Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR INHIBITING NEOPLASTIC CELL
GROWTH
FIELD OF THE INVENTION
The present invention concerns methods and compositions for inhibiting
neoplastic cell growth. In
particular, the present invention concerns antitumor compositions and methods
for the treatment of tumors. The
invention further concerns screening methods for identifying growth
inhibitory, e.g., antitumor compounds.
BACKGROUND OF THE INVENTION
Malignant tumors (cancers) are the second leading cause of death in the United
States, after heart disease
(Boring et al., CA Cancel J. Clin., 43:7 (1993)).
Cancer is characterized by the increase in the number of abnormal, or
neoplastic, cells derived from a
normal tissue which proliferate to 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 mannnalian
patients, preferably humans.
In one aspect, the present invention concerns compositions of matter useful
for the inhibition of neoplastic
cell growtli comprising an effective amount of a PRO polypeptide as herein
defined, or an agonist thereof, in
admixture with a pharmaceutically acceptable carrier. In a preferred
embodiment, the composition of matter
comprises a growth inhibitory amount of a PRO polypeptide, or an agonist
thereof. In another preferred
embodiment, the composition comprises a cytotoxic amount of a PRO polypeptide,
or an agonist thereof.
Optionally, the compositions of matter may contain one or more additional
growth inhibitory and/or cytotoxic
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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 PRO
polypeptide as herein defined, or
an agonist thereof. The tumor is preferably a cancer.
In another aspect, the invention concerns a method for inhibiting the growth
of a tumor cell comprising
exposing the cell to an effective amount of a PRO polypeptide as herein
defined, or an agonist thereof. In a
particular embodiment, the agonist is an anti-PRO agonist antibody. In another
embodiment, the agonist is a
small molecule that mimics the biological activity of a PRO polypeptide. The
method may be performed in. vitro
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.g.,
growth of tumor cells, and the active
agent in the composition is a PRO 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 PRO polypeptide or agonist thereof, for the inhibition of
neoplastic cell growth, wherein the agonist
may be an antibody which binds to the PRO polypeptide.
' In a particular embodiment, the agonist is an anti-PRO agonist antibody. In
another embodiment, the
agonist is a small molecule that mimics the biological activity of a PRO
polypeptide. Similar articles of
manufacture comprising a PRO polypeptide as herein defined, or an agonist
thereof, in an amount that is
therapeutically effective for the treatment of tumor are also within the scope
of the present invention. Also
within the scope of the invention are articles of manufacture comprising a PRO
polypeptide as herein defined,
or an agonist thereof, and a further growth inhibitory agent, cytotoxic agent
or chemotherapeutic agent.
B. Additional Embodiments
In other embodiments of the present invention, the invention provides an
isolated nucleic acid molecule
comprising a nucleotide sequence that encodes a PRO polypeptide.
In one aspect, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% nucleic acid sequence identity, alternatively at least about 81% nucleic
acid sequence identity, alternatively
at least about 82% nucleic acid sequence identity, alternatively at least
about 83% nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence identity, alternatively
at least about 85% nucleic acid
sequence identity, alternatively at least about 86% nucleic acid sequence
identity, alternatively at least about
87% nucleic acid sequence identity, alternatively at least about 88% nucleic
acid sequence identity, alternatively
at least about 89% nucleic acid sequence identity, alternatively at least
about 90% nucleic acid sequence identity,
alternatively at least about 91% nucleic acid sequence identity, alternatively
at least about 92% nucleic acid
sequence identity, alternatively at least about 93% nucleic acid sequence
identity, alternatively at least about
94% nucleic acid sequence identity, alternatively at least about 95% nucleic
acid sequence identity, alternatively
at least about 96% nucleic acid sequence identity, alternatively at least
about 97% nucleic acid sequence identity,
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alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99% nucleic acid
sequence identity to (a) a DNA molecule encoding a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other
specifically defined fragment of the full-length amino acid sequence as
disclosed herein, or (b) the complement
of the DNA molecule of (a).
In other aspects, the isolated nucleic acid molecule comprises a nucleotide
sequence having at least about
80% nucleic acid sequence identity, alternatively at least about 81 % nucleic
acid sequence identity, alternatively
at least about 82% nucleic acid sequence identity, alternatively at least
about 83% nucleic acid sequence identity,
alternatively at least about 84% nucleic acid sequence identity, alternatively
at least about 85% nucleic acid
sequence identity, alternatively at least about 86% nucleic acid sequence
identity, alternatively at least about
87% nucleic acid sequence identity, alternatively at least about 88% nucleic
acid sequence identity, alternatively
at least about 89% nucleic acid sequence identity, alternatively at least
about 90% nucleic acid sequence identity,
alternatively at least about 91% nucleic acid sequence identity, alternatively
at least about 92% nucleic acid
sequence identity, alternatively at least about 93% nucleic acid sequence
identity, alternatively at least about
94% nucleic acid sequence identity, alternatively at least about 95% nucleic
acid sequence identity, alternatively
at least about 96% nucleic acid sequence identity, alternatively at least
about 97% nucleic acid sequence identity,
alternatively at least about 98% nucleic acid sequence identity and
alternatively at least about 99% nucleic acid
sequence identity to (a) a DNA molecule comprising the coding sequence of a
full-length PRO polypeptide
cDNA as disclosed herein, the coding sequence of a PRO polypeptide lacking the
signal peptide as disclosed
herein, the coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without
the signal peptide, as disclosed herein or the coding sequence of any other
specifically defined fragment of the
full-length amino acid sequence as disclosed herein, or (b) the complement of
the DNA molecule of (a).
In a further aspect, the invention concerns an isolated nucleic acid molecule
comprising a nucleotide
sequence having at least about 80% nucleic acid sequence identity,
alternatively at least about 81% nucleic acid
sequence identity, alternatively at least about 82% nucleic acid sequence
identity, alternatively at least about
83% nucleic acid sequence identity, alternatively at least about 84% nucleic
acid sequence identity, alternatively
at least about 85% nucleic acid sequence identity, alternatively at least
about 86% nucleic acid sequence identity,
alternatively at least about 87% nucleic acid sequence identity, alternatively
at least about 88% nucleic acid
sequence identity, alternatively at least about 89% nucleic acid sequence
identity, alternatively at least about
90% nucleic acid sequence identity, alternatively at least about 91% nucleic
acid sequence identity, alternatively
at least abdut 92% nucleic acid sequence identity, alternatively at least
about 93% nucleic acid sequence identity,
alternatively at least about 94% nucleic acid sequence identity, alternatively
at least about 95% nucleic acid
sequence identity, alternatively at least about 96% nucleic acid sequence
identity, alternatively at least about
97% nucleic acid sequence identity, alternatively at least about 98% nucleic
acid sequence identity and
alternatively at least about 99% nucleic acid sequence identity to (a) a DNA
molecule 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).
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Another aspect of the invention provides an isolated nucleic acid molecule
comprising a nucleotide
sequence encoding a PRO polypeptide which is either transmembrane domain-
deleted or transmembrane
domain-inactivated, or is complementary to such encoding nucleotide sequence,
wherein the transmembrane
domain(s) of such polypeptides are disclosed herein. Therefore, soluble
extracellular domains of the herein
described PRO polypeptides are contemplated.
Another embodiment is directed to fragments of a PRO polypeptide coding
sequence, or the complement
thereof, that may find use as, for example, hybridization probes, for encoding
fragments of a PRO polypeptide
that may optionally encode a polypeptide comprising a binding site for an anti-
PRO antibody or as antisense
oligonucleotide probes. Such nucleic acid fragments are usually at least about
20 nucleotides in length,
alternatively at least about 30 nucleotides in length, alternatively at least
about 40 nucleotides in length,
alternatively at least about 50 nucleotides in length, alternatively at least
about 60 nucleotides in length,
alternatively at least about 70 nucleotides in length, alternatively at least
about 80 nucleotides in length,
alternatively at least about 90 nucleotides in length, alternatively at least
about 100 nucleotides in length,
alternatively at least about 110 nucleotides in length, alternatively at least
about 120 nucleotides in length,
alternatively at least about 130 nucleotides in length, alternatively at least
about 140 nucleotides in length,
alternatively at least about 150 nucleotides in length, alternatively at least
about 160 nucleotides in length,
alternatively at least about 170 nucleotides in length, alternatively at least
about 180 nucleotides in length,
alternatively at least about 190 nucleotides in length, alternatively at least
about 200 nucleotides in length,
alternatively at least about 250 nucleotides in length, alternatively at least
about 300 nucleotides in length,
alternatively at least about 350 nucleotides in length, alternatively at least
about 400 nucleotides in length,
alternatively at least about 450 nucleotides in length, alternatively at least
about 500 nucleotides in length,
alternatively at least about 600 nucleotides in length, alternatively at least
about 700 nucleotides in length,
alternatively at least about 800 nucleotides in length, alternatively at least
about 900 nucleotides in length and
alternatively at least about 1000 nucleotides in length, wherein in this
context the term "about" means the
referenced nucleotide sequence length plus or minus 10% of that referenced
length. It is noted that novel
fragments of a PRO polypeptide-encoding nucleotide sequence may be determined
in a routine manner by
aligning the PRO polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any
of a number of well known sequence alignment programs and determining which
PRO polypeptide-encoding
nucleotide sequence fragment(s) are novel. All of such PRO polypeptide-
encoding nucleotide sequences are
contemplated herein. Also contemplated are the PRO polypeptide fragments
encoded by these nucleotide
molecule fragments, preferably those PRO polypeptide fragments that comprise a
binding site for an anti-PRO
antibody.
In another embodiment, the invention provides an isolated PRO polypeptide
encoded by any of the isolated
nucleic acid sequences hereinabove identified.
In a certain aspect, the invention concerns an isolated PRO polypeptide,
comprising an amino acid
sequence having at least about 80% amino acid sequence identity, alternatively
at least about 81% amino acid
sequence identity, alternatively at least about 82% amino acid sequence
identity, alternatively at least about 83%
amino acid sequence identity, alternatively at least about 84% amino acid
sequence identity, alternatively at least
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about 85% amino acid sequence identity, alternatively at least about 86% amino
acid sequence identity,
alternatively at least about 87% amino acid sequence identity, alternatively
at least about 88% amino acid
sequence identity, alternatively at least about 89% amino acid sequence
identity, alternatively at least about 90%
amino acid sequence identity, alternatively at least about 91% amino acid
sequence identity, alternatively at least
about 92% aniino acid sequence identity, alternatively at least about 93%
amino acid sequence identity,
alternatively at least about 94% amino acid sequence identity, alternatively
at least about 95% amino acid
sequence identity, alternatively at least about 96% amino acid sequence
identity, alternatively at least about 97%
amino acid sequence identity, alternatively at least about 98% amino acid
sequence identity and alternatively
at least about 99% amino acid sequence identity to a PRO polypeptide having a
full-length amino acid sequence
as disclosed herein, an amino acid sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other
specifically defined fragment of the full-length amino acid sequence as
disclosed herein.
In a further aspect, the invention concerns an isolated PRO polypeptide
comprising an amino acid sequence
having at least about 80% amino acid sequence identity, alternatively at least
about 81% amino acid sequence
identity, alternatively at least about 82% amino acid sequence ideiitity,
alternatively at least about 83% amino
acid sequence identity, alternatively at least about 84% amino acid sequence
identity, alternatively at least about
85% amino acid sequence identity, alternatively at least about 86% amino acid
sequence identity, alternatively
at least about 87% amino acid sequence identity, alternatively at least about
88% amino acid sequence identity,
alteruatively at least about 89% amino acid sequence identity, alternatively
at least about 90% amino acid
sequence identity, alternatively at least about 91% amino acid sequence
identity, alternatively at least about 92%
amino acid sequence identity, alternatively at least about 93% amino acid
sequence identity, alternatively at least
about 94% amino acid sequence identity, alternatively at least about 95% amino
acid sequence identity,
alternatively at least about 96% amino acid sequence identity, alternatively
at least about 97% amino acid
sequence identity, alternatively at least about 98% amino acid sequence
identity and alternatively at least about
99% amino acid sequence identity to an amino acid sequence encoded by any of
the human protein cDNAs
deposited with the ATCC as disclosed herein.
In a specific aspect, the invention provides an isolated PRO polypeptide
without the N-terminal signal
sequence and/or the initiating methionine and is encoded by a nucleotide
sequence that encodes such an amino
acid sequence as hereinbefore described. Processes for producing the same are
also herein described, wherein
those processes comprise culturing a host cell comprising a vector which
comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of the PRO
polypeptide and recovering the PRO
polypeptide from the cell culture.
Another aspect of the invention provides an isolated PRO polypeptide which is
either transmembrane
domain-deleted or transmembrane domain-inactivated. Processes for producing
the same are also herein
described, wherein those processes comprise culturing a host cell comprising a
vector which comprises the
appropriate encoding nucleic acid molecule under conditions suitable for
expression of the PRO polypeptide
and recovering the PRO polypeptide from the cell culture.
In yet another embodiment, the invention concerns agonists of a native PRO
polypeptide as defined herein.
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In a particular embodiment, the agonist is an anti-PRO antibody or a small
molecule.
In a further embodiment, the invention concerns a method of identifying
agonists to a PRO polypeptide
which comprise contacting the PRO polypeptide with a candidate molecule and
monitoring a biological activity
mediated by said PRO polypeptide. Preferably, the PRO polypeptide is a native
PRO polypeptide.
In a still further embodiment, the invention concerns a composition of matter
comprising a PRO
polypeptide, or an agonist of a PRO polypeptide as herein described, or an
anti-PRO antibody, in combination
with a carrier. Optionally, the carrier is a pharmaceutically acceptable
carrier.
Another embodiment of the present invention is directed to the use of a PRO
polypeptide, or an agonist
thereof as hereinbefore described, or an anti-PRO antibody, for the
preparation of a medicament useful in the
treatment of a condition which is responsive to the PRO polypeptide, an
agonist thereof or an anti-PRO
antibody.
In additional embodiments of the present invention, the invention provides
vectors comprising DNA
encoding any of the herein described polypeptides. Host cells 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 furtlier
provided and coinprises culturing host
cells under conditions suitable for expression of the desired polypeptide and
recovering the desired polypeptide
from the cell culture.
In other embodiments, the invention provides chimeric molecules comprising any
of the herein described
polypeptides fused to a heterologous polypeptide or amino acid sequence.
Example of such chimeric molecules
comprise any of the herein described polypeptides fused to an epitope tag
sequence or a Fc region of an
immunoglobulin.
In yet another embodiment, the invention provides an antibody which
specifically binds to any of the above
or below described polypeptides. Optionally, the antibody is a monoclonal
antibody, humanized antibody,
antibody fragment or single-chain antibody.
In yet other embodiments, the invention provides oligonucleotide probes useful
for isolating genomic and
cDNA nucleotide sequences or as antisense probes, wherein those probes may be
derived from any of the above
or below described nucleotide sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a nucleotide sequence (SEQ ID NO: 1) of a native sequence
PRO943 cDNA, wherein SEQ
ID NO:1 is a clone designated herein as "DNA52192-1369".
Figure 2 shows the amino acid sequence (SEQ ID NO:2) derived from the coding
sequence of SEQ ID
NO: 1 shown in Figure 1.
Figure 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequence
PRO1250 cDNA, wherein
SEQ ID NO:3 is a clone designated herein as "DNA60775-1532".
Figure 4 shows the, amino acid sequence (SEQ ID NO:4) derived from the coding
sequence of SEQ ID
NO:3 shown in Figure 3.
Figure 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequence
PR01337 cDNA, wherein
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SEQ ID NO:5 is a clone designated herein as "DNA66672-1586".
Figure 6 shows the amino acid sequence (SEQ ID NO:6) derived from the coding
sequence of SEQ ID
NO:5 shown in Figure 5.
DETAILED DESCRIPTION OF THE INVENTION
The terms "PRO polypeptide", and "PRO" as used herein and when immediately
followed by a numerical
designation refer to various polypeptides, wherein the complete designation
(i. e., PRO/number) refers to specific
polypeptide sequences as described herein. The terms "PRO/numberpolypeptide"
and "PRO/number" wherein
the term "number" is provided as an actual numerical designation as used
herein encompass native sequence
polypeptides and polypeptide variants (which are further defined herein). The
PRO polypeptides described
herein may be isolated from a variety of sources, such as from human tissue
types or from another source, or
prepared by recombinant or synthetic methods.
A "native sequence PRO polypeptide" comprises a polypeptide having the same
amino acid sequence as
the corresponding PRO polypeptide derived from nature. Such native sequence
PRO polypeptides can be
isolated from nature or can be produced by recombinant or synthetic means. The
term "native sequence PRO
polypeptide" specifically encompasses naturally-occurring truncated or
secreted forms of the specific PRO
polypeptide (e.g., an extracellular domain sequence), naturally-occurring
variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the polypeptide. In
various embodiments of the
invention, the native sequence PRO polypeptides disclosed herein are mature or
full-length native sequence
polypeptides comprising the full-length amino acid sequences shown in the
accompanying figures. Start and
stop codons are shown in bold font and underlined in the figures. However,
while the PRO polypeptide
disclosed in the accompanying figures are shown to begin with methionine
residues designated herein as amino
acid position 1 in the figures, it is conceivable and possible that other
methionine residues located either
upstream or downstream from the amino acid position 1 in the figures may be
employed as the starting amino
acid residue for the PRO polypeptides.
The PRO polypeptide "extracellular domain" or "ECD" refers to a form of the
PRO polypeptide which is
essentially free of the transmembrane and cytoplasmic domains. Ordinarily, a
PRO polypeptide ECD will have
less than 1% of such transmembrane and/or cytoplasmic domains and preferably,
will have less than 0.5% of
such domains. It will be understood that any transmembrane domains identified
for the PRO polypeptides of
the present invention are identified pursuant to criteria routinely employed
in the art for identifying that type
of hydrophobic domain. The exact boundaries of a transmembrane domain may vary
but most likely by no more
than about 5 amino acids at either end of the domain as initially identified
herein. Optionally, therefore, an
extracellular domain of a PRO polypeptide may contain from about 5 or fewer
amino acids on either side of the
transmembrane domain/extracellular domain boundary as identified in the
Examples or specification and such
polypeptides, with or without the associated signal peptide, and nucleic acid
encoding them, are comtemplated
by the present invention.
The approximate location of the "signal peptides" of the various PRO
polypeptides disclosed herein are
shown in the present specification and/or the accompanying figures. It is
noted, however, that the C-terminal
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boundary of a signal peptide may vary, but most likely by no more than about 5
amino acids on either side of
the signal peptide C-terminal boundary as initially identified herein, wherein
the C-terminal boundary of the
signal peptide may be identified pursuant to criteria routinely employed in
the art for identifying that type of
amino acid sequence element (e.g., Nielsen et al., Prot. Eng., 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.
"PRO polypeptide variant" means an active PRO polypeptide as defined above or
below having at least
about 80% amino acid sequence identity with a full-length native sequence PRO
polypeptide sequence as
disclosed herein, a PRO polypeptide sequence lacking the signal peptide as
disclosed herein, an extracellular
domain of a PRO polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of
a full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for
instance, PRO polypeptides wherein one or more amino acid residues are added,
or deleted, at the N- or
C-terminus of the full-length native aniino acid sequence. Ordinarily, a PRO
polypeptide variant will have at
least about 80% amino acid sequence identity, alternatively at least about 81%
amino acid sequence identity,
alternatively at least about 82% aniino acid sequence identity, alternatively
at least about 83% amino acid
sequence identity, alternatively at least about 84% amino acid sequence
identity, alternatively at least about 85%
amino acid sequence identity, alternatively at least about 86% amino acid
sequence identity, alternatively at least
about 87% amino acid sequence identity, alternatively at least about 88% amino
acid sequence identity,
alternatively at least about 89% amino acid sequence identity, alternatively
at least about 90% amino acid
sequence identity, alternatively at least about 91% amino acid sequence
identity, alternatively at least about 92%
amino acid sequence identity, alternatively at least about 93% amino acid
sequence identity, alternatively at least
about 94% amino acid sequence identity, alternatively at least about 95% amino
acid sequence identity,
alternatively at least about 96% amino acid sequence identity, alternatively
at least about 97% amino acid
sequence identity, alternatively at least about 98% aniino acid sequence
identity and alternatively at least about
99% amino acid sequence identity to a full-length native sequence PRO
polypeptide sequence as disclosed
herein, a PRO polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of
a PRO polypeptide, with or without the signal peptide, as disclosed herein or
any other specifically defined
fragment of a full-length PRO polypeptide sequence as disclosed herein.
Ordinarily, PRO variant polypeptides
are at least about 10 amino acids in length, alternatively at least about 20
amino acids in length, alternatively
at least about 30 amino acids in length, alternatively at least about 40 amino
acids in length, alternatively at least
about 50 amino acids in length, alternatively at least about 60 amino acids in
length, alternatively at least about
70 amino acids in length, alternatively at least about 80 amino acids in
length, alternatively at least about 90
amino acids in length, alternatively at least about 100 amino acids in length,
alternatively at least about 150
amino acids in length, alternatively at least about 200 amino acids in length,
alternatively at least about 300
amino acids in length, or more.
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"Percent (%) amino acid sequence identity" with respect to the PRO polypeptide
sequences identified
herein is defined as the percentage of amino acid residues in a candidate
sequence that are identical with the
aniino acid residues in a PRO 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 1 has been filed with user documentation in
the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The
ALIGN-2 program is publicly available through Genentech, Inc., South San
Francisco, California or may be
compiled from the source code provided in Table 1. The ALIGN-2 program should
be compiled for use on a
UNIX operating system, preferably digital UNIX V4.0D. 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
2-3 demonstrate how to
calculate the % amino acid sequence identity of the amino acid sequence
designated "Comparison Protein" to
the amino acid sequence designated "PRO".
Unless specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained
as described above using the ALIGN-2 sequence comparison computer program.
However, % amino acid
sequence identity may also be determined using the sequence comparison program
NCBI-BLAST2 (Altschul
et al., Nucleic Acids Res., 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be
downloaded from http://www.ncbi.nlm.nih.gov, or otherwise obtained from the
National Institute of Health,
Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those
search parameters are set
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to default values including, for example, unmask = yes, strand = all, expected
occurrences =10, minimum low
complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass =
25, dropoff for final gapped
alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino acid
sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B (which
can alternatively be phrased as a given amino acid sequence A that has or
comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of ainino 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 etal., Methods in Enzvmology, 266:460-480 (1996)). Most of
the WU-BLAST-2 search
parameters are set to the default values. Those not set to default values,
i.e., the adjustable parameters, are set
with the following values: overlap span = 1, overlap fraction = 0.125, word
threshold (T) = 11, and scoring
matrix = BLOSUM62. For purposes herein, a % amino acid sequence identity value
is determined by dividing
(a) the number of matching identical amino acids residues between the amino
acid sequence of the PRO
polypeptide of interest having a sequence derived from the native PRO
polypeptide and the comparison amino
acid sequence of interest (i.e., the sequence against which the PRO
polypeptide of interest is being compared
which may be a PRO variant polypeptide) as determined by WU-BLAST-2 by (b) the
total number of amino
acid residues of the PRO polypeptide of interest. For example, in the
statement "a polypeptide comprising an
amino acid sequence A which has or having at least 80% amino acid sequence
identity to the amino acid
sequence B", the amino acid sequence A is the comparison amino acid sequence
of interest and the amino acid
sequence B is the amino acid sequence of the PRO polypeptide of interest.
"PRO variant polynucleotide" or "PRO variant nucleic acid sequence" means a
nucleic acid molecule
which encodes an active PRO polypeptide as defined below and which has at
least about 80% nucleic acid
sequence identity with a nucleic acid sequence encoding a full-length native
sequence PRO polypeptide
sequence as disclosed herein, a full-length native sequence PRO polypeptide
sequence lacking the signal peptide
as disclosed herein, an extracellular domain of a PRO polypeptide, with or
without the signal peptide, as
disclosed herein or any other fragment of a full-length PRO polypeptide
sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about 80% nucleic
acid sequence identity,
alternatively at least about 81% nucleic acid sequence identity, alternatively
at least about 82% nucleic acid
sequence identity, alternatively at least about 83% nucleic acid sequence
identity, alternatively at least about
84% nucleic acid sequence identity, alternatively at least about 85% nucleic
acid sequence identity, alternatively
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at least about 86% nucleic acid sequence identity, alternatively at least
about 87% nucleic acid sequence identity,
alternatively at least about 88% nucleic acid sequence identity, alternatively
at least about 89% nucleic acid
sequence identity, alternatively at least about 90% nucleic acid sequence
identity, alternatively at least about
91% nucleic acid sequence identity, alternatively at least about 92% nucleic
acid sequence identity, alternatively
at least about 93% nucleic acid sequence identity, alternatively at least
about 94% nucleic acid sequence identity,
alternatively at least about 95% nucleic acid sequence identity, alternatively
at least about 96% nucleic acid
sequence identity, alternatively at least about 97% nucleic acid sequence
identity, alternatively at least about
98% nucleic acid sequence identity and alternatively at least about 99%
nucleic acid sequence identity with a
nucleic acid sequence encoding a full-length native sequence PRO polypeptide
sequence as disclosed herein,
a full-length native sequence PRO polypeptide sequence lacking the signal
peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the signal
sequence, as disclosed herein or any other
fragment of a full-length PRO polypeptide sequence as disclosed herein.
Variants do not encompass the native
nucleotide sequence.
Ordinarily, PRO variant polynucleotides are at least about 30 nucleotides in
length, alternatively at least
about 60 nucleotides in length, alternatively at least about 90 nucleotides in
length, alternatively at least about
120 nucleotides in length, alternatively at least about 150 nucleotides in
length, alternatively at least about 180
nucleotides in length, alternatively at least about 210 nucleotides in length,
alternatively at least about 240
nucleotides in length, alternatively at least about 270 nucleotides in length,
alternatively at least about 300
nucleotides in length, alternatively at least about 450 nucleotides in
lengtli, alternatively at least about 600
nucleotides in length, alternatively at least about 900 nucleotides in length,
or more.
"Percent (%) nucleic acid sequence identity" with respect to the PRO
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 PRO 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 alignrnent, including any algorithms needed to achieve maximal
alignment over the full-length of the
sequences being compared. Forpurposes herein, however, % nucleic acid sequence
identity values are obtained
as described below by using the sequence comparison computer program ALIGN-2,
wherein the complete
source code for the ALIGN-2 program is provided in Table 1. The ALIGN-2
sequence comparison computer
program was authored by Genentech, Inc., and the source code shown in Table 1
has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559, where it
is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech, Inc.,
South San Francisco, California or maybe 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 V4.0D. 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
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against a given nucleic acid sequence D (which can alternatively be phrased as
a given nucleic acid sequence
C that has or comprises a certain % nucleic acid sequence identity to, with,
or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by the
sequence alignment program ALIGN-2
in that program's alignment of C and D, and where Z is the total number of
nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence
D, the % nucleic acid sequence identity of C to D will not equal the % nucleic
acid sequence identity of D to
C. As examples of % nucleic acid sequence identity calculations, Tables 4-5
demonstrate how to calculate the
% nucleic acid sequence identity of the nucleic acid sequence designated
"Comparison DNA" to the nucleic acid
sequence designated "PRO-DNA".
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
downloaded from http://www.ncbi.nlm.nih.gov, or otherwise obtained from the
National Institute of Health,
Bethesda, MD. NCBI-BLAST2 uses several search parameters, wherein all of those
search parameters are set
to default values including, for example, unmask = yes, strand = all, expected
occurrences = 10, minimum low
complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass =
25, dropoff for final gapped
alignment = 25 and scoring matrix = BLOSUM62.
In situations where NCBI-BLAST2 is employed for 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 Enzymolojzy, 266:460-480
(1996)). Most of the WU-BLAST-2
search parameters are set to the default values. Those not set to default
values, i.e., the adjustable parameters,
are set with the following values: overlap span = 1, overlap fraction = 0.125,
word threshold (T) = 11, and
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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 fromthe native sequence PRO
polypeptide-encoding nucleic acid and the comparison nucleic acid molecule of
interest (i.e., the sequence
against which the PRO polypeptide-encoding nucleic acid molecule of interest
is being compared which may
be a variant PRO polynucleotide) as determined by WU-BLAST-2 by (b) the total
number of nucleotides of the
PRO polypeptide-encoding nucleic acid molecule of interest. For example, in
the statement "an isolated nucleic
acid molecule comprising a nucleic acid sequence A which has or having at
least 80% nucleic acid sequence
identity to the nucleic acid sequence B", the nucleic acid sequence A is the
comparison nucleic acid molecule
of interest and the nucleic acid sequence B is the nucleic acid sequence of
the PRO polypeptide-encoding
nucleic acid molecule of interest.
In other embodiments, PRO variant polynucleotides are nucleic acid molecules
that encode an active PRO
polypeptide and which are capable of hybridizing, preferably under stringent
hybridization and wash conditions,
to nucleotide sequences encoding the full-length PRO polypeptide shown in the
accompanying figures herein.
PRO variant polypeptides may be those that are encoded by a PRO variant
polynucleotide.
"Isolated", when used to describe the various polypeptides disclosed herein,
means a 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 of N-terminal or internal amino acid sequence by
use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions
using Coomassie blue or,
preferably, silver stain. Isolated polypeptides includes polypeptides in situ
within recombinant cells, since at
least one component of the PRO polypeptide natural environment will not be
present. Ordinarily, however,
isolated polypeptides will be prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a PRO polypeptide or an
"isolated" nucleic acid molecule
encoding an anti-PRO antibody is a nucleic acid molecule that is identified
and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily associated in
the natural source of the PRO-
encoding nucleic acid or the natural source of the anti-PRO-encoding nucleic
acid. Preferably, the isolated
nucleic acid is free of association with all components with which it is
naturally associated. An isolated PRO-
encoding nucleic acid molecule or an isolated anti-PRO-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
PRO-encoding nucleic acid molecule or fromthe anti-PRO-encoding nucleic acid
molecule as it exists in natural
cells. However, an isolated nucleic acid molecule encoding a PRO polypeptide
or an isolated nucleic acid
molecule encoding an anti-PRO antibody includes PRO-nucleic acid molecules or
anti-PRO-nucleic acid
molecules contained in cells that ordinarily express PRO polypeptides or anti-
PRO antibodies where, for
example, the nucleic acid molecule is in a chromosomal location different from
that of natural cells.
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The term "control sequences" refers to DNA sequences necessary for the
expression of an operably linked
coding sequence in a particular host organism. The control sequences that are
suitable for prokaryotes, for
example, include a promoter, optionally an operator sequence, and a ribosome
binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid
sequence. For example, DNA for a presequence or secretory leader is operably
linked to DNA for a PRO
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 syntlletic oligonucleotide
adaptors or linkers are used in accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-PRO
monoclonal antibodies (including agonist antibodies), anti-PRO antibody
compositions with polyepitopic
specificity, single chain anti-PRO antibodies, and fragments of anti-PRO
antibodies (see below). The term
"monoclonal antibody" as used herein refers to an antibody obtained from a
population of substantially
homogeneous antibodies, i.e., the individual antibodies comprising the
population are identical except for
possible naturally-occurring mutations that may be present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when
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 that 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
Biology (Wiley Interscience
Publishers, 1995).
"Stringent conditions" or "high-stringency conditions", as defined herein, may
be identified by those that:
(1) employ low ionic strength and high temperature for washing, for example,
0.015 M sodium chloride/0.0015
M sodium citrate/0.1% sodium dodecyl sulfate at 50 C; (2) employ during
hybridization a denaturing agent,
such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/5OmM 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 NaCl, 0.075 M
sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution,
sonicated salmon sperm DNA (50
Ag/n-A), 0.1% SDS, and 10% dextran sulfate at 42 C, with washes at 42 C in 0.2
x SSC (sodium
chloride/sodium citrate) and 50% formamide at 55 C, followed by a high-
stringency wash consisting of 0.1 x
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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 than 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 NaCl, 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 PRO
polypeptide fused to a"tag polypeptide". The tag polypeptide has enough
residues to provide an epitope against
which an antibody can be made, yet is short enough such that it does not
interfere with activity of the
polypeptide to which it is fused. The tag polypeptide preferably also is
fairly unique so that the antibody does
not substantially cross-react with other epitopes. Suitable tag polypeptides
generally have at least six amino acid
residues and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino
acid residues).
As used herein, the term "immunoadhesin" designates antibody-like molecules
which combine the binding
specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin constant
domains. Structurally, the inununoadhesins comprise a fusion of an amino acid
sequence with the desired
binding specificity which is other than the antigen recognition and binding
site of an antibody (i.e., is
"heterologous"), and an immunoglobulin constant domain sequence. The adhesin
part of an immunoadhesin
molecule typically is a contiguous amino acid sequence comprising at least the
binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin may
be obtained from any
immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including
IgA-1 and IgA-2), IgE, IgD
or IgM.
"Active" or "activity" for the purposes herein refers to form(s) of PRO
polypeptides which retain a
biological and/or an immunological activity of native or naturally-occurring
PRO polypeptides, wherein
"biological" activity refers to a biological function (either inhibitory or
stimulatory) caused by a native or
naturally-occurring PRO polypeptide other than the ability to induce the
production of an antibody against an
antigenic epitope possessed by a native or naturally-occurring PRO polypeptide
and an "immunological" activity
refers to the ability to induce the production of an antibody against an
antigenic epitope possessed by a native
or naturally-occurring PRO polypeptide.
"Biological activity" in the context of an antibody or another agonist that
can be identified by the screening
assays disclosed herein (e.g., an organic or inorganic small molecule,
peptide, etc.) is used to refer to the ability
of such molecules to invoke one or more of the effects listed herein in
connection with the definition of a
"therapeutically effective amount." In a specific embodiment, "biological
activity" is the ability to 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
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cytotoxic activity resulting in the death of the target tumor (e.g., cancer)
cell. Yet another preferred biological
activity is the induction of apoptosis of a target tumor (e.g., cancer) cell.
The phrase "immunological activity" means immunological cross-reactivity with
at least one epitope of
a PRO polypeptide.
"Irnmunological cross-reactivity" as used herein means that the candidate
polypeptide is capable of
competitively inhibiting the qualitative biological activity of a PRO
polypeptide having this activity with
polyclonal antisera raised against the known active PRO 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 binding affinity of
the immunologically cross-reactive molecule (e.g., antibody) identified, to
the corresponding PRO 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 compronuse the well-
being of the patient. This
0
includes, without limitation, abnormal or uncontrollable cell growth,
metastasis, interference with the normal
functioning of neighboring cells, release of cytokines or other secretory
products at abnormal levels, suppression
or aggravation of inflammatory or immunological response, etc.
An "effective amount" of a polypeptide disclosed herein or an agonist thereof,
in reference to inhibition
of neoplastic cell growth, is an amount capable of inhibiting, to some extent,
the growth of target cells. The
term includes an amount capable of invoking a growth inhibitory, cytostatic
and/or cytotoxic effect and/or
apoptosis of the target cells. An "effective amount" of a PRO polypeptide or
an agonist thereof for purposes
of inhibiting neoplastic cell growth may be determined empirically and in a
routine manner.
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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 PRO polypeptide or an agonist thereof for purposes of
treatment of tumor may be
determined empirically and in a routine manner.
A "growth inhibitory amount" of a PRO 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 PRO 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 PRO 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
PRO polypeptide or an agonist thereof for purposes of inhibiting neoplastic
cell growth may be determined
empirically and in a routine manner.
The term "cytotoxic agent" as used herein refers to a substance that inhibits
or prevents the function of
cells and/or causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., II3I IIZS Y90
and Re'$6), 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 Squibb Oncology, Princeton, NJ), and doxetaxel (Taxotere, Rh6ne-Poulenc
Rorer, Antony, Rnace),
toxotere, methotrexate, cisplatin, melphalan, vinblastine, bleomycin,
etoposide, ifosfamide, mitomycin C,
mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin,
carminomycin, aminopterin,
dactinomycin, mitomycins, esperamicins (see, U.S. Patent No. 4,675,187),
melphalan and otherrelated nitrogen
mustards. Also included in this definition are hormonal agents that act to
regulate or inhibit hormone action on
tumors such as tamoxifen and onapristone.
A "growth inhibitory agent" when used herein refers to a compound or
composition which inhibits growth
of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo.
Thus, the growth inhibitory agent is one
which significantly reduces the percentage of the target cells in S phase.
Examples of growth inhibitory agents
include agents that block cell cycle progression (at a place other than S
phase), such as agents that induce Gl
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 Gl also spill over into S-phase arrest, for example, DNA
alkylating agents such as tamoxifen,
prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-
fluorouracil, and ara-C. Further
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information can be found in The Molecular Basis of Cancer, Mendelsohn and
Israel, eds., Chapter 1, entitled
"Cell cycle regulation, oncogens, and antineoplastic drugs" by Murakami et
al., (WB Saunders: Philadelphia,
1995), especially p. 13.
The term "cytokine" is a generic term for proteins released by one cell
population which act on another
cell as intercellular mediators. Examples of such cytokines are lymphokines,
monokines, and traditional
polypeptide hormones. Included among the cytokines are growth hormone such as
human growth hormone, N-
methionyl human growth hormone, and bovine growth hormone; parathyroid
hormone; thyroxine; insulin;
proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle
stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth
factor; fibroblast growth factor;
prolactin; placental lactogen; tumor necrosis factor-a and -(3; mullerian-
inhibiting substance; inouse
gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth
factor; integrin; thrombopoietin
(TPO); nerve growth factors such as NGF-j3; platelet-growth factor;
transforming growth factors (TGFs) such
as TGF-a and TGF-(3; insulin-like growth factor-I and -II; erythropoietin
(EPO); osteoinductive factors;
interferons such as interferon-ca, -(3, 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-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor
necrosis factor such as TNF-a or
TNF-(3; and other polypeptide factors including LIF and kit ligand (KL). As
used herein, the term cytokine
includes proteins from natural sources or from recombinant cell culture and
biologically active equivalents of
the native sequence cytokines.
The term "prodrug" as used in this application refers to a precursor or
derivative form of a
pharmaceutically active substance that is less cytotoxic to tumor cells
compared to the parent drug and is capable
of being enzymatically activated or converted into the more active parent
form. See, e.g., Wilman, "Prodrugs
in Cancer Chemotherapy", Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986)
and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery,"
Directed Drug Delivery,
Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). The prodrugs of
this invention include, but are not
limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs,
glycosylated prodrugs or
optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine
and other 5-fluorouridine
prodrugs which can be derivatized into a prodrug form for use in this
invention include, but are not limited to,
those chemotherapeutic agents described above.
The term "agonist" is used in the broadest sense and includes any molecule
that mimics a biological activity
of a native PRO polypeptide disclosed herein. Suitable agonist molecules
specifically include agonist antibodies
or antibody fragments, fragments or aniino acid sequence variants of native
PRO polypeptides, peptides, small
organic molecules, etc. Methods for identifying agonists of a PRO 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 agent(s) 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.
"Internuttent" administration is treatment that is not consecutively done
without interruption, but rather is cyclic
in nature.
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"Mammal" for purposes of treatment refers to any animal classified as a
mammal, including humans,
domestic and farm animals, and zoo, sports, or pet animals, such as dogs,
cats, cattle, horses, sheep, pigs, goats,
rabbits, etc. Preferably, the manunal 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 weiglit (less than about 10 residues) polypeptides; proteins,
such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose,
mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as TWEENTM,
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 of different immunoglobulin isotypes. Each heavy and
light chain also has regularly
spaced intrachain disulfide bridges. Each heavy chain has at one end a
variable domain (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
througliout the variable domains of
antibodies. It is concentrated in three segments called complementarity-
determining regions (CDRs) or
hypervariable regions both in the light-chain and the heavy-chain variable
domains. The more highly conserved
portions of variable domains are called the framework regions (FR). The
variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a(3-sheet
configuration, connected by three CDRs,
which form loops connecting, and in some cases forming part of, the (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
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"complementarity determining region" or "CDR" (i.e., residues 24-34 (Ll), 50-
56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain;
Kabat et al.; Seauences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institute
of Health, Bethesda, MD. [1991]) and/or those residues from a "hypervariable
loop" (i.e., residues 26-32 (LI),
50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in
the heavy chain 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 Eng., 8(10): 1057-1062
[1995]); single-chain antibody
molecules; and multispecific antibodies formed from antibody fragments.
Papain digestion of antibodies produces two identical antigen-binding
fragments, called "Fab" fragments,
each with a single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to
crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two
antigen-combining sites and is still
capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding site.
This region consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent
association. It is in this configuration that the three CDRs of each variable
domain interact to define an antigen-
binding site on the surface of the VH V, dimer. Collectively, the six CDRs
confer antigen-binding specificity
to the antibody. However, even a single variable domain (or half of an Fv
comprising only tliree CDRs specific
for an antigen) has the ability to recognize and bind antigen, although at a
lower affinity than the entire binding
site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH 1)
of the heavy chain. Fab fragments differ from Fab' fragments by the addition
of a few residues at the carboxy
terminus of the heavy chain CH1 domain including one or more cysteines from
the antibody hinge region. Fab'-
SH is the designation herein for Fab' in which the cysteine residue(s) of the
constant domains bear a free thiol
group. F(ab')2 antibody fragments originally were produced as pairs of Fab'
fragments which have hinge
cysteines between them. Other chemical couplings of antibody fragments are
also known.
The "light chains" of antibodies (innmunoglobulins) from any vertebrate
species can be assigned to one
of two clearly distinct types, called kappa and lambda, based on the amino
acid sequences of their constant
domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, imrnunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several of these may be further divided into subclasses (isotypes),
e.g., IgGl, IgG2, IgG3, IgG4, IgA,
and IgA2.
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
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are highly specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional
(polyclonal) antibody preparations which typically include different
antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are advantageous in
that they are synthesized by the
hybridoma culture, uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the
character of the antibody as being obtained from a substantially homogeneous
population of antibodies, and is
not to be construed as requiring production of the antibody by any particular
method. For example, the
monoclonal antibodies to be used in accordance with the present invention may
be made by the hybridoma
method first described by Kohler etal., Nature, 256:495 [1975], or may be made
by recombinant DNA methods
(see, e.g., U.S. Patent No. 4,816,567). The "monoclonal antibodies" may also
be isolated fromphage antibody
libraries using the techniques described in Clackson et al., Nature, 352:624-
628 [1991] and Marks etal., 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 of the chain(s) 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 etal., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 [1984]).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')Z or
other antigen-binding
subsequences of antibodies) which contain minimal sequence derived froin 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
maxiniize antibody performance.
In general, the humanized antibody will comprise substantially all of at least
one, and typically two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-human
immunoglobulin and all or substantially all of the FR regions are those of a
human immunoglobulin sequence.
The humanized antibody optimally also will comprise at least a portion of an
immunoglobulin constant region
(Fc), typically that of a human immunoglobulin. For further details, see,
Jones et al., Nature, 321:522-525
(1986); Reichmann et al., Nature, 332:323-329 [1988]; and Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992).
The humanized antibody includes a PRIMATIZEDTM antibody wherein the antigen-
binding region of the
antibody is derived from an antibody produced by immunizing macaque monkeys
with the antigen of interest.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a
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polypeptide linker between the VH and VL domains which enables the sFv to form
the desired structure for
antigen binding. For a review of sFv, see, Pluckthun in The Pharmacology of
Monoclonal Antibodies, Vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which fragments
comprise a heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) in the same
polypeptide chain (VH - VL). By using a linker that is too short to allow
pairing between the two domains on
the same chain, the domains are forced to pair with the complementary domains
of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for example, EP
404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified and separated and/or
recovered from a component
of its natural environment. Contaminant components of its natural environment
are materials which would
interfere with diagnostic or therapeutic uses for the antibody, and may
include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred embodiments, the
antibody will be purified (1) to
greater than 95% by weight of antibody as determined by the Lowry method, and
most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-
terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-
PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver stain.
Isolated antibody includes the
antibody in situ within recombinant cells since at least one component of the
antibody's natural environment will
not be present. Ordinarily, however, isolated antibody will be prepared by at
least one purification step.
An antibody that "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a
particular polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide
without substantially binding to any other polypeptide or polypeptide epitope.
The word "label" when used herein refers to a detectable compound or
composition which is conjugated
directly or indirectly to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself
(e.g., radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, may catalyze chemical
alteration of a substrate compound or composition which is detectable. 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 solidphases encompassed herein include those formedpartially or
entirely of glass (e.g., controlled
pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene,
polyvinyl alcohol and silicones. In
certain embodiments, depending on the context, the solid phase can comprise
the well of an assay plate; in
others it is a purification column (e.g., an affinity chromatography column).
This term also includes a
discontinuous solid phase of discrete particles, such as those described in
U.S. Patent No. 4,275,149.
A "liposome" is a small vesicle composed of various types of lipids,
phospholipids and/or surfactant which
is useful for delivery of a drug (such as a PRO polypeptide or antibody
thereto) to a mammal. The components
of the liposome are commonly arranged in a bilayer formation, similar to the
lipid arrangement of biological
membranes.
A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
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As shown below, Table 1 provides the complete source code for the ALIGN-2
sequence comparison
computer program. This source code may be routinely compiled for use on a UNIX
operating system to provide
the ALIGN-2 sequence comparison computer program.
In addition, Tables 2-5 show hypothetical exemplifications for using the above
described method to
determine % amino acid sequence identity (Tables 2-3) and % nucleic acid
sequence identity (Tables 4-5) using
the ALIGN-2 sequence comparison computer program, wherein "PRO" represents the
amino acid sequence of
a hypothetical PRO polypeptide of interest, "Comparison Protein" represents
the amino acid sequence of a
polypeptide against which the "PRO" polypeptide of interest is being compared,
"PRO-DNA" represents a
hypothetical PRO-encoding nucleic acid sequence of interest, "Comparison DNA"
represents the nucleotide
sequence of a nucleic acid molecule against which the "PRO-DNA" nucleic acid
molecule of interest is being
compared, "X", "Y", and "Z" each represent different hypothetical amino acid
residues and "N", "L" and "V"
each represent different hypothetical nucleotides.
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Table 1
*
* C-C increased from 12 to 15
* Z is average of EQ
/* B is average of ND
* match with stop is _M; stop-stop = 0; J(joker) match = 0
#define _M -8 /* value of a match with a stop
int _day[26][26] = {
A B C D EFGHIJKLMNOPQRSTUV WXYZ*/
/* A*/ { 2, 0,-2, 0, 0,-4, 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,-l, 1, 0, 0, 0, 0,-2,-5,
0,-3, 1},
/* C{-2,-4,15,-5,-5,-4,-3,-3,-2, 0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0,
0,-5},
/* D*/ { 0, 3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-l, 0, 0, 0,-2,-7,
0,-4, 2},
/* E*/ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 3},
/* F*/ {-4,-5,-4,-6,-5, 9,-5,-2, 1, 0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-l, 0,
0, 7,-5},
/* G{ 1, 0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-1,-1,-3, 1, 0, 0,-1,-7, 0,-
5, 0},
/* H*/ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0, 3, 2,-1,-1, 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,-l, 1, 3, 0, 0, 0,-2,-3, 0,-
4, 0},
/* L{-2,-3,-6,-4,-3, 2,-4,-2, 2, 0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-
1,-2},
/* M {-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-1, 0,-2,-1, 0, 2,-4, 0,-
2,-1},
/* N{ 0, 2,-4, 2, 1,-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,
0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M},
/* P*/ { 0,-2, 0,-1,-3,-2,-1,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0},
/* Q{ 0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 0,-2,-5, 0,-4, 3},
/* R{-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0, 0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-
4, 0},
/* S*/ { 1, 0, 0, 0, 0,-3, 0, 0,-3,-2, 1,_M, 1,-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, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0, 0},
/* V*/ { 0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-1,-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, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0},
/* Y{-3,-3, 0,-4,-4, 7,-5, 0,-1, 0,-4,-1,-2,-2,_M,-5,-4,-4,-3,-3, 0,-2, 0,
0,10,-4},
/* Z*/ { 0, 1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4}
};
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Table 1 (cont')
#include <stdio.h>
#include <ctype.h>
#define MAXJMP 16 /* max jumps in a diag
#define MAXGAP 24 /* don't continue to penalize gaps larger than this
#define JMPS 1024 /* max jmps in an path */
#define MX 4 /* save if there's at least MX-1 bases since last jmp
#define DMAT 3 /* value of matching bases */
#define DMIS 0 /* penalty for mismatched bases */
#define DINSO 8 /* penalty for a gap
#define DINS1 1 /* penalty per base */
#define PINSO 8 /* penalty for a gap
#define PINS1 4 /* penalty per residue */
struct jmp {
short n[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; /* score at last jmp
long offset; /* offset of prev block
short ijmp; /* current jmp index */
struct jmp jp; /* list of jmps
struct path {
int spc; /* number of leading spaces
short n[JMPS]; /* size of jmp (gap) */
int x[JMPS];/* loc of jmp (last elem before gap)
char *ofile; /* output file name
char *namex[21; /* seq names: getseqs()
char *prog; /* prog name for err msgs
char *seqx[2]; /* seqs: getseqsQ
int dmax; /* best diag: nwQ
int dmaxO; /* final diag
int dna; /* set if dna: mainO
int endgaps; /* set if penalizing end gaps
int gapx, gapy; /* total gaps in seqs
int len0, lenl; /* seq lens */
int ngapx, ngapy; /* total size of gaps
int smax; /* max score: nw()
int *xbm; /* bitmap for matching */
long offset; /* current offset in jmp file
struct diag *dx; /* holds diagonals
struct path pp[2]; /* holds path for seqs
char *callocQ, *mallocQ, *indexQ, *strcpy(;
char *getseqQ, *g_callocQ;
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Table 1 (cont')
/* Needleman-Wunsch alignment program
*
* usage: progs filel file2
* where filel and file2 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 /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650
#include "nw.h"
#include "day.h"
static_dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0
};
static pbval[26]
1, 21 (1 ('D'-'A')) 1 (1 ('N'-'A')), 4, 8, 16, 32, 64,
128, 256, OxFFFFFFF, 1 < < 10, 1 < < 11, 1 < < 12, 1 < < 13, 1 < < 14,
1 15, 1 16, 1 17, 1 18, 1 19, 1 20, 1 21, 1 22,
1 23, 1 24, 1 251 (1 ('E'-'A'))1(1 ('Q'-'A'))
main(ac, av) main
int ac;
char *av[ ];
{
prog = av[0];
if(ac!=3){
fprintf(stderr, "usage: %s filel file2\n", prog);
fprintf(stderr, "where filel and file2 are two dna or two protein
sequences.\n");
fprintf(stderr, "The sequences can be in upper- or lower-case\n");
fprintf(stderr, "Any lines beginning with ';' or '<' are ignored\n");
fprintf(stderr,"Output is in the file \"align.out\"\n");
exit(1);
}
namex[0] = av[I];
namex[l] = av[2];
seqx[0] = getseq(namex[0], &len0);
seqx[1] = getseq(namex[l], &lenl);
xbm = (dna)? dbval : _pbval;
endgaps = 0; /* 1 to penalize endgaps
ofile = "align.out"; /* output file */
nw(); /* fill in the matrix, get the possible jmps
readjmpsQ; /* get the actual jmps */
printQ; /* print stats, alignment */
cleanup(0); /* unlink any tmp files */
}
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Table 1 (cont')
/* 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.
nw() nW
char *px, *py; /* seqs and ptrs
int *ndely, *dely; /* keep track of dely */
int ndelx, delx; /* keep track of delx
int *tmp; /* for swapping rowO, rowl
int mis; /* score for each type
int insO, insl; /* insertion penalties */
register id; /* diagonal index
register ij; /* jmp index */
register *co10, *coll; /* score for curr, last row
register xx, yy; /* index into seqs
dx = (struct diag *)g_calloc("to get diags", len0+len1+1, sizeof(struct
diag));
ndely =(int *)g_calloc("to get ndely", lenl+1, sizeof(int));
dely =(int *)g_calloc("to get dely", len1+1, sizeof(int));
colO =(int *)g_calloc("to get co10", len1+1, sizeof(int));
coll =(int *)g_calloc("to get coll", lenl+1, sizeof(int));
ins0 = (dna)? DINSO : PINSO;
ins l=(dna)? DINS 1: PINS 1;
smax = -10000;
if (endgaps) {
for (co10[0] = dely[0] =-ins0, yy = 1; yy <= lenl; yy++) {
co10[yy] = dely[yy] = co10[yy-1] - insl;
ndely[yy] = yy;
}
co10[0] = 0; /* Waterman Bull Math Bio184 */
}
else
for (yy = 1; yy <= lenl; yy++)
dely[yy] = -insO;
/* fill in match matrix
for (px = seqx[0], xx = 1; xx <= IenO; px++, xx++) {
/* initialize first entry in col
if (endgaps) {
if (xx = = 1)
coll[0] = delx = -(ins0+ins1);
else
coll[0] = delx = co10[0] - insl;
ndelx = xx;
}
else {
col l [0] = 0;
delx = -insO;
ndelx = 0;
}
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Table 1 (cont')
...nw
for (py = seqx[1], yy = 1; yy <= lenl; py++, yy++) {
mis = co10[yy-1];
if (dna)
mis + = (xbm[*px-'A']&xbm[*py-'A'])? DMAT : DMIS;
else
mis + = _day[*px-'A'] [*py-'A'];
/* update penalty for del in x seq;
* favor new del over ongong del
* ignore MAXGAP if weighting endgaps
if (endgaps ndely[yy] < MAXGAP) {
if (co10[yy] - insO > = dely[yy]) {
dely[yy] = co10[yy] - (insO+insl);
ndely[yy] = 1;
} else {
dely[yy] -= insl;
ndely[yy] + +;
}
} else {
if (colO[yy] - (insO+insl) > = dely[yy]) {
dely[yy] = col0[yy] - (insO+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] - insO > = delx) {
delx = coll[yy-1] - (insO+insl);
ndelx = 1;
} else {
delx -= insl;
ndelx+ +;
}
} else {
if (coll[yy-1] - (insO+insl) > = delx) {
delx = coll[yy-1] - (insO+insl);
ndelx = 1;
} else
ndelx++;
}
/* pick the maximum score; we're favoring
* mis over any del and delx over dely
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Table 1 (cont')
...nw
id = xx - yy + lenl - 1;
if (mis > = delx && mis > = dely[yy])
coll [yy] = mis;
else if (delx > = dely[yy]) {
col l [yy] = delx;
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna (ndelx > = MAXJMP
{
&& xx > dx[id].jp.x[ij]+MX) mis > dx[id]. score+ DINSO))
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = ndelx;
dx[id].jp.x[ij] = xx;
dx[id].score = delx;
}
else {
coll[yy] = dely[yy];
ij = dx[id].ijmp;
if (dx[id].jp.n[0] && (!dna ~ I (ndely[yy] > = MAXJMP
{
&& xx > dx[id].jp.x[ij]+MX) mis > dx[id]. score+ DINSO))
dx[id].ijmp++;
if (++ij > = MAXJMP) {
writejmps(id);
ij = dx[id].ijmp = 0;
dx[id].offset = offset;
offset + = sizeof(struct jmp) + sizeof(offset);
}
}
dx[id].jp.n[ij] = -ndely[yy];
dx[id].jp.x[ij] = xx;
dx[id].score = dely[yy];
}
if (xx == len0 && yy < lenl) {
/* last col
*I
if (endgaps)
coll[yy] -= ins0+ins1*(lenl-yy);
if (col l [yy] > smax) {
smax = coll[yy];
dmax = id;
}
}
}
if (endgaps && xx < lenO)
coll[yy-1] -= ins0+ins1*(len0-xx);
if (coll[yy-1] > smax) {
smax = coll[yy-1];
dmax = id;
}
tmp = co10; co10 = coll; coll = tmp;
}
(void) free((char *)ndely);
(void) free((char *)dely);
(void) free((char *)co10);
(void) free((char *)coll); }
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Table 1 (cont')
*
* 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()
* dumpblock() -- dump a block of lines with numbers, stars: pr align()
* nums() -- put out a number line: dumpblock()
* putline() -- put out a line (name, [num], seq, [num]): dumpblockQ
* stars() - -put a line of stars: dumpblockQ
* stripnameQ -- strip any path and prefix from a seqname
#include "nw.h"
#define SPC 3
#define P_LINE 256 /* maximum output line */
#define P SPC 3 /* space between name or num and seq
extern _day[26][26];
int olen; /* set output line length */
FILE *fx; /* output file */
print() print
{
int lx, ly, firstgap, lastgap; /* overlap */
if ((fx = fopen(ofile, "w")) = = 0) {
fprintf(stderr, " % s: can't write %s\n", prog, ofile);
cleanup(1);
}
fprintf(fx, "< first sequence: %s (length =%d)\n", namex[0], len0);
fprintf(fx, "<second sequence: %s (length = %d)\n", namex[l], lenl);
olen = 60;
Ix = 1en0;
ly = lenl;
firstgap = lastgap = 0;
if (dmax < lenl - 1) { /* leading gap in x
pp[0].spc = firstgap = lenl - dmax - 1;
ly - PP[O]=sPc;
}
else if (dmax > lenl - 1) {/* leading gap in y
pp[l].spc = firstgap = dmax - (lenl - 1);
lx -= pp[l].spc;
}
if (dmaxO < lenO - 1) { /* trailing gap in x
lastgap = lenO - dmax0 -1;
lx -= lastgap;
}
else if (dmax0 > len0 - 1) {/* trailing gap in y
lastgap = dmax0 - (len0 - 1);
ly -= lastgap;
}
getmat(lx, ly, firstgap, lastgap);
pr alignQ;
}
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Table 1 (cont')
* trace back the best path, count matches
static
getmat(lx, ly, firstgap, lastgap) getmat
int lx, ly; /* "core" (minus endgaps)
int firstgap, lastgap; /* leading trailing overlap
{
int nm, i0, il, siz0, sizi;
char outx[32];
double pct;
register n0, nl;
register char *p0, *pl;
/* get total matches, score
i0=il=siz0=siz1=0;
p0 = seqx[0] + pp[1].spc;
p1 = seqx[1] + pp[O].spc;
nO = pp[l].spc + 1;
nl = pp[0].spc + 1;
nm0;
while ( *p0 && *pl ) {
if (siz0) {
pl++;
nl++;
siz0--;
}
else if (sizl) {
p0++;
n0++;
sizl--;
}
else {
if (xbm[*p0-'A']&xbm[*pl-'A'])
nm+ +;
if (n0++ = = pp[0].x[i0])
sizO = pp[0].n[i0++];
if (nl++ == pp[1].x[il])
sizl = pp[1].n[il++];
p0++;
pl++;
}
}
/* pet homology:
* if penalizing endgaps, base is the shorter seq
* else, knock off overhangs and take shorter core
if (endgaps)
lx = (lenO < lenl)? lenO : lenl;
else
lx = (lx < ly)? lx : ly;
pct = 100.*(double)nm/(double)lx;
fprintf(fx, "\n");
fprintf(fx, "<%d match%s in an overlap of %d: %.2f percent similarity\n",
nm, (nin 1)? ; "es", lx, pct);
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Table 1 (cont')
fprintf(fx, "<gaps in first sequence: %d", gapx); ...getmat
if (gapx) {
(void) sprintf(outx, " (%d %s%s)",
ngapx, (dna)? "base": "residue", (ngapx 1)? "": "s");
fprintf(fx, " %s", outx);
fprintf(fx, ", gaps in second sequence: %d", gapy);
if (gapy) {
(void) sprintf(outx, " (%d %s%s)",
ngapy, (dna)? "base":"residue", (ngapy == 1)? "":"s");
fprintf(fx, " %s", outx);
}
if (dna)
fprintf(fx,
"\n<score: %d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n",
smax, DMAT, DMIS, DINSO, DINS1);
else
fprintf(fx,
"\n<score: %d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per residue)\n",
smax, PINSO, PINS1);
if (endgaps)
fprintf(fx,
"<endgaps penalized. left endgap: %d %s%s, right endgap: %d %s%s\n",
firstgap, (dna)? "base" : "residue", (firstgap == 1)? "s",
lastgap, (dna)? "base" : "residue", (lastgap == 1)? "s");
else
fprintf(fx, " < endgaps not penalized\n");
}
static nm; /* matches in core -- for checking */
static Imax; /* lengths of stripped file names
static ij[2]; /* jmp index for a path */
static nc[2]; /* number at start of current line */
static ni[2]; /* current elem number -- for gapping
static siz[2];
static char *ps[2]; /* ptr to current element
static char *po[2]; /* ptr to next output char slot */
static char out[2][P_LINE]; /* output line */
static char star[P_LINE]; /* set by stars()
* print alignment of described in struct path pp[ ]
static
pr_align() pr_align
{
int nn; /* char count */
int more;
register i;
for (i = 0, lmax = 0; i< 2; i++) {
nn = stripname(namex[i]);
if (nn > lmax)
Imax = nn;
nc[i] = 1;
ni[i] = 1;
siz[i] = ij[i] = 0;
ps[i] = seqx[i];
po[i] = out[i]; }
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Table 1 (cont')
for (nn = nm = 0, more = 1; more; ) { ...pr_align
for (i = more = 0; i < 2; i++) {
* do we have more of this sequence?
if (!*ps[i])
continue;
more++;
if (pp[i].spc) { /* leading space
*po[i]++
pp[i].spc--;
}
else if (siz[i]) { /* in a gap
*po[i]++
siz[i]--;
}
else { /* we're putting a seq element
*/
*po[i] = *ps[i];
if (islower(*ps[i]))
*ps[i] = toupper(*ps[i]);
po[i]++;
ps[i]++;
* are we at next gap for this seq?
if (ni[i] pp[i].x[ij[i]]) {
* we need to merge all gaps
* at this location
siz[i] = pp[i].n[ij [i] + +];
while (ni[i] == pp[i].x[ij[i]])
siz[i] += pp[i].n[ij[i]++];
}
ni[i] + +;
}
}
if (++nn == olen ~ ~!more && nn) {
dumpblock();
for (i = 0; i < 2; i++)
po[i] = out[i]; nn = 0;
}
}
}
* dump a block of lines, including numbers, stars: pr_align()
static
dumpblock() dumpblock
{
register i;
for(i=0;i<2;i++)
*po[i]-- = '\0';
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Table 1 (cont')
...dumpblock
(void) putc('\n', fx);
for(i=0;i<2;i++){
if (*out[i] && (*out[i] *(po[i]) ! _ ' ')) {
if (i = = 0)
nums(i);
if (i = = 0 && *out[1])
starsQ;
putline(i);
if (i = = 0 && *out[i])
fprintf(fx, star);
if(i==1)
nums(i);
}
}
}
* put out a number line: dumpblock()
*/
static
nums(ix) nums
int ix; /* index in out[ ] holding seq line */
{
char nline[P_LINE];
register i, j;
register char *pn, *px, *py;
for (pn = nline, i 0; i < 1max+P_SPC; i++, pn++)
*pn = "
for (i = nc[ix], py = out[ix]; *py; py++, pn++) {
if (*Py = = , , I I *py
*pn
else {
if (i%10 == 0 (i == 1 &&nc[ix] != 1)) {
j = (i < 0)? -i : i; for (px = pn; j; j / = 10, px--)
*px = j%10 + '0';
if (i < 0)
*px=
}
else
*pn
i++;
}
}
*Pn = '\0';
nc[ix] = i;
for (pn = nline; *pn; pn+ +)
(void) putc(*pn, fx);
(void) putc('\n', fx);
}
* put out a line (name, [num], seq, [num]): dumpblock()
static
putline(ix) putline
int ix; {
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Table 1 (cont')
...putline
int i;
register char *px;
for (px = namex[ix], i = 0; *px && *px px+ +, i++)
(void) putc(*px, fx);
for (; i < 1max+PSPC; i++)
(void) putc(' ', fx);
/* these count from 1:
* ni[ ] is current element (from 1)
* nc[ ] is number at start of current line
for (px = outfix]; *px; px++)
(void) putc(*px&Ox7F, fx);
(void) putc('\n', fx);
}
* put a line of stars (seqs always in out[0], out[1]): dumpblockO
static
stars() stars
f
int i;
register char *p0, *pl, cx, *px;
if (!*out[0] I I (*out[0] == && *(Po[0]) ==
!*out[1] (*out[1] == && *(po[1]) == ' '))
return;
px = star;
for (i = 1max+P_SPC; i; i--)
*px++ = ' ';
for (p0 = out[0], p1 = 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'][*p1-'A'] > 0)
cx =
else
cx=
}
else
cx
*px++ = cx;
}
*px++ = '\n';
*Px = '\0';
}
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Table 1 (cont')
* 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 = 0;
for (px = pn; *px; px+ +)
if (*px = '/')
py = px + 1;
if (py)
(void) strcpy(pn, py);
return(strlen(pn));
}
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Table 1 (cont')
* cleanup() -- cleanup any tmp file
* getseqQ -- read in seq, set dna, len, maxlen
* g_calloc() -- calloc() with error checkin
* readjmps() -- get the good jmps, from tmp file if necessary
* writejmps() -- write a filled array of jmps to a tmp file: nw()
#include "nw.h"
#include <sys/file.h>
char *jname ="/tmp/homgXXXXXX"; /* tmp file for jmps
FILE *fj;
int cleanupQ; /* cleanup tmp file */
long lseek();
* remove any tmp file if we blow
cleanup(i) cleanup
int i;
{
if (fj)
(void) unlink(jname);
exit(i);
}
* read, return ptr to seq, set dna, len, maxlen
* skip lines starting with';', '<', or '>'
* seq in upper or lower case
char *
getseq(file, len) getseq
char *file; /* file name
int *len; /* seq len */
{
char line[1024], *pseq;
register char *px, *py;
int natgc, tlen;
FILE *fp;
if ((fp = fopen(file, "r")) 0) {
fprintf(stderr, " %s: can't read %s\n", prog, file);
exit(1);
}
tlen = natgc = 0;
while (fgets(line, 1024, fp)) {
if (*line = = ' ;' I I *line *line
continue;
for (px = line; *px 1= '\n'; px++)
if (isupper(*px) I I islower(*px))
tlen+ +;
}
if ((pseq = malloc((unsigned)(tlen+6))) 0) {
fprintf(stderr, " %s: malloc() failed to get %d bytes for %s\n", prog, tlen+6,
file);
exit(1);
}
pseq[0] = pseq[1] = pseq[2] = pseq[3] = '\0';
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Table 1 (cont')
...getseq
py = pseq + 4;
*len = tlen;
rewind(fp);
while (fgets(line, 1024, fp)) {
if (*line = = ';' I I *line *line
continue;
for (px = line; *px '\n'; px++) {
if (isupper(*px))
*py++ = *px;
else if (islower(*px))
*py++ = toupper(*px);
if (index("ATGCU", *(py-1)))
natgc+ +;
}
}
*py++ _ '\0';
*PY = t\01 ;
(void) fclose(fp);
dna = natgc > (tlen/3);
return(pseq+4);
}
char *
g_calloc(msg, nx, sz) g_Calloc
char *msg; /* program, calling routine
int nx, sz; /* number and size of elements */
{
char *px, *calloc();
if ((px = calloc((unsigned)nx, (unsigned)sz)) 0) {
if (*msg) {
fprintf(stderr, "%s: g_callocQ failed %s (n=%d, sz=%d)\n", prog, msg, nx, sz);
exit(1);
}
}
return(px);
}
/-k
* get final jmps from dx[ ] or tmp file, set pp[ ], reset dmax: mainQ
*/ .
readjmps() readjmps
{
int fd = -1;
int siz, i0, il;
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 = iO = il = 0, dmax0 = dmax, xx = lenO; ; i++) {
while (1) {
for (j = dx[dmax].ijmp; j > = 0 && dx[dmax].jp.x[j] > xx; j--)
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Table 1 (cont')
...readjmps
if (j < 0 && dx[dmax].offset && fj) {
(void) lseek(fd, dx[dmax].offset, 0);
(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));
dx[dmax].ijmp = MAXJMP-1;
}
else
break;
}
if (i > = JMPS) {
fprintf(stderr, "%s: too many gaps in alignment\n", prog);
cleanup(1);
}
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 + Ienl - 1;
gapy + +;
ngapy -= siz;
/* ignore MAXGAP when doing endgaps
siz = (-siz < MAXGAP endgaps)? -siz : MAXGAP;
i1++;
}
else if (siz > 0) {/* gap in first seq
pp[0j.n[i0] = siz;
pp[0].x[i0] = xx;
gapx+ +;
ngapx + = siz;
/* ignore MAXGAP when doing endgaps */
siz = (siz < MAXGAP endgaps)? siz : MAXGAP;
i0++;
}
}
else
break;
}
/* reverse the order of jmps
for (j = 0, i0--; j < i0; j++, i0--) {
i= pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i;
i= pp[0].x[j]; pp[0].x[j = pp[0].x[i0]; pp[0].x[i0] = i;
}
for (j = 0, i1--; j < il; j++, il--) {
i = pp[1].n[j]; pp[1].n[j] = pp[1].n[il]; pp[1].n[il] = i;
i = PP[ll=xGl; PP[ll=xGl = PP[1].x[il]; pp[1].x[i1] = i;
}
if (fd > = 0)
(void) close(fd);
if (fj) {
(void) unlink(jname);
fj = 0;
offset = 0;
} }
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Table 1 (cont')
* write a filled jmp struct offset of the prev one (if any): nwQ
writejmps(ix) writejmps
int ix;
{
char *mktempQ;
if(!fj){
if (mktemp(jname) < 0) {
fprintf(stderr, "%s: can't mktemp() %s\n", prog, jname);
cleanup(1);
}
if ((fj = fopen(jname, "w")) 0) {
fprintf(stderr, "%s: can't write %s\n", prog, jname);
exit(1);
}
}
(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj);
(void) fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);
}
CA 02390685 2002-06-07
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Table 2
PRO XXXXXXXXXXXXXXX (Length = 15 amino acids)
Comparison Protein XXXXXYYYYYYY (Length = 12 amino acids)
% amino acid sequence identity =
(the number of identically matching amino acid residues between the two
polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid residues of
the PRO polypeptide)
5 divided by 15 = 33.3 %
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Table 3
PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids)
% amino acid sequence identity = .
(the number of identically matching amino acid residues between the two
polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid residues of
the PRO polypeptide) _
5 divided by 10 = 50%
42
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Table 4
PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
% nucleic acid sequence identity =
(the number of identically matching nucleotides between the two nucleic acid
sequences as determined by
ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic
acid sequence) _
6 divided by 14 = 42. 9%
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Table 5
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 %
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II. Compositions and Methods of the Invention
A. Full-length PRO Polypeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PRO polypeptides. In particular,
cDNAs encoding the PRO polypeptide
has been identified and isolated, as disclosed in further detail in the
Examples below.
As disclosed in the Examples below, cDNA clones encoding PRO 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 PRO
polypeptides and encoding
nucleic acids described herein, Applicants have identified what is believed to
be the reading frame best
identifiable with the sequence information available at the time.
B. PRO Variants
In addition to the full-length native sequence PRO polypeptides described
herein, it is contemplated that
PRO variants can be prepared. PRO variants can be prepared by introducing
appropriate nucleotide changes
into the PRO DNA, and/or by synthesis of the desired PRO polypeptide. Those
skilled in the art will appreciate
that amino acid changes may alter post-translational processes of the PRO
polypeptide, such as changing the
number or position of glycosylation sites or altering the membrane anchoring
characteristics.
Variations in the native full-length sequence PRO polypeptide or in various
domains of the PRO
polypeptide 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 PRO polypeptide that results
in a change in the amino acid sequence of the PRO polypeptide as compared with
the native sequence PRO
polypeptide. Optionally the variation is by substitution of at least one amino
acid with any other amino acid in
one or more of the domains of the PRO polypeptide. Guidance in determining
which amino acid residue may
be inserted, substituted or deleted without adversely affecting the desired
activity may be found by comparing
the sequence of the PRO polypeptide with that of homologous known protein
molecules and minimizing the
number of amino acid sequence changes made in regions of high homology. Amino
acid substitutions can be
the result of replacing one amino acid with another amino acid having similar
structural and/or chemical
properties, such as the replacement of a leucine with a serine, i.e.,
conservative amino acid replacements.
Insertions or deletions may optionally be in the range of about 1 to 5 amino
acids. The variation allowed may
be determined by systematically making insertions, deletions or substitutions
of amino acids in the sequence and
testing the resulting variants for activity exhibited by the full-length or
mature native sequence.
PRO 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 arnino acid residues that are not essential for a desired
biological activity of the PRO polypeptide.
PRO fragments may be prepared by any of a number of conventional techniques.
Desired peptide
fragments may be chemically synthesized. An alternative approach involves
generating PRO fragments by
CA 02390685 2002-06-07
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enzymatic digestion, e.g., by treating the protein with an enzyme known to
cleave proteins at sites defined by
particular amino acid residues, or by digesting the DNA with suitable
restriction enzymes and isolating the
desired fragment. Yet another suitable technique involves isolating and
amplifying a DNA fragment encoding
a desired polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired
termini of the DNA fragment are employed at the 5' and 3' primers in the PCR.
Preferably, PRO polypeptide
fragments share at least one biological and/or immunological activity with the
native PRO polypeptide shown
in the accompanying figures.
In particular embodiments, conservative substitutions of interest are shown in
Table 6 under the heading
of preferred substitutions. If such substitutions result in a change in
biological activity, then more substantial
changes, denominated exemplary substitutions in Table 6, or as further
described below in reference to aniino
acid classes, are introduced and the products screened.
Table 6
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) val; leu; ile val
Arg (R) lys; gln; asn lys
Asn (N) gln; his; lys; arg gln
Asp (D) glu glu
Cys (C) ser ser
Gln (Q) asn asn
Glu (E) asp asp
Gly (G) pro; ala ala
His (H) asn; gln; lys; arg arg
Ile (I) leu; val; met; ala; phe;
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala
Ser (S) thr thr
Thr (T) ser ser
Trp (W) tyr; phe tyr
Tyr (Y) trp; phe; thr; ser phe
Val (V) ile; leu; met; phe;
ala; norleucine leu
Substantial modifications in function or immunological identity of the PRO
polypeptide are accomplished
by selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the
polypeptide backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the
charge or hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally occurring
residues are divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
46
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(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
Such substituted residues also may be introduced into the conservative
substitution sites or, more preferably,
into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-
directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis [Carter et al., Nucl.
Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)],
cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA,
317:415 (1986)] or other known techniques can be performed on the cloned DNA
to produce the PRO variant
DNA.
Scanning amino acid analysis can also be employed to identify one or more
amino acids along a contiguous
sequence. Among the preferred scanning amino acids are relatively small,
neutral amino acids. Such amino
acids include alanine, glycine, serine, and cysteine. Alanine is typically a
preferred scanning amino acid among
this group because it eliminates the side-chain beyond the beta-carbon and is
less likely to alter the main-chain
conformation of the variant [Cunninghamand 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 [Creigliton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J.
Mol. Biol., 150:1 (1976)]. If
alanine substitution does not yield adequate amounts of variant, an isoteric
amino acid can be used.
C. Modifications of PRO Polypeptides
Covalent modifications of PRO polypeptides are included within the scope of
this invention. One type
of covalent modification includes reacting targeted amino acid residues of a
PRO polypeptide with an organic
derivatizing agent that is capable of reacting with selected side chains or
the N- or C- terminal residues of the
PRO polypeptide. Derivatization with bifunctional agents is useful, for
instance, for crosslinking PRO
polypeptides to a water-insoluble support matrix or surface for use in the
method for purifying anti-PRO
antibodies, and vice-versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-
phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters
with 4-azidosalicylic acid,
homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate),
bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-
azidophenyl)dithio]propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues
to the corresponding
glutamyl and aspartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
groups of seryl or threonyl residues, methylation of the a-amino groups of
lysine, arginine, and histidine side
chains [T.E. Creighton, Proteins: Structure and Molecular Prooerties, W.H.
Freeman & Co., San Francisco, pp.
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79-86 (1983)], acetylation of.the N-terminal amine, and amidation of any C-
terminal carboxyl group.
Another type of covalent modification of the PRO polypeptide included within
the scope of this invention
comprises altering the native glycosylation pattern of the polypeptide.
"Altering the native glycosylation
pattern" is intended for purposes herein to mean deleting one or more
carbohydrate moieties found in native
sequence PRO polypeptides (either by removing the underlying glycosylation
site or by deleting the
glycosylation by chemical and/or enzymatic means), and/or adding one or more
glycosylation sites that are not
present in the native sequence PRO polypeptide. In addition, the phrase
includes qualitative changes in the
glycosylation of the native proteins, involving a change in the nature and
proportions of the various carbohydrate
moieties present.
Addition of glycosylation sites to the PRO polypeptide may be accomplished by
altering the amino acid
sequence. The alteration may be made, for example, by the addition of, or
substitution by, one or more serine
or threonine residues to the native sequence PRO polypeptide (for 0-linked
glycosylation sites). The PRO
polypeptide amino acid sequence may optionally be altered through changes at
the DNA level, particularly by
mutating the DNA encoding the PRO polypeptide at preselected bases such that
codons are generated that will
translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the PRO
polypeptide is by chemical
or enzymatic coupling of glycosides to the polypeptide. Such methods are
described in the art, e.g., in WO
87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306
(1981).
Removal of carbohydrate moieties present on the PRO polypeptide may be
accomplished chemically or
enzymatically or by mutational substitution of codons encoding for amino acid
residues that serve as targets for
glycosylation. Chemical deglycosylation techniques are known in the art and
described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al.,
Anal. Biochem., 118:131
(1981). 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.
Enzymol., 138:350 (1987).
Another type of covalent modification of PRO polypeptides comprises linking
the PRO polypeptide to one
of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),
polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
The PRO polypeptide of the present invention may also be modified in a way to
form a chimeric molecule
comprising a PRO polypeptide fused to another, heterologous polypeptide or
amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PRO
polypeptide with a tag
polypeptide which provides an epitope to which an anti-tag antibody can
selectively bind. The epitope tag is
generally placed at the amino- or carboxyl- terminus of the PRO polypeptide.
The presence of such epitope-
tagged forms of the PRO polypeptide can be detected using an antibody against
the tag polypeptide. Also,
provision of the epitope tag enables the PRO 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-
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histidine-glycine (poly-His-gly) tags; the flu HA tag polypep6de and its
antibody 12CA5 [Field etal., Mol. Cell.
Biol=, 8:2159-2165 (1988)1; the c-myc tag and the 8F9, 3C7, 6G10, G4, B7 and
9E10 antibodies thereto [Evan
et al., Molecular and Cellular Bioloav, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D(gD)
tag and its antibody [Paborsky etal., Protein Engineering, 3(6):547-553
(1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et a.l., BioTechnology, 6:1204-1210 (1988)]; the KT3
epitope peptide [Martin et al.,
Science. 255:192-194 (1992)]; an a-tubulin epitope peptide [Skinner et al., J.
Biol. Chem., 266:15163-15166
(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc.
Natl. Acad. Sci. USA, 87:6393-
6397 (1990)].
In an alternative embodiment, the chimeric molecule may comprise a fusion of
the PRO polypeptide with
an immunoglobulin or a particular region of an immunoglobulin. For a bivalent
form of the chimeric molecule
(also referred to as an "immunoadhesin"), such a fusion could be to the Fc
region of an IgG molecule. The Ig
fusions preferably include the substitution of a soluble (transmembrane domain
deleted or inactivated) form of
a PRO polypeptide in place of at least one variable region within an Ig
molecule. In a particularly preferred
embodiment, the imrnunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3
regions of an IgGI molecule. For the production of immunoglobulin fusions see
also, U.S. Patent No.
5,428,130 issued June 27, 1995.
D. Preoaration of PRO Polvaentides
The description below relates primarily to production of PRO polypeptides by
culturing cells transformed
or transfected with a vector containing PRO polypeptide nucleic acid. It is,
of course, contemplated that
alternative methods, which are well known in the art, inay be employed to
prepare PROpolypeptides. For
instance, the PRO polypeptide sequence, or portions thereof, may be produced
by direct peptide synthesis using
solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Pe]2tide
Synthesis, W.H. Freeman Co., San
Francisco, CA (1969); Merrifield, J. Am Chem. Soc., 85:2149-2154 (1963)]. In
vitro protein synthesis may
be performed using manual techniques or by automation. Automated synthesis may
be accomplished, for
instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA)
using manufacturer's instructions.
Various portions of the PRO polypeptide may be chemically synthesized
separately and combined using
chemical or enzymatic methods to produce the full-length PRO polypeptide.
1. Isolation of DNA Encoding PRO Polypeytides
DNA encoding PRO polypeptides niay be obtained from a cDNA library prepared
from tissue believed
to possess the PRO mRNA and to express it at a detectable level. Accordingly,
human PRO DNA can be
conveniently obtained from a cDNA library prepared from human tissue, such as
described in the Examples.
The PRO-encoding gene may also be obtained from a genomic library or by known
synthetic procedures (e.g.,
automated nucleic acid synthesis).
Libraries can be screened with probes (such as antibodies to the PRO
polypeptide 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
49
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described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New
York: Cold Spring Harbor
Laboratory Press, 1989). An alternative means to isolate the gene encoding the
PRO polypeptide is to use PCR
methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A
Laboratory Manual (Cold Spring
Harbor Laboratory Press, 1995)].
The Examples below describe techniques for screening a cDNA library. The
oligonucleotide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are
minimized. The oligonucleotide is preferably labeled such that it can be
detected upon hybridization to DNA
in the library being screened. Methods of labeling are well known in the art,
and include the use of radiolabels
like 32P-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
otlier private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across
the full-length sequence can be determined using methods known in the art and
as described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al.,
supra, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into eDNA.
2. Selection and Transformation of Host Cells
Host cells are transfected or transformed with expression or cloning vectors
described herein for PRO
polypeptide 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 Manunalian Cell Biotechnology: a Practical
Approach, 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, CaC12, CaPO4, liposome-mediated and
electroporation. Depending on the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra, or
electroporation is generally used for
prokaryotes. Infection with Agr=obacteriurn turirefaciens is used for
transformation of certain plant cells, as
described by Shaw etal., 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, Virology,
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 etal., J. Bact.,130:946 (1977) and Hsiao etal., Proc.
Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such as by
nuclear microinjection,
CA 02390685 2002-06-07
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electroporation, bacterial protoplast fusion with intact cells, or
polycations, e.g., polybrene, polyornithine, may
also be used. For various techniques for transforming mammalian cells, see,
Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).
Suitable host cells for cloning or expressing the DNA in the vectors herein
include prokaryote, yeast, or
higher eukaryote cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative
or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli.
Various E. coli strains are
publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli
X1776 (ATCC 31,537); E. coli
strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable
prokaryotic host cells include
Enterobacteriaceae such as Escherichia, e.g., E. coli, Etzterobacter,
Erwitiia, Klebsiella, Proteus, Salnzonella,
e.g., Salmonella typhimurium, Serratia, e.g., Serratia inarcescans, and
Shigella, as well as Bacilli such as B.
subtilis and B. liclzeniforrniis (e.g., B. licheniformis 41P disclosed in DD
266,710 published 12 April 1989),
Pseudontonas such as P. aeruginosa, and Streptomyces. These examples are
illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host because it is a
common host strain for
recombinant DNA product fermentations. Preferably, the host cell secretes
miiiimal amounts of proteolytic
enzymes. For example, strain W3110 may be modified to effect a genetic
mutation in the genes encoding
proteins endogenous to the host, with examples of such hosts including E. coli
W3110 strain 1A2, which has
the complete genotype tonA ; E. coli W3 110 strain 9E4, which has the complete
genotype tonA ptr3; E. coli
W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP
ompT kanY; E. coli W3110 strain 37D6, which has the complete genotype tonA
ptr3 phoA E15 (argF-lac)169
degP ompT rbs7 ilvG kan''; E. coli W31 10 strain 40B4, which is strain 37D6
with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant periplasmic
protease disclosed in U.S. Patent No.
4,946,783 issued 7 August 1990. Alternatively, in vitro methods of cloning,
e.g., PCR or other nucleic acid
polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning or
expression hosts for PRO-encoding vectors. Saccharoinyces cerevisiae is a
commonly used lower eukaryotic
host microorganism. Others include Schizosaccharofrzyces ponabe (Beach and
Nurse, Nature, 290: 140 [1981 ];
EP 139,383 published 2 May 1985); Kluyveronayces hosts (U.S. Patent No.
4,943,529; Fleer et al.,
Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683,
CBS4574; Louvencourt etal.,
J. Bacteriol., 737 [1983]), K. fr-agilis (ATCC 12,424), K. bulgaricus (ATCC
16,045), K. wickerainii (ATCC
24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg
et al., Bio/Technology,
8:135 (1990)), K. therinotolerans, and K. marxiauus; yarrowia (EP 402,226);
Pichia pastoris (EP 183,070;
Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]); Candida;
Trichoderma reesia (EP 244,234);
Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263
[1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and
filamentous fungi such as, e.g.,
Neurospora, Penicilliuin, Tolypocladiuin (WO 91/00357 published 10 January
1991), and Asper=gillus hosts
such as A. nidulaus (Ballance et al., Biochem. Biophys. Res. Commun., 112:284-
289 [1983]; Tilburn et al.,
Gene, 26:205-221 [1983]; Yelton etal., Proc. Natl. Acad. Sci. USA, 81: 1470-
1474 [1984]) andA. niger (Kelly
and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable
herein and include, but are not
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limited to, yeast capable of growth on methanol selected from the genera
consisting of Hanseuula, Candida,
Kloeckera, Pichia, Saccharoinyces, Tor-ulopsis, and Rhodotorula. A list of
specific species that are exemplary
of this class of yeasts may be found in C. Anthony, The Biochemistry of
Methylotrophs, 269 (1982).
Suitable host cells for the expression of glycosylated PRO polypeptides are
derived from multicellular
organisms. Examples of invertebrate cells include insect cells such as
Drosophila S2 and Spodoptera Sf9, as
well as plant cells. Examples of useful mammalian host cell lines include
Chinese hamster ovary (CHO) and
COS cells. More specific examples include monkey kidney CV1 line transformed
by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth
in suspension culture, Graham
etal., J. Gen. Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub and Chasin, Proc. Natl.
Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.
Reprod., 23:243-251(1980)); human
lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
mammary tumor (MMT
060562, ATCC CCL51). The selection of the appropriate host cell is deemed to
be within the skill in the art.
3. Selection and Use of a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO polypeptides 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 site(s) using
techniques known in the art. Vector
components generally include, but are not limited to, one or more of a signal
sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a transcription
termination sequence.
Construction of suitable vectors containing one or more of these components
employs standard ligation
techniques which are known to the skilled artisan.
The PRO polypeptide may be produced recombinantly not only directly, but also
as a fusion polypeptide
with a heterologous polypeptide, which may be a signal sequence or other
polypeptide having a specific
cleavage site at the N-terminus of the mature protein or polypeptide. In
general, the signal sequence may be a
component of the vector, or it may be a part of the PRO-encoding DNA that is
inserted into the vector. The
signal sequence may be a prokaryotic signal sequence selected, for example,
from the group of the alkaline
phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For
yeast secretion the signal sequence
may be, e.g., the yeast invertase leader, alpha factor leader (including
Saccharofuyces and Kluyveromyces a-
factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid
phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 April 1990), or the signal
described in WO 90/13646 published
15 November 1990. In mammalian cell expression, mammalian signal sequences may
be used to direct secretion
of the protein, such as signal sequences from secreted polypeptides of the
same or related species, as well as
viral secretory leaders.
Both expression and cloning vectors contain a nucleic acid sequence that
enables the vector to replicate
in one or more selected host cells. Such sequences are well known for a
variety of bacteria, yeast, and viruses.
The origin of replication from the plasmid pBR322 is suitable for most Gram-
negative bacteria, the 2,u plasmid
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origin is suitable for yeast, and various viral origins (SV40, polyoma,
adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, e.g., the gene encoding D-alanine racemase
for Bacilli.
An example of suitable selectable markers for mammalian cells are ttlose that
enable the identification of
cells competent to take up the PRO-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate
host cell when wild-type DHFR is employed is the CHO cell line deficient in
DHFR activity, prepared and
propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216
(1980). A suitable selection gene
for use in yeast is the 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 (1977)].
Expression and cloning vectors usually contain a promoter operably linked to
the PRO-encoding nucleic
acid sequence to direct mRNA synthesis. Promoters recognized by a variety of
potential host cells are well
known. Promoters suitable for use with prokaryotic hosts include the (3-
lactamase and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544
(1979)], alkaline phosphatase, a
tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980);
EP 36,776], and hybrid
promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA,
80:21-25 (1983)]. Promoters
for use in bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA
encoding the PRO polypeptide.
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 Rea., 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.
PRO transcription from vectors in mammalian host cells is controlled, for
example, by promoters obtained
from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504
published 5 July 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma
virus, cytomegalovirus, a retrovirus,
hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian
promoters, e.g., the actin promoter
or an immunoglobulin promoter, and from heat-shock promoters, provided such
promoters are compatible with
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the host cell systems.
Transcription of a DNA encoding the PRO polypeptide by higher eukaryotes may
be increased by inserting
an enhancer sequence into the vector. Enhancers are cis-acting elements of
DNA, usually about from 10 to 300
bp, that act on a promoter to increase its transcription. Many enhancer
sequences are now known from
mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin).
Typically, however, one will use an
enhancer from a eukaryotic cell virus. Examples include the S V40 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 5' or
3' to the PRO polypeptide coding sequence, but is preferably located at a site
5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated
cells from other multicellular organisms) will also contain sequences
necessary for the termination of
transcription and for stabilizing the mRNA. Such sequences are commonly
available from the 5' and,
occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs.
These regions contain nucleotide
segments transcribed as polyadenylated fragments in the untranslated portion
of the mRNA encoding the PRO
polypeptide.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PRO polypeptides
in recombinant vertebrate cell culture are described in Gething et al.,
Nature, 293:620-625 (1981); Mantei et
al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
4. Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample directly, for
example, by conventional
Southern blotting, Northern blotting to quantitate the transcription of rnRNA
[Thomas, Proc. Natl. Acad. Sci.
USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ
hybridization, using an appropriately
labeled probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can
recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA
hybrid duplexes or
DNA-protein duplexes. The antibodies in turn may be labeled and the assay may
be carried out where the
duplex is bound to a surface, so that upon the formation of duplex on the
surface, the presence of antibody
bound to the duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods, such
as
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
inununohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be prepared in
any mammal. Conveniently, the
antibodies may be prepared against a native sequence PRO polypeptide or
against a synthetic peptide based on
the DNA sequences provided herein or against exogenous sequence fused to PRO
DNA and encoding a specific
antibody epitope. 1
5. Purification of PRO Polypeptides
Forms of PRO polypeptides may be recovered from culture medium or from host
cell lysates. If
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membrane-bound, it can be released from the membrane using a suitable
detergent solution (e.g., Triton-X 100)
or by enzymatic cleavage. Cells employed in expression of PRO polypeptides can
be disrupted by various
physical or chen7ical means, such as freeze-thaw cycling, sonication,
mechanical disruption, or cell lysing
agents.
It may be desired to purify PRO polypeptides from recombinant cell proteins or
polypeptides. The
following procedures are exemplary of suitable puriflcation procedures: by
fractionation on an ion-exchange
colunm; ethanol precipitation; reverse phase HPLC; chromatography on silica or
on a cation-exchange resin
such as DEAE; chromatofocusing; SDS-PAGE; aminonium 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 fonns of the PRO polypeptide, Various
methods of protein
purification may be employed and such methods are known in the art and
described for example in Deutscher,
Methods in Enz ymologx,182 (1990); Scopes, Protein Purification: Princinles
and Practice, Springer-Verlag,
New York (1982). The purification step(s) selected will depend, for example,
on the nature of the production
process used and the particular PRO polypeptide 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 PRO polypeptide.
1. Polyclonal Antibodies
Metliods of preparing polyclonal antibodies are known to the skilled artisan.
Polyclonal antibodies can
be raised in a mammal, for example, by one or more injections of an immunizing
agent and, if desired, an
adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in
the mammal by multiple
subcutaneous or intraperitoneal injections. The immunizing agent may include
the PRO polypeptide or a fusion
protein thereof. It may be useful to conjugate the irnnvznizing agent to a
protein known to be immunogenic in
the mammal being immunized. Examples of such immunogenic proteins include but
are not limited to I:eyhole
limpethemocyanin,serumalbumin,bovinethyroglobulin,andsoybeantrypsininhibitor.
Examplesofadjuvants
which rnay be employed include Freund's complete adjuvant and MPL-TDM adjuvant
(monophosphoryl Lipid
A, synthetic trehalose dicorynomycolate). The inununization protocol may be
selected by one skilled in the art
without undue experimentation.
2. Monoclonal Antibodies
The antibodies may, alternatively, be nionoclonal antibodies. Monoclonal
antibodies may be prepared
using hybridonia methods, such as those described by Kohler and Milstein,
Nature. 286:495 (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 orare capa.ble of
producing antibodies that will specifically
bind to the immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
The immunizing agent will typically include the PRO polypeptide or a fusion
protein thereof. Generally,
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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
[Goding, Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Inunortalized
cell lines are usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine and human
origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma
cells may be cultured in a
suitable culture medium that preferably contains one or more substances that
inhibit the growth or survival of
the unfused, immortalized cells. For example, if the parental cells lack the
enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the
hybridomas typically will include
hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances
prevent the growth of HGPRT-
deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of
antibody by the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More
preferred immortalized cell lines are murine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection, Manassas,
Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have
been described for the
production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001
(1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, Marcel Dekker,
Inc., New York, (1987) pp.
51-63].
The culture medium in which the hybridoma cells are cultured can then be
assayed for the presence of
monoclonal antibodies directed against the PRO polypeptide. 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 radioinununoassay (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 [Goding, 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 ifz 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 inununoglobulin 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 murine
antibodies). The hybridoma cells of the
invention serve as a preferred source of such DNA. Once isolated, the DNA may
be placed into expression
vectors, which are then transfected into host cells such as simian COS cells,
Chinese hamster ovary (CHO) cells,
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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
joining to the immuinoglobulin
coding sequence all or part of the coding sequence for a non-immunoglobulin
polypeptide. Such a non-
immunoglobulin polypeptide can be substituted for the constant domains of an
antibody of the invention, or can
be substituted for the variable domains of one antigen-combining site of an
antibody of the invention to create
a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well
known in the art. For example, one method involves recombinant expression of
immunoglobulin light chain
and modified heavy chain. The heavy chain is truncated generally at any point
in the Fc region so as to prevent
heavy chain crosslinking. Alternatively, the relevant cysteine residues are
substituted with another amino acid
residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce
fragments thereof, particularly, Fab fragments, can be accomplished using
routine techniques known in the art.
3. Human and Humanized Antibodies
The antibodies of the invention may further comprise humanized antibodies or
human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, irnmunoglobulin
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.
Humanized antibodies include
human immunoglobulins (recipient antibody) in which residues from a
complementary determining region
(CDR) of the recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as
mouse, rat or rabbit having the desired specificity, affinity and capacity. In
some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding non-human
residues. Humanized
antibodies may also comprise residues which are found neither in the recipient
antibody nor in the imported
CDR or framework sequences. In general, the humanized antibody will comprise
substantially all of at least
one, and typically two, variable domains, in which all or substantially all of
the CDR regions correspond to those
of a non-human immunoglobulin and all or substantially all of the FR regions
are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally also will
comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature,
321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta,
Curr. O. Struct. Biol., 2:593-
596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human. These non-
human amino acid residues are often referred to as "import" residues, which
are typically taken from an "import"
variable domain. Humanization can be essentially performed following the
method of Winter and co-workers
[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-
327 (1988); Verhoeyen et al.,
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Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences
for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric antibodies (U.S. Patent
No. 4,816,567), wherein substantially less than an intact human variable
domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from
analogous sites in rodent antibodies.
Human antibodies can also beproduced using various techniques known in the
art, including phage display
libraries [Hoogenboomand Winter, J. Mol. Biol., 227:381 (1991); Marks etal.,
J. Mol. Biol., 222:581(1991)].
The techniques of Cole et al., and Boerner et al., are also available for the
preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, p. 77 (1985) and Boerner
etal., J. Immuno1.,147 1:86-95 (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/Technology, 10: 779-783
(1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368:
812-13 (1994); Fishwild et al.,
Nature Biotechnology, 14:845-51 (1996); Neuberger, Nature Biotechnology, 14:
826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol., 13 :65-93 (1995).
4. Bispecific 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
PRO polypeptide, the other one is for any other antigen, and preferably for a
cell-surface protein or receptor or
receptor subunit.
Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant production
of bispecific antibodies is based on the co-expression of two inununoglobulin
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 cliromatography steps.
Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in
Traunecker et al., EMBO
J., 10:3655-3659 (1991).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
be fused to immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin
heavy-chain constant domain, comprising at least part of the hinge, CH2, and
CH3 regions. It is preferred to
have the first heavy-chain constant region (CHl) 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
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immunoglobulin light chain, are inserted into separate expression vectors, and
are co-transfected into a suitable
host organism. For further details of generating bispecific antibodies see,
for example, Suresh et al., Methods
in Enzymology, 121:210 (1986).
According to another approach described in WO 96/27011, the interface between
a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers which
are recovered fromrecombinant
cell culture. The preferred interface comprises at least a part of the CH3
region of an antibody constant domain.
In this method, one or more small amino acid side chains from the interface of
the first antibody molecule are
replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory
"cavities" of identical or similar
size to the large side chain(s) are created on the interface of the second
antibody molecule by replacing large
amino acid side chains with smaller ones (e.g., alanine or threonine). This
provides a mechanism for increasing
the yield of the heterodimer over other unwanted end-products such as
homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g., F(ab')Z
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 (1985) describe a procedure wherein intact antibodies
are proteolytically cleaved to
generate F(ab')2 fragments. These fragments are reduced in the presence of the
dithiol complexing agent sodium
arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments
generated are then converted to thionitrobenzoate (TNB) derivatives. One of
the Fab'-TNB derivatives is then
reconverted to the Fab'-thiol by reduction with mercaptoethylaniine 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 inunobilization of enzymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et al., J. Exp. Med., 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately
secreted from E. coli and subjected
to directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus formed was
able to bind to cells overexpressing the ErbB2 receptor and normal human T
cells, as well as trigger the lytic
act'ivity of human cytotoxic lymphocytes against human breast tumor targets.
Various techniques for making and isolating bispecific antibody fragments
directly from recombinant cell
culture have also been described. For example, bispecific antibodies have been
produced using leucine zippers.
Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun
proteins were linked to the Fab' portions of two different antibodies by gene
fusion. The antibody homodimers
were reduced at the hinge region to form monomers and then re-oxidized to form
the antibody heterodimers.
This method can also be utilized for the production of antibody homodimers.
The "diabody" technology
described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)
has provided an alternative
mechanism for making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain
(VH) connected to a light-chain variable domain (V,) by a linker which is too
short to allow pairing between the
two domains on the same chain. Accordingly, the VH and V, domains of one
fragment are forced to pair with
the complementary V, and VH domains of another fragment, thereby forming two
antigen-binding sites. Another
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strategy for making bispecific antibody fragments by the use of single-chain
Fv (sFv) dimers has also been
reported. See, Gruber et al., J. Immunol., 152:5368 (1994).
Antibodies with more than two valencies are contemplated. For example,
trispecific antibodies can be
prepared. Tutt et al., J. Immunol., 147:60 (1991).
Exemplary bispecific antibodies may bind to two different epitopes on a given
PRO polypeptide herein.
Alternatively, an anti-PRO polypeptide arm may be combined with an arm which
binds to a triggering molecule
on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or
B7), or Fc receptors for IgG
(FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus
cellular defense mechanisms
to the cell expressing the particular PRO polypeptide. Bispecific antibodies
may also be used to localize
cytotoxic agents to cells which express a particular PRO polypeptide. These
antibodies possess a PRO-binding
arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such
as EOTUBE, DPTA, DOTA, or
TETA. Another bispecific antibody of interest binds the PRO polypeptide and
further binds tissue factor (TF).
5. Heteroconjugate Antibodies
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies
are composed of two covalently joined antibodies. Such antibodies have, for
example, been proposed to target
immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for
treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may
be prepared in vitro using
known methods in synthetic protein chemistry, including those involving
crosslinking agents. For example,
immunotoxins may be constructed using a disulfide exchange reaction or by
forming a thioether bond.
Examples of suitable reagents for this purpose include iminothiolate and
methyl-4-mercaptobutyrimidate and
those disclosed, for example, in U.S. Patent No. 4,676,980.
6. Effector Function 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 residue(s) 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.
Exp. Med.,176: 1191-1195 (1992) and Shopes, J. Immuno1.,148: 2918-2922 (1992).
Homodimeric antibodies
with enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in
Wolff etal., CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody
can be engineered that has dual
Fc regions and may thereby have enhanced complement lysis and ADCC
capabilities. See, Stevenson et al.,
Anti-Cancer Drug Design, 3: 219-230 (1989).
7. Inununoconiuaates
The invention also pertains to inununoconjugates comprising an antibody
conjugated to a cytotoxic agent
such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant, or animal
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origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above.
Enzymatically active toxins and fragments thereof that can be used include
diphtheria A chain, nonbinding
active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin
A chain, modeccin A chain, alpha-sar.cin, Aleurites fordii proteins, dianthin
proteins, Phytolaca arnericana
proteins (PAPI, PAPII, 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 21zBi,'31I,13'In,
90Y, and 1s6Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters
(such as disuccinimidyl suberate),
aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-
azidobenzoyl) hexanediamine), bis-
diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-
diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-
dinitrobenzene). For example, a
ricin immunotoxin can be prepared as described in Vitetta et al., Science,
238: 1098(1987). Carbon-14-Iabeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacefic 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 thepatient, followed
by removal of unbound conjugate from the circulation using a clearing agent
and then administration of a
"ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
8. Immunoliposomes
The antibodies disclosed herein may also be formulated as immunoliposomes.
Liposomes containing the
antibody are prepared by methods known in the art, such as described in
Epstein et al., Proc. Natl. Acad. Sci.
USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030
(1980); and U.S. Pat. Nos.
4,485,045 and4,544,545. Liposomes with enhanced circulation time are disclosed
in U.S. PatentNo. 5,013,556.
Particularly useful liposomes can be generated by the reverse-phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine
(PEG-PE). Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention can be
conjugated to the liposomes as
described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a
disulfide-interchange reaction. A
chemotherapeutic agent (such as Doxorubicin) is optionally contained within
the liposome. See, Gabizon et al.,
J. National Cancer Inst., 81(19): 1484 (1989).
F. Identification of Proteins Capable of Inhibiting Neoplastic Cell Growth or
Proliferation
The proteins disclosed in the present application have been assayed in a panel
of 60 tumor cell lines
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currently used in the investigational, disease-oriented, in vitro drug-
discovery screen of the National Cancer
Institute (NCI). The purpose of this screen is to identify molecules that have
cytotoxic and/or cytostatic activity
against different types of tumors. NCI screens more than 10,000 new molecules
per year (Monks et al., J. Natl.
Cancer Inst., 83:757-766 (1991); Boyd, Cancer: Princ. Pract. Oncol. Update,
3(10):1-12 ([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 cell-based assays and animal models for tumors (e.g., cancers) can also
be used to verify the findings
of the NCI cancer screen, and to further understand the relationship between
the protein identified herein and
the 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 of the 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
innnunodeficient 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 nu 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,
NFS/N, NZB, NZC, NZW, P, RIII and SJL. In addition, a wide variety of other
animals with inherited
immunological defects other than the nude mouse have been bred and used as
recipients of tumor xenografts.
For further details see, e.g., The Nude Mouse in Oncoloay Research, E. Boven
and B. Winograd, eds., CRC
Press, Inc., 1991.
The cells introduced into such animals can be derived from known tumor/cancer
cell lines, such as, any
of the above-listed tumor cell lines, and, for example, the B104-1-1 cell line
(stable NIH-3T3 cell line
transfected with the ueu protooncogene); ras-transfected NIH-3T3 cells; Caco-2
(ATCC HTB-37); a
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moderately well-differentiated grade II human colon adenocarcinoma cell line,
HT-29 (ATCC HTB-38), or
from tumors and cancers. Samples of tumor or cancer cells can be obtained from
patients undergoing surgery,
using standard conditions, involving freezing and storing in liquid nitrogen
(Karmali et al., Br. J. Cancer,
48:689-696 [1983]).
Tumor cells can be introduced into animals, such as nude mice, by a variety of
procedures. The
subcutaneous (s.c.) space in mice is very suitable for tumor implantation.
Tumors can be transplanted s.c. as
solid blocks, as needle biopsies by use of a trochar, or as cell suspensions.
For solid block or trochar
implantation, tumor 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 ueu-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 of colon 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
"METAMOUSETM" sold by AntiCancer, Inc., (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from
the in vitro cultures can then
be passaged to animals. Such tumors can serve as targets for further testing
or drug screening. Alternatively,
the tumors resulting from the passage can be isolated and RNA from pre-passage
cells and cells isolated after
one or more rounds of passage analyzed for differential expression of genes of
interest. Such passaging
techniques can be performed with any known tumor or cancer cell lines.
For example, Meth A, CMS4, CMS5, CMS21, and WEHI-164 are chemically induced
fibrosarcomas of
BALB/c female mice (DeLeo et al., J. Exp. 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
[1987]). Briefly, tumor cells are propagated in vitro in cell culture. Prior
to injection into the animals, the cell
lines are washed and suspended in buffer, at a cell density of about 10x106 to
10x10' cells/ml. The animals are
then infected subcutaneously with 10 to 100 ,ul of the cell suspension,
allowing one to three weeks for a tumor
to appear.
In addition, the Lewis lung (3LL) carcinoma of mice, which is one of the most
thoroughly studied
experimental tumors, can be used as an investigational tumor model. Efficacy
in this tumor model has been
correlated with beneficial effects in the treatment of human patients
diagnosed with small cell carcinoma of the
lung (SCCL). This tumor can be introduced in normal mice upon injection of
tumor fragments from an affected
mouse or of cells maintained in culture (Zupi et al., Br. J. Cancer, 41,
suppl. 4:309 [1980]), and evidence
indicates that tumors can be started from injection of even a single cell and
that a very high proportion of
infected tumor cells survive. For further information about this tumor model
see, Zacharski, Haemostasis,
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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 converted
into the corresponding volume by using
a matliematical 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 eds.,
Basel, 1989, 301. It is noted, however, that necrosis and inflammatory
responses following treatment may
actually result in an increase in tumor size, at least initially. Therefore,
these changes need to be carefully
monitored, by a combination of a morphometric method and flow cytometric
analysis.
= Recombinant (transgenic) animal models can be engineered by introducing the
coding portion of the genes
identified herein into the genome of animals of interest, using standard
techniques for producing transgenic
animals. Animals that can serve as a target for transgenic manipulation
include, without limitation, mice, rats,
rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g.,
baboons, chimpanzees and monkeys.
Techniques known in the art to introduce a transgene into such animals include
pronucleic microinjection
(Hoppe and Wanger, U.S. Patent No. 4,873,191); retrovirus-mediated gene
transfer into germ lines (e.g., Van
der Putten et al., Proc. Natl. Acad. Sci. USA, 82:6148-615 [1985]); gene
targeting in embryonic stem cells
(Thompson et al., Cell, 56:313-321 [1989]); electroporation of embryos (Lo,
Mol. Cell. Biol., 3:1803-1814
[1983]); sperm-mediated gene transfer (Lavitrano etal., 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
et al., Proc. Natl. Acad. Sci. USA,
89:6232-636 (1992).
The expression of the transgene in transgenic animals can be monitored by
standard techniques. For
example, Southern blot analysis or PCR amplification can be used to verify the
integration of the transgene.
The level of mRNA expression can then be analyzed using techniques such as in
situ hybridization, Northern
blot analysis, PCR, or immunocytochemistry. The animals are further examined
for signs of tumor or cancer
development.
The efficacy of antibodies specifically binding the polypeptides identified
herein and other drug
candidates, can be tested also in the treatment of spontaneous animal tumors.
A suitable target for such studies
is the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly
invasive, malignant tumor that
is the most common oral malignancy of cats, accounting for over 60% of the
oral tumors reported in this species.
It rarely metastasizes to distant sites, although this low incidence of
metastasis may merely be a reflection of
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the short survival times for cats with this tumor. These tumors are usually
not amenable to surgery, primarily
because of the anatomy of the feline oral cavity. At present, there is no
effective treatment for this tumor. Prior
to entry into the study, each cat undergoes complete clinical examination,
biopsy, and is scanned by computed
tomography (CT). Cats diagnosed with sublingual oral squamous cell tumors are
excluded from the study. The
tongue can become paralyzed as a result of such tumor, and even if the
treatment kills the tumor, the animals
may not be able to feed themselves. Each cat is treated repeatedly, over a
longer period of time. Photographs
of the tumors will be taken daily during the treatment period, and at each
subsequent recheck. After treatment,
each cat undergoes another CT scan. CT scans and thoracic radiograins 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, lymphoina,
clirondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of
these mammary adenocarcinoma
in dogs and cats is a preferred model as its appearance and behavior are very
similar to those in humans.
However, the use of this model is limited by the rare occurrence of this type
of tumor in animals.
H. Screening Assays for Drug Candidates
Screening assays for drug candidates are designed to identify compounds that
competitively bind or
complex with the receptor(s) 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
themparticularly 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 inunobilized
component, e.g., the coated surface containing the anchored component. When
the reaction is complete, the
non-reacted components are removed, e.g., by washing, and complexes anchored
on the solid surface are
detected. When the originally non-immobilized component carries a detectable
label, the detection of label
immobilized on the surface indicates that complexing occurred. Where the
originally non-immobilized
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component does not carry a label, complexing can be detected, for example, by
using a labeled antibody
specifically binding the inunobilized complex.
If the candidate compound interacts with but does not bind to a particular
receptor, its interaction with that
polypeptide can be assayed by methods well known for detecting protein-protein
interactions. Such assays
include traditional approaches, such as, cross-linking, co-
immunoprecipitation, and co-purification through
gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using a
yeast-based genetic systemdescribed by Fields and co-workers [Fields and Song,
Nature (London), 340:245-246
(1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)] as
disclosed by Chevray and Nathans
[Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991)]. Many transcriptional
activators, such as yeast GAI,4,
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 of GAIA, and another,
in which candidate activating proteins are fused to the activation domain. The
expression of a GALl-lacZ
reporter gene under control of a GAL4-activated promoter depends on
reconstitution of GAIA activity via
protein=protein interaction. Colonies containing interacting polypeptides are
detected with a chromogenic
substrate for [i-galactosidase. A complete kit (MATCHMAKERTm) for identifying
protein-protein interactions
between two specific proteins using the two-hybrid technique is commercially
available from Clontech. This
system can also be extended to map protein domains involved in specific
protein interactions as well as to
pinpoint amino acid residues that are crucial for these interactions.
I. Pharmaceutical Compositions
The polypeptides of the present invention, agonist antibodies specifically
binding proteins identified
herein, as well as other molecules identified by the screening assays
disclosed herein, can be adniinistered for
the treatment of tumors, including cancers, in the form of pharmaceutical
compositions.
Where antibody fragments are used, the smallest inhibitory fragment which
specifically binds to the
binding domain of the target protein is preferred. For example, based upon the
variable region sequences of an
antibody, peptide molecules can be designed which retain the ability to bind
the target protein sequence. Such
peptides can be synthesized chemically and/or produced by recombinant DNA
technology (see, e.g., Marasco
et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 [1993]).
The formulation herein may also contain more than one active compound as
necessary for the particular
indication being treated, preferably those with complementary activities that
do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an agent that
enhances its function, such as, for
example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-
inhibitory agent. Such molecules are
suitably present in combination in amounts that are effective for the purpose
intended.
Therapeutic formulations of the polypeptides identified herein, or agonists
thereof are prepared for storage
by mixing the active ingredient having the desired degree of purity with
optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 16th
edition, Osol, A. ed. [1980]), in
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the form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed, and include
buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic acid and
methionine; preservatives (such as
octadecyldimetlrylbenzyl ammonium chloride; hexamethonium chloride;
benzalkoniumchloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or
propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about
10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or imrnunoglobulins; hydrophilic
polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine,
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 TWEEN,
PLURONICSTM 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 Remington's Pharmaceutical Sciences, 16t1i
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-glutaniic acid and y ethyl-L-
glutamate, non-degradable ethylene-
vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the
LUPRON DEPOTTM (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
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mechanism is discovered to be intermolecular S-S bond formation through thio-
disulfide interchange,
stabilization may be achieved by modifying sulfhydryl residues, lyophilizing
from acidic solutions, controlling
moisture content, using appropriate additives, and developing specific polymer
matrix compositions.
J. Methods of Treatment
It is contemplated that the polypeptides of the present invention and their
agonists, including antibodies,
peptides, and small molecule agonists, may be used to treat various tumors, e.
g., cancers. Exemplary conditions
or disorders to be treated include benign or malignant tumors (e.g., renal,
liver, kidney, bladder, breast, gastric,
ovarian, colorectal, prostate, pancreatic, lung, vulval, thyroid, hepatic
carcinomas; sarcomas; glioblastomas; and
various head and neck tumors); leukemias and lymphoid malignancies; other
disorders such as neuronal, glial,
astrocytal, hypothalamic and other glandular, macrophagal, epithelial, stromal
and blastocoelic disorders; and
inflammatory, angiogenic and immunologic disorders. The anti-tumor agents of
the present invention (including
the polypeptides disclosed herein and agonists which miniic 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 Chemotheral)y 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 endotlielial 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 adniinistered first,
followed by the administration of
an anti-cancer agent of the present invention. However, simultaneous
administra6on 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 of
effective doses can be performed
following the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in
toxicokinetics" in Toxicoldnetics and New Drue Development, Yacobi et al.,
eds., Pergamon Press, New York
1989, pp. 42-96.
For example, depending on the type and severity of the disease, about 1 ug/kg
to 15 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 1~zg/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated
administrations over several days or longer, depending on the condition, the
treatment is sustained until a desired
suppression of disease symptoms occurs. However, other dosage regimens may be
useful. The progress of this
therapy is easily monitored by conventional techniques and assays. Guidance as
to particular dosages and
methods of delivery is provided in the literature; see, for example, U.S. Pat.
Nos. 4,657,760; 5,206,344; or
5,225,212. It is anticipated that different formulations will be effective for
different treatment compounds and
different disorders, that administration targeting one organ or tissue, for
example, may necessitate delivery in
a manner different from that to another organ 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 inay have a
sterile access port (for example the
container may be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection
needle). The active agent in the composition is an anti-tumor agent of the
present invention. The label on, or
associated with, the container indicates that the composition is used for
diagnosing or treating the condition of
choice. The article of manufacture may further comprise a second container
comprising a pharmaceutically-
acceptable buffer, such as phosphate-buffered saline, Ringer's solution and
dextrose solution. It may further
include other materials desirable from a commercial and user standpoint,
including other buffers, diluents,
filters, needles, syringes, and package inserts with instructions for use.
The following examples are of.fered for illustrative purposes only, and are
not intended to limit the scope
of the present invention in any way.
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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
Culture Collection, Manassas,
VA.
EXAMPLE 1
Extracellular Domain Homology Screening to Identify Novel Polypeptides and
cDNA Encoding Therefor
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public databases (e.g., Dayhoff, GenBank), and proprietary
databases (e.g. LIFESEQ ,
Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the
computer program BLAST or
BLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)) as a
comparison of the ECD protein
sequences to a 6 frame translation of the EST sequences. Those comparisons
with a BLAST score of 70 (or
in some cases, 90) or greater that did not encode known proteins were
clustered and assembled into consensus
DNA sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, WA).
Using this extracellular domain homology screen, consensus DNA sequences were
assembled relative to
the other identified EST sequences using phrap. In addition, the consensus DNA
sequences obtained were often
(but not always) extended using repeated cycles of BLAST or BLAST-2 and phrap
to extend the consensus
sequence as far as possible using the sources of EST sequences discussed
above.
Based upon the consensus sequences obtained as described above,
oligonucleotides were then synthesized
and used to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to
isolate a clone of the full-length coding sequence for a PRO polypeptide.
Forward and reverse PCR primers
generally range from 20 to 30 nucleotides and are often designed to give a PCR
product of about 100-1000 bp
in length. The probe sequences are typically 40-55 bp 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 Biology, 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 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 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; pRK5B is aprecursor of pRK5D that does not contain the SfiI
site; see, Holmes etal., Science,
253:1278-1280 (1991)) in the unique Xhol and NotI sites.
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EXAMPLE 2
Isolation of cDNA Clones Using Signal Algorithm Analvsis
Various polypeptide-encoding nucleic acid sequences were identified by
applying a proprietary signal
sequence finding algorithm developed by Genentech, Inc., (South San Francisco,
CA) upon ESTs as well as
clustered and assembled EST fragments from public (e.g., GenBank) and/or
private (LIFESEQ , Incyte
Pharmaceuticals, Inc., Palo Alto, CA) databases. The signal sequence algorithm
computes a secretion signal
score based on the character of the DNA nucleotides surrounding the first and
optionally the second methionine
codon(s) (ATG) at the 5'-end of the sequence or sequence fragment under
consideration. The nucleotides
following the first ATG must code for at least 35 unambiguous amino acids
without any stop codons. If the first
ATG has the required amino acids, the second is not examined. If neither meets
the requirement, the candidate
sequence is not scored. In order to determine whether the EST sequence
contains an authentic signal sequence,
the DNA and corresponding amino acid sequences surrounding the ATG codon are
scored using a set of seven
sensors (evaluation parameters) known to be associated with secretion signals.
Use of this algorithm resulted
in the identification of numerous polypeptide-encoding nucleic acid sequences.
EXAMPLE 3
Isolation of cDNA Clones Encoding Human PR0943
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described in
EXAMPLE 1 above and is herein designated DNA36360. In some cases, the DNA36360
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 DNA36360 consensus sequence, oligonucleotides were synthesized :
1) to identify by PCR
a cDNA'library that contained the sequence of interest, and 2) for use as
probes to isolate a clone of the
full-length coding sequence for PR0943. 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 bp
in length. The probe sequences
are typically 40-55 bp 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 Biology,
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.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-CGAGATGACGCCGAGCCCCC-3' (SEQ ID NO:7)
reverse PCR primer 5'-CGGT17CGACACGCGGCAGGTG-3' (SEQ ID NO:8)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA36360
sequence which had the following nucleotide sequence:
hybridization probe
5'-TGCTGCTCCTGCTGCCGCCGCTGCTGCTGGGGGCCTTCCCGCCGG-3' (SEQ ID NO:9)
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RNA for construction of the cDNA libraries was isolated from human fetal braiu
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 dTcontaining a NotI
site, linked with blunt to SalI hemikinased adaptors, cleaved witl-- Notl,
sized appropriately by gel
electrophoresis, and cloned in a defined orientation into a suitable cloning
vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site; see, Holmes
et al., Science, 253:1278-1280
(1991)) 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 PR0943 polypeptide (designated herein as DNA52192-1369 [Figure 1,
SEQ ID NO:1]) and the
derived protein sequence for that PR0943 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucelotide positions 150-152 and a stop signal at
nucleotide positions 1662-1664 (Figure 1,
SEQ ID NO:1). The predicted polypeptide precursor is 504 amino acids long
(Figure 2), has a calculated
molecular weight of approximately 54,537 daltons and an estimated pI of
approximately 10.04. Analysis of
the full length PR0943 sequence shown in Figure 2 (SEQ ID NO:2) evidences the
presence of a variety of
iinportant polypeptide domains as shown in Figure 2, wherein the locations
given for those impoltant
polypeptide domains are approximate as described above. Analysis of the full-
length PR0943 sequence shown
in Figure 2 evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino
acid 17; a transmembrane domain from about amino acid 376 to about amino acid
396; tyrosine kinase
phosphorylation sites from about amino acid 212 to about amino acid 220 and
from about amino acid 329 to
about amino acid 337; potential N-glycosylation sites from about amino acid
111 to abbut aniino acid 115, from
about amino acid 231 to about amino acid 235, from about amino acid 255 to
about aniino acid 259, and from
about amino acid 293 to about amino acid 297; and an immunoglobulin and MHC
protein sequence honwlogy
block from about an-ino acid 219 to about amino acid 237. Clone DNA52192-1369
has been deposited with
the ATCC on July 1, 1998 and is assigned ATCC deposit no. 203042.
An analysis of theDayhoff database (version 35.45 SwissProt 35), usingthe
ALIGN-2 sequence alignment
analysis of the full-length sequence shown in Figure 2 (SEQ ID NO:2) evidenced
sequence identity between
the PR0943 amino acid sequence and the following Dayhoff sequences: B49151,
A39752, FGR1_XENLA,
S38579, RATHBFGFRB_1, TVHU2F, FGR2_MOUSE, CEK3_CHICK, P_221080 and A27171_1.
EXAMPLE 4
Isolation of cDNA clones Encoding Human PR01250
Use of the signal algorithm procedure described above in EXAMPLE 2 resulted in
the identification of
an EST cluster sequence from the Incyte database, designated Incyte EST
cluster sequence no. 56523. This
sequence was then compared to a variety of various EST databases as described
under the signal algorithm
procedure above, and further resulted in the identification of Incyte EST
3371784. Further examination and
sequencing of the full-length clone corresponding to this EST sequence
resulted in the isolation of the
full-length DNA sequence DNA60775-1532 (Figure 3, SEQ ID NO:3) and the derived
PR01250 native
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sequence protein (Figure 4, SEQ ID NO:4).
Clone DNA60775-1532 (SEQ ID NO:3) contains a single open reading frame with an
apparent translation
initiation site at nucleotide positions 74-76 and ending at the stop codon
(TAG) at nucleotide positions
2291-2293 (Figure 3). The predicted PRO 1250 polypeptide precursor ( SEQ ID
NO:4) is 739 amino acids long
(Figure 4). The PRO 1250 protein shown in Figure 4 has an estimated molecular
weight of about 82,263 daltons
and a pI of about 7.55. Analysis of PR01250 polypeptide (SEQ ID NO:4)
evidences the presence of the
following: a type II transmembrane domain from about amino acid residues 61 to
about 80, a putative
AMP-binding domain signature sequence from about amino acid residue 314 to
about 326, and. potential
N-glycosylation sites from about amino acid residues 102 to about 106, from
about amino acid residues 588 to
about 594 and from about amino acid residues 619 to about 623. A cDNA clone
containing DNA60775-1250
(SEQ ID NO:3) has been deposited with the ATCC on September 1, 1998 and is
assigned ATCC deposit no.
203173.
EXAMPLE 5
Isolation of cDNA clones Encoding Human PRO 1337
Using the method described in Example 1 above, a single Incyte EST was
identified (EST No.1747546)
and also referred to herein as "DNA4417". To assemble a consensus sequence,
repeated cycles of BLAST and
phrap were used to extend the DNA4417 sequence as far as possible using the
sources of EST sequences
discussed above. The consensus sequence is designated herein as "DNA45669".
Based on the DNA45669
consensus sequence, oligonucleotides were synthesized: 1) to identify by PCR a
cDNA library that contained
the sequence of interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for
PRO1337.
PCR primers (forward and reverse) were synthesized:
forward PCR primer 45699.f1:
5'-CAACCATGCAAGGACAGGGCAGG-3' ( SEQ ID NO:10)
forward PCR primer 45699.f2:
5'-CTTTGCTGTTGGCCTCTGTGCTCCCAACCATGCAAGGACAGGGCAGG-3' (SEQ ID NO: 11)
reverse PCR primer 45669.rl:
5'-TGACTCGGGGTCTCCAAAACCAGC-3' (SEQ ID NO:12)
reverse PCR primer 45669.r2:
5'-GGTATAGGCGGAAGGCAAAGTCGG-3' (SEQ ID NO:13)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus
DNA45669 sequence which had the following nucleotide sequence:
hybridization probe 45669.pl:
5'-GGCATCTTACCTTTATGGAGTACTCTTTGCTGTTGGCCTCTGTGCTCC-3' (SEQ ID NO: 14)
In order to screen several libraries for a source of a full-length clone, DNA
from the libraries was screened
by PCR amplification with the PCR primer pair identified above. A positive
library was then used to isolate
clones encoding the PRO1337 gene using the probe oligonucleotide and one of
the PCR primers. RNA for
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construction of the cDNA libraries was isolated from human tissue.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for
PR01337 (designated herein as DNA66672-1586 [Figure 5, SEQ ID NO:5]; and the
derived protein sequence
for PRO1337.
The entire coding sequence of PR01337 is shown in Figure 5 (SEQ ID NO:5).
Clone DNA66672-1586
contains a single open reading frame with an apparent translational initiation
site at nucleotide positions 60-62
and an apparent stop codon at nucleotide positions 1311-1313. The predicted
polypeptide precursor is 417
anuno acids long. The full-length PRO1337 protein shown in Figure 6 has an
estin--ated molecular weight of
about 46,493 dalt,ons and a pI of about 9.79.
Analysis of PR01337 polypeptide (SEQ ID NO:6) evidences the presence of the
following: a signal
peptide froin about amino acid 1 to about amino acid 20; an N-glycosylation
sites from about amino acid 101
to about amino acid 105 and from about amino acid 390 to about amino acid 394;
a tyrosine kinase
phosphorylation site from about aniino acid 377 to about amino acid 385; and
an N-myristoylation sites from
about amino acid 7 to about amino acid 13, from about amino acid 97 to about
amino acid 103, from about
amino acid 326 to about amino acid 332, and from about amino acid 363 to about
amino acid 369.
An analysis of the Dayhoff database (version 35.45 SwissProt 35), using a WU-
BLAST2 sequence
alignment analysis of the full-length sequence shown in Figure 6(SEQID NO:6)
revealed significant homology
between the PRO 1337 amino acid sequence and the Dayhoff sequence TI1BG_HUMAN.
Homology was also
found between the PR01337 amino acid sequence and the following Dayhoff
sequences: KAIN_HUIvIAN,
HSACTI_l, IPSP_HUMAN, G02081, HAMHPP_1, CPI6_RAT, S31507, AB000547_l, and K.BP
MOUSE.
Clone DNA66672-1586 was deposited with the ATCC on September 22, 1998, and is
assigned ATCC
deposit no. 203265.
EXAMPLE 6
In situ Hybridization
In situ hybridization is a powerful and versatile technique for the detection
and localization of nucleic acid
sequences within cell or tissue preparations. It may be useful, for example,
to identify sites of gene expression,
analyze the tissue distribution of transcription, identify and localize viral
infection, follow changes in spe'cif'ic
mRNA synthesis, and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the
protocol by Lu and Gillett, ell
Vision. i: 169-176 (1994), using PCR-generated s'P-labeled riboprobes.
Briefly, formalin-fixed, paraffin-
embedded human tissues were sectioned, deparaffinized, deproteinated
inproteinase K(20 glml) for 15 minutes
at 37 C, and further processed for in situ hybridization as described by Lu
and Gillett, supra. Ae3-P)UTP-
labeled antisense riboprobe was generated from a PCR product and hybridized at
55 C overnight. The slides
were dipped in Kodak NTB2TM nuclear track emulsion and exposed for 4 weeks.
33P-Riboprobe synthesis
6.0 l (125 mCi) of "P-U'I'P (AmershamBF 1002, SA<2000 Ci/mmol) were speed-
vacuum dried. To
each tube containing dried 33P-UTP, the following ingredients were added:
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2.0 g15x transcription buffer
1.0 i.cl DTT (100 mM)
2.0 gl NTP mix (2.5 mM: 10 gl each of 10 mM GTP, CTP & ATP + 10 l H20)
1.0 gl UTP (50 /,cM)
1.0 gl RNAsin
1.0 ,ul DNA template (1 /.cg)
1.0 l H20
1.0 1cl RNA polymerase (for PCR products T3 = AS, T7 = S, usually)
The tubes were incubated at 37 C for one hour. A total of 1.0 gl RQI DNase was
added, followed by
incubation at 37 C for 15 minutes. A total of 90 ,ul 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-50TM
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 ,ul TE was added, then l,ul
of the final product was pipetted on DE81 paper and counted in 6 ml of
BIOFLUOR IIT"15 The probe was run on a TBE/urea gel. A total of 1-3 gl of the
probe or 5g1 of RNA Mrk III was added
to 3,u1 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 (SARANTM brand) and exposed to XAR film with
an intensifying screen in a
-70 C freezer one hour to overnight.
33P-Hvbridization
A. Pretreati7tent offrozen sections
The slides were removed from the freezer, placed on aluminum trays, and thawed
at room temperature for
5 minutes. The trays were placed in a 55 C incubator for five minutes to
reduce condensation. The slides were
fixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, and
washed in 0.5 x SSC for 5 minutes,
at room temperature (25 m120 x SSC + 975 ml SQ H2O). After deproteination in
0.5 ,ug/ml proteinase K for
10 minutes at 37 C (12.5 /zl of 10 mg/mi stock in 250 ml prewarmed RNAse-free
RNAse buffer), the sections
were washed in 0.5 x SSC for 10 minutes at room temperature. The sections were
dehydrated in 70%, 95%,
and 100% ethanol, 2 minutes each.
B. Pretreatment of paraffira-embedded sections
The slides were deparaffinized, placed in SQ H2O, and rinsed twice in 2 x SSC
at room temperature, for
5 minutes each time. The sections were deproteinated in 20 pg/ml proteinase
K(500 ,ul of 10 mg/ml in 250 ml
RNase-free RNase buffer; 37 C, 15 minutes) for human embryo tissue, or 8 x
proteinase K(100 ,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. Prehybridization
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 1d of hybridization buffer (3.75 g
dextran sulfate + 6 ml SQ HZO),
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vortexed, and heated in the microwave for 2 minutes with the cap loosened.
After cooling on ice, 18.75 ml
formamide, 3.75 m120 x SSC, and 9 ml SQ H2O were added, and the tissue was
vortexed well and incubated
at 42 C for 1-4 hours.
D. Hybridization
1.0 x 106 cpm probe and 1.0 1d tRNA (50 mg/mi stock) per slide were heated at
95 C for 3 minutes. The
slides were cooled on ice, and 48 /A hybridization buffer was added per slide.
After vortexing, 50 /,c133P mix
was added to 50 ,ul prehybridization on the slide. The slides were incubated
overnight at 55 C.
E. Washes
Washing was done for 2x10 minutes with 2xSSC, EDTA at room temperature (400 ml
20 x SSC + 16 ml
0.25 M EDTA, VF4L), followed by RNAseA treatment at 37 C for 30 minutes (500
,ul of 10 mg/ml in 250 ml
Rnase buffer = 20,ug/ml), The slides were washed 2 x10 minutes with 2 x SSC,
EDTA at room temperature.
The stringency wash conditions were as follows: 2 hours at 55 C, 0.1 x SSC,
EDTA (20 ml 20 x SSC + 16 ml
EDTA, V,=4L).
F. Oligonucleotides
In situ analysis was performed on one of the DNA sequences disclosed herein.
The oligonucleotides
employed for these analyses are as follows:
(1) DNA52192-1369
D-269E FGFr p1:
5'-GGATTCTAATACGACTCACTATAGGGCCGCTGACCATGTGGACCAAGG-3' (SEQ ID NO: 15)
C-2571 FGFr p2:
5'-CTATGAAATTAACCCTCACTAAAGGGATCTGGCAGCACGGTGAGGAAG-3' (SEQ ID NO: 16)
G. Results
In situ analysis was performed on the above DNA sequence disclosed herein. The
results from this
analysis are as follows:
DNA52192-1369 (FGF receptor-like molecule)
Expression was observed in fetal skeletal muscle and long bone cartilage.
Elsewhere, a relatively high
background signal is a problem. In one fetal liver, expression appears to
occur at sites of hematopoiesis. The
other fetal liver was negative. Fetal tissues examined (E12-E16 weeks)
included: placenta, umbilical cord, liver,
kidney, adrenals, thyroid, lung, heart, great vessels, esophagus, stomach,
small intestine, spleen, thymus,
pancreas, brain, eye, spinal cord, body wall, pelvis and lower limb. Adult
human tissues examined included:
liver, kidney, adrenals, myocardium, aorta, spleen, lung, skin,
chondrosarcoma, eye, stomach, gastric carcinoma,
colon, colonic carcinoma, renal cell carcinoma, prostate, bladder mucosa and
gall bladder. Acetominophen
induced liver injury and hepatic cirrhosis. Rhesus tissues examined included
cerebral cortex (rm) and
hippocampus (rm). Chimp tissues examined included: thyroid, parathyroid,
ovary, nerve, tongue, thymus,
adrenals, gastric mucosa and salivary gland.
EXAMPLE 7
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Use of PRO as a Hybridization Probe
The following method describes use of a nucleotide sequence encoding PRO as a
hybridization probe.
DNA comprising the coding sequence of full-length or mature PRO as disclosed
herein or a fragment
thereof is employed as a probe to screen for homologous DNAs (such as those
encoding naturally-occurring
variants of PRO) in human tissue cDNA libraries or human tissue genomic
libraries.
Hybridization and washing of filters containing either library DNAs is
performed under the following high-
stringency conditions. Hybridization of radiolabeled probe derived from the
gene encoding a PRO polypeptide
to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS,
0.1% sodium pyrophosphate,
50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and 10% dextran
sulfate at 42 C for 20 hours.
Washing of the filters is performed in an aqueous solution of 0.1x SSC and
0.1% SDS at 42 C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence PRO can
then be identified using standard techniques known in the art.
EXAMPLE 8
Expression of PRO in E. coli
This example illustrates preparation of an unglycosylated form of PRO by
recombinant expression in E.
coli.
The DNA sequence encoding PRO is initially amplified using selected PCR
primers. The primers should
contain restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression
vector. A variety of expression vectors may be employed. An example of a
suitable vector is pBR322 (derived
from E. coli; see, Bolivar et al., Gene, 2:95 (1977)) which contains genes for
ampicillin and tetracycline
resistance. The vector is digested with restriction enzyme and
dephosphorylated. The PCR amplified sequences
are then ligated into the vector. The vector will preferably include sequences
which encode for an antibiotic
resistance gene, a trp promoter, a poly-His leader (including the first six
STII codons, poly-His sequence, and
enterokinase cleavage site), the PRO coding region, lambda transcriptional
terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. coli strain using
the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow
on LB plates and antibiotic
resistant colonies are then selected. Plasmid DNA can be isolated and
confirmed by restriction analysis and
DNA sequencing.
Selected clones can be grown overnight in liquid culture medium such as LB
broth supplemented with
antibiotics. The overnight culture may subsequently be used to inoculate a
larger scale culture. The cells are
then grown to a desired optical density, during which the expression promoter
is turned on.
After culturing the cells for several more hours, the cells can be harvested
by centrifugation. The cell
pellet obtained by the centrifugation can be solubilized using various agents
known in the art, and the solubilized
PRO protein can then be purified using a metal chelating column under
conditions that allow tight binding of
the protein.
PRO may be expressed in E. coli in a poly-His tagged form, using the following
procedure. The DNA
encoding PRO is initially amplified using selected PCR primers. The primers
will contain restriction enzyme
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sites which correspond to the restriction enzyme sites on the selected
expression vector, and other useful
sequences providing for efficient and reliable translation initiation, rapid
purification on a metal chelation
column, and proteolytic removal with enterokinase. The PCR-amplified, poly-His
tagged sequences are then
ligated into an expression vector, which is used to transform an E. coli host
based on strain 52 (W3110
fuhA(tonA) lon galE rpoHts(htpRts) c1pP(lacIq). Transformants are first grown
in LB containing 50 mg/ml
carbenicillin at 30 C with shaking until an OD6oo of 3-5 is reached. Cultures
are then diluted 50-100 fold into
CRAP media (prepared by mixing 3.57 g(NH4)ZSO4, 0.71 g sodium citrate=2H20,
1.07 g KCl, 5.36 g Difco
yeast extract, 5.36 g Sheffield hycase SF in 500 ml water, as well as 110 mM
MPOS, pH 7.3, 0.55% (w/v)
glucose and 7 mM MgSO4) 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. 1M and 0.02 M, respectively, and the solution is stirred
overnight at 4 C. This step results
in a denatured protein with all cysteine residues blocked by sulfitolization.
The solution is centrifuged at 40, 000
rpm in a Beckman Ultracentifuge for 30 min. The supernatant is diluted with 3-
5 volumes of metal chelate
column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22
micron filters to clarify. The
clarified extract is loaded onto a 5 n-A Qiagen Ni'+-NTA metal chelate column
equilibrated in the metal chelate
column buffer. The column is washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol
grade), pH 7.4. The protein is eluted with buffer containing 250 mM imidazole.
Fractions containing the
desired protein are pooled and stored at 4 C. Protein concentration is
estimated by its absorbance at 280 nm
using the calculated extinction coefficient based on its amino acid sequence.
The proteins are refolded by diluting the sample slowly into freshly prepared
refolding buffer consisting
of: 20 mM Tris, pH 8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine
and 1 mM EDTA. Refolding
volumes are chosen so that the final protein concentration is between 50 to
100 micrograms/ml. The refolding
solution is stirred gently at 4 C for 12-36 hours. The refolding reaction is
quenched by the addition of TFA
to a final concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution
is filtered through a 0.22 micron filter and acetonitrile is added to 2-10%
final concentration. The refolded
protein is chromatographed on a Poros R1/H reversed phase column using a
mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of fractions
with A280 absorbance are analyzed
on SDS polyacrylamide gels and fractions containing homogeneous refolded
protein are pooled. Generally, the
properly refolded species of most proteins are eluted at the lowest
concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors shielded from
interaction with the reversed phase
resin. Aggregated species are usually eluted at higher acetonitrile
concentrations. In addition to resolving
misfolded forms of proteins from the desired form, the reversed phase step
also removes endotoxin from the
samples.
Fractions containing the desired folded PRO polypeptide are pooled and the
acetonitrile removed using
a gentle stream of nitrogen directed at the solution. Proteins are formulated
into 20 m1VI Hepes, pH 6.8 with
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0.14 M sodium chloride and 4% mannitol by dialysis or by gel filtration using
G25 Superfine (Pharmacia) resins
equilibrated in the formulation buffer and sterile filtered.
Many of the PRO polypeptides disclosed herein were successfully expressed as
descibed above.
EXAMPLE 9
Expression of PRO in mannnalian cells
This example illustrates preparation of a potentially glycosylated form of PRO
by recombinant expression
in mammalian cells.
The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as
the expression vector.
Optionally, the PRO DNA is ligated into pRK5 with selected restriction enzymes
to allow insertion of the PRO
DNA using ligation methods such as described in Sambrook et al., supra. The
resulting vector is called pRK5-
PRO.
In one embodiment, the selected host cells may be 293 cells. Human 293 cells
(ATCC CCL 1573) are
grown to confluence in tissue culture plates in medium such as DMEM
supplemented with fetal calf serum and
optionally, nutrient components and/or antibiotics. About 10 ,ug pRK5-PRO DNA
is mixed with about 1,ug
DNA encoding the VA RNA gene [Thimmappaya et al., Cel1, 31:543 (1982)] and
dissolved in 500 /A of 1 mM
Tris-HC1, 0.1 mM EDTA, 0.227 M CaCl,. To this mixture is added, dropwise, 500
,ul of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO4, 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 /'cCi/m135S-cysteine and 200
kCi/m135S-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
PRO polypeptide. The cultures containing transfected cells may undergo further
incubation (in serum free
medium) and the medium is tested in selected bioassays.
In an alternative technique, PRO may be introduced into 293 cells transiently
using the dextran sulfate
method described liy Somparyrac etal., Proc. Natl. Acad. Sci.,12:7575 (1981).
293 cells are grown to maximal
density in a spinner flask and 700 ,ug pRK5-PRO DNA is added. The cells are
first concentrated from the
spinner flask by centrifugation and washed with PBS. The DNA-dextran
precipitate is incubated on the cell
pellet for four hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture
medium, and re-introduced into the spinner flask containing tissue culture
medium, 5/,tg/ml bovine insulin and
0.1 /.cg/ml bovine transferrin. After about four days, the conditioned media
is centrifuged and filtered to remove
cells and debris. The sample containing expressed PRO can then be concentrated
and purified by any selected
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method, such as dialysis and/or column chromatography.
In another embodiment, PRO can be expressed in CHO cells. The pRK5-PRO can be
transfected into
CHO cells using known reagents such as CaPO4 or DEAE-dextran. As described
above, the cell cultures can
be incubated, and the medium replaced with culture medium (alone) or medium
containing a radiolabel such
as 35S-methionine. After determining the presence of a PRO polypeptide, the
culture medium may be replaced
with serum free medium. Preferably, the cultures are incubated for about 6
days, and then the conditioned
medium is harvested. The medium containing the expressed PRO polypeptide can
then be concentrated and
purified by any selected method.
Epitope-tagged PRO may also be expressed in host CHO cells. The PRO may be
subcloned out of the
pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a
selected epitope tag such as a poly-
His tag into a Baculovirus expression vector. The poly-His tagged PRO insert
can then be subcloned into a
SV40 driven vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the
CHO cells can be transfected (as described above) with the S V40 driven
vector. Labeling may be performed,
as described above, to verify expression. The culture medium containing the
expressed poly-His tagged PRO
can then be concentrated and purified by any selected method, such as by NiZ+-
chelate affinity chromatography.
PRO may also be expressed in CHO and/or COS cells by a transient expression
procedure or in CHO cells
by another stable expression procedure.
Stable expression in CHO cells is performed using the following procedure. The
proteins are expressed
as an IgG construct (immunoadhesin), in which the coding sequences for the
soluble forms (e.g., extracellular
domains) of the respective proteins are fused to an IgG1 constant region
sequence containing the hinge, CH2
and CH2 domains and/or as a poly-His tagged form.
Following PCR amplification, the respective DNAs are subcloned in a CHO
expression vector using
standard techniques as described in Ausubel et al., Current Protocols of
Molecular Biology, Unit 3.16, John
Wiley and Sons (1997). CHO expression vectors are constructed to have
compatible restriction sites 5' and 3'
of the DNA of interest to allow the convenient shuttling of cDNA's. The vector
used in expression in CHO
cells is as described in Lucas et al., Nucl. Acids Res., 24:9 (1774-1779
(1996), and uses the SV40 early
promoter/enhancer to drive expression of the cDNA of interest and
dihydrofolate reductase (DHFR). DHFR
expression permits selection for stable maintenance of the plasmid following
transfection.
Twelve micrograms of the desired plasmid DNA is introduced into approximately
10 million CHO cells
using commercially available transfection reagents Superfect (Qiagen), Dosper
or Fugene (Boehringer
Mannheim). The cells are grown as described in Lucas et al., supra.
Approximately 3 x 10' cells are frozen
in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into a water
bath and mixed by
vortexing. The contents are pipetted into a centrifuge tube containing 10 ml
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 t.cm diafiltered fetal bovine serum). The cells
are then aliquoted into a 100 ml
spinner containing 90 ml of selective media. After 1-2 days, the cells are
transferred into a 250 ml spinner
filled with 150 mi selective growth medium and incubated at 37 C. After
another 2-3 days, 250 nil, 500 ml and
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2000 ml spinners are seeded with 3 x 105 cells/ml. The cell media is exchanged
with fresh media by
centrifugation and resuspension in production medium. Although any suitable
CHO media may be employed,
a production medium described in U.S. Patent No. 5,122,469, issued June 16,
1992 may actually be used. A
3L production spinner is seeded at 1.2 x 106 cells/ml. On day 0, the cell
number and pH is determined. On day
1, the spinner is sampled and sparging 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 365Medical Grade Emulsion) taken.
Tliroughout theproduction,
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
gm 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 condidoned media to a concentration of
5 mM. The conditioned niedia
is pumped onto a 6 ml Ni Z*-NTA column equilibrated in 20 mM Hepes, pH 7.4,
buffer containing 0.3 M NaC1
and 5 mM imidazole at a flow rate of 4-5 mUmin. at 4 C. After loading, the
column is washed with additional
equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M in-udazole. The highly
purified protein is subsequently desalted into a storage buffer containing 10
mM Hepes, 0.14 M NaCl and 4%
mannitol, pH 6.8, with a 25 inl G25 Superfine (Pharmacia) column and stored at
-80 C.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned
media as follows. The
conditioned medium is pumped onto a 5 ml Protein A column (Pharmacia) which
has been equilibrated in 20
nilvl Na phosphate buffer, pH 6.8. After loading, the column is washed
extensively with equilibration buffer
before el.ution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting I
ml fractions into tubes containing 275 gl of I M Tris buffer, pH 9. The highly
purified proteiu 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-tsrminal amino acid sequencing by Edman
degradation.
Many of the PRO polypeptides disclosed herein were successfully expressed as
descibed above.
EXAMPLE 10
Expression of PRO in Yeast
The following method describes recombinant expression of PRO in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PRO from the
ADH2/GAPDH promoter. DNA encoding PRO and the pronroter is inserted into
suitable restriction enzyme
sites in the selected plasmid to direct intracellular expression of PRO. For
secretion, DNA encoding PRO can
be cloned into the selected plasmid, together with DNA encoding the ADH2IGAPDH
promoter, a native PRO
signal peptide or other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory
signal/leader sequence, and linker sequences (if needed) for expression of
PRO.
Yeast cells, such as yeast strain AB 110, can then be transformed with the
expression plasmids described
above and cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by
precipitation with 10% trichloroacetic acid and separation by SDS-PAGE,
followed by staining of the gels with
*-trademark 81
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Coomassie Blue stain.
Recombinant PRO can subsequently be isolated and purified by removing the
yeast cells from the
fermentation medium by centrifugation and then concentrating the medium using
selected cartridge filters. The
concentrate containing PRO may further be purified using selected column
chromatography resins.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 11
Expression of PRO in Baculovirus-Infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected
insect cells.
The sequence coding for PRO is fused upstream of an epitope tag contained
within a baculovirus
expression vector. Such epitope tags include poly-His tags and immunoglobulin
tags (like Fc regions of IgG).
A variety of plasmids may be employed, including plasmids derived from
commercially available plasmids such
as pVL1393 (Novagen). Briefly, the sequence encoding PRO or the desired
portion of the coding sequence of
PRO (such as the sequence encoding the extracellular domain of a transmembrane
protein or the sequence
encoding the mature protein if the protein is extracellular) is amplified by
PCR with primers complementary to
the 5' and 3' regions. The 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 plasmitd and
BaculoGoldTM virus DNA
(Pharmingen) into Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially
available from GIBCO-BRL). After 4 - 5 days of incubation at 28 C, the
released viruses are harvested and
used for further 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 PRO can then be purified, for example, by Ni2+-
chelate affinity chromatography
as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al.,
Nature, 362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in
sonication buffer (25 ml Hepes, pH
7.9; 12.5 rnM MgClz, 0.1 mIvl EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCI), 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 NaCl, 10% glycerol, pH 7.8) and filtered
through a 0.45 ,um filter. A Ni2+-
NTA agarose column (commercially available from Qiagen) is prepared with a bed
volume of 5 ml, washed with
25 ml of water and equilibrated with 25 ml of loading buffer. The filtered
cell extract is loaded onto the column
at 0.5 ml per minute. The column is washed to baseline A280 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
NaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein. After
reaching A280 baseline again,
the column is developed with a 0 to 500 mM imidazole gradient in the secondary
wash buffer. One ml fractions
are collected and analyzed by SDS-PAGE and silver staining or Western blot
with Niz+-NTA-conjugated to
alkaline phosphatase (Qiagen). Fractions containing the eluted Hislo-tagged
PRO are pooled and dialyzed
against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PRO can be
performed using known
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chromatography techniques, including for instance, Protein A or protein G
column chromatography.
Many of the PRO polypeptides disclosed herein were successfully expressed as
described above.
EXAMPLE 12
Preparation of Antibodies that Bind PRO
This example illustrates preparation of monoclonal antibodies which can
specifically bind the PRO
polypeptide or an epitope on the PRO polypeptide without substantially binding
to any otlier polypeptide or
polypeptide epitope.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for instance,
in Goding, supra. Immunogens that may be employed include purified PRO, fusion
proteins containing PRO,
and cells expressing recombinant PRO on the cell surface. Selection of the
immunogen can be made by the
skilled artisan without undue experimentation.
Mice, such as Balb/c, are immunized with the PRO immunogen emulsified in
complete Freund's adjuvant
and injected subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the
immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research,
Hamilton, MT) and injected
into the animal's hind foot pads. The immunized mice are then boosted 10 to 12
days later with additional
immunogen emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted
with additional immunization injections. Serum samples may be periodically
obtained from the mice by retro-
orbital bleeding for testing in ELISA assays to detect anti-PRO antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected with
a final intravenous injection of PRO. Three to four days later, the mice are
sacrificed and the spleen cells are
harvested. The spleen cells are then fused (using 35% 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 PRO.
Detern-ftation of "positive"
hybridoma cells secreting the desired monoclonal antibodies against PRO is
within the skill in the art.
The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balb/c mice to produce
ascites containing the anti-PRO monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in
tissue culture flasks or roller bottles. Purification of the monoclonal
antibodies produced in the ascites can be
accomplished using ammonium sulfate precipitation, followed by gel exclusion
chromatography. Alternatively,
affinity chromatography based upon binding of antibody to protein A or protein
G can be employed.
EXAMPLE 13
Purification of PRO Polyeeptides Using Specific Antibodies
Native or recombinant PRO polypeptides may be purified by a variety of
standard techniques in the art of
protein purification. For example, pro-PRO polypeptide, mature PRO
polypeptide, or pre-PRO polypeptide is
purified by immunoaffinity chromatography using antibodies specific for the
PRO polypeptide of interest. In
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general, an immunoaffinity column is constructed by covalently coupling the
anti-PRO polypeptide antibody
to an activated chromatographic resin.
Polyclonal immunoglobulins are prepared fromimmune sera either by
precipitation with ammonium sulfate
or by purification on immobilized Protein A (Pharmacia LKB Biotechnology,
Piscataway, N.J.). Likewise,
monoclonal antibodies are prepared from mouse ascites fluid by ammonium
sulfate precipitation or
chromatography on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a
chromatographic resin such as CnBr-activated SEPHAROSETM (PharinaciaLKB
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 PRO
polypeptide by preparing a
fraction from cells containing the PRO polypeptide in a soluble form. This
preparation is derived by
solubilization of the whole cell or of a subcellular fraction obtained via
differential centrifugation by the
addition of detergent or by other methods well known in the art.
Alternatively, soluble PRO polypeptide
containing a signal sequence may be secreted in useful quantity into the
medium in which the cells are grown.
A soluble PRO polypeptide-containing preparation is passed over the
immunoaffinity column, and the
column is washed under conditions that allow the preferential absorbance of
the PRO polypeptide (e.g., high
ionic strength buffers in the presence of detergent). Then, the column is
eluted under conditions that disrupt
antibody/PRO polypeptide binding (e.g., a low pH buffer such as approximately
pH 2-3, or a high concentration
of a chaotrope such as urea or thiocyanate ion), and the PRO polypeptide is
collected.
EXAMPLE 14
Drug Screening
This invention is particularly useful for screening compounds by using PRO
polypeptides or a binding
fragment thereof in any of a variety of drug screening techniques. The PRO
polypeptide or fragment employed
in such a test may either be free in solution, affixed to a solid support,
borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic or
prokaryotic host cells which are stably
transformed with recombinant nucleic acids expressing the PRO polypeptide or
fragment. Drugs are screened
against such transformed cells in competitive binding assays. Such cells,
either in viable or fixed form, can be
used for standard binding assays. One may measure, for example, the formation
of complexes between a PRO
polypeptide or a fragment and the agent being tested. Alternatively, one can
examine the diminution in complex
formation between the PRO polypeptide and its target cell or target receptors
caused by the agent being tested.
Thus, the present invention provides methods of screening for drugs or any
other agents which can
affect a PRO polypeptide-associated disease or disorder. These methods
comprise contacting such an agent with
a PRO polypeptide or fragment thereof and assaying (i) for the presence of a
complex between the agent and
the PRO polypeptide or fragment, or (ii) for the presence of a complex between
the PRO polypeptide or
fragment and the cell, by methods well known in the art. In such competitive
binding assays, the PRO
polypeptide or fragment is typically labeled. After suitable incubation, the
free PRO polypeptide or fragment
is separated from that present in bound form, and the amount of free or
uncomplexed label is a measure of the
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ability of the particular agent to bind to the PRO polypeptide or to interfere
with the PRO polypeptide/cell
complex.
Another technique for drug screening provides high throughput screening for
compounds having
suitable binding affinity to a polypeptide and is described in detail in WO
84/03564, published on September
13, 1984. Briefly stated, large numbers of different small peptide test
compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied to a PRO
polypeptide, the peptide test
compounds are reacted with the PRO polypeptide and washed. Bound PRO
polypeptide is detected by methods
well known in the art. Purified PRO polypeptide can also be coated directly
onto plates for use in the
aforementioned drug screening techniques. In addition, non-neutralizing
antibodies can be used to capture the
peptide and immobilize it on the solid support.
This invention also contemplates the use of competitive drug screening assays
in which neutralizing
antibodies capable of binding a PRO polypeptide specifically compete with a
test compound for binding to the
PRO polypeptide or fragments thereof. In this manner, the antibodies can be
used to detect the presence of any
peptide which shares one or more antigenic determinants with a PRO
polypeptide.
EXAMPLE 15
Rational Druz Design
The goal of rational drug design is to produce structural analogs of a
biologically active polypeptide
of interest (i.e., a PRO polypeptide) or of small molecules with which they
interact, e.g., agonists, antagonists,
or inhibitors. Any of these examples can be used to fashion drugs which are
more active or stable forms of the
PRO polypeptide or which enhance or interfere with the function of the PRO
polypeptide in vivo (c. f., Hodgson,
Bio/Technologv, 9: 19-21 (1991)).
In one approach, the three-dimensional structure of the PRO polypeptide, or of
a PRO
polypeptide-inhibitor complex, is determined by x-ray crystallography, by
computer modeling or, most typically,
by a combination of the two approaches. Both the shape and charges of the PRO
polypeptide must be
ascertained to elucidate the structure and to determine active site(s) of the
molecule. Less often, useful
information regarding the structure of the PRO polypeptide may be gained by
modeling based on the structure
of homologous proteins. In both cases, relevant structural information is used
to design analogous PRO
polypeptide-like molecules or to identify efficient inhibitors. Useful
examples of rational drug design may
include molecules which have improved activity or stability as shown by
Braxton and Wells, Biochemistry,
31:7796-7801(1992) or which act as inhibitors, agonists, or antagonists of
native peptides as shown by Athauda
et al., J. Biochem., 113:742-746 (1993).
It is also possible to isolate a target-specific antibody, selected by
functional assay, as described above,
and then to solve its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent
drug design can be based. It is possible to bypass protein crystallography
altogether by generating anti-idiotypic
antibodies (anti-ids) to a functional, pharmacologically active antibody. As a
mirror image of a mirror image,
the binding site of the anti-ids would be expected to be an analog of the
original receptor. The anti-id could then
be used to identify and isolate peptides from banks of chemically or
biologically produced peptides. The
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isolated peptides would then act as the pharmacore.
By virtue of the present invention, sufficient amounts of the PRO polypeptide
may be made available
to perform such analytical studies as X-ray crystallography. In addition,
knowledge of the PRO polypeptide
amino acid sequence provided herein will provide guidance to those employing
computer modeling techniques
in place of or in addition to x-ray crystallography.
EXAMPLE 16
In Vitro Antitumor Assay
The antiproliferative activity of the PR0943, PR01250, and PR01337 polypeptide
was determined
in the investigational, disease-oriented in vitro anti-cancer drug discovery
assay of the National Cancer Institute
(NCI), using a sulforhodamine B (SRB) dye binding assay essentially as
described by Skehan et al., J. Natl.
Cancer Inst., 82:1107-1112 (1990). The 60 tumor cell lines employed in this
study ("the NCI panel"), as well
as conditions for their maintenance and culture in vitro have been described
by Monks et al., J. Natl. Cancer
Inst., 83:757-766 (1991). The purpose of this screen is to initially evaluate
the cytotoxic and/or cytostatic
activity of the test compounds against different types of tumors (Monks et al.
, supra=, Boyd, Cancer: Princ. Pract.
Oncol. Update, 3(10):1-12 [1989]).
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 gl 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 of 24
hours at 37 C for stabilization. Dilutions at twice the intended test
concentration were added at time zero in
100 ,ul 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% COZ atmosphere
and 100% humidity.
After incubation, the medium was removed and the cells were fixed in 0.1 ml of
10% trichloroacetic
acid at 40 C. The plates were rinsed five times with deionized water, dried,
stained for 30 minutes with 0.1 ml
of 0.4% sulforhodamine B dye (Sigma) dissolved in 1% acetic acid, rinsed four
times with 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. The absorbance (OD) of
sulforhodamine B at 492 nm was
measured using a computer-interfaced, 96-well microtiter plate reader.
A test sample is considered positive if it shows at least 40% growth
inhibitory effect at one or more
concentrations. The results are shown in the following Table 7, where the.
tumor cell type abbreviations are as
follows:
NSCL = non-small cell lung carcinoma; CNS = central nervous system
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Table 7
Test compound Days Tumor Cell Line Type Cell Line Designation
PR0943 N/A NSCL HOP-92, NCI-H522
PR0943 N/A Colon KM12
PR0943 N/A Breast HS578T
PR0943 N/A Ovarian OVCAR-3
PRO943 N/A Leukemia CCRF-CEM, HL-60(TB),
MOLT-4, RPMI-8226
PRO943 N/A Melanoma LOXIMVI
PR0943 N/A CNS SNB-19
PR01250 N/A NSCL NCI-H23, NCI-H522
PR01250 N/A CNS SF-268, SNB-19, U251
PR01337 N/A Leukemia RPMI-8226
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
DNA52192-1369 203042 July 1, 1998
DNA60775-1532 203173 September 1, 1998
DNA66672-1586 203265 September 22, 1998
These deposits were made under the provisions of the Budapest Treaty on the
International Recognition
of the Deposit of Microorganisms for the Purpose of PatentProcedure and the
Regulations thereunder (Budapest
Treaty). This assures maintenance of a viable culture of the deposit for 30
years from the date of deposit. The
deposits will be made available by ATCC under the terms of the Budapest
Treaty, and subject to an agreement
between Genentech, Inc., and ATCC, which assures permanent and unrestricted
availability of the progeny of
the culture of the deposit to the public upon issuance of the pertinent U.S.
patent or upon laying open to the
public of any U.S. or foreign patent application, whichever comes first, and
assures availability of the progeny
to one determined by the U.S. Commissioner of Patents and Trademarks to be
entitled thereto according to 35
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
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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.
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PCT P3134R1
Original (for SUBMISSION) - printed on 08.11.2000 02:18:25 PM
0-1 Form - PCT/RO/134 (EASY)
Indications Relating to Deposited
Microorganism(s) or Other Biological
Material (PCT Rule 13bis)
0-1-1 Prepared using PCT-EASY Version 2.91
(updated 10.10.2000)
0-2 International Application No.
0-3 Applicant's or agent's file reference P3134R1
I The indications made below relate to
the deposited microorganism(s) or
other biological material referred to in
the description on:
1-1 page 87
1-2 line 19
1-3 Identification of Deposit
1-3-1 Name of depositary institution p,merican Type Culture Collection
1-3-2 Address of depositary institution 10801 University Blvd., Manassas,
Virginia 20110-2209United States of
America
1-3-3 Date of deposit 01 July 1998 (01.07.1998)
1-3-4 Accession Number ATCC 203042
1-4 Additional Indications NONE
1-5 Designated States for Which all designated States
Indications are Made
1-6 Separate Furnishing of Indications NONE
These indications will be submitted to
the International Bureau later
2 The indications made below relate to
the deposited microorganism(s) or
other biological material referred to in
the description on:
2-1 page 87
2-2 line 20
2-3 Identification of Deposit
2-3-1 Name of depositary institution American Type Culture Collection
2-3-2 Address of depositary institution 10801 University Blvd., Manassas,
Virginia 20110-2209United States of
America
2-3-3 Dateofdeposit 01 September 1998 (01.09.1998)
2-3-4 Accession Number ATCC 203173
2-4 Additional Indications NONE
2-5 Designated States for Which all designated States
Indications are Made
2-6 Separate Furnishing of Indications NONE
These indications will be submitted to
the International Bureau later
3 The indications made below relate to
the deposited microorganism(s) or
other biological material referred to in
the description on:
3-1 page 87
3-2 line 21
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PCT P3134R1
Original (for SUBMISSION) - printed on 08.11.2000 02:18:25 PM
3-3 Identification of Deposit
3-3-1 Name of depositary institution American Type Culture Collection
3-3-2 Address of depositary institution 10801 University Blvd., Manassas,
Virginia 20110-2209United States of
America
3-3-3 Date of deposit 22 September 1998 (22.09.1998)
3-3-4 Accession Number ATCC 203265
3-4 Additional Indications NONE
3-5 Designated States for Which all designated States
Indications are Made
3-6 Separate Furnishing of Indications NONE
These indications will be submitted to
the International Bureau later
FOR RECEIVING OFFICE USE ONLY
0-4 This form was received with the ~
international application: yes r no)
0-4-1 uthorized officer /
FOR INTERNATIONAL BUREAU USE ONLY
0-5 This form was received by the
internationai Bureau on: 04 . Q l= Q
0-5-1 Authorized officer
CA 02390685 2002-10-22
Sequence Listing
<110> Genentec:h, inc.
Ashkenazi,Avi J.
Goddard, Audrey
Curney,Austin L.
Napier, Mary A.
Watanabe, Coiin K.
Wood,6Vil'iarn I.
<120> METHODS AND COMPOSITIONS FOR INHIY,ITINC NEOPLASTIC
CELL GROWTH
<130> P3134R1?C7'
<140> PCT/USOO/30592
<141> 2000-11-08
<150> PCT/US00/00376
<151> 2000-01-06
<150> ?CT/US00/04342
<151> 2000-02-18
<150> PCT/US00/05841
<151> 2000-03-02
<150> PCT/USOO/08439
<151> 2000-03-30
<150> PCT/USOO/20710
<151> 2000-07-28
<160> 16
<210> 1
<211> 3402
<212> DNA
<213> Homo Sapion
<400> 1
gccgccccgc cccqagaccg qgcccggggq cgc:ggggcgc cgggat;gcgg 50
cgcccggggc ggcgatgacc gcggagcgca cgc;cgagggc cc<lgccc.tga 100
ccccgccgcc cgcccgctga qccccccgcc gaggtacaga caqgccgaga 150
tgacgccgag ccccctgt:tg ctgctcctgc tgc:cg;:cgct_ gctgctgggg 200
gccttcccac cggccgccgc cgcccgaggc cccccaaago tggcggacaa 250
ggtggtccca cggcaggtgg cccggctggg ccgcaatgtq cgqctgcagt 300
gcccagtgga gggggacccg ccgccgctga ccatgtggac caaggatggc 350
cgcaccatcc acagcggctg gagccgcttc cgc.gtgctgc cgaaggggct 400
gaaggtgaag caggtggagc gggaggatgc cggcgtgr_ac gtgtgcaagg 450
ccaccaacgg cttcggcagc ctgac.;cgtca act:acaccc', c:gtcgtgctg 500
gatgacatta qcccagggaa ggagagcctg gggcc=c~gac<i c,Ictcc:c.tgcr 550
gggtcaagag gaccccgcca gccagcagcg ggc.acgaccq cgcttcacac 600
agccctccaa gatgaggcgc cgggtgatcg cacggcccg~ gggtagctcc 650
CA 02390685 2002-10-22
gtgcggctca agtgcgtggc cagcgggcac cctcggcccg acatcacgtg 700
gatgaaggac gaccaggcct tgacgcgccc agaggccgct gagcccagga 750
agaagaagtg gacactgagc cLqaagaacc tgcgc,_cgga ggacagcggc 800
aaatacacct gccgcgtgtc gaaccgcgcg ggcgccatca acqccaccta 850
caaggtggat gtgatccagc ggacccgttc caagcccgtq c.tcacaqgca 900
cgcaccccgt gaacacgacg gtggact,tcg gggggaccac gtccttccag 950
tgcaaggtgc gcagcgacgt gaagccggtg atc:.ca:ltggc tgaagcgcgt 1000
ggagtacggc gccgagggcc gccacaactc caccatcga; gtgggcggcc 1050
agaagtttgt ggtgctgccc acgggtgacg t.gtggtcgcg gcccgacggc 1100
tcctacctca ataagctgct catcacccgt gcccgccagg acgatgcggg 1150
catgtacatc tgccttggcg ccaacaccat gggctacagc ttccgcagcg 1200
ccttcctcac cgtgctgcca gacccaaaac cgccagggcc acctgtggcc 1250
tcctcgtcct cggccactag cctgccgtgg cccgtqgtc<, tcggcatccc 1300
agccggcgct gtcttcatcc tgggcaccct gctcc~qtgq ctttgccagg 1350
cccagaagaa gccgtgcacc cccgcc,tcctg cccct"ccct gcctgggcac 1400
cgcccgccgg ggacggcccg cgawqcagc ggagaaagg accttccctc 1450
gttggccgcc ctcagcgctg gccctggtgt ggggc~gtgt gacrgagcatg 1500
ggtctccggc agccccccag cacitactgg gcccaggccc agttgctggc 1550
cctaagttgt accccaaact ctacacagac atccacacac acacacacac 1600
acactctcac acacactcac acgtggaggg caagg,ccac c.agcacatcc 1650
actatcagtg ctagacggca ccgtatctgc agtggcjcacg ggggggccgg 1700
ccagacaggc agactgggag gatggaggac: ggagcigcag acgaaggcag 1750
gggacccatg gcgaggagga atgqccagca ccc.caggcag tctgtgtgtg 1800
aggcatagcc cctqgacaca cacocacaga cacacocact acctggatgc 1850
atgtatgcac acacatgcgc gcacacgtgc tccctqaagg cacacgtacg 1900
cacacgcaca tgcacagata tgccgcctgq gcacacagat aagctgccca 1950
aatgcacgca cacgcacaga gacatgccat7 aacat"caaa gacatgctgc 2000
ctgaacatac acacgcacac ccatgcgcaq atctgctgcc tggacacaca 2050
cacacacacg gatatgctgt ctggacgcac acacg:gcag atatggtatc ?100
cggacacaca cgtgcacaga tatqctgcct ggacacacag ataatgctgc 2150
cttgacacac acatgcacgg atattgcctg gacacacaca cacacacacg 2200
cgtgcacaga tatgctgtct ggacacgcac acacatccag atatgctgcc 2250
tggacacaca cttccagaca cacqigcaca ggcgc,cata Lgctgcctgg 2300
CA 02390685 2002-10-22
acacacgcag atatgctgtc tagtcacaca cacacclcaga catgctgtcc 2350
ggacacacac acgcatgcac agatat_gctg tccggacaca cacacgcacg 2400
cagatatgct gcctggacac acacacagat aat.gc~gcct caacactcac 2450
acacgtgcag atattgcctg gacacacaca tgt:gcacaga tatgctgtct 2500
ggacatgcac acacgtgcag atatgctgtc cggatAcaca cgcacgcaca 2550
cacgcagata tgctgcctgg gcacacactt ccgganacac atgcacacac 2600
aggtgcagat atgctgcctg gacacacaca cagataatgc tgcctcaaca 2650
ctcacacacg tgcagatatt qcctggacac acaca,gtgc acagatatgc 2700
tgtctggaca tgcacacacg tgcagatatg ctgtcaggat acacacgcac 2750
gcacacatgc agatatgctg cctgggcaca cacttacgga cacacatgca 2800
cacacaggtg cagatatgct gcctggacac acgcaqactg acgtgctttt 2850
gggagggtgt gccqtgaagc ctgcagtacg tgtgccgtga ggctcatagL 2900
tgatgaggga ctttccctgc tccaccgtca ctccc~caac tctgcccgcc 2950
tctgtccccg cctcagtccc cgcctccatc cccgcctct.y icccctggcc 3000
ttggcggcta tttttgccac ctgccttggg tgcccaggaq tcccctactg 3050
ctgtgggctg gggttggggg cacagcagcc ccaagactga gaqgctggag 3100
cccatggcta gtggctcatc cccagigcat tctcc:_ccty acacagagaa 3150
ggggccttgg tatttatatt taagaaatga agataatatt aataatgatg 3200
gaaggaagac tgggttgcag ggact<1-tggt; ctctcat_ggc, gcccgggacc 3250
cgcctggtct ttcagccatg ctgatgacca caccccgtcc aggccagaca 3300
ccacccccca ccccactgtc qt.ggtggccc cagatatctcr taattttatg 3350
~agagtttga gct,;7aagccc cgtatattta atttartttcr ttaaacacaa 3400
aa 3402
<210> 2
<211> 504
<212> PPT
<213> Homo Sapieri
<400> 2
Net "'hr Pro Ser Pro Leu Leu Leu Leu Lez Len Pro Pro Leli Leu
1 5 1,) 15
Leu Gly Ala Phe Pro Pro A1a Ala Ala Ala Arg_ Gly Pro Prc~ Lys
20 2) 30
N:'et Ala Asp Lys Val. Va'w Pro Arg Gln Val. Ala Arg Leu Gly Arg
35 4) 45
Thr Val Arg Lea Gin Cys Pro Val Glu GLy Asp Pro Pro Pro Leu
50 55 60
Thr Met Trp Thr Lys Asp GLy Arg 7'hr 11n HiL Ser Gly Trp Ser
6ti 7') 75
CA 02390685 2002-10-22
Arg Phe Arg Vai Leu Pro GLn Gly L,eu Lya Va1 Lys Gin Val Glu
80 8'~ 90
Arg G'lu Asp Ala Giy Vai. T,,,7r Val Cys Lya Ala Thr Asn Gly Phe
95 100 L05
(3ly Ser Leu Ser Val Asn Tyr Thr Leu Va Val Leu Asp Asp Ile
110 11') 120
Ser Pro Gly Lys Glu Ser Leu Gly Pro Asi) Ser Ser Ser Gly Gly
125 130 135
Gln Glu Asp Pro Ala Ser Gln Gln Trp AL,a Arg Pro Arg Phe Thr
140 14'; 150
Gln Pro Ser Lys Met Arg Arg Arg Val 11i-, Ala Arg Pro Va1 Gly
155 160 165
Ser Ser Val Arg Leu Lys Cys Val Ala Se. Gly His Pro Arcq Pro
170 17~ L80
Asp Ile Thr Tr~) Met Lys Asp Asp Gl.n Al, i Leu Thi Arg Pro Glu
185 19=) 195
Ala Ala Glu Pro Arg Lys Lys Lys "'rp Thr Leu Ser Leu Ly~. Asn
200 20'> 210
Leu Arg Pro G1u Asp Ser G1y Lys Tyr Th_~ Cys Arg Val Ser Asn
215 22;? 225
Arg Ala Gly Ala Ile Asri A1a Thr Tyr Ly:; Val Asp Val 11< Gin
230 23'a 240
Arg Thr Arg Ser Lys Pro Val Leu 'I'hr Gly Thr His Pro Val Asn
245 250 255
Thr 7'hrVal Asp Phe Gly GLy Thr Thr Ser Phe Gln Cys Lys Val
260 265 270
Arg Ser Asp VaL Lys Pro V,il Ile Gln Trl) Let: Lys Arg Val Glu
275 28i) 285
'Pyr Gly Ala Glu Gly Arg Y.is Asn Ser Th_-Ile Asp Val Gly Gly
290 29) 300
G1n Lys Phe Val Val Leu Pro Thr Gly AsI) Va1 Trp Ser Arcr Pro
305 311J 315
Asp (31y Ser Tyr Leu Asri Lys Leu Leu I1+, ThrArq_ Ala Arcf Gln
320 32) 330
Asp Asp Ala Gly Met Tyr I':.e Cys Leu Gly Ala Asn Thr MeTG1y
335 34~) 345
'I'yr Ser Phe Arg Ser Al a Phe Leu Thr Va L Let.: Pro Asp Pro Lys
350 35') 360
Pro Pro Gly Pro Pro Val A1a Ser Ser Ser Ser Ala Thr Ser Leu
365 37J 375
Pro Trp Pro Val Val Ile Gly Ile Pro Alt.i Gly Ala Val Phe Ile
380 38) 390
Leu Gly Thr Leu Leu Leu Trp Leu C:ys Gl~i Alr_ Gin Lys Lys Pro
395 40) 405
Cys '~'hr Pro Ala Pro Ala Pro Pro Leu Pr~) G7.y His Arg Pro Pro
CA 02390685 2002-10-22
410 415 420
Gly Thr Ala Arg Asp Arg Ser Gly Asp Lys Asp Leu Pro Ser Leu
425 430 435
Ala Ala Leu Ser Ala Gly Pro Gly Val Gly Leu Cys Glu GlA His
440 44-) 450
Gly Ser Pro Ala Ala Pr_ oClr His leu Leu Gly Pro Gly Pr~ Val
455 460 465
Ala Gly Pro Lys Leu Tyr Pro Lys Leu '1yr Thr Asp Ile Hi:_Thr
470 ; R) 480
His Thr His Thr His Ser His Thr His Ser His Val Glu Gly Lys
485 49J 495
Val His Gln His Ile His Tyr Gln Cys
500
<210> 3
<211> 3316
<212> DNA
<213> Hor,zo Sapien
<400> 3
ctgacatggc ctgactcggg acagctcaga gcagggcaga actggggaca 50
ctctgggccg gccttctgcc tgcatggacg ctctgaagcc accctqtctc 100
tggaggaacc acgagcgagg gaagaaggac agggaczcgt_ gtggcaggaa 150
qaactcagag ccgggaagcc cccatwact agaagcactg agagatgcgg 200
ccccctcgca gggtctgaat _tcctclctgc tgttc icaaa gatgcttttt 250
atctttaact ttttgttttc c.ccacttccg accccqgcgt tgatctgcat 300
cctgacattt ggagctgcca tcttcttgtg gctgatcacc agacctcaac 350
ccgtcttacc tcttcttgac c:tgaacaatc agtctgtggg aattgaggga 400
ggagcacgga agggggtttc cca:3aagaac aat:gacctaa ca<..gttgctg 450
cttctcagat gccaagacta tgtatgaggt tttccaaaga ggactcgctg 500
tgtct.gacaa tgggccctgc ttgggatata gaaaaccaaa ccagccctac 550
agatqgctat cttacaaaca ggtgtctgat agagc<xgagt acctgggttc 600
ctgtctcttg cataaaggtt ataaatcatc acc:agaccag tttgtcggca 650
tctttgctca gaataggcca gagtggatca tctccqaatt ggcttgttac 700
acgtactcta tggtagctgt accnctgtat gacaccttgg gaccagaagc 750
catcgtacat attqtcaaca aggctgatat cgc=catqgtg atctgtgaca 800
caccccaaaa ggcattgqtg c.tgataggga atgtanagaa aggctt.cacc 350
ccgagcctga aggtgatcat cctta7ggac cccttigatgatgacctgaa 900
gcaaagaggg gagaagagtg aaattgagat cttatcccta tatgatgczg 950
agaacctagg caaagagcac ttcagaaaac ctgtgcctcc tagcccagaa L000
gacctgagcg tcatctgctt caccagtggg accac<ic;gtg accccaaagg 1050
CA 02390685 2002-10-22
agccatgata acccatcaaa atattgtttc aaatgctgct gcctttctca 1100
aatgtgtgga gcatgcttat gacrcccactc ctgatgat.gt ggccatatcc 1150
tacctccctc tggctcatat gtttgagagg attgtacagg ctgttgtgta 1200
cagct:gtgga gccagagttg gattcttcca aggggatatt, cggt.tgctgg 1250
ctgacgacat gaagactttg aagcccacat tgtttccc.gc ggtgcct:cga 1300
ctccLtaaca ggatctacga taaggtacaa aatgaggcca agac.accctt 1350
gaagaagttc ttgttgaagc tggctgtttc cagtaaatt_c aaagagcttc 1400
aaaaqggtat catcaggcat gatagtttct gggacaagcn catctttgca 1450
aagatccagg acagcctggg cggaagggtt cgtgtaattg tcactggagc 1500
tgcccccatg Lccacttcag tcatgacatt cttccgggca gcaatgggat 1550
gtcaggtgta tgaagcttat ggtcaaacag aatgcacagg tggctgtaca 1600
tttacattac ctggggactg gacatcaggt cacgttgggg tgcccctggc 1650
ttgcaattac gtgaagctgg aagatgtggc tgacatgaac tactttacag 1700
tgaar.aatga aggagaggtc tgcatcaagq gtacaaacgt gtncaaagga 1750
tacctgaagg accctgagaa gacacaggaa gccctcxgaca gtclatggctg 1800
gcttcacaca ggagacattg gtcgctggct cccgaatgga actctgaaga 1850
tcatcgaccg taaaaagaac attttcaagc tggcccaagq agaatacatt 1900
gcaccagaga agatagaaaa tatctacaac aggagtcaaa cagtgttaca 1950
aatt~ttgta cacggggaga gcttacggtc atc.cttagta ggagtggtgg 2000
ttcctgacac agatgtactt ccctcatttg cagccaaqcv tggggtgaag 2050
ggctc.ctttg aggaactgtg ccaaaaccaa gtigtoaggg aagccatttt 2100
agaagacttg cagaaaattg ggaaagaaag tggccttaaa acctttgaac 2150
aggtcaaagc catttttctt catccagagc cattttccat tgaaaatggg 2200
ctcttgacac caacattgaa agcaaagcga ggagagcttt:: ccaaatactt 2250
tcggacccaa attgacagcc tgtatgagca catccaggat: taggataagg 2300
tacttaagta cctgccggcc cactgtgcac tgcttqigag aaaatggatt 2350
aaaaactatt cttacatttg ttttgccttt cctcctattt: ttt:tttaacc 2400
tgttaaactc taaagccata gcttttgttt.: tatatVgaga catataatgt 2450
gtaaacttag ttcccaaata aatcaatcct: gtctttccca tcttcgatgt 2500
tgctaatatt aaggcttcag ggctactttt atcaacatgr ctqtcttcaa 2550
gatcccagtt tatgtt:ctgt gtccttcctc atgat!_t:cca accttaatac 2600
tattagtaac cacaagttca agggtcaaag ggacc cnctq tgccttcttc 2650
tttgttttgt gataaacata acttgccaac ag;.ct~natg cttatttaca 2700
CA 02390685 2002-10-22
tcttctactg ttcaaactaa gaqat.tttta aatt,c:tgaaa aactgct.t:ac 2750
aattcatgtt ttctagccac tccacaaacc actaaaattt tagttttagc 2800
ctatc:actca tgtcaatcat atrtatgaga caaat:Jtctc cgatgctctt 2850
ctgcgtaaat taaat.tgtgt actgaaggga aaagt.tga!: cataccaaac 2900
atttcctaaa ctctctagtt agatatctga ctigggagta ttaaaaattg 2950
ggtctatgac atactgtcca aaaggaatgc tgtt:cttaaa gcattat.tta 3000
cagtaggaac tggggagtaa atctgttccc tac.agt.ttgc t.gc,tgagctg 3050
gaagctgtgg gggaaggagt tgacaggtgg gcccagtgaa ctr_ttccagt 3100
aaatgaagca agcactgaat aaaaacctcc tgaactggga acaaagatct 3150
acaggcaagc aagatgccca cac=aacaggc ttatt?..ctg tgaaggaacc 3200
aactgatctc ccccaccctt ggattagagt: tcctgcicta cct:tacccac 3250
agataacaca tgttgtttct acttgt.aaat: gtaaaqcctt taaaataaac 3300
tattacagat aaaaaa 3316
<210> 4
<211> 739
<212> PR'I'
<213> Homo Sapien
<400> 4
Met Asp Ala Leu Lys Pro Pro Cys Leu Trp Arg Asr. His Glu Arg
1 5 li~ 15
Gly Lys Lys Asp Arg Asp Ser Cys Gly Atcl Lys Asr: Ser G1o Pro
20 ;?'> 30
Gly Ser Pro His Ser Leu GLu Ala Leu Arq Asp Ala Ala Pro Ser
35 4() 45
Gln Gly Leu Asn Phe Leu Leu Leu Phe Thr Lys Met Leu Phe Ile
50 cii, 60
Phe Asn Phe Leu Phe Ser Pro Leu Pro Thr Pro Ala Leu 11e Cys
65 7l1 75
Ile Leu Thr Phe Gly Ala Ala Ile Phe Leu Trp Leu Tle Thr Arg
80 8~- 90
Pro Gln Pro Val Leu Pro Leu Leu Asp Let; Asn Asn c;,ln Ser Val
95 10(; 105
Gly Iie Glu Gly Gly Ala Arg Lys Gly Val Ser Gin Lys Asn Asn
110 11 ~: 120
Asp Leu Thr Ser Cys Cys Phe Ser Asp Ala Lys Thr Met Tyr Glu
125 1.30 135
Val Phe Gln Arg Gly Leu Ala Val Ser Asp Asn Gly Pro Cys Leu
140 1.Q, 150
Gly Tyr Arg Lys Pro Asn Gln Pro Tyr Azg Trp Leu Ser Tyr Lys
155 160 165
Gl.n Val Ser Asp Arg Ala G1u Tyr Leu Gl.y Ser Cys Leu Leu His
CA 02390685 2002-10-22
170 , 5 180
Lys Gly Tyr Lys Ser Ser Pro Asp Gin Phe Val Gly Ile Phe Ala
185 190 195
Gln Asn Arg Pro Glu Trp Ile Ile Ser Glu Leu Ala Cys Tyr Thr
200 205 210
Tyr Ser Met Val Ala Val Pro Lea Tyr Asp Th:r Leu Gly Pr" Glu
215 :?2 0 225
Ala Ile Val His Ile Val Asn Lys Ala Asp Ile Ala Met Val Ile
230 2K) 240
Cys Asp Thr Pro Gln Lys Ala Leu Val Leu Ile Gly Asn Val G1u
245 250 255
Lys Gly Phe Thr Pro Ser Leu Lys Val Z 1 e I le L,eu Met Asp Pro
260 26':i 270
Phe Asp Asp Asp Leu Lys Gln Arg G1y Glu Lys Ser Gly Il~:._~ Glu
275 280 285
Ile Leu Ser Leu Tyr Asp Ala Glu Asn Leu Glv Lys Glu His Phe
290 295 300
Arg Lys Pro Val Pro Pro Ser Pro Glu Asp Leu Ser Va1 Ile Cys
305 310 315
Phe Thr Ser Gly Thr Thr Gly Asp Pro Lys G1y Ala Met Ile Thr
320 325 330
His Gln Asn Ile Val Ser Asn Ala Ala Ala Phe Lea Lys Cys Val
335 340 345
Glu His Ala Tyr Glu Pro Thr Pro Asp Asp Val Ala Ile Ser Tyr
350 355 360
i.,eu Pro Leu Ala His Me7 Phe G1u Arg I le Va'_ Gln Ala Va L Val
365 370 375
Tyr Ser Cys Gly Ala Arg Val Gly Phe Phe Gin Gly Asp Ila Arg
380 385 390
Leu Leu Ala Asp Asp Met Lys Thr Leu Lys Pro Thr Leu Phe Pro
395 400 405
Ala Val Pro Arg Leu Leu Asn Arg Ae Tyr Asp Lys Val Gln Asn
410 417 420
G'lu Ala Lys Thr Pro Leu Lys Lys Phe L:e a Ler.:: Lys Leu Ala Val
425 43>> 435
Ser Ser Lys Phe Lys Glu Leu Gln Lys Gly Ile Ile Arg His Asp
440 445 450
Ser Phe Trp Asp Lys Leu Ile Phe Ala Lys Ile Gln Asp Sei Leu
455 46') 465
Gly Gly Arg Val Arg Val Ile Val Thr G1; Ala Ala Pro Met Ser
470 47) 480
Thr Ser Val Met Thr Phe Phe Arg Ala Ala Met Gly Cys Gln Val
485 49) 495
Tyr Glu Ala Tyr Gly Gin Ttir Glu Cys Thn Gly Gly Cys Thr Phe
500 5 05 510
CA 02390685 2002-10-22
Thr Leu Pro Gly Asp 'Prp Thr Ser Gly His Va1 Gly Vai Pro Leu
515 520 525
Ala r'ys Asn Tyr Val Lys Leu Glu Asp Val. Ala Asp Met As:t Tyr
530 ',35 540
Phe 'I'hr Val Asn Asn Glu Gly Glu Val Cys -_r 1t~ Lys Gly Th; Asn
545 550 555
Val Phe Lys Gl y Tyr Leu I:ys Asp Pro Crl u Lys 1'h r G 1n G1 ~,_ Ala
560 1) 65 570
Leu Asp Ser Asp Gly Trp Leu His Thr Gly Asp Ile Gly Ary Trp
575 580 585
Leu Pro Asn Gly Thr Leu Lys Ile Ile Asp Arc_) Lys Lys Asn Ile
590 59'i 600
Phe Lys Leu Ala Gln Gly G1u Tyr Ll.e Ala Pro Glu Lys Ile Glu
605 610 615
Asn Ile Tyr Asn Arg Ser (j'ln Pro Val Le.i G1ti Ile Phe Va] His
620 62'> 630
Gly Glu Ser Leu Arg Ser Ser Leu Val Gl,r Va- Vai Val Pro Asp
635 64.) 645
Thr Asp Val Leu Pro Ser Phe Ala Ala Lys Leu Gly Val Lys Gly
650 65) 660
Ser Phe Glu Gl.u Leu Cys Gln Asn Gln Va; Val Arg_ Glu Ala Ile
665 67J 675
Lea Glu Asp Leu Gln Lys 11e Gly Lys Glu Ser Gly Leu Lys 'Phr
680 68> 690
Phe Glu G1n Va1 Lys Ala Iie Phe Leu His Pro Glu Pro Ph(,- Ser
695 70i) 705
Ile Glu Asn Gly Leu Leu Thr Pro Thr Le..j Lys Ala Lys Ar(r Gly
710 71') 720
Glu Leu Ser Lys Tyr Phe Arg Thr Gln Ilr> Asp Sei Leu 'Tyr Glu
725 73,.1 735
His I:le Gln Asp
<210> 5
<211> 1743
<212> DNA
<213> Homo Sapien
<400> 5
caaccatgca aggacagggc aggagaagag gaacctgcaa agacatattt 50
tgttccaaaa tggcatctta cctttatgga gtactctttg ctgttggcct L00
ctgtgctcca atctactgtg tgtccccggc caatclccccc agtgcatacc L50
cccgcccttc ctccacaaag agcacccctg cctcacaggt gtattccctc 200
aacaccgact ttgccttccg cctataccgc aggctgattt tggagacccc :?50
gagtcagaac atcttcttct cccctctgag tgtcl-ccact tccctggcca 300
CA 02390685 2002-10-22
tgctctccct tggggcccac icagt.cacca agacc~agat. tctccaqggc 350
ctgggcttca acctcacaca cacaccagag tczgccatcc accagggctt 400
ccagcacctg gttcactcac tgactgttcc cagcaaagac ctgacct.tga 450
agatgggaag tgccctcttc gtcaagaagg agctgcagcL gcaggcaaat 500
ttctigggca atgtcaagag gctgtatgaa gc.agaagtcc tttctacaga 550
tttciccaac ccctccattg c.ccaggcgag ga*_caacagc catgtgaaaa 600
agaagaccca agggaaggtt gtagacataa tccaaggcct tgaccttctg 650
acggccatgg ttctggtgaa tcacattt.tc tttaaagcca agtgggagaa 700
gcccittcac cttgaatata caagaaagaa cttcccattc ctggtgggcg 750
agcaggtcac tgtgcaagtc cccatqatgc accagaaaga crcagttcgct 800
tttggggtgg atacagagct gaactgcttt gtgctt3caga tggattacaa 850
gggagatgcc gtggccttct ttgtcctccc tagcaagggc aagatgaggc 900
aactggaaca ggccttgtca gccagaacac tgataaagtg gagccactca 950
ctccagaaaa ggtggataga ggtgttcatc cccagatttt ccatttctgc 1000
ctcctacaat ctggaaacca tcctcccgaa gatgggc.atc c.aaaatgcct 1050
ttgacaaaaa tgctgatttt tctggaattg caaagagaga ctccctgcag 1100
gtttctaaag caacccacaa ggctgtgctg gargtcagt.g aagagggcac 1150
tgaggccaca gcagctacca ccaccaagtt catagtccga tcgaaggatg 1200
gtccc.tctta cttcactgtc tccttcaata ggaccttcct qatgatgatt 1250
acaaataaag ccacagacgg tattctcttt ctagggaaaq tggaaaatcc 1300
cactaaatcc taggtgggaa atggcctgtt aactga.tggc acattgctaa 1350
tgcacaagaa ataacaaacc acatccctct ttctgctctq agggtgcatt 1400
tgaccccagt ggagctggat tcctctggcag ggatgccact tccaaggctc 1450
aatcaccaaa ccatcaacag ggaccccagt cacaagccaa cacccattaa 1500
ccccagtcag tgcccttttc cacaaattct cccagqtaac tagcttcatg 1550
ggatgttgct gggttaccat atttccattc ct~qgjgctc ccaggaatgg 1600
aaatacgcca acccaggtta ggcacctcta ttgcalaatr_ acaataacac 1650
atccaataaa actaaaatat gaatt~aaaa aaaaaaaaaa aaaaaaaaaa 1700
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa<i 1743
<210> 6
<211> 417
<212> PRS."
<213> Homo Sapien
<400> 6
Met Ala Ser Tyr Leu Tyr Gly Vai Leu Phe Ala Va i Gly Le~ Cys
1 5 1 (') 15
CA 02390685 2002-10-22
Ala Pro Ile Tyr Cys VaL Ser Pro Ala Asn Ala Pro Ser Alta Tyr
20 25 30
Pro Arg Pro Ser Ser Thr Lys Ser Thr Pro Ala Ser Gln Va Tyr
35 40 45
Ser Leu Asn Thr Asp Phe Ala Phe Arg Led Tyr Arg Arg Le;~ Val
50 55 60
Leu Glu Thr Pro Ser Gln Asn Ile Phe Phe Ser Pro Val Ser Val
65 70 75
Ser Thr Ser Leu Ala Met Leu Ser Leu Gly Ala His Ser Va! Thr
80 85 90
Lys Thr Gin Ile Leu Gln G1y Leu Gly Phe Asn Leu Thr His Thr
95 100 105
Pro Glu Ser Ala Ile His Gln Gly Phe Gln His Leu Val His Ser
110 115 120
Leu Thr Val Pro Ser Lys Asp Leu Thr Leu Lys Met Gly Ser Ala
125 130 135
Leu ?he Val Lys Lys Glu Leu Gln heu Gln. Ala Asn Phe Leu Gly
140 245 150
Asn Val Lys Arg Leu Tyr Glu Ala Glu Va' Phe Ser Thr Asp Phe
155 N0 165
Ser Asn Pro Ser Ile Ala Gln Ala Arg Ile Asn Se.Y His Va_ Lys
170 175 180
Lys Lys Thr Gln Gly Lys Val Val Asp Ile Ile Gln Gly Lea Asp
185 190 195
Leu Leu Thr Ala Met Va1 Leu Val Asn His Ile Phe Phe Lys Ala
200 205 210
Lys Trp Glu Lys Pro Phe His Leu Glu Tyr Thr F,rg_ Lys Asn Phe
215 220 225
Pro Phe Leu Val Gly Glu Gln Va1 Thr VaL Gln Val Pro Met Met
230 235 240
His Gln Lys Glu Gln Phe Ala Phe Gly VaL Asp Thr Glu Len Asn
245 250 255
Cys Phe Val Leu Gln Met Asp Tyr Lys Gly Asp Ala Val Ala Phe
260 265 270
Phe Val Leu Pro Ser Lys Gly Lys Met Arg Gln Leu Glu Gln Ala
275 280 285
Leu Ser Ala Arg Thr Leu Ile Lys Trp Ser His Ser Leu GLn Lys
290 295 300
Arg Trp 11e Glu Va1 Phe Ile Pro Arg Phe Ser Ile Ser Ala Ser
305 311) 315
Tyr Asn Leu Glu Thr Ile Leu Pro Lys Met Gly 11e G1n Asn Ala
320 325 330
Phe Asp Lys Asn Ala Asp Phe Ser Gly 11e Ala Lys Arg As}.Ser
335 340 345
CA 02390685 2002-10-22
Leu Gin Val Ser Lys Ala Thr His Lys Ala Va!L Le..i Asp Val Ser
350 355 360
Glu Glu Gly Thr Glu Ala Thr Ala Ala Thr Thr Thr Lys Phu Ile
365 370 375
Val Arg Ser Lys Asp Gly Pro Ser Tyr Phe Thr Va1 Ser Phe Asn
380 385 390
Arg Thr Phe Leu Met Met ] le Thr Asn Lys Ala Thr Asp G1y Ile
395 400 405
Leu Phe Leu Gly Lys Val Glu Asn Pro Thr Lys Ser
410 415
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic 0ligonucleotide Probe
<400> 7
cqagatgacg ccgagccccc 20
<210> 8
<211> 27~
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonuc.leotide Probe
<400> 8
cggtvcgaca cgcggcaggt g 21
<210> 9
<211> 45
<212> DNA
<213> Artific:ial Sequence
<220>
<223> Syntheti.c Oligonucleotide Probe
<400> 9
tgctgctcct gctgccgccg ctgctgctgg gggccttccc gccgg 45
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide Probe
<400> 10
caaccatgca aggacagggc agg 23
<210> 11
<211> 47
<212> DNA
<213> Ar.tificial Sequence
<220>
CA 02390685 2002-10-22
<223> Synthetic Oligonacleotide Pr.ob=
<400> 11
ctttgctgtt ggcctctgtg ctcccaacca tgcaaggaca gggcagg 47
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide Probe.
<400> 12
tgac~cgggg t:ctccaaaac cagc 24
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide Probe
<400> 13
ggtataggcg gaaggcaaag tcc,tg 24
<210> 14
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide Probe
<400> 14
ggcatcttac ctttatggag tactctttgc t:gttggcctc t::gtgctcc ?3
<210> 15
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide Probe.
<400> 15
ggattctaat acgactcact atagggccgc tgaccatgtg gaccaagg 48
<210> 16
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic Oligonucleotide Probe
<400> 16
ctatgaaatt aaccctcact aaagggatc.t ggcag_acgcf t.gaggaag U
