METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL GROWTH

METHODES ET COMPOSITIONS PERMETTANT D'INHIBER LA CROISSANCE DE CELLULES NEOPLASIQUES

English Abstract

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. 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.

French Abstract

L'invention concerne des méthodes et des compositions permettant d'inhiber la croissance de cellules néoplasiques. Cette invention concerne plus spécialement des compositions antinéoplasiques et des méthodes de traitement des tumeurs. Elle concerne encore des méthodes de criblage permettant d'identifier les inhibiteurs de croissance, par exemple des composés antinéoplasiques. La présente invention a en outre trait à des nouveaux polypeptides et à des molécules d'acide nucléiques codant pour ces polypeptides. L'invention porte encore sur: des vecteurs et des cellules hôtes comprenant ces séquences d'acides nucléiques, des molécules de polypeptides chimériques où les polypeptides obtenus selon l'invention sont fusionnés à des séquences de polypeptides hétérologues, des anticorps qui se lient aux polypeptides obtenus selon l'invention, et des méthodes de préparation de ces polypeptides.

Claims

WHAT IS CLAIMED IS:
I. A composition of matter useful for the inhibition of neoplastic cell
growth, said composition
comprising an effective amount of a PRO655. PRO364 or PRO344 polypeptide, or
an monist thereof. in admixture
with a pharmaceutically acceptable carrier.
2. The composition of matter of Claim 1 comprising a growth inhibitory amount
of a PRO655.
PRO364 or PRO344 polypeptide, or an agonist thereof.
3. The composition of matter of Claim I comprising a cytotoxic amount of a
PRO655, PRO364
or PRO344 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 PRO655. PRO364 or PRO344
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 PRO655, PRO364 or PRO344 polypeptide, or an agonist
thereof.
9. The method of Claim 8, wherein said agonist is an anti-PRO655, anti-PRO364
or anti-PRO344
agonist antibody.
10. The method of Claim 8, wherein said agonist is a small molecule mimicking
the biological
activity of a PRO655, PRO364 or PRO344 polypeptide.
l I . 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:
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a container; and
a composition comprising an active agent contained within the container;
wherein said active agent in the
composition is a PRO655, PRO364 or PRO344 polypeptide, or an monist therof.
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-
PRO655, anti-PRO364
or anti-PRO344 agonist antibody.
16. The article of manufacture of Claim 13, wherein said agonist is a small
molecule mimicking the
biological activity of a PRO655, PRO364 or PRO344 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 3l,wherein said composition
additionaliycomprisesa 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), and Figure 6 (SEQ ID NO:17).
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), and Figure 5 (SEQ ID NO:16).
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), and Figure S (SEQ ID NO:16).
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 209509, 209436 or
209492.
23. A vector comprising the nucleic acid of any one of Claims 19 to 22.
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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 PRO655, PRO364 or PRO344 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.
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), and Figure 6 (SEQ ID NO:17).
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), and Figure 6 (SEQ ID NO:17).
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
209509, 209436 or 209492.
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 heterologous amino acid
sequence is a Fc region
of an immunoglobulin.
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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), or Figure 6 (SEQ ID NO:17), lacking its associated signal
peptide;
(b) a nucleotide sequence encoding an extracellular domain of the polypeptide
shown in Figure 2
(SEQ ID NO:2), Figure 4 (SEQ ID NO:7), or Figure 6 (SEQ ID NO:17), 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), or Figure 6 (SEQ ID NO:17), lacking its
associated signal peptide.
40. An isolated poiypeptide having at least 80% amino acid sequence identity
to:
(a) the polypeptide shown in Figure 2 (SEQ ID NO:2), Figure 4 (SEQ 1D NO:7),
or Figure 6 (SEQ
ID NO:17), 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), or Figure 6 (SEQ ID NO:17), 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), or Figure 6 (SEQ ID NO:17), lacking its associated signal peptide.
100

Description

CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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 ul., 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 form 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 of matter useful
for the inhibition of neoplastic
cell growth comprising an effective amount of a PR065~, PR0364 or PR0344
polypeptide as herein defined, or
2S 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 PR0655, PR0364
or PR0344 polypeptide, or
an agonist thereof. In another preferred embodiment, the composition comprises
a cytotoxic amount of a PR0655,


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PR0364 or PR0344 polypeptide, or an agonist thereof. Optionally. the
compositions of matter may contain one
or more additional ;rowth 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 PR0655.
PR0364 or PR0344 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 Qrowth
of a tumor cell comprising
exposing the cell to an effective amount of a PR0655, PR0364 or PR0344
polypeptide as herein defined, or an
agonist thereof. !n a particular embodiment, the aaonist is an anti-PR0655,
anti-PR0364 or anti-PR0344 agonist
antibody. In another embodiment, the agonist is a small molecule that mimics
the biological activity of a PR0655,
PR0364 or PR0344 polypeptide. The method may be performed in vilro or in vivo.
In a still further embodiment, the invention concerns 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. ~..
growth of tumor cells, and the active agent
in the composition is a PR0655, PR0364 or PR0344 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 of said PR0655, PR0364 or PR0344 polypeptide or agonist thereof, for the
inhibition of neoplastic cell
growth, wherein the agonist may be an antibody which binds to the PR0655,
PR0364 or PR0344 polypeptide.
!n a particular embodiment, the agonist is an anti-PR0655. anti-PR0364 or anti-
PR0344 agonist antibody. In
another embodiment, the agonist is a small molecule that mimics the biological
activity of a PR0655, PR0364 or
PR0344 polypeptide. Similar articles of manufacture comprising a PR0655,
PR0364 or PR0344 polypeptide as
herein defined, or an agonist thereof in an amount that is therapeutical ly
effective for the treatment oftumor are also
within the scope of the present invention. Also within the scope of the
invention are articles of manufacture
comprising a PR0655, PR0364 or PR0344 polypeptide as herein defined, or an
agonist thereof, and a further
growth inhibitory agent, cytotoxic agent or chemotherapeutic went.
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 PR0655, PR036.1 or PR0344
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 feast 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%
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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 PR0655, PR0364 or PR0344 polypeptide having a ful I-length am ino acid
sequence as disclosed herein, an amino
acid sequence lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane protein,
with or without the signal peptide, as disclosed herein or any other
specifically defined fragment of the full-length
amino acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% 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 of a full-length PR0655, PR0364 or PR034.1 polypeptide
cDNA as disclosed herein, the
coding sequence ofa PR0655, PR0364 or PR0344 polypeptide lacking the signal
peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane PR0655, PR0364
or PR0344 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).
!n 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 of the
human protein cDNAs deposited
with the ATCC as disclosed herein, or (b) the complement of the DNA molecule
of (a).
Anotheraspect the invention provides an isolated nucleic acid molecule
comprising a nucleotide sequence
3


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encoding a PR065~. PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptides are contemplated.
Another embodiment is directed to fragments of a PR065~, PR0364 or PR0344
polypeptide coding
sequence, or the complement thereof, that may find use as, for example,
hybridization probes, for encoding
fragments of a PR0655, PR0364 or PR0344 polypeptide that may optionally encode
a polypeptide comprising
a binding site for an anti-PR0655, anti-PR0364 or anti-PR0344 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 preferably
at least about I 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 150 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 350
nucleotides in length, yet more preferably
at least about 400 nucleotides in length, yet more preferably at least about
450 nucleotides in length, yet more
preferably at least about 500 nucleotides in length, yet more preferably at
least about 600 nucleotides in length, yet
more preferably at least 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
PR0655, PR0364 or PR0344
polypeptide-encoding nucleotide sequence may be determined in a routine manner
by aligning the PR0655,
PR0364 or PR0344 potypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any
of a number ofweil known sequence alignment programs and determining which
PR065.i, PR0364 or PR0344
polypeptide-encoding nucleotide sequence fragments) are novel. All of such
PR0655, PR0364 or PR0344
polypeptide-encoding nucleotide sequences are contemplated herein. Also
contemplated are the PR0655, PR0364
or PR0344 polypeptide fragments encoded by these nucleotide molecule
fragments, preferably those PR0655,
PR0364 or PR0344 polypeptide fragments that comprise a binding site for an
anti-PR065~, anti-PR0364 or anti-
PR0344 antibody.
!n another embodiment, the invention provides isolated PR0655, PR0364 or
PR0344 polypeptide
encoded by any of the isolated nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PR0655, PR0364 or
PR0344 polypeptide,
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comprising an amino acid sequence havin~_ 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 PR0655. PR0364 or PR0344 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
ofa transmembrane protein, with or without the signal peptide, as disclosed
herein or any other specifically defined
fragment of the full-length amino acid sequence as disclosed herein.
In a further aspect, the invention concerns an isolated PR0655, PR0364 or
PR0344 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 PR0655, PR0364 or
PR0344 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


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with the amino acid sequence of a PRO65~. PR0364 or PR034-I 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 ofatransmembrane protein, with or without the signal peptide, as
disclosed herein or any other specifically
defined fragment of the full-length amino acid sequence as disclosed herein.
In a specific aspect, the invention provides an isolated PR06». PR0364 or
PR0344 polypeptide without
the N-terminal signal sequence and/or the initiating methionine and is encoded
by a nucleotide sequence that
encodes such an amino acid sequence as hereinbefore described. Processes for
producing the same are also herein
described, wherein those processes comprise culturing a host cell comprising a
vector which comprises the
appropriate encoding nucleic acid molecule under conditions suitable for
expression of the PR0655, PR0364 or
PR0344 polypeptide and recovering the PR065~, PR0364 or PR03.~4 polypeptide
from the cell culture.
Another aspect of the invention provides an isolated PR0655. PR0364 or PR0344
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 PR0655,
PR0364 or PR0344 polypeptide and recovering the PR0655. PR0364 or PR0344
polypeptide from the cell
culture.
In yet another embodiment, the invention concerns agonists of a native PR0655,
PR0364 or PR0344
polypeptide as defined herein. In a particular embodiment, the agonist is an
anti-PR065.i, anti-PR0364 or anti-
PR0344 agonist antibody or a small molecule.
In a further embodiment, the invention concerns a method of identifying
agonists to a PR0655, PR0364
or PR0344 polypeptide which comprise contacting the PR0655, PR0364 or PR0344
polypeptide with a candidate
molecule and monitoring a biological activity mediated by said PR0655, PR0364
or PR0344 polypeptide.
Preferably, the PR0655, PR0364 or PR0344 polypeptide is a native PR0655,
PR0364 or PR0344 polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PROb55,
PR0364 or PR0344 polypeptide, or an agonist of a PR0655, PR0364 or PR0344
polypeptide as herein described,
or an anti-PR0655, anti-PR0364 or anti-PR0344 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
PR0655. PR0364 or PR0344
polypeptide, or an agonist thereof as hereinbefore described, or an anti-
PR0655, anti-PR0364 or anti-PR0344
agonist antibody, for the preparation of a medicament useful in the treatment
of a condition which is responsive to
the PR0655, PR0364 or PR0344 polypeptide, an agonist thereof or an anti-
PR065~, anti-PR0364 or anti-PR0344
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
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culture.
In other embodiments, the invention provides chimeric molecules comprising any
ofthe 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 ta;
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. Optional ly, the antibody is a monoclonal
antibody, humanized antibody, antibody
fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucieotide 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 a nucleotide sequence (SEQ ID NO: I ) of a native sequence
PR065~ cDNA, wherein SEQ
ID NO:1 is a clone designated herein as "DNA50960-1224".
Figure 2 shows the amino acid sequence (SEQ ID N0:2) derived from the coding
sequence of SEQ ID
NO:I shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ I D N0:6) of a native sequence
PR0364 cDNA, wherein SEQ
ID N0:6 is a clone designated herein as "DNA47365-1206".
Figure 4 shows the amino acid sequence (SEQ 1D N0:7) derived from the coding
sequence of SEQ ID
N0;6 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ lD N0:16) of a native sequence
PR0344 cDNA, wherein
SEQ ID N0:16 is a clone designated herein as "DNA40592-1242".
Figure 6 shows the amino acid sequence (SEQ ID N0:17) derived from the coding
sequence of SEQ ID
N0:16 shown in Figure 5.
DETAILED DESCRIPTION OF THE INVENTION
The terms "PR0655", "PR0364" or"PR0344" polypeptide or protein when used
herein encompass native
sequence PR0655, PR0364 or PR0344 and PR0655, PR0364 or PR0344 variants (which
are further defined
herein). The PR0655, PR0364 or PR0344 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 PR0655", ''native sequence PR0364" or "native sequence
PR0344" comprises a
polypeptide having the same amino acid sequence as the PR0655. PR0364 or
PR0344 polypeptide as derived from
nature. Such native sequence PR0655, PR0364 or PR0344 polypeptide can be
isolated from nature or can be
produced by recombinant and/or synthetic means. The term "native sequence"
PR0655. PR0364 or PR0344
specifically encompasses naturally-occurring truncated 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


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
PR0655. PR0364 and PR0344 polypeptides. In one embodiment of the invention,
the native sequence PR0655,
PR0364 or PR0344 polypeptide is a mature or full-length native sequence
PR0655, PR0364 or PR0344
polypeptide as shown in Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7), or
Figure 6 (SEQ ID NO:I7),
respectively. Also, while the PR0655, PR0364. and PR0344 polypeptides
disclosed in Figure 2 (SEQ ID N0:2),
Figure 4 (SEQ ID N0:7), or Figure 6 (SEQ ID N0:17), respectively, are shown to
begin with the methionine
residue designated therein as amino acid position I, it is conceivable and
possible that another methionine residue
located either upstream or downstream from amino acid position I in Figure 2
(SEQ ID N0:2), Figure 4 (SEQ !D
N0:7), or Figure 6 (SEQ ID N0:17), respectively, may be employed as the
starting amino acid residue for the
PR0655, PR0364 or PR0344 polypeptide.
IO The "extracellular domain" or "ECD" ofa polypeptide disclosed herein refers
to a form ofthe polypeptide
which is essentially free ofthe transmembrane and cytoplasmic domains.
Ordinarily, a polypeptide ECD will have
less than about I% 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
IS 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 of the present invention, the extracel lular domain of
a polypeptide ofthe present invention
comprises amino acids I to X ofthe mature amino acid sequence, wherein X is
any amino acid within 5 amino acids
on either side of the extracellular domain/transmembrane domain boundary.
20 The approximate location of the "signal peptides" of the 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 e~ al.,
2S Prot. Ene., 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.
30 "PR0655 variant polypeptide" means an active PR0655 polypeptide (other than
a native sequence
PR0655 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues I or about 22 to 208 of the PR0655 polypeptide
shown in Figure 2 (SEQ ID N0:2),
(b) X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2),
wherein X is any amino acid residue
from 17 to 26 of Figure 2 (SEQ 1D N0:2), or (c) another specifically derived
fragment of the amino acid sequence
3S shown in Figure 2 (SEQ ID N0:2).
"PR0364 variant polypeptide" means an active PR0364 polypeptide (other than a
native sequence
PR0364 polypeptide) as defined below. having at least about 80% amino acid
sequence identity with the amino
8


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
acid sequence of (a) residues I or about 26 to 241 of the PR0364 polypeptide
shown in Figure 4 (SEQ ID N0:7),
(b) X to 24 I of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7),
wherein X is any amino acid residue
from 21 to 30 of Figure 4 (SEQ ID N0:7), (c) 1 or about 26 to X of Figure 4
(SEQ ID N0:7), wherein X is any
amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID N0:7) or
(d) another specifically derived
fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
"PR0344 variant polypeptide" means an active PR0344 polypeptide (other than a
native sequence
PR0344 polypeptide) as defined below, having at least about 80% amino acid
sequence identity with the amino
acid sequence of (a) residues I or about 16 to 243 of the PR0344 polypeptide
shown in Figure 6 (SEQ ID N0:17),
(b) X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17),
wherein X is any amino acid residue
from 11 to 20 of Figure 6 (SEQ ID NO: ! 7), or (c) another specifically
derived fragment of the amino acid sequence
shown in Figure 6 (SEQ ID N0:17).
Such PR0655, PR0364 and PR0344 variants include, for instance, PR0655, PR0364
and PR0344
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 PR0655 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 feast 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 22 to 208 of the PR0655
poiypeptide shown in Figure 2 (SEQ ID
N0:2), (b) X to 208 of the PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2),
wherein X is any amino acid
residue from 17 to 26 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).
Ordinarily. a PR0364 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 identiy. 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
9


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WO 00/32778 PCT/US99/28409
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 26 to 241 of the PR0364
polypeptide shown in Figure 4 (SEQ ID
N0:7), (b) X to 241 of the PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7),
wherein X is any amino acid
residue from 21 to 30 of Figure 4 (SEQ ID N0:7), (c) 1 or about 26 to X of
Figure 4 (SEQ ID N0:7), wherein X
is any amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID
N0:7) or (d) another specifically
derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
Ordinarily, a PR0344 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 16 to 243 of the PR0344
polypeptide shown in Figure 6 (SEQ ID
N0:17), (b) X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID
N0:17), wherein X is any amino acid
residue from 11 to 20 of Figure 6 (SEQ ID N0:17), or (c) another specifically
derived fragment of the amino acid
sequence shown in Figure 6 (SEQ ID N0:17).
Ordinarily, PR0655, PR0364 and PR0344 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 ~0 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 l00 amino acids in
length, more often at least about I50 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 I provides the complete source code for the ALIGN-3
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-3B) and °% nucleic
acid sequence identity (Tables 2C-2D)


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
using the ALIGN-2 sequence comparison computer program, wherein "PRO"
represents the amino acid sequence
of a hypothetical PR0655, PR0364 or PR0344 polypeptide of interest.
"Comparison Protein" representstheamino
acid sequence of a polypeptide against which the "PRO" polypeptide of interest
is being compared, "PRO-DNA"
represents a hypothetical PR0655-, PR0364-or PR0344-encoding nucleic acid
sequence of interest,"Comparison
S DNA" represents the nucleotide sequence of a 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.
11


CA 02348157 2001-04-23
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/_
Table 1
* C-C increased from 12 to 15
* Z is average of EQ
* B is average of ND
* match with stop is M; stop-stop = 0; 1 (joker) match = 0
*%
A'defne 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,-1, 1, 0, 0. 0, 0,-2,-5,
0,-3, 1},
/* C *I {-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,-1,
0,-4, 2},
/* E */ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3.-2, l,_M,-I. 2.-1, 0, 0, 0,-Z,-7,
0,-4, 3},
/* F */ {-4,-5,-4,-6,-5. 9,-5,-2, 1, 0,-5, 2, 0,-4, M,-5,-S,-4,-3,-3, 0,-1, 0,
0, 7,-5}.
1* G */ { 1. 0,-3, l, 0,-5, 5,-2,-3, 0,-2,-4,-3, O, M,-1,-1,-3. I, 0, 0,-1,-7,
0,-5, 0}.
/* H *1 {-1. 1,-3. 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2, M, 0, 3, 2.-1,-l, 0,-2,-3.
0, 0, 2},
/* I */ {-1,-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, 0, 0, M, 0. 0, 0. 0, 0, 0. 0, 0,
0, 0, 0},
/* 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}.
I* L */ {-2.-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3.-3,-I, 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,-0,
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,-1,-3,-l,-1,-5,-1, 0,-2, 0,-1,-3,-2,-1, M, 6, 0, 0, 1, 0, 0,-1.-6,
0,-5, 0},
I* Q *1 { 0, 1,-5. 2, 2,-S,-1, 3,-2, 0. 1,-2,-1, 1, M, 0, 4, 1,-1,-1, 0,-2,-5,
0,-4, 3},
/* R *1 {-2. 0,-4,-l,-l.-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0. 1, 6, 0.-1, 0,-2, 2,
0,-4, 0},
/* S */ { 1, 0, 0, 0, 0,-3, 1,-1,-1, 0, 0,-3,-2, 1, M, l,-1, 0. 2. 1, 0,-1,-2,
0,-3, 0},
/* T */ { 1, 0,-2. 0, 0,-3, 0,-l, 0, 0, 0,-1,-1, 0, M. 0,-1,-1, 1, 3, 0, 0,-5,
0,-3, 0},
/* U */ { 0, 0, 0, 0, 0, 0, 0, 0, 0. 0, 0, 0, 0, O, M, 0, 0, 0, 0, 0, 0. 0. 0.
0, 0, 0},
/* V */ { 0,-2,-2,-2,-2,-1,-I,-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}.
/* X */ { 0, 0, 0, 0, 0, 0, 0, 0, 0. 0, 0, 0, 0. O, M, 0. 0. 0. 0, 0, 0. 0, 0,
0, 0, 0},
1* Y *I {-3,-3, 0,-4,-4, 7,-5, 0,-I, 0,-4,-I,-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,-I, 1, M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4}
};
Page 1 of day.h
12


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/*


*I


#include< >
stdio.h


#include<
ctype.h
>


#defineMAXJMP 16 l* max jumps in a diag *I


#defineMAXGAP l* don't continue to penalize
24 gaps larger than this *I


#defineJMPS 1024 /* max jmps in an path */


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


#defineDMAT 3 /* value of matching bases
*/


#detineDMIS 0 /* penalty for mismatched
bases */


#de6neDINSO8 /* penalty for a gap *1


#defineDINSI1 !* penalty per base */


#defmePINSO8 /* penalty for a gap */


#definePINS14 /* penalty per residue */


struct
jmp
{


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


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


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


struct
diag
{


int score; 1* score at last jmp */


long offset; /* offset of prev block
*/


shortijmp; l* current jmp index */


strnct l* list of jmps */
jmp
jp;



struct
path
{


int spc; /* number of leading spaces
*/


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


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


}:


char *ofile; /* output file name */


char *namex(2];/* seq names: getseqsp *1


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


char *seqx(2]; /* seqs: getseqsQ *1


int dmax: /* best diag: nwp */


int dmax0; /* final diag */


int dna; /* set if dna: main() *1


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


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


int IenO, lent;l* seq lens */


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


int smax; /' max score: nwp */


int *xbm; /* bitmap for matching */


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


structdiag *dx; /* holds diagonals *1


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


char *callocQ, Q, *indexQ, *strcpyQ;
*mailoc


char *getseqQ,
*g callocQ;



Page 1 of nw.h
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/* Needleman-Wunsch alignment program
* usage: props 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 file in Itmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
*/
~ittclude "nw.h"
#inciude "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 < < I 1, 1 < < 12, 1 < < 13, I < < 14,
l«15, 1«l6, 1«17, 1«18, 1«19, 1«20, 1«21, 1«22,
1«23. 1«24, 1«25(1«('E'-'A'))~(1«('Q'-'A'))
main(ac, av) I>t181I1
int ac;
sitar *avp;
prop = av(0];
if (ac ! = 3) {
fprintf(stderr,"usage: %s filet filet\n", prop);
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 \"align.out\"1n");
exit( I );
namex[0] = av[1];
namex[I] = av(2];
seqx[0] = getseq(namex[0], &len0):
seqx[1] = getseq(namex[1], &lenl);
xbm = (dna)? dbval : pbval;
endgaps = 0; /* 1 to penalize endgaps */
ofile = "align.out"; /* output file */
nwQ; l* fill in the matrix, get the possible jmps */
readjmpsQ; /* get the actual jmps */
print(); /* print stars, aligtunent */
cleanup(0); I* unlink any tmp files *1
Page 1 of nw.c
14


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WO 00/32778 PCT/US99/28409
I* 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 IIW


{


char *px, *py; /* seqs and ptrs *!


int *ndely, I* keep track of dely *I
*dely;


int ndelx, delx;/* keep track of delx */


int *tmp; /* for swapping row0, cowl */


int mis; /* score for each type */


int ins0, insl;/* insertion penalties */


register id; I* diagonal index *I


register ij; /* jmp index */


register *col0, *coll;/* score for curt, last row */


register xx, yy; /* index into seqs *1


dx = (struct diag *)g calloc("to get diags", IenO+lenl+I, 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 get col0", lenl + 1, sizeof(int));
coi l = (int *)g calloc("to get coi l ", lenl + 1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
insl = (dna)? DINS1 : PINS1;
smax = -10000;
if (endgaps) {
for (col0[0] = dely[O) _ -ins0, yy = 1; yy < = lenl; yy++) {
col0[yy] = dely(yy] = col0[yy-1] - insl;
ndely[yYl = YY:
col0[0] = 0; /* Waterman Bull Math Biol 84 */
else
for (yy = 1; yy < = lent; yy++)
dely(yy] _ -ins0;
/* fill in match matrix
*/
for (px = seqx(0], xx = 1; xx < = len0; px++, xx++) {
/* initialize tirst entry in col
*I
if (endgaps) {
if (xx = = I )
coil[0] = delx = -(ins0+insl);
else
coil[0] = delx = col0[0] - insl;
ndelx = xx;
else {
col l (0] = 0;
delx = -ins0;
ndelx = 0;
Page 2 of nw.c


CA 02348157 2001-04-23
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...nw
for (py = seqx[1], yy = 1: yy < = lent: py++, yy++) {
mis = col0[yy-1];
if (dna)
mss + _ (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis += day[*px-'A'][*py-'A'j;
/* 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 {
dely[yy] -= insl;
ndely[yy] + +;
}
} else {
if (col0[yy] - (ins0+insl) > = dely[yy]) {
dely[yy] = col0[yy] - (ins0+insl);
ndely[yy] = 1;
} else
ndely[yy] ++;
}
/* update penalty for del in y seq;
* favor new del over ongong del
*/
if (endgaps ~ ~ ndelx < MAXGAP) {
if (coll[yy-1] - ins0 > = delx) {
delx = coll[yy-1] - (ins0+insl);
ndelx = 1;
} else {
deix -= insl;
ndelx+ +;
}
} else {
if (coil[yy-1] - (ins0+insl) > = delx) {
deli = colt[yy-1] - (ins0+insl);
ndelx = 1;
} eLse
ndelx++;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
*/
Page 3 of nw.c
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id = xx - yy + lent - l ;
if (mis > = delx && mis > = dely[yy])
...nw
cot l [yyJ = mis;
else if (delx > = dely[yy]) {
colt[yy] = deli;
ij = dx[id].ijmp;
if (dx[id].jp.n(O] && (!dna ~ ~ (ndelx > = MAXJMP
&& xx > dx[id].jp.x[ijJ+MX) ~ ~ mis > dx[id].score+DINSO)) {
dx(idJ.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] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
else {
colt[yY1 = 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[yYl;
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
if (xx = = IenO && yy < ten 1 ) {
/* last cot
x~
if (endgaps)
coll(yy] -= ins0+insl*(lenl-yy);
if (cot 1 [yy] > smax) {
smax = coll(yyJ;
dmax = id;
if (endgaps && xx < IenO)
coll[yy-I] -= ins0+insl*(len0-xx);
if (coll[yy-1] > smax) {
smax = coll(yy-1];
dmax = id;
tmp = col0; col0 = cot 1; cot l = tmp;
(void) free((char *)ndely);
(void) free((c6ar *)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:
* getmat() -- 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 align()
* numsp -- put out a number line: dumpblockp
* putlineQ -- put out a line (name, [num], seq, [num]): dumpblockQ
* starsp - -put a line of stars: dumpblockp
* stripnameQ -- strip any path and prefix from a seqname
*/
A~include "nw.h"
define SPC 3
Atdefine P LINE 256 /* maximum output line *1
A~define P SPC 3 !* space between name or num and seq */
extern day[26][26];
int oleo; I* set output line length *I
FILE *fx; /* output file *1
primp p)rlIlt
{
int lx, ly, firstgap, lastgap; I* overlap *I
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)\n", namex[1], ienl);
oleo = 60;
Ix = IenO:
ly = lenl;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x */
pp[O].spc = firstgap = lenl - dmax - 1;
ly __ ~[Ol,spc:
else if (dmax > lenl - I) { /* leading gap in y */
pp[i].spc = firstgap = dmax - (lenl - 1);
Ix -= PP[ll.sPc:
if (dmax0 < IenO - I) { /* trailing gap in x */
lastgap = IenO - dmax0 -I;
Ix -= lastgap;
else if (dmax0 > len0 - 1) { 1* trailing gap in y */
lastgap = dmax0 - (IenO - 1 );
ly _= lastgap;
getmat(lx, ly, firstgap, lastgap);
pr alignQ;
Page 1 of nwprint.c
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/*
* trace back the best path, count matches
*/
static
getmat(Ix, ly, firstgap, lastgap) g8ltlIlat
int Ix, ly; /* "core" (minus endgaps) */
int firstgap, lastgap; /* leading trailing overlap */
{
int tun, i0, il, siz0, sizl;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
I* get total matches, score
*/
i0 = il = siz0 = sizl = 0;
p0 = seqx[0] + pp[lJ.spc;
pl = seqx[1] + pp[0].spc;
n0 = pp[1].spc + 1;
nl = pp[0].spc + l;
nm = 0;
while ( *p0 8t& *pl ) {
if (siz0) {
pl++;
nl++;
siz0--;
else if (sizl) {
p0++;
n0++;
siz 1--;
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm++;
if (n0++ _=pp[0].x[i0])
siz0 = pp[0].n[i0++);
if(nl++ _= pp[1].x[ilJ)
sizf = pp[1].n[il++];
p0++;
pl++;
I* pct homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
i/
d (eAdga~)
Ix = (len0 < lenl)? IenO : lenl;
else
Ix = (Ix < ly)? !x : ly;
pct = 100.*(double)tun/(double)Ix;
fprimf(fx, "1n");
fprintf(fx, "< 96d match%s in an overlap of %d: %.2f percent similarityln",
tun, (tun == 1)? "" . "es". lx, pct):
Page 2 of nwprint.c
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fprintf(fx, " < gaps in first sequence: %d", gapx); ...gCtIllat
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");
tprintf(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, DINSI);
else
fprintf(fx.
"\n < score: %d (Dayhoff PAM 250 matrix, gap penalty = % d + %d per
residue)\n",
stnax, PINSO, PINSI);
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");
sGttic nm; l* matches in core
-- for checking */


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


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


static nc[2]; 1* number at start
of current line */


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


static siz[2];


static char *ps[2]: /* ptr to current
element *1


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


static char out[2][P I* output line *I
LINE];


static char star[P LINE];/* set by stars()
*!


/*
* print aligtunent of described instruct path pp[]
*/
static
pr alignQ
pr align
~t nn: /* char count */
int more;
register i;
for (i = 0, Imax = 0; i < 2; i++) {
nn = stripname(namex[i]);
if (nn > Imax)
lmax = nn;
~fi] = I;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps(i] = seqx[i];
po[i] = aut[i];
Page 3 of nwprint.c


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for (nn = nm = 0, more = 1; more: ) { ...pT align
for (i = more = 0; i < 2; i++) {
/*
* do we have more of this sequence'?
*/
if (!*ps[il)
continue;
more++;
if (pp[i].spc) { /* leading space */
*po[i]++ _ ,
PP[i].spc__;
else if (siz[i]) { I* in a gap *I
*po[i] + + _
siz[i]__;
else { l* we're puuing a seq element
*/
*Pofil = *Ps[i];
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i]++;
/*
* are we at next gap for this seq?
*/
if (ni[i] _= pp[i].x[ij[i]]) {
l*
* we need to merge all gaps
* at this location
*/
siz[i] = pp[i].n[ij[i]++];
white (ni[i] _- pp[i].x[ijfilD
siz(i] += pp[i].n[ij[i]++];
ni[i]++;
if (++nn == olen ~ ~ !more 8r.8c nn) {
dumpblockQ;
for (i = 0; i < Z; i++)
po[i] = out[i];
nn = 0;
/*
* dump a block of lines, including numbers, stars: pr align()
*/
static
dttmpblackp dumpblock
{
renter i;
for (i = 0; i < 2; i++)
*po[i]__ ~ '10':
Page 4 of nwprint. c
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(void) putc('\n', fx);
for(i=O:i<2;i++){
if (*out[i] && (*out[i] ! _ ' ' I I *(po(i]) ! _ ' ')) {
if (i == 0)
nums(i);
if (i == 0 && *out[1])
starsQ;
putline(i);
if (i = = 0 && *out[ 1 ])
fprintf(fx, star);
if (i == 1)
nums(i);
...dumpblock
/*
* pttt out a number line: dumpblockQ
*I
static
nums(ix) nUlriS
int ix; /* index in out[] holding seq line */
{
char nline[P LINE];
register i, j:
register char *pn, *px, *py;
for (pn = mine, i = 0; i < Imax+P SPC: i++, pn++)
*pn = ,
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
~(*PY =- ' I I *PY =- '-7
*Pn = ,
else {
if (i%10 == 0 I I (i == 1 && nc[ix] != I)) {
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';
ttc[ix] = i;
for (pn = nline; *pn; pn++)
(void) putc(*pn, fx);
(void) putc('\n', fx);
/*
* put out a line (name, [num], seq, [num]): dumpblockQ
*I
static
P~u~(~) putline
iot ix;
{
Page 5 of nwprint.c
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...putline
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 I:
* niQ 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);
/*
* put a line of stars (seqs always in out[O], out[1]): dumpblockQ
*/
static
stars()
Stll'S
{
int i;
register char *p0, *pl, cx, *px;
if (!*out(0] ~ ~ (*out[0] _- ' && *(po[O]) _- ' ') ~ ~
!*out(1] ~ ~ (*out[I] _ _ ' && *(Poll]) _- ' '))
return;
px = star;
for (i = Imax+P SPC; i; i--)
*px++ _ ,
for (p0 = out[O], 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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/*
* strip path or prefix from pn, return len: pr align()
*/
static
stripname(pn) stripname
char *pn; /* file name (may be path) */
register char *px, *py;
PY = ~:
for (px = pn; *px; px++)
if (*px = _ '/')
py=px+ 1;
(PY)
(void) strcpy(pn, py);
return(strlen(pn));
Page 7 of nwprint.c
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/*
* cleanup() -- cleanup any tmp tile
* getseqQ -- read in seq, set dna, len. maxlen
* g callocQ -- callocp with error checkin
* readjmpsQ -- get the good jmps, from tmp fife if necessary
* writejmpsQ -- write a f-filled array of jmps to a tmp file: nwp
*/
Ninclude "nw.h"
~linclude < sys/file.h >
char *jname = "/tmp/homgXXXXXX"; !* tmp file for jmps */
FILE *tj;
int cleanupQ; /* cleanup tmp file */
long IseekQ:
/*
* remove any tmp file if we blow
*/
cleanup(i) cleanup
int i:
{
if (tj)
(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) geltSeq
char *file: I* file name */
int *len; l* seq fen */
{
char line[1024], *pseq;
register char *px, *py:
int natgc, tlen;
FILE *tp;
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 =- ' <' ~ ~ *line =_ ' >')
continue;
for (px = line; *px !_ '1n': 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);
P~qI01 = P~qll] = Pseq[21 = 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 ! _ '1n'; 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 calloc(msg, nx, sz)
char *msg; /* program, calling routine */
int nx, sz; /* number and size of elements *l
{
char *px, *callocp;
if ((px = calloc((unsigned)nx. (unsigned}sz)) _ = 0) {
if (*msg) {
fprintf(stderr. "%as: g callocQ failed %s (n=%d, sz=%d)1n", prog, msg, nx,
sz);
exit(1);
return(px);
l*
* get final jmps from dx[] or tmp file, set pp[], reset dmax: main()
*/
readjmpsQ readjmps
{
int fd = -I:
int siz, i0, i 1;
register i. j, xx;
if (fj) {
(void) fclose(fj);
if ((fd = open(jname. O_RDONLY, 0)) < 0) {
fprintf(stderr. %s: can't open() %s\n", prog, jname);
cleanup(1);
for (i = i0 = il = 0, dmax0 = dmax, xx = len0; : i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x(j] > = xx; j--)
Page 2 of nwsubr.c
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...readjmps
if (j < 0 && dx[dmax].offset && tj) {
(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, "9os: too many gaps in alignment\n", prog):
cleanup( I );
if (j > = 0) {
siz = dx[dmax].jp.n[j];
xx = dx[dmax).jp.x(j];
dmax += siz;
if (siz < 0) { /* gap in second seq */
PP[1].n[il] _ -siz;
xx + = siz:
/* id = xx - yy + lenl - 1
*/
pp[1].x[il] = xx - dmax + lent - l;
gapy + + ;
ngapy -= siz;
I* ignore MAXGAP when doing endgaps *I
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--) {
i = PP[0l.nGl; PP[Ol.nU1 = PP[0].n[i0]; PP[Ol.n[i0] = i:
i = PP[OI~xG]; PP[OI.xG] = PP[0].x[i0); PP[0].x[i0] = i;
for (j = 0. il--; j < il; j++, il--) {
i = PP[1].nU]; PP[ll.nLl] = PP[ll.nlil]; PP[1].n[il] = i:
i = PP[1].x[j]; PP[ll.xLJ] = PP[11.x[il]: PP[ll.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 tilled jmp struct offset of the prev one (if any): nwp
*/
writejmps(ix) writejmps
int ix;
char *mktempQ;
if (!fj) {
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktemp() %s\n", prog, jname);
cleanup(/);
if ((fj = fopen(jname, "w")) _ = 0) {
fprintf(stderr, " % s: can't write % stn", prog, jname);
exit( 1 );
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset. sizeof(dx[ixJ.offset), l, fj);
Page 4 of nwsubr.c
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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
poiypeptide) _
divided by 15 = 33.3 %
29


CA 02348157 2001-04-23
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Table 2B
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = IS 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 LO = 50%


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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%
31


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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
32


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"Percent (°'°) amino acid sequence identity" with respect to the
PR06S5. PR0364 and PR034.1
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 PRO6SS, PR0364 or PR0344
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 1. The ALIGN-2 sequence comparison computer
program was authored by
Genentech, Inc., and the source code shown in Table I 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 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 othenvise, 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 NCB/-BLAST2 sequence comparison program
may be downloaded from
http://www.ncbi.nlm.nih.gov. NCB/-BLAST2 uses several search parameters,
wherein all of those search
33


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parameters are set to default values includin=, for example. unmask = yes,
strand = all, expected occurrences = 10.
minimum low complexity length = I S!S, multi-pass e-value = 0.01, constant for
multi-pass = 2S. 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 Enzvmoloav, 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 % 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 poiypeptide 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 am ino 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 iriterest and the amino acid
sequence B is the amino acid sequence of
the PRO polypeptide of interest.
"PR0655 variant polynucleotide" or "PR06S5 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR06SS 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 22 to 208 of the
PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), (b) a nucleic acid
sequence which encodes amino acids
X to 208 ofthe PR0655 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X
is any amino acid residue from
17 to 26 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 PR0655 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
34


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at least about 83% nucleic acid sequence identity, more preferably at feast
about 84°ro nucleic acid sequence identir<~,
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 I or about 22 to 208
of the PR0655 polypeptide shown
in Figure 2 (SEQ ID N0:2), (b) a nucleic acid sequence which encodes amino
acids X to 208 of the PR0655
polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X is any amino acid
residue from 17 to 26 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). PR0655 polynucleotide variants
do not encompass the native
PR0655 nucleotide sequence.
"PR0364 variant polynucleotide" or "PR0364 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0364 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 26 to 241 of the
PR0364 polypeptide shown in Figure 4 (SEQ ID N0:7), (b) a nucleic acid
sequence which encodes amino acids
X to 241 ofthe PR0364 poiypeptide shown in Figure 4 (SEQ ID N0:7), wherein X
is any amino acid residue from
21 to 30 of Figure 4 {SEQ ID N0:7), (c) a nucleic acid sequence which encodes
amino acids I or about 26 to X
of Figure 4 (SEQ ID N0:7), wherein X is any amino acid from amino acid 158 to
amino acid I67 of Figure 4 (SEQ
ID N0:7) or (d) a nucleic acid sequence which encodes another specifically
derived fragment of the amino acid
sequence shown in Figure 4 (SEQ 1D N0:7). Ordinarily, a PR0364 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


CA 02348157 2001-04-23
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acid sequence which encodes residues I or about 26 to 2:1 I of the PR0364
polypeptide shown in Figure 4 (SEQ
ID N0:7), (b) a nucleic acid sequence which encodes amino acids X to 241 of
the PR0364 polypeptide shown in
Figure 4 (SEQ ID N0:7), wherein X is any amino acid residue from 21 to 30 of
Figure 4 (SEQ ID N0:7), (c) a
nucleic acid sequence which encodes amino acids 1 or about 26 to X of Figure 4
(SEQ ID N0:7), wherein X is any
amino acid from amino acid 158 to amino acid 167 of Figure 4 (SEQ ID N0:7) or
(d) a nucleic acid sequence which
encodes another specifically derived fragment of the amino acid sequence shown
in Figure 4 (SEQ ID N0:7).
PR0364 polynucleotide variants do not encompass the native PR0364 nucleotide
sequence.
"PR0344 variant polynucleotide" or "PR0344 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0344 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 16 to 243 of the
PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), (b) a nucleic acid
sequence which encodes amino acids
X to 243 of the PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), wherein X
is any amino acid residue
from 11 to 20 of Figure 6 (SEQ ID N0:17),or (c) a nucleic acid sequence which
encodes another specifically
derived fragment of the amino acid sequence shown in Figure 6 (SEQ ID N0:17).
Ordinarily, a PR0344 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 16 to 243
of the PR0344 poiypeptide shown
in Figure 6 (SEQ ID N0:17), (b) a nucleic acid sequence which encodes amino
acids X to 243 of the PR0344
poiypeptide shown in Figure 6 (SEQ ID N0:17), wherein X is any amino acid
residue from I 1 to 20 of Figure 6
(SEQ ID N0:17),or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the amino
acid sequence shown in Figure 6 (SEQ ID N0:17). PR0344 polynucleotide variants
do not encompass the native
PR0344 nucleotide sequence.
Ordinarily, PR0655, PR0364 and PR0344 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 150
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
36


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WO 00/32778 PCT/US99/28409
in length, more often at least about 450 nucleotides in ien~th. 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 PR065~,
PR0364 and PR0344
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 PR0655, PR0364
or PR0344 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 I
IS 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. TXUSi0087. The ALIGN-2
program is publicly available
through Genentech, Inc., South San Francisco, California or may be compiled
from the source code provided
inTable I.' 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
nucleicacidsequenceidentitycalculations,Tables 2C-2D demonstrate how to
calculate the%nucleicacid sequence
identity ofthe 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 NCBI-BLAST2 sequence
comparison program may be
37


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downloaded from http:/iwww.ncbi.nlm.nih.gov. NCB/-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 will 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 Enzymolosy, 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 = l, overlap fraction = 0.125,
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
pofynucleotide) 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 molecute of interest.
In other embodiments, PR0655, PR0364 and PR0344 variant polynucleotides are
nucleic acid molecules
that encode an active PR0655, PR0364 or PR0344 polypeptide, respectively, and
which are capable of
hybridizing, preferably under stringent hybridization and wash conditions, to
nucleotide sequences encoding the
full-length PR065~ polypeptide shown in Figure 2 (SEQ ID N0:2), to nucleotide
sequences encoding the full-
length PR0364 poiypeptide shown in Figure 4 (SEQ ID N0:7), to nucleotide
sequences encoding the full-length
PR0344 polypeptide shown in Figure 6 (SEQ ID N0:17), respectively. PR0655,
PR0364 and PR0344 variant
polypeptides may be those that are encoded by a PR0655, PR0364 or PR0344
variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity
comparisons performed as
38


CA 02348157 2001-04-23
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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.
Por 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
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 enzymes, hormones, and other
proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the polypeptide will be purified ( 1 ) to a
degree sufficient to obtain at least 15
residues ofN-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 sire within recombinant cells,
since at least one component of the
PR0655, PR0364 or PR0344 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 PR0655, PR0364 or
PR0344polypeptide or an "isolated"
nucleic acid molecule encoding an anti-PR0655, anti-PR0364 or anti-PR0344
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 ofthe PR0655-, PR0364- or PR0344-encoding
nucleic acid or the anti-PR0655-,
anti-PR0364- or anti-PR0344-encoding nucleic acid. Preferably, the isolated
nucleic acid is free of association
with all components with which it is naturally associated. An isolated PR0655-
, PR0364- or PR0344-encoding
nucleic acid molecule or an isolated anti-PR065 ~-, anti-PR0364- or anti-
PR0344-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 PR0655-, PR0364- or PR0344-encoding nucleic acid
molecule or from the anti-PR0655-,
anti-PR0364- or anti-PR0344-encoding nucleic acid molecule as it exists in
natural cells. However, an isolated
nucleic acid molecule encoding a PR0655, PR0364 or PR0344 polypeptide or an
isolated nucleic acid molecule
encodingan anti-PR0655, anti-PR0364 or anti-PR0344 antibody includes PR0655-
,PR0364-orPR0344-nucleic
39


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WO 00/32778 PCT/US99/28409
acid molecules or anti-PR0655-, anti-PR0364- or anti-PR0344-nucleic acid
molecules contained in cells that
ordinarily express PR065S, PR0364 or PR0344 polypeptides or anti-PR06S5, anti-
PR0364 or anti-PR0344
antibodies where, for example, the nucleic acid molecule is in a chromosomal
location different from that ofnatural
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 finked" when it is placed into a functional
relationship with another nucleic acid
sequence. For example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide
if it is expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the transcription of the
sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the case of
a secretory leader: contiguous and
in reading phase. However, enhancers do not have to be contiguous. Linking is
accomplished by 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-
PR06SS, anti-PR0364 and anti-PR0344 monoclonal antibodies (including
agonistantibodies),anti-PR0655,anti-
PR0364 and anti-PR0344 antibody compositions with polyepitopic specificity,
single chain anti-PR0655, anti-
PR0364 and anti-PR0344 antibodies, and fragments of anti-PR06S5, anti-PR0364
and anti-PR0344 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
et al., Current Protocols in Molecular Biolo v Wiley Interscience Publishers,
(1995).
"Stringent conditions" or "high stringency conditions", as defined herein, may
be identified by those that:
( l ) employ low ionic strength and high temperature for washing, for example
0.015 M sodium chioride/0.001 S M
sodium citrate/0.1% sodium dodecyl sulfate at SO°C; (2) employ during
hybridization a denaturing agent, such as
formamide, for example, 50% (v/v) formamide with 0.1% bovine serum
albumin/O.I% Ficoll/0.1%


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
polyvinylpyrrolidone/~OmM sodium phosphate buffer at pH 6.5 with 750 mM sodium
chloride. 75 mM sodium
citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI,
0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, ~ 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 of0.1 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 PR0655,
PR0364 or PR0344 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 am ino 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 lgM.
"Active" or "activity" for the purposes herein refers to forms) of PR0655,
PR0364 or PR0344 which
retain a biological andlor an immunological activity of native or naturally-
occurring PR0655, PR0364 or PR0344,
wherein "biological" activity refers to a biological function (either
inhibitory or stimulatory) caused by a native or
naturally-occurring PR0655, PR0364 or PR0344 other than the ability to induce
the production of an antibody
against an antigenic epitope possessed by a native or naturally-occurring
PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344.
"Biological activity" in the context of an antibody or another agonist that
can be identified by the screening
41


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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
"therapeuticallyeffective amount." In a specific embodiment, "biological
activity" is the ability to inhibit neoplastic
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 "immunologicai activity" means immunological cross-reactivity with
at least one epitope of
a PR0655, PR0364 or PR0344 polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate
polypeptide is capable of
competitively inhibiting the qualitative biological activity of a PR0655,
PR0364 or PR0344 polypeptide having
this activity with polyclonal antisera raised against the known active PR0655,
PR0364 or PR0344 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 subcutaneous
injection in incomplete Freunds. The immunological cross-reactivity preferably
is "specific". which means that
the bindingaffinity ofthe immunologically cross-reactive molecule (e. g.,
antibody) identified, to the corresponding
PR0655, PR0364 or PR0344 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 affinity of that molecule to any other known native
polypeptide.
"Tumor", as used herein, refers to all 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
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
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CA 02348157 2001-04-23
WO 00132778 PCT/US99/28409
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 andlor
cytotoxic effect and/or apoptosis of
the target cells. An "effective amount" of a PR0655, PR0364 or PR0344
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 PR0655, PR0364 or PR0344 polypeptide or an agonist thereof for purposes
of treatment of tumor may be
1S determined empirically and in a routine manner.
A "growth inhibitory amount" of a PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide or an
agonist thereof for purposes of
inhibiting neoplastic cell growth may be determined empirically and in a
routine manner.
A "cytotoxic amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist
thereof is an amount
capable of causing the destruction of a cell, especially tumor, e.g., cancer
cell, either in vitro or in vivo. A
"cytotoxic amount" of a PR0655, PR0364 or PR0344 polypeptide or an agonist
thereof for purposes of inhibiting
neoplastic cell growth may be determined empirically and in a routine manner.
The term "cytotoxic anent" 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'=', Y9° and
Re'e°), 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-
Myers SquibbOncology, Princeton, NJ), and doxetaxel (Taxotere, Rhdne-Poulenc
Rorer, Antony, Rnace), toxotere,
methotrexate, cisplatin, meiphalan, 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
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WO 00/32778 PCT/US99/28409
of a cell, especially tumor, e.g., cancer cell. either in vitro or in viva.
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 1 arrest and M-
phase arrest. Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxol, and topo II
inhibitors such as doxorubicin, epirubicin. daunorubicin, etoposide, and
bleomycin. Those agents that arrest G 1
also spill over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, S-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;
proiactin; placental lactogen; tumor necrosis factor-a and -p; mullerian-
inhibiting substance; mouse gonadotropin-
associated peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-(i; platelet-growth factor; transforming growth
factors (TGFs) such as TGF-a and
TGF-p; insulin-like growth factor-1 and -fI; 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-1, IL-la, IL-2, IL-3,
IL-4, IL-S, IL-6, IL-7, IL-8, IL-9, IL-11, IL-I2; 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, 6lSth
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, S-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 PR06SS, PR0364 or PR0344 poiypeptide disclosed herein.
Suitable agonist molecules
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WO 00/32778 PCT/US99/28409
specifically include agonist antibodies or antibody fragments, fragments or
amino acid sequence variants of native
PR0655, PR0364 or PR0344 polypeptides, peptides, small organic molecules, etc.
Methods for identifying
agonists of a PR0655, PR0364 or PR0344 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, hut rather is cyclic in nature.
"Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans,
domestic and fatrrt animals, and zoo, sports, or pet animals, such as dogs,
cats, cattle, horses, sheep, pigs, goats,
rabbits, etc. Preferably, the mammal is human.
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which are
nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the
physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically acceptable
carriers include buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid;
low molecular weight (less than about 10 residues) polypeptide; proteins, such
as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpytroiidone; amino
acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose,
mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as TWEENT"',
polyethylene glycol (PEG), and
PLURONICSTM
"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 ofdifferent immunoglobulin isotypes. Each heavy and light chain
also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at one end a variable domain (VH)
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 of the 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 complementarily-detetmtining 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


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/Z8409
FR regions, largely adopting a (i-sheet configuration, connected by three
CDRs, which form loops connecting, and
in some cases forming part of, the (3-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
"complementarily determining region" or "CDR" (i.e., residues 24-34 (L 1 ), SO-
S6 (L2) and 89-97 (L3) in the light
chain variable domain and 31-3S (H1), SO-65 (H2) and 9S-102 (H3) in the heavy
chain variable domain; Kabat et
al., Seauences of Proteins of Immunolo~,ical Interest, Sth Ed. Public Health
Service, National Institute of Health,
Bethesda, MD. [ 1991 j) 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 (H 1 ), 53-SS (H2) and
96-101 (H3) in the heavy chain
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')z, and Fv fragments;
diabodies; linear antibodies (Zapata et al., Protein Ens;., 8 10 : 1057-1062
[1995]); single-chain antibody
molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments,
each with a single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to
crystallize readily. Pepsin treatment yields an F(ab')Z fragment that has two
antigen-combining sites and is still
capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding site.
This region consists of a dimer ofone heavy- and one tight-chain variable
domain in tight, non-covalent association.
It is in this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on
the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-
binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising only three
CDRs specific for an antigen) has
the ability to recognize and bind antigen, although at a lower affinity than
the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH I )
of the heavy chain. Fab fragments differ from Fab' fragments by the addition
of a few residues at the carboxy
terminus ofthe 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')z antibody fragments originally were produced as pairs of Fab' fragments
which have hinge cysteines between
them. Outer 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 of their constant domains.
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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, lgE, IgG, and
IgM, and several ofthese may be further divided into subclasses (isotypes),
e.g., IgG I, IgG2, IgG3, IgG4, 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. Furthetzttore, in
contrast to conventional (polyclonal)
antibody preparationswh ichtypically includedifferent 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
the antibody by any particular method. For example, the monocional 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],
ormay 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,
x:624-628 [1991] and Marks et al., J. Mol. Biol.. 222:581-597 (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 ofthe
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 er al.,
Proc. Natl. Acad. Sci. USA, 81:6851
6855 [ 1984J).
"Humanized" fotrns of non-human (e.g., murine) antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 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
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CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
optimally also will comprise at least a portion of an immunoelobulin constant
region (Fc), typically that ofa human
immunoglobulin. For further details, see, Jones et al., Nature. X21:522-525
(1986); Reichmann et al., Nature.
X32:323-329 [1988]; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). The
humanized antibody includes a
PR1MATIZEDTMantibodywhereinthe 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 V" and V~ 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 Pharmacoloay of Monoclonal
Antibodies, Vol. 113. Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which fragments
comprise a heavy-chain variable domain (V") connected to a light-chain
variable domain (V~) in the same
polypeptide chain (VH - V~). By using a linker that is too short to allow
pairing between the two domains on the
same chain, the domains are forced to pair with the complementary domains of
another chain and create two
antigen-binding sites. Diabodies are described more fully in, for example, EP
404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated andlor
recovered from a component
of its natural environment. Contaminant components of its natural environment
are materials which would interfere
with diagnostic or therapeutic uses for the antibody, and may include enrymes,
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-tenminal 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
(eg., 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.
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A "liposome" is a small vesicle composed ofvarious types of lipids,
phospholipids and/or surfactant which
is useful for delivery of a drug (such as a PR0655, PR0364 or PR0344
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.
S A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
II. Comt~ositions and Methods of the Invention
A. Full-len th h PR0655. PR0364 and PR0344 Polvpeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PR0655, PR0364 and PR0344. In
particular, cDNAs encoding PR0655,
PR0364 and PR0344 polypeptides have been identified and isolated, as disclosed
in further detail in the Examples
below.
As disclosed in the Examples below, cDNA clones encoding PR0655, PR0364 and
PR0344 polypeptides
have been deposited with the 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 PR0655, PR0364 and
PR0344 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 lime.
B. PR0655. PR0364 and PR0344 Variants
In addition to the full-length native sequence PR0655, PR0364 and PR0344
polypeptides described
herein, it is contemplated that PR0655, PR0364 and PR0344 variants can be
prepared. PR0655, PR0364 and
PR0344 variants can be prepared by introducing appropriate nucleotide changes
into the PR0655, PR0364 or
PR0344 DNA, and/or by synthesis of the desired PR0655, PR0364 or PR0344
polypeptide. Those skilled in the
art will appreciate that amino acid changes may alter post-translational
processes of the PR0655, PR0364 or
PR0344 polypeptide, such as changing the number or position of glycosylation
sites or altering the membrane
anchoring characteristics.
Variations in the native full-length sequence PR0655, PR0364 or PR0344 or in
various domains of the
PR0655, PR0364 or PR0344 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 PR0655, PR0364 or
PR0344 that results in a change in the amino acid sequence of the PR0655,
PR0364 or PR0344 as compared with
the native sequence PR0655, PR0364 or PR0344. Optionally the variation is by
substitution of at least one amino
acid with any other amino acid in one or more of the domains of the PR0655,
PR0364 or PR0344. 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 PR0655, PR0364
or PR0344 with that of
homologous known protein molecules and minimizing the number of amino acid
sequence changes made in regions
49


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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 I to S amino
acids. The variation allowed may be determined by systematically making
insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for activity
exhibited by the full-length or mature
native sequence.
PR0655, PR0364 and PR0344 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 PR0655, PR0364 or PR0344 polypeptide.
PR0655, PR0364 and PR0344 fragments may be prepared by any of a number of
conventional
techniques. Desired peptide fragments may be chemically synthesized. An
alternative approach involves
generating PR0655, PR0364 and PR0344 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 enrymes and isolating the desired fragment. Yet another
suitable technique involves isolating
and amplifying a DNA Fragment encoding a desired polypeptide fragment, by
polymerase chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA fragment are
employed at the S' and 3' primers in the
PCR. Preferably, PR0655, PR0364 and PR0344 polypeptide fragments share at
least one biological and/or
immunological activity with the native PR06SS, PR0364 or PR0344 polypeptide
shown in Figure 2 (SEQ ID
N0:2), Figure 4 (SEQ ID N0:7), and Figure 6 (SEQ 1D N0:17), 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.


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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


10Glu (E) asp asp


Gly (G) pro; ala ala


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


lle (I) leu; val: met; ala; phe;


norleucine leu


15Leu (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


20Pro (P) ala ala


Ser (S) thr thr


Thr (T) ser ser


Trp (W) tyr; phe tyr


Tyr (Y) trp; phe; thr; ser phe


25Val (V) iie; ieu; met; phe;


ala; norleucine leu


Substantial modifications in function or immunological identity of the PR0655,
PR0364 or PR0344
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
30 conformation, (b) the charge or hydrophobicity of the molecule at the
target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on common side-
chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
35 (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
40 the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
ofigonucleotide-mediated (site-
S1


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
directed) mutagenesis, alanine scanning; and PCR mutaoenesis. Site-directed
mutagenesis [Carter et al., Nucl.
Acids Res.. 13:4331 ( 1986); Zoller et al., Nucl. Acids Res.. 10:6487 (
1987)], cassette mutagenesis [Wells er al.,
Gene. 34:315 ( 1985)], restriction selection mutagenesis [Wells et a/.,
Philos. Traps. R. Soc. London SerA _317:415
( 1986)] or other known techniques can be performed on the cloned DNA to
produce the PR0655, PR0364 or
PR0344 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 [Cunnineham 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 PR0655 PR0364 and PR0344
Covalent modifications of PR0655, PR0364 and PR0344 are included within the
scope of this invention.
One type of covalent modification includes reacting targeted amino acid
residues of a PR0655, PR0364 or
PR0344 polypeptide with an organic derivatizing agent that is capable of
reacting with selected side chains or the
N- or C- terminal residues of the PR0655, PR0364 or PR0344. Derivatization
with bifunctional agents is useful,
for instance, for crosslinking PR0655, PR0364 or PR0344 to a water-insoluble
support matrix or surface for use
in the method for purifying anti-PR0655, anti-PR0364 or anti-PR0344
antibodies, and vice-versa. Commonly
used crosslinkingagents include, e.g., I,1-bis(diazoacetyl)-2-
phenylethaneglutaraldehyde,N-hydroxysuccinimide
esters, forexampie, esters with 4-azidosalicylicacid, homobifunctional
imidoesters, inciuding 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 ofseryl orthreonyl residues, methylation of the a-amino groups of
lysine, arginine, and histidine side chains
[T.E. Creighton, Proteins: Structure and Molecular Properties W.H. Freeman &
Co., San Francisco, pp. 79-86
( 1983)], acetylation of the N-terminal amine, and amidation of any C-terminal
carboxyl group.
Another type of covalent modification of the PR0655, PR0364 or PR0344
polypeptide included within
the scope of this invention comprises altering the native glycosylation
pattern of the polypeptide. "Altering the
native giycosylation pattern" is intended for purposes herein to mean deleting
one or more carbohydrate moieties
found in native sequence PR0655, PR0364 or PR0344 (either by removing the
underlying glycosylation site or
by deleting the glycosylation by chemical and/or enzymatic means), and/or
adding one or more glycosylation sites
that are not present in the native sequence PR0655, PR0364 or PR0344. In
addition, the phrase includes
qualitative changes in the glycosylation of the native proteins, involving a
change in the nature and proportions of
52


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
the various carbohydrate moieties present.
Addition of glycosylation sites to the PR0655, PR0364 or PR0344 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 PR0655,
PR0364 or PR0344 (for O-linked
glycosylation sites). The PR0655, PR0364 or PR0344 amino acid sequence may
optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding the
PR0655, PR0364 or PR0344
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 PR0655,
PR0364 or PR0344
polypeptide is by chemical or enzymatic coupling of glycosides to the
pofypeptide. Such methods are described
in the art, e.g., in WO 87/05330 published I 1 September 1987, and in Aplin
and Wriston, CRC Crit. Rev. Biochem..
pp. 259-306 ( 1981 ).
Removal of carbohydrate moieties present on the PR0655, PR0364 or PR0344
polypeptide may be
accompl fished chem ically or enzymatically or by mutational substitution of
codons encoding for am ino 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. Biophvs., 259:52 ( 1987)
and by Edge et al., Anal. Biochem..
I 18:131 ( 1981 ). Enzymatic cleavage of carbohydrate moieties on polypeptides
can be achieved by the use of a
variety of endo- and exo-glycosidases as described by Thotakura et al., Meth.
Enzvmol.. 138:350 (1987).
Another type of covalent modification of PR0655, PR0364 or PR0344 comprises
linking the PR0655,
PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide of the present invention may also be
modified in a way
to form a chimeric molecule comprising PRO655, PR0364 or PR0344 fused to
another, heterologous polypeptide
or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PR0655,
PR0364 or PR0344
polypeptide with a tag polypeptide which provides an epitope to which an anti-
tag antibody can selectively bind.
The epitope tag is generaliy placed at the amino- or carboxyl- terminus of the
PR0655, PR0364 or PR0344
polypeptide. The presence of such epitope-tagged forms of the PR0655, PR0364
or PR0344 polypeptide can be
detected using an antibody against the tag polypeptide. Also, provision of the
epitope tag enables the PR0655,
PR0364 or PR0344 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 er al., Mol. Cell.
Biol., 8:2159-2165 ( 1988)]; the c-myc
tag and the SF9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto (Evan et al.,
Molecular and Cellular Bioloev.
5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and
its antibody [Paborsky e~ al.,
Protein EnQineerinQ. 'y6 :547-553 (1990)]. Other tag polypeptides include the
Flag-peptide [Hopp er al.,
Bio'TechnoloQV. 6: I 204-1210 ( 1988)]; the KT3 epitope peptide [Martin et
al., Science. 255: I 92-194 ( 1992)]; an
53


CA 02348157 2001-04-23
WO 00/32778 PC'T/US99/28409
a-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266: I 5 I 63-1 S I
66 ( 1991 )]; and the T7 gene ! 0 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 PR0655, PR0364 or
PR0344 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 Lhe Fc region of an
IgG molecule. The Ig fusions preferably include the substitution of a soluble
(transmembrane domain deleted or
inactivated) form of a PR0655, PR0364 or PR0344 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, CH I, CH2 and CH3 regions of an (gG I molecule. For the
production of immunoglobulin fusions see
also, US Patent No. 5,428,130 issued June 27, 1995.
D. Preuaration of PR0655, PR0364 and PR0344
The description below relates primarily to production of PR0655, PR0364 or
PR0344 by culturing cells
transformed or transfected with a vector containing PR0655, PR0364 or PR0344
nucleic acid. it is, of course,
contemplated that alternative methods, which are well known in the art, may be
employed to prepare PR0655,
PR0364 or PR0344. For instance, the PR0655, PR0364 or PR0344 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 PR0655, PR0364 or
PR0344 polypeptide may be
chemically synthesized separately and combined using chemical or enzymatic
methods to produce the full-length
PR0655, PR0364 or PR0344 polypeptide.
Isolation of DNA Encoding PR0655, PR0364 or PR0344
DNA encoding PR0655, PR0364 or PR0344 may be obtained from a cDNA library
prepared from tissue
believed to possess the PRO655, PR0364 or PR0344 m RNA and to express it at a
detectable level. Accordingly,
human PR0655, human PR0364 or human PR0344 DNA can be conveniently obtained
from a cDNA library
prepared from human tissue, such as described in the Examples. The PR0655-,
PR0364- or PR0344-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 PR0655,
PR0364 or PR0344 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
PR0655, PR0364 or PR0344 is to
use PCRmethodology [Sambrook er al., supra; Dieffenbach etal.. PCR Primer: A
Laboratory Manual (Cold Spring
54


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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'=P-labeled ATP,
biotinylation or enzyme 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
sequences deposited and available in public databases such as GenBank or other
private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across the
full-length sequence can be determined using methods known in the art and as
described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook e~ al.,
supra, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
2. Selection and Transformation of Host Cells
Host cells are uansfected or transformed with expression or cloning vectors
described herein for PR0655,
PR0364 or PR0344 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 Apuroach,
M. Butler, ed. (IRL Press, 1991 )
and Sambrook et al., supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCI=, CaPO" liposome-mediated and
electroporation. Depending on the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sambrook er al., supra, or
electroporation is generally used for
prokaryotes. Infection with Agrobacterium tnmefaciens is used for
transformation of certain plant cells, as
described by Shaw er 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, Viroloev, 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 ( I 977) and Hsiao et al., Proc. Natl.
Acad. Sci. (USA). 76:3829 ( 1979). However,
other methods for introducing DNA into cells, such as by nuclear
microinjection, electroporation, bacterial
3S protoplast fusion with intact cells, or polycations, e.g., polybrene,
polyomithine, may also be used. For various
techniques for transforming mammalian cells, see, Keown et al., Methods in
Enzvmolow, I 85:527-537 ( I 990) and
5$


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
Mansour et al., Nature. 336:348-352 ( I 988).
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 K 12 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 Enterobacteriaceae such
as Escherichia, e.g., E. toll, Enterobacter, Erwinia, Klebsiella, Proteus.
Salmonella, e.g.. Salmonellatvphimuritrm,
Serratia, e.g., Serratia nrarcescans, and Shigella, as well as Bacilli such as
B. subtilis and B. licheniformis (e.g.,
B. licheniformis 41 P disclosed in DD ?66,710 published 12 April 1989),
Pseudomonas such as P. aertrginosa, and
Streptomyces. These examples are illustrative rather than limiting. Strain W31
I 0 is one particularly preferred host
or parent host because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host
cell secretes minimal amounts of proteolytic enzymes. For example, strain
W3110 may be modified to effect a
genetic mutation in the genes encoding proteins endogenous to the host, with
examples of such hosts including E.
toll W31 10 strain I A2, which has the complete genotype tonA ; E. toll W31 10
strain 9E4, which has the complete
genotype tonA ptr3; E. toll W31 t0 strain 27C7 (ATCC 55,244). which has the
complete genotype tonAptr3 phoA
EI S (argF lac) 169 degP ompT karf; E. toll W3110 strain 37D6, wh ich has the
complete genotype tonA ptr3 phoA
EIS (argF lac)169 degP ompT rbs7 ilvG karr ; E. toll W3110 strain 4084, 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 polymerise reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning or
expression hosts for PR0655-, PR0364- or PR0344-encoding vectors.
Saccharomyces cerevisiae is a commonly
used lower eukaryotic host microorganism. Others include Schcosaccharomyces
pombe (Beach and Nurse,
Nature. 290: 140 [1981]; EP 139,383 published 2 May 1985); Kluymeromyces hosts
(U.S. Patent No. 4,943,529;
Fleer et al., Bio/Technolow, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-
8C, CBS683, CBS4574;
Louvencourt et al., J. Bacteriol.. 737 [ 1983]), K. fragilis (ATCC 12,424), K.
bulgaricrrs (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilurum (ATCC
36,906; Van den Berg et al.,
Bio/Technolosy. 8: I 35 ( 1990)), K. thermotolerans, and K. marxiantrs;
yarrowia (EP 402.236); 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. Acid. Sci. USA, 76:259-5263
[1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 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. nidnlans (Ballance et al., Biochem. Biophvs. Res. Commun., I 12:284-289
[1983]; Tilburn et al., Gene,
26:205-221 [1983]: Yelton et al., Proc. Natl. Acid. Sci. USA 81: 1470-1474
[1984]) and A. niger (Kelly and
Hynes, EMBO J.. ~t:47S-479 [ 1985]). Methylotropic yeasts are suitable herein
and include, but are not limited to,
yeast capable of growth on methanol selected from the genera consisting of
Hansenula, Candida. Kloeckera,
Pichia. Sacclurronrrces, Torrrlopsis, and Rhodotorula. A list of specific
species that are exemplary of this class of
56


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
yeasts may be found in C. Anthony, The Biochemistry of Methvlotrophs. 269
(1982).
Suitable host cells for the expression of glycosylated PR0655, PR0364 or
PR0344 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}
S and COS cells. More specific examples include monkey kidney CV I line
transformed by SV40 (COS-7, ATCC
CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth
in suspension culture, Graham
et al., J. Gen. Virol.. 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA, 77:4216 ( 1980)); mouse sertoli cells (TM4, Masher. Biol.
Renrod., 23:243-251 ( 1980)); human
lung cells (W 138, ATCC CCL ?5); human liver cells (Hep G2, HB 8065); and
mouse mammary tumor (MMT
060562, ATCC CCL51 ). The selection of the appropriate host cell is deemed to
be within the skill in the art.
Selection and Use of a Reolicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PR0655, PR0364 or PR0344
may be inserted
into a replicable vector for cloning (amplification of the DNA) or for
expression. Various vectors are publicly
available. The vector may, for example, be in the form of a plasmid, cosmid,
viral particle, or phage. The
appropriate nucleic acid sequence may be inserted into the vector by a variety
of procedures. In general, DNA is
inserted into an 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 term
ination sequence. Construction of suitable
vectors containing one or more of these components employs standard ligation
techniques which are known to the
skilled artisan.
The PR0655, PR0364 or PR0344 may be produced recombinantly not only directly,
but also as a fusion
polypeptide with a heterologous polypeptide, which may be a signal sequence or
other polypeptide having a specific
cleavage site at the N-terminus of the mature protein or polypeptide. In
general, the signal sequence may be a
component of the vector, or it may be a part of the PR0655-. PR036d- or PR0344-
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 Saccharomvces and Kluyveromvces
a-factor leaders, the latter described in U.S. Patent No. 5.010,182), or acid
phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 April 1990), or the signal
described in WO 90/13646 published 15
November 1990. In mammalian cell expression, mammalian signal sequences may be
used to direct secretion of
the protein, such as signal sequences from secreted 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 2u plasmid origin
is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus.
VSV or BPV} are useful for cloning
57


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/Z8409
vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker_
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, c.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 PR0655-, PR0364- or PR0344-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 trpl 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 ( t 977)].
Expression and cloning vectors usually contain a promoter operably finked to
the PR0655-, PR0364- or
PR0344-encoding nucleic acid sequence to direct mRNA synthesis. Promoters
recognized by a variety ofpotential
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, 28 I :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. 80:21-35 ( t 983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding PR0655, PR0364 or PR0344.
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, Biochemistry, 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
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.
PR0655. PR0364 or PR0344 transcription from vectors in mammalian host cells is
controlled, for
example, by promoters obtained from the genomes of viruses such as polyoma
virus, fowipox virus (UK 2,211,504
published S July 1989), adenovirus (such as Adenovirus 3), 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
58


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
promoters are compatible with the host cell systems.
Transcription of a DNA encoding the PR0655, PR0364 or PR0344 by higher
eukaryotes may be
increased by inserting an enhancer sequence into the vector. Enhancers are cis-
acting elements of DNA, usually
about from 10 to 300 bp, that act on a promoter to increase its transcription.
Many enhancer sequences are now
known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and
insulin). Typically, however, onewill
use an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter enhancer,
the polyoma enhancer on the late
side of the replication origin, and adenovirus enhancers. The enhancer may be
spliced into the vector at a position
S' or 3' to the PR0655, PR0364 or PR0344 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 oftranscription
and for stabilizing the mRNA. Such sequences are commonly available from the
5' and, occasionally 3',
untranslatedregionsofeukaryoticorviraIDNAsorcDNAs.
Thesere~ionscontainnucleotidesegmentstranscribed
I 5 as polyadenylated fragments in the untranslated portion of the mRNA
encoding PRO655, PR0364 or PR0344.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PR0655, PR0364
or PR0344 in recombinant vertebrate cell culture are described in Gething et
al., Nature, 293:620-625 (1981);
Mantel et al., Nature, 281:40-46 ( 1979); EP 117,060; and EP I 17,058.
4. Detecting Gene Am~ificationlExoression
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 siW hybridization,
using an appropriately labeled probe,
based on the sequences provided herein. Alternatively, antibodies may be
employed that can recognize specific
duplexes, including DNA duplexes, RNA duplexes. and DNA-RNA hybrid duplexes or
DNA-protein duplexes.
The antibodies in turn may be labeled and the assay may be carried out where
the duplex is bound to a surface, so
that upon the formation of duplex on the surface, the presence of antibody
bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as
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 PR0655, PR0364 or PR0344
polypeptide or against a
synthetic peptide based on the DNA sequences provided herein or against
exogenous sequence fused to PR0655,
PR0364 or PR0344 DNA and encoding a specific antibody epitope.
5. Purification of Polvpeptide
Forms of PR0655, PR0364 or PR0344 may be recovered from culture medium or from
host cell lysates.
59


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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 PR0655, PR0364 or
PR0344 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 PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344. Various methods
of protein purification may
be employed and such methods are known in the art and described for example in
Deutscher, Methods in
Enzvmoloev, I 82 ( 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 PR06S5, PR0364 or PR0344 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 PR065S, PR0364 or
PR0344 polypeptide.
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 PR06S5,
PR0364 or PR0344 polypeptide or
a fusion protein thereof. It may be useful to conjugate the immunizing agent
to a protein known to be immunogenic
in the mammal being immunized. Examples of such immunogenic proteins include
but are not limited to keyhole
limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin
inhibitor. Examples of adjuvants
which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid A,
synthetic trehalose dicorynomycolate). The immunization protocol may be
selected by one skilled in the art without
undue experimentation.
2. Monoclonal Antibodies
The 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.


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
The immunizing agent will typically include the PR06~5. PR0364 or PR0344
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: Principles
and Practice. Academic Press, ( t 986)
pp. 59-103]. Immortalized cell lines are usually transformed mammaliancel(s,
particularly myelomacells ofrodent,
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 marine myeloma lines. which can be
obtained, for instance. from the Salk
Institute Cell Distribution Center, San Die_o, California and the American
Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have
been described forthe production
of human monoclonal antibodies [Kozbor, J. Immunoi., 133:3001 ( 1984); Brodeur
et al., Monoclonal Antibody
Production Techniaues and Applications, Marcel Dekker. Inc., New York, (1987)
pp. S1-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of
monoclonal antibodies directed against PR0655, PR036.1 or PR0344. Preferably,
the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined by
immunoprecipitation or by an in vitro
binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent
assay (ELISA). Such
techniques and assays are known in the art. The binding affinity of the
monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem.,
107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution
procedures and grown by standard methods [coding, 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
or ascites fluid by conventional immunoglobulin purification procedures such
as, for example, protein A-
Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or
affinity chromatography.
The monoclonal antibodies may also be made by recombinant DNA methods, such as
those described in
U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the
invention can be readily isolated and
sequenced using conventional procedures (e.g., by using oligonucleotide probes
that are capable of binding
specifically to genes encoding the heavy and light chains of marine
antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression vectors,
61


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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.
Patent No. 4,816,567: Morrison et al., supra) or by covalently joinin; to the
immunoglobulin coding sequence all
or pan 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 ofantibodiesto produce
fragments thereof, particularly, Fab fragments, can be accomplished using
routine techniques known in the art.
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) ofthe
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 substantially all of at least
one, and typical ly two, variable domains.
in which all or substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and al!
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 ul., Nature, 321:522-~2~ ( 1986);
Riechmann e~ al., Nature, 332:323-329
( 1988); and Presta, Curr. Op. Struct. Biol.. ?:593-596 ( 1993)].
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, 331:522-525 (1986}: Riechmann er al., Nature, 332:323-
327 (1988); Verhoeyen et al.,
62


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
Science, 339: I 534-1536 ( 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, J. Mol. Biol.. 227:381 (1991): Marks et al.,
J. Mol. Biol.. 222:581 (1991)].
The techniques of Cole et al., and Boerner et al., are also available for the
preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Theranv. Alan R.
Liss, p. 77 ( 1985) and Boerner et al.,
J. lmmunol., 147 l :86-95 (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/Technolow, 10: 779-783
( 1992); Lonberg et al., Nature. 368: 856-859 ( 1994); Morrison. Nature, 368:
812-13 ( I 994); Fishwild et al., Nature
Biotechnoloev, 14:845-51 ( 1996); Neuberger, Nature Biotechnoloav. 14: 826 (
1996); Lonberg and Huszar, Intern.
Rev. Immunol., I 3 :6S-93 ( 1995).
4. Bisnecific 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
PR065S, PR0364 or PR0344, 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
ofbispecific antibodies is based on the co-expression of two immuno~lobulin
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., I 0:3655-3659 ( t 991 ).
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. CH?, 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
63


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
chain, are inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further
detailsofgeneratingbispecificantibodiessee, for example, Suresh erul., Methods
in Enzvmoloev 121:210(1986).
According to another approach described in WO 96/2701 1. 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 pan 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'),
bispecific antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared using chemical linkage. Brennan
et al., Science, 229:81 ( I 985) describe a procedure wherein intact
antibodies are proteolytically cleaved to generate
F(ab')= fragments. These fragments are reduced in the presence of the dithio)
complexing agent sodium arsenite
to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then
converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the
Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced
can be used as agents for the
selective immobilization of enzymes.
Fab' fragments may be directly recovered from E. toll and chemically coupled
to form bispecific
antibodies. Shalaby e~ al., J. Ex~. Med., 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')_ molecule. Each Fab' fragment was separately
secreted from E. toll and subjected to
directed chemical coupling in virro to form the bispecific antibody. The
bispecific antibody thus formed was able
to bind to cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity
of human cytotoxic lymphocytes against human breast tumor tar_ets.
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 e~ al., J. Immunol., 148(5):1547-1553 ( 1992). The leucine zipper
peptides from the Fos and Jun proteins
were linked to the Fab' portions of two different antibodies by gene fusion.
The antibody homodimers were reduced
at the hinge region to form monomers and then re-oxidized to form the antibody
heterodimers. This method can
also be utilized for the production of antibody homodimers. The wdiabody"
technology described by Hollinger et
aL, Proc. Natl. Acad. Sci. USA 90:6444-6448 ( 1993) has provided an
alternative mechanism for making bispecific
antibody fragments. The fragments comprise a heavy-chain variable domain (VH)
connected to a light-chain
variable domain (V~) by a linker which is too short to allow pairin; between
the two domains on the same chain.
Accordingly, the V" and V~ domains of one fragment are forced to pair with the
complementary V~ and V" domains
of another fragment, thereby forming two antigen-binding sites. Another
strategy for making bispecific antibody
64


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
fragments by the use of single-chain Fv (sFv) dimers has also been reported.
See. Gruber et al.. J. Immunol..
I X2:5368 ( 1994 ).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be
prepared. Tutt et aL, J. Immunol., Id7:60 ( 1991 ).
Exemplarv_ bispecific antibodies may bind to two different epitopes on a given
PR0655, PR0364 or
PR0344 polypeptide herein. Alternatively, an anti-PR06~.i. anti-PR0364 or anti-
PR0344 polypeptide arm may
be combined with an arm which binds to a triggering molecule on a leukocyte
such as a T-cell receptor molecule
(e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI
(CD64), FcyRl1 (CD32) and
FcyRl l l {CD 16) so as to focus cellular defense mechanisms to the cel I
expressing the particular PR0655, PR0364
or PR0344 polypeptide. Bispecific antibodies may also be used to localize
cytotoxic agents to cells which express
a particular PR0655, PR0364 or PR0344 polypeptide. These antibodies possess a
PR0655-, PR0364- or
PR0344-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 PR0655,
PR0364 or PR0344
polypeptide and further binds tissue factor (TF).
1$ 5. Heteroconiugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies
are composed of two covalently joined antibodies. Such antibodies have, for
example, been proposed to target
immune system cells to unwanted cells [U.S. Patent No. 1,676,980], 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
involvingcrosslinkingagents. 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-d-
mercaptobutyrimidate and those disclosed, for
example, in U.S. Patent No. 4,676,980.
6. Effector Function Engineering
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to
enhance, e.g., the effectiveness of the antibody in treating cancer. For
example, cysteine residues) may be
introduced into the Fc region, thereby allowing interchain disulfide bond
formation in this region. The
homodimeric antibody thus generated may have improved internalization
capability and/or increased complement-
mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See, Caron et al., J. Ex~. Med., 176:
I 191-1195 ( 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. Sc~e, Stevenson
et al., Anti-Cancer Drug Design,
3: 219-230 ( 1989).
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CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
Immunoconiugates
The invention also pertains to immunoconjugates comprising an antibody
conjugated toacytotoxic 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.
Enrymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding active
fragments of diphtheria toxin, exotoxin A chain (from Pseudomonus aeruginosa),
ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phyrolaca americarra proteins (PAPI,
PAPI1, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A
variety of radionuclides are available for
the production of radioconjugated antibodies. Examples include ='=Bi. "'l,
"'In, ~°Y, and'B6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling
agents such asN-succinimidyl-3-(2-pyridyldithiol)propionate(SPDP).
iminothiolane(IT),bifunctionalderivatives
of imidoesters (such as dimethyladipimidate 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,S-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be
prepared as described in Vitetta et al., Science, 238: 1098(1987). Carbon-14-
labeled 1-isothiocyanatobenryl-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. Immunolinosomes
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 er aL, Proc. Natl. Acad. Sci. USA
82: 3688 (1985); Hwang e~ aL, 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: 386-288 (1982) via a disulfide-interchange reaction. A
chemotherapeutic agent (such as
Doxorubicin) is optionally contained within the liposome. .See, Gabizon e~ al,
J. National Cancer Inst.. 81 ( 19):
1484 ( 1989).
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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 vi~ro drug-
discovery screen of the National Cancer
Institute (NCI). The purpose of this screen is to identify molecules that have
cytotoxic andlor cytostatic activity
$ against different types of tunl,ors. NCI screens more than 10,000 new
molecules per year (Monks el al., J. Natl.
Cancer Inst., 83:757-766 ( 1991 ): Boyd, Cancer: Princ. Pract. Oncol. Update,
3 10 : I-12 ([ 1989]). The tumor cell
lines employed in this study have been described in Monks et al.. supra. The
cell lines the growth of which has
been significantly inhibited by the proteins of the present application are
specified in the Examples.
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 cel I-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
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 well 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 ntr 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, B10.LP, C17, C3H, C57BL, C57, CBA, DBA, DDD. I/st, NC, NFR,
NFS, NFSM, 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 Oncoloev Research, E. Boven and B. WinoVrad, eds., CRC
Press. lnc., 199(.
The cells introduced into such animals can be derived from known tumor/cancer
cell lines. such as, any
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of the above-listed tumor cell lines, and, for example. the 8104- I-1 cell
line (stable NIH-3T3 cell line transfected
with the ne:r protooncogene); ras-transfected NIH-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
S 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. 'Fhe
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
sol id block or trochar implantation, tumor
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 virro 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 WEHI-164 are chemically induced
fibrosarcomas of
BALB/c female mice (DeLeo et aL, J. Exo. Med., 146:720 [ 1977]), which provide
a highly controllable model
system for studying the anti-tumor activities of various agents (Palladino et
al., J. Immunol.. 138:4023-4032
[ t 987]). 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 1 Ox 10°
to I Ox 10' cells/ml. The animals are then
infected subcutaneously with 10 to I 00 ,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 benefcial effects in the treatment ofhuman patients diagnosed
with small cell carcinoma ofthe 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
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WO 00/32778 PCT/US99/28409
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 on an
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 convened
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
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 gds., 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) animal models can be engineered by introducing the
coding portion ofthe genes
identified herein into
thegenomeofanimalsofinterest,usingstandardtechniquesforproducingtransgenicanima
ls.
Animals that can serve as a target for transeenic 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 etal., C~ 56:313-321
[1989]); electroporation of embryos (Lo, Mol. Cell. Biol.. 3_:1803-18t4
(1983]); sperm-mediated gene transfer
(Lavitrano et al., Cell, 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 er
al., Proc. Natl. Acad. Sci. USA. _89:6232-
636 ( 1992).
The expression of the trans~ene 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 ofcats, 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
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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 squarnous 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. A fter
treatment. each cat undergoes another CT
scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter.
The data are evaluated for
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 rumor
in animals.
H. Screenine Assavs for Drue 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 sol id 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


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
be detected, for example, by using a labeled antibody specifically binding the
immobilized complex.
Ifthe candidate compound interacts with but does not bind to a particular
receptor, its interaction with that
polypeptide can be assayed by methods wel I known for detectin_ 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 Sone. Nature
(London), 340:245-246 ( 1989); Chien
et 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
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 GAL t-lacZ
reporter gene under control of a
GAL4-activated promoter depends on reconstitution of GALA activity via protein-
protein interaction. Colonies
1 S containing interacting poiypeptides 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 potypeptides 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 er
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 _rowth-
inhibitory agent. Such molecules are
suitably present in combination in amounts that are effective for the purpose
intended.
Therapeutic formulations ofthe polypeptides identified herein, oragonists
thereof are prepared for storage
by mixing the active ingredient having the desired degree of purity with
optional pharmaceutically acceptable
carriers, excipients or stabilizers (Renrinmnn's PHarmaceurica! Sciences 16th
edition, Osol, A. ed. [ 1980]), in the
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form of lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic
to recipients at the dosages and concentrations employed, and include buffers
such as phosphate, citrate, and other
organic acids;antioxidants including ascorbic acid
andmethionine:preservatives(suchasoctadecyldimethylbenzyl
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)
polvpeptides: proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparasine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates
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-particles and nanocapsules) or in
macroemulsions. Such techniques
are disclosed in ReminQton'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
lyophilization and reconstitution.
Therapeutic compositions herein generally are placed into a container having a
sterile access port, for
example, an intravenous solution bag or vial having a stopper pierceable by a
hypodermic injection needle.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in the
form of shaped articles, e.g., films, or microcapsules. Examples of sustained-
release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No.
3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-
degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON 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
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sulfhydryl residues, lyophilizing from acidic solutions, controllin~z moisture
content. using appropriate additives,
and developing specific polymer matrix compositions.
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
various head and neck tumors); leukemias and lymphoid malignancies; other
disorders such as neuronal, glial,
astrocytai, 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 Chemotheraov 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
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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 of the 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 for human 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 I ,ug/kg
to (5 mg/kg (e.g., 0.1-20
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
I ~cg/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 ofdisease
symptoms occurs. However, other dosage regimens may be useful. The progress
ofthis 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
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 phatntaceutically-
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, f Iters, 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.
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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
CultureCollection.Manassas, VA.
EXAMPLE 1
Isolation of cDNA clones Encoding PR065~. PR0364 and PR0344
(A) PR0655
An expressed sequence tag (EST) DNA database (Ll FESEQh . ! ncyte
Phamaceuticals, Palo Alto. CA) was
searched and an EST was identified which showed homology to interferon.
Possible homology was noted between
Incyte EST 3728969 (subsequently renamed as DNA49668) and mammalian alpha
interferons. The homology was
confirmed by inspection.
RNA for construction of cDNA libraries was then isolated from human small
intestine (LIB 99). The
cDNA libraries used to isolate the cDNA clones encoding human PR0655 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 Sall hemikinased
adaptors, cleaved with Noti, sized
appropriately by gel electrophoresis, and cloned in a defined orientation into
a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor of pRKSD that does not contain the SfiI
site; see, Holmes ei al., Science,
253:1278-1280 ( 1991 )) in the unique Xhol and Notl.
Oligonucleotides 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 PRO655. 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 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 Bioloey, 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 probes employed were as follows:
reverse PCR primer 1:
5'-TCTCTGCTTCCAGTCCCATGAGTGC-3' (SEQ ID N0:3)
reverse PCR primer 2:
5'-GCTTCCAGTCCCATGAGTGCTTCTAGG-3' (SEQ 1D N0:4)
hvbridization probe
5'-GGCCATTCTCCATGAGATGCTTCAGCAGATCTTCAGCCTCTTCAGGGCAA-3' (SEQ ID NO:S)
A full length clone was identified that contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 621-623 and a stop signal at
nucleotide positions 1245-1247 (Figure I, SEQ
ID NO:1 ). The predicted polypeptide precursor is 208 amino acids long, and
has a calculated molecular weight of


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
approximately 24,414 daltons and an estimated p1 of approximately 8.92.
Analysis of the full-length PR0655
sequence shown in Figure 2 (SEQ ID N0:2) 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 PR0655 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 31: N-glycosylation sites
from about amino acid 95 to about
amino acid 99, and from about am ino acid I 04 to about amino acid I 08: a
casein kinase 11 phosphorylation site from
about amino acid l81 to about amino acid I 85; an N-myristoylation site from
about amino acid 133 to about amino
acid 139; and an interferon alpha, beta and delta family signature from about
amino acid 147 to about amino acid
166. Clone DNA50960-1224 has been deposited with ATCC on December 3, 1997 and
is assigned ATCC deposit
no.209509.
An analysis ofthe Dayhoffdatabase (version35.45 SwissProt 35), using the ALIGN-
2 sequence alignment
analysis ofthe full-length sequence shown in Figure 2 (SEQ ID N0:2), evidenced
about 35-40% sequence identity
between the PR0655 amino acid sequence and various human IFN-a species. The
homology is highest within the
22-189 amino acid region of the sequence shown in Figure 2 (SEQ ID N0:2). At
the nucleotide level, the
homology with the coding sequence of IFN-a is about 60%.
(B) PR0364
An expressed sequence tag (EST) DNA database and a proprietary EST database
(LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA) was searched and an EST (Incyte ESTno.
3003460) was identified which showed
homology to members of the tumor necrosis factor receptor (TNFR) family of
polypeptides.
A consensus DNA sequence was then assembled relative to the Incyte EST no.
3003460 and other EST
sequences using repeated cycles of BLAST (Altshul et al., Methods in
Enzvmoloev, 266:460-480 (1996)) and
"phrap" (Phil Green, University of Washington, Seattle, Washington). This
consensus sequence is herein
designated DNA44825.
Based upon the DNA44825 consensus sequence, oligonucleotides probes 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 PR0364. 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 order to screen several libraries for a
full-length clone, DNA from the libraries
was screened by PCR amplification, as per Ausubel e~ al.. Current Protocols in
Molecular Biolosv, 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 probes employed were as follows:
forward PCR urimer 1:
5'-CACAGCACGGGGCGATGGG-3' (SEQ ID N0:8)
forward PCR primer 2:
5'-GCTCTGCGTTCTGCTCTG-3' (SEQ ID N0:9)
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forward PCR primer 3:
5'-GGCACAGCACGGGGCGATGGGCGCGTTT-3' (SEQ ID NO:10)
reverse PCR primer 1:
5'-CTGGTCACTGCCACCTTCCTGCAC-3' (SEQ ID NO: I t )
reverse PCR primer 2:
5'-CGCTGACCCAGGCTGAG-3' (SEQ ID N0:12)
reverse PCR primer 3:
5'-GAAGGTCCCCGAGGCACAGTCGATACA-3' (SEQ ID N0:13)
hybridization probe I
5'-GAGGAGTGCTGTTCCGAGTGGGACTGCATGTGTGTCCAGC-3' (SEQ ID NO: I4)
hybridization arobe 2:
5'-AGCCTGGGTCAGCGCCCCACCGGGGGTCCCGGGTGCGGCC-3' (SEQ ID NO: l5)
RNA for construction ofcDNA libraries was then isolated from human small
intestine tissue. The cDNA
libraries used to isolate the cDNA clones encoding human PR0364 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 Nott site, linked with blunt to Sall 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 Xhol and Notl.
A full length clone for DNA47365-1206 was identified that contained a single
open reading frame with
an apparent translational initiation site at nucleotide positions 121-123 and
a stop signal at nucleotide positions 844-
846 (Figure 3, SEQ ID N0:6). The predicted polypeptide precursor is 241 amino
acids long, and has a calculated
molecular weight of approximately 26,000 daltons and an estimated pl of
approximately 6.34.
Analysis of the full-length PR0364 sequence shown in Figure 4 (SEQ ID N0:7)
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 PR0364
sequence evidenced the following: a
signal peptide from about amino acid 1 to about amino acid 25; a potential
transmembrane domain from about
amino acid 163 to about amino acid 183: an N-glycosylation site from about
amino acid 146 to about amino acid
1 S0; N-myristoylation sites from about amino acid ~ to about amino acid I I,
from about amino acid 8 to about
amino acid 14, from about amino acid 25 to about amino acid 31, from about
amino acid 30 to about amino acid
36, from about amino acid 33 to about amino acid 39, from about amino acid 118
to about amino acid 124, from
about amino acid 122 to about amino acid 128, and from about amino acid 156 to
about amino acid 162; a
prokaryotic membrane lipoprotein lipid attachment site from about amino acid
166 to about amino acid 177; and
a leucine zipper pattern from about amino acid 171 to about amino acid 193..
Clone DNA47365-1206 has been deposited with ATCC on November 7, 1997 and is
assigned ATCC
deposit no. 209436.
An analysisofthe Dayhoff database (version 35.45 SwissProt 35), using the
ALIGN-2 sequence alignment
77


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analysis of the full-length sequence shown in Figure d (SEQ ID N0:7),
evidenced sequence identity between the
PR0364 amino acid sequence and members of the tumor necrosis factor receptor
family, thereby indicating that
PR0364 may be a novel member of the tumor necrosis factor receptor family.
A detailed review of the amino acid sequence of the full-length native PR0364
polypeptide and the
nucleotide sequence that encodes that amino acid sequence evidences sequence
homology with the mouse GITR
protein reported by Nocentini et al., Proc. Natl. Acad. Sci. USA, 91:6216-6221
(1997). It is possible, therefore,
that PR0364 represents the human counterpart to the mouse GITR protein
reported by Nocentini et al.
(C) PR0344
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 EST databases (e.g., GenBank), and a proprietary EST
database (LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA}. The search was performed using the computer
program BLAST or BLAST2
[Altschul et aL, Methods in Enzvmolopyy, 266:460-480 ( 1996)) as a comparison
of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
1S 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).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA34398. tn some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed
above.
Based on the DNA34398 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 PR0344. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about I 00-1000 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 Bioloev s:rpra, 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.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 1:
5'-TACAGGCCCAGTCAGGACCAGGGG-3' (SEQ ID N0:18)
forward PCR primer 2:
5'-AGCCAGCCTCGCTCTCGG-3' (SEQ ID N0:19)
forward PCR primer 3:
78


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
5'-GTCTGCGATCAGGTCTGG-3' (SEQ ID N0:20)
reverse PCR primer I
5'-GAAAGAGGCAATGGATTCGC-3' (SEQ ID N0:21 )
reverse PCR primer 2:
S 5'-GACTTACACTTGCCAGCACAGCAC-3' (SEQ ID N0:22)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA34398
sequence which had the following nucleotide sequence:
hybridization probe:
5'-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3' (SEQ ID N0:23)
RNA for construction of the cDNA libraries was isolated from human fetal lung
tissue. 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 Sfil site; see, Holmes et al.. Science,
253:1278- t 280 ( I 991 )) in the unique Xhol
and Notl sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for a
full-length PR0344 polypeptide (designated herein as DNA40592-1242 [Figure S,
SEQ ID NO: 16)) and the
derived protein sequence for that PR0344 polypeptide.
The full length clone identifced above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 227-229 and a stop signal at
nucleotide positions 956-958 (Figure 5, SEQ ID
N0:16). The predicted polypeptide precursor is 243 amino acids long, and has a
calculated molecular weight of
approximately 25,298 daltons and a pI ofabout 6.44. Analysis ofthe full-length
PR0344 sequence shown in Figure
6 (SEQ 1D N0:17) 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
PR0344 sequence evidenced the following: a signal peptide from about amino
acid 1 to about amino acid 15; N-
myristoylation sites from about amino acid I f to about amino acid 17, from
about amino acid 68 to about amino
acid 74, and from about amino acid 216 to about amino acid 223: and a cell
attachment sequence from about amino
acid 77 to about amino acid 80.
Clone DNA40592-1242 has been deposited with ATCC on November 2l, 1997 and is
assigned ATCC
deposit no. 209492.
EXAMPLE 3
In sitzr Hybridization
In suu 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
79


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mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version ofthe
protocol by Lu and Gillett, Cell
Vision. I: 169-176 (1994), using PCR-generated '3P-labeled riboprobes.
Briefly, formalin-fixed, paraffin-
embedded human tissues were sectioned, deparaffinized, deproteinated in
proteinase K (20 g/ml) for IS minutes
at 37°C, and further processed for in sinr hybridization as described
by Lu and Gillett, srrpra. A ("-P)UTP-labeled
antisense riboprobe was generated from a PCR product and hybridized at 55
°C overnight. The slides were dipped
in Kodak NTB2T"' nuclear track emulsion and exposed for 4 weeks.
"P-Ribonrobe svnthesis
6.0 ul (125 mCi) of "P-UTP (Amersham BF 1002. SA<2000 Ci/mmol) were speed-
vacuum dried. To
each tube containing dried "P-UTP, the following ingredients were added:
2.0Ic1 Sx transcription buffer
1.0 ~I DTT (100 mM)
2.0 ,ul NTP mix (2.5 mM: 10 tcl each of 10 mM GTP, CTP & ATP + 10 ~1 H=O)
t.0 ul UTP (50 ~cM)
1.0 ~cl RNAsin
I.0 ~cl DNA template ( 1 fig)
1.0 ~I H,O
1.0 ~cl RNA polymerise (for PCR products T3 = AS, T7 = S, usually)
The tubes were incubated at 37°C for one hour. A total of 1.0 ul RQ1
DNase was added, followed by
incubation at 37°C for 15 minutes. A total of 90 ~cl 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-SOT"'
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 ~I TE was added, then t ~I 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 ul of the probe or 5 ul of
RNA Mrk III was added
to 3 ~l 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-Hybridization
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 (2S ml 20 x SSC + 975 ml SQ H=O). After deproteination in 0.5
~g/ml proteinase K for 10
minutes at 37°C (12.5 ul of 10 mglml 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%


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
ethanol, 2 minutes each.
B. Pretreatment oJparajfrn-embedded sections
The slides were deparaffinized, placed in SQ H,O, and rinsed twice in 2 x SSC
at room temperature, for
minutes each time. The sections were deproteinated in 20 ~g/ml proteinase K
(500 ul of l0 mg/ml in 250 ml
5 RNase-free RNase buffer; 37°C, 15 minutes) for human embryo tissue.
or 8 x proteinase K ( l00 ul in 250 ml Rnase
buffer, 37°C, 30 minutes) for formalin tissues. Subsequent rinsing in
0.5 x SSC and dehydration were performed
as described above.
C. Prehybridi=anon
The slides were laid out in a plastic box lined with Box buffer (4 x SSC, 50%
formamide) - saturated filter
paper. The tissue was covered with 50 ~1 of hybridization buffer (3.75 g
dextran sulfate+6 ml SQ H,O), vortexed,
and heated in the microwave for 2 minutes with the cap loosened. After cooling
on ice, 18.75 ml fotrnamide, 3.75
ml 20 x SSC, and 9 ml SQ H=O were added, and the tissue was vortexed well and
incubated at 42°C for 1-4 hours.
D. Hybridisation
1.0 x 10° cpm probe and I .0 ul tRNA (50 mg/ml stock) per slide were
heated at 95 °C for 3 minutes. The
slides were cooled on ice, and 48 ~cl hybridization buffer was added per
slide. After vortexing, 50 ul "P mix was
added to 50 ul prehybridization on the slide. The slides were incubated
overnight at 55°C.
E. Washes
Washing was done for 2x 10 minutes with 2xSSC, EDTA at room temperature (400
ml 20 x SSC + 16 ml
0.25 M EDTA, Vr4L), followed by RNAseA treatment at 37°C for 30 minutes
(500 ~I of 10 mg/ml in 250 ml
Rnase buffer=20 ~cg/ml), The slides were washed 2 x 10 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,
V f=4L).
F. Oligon:~cleotides
In situ analysis was performed on one of the DNA sequences disclosed herein.
The oligonucleotides
employed for this analysis are as follows:
( 1 ) DNA47365-1206 (PR0364)
pl:
5'-GGA TTC TAA TAC GAC TCA CTA TAG GGC AAC CCG AGC ATG GCA CAG CAC-3' (SEQ 1D
N0:24)
p2:
5'-CTA TGA AAT TAA CCC TCA CTA AAG GGA TCT CCC AGC CGC CCC TTC TC-3' (SEQ ID
N0:25)
(G) Results
!n situ analysis was performed on the above DNA sequence disclosed herein. The
results from this
analysis is as follows:
(I) DNA47365-1206 (PR0364) (novel TNF-receptor Homoloe)
In the fetus, expression was observed in the fascia lining the anterior
surface of the vertebral body. In
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CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
addition, expression was seen over the fetal retina. Low level expression was
seen over fetal neurones. All other
tissues were negative.
EXAMPLE 3
Use of PR0655, PR0364 or PR0344 as a Hybridization Probe
The following method describes use ofa nucleotide sequence encoding PR0655,
PR0364 or PR0344 as
a hybridization probe.
DNA comprising the coding sequence of full-length or mature PR0655, PR0364 or
PR0344 (as shown
in Figure I, 3, and 5, respectively, SEQ ID NOS: I, 6, and 16, respectively)
or a fragment thereof is employed as
a probe to screen for homologous DNAs (such as those encoding naturally-
occurring variants of PR0655, PR0364
or PR0344) 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 radioiabeled probe derived from
the gene encoding a PR0655,
PR0364 or PR0344 polypeptide to the filters is performed in a solution of 50%
formamide, Sx SSC, 0.1% SDS,
0. I % 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 O. l x SSC and 0.1% 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 4
Expression of PR0655, PR0364 or PR0344 in E. coli
This example illustrates preparation of an unglycosylated form of PR0655,
PR0364 or PR0344 by
recombinant expression in E. toll.
The DNA sequence encoding PR0655, PR0364 or PR0344 is initially amplified
using selected PCR
primers. The primers should contain restriction enzyme sites which correspond
to the restriction enryme 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. toll; 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-H is sequence,
and enterokinase cleavage site), the PR0655, PR0364 or PR0344 coding region,
lambda transcriptionalterminator,
and an argU gene.
The ligation mixture is then used to transform a selected E. toll strain using
the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow
on LB plates and antibiotic resistant
colonies are then selected. Plasmid DNA can be isolated and confirmed by
restriction analysis and DNA
sequencing.
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Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with
antibiotics. The overnight culture may subsequently be used to inoculate a
larger scale culture. The cells are then
grown to a desired optical density, during which the expression promoter is
turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell
pellet obtained by the centrifugation can be solubilized using various agents
known in the art, and the solubilized
PR0655, PR0364 or PR0344 protein can then be purified using a metal chelating
column under conditions that
allow tight binding of the protein.
PR0655, PR0364 or PR0344 may be expressed in E. coli in a poly-His tagged
form, using the following
procedure. The DNA encoding PR0655, PR0364 or PR0344 is initially amplified
using selected PCR primers.
The primers will contain restriction enzyme sites which correspond to the
restriction enzyme sites on the selected
expression vector, and other useful sequences providing for efficient and
reliable translation initiation, rapid
purification on a metal chelation column, and proteolytic removal with
enterokinase. The PCR-amplified, poly-His
tagged sequences are then ligated into an expression vector, which is used to
transform an E. coli host based on
strain 52 (W31 l0 fuhA(tonA) Ion galE rpoHts(htpRts) clpP(laclq).
Transfotmants are first grown in LB containing
50 mg/ml carbenicillin at 30°C with shaking until an OD6~ of 3-5 is
reached. Cultures are then diluted 50-I00 fold
into CRAP media (prepared by mixing 3.57 g (NH,,),SO,, 0.71 g sodium
citrate~2H20, 1.07 g KCI, 5.36 g Difco
yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 1 10 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
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 0.33 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 SO 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. ?0 mM glycine
and I mM EDTA. Refolding
volumes are chosen so that the final protein concentration is between 50 to
100 micrograms/ml. The refolding
solution is stirred gently at 4 °C for 12-36 hours. The refolding
reaction is quenched by the addition of TFA to a
final concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is
filtered through a 0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein
83


CA 02348157 2001-04-23
WO 00132778 PCT/US99/28409
is chromatographed on a Poros R i /H reversed phase column using a mobile
buffer of 0. I % TFA with elution with
a gradient of acetonitrile from 10 to 80%. Aliquots of fractions with
A=a° absorbance are analyzed on SDS
polyacrylamide gels and fractions containing homogeneous refolded protein are
pooled. Generally, the property
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 higheracetonitrile concentrations. In addition
to resolving misfolded forms ofproteins
from the desired form, the reversed phase step also removes endotoxin from the
samples.
Fractions containing the desired folded PR0655, PR0364 or PR0344 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 Supe~ne
(Pharmacia) resins equilibrated in the formulation buffer and sterile
ftltered.
PR0655 and PR0364 were successfully expressed in E. coli in a poly-His tagged
form by the above
procedure.
EXAMPLE 5
Expression of PR0655, PR0364 or PR0344 in mammalian cells
This example illustrates preparation of a potentially glycosylated form of
PR0655, PR0364 or PR0344
by recombinant expression in mammalian cells.
The vector, pRKS (see EP 307,247, published March I5, 1989), is employed as
the expression vector.
Optionally, the PR0655, PR0364 or PR0344 DNA is ligated into pRKS with
selected restriction enzymes to allow
insertion of the PR0655, PR0364 or PR0344 DNA using ligation methods such as
described in Sambrook et al.,
supra. The resulting vector is called pRKS-PR0655, pRKS-PR0364 or pRKS-PR0344.
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 componentsand/orantibiotics. About I 0 ~g pRKS-PR0655,
pRKS-PR0364 or pRKS-PR0344
DNA is mixed with about I beg DNA encoding the VA RNA gene [Thimmappaya et
al., Cell, 31:543 (1982)] and
dissolved in 500 ul of 1 mM Tris-HC1, 0. l mM EDTA, 0.227 M CaCI=. To this
mixture is added, dropwise, 500
~cl of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO,, and a precipitate is
allowed to form for 10 minutes
at 25 °C. The precipitate is suspended and added to the 293 cells and
allowed to settle for about four hours at 37°C.
The culture medium is aspirated off and 2 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 ~eCi/ml "S-cysteine and 200
~cCi/ml "S-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
PR0655, PR0364 or PR0344 polypeptide. The cultures containing transfected
cells may undergo further
incubation (in serum free medium) and the medium is tested in selected
bioassays.
84


CA 02348157 2001-04-23
WO 00132778 PCT/US99/28409
In an alternative technique, PR065~. PR0364 or PR0344 may be introduced into
293 cells transiently
using the dextran sulfate method described by Somparyrac er al.. Proc. Natl.
Acad. Sci.. 12:7575 ( 1981 ). 293 cells
are grown to maximal density in a spinner flask and 700 ~g pRKS-PR0655, pRKS-
PR0364 or pRKS-PR0344
DNA is added. The cells are first concentrated from the spinner flask by
centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four hours. The
cells are treated with 20% glycerol for
90 seconds, washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture
medium, 5 ~g/ml bovine insulin and 0. I ug/ml bovine transferrin. After about
four days, the conditioned media
is centrifuged and filtered to remove cells and debris. The sample containing
expressed PR0655, PR0364 or
PR0344 can then be concentrated and purified by any selected method, such as
dialysis and/or column
chromatography.
In another embodiment, PR065~. PR0364 or PR034a can be expressed in CHO cells.
The pRKS-
PR0655, pRKS-PR0364 or pRKS-PR0344 can be transfected into CHO cells using
known reagents such as CaP04
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 ''S-methionine. After
determining the presence of a
PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide can then be
concentrated and purified by any
selected method.
Epitope-tagged PR0655, PR0364 or PR0344 may also be expressed in host CHO
cells. The PR0655,
PR0364 or PR0344 may be subcloned out of the pRKS vector. The subclone insert
can undergo PCR to fuse in
frame with a selected epitope tag such as a poly-His tag into a Baculovirus
expression vector. The poly-His tagged
PR0655, PR0364 or PR0344 insert can then be subcloned into a SV~FO 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 PR0655, PR0364 or PR0344 can then be
concentrated and purified by
any selected method, such as by Ni='-chelate affinity chromatosraphy.
PR0655, PR0364 or PR0344 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 fotitt.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using
standard techniques as described in Ausubel w al., Current Protocols of
Molecular Biolow, Unit 3.16, John Wiley
and Sons ( I 997). CHO expression vectors are constructed to have compatible
restriction sites 5' and 3' of the DNA
of interest to allow the convenient shuttl in, 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


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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~ (8oehringer
Mannheim). The cells are grown as described in Lucas er 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 I 0 mls
of media and centrifuged at 1000 rpm
for 5 minutes. The supernatant is aspirated and the cells are resuspended in
10 ml of selective media (0.2 um
filtered PS20 with 5% 0.2 ~cm diafiltered fetal bovine serum). The cells are
then aliquoted into a 100 ml spinner
containing 90 ml of selective media. After I-2 days, the cells are transferred
into a 250 ml spinner filled with I50
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 10° cells/ml. On day 0, the cell number and pH is determined. On day
I, the spinner is sampled and sparging
with filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33°C, and 30 ml of 500
g/L glucose and 0.6 ml of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical
Grade Emulsion) taken. Throughout the production. the pH is adjusted as
necessary to keep it at around 7.2. After
10 days, or until the viability drops below 70%, the cell culture is harvested
by centrifugation and filtering through
a 0.22 gem 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 '-'-
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 (Pharcnacia) 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 S mi Protein A column (Phatmacia) 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 i 00 mM citric acid, pH 3.5. The eluted protein is immediately
neutralized by collecting 1 ml fractions
into tubes containing 275 ~cl of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into
storage buffer as described above for the poly-His tagged proteins. The
homogeneity is assessed by SDS
polyacrylamide Gels and by N-terminal amino acid sequencing by Edman
degradation.
PR0364 was stably expressed in CHO cells by the above described method. In
addition, PR0364 was
expressed in CHO cells by the transient expression procedure.
86


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EXAMPLE 6
Expression of PR065~. PR036-l or PR03d4 in Yeast
The following method describes recombinant expression of PR0655. PR0364 or
PR0344 in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PR0655,
PR0364 or PR0344 from the ADH2/GAPDH promoter. DNA encoding PR0655. PR0364 or
PR0344 and the
promoter is inserted into suitable restriction enzyme sites in the selected
plasmid to direct intracellular expression
of PR0655, PR0364 or PR0344. For secretion, DNA encoding PR065~, PR0364 or
PR0344 can be cloned into
the selected plasmid, together with DNA encoding the ADH2!GAPDH promoter, a
native PR0655, PR0364 or
PR0344 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 PR0655, PR0364 or PR0344.
Yeast cells, such as yeast strain ABl 10, 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 PR0655, PR0364 or PR0344 can subsequently be isolated and purified
by removing the
yeast cells from the fermentation medium by centrifugation and then
concentrating the medium using selected
carnidge filters. The concentrate containing PR0655, PR0364 or PR0344 may
further be purified using selected
column chromatography resins.
EXAMPLE 7
Expression of PR0655, PR0364 or PR0344 in Baculovirus-Infected Insect Cells
The fotlowing method describes recombinant expression in Baculovirus-infected
insect cells.
The sequence coding for PR0655, PR0364 or PR0344 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 IQG). A variety of plasmids may be employed, including plasmids
derived from commercially available
2S plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding PR0655,
PR0364 or PR0344 or the
desired portion of the coding sequence of PR0655, PR0364 or PR0344 (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 plasmidand
BaculoGoIdT" virus DNA
(Pharmingen)intoSpodopterafr:rgiperda("Sf~9")cells(ATCCCRL
1711)usinglipofectin(commerciallyavailable
from GIBCO-BRL). After 4 - 5 days of incubation at 28°C, the released
viruses are harvested and used for further
amplifications. Viral infection and protein expression are performed as
described by O'Reilley et al., Baculovirus
expression vectors: A Laboratory Manual. Oxford: Oxford University Press (
1994).
Expressed poly-His tagged PR0655, PR0364 or PR0344 can then be purified, for
example, by Ni='-
87


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
chelate affinity chromatography as follows. Extracts are prepared from
recombinant virus-infected Std cells as
described by Rupert et al., Nature, 362: i 75-179 ( 1993). Briefly. Sf9 cells
are washed, resuspended in sonication
buffer (25 ml Hepes, pH 7.9; 12.5 mM MgCf,; O.l mM EDTA; 10°.%
glycerol; 0.1% NP-40; 0.4 M KC1), 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 Ni='-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 A,s°
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 A:e° 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 Ni='-NTA-
conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted H is,o tagged PR0655, PR0364 or
PR0344, respectively, are pooled and
dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR0655, PR0364 or
PR0344 can be
performed using known chromatography techniques, including for instance,
Protein A or protein G column
chromatography.
FollowingPCRamplification,the respective coding sequences 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~
baculovirvs DNA (Pharmingen) are co-transfected into 105 Spodoptera frugiperda
("Sf9") cells (ATCC CRL
l7l 1), 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 Sf9 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 '-'-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 first vita! amplification supernatant is used to infect a spinner culture
(500 ml) of Sf9 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 ='-NTA column (Qiagen). Before purification, imidazole is
added to the conditioned media to
88


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
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 ~ 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 G2~ 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 ~ 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 mi 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.
PR0344 was expressed in baculovirus infected Sf9 insect cells.
Alternatively, a modified baculovirus procedure may be used incorporating high-
5 cells. In this procedure,
the DNA encoding the desired sequence is amplified with suitable systems, such
as Pfu (Stratagene), or fused
upstream (5'-00 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 1-1
(Novagen). The pIE 1-1 and pIE 1-2 vectors
are designed for constitutive expression of recombinant proteins from the
baculovirus ie I 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
enzyme sites. The product is then digested
with those selected restriction enzymes and subcloned into the expression
vector. For example, derivatives of
plEl-I 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 150 mm plate, 30 ~cg of pIE based vector containing the sequence is mixed
with I 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 ofCeIIFectin (CeIIFECTIN (GibcoBRL # t 0362-O I 0) (vortexed to
mix)) is mixed with I ml of Ex-Cell
medium. The two solutions are combined and allowed to incubate at room
temperature for 15 minutes. 8 ml of
3S Ex-Cell media is added to the 2 ml of DNA/CeIIFECTIN mix and this is
layered on high-~ 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
89


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
CeIIFECT1N, 30 m I of fresh Ex-Cel I media is added and the cel Is are
incubated for 3 days at 28°C. The supernatant
is harvested and the expression ofthe sequence in the baculovirus expression
vector is determined by batch binding
of 1 ml ofsupernatent to 25 ml ofNi ='-NTA beads (QIAGEN) forhistidine tagged
proteins or Protein-A Sepharose
CL-4B beads (Pharmacia) for IeG 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 ta~~ed
constructs, the protein comprising the
sequence is purified using a Ni ='-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned
media to a concentration of 5 mM. The conditioned media is pumped onto a 6 ml
Ni ='-NTA column equilibrated
in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a
flow rate of4-5 mUmin. at48°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
had been equilibrated in 20
mM Na phosphate buffer, pH 6.8. After loading, the column is washed
extensively with equilibration buffer before
elution with ! 00 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 sequence is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation and other analytical
procedures as desired or necessary.
PR0364 and PR0344 were expressed using the above baculovirus procedure
employing high-5 cells.
EXAMPLE 8
Preparation of Antibodies that Bind PR0655 PR0364 or PR0344
This example illustrates preparation of monoclonal antibodies which can
specifically bind PR0655,
PR0364 or PR0344.
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 PR0655,
PR0364 or PR0344, fusion
proteins containing PR0655, PR03b4 or PR0344, and cellsexpressingrecombinant
PR0655. PR0364 or PR0344
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 PR0655, PR0364 or PR0344
immunogen emulsified in
complete Freund's adjuvant and injected subcutaneously or intraperitoneally in
an amount from 1-100 micrograms.
Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi
Immunochemical Research, Hamilton,
MT) and injected into the animal's hind foot pads. The immunized mice are then
boosted 10 to 12 days later with
additional immunogen emulsified in the selected adjuvant. Thereafter, for
several weeks, the mice may also be


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
boosted with additional immunization injections. Serum samples may be
periodically obtained from the mice by
retro-orbital bleeding for testing in EL1SA assays to detect anti-PR065~, anti-
PR0364 or anti-PR0344 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected with
a final intravenous injection of PR0655, PR0364 or PR0344. 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 murine
myeloma cell line such as P3X63AgU.1, available from ATCC. No. CRL 1597. The
fusions generate hybridoma
cells which can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and
thymidine) medium to inhibit proliferation of non-fused cells, myeloma
hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an ELISA for reactivity against
PR0655, PR0364 or PR0344.
Determination of"positive" hybridoma cells secreting the desired monoclonal
antibodies against PR0655, PR0364
or PR0344 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-PR0655, anti-PR0364 or anti-PR03~1-t monoclonal
antibodies. Alternatively, the
hybridoma cells can be grown in tissue culture flasks or roller bottles.
Purification of the monoclonal antibodies
produced in the ascites can be accomplished using ammonium sulfate
precipitation, followed by gel exclusion
chromatography. Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein
G can be employed.
EXAMPLE 9
Purification of PR0655, PR0364 or PR0344 Polvoentides Using, Specific
Antibodies
Native or recombinant PR0655, PR0364 or PR0344 polypeptides may be purified by
a variety of
standard techniques in the art of protein purification. For example, pro-
PR0655, pro-PR0364 or pro-PR0344
polypeptide, mature PR0655, mature PR0364 or mature PR034.1 polypeptide, or
pre-PR0655, pre-PR0364 or
pre-PR0344 polypeptide is purified by immunoaffinity chromatography using
antibodies specific for the PR0655,
PR0364 or PR0344 polypeptide of interest. In general, an immunoaffinity column
is constructed by covalently
coupling the anti-PR0655, anti-PR0364 or anti-PR0344 polypeptide antibody to
an activated chromatographic
resin.
Polyclonal immunoglobulins are prepared from immune sera either by
precipitation with ammonium
sulfate or by purification on immobilized Protein A (Pharmacia LKB
Biotechnology, Piscataway, N.J.). Likewise,
monoclonal antibodiesare prepared from mouse ascites fluid by ammonium sulfate
precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is covalentiy
attached to a chromatographic resin
such as CnBr-activated SEPHAROSET'" (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin,
the resin is blocked, and the derivative resin is washed according to the
manufacturer's instructions.
Such an immunoaffinity column is utilized in the purification of the PR0655,
PR0364 or PR0344
polypeptide by preparing a fraction from cel Is containing the PR06>j, PR0364
or PR0344 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,
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WO 00/32778 PCT/US99/28409
soluble PR0655. PR0364 or PR0344 polypeptide containing a signal sequence may
be secreted in useful quantity
into the medium in which the cells are grown.
A soluble PR0655, PR0364 or PR0344 polypeptide-containing preparation is
passed over the
immunoaffinity column, and the column is washed under conditions that allow
the preferential absorbance of the
PR0655, PR0364 or PR0344 polypeptide (e.g., high ionic strength buffers in the
presence of detergent). Then,
the column is eluted under conditions that disrupt antibody/PR06»,
antibody/PR0364 or antibody/PR0344
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 PR0655, PR0364 or PR0344 polypeptide
is collected.
EXAMPLE 10
Drua Screenine
This invention is particularly useful for screening compounds by using PR065~.
PR0364 or PR0344
polypeptides or a binding fragment thereof in any of a variety of drug
screening techniques. The PR0655, PR0364
or PR0344 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 PR0655,
PR0364 or PR0344 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 PR0655, PR0364 or
PR0344 polypeptide or a
fragment and the agent being tested. Alternatively, one can examine the
diminution in complex formation between
the PR0655, PR0364 or PR0344 polypeptide and its target cell or target
receptors caused by the agent being
tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can affect
a PR0655, PR0364 or PR0344 polypeptide-associated disease or disorder. These
methods comprise contacting
such an agent with a PR0655, PR0364 or PR0344 polypeptide or fragment thereof
and assaying (i) for the
presence of a complex between the agent and the PR0655, PR0364 or PR0344
polypeptide or fragment, or (ii)
for the presence of a complex between the PR0655, PR0364 or PR0344 polypeptide
or fragment and the cell, by
methods well known in the art. In such competitive binding assays, the PR0655,
PR0364 or PR0344 polypeptide
or fragment is typically labeled. After suitable incubation, the free PR0655,
PR0364 or PR0344 polypeptide or
fragment is separated from that present in bound form, and the amount of free
or uncomplexed label is a measure
of the ability of the particular agent to bind to the PR065~, PR0364 or PR0344
polypeptide or to interfere with
the PR0655, PR0364 or PR0344 polypeptide/cell complex.
Another technique for drug screening provides high throughput screening for
compounds having suitable
binding affinity to a polypeptide and is described in detail in WO 84/03564,
published on September l3, 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 PR0655. PR0364 or
PR0344 polypeptide, the peptide test
compounds are reacted with the PR0655, PR0364 or PR0344 polypeptide and
washed. Bound PR0655, PR0364
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WO 00/32778 PC'f/US99/28409
or PR0344 polypeptide is detected by methods well known in the art. Purified
PR0655. PR0364 or PR0344
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 PR0655, PR0364 or PR0344 polypeptide
specifically compete with a test
compound for binding to the PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344 polypeptide.
EXAMPLE 11
Rational Drug Desien
The goal of rational drug design is to produce structural analogs of a
biologically active polypeptide of
interest (i.e., a PR0655, PR0364 or PR0344 poiypeptide) or of small molecules
with which they interact, e.g.,
agonists, antagonists, or inhibitors. Any of these examples can be used to
fashion drugs which are more active or
stable forms of the PR0655, PR0364 or PR0344 polypeptide or which enhance or
interfere with the function of
the PR0655, PR0364 or PR0344 polypeptide in vivo (cf., Hodason.
Bio/Technoloey, 9: 19-21 ( (991 )).
In one approach, the three-dimensional structure of the PR0655, PR0364 or
PR0344 polypeptide, or of
a PR0655, PR0364 or PR0344 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
PR0655, PR0364 or PR0344 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 PR0655, PR0364 or PR0344
polypeptide may be gained by modeling based on the structure of homologous
proteins. In both cases, relevant
structural information is used to design analogous PR0655, PR0364 or PR0344
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. 3 I :7796-
7801 ( 1992) or which act as inhibitors.
agonists, or antagonists of native peptides as shown by Athauda et ul., J.
Biochem.. 1 13:742-746 ( 1993).
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above,
and then to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug
design can be based. It is possible to bypass protein crystallography
altogether by generating anti-idiotypic
antibodies (anti-ids) to a functional, pharmacologically active antibody. As a
minor 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 PR0655, PR0364
or PR0344 polypeptide
may be made available to perform such analytical studies as X-ray
crystallography. In addition, knowledge ofthe
PR0655, PR0364 or PR0344 polypeptide amino acid sequence provided herein wilt
provide guidance to those
employing computer modeling techniques in place of or in addition to x-ray
crystallography.
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WO 00/32778 PCT/US99/28409
EXAMPLE 12
In G'irro Antitumor Assav
The antiproliferative activity of the PR0655, PR0364 and PR0344 polypeptides
was determined in the
investigational, disease-oriented in vitro anti-cancer drub discovery, assay
of the National Cancer Institute (NCI),
using a sulforhodam ine B (SRB) dye binding assay essentially as described by
Skehan et al., J. Natl. Cancer Inst.,
82:1107-I 112 ( 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 lnst.. 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
~:1-12 [1989]).
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
~cl 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. Ditutions at twice the intended test concentration were added
at time zero in 100 ~I 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 100% humidity.
After incubation, the medium was removed and the cells were fixed in 0. t 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 I% acetic acid, rinsed four times
with 1% 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.
Theabsorbance(OD)ofsulforhodamineBat492nmwasmeasured
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
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WO 00/32778 PCT/US99/28409
Table 4
CompoundConcentrationDays Tumor Cell Tvpe Desi nQ anon


PR0655 22.2 nM 2 Ovarian OVCAR-4


PR0655 22.2 nM 6 Colon HCT-15


PR0655 22.2 nM 6 Melanoma UACC-257


PR0655 18.0 nM 6 Melanoma LOX IMVI


PR0655 18.0 nM 6 Colon KM-12


PR0364 27.23 nM 6 NSCL HOP62


PR0364 27.33 nM 6 Colon KM-12


PR0364 27.23 nM 6 CNS SF295


PR0364 27.23 nM 6 Melanoma LOX 1MVI


PR0364 27.23 nM 6 Renal UO-31


PR0364 135.00 nM 2 Colon KM-12


PR0364 135.00 nM 6 NSCL HOP62


PR0364 135.00 nM 6 Melanoma LOX IMVI


PR0364 135.00 nM 6 Colon HT-29


PR0364 135.00 nM 6 Ovarian IGROVI


PR0364 135.00 nM 6 Breast MDA-MB 435


PR0344 1.2 nM 2 Leukemia HL-60 (TB)


PR0344 1.2 nM 6 Renal UO-31 and
CAKI-1


PR0344 14.9 nM 2 Colon KM-12


PR0344 14.9 nM 2 CNS SF-268


PR0344 14.9 nM 2 Ovarian OVCAR-4


PR0344 14.9 nM 2 Renal CAKI-1


PR0344 14.9 nM 2 Breast MDA-MB-435


PR0344 14.9 nM 6 Leukemia HL-60 (TB)


PR0344 14.9 nM 6 Colon KM-12


PR0344 14.9 nM 6 CNS SF-295


PR0344 14.9 nM 6 NSCL HOP62


Deposit of Material
The following materials have been deposited with the American Type Culture
Collection, 10801
University Blvd., Manassas. VA 20110-2209, USA (ATCC):
Material ATCC Dep. No. Deposit Date_
DNA50960-1224 209509 December 3, 1997
DNA47365-1206 209436 November 7, 1997
DNA40592-1242 209492 November 21, 1997
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


CA 02348157 2001-04-23
WO 00/32778 PCT/US99/28409
culture of the deposit to the public upon issuance of the pertinent U.S.
patent or upon laying open to the public of
any U.S. or foreign patent application, whichever comes first, and assures
availability of the progeny to one
determined by the U.S. Commissioner of Patents and Trademarks to be entitled
thereto according to 35 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 of the 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.
96