = col0[yy-1];


if (dna)


S mis += (xbm[*px-'A']&xbm[*py-'A'])? DMAT :
DMIS;


else


mis += day[*px-'A'][*py-'A'];


/* update penalty for del in x seq;


* favor ~w del over ongong del


* ignore MAXGAP if weighting endgaps


*/


if (endgaps ~ ~ ndely[yy] < MAXGAP) {


if (col0[yy] - ins0 > = defy[yy]) {


defy[yy] = col0[yy] - (ins0+insl);


ndely[yy] = 1;


} ~e {


defy[yy] -= insl;


ndely[yy]++;


}


} else {


if (col0[yy] - (ins0+insl) >'= dely[yy]) {


dely[yy] = colOjyy] - (ins0+insl);


ndely[yy] = 1;


} else


ndely[yy]++;


}


/* update penalty for del in y seq;


* favor new del over ongong del


*/


if (endgaps ~ ~ ndelx < MAXGAP) {


if (coll(yy-1] - ins0 > = deli) {


deli = coll(yy-1] - (ins0+insl);


ndelx = 1;


} else {


deli -= insl;


ndelx++;


}


} else {


if (coll[yy-1] - (ins0+insl) > = deli) {


delx = coll[yy-1] - (ins0+insl);


ndelx = 1;


} else


ndelx++;


}


/* pick the maximum score; we're favoring


* mis over any del and deli over defy


*/


60
...nw
54


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
Table 1 (cony)
...nw
id=xx-yy+Ienl-1;
if (mis > = delx && mis > = dely[yy])
coll[yy] = mis;
else ff (delx > = dely[yy]) {
coll[yy] = deli;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (ldna ~ ~ (ndelx > = MA3~TMP
&& xx > dx[id].jp.x[ij]+M30 ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXrMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx(id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
1
1
dx[id].jp.n[ij] = ndelz;
dx[id].jp.x[ij] = xx; ,
dx[id].score = delx;
else {
coll(py] = dely[yy];
ij = dx[id].ijmp;
~5 if (dx[id].jp.n[0] && (!dna ~ ~ (ndely[yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MJQ ~ ~ mis > dx[id]acore+DINSO)) {
dx[id].ijmp++;
if (++ij > = MA?fJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += sizeof(struct jmp) + sizeof(offset);
1
dx[id].jp.n[ij] _ -ndely[yy];
dx[id].jp.x[ij] = zx;
dx[id].score = dely[yy];
if (xx == lei && yy < lent) {
. /* last col
*/
if (endgaps)
coll[yy] -= ins0+insl*(lenl-yy);
if (coll[yy] > smax) {
smax = coll[yy];
dmaz = id;
1
if (endgaps && xx < len0)
toll[yy-1] -= ins0+insl*(len0-xx);
if (coll[yy-1] > smax) {
smax = coil[yy-1];
dmax = id;
tmp = col0; col0 = coil; cull = tmp;
1
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)coll); }


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 1 (cony)
/*
*
* 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[]; print0
* dumpblockQ -- dump a block of lines with numbers, stars: pr align()
* nums() - put out a number line: dumpblockQ
* putline0 -- put out a line (name, [num], seq, [num]): dumpblock0
* stars0 - -put a line of stars: dumpblockQ
* stripnameQ - strip any path and prefix from a seqname
*1
ZS #include "nw.h"
#define SPC 3
#detlne P LINE 256 I* maximum output line *I
#defme P SPC 3 /* space between name or num and seq */
-
extern _day[26][26j;
int olen; /* set output line length */
FILE *fx; /* output file */
print
print() '
{
int lx, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup(I);
fprintf(fx, "<first sequence: %s (length = 96d)1n", namex[Oj, len0);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[Ij, lenl);
olen = 60;
Ix = len0;
ly = lent;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x *I
pp[0].spc = firstgap = lenl - dmax - 1;
ly _= pP[0].spc;
else if (dmax > lenl - 1) { /* leading gap in y */
pp[1].spc = firstgap = dmax - (lenl - 1);
Ix -= pp[I].spc;
if (dmax0 < len0 - I) { /* trailing gap in x *!
lastgap = len0 - dmax0 -1;
lx -= lastgap;
SO )
else if (dmax0 > IenO - 1) { /* trailing gap in y */
lastgap = dmax0 - (len0 - I);
ly -= lastgap;
getmat(Ix, ly, firstgap, lasigap);
pr align();
56


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
Table 1 (cony)
/*
* trace back the best path, count matches
*/
static
S getmat(lx, ly, firstgap, lastgap) getlriat
int lx, ly; /* "core" (minus endgaps) *!
int firstgap, lastgap; /* leading trailing overlap */
{
int nm, i0, il> siz0, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
IS /* get total matches, score
*/
i0 = il = siz0 = sizl = 0;
p0 = seqx[0] + pp[I].spc;
pl = seqx[1] + pp[0].spc;
n0 = pp[1].spc + 1;
nl = pp[0].spc + 1;
nm = 0;
while ( *p0 && *pl ) {
if (siz0) {
pl++;
nl + +;
siz0-;
else if (sizl) {
p0++;
n0++;
sizl--;
else {
if (xbm(*p0-'A']&xbm(*pl-'A'])
nm++;
if (n0++ == pp[0].x[i0])
siz0 = pp[0].n[i0++];
if (nl++ _= pp[1].x[il])
sizl = pp[1].n[il++];
p0++;
pl++;
1* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, (mock off overhangs and take shorter core
*/
if (endgaps)
lx = pen0 < lenl)? len0 : lenl;
else
lx = (lx < ly)? lx : ly;
pct = 100. *(double)nm/(double)lx;
' fprintf(fx, non");
fprintf(fx, " < % d match %s in an overlap of % d: % .2f percent
similarityln",
~ =0 1)? °" : n~"~. 17C, pCt);
57


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/U6520
Table 1 (cony)
fprintf(fx, " < gaps in first sequence: 'Y d", gapx); ... getfnAt
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base":"residue", (ngapx == I)? "~:"s");
fprintt(fx,"Yos", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
(gaPY) {
(void) sprintf(outx, " (%d ~s%s)",
ngapy, (dna)? "base":"residue", (ngapy == I)7 "":"s");
fprintf(fx," ~s", outx);
if (dna)
fprintf(fx,
"\n<score: ~d (match = 96d, mismatch = ~d, gap penalty = ~d + 9bd per
base)\n",
smax, DMAT, DMIS, DINSO, DINSI);
else
fprintf(fx,
"\n<score: ?6d (Dayhoff PAM 250 matrix, gap penalty = ~d + hod per
residue)\n",
smax, PINSO, PINSl);
if (endgaps)
fPT~~.
"<endgaps penalized. left endgap: %d %s~s, right endgap: ~d 96s96s\n",
firstgap, (dna)? "base" : "residue", (ftrstgap == I)? "" : "s",
lastgap, (dna)7 "base" : "residue", (lastgap == 1)? "" : "s");
else
fprintf(&, "<endgapsnotpenalizedln");
static nm; /* matches in core -- for checlang */


static Imax; /* lengths of stripped file names */


static ij[2]; /* jmp index for a path */


static nc[2]; . /* number at start of current line *!


static ni[2]; /* current elem number -- for gapping */


static siz[2];


static *ps[2]; /* ptr to current element */
char


static *po[2]; /* ptr to next output char slot */
char


static out[2][P_LINE]; l* output line *!
char


static star[P_LINE]; /* set by stars() */
char



/*


* print of described in struct path pp[]
alignment


*l


static


pr align()13P ahgtl



int nn; /* char count */


i~ more;


register i;



for (i 0, lmax = 0; i < 2; i++) {
=


nn = stripname(namex[i]);


if (nn > lmax)


lmax = nn;


nc[iJ = I;
ni[i] = l; .
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[iJ = out[i]; }
58

CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
Table 1 (cony)
for (nn = nm = 0, more = 1; more; ) { ...pr align
for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence?
*/
if (!*ps[i])
continue;
more++;
if (pp[i].spc) { /* leading space */
*po[i]++ _ ' ';
PPfi].sPc--;
1$ }
else if (siz[i]) { /* in a gap */
*po[i]++ _ ,
siz[i]--;
else { l* we're putting a seq element
*/
*po[i] _ *ps[i];
if (islower(*ps(i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps(i]++;
/*
* are we at next gap for this seq?
*/
(~[7 ° ° PP[i].x[ij[i]]) {
/*
* we need to merge all gaps
* at this location
*/
siz[i] = pp[i].n[ij[i]++];
while (ni[i] _= pp[i].x[ij[i]])
siz[i] += pp[i].n[ij[i]++];
ni[i]++;
}
if (++nn == olen ~ ~ !more && nn) {
dumpblockQ;
for (i = 0; i < 2; i++)
po[i] = out[i];
nn = 0;
}
}
SO }
/*
* dump a block of lines, including numbers, stars: pr align
*/ .
static
dumpblockQ dumpblock
{
register i;
f0 for (i = 0; i < 2; i++)
. *po[i]- _ '\0';
59

CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 1 (cony)
... dumpb]ock
(void) putc('\n', fx);


for (i = 0; i < 2; i++) {


If .(*out[i] &&. (*out[i] !_ ' ' I I *(po[il) ! _
' ')) {


if (i == 0)


nums(i);


if (i == 0 && *out[1])


stars0;


putiine(i);


if (i == 0 && *out[1P


fprintf(fx, star);


if (i == 1)


nums(i); _


}


}


}


/*


* put
out
a
number
line:
dumpbloclcQ


*/


static


nums(ix)IlilrilS


int ix; /* index in out[] holding seq line */


{


char nline[P_LINE];


register i, j;


register char *pn, *px, *py;


for (pn = mine, i = 0; i < lmax+P SPC; i++, pn++)


*pn = , ~;


for (i = ncjix], py = out[ix]; *py; py++, pn++) {


~ (*~Y = _ ' ' I I *~Y = _ '-')


*pn = ~ ~,


else {


if (i % 10 == 0 I I (i == 1 && nc[ix] ! = 1)) {


j = (i < 0)? -i : i;


for (px = pn; j; j /= 10, px--)


*px = j9~10 + '0';


if (i < o)


*px = , , ;


}


else


*pn _-. ~ ~:


i++;


}


}


*pn = ~\0~.
~


nc[ix] = i;


for (pn = mine; *pn; pn++)


(void) putc(*pn, fx);


(void) putc('1n', fx);



/*


* put
out
a
line
(name,
[num],
seq,
[num]):
dumpblockp


*/


static


putline(ix)
puthlle


int ix; {



CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
Table 1 (coast')
...putline
int i;


register char *px;



for (px = namex[ix], i = 0; *px && *px ! _ ':'; px++,
i++)


(void) putc(*px, fx);


for (; i < lmax+P SPC; i++)


(void) putc(' ', fx);



/* these count from i: '


* nib is current element (from 1)


* nc[] is number at start of current line


*/ .


for (px = out[ix]; *px; px++)


(void) putc(*px&Ox7F, fx);


(void) putc('\n', fx);


}



I*


* line of stars (seqs always in out[O], out[1]): dumpblock0
put
a


*/


static


stars()
stars


{


int i;


register char *p0, *pl, cx, *px;


if (!*out(0] I I (*out[0] _ _ ' ' &8c *(po[o]) _ _
' ') I I


!*OUt(1] I I (*OUt[1] __ ' ' BLBC *(p0[1]) _- ' '))


return;


px = star;


for (i = lmax+P SPC; i; i--)


*px++ _ ' ',


for (p0 = out(0], pl = out[1]; *p0 && *pl; p0++, pl++)
{


if (isalpha(*p0) && isalpha(*pl)) {


if (xbm[*p0-'A']&xbm[*pl-'A']) {


cx = '*';


nm++;


}


else if (!dna && day[*p0-'A'][*pl-'A'] > 0)


cx = '.~;


else


cx = ";


}


else


cx = ,


*px++ = cx;


}


*px++ _ '\n';


*px = '\0';


}


61


CA 02534018 2001-02-28
WO 01/G8848 PCT/USO1/OG520
Table 1 (cony)
/*
* strip path or prefix from pn, return len: pr alignQ
*%
static
S stripname(pn) stripname
char *pn; I * file name (may be path) *I
register char *px, *py;
py = 0;
for (px = pn; *px; px++)
if (*px -- '/')
py=px+l;
(PY)
1$ (void) strcpy(pn, py);
return(strlen(pn));
25
35
45
55
62


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/06520
Table 1 (cony)
/*
* cleanupQ - cleanup any tmp file
* getseqQ - read in seq, set dna, len, maxlen
* g callocp -- callocp with error checldn
$ * readjmpsQ -- get the good jmps; from tmp file if necessary
* writejmpsQ - write a filled array of jmps to a tmp file: nwQ
*/
/finclude "nw.h"
#include < syslfile.h >
char *jname = "/tmp/homg3~7LX3fX"; . . . /* ~p ~e for jmps */
FILE *fj;
int cleanup(); l* cleanup tmp file *I
long lseelcQ;
/*
* remove any tmp file if we blow
*/
cleanup(i) cleanup
int i;
{
if (fj)
(void) unlinl.(jname);
ezit(i);
1
/*
* read, return ptr to seq, set dna, len, maiden
* skip lines starting with ';', ' <', or ' >'
* seq in upper or lower case
*/
char
getseq(file, len) getSe(1
3S char *file; /* file name */
int *len; /* seq len */
f
char , line[1024], *pseq;
register char *px, *py;
40 int natgc, lien;
FILE *fp;
if ((fp = fopen(fde, "r")) _ = 0) {
fprintf(stderr,"%"s: can't read %s~n", prog, file);
45 exit(1);
1
lien = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =_ '~' ~ ~ *line == ' <' ~ ~ *line =_ ' >')
S0 continue;
for (px = line; *px ! _ '\n'; px++)
if (isupper(*px) ~ ~ islower(*px))
lien++;
1
55 if ((pseq = malloc((unsigned)(tlen+G))) _= 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for ~s\n", prog, lien+6,
file);
exit(1);
60 Pseq[0] = pseq[1] = pseq(2] = pseq[3] _ '\0';
63

CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 1 (cony)
...getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 102,4, fp)) {
if (*line =- ' ~ ~ *line =- ' <' ~ ~ *line =- ' >')
continue;
for (px = line; *px ! _ '\n'; px++) {
if (isupper(*px))
*py++ = *px;
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU",*(py-1)))
natgc+ +;
*py++ _ '\o';
*Py = '\0,;
(void) fclose(fp);
dna = natgc > (tlen/3); '
return(pseq+4);
char
g~calloc(msg, nx, sz) g-CallOC
char *msg; /* program, calling routine */
int nx, sz; /* number and size of elements */
{
char *px, *calloc0; '
if ((px = calloc((unsigned)nx, (unsigned)sz)) _ = 0) {
if (*msg) {
fprintf(stderr, "%s: g-callocQ failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(1);
return(px);
j
/*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: mainQ
*l
readjmps() readjmps
{
int fd = -1;
int siz, i0, il;
register i, j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname, O ItDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't openQ %s\n", prog, jname);
cleanup(1);
}
for (i = i0 = il = 0, dmax0 = dmax, xx = len0; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j-)
,
64

CA 02534018 2001-02-28
WO O1/G8848 PCT/US01/06520
Table 1 (cony)
...readjmps
if (j < 0 && dx[dmax].offset && fj) {
(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;
}
if (i > = JMPS) { .
fprintf(stderr, "%s: too many gaps in alignmentln", prog);
cleanup(1);
1 s if (j > = o) {
siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x[jl;
dmax += siz;
if (siz < 0) { /* gap in second seq */
pp[1].n[il] _ -siz;
xx += siz;
/*id=xx-yy+lenl-1
*/
Pa[1]++11] = xx - dmax + lenl - 1;
g PY
~aPY -= s~>
/* ignore MAXGAP when doing endgaps */
siz = (-siz < MAXGAP ~ ) endgaps)? -siz : MAXGAP;
il+'+;
}
else if (siz > 0) { /* gap in first seq */
pp[0].n[i0] = siz;
PP[0] ~ x[i0] = xx;
gapx++;
ngapx += siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
}
}
else
break; ,
}
/* reverse the order of jmps
*/
for (j = 0, i0--; j < i0; j++, i0--) {
i = PP[OI.nU]; PP[Ol.nLll = PP[0].n[i0]; pP[0].n[i0] = i;
' - PP[0].xClI;.PP[0].xLl] = PP[0].x[i0]; pPCOI.x[i0] = i;
}
for (j = 0, il--; j < il; j++, il--) {
i = pp[1].n[j]; pp[1].n[j] = pp(1].n[il]; pp[1].n[il] = i;
i = pp[1].x[j]; pp[1].x[j] = pp[1].x[il]; pp[1].x[il] = i;
~ (fd > = o)
(void) close(fd);
~(f){
(void) unlink(jname);
fj = 0;
offset = 0;
} }


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 1 (cont'1
/*
* write a filled jmp struct offset of the prev one (if any): nwQ
*!
S writejmps(ix) wTitejmps
int ix;
char *mktempQ;
if (!fj) f
if (mlrtemp(jname) < 0) {
fprintt(stderr, "96s: can't mlttemp0 ~s~n", prog, jname);
cleanup(1);
if ((fj = fopen(jname, "w")) _ = 0) f
fprintf(stderr, "%s: can't write ~s~n", prog, jname);
exit(1);
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, C7);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
30
3S
4S
55
66

CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 2
PRO XXXXXX?~XXXX (Length = 15 amino acids)
Comparison Protein XX~~~'YYYYYYY (Length = 12 amino acids)
% amino acid sequence identify =
(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
Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXX7~~YYYYZZYZ (Length = 15 amino acids)
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 10 = 50%
Table 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 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 number of nucleotides of the PRO-DNA nucleic acid
sequence) _
6 divided by 14 = 42.9%
67


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 5
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLW (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 number of nucleotides of the PRO-DNA nucleic acid
sequence) _
4 divided by 12 = 33.3 3&
II. Compositions and Methods of the Invention
A. F~11-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
polypeptides have been identified and isolated, as disclosed in farther 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 bin 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 pokypeptides described
herein, it is contemplated that
PRO variants 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 glycosykation sites or altering the membrane anchoring characteristics.
. Variations in the native full-kength sequence PRO or in various domains of
the PRO described herein,
can be made, for example, using any of the techniques and guidelines for
conservative and non-conservative
68


CA 02534018 2001-02-28
WO 01168848 PCT/USO1/06520
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 codons 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
S 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
and/or chemical properties, such
as the replacement of a leucine with a serine, i.e., conservative amino acid
replacements. Insertions or deletions
may optionally be in the range of about 1 to S amino acids. The variation
allowed may be 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.
PRO polypepdde fragments are provided herein. Such fragments may be truncated
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 biologicai activity of the PRO
1S 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 lmown 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 polymerase chain reaction (PCR). Oligonucleotides
that define the desired 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 and/or immunological activity with the native
PRO polypeptide disclosed herein.
In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the heading
2S 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.
69


CA 02534018 2001-02-28
WO O1/G88.~8 PCT/USO1/06520
Table 6
Original Exemplary Preferred


Residue Substitutions Substitutions


Ala (A) val; leu; ile val


Arg (R) lys; gln; asn lys


Asn (I~ gln; his; lys; arg gln


Asp (D) glu glu


Cys (C) ser ser


Gln (Q) asn asn


Glu (E) asp asp


Gly (G) pro; ala ala


His (H) asn; gln; lys; arg arg


Ile (I) leu; val; met; ala;
phe;


norleucine leu


Leu (L) norleucine; ile; val;


met; ala; phe ile


Lys (I~ arg; gln; asn arg


Met (M) leu; phe; ile leu


Phe (F) leu; val; ile; ala; leu
tyr


Pro (P) ala ala


Ser (S) thr thr


Thr (T) ser Ser


Trp (R~ tyr; phe tyr


Tyr ('S~ trp; phe; thr; ser phe


Val (V) ile; leu; met; phe;


ala; norleucine leu


Substantial modifications in function or immunological identity of the PRO
polypeptide are accomplished
by selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide
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, leu, 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
oligonucleotide-mediated (site
directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis [Carter et al., Nucl.
Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)],
cassette mutagenesis [Wells et al.,


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Gene, 34:315 (I985)], restriction selection mutagenesis [Wells et al., Philos.
Traps. R. Soc. London SerA,
317:415 (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 alanine, glycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the beta-carbon
and is less likely to alter the main-
chain conformation of the variant [G~mningham 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 Proteins, (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol., 150:1
(1976)]. 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
cmsslinlang agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-hydroxysuccinimide
esters, for example, esters with 4-azidosalicylic acid, homobifunctional
imidoesters, including disuccinimidyl
esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1, 8-octane
and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding
glutamyl and aspariyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
groups of Beryl or threonyl residues, methylation of the a-amino groups of
lysine, arginine, and histidine side
chains [T.E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco,
pp. 79-86 (1983)], 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 polypeptide.
"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 and/or
enzymatic means), and/or adding one or more glycosylation sites that are not
present in the native sequence PRO.
In addition, the phrase includes qualitative changes in the glycosylation of
the native proteins, involving a change
in the nature and proportions of the various carbohydrate moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished 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 threonine residues to the native sequence PRO (for O-linked glycosylation
sites). The PRO amino acid
sequence may optionally be altered through changes at the DNA Level,
particularly by mutating the DNA encoding
71


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
the PRO polypeptide at preselected bases such that codons are generated that
will translate into the 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
described in the art, e.g., in WO
87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306
(1981).
Removal of carbohydrate moieties present on the PRO pokypeptide may be
accomplished chemically or
enzymatically or by mutational substitution of codons encoding for amino acid
residues that serve as targets for
glycosylation. Chemical deglycosylation techniques are known in the art and
described, for instance, by
Haldmuddin, et al., Arch. Biochem. Bioohvs., 259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety of
endo- and exo-glycosidases as described by Thotakura et ak., Meth. E ,nz~mol.,
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 tag 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 the 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 et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc
tag and the 8F9, 3C7, 6E10, G4,
B7 and 9E10 antibodies thereto [Evan et ak., Molecular and Cellular Bioloey,
5:3610-3616 (1985)]; and the
Herpes Simplex virus gkycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein En ing eJg, 3(6):547-
553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al. ,
BioTechnolo~v, 6_:1204-1210 ( 1988)];
the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an a-
tubulin epitope peptide [Skinner et
al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein
peptide tag [Lutz-Freyermuth et al.,
Proc. Natl. Acad. Sci. USA, 87:6393-6397 (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 inactivated) form of a PRO
polypeptide in place of at least one variable region within an Ig molecule. In
a particularly preferred embodiment,
the immunoglobukin fusion includes the hinge, CH2 and CH3, or the hinge, CH1,
CH2 and CH3 regions of an
IgGl molecule. For the production of immunoglobulin fusions see also US Patent
No. 5,428,130 issued June 27,
72


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
1995.
D. Preyaration 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-
Phase 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 enzymatic methods to produce the
full-length PRO.
1. Isolation of DNA Encoding
DNA encoding PRO 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 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 genomic library or by known synthetic procedures
(e.g., automated nucleic acid
synthesis).
Libraries can be screened with probes (such as antibodies to the PRO or
oligonucleotides of at least about
20-80 bases) designed to identify the gene of interest or the protein encoded
by it. Screening the cDNA or
genomic library with the selected probe may be conducted using standard
procedures, such as described in
Sambrook et al., Molecular Cloning A Laboratory Manual (New York: Cold Spring
Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding PRO is to use PCR
methodology [Sambrook et al.,
' su ra; Dieffenbach et al., PCR Primer: A Laboratory.Manual (Cold Spring
Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The
oligonucleotide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are minimized.
The oligonucleotide is preferably labeled such that if can be detected upon
hybridization to DNA in the library
being screened. Methods of labeling are well known in the art, and include the
use of radiolabels like'ZP-labeled
ATP, biotinylation or enzyme labeling. Hybridization conditions, including
moderate stringency and high
stringency, are provided in Sambrook et al., supra.
Sequences identiEed in such library screening methods can be compared and
aligned to other lmown
sequences deposited and available in public databases such as GenBank or other
private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across
the full-length sequence can be determined using methods known in the art and
as described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al.,
supra, to detect precursors and
73


CA 02534018 2001-02-28
WO O1ZG8848 PCTlUSOIl06520
processing intermediates of mRNA that may not have been reverse-izanscribed
into eDNA.
2. Selection and Transformation 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
for inducing promoters, selecting
transformants, oz 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. Tn general,
principles, protocols, and practical techniques for maximizing the
productivity of cell cultures can be found in
Mammalian Cell Biotechnology: a Practical A,.poroach, 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, CaCl2, CaPO~, liposome-mediated and
electroporation. Depending on the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., ssunra, or
electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as
described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29
June 1989. For mammalian cells
without such cell walls, the calcium phosphate precipitation method of Graham
and van der )rb, Virolo , 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., 130:946 (1977) and Hsiao et al., Proc. Natl.
Acad. Sci. (USA), 76:3829 (1979).
However, other methods for introducing DNA into cells, such as by nuclear
microinjection, electroporation,
bacterial protoplast fusion with intact cells, or polycations, e.g.,
polybrene, polyornithine, may also be used. For
various techniques for transforming mammalian cells, see Keown et al., Methods
in Enzymoloev, 185:527-537
(1990) and Mansour et al., Nature, 336:348-352 (1988).
Suitable host cells for 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. coli.
Various E. coli strains are publicly
available, such as E. coIi K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC
31,537); E. coti strain
W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host
cells include
F.nterobacteriaceae such as Escheric)tia, e.g., E. coll, Enterobacter,
Erwinia, Klebsiella, Proteus, Salmonella,
e.g., Salmonella typhirnurium, Serratia, e.g., Serratia rnarcescans, and
Shigella, as well as Bacillt such as B.
subtilis and B. licheniformis (e.g., B, Zlchenifonnis 41P disclosed in DD
266,710 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 examples of such hosts including E. coli W3110 strain 1A2,
which has the complete genotype
tonA ; E. coli W3110 strain 9FA~, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC
74


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/06520
55,244), which has the complete genotype tonA ptr3 phoA E15 (argF lac)169 degP
ompT kan'; E. coli W3110
strain 37D6, which has the complete genotype tonA ptr3 phoA EI S (argF lac)169
degP ompT rbs7 ilvG kan';
E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an
E. coli strain having mutant periplasmic 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. Saccharonryces cerevisiae is a
commonly used lower eukaryotic
host microorganism. Others include Schizosaccharomyces pombe (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.,
BiolTechnolo~y, 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), K.
bulgaricus (ATCC 16,045), K. wickeramii
(ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den
Berg et al.,
Bio/Technolosy, 8:135 (1990)), K. thermotolerans, and K. marxianus; yarrowia
(EP 402,226); Pichia pastoris
(EP 183,070; Sreekrishna et al., J. 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]); Schwanniomyces
such as Schwannionryces occidentalis (EP 394,538 published 31 October 1990);
and 6lamentous fungi such as,
e.g., Neurospora, Penicilliutn, Tolypocladium (VJO 91/00357 published 10
January 1991), andAspergillus hosts
such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-
289 [1983]; Tilburn et al.,
Gene, 26:205-221 (1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-
1474 (1984]) and A. niger (Kelly
and Hynes, EMBO J., 4:475-479 [1985]).~ Methylotropic yeasts are suitable
herein and include, but are not
limited to, yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida,
Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of
specific species that are exemplary
of this class of yeasts may be found in C. Anthony, The Biochemistry of
Methvlotrophs, 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 DrosQphila S2 and
Spodoptera Sf9, 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 CVl line transformed by SV40 (COS-7,
ATCC CRL 1651); 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 cells/-DHFR (CHO, Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA,
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251
(1980)); human lung cells (W138,
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 Re licable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may be inserted into
a replicable vector
for cloning (amplification of the DNA) or for expression. Various vectors are
publicly available. The vector
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


CA 02534018 2001-02-28
WO 01/68848 PCT1i7S01/06520
appropriate restriction endonuclease sites) using techniques known in the art.
Vector components generally
include, but are not 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 recombinantly not only directly, but also as a fusion
polypeptide 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 II leaders. For yeast secretion
the signal sequence may be, e.g., the
yeast invertase leader, alpha factor leader (including Saccharomyces and
Kluyveromyces a-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 1990), or the signal described in WO 90/13646
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 polypepddes of the same or related species, as
well as viral 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 the plasnud pBR322 is suitable for most Gram-
negative bacteria, the 2tc plasmid
origin is suitable for yeast, and various viral origins (SV40, polyoma,
adenovirus, VSV or BPV) are useful 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
thymidine 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. Natl. Acad. Sci. USA. 77:4216
(1980). A suitable selection gene
for use in yeast is the trill gene present in the yeast plasmid 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 [Jones, Genetics, 85:12 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to
the PRO-encoding nucleic
acid sequence to direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well
known. Promoters suitable for use with prokaryotic hosts include the (3-
lactamase and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544
(1979)], alkaline phosphatase, a
tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776], and hybrid
76


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sei. 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 ldnase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or
other glycolytic enzymes [Hess
et al., J. Adv. Enz~ne Re~~, 7:149 (1968); Holland, Biochemistry, 17:4900
(1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase,hexokinase,pyruvate
decarboxylase,phosphofructoldnase,glucose-6-
phosphate isomerase, 3-phosphoglycerate mutase, pyruvate ldnase,
triosephosphate isomerase, phosphoglucose
isomerase, and glucoldnase.
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
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 300 bp,
that act on a promoter to increase its transcription. Many enhancer sequences
are now known 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 enhaneer on the late side
of the replication 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 5' 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 from 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 et al., Nature, 293:620-625
(1981); Mantel et al., Nature, 281:40-
46 (I979); EP 117,060; and EP 117,058.
4. Detecting Gene AmplificationlExpression
77


CA 02534018 2001-02-28
wo oo~xx-tx PcT/uso~/o~;2u
Gene amplification and/or expression may be measured in a sample directly, for
example, by
conventional Southern blotting, Northern blotting to quantitate the
transcription of mRNA [Thomas, Proc. Natl.
Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ
hybridization, using an
appropriately labeled probe, based on the 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
inununohistochemical 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 and/or 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
antibody epitope.
5. Purification of Polypeptide
Forms of PRO may be recovered from culture medium or from host cell lysates.
If membrane-bound,
it can be released from the membrane using a suitable detergent solution (e.
g. Triton-X 100) or by enzymatic
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 from 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 on silica or on a cation-
exchange resin such as DEAF;
chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration
using, for example, Sephadex G-75;
protein A Sepharose columns to remove contaminants such as IgG; and metal
chelating 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, Methods in EnzymOlo~y, 182
(1990); Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York (1982). The
purification steps) selected will
depend, for example, on the nature of the production process used and the
particular PRO produced.
E. Uses for PRO
Nucleotide sequences (or their complement) encoding PRO have various
applications in the art of
molecular biology, including uses as hybridization probes, in chromosome 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.
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
*-t:rademarit ~$


CA 02534018 2001-02-28
WO OI/G8848 PCT/US01/06520
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'~P or 355, or enzymatic labels such as alkaline
phosphatase coupled to the probe via
avidin/biotin 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 mRIVA (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 14
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 Krol et al. (BioTechniques
6:958, 1988).
Binding of antisense or sense oligonucleotides to target nucleic acid
sequences results in the formation
of duplexes that block transcription or translation 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 covalently
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.
Antisense or sense oligonucleotides may be introduced into a cell containing
the target nucleic acid
sequence by any gene transfer method, including, for example, CaP04-mediated
DNA transfection,
electroporation, or by using gene transfer vectors such as Epstein-Burr virus.
In a preferred procedure, an
79


CA 02534018 2001-02-28
WO 01/G8848 PCT/USO1/OG520
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 in
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
90/13641).
Sense or antisense oligonucleotides also may be introduced into a cell
containing the target nucleotide
sequence by formation of a conjugate with a ligand binding molecule, as
described in WO 91/04753. Suitable
ligand binding molecules include, but are not limited to, cell surface
receptors, growth factors, other cytoldnes,
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 oligonucleotide may be introduced into
a cell containing the target
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 least about 5 bases
in length, about 10 bases
in length, about 15 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 45 bases in length,
about 50 bases in length, about 55
bases 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 85 bases is 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 PRO 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 in situ hybridization, linkage analysis
against known chromosomal markers, and
hybridization screening with libraries.
When the coding sequences for PRO 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 receptor/ligand
binding interaction can be identified.
Proteins involved in such binding interactions can also be used to screen for
peptide or small molecule inhibitors
or agonists of the binding interaction. Also, the receptor PRO can be used to
isolate correlative ligand(s).
Screening assays can be designed to fmd lead compounds that mimic the
biological activity of a native PRO ox
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 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.


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
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 transgenic 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 contain
cells which express DNA encoding PRO. Methods for generating transgenic
animals, particularly 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 overexpression. 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
"knock 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 electroporation) and
cells in which the introduced DNA
has homologously recombined with the endogenous DNA are selected [see e.g., Li
et al., Cell, 69:915 (1992)].
The selected cells are then injected into a blastocyst 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. 113-152]. 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. Progeny
harboring the homologously recombined DNA in their germ cells can be
identified by standard techniques and
used to breed animals in which all cells 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 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 fox replacement of a defective gene. "Gene
therapy" includes both conventional
81


CA 02534018 2001-02-28
WO O1/C8848 PCT/USO1/OG520
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 oligonuckeotides 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
oligonucleotides 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 vitro, 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 liposomes, 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 BiotechnoloQy
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 kigand for
a receptor on the target cell, etc. Where liposomes are employed, proteins
which bind to a cell surface membrane
protein associated with endocytosis may be used for targeting and/or 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., J. Biol. 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 identification. 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
diagnostically for tissue typing, wherein the PRO polypeptides of the 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 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 lmown
methods to prepare pharmaceutically
useful compositions, whereby the PRO product hereof is combined in admixture
with a pharmaceutically
acceptable carrier vehicle. Therapeutic formulations are prepared for storage
by mixing the active ingredient
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CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
having the desired degree of purity with optional physiologically acceptable
carriers, excipients or stabilizers
(Reminaton'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 immunoglobulins; hydrophilic polymers such as
polyvinylpyrrofidone, 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; and/or nonionic surfactants
such as TWEENTM, PLURONICS'~"''
or PEG.
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
lyophilization 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 with lmown methods, e.g. injection or
infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or
intralesional mutes, topical
administration, or by sustained release systems.
Dosages and desired drug concentrations of pharmaceutical compositions of the
present invention may
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 guidance
for the determination of effective doses for human therapy, hzterspecies
scaling of effective 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 ng/kg to up to 100 mg/kg of mammal body
weight or more .per day,
preferably about 1 ~g/kg/day to 10 mg/kg/day, 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.
4,657,760; 5,206,344; or 5,225,212. It is anticipated that different
formulations will be effective for different
treatment compounds and different disorders, 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 administration of the PRO
polypeptide, microencapsulation of the PRO polypeptide is contemplated.
Microencapsulation of recombinant
proteins for sustained release has been successfully performed with human
growth hormone (rhGH), interferon-
3S (rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat. Med., 2:795-
799 (1996); Yasuda, Biomed. Ther.,
27:1221-1223 (1993); Hora et al., Bio/Technolosy. 8:755-758 (1990); Cleland,
"Design and Production of Single
Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in
Vaccine Desire Subunit
83


CA 02534018 2001-02-28
WO 01/688:18 PCT/USO1/06520
and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995),
pp. 439-462; WO
97/03692, WO 96/40(?72, WO 96/07399; and U.S. Pat. No. 5,654,010.
The sustained-release formulations of these proteins were developed using poly-
lactic-coglycolic acid
(PLGA) polymer due to its biocompatibility and wide range of biodegradable
properties. The degradation products
of PLGA; lactic and glycolic acids, can be cleared quickly within the human
body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on its
molecular weight and composition. Lewis,
~Controlled release of bioactive agents from lactide/glycolide polymer," in:
M. Chasin and R. Langer (Eds.),
Biodegradable Polymers as Dru De~li'very Systems (Marcel Deld<er: New York,
1990), pp. 1-41.
This invention encompasses methods of screening compounds to identify those
that mimic the PRO
polypeptide (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 chenucal
libraries, making them 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 complex 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 anh'body, speck
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
label, 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-immobilized component carries a detectable label, the
detection of label immobilized on
the surface indicates that complexing occurred. Where 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.
If the candidate compound interacts with but does not bind to a particular PRO
polypeptide encoded by
a gene identified herein, its .interaction with that polypeptide can be
assayed by methods well lmown for detecting
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 lLondon~, 340:245-246 (1989); Chien et al., Pros.
Natl. Acad. Sci. USA, 88:9578-9582
84


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
(1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:
5789-5793 (1991). Many
transcriptional activators, such as yeast GAL4, 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
to as the "two-hybrid system") takes
advantage of this property, and employs two hybrid proteins, one in which the
target protein is fused to the DNA-
binding domain of GALA, and another, in which candidate activating proteins
are fused to the activation domain.
The expression of a GALL-lacZ reporter gene under control of a GAIA-activated
promoter depends on
reconstitution of GAL4 activity via protein-protein interaction. Colonies
containing interacting polypeptides are
detected with a chromogenic substrate for p-galactosidase. A complete Idt
(MATCHMAKERT"') 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 identified 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
1S 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 control. 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,
2S 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 PRO polypeptide can be labeled, such as by radioactivity, such that
the number of PRO polypeptide
molecules bound to the receptor can be used to determine the effectiveness of
the potential antagonist. 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. Coligan et al., C~rrrent Protocols in Immun.,
1(2): Chapter 5 (1991).
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.
Transfected cells that are grown on glass
slides are exposed to labeled PRO polypeptide. The PRO polypeptide can be
labeled by a variety of means
including iodination or inclusion of a recognition site for a site-specific
protein kinase. Following fixation and
incubation, the slides are subjected to autoradiographic 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,
8S


CA 02534018 2001-02-28
WO 01168848 PCT/USO1/06520
clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PRO
polypeptide can be photoaffmity-
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
S from micro- sequencing would be used to design a set of degenerate
oligonucleotide 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 interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide
that binds to the fusions of
immunoglobulin with PRO polypeptide, and, in particular, antibodies including,
without limitation, poly- and
monoclonal antibodies and antibody fragments, single-chain antibodies, anti-
idiotypic antibodies, and chimeric
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 antisense RNA or DNA
construct prepared using
antisense 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 oligoiiucleotide
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., Science, 251:1360 (1991)),
thereby preventing transcription and the
production of the PRO polypeptide. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the PRO polypeptide (antisense -
Okano, Neurochem., 56:560
(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC
Press: Boca Raton, PL, 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 polypeptide. When antisense
DNA is used,
oligodeoxyribonucleotides derived from the translation-initiation site, e.g.,
between about -10 and + 10 positions
of the target gene nucleotide sequence, are preferred.
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.
ltibozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of RNA.
86


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Ribozymes act by sequence-speck 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 further details see, e.g., Rossi, Current Bioloay, 4:469-471 (1994), and
PCT publication No. WO 97/33551
(published September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded
and composed of deoxynucleotides. The base composition of these
oligonueleotides 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 and/or by any other screening techniques well lmown 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. Polyclonal Antibodies
The anti-PRO antibodies may comprise polyclonal antibodies. Methods of
preparing polyclonal
antibodies are known to the skilled artisan. Polyclonal 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
and/or adjuvant will be injected in the mammal 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
thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may
be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic
trehalose dicorynomycolate).
The immunization protocol may be selected by one skilled in the art without
undue experimentation.
2. Monoclonal 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, 256:495 (1975).
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
87


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/OG520
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
[Goding, Monoclonal Antibodies: Principles 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 hybridoma
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 medium. More
preferred immortalized cell lines are marine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection, Manassas,
Virginia. Human 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, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of
monoclonal antibodies directed against 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
known in the art. The binding affinity of the monoclonal antibody can, for
example, be determined by the
Scatchard analysis of Munson and Pollard, Anal. 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.
The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture medium
or ascites fluid by conventional immunoglobulin purification procedures such
as, for example, protein A-
Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
The monoclonal antibodies 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 canbe 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
88


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
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 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
Imown in the art. For example, one method involves recombinant expression of
immunoglobulin light chain and
modified heavy chain. The heavy chain is truncated generally at any point in
the Fc region so as to prevent heavy
chain crosslinldmg. Alternatively, the relevant cysteine residues are
substituted with another amino acid residue
or are deleted so as to prevent crosslinldng.
IO In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to
produce fragments thereof, particularly, Fab fragments, can be accomplished
using routine techniques kaown in
the art.
3. Human and Humanized Antibodies
The anti-PRO antibodies of the invention may further comprise humanized
antibodies or human
antibodies. Humanized forms of non-human (e.g., marine) antibodies are
chimeric immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Pab, Fab', F(ab')2 or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived from non-
human immunoglobulin.
Humanized antibodies include human itnmunoglobulins (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
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 itnmunoglobulin 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 regions
correspond to those of a non-human immunoglobulin and all or substantially all
of the FR regions are those of a
human immunoglobulin consensus sequence. The humanized antibody optimally also
will comprise at least a
portion of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al.,
Nature, 321:522-525 (I986); Riechmann et al., Nature, 332:323-329 (1988); and
Presto, Curr. Op. 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 refezred to as "import" residues, which
are typically taken from an "import"
variable domain. Humanization can be essentially performed following the
method of Winter and co-workers
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-
327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences
for the corresponding sequences
of a human anri'body. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Patent No.
s9


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
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, J. Mol. Biol., 227:381 (1991); Maria
et al., J. Mol. Biol., 222:581
(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 Therapy,
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
IO 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., Bio/Technolo~y 10,
779-783 (1992); Lonberg et ad., Nature 368 856-859 (1994); Morrison, Nature
368, 812-13 (1994); Fishwild et
1S al., Nature Biotechnoloev 14, 845-51 (1996); Neuberger, Nature
Biotechnolo~y 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 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
20 human) from which the matured antibody is prepared.
4. Bispecific Antibodies
Bispecii~ic antibodies are monoclonal, preferably human or humanized,
antibodies that have binding
specificities for at least two different antigens. In the present case, one of
the binding speci~cities is for the PRO,
25 the other one is for any other antigen, and preferably for a cell-surface
protein or receptor or receptor subunit.
Methods for making bispecific antibodies are lrnown in the art. Traditionally,
the recombinant production
of bispecific antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where
the two heavy chains have different specificities [Milstein and G~ello,
Nature, 305:537-539 (1983)]. Because of
the random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a
30 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 affuuty
chromatography steps. Similar procedures
are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al.,
fiMBO J., 10:3655-3659
(1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
35 be fused to immunoglobulin constant domain sequences. The fusion preferably
is with an immunoglobulin heavy-
chain constant domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the
first heavy-chain constant region (CH1) containing the site necessary for
light-chain binding present in at least


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and,
if desired, the immunoglobulin
light chain, are inserted into separate expression vectors, and are co-
transfected into a suitable host organism.
For further details of generating bispecific antibodies see, for example,
Suresh et al., Methods in Enzvmoloav,
121:210 (1986).
According to another approach described in VJO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maacimize 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
replaced with larger side chains (e.g, tyrosine or tryptophan). Compensatory
"cavities" of identical or similar
size to the large side chains) are created on the interface of the second
antibody molecule by replacing large
amino acid side chains with smaller ones (e.g. alanine or threonine). This
provides a mechanism for increasing
the yield of the heterodimer over other unwanted end-products such as
homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g. F(ab')2
bispecific antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared 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 directly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et al., J. Ex~. Med. 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')z molecule. Each Fab' fragment was separately
secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity
of human cytotoxic lymphocytes against human breast tumor targets.
Various technique for making and isolating bispecific antibody fragments
directly from recombinant cell
culture have also been described. For example, bispecific antibodies have been
produced using leucine zippers.
Kostelny et al. , J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun proteins
were linked to the Fab' portions of two different antibodies by gene fusion.
The antibody homodimers were
reduced at the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This
method can also be utilized for the production of antibody homodimers. The
"diabody" technology described by
Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444.-6448 (1993) has provided
an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a heavy-chain
variable domain (VH) connected
to a light-chain variable domain (V,~ by a linker which is too short to allow
pairing between the two domains on
the same chain. Accordingly, the VH and VL domains of one fragment are forced
to pair with the complementary
91


CA 02534018 2001-02-28
WO 01!68848 PCT/US01I06520
V~ and VH domains of another fragment, thereby forming two antigen-binding
sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has
also been reported. See, Gruber et
al., J. Immunol. 152;5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispeci~c antibodies can be prepared.
Tutt 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
(Fc~yR), such as Fc~yRI (CD64), FcyRII (GD32) and FcyRIII (CD16) so as to
focus cellular defense mechanisms
to the cell expressing the particular PRO polypeptide. Bispecific antibodies
may also be used to localize cytotoxic
agents to cells which express a particular PRO polypeptide. 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. Heteroconj_gate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies
are composed of two covalently joined antibodies. Such antibodies have, for
example, been proposed to target
immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for
treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may
be prepared in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinldng 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 those disclosed,
for example, in U.S. Patent No. 4,676,980.
6. Effector Function Engineering
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 cancer. For
example, cysteine residues) may be
introduced into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability and/or increased complement-
mediated call lolling 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 et al. 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 Drug Design.
3: 219-230 (1989).
'
7. Imtnunoconju ag tes
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent
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CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/OG520
such as a chemotherapeutic agent, toxin (e. g. , an enzymatically active toxin
of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins (PAPI,
PAPA, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of zadionuclides are available
for the production of radioconjugated antibodies. Examples include zl2Bi,
'31I, '3~In, 9°Y, and lssRe.
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 compounds (such as 1,S-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 exemplary chelating
agent for conjugation of
radionucleotide to the antibody. See W094/11026.
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).
8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing
the antibody are prepared by methods laiown in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci.
USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos.
4,485,045 and 4,544,S4S. 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 ., 1. Biol. Chem. , 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such
as Doxorubicin) is optionally contained within the liposome. See Gabizon et
al. ,1. National Cancer Inst., 81(19):
1484 (1989).
9. Pharmaceutical Compositions of Antibodies
Antibodies specifically binding a PRO polypeptide identified herein, as well
as other molecules identified
93


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
by the screening assays disclosed hereinbefore, can be administered for the
treatment 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. However, lipofections or liposomes can also >ie used
to deliver the antibody, or 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 the target protein
sequence. Such peptides can be synthesized chemically and/or produced by
recombinant DNA technology. See,
e.g., Marasco et al., Proc. Natl. Acid. Sci. USA, 90: 7889-7893 (1993). The
formulation 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 cytotoxic agent, cytoldne,
chemotherapeutic 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 microcapsules prepared, for
example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal drug
delivery systems (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 membranes.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in
the form of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include
polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S.
Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate,
non-degradable ethylene-vinyl
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-D-(-)-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 long time, 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
aggregation mechanism is discovered
to be intermolecular S-S bond formation through thio-disulfide interchange,
stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling
moisture content, using appropriate
additives, and developing specific polymer matrix compositions.
94


CA 02534018 2001-02-28
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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, lnc.
(1987) pp. 147-158]. 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,'4C,'zP,'sS, or'ZSI, a
fluorescent or chemiluminescent
compound, such as fluorescein isothiocyanate, rhodamine, 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., Biochemistrv, 13:1014 (1974); Pain et al., J.
Immunol. Meth., 40:219 (1981); and
Nygren, J. Histochem. and Cytochem., 30:407 (1982).
Anti-PRO antibodies also are useful for the affinity purification of PLO 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 filter 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.
All patent and literature references cited in the present specification are
hereby incorporated by reference
in their entirety.
EXAMPLES
Commercially available reagents referred to in the examples were used
according to manufacturer's
instructions 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 Homoloay Screening to Identify Novel
Poly~peptides 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, GenBank), and proprietary
databases (e.g. LIFESEQTM,
Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the
computer program BLAST or


CA 02534018 2001-02-28
WO O1/G8848 PCTIUS01/06520
BLAST-2 (Altschul et al., Methods in Enzvmology, 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
S 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 phrap
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 PRO polypeptide.
Forward and reverse PCR primers
generally 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-SS by in length. In some
cases, additional oligonucleotides are
synthesized when the consensus sequence is greater than about 1-l.Skbp. In
order to screen several libraries for
a full-length clone, DNA from the libraries was screened by PCR amplification,
as per Ausubel et al., Current
1S Protocols in Molecular Bioloey, 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 SaII hemilanased
adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined orientation into
a suitable cloning vector (such as
pRKB or pRICD; pRKSB is a precursor of pRKSD that does not contain the SfiI
site; see, Hohnes et al. , Science,
253:1278-1280 (1991)) in the unique XhoI and NotI sites.
EXAMPLE 2: Isolation of cDNA clones b~Amylase Screening
2S 1. Preparation of oligo dT grimed cDNA library
mRNA was isolated from a human tissue of interest using reagents and protocols
from Invitrogen, San
Diego, CA (Fast Track 2). This RNA was used to generate an oligo dT primed
cDNA 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/NotI Tinkered cDNA
was cloned into XhoT/NotI cleaved vector, pRKSD is a cloning vector that has
an sp6 transcription initiation site
followed by an SfiI restriction enzyme site preceding the XhoI/Notl cDNA
cloning sites.
2. Preparation of random_primed cDNA library
A secondary cDNA library was generated in order to preferentially represent
the S' ends of the primary
3S cDNA 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 pSST-AMY.O using reagents
and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In this
procedure the double stranded cDNA was
96


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
sized to 500-1000 bp, tinkered with blunt to NotI adaptors, cleaved with SfiI,
and cloned into SfiI/NotI 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 dehydrogenase 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 appropriately 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 DH10B bacteria (Life Technologies, 20 ml). The bacteria and
vector mixture was then
electroporated as recommended by the manufacturer. Subsequently, SOC media
(Life Technologies, 1 ml) was
added and the mixture was incubated at 37°C for 30 minutes. The
transformants were then plated onto 20
standard 150 mm LB plates containing ampicillin and incubated fox 16 hours
(37°C). Positive colonies were
scraped off the plates and the DNA was isolated from the bacterial pellet
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
plasmid/cDNA 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 HD56-5A (ATCC-90785). This strain has the following
genotype: MAT
alpha, ura3-52, leu2-3, leu2-112, his3-11, his3-15, MAL+, SUC+, GAL+.
Preferably, yeast mutants can be
employed that have deficient post-translational pathways. Such mutants may
have translocation deficient alleles
in sec71, sec72, sec62, with truncated sec71 being most preferred.
Alternatively, antagonists (including antisense
nucleotides and/or ligands) which interfere with the normal operation of these
genes, other proteins implicated
in this post translation pathway (e.g., SEC6lp, SEC72p, SEC62p, SEC63p, TDJlp
or SSAlp-4p) or the complex
formation of 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. , 20:1425
(1992). Transformed cells were then inoculated from agar into YEPD complex
media broth (100 ml) and grown
overnight at 30°C. The YEPD broth was prepared as described in Kaiser
et al. , Methods in Yeast Genetics, Cold
Spring Harbor Press, Cold Spring Harbor, NY, p. 207 (1994). The overnight
culture was then diluted to about
2 x 106 cells/ml (approx. OD6oo=0.1) into fresh YEPD broth (500 ml) and
regrown to 1 x 10' cells/ml (approx.
ODboo=0.4-0.5).
The cells were then harvested and prepared for transformation by transfer into
GS3 rotor bottles in a
Sorval GS3 rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water,
and centrifuged again in SO ml falcon tubes at 3,500 rpm in a Beckman GS-6KR
centrifuge. The supernatant was
discarded and the cells were subsequently washed with LiAc/TE (10 ml, 10 mM
Tris-HCI, 1 mM EDTA pH 7.5,
100 mM LizOOCCH3), and resuspended into LiAc/TE (2.5 ml).
Transformation took place by mixing the prepared cells (100 pl) with freshly
denatured single stranded
97


CA 02534018 2001-02-28
WO O1/~8848 PCT/USO1/06520
salmon testes DNA (Lofstrand Labs, Gaithersburg, MD) and transforming DNA (1
~,g, vol. < 10 pl) in
microfuge tubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE
(600 pl, 40% polyethylene
glycol-4000, 10 mM Tris-HCI, 1 mM EDTA, 100 mM LiZOOCCH3, pH 7.5) was added.
This mixture was
gently mixed and incubated at 30°C while agitating for 30 minutes. The
cells were then heat shocked at 42°C
for 15 minutes, and the reaction vessel centrifuged in a microfuge at 12,000
rpm for 5-10 seconds, decanted and
resuspended into TE (500 pl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followed by
recentrifugation. The cells
were then diluted into TE (1 ml) and aliquots (200 p,l) 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 synthetic complete dextrose agar lacking uracil
(SCD-Ura) prepared as
described in Kaiser et al. , Methods 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 performed 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
incorporated 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 were picked and streaked across fresh selective media
(onto 150 mm plates) in
order to obtain well isolated and identifiable single colonies. Well isolated
single colonies positive for amylase
secretion were detected by direct incorporation of red starch into buffered
SCD-Ura agar. Positive colonies were
determined by their ability to break down starch resulting in a clear halo
around the positive colony visualized
directly.
4. Isolation of DNA by PCR Amplification
When a positive colony was isolated, a portion of it was picked by a toothpick
and diluted into sterile
water (30 pl) 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 ~.l) was used as a
template for the PCR reaction in a
25 ~.1 volume containing: 0.5 p,l Klentaq (Clontech, Palo Alto, CA); 4.0 pl 10
mM dNTP's (Perkin Elmer-Cetus);
2.5 wl Kentaq buffer (Clontech); 0.25 ~.1 forward oligo 1; 0.25 ~1 reverse
oligo 2; 12.5 pl distilled water. The
sequence of the forward oligonucleotide 1 was:
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID N0:611)
The sequence of reverse oligonucleotide 2 was:
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID N0:612)
PCR was then performed as follows:
a. Denature 92°C, 5 minutes
b. 3 cycles of: Denature 92°C, 30 seconds
Anneal 59°C, 30 seconds
98


CA 02534018 2001-02-28
WO 01/68848 PCT/US01106520
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: Denature 92°C, 30 seconds
Anneal 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 pSST-AMY.O
when no insert was present.
Typically, the first 18 nucleotides of the 5' end of these oligonucleotides
contained annealing sites for the
sequencing primers. Thus, the total product of the PCR reaction from 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 ~,1) was examined by agarose
gel electrophoresis in a
1'~ agarose gel using a Tris-Borate-EDTA (TBE) buffering system as described
by Sambrook et ad., supra.
Clones resulting 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 Inc.,
Chatsworth, CA).
EXAMPLE 3: Isolation of cDNA Clones UsingS_ianal Algorithm Analvsis
Various polypeptide-encoding nucleic acid sequences were identified by
applying a proprietary signal
sequence finding algorithm developed by Genentech, Inc. (South San Francisco,
CA) upon ESTs as well as
clustered and assembled EST fragments from public (e.g., GenBank) and/or
private (LIFESEQ~, Incyte
Pharmaceuticals, Tnc., 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
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 35 unambiguous amino acids
without any stop codons. 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 signal sequence,
the DNA and corresponding amino acid sequences surrounding the ATG codon 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 Polypeptides
Using the techniques described in Examples 1 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.
99


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/OG520
Table 7
Material ATCC Dep. No. Deposit Date


DNA16435-1208209930 June 2, 1998


DNA23318-1211209787 April2l, 1998


DNA23322-1393203400 October 27, 1998


DNA23334-1392209918 June 2, 1998


DNA26843-1389203099 August 4, 1998


DNA 26844-1394209926 June 2, 1998


DNA30867-1335209807 April 28, 1998


DNA33470-1175209398 October 17, 1997


DNA34436-1238209523 December 10,
1997


DNA35557-1137209255 September 16,
1997


DNA35599-1168209373 October 16, 1997


DNA35668-1171209371 October 16, 1997


DNA36992-1168209382 October 16, 1997


DNA39423-1182209387 October 17, 1997


DNA39427-1179209395 October 17, 1997


DNA39510-1181209392 October 17, 1997


DNA39518-1247209529 December 10,
1997


DNA39975-1210209783 Apri121, 1998


DNA39976-1215209524 December 10,
1997


DNA39979-1213209789 April 21, 1998


DNA40594-1233209617 February 5, 1998


DNA40603-1232209486 November 21,
1997


DNA40604-1187209394 October 17, 1997


DNA40625-1189209788 April2l, 1998


DNA41225-1217209491 November 21,
1997


DNA41379-1236209488 November 21,
1997


DNA41386-1316209703 March 26, 1998


DNA44161-1434209907 May 27, 1998


DNA44179-1362209851 May 6, 1998


DNA44192-1246209531 December 10,
1997


DNA44694-1500203114 August 11, 1998


DNA45234-1277209654 March 5, 1998


DNA45409-2511203579 January 12, 1999


DNA45415-1318209810 April 28, 1998


DNA45417-1432209910 May 27, 1998


DNA45493-1349209805 April 28, 1998


100


CA 02534018 2001-02-28
WO O1JG8848 . PCT/USO1JOG520
Table 7 fcont')


Material ATCC Deb Deposit Date
No.


DNA46776-1284209721 March 31, 1998


DNA48296-1292209668 March 11, 1998


DNA48306-1291209911 May 27, 1998


DNA48328-1355209843 May 6, 1998


DNA48329-1290209785 Apri121, 1998


DNA48334-1435209924 June 2, 1998


DNA49141-1431203003 June 23, 1998


DNA49624-1279209655 March 5, 1998


DNA49647-1398209919 June 2, 1998


DNA49819-1439209931 June 2, 1998


DNA50911-1288209714 March 31, 1998


DNA50914-1289209722 March 31, 1998


DNA50919-1361209848 May 6, 1998
'


DNA50980-1286209717 March 31, 1998


DNA52185-1370209861 May 14, 1998


DNA53906-1368209747 April7, 1998


DNA53912-1457209870 May 14, 1998


DNA53913-1490203162 August 25, 1998


DNA53977-1371209862 May 14, 1998


DNA53978-1443209983 June 16, 1998


DNA53996-1442209921 June 2, 1998


DNA54002-1367209754 April 7, 1998


DNA55737-1345209753 Apri17, 1998


DNA56050-1455203011 June 23, 1998


DNA56052-1454203026 June 23, 1998


DNA56107-1415203405 October 27, 1998
1


DNA56110-1437203113 August 11, 1998


DNA56406-1704203478 November 17, 1998


DNA56409-1377209882 May 20, 1998


DNA56410-1414209923 June 2, 1998


DNA56436-1448209902 May 27, 1998


DNA56529-1647203293 September 29, 1998


DNA56855-1447203004 June 23, 1998


DNA56859-1445203019 June 23, 1998


DNA56860-1510209952 June 9, 1998


DNA56865-1491203022 June 23, 1998


101


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 7 (coat')


Material ATCC Dep. Deposit Date
No.


DNA56868-1478203024 June 23, 1998


DNA56869-1545203161 August 25, 1998


DNA56870-1492209925 June 2, 1998


DNA57039-1402209777 April 14, 1998


DNA57253-1382209867 May 14, 1998


DNA57254-1477203289 September 29, 1998


DNA57699-1412203020 June 23, 1998


DNA57704-1452209953 June 9, 1998


DNA57710-1451203048 July 1, 1998


DNA57827-1493203045 July 1, 1998


DNA57844-1410203010 June 23, 1998


DNA58723-1588203133 August 18, 1998


DNA58727-1474203171 September 1, 1998


DNA58730-1607203221 September 15, 1998


DNA58732-1650203290 September 29, 1998


DNA58737-1473203136 August 18, 1998


DNA58743-1609203154 August 25, 1998


DNA58747-1384209868 May 14, 1998


DNA58828-1519203172 September 1, 1998


DNA58846-1409209957 June 9, 1998


DNA58848-1472209955 June 9, 1998


DNA58849-1494209958 June 9, 1998


DNA58850-1495209956 June 9, 1998


DNA58852-1637203271 September 22, 1998


DNA58853-1423203016 June 23, 1998


DNA58855-1422203018 June 23, 1998


DNA59211-1450209960 June 9, 1998


DNA59212-1627203245 September 9, 1998


DNA59213-1487209959 June 9, 1998


DNA59219-1613203220 September 15, 1998


DNA59497-1496209941 June 4, 1998


DNA59602-1436203051 July 1, 1998


DNA59603-1419209944 June 9, 1998


DNA59605-1418203005 June 23, 1998


DNA59607-1497209946 June 9, 1998


DNA59610-1556209990 June 16, 1998


102


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Table 7 (cony)


Material ATCC Dep. Deposit Date
No.


DNAS9612-1466209947 June 9, 1998


DNA59613-1417203007 June 23, 1998


DNA59616-1465209991 June 16, 1998


DNA59619-1464203041 July 1, 1998


DNA59625-1498209992 ' June 16, 1998


DNA59817-1703203470 November 17, 1998


DNA59827-1426203089 August 4, 1998


DNA59828-1608203158 August 25, 1998


DNA59837-2545203658 February 9, 1999


DNAS9844-2542203650 February 9, 1999


DNA59853-1505209985 June 16, 1998


DNA59854-1459209974 June 16, 1998


DNA59855-1485209987 June 16, 1998


DNA60278-1530203170 September 1, 1998


DNA60283-1484203043 July 1, 1998


DNA60608-1577203126 August 18, 1998


DNA60611-1524203175 September 1, 1998


DNA60619-1482209993 June 16, 1998


DNA6062S-1507209975 June 16, 1998


DNA60629-1481209979 June 16, 1998


DNA60740-1615203456 November 3, 1998


DNA61608-1606203239 September 9, 1998


DNA61755-1554203112 August 11, 1998


DNA62809-1531203237 September 9, 1998


DNA62812-1594203248 September 9, 1998


DNA62813-2544203655 February 9, 1999


DNA62845-1684203361 October 20, 1998


DNA64849-1604203468 November 17, 1998


DNA64852-1589203127 August 18, 1998


DNA64863-1573203251 September 9, 1998


DNA64881-1602203240 September 9, 1998


DNA64902-1667203317 October 6, 1998


DNA64952-1568203222 September 15, 1998


DNA6S403-1565203230 September 15, 1998


DNA65413-1534203234 September 15, 1998


DNA65423-1595203227 September 15, 1998


103


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 7 (cony)


Material ATCC Dep. De~sit Date
No.


DNA66304-1546203321 October 6, 1998


DNA66308-1537203159 August 25, 1998


DNA66511-1563203228 , September 15, 1998


DNA66512-1564203218 September 15, 1998


DNA66519-1535203236 September 15, 1998


DNA66521-1583203225 September 15, 1998


DNA66658-1584203229 September 15, 1998


DNA66660-1585203279 September 22, 1998
,


DNA66669-1597203272 September
22, 1998


DNA66674-1599203281 September 22, 1998


DNA68836-1656203455 November 3, 1998


DNA68862-2546203652 February 9, 1999


DNA68866-1644203283 September 22, 1998


DNA68869-1610203164 August 25, 1998


DNA68871-1638203280 September 22, 1998


DNA68879-1631203274 September 22, 1998


DNA68880-1676203319 October 6, 1998


DNA68882-1677203318 October 6, 1998


DNA68883-1691203535 December 15, 1998


DNA68885-1678203311 October 6, 1998


DNA71180-1655203403 October 27, 1998


DNA71184-1634203266 September 22, 1998


DNA71213-1659203401 October 27, 1998


DNA71234-1651203402 October 27, 1998


DNA71269-1621203284 September 22, 1998


DNA71277-1636203285 September 22, 1998


DNA71286-1687203357 October 20, 1998


DNA71883-1660203475 November 17, 1998


DNA73401-1633203273 September 22, 1998


DNA73492-1671203324 October 6, 1998


DNA73730-1679203320 October 6, 1998


DNA73734-1680203363 October 20, 1998


DNA73735-1681203356 October 20, 1998


DNA73742-1662203316 October 6, 1998


DNA73746-1654203411 October 27, 1998


DNA73760-1672203314 October 6, 1998


104


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 7 (cony)


Material ATCC Dep. Deposit Date
No.


DNA76393-1664203323 October 6, 1998


DNA76398-1699203474 November 17, 1998


DNA76399-1700203472 November 17, 1998


DNA76522-2500203469 November 17, 1998


DNA76533-1689203410 October 27, 1998


DNA77303-2502203479 November 17, 1998


DNA77626-1705203536 December 15, 1998


DNA77648-1688203408 October 27, 1998


DNA81754-2532203542 December 15, 1998


DNA81757-2512203543 December 15, 1998


DNA82302-2529203534 December 15, 1998


DNA82340-2530203547 December 22, 1998


DNA87991-2540203656 February 9, 1999


DNA92238-2539203602 January 20, 1999


DNA115291-2681PTA-202 June 8, 1999


DNA23336-2861PTA-1673 April 11, 2000


DNA30862-1396209920 June 2, 1998


DNA30871-1157209380 October 16, 1997


DNA32279-1131209259 September 16, 1997


DNA33206-1165209372 October 16, 1997


DNA35673-1201209418 October 28, 1997


DNA47361-1154-2209431 November 7, 1997


DNA49631-1328209806 April 28, 1998


DNA52594-1270209679 March 17, 1998


DNA55800-1263209680 March 17, 1998


DNA56531-1648203286 September 29, 1998


DNA56965-1356209842 May 6, 1998


DNA57037-1444209903 May 27, 1998


DNA57695-1340203006 June 23, 1998


DNA57834-1339209954 June 9, 1998


DNA57841-1522203458 November 3, 1998


DNA58847-1383209879 May 20, 1998


DNA59493-1420203050 July 1, 1998


DNA59586-1520203288 September 29, 1998


DNA59608-2577203870 March 23, 1999


DNA59849-1504209986 June 16, 1998


105


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 7 (cont'1


Material ATCC Dep. Deposit Date
No.


DNA60292-1506203540 December 15, 1998


DNA62377-1381-1203552 December 22, 1998


DNA62880-1513203097 August 4, 1998


DNA66672-1586203265 September 22, 1998


DNA67962-1649203291 September 29, 1998


DNA69555-2867PTA-1632 April 4, 2000


DNA71162-2764PTA-860 October 19, 1999


DNA71290-1630203275 September 22, 1998


DNA76401-1683203360 October 20, 1998


DNA76541-1675203409 October 27, 1998


DNA76788-2526203551 December 22, 1998


DNA77623-2524203546 December 22, 1998


DNA80136-2503203541 December 15, 1998


DNA83568-2692PTA-386 July 20, 1999


DNA84210-2576203818 March 2, 1999


DNA86576-2595203868 March 23, 1999


DNA87976-2593203888 March 30, 1999


DNA92256-2596203891 March 30, 1999


DNA92289-2598PTA-131 May 25, 1999


DNA96850-2705PTA-479 August 3, 1999


DNA96855-2629PTA-18 May 4, 1999


DNA96857-2636PTA-17 May 4, 1999


DNA96860-2700PTA-478 August 3, 1999


DNA96861-2844PTA-1436 March 2, 2000


DNA96866-2698PTA-491 August 3, 1999


DNA96870-2676PTA-254 June 22, 1999


DNA96872-2674PTA-550 August 17, 1999


DNA96878-2626PTA-23 May 4, 1999


DNA96879-2619203967 April 27, 1999


DNA96889-2641PTA-119 May 25, 1999


DNA96893-2621PTA-12 May 4, 1999


DNA96897-2688PTA-379 July 20, 1999


DNA98564-2643PTA-125 May 25, 1999


DNA107443-2718PTA-490 August 3, 1999


DNA107786-2723PTA-474 August 3, 1999


DNA108682-2712PTA-486 August 3, 1999


106


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/06520
. Table 7 (cony)


Material ATCC Dep. Deposit Date
No.


DNA108684-2761PTA-653 September 14, 1999


DNA108701-2749PTA-554 August 17, 1999


DNA108720-2717PTA-511 August 10, 1999


DNA108726-2729PTA-514 August 10, 1999


DNA108728-2760PTA-654 September 14, 1999


DNA108738-2767PTA-862 October 19, 1999


DNA 108743-2722PTA-508 August 10, 1999


DNA108758-2759PTA-655 September 14, 1999


DNA108765-2758PTA-657 September 14, 1999


DNA108783-2747PTA-616 August 31, 1999


DNA108789-2748PTA-547 August 17, 1999


DNA108806-2724PTA-610 August 31, 1999


DNA108936-2719PTA-519 August 10, 1999


DNA119510-2771PTA-947 November 9, 1999


DNA119517-2778PTA-951 November 16, 1999


DNA119535-2756PTA-613 August 31, 1999


DNA119537-2777PTA-956 November 16, 1999


DNA119714-2851PTA-1537 March 21, 2000


DNA125170-2780PTA-953 November 16, 1999


DNA129594-2841PTA-1481 March 14, 2000


DNA129793-2857PTA-1733 April 18, 2000


DNA130809-2769PTA-949 November 9, 1999


DNA131639-2874PTA-1784 April 25, 2000


DNA131649-2855PTA-1482 March 14, 2000
~


DNA131652-2876PTA-1628 April 4, 2000


DNA131658-2875PTA-1671 April 11, 2000


DNA132162-2770PTA-950 November 9, 1999


DNA136110-2763PTA-652 September 14, 1999


DNAi39592-2866PTA-1587 March 28, 2000


DNA139608-2856PTA-1581 March 28, 2000


DNA143292-2848PTA-1778 April 25, 2000


DNA144844-2843PTA-1536 March 21, 2000


DNA144857-2845PTA-1589 March 28, 2000


DNA145841-2868PTA-1678 April 11, 2000


DNA148004-2882PTA-1779 April 25, 2000


DNA149893-2873PTA-1672 April 11, 2000


107


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/OG520
Table 7 (cony)


Material ATCC Dep. Deposit Date
No.


DNA149930-2884 PTA-1668 April 11, 2000


DNA150157-2898 PTA-1777 April 25, 2000


DNA150163-2842 PTA-1533 March 21, 2000


DNA153579-2894PTA-1729 April 18, 2000


DNA164625-2890 PTA-1535 March 21, 2000


DNA57838-1337 203014 June 23, 1998


DNA59777-1480 203111 August 11, 1998


DNA66675-1587 203282 September 22, 1998


DNA76532-1702203473 November 17, 1998


DNA105849-2704 PTA-473 August 3, 1999


DNA83500-2506 203391 October 29, 1998


These deposits were made under the provisions of the Budapest Treaty on the
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 deposit for 30
years from the date of deposit. The
deposits will be made available by ATCC under the terms 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
culture 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 to be entitled
thereto according to 35 USC ~
122 and the Commissioner's rules pursuant thereto (including 37 CFR ~ 1.14
with particular reference to 886 OG
638).
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 government in
accordance with its patent 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.
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 filters is performed in a
solution of 50% formamide, 5x SSC, 0.1 % SDS, 0.1 % sodium pyrophosphate, 50
mM sodium phosphate, pH
108


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
6.8, 2x Denhardt's solution, and 10% dextran sulfate at 42°C for 20
hours. Washing of the filters is performed
in an aqueous solution of 0. lx SSC and 0.1 % 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 lmown in the art.
EXAMPLE 6: Expression of PRO in E. coli
This example illustrates preparation of an unglycosylated form of PRO by
recombinant expression in E.
coli.
The DNA sequence encoding PRO is initially amplified using selected PCR
primers. 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.
coli; 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 PCR
amplified sequences are then ligated
into the vector. The vector will preferably include sequences which, encode
for an antibiotic resistance gene, a
tzp promoter, a polyhis leader (including the first six STII codons, polyhis
sequence, and enterokinase cleavage
site), the PRO coding region, lambda transcriptional terminator, and an argU
gene.
The ligarion mixture is then used to transform a selected E. coli strain using
the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow
on LB plates and antibiotic resistant
colonies are then selected. Plasmid DNA can be isolated and confirmed 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 may 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 solubilized using various agents
lrnown in the art, and the solubilized
PRO protein can then be purified using a metal chelating column under
conditions that allow tight binding of the
protein.
PRO may be expressed in E. coli in a poly-His tagged form, using the following
procedure. The DNA
encoding PRO is initially amplified using selected PCR primers. The primers
will 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. coli host based on strain
52 (W3110 fuhA(tonA) lon gakE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB containing 50
mglml carbenicillin 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 3.57 g (NH4)ZS04, 0.71 g sodium citrate~2H20, 1.07 g KCl, 5.36 g
Difco yeast extract, 5.36 g
Sheffield hycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, O.SS%
(w/v) glucose and 7 mM
MgS04) and grown for approximately 20-30 hours at 30°C with shaking.
Samples are removed to verify
109


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen
until purification and refolding.
E, coli paste from 0.5 to 1 L fermentations (6-10 g pellets) is resuspended in
10 volumes (w/v) in 7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulEte and sodium
tetrathionate is added to make final
concentrations of O.1M and 0.02 M, respectively, and the solution is stirred
overnight at 4°C. This step results
in a denatured protein with all 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 mM 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 coefficient based on its amino acid sequence.
The proteins are refolded 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 i mM EDTA. Refolding
volumes are chosen so that the final protein concentration is between 50 to
100 micrograms/ml. The refolding
solution is stirred gently at 4°C for 12-36 hours. The refolding
reaction is quenched by the addition of TFA to
a final concentration 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 Rl/H reversed phase column using a
mobile buffer of 0.1 % TFA with
elution with a gradient of acetonitrile from 10 to 80% . Aliquots of fractions
with A280 absorbance 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 acetonitrile since those species
are the most compact with their hydrophobic interiors shielded from
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 4% mannitol by dialysis or by gel filtration using G25
Superfine (Pharmacia) resins
equilibrated in the formulation buffer and sterile filtered.
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 potentially glycosylated form of FRO
by recombinant expression
in mammalian cells.
The vector, pRKS (see EP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the PRO DNA is ligated into pRKS 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 pRKS-
110


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
PRO.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells
(ATCC CCL 1573) are
grown to confluence in tissue culture plates in medium such as DMEM
supplemented with fetal calf serum and
optionally, nutrient components and/or antibiotics. About 10 wg pRKS-PRO DNA
is mixed with about 1 ~.g DNA
encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and
dissolved in 500 ~sl of 1 mM Tris-
HCI, 0.1 mM EDTA, 0.227 M CaClz. To this mixture is added, dropwise, 500 pl of
50 mM HEPES (pH 7.35),
280 mM NaCI, 1.5 mM NaP04, 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 ml 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 transfections, the culture medium is removed
and replaced with culture
medium (alone) or culture medium containing 200 ~cCiJml 35S-cysteine and 200
~Ci/ml 35S-methionine. After a
12 hour incubation, the conditioned medium is collected, concentrated on a
spin filter, and loaded onto a 15
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 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., Proc. Natl. Acad. Sci., 12:7575 (1981).
293 cells are grown to maximal
density in a spinner flask and 700 wg pRKS-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 pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds, washed with
tissue culture medium, and re-
introduced into the spinner flask containing tissue culture medium, 5 ~g/ml
bovine insulin and 0.1 ~.g/ml 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 CaP04 or DEAE-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 is
harvested. The medium containing the expressed PRO can then be concentrated
and purified by any selected
method.
Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be
subcloned out of the
pRKS vectox. 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 PRO 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 tagged PRO can then be
111


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
concentrated and purified by any selected method, such as by NiZ+-chelate
affinity chromatography.
PRO may also be expressed in CHO and/or COS cells by a transient expression
procedure or in CHO
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 IgGI 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 Biolo~v, Unit 3.16, John Wiley
and Sons (1997). CHO expression vectors are constructed to have compatible
restriction sites 5' and 3' of the
DNA of interest to allow the convenient shuttling of cDNA's. The vector used
expression in CHO cells is as
described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses
the SV40 early promoter/enhancer
to drive expression of the cDNA of interest and dihydrofolate reductase
(DHFR). DHFR expression permits
selection for stable maintenance of the plasmid following transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO cells
using commercially available transfection reagents Superfect° (Qiagen),
Dosper or Fugene° (Boehringer
Mannheim). The cells are grown as described in Lucas et al., supra.
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 cells/mL. 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 cells/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 temperature shifted
to 33°C, and 30 mL of 500 g/L glucose and 0.6 mL 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 ~cm 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 ml/min. at 4°C. After loading, the
column is washed with additional
112


CA 02534018 2001-02-28
WO O1/G8848 PCT/US01/06520
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 NaCl and 4%
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 pumped onto a 5 ml 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 ~L 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.
EXAMPLE 8: Expression of PRO in Yeast
The following method describes recombinant expression 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 ADH2/GAPDH
promoter, a native PRO signal
peptide or other mammalian signal peptide, or, for example, a yeast alpha-
factor or invertase secretory
signal/leader sequence, and linker sequences (if needed) for expression of
PRO.
Yeast cells, such as yeast strain AB110, can then be transformed with the
expression plasmids described
above and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by
precipitation with 10 % trichloroacetic 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 purifted using selected column
chromatography resins.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 9: Expression 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 is fused upstream of an epitope tag contained
within a baculovirus
expression vector. Such epitope tags include poly-his tags and immunoglobulin
tags (like Fc regions of IgG).
A variety of plasmids may be employed, including plasmids derived from
commercially available plasmids such
as pVL1393 (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 transmembrane
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 S' primer may incorporate flanking (selected) restriction
enzyme sites. The product is then
113


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digested with those selected restriction enzymes and subcloned into the
expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoldT"'virus DNA
(Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially
available from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the
released viruses are harvested and used
for further ampliftcations. 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 PRO can then be purified, for example, by Ni2+-
chelate affinity
chromatography as follows. Extracts are prepared from recombinant virus-
infected Sf9 cells as described by
Rupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed,
resuspended in sonication buffer (25
mL Hepes, pH 7.9; 12.5 mM MgCl2; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M
KCk), and sonicated
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, 10% glycerol, pH 7.8) and
filtered through a 0.45 um filter.
A Ni2+-NTA agarose column (commercially available from Qiagen) 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 minute. The column is washed to baseline AZeo
with loading buffer, at which point
fraction collection is started. Next, the column is washed with a secondary
wash buffer (50 mM phosphate; 300
mM NaCI, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.
After reaching AZao baseline
again, the column is developed with a 0 to 500 mM Imidazole gradient in the
secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver staining or
Western blot with Niz+-NTA-conjugated
to alkaline phosphatase (Qiagen). Fractions containing the eluted His,o tagged
PRO are pooled and dialyzed
against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be
performed using lrnown
chromatography techniques, including for instance, Protein A or protein G
column chromatography.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 10: Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can
specifically bind PRO.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for instance,
in Goding, supra. Immunogens that may be employed include purified PRO, fusion
proteins containing PRO,
and cells expressing recombinant PRO on the cell surface. Selection of the
immunogen can be made by the skilled
artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in
complete Freund's adjuvant
and injected subcutaneously or intraperitoneally 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 12
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 mice by retro-orbital
bleeding for testing in ELTSA assays to detect anti-PRO antibodies.
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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 % 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 hybridoma cells can be injected intraperitoneally into syngeneic
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,
affinity chromatography based upon binding of antibody to protein A or protein
G can be employed.
EXAMPLE 11: Purification of PRO Polyrx~tides 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 polypeptide
is purified by immunoaffmity chromatography using antibodies specific for the
PRO polypeptide of interest. In
general, an immunoaffmity column is constructed by covalently 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, Piscataway, N.J.). Likewise,
monoclonal antibodies are prepared from mouse ascites fluid by ammonium
sulfate precipitation or
chromatography on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a
chromatographic resin such as CnBr-activated SEPHAROSET"' (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 immunoaffmity column is utilized in the purification 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 subcellular fraction obtained via differential
centrifugation by the addition of detergent or by
other methods well known in the art. Alternatively, 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
immunoaffmity 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
antibody/PRO 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.
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EXAMPLE 12: Drug Screening
This invention is particularly useful for screening compounds by using PRO
polypeptides or binding
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 recombinant 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 diminution in complex
formation between the PRO polypeptide and its target cell or target receptors
caused by the 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 an PRO
polypeptide 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 polypeptide/cell
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 polypeptide, the
peptide test compounds are reacted
with PRO polypeptide and washed. Bound PRO polypeptide is detected by methods
well known in the art.
Purred 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 PRO
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 Drug Design
The goal of rational drug design is to produce structural analogs of
biologically active polypeptide of
interest (i. e. , a PRO polypeptide) 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 (c.f., Hodgson,
Bio/Technologv, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of
an PRO
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WO O1/G8848 PCT/US01106520
polypeptide-inhibitor complex, is determined 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 ascertained
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, Biochemistry, 31:7796-
7801 (1992) or which act as inhibitors,
agonists, or antagonists of native peptides as shown by Athauda et al., J.
Biochem., 113:742-746 (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
antibodies (anti-ids) to a functional, pharmacologically active antibody. As a
mirror 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 to
perform such analytical studies as X-ray crystallography. In addition,
Imowledge of the PRO polypeptide amino
acid sequence prnvided herein will provide guidance to those employing
computer modeling techniques in place
of or in addition to x-ray crystallography.
EXAMPLE 14: Identification of PRO Polypeptides That Stimulate TNF-a Release In
Human Blood (Assay x28)
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 p,l of human blood
supplemented with SOmM Hepes buffer (pH 7.2) is aliquoted per well in a 96
well test plate. To each well is then
added 3001 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 SOp,l 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.
The following PRO polypep2ides tested positive in this assay:
PR01079, PR0827, PR0791, PR01131, PR01316, PR01183, PR01343, PR01760, PR01567,
and PR04333.
EXAMPLE 15: Promotion of Chondrocvte Redifferentiation (Assay 129)
This assay is designed to determine whether PRO polypeptides of the present
invention show the ability
to induce the proliferation and/or redifferentiation of chondrocytes in
culture. PRO polypeptides testing positive
in this assay would be expected to be useful for the therapeutic treatment of
various bone and/or cartilage
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disorders such as, for example, sports injuries and arthritis.
Porcine chondrocytes are isolated by overnight collagenase digestion of
articular cartilage of the
metacarpophalangeal joint of 4-6 month old female pigs. The isolated cells are
then seeded at 25,000 cells/em2
in Ham F-12 containing 10% FBS and 4 ~,g/ml gentamycin. The culture media is
changed every third day. On
day 12, the cells are seeded in 96 well plates at 5,000 cells/well in 100,1 of
the same media without serum and
100 ~I of either serum-free medium (negative control), staurosporin (final
concentration of 5 nM; positive control)
or the test PRO polypeptide are added to give a final volume of 200 ~,1/well.
After.5 days at 37°C, 22 p,l of
media comtaining 100~.g/ml Hoechst 33342 and 50 pg/ml 5-CFDA is added to each
well and incubated for an
additional 10 minutes at 37°C. A picture of the green fluorescence is
taken for each well and the differentiation
state of the chondrocytes is calculated by morphometric analysis. A positive
result in the assay is obtained when
the >50% of the PRO polypeptide treated cells are differentiated (compared to
the background obtained by the
negative control).
PR06029 polypeptide tested positive in this assay.
EXAMPLE 16: Microarrav Analysis to Detect Overezpression of PRO Polypentides
in Cancerous Tumors
' Nucleic acid microarrays, often containing thousands of gene sequences, are
useful for identifying
differentially expressed genes in diseased tissues as compared to their normal
counterparts. Using nucleic acid
microarrays, test and control mRNA samples from test and control tissue
samples are reverse transcribed and
labeled to generate eDNA probes. The cDNA probes are then hybridized to an
array of nucleic acids immobilized
on a solid support. The array is configured such that the sequence and
position of each member of the array is
lvaown. For example, a selection of genes known to be expressed in certain
disease states may be arrayed on a
solid support. Hybridization of a labeled probe with a particular array member
indicates that the sample from
which the probe was derived expresses that gene. If the hybridization signal
of a probe from a test (disease tissue)
sample is greater than hybridization signal of a probe from a control (normal
tissue) sample, the gene or genes
overexpressed in the disease tissue are identified. The implication of this
result is that an overexpressed protein
in a diseased tissue is useful not only as a diagnostic marker for the
presence of the disease condition, but also
as a therapeutic target for treatment of the disease condition.
The methodology of hybridization of nucleic acids and microarray technology is
well known in the art.
In the present example, the specific preparation of nucleic acids for
hybridization and probes, slides, and
hybridization conditions
In the present example, cancerous tumors derived from various human tissues
were studied for PRO
polypeptide-encoding gene expression relative to non-cancerous human tissue in
an attempt to identify those PRO
polypeptides which are overexpressed in cancerous tumors. Two sets of
experimental data were generated. In
one set, cancerous human colon tumor tissue and matched non-cancerous human
colon tumor tissue from the same
patient ("matched colon control") were obtained and analyzed for PRO
polypeptide expression using the above
described microarray technology. In the second set of data, cancerous human
tumor tissue from any of a variety
of different human tumors was obtained and compared to a "universal"
epithelial control sample which was
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prepared by pooling non-cancerous human tissues of epithelial origin,
including liver, kidney, and lung, mRNA
isolated from the pooled tissues represents a mixture of expressed gene
products from these different tissues.
Microarray hybridization experiments using the pooled control samples
generated a linear plot in a 2-color
analysis. The slope of the line generated in a 2-color analysis was then used
to normalize the ratios of (test:control
detection) within each experiment. The normalized ratios from various
experiments were then compared and used
to identify clustering of gene expression. Thus, the pooled "universal
control" sample not only alkowed effective
relative gene expression determinations in a simple 2-sample comparison, it
also allowed mufti-sample
comparisons across several experiments.
In the present experiments, nucleic acid probes derived from the herein
described PRO polypeptide-
encoding nucleic acid sequences were used in the creation of the microarray
and RNA from the tumor tissues
listed above were used for the hybridization thereto. A value based upon the
normalized ratio:experimental ratio
was designated as a "cutoff ratio". Only values that were above this cutoff
ratio were determined to be
significant. Table 8 below shows the results of these experiments,
demonstrating that various PRO polypeptides
of the preent invention are significantly overexpressed in various human tumor
tissues as compared to a non-
cancerous human tissue control. As described above, these data demonstrate
that the PRO polypeptides of the
present invention are useful not only as diagnostic markers for the presence
of one or more cancerous tumors,
but also serve as therapeutic targets for the treatment of those tumors.
Table 8
Molecule is overexgessed in: as compared to:


PR0276 lung tumor universal normal
control


PR0284 colon tumor universal normal
control


PR0284 lung tumor universal normal
control


PR0284 breast tumor universal normal
control


PR0193 cplon tumor universal normal
control


PR0193 lung tumor universal normal
control


PR0193 breast tumor universal normal
control


PR0193 prostate tumor universal normal
control


PR0190 colon tumor universal normal
control


PR0190 lung tumor universal normal
control


PR0190 breast tumor universal normal
control


PR0180 colon tumor universal normal
control


PR0180 kung tumor universal normal
control


PR0180 breast tumor universal normal
control


PR0194 colon tumor universal normal
control


PR0194 lung tumor universal normal
control


PR0194 breast tumor universal normal
control


PR0194 cervical tumor universal normal
control


PR0218 colon tumor universal normal
control


PR0218 lung tumor universal normal
control


PR0260 colon tumor universal normal
control


PR0260 lung tumor universal normal
control


PR0260 breast tumor universal normal
control


PR0260 rectal tumor universal normal
controk


PR0233 colon tumor universal normal
control


PR0233 lung tumor universal normak
control


PR0233 breast tumor universal normal
control


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Table. 8 (cony)


Molecule is overexpressed in: as compared
to:


PR0234 colon tumor universal normal
control


PR0234 lung tumor universal normal
control


PR0234 breast tumor universal normal
control


PR0234 liver tumor universal normal
control


PR0236 colon tumor universal normal
control


PR0236 lung tumor universal normal
control


PR0236 breast tumor universal normal
control


PR0244 colon tumor universal normal
control


PR0244 lung tumor universal normal
control


PR0262 colon tumor universal normal
control


PR0262 lung tumor universal normal
control


PR0262 breast tumor universal normal
control


PR0271 colon tumor universal normal
control


PR0271 lung tumor universal normal
control


PR0268 colon tumor universal normal
control


PR0268 lung tumor universal normal
control


PR0268- breast tumor universal normal
control


PR0270 colon tumor universal normal
control


PR0270 lung tumor universal normal
control


PR0270 breast tumor universal normal
control


PR0270 liver tumor universal normal
control


PR0355 lung tumor universal normal
control


PR0355 breast tumor universal normal
control


PR0355 prostate tumor universal normal
control


PR0298 colon tumor universal normal
control


PR0298 lung tumor universal normal
control


PR0298 breast tumor universal normal
control


PR0299 colon tumor universal normal
control


PR0299 lung tumor universal normal
control


PR0299 breast tumor universal normal
control


PR0296 colon tumor universal normal
control


PR0296 breast tumor universal normal
control


PR0329 colon tumor universal normal
control


PR0329 lung tumor universal normal
control


PR0329 breast tumor universal normal
control


PR0330 colon tumor universal normal
control


PR0330 lung tumor universal normal
control


PR0294 lung tumor universal normal
control


PR0294 breast tumor universal normal
control


PR0300 colon tumor universal normal
control


PR0300 lung tumor universal normal
control


PR0300 breast tumor universal normal
control


PR0307 lung tumor universal normal
control


PR0334 colon tumor universal normal
control


PR0334 lung tumor universal normal
control


PR0334 breast tumor universal normal
control


PR0334 prostate tumor universal normal
control


PR0352 colon tumor universal normal
control


PR0352 lung tumor universal normal
control


PR0352 breast tumor universal normal
control


PR0352 liver tumor universal normal
control


PR0710 breast tumor universal normal
control


PR0873 colon tumor universal normal
control


PR0873 lung tumor universal normal
control


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Table 8 (cont'1


Molecule is overexpressed in: as compared
to:


PR0873 breast.tumor universal normal
control


PR0873 prostate tumor universal normal
control


PR0354 colon tumor universal normal
control


PR0354 lung tumor universal normal
control


PR0354 breast tumor universal normal
control


PRO1ISI lung tumor universal normal
control


PR01151 breast tumor universal normal
control


PR0382 colon tumor universal normal
control


PR0382 lung tumor universal normal
control


PR0382 breast tumor universal normal
control


PR01864 lung tumor universal normal
control


PR01864 breast tumor universal normal
control


PR01864 liver tumor universal normal
control


PR0386 colon tumor universal normal
control


PR0386 lung tumor universal normal
control


PR0386 prostate tumor universal normal
control


PR0541 colon tumor universal normal
control


PR0541 lung tumor universal normal
control


PR0541 breast tumor universal normal
control


PR0852 breast tumor universal normal
control


PR0700 colon tumor universal normal
control


PR0700 lung tumor universal normal
control


PR0700 breast tumor universal normal
control


PR0700 rectal tumor universal normal
control


~PR0708 colon tumor universal normal
control


PR0708 lung tumor universal normal
control


PR0708 breast tumor universal normal
control


PR0707 colon tumor universal normal
control


PR0707 lung tumor universal normal
control


PR0864 colon tumor universal normal
control


PR0864 lung tumor universal normal
control


PR0864 breast tumor universal normal
control


PR0706 colon tumor universal normal
control


PR0706 lung tumor universal normal
control


PRb706 breast tumor universal normal
control


PR0706 liver tumor universal normal
control


PR0732 lung tumor universal normal
control


PR0732 breast tumor universal normal
control


PR0732 cervical tumor universal normal
control


PR0537 colon tumor universal normal
control


PR0537 lung tumor universal normal
control


PR0537 breast tumor universal normal
control


PR0545 lung tumor universal normal
control


PR0545 breast tumor universal normal
control


PR0718 lung tumor universal normal
control


PR0718 breast tumor universal normal
control


PR0872 lung tumor universal normal
control


PR0872 breast tumor universal normal
control


PR0872 liver tumor universal normal
control


PR0704 colon tumor universal normal
control


PR0704 lung tumor universal normal
control


PR0704 ~ breast tumor universal normal
control


PR0705 lung tumor universal normal
control


PR0705 breast tumor universal normal
control


121


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Table 8 (cony)


Molecule is overex~ressed in: as compared
to:


PR0871 lung tumor universal normal
control


PR0871 breast tumor universal normal
control


PR0871 liver tumor universal normal
control


PR0702 lung tumor universal normal
control


PR0944 colon tumor universal normal
control


PR0944 lung tumor universal normal
control


PR0944 rectal tumor universal normal
control


PR0739 lung tumor universal normal
control


PR0739 breast tumor universal normal
control


PR0739 prostate tumor universal normal
control


PR0941 colon tumor universal normal
control


PR0941 lung tumor universal normal
control


PR0941 breast tumor universal normal
control


PR0941 rectal tumor universal normal
control


PR01082 lung tumor universal normal
control


PR01082 breast tumor universal normal
control


PR01133 colon tumor universal normal
control


PR01133 lung tumor universal normal
control


PR0983 colon tumor universal normal
control


PR0983 lung tumor universal normal
control


PR0983 breast tumor universal normal
control


PR0784 colon tumor universal normal
control


PR0784 lung tumor universal normal
control


PR0784 breast tumor universal normal
control


PR0784 prostate tumor universal normal
control


PR0783 colon tumor universal normal
control


PR0783 lung tumor universal normal
control


PR0783 breast tumor universal normal
control


PR0783 liver tumor universal normal
control


PR0940 colon tumor universal normal
control


PR0940 lung tumor universal normal
control


PR0940 breast tumor universal normal
control


PR0768 colon tumor universal normal
control


PR0768 lung tumor universal normal
control


PR0768 breast tumor universal normal
control


PR01079 colon tumor universal normal
control


PR01079 lung tumor universal normal
control


PR01079 breast tumor universal normal
control


PR01079 rectal tumor universal normal
control


PR01078 colon tumor universal normal
control


PR01078 lung tumor universal normal
control


PR01018 colon tumor universal normal
control


PR01018 lung tumor universal normal
control


PR01018 breast tumor universal normal
control


PR0793 colon tumor universal normal
control


PR0793 lung tumor universal normal
control


PR0793 breast tumor universal normal
control


PR0793 rectal tumor universal normal
control


PR01773 colon tumor universal normal
control


PR01773 lung tumor universal normal
control


PR01773 prostate tumor universal normal
control


PR01014 lung tumor universal normal
control


PR01014 breast tumor universal normal
control


PR01013 colon tumor universal normal
control


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Table 8~cont~


Molecule is oyerexpressed in: as comQ,ared
to:


PR01013 lung tumor universal normal
control


PR01013 breast tumor universal normal
control


PR01013 liver tumor universal normal
control


PR0937 colon tumor universal normal
control


PR0937 lung tumor universal normal
control


PR0937 breast tumor universal normal
control


PR0937 cervical tumor universal normal
control


PR0937 rectal tumor universal normal
control


PR01477 lung tumor universal normal
control


PR01477 breast tumor universal normal
control


PR01477 rectal tumor universal normal
control


PR0842 colon tumor universal normal
control


PR0842 lung tumor universal normal
control


PR0842 breast tumor universal normal
control


PR0839 colon tumor universal normal
control


PR01180 colon tumor universal normal
control


PR01180 lung tumor universal normal
control


PR01180 liver tumor universal normal
control


PR01134 lung tumor universal normal
control


PR01134 breast tumor universal normal
control


PR01134 prostate tumor universal normal
control


PR01115 colon tumor universal normal
control


PRO1115 lung tumor universal normal
control


PRO1115 breast tumor universal normal
control


PR01277 colon tumor universal normal
control


PR01277 lung tumor universal normal
control


PR01135 lung tumor universal normal
control


PR01135 breast tumor universal normal
control


PR01135 cervical tumor universal normal
control


PR0827 colon tumor universal normal
control


PR0827 lung tumor universal normal
control


PR0827 prostate tumor universal normal
control


PR0827 cervical tumor universal normal
control


PR01057 lung tumor universal normal
control


PR01057 breast tumor universal normal
contxol


PR01113 colon tumor universal normal
control


PR01113 lung tumor universal normal
control


PR01006 colon tumor universal normal
control


PR01006 lung tumor universal normal
control


PR01006 breast tumor universal normal
control


PR01006 rectal tumor universal normal
control


PR01074 lung tumor universal normal
control


PR01074 rectal tumor universal normal
control


PR01073 lung tumor universal normal
control


PR01073 breast tumor universal normal
control


PR01136 colon tumor universal normal
control


PR01136 lung tumor universal normal
control


PR01136 breast tumor universal normal
control


PR01004 lung tumor universal normal
control


PR01344 colon tumor universal normal
control


PR01344 lung tumor universal normal
control


PR01344 breast tumor universal normal
control


PR01344 rectal tumor universal normal
control


PRO111.0 colon tumor universal normal
control


123


CA 02534018 2001-02-28
WO 01/68848 PCTlUS01/06520
Table 8 fcont')


Molecule is overexpressed in: as compared
to:


PRO1110 lung tumor universal normal
control


PRO1110 breast tumor universal normal
control


PR01378 colon tumor universal normal
control


PR01378 lung tumor universal normal
control


PR01378 prostate tumor universal normal
control


PR01378 cervical tumor universal normal
control


PR01481 colon tumor universal normal
control


PR01481 lung tumor universal normal
control


PRO 1109 lung tumor universal normal
control


PR01109 breast tumor universal normal
control


PR01383 colon tumor universal normal
control


PR01383 lung tumor universal normal
control


PR01383 breast tumor universal normal
control


PR01072 lung tumor universal normal
control


PR01189 colon tumor universal normal
control


PR01189 lung tumor universal normal
control


PR01189 breast tumor universal normal
control


PR01189 prostate tumor universal normal
control


PR01003 colon tumor universal normal
control


PR01003 lung tumor universal normal
control


PR01003 breast tumor universal normal
control


PR01003 liver tumor universal normal
control


PR01003 rectal tumor universal normal
control


PR01108 colon tumor universal normal
control


PR01108 lung tumor universal normal
control


PR01108 breast tumor universal normal
control


PR01137 colon tumor universal normal
control


PR01137 lung tumor universal normal
control


PR01137 breast tumor universal normal
control


PR01138 colon tumor w niversal normal
control


PR01138 lung tumor universal normal
control


PR01138 breast tumor universal normal
control


PR01415 colon tumor universal normal
control


PR01415 lung tumor universal normal
control


PR01415 prostate tumor universal normal
control


PR01054 lung tumor universal normal
control


PR01054 breast tumor universal normal
control


PR0994 colon tumor universal normal
control


PR0994 lung tumor universal normal
control


PR0994 rectal tumor universal normal
control


PR01069 lung tumor universal normal
control


PR01069 breast tumor universal normal
control


PR01411 colon tumor universal normal
control


PR01411 lung tumor universal normal
control


PR01129 lung tumor universal normal
control


PR01129 rectal tumor universal normal
control


PR01359 colon tumor universal normal
control


PR01359 lung tumor universal normal
control


PR01359 breast tumor universal normal
control


PR01359 prostate tumor universal normal
control


PR01139 lung tumor universal normal
control


PR01065 lung tumor universal normal
control


PR01028 colon tumor universal normal
control


PR01028 lung tumor universal normal
control


124


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 8 (cony)


Molecule is overexeressed in: as co~ared to:


PR01028 breast tumor universal normal
control


PR01028 cervical tumor universal normal
control


PR01027 colon tumor universal normal
control


PR01027 lung tumor universal normal
control


PR01027 breast tumor universal normal
control


PR01140 colon tumor universal normal
control


PR01140 breast tumor universal normal
control


PR01291 colon tumor universal normal
control


PR01291 breast tumor universal normal
control


PR01105 colon tumor universal normal
control


PRO1105 lung tumor universal normal
control


PR01026 lung tumor universal normal
control


PR01026 prostate tumor universal normal
control


PR01104 colon tumor universal normal
control


PR01104 lung tumor universal normal
control


PR01104 breast tumor universal normal
control


PRO1100 colon tumor universal normal
control


PRO1100 lung tumor universal normal
control


PRO1100 breast tumor universal normal
control


PRO1100 rectal tumor universal normal
control


PR01141 lung tumor universal normal
control


PR01772 colon tumor universal normal
control


PR01772 lung tumor universal normal
control


PR01772 breast tumor universal normal
control


PR01772 cervical tumor universal normal
control


PR01064 colon tumor universal normal
control


PR01064 lung tumor universal normal
control


PR01379 colon tumor universal normal
control


PR01379 lung tumor universal normal
control


PR01379 cervical tumor universal normal
control


PR03573 lung tumor universal normal
control


PR03573 breast tumor universal normal
control


PR03566 colon tumor universal normal
control


PR03566 lung tumor universal normal
control


PR01156 lung tumor universal normal
control


PR01156 breast tumor universal normal
control


PR01156 prostate tumor universal normal
~ control


PR01098 colon tumor universal normal
control


PR01098 lung tumor universal normal
control


PR01098 rectal tumor universal normal
control


PR01128 colon tumor universal normal
control


PR01128 lung tumor universal normal
control


PR01128 breast tumor universal normal
control


PR01248 lung tumor universal normal
control


PR01248 breast tumor universal normal
control


PR01127 colon tumor universal normal
control


PR01127 lung tumor universal normal
control


PR01127 breast tumor universal normal
control


PR01316 colon tumor . universal normal
control


PR01316 lung tumor universal normal
control


PR01316 breast tumor universal normal
control


PR01197 colon tumor , universal normal
control


PR01197 lung tumor universal normal
control


PR01197 breast tumor universal normal
control


125


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06s20
Table 8 lcont')


Molecule is overexpressed in: as compared
to:


PR01125 lung tumor universal normal
control


PR01158 breast tumor universal normal
control


PR01124 colon tumor universal normal
control


PR01124 lung tumor universal normal
control


PR01380 colon tumor universal normal
control


PR01380 lung tumor universal normal
control


PR01380 breast tumor universal normal
control


PR01380 liver tumor universal normal
control


PR01377 colon tumor universal normal
control


PR01377 lung tumor universal normal
control


PR01287 lung tumor universal normal
control


PR01287 breast tumor universal normal
control


PR01249 lung tumor universal normal
control


PR01249 breast tumor universal normal
control


PR01335 colon tumor universal normal
control


PR01335 lung tumor universal normal
control


PR01335 breast tumor universal normal
control


PR03572 lung tumor universal normal
control


PR01599 colon tumor universal normal
control


PR01599 lung tumor universal normal
control


PR01599 breast tumor universal normal
control


PR01374 lung tumor universal normal
control


PR01374 breast tumor universal normal
control


PR01345 lung tumor universal normal
control


PR01345 breast tumor universal normal
control


PR01311 lung tumor universal normal
control


PR01311 breast tumor universal normal
control


PR01357 colon tumor universal normal
control


PR01357 lung tumor universal normal
control


PR01557 colon tumor universal normal
control


PR01557 lung tumor universal normal
control


PR01557 breast tumor universal normal
control


PROI305 colon tumor universal normal
control


PR01305 lung tumor universal normal
control


PR01305 breast tumor universal normal
control


PR01302 colon tumor universal normal
control


PR01302 lung tumor universal normal
control


PR01302 breast tumor universal normal
control


PR01302 rectal tumor universal normal
control


PR01266 colon tumor universal normal
control


PR01336 colon tumor universal normal
control


PR01336 lung tumor universal normal
control


PR01336 breast tumor universal normal
control


PR01278 colon tumor universal normal
control


PR01278 lung tumor universal normal
control


PR01270 breast tumor universal normal
control


PR01298 colon tumor universal normal
control


PR01298 lung tumor universal normal
control


PR01301 lung tumor universal normal
control


PR01301 breast tumor universal normal
control


PR01268 colon tumor universal normal
control


PR01268 breast tumor universal normal
control


PR01327 lung tumor universal normal
control


PR01327 breast tumor universal normal
control


126


CA 02534018 2001-02-28
WO O1/G8848 PCT/USO1/06520
Table 8 (cony)


Molecule is overexpressed in: as compared to:


PR01328 colon tumor universal normal
control


PR01328 lung tumor universal normal
control


PR01328 breast tumor universal normal
control


PR01329 colon tumor universal normal
control


PR01329 lung tumor universal normal
control


PR01329 breast tumor universal normal
control


PR01339 colon tumor universal normal
control


PR01339 lung tumor universal normal
control


PR01342 colon tumor ~ w niversal normal
control


PR01342 lung tumor universal normal
control


PR01342 breast tumor universal normal
control


PR01342 rectal tumor universal normal
control


PR01487 colon tumor universal normal
control


PR01487 breast tumor universal normal
control


PR03579 lung tumor universal normal
control


PR03579 breast tumor universal normal
control


PR01472 colon tumor universal normal
control


PR01472 lung tumor universal normal
control


PR01385 lung tumor universal normal
control


PR01385 breast tumor universal normal
control


PR0146I colon tumor universal normal
. control


PR01461 lung tumor universal normal
control


PR01461 breast tumor universal normal
control


PR01429 colon tumor universal normal
control


PR01429 lung tumor universal normal
control


PR01429 breast tumor universal normal
control


PR01568 lung tumor universal normal
control


PR01568 breast tumor universal normal
control


PR01569 colon tumor universal nozmal
control


PR01569 lung tumor universal normal
control


PR01569 breast tumor universal normal
control


PR01753 colon tumor universal normal
control


PR01753 lung tumor universal normal
control


PR01570 colon tumor universal normal
control


PR01570 lung tumor universal normal
control


PR01570 breast tumor universal normal
control


PR01570 prostate tumor universal normal
control


PR01570 rectal tumor universal normal
control


PR01559 colon tumor universal normal
control


PR01559 lung tumor universal normal
control


PROI559 breast tumor universal normal
control


PR01486 lung tumor universal normal
control


PR01486 breast tumor universal normal
control


PR01433 colon tumor universal normal
control


PR01433 lung tumor universal normal
control


PR01433 breast tumor universal normal
control


PR01433 rectal tumor universal normal
control


PR01490 lung tumor universal normal
control


PR01490 breast tumor universal normal
control


PR01482 lung tumor universal normal
control


PR01482 breast tumor universal normal
control


PR01409 colon tumor universal normal
control


PR01409 lung tumor universal normal
control


SS PR01409 breast tumor universal normal
control


127


CA 02534018 2001-02-28
WO 01/68848 PCT/US01/06520
Table 8 (cony)


Molecule is overexpressed in: as compared
to:


PR01446 colon tumor universal normal
control


PR01446 lung tumor universal normal
control


PR01446 breast tumor universal normal
control


PR01446 prostate tumor universal normal
control


PR01604 colon tumor universal normal
control


PR01604 lung tumor universal normal
control


PR01604 breast tumor universal normal
control


PR01491 colon tumor universal normal
control


PR01491 lung tumor universal normal
control


PR01491 breast tumor universal normal
control


PR01431 colon tumor universal normal
control


PR01431 lung tumor universal normal
control


PR01563 colon tumor universal normal
control


PR01563 lung tumor universal normal
control


PR01563 breast tumor universal normal
control


PRO1S71 colon tumor universal normal
control


PR01571 lung tumor universal normal
control


PR01571 breast tumor universal normal
control


PR01572 lung tumor universal normal
control


PR01572 prostate tumor universal normal
control


PR01573 lung tumor universal normal
control


PR01573 breast tumor universal normal
control


PR01508 lung tumor universal normal
control


PR01508 breast tumor ~ universal normal
control


PR01485 colon tumor universal normal
control


PR01485 lung tumor universal normal
control


PR01564 colon tumor universal normal
control


PR01564 lung tumor universal normal
control


PR01564 breast tumor universal normal
control


PRO15S0 colon tumor universal normal
control


PR01550 lung tumor universal normal
control


PR01550 breast tumor universal normal
control


PR01757 lung tumor universal normal
control


PR01757 breast tumor universal normal
control


PR01757 prostate tumor universal normal
control


PR01758 lung tumor universal normal
control


PR01781 colon tumor universal normal
control


PR01781 lung tumor universal normal
control


PR01781 breast tumor universal normal
control


PR01606 lung tumor universal normal
control


PR01606 breast tumor universal normal
control


PR01784 colon tumor universal normal
control


PR01784 lung tumor universal normal
control


PR01784 breast tumor universal normal
control


PR01774 colon tumor universal normal
control


PR01774 lung tumor universal normal
control


PR01774 breast tumor universal normal
control


PR01605 colon tumor universal normal
control


SO PR01605 lung tumor universal normal
control


PR01605 prostate tumor universal normal
control


PR01928 colon tumor universal normal
control


PR01928 lung tumor universal normal
control


PR01928 cervical tumor universal normal
control


PR0186S lung tumor universal normal
control


128


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 8 (cont~


Molecule is overex~essed in: as compared
to:


PR01865 liver tumor universal normal
control


PR01925 lung tumor universal normal
control


PR01926 liver tumor universal normal
control


PR02630 colon tumor universal normal
control


PR02630 lung tumor universal normal
control


PR02630 breast tumor universal normal
control


PR02630 liver tumor universal normal
control


PR03443 colon tumor universal normal
control


PR03443 lung tumor universal normal
control


PR03443 breast tumor universal normal
control


PR03301 colon tumor universal normal
control


PR03301 lung tumor universal normal
control


PR03301 breast tumor universal normal
control


PR03301 rectal tumor universal normal
control


PR03442 colon tumor universal normal
control


PR03442 lung tumor universal normal
control


PR03442 rectal tumor universal normal
control


PR04978 colon tumor universal normal
control


pR04978 lung tumor universal normal
control


PR04978 breast tumor universal normal
control


PR04978 rectal tumor universal normal
control


PR05801 colon tumor universal normal
control


PR05801 ' breast tumor universal normal
control


PR019630 colon tumor universal normal
control


PR0203 colon tumor universal normal
control


PR0204 colon tumor universal normal
control


PR0204 lung tumor universal normal
control


PR0204 breast tumor . universal normal
control


PR0204 prostate tumor universal normal
control


PR0210 colon tumor universal normal
control


PR0210 lung tumor universal normal
control


PR0223 lung tumor universal normal
control


PR0223 breast tumor universal normal
control


PR0247 colon tumor universal normal
control


PR0247 lung tumor universal normal
control


PR0247 breast ~ universal
normal control


PR0358 lung tumor universal normal
control


PR0358 breast tumor universal normal
control


PR0358 prostate tumor universal normal
control


PR0724 lung tumor universal normal
control


PR0868 colon tumor universal normal
control


PR0868 lung tumor universal normal
control


PR0868 prostate tumor universal normal
control


PR0868 rectal tumor universal normal
control


pR0740 colon tumor universal normal
control


PR01478 colon tumor universal normal
control


PR01478 lung tumor universal normal
control


PR0162 colon tumor universal normal
control


PR0162 lung tumor universal normal
control


PR0162 breast tumor universal normal
control


PR0828 colon tumor universal normal
control


PR0828 lung tumor universal normal
control


PR0828 breast tumor universal normal
control


PR0828 cervical tumor universal normal
control


129


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 8 (cont'1


Molecule is overexpressed in: as compared
to:


PR0828 liver tumor universal normal
control


PR0819 lung tumor universal normal
control


PR0819 breast tumor universal normal
control


PR0819 rectal tumor universal normal
control


PR0813 colon tumor universal normal
control


PR0813 lung tumor universal normal
control


PR0813 breast tumor universal normal
control


PR0813 prostate tumor universal normal
control


PR01194 colon tumor universal normal
control


PR01194 lung tumor universal normal
control


PR01194 breast tumor universal normal
control


PR0887 colon tumor universal normal
control


PR0887 lung tumor universal normal
control


PR0887 rectal tumor universal normal
control


PR01071 colon tumor universal normal
control


PR01071 lung tumor universal normal
control


PR01071 breast tumor universal normal
control


PR01029 colon tumor universal normal
control


PR01029 lung tumor universal normal
control


PROI029 breast tumor universal normal
control


PR01190 lung tumor universal normal
control


PR01190 breast tumor universal normal
control


PR04334 lung tumor universal normal
control


PROi155 colon tumor universal normal
control


PR01155 lung tumor universal normal
control


PR01157 breast tumor universal normal
control


PROI157 cervical tumor universal normal
control


PR01122 lung tumor universal normal
control


PR01122 breast tumor universal normal
control


PR01183 colon tumor universal normal
control


PR01183 lung tumor universal normal
control


PR01183 breast tumor universal normal
control


PR01337 colon tumor universal normal
control


PR01337 lung tumor universal normal
control


PR01337 breast tumor universal normal
control


PR01480 colon tumor universal normal
control


PR01480 lung tumor universal normal
control


PR01480 breast tumor universal normal
control


PR019645 colon tumor universal normal
control


PR09782 colon tumor universal normal
control


PR01419 colon tumor universal normal
control


PR01575 colon tumor universal normal
control


PR01575 lung tumor universal normal
control


PR01567 colon tumor universal normal
control


PR01567 lung tumor universal normal
control


PR01567 breast tumor universal normal
control


PR01891 colon tumor universal normal
control


PR01889 colon tumor universal normal
control


PR01889 lung tumor universal normal
control


PR01785 lung tumor universal normal
control


PR01785 prostate tumor universal normal
control


PR06003 colon tumor universal normal
control


PR04333 colon tumor universal normal
control


PR04356 colon tumor universal normal
control


130


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 8 (cony)


Molecule is overexpressed in: as compared
to:


PR04352 colon tumor universal normal
control


PR04354 colon tumor universal normal
control


PR04354 lung tumor universal normal
control


PR04354 prostate tumor universal normal
control


PR04369 colon tumor universal normal
control


PR06030 colon tumor universal normal
control


PR04433 colon tumor universal normal
control


PR04424 colon tumor universal normal
control


PR04424 breast tumor universal normal
control


PR06017 colon tumor universal normal
control


PR019563 colon tumor universal normal
control


PR06015 colon tumor universal normal
control


PR05779 colon tumor universal normal
control


PR05776 colon tumor universal normal
control


PR04430 lung tumor universal normal
control


PR04421 colon tumor universal normal
control


PR04499 colon tumor universal normal
control


PR04423 colon tumor universal normal
control


PR05998 colon tumor universal normal
control


PR05998 lung tumor universal normal
control


PR04501 colon tumor universal normal
control


PR06240 colon tumor universal normal
control


PR06245 colon tumor universal normal
control


2S PR06175 colon tumor universal normal
control


PR09742 colon tumor universal normal
control


PR07179 colon tumor universal normal
control


PR06239 colon tumor universal normal
control


PR06493 colon tumor universal normal
control


PR09741 colon tumor universal normal
control


PR09822 colon tumor universal normal
control


PR06244 colon tumor universal normal
control


PR09740 colon tumor universal normal
control


PR09739 colon tumor universal normal
control


PR07177 colon tumor universal normal
control


PR07178 colon tumor universal normal
control


PR06246 colon tumor universal normal
control


PR06241 colon tumor universal normal
control


PR09835 colon tumor universal normal
control


PR09857 colon tumor universal normal
control


PR07436 colon tumor universal normal
control


PR09856 colon tumor universal normal
control


PR019605 colon tumor universal normal
control


PR09859 colon tumor universal normal
control


PR012970 colon tumor universal normal
control


PR019626 colon tumor universal normal
control


PR09883 colon tumor universal normal
control


PR019670 colon tumor universal normal
control


PR019624 colon tumor universal normal
control


PR019680 colon tumor universal normal
control


PR019675 colon tumor universal normal
control


PR09834 colon tumor universal normal
control


PR09744 colon tumor universal normal
control


PR019644 colon tumor universal normal
control


SS PR019625 colon tumor universal normal
control


131


CA 02534018 2001-02-28
WO 01/68848 PCT/USO1/06520
Table 8 (coot')


Molecule is overex~essed in: as comRared
to:


PR019597 colon tumor universal normal
control


PR016090 colon tumor universal normal
control


PR019576 colon tumor w niversal normal
control


PR019646 colon tumor universal normal
control


PR019814 colon tumor universal normal
control


PR019669 colon tumor universal normal
control


PR019818 colon tumor universal normal
control


PR020088 colon tumor universal normal
control


PR016089 colon tumor ~ ~ wniversal normal
control


PR020025 colon tumor universal normal
control


PR020040 colon tumor universal normal
control


PR01760 adrenal tumor universal normal
control


PR01760 breast tumor universal normal
control


PR01760 cervical tumor universal normal
control


PR01760 colon tumor universal normal
control


PR01760 liver tumor universal normal
control


PR01760 lung tumor universal normal
control


PR01760 prostate tumor universal normal
control


PR01760 rectal tumor universal normal
control


PR06029 adrenal tumor universal normal
control


PR06029 colon tumor universal nozxnal
control


PR06029 prostate tumor universal normal
control


PRO1801 colon tumor universal normal
control


PR01801 lung tumor universal normal
control


132


CA 02534018 2001-02-28
LA PRESENTS P:~RTIE DE CETTE DEI~L~.NDE OU CE BREVETS
CO1~IPREND PLUS D'U'N TOIYIE.
CECI EST LE TOiYIE ~ DE
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
~UlVI~~ APPEICATIOiVS / PATENTS
TIiIS SECTION OF THE APPLICATION / P ATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUI~tE OF
NOTE: For additional volumes please contact the Canadian Patent Office.