METHODES PERMETTANT D'INHIBER LA CROISSANCE DE CELLULES NEOPLASIQUES

METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL GROWTH

Abrégé français

Cette invention a trait à des méthodes, ainsi qu'aux compositions correspondantes, permettant d'inhiber la croissance de cellules néoplasiques. Elle concerne, notamment, des compositions antitumorales et des méthode de traitemente tumeurs. Elle porte, en outre, sur des méthodes de criblage aux fins de l'identification d'inhibiteurs de croissance, c'est-à-dire, des composés antitumoraux. Elle a trait, de surcroît, à de nouveaux polypeptides et à des molécules d'acide nucléique les codant. Il est également question, dans cette invention, de vecteurs et de cellules hôtes comportant ces séquences nucléotidiques, de molécules polypeptidiques chimères comportant les polypeptides susmentionnés fusionnés avec des séquences polypeptidiques hétérologues, d'anticorps se liant à ces polypeptides et de procédés de production desdits polypeptides.

Abrégé anglais

The present invention concerns methods and compositions for inhibiting
neoplastic cell growth. In particular, the present invention concerns
antitumor compositions and methods for the treatment of tumors. The invention
further concerns screening methods for identifying growth inhibitory, e.g.,
antitumor compounds. In addition, 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.

Revendications

WHAT IS CLAIMED IS:
1. A composition of matter useful for the inhibition of neoplastic cell
growth, said composition
comprising an effective amount of a PRO179, PRO207. PRO320, PRO219, PRO221,
PRO224, PRO328, PRO301,
PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, or an agonist thereof,
in admixture with a
pharmaceutically acceptable carrier.
2. The composition of matter of Claim 1 comprising a growth inhibitory amount
of a PRO179,
PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362,
PRO356, PRO509 or
PRO866 polypeptide, or an agonist thereof.
3. The composition of matter of Claim 1 comprising a cytotoxic amount of a
PRO179, PRO207,
PRO320, PRO219, PRO221, PRO224, PRO328, PRO301, PRO526, PRO362, PRO356, PRO509
or PRO866
polypeptide, or an agonist thereof.
4. The composition of matter of Claim 1 additionally comprising a further
growth inhibitory agent,
cytotoxic agent or chemotherapeutic agent.
5. A composition of matter useful for the treatment of a tumor in a mammal,
said composition
comprising a therapeutically effective amount of a PRO179, PRO207, PRO320,
PRO219, PRO221, PRO224,
PRO328, PRO301, PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, or an
agonist thereof.
6. The composition of matter of Claim 5, wherein said tumor is a cancer.
7. The composition of matter of Claim 6, wherein the cancer is selected from
the group consisting
of breast cancer, ovarian cancer, renal cancer, colorectal cancer, uterine
cancer, prostate cancer, lung cancer, bladder
cancer, central nervous system cancer, melanoma and leukemia.
8. A method for inhibiting the growth of a tumor cell comprising exposing said
tumor cell to an
effective amount of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328,
PRO301, PRO526,
PRO362, PRO356, PRO509 or PRO866 polypeptide, or an agonist thereof.
9. The method of Claim 8, wherein said agonist is an anti-PRO179, anti-PRO207,
anti-PRO320,
anti-PRO219, anti-PRO221, anti-PRO224, anti-PRO328, anti-PRO301,anti-
PRO526,anti-PRO362, anti-PRO356,
anti-PRO509 or anti-PRO866 agonist antibody.
10. The method of Claim 8, wherein said agonist is a small molecule mimicking
the biological
activity of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224, PRO328, PRO301,
PRO526, PRO362,
140

PRO356, PRO509 or PRO866 polypeptide.
11. The method of Claim 8, wherein said step of exposing occurs in vitro.
12. The method of Claim 8, wherein said step of exposing occurs in vivo.
13. An article of manufacture comprising:
(a) a container; and
(b) a composition comprising an active agent contained within the container;
wherein said active
agent in the composition is a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224,
PRO328, PRO301,
PRO526, PRO362, PRO356, PRO509 or PRO866 polypeptide, or an agonist thereof.
14. The article of manufacture of Claim 13, further comprising a label affixed
to said container, or
a package insert included in said container, referring to the use of said
composition for the inhibition of neoplastic
cell growth.
15. The article of manufacture of Claim 13, wherein said agonist is an anti-
PRO179, anti-PRO207,
anti-PRO320, anti-PRO219, anti-PRO221, anti-PRO224,anti-PRO328,anti-
PRO301,anti-PRO526, anti-PRO362,
anti-PRO356, anti-PRO509 or anti-PRO866 agonist antibody.
16. The article of manufacture of Claim 13, wherein said agonist is a small
molecule mimicking the
biological activity of a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224,
PRO328, PRO301, PRO526,
PRO362, PRO356, PRO509 or PRO866 polypeptide.
17. The article of manufacture of Claim 13, wherein said active agent is
present in an amount that
is effective for the treatment of tumor in a mammal.
18. The article of manufacture of Claim l3, wherein said composition
additionally comprises a further
growth inhibitory agent, cytotoxic agent or chemotherapeutic agent.
19. Isolated nucleic acid having at least 80% nucleic acid sequence identity
to a nucleotide sequence
that encodes an amino acid sequence selected from the group consisting of the
amino acid sequence shown in
Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ 1D NO:10),
Figure 8 (SEQ ID NO:15), Figure
(SEQ ID NO:20), Figure 12 (SEQ ID NO:25), Figure 14 (SEQ ID NO:30), Figure 16
(SEQ ID NO:35), Figure
18 (SEQ ID NO:43), Figure 20 (SEQ ID NO:48), Figure 22 (SEQ ID NO:55), Figure
24 (SEQ ID NO:60), and
Figure 26 (SEQ ID NO:62).
141

20. Isolated nucleic acid having at least 80% nucleic acid sequence identity
to a nucleotide sequence
selected from the group consisting of the nucleotide sequence shown in Figure
1 (SEQ ID NO:1), Figure 3 (SEQ
ID NO:6), Figure 5 (SEQ ID NO:9), Figure 7 (SEQ ID NO:14), Figure 9 (SEQ ID
NO:19), Figure 11 (SEQ ID
NO:24), Figure 13 (SEQ ID NO:29), Figure 15 (SEQ ID NO:34), Figure 17 (SEQ ID
NO:42), Figure 19 (SEQ ID
NO:47), Figure 21 (SEQ ID NO:54), Figure 23 (SEQ ID NO:59), and Figure 25 (SEQ
ID NO:61).
21. Isolated nucleic acid having at least 80% nucleic acid sequence identity
to a nucleotide sequence
selected from the group consisting of the full-length coding sequence of the
nucleotide sequence shown in Figure
1 (SEQ ID NO:1), Figure 3 (SEQ ID NO:6), Figure 5 (SEQ ID NO:9), Figure 7 (SEQ
ID NO: 14), Figure 9 (SEQ
ID NO:19), Figure 11 (SEQ 1D NO:24), Figure 13 (SEQ ID NO:29), Figure 15 (SEQ
ID NO:34), Figure 17 (SEQ
ID NO:42), Figure 19 (SEQ ID NO:47), Figure 21 (SEQ ID NO:54), Figure 23 (SEQ
ID NO:59), and Figure 25
(SEQ ID NO:61).
22. 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 209281, 209358,
209670, 209384, 209262,
209263, 209438, 209432, 209704, 209620, 209422, or 209750.
23. A vector comprising the nucleic acid of any one of Claims 19 to 22.
24. The vector of Claim 23 operably linked to control sequences recognized by
a host cell
transformed with the vector.
25. A host cell comprising the vector of Claim 23.
26. The host cell of Claim 25, wherein said cell is a CHO cell.
27. The host cell of Claim 25, wherein said cell is an E. coli.
28. The host cell of Claim 25, wherein said cell is a yeast cell.
29. The host cell of Claim 25, wherein said cell is a Baculovirus-infected
insect cell.
30. A process for producing a PRO179, PRO207, PRO320, PRO219, PRO221, PRO224,
PRO328,
PRO301, PRO526, PRO362, PRO356, PRO509or PRO866 polypeptide comprising
culturing the host cell of Claim
25 under conditions suitable for expression of said polypeptide and recovering
said polypeptide from the cell
culture.
142

31. An isolated polypeptide having at least 80% amino acid sequence identity
to an amino acid
sequence selected from the group consisting of the amino acid sequence shown
in Figure 2 (SEQ ID NO:2), Figure
4 (SEQ ID NO:7), Figure 6 (SEQ ID NO:10), Figure 8 (SEQ ID NO:15), Figure 10
(SEQ ID NO:20), Figure 12
(SEQ ID NO:25), Figure 14 (SEQ ID NO:30), Figure 16 (SEQ ID NO:35), Figure 18
(SEQ ID NO:43), Figure 20
(SEQ ID NO:48), Figure 22 (SEQ ID NO:55), Figure 24 (SEQ ID NO:60), and Figure
26 (SEQ ID NO:62).
32. An isolated polypeptide scoring at least 80% positives when compared to an
amino acid sequence
selected from the group consisting of the amino acid sequence shown in Figure
2 (SEQ ID NO:2), Figure 4 (SEQ
ID NO:7), Figure 6 (SEQ ID NO:10), Figure 8 (SEQ ID NO:15), Figure 10 (SEQ ID
NO:20), Figure 12 (SEQ ID
NO:25), Figure 14 (SEQ ID NO:30), Figure 16 (SEQ ID NO:35), Figure 18 (SEQ ID
NO:43), Figure 20 (SEQ ID
NO:48), Figure 22 (SEQ ID NO:55), Figure 24 (SEQ ID NO:60), and Figure 26 (SEQ
ID NO:62).
33. 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
209281, 209358, 209670, 209384, 209262, 209263, 209438, 209432, 209704,
209620, 209422, or 209750.
34. A chimeric molecule comprising a polypeptide according to any one of
Claims 31 to 33 fused
to a heterologous amino acid sequence.
35. The chimeric molecule of Claim 34, wherein said heterologous amino acid
sequence is an epitope
tag sequence.
36. The chimeric molecule of Claim 34, wherein said heterologousamino acid
sequence is a Fc region
of an immunoglobulin.
37. An antibody which specifically binds to a polypeptide according to any one
of Claims 31 to 33.
38. The antibody of Claim 37, wherein said antibody is a monoclonal antibody,
a humanized antibody
or a single-chain antibody.
39. Isolated nucleic acid having at least 80% nucleic acid sequence identity
to:
(a) a nucleotide sequence encoding the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4
(SEQ ID NO:7), Figure 6 (SEQ ID NO:10), Figure 8 (SEQ ID NO:15), Figure 10
(SEQ ID NO:20), Figure 12
(SEQ ID NO:25), Figure 14 (SEQ ID NO:30), Figure 16 (SEQ ID NO:35), Figure 18
(SEQ ID NO:43), Figure 20
(SEQ ID NO:48), Figure 22 (SEQ ID NO:55), Figure 24 (SEQ ID NO:60), or Figure
26 (SEQ ID NO:62), lacking
its associated signal peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown in Figure 2
143

(SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO:10), Figure 8 (SEQ
ID NO:15), Figure 10 (SEQ
ID NO:20), Figure 12 (SEQ ID NO:25), Figure 14 (SEQ ID NO:30), Figure 16 (SEQ
ID NO:35), Figure 18 (SEQ
ID NO:43), Figure 20 (SEQ ID NO:48), Figure 22 (SEQ ID NO:55), Figure 24 (SEQ
ID NO:60), or Figure 26
(SEQ ID NO:62), with its associated signal peptide; or
(c) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:7), Figure 6 (SEQ ID NO:10), Figure 8 (SEQ
ID NO:15), Figure 10 (SEQ
ID NO:20), Figure 12 (SEQ ID NO:25), Figure 14 (SEQ ID NO:30), Figure 16 (SEQ
ID NO:35), Figure 18 (SEQ
ID NO:43), Figure 20 (SEQ ID NO:48), Figure 22 (SEQ ID NO:55), Figure 24 (SEQ
ID NO:60), or Figure 26
(SEQ ID NO:62), lacking its associated signal peptide.
40. An isolated polypeptide having at least 80% amino acid sequence identity
to:
(a) the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ ID NO:7),
Figure 6 (SEQ ID
NO:10), Figure 8 (SEQ ID NO: 15), Figure 10 (SEQ ID NO:20), Figure 12 (SEQ ID
NO:25), Figure 14 (SEQ ID
NO:30), Figure 16 (SEQ ID NO:35), Figure 18 (SEQ ID NO:43), Figure 20 (SEQ ID
NO:48), Figure 22 (SEQ ID
NO:55), Figure 24 (SEQ ID NO:60), or Figure 26 (SEQ ID NO:62), lacking its
associated signal peptide;
(b) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4 (SEQ ID
NO:7), Figure 6 (SEQ ID NO:15), Figure 10 (SEQ ID NO:24), Figure 14 (SEQ ID
NO:32), Figure 16 (SEQ ID
NO:37), Figure 18 (SEQ ID NO:42), Figure 20 (SEQ ID NO:50), Figure 22 (SEQ ID
NO:55), Figure 24 (SEQ ID
NO:61), Figure 26 (SEQ ID NO:69), Figure 28 (SEQ ID NO:76), Figure 30 (SEQ ID
NO:78), Figure 32 (SEQ ID
NO:83) or Figure 34 (SEQ ID NO:91), with its associated signal peptide; or
(c) an extracellular domain of the polypeptide shown in Figure 2 (SEQ ID
NO:2), Figure 4 (SEQ ID
NO:7), Figure 6 (SEQ ID NO: 15), Figure 10 (SEQ ID NO:24), Figure 14 (SEQ ID
NO:32), Figure 16 (SEQ ID
NO:37), Figure 18 (SEQ ID NO:42), Figure 20 (SEQ ID NO:50), Figure 22 (SEQ ID
NO:55), Figure 24 (SEQ ID
NO:61), Figure 26 (SEQ ID NO:69), Figure 28 (SEQ ID NO:76), Figure 30 (SEQ ID
NO:78), Figure 32 (SEQ ID
NO:83) or Figure 34 (SEQ ID NO:91), lacking its associated signal peptide.
144

Description

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL
GROWTH
FIELD OF THE INVENTION
The present invention concerns methods and compositions for inhibiting
neoplastic cell growth. In
particular, the present invention concerns antitumor compositions and methods
for the treatment of tumors. The
invention further concerns screening methods for identifying growth
inhibitory, e. g., antitumor compounds.
BACKGROUND OF THE INVENTION
Malignant tumors (cancers) are the second leading cause of death in the United
States, after heart disease
(Boring et al., CA Cancel J. Clin.. 43:7 ( 1993)).
Cancer is characterized by the increase in the number of abnormal, or
neoplastic, cells derived from a
normal tissue which proliferate to fonm a tumor mass, the invasion of adjacent
tissues by these neoplastic tumor
cells, and the generation of malignant cells which eventually spread via the
blood or lymphatic system to regional
lymph nodes and to distant sites (metastasis). In a cancerous state a cell
proliferates under conditions in which
normal cells would not grow. Cancer manifests itself in a wide variety of
forms, characterized by different degrees
of invasiveness and aggressiveness.
Despite recent advances in cancer therapy, there is a great need for new
therapeutic agents capable of
inhibiting neoplastic cell growth. Accordingly, it is the objective of the
present invention to identify compounds
capable of inhibiting the growth of neoplastic cells, such as cancer cells.
SUMMARY OF THE INVENTION
A. Embodiments
The present invention relates to methods and compositions for inhibiting
neoplastic cell growth. More
particularly, the invention concerns methods and compositions for the
treatment of tumors, including cancers, such
as breast, prostate, colon, lung, ovarian, renal and CNS cancers, leukemia,
melanoma, etc., in mammalian patients,
preferably humans.
In one aspect, the present invention concerns compositions ofmatter useful for
the inhibition of neoplastic
cell growth comprising an effective amount of a PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide as herein
defined, or an agonist
thereof, in admixture with a pharmaceutically acceptable carrier. In a
preferred embodiment, the composition of
matter comprises a growth inhibitory amount of a PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide, oran
agonistthereof. In another
preferred embodiment, the composition comprises a cytotoxic amount of a
PR0179, PR0207, PR0320, PR0219,
PR022 I , PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866
polypeptide, or an
agonist thereof. Optionally, the compositions of matter may contain one or
more additional growth inhibitory
and/or cytotoxic and/or other chemotherapeutic agents.
In a further aspect, the present invention concerns compositions of matter
useful for the treatment of a
tumor in a mammal comprising a therapeutically effective amount of a PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866
polypeptide as herein
defined, or an agonist thereof. The tumor is preferably a cancer.
In another aspect, the invention concerns a method for inhibiting the growth
of a tumor cell comprising
exposing the cell to an effective amount of a PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide as herein
defined, or an agonist thereof.
In a particular embodiment, the agonist is an anti-PR0179, anti-PR0207, anti-
PR0320, anti-PR0219, anti-
PR0221, anti-PR0224, anti-PR0328, anti-PR0301, anti-PR0526, anti-PR0362, anti-
PR0356, anti-PR0509 or
anti-PR0866 agonist antibody. In another embodiment, the agonist is a small
molecule that mimics the biological
activity of a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 polypeptide. The method may be performed in vitro or
in vivo.
In a still further embodiment, the present invention provides an article of
manufacture comprising:
(a) a container;
(b) a composition comprising an active agent contained within the container;
wherein the
composition is effective for inhibiting the neoplastic cell growth, e.g.,
growth of tumor cells, and the active agent
in the composition is a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide as herein defined, or an agonist
thereof; and
(c) a label affixed to said container, or a package insert included in said
container referring to the use
ofsaid PR0179, PR0207,PR0320,PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide or agonist thereof, for the inhibition of
neoplastic cell growth, wherein the agonist
may be an antibody which binds to the PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide. In a particular
embodiment, the agonist
is an anti-PROI79, anti-PR0207, anti-PR0320, anti-PR0219, anti-PR0221, anti-
PR0224, anti-PR0328, anti-
PR0301; anti-PR0526, anti-PR0362, anti-PR0356, anti-PR0509 or anti-PR0866
agonist antibody. In another
embodiment, the agonist is a small molecule that mimics the biological
activity of a PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide.
Similar articles ofmanufacture comprising a PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide as herein
defined, or an agonist thereof
in an amount that is therapeutically effective for the treatment of tumor are
also within the scope of the present
invention. Also within the scope of the invention are articles of manufacture
comprising a PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
2

CA 02353799 2001-06-08
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polypeptide as herein defined, or an agonist thereof, and a further growth
inhibitory agent, cytotoxic agent or
chemotherapeutic agent.
B. Additional Embodiments
In other embodiments of the present invention, the invention provides an
isolated nucleic acid molecule
comprising a nucleotide sequence that encodes a PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% sequence identity, preferably at least about 81 % sequence identity, more
preferably at least about 82%
sequence identity, yet more preferably at least about 83% sequence identity,
yet more preferably at least about 84%
sequence identity, yet more preferably at least about 85% sequence identity,
yet more preferably at least about 86%
sequence identity, yet more preferably at least about 87% sequence identity,
yet more preferably at least about 88%
sequence identity, yet more preferably at least about 89% sequence identity,
yet more preferably at least about 90%
sequence identity, yet more preferably at least about 91 % sequence identity,
yet more preferably at least about 92%
sequence identity, yet more preferably at least about 93% sequence identity,
yet more preferably at least about 94%
sequence identity, yet more preferably at least about 95% sequence identity,
yet more preferably at least about 96%
sequence identity, yet more preferably at least about 97% sequence identity,
yet more preferably at least about 98%
sequence identity and yet more preferably at least about 99% sequence identity
to (a) a DNA molecule encoding
a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 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 ofthe 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% sequence identity, preferably at least about 81 % sequence identity, more
preferably at least about 82%
sequence identity, yet more preferably at least about 83% sequence identity,
yet more preferably at least about 84%
sequence identity, yet more preferably at least about 85% sequence identity,
yet more preferably at least about 86%
sequence identity, yet more preferably at least about 87% sequence identity,
yet more preferably at least about 88%
sequence identity, yet more preferably at least about 89% sequence identity,
yet more preferably at least about 90%
sequence identity, yet more preferably at least about 91 % sequence identity,
yet more preferably at least about 92%
sequence identity, yet more preferably at least about 93% sequence identity,
yet more preferably at least about 94%
sequence identity, yet more preferably at least about 95% sequence identity,
yet more preferably at least about 96%
sequence identity, yet more preferably at least about 97% sequence identity,
yet more preferably at least about 98%
sequence identity and yet more preferably at least about 99% sequence identity
to (a) a DNA molecule comprising
the coding sequence ofa full-length PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide cDNA as disclosed herein,
the coding sequence of
a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
3

CA 02353799 2001-06-08
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PR0509 or PR0866 polypeptide lacking the signal peptide as disclosed herein,
the coding sequence of an
extracellular domain of a transmembrane PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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).
In a further aspect, the invention concerns an isolated nucleic acid molecule
comprising a nucleotide
sequence having at least about 80% sequence identity, preferably at least
about 81% sequence identity, more
preferably at least about 82% sequence identity, yet more preferably at least
about 83% sequence identity, yet 'more
preferably at least about 84% sequence identity, yet more preferably at least
about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more preferably at least
about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more preferably at least
about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more preferably at least
about 91 % sequence identity, yet more
preferably at least about 92% sequence identity, yet more preferably at least
about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more preferably at least
about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more preferably at least
about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more preferably at
least about 99% sequence identity to (a)
a DNA molecule that encodes the same mature polypeptide encoded by any ofthe
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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 polypeptide which is either transmembrane domain-
deleted or transmembrane
domain-inactivated, or is complementary to such encoding nucleotide sequence,
wherein the transmembrane
domains) of such polypeptide are disclosed herein. Therefore, soluble
extracellular domains of the herein
described PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 polypeptides are contemplated.
Another embodiment is directed to fragments of a PR0179, PR0207, PR0320,
PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
coding sequence, or
the complement thereof, that may find use as, for example, hybridization
probes, for encoding fragments of a
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide that may optionally encode a polypeptide
comprising a binding site for an anti-
PR0179,anti-PR0207,anti-PR0320,anti-PR0219,anti-PR0221, anti-PR0224, anti-
PR0328, anti-PR0301, anti-
PR0526, anti-PR0362, anti-PR0356, anti-PR0509 or anti-PR0866 antibody or as
antisense oligonucleotide
probes. Such nucleic acid fragments are usually at least about 20 nucleotides
in length, preferably at least about
30 nucleotides in length, more preferably at least about 40 nucleotides in
length, yet more preferably at least about
50 nucleotides in length, yet more preferably at least about 60 nucleotides in
length, yet more preferably at least
about 70 nucleotides in length, yet more preferably at least about 80
nucleotides in length, yet more preferably at
least about 90 nucleotides in length, yet more preferably at least about 100
nucleotides in length, yet more
4

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preferably at least about 1 10 nucleotides in length, yet more preferably at
least about 120 nucleotides in length, yet
more preferably at least about 130 nucleotides in length, yet more preferably
at least about 140 nucleotides in
length, yet more preferably at least about 1 SO nucleotides in length, yet
more preferably at least about 160
nucleotides in length, yet more preferably at least about 170 nucleotides in
length, yet more preferably at least about
180 nucleotides in length, yet more preferably at least about 190 nucleotides
in length, yet more preferably at least
about 200 nucleotides in length, yet more preferably at least about 250
nucleotides in length, yet more preferably
at least about 300 nucleotides in length, yet more preferably at least about
3S0 nucleotides in length, yet more
preferably at least about 400 nucleotides in length, yet more preferably at
least about 4S0 nucleotides in length, yet
more preferably at least about S00 nucleotides in length, yet more preferably
at least about 600 nucleotides in
length, yet more preferably at (east about 700 nucleotides in length, yet more
preferably at least about 800
nucleotides in length, yet more preferably at least about 900 nucleotides in
length and yet more preferably 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 PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PROS26, PR0362, PR0356, PROS09
or PR0866
polypeptide-encoding nucleotide sequence may be determined in a routine manner
by aligning the PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PROS26, PR0362,
PR0356, PROS09 or
PR0866 polypeptide-encoding nucleotide sequence with other known nucleotide
sequences using any of a number
of well known sequence alignment programs and determining which PR0179,
PR0207, PR0320, PR0219,
PRO221, PR0224, PR0328, PR0301, PROS26, PR0362, PR03S6, PR0509 or PR0866
polypeptide-encoding
nucleotide sequence fragments) are novel. All of such PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PROS26, PR0362, PR03S6, PROS09 or PR0866 polypeptide-encoding
nucleotide sequences
are contemplated herein. Also contemplated are the PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PROS09 or PR0866 polypeptide fragments
encoded by these
nucleotide molecule fragments, preferably those PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR03S6, PROS09 or PR0866 polypeptide fragments
that comprise a
binding site for an anti-PR0179, anti-PR0207, anti-PR0320, anti-PR0219, anti-
PR0221, anti-PR0224, anti-
PR0328, anti-PR0301, anti-PR0526, anti-PR0362, anti-PR03S6, anti-PROS09 or
anti-PR0866 antibody.
In another embodiment, the invention provides isolated PR0179, PR0207, PR0320,
PR0219, PR0221,
PR0224, PR0328, PR0301, PROS26, PR0362, PR03S6, PROS09 or PR0866 polypeptide
encoded by any ofthe
isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PROS26, PR0362, PR03S6, PROS09 or PR0866 polypeptide,
comprising an amino
acid sequence having at least about 80% sequence identity, preferably at least
about 81 % sequence identity, more
preferably at least about 82% sequence identity, yet more preferably at least
about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more preferably at least
about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more preferably at least
about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more preferably at least
about 89% sequence identity, yet more
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CA 02353799 2001-06-08
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preferably at least about 90% sequence identity, yet more preferably at least
about 91 % sequence identity, yet more
preferably at least about 92% sequence identity, yet more preferably at least
about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more preferably at least
about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more preferably at least
about 97% sequence identity, yet more
S preferably at least about 98% sequence identity and yet more preferably at
least about 99% sequence identity to a
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 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 ofthe full-length amino
acid sequence as disclosed herein.
In a further aspect, the invention concerns an isolated PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
comprising an amino
acid sequence having at least about 80% sequence identity, preferably at least
about 81 % sequence identity, more
preferably at least about 82% sequence identity, yet more preferably at least
about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more preferably at least
about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more preferably at least
about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more preferably at least
about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more preferably at least
about 91 % sequence identity, yet more
preferably at least about 92% sequence identity, yet more preferably at least
about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more preferably at least
about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more preferably at least
about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more preferably at
least about 99% sequence identity to an
amino acid sequence encoded by any of the human protein cDNAs deposited with
the ATCC as disclosed herein.
In a further aspect, the invention concerns an isolated PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
comprising an amino
acid sequence scoring at least about 80% positives, preferably at least about
81 % positives, more preferably at least
about 82% positives, yet more preferably at least about 83% positives, yet
more preferably at least about 84%
positives, yet more preferably at least about 85% positives, yet more
preferably at least about 86% positives, yet
more preferably at least about 87% positives, yet more preferably at least
about 88% positives, yet more preferably
at least about 89% positives, yet more preferably at least about 90%
positives, yet more preferably at least about
91% positives, yet more preferably at least about 92% positives, yet more
preferably at least about 93% positives,
yet more preferably at least about 94% positives, yet more preferably at least
about 95% positives, yet more
preferably at least about 96% positives, yet more preferably at least about
97% positives, yet more preferably at
least about 98% positives and yet more preferably at least about 99% positives
when compared with the amino acid
sequence ofa PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 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,
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CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
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 specific aspect, the invention provides an isolated PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356,
PR0509orPR0866polypeptidewithouttheN-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
PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide
and recovering the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide from the cell culture.
Another aspect of the invention provides an isolated PR0179, PR0207, PR0320,
PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide and recovering the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists of a native PR0179,
PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide
as defined herein. In a particular embodiment, the agonist is an anti-PR0179,
anti-PR0207, anti-PR0320, anti-
PR0219, anti-PR0221, anti-PR0224, anti-PR0328,anti-PR0301,anti-PR0526,anti-
PR0362,anti-PR0356, anti-
PR0509 or anti-PR0866 agonist antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying
agonists to a PROI 79, PR0207,
PRO320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide which comprise contacting the PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide with a candidate
molecule and monitoring
a biological activity mediated by said PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide. Preferably, the
PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide is a native PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PROI79,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptide, or an agonist of a PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide as herein
described, or an anti-PRO 179,
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CA 02353799 2001-06-08
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anti-PR0207, anti-PR0320, anti-PR0219, anti-PR0221,anti-PR0224,anti-
PR0328,anti-PR0301, anti-PR0526,
anti-PR0362, anti-PR0356, anti-PR0509 or anti-PR0866 agonist 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
PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide,
or an agonist thereof as hereinbefore described, or an anti-PR0179, anti-
PR0207, anti-PR0320, anti-PR0219,
anti-PR022 I , anti-PR0224, anti-PR0328, anti-PR0301,anti-PR0526, anti-PR0362,
anti-PR0356, anti-PR0509
or anti-PR0866 agonist antibody, for the preparation of a medicament useful in
the treatment of a condition which
is responsive to the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide, an agonist thereof or an anti-
PR0179, anti-I?R0207, anti-
PR0320,anti-PR0219,anti-PR0221,anti-PR0224, anti-PR0328, anti-PR0301, anti-
PR0526, anti-PR0362,anti-
PR0356, anti-PR0509 or anti-PR0866 agonist 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, yeast, or Baculovirus-
infected insect cells. A process for
producing any of the herein described polypeptides is further provided and
comprises culturing host cells under
conditions suitable for expression of the desired polypeptide and recovering
the desired polypeptide from the cell
culture.
In other embodiments, the invention provides chimeric 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 specifically
binds to any of the above
or below described polypeptides. Optionally, the antibody is a monoclonal
antibody, humanized antibody, antibody
fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful
for isolating genomic and
cDNA nucleotide sequences or as antisense probes, wherein those probes may be
derived from any of the above
or below described nucleotide sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the nucleotide sequence (SEQ ID NO:1 ) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0179, wherein the nucleotide sequence (SEQ ID NO:1
) is a clone designated herein
as DNA 16451-1078. Also presented in bold font and underlined are the
positions of the respective start and stop
codons.
Figure 2 shows the amino acid sequence (SEQ ID N0:2) of a native sequence
PR0179 polypeptide as
derived from the coding sequence of SEQ ID NO:1 shown in Figure 1.
Figure 3 shows the nucleotide sequence (SEQ ID N0:6) of a cDNA containing a
nucleotide sequence
8

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
encoding native sequence PR0207, wherein the nucleotide sequence (SEQ ID N0:6)
is a clone designated herein
as DNA30879-1152. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 4 shows the amino acid sequence (SEQ ID N0:7) of a native sequence
PR0207 polypeptide as
derived from the coding sequence of SEQ ID N0:6 shown in Figure 3.
Figure 5 shows the nucleotide sequence (SEQ ID N0:9) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0320, wherein the nucleotide sequence (SEQ ID N0:9)
is a clone designated herein
as DNA32284-1307. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 6 shows the amino acid sequence (SEQ ID NO:10) of a native sequence
PR0320 polypeptide as
derived from the coding sequence of SEQ ID N0:9 shown in Figure 5.
Figure 7 shows the nucleotide sequence (SEQ ID N0:14) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0219, wherein the nucleotide sequence (SEQ ID
N0:14) is a clone designated herein
as DNA32290-1164. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 8 shows the amino acid sequence (SEQ ID NO:15) of a native sequence
PR0219 polypeptide as
derived from the coding sequence of SEQ ID N0:14 shown in Figure 7.
Figure 9 shows the nucleotide sequence (SEQ ID N0:19) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0221, wherein the nucleotide sequence (SEQ ID
N0:19) is a clone designated herein
as DNA33089-1132. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 10 shows the amino acid sequence (SEQ ID N0:20) of a native sequence
PR0221 polypeptide as
derived from the coding sequence of SEQ ID N0:19 shown in Figure 9.
Figure 11 shows the nucleotide sequence (SEQ ID N0:24) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0224, wherein the nucleotide sequence (SEQ 1D
N0:24) is a clone designated herein
as DNA33221-1133. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 12 shows the amino acid sequence (SEQ ID N0:25) of a native sequence
PR0224 polypeptide as
derived from the coding sequence of SEQ ID N0:24 shown in Figure 11.
Figure 13 shows the nucleotide sequence (SEQ ID N0:29) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0328, wherein the nucleotide sequence (SEQ ID
N0:29) is a clone designated herein
as DNA40587-1231. Also presented in bold font and underlined are the positions
ofthe respective start and stop
codons.
Figure 14 shows the amino acid sequence (SEQ ID N0:30) of a native sequence
PR0328 polypeptide as
derived from the coding sequence of SEQ ID N0:29 shown in Figure 13.
Figure I S shows the nucleotide sequence (SEQ ID N0:34) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0301, wherein the nucleotide sequence (SEQ ID
N0:34) is a clone designated herein
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CA 02353799 2001-06-08
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as DNA40628-1216. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 16 shows the amino acid sequence (SEQ ID N0:35) of a native sequence
PR0301 polypeptide as
derived from the coding sequence of SEQ 1D N0:34 shown in Figure 15.
Figure 17 shows the nucleotide sequence (SEQ ID N0:42) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0526, wherein the nucleotide sequence (SEQ ID
N0:42) is a clone designated herein
as DNA44184-1319. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 18 shows the amino acid sequence (SEQ ID N0:43) of a native sequence
PR0526 polypeptide as
derived from the coding sequence of SEQ ID N0:42 shown in Figure 17.
Figure 19 shows the nucleotide sequence (SEQ ID N0:47) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0362, wherein the nucleotide sequence (SEQ ID
N0:47) is a clone designated herein
as DNA45416-1251. Also presented in bold font and underlined are the positions
ofthe respective start and stop
codons.
Figure 20 shows the amino acid sequence (SEQ ID N0:48) of a native sequence
PR0362 polypeptide as
derived from the coding sequence of SEQ ID N0:47 shown in Figure 19.
Figure 21 shows the nucleotide sequence (SEQ ID N0:54) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0356, wherein the nucleotide sequence (SEQ ID
N0:54) is a clone designated herein
as DNA47470-I 130-P1. Also presented in bold font and underlined are the
positions of the respective start and
stop codons.
Figure 22 shows the amino acid sequence (SEQ ID NO:55) of a native sequence
PR0356 polypeptide as
derived from the coding sequence of SEQ ID N0:54 shown in Figure 21.
Figure 23 shows the nucleotide sequence (SEQ ID N0:59) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0509, wherein the nucleotide sequence (SEQ ID
N0:59) is a clone designated herein
as DNA50148-1068. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 24 shows the amino acid sequence (SEQ ID N0:60) of a native sequence
PR0509 polypeptide as
derived from the coding sequence of SEQ ID N0:59 shown in Figure 23.
Figure 25 shows the nucleotide sequence (SEQ ID N0:61 ) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0866, wherein the nucleotide sequence (SEQ ID N0:61
) is a clone designated herein
as DNA53971-1359. Also presented in bold font and underlined are the positions
ofthe respective start and stop
codons.
Figure 26 shows the amino acid sequence (SEQ ID N0:62) of a native sequence
PR0866 polypeptide as
derived from the coding sequence of SEQ ID N0:61 shown in Figure 25.
DETAILED DESCRIPTION OF THE INVENTION
The terms "PR0179", "PR0207", "PR0320", "PR0219", "PR0221 ","PR0224","PR0328",
"PR0301 ",
"PR0526", "PR0362", "PR0356", "PR0509" or "PR0866" polypeptide or protein when
used herein encompass

CA 02353799 2001-06-08
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native sequence PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362,
PR0356, PR0509 and PR0866 polypeptides and PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866 variants (which are
further defined herein).
The PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide may be isolated from a variety of sources, such
as from human tissue types or
from another source, or prepared by recombinant and/or synthetic methods.
A "native sequence PR0179", "native sequence PR0207", "native sequence
PR0320", "native sequence
PR0219", "native sequence PR0221 ", "native sequence PR0224", "native sequence
PR0328", "native sequence
PR0301 ", "native sequence PR0526", "native sequence PR0362", "native sequence
PR0356", "native sequence
PR0509", or "native sequence PR0866" comprises a polypeptide having the same
amino acid sequence as the
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide as derived from nature. Such native sequence
PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide
can be isolated from nature or can be produced by recombinant and/or synthetic
means. The term "native sequence"
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 specifically encompasses naturally-occurringtruncated or
secreted forms (e.g., an extracellular
domain sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring
allelic variants of the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526,
PR0362, PR0356, PR0509 and PR0866 polypeptides. In one embodiment ofthe
invention, the native sequence
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide is a mature or full-length native sequence PRO
179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866
polypeptide as shown
in Figure 2 (SEQ ID N0:2}, Figure 4 (SEQ ID N0:7), Figure 6 (SEQ ID NO:10),
Figure 8 (SEQ ID N0:15),
Figure 10 (SEQ ID N0:20), Figure 12 (SEQ ID N0:25), Figure 14 (SEQ ID N0:30),
Figure 16 (SEQ ID N0:35),
Figure 18 (SEQ ID N0:43), Figure 20 (SEQ ID N0:48), Figure 22 (SEQ ID N0:55),
Figure 24 (SEQ ID N0:60)
or Figure 26 (SEQ ID N0:62), respectively. Also, while the PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866 polypeptides
disclosed in Figure
2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7), Figure 6 (SEQ ID NO:10), Figure 8
(SEQ ID NO:15), Figure 10
(SEQ ID N0:20), Figure 12 (SEQ ID N0:25), Figure 14 (SEQ ID N0:30), Figure 16
(SEQ ID N0:35), Figure 18
(SEQ ID N0:43), Figure 20 (SEQ ID N0:48), Figure 22 (SEQ ID N0:55), Figure 24
(SEQ ID N0:60) or Figure
26 (SEQ ID N0:62), respectively, are shown to begin with the methionine
residue designated therein as amino acid
position 1, it is conceivable and possible that another methionine residue
located either upstream or downstream
from amino acid position 1 in Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7),
Figure 6 (SEQ ID NO:10),
Figure 8 (SEQ ID NO:15), Figure 10 (SEQ ID N0:20), Figure 12 (SEQ ID N0:25),
Figure 14 (SEQ ID N0:30),
Figure 16 (SEQ ID N0:35), Figure 18 (SEQ ID N0:43), Figure 20 (SEQ ID N0:48),
Figure 22 (SEQ ID N0:55),
Figure 24 (SEQ ID N0:60) or Figure 26 (SEQ ID N0:62), respectively, may be
employed as the starting amino
acid residue for the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 potypeptide.
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The "extracellulardomain" or"ECD" ofa polypeptide disclosed herein refers to a
form ofthe polypeptide
which is essentially free of the transmembrane and cytoplasmic domains.
Ordinarily, a polypeptide ECD will have
less than about 1 % of such transmembrane and/or cytoplasmic domains and
preferably, will have less than about
0.5% of such domains. It will be understood that any transmembrane domains)
identified for the 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 and as
shown in the appended figures. As
such, in one embodiment ofthe present invention, the extracellular domain of a
polypeptide of the present invention
comprises amino acids 1 to X of the mature amino acid sequence, wherein X is
any amino acid within 5 amino acids
on either side of the extracellular domain/transmembrane domain boundary.
The approximate location ofthe "signal peptides" ofthe various PRO
polypeptides disclosed herein are
shown in 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 ofthe
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. Ena., 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 peptide as identified
herein, and the polynucleotides encoding them, are contemplated by the present
invention.
"PR0179 variant polypeptide" means an active PR0179 polypeptide (other than a
native sequence
PR0179 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 17 to 460 of the PR0179 polypeptide
shown in Figure 2 (SEQ ID N0:2),
(b) X to 460 of the PR0179 polypeptide shown in Figure 2 (SEQ ID N0:2),
wherein X is any amino acid residue
from 12 to 21 of Figure 2 (SEQ ID N0:2), or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure 2 (SEQ ID N0:2).
"PR0207 variant polypeptide" means an active PR0207 polypeptide (other than a
native sequence
PR0207 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 41 to, 249 of the PR0207 polypeptide
shown in Figure 4 (SEQ ID N0:7),
(b) X to 249 of the PR0207 polypeptide shown in Figure 4 (SEQ ID N0:7),
wherein X is any amino acid residue
from 36 to 45 of Figure 4 (SEQ ID N0:7), or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure 4 (SEQ ID N0:7).
"PR0320 variant polypeptide" means an active PR0320 polypeptide (other than a
native sequence
PR0320 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 22 to 338 of the PR0320 polypeptide
shown in Figure 6 (SEQ 1D NO:10),
(b) X to 338 ofthe PR0320 polypeptide shown in Figure 6 (SEQ ID NO:10),
wherein X is any amino acid residue
from 17 to 26 of Figure 6 (SEQ ID NO:10), or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure 6 (SEQ ID NO:10).
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"PR0219 variant polypeptide" means an active PR0219 polypeptide (other than a
native sequence
PR0219 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 24 to 1005 of the PR0219 polypeptide
shown in Figure 8 (SEQ ID NO:15),
(b) X to 1005 ofthe PR0219 polypeptide shown in Figure 8 (SEQ ID NO:15),
wherein X is any amino acid residue
from 19 to 28 of Figure 8 (SEQ ID NO:15), or (c) another specifically derived
fragment ofthe amino acid sequence
shown in Figure 8 (SEQ ID NO:15).
"PR0221 variant polypeptide" means an active PR0221 polypeptide (other than a
native sequence
PR0221 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 34 to 259 ofthe PR0221 polypeptide
shown in Figure 10 (SEQ ID N0:20),
(b) X to 259 ofthe PR0221 polypeptide shown in Figure 10 (SEQ ID N0:20),
wherein X is any amino acid residue
from 29 to 38 of Figure 10 (SEQ ID N0:20), (c) 1 or about 34 to X of Figure 10
(SEQ ID N0:20), wherein X is
any amino acid from amino acid 199 to amino acid 208 of Figure 10 (SEQ ID
N0:20), or (d) another specifically
derived fragment of the amino acid sequence shown in Figure 10 (SEQ ID N0:20).
"PR0224 variant polypeptide" means an active PR0224 polypeptide (other than a
native sequence
PR0224 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 31 to 282 of the PR0224 polypeptide
shown in Figure 12 (SEQ ID N0:25),
(b) X to 282 ofthe PR0224 polypeptide shown in Figure 12 (SEQ ID N0:25),
wherein X is any amino acid residue
from 26 to 35 of Figure 12 (SEQ 1D N0:25), (c) 1 or about 31 to X of Figure 12
(SEQ ID N0:25}, wherein X is
any amino acid from amino acid 226 to amino acid 235 of Figure 12 (SEQ ID
N0:25), or (d) another specifically
derived fragment of the amino acid sequence shown in Figure 12 (SEQ ID N0:25).
"PR0328 variant polypeptide" means an active PR0328 polypeptide (other than a
native sequence
PR0328 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 23 to 463 of the PR0328 polypeptide
shown in Figure 14 (SEQ 1D N0:30),
(b) X to 463 of the PR0328 polypeptide shown in Figure 14 (SEQ ID N0:30),
wherein X is any amino acid residue
from 18 to 27 of Figure 14 (SEQ ID N0:30), or (c) another specifically derived
fragment of the amino acid
sequence shown in Figure 14 (SEQ ID N0:30).
"PR0301 variant polypeptide" means an active PR0301 polypeptide (other than a
native sequence
PR0301 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 28 to 299 of the PR0301 polypeptide
shown in Figure 16 (SEQ ID N0:35),
(b} X to 299 of the PR0301 polypeptide shown in Figure 16 (SEQ ID N0:35),
wherein X is any amino acid residue
from 23 to 32 of Figure 16 (SEQ ID N0:35), (c) 1 or about 28 to X of Figure 16
(SEQ ID N0:35), wherein X is
any amino acid from amino acid 230 to amino acid 239 of Figure 16 (SEQ ID
N0:35), or (d) another specifically
derived fragment of the amino acid sequence shown in Figure 16 (SEQ ID N0:35).
"PR0526 variant polypeptide" means an active PR0526 polypeptide (other than a
native sequence
PR0526 polypeptide) as defined below, having at feast about 80% amino acid
sequence identity with the amino
acid sequence of(a) residues 1 or about27 to 473 ofthe PR0526 polypeptide
shown in Figure 18 (SEQ ID N0:43),
(b) X to 473 ofthe PR0526 polypeptide shown in Figure 18 (SEQ ID N0:43),
wherein X is any amino acid residue
from 22 to 31 of Figure 18 (SEQ ID N0:43), or (c) another specifically derived
fragment of the amino acid
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sequence shown in Figure 18 (SEQ ID N0:43).
"PR0362 variant polypeptide" means an active PR0362 polypeptide (other than a
native sequence
PR0362 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 20 to 321 of the PR0362 polypeptide
shown in Figure 20 (SEQ ID N0:48),
(b) X to 321 of the PR0362 polypeptide shown in Figure 20 (SEQ ID N0:48),
wherein X is any amino acid residue
from 15 to 24 of Figure 20 (SEQ ID N0:48), (c) 1 or about 20 to X of Figure 20
(SEQ ID N0:48), wherein X is
any'amino acid from amino acid 276.to amino acid 285 of Figure 20 (SEQ ID
N0:48), or (d) another specifically
derived fragment of the amino acid sequence shown in Figure 20 (SEQ ID N0:48).
"PR0356 variant polypeptide" means an active PR0356 polypeptide (other than a
native sequence
PR0356 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 27 to 346 of the PR0356 polypeptide
shown in Figure 22 (SEQ ID NO:55),
(b) X to 346 of the PR0356 polypeptide shown in Figure 22 (SEQ ID NO:55),
wherein X is any amino acid residue
from 22 to 31 of Figure 22 (SEQ ID NO:SS), or (c) another specifically derived
fragment of the amino acid
sequence shown in Figure 22 (SEQ ID NO:55).
"PR0509 variant polypeptide" means an active PR0509 polypeptide (other than a
native sequence
PR0509 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 37 to 283 of the PR0509 polypeptide
shown in Figure 24 (SEQ ID N0:60),
(b) X to 283 of the PR0509 polypeptide shown in Figure 24 (SEQ ID N0:60),
wherein X is any amino acid residue
from 32 to 41 of Figure 24 (SEQ ID N0:60), (c) 1 or about 37 to X of Figure 24
(SEQ ID N0:60), wherein X is
any amino acid from amino acid 200 to amino acid 209 of Figure 24 (SEQ ID
N0:60), or (d) another specifically
derived fragment of the amino acid sequence shown in Figure 24 (SEQ ID N0:60).
"PR0866 variant polypeptide" means an active PR0866 polypeptide (other than a
native sequence
PR0866 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues 1 or about 27 to 331 of the PR0866 poiypeptide
shown in Figure 26 (SEQ ID N0:62),
(b) X to 331 of the PR0866 polypeptide shown in Figure 26 (SEQ ID N0:62),
wherein X is any amino acid residue
from 22 to 31 of Figure 26 (SEQ ID N0:62), or (c) another specifically derived
fragment of the amino acid
sequence shown in Figure 26 (SEQ ID N0:62).
Such PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 and PR0866 variants include, for instance, PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866 polypeptides
wherein one or
more amino acid residues are added, or deleted, at the N- or C-terminus, as
well as within one or more internal
domains of the native sequence.
Ordinarily, a PR0179 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
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sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
S 96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 17 to 460 of the PR0179
polypeptide shown in Figure 2 (SEQ ID
N0:2), (b) X to 460 of the PR0179 polypeptide shown in Figure 2 (SEQ ID N0:2),
wherein X is any amino acid
residue from 12 to 21 of Figure 2 (SEQ ID N0:2), or (c) another specifically
derived fragment of the amino acid
sequence shown in Figure 2 (SEQ 1D N0:2).
Ordinarily, a PR0207 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity. more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 41 to 249 of the PR0207
polypeptide shown in Figure 4 (SEQ ID
N0:7), (b) X to 249 ofthe PR0207 polypeptide shown in Figure 4 (SEQ ID N0:7),
wherein X is any amino acid
residue from 36 to 45 of Figure 4 (SEQ ID N0:7), or (c) another specifically
derived fragment of the amino acid
sequence shown in Figure 4 (SEQ ID N0:7).
Ordinarily, a PR0320 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid

CA 02353799 2001-06-08
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sequence identity with (a) residues 1 or about 22 to 338 of the PR0320
polypeptide shown in Figure 6 (SEQ ID
NO:10), (b) X to 338 of the PR0320 polypeptide shown in Figure 6 (SEQ ID
NO:10), wherein X is any amino acid
residue from 17 to 26 of Figure 6 (SEQ ID NO:10), or (c) another specifically
derived fragment of the amino acid
sequence shown in Figure 6 (SEQ ID NO:10).
Ordinarily, a PR0219 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence idemity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 24 to 1005 of the PR0219
polypeptide shown in Figure 8 (SEQ ID
NO: I5), (b) X to 1005 of the PR0219 polypeptide shown in Figure 8 (SEQ ID
N0:15), wherein X is any amino
acid residue from 19 to 28 of Figure 8 (SEQ ID N0:15), or (c) another
specifically derived fragment of the amino
acid sequence shown in Figure 8 (SEQ ID N0:15).
Ordinarily, a PR0221 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 34 to 259 of the PR0221
polypeptide shown in Figure 10 (SEQ ID
N0:20), (b) X to 259 of the PR0221 polypeptide shown in Figure 10 (SEQ ID
N0:20), wherein X is any amino
acid residue from 29 to 38 of Figure 10 (SEQ ID N0:20), (c) 1 or about 34 to X
of Figure 10 (SEQ ID N0:20),
wherein X is any amino acid from amino acid 199 to amino acid 208 of Figure 10
(SEQ ID N0:20), or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 10
(SEQ 1D N0:20).
Ordinarily, a PR0224 variant will have at least about 80% amino acid sequence
identity, more preferably
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at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues l or about 31 to 282 of the PR0224
polypeptide shown in Figure 12 (SEQ ID
N0:25), (b) X to 282 of the PR0224 polypeptide shown .in Figure l2 (SEQ ID
N0:25), wherein X is any amino
acid residue from 26 to 35 of Figure 12 (SEQ ID N0:25), (c) 1 or about 31 to X
of Figure 12 (SEQ ID N0:25),
wherein X is any amino acid from amino acid 226 to amino acid 235 of Figure 12
(SEQ ID N0:25), or (d) another
specifically derived fragment ofthe amino acid sequence shown in Figure 12
(SEQ ID N0:25).
Ordinarily, a PR0328 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at (east about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 23 to 463 of the PR0328
polypeptide shown in Figure 14 (SEQ ID
N0:30), (b) X to 463 of the PR0328 polypeptide shown in Figure 14 (SEQ ID
N0:30), wherein X is any amino
acid residue from I 8 to 27 of Figure 14 (SEQ ID N0:30), or (c) another
specifically derived fragment of the amino
acid sequence shown in Figure 14 (SEQ ID N0:30).
Ordinarily, a PR0301 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% am ino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
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sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 28 to 299 of the PR0301
polypeptide shown in Figure 16 (SEQ ID
N0:35), (b) X to 299 of the PR0301 polypeptide shown in Figure 16 (SEQ ID
N0:35), wherein X is any amino
acid residue from 23 to 32 of Figure 16 (SEQ ID N0:35), (c) 1 or about 28 to X
of Figure 16 (SEQ ID N0:35),
wherein X is any amino acid from amino acid 230 to amino acid 239 of Figure 16
(SEQ ID N0:35), or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 16
(SEQ ID N0:35).
Ordinarily, a PR0526 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferabiy at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 27 to 473 of the PR0526
polypeptide shown in Figure 18 (SEQ ID
N0:43), (b) X to 473 of the PR0526 polypeptide shown in Figure 18 (SEQ 1D
N0:43), wherein X is any amino
acid residue from 22 to 31 of Figure 18 (SEQ ID N0:43), or (c) another
specifically derived fragment of the amino
acid sequence shown in Figure 18 (SEQ ID N0:43).
Ordinarily, a PR0362 variant wilt have at least about 80% amino acid sequence
identity, more preferably
at least about 81% amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
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preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 20 to 321 of the PR0362
polypeptide shown in Figure 20 (SEQ ID
N0:48), (b) X to 321of the PR0362 polypeptide shown in Figure 20 (SEQ ID
N0:48), wherein X is any amino
acid residue from 15 to 24 of Figure 20 (SEQ ID N0:48), (c) 1 or about 20 to X
of Figure 20 (SEQ ID N0:48),
wherein X is any amino acid from amino acid 276 to amino acid 285 of Figure 20
(SEQ ID N0:48), or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 20
(SEQ ID N0:48).
Ordinarily, a PR0356 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues 1 or about 27 to 346 of the PR0356
polypeptide shown in Figure 22 (SEQ ID
N0:55), (b) X to 346 of the PR0356 polypeptide shown in Figure 22 (SEQ ID
N0:55), wherein X is any amino
acid residue from 22 to 31 of Figure 22 (SEQ ID N0:55), or (c) another
specifically derived fragment of the amino
acid sequence shown in Figure 22 (SEQ ID N0:55).
Ordinarily, a PR0509 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81 % amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least abort 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues I or about 37 to 283 of the PR0509
polypeptide shown in Figure 24 (SEQ ID
N0:60), (b) X to 283of the PR0509 polypeptide shown in Figure 24 (SEQ ID
N0:60), wherein X is any amino
acid residue from 32 to 41 of Figure 24 (SEQ ID N0:60), (c) 1 or about 37 to X
of Figure 24 (SEQ ID N0:60),
wherein X is any amino acid from amino acid 200 to amino acid 209 of Figure 24
(SEQ ID N0:60), or (d) another
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specifically derived fragment of the amino acid sequence shown in Figure 24
(SEQ ID N0:60).
Ordinarily, a PR0866 variant will have at least about 80% amino acid sequence
identity, more preferably
at least about 81% amino acid sequence identity, more preferably at least
about 82% amino acid sequence identity,
more preferably at least about 83% amino acid sequence identity, more
preferably at least about 84% amino acid
sequence identity, more preferably at least about 85% amino acid sequence
identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more
preferably at least about 88% amino acid sequence identity, more preferably at
least about 89% amino acid
sequence identity, more preferably at least about 90% amino acid sequence
identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more
preferably at least about 93% amino acid sequence identity, more preferably at
least about 94% amino acid
sequence identity, more preferably at least about 95% amino acid sequence
identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more
preferably at least about 98% amino acid sequence identity and yet more
preferably at least about 99% amino acid
sequence identity with (a) residues I or about 27 to 331 of the PR0866
polypeptide shown in Figure 26 (SEQ ID
N0:62), (b) X to 331of the PR0866 polypeptide shown in Figure 26 (SEQ ID
N0:62), wherein X is any amino
acid residue from 22 to 31 of Figure 26 (SEQ ID N0:62), or (c) another
specifically derived fragment of the amino
acid sequence shown in Figure 26 (SEQ ID N0:62).
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 and PR0866 variant polypeptides do not encompass the native
PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PROS26, PR0362, PR03S6, PR0509 and
PR0866 polypeptide
sequence. Ordinarily, PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 and PR0866 variant polypeptides are at least about 10
amino acids in length, often
at least about 20 amino acids in length, more often at least about 30 amino
acids in length, more often at least about
40 amino acids in length, more often at least about 50 amino acids in length,
more often at least about 60 amino
acids in length, more often at least about 70 amino acids in length, more
often at least about 80 amino acids in
length, more often at least about 90 amino acids in length, more often at
least about 100 amino acids in length, more
often at least about 1 SO amino acids in length, more often at least about 200
amino acids in length, more often at
least about 250 amino acids in length, more often at least about 300 amino
acids in length, or more.
As shown below, Table 1 provides the complete source code for the ALIGN-2
sequence comparison
computer program. This source code may be routinely compiled for use on a UNIX
operating system to provide
the ALIGN-2 sequence comparison computer program.
In addition, Tables 2A-2D show hypothetical exemplifications for using the
below described method to
determine % amino acid sequence identity (Tables 2A-2B) and % nucleic acid
sequence identity (Tables 2C-2D)
using the ALIGN-2 sequence comparison computer program, wherein "PRO"
represents the amino acid sequence
ofa hypothetical PR0179, PR0207,PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PROS26, PR0362,
PR03S6, PR0509 or PR0866 polypeptide of interest, "Comparison Protein"
represents the amino acid sequence
of a polypeptide against which the "PRO" polypeptide of interest is being
compared, "PRO-DNA" represents a
hypothetical PR0179-, PR0207-, PR0320-, PR0219-, PR0221-, PR0224-, PR0328-,
PR0301-, PROS26-,

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PR0362-, PR0356-, PR0509- or PR0866-encoding nucleic acid sequence of
interest, "Comparison DNA"
represents the nucleotide sequence ofa nucleic acid molecule against which the
"PRO-DNA" nucleic acid molecule
of interest is being compared, "X", "Y", and "Z" each represent different
hypothetical amino acid residues and "N",
"L" and "V" each represent different hypothetical nucleotides.
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/*
Table 1
* C-C increased from 12 to IS
* Z is average of EQ
* B is average of ND
* match with stop is M; stop-stop = 0; J (joker) match = 0
*/
Ifdefine M -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 */
/* A */ { 2, 0,-2. 0, 0,-4, 1,-1,-1, 0,-1,-2,-1, 0, M, 1, 0,-2, 1, 1, 0, 0,-6,
0,-3, 0},
/* B */ { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2, M,-I, 1, 0, 0, 0, 0,-2,-5,
0,-3, 1},
/* C */ {-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8,
0, 0,-5},
/* D */ { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2, M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 2},
/* E *l { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1, M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 3},
/* F */ {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-5,-4,-3,-3, 0,-1, 0,
0, 7,-5},
/* G */ { 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, O, M,-1,-I,-3, 1, 0, 0,-1,-7,
0,-5, 0},
/* H */ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2,-I,-1, 0,-2,-3,
0, 0, 2},
/* I */ {-I,-2,-2,-2,-2, 1,-3,-2, 5, 0,-2, 2, 2,-2, M,-2,-2,-2,-1, 0, 0, 4,-5,
0,-1,-2},
/*J*/ {0,0,0,0,0,0,0,0,0,0,0,0,O,O, M,0,0,0,0,0,0,0,0,0,0,0},
1* K */ {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1, M,-1, 1, 3, 0, 0, 0,-2,-3,
0,-4, 0},
/* L */ {-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1, 0, 2,-2,
0,-1,-2},
/* M */ {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2, M,-2,-1, 0,-2,-1, 0, 2,-4,
0,-2,-1},
/* N */ { 0, 2,-4, 2, I,-4, 0, 2,-2, 0, 1,-3,-2, 2, M,-1, 1, 0, 1, 0, 0,-2,-4,
0,-2, 1},
/* O */ { M,_M,_M,_M, M, M, M, M, M, M, M, M, M, M, O, M, M,_M,
M,_M,_M,_M,_M,_M,_M,_M},
/* P */ { 1,-I,-3,-1,-I,-5,-1, 0,-2, 0,-1,-3,-2,-I, M, 6, 0, 0, 1, 0, 0,-1,-6,
0,-5, 0},
/* Q */ { 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1, M, 0, 4, I,-1,-1, 0,-2,-5,
0,-4, 3},
/* R */ {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2. 2,
0,-4, 0},
I* S */ { 1, 0, 0, 0, 0,-3, I,-I,-1, 0, 0,-3,-2, I, M, I,-1, 0, 2, 1, 0,-1,-2,
0.-3, 0},
/* T */ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-I,-1, 0, M, 0,-I,-1, 1, 3, 0, 0,-5,
0,-3, 0},
/* a */ { o, o, o, o, o, o, o, o, o, o, o, o, o, o,_ivl, o, o, o, o, o, o, o,
o, o, o, o},
/* V */ { 0,-2,-2,-Z,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2, M,-I,-2,-2,-1, 0, 0, 4,-6,
0,-2,-2},
/* W */ {-6,-5,-8,-7,-7, 0,-7,-3,-5, 0,-3,-2,-4,-4,_M,-6,-5, 2,-2,-5, 0,-6,17,
0, 0,-6},
I*X*l {0,0,0,0,0,0,0,0,0,0,0,0,0,0, M,0,0,0,0,0,0,0,0,0,0,0},
/* Y */ {-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2,_M,-5,-4,-4,-3,-3, 0,-2, 0,
0,10,-4},
/* Z */ { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, I =M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4}
};
Page 1 of day.h
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/*
*/
#include
<
stdio.h
>
#include h
< >
ctype.
#defineMAXJMP 16 I* max jumps in a diag */
#defineMAXGAP 24 /* don't continue to penalize gaps larger
than this */
#defmeJMPS 1024!* max jmps in an path */
#defineMX 4 I* save if there's at least MX-1 bases
since last jmp *l
#detineDMAT 3 !* value of matching bases */
#defineDMIS 0 /* penalty for mismatched bases */
#defineDINSO 8 /* penalty for a gap */
#defineDINS 1 /* penalty per base */
1
#definePINSO 8 /* penalty for a gap */
#defmePINS1 4 /* penalty per residue */
struct
jmp
{
short n[MAXJMP];
/*
size
of
jmp
(neg
for
dely)
*/
unsigned x[MAXJMP];
short /*
base
no.
of
jmp
in
seq
x
*/
}; /* limits seq to 2"16 -1 *l
struct
diag
{
int score;I* score at last jmp *I
long offset;/* offset of prey block *!
short ijmp;/* current jmp index */
struct jp; /* list of jmps */
jmp
};
struct
path
{
int spc; /* number of leading spaces */
shortn(JMPS]; /* size of jmp (gap) */
int x[JMPS]; /* loc of jmp (last elem before gap) */
};
char *ofile; /* output file name */
char *namex[2]; /* seq names: getseqsQ */
char *prog; /* prog name for err msgs */
char *seqx[2]; /* seqs: getseqsQ */
int dmax; /* best diag: nwQ */
int dmax0; /* final diag */
int dna; /* set if dna: main() */
int endgaps; l* set if penalizing end gaps */
int gapx, 1* total gaps in seqs */
gapy;
int len0,; /* seq lens */
lent
int ngapx, 1* total size of gaps */
ngapy;
int smax; /* max score: nwQ */
int *xbm; 1* bitmap for matching */
long offset; /* current offset in jmp file */
structdiag *dx; /* holds diagonals */
structpath pp[2]; /* holds path for seqs *1
char *callocQ,*mallocQ, *indexp, *strcpyp;
char *getseqQ,*g
callocQ;
Page 1 of nw.h
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/* Needleman-Wunsch alignment program
* usage: progs filet filet
* where filet and filet are two dna or two protein sequences.
* The sequences can be in upper- or lower-case an may contain ambiguity
* Any lines beginning with '; ' > ' or ' < ' are 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 tmp tile in ltmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*/
t/include "nw.h"
1/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
};
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, I«19, 1«20, l«21, 1«22,
1«23, I«24, 1«25(1«('E'-'A'))~(1«('Q'-'A'))
};
main(ac, av) main
int ac;
char *av[];
prog = av[0];
if (ac ! = 3) {
fprintf(stderr,"usage: %s filet filet\n", prog);
fprintf(stderr,"where filet and filet are two dna or two protein
sequences.ln");
fprintf(stderr, "The sequences can be in upper- or lower-case\n");
fprintf(stderr,"Any lines beginning with ';' or ' <' are ignored\n");
fprintf(stderr,"Output is in the file 1"align.outl"\n");
exit(I);
namex[0] = av[1];
namex(1] = av[2];
seqx[O] = getseq(namex[0], &IenO);
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : pbval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; /* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps *I
printQ; I* print scats, alignment *I
cleanup(0); /* unlink any tmp files */
Page 1 of nw.c
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/* do the alignment, return best score: main()
* 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.
*/
nwQ L1W
{
char *px, *py; I* seqs and ptrs */
int *ndely, *dely;l* keep track of dely */
int ndelx, delx;I* keep track of delx *I
int *tmp; /* for swapping row0, rowl */
int mis; /* score for each type */
int ins0, insl; /* insertion penalties */
register id; 1* diagonal index */
register ij; /* jmp index */
register *col0, *coll;/* score for curr, last row */
register xx, yy; I* index into seqs *I
dx = (struct diag *)g calloc("to get diags", IenO+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 calloc("to getcol0", lenl+1, sizeof(int));
col l = (int *)g calloc("to get col l ", lenl + 1, sizeof(int));
ins0 = {dna)? DINSO : PINSO;
ins 1 = (dna)? DINS 1 : PINS 1;
smax = -10000;
if (endgaps) {
for (col0[0) = dely[0) _ -ins0, yy = 1; yy < = lenl; yy++) {
col0[yy] = dely[yy] = col0[yy-1] - insl;
ndely[yyJ = yy;
col0[0] = 0; /* Watetman Bull Math Biol 84 */
j
else
for (yy = 1; yy < = lenl; yy++)
dely[yyJ = -ins0;
/* fill in match matrix
*/
for (px = seqx[OJ, xx = 1; xx < = IenO; px++, xx++) {
/* initialize first entry in col
*/
if (endgaps) {
if (xx == 1)
toll[0] = delx = -(ins0+insl);
else
coil[O] = delx = col0[0] - insl;
ndelx = xx;
else {
col 1 [0] = 0;
delx = -ins0;
ndelx = 0;
Page 2 of nw.c

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for (py = seqx[1J, yy = 1; yy <= lent; py++, yy++) {
mis = col0[yy-1];
if (dna)
mis +_ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis += day[*px-'A'][*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
*/
if (endgaps ~ ~ ndely[yy] < MAXGAP) {
if (col0[yy] - ins0 > = dely[yy]) {
dely[yy] = col0[yy] - (ins0+insl);
ndely[yy] = 1;
} else {
delyjyy] -= insl;
ndely[yy] + + ;
}
} else {
if (col0[yy] - (ins0+insl) > = dely[yy]) {
dely[yy] = col0(yy] - (ins0+insI);
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 > = delx) {
delx = coil[yy-1] - (ins0+insl);
ndelx = 1;
} else {
delx - = insl;
ndelx+ + ;
}
} else {
if (toll[yy-I] - (ins0+insl) > = delx) {
delx = coil[yy-1] - (ins0+insl);
ndelx = 1;
} else
ndelx+ + ;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
*/
...nw
Page 3 of nw.c
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id=xx-yy+lenl-1;
if (mis > = delx && mis > = dely[yy])
...nw
coll.[yy] = mis;
else if (delx > = dely[yy]) {
coi l [yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ ~ (ndelx > = MAXJMP
&& xx > dx(id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset += slzeof(struct jmp) + sizeof(offset);
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
else {
coll[yy] = dely[yy);
ij = dx[id].ijmp;
if (dx[id].jp.n[0) && (!dna ~ ~ (ndely(yy] > = MAXJMP
&& xx > dx[id].jp.x[ij]+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
dx[id].jp.n[ij] _ -ndely[yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
if (xx = = IenO && yy < len 1 ) {
/* last col
*/
if (endgaps)
coll[yy] -= ins0+insl*(lenl-yy);
if (col l [yy] > smax) {
smax = coll[yy];
dmax = id;
if (endgaps && xx < IenO)
coll[yy-1] -= ins0+insl*(IenO-xx);
if (col l (yy-I ] > smax) {
smax = coll[yy-1];
dmax = id;
tmp = col0; col0 = colt; coil = tmp;
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)col0);
(void) free((char *)coll);
Page 4 of nw.c
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/*
* print() -- only routine visible outside this module
* static:
* getmatQ -- trace back best path, count matches: print()
* pr align() -- print alignment of described in array p[]: print()
* dumpblockQ -- dump a block of lines with numbers, stars: pr alignQ
* numsQ -- put out a number line: dumpblockQ
* putline() -- put out a line (name, [num], seq, (num]): dumpblock()
* stars() - -put a line of stars: dumpblockQ
* stripnamep -- strip any path and prefix from a seqname
*/
~Jinclude "nw.h"
!t'define SPC 3
I~define P_LINE 256 /* maximum output line */
lfdefine P-SPC 3 /* space between name or num and seq */
extern _day[26][26];
int olen; /* set output line length */
FILE *fx; /* output file */
print()
print
{
int Ix, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) _ = 0) {
fprintf(stderr,"%s: can't write %s\n", prog, ofile);
cleanup(1);
fprintf(fx, "<first sequence: %s (length = %d)\n", namex[0], IenO);
fprintf(fx, "<second sequence: %s (length = %d)1n", namex[1], lenl);
olen = 60;
lx = IenO;
ly = lent;
firstgap = lastgap = 0;
if {dmax < lenl - 1) { /* leading gap in x */
pp[0].spc = firstgap = lent - dmax - 1;
ly _= pP[ol~sP~;
else if (dmax > lenl - 1) { /* leading gap in y */
pp[1].spc = firstgap = dmax - (lenl - 1);
lx -= pp[1].spc;
if (dmax0 < len0 - 1) { /* trailing gap in x */
lastgap = IenO - dmax0 -1;
lx -= lastgap;
else if (dmax0 > IenO - 1) { /* trailing gap in y */
lastgap = dmax0 - (len0 - 1);
ly -= lastgap;
getmat(Ix, ly, firstgap, lastgap);
pr alignQ;
Page 1 of nwprint.c
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/*
* trace back the best path, count matches
*/
static
getmat(lx, ly, firstgap, lastgap) getmat
int Ix, 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;
/* get total matches, score
*/
i0 = i 1 = siz0 = siz I = 0;
p0 = seqx[O] + pp[1].spc;
pl = seqx[1] + pp[0].spc;
n0 = pp(1].spc + 1;
nl = pp[O].spc + 1;
nm = 0;
while ( *p0 && *p 1 ) {
if (siz0) {
pl++;
nl++;
siz0--;
else if (sizl) {
p0++;
n0+ +;
sizl--;
else {
if (xbm[*p0-'A']&xbm[*pl-'A'
nm++;
if (n0++ _= pp[O].x[i0]}
siz0 = pp[O].n[i0++];
if (nl++ _= pp[1].x[il])
sizl = pp[1].n[il++];
p0++;
pl++;
/* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
*l
if (endgaps)
lx = (len0 < lenl)? IenO : lenl;
else
lx = (lx < ly)? lx : ly;
pct = 100.*(double)nm/(double)lx;
fprintf(fx, "\n");
fprintf(fx, " < % d match % s in an overlap of %d: % .2f percent
similarity\n",
nm, (nm == 1)? "" . "es", lx, pct);
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fprintf(fx, "<gaps in first sequence: %d", gapx); ...getlllat
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base":"residue", (ngapx == 1)? "":"s");
fprintf(fx,"%s", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gapY) {
(void) sprintf(outx, " (%d %s%s)",
ngapy, (dna)? "base":"residue", (ngapy = = 1)? "~:"s");
fprintf(fx,"%s", outx);
if (dna)
fprintf(fx,
"\n < score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per
base)\n",
smax, DMAT, DMIS, DINSO, DINS1);
else
fprintf(fx,
"\n < score: % d (Dayhoff PAM 250 matrix, gap penalty = % d + % d per
residue)\n",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
" < endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n",
firstgap, (dna)? "base" : "residue", (firstgap == 1)? "' . "s",
lastgap, (dna)? "base" : "residue", (lastgap == I)? "" . "s");
else
fprintf(fx, " < endgaps not penalized\n");
static nm; /* matches in core
-- for checking */
static lmax; /* 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]; I * ptr to next output
char char slot *I
static out[2][P /* output line */
char LINE];
static star[P LINE];I* set by stars() *f
char
/*
* print alignment of described in struct path pp[]
*/
static
pr align() pr ~lgI1
{
int nn; I * char count *I
int more;
register i;
for (i = 0, lmax = 0; i < 2; i++) {
nn = stripname(namex[i]};
if (nn > Imax)
Imax = nn;
nc[i] = 1;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[i] = out[i];
Page 3 of nwprint. c

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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]++ _ ,
PP[il ~ sPc":
else if (siz(i]) { 1* in a gap */
*po[i]++ _ ,
siz[i]__;
else { l* we're putting a seq element
*/
*po(il = *Psti]:
if (islower(*ps(i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i]++;
I*
* are we at next gap for this seq?
*I
if (ni[i] _= 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;
/*
* dump a block of lines, including numbers, stars: pr align()
*l
static
aumpblock() dumpblock
{
register i;
for (i = 0; i < 2; i++)
*po[i]__ _ '\0';
Page 4 of nwprint.c
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(void) putc('\n', fx);
for (i = 0; i < 2; i++) {
if (*out[i] && (*out[i] ! _ ' ' ~ I *(po[i]) ! _ ' ')) {
if (i == 0)
nums(i);
if (i == 0 && *out[1])
starsp;
putline(i);
if (i == 0 && *out[1])
fprintf(fx, star);
if (i == 1)
nums(i);
...dumpblock
/*
* put out a number line: dumpblockQ
*/
static
nums(ix) riumS
int ix; /* index in out[] holding seq line */
{
char nline[P LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline, i = 0; i < Imax+P SPC; i++, pn++)
*Pn = ,
for (i = nc[ix], py = out(ix]; *py; py++, pn++) {
if (*py =- ' I I *PY =- '-')
*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 = j%10 + '0';
if (i < 0)
*Px = ,
else
*Pn = ,
i++;
*Pn = '\0';
nc[ix] = i;
for (pn = nline; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
/*
* put out a line (name, [num], seq, [num]): dumpblockQ
*/
static
putline(ix) putllrie
int ix;
{
Page 5 of nwprint.c
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int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px ! _ '~'; px++, i++)
(void) putc(*px, fx);
for (; i < Imax+P SPC; i++)
(void) putc(' ', fx);
/* these count from 1:
* ni[] 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);
...putline
/*
* put a line of stars (seqs always in out[0], out[1]): dumpblockQ
*/
static
starsQ Stal'S
{
int i;
register char *p0, *pl, cx, *px;
if (!*out[0] ~ ~ (*out[0] _ _ ' && *(po(0]) _ - ' ') ~ ~
!*out(1] ( ~ (*out[1] _- ' && *(po[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
else
cx = ,
*px++ = cx;
*px++ _ '1n';
*Px = ~\0';
cx=' ,
Page 6 of nwprint.c
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l*
* strip path or prefix from pn, return len: pr align()
*/
static
stripname(pn) stripname
char *pn; l* file name (may be path) *I
register char *px, *py;
PY = ~:
for (px = pn; *px; px++)
if (*px =_ '/')
py=px+1;
if (PY)
(void) strcpy(pn, py);
return(strlen(pn));
Page 7 of nwprint. c
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I*
* cleanup() -- cleanup any tmp file
* getseqQ -- read in seq, set dna, len, maxlen
* g callocQ -- callocQ with error checkin
* readjmpsQ -- get the good jmps, from tmp file if necessary
* writejmpsQ -- write a filled array of jmps to a tmp file: nwQ
*I
include "nw.h"
#include < sys/file.h >
char *jname = "Itmp/homgXXXXXX"; /* tmp file for jmps *1
FILE *fj;
int cleanupQ; /* cleanup tmp file */
long lseekQ;
/*
* remove any tmp file if we blow
*/
cleanup(i) cleanup
int i;
{
if (fj)
(void) unlink(jname);
exit(i);
/*
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with '; , ' <', or ' >'
* seq in upper or lower case
*/
char
getseq(file, len) getseq
char *file; /* file name */
int *len; /* seq len */
{
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file,"r")) _ = 0) {
fprintf(stderr,"%s: can't read %s\n", grog, file);
exit(1);
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line =_ ''' ~ ~ *line =_ ' <' ~ ~ *iine =- ' >')
continue;
for (px = line; *px !_ '\n'; px++)
if (isupper(*px) ~ ~ islower(*px))
tlen+ +;
if ((pseq = malloc((unsigned)(tlen+6))) _= 0) {
fprintf(stderr,"%s: mallocQ failed to get %d bytes for %s\n", prog, tlen+6,
file);
exit(1);
pseq[0] = pseq[1] = pseq[2] = pseq[3] _ '\0';
Page 1 of nwsubr.c

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...getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, 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++ = '\0';
*PY = '\0'~
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
char
g caUoc(msg, nx, sz) g_Ca~IOC
char *msg; /* program, calling routine *l
int nx, sz; I * number and size of elements *I
{
char *px, *callocQ;
if ((px = calloc((unsigned)nx, (unsigned)sz)) _ = 0) {
if (*msg) {
fprintf(stderr, "%s: g calloc() failed %s (n=%d, sz=%d)\n", prog, msg, nx,
sz);
exit(1 );
return(px);
1*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main()
*/
readjmpsQ
readjmps
{
int fd = -1;
int siz, i0, il;
register i, j, xx;
if (fj) {
(void) fclose(tj);
if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintf(stderr, " % s: can't open() % stn", prog, jname);
cleanup(1);
for (i = i0 = il = 0, dmax0 = dmax, xx = IenO; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > = xx; j--)
Page 2 of nwsubr.c
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...readjmps
if (j < 0 && dx[dmax].offset && fj) {
(void) Iseek(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 alignment\n", prog);
cleanup(1);
if (j > = o) {
siz = dx[dmax].jp.n[j];
xx = dx[dmax].jp.x(j];
dmax += siz;
if (siz < 0) { /* gap in second seq */
PP[1].nfil] _ -siz;
xx + = siz;
/*id=xx-yy+lenl-1
*/
pp[1).x[il] = xx - dmax + lenl - 1;
gapy++;
ngapy -= siz;
/* 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;
I* ignore MAXGAP when doing endgaps *I
siz = (siz < MAXGAP ~ ~ endgaps)? siz : MAXGAP;
i0++;
else
break;
/* reverse the order of jmps
*/
for (j = 0, i0--; j < i0; j++, i0--) {
~ = PP[0].n(j]; PP[0].n(j] = PP[Ol.n[i0]; pp(0].n[i0] = i;
~ = PP[Ol.xGl; PP[OI.xG] = PP[0]~x[i0]; PP[Ol.x[i0] = i;
for (j = 0, il--; j < il; j++, il--) {
i = PP[1].nU]; PP[1].nGl = PP[ll.n[il]; PP[ll.nfil] = i;
~ = PP[1].xU]> PP[1].xG] = PP[1Lx[il]; pP[Il.x[il] = i:
if (fd > = 0)
(void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0;
Page 3 of nwsubr.c
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/*
* write a filled jmp struct offset of the prev one (if any): nwQ
*l
writejmps(ix) W1'ItB,)IIllpS
int ix;
{
char *mktempQ;
if (?fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, " % s: can't mktemp() % s\n", prog, jname);
cleanup(I);
if ((fj = fopen(jname, "w")) _ = 0) {
fprintf(stderr, "%s: can't write %s\n", prog, jname);
exit(1);
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
Page 4 of nwsubr. c
38

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Table 2A
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 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) _
divided by 15 = 33.3 %
39

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Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (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) _
divided by 10 = 50

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Table 2C
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%
41

CA 02353799 2001-06-08
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Table 2D
PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides)
Comparison DNA NNNNLLLVV (Length = 9 nucleotides)
nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
4 divided by 12 = 33.3
42

CA 02353799 2001-06-08
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"Percent (%) amino acid sequence identity" with respect to the PR0179, PR0207,
PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866
polypeptide sequences
identified herein is defined as the percentage of amino acid residues in a
candidate sequence that are identical with
the amino acid residues in a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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, ALIGN-2 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 obtained as described below by using the
sequence comparison computer program
ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided
in Table I . The ALIGN-2
sequence comparison computer program was authored by Genentech, Inc., and the
source code shown in Table I
has been flied 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. 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.
For purposes herein, 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, Tables 2A-2B
demonstrate how to calculate
the % amino acid sequence identity of the amino acid sequence designated
"Comparison Protein" to the amino acid
sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained
as described above using the ALIGN-2 sequence comparison computer program.
However, % 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)). The NCBI-BLAST2 sequence comparison program
may be downloaded from
43

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
http://www.ncbi.nlm.nih.gov. NCB1-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 % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of B
to A.
In addition, % amino acid sequence identity may also be determined using the
WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymolosv. 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) = 1 l, and scoring matrix =
BLOSUM62. For purposes herein, a % amino acid sequence identity value is
determined by dividing (a) the
number of matching identical amino acids 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 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 amino acid
sequence B is the amino acid sequence of
the PRO polypeptide of interest.
"PR0179 variant polynucleotide" or "PR0179 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PRO 179 polypeptide as defined below and
which has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 17 to 460 of the
PR0179 polypeptide shown in Figure 2 (SEQ ID N0:2), (b) a nucleic acid
sequence which encodes amino acids
X to 460 ofthe PRO 179 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X
is any amino acid residue from
12 to 21 of Figure 2 (SEQ ID N0:2), or (c) a nucleic acid sequence which
encodes another specifically derived
fragment of the amino acid sequence shown in Figure 2 (SEQ ID N0:2).
Ordinarily, a PR0179 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
44

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 17 to 460
of the PR0179 polypeptide shown
in Figure 2 (SEQ ID N0:2), (b) a nucleic acid sequence which encodes amino
acids X to 460 of the PR0179
polypeptide shown in Figure 2 (SEQ 1D N0:2), wherein X is any amino acid
residue from 12 to 21 of Figure 2
(SEQ ID N0:2), or (c) a nucleic acid sequence which encodes another
specifically derived fragment ofthe amino
acid sequence shown in Figure 2 (SEQ ID N0:2). PR0179 polynucleotide variants
do not encompass the native
PR0179 nucleotide sequence.
"PR0207 variant polynucleotide" or "PR0207 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0207 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 41 to 249 of the
PR0207 polypeptide shown in Figure 4 (SEQ ID N0:7), (b) a nucleic acid
sequence which encodes amino acids
X to 249 of the PR0207 polypeptide shown in Figure 4 (SEQ ID N0:7), wherein X
is any amino acid residue from
36 to 45 of Figure 4 (SEQ ID N0:7), or (c) a nucleic acid sequence which
encodes another specifically derived
fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
Ordinarily, a PR0207 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 41 to 249
of the PR0207 polypeptide shown

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
in Figure 4 (SEQ ID N0:7), (b) a nucleic acid sequence which encodes amino
acids X to 249 of the PR0207
polypeptide shown in Figure 4 (SEQ ID N0:7), wherein X is any amino acid
residue from 36 to 45 of Figure 4
(SEQ ID N0:7), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the amino
acid sequence shown in Figure 4 (SEQ ID N0:7). PR0207 polynucleotide variants
do not encompass the native
PR0207 nucleotide sequence.
"PR0320 variant polynucleotide" or "PR0320 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0320 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 22 to 338 of the
PR0320 polypeptide shown in Figure 6 (SEQ ID NO:10), (b) a nucleic acid
sequence which encodes amino acids
X to 338 of the PR0320 polypeptide shown in Figure 6 (SEQ ID NO:10), wherein X
is any amino acid residue
from 17 to 26 of Figure 6 (SEQ ID NO:10), or (c) a nucleic acid sequence which
encodes another specifically
derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID NO:10).
Ordinarily, a PR0320 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at (east about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 22 to 338
of the PR0320 polypeptide shown
in Figure 6 (SEQ ID NO:10), (b) a nucleic acid sequence which encodes amino
acids X to 338 of the PR0320
polypeptide shown in Figure 6 (SEQ ID NO:10), wherein X is any amino acid
residue from 17 to 26 of Figure 6
(SEQ ID NO:10), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the amino
acid sequence shown in Figure 6 (SEQ ID NO:10). PR0320 polynucleotide variants
do not encompass the native
PR0320 nucleotide sequence.
"PR0219 variant polynucleotide" or "PR0219 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0219 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 24 to 1005 of the
PR0219 polypeptide shown in Figure 8 (SEQ ID NO:15), (b) a nucleic acid
sequence which encodes amino acids
X to 1005 of the PR0219 polypeptide shown in Figure 8 (SEQ ID NO:1 S), wherein
X is any amino acid residue
from 19 to 28 of Figure 8 (SEQ ID NO:15), or (c) a nucleic acid sequence which
encodes another specifically
derived fragment of the amino acid sequence shown in Figure 8 (SEQ ID NO:15).
Ordinarily, a PR0219 variant
46

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polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 9l% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 24 to
1005 ofthe PR0219 polypeptide shown
in Figure 8 (SEQ ID NO:15), (b) a nucleic acid sequence which encodes amino
acids X to 1005 of. the PR0219
polypeptide shown in Figure 8 (SEQ ID NO:15), wherein X is any amino acid
residue from 19 to 28 of Figure 8
(SEQ ID NO: I5), or (c) a nucleic acid sequence which encodes another
specifically derived fragment ofthe amino
acid sequence shown in Figure 8 (SEQ ID NO:15). PR0219 polynucleotide variants
do not encompass the native
PR0219 nucleotide sequence.
"PR0221 variant polynucleotide" or "PR0221 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0221 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 34 to 259 of the
PR0221 polypeptide shown in Figure 10 (SEQ ID N0:20), (b) a nucleic acid
sequence which encodes amino acids
X to 259 of the PR0221 polypeptide shown in Figure 10 (SEQ ID N0:20), wherein
X is any amino acid residue
from 29 to 38 of Figure 10 (SEQ ID N0:20), (c) 1 or about 34 to X of Figure 10
(SEQ ID N0:20), wherein X is
any amino acid from amino acid 199 to amino acid 208 of Figure 10 (SEQ ID
N0:20), or (d) a nucleic acid
sequence which encodes another specifically derived fragment of the amino acid
sequence shown in Figure 10
(SEQ ID N0:20). Ordinarily, a PR0221 variant polynucleotide will have at least
about 80% nucleic acid sequence
identity, more preferably at least about 81 % nucleic acid sequence identity,
more preferably at least about 82%
nucleic acid sequence identity, more preferably at least about 83% nucleic
acid sequence identity, more preferably
at least about 84% nucleic acid sequence identity, more preferably at least
about 85% nucleic acid sequence identity,
more preferably at least about 86% nucleic acid sequence identity, more
preferably at least about 87% nucleic acid
sequence identity, more preferably at least about 88% nucleic acid sequence
identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about 90% nucleic
acid sequence identity, more
preferably at least about 91% nucleic acid sequence identity, more preferably
at least about 92% nucleic acid
sequence identity, more preferably at least about 93% nucleic acid sequence
identity, more preferably at least about
94% nucleic acid sequence identity, more preferably at least about 95% nucleic
acid sequence identity, more
preferably at least about 96% nucleic acid sequence identity, more preferably
at least about 97% nucleic acid
47

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sequence identity, more preferably at least about 98% nucleic acid sequence
identity and yet more preferably at least
about 99% nucleic acid sequence identity with either (a) a nucleic acid
sequence which encodes residues 1 or about
34 to 259 of the PR0221 polypeptide shown in Figure 10 (SEQ ID N0:20), (b) a
nucleic acid sequence which
encodes amino acids X to 259 of the PR0221 polypeptide shown in Figure 10 (SEQ
ID N0:20), wherein X is any
S amino acid residue from 29 to 38 of Figure 10 (SEQ 1D N0:20), (c) 1 or about
34 to X of Figure 10 (SEQ ID
N0:20), wherein X is any amino acid from amino acid 199 to amino acid 208 of
Figure 10 (SEQ ID N0:20), or
(d) a nucleic acid sequence which encodes another specifically derived
fragment of the amino acid sequence shown
in Figure 10 (SEQ ID N0:20). PR0221 polynucleotide variants do not encompass
the native PR0221 nucleotide
sequence.
"PR0224 variant polynucleotide" or "PR0224 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0224 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 31 to 282 of the
PR0224 polypeptide shown in Figure 12 (SEQ ID N0:25), (b) a nucleic acid
sequence which encodes amino acids
X to 282 of the PR0224 polypeptide shown in Figure 12 (SEQ ID N0:25), wherein
X is any amino acid residue
1S from 26 to 35 of Figure 12 (SEQ ID N0:25), (c) 1 or about 31 to X of Figure
I2 (SEQ ID N0:25), wherein X is
any amino acid from amino acid 226 to amino acid 235 of Figure 12 (SEQ ID
N0:25), or (d) a nucleic acid
sequence which encodes another specifically derived fragment of the amino acid
sequence shown in Figure 12
(SEQ ID N0:25). Ordinarily, a PR0224 variant polynucleotide will have at least
about 80% nucleic acid sequence
identity, more preferably at least about 8l% nucleic acid sequence identity,
more preferably at least about 82%
nucleic acid sequence identity, more preferably at least about 83% nucleic
acid sequence identity, more preferably
at least about 84% nucleic acid sequence identity, more preferably at least
about 85% nucleic acid sequence identity,
more preferably at least about 86% nucleic acid sequence identity, more
preferably at least about 87% nucleic acid
sequence identity, more preferably at least about 88% nucleic acid sequence
identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about 90% nucleic
acid sequence identity, more
2S preferably at least about 91% nucleic acid sequence identity, more
preferably at least about 92% nucleic acid
sequence identity, more preferably at least about 93% nucleic acid sequence
identity, more preferably at least about
94% nucleic acid sequence identity, more preferably at least about 95% nucleic
acid sequence identity, more
preferably at least about 96% nucleic acid sequence identity, more preferably
at least about 97% nucleic acid
sequence identity, more preferably at least about 98% nucleic acid sequence
identity and yet more preferably at least
about 99% nucleic acid sequence identity with either (a) a nucleic acid
sequence which encodes residues 1 or about
31 to 282 of the PR0224 polypeptide shown in Figure 12 (SEQ 1D N0:25), (b) a
nucleic acid sequence which
encodes amino acids X to 282 of the PR0224 polypeptide shown in Figure 12 (SEQ
1D N0:25), wherein X is any
amino acid residue from 26 to 35 of Figure 12 (SEQ ID N0:25}, (c) 1 or about
31 to X of Figure 12 (SEQ ID
N0:25), wherein X is any amino acid from amino acid 226 to amino acid 235 of
Figure 12 (SEQ ID N0:25), or
(d) a nucleic acid sequence which encodes another specifically derived
fragment ofthe amino acid sequence shown
in Figure 12 (SEQ ID N0:25). PR0224 polynucleotide variants do not encompass
the native PR0224 nucleotide
sequence.
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"PR0328 variant polynucleotide" or "PR0328 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0328 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 23 to 463 of the
PR0328 polypeptide shown in Figure 14 (SEQ ID N0:30), (b) a nucleic acid
sequence which encodes amino acids
S X to 463 of the PR0328 polypeptide shown in Figure 14 (SEQ ID N0:30),
wherein X is any amino acid residue
from 18 to 27 of Figure 14 (SEQ ID N0:30), or (c) a nucleic acid sequence
which encodes another specifically
derived fragment of the amino acid sequence shown in Figure 14 (SEQ ID N0:30).
Ordinarily, a PR0328 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 23 to 463
of the PR0328 polypeptide shown
in Figure 14 (SEQ ID N0:30), (b) a nucleic acid sequence which encodes amino
acids X to 463 of the PR0328
polypeptide shown in Figure 14 (SEQ ID N0:30), wherein X is any amino acid
residue from 18 to 27 of Figure
14 (SEQ ID N0:30), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the
amino acid sequence shown in Figure 14 (SEQ ID N0:30). PR0328 polynucleotide
variants do not encompass
the native PR0328 nucleotide sequence.
"PR0301 variant polynucleotide" or "PR0301 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0301 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 28 to 299 of the
PR0301 polypeptide shown in Figure 16 (SEQ ID N0:35), (b) a nucleic acid
sequence which encodes amino acids
X to 299 of the PR0301 polypeptide shown in Figure 16 (SEQ ID N0:35), wherein
X is any amino acid residue
from 23 to 32 of Figure 16 (SEQ ID N0:35), (c) I or about 28 to X of Figure 16
(SEQ ID N0:35), wherein X is
any amino acid from amino acid 230 to amino acid 239 of Figure 16 (SEQ ID
N0:35), or (d) a nucleic acid
sequence which encodes another specifically derived fragment of the amino acid
sequence shown in Figure 16
(SEQ ID N0:35). Ordinarily, a PR0301 variant polynucleotide will have at least
about 80% nucleic acid sequence
identity, more preferably at least about 81% nucleic acid sequence identity,
more preferably at least about 82%
nucleic acid sequence identity, more preferably at least about 83% nucleic
acid sequence identity, more preferably
at least about 84% nucleic acid sequence identity, more preferably at least
about 85% nucleic acid sequence identity,
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more preferably at least about 86% nucleic acid sequence identity, more
preferably at least about 87% nucleic acid
sequence identity, more preferably at least about 88% nucleic acid sequence
identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about 90% nucleic
acid sequence identity, more
preferably at least about 91% nucleic acid sequence identity, more preferably
at least about 92% nucleic acid
sequence identity, more preferably at least about 93% nucleic acid sequence
identity, more preferably at least about
94% nucleic acid sequence identity, more preferably at least about 95% nucleic
acid sequence identity, more
preferably at least about 96% nucleic acid sequence identity, more preferably
at least about 97% nucleic acid
sequence identity, more preferably at least about 98% nucleic acid sequence
identity and yet more preferably at least
about 99% nucleic acid sequence identity with either (a) a nucleic acid
sequence which encodes residues 1 or about
28 to 299 of the PR0301 polypeptide shown in Figure 16 (SEQ ID N0:35), (b) a
nucleic acid sequence which
encodes amino acids X to 299 of the PR0301 polypeptide shown in Figure 16 (SEQ
ID N0:35), wherein X is any
amino acid residue from 23 to 32 of Figure 16 (SEQ ID N0:35), (c) 1 or about
28 to X of Figure 16 (SEQ ID
N0:35), wherein X is any amino acid from amino acid 230 to amino acid 239 of
Figure 16 (SEQ iD N0:35), or
(d) a nucleic acid sequence which encodes another specifically derived
fragment ofthe amino acid sequence shown
in Figure 16 (SEQ ID N0:35). PR0301 polynucleotide variants do not encompass
the native PR0301 nucleotide
sequence.
"PR0526 variant polynucleotide" or "PR0526 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0526 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues I or about 27 to 473 of the
PR0526 polypeptide shown in Figure 18 (SEQ ID N0:43), (b) a nucleic acid
sequence which encodes amino acids
X to 473 of the PR0526 polypeptide shown in Figure 18 (SEQ ID N0:43), wherein
X is any amino acid residue
from 22 to 3 I of Figure 18 (SEQ ID N0:43), or (c) a nucleic acid sequence
which encodes another specifically
derived fragment of the amino acid sequence shown in Figure 18 (SEQ ID N0:43).
Ordinarily, a PR0526 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 8l%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 27 to 473
of the PR0526 polypeptide shown
in Figure 18 (SEQ ID N0:43), (b) a nucleic acid sequence which encodes amino
acids X to 473 of the PR0526

CA 02353799 2001-06-08
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polypeptide shown in Figure 18 (SEQ ID N0:43), wherein X is any amino acid
residue from 22 to 31 of Figure
18 (SEQ ID N0:43), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the
amino acid sequence shown in Figure 18 (SEQ ID N0:43). PR0526 polynucleotide
variants do not encompass
the native PR0526 nucleotide sequence.
"PR0362 variant polynucleotide" or "PR0362 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0362 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 20 to 321 of the
PR0362 polypeptide shown in Figure 20 (SEQ ID N0:48), (b) a nucleic acid
sequence which encodes amino acids
X to 321 of the PR0362 polypeptide shown in Figure 20 (SEQ ID N0:48), wherein
X is any amino acid residue
from 1 S to 24 of Figure 20 (SEQ ID N0:48), (c) 1 or about 20 to X of Figure
20 (SEQ lD N0:48), wherein X is
any amino acid from amino acid 276 to amino acid 285 of Figure 20 (SEQ ID
N0:48), or (d) a nucleic acid
sequence which encodes another specifically derived fragment of the amino acid
sequence shown in Figure 20
(SEQ ID N0:48). Ordinarily, a PR0362 variant polynucieotide will have at least
about 80% nucleic acid sequence
identity, more preferably at least about 81 % nucleic acid sequence identity,
more preferably at least about 82%
nucleic acid sequence identity, more preferably at least about 83% nucleic
acid sequence identity, more preferably
at least about 84% nucleic acid sequence identity, more preferably at least
about 85% nucleic acid sequence identity,
more preferably at least about 86% nucleic acid sequence identity, more
preferably at least about 87% nucleic acid
sequence identity, more preferably at least about 88% nucleic acid sequence
identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about 90% nucleic
acid sequence identity, more
preferably at least about 91 % nucleic acid sequence identity, more preferably
at least about 92% nucleic acid
sequence identity, more preferably at least about 93% nucleic acid sequence
identity, more preferably at least about
94% nucleic acid sequence identity, more preferably at least about 95% nucleic
acid sequence identity, more
preferably at least about 96% nucleic acid sequence identity, more preferably
at least about 97% nucleic acid
sequence identity, more preferably at least about 98% nucleic acid sequence
identity and yet more preferably at least
about 99% nucleic acid sequence identity with either (a) a nucleic acid
sequence which encodes residues 1 or about
20 to 321 of the PR0362 polypeptide shown in Figure 20 (SEQ ID N0:48), (b) a
nucleic acid sequence which
encodes amino acids X to 321 of the PR0362 polypeptide shown in Figure 20 (SEQ
ID N0:48), wherein X is any
amino acid residue from I S to 24 of Figure 20 (SEQ ID N0:48), (c) 1 or about
20 to X of Figure 20 (SEQ ID
N0:48), wherein X is any amino acid from amino acid 276 to amino acid 285 of
Figure 20 (SEQ ID N0:48), or
(d) a nucleic acid sequence which encodes another specifically derived
fragment of the amino acid sequence shown
in Figure 20 (SEQ ID N0:48). PR0362 polynucleotide variants do not encompass
the native PR0362 nucleotide
sequence.
"PR0356 variant polynucleotide" or "PR0356 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0356 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 27 to 346 of the
PR0356 polypeptide shown in Figure 22 (SEQ ID NO:55), (b) a nucleic acid
sequence which encodes amino acids
X to 346 of the PR0356 polypeptide shown in Figure 22 (SEQ ID NO:55), wherein
X is any amino acid residue
51

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from 22 to 31 of Figure 22 (SEQ ID NO:55), or (c) a nucleic acid sequence
which encodes another specifically
derived fragment of the amino acid sequence shown in Figure 22 (SEQ ID NO:55).
Ordinarily, a PR0356 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 27 to 346
of the PR0356 polypeptide shown
in Figure 22 (SEQ ID NO:55), (b) a nucleic acid sequence which encodes amino
acids X to 346 of the PR0356
polypeptide shown in Figure 22 (SEQ ID NO:55), wherein X is any amino acid
residue from 22 to 31 of Figure
22 (SEQ ID NO:55), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the
amino acid sequence shown in Figure 22 (SEQ ID NO:55). PR0356 polynucleotide
variants do not encompass
the native PR0356 nucleotide sequence.
"PR0509 variant polynucleotide" or "PR0509 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0509 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 37 to 283 of the
PR0509 polypeptide shown in Figure 24 (SEQ ID N0:60), (b) a nucleic acid
sequence which encodes amino acids
X to 283 of the PR0509 polypeptide shown in Figure 24 (SEQ ID N0:60), wherein
X is any amino acid residue
from 32 to 41 of Figure 24 (SEQ ID N0:60), (c) 1 or about 37 to X of Figure 24
(SEQ ID N0:60), wherein X is
any amino acid from amino acid 200 to amino acid 209 of Figure 24 (SEQ ID
N0:60), or (d) a nucleic acid
sequence which encodes another specifically derived fragment of the amino acid
sequence shown in Figure 24
(SEQ ID N0:60). Ordinarily, a PR0509 variant polynucleotide will have at least
about 80% nucleic acid sequence
identity, more preferably at least about 81% nucleic acid sequence identity,
more preferably at least about 82%
nucleic acid sequence identity, more preferably at least about 83% nucleic
acid sequence identity, more preferably
at least about 84% nucleic acid sequence identity, more preferably at least
about 85% nucleic acid sequence identity,
more preferably at least about 86% nucleic acid sequence identity, more
preferably at least about 87% nucleic acid
sequence identity, more preferably at least about 88% nucleic acid sequence
identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about 90% nucleic
acid sequence identity, more
preferably at (east about 91% nucleic acid sequence identity, more preferably
at least about 92% nucleic acid
sequence identity, more preferably at least about 93% nucleic acid sequence
identity, more preferably at least about
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94% nucleic acid sequence identity, more preferably at least about 95% nucleic
acid sequence identity, more
preferably at least about 96% nucleic acid sequence identity, more preferably
at least about 97% nucleic acid
sequence identity, more preferably at least about 98% nucleic acid sequence
identity and yet more preferably at least
about 99% nucleic acid sequence identity with either (a) a nucleic acid
sequence which encodes residues 1 or about
37 to 283 of the PR0509 polypeptide shown in Figure 24 (SEQ ID N0:60), (b) a
nucleic acid sequence which
encodes amino acids X to 283 of the PR0509 polypeptide shown in Figure 24 (SEQ
ID N0:60), wherein X is any
amino acid residue from 32 to 41 of Figure 24 (SEQ 1D N0:60), (c) 1 or about
37 to X of Figure 24 (SEQ ID
N0:60), wherein X is any amino acid from amino acid 200 to amino acid 209 of
Figure 24 (SEQ ID N0:60), or
(d) a nucleic acid sequence which encodes another specifically derived
fragment ofthe amino acid sequence shown
in Figure 24 (SEQ ID N0:60). PR0509 polynucleotide variants do not encompass
the native PR0509 nucleotide
sequence.
"PR0866 variant polynucleotide" or "PR0866 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0866 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 27 to 331 of the
PR0866 polypeptide shown in Figure 26 (SEQ ID N0:62), (b) a nucleic acid
sequence which encodes amino acids
X to 331 of the PR0866 polypeptide shown in Figure 26 (SEQ ID N0:62), wherein
X is any amino acid residue
from 22 to 31 of Figure 26 (SEQ ID N0:62), or (c) a nucleic acid sequence
which encodes another specifically
derived fragment of the amino acid sequence shown in Figure 26 (SEQ ID N0:62).
Ordinarily, a PR0866 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 27 to 331
of the PR0866 polypeptide shown
in Figure 26 (SEQ ID N0:62), (b) a nucleic acid sequence which encodes amino
acids X to 331 of the PR0866
polypeptide shown in Figure 26 (SEQ ID N0:62), wherein X is any amino acid
residue from 22 to 31 of Figure
26 (SEQ ID N0:62), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the
amino acid sequence shown in Figure 26 (SEQ ID N0:62). PR0866 polynucleotide
variants do not encompass
the native PR0866 nucleotide sequence.
Ordinarily, PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526,
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PR0362, PR0356, PR0509 and PR0866 variant polynucleotides are at least about
30 nucleotides in length, often
at least about 60 nucleotides in length, more often at least about 90
nucleotides in length, more often at least about
120 nucleotides in length, more often at least about 1 SO nucleotides in
length, more often at least about 180
nucleotides in length, more often at least about 210 nucleotides in length,
more often at least about 240 nucleotides
in length, more often at least about 270 nucleotides in length, more often at
least about 300 nucleotides in length,
more often at least about 450 nucleotides in length, more often at least about
600 nucleotides in length, more often
at least about 900 nucleotides in length, or more.
"Percent (%) nucleic acid sequence identity" with respect to the PRO 179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866
polypeptide-encoding
nucleic acid sequences identified herein is defined as the percentage of
nucleotides in a candidate sequence that are
identical with the nucleotides in a PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide-encoding nucleic acid
sequence, 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,
ALIGN-2 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, % nucleic acid
sequence identity values are obtained
as described below by using the sequence comparison computer program ALIGN-2,
wherein the complete source
code for the ALIGN-2 program is provided in Table 1. The ALIGN-2 sequence
comparison computer program
was authored by Genentech, Inc., and the source code shown in Table 1 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 Genentech,
Inc., South San Francisco,
California or may be compiled from the source code provided inTable 1. 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.
For purposes herein, 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
54

CA 02353799 2001-06-08
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nucleic acid sequence identitycalculations,Tables 2C-2D 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".
Unless specifically stated otherwise, all % nucleic acid sequence identity
values used herein are obtained
as described above using the ALIGN-2 sequence comparison computer program.
However, % 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)). The NCB/-BLAST2 sequence
comparison program may be
downloaded from http://www.ncbi.nlm.nih.gov. 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 NCB/-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 wilt not equal the % nucleic
acid sequence identity of D to C.
In addition, % nucleic acid sequence identity values may also be generated
using the WU-BLAST-2
computer program (Altschul et al., Methods in Enzvmolo~v, 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 = O.I25,
word threshold (T) = 11, and scoring
matrix = BLOSUM62. For purposes herein, 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.
In other embodiments, PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301,

CA 02353799 2001-06-08
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PR0526, PR0362, PR0356, PR0509 and PR0866 variant polynucleotides are nucleic
acid molecules that encode
an active PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 polypeptide, respectively, and which are capable of
hybridizing, preferably under
stringent hybridization and wash conditions, to nucleotide sequences encoding
the full-length PRO 179 polypeptide
shown in Figure 2 (SEQ ID N0:2), to nucleotide sequences encoding the full-
length PR0207 polypeptide shown
in Figure 4 (SEQ ID N0:7), to nucleotide sequences encoding the full-length
PR0320 polypeptide shown in Figure
6 (SEQ ID NO:10), to nucleotide sequences encoding the full-length PR0219
polypeptide shown in Figure 8 (SEQ
ID NO:15), to nucleotide sequences encoding the full-length PR0221 polypeptide
shown in Figure 10 (SEQ ID
N0:20), to nucleotide sequences encoding the full-length PR0224 polypeptide
shown in Figure 12 (SEQ ID
N0:25), to nucleotide sequences encoding the full-length PR0328 polypeptide
shown in Figure 14 (SEQ ID
N0:30), to nucleotide sequences encoding the full-length PR0301 polypeptide
shown in Figure 16 (SEQ ID
N0:35), to nucleotide sequences encoding the full-length PR0526 polypeptide
shown in Figure 18 (SEQ ID
N0:43), to nucleotide sequences encoding the full-length PR0362 polypeptide
shown in Figure 20 (SEQ ID
N0:48), to nucleotide sequences encoding the full-length PR0356 polypeptide
shown in Figure 22 (SEQ ID
NO:55), to nucleotide sequences encoding the full-length PR0509 polypeptide
shown in Figure 24 (SEQ ID
N0:60), to nucleotide sequences encoding the full-length PR0866 polypeptide
shown in Figure 26 (SEQ ID
N0:62), respectively. PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 and PR0866 variant polypeptides may be those that are
encoded by a PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity
comparisons performed as
described above, includes amino acid residues in the sequences compared that
are not only identical, but also those
that have similar properties. Amino acid residues that score a positive value
to an amino acid residue of interest
are those that are either identical to the amino acid residue of interest or
are a preferred substitution (as defined in
Table 3 below) of the amino acid residue of interest.
For purposes herein, the % value of positives 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 % positives 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 scoring a positive value as
defined above 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 % positives of A to B will not equal the % positives of B to
A.
"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. Preferably, the
56

CA 02353799 2001-06-08
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isolated polypeptide is free of association with all components with which it
is naturally associated. Contaminant
components of its natural environment are materials that would typically
interfere with diagnostic or therapeutic
uses for the polypeptide, and may include enrymes, hormones, and other
proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the polypeptide will be purified ( I ) to a
degree sufficient to obtain at least 1 S
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 component of the
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR03S6,
PR0509 or PR0866 natural environment will not be present. Ordinarily, however,
isolated polypeptide will be
prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a PR0179, PR0207, PR0320,PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PROS09 or PR0866 polypeptide or an
"isolated" nucleic acid
molecule encoding an anti-PR0179, anti-PR0207, anti-PR0320, anti-PR0219, anti-
PR0221, anti-PR0224, anti-
PR0328, anti-PR0301, anti-PR0526,anti-PR0362, anti-PR0356, anti-PR0509 or anti-
PR0866 antibody 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 PR0179-, PR0207-
, PR0320-, PR0219-, PR0221-,
PR0224-, PR0328-, PR0301-, PR0526-, PR0362-, PR0356-, PR0509- or PR0866-
encoding nucleic acid or
the anti-PRO 179-,anti-PR0207-,anti-PR0320-, anti-PR0219-, anti-PR0221-, anti-
PRO224-, anti-PR0328-, anti-
PR0301-, anti-PR0526-,anti-PR0362-, anti-PR0356-, anti-PR0509- or anti-PR0866-
encoding nucleic acid.
Preferably, the isolated nucleic acid is free of association with all
components with which it is naturally associated.
An isolated PR0179-, PR0207-, PR0320-, PR0219-, PR0221-, PR0224-, PR0328-,
PR0301-, PR0526-,
PR0362-, PR0356-, PR0509- or PR0866-encoding nucleic acid molecule or an
isolated anti-PR0179-, anti-
PR0207-, anti-PR0320-, anti-PR0219-, anti-PR0221-, anti-PR0224-, anti-PR0328-,
anti-PR0301-, anti-
PR0526-,anti- PR0362-, anti-PR0356-, anti-PR0509- or anti-PR0866-encoding
nucleic acid molecule is other
than in the form or setting in which it is found in nature. Isolated nucleic
acid molecules therefore are distinguished
from the PR0179-, PR0207-, PR0320-, PR0219-, PR0221-, PR0224-, PR0328-,PR0301-
,PR0526-,PR0362-,
PR0356-, PR0509- or PR0866-encoding nucleic acid molecule or from the anti-
PR0179-, anti-PR0207-, anti-
PR0320-, anti-PR0219-, anti-PR0221-, anti-PR0224-, anti-PR0328-, anti-PR0301-,
anti-PR0526-,anti-
PR0362-, anti-PR0356-, anti-PR0509-oranti-PR0866-encoding nucleic acid
molecule as it exists in natural cells.
However, an isolated nucleic acid molecule encoding a PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR03S6, PR0509 or PR0866 polypeptide or an
isolated nucleic acid
molecule encoding an anti-PR0179, anti-PR0207, anti-PR0320, anti-PR0219, anti-
PR0221, anti-PR0224, anti-
PR0328, anti-PR0301,anti-PR0526,anti-PR0362, anti-PR0356, anti-PR0509 or anti-
PR0866 antibody includes
PR0179-, PR0207-, PR0320-, PR0219-, PR0221-, PR0224-, PR0328-, PR0301-, PR0526-
, PR0362-,
PR0356-, PR0509- or PR0866-nucleic acid molecules or anti-PR0179-, anti-PR0207-
, anti-PR0320-, anti-
PR0219-, anti-PR0221-, anti-PR0224-, anti-PR0328-, anti-PR0301-, anti-PR0526-
,anti- PR0362-, anti-
PR0356-, anti-PR0509-oranti-PR0866-nucleicacid molecules contained in cells
that ordinarily express PRO 179,
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CA 02353799 2001-06-08
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PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptides or anti-PR0179, anti-PR0207, anti-PR0320, anti-PR0219,
anti-PR0221, anti-PR0224,
anti-PR0328, anti-PR0301, anti-PR0526,anti- PR0362, anti-PR0356, anti-PR0509
or anti-PR0866 antibodies
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 known 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
l5 in reading phase. However, enhancers do not have to be contiguous. Linking
is accomplished by ligation at
convenient restriction sites. If such sites do not exist, the synthetic
oligonucleotide adaptors or linkers are used in
accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-
PRO 179, anti-PR0207, anti-PR0320, anti-PR0219, anti-PR0221,anti-PR0224,anti-
PR0328,anti-PR0301,anti-
PR0526,anti- PR0362, anti-PR0356, anti-PR0509 and anti-PR0866 monoclonal
antibodies (including agonist
antibodies), anti-PR0179, anti-PR0207, anti-PR0320, anti-PR0219, anti-PR0221,
anti-PR0224, anti-PR0328,
anti-PR0301, anti-PR0526,anti-PR0362, anti-PR0356, anti-PR0509 and anti-PR0866
antibody compositions
with polyepitopic specificity, single chain anti-PR0179, anti-PR0207, anti-
PR0320, anti-PR0219, anti-PR0221,
anti-PR0224, anti-PR0328, anti-PR0301, anti-PR0526,anti-PR0362, anti-PR0356,
anti-PR0509 and anti-
PR0866 antibodies, and fragments ofanti-PRO 179,anti-PR0207, anti-PR0320, anti-
PR0219, anti-PR0221, anti-
PR0224, anti-PR0328, anti-PR0301, anti-PR0526,anti-PR0362, anti-PR0356, anti-
PR0509 and anti-PR0866
antibodies (see below). The term "monoclonal antibody" as used herein refers
to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are
identical except for possible naturally-occurring mutations that may be
present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when 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
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CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
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/50mM 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), SO mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution,
sonicated salmon sperm DNA (50
pg/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 50% 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 ( 150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 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 PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 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
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 refers to forms) of PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 which
retain a biological
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CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
and/or an immunological activity ofnative or naturally-occurring PRO 179,
PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866, wherein
"biological" activity
refers to a biological function (either inhibitory or stimulatory) caused by a
native or naturally-occurring PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 other than the ability to induce the production of an antibody against
an antigenic epitope possessed by
a native or naturally-occurring PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866.
"Biological activity" in the context of an antibody or another agonist that
can be identified by the screening
assays disclosed herein (e.g., an organic or inorganic small molecule,
peptide, etc.) is used to refer to the ability of
such molecules to invoke one or more of the effects listed herein in
connection with the definition of a
"therapeutically effective amount." In a specific embodiment, "biological
activity" is the ability to inhibitneoplastic
cell growth or proliferation. A preferred biological activity is inhibition,
including slowing or complete stopping,
of the growth of a target tumor (e.g., cancer) cell. Another preferred
biological activity is cytotoxic activity
resulting in the death ofthe target tumor (e.g., cancer) cell. Yet another
preferred biological activity is the induction
of apoptosis of a target tumor (e.g., cancer) cell.
The phrase "immunological activity" means immunological cross-reactivity with
at least one epitope of
a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate
polypeptide is capable of
competitively inhibiting the qualitative biological activity of a PR0179,
PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
having this activity
with polyclonal antisera raised against the known active PRO 179, PR0207,
PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide. Such
antisera are prepared in
conventional fashion by injecting goats or rabbits, for example,
subcutaneously with the known active analogue
in complete Freund's adjuvant, followed by booster intraperitoneal or
subcutaneousinjectionin incomplete Freunds.
The immunological cross-reactivity preferably is "specific", which means that
the binding affinity of the
immunologically cross-reactive molecule (e.g., antibody) identified, to the
corresponding PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide is significantly higher (preferably at least about 2-times, more
preferably at least about 4-times, even
more preferably at least about 6-times, most preferably at least about 8-times
higher) than the binding affnity of
that molecule to any other known native potypeptide.
"Tumor", as used herein, refers to atl neoplastic cell growth and
proliferation, whether malignant or
benign, and all pre-cancerous and cancerous cells and tissues.
The terms "cancer" and "cancerous" refer to or describe the physiological
condition in mammals that is

CA 02353799 2001-06-08
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typically characterized by unregulated cell growth. Examples of cancer include
but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such
cancers include breast cancer,
prostate cancer, colon cancer, squamous cell cancer, small-cell lung cancer,
non-small cell lung cancer, ovarian
cancer, cervical cancer, gastrointestinal cancer, pancreatic cancer,
glioblastoma, liver cancer, bladder cancer,
hepatoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma,
kidney cancer, vulval cancer,
thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
"Treatment" is an intervention performed with the intention of preventing the
development or altering the
pathology of a disorder. Accordingly, "treatment" refers to both therapeutic
treatment and prophylactic or
preventative measures. Those in need of treatment include those already with
the disorder as well as those in which
the disorder is to be prevented. In tumor (e.g., cancer) treatment, a
therapeutic agent may directly decrease the
pathology of tumor cells, or render the tumor cells more susceptible to
treatment by other therapeutic agents, e.g.,
radiation and/or chemotherapy.
The "pathology" of cancer includes all phenomena that compromise the well-
being of the patient. This
'includes, without limitation, abnormal or uncontrollable cell growth,
metastasis, interference with the normal
functioning of neighboring cells, release of cytokines or other secretory
products at abnormal levels, suppression
or aggravation of inflammatory or immunological response, etc.
An "effective amount" of a polypeptide disclosed herein or an agonist thereof,
in reference to inhibition
of neoplastic cell growth, is an amount capable of inhibiting, to some extent,
the growth of target cells. The term
includes an amount capable of invoking a growth inhibitory, cytostatic and/or
cytotoxic effect and/or apoptosis of
the target cells. An "effective amount" of a PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide or an agonist
thereof for purposes of
inhibiting neoplastic cell growth may be determined empirically and in a
routine manner.
A "therapeutically effective amount", in reference to the treatment of tumor,
refers to an amount capable
of invoking one or more of the following effects: (1) inhibition, to some
extent, of tumor growth, including,
slowing down and complete growth arrest; (2) reduction in the number of tumor
cells; (3) reduction in tumor size;
(4) inhibition (i.e., reduction, slowing down or complete stopping) of tumor
cell infiltration into peripheral organs;
(5) inhibition (i.e., reduction, slowing down or complete stopping) of
metastasis; (6) enhancement of anti-tumor
immune response, which may, but does not have to, result in the regression or
rejection of the tumor; and/or (7)
relief, to some extent, of one or more symptoms associated with the disorder.
A "therapeutically effective amount"
of a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide or an agonist thereof for purposes of treatment
of tumor may be determined
empirically and in a routine manner.
A "growth inhibitory amount" of a PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide or an agonist
thereof is an amount capable
of inhibiting the growth of a cell, especially tumor, e.g., cancer cell,
either in vitro or in vivo. A "growth inhibitory
amount" of a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 polypeptide or an agonist thereof for purposes of
inhibiting neoplastic cell growth
61

CA 02353799 2001-06-08
WO 00137638 PCT/US99/28565
may be determined empirically and in a routine manner.
A "cytotoxic amount" ofa PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide or an agonist thereof is
an amount capable of
causing the destruction of a cell, especial ly tumor, e.g., cancer cell,
either in vitro or in vivo. A "cytotoxic amount"
ofa PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide or an agonist thereof for purposes of inhibiting
neoplastic cell growth may be
determined empirically and in a routine manner.
The term "cytotoxic agent" as used herein refers to a substance that inhibits
or prevents the function of
cells and/or causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., I"', I''-5, y9o and
Re'8°), chemotherapeutic agents, and toxins such as enzymatically
active toxins of bacterial, fungal, plant or animal
origin, or fragments thereof.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of
tumor, e.g., cancer.
Examples of chemotherapeutic agents include adriamycin, doxorubicin,
epirubicin, 5-fluorouracil, cytosine
arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids,
e.g., paclitaxel (Taxol, Bristol-
MyersSquibbOncology, Princeton, NJ), and doxetaxel (Taxotere, Rhone-Poulenc
Rorer, Antony, Rnace),toxotere,
methotrexate, cisplatin, melphalan, vinblastine, bleomycin, etoposide,
ifosfamide, mitomycin C, mitoxantrone,
vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin,
aminopterin, dactinomycin,
mitomycins, esperamicins (see, U.S. Patent No. 4,675,187), melphalan and other
related nitrogen mustards. Also
included in this definition are hormonal agents that act to regulate or
inhibit hormone action on tumors such as
tamoxifen and onapristone.
A "growth inhibitory agent" when used herein refers to a compound or
composition which inhibits growth
of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo.
Thus, the growth inhibitory agent is one which
significantly reduces the percentage of the target cells in S phase. Examples
of growth inhibitory agents include
agents that block cell cycle progression (at a place other than S phase), such
as agents that induce G I arrest and M-
phase arrest. Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxol, and topo I1
inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and
bleomycin. Those agents that arrest GI
also spill over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in The
Molecular Basis of Cancer. Mendelsohn and Israel, eds., Chapter I , entitled
"Cell cycle regulation, oncogens, and
antineoplastic drugs" by Murakami et al., (WB Saunders: Philadelphia, 1995),
especially p. 13.
The term "cytokine" is a generic term for proteins released by one cell
population which act on another
cell as intercellular mediators. Examples of such cytokines are lymphokines,
monokines, and traditional
polypeptide hormones. Included among the cytokines are growth hormone such as
human growth hormone, N-
methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin;
proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle
stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth
factor; fibroblast growth factor;
prolactin; placental lactogen; tumor necrosis factor-a and -a; mullerian-
inhibiting substance; mouse gonadotropin-
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WO 00137638 PCT/US99/28565
associated peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-Vii; platelet-growth factor; transforming growth
factors (TGFs) such as TGF-a and
TGF-Vii; insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as
interferon-a, -Vii, and -y; colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF); granulocyte-
macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such
as IL-I, IL-1 a, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such
as TNF-a or TNF-Vii; and other
polypeptide factors including LIF and kit ligand (KL). As used herein, the
term cytokine includes proteins from
natural sources or from recombinant cell culture and biologically active
equivalents of the native sequence
cytokines.
The term "prodrug" as used in this application refers to a precursor or
derivative form of a
pharmaceutically active substance that is less cytotoxic to tumor cells
compared to the parent drug and is capable
of being enzymatically activated or converted into the more active parent
form. See, e.g., Wilman, "Prodrugs in
Cancer Chemotherapy", Biochemical Society Transactions, 14, pp. 375-382, 615th
Meeting Belfast (1986) and
Stella et aL, "Prodrugs: A Chemical Approach to Targeted Drug Delivery,"
Directed Drug Delivery. Borchardt et
al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of this invention
include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs, glycosylated
prodrugs or optionally
substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-
fluorouridine prodrugs which can
be derivatized into a prodrug form for use in this invention include, but are
not limited to, those chemotherapeutic
agents described above.
The term "agonist" is used in the broadest sense and includes any molecule
that mimics a biological
activity of a native PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide disclosed herein. Suitable
agonist molecules specifically
include agonist antibodies or antibody fragments, fragments or amino acid
sequence variants of native PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptides, peptides, small organic molecules, etc. Methods for
identifying agonists of a PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptide may comprise contacting a tumor cell with a candidate
agonist molecule and measuring the
inhibition of tumor cell growth.
"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
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nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the
physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically acceptable
carriers include buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid;
low molecular weight (less than about l0 residues) polypeptides; proteins,
such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose,
mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitoi or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as TWEENT"',
polyethylene glycol (PEG), and
PLURONICST"'
"Native antibodies" and "native immunoglobulins" are usually heterotetrameric
glycoproteins of about
150,000 daltons, composed of two identical light (L) chains and two identical
heavy (H) chains. Each light chain
is linked to a heavy chain by one covalent disulfide bond, while the number of
disulfide linkages varies among the
heavy chains of different immunoglobulin isotypes. Each heavy and light chain
also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable domain (V")
followed by a number of constant
domains. Each light chain has a variable domain at one end (V~) and a constant
domain at its other end; the
constant domain of the light chain is aligned with the first constant domain
of the heavy chain, and the light-chain
variable domain is aligned with the variable domain ofthe heavy chain.
Particular amino acid residues are believed
to form an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable
domains differ extensively in
sequence among antibodies and are used in the binding and specificity of each
particular antibody for its particular
antigen. However, the variability is not evenly distributed throughout the
variable domains of antibodies. It is
concentrated in three segments called complementarity-determining regions
(CDRs) or hypervariable regions both
in the light-chain and the heavy-chain variable domains. The more highly
conserved portions of variable domains
are called the framework regions (FR). The variable domains of native heavy
and light chains each comprise four
FR regions, largely adopting a (3-sheet configuration, connected by three
CDRs, which form loops connecting, and
in some cases forming part of, the (i-sheet structure. The CDRs in each chain
are held together in close proximity
by the FR regions and, with the CDRs from the other chain, contribute to the
formation of the antigen-binding site
of antibodies (see, Kabat et al., NIH Publ. No.91-3242, Vol. I, pages 647-669
( 1991 )). The constant domains are
not involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as
participation of the antibody in antibody-dependent cellular toxicity.
The term "hypervariable region" when used herein refers to the amino acid
residues of an antibody which
are responsible for antigen-binding. The hypervariable region comprises amino
acid residues from a
"complementarity determining region" or "CDR" (i.e., residues 24-34 (L 1 ), 50-
56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy
chain variable domain; Kabat et
al., Se4uences of Proteins of ImmunoloQical Interest. 5th Ed. Public Health
Service, National Institute of Health,
Bethesda, MD. [ 1991 ]) and/or those residues from a "hypervariable loop" (i.
e., residues 26-32 (L 1 ), SO-52 (L2) and
91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the heavy chain
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variable domain; Clothia and Lesk, J. Mol. Biol., 196:901-917 [ 1987]).
"Framework" or "FR" residues are those
variable domain residues other than the hypervariable region residues as
herein defined.
"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'),, and Fv fragments;
diabodies; linear antibodies (Zapata et al., Protein Ene., 8~: 1057-1062
[1995]); single-chain antibody
molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion ofantibodies 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'), 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 ofone 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 V"-V~ 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 (CH 1 )
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 CH 1 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')2 antibody fragments originally were produced as pairs of Fab' fragments
which have hinge cysteines between
them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one
oftwo clearly distinct types, called kappa and lambda, based on the amino acid
sequences oftheir 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
lgM, and several of these may be further divided into subclasses (isotypes),
e.g., IgG 1, IgG2, IgG3, lgG4, IgA, and
IgA2.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of
substantially homogeneous antibodies, i. e., the individual antibodies
comprising the population are identical except
for possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site. Furthermore, in
contrast to conventional (polyclonal)
antibodypreparationswhich typically include different antibodies directed
against different determinants (epitopes),
each monoclonal antibody is directed against a single determinant on the
antigen. In addition to their specificity,
the monoclonal antibodies are advantageous in that they are synthesized by the
hybridoma culture, uncontaminated
by other immunoglobulins. The modifier "monoclonal" indicates the character of
the antibody as being obtained
from a substantially homogeneous population of antibodies, and is not to be
construed as requiring production of

CA 02353799 2001-06-08
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the antibody by any particular method. For example, the monoclonal antibodies
to be used in accordance with the
present invention may be made by the hybridoma method first described by
Kohler et aL, Nature, 256:495 [ 1975],
or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature,
352:624-628 [1991 ] and Marks et al., J. Mol. Biol., 222:581-S97 (1991 ), for
example.
The monoclonal antibodies herein specifically include "chimeric" antibodies
(immunoglobulins) in which
a portion of the heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody class
or subclass, while the remainder of the
chains) is identical with or homologous to corresponding sequences in
antibodies derived from another species
or belonging to another antibody class or subclass, as well as fragments of
such antibodies, so long as they exhibit
the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-
6855 [ 1984]).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or
other antigen-binding subsequences
of antibodies) which contain minimal sequence derived from non-human
immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody) in which
residues from a 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
FR residues of the human
immunoglobulin are replaced by corresponding non-human residues. Furthermore,
humanized antibodies may
comprise residues which are found neither in the recipient antibody nor in the
imported CDR or framework
sequences. These modifications are made to further refine and maximize
antibody performance. 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
sequence. The humanized antibody
optimally also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human
immunoglobulin. For further details, see, Jones et al., Nature, 321:522-S2S
(1986); Reichmann et al., Nature,
332:323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The
humanized antibody includes a
PRIMATIZEDTMantibodywhereinthe antigen-binding region ofthe antibody is
derived from an antibody produced
by immunizing macaque monkeys with the antigen of interest.
"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 Pharmacoloey of Monoclonal
Antibodies Vol. 113, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-31 S (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
polypeptide chain (VH - V~). By using a linker that is too short to allow
pairing between the two domains on the
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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
S 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.
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. The
label may also be a non-detectable
entity such as a toxin.
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 PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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.
II. Compositions and Methods of the Invention
A. Full-leneth PR0179, PR0207, PR0320, PR0219. PR0221. PR0224. PR0328, PR0301,
PR0526.
PR0362. PR0356, PR0509 and PR0866 Polvneptides
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866. In particular, cDNAs
encoding PR0179, PR0207,
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PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
and PR0866
polypeptides have been identified and isolated, as disclosed in further detail
in the Examples below.
As disclosed in the Examples below, cDNA clones encoding PR0179, PR0207,
PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, and PR0866
polypeptides have been
deposited with the ATCC [with the exception of clone PR0509 which was not
deposited with ATCC]. The actual
nucleotide sequences of the clones can readily be determined by the skilled
artisan by sequencing of the deposited
clones using routine methods in the art. The predicted amino acid sequences
can be determined from the nucleotide
sequences using routine skill. For the PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866 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. PR0179 PR0207 PR0320 PR0219 PR0221 PR0224 PR0328 PR0301 PR0526 PR0362
PR0356, PR0509 and PR0866 Variants
tn addition to the full-length native sequence PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866 polypeptides
described herein, it is
contemplated that PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 and PR0866 variants can be prepared. PR0179, PR0207,
PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866
variants can be
prepared by introducing appropriate nucleotide changes into the PR0179,
PR0207, PR0320, PR02I9, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 DNA, and/or
by synthesis of the
desiredPR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide. Those skilled in the art will appreciate that
amino acid changes may alter post-
translational processes of the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide, such as changing the
number or position of
glycosylation sites or altering the membrane anchoring characteristics.
Variations in the native full-length sequence PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 or in various domains
of the PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 described herein, can be made, for example, using any of the techniques
and guidelines for conservative
and non-conservative mutations set forth, for instance, in U.S. Patent No.
5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding the PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 that
results in a change
in the amino acid sequence ofthe PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 as compared with the native sequence
PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866.
Optionally the variation is by substitution of at least one amino acid with
any other amino acid in one or more of
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the domains of the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866. Guidance in determining which amino acid
residue may be inserted,
substituted or deleted without adversely affecting the desired activity may be
found by comparing the sequence of
the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 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 5 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.
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 and PR0866 polypeptide 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 biological activity of the
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR030I, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide.
PR0179, PR0207, PR0320, PR0219, PR022I, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 and PR0866 fragments may be prepared by any of a number of
conventional techniques.
Desired peptide fragments may be chemically synthesized. An alternative
approach involves generating PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 and
PR0866 fragments by enzymatic digestion, e.g., by treating the protein with an
enzyme known to cleave proteins
at sites defined by particular amino acid residues, or by digesting the DNA
with suitable restriction enzymes and
isolating the desired fragment. Yet another suitable technique involves
isolating and amplifying a DNA fragment
encoding a desired polypeptide fragment, by polymerise chain reaction (PCR).
Oligonucleotides that define the
desired termini of the DNA fragment are employed at the 5' and 3' primers in
the PCR. Preferably, PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 and
PR0866 polypeptide fragments share at least one biological andlor
immunological activity with the native PRO 179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptides shown in Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7),
Figure 6 (SEQ ID NO:10),
Figure 8 (SEQ ID NO:15), Figure 10 (SEQ ID N0:20), Figure 12 (SEQ ID N0:25),
Figure 14 (SEQ ID N0:30),
Figure 16 (SEQ ID N0:35), Figure 18 (SEQ ID N0:43), Figure 20 (SEQ ID N0:48),
Figure 22 (SEQ ID NO:55),
Figure 24 (SEQ ID N0:60), or Figure 26 (SEQ ID N0:62), respectively.
In particular embodiments, conservative substitutions of interest are shown in
Table 3 under the heading
of preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial
changes, denominated exemplary substitutions in Table 3, or as further
described below in reference to amino acid
classes, are introduced and the products screened.
69

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Table 3
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys
Asn (N) 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
lle (1) leu; val; met; ala; phe;
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr ~r
Thr (T) ser ser
Trp (W) h'r~ phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
ala; norleucine leu
Substantial modifications in function or immunological identity of the PROI79,
PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 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:
(I) 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.

CA 02353799 2001-06-08
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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.. I0:6487 (1987)],
cassette mutagenesis [Wells et al.,
Gene. 34:315 ( 1985)), restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA, 317:41 S
( 1986)] or other known techniques can be performed on the cloned DNA to
produce the PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
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 [Cunningham and Wells, Science. 244: 1081-
1085 (1989)]. Alanine is also
typically preferred because it is the most common amino acid. Further, it is
frequently found in both buried and
exposed positions [Creighton, The 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 PR0179, PR0207, PR0320, PR0219, PR0221 PR0224 PR0328
PR0301
PROS26. PR0362, PR0356, PR0509 and PR0866
Covalent modifications of PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PROS26, PR0362, PR03S6, PROS09 and PR0866 are included within the scope of
this invention. One type of
covalent modification includes reacting targeted amino acid residues of a
PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PROS26, PR0362, PR0356, PROS09 or PR0866
polypeptide with an
organic derivatizing agent that is capable of reacting with selected side
chains or the N- or C- terminal residues of
the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR03S6,
PR0509 or PR0866. Derivatization with bifunctional agents is useful, for
instance, for crosslinking PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 to a water-insoluble support matrix or surface for use in the method
for purifying anti-PR0179, anti-
PR0207,anti-PR0320, anti-PR0219, anti-PR0221, anti-PR0224, anti-PR0328,anti-
PR0301,anti-PROS26,anti-
PR0362, anti-PR03S6, anti-PR0509 or anti-PR0866 antibodies, and vice-versa.
Commonly used crosslinking
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 aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
groups of Beryl or threonyl residues, methylation ofthe 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
71

CA 02353799 2001-06-08
WO 00137638 PCT/US99/28565
(1983)], acetylation of the N-terminal amine, and amidation of any C-terminal
carboxyl group.
Another type of covalent modification of the PROI79, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide included
within the scope ofthis
invention comprises altering the native glycosylation pattern ofthe
polypeptide. "Altering the native glycosylation
pattern" is intended for purposes herein to mean deleting one or more
carbohydrate moieties found in native
sequence PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 (either by removing. the underlying glycosylation
site or by deleting the
giycosylation by chemical and/or enzymatic means), and/or adding one or more
glycosylation sites that are not
present in the native sequence PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866. 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 ofglycosylation sites to the PRO 179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
1'R0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 (for O-linked
glycosylation sites). The
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 amino acid sequence may optionally be altered through changes
at the DNA level, particularly
by mutating the DNA encoding the PR0179, PR0207, PR0320, PR0219, PR022 l,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179,
PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 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 PROI?9, PR0207, PR0320,
PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
may be accomplished
chemically or enzymatically or by mutational substitution of codons encoding
for amino acid residues that serve
as targets for glycosylation. Chemical deglycosylation techniques are known in
the art and described, for instance,
by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et
al., Anal. Biochem., 118:131
( 1981 ). Enrymatic 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 al., Meth. Enzvmol.,
138:350 (1987).
Another type of covalent modification of PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 comprises linking the
PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
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CA 02353799 2001-06-08
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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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 or PR0866 polypeptide of the present invention may also be
modified in a way to form a
chimeric molecule comprising PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 fused to another, heterologous
polypeptide or amino acid
sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PR0179,
PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide
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 ofthe PR0179,
PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR030 I , PR0526, PR0362, PR0356, PR0509 or PR0866
polypeptide. The presence of such
epitope-tagged forms of the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide can be detected using an
antibody against the tag
polypeptide. Also, provision of the epitope tag enables the PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
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 9EI0 antibodies
thereto [Evan et al., Molecular and Cellular Bioloey 5:3610-3616 (1985)]; and
the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Eneineerine, x:547-553 ( 1990)). Other tag
polypeptides include the Flag-peptide [Hopp etal., BioTechnoloev, 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-I 5166 (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 PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide 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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide in place of at least one variable
region within an Ig molecule.
In a particularly preferred embodiment, the immunoglobulin fusion includes the
hinge, CH2 and CH3, or the hinge,
CH1, CH2 and CH3 regions of an IgGI molecule. For the production of
immunoglobulin fusions see also, US
Patent No. 5,428,130 issued June 27, 1995.
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CA 02353799 2001-06-08
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D. PreoarationofPR0179.PR0207 PR0320 PR0219 PR0221 PR0224 PR0328 PR0301 PR0526
PR0362. PR0356, PR0509 and PR0866
The description below relates primarily to production of PRO 179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 by culturing
cells transformed or
transfected with a vector containing PRO 179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 nucleic acid. tt is, of course,
contemplated that alternative
methods, which are well known in the art, may be employed to prepare PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866. For
instance, the
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide 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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide may be chemically synthesized separately and
combined using chemical or
enzymatic methods to produce the full-length PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide.
1. Isolation of DNA Encodine PRO 179 PR0207 PR0320 PR0219 PR0221 PR0224
PR0328. PR0301, PR0526, PR0362 PR0356 PR0509 or PR0866
DNA encoding PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526,
PR0362, PR0356, PR0509 or PR0866 may be obtained from a cDNA library prepared
from tissue believed to
possess the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 mRNA and to express it at a detectable level.
Accordingly, human PR0179,
PR0207, PR0320, PR0219, PR022I, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 DNA can be conveniently obtained from a cDNA library prepared from
human tissue, such as described
in the Examples. The PR0179-, PR0207-, PR0320-, PR0219-, PR0221-, PR0224=,
PR0328-, PR0301-,
PR0526-, PR0362-, PR0356-, PR0509- or PR0866-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 PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 Clonine: A Laboratory Manual (New York: Cold
Spring Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 is to use PCR
methodology
~4

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
[Sambrook et al., supra; 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 it 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 enryme labeling. Hybridization conditions, including moderate
stringency and high stringency,
are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known
IO 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 ofthe 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
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for PRO 179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 production and cultured in conventional nutrient media modified as
appropriate for inducing promoters,
selecting transformants, or amplifying the genes encoding the desired
sequences. The culture conditions, such as
media, temperature, pH and the like, can be selected by the skilled artisan
without undue experimentation. In
general, principles, protocols, and practical techniques for maximizing the
productivity of cell cultures can be found
in Mammalian Cell Biotechnoloev: a Practical Approach. M. Butler, ed. (IRL
Press, 1991) and Sambrook et al.,
supra.
Methods of eukaryotic cell transfectioit and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCl2, CaP04, 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., supra, 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 Eb, Viroloey, 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 etal., Proc. Natl. Acad.
Sci. fUSA). 76:3829 (19?9). However,
other methods for introducing DNA into cells, such as by nuclear
microinjection, electroporation, bacterial

CA 02353799 2001-06-08
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protoplast fusion with intact cells, or polycations, e.g., polybrene,
polyoznithine, may also be used. For various
techniques for transforming mammalian cells, see, Keown et al., Methods in
EnzvmoloQV 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. toll strains are publicly
available, such as E. toll K12 strain MM294 (ATCC 31,446); E. toll X 1776
(ATCC 31,537); E. toll strain W3110
(ATCC 27,325) and KS 772 (ATCC 53,635). Other suitable prokaryotic host cells
include Enterobaeteriaceae such
as Escherichia, e.g., E. toll, Enterobacter, Erwinia, Klebsiella, Proteus,
Salmonella, e.g., Salmonella typhimurizrm,
Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as
B. subtilis and B. lichenrformis (e.g.,
B. licheniformis 41 P 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 W31
10 may be modified to effect a
genetic mutation in the genes encoding proteins endogenous to the host, with
examples of such hosts including E.
toll W3110 strain 1A2, which has the complete genotype tonA ; E. toll W3110
strain 9E4, which has the complete
genotype tonA ptr3; E. toll W3110 strain 27C7 (ATCC SS,244), which has the
complete genotype tonA ptr3 phoA
EI S (argF lac) 169 degP ompTkan ; E. toll W31 I 0 strain 37D6, which has the
complete genotype tonA ptr3 phoA
El5 (argF-lac)169 degP ompT rbs7 ilvG kan'; E. toll W3110 strain 40B4, which
is strain 37D6 with a non-
kanamycin resistant degP deletion mutation; and an E. toll 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 PR0179-, PR0207-, PR0320-, PR0219-, PR0221-, PR0224-,
PR0328-, PR0301-,
PROS26-, PR0362-, PR03S6-, PROS09- or PR0866-encoding vectors. Saccharomyces
cerevisiae is a commonly
used lower eukaryotic host microorganism. Others include
Schizosaccharomycespombe (Beach and Nurse, Nature.
290: 140 [1981]; EP 139,383 published 2 May 1985); Klzryveromyces hosts (U.S.
Patent No. 4,943,529; Fleer et
al., Bio/Technoloey, 9:968-97S (1991)) such as, e.g., K. lactis (MW98-8C,
CBS683, CBS4S74; Louvencourt et
al., J-Bacteriol., 737 [1983]), X. Jragilis (ATCC 12,424), K. bulgaricztr
(ATCC 16,045), K. wickeramii (ATCC
24,178), K. waltii (ATCC S6,S00), K. drosophilarum (ATCC 36,906; Van den Berg
et al., Bio/Technoloey, 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 Schwanniomyces
occidentalis (EP394,S38published 31 October 1990); and filamentous fungi such
as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts
such as A. nidulans (Ballance
et al., Biochem. Bioahvs. 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
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CA 02353799 2001-06-08
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[ 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 Biochemistrv of Methylotrophs, 269 (1982).
Suitable host cells for the expression of glycosylated PR0179, PR0207, PR0320,
PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 are derived
from multicellular
organisms. Examples of invertebrate cells include insect cells such as
Drosophila 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 CV 1 line transformed by SV40
(GOS-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 CCLS l ). The
selection of the appropriate host cell is deemed to be within the skill in the
art.
3. Selection and Use of a Renlicable Vector
The nucleic acid (e.g., cDNA orgenomic DNA) encoding PR0179, PR0207, PR0320,
PR0219,PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 may be
inserted into a replicable
vector for cloning (amplification ofthe 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 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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 or PR0866 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
ofthe vector, or it may be a part ofthe PR0179-, PR0207-, PR0320-, PR0219-,
PR0221-, PR0224-, PR0328-,
PR0301-, PR0526-, PR0362-, PR0356-, PR0509- or PR0866-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, Ipp, 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/
I 3646 published 15 November 1990.
In mammalian cell expression, mammalian signal sequences may be used to direct
secretion of the protein, such
77

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
as signal sequences from secreted polypeptides 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 plasmid pBR322 is suitable for most Gram-
negative bacteria, the 2~ 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., ampiciliin,
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 179-, PR0207-, PR0320-, PR0219-, PR0221-,
PR0224-, PR0328-, PR0301-,
PR0526-, PR0362-, PR0356-, PR0509- or PR0866-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 trp t 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 trpl 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 PR0179-, PR0207-,
PR0320-, PR0219-, PR0221-, PR0224-, PR0328-, PR0301-, PR0526-, PR0362-, PR0356-
, PR0509- or
PR0866-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 promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad.
Sci. USA 80:21-25 ( 1983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-
phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem.. 255:2073 ( 1980)] or
other glycolytic enzymes [Hess,et
al., J. Adv. Enzyme Red 7:149 ( 1968); Holland, Biochem istry, 17:4900 (
1978)], such as enolase, glyceraldehyde-
3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase,
and glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription
controlled by growth conditions, are the promoter regions for alcohol
dehydrogenase 2, isocytochrome C, acid
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CA 02353799 2001-06-08
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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.
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 or PR0866 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 promotersare compatible
with the host cell systems.
Transcription ofa DNA encoding the PRO 179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 by higher eukaryotes may be
increased by inserting
an enhances 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 enhances
sequences are now known from mammalian
genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically,
however, one will use an enhances from
a eukaryotic cell virus. Examples include the SV40 enhances on the late side
ofthe replication origin (bp 100-270),
the cytomegalovirus early promoter enhances, the polyoma enhances on the late
side of the replication origin, and
adenovirus enhancers. The enhances may be spliced into the vector at a
position 5' or 3' to the PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
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 regionsof eukaryotic or viral DNAs or cDNAs. These regions
contain nucleotide segments transcribed
as polyadenylated fragments in the untranslated portion of the mRNA encoding
PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866 in
recombinant vertebrate cell culture are described in Gething et al., Nature,
293:620-625 ( 1981 ); Mantei et al.,
Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
4. Detective Gene Amplification/Expression
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.
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CA 02353799 2001-06-08
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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
immunohistochemical staining of cells or tissue sections and assay of cell
culture or body fluids, to quantitate
directly the expression of gene product. Antibodies useful for
immunohistochemical staining 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 PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide or
against a synthetic peptide
based on the DNA sequences provided herein or against exogenous sequence fused
to PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 DNA and
encoding a specific antibody epitope.
5. Purification of Polvveutide
Forms of PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526,
PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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 canon-
exchange resin such as DEAE;
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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866. Various methods of protein purification may
be employed and such
methods are known in the art and described for example in Deutscher, Methods
in Enzvmology, 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 PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 produced.
E. Antibodies
Some drug candidates for use in the compositions and methods of the present
invention are antibodies and
antibody fragments which mimic the biological activity of a PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide.

CA 02353799 2001-06-08
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Polvclonal 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 PR0179,
PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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.
Monoclonal Antibodies
The 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 immunizingagent wil I typical ly include the PRO I 79, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide or a
fusion protein thereof.
Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of
human origin are desired, or spleen
cells or lymph node cells are used if non-human mammalian sources are desired.
The lymphocytes are then fused
with an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell
[coding, Monoclonal Antibodies: Princ~les and Practice, Academic Press, (
1986) pp. 59-103]. Immortalized cell
lines are usually transformed mammalian cells, particularly myeloma cells of
rodent, bovine and human origin.
Usually, rat or mouse myeloma cell lines are employed. The 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 enryme
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 murine 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-humanheteromyeloma cell lines also have been
described forthe production
of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 ( 1984); Brodeur
et al., Monoclonal Antibody
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CA 02353799 2001-06-08
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Production Techniques and Annlications 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 PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866. Preferably, the binding
specificity of monoclonal
S antibodies produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay,
such as radioimmunoassay (RIA) or enryme-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, supra]. 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
orascites fluid by conventional immunoglobulin purificationproceduressuch as,
for example, protein A-Sepharose,
1S 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 can be readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide probes
that are capable of binding
specifically to genes encoding the heavy and light chains of murine
antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression vectors,
which are then transfected into host cells such as simian COS cells, Chinese
hamster ovary {CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein, to obtain
the synthesis of monoclonal
antibodies in the recombinant host cells. The DNA also may be modified, for
example, by substituting the coding
sequence for human heavy and light chain constant domains in place of the
homologous murine sequences [U.S.
2S Patent No. 4,816,567; Morrison et al., supra] 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
known 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 crosslinking. Alternatively, the relevant cysteine residues are
substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce
3S fragments thereof, particularly, Fab fragments, can be accomplished using
routine techniques known in the art.
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3. Human and Humanized Antibodies
The antibodies of the invention may further comprise humanized antibodies or
human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains
or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding
subsequences of antibodies) which
contain minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human
immunoglobulins (recipient antibody) in which residues from a complementary
determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human
immunoglobulin are replaced by corresponding non-human residues. Humanized
antibodies may also comprise
residues which are found neither in the recipient antibody nor in the imported
CDR or framework sequences. In
general, the humanized antibody will comprise substantial ly al l 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 ( 1986);
Riechmann et al., Nature, 332:323-329
( 1988); and Presta, Cun . Oo. Struct. Biol 2:593-596 ( 1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human. These non-
human amino acid residues are often referred to as "import" residues, which
are typically taken from an "import"
variable domain. Humanization 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 antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Patent No.
4,816,567), wherein substantially less than an intact human variable domain
has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from analogous
sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage display
libraries [Hoogenboom and Winter, JJ Mol. Biol., 227:381 (1991); Marks et al.,
JJ 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 Theranv Alan R.
Liss, p. 77 ( 1985) and Boerner et al.,
J. Immunol., 147(1):86-9S (1991)]. Similarly, human antibodies can be made by
the introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody production
is observed, which closely
resembles that seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016, and in the following scientific publications: Marks et
al., Bio/Technoloey, 10: 779-783
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CA 02353799 2001-06-08
WO 00137638 PCT/US99/28565
(1992); Lonbergetal., Nature. 368: 856-859 ( 1994); Morrison, Nature, 368: 812-
13 (1994); Fishwild et al., Nature
Biotechnoloev. 14:845-51 ( t 996); Neuberger, Nature Biotechnoloey 14: 826 (
1996); Lonberg and Huszar, Intern.
Rev. Immunol., 13 :65-93 (1995).
4. Bisuecific Antibodies
Bispecific 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 specificities is for the
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PRO301, PR0526,
PR0362, PR0356,
PR0509 or PR0866, 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 known 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 Cuello,
Nature, 305:537-539 (1983)]. Because of
the random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a
potential mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The
purification of the correct molecule is usually accomplished by affinity
chromatography steps. Similar procedures
are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al.,
EMBO J.. 10:3655-3659 (1991 ).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
be fused to immunoglobulin constant domain sequences. The fusion preferably is
with an 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 (CH 1 ) containing the site necessary for light-
chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if
desired, the immunoglobulin light
chain, are inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further
details of generating bispecific antibodies see, for example, Suresh et al.,
Methods in Enzymolosy 121:210 ( 1986).
According to another approach described in WO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers 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 ofthe 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, bispecifc antibodies can be prepared
using chemical linkage. Brennan
et al., Science. 229:81 ( 1985) describe a procedure wherein intact antibodies
are proteolytically cleaved to generate
F(ab'), fragments. These fragments are reduced in the presence of the dithiol
complexing agent sodium arsenite
84

CA 02353799 2001-06-08
WO 00137638 PCT/US99/28565
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. Exp. Med., 175:217-225 ( 1992) describe the
production of a fully humanized
bispecific antibody F(ab')Z molecule. Each Fab' fragment was separately
secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human T cells,
as welt as trigger the lytic activity
of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques 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.. 14_ 8(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 (V")
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 V" and VL domains of one fragment are forced to pair with the
complementary 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,
trispecific antibodies can be
prepared. Tutt et al., J. Immunol.. 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given
PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide herein. Alternatively, an anti-PROI 79, anti-PR0207, anti-PR0320,
anti-PR0219, anti-PR022I , anti-
PR0224, anti-PR0328, anti-PR0301, anti-PR0526, anti-PR0362, anti-PR0356, anti-
PR0509 or anti-PR0866
polypeptide arm may be combined with an arm which binds to a triggering
molecule on a leukocyte such as a T-cell
receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG
(FcyR), such as FcyRl (CD64), FcyRII
(CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the
cell expressing the particular
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which
express a particular PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 polypeptide. These antibodies possess a
PR0179-, PR0207-, PR0320-,

CA 02353799 2001-06-08
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PR0219-, PR0221-, PR0224-, PR0328-, PR0301-, PR0526-, PR0362-, PR0356-, PR0509-
or PR0866-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 179, PR0207,
PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide and
further binds tissue factor
S (TF).
5. Heteroconju~ate Antibodies
Heteroconjugate antibodies are also within the scope ofthe 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 crosslinking
agents. For example, immunotoxins
may be constructed using a disulfide exchange reaction or by forming a
thioether bond. Examples of suitable
reagents for this purpose include iminothiolate and methyl-4-
mercaptobutyrimidate and those disclosed, for
example, in U.S. Patent No. 4,676,980.
6. Effector Function E~ineerine
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 andlor increased complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See, Caron et al., J. Exo. 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 Druo Design
3: 219-230 ( 1989).
7. Immunoconjuga'
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent
such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
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,
86

CA 02353799 2001-06-08
WO 00137638 PCT/US99/28565
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for
the production of radioconjugated antibodies. Examples include 2'-'Bi, "'l,
"'In, ~°Y, and '86Re.
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 I,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be
prepared as described in Vitetta et al., Science, 238: 1098(1987). Carbon-14-
labeled 1-isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (MX-DTPA) is an 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 as
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 known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci. USA
82: 3688 (1985); Hwang etal., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and
U.S. Pat. Nos. 4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed in U.S.
Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-
PE). Liposomes are extruded through filters ofdefined pore size to yield
liposomes with the desired diameter. Fab'
fragments of the antibody of the present invention can be conjugated to the
liposomes as described in Martin et al.,
J. Biol. Chem., 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.,
J. National Cancer Inst. 81 19
1484 ( 1989).
F. Identification of Proteins Capable of Inhibiting-Neoplastic Cell Growth or
Proliferation
The proteins disclosed in the present application have been assayed in a panel
of 60 tumor cell lines
currently used in the investigational, disease-oriented, in vitro drug-
discovery screen of the National Cancer
Institute (NCI). The purpose of this screen is to identify molecules that have
cytotoxic and/or cytostatic activity
against different types of tumors. NCI screens more than 10,000 new molecules
per year (Monks et al., J. Natl.
Cancer Inst., 83:757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol Update 3~:1-
12 ([1989]). The tumor cell
lines employed in this study have been described in Monks et al., supra. The
cell tines the growth of which has
been significantly inhibited by the proteins of the present application are
specified in the Examples.
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The results have shown that the proteins tested show cytostatic and, in some
instances and concentrations.
cytotoxic activities in a variety of cancer cell lines, and therefore are
useful candidates for tumor therapy.
Other cell-based assays and animal models for tumors (e.g., cancers) can also
be used to verify the findings
of the NCI cancer screen, and to further understand the relationship between
the protein identified herein and the
S development and pathogenesis of neoplastic cell growth. For example, primary
cultures derived from tumors in
transgenic animals (as described below) can be used in the cell-based assays
herein, although stable cell lines are
preferred. Techniques to derive continuous cell lines from transgenic animals
are well known in the art (see, e.g.,
Small et al., Mol. Cell. Biol.. 5:642-648 [ 1985]).
G. Animal Models
A variety of wel! known animal models can be used to further understand the
role of the molecules
identified herein in the development and pathogenesis of tumors, and to test
the efficacy of candidate therapeutic
agents, including antibodies, and other agonists ofthe native polypeptides,
including small molecule agonists. The
in vivo nature of such models makes them particularly predictive of responses
in human patients. Animal models
of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer,
lung cancer, etc.) include both non-
recombinant and recombinant (transgenic) animals. Non-recombinant animal
models include, for example, rodent,
e.g., murine models. Such models can be generated by introducing tumor cells
into syngeneic mice using standard
techniques, e.g., subcutaneous injection, tail vein injection, spleen
implantation, intraperitoneal implantation,
implantation under the renal capsule, or orthopin implantation, e.g., colon
cancer cells implanted in colonic tissue.
(See, e.g., PCT publication No. WO 97/33551, published September 18, 1997).
Probably the most often used animal species in oncological studies are
immunodeficient mice and, in
particular, nude mice. The observation that the nude mouse with hypo/aplasia
could successfully act as a host for
human tumor xenografts has lead to its widespread use for this purpose. The
autosomal recessive m~ gene has been
introduced into a very large number of distinct congenic strains of nude
mouse, including, for example, ASW,
A/He, AKR, BALB/c, BIO.LP, CI7, C3H, C57BL, C57, CBA, DBA, DDD, I/st, NC, NFR,
NFS, NFS/N, NZB,
NZC, NZW, P, RIII and SJL. In addition, a wide variety of other animals with
inherited immunological defects
other than the nude mouse have been bred and used as recipients of tumor
xenografts. For further details see, e.g.,
The Nude Mouse in Oncolo y Research E. Boven and B. Winograd, eds., CRC Press,
Inc., 1991.
The cells introduced into such animals can be derived from known tumor/cancer
cell lines, such as, any
of the above-listed tumor cell lines, and, for example, the B104-1-I cell line
(stable NIH-3T3 cell line transfected
with the neu protooncogene); ras-transfected N1H-3T3 cells; Caco-2 (ATCC HTB-
37); a moderately well
differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-
38), or from tumors and cancers.
Samples of tumor or cancer cells can be obtained from patients undergoing
surgery, using standard conditions,
involving freezing and storing in liquid nitrogen (Karmali et al., Br. J.
Cancer. 48:689-696 [ 1983]).
Tumor cells can be introduced into animals, such as nude mice, by a variety of
procedures. The
subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
Tumors can be transplanted s.c. as solid
blocks, as needle biopsies by use of a trochar, or as cell suspensions. For
solid block or trochar implantation, tumor
88

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tissue fragments of suitable size are introduced into the s.c. space. Cell
suspensions are freshly prepared from
primary tumors or stable tumor cell lines, and injected subcutaneously. Tumor
cells can also be injected as
subdermal implants. In this location, the inoculum is deposited between the
lower part of the dermal connective
tissue and the s.c. tissue. Boven and Winograd ( 1991 ), supra. Animal models
of breast cancer can be generated,
for example, by implanting rat neuroblastoma cells (from which the neu oncogen
was initially isolated), or neu-
transformed NIH-3T3 cells into nude mice, essentially as described by Drebin
et al., Proc. Natl. Acad. Sci. USA
83:9129-9133(1986).
Similarly, animal models ofcolon cancer can be generated by passaging colon
cancer cells in animals, e.g.,
nude mice, leading to the appearance of tumors in these animals. An orthotopic
transplant model of human colon
cancer in nude mice has been described, for example, by Wang et al., Cancer
Research, 54:4726-4728 ( 1994) and
Too et al., Cancer Research, 55:681-684 (1995). This model is based on the so-
called"METAMOUSE" sold by
Anticancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from
the in vitro cultures can then .
be passaged to animals. Such tumors can serve as targets for further testing
or drug screening. Alternatively, the
tumors resulting from the passage can be isolated and RNA from pre-passage
cells and cells isolated after one or
more rounds of passage analyzed for differential expression of genes of
interest. Such passaging techniques can
be performed with any known tumor or cancer cell lines.
For example, Meth A, CMS4, CMSS, CMS21, and WEH1-164 are chemically induced
fibrosarcomas of
BALB/c female mice (DeLeo et al, J. Exn. Med., 146:720 [1977]), which provide
a highly controllable model
system for studying the anti-tumor activities of various agents (Palladino et
al., J. lmmunol., 138:4023-4032
[ 1987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior
to injection into the animals, the cell lines
are washed and suspended in buffer, at a cell density of about l Ox 106 to l
Ox 10' cells/ml. The animals are then
infected subcutaneously with 10 to 100 ul of the cell suspension, allowing one
to three weeks for a tumor to appear.
In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most
thoroughly studied
experimental tumors, can be used as an investigational tumor model. Efficacy
in this tumor model has been
correlated with beneficial effects in the treatment of human patients
diagnosed with small cell carcinoma of the lung
(SCCL). This tumor can be introduced in normal mice upon injection of tumor
fragments from an affected mouse
or of cells maintained in culture (Zupi et al., Br. J. Cancer, 41, suppl.
4:309 [1980]), and evidence indicates that
tumors can be started from injection of even a single cell and that a very
high proportion of infected tumor cells
survive. For further information about this tumor model see, Zacharski,
Haemostasis, 16:300-320 [ 1986]).
One way of evaluating the efficacy of a test compound in an animal model is
implanted tumor is to
measure the size of the tumor before and after treatment. Traditionally, the
size of implanted tumors has been
measured with a slide caliper in two or three dimensions. The measure limited
to two dimensions does not
accurately reflect the size of the tumor, therefore, it is usually converted
into the corresponding volume by using
a mathematical formula. However, the measurement of tumor size is very
inaccurate. The therapeutic effects of
a drug candidate can be better described as treatment-induced growth delay and
specific growth delay. Another
important variable in the description of tumor growth is the tumor volume
doubling time. Computer programs for
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the calculation and description of tumor growth are also available, such as
the program reported by Rygaard and
Spang-Thomsen, Proc. 6th Int. Workshop on Immune-Deficient Animals Wu and
Sheng eds., Basel, 1989, 301.
It is noted, however, that necrosis and inflammatory responses following
treatment may actually result in an
increase in tumor size, at least initially. Therefore, these changes need to
be carefully monitored, by a combination
of a morphometric method and flow cytometric analysis.
Recombinant (transgenic) anima( models can be engineered by introducing the
coding portion ofthe genes
identified herein into the genome ofanimals of interest, using standard
techniques for producing transgenic animals.
Animals that can serve as atarget fortransgenic manipulation include, without
limitation, mice, rats, rabbits, guinea
pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees
and monkeys. Techniques known
in the art to introduce a transgene into such animals include pronucleic
microinjection (Hoppe and Wanger, U.S.
Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines
(e.g., Van der Putten et al., Proc. Natl.
Acad. Sci. USA. 82:6148-615 [ 1985]); gene targeting in embryonic stem cells
(Thompson et al., Cell, 56:313-321
[1989]); electroporation of embryos (Lo, Mol. Cell. Biol.. 3:1803-1814
[1983]); sperm-mediated gene transfer
(Lavitrano et al., C~ 57:717-73 [1989]). For review, see, for example, U.S.
Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those
that carry the transgene only
in part of their cells ("mosaic animals"). The transgene can be integrated
either as a single transgene, or in
concatamers, e.g., head-to-head or head-to-tail tandems. Selective
introduction of a transgene into a particular cell
type is also possible by following, for example, the technique of Lasko et aL,
Proc. Natl. Acad. Sci. USA. 89:6232-
636 ( I 992).
The expression of the transgene in transgenic animals can be monitored by
standard techniques. For
example, Southern blot analysis or PCR amplification can be used to verify the
integration of the transgene. The
level of mRNA expression can then be analyzed using techniques such as in situ
hybridization, Northern blot
analysis, PCR, or immunocytochemistry. The animals are further examined for
signs of tumor or cancer
development.
The efficacy of antibodies specifically binding the polypeptides identified
herein and other drug
candidates, can be tested also in the treatment of spontaneous animal tumors.
A suitable target for such studies is
the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly
invasive, malignant tumor that is the
most common oral malignancy of cats, accounting for over 60% of the oral
tumors reported in this species. It rarely
metastasizes to distant sites, although this low incidence of metastasis may
merely be a reflection of the short
survival times for cats with this tumor. These tumors are usually not amenable
to surgery, primarily because of the
anatomy of the feline oral cavity. At present, there is no effective treatment
for this tumor. Prior to entry into the
study, each cat undergoes complete clinical examination, biopsy, and is
scanned by computed tomography (CT).
Cats diagnosed with sublingual oral squamous cell tumors are excluded from the
study. The tongue can become
paralyzed as a result of such tumor, and even if the treatment kills the
tumor, the animals may not be able to feed
themselves. Each cat is treated repeatedly, over a longer period of time.
Photographs of the tumors will be taken
daily during the treatment period, and at each subsequent recheck. After
treatment, each cat undergoes another CT
scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter.
The data are evaluated for

CA 02353799 2001-06-08
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differences in survival, response and toxicity as compared to control groups.
Positive response may require
evidence of tumor regression, preferably with improvement of quality of life
and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma,
adenocarcinoma, lymphoma,
chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of
these mammary adenocarcinoma
in dogs and cats is a preferred model as its appearance and behavior are very
similar to those in humans. However,
the use of this model is limited by the rare occurrence of this type of tumor
in animals.
H. Screening Assays for Drug Candidates
Screening assays for drug candidates are designed to identify compounds that
competitively bind or
complex with the receptors) of the polypeptides identified herein, or
otherwise signal through such receptor(s).
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, including peptides, preferably
soluble peptides, (poly)peptide-
immunoglobulin fusions, 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. 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.
In binding assays, the interaction is binding and the complex formed can be
isolated or detected in the
reaction mixture. In a particular embodiment, a receptor of a 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 polypeptide and drying. Alternatively, an immobilized antibody, e.g., a
monoclonal antibody, specific for the
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
receptor, its interaction with that
polypeptide can be assayed by methods well known for detecting protein-protein
interactions. Such assays include
traditional approaches, such as, 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 f
London). 340:245-246 ( 1989}; Chien
er al., Proc. Natl. Acad. Sci. USA 88:9578-9582 ( 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
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discrete modular domains, one acting as the DNA-binding domain, while 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 ofGAL4, and
another, in which candidate activating
proteins are fused to the activation domain. The expression of a GALL-IacZ
reporter gene under control of a
GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-
protein interaction. Colonies
containing interacting polypeptides are detected with a chromogenic substrate
for (i-galactosidase. A complete kit
(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.
I. Pharmaceutical Compositions
The polypeptides of the present invention, agonist antibodies specifically
binding proteins identified
herein, as well as other molecules identified by the screening assays
disclosed herein, can be administered for the
treatment of tumors, including cancers, in the form of pharmaceutical
compositions.
Where antibody fragments are used, the smallest inhibitory fragment which
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 which 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. Acad. 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, cytokine, chemotherapeutic agent, or growth-
inhibitory agent. Such molecules are
suitably present in combination in amounts that are effective for the purpose
intended.
Therapeutic formulations ofthe polypeptides identified herein, or agonists
thereof are prepared for storage
by mixing the active ingredient having the desired degree of purity with
optional pharmaceutically acceptable
carriers, excipients or stabilizers (ReminQton'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 acidand methionine;
preservatives (such as octadecyldimethylbenzyl
ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol;
resorcinol; cyclohexanol; 3-pentanol;
and m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates
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including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars
such as sucrose, mannitol,
trehalose or sorbitol; salt-forming counter-ions such as sodium; metal
complexes (e.g., Zn-protein complexes);
and/or non-ionic surfactants such as TWEENT"', PLURONICST"' or polyethylene
glycol (PEG).
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 a cytotoxic agent,
cytokine 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-panicles and nanocapsules) or in
macroemulsions. Such techniques
are disclosed in Remineton's Pharmaceutical Sciences 16th edition, Osol, A.
ed. (1980).
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
Iyophilization 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.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipenmeable 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 DEPOTT"'
(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.
J. Methods of Treatment
It is contemplated that the polypeptides of the present invention and their
agonists, including antibodies,
peptides, and small molecule agonists, may be used to treat various tumors,
e.g., cancers. Exemplary conditions
or disorders to be treated include benign or malignant tumors (e.g., renal,
liver, kidney, bladder, breast, gastric,
ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic
carcinomas; sarcomas; glioblastomas; and
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various head and neck tumors); leukemias and lymphoid malignancies; other
disorders such as neuronal, glial,
astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal
and blastocoelic disorders; and
inflammatory, angiogenic and immunologic disorders. The anti-tumor agents of
the present invention (including
the polypeptides disclosed herein and agonists which mimic their activity,
e.g., antibodies, peptides and small
organic molecules), are administered to a mammal, preferably a human, in
accord with known methods, such as
intravenous administration as a bolus or by continuous infusion over a period
of time, or by intramuscular,
intraperitoneal,intracerobrospinal,intraocular, intraarterial,
intralesional,subcutaneous,intraarticular,intrasynovial,
intrathecal, oral, topical, or inhalation routes.
Other therapeutic regimens may be combined with the administration of the anti-
cancer agents of the
instant invention. For example, the patient to be treated with such anti-
cancer agents may also receive radiation
therapy. Alternatively, or in addition, a chemotherapeutic agent may be
administered to the patient. Preparation
and dosing schedules for such chemotherapeutic agents may be used according to
manufacturers' instructions or
as determined empirically by the skilled practitioner. Preparation and dosing
schedules for such chemotherapy are
also described in Chemotherapy Service ed., M.C. Perry, Williams & Wilkins,
Baltimore, MD (1992). The
chemotherapeutic agent may precede, or follow administration of the anti-tumor
agent of the present invention, or
may be given simultaneously therewith. The anti-cancer agents of the present
invention may be combined with an
anti-oestrogen compound such as tamoxifen or an anti-progesterone such as
onapristone (see, EP 616812) in
dosages known for such molecules.
It may be desirable to also administer antibodies against tumor associated
antigens, such as antibodies
which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular endothelial factor
(VEGF). Alternatively, or in
addition, two or more antibodies binding the same or two or more different
cancer-associated antigens may be co-
administered to the patient. Sometimes, it may be beneficial to also
administer one or more cytokines to the patient.
In a preferred embodiment, the anti-cancer agents herein are co-administered
with a growth inhibitory agent. For
example, the growth inhibitory agent may be administered first, followed by
the administration of an anti-cancer
agent of the present invention. However, simultaneous administration or
administration of the anti-cancer agent
of the present invention first is also contemplated. Suitable dosages for the
growth inhibitory agent are those
presently used and may be lowered due to the combined action (synergy) of the
growth inhibitory agent and the
antibody herein.
For the prevention or treatment of disease, the appropriate dosage of an anti-
tumor agent herein will
depend on the type of disease to be treated, as defined above, the severity
and course of the disease, whether the
agent is administered for preventive or therapeutic purposes, previous
therapy, the patient's clinical history and
response to the agent, and the discretion ofthe attending physician. The agent
is suitably administered to the patient
at one time or over a series of treatments. Animal experiments provide
reliable guidance for the determination of
effective doses forhuman therapy. Interspecies scaling ofeffective 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 Drus Development Yacobi et al., eds., Pergamon Press, New York 1989,
pp. 42-96.
For example, depending on the type and severity of the disease, about 1 uglkg
to 15 mg/kg (e.g., 0.1-20
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CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
mg/kg) of an antitumor agent is an initial candidate dosage for administration
to the patient, whether, for example,
by one or more separate administrations, or by continuous infusion. A typical
daily dosage might range from about
1 ~g/kg to 100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over
several days or longer, depending on the condition, the treatment is sustained
until a desired suppression of disease
symptoms occurs. However, other dosage regimens may be useful. The progress of
this therapy is easily monitored
by conventional techniques and assays. 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
1~ or tissue.
K. Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing
materials useful for the
diagnosis or treatment of the disorders described above is provided. The
article of manufacture comprises a
container and a label. Suitable containers include, for example, bottles,
vials, syringes, and test tubes. The
containers may be formed from a variety of materials such as glass or plastic.
The container holds a composition
which is effective for diagnosing or treating the condition and may have a
sterile access port (for example the
container may be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection
needle). The active agent in the composition is an anti-tumor agent of the
present invention. The label on, or
associated with, the container indicates that the composition is used for
diagnosing or treating the condition of
choice. The article of manufacture may further comprise a second container
comprising a pharmaceutically-
acceptable buffer, such as phosphate-buffered saline, Ringer's solution and
dextrose solution. It may further include
other materials desirable from a commercial and user standpoint, including
other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
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
CultureCol lection, Manassas, VA.

CA 02353799 2001-06-08
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EXAMPLE 1: Extracellular Domain Homolo~y Screenine to Identify Novel
Polypeptides 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. LIFESEQ~, lncyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST-2
(Altschul et al., Methods in Enzymoloev. 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,
Washington).
Using this extracellular domain homology screen, consensus DNA sequences were
assembled relative to
the other identified EST sequences using phrap. In addition, the consensus DNA
sequences obtained were often
(but not always) extended using repeated cycles of BLAST or BLAST-2 and 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 40-55 by in length. In some cases, additional
oligonucleotides are synthesized when
the consensus sequence is greater than about 1-1.5 kbp. 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 Protocols in Molecular
Biolosy, 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 Notl site, linked with blunt to SaII hemikinased adaptors,
cleaved with Notl, sized appropriately
by gel electrophoresis, and cloned in a defined orientation into a suitable
cloning vector (such as pRKB or pRKD;
pRKSB is a precursor of pRKSD that does not contain the SfiI site; see, Holmes
et al., Science. 253:1278-1280
( 1991 )) in the unique XhoI and NotI sites.
EXAMPLE 2
Isolation of cDNA clones Encoding Human PR0179
A cDNA clone (DNA16451-1078) encoding a native human PR0179 polypeptide was
identified using
a yeast screen, in a human fetal liver library that preferentially represents
the 5' ends of the primary cDNA clones.
The primers used for the identification of DNA16451-1078 are as follows:
OLI114:
5'-CCACGTTGGCTTGAAATTGA-3'
(SEQ ID N0:3)
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OLI115:
5'-CCTTTAGAATTGATCAAGACAATTCATGATTTGATTCTCTATCTCCAGAG-3' (SEQ ID N0:4)
OLl l 16:
5'-TCGTCTAACATAGCAAATC-3'
(SEQ ID N0:5)
S Clone DNA 16451-1078 contains a single open reading frame with an apparent
translational initiation site
at nucleotide positions 37-39, and an apparent stop codon at nucleotide
positions 1417-1419 (Figures 1; SEQ ID
NO:1). The predicted polypeptide precursor is 460 amino acids long. The full-
length PR0179 protein is shown
in Figure 2 (SEQ ID N0:2).
Analysis of the full-length PR0179 sequence shown in Figure 2 (SEQ ID N0:2)
evidences the presence
of important polypeptide domains as shown in Figure 2, wherein the locations
given forthose important polypeptide
domains are approximate as described above. Analysis of the full-length PR0179
sequence (Figure 2; SEQ ID
N0:2) evidences the presence of the following: a signal peptide from about
amino acid 1 to about amino acid 16;
N-glycosylation sites from about amino acid 23 to about amino acid 27, from
about amino acid 115 to about amino
acid 119, from about amino acid 296 to about amino acid 300, and from about
amino acid 357 to about amino acid
361; cAMP- and cGMP-dependent protein kinase phosphorylation sites from about
amino acid 100 to about amino
acid 104 and from about amino acid 204 to about amino acid 208; a tyrosine
kinase phosphorylation site from about
amino acid 342 to about amino acid 351; N-myristoylation sites from about
amino acid 279 to about amino acid
285, from about amino acid 352 to about amino acid 358, and from about amino
acid 367 to about amino acid 373;
and leucine zipper patterns from about amino acid 120 to about amino acid 142
and from about amino acid 127 to
about amino 149.
Clone DNA16451-1078 has been deposited with ATCC on September 18, 1997 and is
assigned ATCC
deposit no. 209281. The ful I-length PRO 179 protein shown in Figure 2 has an
estimated molecular weight of about
53,637 daltons and a pl of about 6.61.
An analysis of the Dayhoff database (version 35.45 SwissProt 35) of the full-
length sequence shown in
2S Figure 2 (SEQ ID N0:2), evidenced the presence of a fibrinogen-like domain
exhibiting a high degree of sequence
homology with the two known human ligands ofthe TIE-2 receptor (h-TIE-2L1 and
h-TIE-2L2). The abbreviation
"TIE" is an acronym which stands for "tyrosine kinase containing Ig and EGF
homology domains" and was coined
to designate a new family of receptor tyrosine kinases. Accordingly, PR0179
has been identified as a novel
member of the TIE ligand family.
EXAMPLE 3
Isolation of cDNA clones Encodine Human PR0207
An expressedsequence tag (EST) DNA database (LIFESEQ~, Incyte Pharmaceuticals,
Palo Alto, CA) was
searched and an EST was identified which showed homology to human Apo-2
ligand. A human fetal kidney cDNA
library was then screened. mRNAisolated from human fetal kidney tissue
(Clontech) was used to prepare the
3S cDNA library. This RNA was used to generate an oligo dT primed cDNA library
in the vector pRKSD using
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CA 02353799 2001-06-08
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reagents and protocols from Life Technologies, Gaithersburg, MD (Super Script
Plasmid System). (n this
procedure, the double stranded cDNA was sized to greater than 1000 by and the
SaII/NotI tinkered cDNA was
cloned into XhoI/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/NotI cDNA
cloning sites. The library was screened
by hybridization with a synthetic oligonucleotide probe:
5'-CCAGCCCTCTGCGCTACAACCGCCAGATCGGGGAGTTTATAGTCACCCGG-3' (SEQ ID N0:8)
based on the EST.
A cDNA clone was sequenced in entirety. A nucleotide sequence ofthe full-
length DNA30879-1152 is
shown in Figure 3 (SEQ ID N0:6). Clone DNA30879-1152 contains a single open
reading frame with an apparent
translational initiation site at nucleotide positions 58-60 (Figure 3; SEQ ID
N0:6) and an apparent stop codon at
nucleotide positions 805-807. The predicted polypeptide precursor is 249 amino
acids long.
Analysis ofthe fall-length PR0207 sequence shown in Figure 4 (SEQ ID N0:7)
evidences the presence
of important polypeptide domains as shown in Figure 4, wherein the locations
given forthose important poiypeptide
domains are approximate as described above. Analysis of the full-length PR0207
sequence (Figure 4; SEQ ID
N0:7) evidences the presence of the following: a signal peptide from about
amino acid 1 to about amino acid 40;
an N-glycosylation site from about amino acid 139 to about amino acid 143; N-
myristoylation sites from about
amino acid 27 to about amino acid 33, from about amino acid 29 to about amino
acid 35, from about amino acid
36 to about amino acid 42, from about amino acid 45 to about amino acid 51,
from about amino acid 118 to about
amino acid 124, from about amino acid 121 to about amino acid 127, from about
amino acid 125 to about amino
acid 131, and from about amino acid 128 to about amino acid 134; amidation
sites from about amino acid 10 to
about amino acid 14 and from about amino acid 97 to about amino acid 101; and
a prokaryotic membrane
lipoprotein lipid attachment site from about amino acid 24 to about amino acid
35. Clone DNA30879-1152 has
been deposited with ATCC on October 10, 1997 and is assigned ATCC deposit no.
209358. The full-length
PR0207 protein shown in Figure 4 has an estimated molecular weight of about
27,216 daltons and a pI of about
9.61.
Based on a BLAST and FastA sequence alignment analysis (using the ALIGN-2
computer program) of
the full-length PR0207sequence shown in Figure 4 (SEQ ID N0:7), PR0207 shows
amino acid sequence identity
to several members of the TNF cytokine family, and particularly, to human
lymphotoxin-beta (23.4%) and human
CD40 ligand (19.8%).
EXAMPLE 4
Isolation of cDNA clones Encoding Human PR0320
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA28739. Based on the
DNA28739 consensus sequence, oligonucleotides were synthesized: I) to identify
by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0320.
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A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer:
S'-CCTCAGTGGCCACATGCTCATG-3' (SEQIDNO:11)
reverse PCR primer:
S 5'-GGCTGCACGTATGGCTATCCATAG-3' (SEQ ID N0:12)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28739
sequence which had the following nucleotide sequence:
hybridization probe:
5'-GATAAACTGTCAGTACAGCTGTGAAGACACAGAAGAAGGGCCACAGTGCC-3' (SEQ ID N0:i3)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0320 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal lung tissue (LIB025).
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA32284-1307 [Figure 5, SEQ ID N0:9]; and the derived protein sequence
for PR0320.
The entire coding sequence of DNA32284-1307 is included in Figure 5 (SEQ ID
N0:9). Clone
DNA32284-1307 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 135-137, and an apparent stop codon at nucleotide positions 1149-
1151. The predicted polypeptide
precursor is 338 amino acids long. Analysis of the full-length PR0320 sequence
shown in Figure 6 (SEQ ID
NO:10) evidences the presence of a variety of important polypeptide domains,
wherein the locations given forthose
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0320
polypeptide shown in Figure 6 evidences the presence of the following: a
signal peptide from about amino acid 1
to about amino acid 21; an amidation site from about amino acid 330 to about
amino acid 334; aspartic acid and
asparagine hydroxylation sites from about amino acid 109 to about amino acid
121, from about amino acid 191 to
about amino acid 203, and from about amino acid 236 to about amino acid 248;
an EGF-like domain cysteine
pattern signature from about amino acid 80 to about amino acid 91; calcium-
binding EGF-like domains from about
amino acid I03 to about amino acid 125, from about amino acid 230 to about
amino acid 252, and from about
amino acid 185 to about amino acid 207. Clone DNA32284-1307 has been deposited
with the ATCC on March
11, 1998 and is assigned ATCC deposit no. 209670. The full-length PR0320
protein shown in Figure 6 has an
estimated molecular weight of about 37,143 daltons and a pI of about 8.92.
EXAMPLE 5
Isolation of cDNA clones Encodin Human PR0219
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA28729. Based on the
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DNA28729 consensus sequence, oligonucleotides were synthesized: 1 ) to
identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0219.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer:
S'-GTGACCCTGGTTGTGAATACTCC-3'
(SEQ ID N0:16)
reverse PCR primer:
5'-ACAGCCATGGTCTATAGCTTGG-3' (SEQ ID NO:I7)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28729
sequence which had the following nucleotide sequence:
hybridization probe:
S'-GCCTGTCAGTGTCCTGAGGGACACGTGCTCCGCAGCGATGGGAAG-3' (SEQ ID N0:18)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0219 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal kidney tissue.
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA32290-1164 [Figure 7, SEQ ID N0:14]; and the derived protein sequence
for PR0219.
The entire coding sequence of DNA32290-1164 is included in Figure 7 (SEQ ID
N0:14). Clone
DNA32290-1164 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 204-206, and an apparent stop codon at nucleotide positions 2949-
2951. The predicted polypeptide
precursor is 1005 amino acids long. Analysis of the full-length PR0219
sequence shown in Figure 8 (SEQ ID
NO:15) evidences the presence of a variety of important polypeptide domains,
wherein the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0219
polypeptide shown in Figure 8 evidences the presence of the following: a
signal peptide from about amino acid 1
to about amino acid 23; an N-glycosylation site from about amino acid 221 to
about amino acid 225; cAMP- and
cGMP-dependent protein kinase phosphorylation sites from about amino acid 11 S
to about amino acid 119, from
about amino acid 606 to about amino acid 610, and from about amino acid 892 to
about amino acid 896; N-
myristoylation sites from about amino acid 133 to about amino acid 139, from
about amino acid 258 to about amino
acid 264, from about amino acid 299 to about amino acid 305, from about amino
acid 340 to about amino acid 346,
from about amino acid 4S3 to about amino acid 459, from about amino acid 494
to about amino acid 500, from
about amino acid 639 to about amino acid 645, from about amino acid 690 to
about amino acid 694, from about
amino acid 7S2 to about amino acid 758, and from about amino acid 792 to about
amino acid 798; amidation sites
from about amino acid 314 to about amino acid 318, from about amino acid S60
to about amino acid 564, and from
about amino acid 601 to about amino acid 605; and aspartic acid and asparagine
hydroxylation sites from about
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CA 02353799 2001-06-08
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amino acid 253 to about amino acid 265, from about amino acid 294 to about
amino acid 306, from about amino
acid 335 to about amino acid 347, from about amino acid 376 to about amino
acid 388, from about amino acid 417
to about amino acid 429, from about amino acid 458 to about amino acid 470,
from about amino acid 540 to about
amino acid 552, and from about amino acid 581 to about amino acid 593. Clone
DNA32290-1164 has been
deposited with the ATCC on October 17,1997 and is assigned ATCC deposit no.
209384. The full-length PR0219
protein shown in Figure 8 has an estimated molecular weight of about 102,233
daltons and a p1 of about 6.02.
An analysis of the full-length PR0219 sequence shown in Figure 8 (SEQ ID
N0:15), suggests that
portions of it possess significant homology to the mouse and human matrilin-2
precursor polypeptides.
EXAMPLE 6
Isolation of cDNA clones Encoding Human PR0221
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA28756. Based on the
DNA28756 consensus sequence, oligonucleotides were synthesized: 1) to identify
by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0221.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR~rimer:
5'-CCATGTGTCTCCTCCTACAAAG-3' (SEQ ID N0:21)
reverse PCR primer:
5'-GGGAATAGATGTGATCTGATTGG-3' (SEQ ID N0:22)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28756
sequence which had the following nucleotide sequence:
hybridization probe:
5'-CACCTGTAGCAATGCAAATCTCAAGGAAATACCTAGAGATCTTCCTCCTG-3' (SEQ ID N0:23)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0221 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal lung tissue.
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA33089-1132 [Figure 9, SEQ ID N0:19]; and the derived protein sequence
for PR0221.
The entire coding sequence of DNA33089-1132 is included in Figure 9 (SEQ ID
N0:19). Clone
DNA33089-1132 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 179-181, and an apparent stop codon at nucleotide positions 956-958.
The predicted polypeptide
precursor is 259 amino acids long. Analysis of the full-length PR0221 sequence
shown in Figure 10 (SEQ ID
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N0:20) evidences the presence of a variety of important polypeptide domains,
wherein the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0221
polypeptide shown in Figure 10 evidences the presence of the following: a
signal peptide from about amino acid
1 to about amino acid 33; a transmembrane domain from about amino acid 204 to
about amino acid 219; N-
glycosylation sites from about amino acid 47 to about amino acid 51 and from
about amino acid 94 to about amino
acid 98; a cAMP- and cGMP-dependent protein kinase phosphorylation site from
about amino acid 199 to about
amino acid 203; and N-myristoylation sites from about amino acid 37 to about
amino acid 43, from about amino
acid 45 to about amino acid S 1, and from about amino acid 110 to about amino
acid 116. Clone DNA33089-1132
has been deposited with the ATCC on September 16, 1997 and is assigned ATCC
deposit no. 209262. The full-
length PR0221 protein shown in Figure 10 has an estimated molecular weight of
about 29,275 daltons and a pl of
about 6.92.
An analysis of the full-length PR0221sequence shown in Figure 10 (SEQ 1D
N0:20), shows it has
homology to members of the leucine rich repeat protein superfamily, including
SLIT protein.
EXAMPLE 7
Isolation of cDNA clones Encodine Human PR0224
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA3084S. Based on the
DNA30845 consensus sequence, oligonucleotides were synthesized: 1) to identify
by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0224.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer:
S'-AAGTTCCAGTGCCGCACCAGTGGC-3'
(SEQ ID N0:26)
reverse PCR primer:
S'-TTGGTTCCACAGCCGAGCTCGTCG-3' (SEQ ID N0:27)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA30845
sequence which had the following nucleotide sequence:
hybridization probe:
S'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAATGCCCACC-3' (SEQ ID N0:28)
In order to screen several libraries for a source ofa full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0224 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal liver tissue.
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
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for DNA33221-1133 [Figure 11, SEQ ID N0:24]; and the derived protein sequence
for PR0224.
The entire coding sequence of DNA33221-1133 is included in Figure I1 (SEQ ID
N0:24). Clone
DNA33221-1133 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 33-35, and an apparent stop codon at nucleotide positions 879-881.
The predicted polypeptide precursor
is 282 amino acids long. Analysis of the full-length PR0224 sequence shown in
Figure 12 (SEQ ID N0:25)
evidences the presence of a variety of important polypeptide domains, wherein
the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0224
polypeptide shown in Figure 12 evidences the presence of the following: a
signal peptide from about amino acid
1 to about amino acid 30; a transmembrane domain from about amino acid 231 to
about amino acid 248; N-
glycosylation sites from about amino acid 126 to about amino acid 130, from
about amino acid 195 to about amino
acid 199, and from about amino acid 213 to about amino acid 217; N-
myristoylation sites from about amino acid
3 to about amino acid 9, from about amino acid 10 to about amino acid 16, from
about amino acid 26 to about
amino acid 32, from about amino acid 30 to about amino acid 36, from about
amino acid 112 to about amino acid
118, from about amino acid 166 to about amino acid 172, from about amino acid
212 to about amino acid 218, from
about amino acid 224 to about amino acid 230, from about amino acid 230 to
about amino acid 236, and from about
amino acid 263 to about amino acid 269; a prokaryotic membrane lipoprotein
lipid attachment site from about
amino acid 44 to about amino acid 55; and a leucine zipper pattern from about
amino acid 17 to about amino acid
39. Clone DNA33221-1133 has been deposited with the ATCC on September 16, 1997
and is assigned ATCC
deposit no. 209263. The full-length PR0224 protein shown in Figure 12 has an
estimated molecular weight of
about 28,991 daltons and a pI of about 4.62.
An analysis ofthe full-length PR0224 sequence shown in Figure 12 (SEQ ID
N0:25), suggests that it has
homology to very low-density lipoprotein receptors, apolipoprotein E receptor
and chicken oocyte receptor P95.
Based on a BLAST and FastA sequence alignment analysis of the full-length
sequence, PR0224 has amino acid
sequence identity to portions of these proteins in the range from 28% to 45%,
and overall identity with these
proteins in the range from 33% to 39%.
EXAMPLE 8
Isolation of cDNA clones Encodine Human PR0328
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA35615. Based on the
DNA35615 consensus sequence, oligonucleotides were synthesized: 1 ) to
identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0328.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer'
5'-TCCTGCAGTTTCCTGATGC-3'
(SEQ ID N0:31)
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reverse PCR primer:
5'-CTCATATTGCACACCAGTAATTCG-3' (SEQ ID N0:32)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA35615
sequence which had the following nucleotide sequence:
hybridization probe:
5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3' (SEQ ID N0:33)
In order to screen several libraries for a source ofa full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0328 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal kidney tissue.
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA40587-1231 [Figure 13, SEQ ID N0:29]; and the derived protein sequence
for PR0328.
The entire coding sequence of DNA40587-1231 is included in Figure 13 (SEQ ID
N0:29). Clone
DNA40587-1231 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 15-17, and an apparent stop codon at nucleotide positions 1404-1406.
The predicted polypeptide
precursor is 463 amino acids long. Analysis of the full-length PR0328 sequence
shown in Figure 14 (SEQ ID
N0:30) evidences the presence of a variety of important polypeptide domains,
wherein the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0328
polypeptide shown in Figure 14 evidences the presence of the following: a
signal peptide from about amino acid
1 to about amino acid 22; N-glycosylation sites from about amino acid 1 14 to
about amino acid 118, from about
amino acid 403 to about amino acid 407, and from about amino acid 409 to about
amino acid 413; a
glycosaminoglycan attachment site from about amino acid 439 to about amino
acid 443; N-myristoylation sites
from about amino acid 123 to about amino acid 129, from about amino acid 143
to about amino acid 149, from
about amino acid 152 to about amino acid 158, from about amino acid 169 to
about amino acid 175, from about
amino acid 180 to about amino acid 186, from about amino acid 231 to about
amino acid 237, and from about
amino acid 250 to about amino acid 256; amidation sites from about amino acid
82 to about amino acid 88 and from
about amino acid 172 to about amino acid 176; a peroxidase proximal heme-
ligand signature from about amino acid
287 to about amino acid 298; an extracellular protein SCP/Tpx-1/Ag5/PR-1/Sc7
signature 1 domain from about
amino acid 127 to about amino acid 138; and art extracellularprotein SCP/Tpx-
1/Ag5/PR-1/Sc7 signature 2 domain
from about amino acid 160 to about amino acid 172. Clone DNA40587-1231 has
been deposited with the ATCC
on November 7, 1997 and is assigned ATCC deposit no. 209438. The full-length
PR0328 protein shown in Figure
14 has an estimated molecular weight of about 49,471 daltons and a pI of about
5.36.
An analysis of the full-length PR0328sequence shown in Figure 14 (SEQ ID
N0:30), suggests that
portions of it possess significant homology to the human glioblastoma protein,
and to the cysteine rich secretory
protein thereby indicating that PR0328 may be a novel glioblastoma protein or
cysteine rich secretory protein.
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EXAMPLE 9
Isolation of cDNA clones Encodine Human PR0301
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA35936. Based on the
DNA35936 consensus sequence, oligonucleotides were synthesized: 1) to identify
by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0301.
The oligonucleotides used in the above procedure were the following:
forward PCR primer 1:
5'-TCGCGGAGCTGTGTTCTGTTTCCC-3' (SEQ ID N0:36)
forward PCR primer 2:
5'-ACACCTGGTTCAAAGATGGG-3' (SEQ ID N0:37)
forward PCR primer 3:
5'-TTGCCTTACTCAGGTGCTAC-3' (SEQ ID N0:38)
reverse PCR primer 1:
5'-TAGGAAGAGTTGCTGAAGGCACGG-3' (SEQ ID N0:39)
reverse PCR primer 2:
5'-ACTCAGCAGTGGTAGGAAAG-3' (SEQ ID N0:40)
hybridization~robe 1:
S'-TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT-3'
(SEQ ID N0:41 )
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0301 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal kidney tissue.
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA40628-1216 [Figure 15, SEQ ID N0:34]; and the derived protein sequence
for PR0301.
The entire coding sequence of DNA40628-1216 is included in Figure 15 (SEQ ID
N0:34). Clone
DNA40628-1216 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 52-54, and an apparent stop codon at nucleotide positions 949-951.
The predicted polypeptide precursor
is 299 amino acids long. Analysis of the full-length PR0301 sequence shown in
Figure 16 (SEQ ID N0:35)
evidences the presence of a variety of important polypeptide domains, wherein
the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0301
polypeptide shown in Figure 16 evidences the presence of the following: a
signal peptide from about amino acid
1 to about amino acid 27; a transmembrane domain from about amino acid 235 to
about amino acid 256; an N-
glycosylation site from about amino acid 185 to about amino acid 189; a cAMP-
and cGMP-dependent protein
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kinase phosphorylation site from about amino acid 270 to about amino acid 274;
and N-myristoylation sites from
about amino acid 105 to about amino acid 111, from about amino acid 1 16 to
about amino acid 122, from about
amino acid 158 to about amino acid 164, from about amino acid 219 to about
amino acid 225, from about amino
acid 237 tq about amino acid 243, and from about amino acid 256 to about amino
acid 262. Clone DNA40628-
1216 has been deposited with the ATCC on November 7, 1997 and is assigned ATCC
deposit no. 209432. The
full-length PR0301 protein shown in Figure 16 has an estimated molecular
weight of about 32,583 daltons and a
pl of about 8.29.
Based on a BLAST and FastA sequence alignment of the full-length PR0301
sequence shown in Figure
16 (SEQ ID N0:35), PR0301 shows amino acid sequence identity to the A33
antigen precursor (30%) and the
coxsackie and adenovirus receptor protein (29%).
EXAMPLE 10
isolation of cDNA clones Encodin Human PR0526
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. An iniiial consensus sequence was identified
designated herein as DNA39626.init.
In addition, the initial consensus DNA sequence was extended using repeated
cycles of BLAST and phrap to extend
the initial consensus sequence as far as possible using the sources of EST
sequences discussed above. The
assembled consensus sequence is designated herein as <consen0l >. Based on the
<consen0l > consensus sequence,
oligonucleotides were synthesized: I) to identify by PCR a cDNA library that
contained the sequence of interest,
and 2) for use as probes to isolate a clone of the full-length coding sequence
for PR0526.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer:
S'-TGGCTGCCCTGCAGTACCTCTACC-3'
(SEQ ID N0:44)
reverse PCR primer:
5'-CCCTGCAGGTCATTGGCAGCTAGG-3'
(SEQ ID N0:45)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the <consen0l> consensus
sequence which had the following nucleotide sequence:
hybridization probe:
S'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCACAC-3' (SEQ ID N0:46)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PROS26 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal Liver tissue (LIB228).
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA44184-1319 [Figure 17, SEQ ID N0:42]; and the derived protein sequence
for PR0526.
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The entire coding sequence of DNA44184-1319 is included in Figure 17 (SEQ ID
N0:42). Clone
DNA44184-1319 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions S14-516, and an apparent stop codon at nucleotide positions 1933-
1935. The predicted polypeptide
precursor is 473 amino acids long. Analysis of the full-length PR0526 sequence
shown in Figure 18 (SEQ ID
N0:43 ) evidences the presence of a variety of important polypeptide domains,
wherein the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0526
polypeptide shown in Figure 18 evidences the presence of the following: a
signal peptide from about amino acid
1 to about amino acid 26; a leucine zipper pattern from about amino acid 135
to about amino acid 157; a
glycosaminoglycan attachment site from about amino acid 436 to about amino
acid 440; N-glycosylation sites from
about amino acid 82 to about amino acid 86, from about amino acid 179 to about
amino acid l 83, from about amino
acid 237 to about amino acid 241, from about amino acid 372 to about amino
acid 376, and from about amino acid
423 to about amino acid 427; and a von Willebrand factor (VWF) type C domain
from about amino acid 411 to
about amino acid 427. Clone DNA44184-1319 has been deposited with the ATCC on
March 26, 1998 and is
assigned ATCC deposit no. 209704. The full-length PR0526 protein shown in
Figure 18 has an estimated
molecular weight of about 50,708 daltons and a p1 of about 9.28.
An analysis of the full-length PROS26 sequence shown in Figure 18 (SEQ ID
N0:43), suggests that
portions of it possess significant homology to leucine repeat rich proteins
including ALS, SLIT, carboxypeptidase
and platelet glycoprotein V thereby indicating that PROS26 is a novel protein
which is involved in protein-protein
interactions.
EXAMPLE 11
Isolation of cDNA clones Encoding Human PR0362
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as
described in Example 1 above. This consensus sequence is designated herein as
DNA422S7. Based on the
DNA42257 consensus sequence, oligonucleotides were synthesized: 1) to identify
by PCR a eDNA library that
contained the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for
PR0362.
A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 1:
S'-TATCCCTCCAATTGAGCACCCTGG-3' ~ (SEQ ID N0:49)
forward PCR primer 2:
S'-GTCGGAAGACATCCCAACAAG-3' (SEQ ID NO:SO)
reverse PCR primer 1:
S'-CTTCACAATGTCGCTGTGCTGCTC-3' (SEQ ID NO:S 1 )
reverse PCR primer 2:
S'-AGCCAAATCCAGCAGCTGGCTTAC-3' (SEQ ID N0:52)
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Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the DNA42257 consensus
sequence which had the following nucleotide sequence:
_hybridization probe:
5'-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCTGAT-3' (SEQ ID N0:53)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0362 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal brain tissue (L1B153).
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
for DNA45416-1251 [Figure 19, SEQ ID N0:47]; and the derived protein sequence
for PR0362.
The entire coding sequence of DNA45416-1251 is included in Figure 19 (SEQ ID
N0:47). Clone
DNA45416-1251 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 119-121, and an apparent stop codon at nucleotide positions 1082-
1084. The predicted polypeptide
precursor is 321 amino acids long. Analysis of the full-length PR0362 sequence
shown in Figure 20 (SEQ ID
N0:48) evidences the presence of a variety of important polypeptide domains,
wherein the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0362
polypeptide shown in Figure 20 evidences the presence of the following: a
signal peptide from about amino acid
1 to about amino acid 19; a transmembrane domain from about amino acid.281 to
about amino acid 300; a
glycosaminoglycan attachment site from about amino acid 149 to about amino
acid 153; a cAMP- and cGMP-
dependent protein kinase phosphorylation site from about amino acid 308 to
about amino acid 312; and N-
myristoylation sites from about amino acid 2 to about amino acid 8, from about
amino acid 148 to about amino acid
154, from about amino acid 158 to about amino acid 164, from about amino acid
207 to about amino acid 213, and
from about amino acid 215 to about amino acid 221. Clone DNA45416-1251 has
been deposited with the ATCC
on February 5, 1998 and is assigned ATCC deposit no. 209620. The full-length
PR0362 protein shown in Figure
20 has an estimated molecular weight of about 35,544 daltons and a pI of about
8.51.
An analysis of the full-length PR0362 sequence shown in Figure 20 (SEQ ID
N0:48), suggests that it
possesses significant similarity to the A33 antigen protein and the HCAR
protein. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced significant
homology between the PR0362 amino
acid sequence and the following Dayhoff sequences: AB002341-1, HSU55258_I,
HSC7NRCAM_1,
RNU81037_1, A33 HUMAN, P W 14158, NMNCAMRI_1, HSTITINN2_1, S71824_l, and
HSU63041_1.
EXAMPLE 12
Isolation of cDNA clones Encodine Human PR0356
An expressed sequence tag (EST) DNA database (LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA)
was searched and an EST (#2939340) was identified that had homology to PR0179
[identified in EXAMPLE 2
above and designated DNA 16451-1078 (Figure 1; SEQ ID NO:1 )]. To clone
PR0356, a human fetal lung library
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prepared from mRNA purchased from Clontech, lnc., (Palo Alto, CA), catalog #
6528-1 was used, following the
manufacturer's instructions.
The cDNA libraries used to isolate the cDNA clones encoding human PR0356 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 Not1 site, linked with blunt to SaII hem
ikinased adaptors, cleaved with Nott,
sized appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such
as pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the Sfil
site; see, Holmes et al., Science,
253:1278-1280 (1991)) in the unique Xhol and Notl.
Oligonucleotide probes based upon the above described EST sequence were then
synthesized: 1 ) to
identify by PCR a cDNA library that contained the sequence of interest, and 2)
for use as probes to isolate a clone
of the full-length coding sequence for PR0356. Forward and reverse PCR primers
generally range from 20-30
nucleotides and are often designed to give a PCR product of about 100-1000 by
in length. The probe sequences
are typically 40-55 by in length. In 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 Protocols in
Molecular Bioloev, supra, 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 oligonucleotide sequences used were as follows:
5'-TTCAGCACCAAGGACAAGGACAATGACAACT-3' (SEQ ID N0:56)
5'-TGTGCACACTTGTCCAAGCAGTTGTCATTGTC-3' (SEQ ID N0:57)
ZO S'-GTAGTACACTCCATTGAGGTTGG-3' (SEQ ID N0:58)
A cDNA clone was identified and sequenced in entirety. The entire nucleotide
sequence of
DNA47470-1130-P1 is shown in Figure 21 (SEQ ID N0:54). Clone DNA47470-1130-PI
contains a single open
reading frame with an apparent translational initiation site at nucleotide
positions 215-217, and a stop codon at
nucleotide positions 1253-1255 (Figure 21; SEQ ID N0:54). The predicted
polypeptide precursor is 346 amino
acids long, and has a calculated molecular weight of approximately 40,018
daltons and an estimated pl of about
8.19. The full-length PR0356 protein is shown in Figure 22 (SEQ ID NO:55).
Analysis ofthe full-length PR0356 sequence shown in Figure 22 (SEQ IDNO:55)
evidences the presence
of important polypeptide domains as shown in Figure 22, wherein the locations
given for those important
polypeptide domains are approximate as described above. Analysis of the full-
length PR0356 sequence (Figure
22; SEQ 1D NO:55) evidences the presence of the following: a signal peptide
from about amino acid 1 to about
amino acid 26; N-glycosylation sites from about amino acid 58 to about amino
acid 62, from about amino acid 253
to about amino acid 257, and from about amino acid 267 to about amino acid
271; a glycosaminoglycan attachment
site from about amino acid 167 to about amino acid 171; a CAMP- and cGMP-
dependent protein kinase
phosphorylation site from about amino acid 176 to about amino acid 180; N-
myristoylation sites from about amino
acid 168 to about amino acid 174, from about amino acid 196 to about ammo acid
202, from about amino acid 241
to about amino acid 247, from about amino acid 252 to about amino acid 258,
from about amino acid 256 to about
amino acid 262, and from about amino acid 327 to about amino acid 333; and a
cell attachment sequence from
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about amino acid 199 fo about amino acid 202.
Clone DNA47470-1130-P1 has been deposited with ATCC on October 28, 1997 and is
assigned ATCC
deposit no. 209422. It is understood that the deposited clone has the actual
correct sequence rather than the
representations provided herein.
An analysis ofthe Dayhoffdatabase (version 35.45 SwissProt35), using the ALIGN-
2 sequence alignment
analysis of the full-length sequence shown in Figure 22 (SEQ ID NO:55), shows
amino acid sequence identity
between the PR0356 amino acid sequence and both TIE-2L 1 (32%) and TIE-2L2
(34%). The abbreviation "TIE"
is an acronym which stands for "~rosine kinase containing 1~ and EGF homology
domains" and was coined to
designate a new family of receptor tyrosine kinases.
EXAMPLE 13
Isolation of cDNA clones Encoding Human PR0509
To isolate a cDNA for DNA50148-1068, a bacteriophage library of human retinal
cDNA (commercially
available from Clontech) was screened by hybridization with a synthetic
oligonucleotide probe based on an EST
sequence (GenBank locus AA021617), which showed some degree ofhomology to
members ofthe TNFR family.
The oligonucleotide probe employed in the screening was 60 by long. Five
positive clones (containing cDNA
inserts of 1.8-1.9 kb) were identified in the cDNA library, and the positive
clones were confirmed to be specific
by PCR using the above hybridization probe as a PCR primer. Single phage
plaques containing each of the five
positive clones were isolated by limiting dilution and the DNA was purified
using a Wizard Lambda Prep DNA
purification kit (commercially available from Promega).
The cDNA inserts from three of the five bacteriophage clones were excised from
the vector arms by
digestion with EcoRI, gel-purified, and subcloned into pRKS and sequenced on
both strands. The three clones
contained an identical open reading frame (with the exception of an intron
found in one of the clones).
The entire nucleotide sequence of DNA50148-1068 is shown in Figure 23 (SEQ ID
NO: 59). The cDNA
contained one open reading frame with a translational initiation site assigned
to the ATG codon at nucleotide
positions 82-84. The open reading frame ends at the termination codon TGA at
nucleotide positions 931-933.
The predicted amino acid sequence of the full length PR0509 polypeptide
sequence contains 283 amino
acids. The full-length PR0509 protein is shown in Figure 24 (SEQ ID N0:60) and
has an estimated molecular
weight of approximately 30,420 and a pl of about 7.34.
Analysis ofthe full-length PR0509 sequence shown in Figure 24 (SEQ ID N0:60)
evidences the presence
of important polypeptide domains as shown in Figure 24, wherein the locations
given for those important
polypeptide domains are approximate as described above. Analysis of the full-
length PR0509 sequence (Figure
24; SEQ 1D N0:60) evidences the presence of the following: a signal peptide
from about amino acid 1 to about
amino acid 36; a transmembrane domain from about amino acid 205 to about amino
acid 221; N-glycosylation sites
from about amino acid 110 to about amino acid 114 and from about amino acid
173 to about amino acid 177; N
myristoylation sites from about amino acid 81 to about amino acid 87, from
about amino acid 89 to about amino
acid 95, from about amino acid 104 to about amino acid 110, from about amino
acid 120 to about amino acid 126,
110

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,~;.~cm.~'t;C~,2,'<:y
CA 02353799 2001 06-08~~~~~ .. .... ra
t _ i,~~7v7VVVTT J~ .. P~ DESCf'.AMD'
, VVv7a7 GVJV~J!
J.
t"~..a~ r ..ia "s.l.,.-..:...;. . . 1
l~s~::a;<riu.w:~a'<f~7'd~#t~&
v.,:;:;z.3..f _ ,_ :, ~. ,. ~.N~_'=:e;
~s.. r.. ~~ .ss.,= - l:,n:zs.~..,..:
. 1 . .
.
. . . . . . .
.
1 1 1 1
' .
from about amino acid 153 to about amino acid 159, from about amino acid 193
to about amino acid 199, from
about amino acid 195 to about amino acid 201, and from about amino acid 220 to
about amino acid 226; and a cell
attachment sequence from about amino acid 23I to about amino acid 234.
An alignment (using the ALIGN~'~' computer program) of a 58 amino acid long
cytopIasmic region of
PR0509 with other known members of the human TNF receptor family showed some
sequence similarity, and in
particular to CD40 (12 identities) and LT-beta receptor (11 identities).
EXAMPLE 14
Isolation of cDNA clones Encoding Human PR0866
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described in
Example 1 above. This consensus sequence is designated herein as DNA44708.
Based on the DNA44708
consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a
cDNA library that contained the
sequence of interest, and 2) for use as probes to isolate a clone of the full-
length coding sequence for PR0866.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 1:
5'-CAGCACTGCCAGGGGAAGAGGG-3' (SEQ ID N0:63)
forward PCR primer 2:
5'-GAGGACZ,CGCTACGT'CCG-3' (SEQ ID N0:64)
forward PCR primer 3:
5'-CAGCCCCTTCTCCTCCTTTCTCCC-3' (SEQ ID N0:65)
reverse PCR primer 1:
5'-GCAGTTATCAGGGACGCACTCAGCC-3' (SEQ ID N0:66)
reverse PCRprimer 2:
5'-CCAGCGAGAGGCAGATAG-3' (SEQ ID N0:67)
reverse PCR~rimer 3:
5'-CGGTCACCGTGTCCTGCGGGATG-3' (SEQ ID N0:68)
f
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the DNA44708 consensus
sequence which had the following nucleotide sequence:
hybridization probe:
5'-CAGCCCGTTCTCCTCCTTTCZ'CCCACGTCCTATCTGCCTCTC-3' (SEQ >D N0:69)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones
encoding the PR0866 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction
of the cDNA libraries was isolated from human fetal kidney tissue (ISB228).
DNA sequencing of the isolated clones isolated as described above gave the
full-length DNA sequence
111
AMENDED SHEET

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. . -;.:..
'~
'
. . : . . . ..
.
: .
. . . . . ..
..
.. ... .. . . .. ..
for DNA53971-1359 [Figure 25, SEQ ID N0:61]; and the derived protein sequence
for PR0866.
The entire coding sequence of DNA53971-1359 is included in Figure 25 (SEQ 1D
N0:61). Clone
DNA53971-1359 contains a single open reading frame with an apparent
translational initiation site at nucleotide
positions 275-277, and an apparent stop codon at nucleotide positions 1268-
1270. The predicted polypeptide
precursor is 331 amino acids long. Analysis of the full-length PR0866 sequence
shown in Figure 26 (SEQ 1D
N0:62) evidences the presence of a variety of important polypeptide domains,
wherein the locations given for those
important polypeptide domains are approximate as described above. Analysis of
the full-length PR0866
polypeptide shown in Figure 26 evidences the presence of the following: a
signal peptide fmm about amino acid
1 to about amino acid 26; a glycosaminoglycan attachment site from about amino
acid 131 to about amino acid 135;
a cAMP- and cGMP-dependent protein kinase phosphorylation site from about
amino acid 144 to about amino acid
148; and N-myristoylation sites from about amino acid 26 to about amino acid
32, from about amino acid 74 to
about amino acid 80, from about amino acid 132 to about amino acid 138, from
about amino acid 134 to about
amino acid 140, from about amino acid 190 to about amino acid 196, from about
amino acid 287 to about amino
acid 293, and from about amino acid 290 to about amino acid 296. Clone
DNA53971-1359 has been deposited with
the ATCC on April 7, 1998 and is assigned ATCC deposit no. 209750. The full-
length PR0866 protein shown
in Figure 26 has an estimated molecular weight of about 35,844 daltons and a
pI of about 5.45.
An analysis of the full-length PR0866 sequence shown in Figure 26 (SEQ ID
N0:62}, suggests that it
possesses significant similarity to the mindin/spondin family of proteins,
thereby indicating that PR0866 may be
a novel mindin homolog. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35)
evidenced significant homology between the PR0866 amino acid sequence and the
following Dayhoff sequences:
AB006085_I, AB006084_l, AB006086_l, AF017267_l, CWU42213_l, AC004160_l,
CPMICRP_l, S49108,
A48569 and I46687.
EXAMPLE 15
In situ Hybridization
In situ hybridization is a powerful and versatile technique for the detection
and localization of nucleic acid
sequences within cell or tissue preparations. It may be useful, for example,
to identify sites of gene expression,
analyze the tissue distribution of transcription, identify and localize viral
infection, follow changes in specific
mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the
protocol by Lu and Gillett, elI
Vision.1_:169-176 (1994), using PCR-generated ~'P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded
human tissues were sectioned, deparaffinized, deproteinated in proteinase K
(20 g/ml) for 15 minutes at 37 °C, and
further processed for in situ hybridization as described by Lu and Gillett,
supra. A (33-P)UTP-labeled antisense
riboprobe was generated from a PCR product and hybridized at 55 °C
overnight. The slides were dipped in Kodak
NTB2'M nuclear track emulsion and exposed for 4 weeks.
~'P-Ribonrobe synthesis
6.0 ~1 (125 mCi) of 3'P-ITTP (Amersham BF 1002, SAQ,000 Cilmmol) were speed-
vacuum dried. To
112
AMENDED SHEET ~3ri

CA 02353799 2001-06-08
WO 00/37638 PCT/US99I28565
each tube containing dried "P-UTP, the following ingredients were added:
2.0 ul Sx transcription buffer
1.0 ul DTT (100 mM)
2.0 ~cl NTP mix (2.5 mM: 10 ~cl each of 10 mM GTP, CTP & ATP + 10 pl H,O)
1.0 ul UTP (50 pM)
1.0 ~1 RNAsin
1.0 ~I DNA template ( 1 ~cg)
1.0 E.cl HZO
1.0 uI RNA polymerase (for PCR products T3 = AS, T7 = S, usually}
The tubes were incubated at 37°C for one hour. A total of 1.0 pl RQ1
DNase was added, followed by
incubation at 37°C for 15 minutes. A total of90 ~l TE (10 mM Tris pH
7.6/1 mM EDTA pH 8.0) was added, and
the mixture was pipetted onto DE81 paper. The remaining solution was loaded in
a MICROCON-50 T"'
ultrafiltration unit, and spun using program 10 (6 minutes). The filtration
unit was inverted over a second tube and
spun using program 2 (3 minutes). After the final recovery spin, a total of
100 ~l TE was added, then 1 ~1 of the
final product was pipetted on DE81 paper and counted in 6 ml of BIOFLUOR
IIT"'.
The probe was run on a TBE/urea gel. A total of 1-3 ~I of the probe or 5 ul of
RNA Mrk III was added
to 3 ~1 of loading buffer. After heating on a 95°C heat block for three
minutes, the gel was immediately placed
on ice. The wells of gel were flushed, and the sample was loaded and run at
180-250 volts for 45 minutes. The
gel was wrapped in plastic wrap (SARANT"' brand) and exposed to XAR film with
an intensifying screen in a -
70°C freezer one hour to overnight.
"P-Hvbridization
A. Pretreatment of frozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed
at room temperature for
5 minutes. The trays were placed in a 55 °C incubator for five minutes
to reduce condensation. The slides were
fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and
washed in 0.5 x SSC for 5 minutes, at
room temperature (25 ml 20 x SSC + 975 ml SQ Hz0). After deproteination in 0.5
pg/ml proteinase K for 10
minutes at 37°C (12.5 ~l of 10 mg/ml stock in 250 ml prewarmed RNAse-
free RNAse buffer), the sections were
washed in 0.5 x SSC for 10 minutes at room temperature. The sections were
dehydrated in 70%, 95%, and 100%
ethanol, 2 minutes each.
B. Pretreatment of para~n-embedded sections
The slides were deparaffinized, placed in SQ H,O, and rinsed twice in 2 x SSC
at room temperature, for
5 minutes each time. The sections were deproteinated in 20 ,ug/ml proteinase K
(500 ~I of 10 mg/ml in 250 ml
RNase-free RNase buffer; 37 °C, 15 minutes) for human embryo tissue, or
8 x proteinase K ( 100 pl in 250 ml Rnase
buffer, 37°C, 30 minutes) for formaIin tissues. Subsequent rinsing in
0.5 x SSC and dehydration were performed
as described above.
113

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G ~ "~ ,.~ ~ ~ ~, J
3'!. 07 2000' ~9~~,°,~3"~'-:~,?° ~~°:,~ ~'856~': .. ....
.. P~ DES.CPAMD
. . . . . . . . . ..
. . . . . ..
..
.. ... .. . . .. ..
C. Preltybridization
The slides were laid out in a plastic box lined with Box buffer (4 x SSC, SO~o
formamide) - saturated filter
paper. The tissue was covered with 50 ~1 of hybridization buffer (3.75 g
dextran sulfate + 6 ml SQ H20), vortexed,
aad heated in the microwave for 2 minutes with the cap loosened. After cooling
on ice,18.75 ml fora~amide, 3.75
ml 20 x SSC, and 9 ml SQ HZO were added, and the tissue was vartexed well and
incubated at 42°C for 1-4 hours.
D. Hybridizasion
1.0 x 106 cpm probe and 1.0 ~1 tRNA (50 mglml stock) per slide were heated at
95 °C for 3 minutes. The
slides were cooled on ice, and 48 ~1 hybridization buffer was added per slide.
After vortexing, 50 ul ~P mix was
added to 50 ~d prehybridization on the slide. The slides were incubated
overnight at 55°C.
E. Washes
Washing was done for 2z 10 minutes with 2xSSC, EDTA at room temperature (400
ml 20 x SSC + 16 ml
0.25 M EDTA, Vt=4L), followed by RNAseA treatment at 37 °C for 30
minutes (500 ~cl of 10 mglml in 250 ml
Rnase buffer = 20 ~cg/ml), The slides were washed 2 x10 minutes with 2 x SSC,
EDTA at room temperature. The
stringency wash conditions were as follows: 2 hours at 55 °C, 0.1 x
SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA,
vt-4L).
F. Oligonucleotides
In situ analysis was performed on 5 of the DNA sequences disclosed herein. The
oligonucleotides
employed for these analyses are as follows:
(1) DNA30879-1152 fPR02071
pl:
i 5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC TCC TGC GCC TTT CCT GAA CC-3' (SEQ m
N0:70)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GAC CCA TCC TTG CCC ACA GAG-3' (SEQ m
N0:71)
(2) DNA33089-1132 (PR0221 )
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC TGT GCT TTC ATT CTG CCA GTA-3' (SEQ m
N0:72)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA GGG TAC AAT TAA GGG GTG GAT 3' (SEQ m
N0:73)
(3) DNA3322I-1133 (PR0224)
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GCA GCG ATG GCA GCG ATG AGG-3' (SEQ ID
N0:74)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CAG ACG GGG CAG CAG GGA GTG-3' (SEQ m
N0:75)
114
°~Y, ~ ~ ..a.~ ~; '."'.i~.
,Pnnte~ ~fi 08 2000' AMENDED SHEET ,;,

.~ x~>_y , ~~.,~".,:~,wt.:~ -02353799~2001-06-08 r"'' .#°"~L'~!>r, ;r,
..g ~.;~
'81 0~.200D"cg~J~~,.,.,-,.,,:-.~,~~a,285fi5:' ~~ ~..~ .~ ~ P~DESCPAMD
~a..w..~,a~<-:....~w..r,...>.i.:~r:.. 4:~fe~.~.r;~Wkf-.ti<~~s~.xk:-
.~a:'°~a,/,~..,s.<.-~:.as(~:.J ~ ~ ~ ~~!~:r;sa~ m,s »~» __ ~,x.>
t ~ d_
~ ~ ! ~ ~ ~ ~ ~ ~
~ r 1 ~ 1 ~ ~ ~ ~ ~
~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~ ~ ~~ ~~~ r~ ~ ~ ~~ ~"
(4) DNA40628-1216 (PR0301)
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC GAG TCC TTC GGC GGC TGT T 3' (SEQ 1D
N0:76)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CGG GTG CTT TTG GGA TTC GTA-3' (SEQ ID
N0:77)
(5) 17NA45416-1251 (PR0362)
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC CTC CAA GCC CAC AGT GAC AA-3' (SEQ ID
N0:78)
p2:
1 O 5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA CCT CCA CAT TTC CTG CCA GTA-3' (SEQ
ID N0:79)
G. Results
In situ analysis was performed on the above 5 DNA sequences disclosed herein.
The results from these
analyses are as follows:
(1) DNA30879-1152 (PR0207) (Apo2L Homoloe)
Low-level expression was observed over a chondrosarcoma, and over one other
soft-tissue sarcoma. All
other tissues were negative.
Human fetal tissues examined (E12-E16 weeks) included: placenta, umbilical
cord, liver, kidney, adrenals,
thyroid, lungs, heart, great vessels, esophagus, stomach, small intestine,
spleen, thymus, pancreas, brain, eye, spinal
cord, body wall, pelvis and lower limb.
> Adult tissues examined included: kidney (normal and end-stage), adrenal,
myocardium, spleen, lymph
node, pancreas, lung, skin, eye (including retina), bladder, and liver
(normal, cirrhotic, acute failure).
Non-human primate tissues examined included:
Chimp tissues: salivary gland, stomach, thyroid, parathyroid, tongue, thymus,
ovary, and lymph
node.
Rhesus monkey tissues: cerebral cortex, hippocampus, cerebellum, penis
(2) 1JNA33089-1132 LPR0221) (1 TM receptor)
Specific expression was observed over fetal cerebral white and grey matter, as
well as over neurones in
the spinal cord. The probe appears to cross react with rat. Low level
expression was seen over cerebellar neurones
in adult rhesus brain All other tissues were negative.
Fetal tissues examined (E12-E16 weeks) included placenta, umbilical cord,
liver, kidney, adrenals, thyroid,
lungs, heart, great vessels, esophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord,
body wall, pelvis and lower limb.
Adult tissues examined included: liver, kidney, adrenal, myocardium, aorta,
spleen, lymph node, pancreas,
115
AMENDED SHEET ~~=

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/Z8565
lung, skin, cerebral cortex (rm), hippocampus (rm), cerebellum (rm), penis,
eye, bladder, stomach, gastric
carcinoma, colon, colonic carcinoma, and chondrosarcoma; also acetominophen
induced liver injury and hepatic
cirrhosis.
(3) DNA33221-1133 (PR0224) (LDLR homology - 1 TMl
S Observed expression was limited to vascular endothelium in fetal spleen,
adult spleen, fetal liver, adult
thyroid and adult lymph node (chimp). Additional site of expression was seen
in the developing spinal ganglia.
All other tissues were negative.
Human fetal tissues examined (E 12-E 16 weeks) included: placenta,
umbilicalcord, liver, kidney, adrenals,
thyroid, lungs, heart, great vessels, esophagus, stomach, small intestine,
spleen, thymus, pancreas, brain, eye, spinal
cord, body wall, pelvis and tower limb.
Adult tissues examined included: kidney, (normal and end-stage), adrenal,
myocardium, aorta, spleen,
lymph node, pancreas, lung, skin, eye (including retina), bladder, and liver
(normal, cirrhotic, acute failure).
Non-human tissues examined included:
Chimp tissues: salivary gland, stomach, thyroid, parathyroid, skin, thymus,
ovary, lymph node.
Rhesus monkey tissues: cerebral cortex, hippocampus, cerebellum, penis.
(4) DNA40628-1216 (PR0301) (CD22 homoloe (JAM hloe, A33 AQ hlo~)
Expression in inflamed human tissues (psoriasis, IBD, inflamed kidney,
in,Jlamed lung, hepatitis, normal tonsil,
adult and chimp multiblocks):
Expression was evaluated in predominantly inflamed human tissue with a few
normal human and non-
human primate tissues. Expression was seen in every epithelial structure
evaluated including the mucosal
epithelium ofthe colon, bronchial large airway epithelium, oral mucosa
(tongue), tonsillar crypt mucosa, placental
mucosa, prostatic mucosa, glandular stomach mucosa, epithelial cells of thymic
Hassall's corpuscles, hepatocytes,
biliary epithelium, and placental epithelium. The only evidence of expression
outside of an epithelial structure was
weak low, inconsistent expression in the germinal centers of follicles in a
tonsil with reactive hyperplasia.
2S In non-human primate tissues the following was observed:
Chimp tissues: weak diffuse expression was observed in the epidermis of the
tongue epithelium; in the
thymus, weak specific expression was seen in thymic epithelium of Hassall's
corpuscles; in the stomach, mild
diffuse expression was observed in the epithelium of the glandular mucosa.
In human tissues: In the liver (multiblock including: chronic cholangitis,
lobular hyperplasia,
acetominophen toxicity): there was diffuse low to moderate expression in
hepatocytes and biliary epithelium.
Expression was most prominent in perilobular/periportal hepatocytes. It was
most prominent in biliary eptithelium
in sections of the liver with chronic sclerosing choiangitis. Expression was
not present in all samples present; this
may reflect sample quality more than expression variability.
In psoriasis: weak expression in the epidermis was seen.
In the lung with chronic interstitial pneumonia or chronic bronchitis: low
diffuse expression was seen in
116

CA 02353799 2001-06-08
WO 00137638 PCT/US99/28565
the mucosal epithelium of large airways; weak diffuse expression was also seen
in alveolar epithelium. There was
no expression in the epithelium of the submucosal glands of
bronchi/bronchioles.
In placenta: there was moderate diffuse expression in placental epithelium.
In the prostate: there was low diffuse expression in prostatic epithelium.
In the gall bladder: there was moderate diffuse expression in the mucosal
epithelium.
In the tonsil with reactive hyperplasia: high diffuse expression was seen in
the epithelium of the tonsillar
mucosa and crypts; the signal was highest in the mucosal cells which line the
tonsillar crypts. There was weak
inconsistent diffuse expression in the germinal centers of cortical follicles
(B lymphocyte areas); however, in no
other tissue evaluated with lymphoid structures or lymphocytic inflammation
was there any expression in B
lymphocytes.
In the colon with inflammatory bowel disease and polyp/adenomatous changes:
low expression was
observed in the mucosal epithelium; expression was greatest in the villi tips.
In the one specimen with a polyp,
there was no evidence of increased expression ofthe dysplastic epithelium
ofthe polyp as compared to the adjacent
mucosa. There was no apparent expression in reactive mucosal lymphoid tissue
that was present in many of the
sections.
(5) DNA45416-1251 1PR0362) (I~ domain homology)
Expression in in,Jlamed human tissues (psoriasis, IBD, inflamed kidney,
inflamed lung, hepatitis, normal tonsil,
adult and chimp multiblocks):
The expression of this novel protein was evaluated in a variety of human and
non-human primate tissues
and was found to be highly restricted. Expression was present only in alveolar
macrophages in the lung and
Kupffer cells of the hepatic sinusoids. Expression in these cells was
significantly increased when these distinct cell
populations were activated. Although these two subpopulations of tissue
macrophages are located in different
organs, they have similar biological functions. Both types of these phagocytes
act as biological filters to remove
material from the blood stream or airways including pathogens, senescent cells
and proteins and both are capable
of secreting a wide variety of important proinflammatory cytokines.
In inflamed lung (seven patient samples), expression was prominent in reactive
alveolar macrophage cell
populations defined as large, pale often vacuolated cells present singly or in
aggregates within alveoli and was weak
to negative in normal, non-reactive macrophages (single scattered cells of
normal size). Expression in alveolar
macrophages was increased during inflammation when these cells were both
increased in numbers and size
(activated). Despite the presence of histocytes in areas of interstitial
inflammation and peribronchial lymphoid
hyperplasia in these tissues, expression was restricted to alveolar
macrophages. Many of the inflamed lungs also
had some degree of suppurative inflammation; expression was not present in
neutrophilic granulocytes.
In liver, there was strong expression in reactive/activated Kupffer cells in
livers with acute centrilobular
necrosis (acetominophen toxicity) or fairly marked periportal inflammation.
However, there was weak or no
expression in Kupffer cells in normal liver or in liver with only mild
inflammation or mild to moderate lobular
hyperplasia/hypernophy. Thus, as in the lung, there was increased expression
in activated/reactive cells.
117

CA 02353799 2001-06-08
WO 00/37638 PCT/US99l28565
There was no expression of this molecule in histiocytes/macrophages present in
the inflammed bowel,
hyperplastic/reactive tonsil or normal lymph node. The lack of expression in
these tissues which all contain
histiocytic inflammation or resident macrophage populations strongly supports
restricted expression to the unique
macrophage subset populations defined as alveolar macrophage and hepatic
Kupffer cells.
Human tissues evaluated which had no detectable expression included:
infammatory bowel disease (seven
patient samples with moderate to severe disease), tonsil with reactive
hyperplasia, peripheral lymph node, psoriatic
skin (two patient samples with mild to moderate disease), heart, and
peripheral nerve.
Chimp tissues evaluated which had no detectable expression included: tongue,
stomach, and thymus.
EXAMPLE 16
Use of PR0179, PR0207, PRO320, PR0219, PR0221, PR0224 PR0328 PR0301 PR0526
PR0362
PR0356, PR0509 or PR0866 as a Hybridization Probe
The following method describes use of a nucleotide sequence encoding PR0179,
PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 as a
hybridization probe.
DNA comprising the coding sequence of full-length or mature PR0179, PR0207,
PR0320, PR0219,
PR0221, PR0224, PR0328, PR030I, PR0526, PR0362, PR0356, PR0509 or PR0866 (as
shown in Figure 1,
3, 5, 7, 9, I 1, 13, 15, 17, 19, 21, 23, and 25, respectively, SEQ ID NOS: 1,
6, 9, 14, 19, 24, 29, 34, 42, 47, 54, 59,
and 61, respectively) or a fragment thereof is employed as a probe to screen
for homologous DNAs (such as those
encoding naturally-occurring variants of PR0179, PR0207, PR0320, PR02I9,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866) 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 probe derived from
the gene encoding a PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptide to the filters is performed in a solution of 50% formamide,
Sx SSC, 0.1% SDS, 0.1 % sodium
pyrophosphate, 50 mM sodium phosphate, pH 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 0.1 x SSC
and 0. I % SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence can then
be identified using standard techniques known in the art.
EXAMPLE 17
Expression of PR0179 PR0207 PR0320 PR0219 PR0221 PR0224 PR0328 PR0301 PR0526
PR0362, PR0356, PR0509 or PR0866 in E. coli
This example illustrates preparation of an unglycosylated form of PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 by
recombinant
expression in E. coli.
118

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
The DNA sequence encoding PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 trp promoter, a poly-His leader (including the first six
STII codons, poly-His sequence, and
enterokinase cleavage site), the PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
IO PR0526, PR0362, PR0356, PR0509 or PR0866 coding region, lambda
transcriptional terminator, and an argU
gene.
The ligation 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
1S 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
20 pellet obtained by the centrifugation can be solubilized using various
agents known in the art, and the solubilized
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 protein can then be purified using a metal chelating column
under conditions that allow tight
binding of the protein.
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
25 PR0356, PR0509 or PR0866 may be expressed in E. coli in a poly-His tagged
form, using the following
procedure. The DNA encoding PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 is initially amplified using selected
PCR primers. The primers
will contain restriction enzyme sites which correspond to the restriction
enryme sites on the selected expression
vector, and other useful sequences providing for efficient and reliable
translation initiation, rapid purification on
30 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 galE rpoHts(htpRts) clpP(lacIq). Transformants are first
grown in LB containing 50
mg/m1 carbenicillin at 30°C with shaking until an ODboo of 3-5 is
reached. Cultures are then diluted 50-100 fold
into CRAP media (prepared by mixing 3.57 g (NH4),SO,, 0.71 g sodium
citrate~2H20, 1.07 g KCI, 5.36 g Difco
35 yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 110
mM MPOS, pH 7.3, 0.55% (w/v) glucose
and 7 mM MgSO,) and grown for approximately 20-30 hours at 30 °C with
shaking. Samples are removed to verify
expression by SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen
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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 sulfite and sodium
tetrathionate is added to make final
concentrations of 0.1 M 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 1 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 R I /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
AZx° 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 m isfolded foams of proteins
from the desired form, the reversed phase step also removes endotoxin from the
samples.
Fractions containing the desired folded PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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.
PR0207, PR0224, and PR0301 were successfully expressed in E. coli in a poly-
His tagged form by the
above procedure.
EXAMPLE 18
Expression of PR0179, PR0207, PR0320, PR0219, PR0221. PR0224, PR0328, PR0301,
PR0526
PR0362, PR0356. PR0509 or PR0866 in mammalian cells
This example illustrates preparation of a potentially glycosylated form of
PR0179, PR0207, PR0320,
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CA 02353799 2001-06-08
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PR0219, PR022 I , PR0224, PR0328, PR0301, PR0526, PR0362, PR0356,PR0509 or
PR0866 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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362,
PR03S6, PR0509 or PR0866 DNA is ligated into pRKS with selected restriction
enrymes to allow insertion of
the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PROS09 or PR0866 DNA using ligation methods such as described in Sambrook et
al., supra. The resulting vector
is called pRKS-PR0179,pRK5-PR0207,pRK5-PR0320,pRKS-PR0219,pRK5-PR0221,pRK5-
PR0224, pRKS
PR0328, pRKS-PR0301, pRKS-PR0526, pRKS-PR0362, pRKS-PR0356, pRKS-PR0509 or
pRKS-PR0866,
respectively.
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 ~g pRKS-PRO 179,
pRKS-PR0207, pRKS-PR0320,
pRKS-PR0219, pRKS-PR0221,pRKS-PR0224,pRK5-PR0328,ARKS-PR0301,pRKS-PR0526,pRK5-
PR0362,
ARKS-PR0356, pRKS-PROS09 or pRKS-PR0866 DNA is mixed with about 1 ~g DNA
encoding the. VA RNA
gene [Thimmappaya et al., Cell 31:543 ( 1982)] and dissolved in 500 ~1 of 1 mM
Tris-HCI, 0.1 mM EDTA, 0.227
M CaClz. To this mixture is added, dropwise, 500 ~l of 50 mM HEPES (pH 7.35),
280 mM NaCI, 1.5 mM NaPO,,
and a precipitate is allowed to form for 10 minutes at 2S°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 ~cCi/ml'SS-cysteine and 200
~Cilml'SS-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 the
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide. T'he cultures containing transfected cells may
undergo further incubation (in
serum free medium) and the medium is tested in selected bioassays.
In an alternative technique, PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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 tcg pRKS-PR0179, pRKS-PR0207, pRKS-
PR0320, pRKS-PR0219,
pRKS-PR0221, pRKS-PR0224,pRK5-PR0328,pRK5-PR0301,pRKS-PR0526,pRKS-PR0362,pRK5-
PR0356,
pRKS-PROS09 or pRKS-PR0866 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 ~glml bovine insulin and
0.1 ~eg/ml bovine transferrin. After
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about four days, the conditioned media is centrifuged and filtered to remove
cells and debris. The sample
containing expressed PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 can then be concentrated and purified by any
selected method, such as
dialysis and/or column chromatography.
In another embodiment, PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 can be expressed in CHO cells. The
pRKS-PR0179, pRKS-
PR0207, pRKS-PR0320, pRKS-PR0219, pRKS-PR0221, pRKS-PR0224, pRKS-PR0328, pRKS-
PR0301,
pRKS-PR0526, pRKS-PR0362, ARKS-PR0356, pRKS-PR0509 or pRKS-PR0866 can be
transfected into CHO
cells using known reagents such as CaPO, or DEAF-dextran. As described above,
the cell cultures can be
incubated, and the medium replaced with culture medium (alone) or medium
containing a radiolabel such as'SS-
methionine. After determining the presence of a PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide
can then be
concentrated and purified by any selected method.
Epitope-tagged PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR030I,
PR0526,
PR0362, PR0356, PR0509 or PR0866 may also be expressed in host CHO cells. The
PR0179, PR0207,
PR0320, PR0219, PR022 l , PR0224, PRO328, PR0301, PR0526, PR0362, PR0356,
PR0509 or PR0866 may
be subcloned out ofthe pRKS vector. The subclone insert can undergo PCR to
fuse in frame with a selected epitope
tag such as a poly-His tag into a Baculovirus expression vector. The poly-His
tagged PRO 179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 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
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 can then be concentrated and purified by any selected method,
such as by Ni2'-chelate affinity
chromatography.
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362,
PR0356, PR0509 or PR0866 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 IgG 1 constant region
sequence containing the hinge, CH2 and
CH2 domains and/or as 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 y Unit 3. I6, John Wiley
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CA 02353799 2001-06-08
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and Sons ( t 997). 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 in
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~ (Quiagen),
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 a water
bath and mixed by
vortexing. The contents are pipetted into a centrifuge tube containing 10 m Is
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 ~m
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 and pH is determined. On day 1, the
spinner is sampled and sparging
2U 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 drops below 70%, the cell culture is harvested
by centrifugation and filtering through
a 0.22 ~m filter. The filtrate is 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 Z'-
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 2'-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
equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly
purified protein is subsequently desalted into a storage buffer containing 10
mM Hepes, 0.14 M NaCI and 4%
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
has been equilibrated in 20 mM
Na phosphate buffer, pH 6.8. After loading, the column is washed extensively
with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is immediately
neutralized by collecting 1 ml fractions
into tubes containing 275 ,ul of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into
storage buffer as described above for the poly-His tagged proteins. The
homogeneity is assessed by SDS
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polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation.
PRO 179, PR0320, PR0219, PR0221, PR0224, PR0328, PR030I , PR0356, PR0509, and
PR0866 were
stably expressed in CHO cells by the above described method. In addition,
PR0224, PR0328, PR0301, and
PR0356 were expressed in CHO cells by the transient expression procedure.
EXAMPLE 19
Expression of PRO 179, PR0207 PR0320 PR0219 PR0221 PR0224 PR0328 PR0301 PR0526
PR0362, PR0356, PR0509 or PR0866 in Yeast
The following method describes recombinant expression of PR0179, PR0207,
PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 in
yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PROI79,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 from the ADH2/GAPDH promoter. DNA encoding PR0179, PR0207, PR0320,
PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 and the
promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224; PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866. For
secretion, DNA encoding PRO 179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 can be cloned into the selected plasmid,
together with DNA encoding the
ADH2/GAPDH promoter, a native PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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 I 79, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362,
PR0356, PR0509 or PR0866.
Yeast cells, such as yeast strain AB 110, can then be transformed with the
expression plasmids described
above and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by
precipitation with 10% trichloroacetic acid and separation by SDS-PAGE,
followed by staining of the gels with
Coomassie Blue stain.
Recombinant PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526,
PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 may further be purified using selected column
chromatography resins.
EXA- MPLE 20
Expression of PR0179 PR0207 PR0320 PR0219 PR0221 PR0224 PR0328 PR0301 PR0526
PR0362 PR0356 PR0509 or PR0866 in Baculovirus-Infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected
insect cells.
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The sequence coding for PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR052b, PR0362, PR0356, PR0509 or PR0866 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 PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 or
the desired portion
ofthe coding sequence ofPR0179, PR0207, PR0320, PR02I9, PR0221, PR0224,
PR0328, PR0301, PR0526,
PR0362, PR0356, PR0509 or PR0866 (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 5' primer may
incorporate flanking (selected)
restriction enzyme sites. The product is then digested with those selected
restriction enzymes and subcloned into
the expression vector.
Recombinant baculovirus is generated by co-transfecting the above plasmid and
BaculoGoIdT"'virus DNA
(Pharmingen) into Spodopterafrugiperda ("Sf9") cells (ATCCCRL 1711 )using
lipofectin (commercially available
from G1BC0-BRL). After 4 - 5 days of incubation at 28°C, the released
viruses are harvested and used for further
amplifications. Viral infection and protein expression are performed as
described by O'Reilley et al., Baculovirus
expression vectors: A Laboratory Manual Oxford: Oxford University Press
(1994).
Expressed poly-His taggedPR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 can then be purified, for example, by
Ni'-'-chelate affinity
chromatography as follows. Extracts are prepared from recombinant virus-
infected Sf~ 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 MgCIZ; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), 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 mm 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 AZa° 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
A28° 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,°-tagged PRO 179, PR0207,
PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866, respectively,are
pooled and dialyzed against
loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR0179, PR0207,
PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 can
be performed using
known chromatography techniques, including for instance, Protein A or protein
G column chromatography.
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Following PCR amplification, the respectivecodingsequences are subcloned into
a baculovirus expression
vector (pb.PH.IgG for IgG fusions and pb.PH.His.c for poly-His tagged
proteins), and the vector and Baculogold~
baculovirus DNA (Pharmingen) are co-transfected into 105 Spodoptera frugiperda
("Sf9") cells (ATCC CRL
1711 ), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are
modifications of the commercially available
baculovirus expression vector pVL 1393 (Pharmingen), with modified polylinker
regions to include the His or Fc
tag sequences. The cells are grown in Hink's TNM-FH medium supplemented with
10% FBS (Hyclone). Cells
are incubated for 5 days at 28°C. The supernatant is harvested and
subsequently used for the first viral
amplification by infecting Sfi7 cells in Hink's TNM-FH medium supplemented
with 10% FBS at an approximate
multiplicity of infection (MOI) of 10. Cells are incubated for 3 days at
28°C. The supernatant is harvested and
the expression of the constructs in the baculovirus expression vector is
determined by batch binding of 1 ml of
supernatant to 25 ml ofNi Z'-NTA beads (QIAGEN) for histidine tagged proteins
or Protein-A Sepharose CL-4B
beads (Pharmacia) for 1gG tagged proteins followed by SDS-PAGE analysis
comparing to a known concentration
of protein standard by Coomassie blue staining.
The first viral amplification supernatant is used to infect a spinner culture
(500 ml) of Sfi9 cells grown in
ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells
are incubated for 3 days at
28 °C. The supernatant is harvested and filtered. Batch binding and SDS-
PAGE analysis is repeated, as necessary,
until expression of the spinner culture is confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) is harvested by
centrifugation to remove
the cells and filtered through 0.22 micron filters. For the poly-His tagged
constructs, the protein construct is
purified using a Ni Z*-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 equilibration buffer and the
protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing
10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine
(Pharmacia) column and stored
at -80°C.
Immunoadhesin (Fc containing) constructs ofproteins are purified from the
conditioned media as follows.
The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which
has 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 ml 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 of the proteins is verified by
SDS polyacrylamide gel (PEG) electrophoresis and N-terminal amino acid
sequencing by Edman degradation.
PR030I, PR0362, PR0356, PR0509 and PR0866 were expressed in baculovirus
infected Sf9 insect
cells.
Alternatively, a modified baculovirus procedure may be used incorporating high-
S cells. In this procedure,
the DNA encoding the desired sequence is amplified with suitable systems, such
as Pfu (Stratagene), or fused
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upstream (5'-of) of an epitope tag contained with 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 pIE I-1
(Novagen). The pIE 1-I and p1E 1-2 vectors
are designed for constitutive expression of recombinant proteins from the
baculovirus ie 1 promoter in stably-
transformed insect cells ( I ). The plasmids differ only in the orientation of
the multiple cloning sites and contain all
promoter sequences known to be important for iel-mediated gene expression in
uninfected insect cells as well as
the hr5 enhancer element. pIE 1-1 and pIE 1-2 include the translation
initiation site and can be used to produce fusion
proteins. Briefly, the desired sequence or the desired portion of the sequence
(such as the sequence encoding the
extracellular domain of a transmembrane protein} is amplified by PCR with
primers complementary to the 5' and
3'regions. The 5' primer may incorporate flanking (selected) restriction
enryme sites. The product is then digested
with those selected restriction enzymes and subcloned into the expression
vector. For example, derivatives of
plEl-1 can include the Fc region of human IgG (pb.PH.IgG) or an 8 histidine
(pb.PH.His) tag downstream (3'-of)
the desired sequence. Preferably, the vector construct is sequenced for
confirmation.
High-5 cells are grown to a confluency of 50% under the conditions of,
27°C, no CO,, NO pen/strep. For
each 1 SO mm plate, 30,ug ofpIE based vector containing the sequence is mixed
with 1 ml Ex-Cell medium (Media:
Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media is
light sensitive)), and in a separate
tube, 100 ~I ofCellFectin (CeIIFECTIN (GibcoBRL # 10362-O 10) (vortexed to
mix)) is mixed with 1 ml ofEx-Cell
medium. The two solutions are combined and allowed to incubate at room
temperature for I S minutes. 8 ml of
Ex-Cell media is added to the 2ml of DNA/CeIIFECTIN mix and this is layered on
high-5 cells that have been
washed once with Ex-Cell media. The plate is then incubated in darkness for 1
hour at room temperature. The
DNA/CeIIFECTIN mix is then aspirated, and the cells are washed once with Ex-
Cell to remove excess
CeIIFECTIN, 30 m 1 of fresh Ex-Cell media is added and the cells are incubated
for 3 days at 28°C. The supernatant
is harvested and the expression of the sequence in the baculovirus expression
vector is determined by batch binding
of 1 ml of supernatent to 25 ml ofNi 2*-NTA beads (QIAGEN) for histidine
tagged proteins or Protein-A Sepharose
CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE analysis
comparing to a known
concentration of protein standard by Coomassie blue staining.
The conditioned media from the transfected cells (0.5 to 3 L) is harvested by
centrifugation to remove the
cells and filtered through 0.22 micron filters. For the poly-His tagged
constructs, the protein comprising the
sequence is purified using a Ni 2+-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 2'-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 48°C.
After loading, the column is washed with additional equilibration buffer and
the protein eluted with equilibration
buffer containing 0.25 M imidazole. The highly purified protein is then
subsequently desalted into a storage buffer
containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25
Superfine (Pharmacia) column
and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins are purified from the
conditioned media as follows.
The conditioned media is pumped onto a 5 ml Protein A column (Pharmacia) which
has been equilibrated in 20 mM
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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 I ml fractions
into tubes containing 275 ml 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 of the sequence is assessed
S by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation and other analytical
procedures as desired or necessary.
PRO 179, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, and PR0356 were
expressed using
the above baculovirus procedure employing high-5 cells.
EXAMPLE 21
Preparation of Antibodies that Bind PRO 179 PR0207 PR0320 PR0219 PR0221
PR0224, PR0328
PR0301, PR0526, PR0362. PR0356 PR0509 or PR0866
This example illustrates preparation of monoclonal antibodies which can
specifically bind PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866.
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 PR0179,
PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866
fusion proteins
containing PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 and cells expressing recombinant PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 lmmunochemical 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 ELISA assays to detect anti-PR0179, anti-PR0207, anti-
PR0320, anti-PR0219, anti-
PR0221, anti-PR0224, anti-PR0328, anti-PR0301, anti-PR0526, anti-PR0362, anti-
PR0356, anti-PR0509 or
anti-PR0866 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected with
a final intravenous injection of PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866. 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.1, available from ATCC, No. CRL 1597. The fusions
generate hybridoma cells which
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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
PR0179, PR0207, PR0320,
PR0219, PR022 I , PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866. Determination
of "positive" hybridoma cells secreting the desired monoclonal antibodies
against PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 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 179, anti-PR0207, anti-PR0320, anti-PR0219,
anti-PR0221, anti-PR0224, anti-
PR0328, anti-PR0301, anti-PR0526, anti-PR0362, anti-PR0356, anti-PR0509 or
anti-PR0866 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 ofantibody
to protein A or protein G can be employed.
EXAMPLE 22
Purification of PR0179 PR0207 PR0320 PR0219 PR0221 PR0224 PR0328 PR0301 PR0526
PR0362. PR0356, PR0509 or PR0866 Polvneptides Usine Saecific Antibodies
Native or recombinant PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptides may be purified by a
variety of standard techniques
in the art of protein purification. For example, pro-PROI79, pro-PR0207, pro-
PR0320, pro-PR0219, pro-
PR022I , pro-PR0224, pro-PR0328, pro-PR0301, pro-PR0526, pro-PR0362, pro-
PR0356, pro-PR0509 or pro-
PR0866 polypeptide, mature PRO 179, mature PR0207, mature PR0320,
maturePR0219,mature PR0221, mature
PR0224, mature PR0328, mature PR0301, mature PR0526, mature PR0362, mature
PR0356, mature PR0509
or mature PR0866 polypeptide, or pre-PR0179, pre-PR0207, pre-PR0320, pre-
PR0219, pre-PR0221, pre-
PR0224, pre-PR0328, pre-PR0301, pre-PR0526, pre-PR0362, pre-PR0356, pre-PR0509
or pre-PR0866
polypeptide is purified by immunoaffinity chromatography using antibodies
specific for the PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide of interest. In general, an immunoaffinity column is constructed
by covalentiy coupling the
anti-PR0179,anti-PR0207,anti-PR0320,anti-PR0219, anti-PR0221, anti-PR0224,
anti-PR0328, anti-PR0301,
anti-PR0526, anti-PR0362, anti-PR0356, anti-PR0509 or anti-PR0866 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,
monoclonalantibodies 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"" (Pharcnacia LKB Biotechnology). The
antibody is coupled to the resin,
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the resin is blocked, and the derivative resin is washed according to the
manufacturer's instructions.
Such an immunoaffinity column is utilized in the purification ofthe PR0179,
PR0207, PR0320, PR0219,
PR022 I , PR0224, PR0328, PR030I , PR0526, PR0362, PR0356, PR0509 or PR0866
polypeptide by preparing
a fraction from cells containing the PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR0356, PR0509 or PR0866 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
PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptide
containing a signal sequence may be secreted in useful quantity into the
medium in which the cells are grown.
A soluble PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526,
PR0362, PR0356, PR0509 or PR0866 palypeptide-containing preparation is passed
over the immunoaffinity
column, and the column is washed under conditions that allow the preferential
absorbance ofthe PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509
or PR0866
polypeptide (e.g., high ionic strength buffers in the presence of detergent).
Then, the column is eluted under
conditions that disrupt antibody/PROl79, antibody/PRO207, antibody/PR0320,
antibody/PR0219,
antibody/PR0221,
antibody/PR0224,antibody/PR0328,antibody/PR0301,antibody/PR0526,
antibody/PR0362,
antibody/PR0356, antibody/PR0509 or antibody/PR0866 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 the PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptide is collected.
EXAMPLE 23
Drug Screening
This invention is particularly useful for screening compounds by using PROI79,
PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 polypeptides
or a binding fragment thereof in any of a variety of drug screening
techniques. The PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301; PR0526, PR0362, PR0356, PR0509 or
PR0866 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 PR0179,
PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 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 a
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 polypeptide or a fragment and the agent being tested.
Alternatively, one can examine the
diminution in complex formation between the PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide and its target
cell or target receptors
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caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can affect
a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PROS26,
PR0362, PR0356,
PR0509 or PR0866 polypeptide-associated disease or disorder. These methods
comprise contacting such an agent
S with a PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PROS26, PR0362, PR03S6,
PR0509 or PR0866 polypeptide or fragment thereof and assaying (i) for the
presence of a complex between the
agent and the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PROS26, PR0362,
PR03S6, PROS09 or PR0866 polypeptide or fragment, or (ii) for the presence of
a complex between the PRO 179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR03S6, PROS09 or
PR0866 polypeptide or fragment and the cell, by methods well known in the art.
In such competitive binding
assays, the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PROS26, PR0362,
PR0356, PROS09 or PR0866 polypeptide or fragment is typically labeled. After
suitable incubation, the free
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PROS26,
PR0362, PR03S6,
PR0509 or PR0866 polypeptide or fragment is separated from that present in
bound fonm, and the amount of free
1S or uncomplexed label is a measure of the ability of the particular agent to
bind to the PRO 179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PROS26, PR0362, PR0356, PR0509 or
PR0866 polypeptide
or to interfere with the PR0179, PR0207, PR0320, PR0219, PR0221, PR0224,
PR0328, PR0301, PROS26,
PR0362, PR03S6, PR0509 or PR0866 polypeptide/cel) 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 PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224,
PR0328, PR0301, PR0526, PR0362, PR0356, PROS09 or PR0866 polypeptide, the
peptide test compounds are
reacted with the PRO I 79, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362,
2S PR0356, PR0509 or PR0866 polypeptide and washed. Bound PROI?9, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 poiypeptide
is detected by methods
well known in the art. Purified PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PROS26, PR0362, PR03S6, PR0509 or PR0866 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 a PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301,
PR0526, PR0362, PR03S6, PROS09 or PR0866 polypeptide specifically compete with
a test compound for
binding to the PR0179, PR0207, PR0320, PR02I9, PR0221, PR0224, PR0328, PR0301,
PROS26, PR0362,
3S PR03S6, PR0509 or PR0866 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 a PR0179, PR0207,
PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PROS26, PR0362, PR03S6, PROS09
or PR0866
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polypeptide.
EXAMPLE 24
Rational Dru~ Desien
The goal of rational drug design is to produce structural analogs of a
biologically active polypeptide of
interest (i.e., a PR0179, PR0207, PR0320, PR0219, PR022I, PR0224, PR0328,
PR0301, PR0526, PR0362,
PR0356, PR0509 or PR0866 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 PROI79, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301,
PR0526, PR0362,
PR0356, PR0509 or PR0866 polypeptide or which enhance or interfere with the
function of the PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptide in vivo (cf., Hodgson, Bio/TechnoloQV 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PR0179, PR0207,
PR0320, PR0219, PR0221,
PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide,
or of a PR0179,
PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 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 PR0179, PR0207, PR0320,
PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362, PR0356, PR0509 or
PR0866 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 PR0179, PR0207, PR0320, PR0219,
PR0221, PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 or PR0866 polypeptide may be gained by
modeling based on the
structure of homologous proteins. In both cases, relevant structural
information is used to design analogous
PR0179, PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526,
PR0362, PR0356,
PR0509 or PR0866 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 PR0179, PR0207,
PR0320, PR0219,
PR0221,PR0224,PR0328,PR0301,PR0526,PR0362,PR0356,PR0509orPR0866polypeptidemaybe
made
available to perform such analytical studies as X-ray crystallography. In
addition, knowledge of the PR0179,
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PR0207, PR0320, PR0219, PR0221, PR0224, PR0328, PR0301, PR0526, PR0362,
PR0356, PR0509 or
PR0866 polypeptide amino acid sequence provided herein will provide guidance
to those employing computer
modeling techniques in place of or in addition to x-ray crystallography.
EXAMPLE 25
In Vitro Antitumor Assav
The antiproliferative activity of the PR0179, PR0207, PR0320, PR0219, PR0221,
PR0224, PR0328,
PR0301, PR0526, PR0362, PR0356, PR0509 and PR0866 polypeptides was determined
in the investigational,
disease-oriented in vitro anti-cancer drug discovery assay of the National
Cancer Institute (NCI), using a
sulforhodamine B (SRB) dye binding assay essentially as described by Skehan et
al., J. Natl. Cancer Inst. _82:1107-
1112 (1990). The 60 tumor cell lines employed in this study ("the NCI panel"),
as well as conditions for their
maintenance and culture in vitro have been described by Monks et al., J. Natl.
Cancer Inst. 83:757-766 ( 1991 ).
The purpose of this screen is to initially evaluate the cytotoxic and/or
cytostatic activity of the test compounds
against different types of tumors (Monks et al., supra; Boyd, Cancer: Princ.
Pract. Oncol Update 3 10 :1-12
[ 1989]).
Z 5 Cells from approximately 60 human tumor cell lines were harvested with
trypsin/EDTA (Gibco), washed
once, resuspended in IMEM and their viability was determined. The cell
suspensions were added by pipet (100
pl volume) into separate 96-well microtiter plates. The cell density for the 6-
day incubation was less than for the
2-day incubation to prevent overgrowth. Inoculates were allowed a
preincubation period of24 hours at 37°C for
stabilization. Dilutions at twice the intended test concentration were added
at time zero in 100 pl aliquots to the
microtiter plate wells (1:2 dilution). Test compounds were evaluated at five
half log dilutions (1000 to 100,000-
fold). Incubations took place for two days and six days in a 5% CO= atmosphere
and I 00% humidity.
After incubation, the medium was removed and the cells were fxed in 0.1 ml of
10% trichloroacetic acid
at 40°C. The plates were rinsed five times with deionized water, dried,
stained for 30 minutes with 0.1 ml of 0.4%
sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsed four times
with I% acetic acid to remove
unbound dye, dried, and the stain was extracted for five minutes with 0.1 ml
of 10 mM Tris base
(tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD)
ofsulforhodamineB at 492 nm was measured
using a computer-interfaced, 96-well microtiter plate reader.
A test sample is considered positive if it shows at least 40% growth
inhibitory effect at one or more
concentrations. The results are shown in the following Table 4, where the
tumor cell type abbreviations are as
follows:
NSCL = non-small cell lung carcinoma; CNS = central nervous system
133

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Table 4
Compound Tumor Cell Tvpe Desienation
PRO 179 Leukemia CCRF-CEM
PR0179 Breast HS 578T
PR0179 NSCL SR
PRO 179 Breast NCI/ADR-RES
PR0179 Leukemia HL-60 (TB); SR
PR0179 NSCL HOP-62; NCI-H460
PRO 179 Breast MDA-N
PR0179 NSCL NCI-H522
PR0179 Colon COLO 205; HCC-2998
PRO 179 CNS SF-295
PRO 179 Breast MDA-MB-435
PRO 179 Prostate PC-3
PR0179 Leukemia MOLT-4
PR0179 Melanoma SK-MEL-5; SK-MEL-2
PR0179 Breast MDA-MD-435; T-47D
PR0179 Melanoma MALME-3M
PRO 179 NSCL NCI-H322
PR0179 Colon HCT-15
PRO 179 Ovarian OVCAR-3
PRO 179 NSCL NCI-H226
PR0179 Renal RXF-393
PR0207 Renal CAKI-1; RXF-393
PR0207 Leukemia MOLT-4; SR
PR0207 NSCL NCI-H322M; NCI-H522
PR0207 NSCL HOP-62
PR0207 Colon COLO 205
PR0207 Melanoma LOX IMVI
PR0207 Ovarian IGROV 1
PR0207 Renal ACHN
PR0207 Prostate PC-3
PR0207 Breast MDA-MB-231 /ATCC
PR0320 Leukemia CCRF-CEM; RPMI-8226
PR0320 NSCL HOP62; NCI H322M
PR0320 Colon HCT-116
PR0320 Renal SN 12C
PR0320 Breast MDA-N
PR0320 Ovarian OVCAR-3
PR0320 Melanoma MALME-3M
PR0219 Leukemia SR
PR0219 NSCL NCI-H5222
PR0219 Breast MCF7
PR0219 Leukemia K-562; RPMI-8226
PR0219 NSCL HOP-62; NCI-H322M
PR0219 NSCL NCI -H460
PR0219 Colon HT29; KM12; HCT-116
PR0219 CNS SF-539; U251
PR0219 Prostate DU-145
PR0219 Breast MDA-N
134

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Table 4 Continued
Compound Tumor Cell Type Desisnation
PR0219 Ovarian IGROV 1
PR0219 NSCL NCI-H226
PR0219 Leukemia MOLT-4
PR0219 NSCL AS49/ATCC; EKVX; NCI-H23
PR0219 Colon HCC-2998
PR0219 CNS SF-295; SNB-19
PR0219 Melanoma SK-MEL-2; SK-MEL-5
PR0219 Melanoma UACC-257; UACC-62
PR0219 Ovarian OCAR-4; SK-OV-3
PR0219 Renal 786-0; ACHN; CAKI-1; SN
12C
PR0219 Renal TK-10; UO-31
PR0219 Breast NCI/ADR-RES;BT-S49;T-47D
PR0219 Breast MDA-MB-43S
PR022 I Leukemia CCRF-CEM
PR0221 Leukemia MOLT-4
PR0221 NSCL HOP-62
PR022 l Breast M DA-N
PR0221 Leukemia RPMI-8226; SR
PR0221 NSCL NC I-H460
PR0221 Colon HCC-2998
PR0221 Ovarian IGROV 1
PR0221 Renal TK-10
PR0221 Breast MCF7
PR0221 Leukemia K-S62
PR0221 Breast MDA-MB-435
PR0224 Ovarian OVCAR-4
PR0224 Renal RXF 393
PR0224 Prostate DU-145
PR0224 NSCL HOP-62; NCI-H322M
PR0224 Melanoma LOX IMVI
PR0224 Ovarian OVCAR-8
PR0224 Leukemia SR
PR0224 NSCL NCI-H460
PR0224 CNS SF-295
PR0224 Leukemia RPMI-8226
PR0224 Breast BT-549
PR0224 Leukemia CCRF-CEM; LH-60 (TB)
PR0224 Colon HCT-116
PR0224 Breast MDA-MB-435
PR0224 Leukemia HL-60 (TB)
PR0224 Colon HCC-2998
PR0224 Prostate PC-3
PR0224 CNS U251
PR0224 Colon HCT-1 S
PR0224 CNS SF-S39
PR0224 Renal ACHN
PR0328 Leukemia RPMI-8226
SO PR0328 NSCL A549/ATCC; EKVX; HOP-62
135

CA 02353799 2001-06-08
WO 00/37638 PCT/US99/28565
Table 4 Continued
Compound Tumor Cell Tvpe Desisnation
PR0328 NSCL NCI-H23; NCI-H322M
PR0328 Colon HCT-15; KM12
PR0328 CNS SF-295; SF-539; SNB-19;
0251
PR0328 Melanoma M 14; UACC-257; UCAA-62
PR0328 Renal 786-0; ACHN
PR0328 Breast MCF7
PR0328 Leukemia SR
PR0328 Colon NCI-H23
PR0328 Melanoma SK-MEL-5
PR0328 Prostate DU-145
PR0328 Melanoma LOX IMVI
PR0328 Breast MDA-MB-435
PR0328 Ovarian OVCAR-3
PR0328 Breast T-47D
PR0301 NSCL NCI-H322M
PR0301 Leukemia MOLT-4; SR
PR0301 NSCL A549/ATCC; EKVX;
PR0301 NSCL NCI-H23; NCI-460; NCI-H226
PR0301 Colon COLO 205; HCC-2998;
PR0301 Colon HCT-15; KM12; HT29;
PR0301 Colon HCT-116
PR0301 CNS SF-268; SF-295; SNB-19
PR0301 Melanoma MALME-3M; SK-MEL-2;
PR0301 Melanoma SK-MEL-S;UACC-257
PR0301 Melanoma UACC-62
PR0301 Ovarian IGROVI; OVCAR-4
PR0301 Ovarian OVCAR-5
PR0301 Ovarian OVCAR-8; SKOOV-3
PR0301 Renal ACHN;CAKI-1;TK-10; UO-31
PR0301 Prostate PC-3; DU-145
PR0301 Breast NCI/ADR-RES; HS 578T
PR0301 Breast MDA-MB-435;MDA-N;T-47D
PR0301 Melanoma M 14
PR0301 Leukemia CCRF-CEM;HL-60(TB);K-562
PR0301 Leukemia RPMI-8226
PR0301 Melanoma LOX IMVI
PR0301 Renal 786-0; SN12C
PR0301 Breast MCF7; MDA-MB-231/ATCC
PR0301 Breast BT-549
PR0301 NSCL HOP-62
PR030I CNS SF-539
PR0301 Ovarian OVCAR-3
PR0526 NSCL HOP-62; NCI-H322M
PR0526 Colon HCT-116
PR0526 Melanoma LOX IMVI; SK-MEL-2
136

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Table 4 Continued
Compound Tumor Cell Tvne Desi nation
PR0526 Ovarian OVCAR-3
PR0526 Prostate PC-3
PR0526 NSCL NCI-H226
PR0526 CNS SF-539
PR0526 Renal CAKI-1; RXF 393
PR0362 NSCL NCI-H322M
PR0362 Colon HCT-116
PR0362 CNS SF-295
PR0362 Melanoma LOX IMVI
PR0362 Leukemia MOLT-4; RPMI-8226; SR
PR0362 Colon COLD 205
PR0362 Breast HS 578T; MDA-N
PR0362 Prostate PC-3
PR0362 Leukemia HL-60 (TB); K-562
PR0362 NSCL EKVX; NCI-H23
PR0362 Colon HCC-2998
PR0362 CNS U251
PR0362 Melanoma UACC-257; UACC-62
PR0362 Ovarian OVCAR-8
PR0362 Breast T-47D
PR0362 NSCL NCI-H522
PR0362 Renal RXF 393; UO-31
PR0362 Breast MDA-MB-435
PR0362 NSCL HOP-62; NCI-H522
PR0362 Colon KM 12
PR0362 Melanoma MALME-3M; SK-MEL-2
PR0362 Melanoma SK-MEL-28; SK-MEL-5
PR0362 Ovarian OVCAR-3; OVCAR-4
PR0362 Breast MCF7
PR0356 Leukemia CCRF-CEM; MOLT-4; SR
PR0356 NSCL NCI-H23; NCI-H322M
PR0356 NSCL NCI-H460
PR0356 NSCL A549/ATCG
PR0356 Colon HCT-116; HCT-15; H29;
KM 12
PR0356 CNS SF-268; SF-295; SF-539
PR0356 CNS SNB-19
PR0356 Melanoma LOX IMVI; SK-MEL-5
PR0356 Melanoma UACC-257
PR0356 Melanoma UACC-62
PR0356 Ovarian OVCAR-8; OVCAR-5
PR0356 Renal SN 12C
PR0356 Prostate DU-145
PR0356 Leukemia K-562
PR0356 Leukemia HL-60 (TB)
PR0356 Breast MDA-N
PR0356 NSCL EKVX; HOP-92
PR0356 Colon COLO 205; SW-620
PR0356 CNS SNB-75; 0251
137

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Table 4 Continued
Compound Tumor Cell Tvoe Designation
PR0356 Melanoma M 14
PR0356 Ovarian IGROV 1; OVCAR-4;
PR0356 Renal RXF 393
PR0356 Breast BT-549
PR0356 NSCL NCI-H226
PR0356 Breast MDA-MB-435
PR0356 NSCL HOP-62
10PR0356 Renal UO-31
PR0356 Leukemia RPMI-8226
PR0509 Leukemia K-562; MOLT-4
PR0509 NSCL HOP-92
PR0509 Colon S W-620
15PR0509 CNS 0251
PR0509 Melanoma SK-MEL-28
PR0509 Renal A498
PR0509 Breast MDA-MB-435
PR0509 Leukemia RPMI-8226
20PR0509 Melanoma SK-MEL-2
PR0509 Ovarian OVCAR-3
PR0509 Renal CAKI-1
PR0866 Leukemia HL-60 (TB); MOLT-4; SR
PR0866 NSCL HOP-62
25PR0866 NSCL HOP-92
PR0866 Colon KM 12
PR0866 CNS SF-295
PR0866 Ovarian IGROV 1
PR0866 Breast MDA-MB-435
30PR0866 Melanoma LOX IMVI
Deposit of Material
The following
materials have
been deposited
with the American
Type Culture
Collection, 10801
University Btvd.,, VA 20110-2209, USA
Manassas (ATCC):
Material ATCC Dep. No. Deposit Date
35DNA16451-1078 209281 September 18, 1997
DNA30879-1152 209358 October 10, 1997
DNA32284-1307 209670 March I1, 1998
DNA32290-1164 209384 October 17, 1997
DNA33089-1132 209262 September 16, 1997
40DNA33221-1133 209263 September 16, 1997
DNA40587-1231 209438 November 7, 1997
DNA40628-1216 209432 November 7, 1997
DNA44184-1319 209704 March 26, 1998
138

CA 02353799 2001-06-08
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DNA45416-1251 209620 February 5,
1998
DNA47470-1130-P1 209422 October 28,
1997
DNA53971-1359 209750 April 7, 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
IO 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 U.S.C. ~
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.
The foregoing written specification is considered to be sufficient to enable
one skilled in the art to practice
the invention. The present invention is not to be limited in scope by the
construct deposited, since the deposited
embodiment is intended as a single illustration of certain aspects of the
invention and any constructs that are
functionally equivalent are within the scope of this invention. The deposit of
material herein does not constitute
an admission that the written description herein contained is inadequate to
enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be construed as
limiting the scope ofthe claims to the specific
illustrations that it represents. Indeed, various modifications of the
invention in addition to those shown and
described herein will become apparent to those skilled in the art from the
foregoing description and fall within the
scope of the appended claims.
139