Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR THE TREATMENT OF TUMOR
Field of the Invention
The present invention relates to compositions and methods for the diagnosis
and treatment of tumor.
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 an 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.
Alteration of gene expression is intimately related to the uncontrolled cell
growth and de-differentiation
which are a common feature of all cancers. The genomes of certain well studied
tumors have been found to show
decreased expression of recessive genes, usually referred to as tumor
suppression genes, which would normally
function to prevent malignant cell growth, and/or overexpression of certain
dominant genes, such as oncogenes,
that act to promote malignant growth. Each of these genetic changes appears to
be responsible for importing some
of the traits that, in aggregate, represent the full neoplastic phenotype
(Hunter, C~ 64:1129 [ 1991 ] and Bishop,
C~ 64:235-248 [ 1991 ]).
A well known mechanism of gene (e.g., oncogene) overexpression in cancer cells
is gene amplification.
This is a process where in the chromosome of the ancestral cell multiple
copies of a particular gene are produced.
The process involves unscheduled replication of the region of chromosome
comprising the gene, followed by
recombination ofthe replicated segments back into the chromosome (Alitalo et
al., Adv. Cancer Res., 47:235-281
[ 1986]). It is believed that the overexpression of the gene parallels gene
amplification, i.e., is proportionate to the
number of copies made.
Proto-oncogenes that encode growth factors and growth factor receptors have
been identified to play
important roles in the pathogenesis of various human malignancies, including
breast cancer. For example, it has
been found that the human ErbB2 gene (erbB2, also known as her2, or c-erbB-2),
which encodes a 185-kd
transmembrane glycoprotein receptor (p185~'u; HER2) related to the epidermal
growth factor receptor EGFR),
is overexpressed in about 25% to 30% of human breast cancer (Slamon et al., Sc
fence. 235:177-182 [ 1987]; Slamon
etal., Science, 244:707-712 [1989]).
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It has been reported that gene amplification of a proto-oncogene is an event
typically involved in the more
malignant forms of cancer, and could act as a predictor of clinical outcome
(Schwab e~ al., Genes Chromosomes
Cancer. 1_:181-193 [ 1990]; Alitalo et al., supra). Thus, erbB2 overexpression
is commonly regarded as a predictor
of a poor prognosis, especially in patients with primary disease that involves
axillary lymph nodes (Slamon et al.,
S [ 1987] and [ 1989], supra; Ravdin and Chamness, Gene, 159:19-27 [ 1995]:
and Hynes and Stern, Biochim. Bionhvs.
Acta, 1198:165-l84 [1994]), and has been linked to sensitivity and/or
resistance to hormone therapy and
chemotherapeutic regimens, including CMF (cyclophosphamide, methotrexate, and
fluoruracil) and anthracycl fines
(Baselga et al., Oncoloev, 1 l (3 Suppl t):43-48 [ 1997]). However, despite
the association of erbB2 overexpression
with poor prognosis, the odds of HER2-positive patients responding clinically
to treatment with taxanes were
greaterthan three times those of HER2-negative patients (lbia~. A
recombinanthumanized anti-ErbB2 (anti-HER2)
monoclonal antibody (a humanized version of the murine anti-~rbB2 antibody
4D5, referred to as rhuMAb HER2
or Herceptin"') has been clinically active in patients with ErbB2-
overexpressing metastatic breast cancers that had
received extensive prior anticancer therapy. (Baselga et al., J. Clin. Oncol..
14:737-744 [ 1996]).
In light of the above, there is obvious interest in identifying novel methods
and compositions which are
l5 useful for diagnosing and treating tumors which are associated with gene
amplification.
Summary of the Invention
The present invention concerns compositions and methods for the diagnosis and
treatment of neoplastic
cell growth and proliferation in mammals, including humans. The present
invention is based on the identification
of genes that are amplified in the genome of tumor cells. Such gene
amplification is expected to be associated with
the overexpression of the gene product and contribute to tumorigenesis.
Accordingly, the proteins encoded by the
amplified genes are believed to be useful targets for the diagnosis and/or
treatment (including prevention) ofcertain
cancers, and may act as predictors of the prognosis of tumor treatment.
In one embodiment, the present invention concerns an isolated antibody which
binds to a polypeptide
designated herein as a PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230,
PR0261, PR0246 or PR0317
polypeptide. In one aspect, the isolated antibody specifically binds to a
PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide. In another aspect, the
antibody induces the death
of a cell which expresses a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR0317 polypeptide. Often, the cell that expresses the PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230,
PR0261, PR0246 or PR0317 polypeptide is a tumor cell that overexpresses the
polypeptide as compared to a
normal cell of the same tissue type. In yet another aspect, the antibody is a
monoclonal antibody, which preferably
has non-human complementarity determining region (CDR) residues and human
framework region {FR) residues.
The antibody may be labeled and may be immobilized on a solid support. In yet
another aspect, the antibody is an
antibody fragment, a single-chain antibody, or a humanized antibody which
binds, preferably specifically, to a
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide.
In another embodiment, the invention concerns a composition of matter which
comprises an antibody
which binds, preferably specifically, to a PR0187, PR0533, PR0214, PR0240,
PR021 I, PR0230, PR0261,
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PR0246 or PR0317 polypeptide in admixture with a pharmaceutically acceptable
carrier. In one aspect, the
composition of matter comprises a therapeutically effective amount of the
antibody. In another aspect, the
composition comprises a further active ingredient, which may, for example, be
a further antibody or a cytotoxic
or chemotherapeutic agent. Preferably, the composition is sterile.
In a further embodiment, the invention concerns isolated nucleic acid
molecules which encode anti-
PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-
PR0261, anti-PR0246 or
anti-PR0317 antibodies, and vectors and recombinant host cells comprising such
nucleic acid molecules.
In a still further embodiment, the invention concerns a method for producing
an anti-PR0187, anti-
PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-PR0261, anti-
PR0246 or anti-PR0317
antibody, wherein the method comprises culturing a host cell transformed with
a nucleic acid molecule which
encodes the antibody under conditions sufficient to allow expression of the
antibody, and recovering the antibody
from the cell culture.
The invention further concerns antagonists ofa PRO 187, PR0533, PR0214,
PR0240, PR0211, PR0230,
PR0261, PR0246 or PR0317 polypeptide that inhibit one or more of the
biological and/or immunological
functions or activities of a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR0317 polypeptide.
In a further embodiment, the invention concerns an isolated nucleic acid
molecule that hybridizes to a
nucleic acid molecule encoding a PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246
or PR0317 polypeptide or the complement thereof. The isolated nucleic acid
molecule is preferably DNA, and
hybridization preferably occurs under stringent hybridization and wash
conditions. Such nucleic acid molecules
can act as antisense molecules of the amplified genes identified herein,
which, in turn, can find use in the
modulation of the transcription and/or translation of the respective amplified
genes, or as antisense primers in
amplification reactions. Furthermore, such sequences can be used as part of a
ribozyme and/or a triple helix
sequence which, in turn, may be used in regulation of the amplified genes.
In another embodiment, the invention provides a method for determining the
presence of a PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide
in a sample suspected
of containing a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317
polypeptide, wherein the method comprises exposing the sample to an anti-
PR0187, anti-PR0533, anti-PR0214,
anti-PR0240, anti-PR021 I, anti-PR0230, anti-PR0261, anti-PR0246 or anti-
PR0317 antibody and determining
binding of the antibody to a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR0317 polypeptide in the sample. In another embodiment, the invention
provides a method for determining the
presenceofa PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or
PR0317 polypeptide
in a cell, wherein the method comprises exposing the cell to an anti-PR0187,
anti-PR0533, anti-PR0214, anti-
PR0240, anti-PR0211, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317
antibody and detetlrtining
binding of the antibody to the cell.
In yet another embodiment, the present invention concerns a method of
diagnosing tumor in a mammal,
comprising detecting the level of expression of a gene encoding a PR0187,
PR0533, PR0214, PR0240, PR0211,
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PR0230, PR0261, PR0246 or PR0317 polypeptide (a) in a test sample of tissue
cells obtained from the mammal,
and (b) in a control sample of known normal tissue cells of the same cell
type, wherein a higher expression level
in the test sample as compared to the control sample, is indicative of the
presence of tumor in the mammal from
which the test tissue cells were obtained.
In another embodiment, the present invention concerns a method of diagnosing
tumor in a mammal,
comprising(a) contactingan anti-PR0187,anti-PR0533, anti-PR0214, anti-PR0240,
anti-PR0211, anti- PR0230,
anti-PR0261, anti-PR0246 or anti-PR0317 antibody with a test sample oftissue
cells obtained from the mammal,
and (b) detecting the formation of a complex between the anti-PROI 87, anti-
PR0533, anti-PR0214, anti-PR0240,
anti-PR0211, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody and
a PR0187, PR0533,
l0 PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide in the
test sample, wherein the
formation of a complex is indicative of the presence of a tumor in said
mammal. The detection may be qualitative
or quantitative, and may be performed in comparison with monitoring the
complex formation in a control sample
of known normal tissue cells of the same cell type. A larger quantity of
complexes formed in the test sample
indicates the presence of tumor in the mammal from which the test tissue cells
were obtained. The antibody
1 S preferably carries a detectable label. Complex formation can be monitored,
for example, by light microscopy, flow
cytometry, fluorimetry, or other techniques known in the art.
The test sample is usually obtained from an individual suspected to have
neoplastic cell growth or
proliferation (e.g. cancerous cells).
In another embodiment, the present invention concernsa cancerdiagnostickit
comprising an anti-PRO 187,
20 anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-
PR0261, anti-PR0246 or anti
PR0317 antibody and a carrier (e.g., a buffer) in suitable packaging. The kit
preferably contains instructions for
using the antibody to detect the presence of a PR0187, PR0533, PR0214, PR0240,
PR02 I 1, PR0230, PR0261,
PR0246 or PR0317 polypeptide in a sample suspected of containing the same.
In yet another embodiment, the invention concerns a method for inhibiting the
growth of tumor cells
25 comprising exposing tumor cells which express a PR0187, PR0533, PR0214,
PR0240, PRO211, PR0230,
PR0261, PR0246 or PR0317 polypeptide to an effective amount of an agent which
inhibits a biological and/or
immunological activity and/or the expression of a PR0187, PR0533, PR0214,
PR0240, PR021 l, PR0230,
PR0261, PR0246 or PR0317 polypeptide, wherein growth of the tumor cells is
thereby inhibited. The agent
preferably is an anti-PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-
PR0211, anti-PR0230, anti
30 PR0261,anti-PR0246or anti-PR0317 antibody, a small organic and inorganic
molecule, peptide, phosphopeptide,
antisense or riboryme molecule, or a triple helix molecule. In a specific
aspect, the agent, e.g., the anti-PR0187,
anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-PR0261,
anti-PR0246 or anti-
PR0317 antibody, induces cell death. In a further aspect, the tumor cells are
further exposed to radiation treatment
and/or a cytotoxic or chemotherapeutic agent.
35 In a further embodiment, the invention concerns an article of manufacture,
comprising:
a container;
a label on the container; and
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a composition comprising an active agent contained within the container;
wherein the composition is
effective for inhibiting the growth of tumor cells and the label on the
container indicates that the composition can
be used for treating conditions characterized by overexpression of a PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide as compared to a normal
cell ofthe same tissue type.
In particular aspects, the active agent in the composition is an agent which
inhibits an activiiy and/or the expression
of a PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317
polypeptide. In
preferred aspects, the active agent is an anti-PR0187, anti-PR0533, anti-
PR0214, anti-PR0240, anti-PR0211,
anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 antibody or an antisense
oligonucleotide.
The invention also provides a method for identifying a compound that inhibits
an activity of a PRO 187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide,comprisingcontacting
a candidate compound with a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR031? polypeptide under conditions and for a time sufficient to allow these
two components to interact and
determining whether a biological and/or immunological activity of the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide is inhibited. In a
specific aspect, eitherthe candidate
compound or the PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261,
PR0246 or PR0317
polypeptide is immobilized on a solid support. In another aspect, the non-
immobilized component carries a
detectable label. In a preferred aspect, this method comprises the steps of
(a) contacting cells and a candidate
compound to be screened in the presence of PR0187, PR0533, PR0214, PR0240,
PR021 l, PR0230, PR0261,
PR0246 or PR0317 polypeptide under conditions suitable for the induction of a
cellularresponse normally induced
by a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide and (b)
determining the induction of said cellular response to deterrrrine if the test
compound is an effective antagonist.
In another embodiment, the invention provides a method for identifying a
compound that inhibits the
expression of a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317
polypeptide in cells that express the polypeptide, wherein the method
comprises contacting the cells with a
candidate compound and determining whether the expression of the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide is inhibited. In a
preferred aspect, this method
comprises the steps of (a) contacting cells and a candidate compound to be
screened under conditions suitable for
allowing expression of the PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR0317 polypeptide and (b) determining the inhibition of expression of said
polypeptide.
Brief Description of the Fisures
Figure 1 shows the nucleotide sequence (SEQ ID NO:1 ) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0187, wherein the nucleotide sequence (SEQ ID NO:1
) is a clone designated herein
as DNA27864-11 S5. Also presented in bold font and underlined are the
positions of the respective start and stop
codons.
Figure 2 shows the amino acid sequence (SEQ ID N0:2) of a native sequence
PR0187 polypeptide as
derived from the coding sequence of SEQ ID NO:1.
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Figure 3 shows the nucleotide sequence (SEQ 1D N0:6) of a cDNA containing a
nucleotide sequence
encoding native sequence PROS33, wherein the nucleotide sequence {SEQ ID N0:6)
is a clone designated herein
as DNA49435-1219. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 4 shows the amino acid sequence (SEQ ID N0:7) of a native sequence
PROS33 polypeptide as
derived from the coding sequence of SEQ ID N0:6.
Figure S shows the nucleotide sequence (SEQ ID NO:11 ) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0214, wherein the nucleotide sequence (SEQ ID NO:11
) is a clone designated herein
as DNA32286-1191. Also presented in bold font and underlined are the positions
of the respective start and stop
l0 codons.
Figure 6 shows the amino acid sequence (SEQ ID N0:12) of a native sequence
PR0214 polypeptide as
derived from the coding sequence of SEQ ID NO:11.
Figure 7 shows the nucleotide sequence (SEQ ID N0:16) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0240, wherein the nucleotide sequence (SEQ ID
N0:16) is a clone designated herein
1S as DNA34387-1138. Also presented in bold font and underlined are the
positions ofthe respective start and stop
codons.
Figure 8 shows the amino acid sequence (SEQ ID N0:17) of a native sequence
PR0240 poiypeptide as
derived from the coding sequence of SEQ ID N0:16.
Figure 9 shows the nucleotide sequence (SEQ 1D N0:21) of a cDNA containing a
nucleotide sequence
20 encoding native sequence PR0211, wherein the nucleotide sequence (SEQ ID
N0:21 ) is a clone designated herein
as DNA32292-1131. Also presented in bold font and underlined are the positions
ofthe respective start and stop
codons.
Figure 10 shows the amino acid sequence (SEQ ID N0:22) of a native sequence
PR021 l polypeptide as
derived from the coding sequence of SEQ 1D N0:21.
2S Figure 11 shows the nucleotide sequence (SEQ ID N0:26) of a cDNA containing
a nucleotide sequence
encoding native sequence PR0230, wherein the nucleotide sequence (SEQ ID
N0:26) is a clone designated herein
as DNA33223-l 136. Also presented in bold font and underlined are the
positions of the respective start and stop
codons.
Figure l2 shows the amino acid sequence (SEQ ID N0:27) of a native sequence
PR0230 polypeptide as
30 derived from the coding sequence of SEQ ID N0:26.
Figure 13 shows the nucleotide sequence (SEQ ID N0:31 ) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0261, wherein the nucleotide sequence (SEQ ID N0:31
) is a clone designated herein
as DNA33473-1176. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
35 Figure 14 shows the amino acid sequence (SEQ ID N0:32) of a native sequence
PR0261 polypeptide as
derived from the coding sequence of SEQ ID N0:31.
Figure 1 S shows the nucleotide sequence (SEQ ID N0:36) of a cDNA containing a
nucleotide sequence
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encoding native sequence PR0246, wherein the nucleotide sequence (SEQ ID
N0:36) is a clone designated herein
as DNA35639-1172. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure 16 shows the amino acid sequence (SEQ ID N0:37) of a native sequence
PR0246 polypeptide as
~ derived from the coding sequence of SEQ ID N0:36.
Figure 17 shows the nucleotide sequence (SEQ ID N0:41 ) of a cDNA containing a
nucleotide sequence
encoding native sequence PR0317, wherein the nucleotide sequence (SEQ ID N0:41
) is a clone designated herein
as DNA33461-1199. Also presented in bold font and underlined are the positions
of the respective start and stop
codons.
Figure I8 shows the amino acid sequence (SEQ ID N0:42) of a native sequence
PR0317 polypeptide as
derived from the coding sequence of SEQ 1D N0:41.
Figures 19A through 19D show hypothetical exemplifications for using the below
described method to
determine % amino acid sequence identity (Figures 19A-B) and % nucleic acid
sequence identity (Figures 19C-
D) using the ALIGN-2 sequence comparison computer program, wherein "PRO"
represents the amino acid
IS sequence of a hypothetical PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR0317 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
PROI 87-, PR0533-, PR0214-, PR0240-, PR0211-, PR0230-, PR0261-, PR0246- or
PR0317-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.
Figures 20A through 20Q provide 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.
Figure 21 is a map of Chromosome 8 showing the mapping region of DNA27864-1
I55.
Figure 22 is a map of Chromosome 2 showing the mapping region of DNA34387-
1138.
Figure 23 is a map of Chromosome 1 showing the mapping region of DNA33223-
1136.
Detailed Description of the Invention
Definitions
The phrases "gene amplification" and "gene duplication" are used
interchangeably and refer to a process
by which multiple copies of a gene or gene fragment are formed in a particular
cell or cell line. The duplicated
region (a stretch of amplified DNA) is often referred to as "amplicon."
Usually, the amount of the messenger RNA
(mRNA) produced, i. e., the level of gene expression, also increases in the
proportion of the number of copies made
of the particular gene expressed.
"Tumor", as used herein, refers to all neoplastic cell growth and
proliferation, whethermalignant or benign,
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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,
gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer,
ovarian cancer, liver cancer, bladder
cancer, hepatoma, colorectal cancer, endometrial carcinoma, salivary gland
carcinoma, kidney cancer, liver 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 welt as those in which
the disorder is to be prevented. In tumor (e.g., cancer) treatment, a
therapeutic agent may directly decrease the
pathology of tumor cells, or render the tumor cells more susceptible to
treatment by other therapeutic agents, e.g.,
radiation and/or chemotherapy.
The "pathology" of cancer includes all phenomena that compromise the well-
being of the patient. This
includes, without limitation, abnormal or uncontrollable cell growth,
metastasis, interference with the normal
functioning of neighboring cells, release of cytokines or other secretory
products at abnormal levels, suppression
or aggravation of inflammatory or immunological response, etc.
"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,
horses, cats, cattle, pigs, sheep, etc.
Preferably, the mammal is human.
"Carriers" as used herein include pharmaceutically acceptable carriers,
excipients, or stabilizers which are
nontoxic to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the
physiologically acceptable carrier is an aqueous pH buffered solution.
Examples of physiologically acceptable
carriers include buffers such as phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid;
low molecular weight (less than about 10 residues) polypeptide; proteins, such
as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine,
asparagine, arginine or lysine; monosaccharides, 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 TWEENrM,
polyethylene glycol (PEG), and
PLURONICST'"
Administration "in combination with" one or more further therapeutic agents
includes simultaneous
(concurrent) and consecutive administration in any order.
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., l"', I'Z5, Y'° and
Re's), chemotherapeutic agents, and toxins such as enzymatically active toxins
of bacterial, fungal, plant or animal
origin, or fragments thereof.
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A "chemotherapeutic agent" is a chemical compound useful in the treatment of
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-PoulencRorer, 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. Pat. No. 4,675,187), 5-FU, 6-thioguanine, 6-
mercaptopurine, actinomycin D, VP-16,
chlorambucil, melphalan, and other related nitrogen mustards. Also included in
this definition are hormonal agents
that act to regulate or inhibit hormone action on tumors such as tamoxifen and
onapristone.
A "growth inhibitory agent" when used herein refers to a compound or
composition which inhibits growth
of a cell, especially cancer cell overexpressing any of the genes identified
herein, either in vitro or in vivo. Thus,
the growth inhibitory agent is one which significantly reduces the percentage
of cells overexpressing such genes
in S phase. Examples of growth inhibitory agents include agents that block
cell cycle progression (at a place other
than S phase), such as agents that induce G 1 an-est and M-phase arrest.
Classical M-phase blockers include the
IS vincas (vincristine and vinblastine), taxol, and topo II inhibitors such as
doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G 1 also spill over into S-
phase anrest, for example, DNA
alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-
fluorouracil, and ara-C. Further information can be found in The Molecular
Basis ojCancer, Mendelsohn and
Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogens, and
antineoplastic drugs" by Murakami er al., ( WB
Saunders: Philadelphia, 1995), especially p. 13.
"Doxorubicin" is an anthracycline antibiotic. The full chemical name of
doxorubicin is (8S-cisrl0-[(3-
amino-2,3,6-trideoxy-a-L-lyxo-hexapyranosyl)oxy]-7,8,9, IO-tetrahydro-6,8,1 I-
trihydroxy-8-(hydroxyacetyl~l-
methoxy-5,12-naphthacenedione.
The tenor "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; prorefaxin; 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; mouse gonadotropin-
associated peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-Vii; platelet-growth factor; transforming growth
factors (TGFs) such as TGF-a and
TGF-Vii; insulin-like growth factor-I and -II; erythropoietin (EPO);
osteoinductive factors; interferons such as
interferon -a, -Vii, and -y; colony stimulating factors (CSFs) such as
macrophage-CSF (M-CSF); granulocyte-
macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such
as IL-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
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CA 02341304 2001-03-02
WO 00115666 PCT/US99/20594
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 enrymatically activated or converted into the more active parent
form. See, e.g., Wilman, "Prodrugs in
Cancer Chemotherapy", Biochemical Society Transactions, 14:375-382, 615th
Meeting, Belfast ( 1986), and Stella
et al., "Prodtvgs: A Chemical Approach to Targeted Drug Delivery", Directed
Drue Delivery. Borchardt et al.,
(ed.), pp. 147-267, Humans Press ( 1985). The prodrugs of this invention
include, but are not limited to, phosphate-
containingprodrugs,thiophosphate-containing prodrugs, sulfate-containing
prodrugs,peptide-containin~rodrugs,
D-amino acid-modified prodrugs, glysocylated prodrugs, 13-lactam-containing
prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-
containing prodrugs, 5-
fluorocytosine and other 5-fluorouridine prodrugs which can be converted into
the more active cytotoxic free drug.
Examples of cytotoxic drugs that can be derivatized into a prodrugs form for
use in this invention include, but are
not limited to, those chemotherapeutic agents described above.
I 5 An "effective amount" of a polypeptide disclosed herein or an antagonist
thereof, in reference to inhibition
of neoplastic cell growth, tumor growth or cancer 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 PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide antagonist for
purposes of inhibiting
20 neoplastic cell growth, tumor growth or cancer cell growth, may be
determined empirically and in a routine manner.
A "therapeutically effective amount", in reference to the treatment of tumor,
refers to an amount capable
of invoking one or more of the following effects: ( 1 ) inhibition, to some
extent, oftumor 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)
25 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 PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide
antagonist for purposes of treatment of tumor may be determined empirically
and in a routine manner.
30 A "growth inhibitory amount" of a PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261,
PR0246 or PR0317 antagonist 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
PROI 87, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 antagonist for purposes of inhibiting
neoplastic cell growth may
be determined empirically and in a routine manner.
35 A "cytotoxic amount" ofa PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230,
PR0261, PR0246
or PR0317 antagonist 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 PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230,
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WO 00/15666 PCT/US99/20594
PR0261, PR0246 or PR0317 antagonist for purposes of inhibiting neoplastic cell
growth may be determined
empirically and in a routine manner.
The terms "PRO 187", "PR0533 ", "PR02 I 4", "PR0240", "PR0211 ", "PR0230",
"PR0261 ", "PR0246"
or "PR0317", "PR0187 polypeptide", "PR0533 polypeptide", "PR0214 polypeptide".
"PR0240 polypeptide",
"PR0211 polypeptide", "PR0230 polypeptide", "PR0261 polypeptide", "PR0246
polypeptide" or "PR0317
polypeptide" when used herein encompass native sequence PR0187, PR0533,
PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 poiypeptides and PR0187, PR0533, PR0214,
PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide variants (which are further
defined herein). The PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide
may be isolated from
a variety of sources, such as from human tissue types or from another source,
or prepared by recombinant and/or
synthetic methods.
A "native sequence" PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or
PR0317 polypeptide comprises a polypeptide having the same amino acid sequence
as a PR0187, PR0533,
PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide derived
from nature. Such
native sequence PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317
polypeptide can be isolated from nature or can be produced by recombinant
and/or synthetic means. The term
"native sequence" PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261,
PR0246 or PR0317
polypeptide specifically encompasses naturally-occurringtruncated or secreted
forms (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively spliced
forms) and naturally-occurring allelic
variants of the PR0187, PR0533, PR0214, PR0240, PR02I1, PR0230, PR0261, PR0246
and PR0317
polypeptide. In certain embodiments of the invention, the native sequence PROI
87, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide is a mature or full-
length native sequence PR0187,
PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317 polypeptide
comprising the amino
acid sequence of Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7), Figure 6 (SEQ
ID N0:12), Figure 8 (SEQ
ID N0:17), Figure 10 (SEQ ID N0:22), Figure 12 (SEQ ID N0:27), Figure 14 (SEQ
ID N0:32), Figure 16 (SEQ
ID N0:37), or Figure 18 {SEQ ID N0:42), respectively. Fragments of the
respective native polypeptides herein
include, but are not lim ited, to polypeptide variants from which the native N-
terminal signal sequence has been fully
or partially deleted or replaced by another sequence, and extracellular
domains of the respective native sequences,
regardless whether such truncated (secreted) foams occur in nature. Fragments
are preferably sufficient in length
for the production of an antibody specifically binding the corresponding
native "PRO" polypeptide.
"PR0187 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 23 to 205 of the PR0187
polypeptide shown in Figure 2 (SEQ ID N0:2), (b) X to 205 of the PR0187
polypeptide shown in Figure 2 (SEQ
1D N0:2), wherein X is any amino acid residue from 18 to 27 of Figure 2 (SEQ
ID N0:2) or (c) another specifically
derived fragment of the amino acid sequence shown in Figure 2 (SEQ ID N0:2).
"PR0533 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 23 to 216 of the PR0533
CA 02341304 2001-03-02
WO 00/15666 PCT/US99/20594
polypeptide shown in Figure 4 (SEQ ID N0:7), (b) X to 216 of the PR0533
polypeptide shown in Figure 4 (SEQ
ID N0:7), wherein X is any amino acid residue from 18 to 27 of Figure 4 (SEQ
1D N0:7) or (c) another specifically
derived fragment of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
"PR0214 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 30 to 420 of the PR0214
poiypeptide shown in Figure 6 (SEQ ID N0:12), (b) X to 420 of the PR0214
polypeptide shown in Figure 6 (SEQ
ID N0:12), wherein X is any amino acid residue from 25 to 34 of Figure 6 (SEQ
ID N0:12), (c) 1 or about 30 to
X of Figure 6 (SEQ ID N0:12), wherein X is any amino acid from amino acid 367
to amino acid 376 of Figure 6
(SEQ ID N0:12) or (d) another specifically derived fragment of the amino acid
sequence shown in Figure 6 (SEQ
ID N0:12).
"PR0240 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues I or
about 31 to 229 of the PR0240
polypeptide shown in Figure 8 (SEQ ID N0:17), (b) X to 229 of the PR0240
polypeptide shown in Figure 8 (SEQ
ID NO: l7), wherein X is any amino acid residue from 26 to 35 of Figure 8 (SEQ
ID NO: I7), (c) 1 or about 31 to
1 S X of Figure 8 (SEQ ID N0:17), wherein X is any amino acid from amino acid
193 to amino acid 202 of Figure 8
(SEQ ID N0:17) or (d) another specifically derived fragment of the amino acid
sequence shown in Figure 8 (SEQ
ID N0:17).
"PR0211 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 25 to 353 of the PR0211
polypeptide shown in Figure 10 (SEQ ID N0:22), (b) X to 353 of the PR02I 1
poiypeptide shown in Figure 10
(SEQ ID N0:22), wherein X is any amino acid residue from 20 to 29 of Figure 10
(SEQ ID N0:22) or (c) another
specifically derived fragment of the amino acid sequence shown in Figure 10
(SEQ ID N0:22).
"PR0230 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 22 to l64 of the PR0230
polypeptide shown in Figure I2 (SEQ ID N0:27), (b) X to l64 of the PR0230
polypeptide shown in Figure 12
(SEQ ID N0:27), wherein X is any amino acid residue from 17 to 26 of Figure I2
(SEQ ID N0:27) or (c) another
specifically derived fragment of the amino acid sequence shown in Figure 12
(SEQ ID N0:27).
"PR0261 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 24 to 250 of the PR0261
polypeptide shown in Figure l4 (SEQ ID N0:32), (b) X to 250 of the PR0261
polypeptide shown in Figure 14
(SEQ ID N0:32), wherein X is any amino acid residue from 19 to 28 of Figure 14
(SEQ ID N0:32) or (c) another
specifically derived fragment of the amino acid sequence shown in Figure 14
(SEQ ID N0:32).
"PR0246 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about 30 to 390 of the PR0246
polypeptide shown in Figure 16 (SEQ ID N0:37), (b) X to 390 of the PR0246
polypeptide shown in Figure 16
(SEQ ID N0:37), wherein X is any amino acid residue from 25 to 34 of Figure 16
(SEQ ID N0:37), (c) 1 or about
30 to X of Figure 16 (SEQ ID N0:37), wherein X is any amino acid from amino
acid 242 to amino acid 251 of
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WO 00/15666 PCT/US99/20594
Figure 16 (SEQ ID N0:37) or (d) another specifical ly derived fragment of the
amino acid sequence shown in Figure
16 (SEQ ID N0:37).
"PR0317 variant polypeptide" means an active polypeptide as defined below
having at least about 80%
amino acid sequence identity with the amino acid sequence of (a) residues 1 or
about l9 to 366 of the PR0317
polypeptide shown in Figure 18 (SEQ ID N0:42), (b) X to 366 of the PR0317
polypeptide shown in Figure 18
(SEQ ID N0:42), wherein X is any amino acid residue from 14 to 23 of Figure 18
(SEQ ID N0:42) or (c) another
specifically derived fragment of the amino acid sequence shown in Figure 18
(SEQ ID N0:42).
Such PROI 87, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 and
PR0317 variant
polypeptides include, for instance, PR0187, PR0533, PR0214, PR0240, PR021 l,
PR0230, PR0261, PR0246
or PR0317 polypeptides wherein one or more amino acid residues are added, or
deleted, at the N- and/or C-
tettrtinus, as well as within one or more internal domains, of the sequence of
Figure 2 (SEQ ID N0:2), Figure 4
(SEQ ID N0:7), Figure 6 (SEQ ID N0:12), Figure 8 (SEQ ID N0:17), Figure l0
(SEQ ID N0:22), Figure l2
(SEQ ID N0:27), Figure l4 (SEQ ID N0:32), Figure 16 (SEQ ID N0:37), or Figure
IS (SEQ ID N0:42),
respectively.
Ordinarily, a PRO 187 variant polypeptide wi II have at least about 80% am ino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferabty at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues 1 or about 23 to 205
of the PR0187 polypeptide shown
in Figure 2 (SEQ ID N0:2), (b) X to 205 of the PRO187 polypeptide shown in
Figure 2 (SEQ ID N0:2), wherein
X is any amino acid residue from 18 to 27 of Figure 2 (SEQ ID N0:2) or (c)
another specifically derived fragment
of the amino acid sequence shown in Figure 2 (SEQ ID N0:2).
Ordinarily, a PR0533 variant polypeptide wil I have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
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WO 00/15666 PCT/US99/20594
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino 'acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues t or about 23 to 216
of the PR0533 polypeptide shown
in Figure 4 (SEQ ID N0:7), (b) X to 216 of the PR0533 polypeptide shown in
Figure 4 (SEQ ID N0:7), wherein
X is any amino acid residue from 18 to 27 of Figure 4 (SEQ ID N0:7) or (c)
another specifically derived fragment
of the amino acid sequence shown in Figure 4 (SEQ ID N0:7).
Ordinarily, a PR0214 variant polypeptide will have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues l or about 30 to 420
of the PR0214 polypeptide shown
in Figure 6 (SEQ 1D N0:12), (b) X to 420 of the PR0214 polypeptide shown in
Figure 6 (SEQ ID N0:12), wherein
X is any amino acid residue from 25 to 34 of Figure 6 (SEQ ID NO: l2), (c) 1
or about 30 to X of Figure 6 (SEQ
ID N0:12), wherein X is any amino acid from amino acid 367 to amino acid 376
of Figure 6 (SEQ ID N0:12) or
(d) another specifically derived fragment of the amino acid sequence shown in
Figure 6 (SEQ ID N0:12).
Ordinarily, a PR0240 variant po lypeptide wil l have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
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sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues 1 or about 31 to 229
of the PR0240 polypeptide shown
in Figure 8 (SEQ ID N0:17), (b) X to 229 of the PR0240 polypeptide shown in
Figure 8 {SEQ ID NO: I 7), wherein
X is any amino acid residue from 26 to 35 of Figure 8 (SEQ ID N0:17), (c) 1 or
about 31 to X of Figure 8 (SEQ
S ID N0:17), wherein X is any amino acid from amino acid 193 to amino acid 202
of Figure 8 (SEQ ID N0:17) or
(d) another specifically derived fragment of the amino acid sequence shown in
Figure 8 (SEQ ID N0:17).
Ordinarily, a PR0211 variant polypeptide will have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues 1 or about 25 to 353
of the PR0211 polypeptide shown
in Figure 10 (SEQ ID N0:22), (b) X to 353 of the PR0211 polypeptide shown in
Figure 10 (SEQ ID N0:22),
wherein X is any amino acid residue from 20 to 29 of Figure 10 (SEQ ID N0:22)
or {c) another specifically derived
fragment ofthe amino acid sequence shown in Figure i0 (SEQ ID N0:22).
Ordinarily, a PR0230 variant polypeptide will have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues I or about 22 to 164
of the PR0230 polypeptide shown
in Figure 12 (SEQ ID N0:27), (b) X to 164 of the PR0230 polypeptide shown in
Figure 12 (SEQ ID N0:27),
wherein X is any amino acid residue from 17 to 26 of Figure 12 (SEQ ID N0:27)
or (c) another specifically derived
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fragment of the amino acid sequence shown in Figure 12 (SEQ ID N0:27).
Ordinarily, a PR0261 variant polypeptide will have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
l0 sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues 1 or about 24 to 250
of the PR0261 polypeptide shown
in Figure 14 (SEQ ID N0:32), (b) X to 250 of the PR0261 polypeptide shown in
Figure l4 (SEQ ID N0:32),
wherein X is any amino acid residue from 19 to 28 of Figure 14 (SEQ ID N0:32)
or (c) another specifically derived
fragment of the amino acid sequence shown in Figure 14 (SEQ ID N0:32).
Ordinarily, a PR0246 variant polypeptide will have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues 1 or about 30 to 390
of the PR0246 polypeptide shown
in Figure 16 (SEQ ID N0:37), (b) X to 390 of the PR0246 polypeptide shown in
Figure 16 (SEQ ID N0:37),
wherein X is any amino acid residue from 25 to 34 of Figure l6 (SEQ ID N0:37),
(c) 1 or about 30 to X of Figure
16 {SEQ ID N0:37), wherein X is any amino acid from amino acid 242 to amino
acid 251 of Figure 16 (SEQ ID
N0:37) or (d) another specifically derived fragment of the amino acid sequence
shown in Figure 16 (SEQ ID
N0:37).
Ordinarily, a PR0317 variant polypeptide will have at least about 80% amino
acid sequence identity, more
preferably at least about 81% amino acid sequence identity, more preferably at
least about 82% amino acid
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sequence identity, more preferably at least about 83% amino acid sequence
identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more
preferably at least about 86% amino acid sequence identity, more preferably at
least about 87% amino acid
sequence identity, more preferably at least about 88% amino acid sequence
identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more
preferably at least about 91% amino acid sequence identity, more preferably at
least about 92% amino acid
sequence identity, more preferably at least about 93% amino acid sequence
identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more
preferably at least about 96% amino acid sequence identity, more preferably at
least about 97% amino acid
sequence identity, more preferably at least about 98% amino acid sequence
identity and yet more preferably at least
about 99% amino acid sequence identity with (a) residues l or about 19 to 366
of the PR0317 polypeptide shown
in Figure I8 (SEQ ID N0:42), (b) X to 366 of the PR0317 polypeptide shown in
Figure 18 (SEQ ID N0:42),
wherein X is any amino acid residue from 14 to 23 of Figure 18 (SEQ ID N0:42)
or (c) another specifically derived
fragment of the amino acid sequence shown in Figure 18 (SEQ ID N0:42).
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 and PR0317
variant
polypeptides do not encompass the native PROl87, PR0533, PR0214, PR0240, PR021
l, PR0230, PR0261,
PR0246 or PR0317 polypeptide sequence. Ordinarily, PRO 187, PR0533, PR0214,
PR0240, PR0211, PR0230,
PR0261, PR0246 or PR0317 variant polypeptides are at least about 10 amino
acids in length, often at least about
amino acids in length, more often at least about 30 amino acids in length,
more often at least about 40 amino
20 acids in length, more often at least about 50 amino acids in length, more
often at least about 60 amino acids in
length, more often at least about 70 amino acids in length, more often at
least about 80 amino acids in length, more
often at least about 90 amino acids in length, more often about 100 amino
acids in length, more often at least about
150 amino acids in length, more often at least about 200 amino acids in
length, more often at least about 300 amino
acids in length, or more.
"Percent (%) amino acid sequence identity" with respect to the PR0187, PR0533,
PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 and PR0317 polypeptide sequences identified
herein is defined as the
percentage of amino acid residues in a candidate sequence that are identical
with the amino acid residues in a
PRO ( 87, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
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 Figures 20A-Q. The ALIGN-2
sequence comparison computer
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program was authored by Genentech, Inc. , and the source code shown in Figures
20A-Q 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 Figures 20A-Q. The
ALIGN-2 program should be compiled for use on a UNIX operating system,
preferably digital UNIX V4.OD.
All sequence comparison parameters are set by the ALIGN-2 program and do not
vary.
For purposes herein, the % amino acid sequence identity of a given amino acid
sequence A to, with,
or against a given amino acid sequence B (which can alternatively be phrased
as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity to, with, or
against a given amino acid
sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues in
B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not equal the %
amino acid sequence identity of
B to A. As examples of % amino acid sequence identity calculations, Figures
19A-B 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. 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, mufti-pass e-
value = 0.01, constant for
mufti-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix =
BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino
acid sequence identity of a given amino acid sequence A to, with, or against a
given amino acid sequence B
(which can alternatively be phrased as a given amino acid sequence A that has
or comprises a certain % amino
acid sequence identity to, with, or against a given amino acid sequence B) is
calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical matches by
the sequence alignment program
NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total
number of amino acid residues
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in B. It will be appreciated that where the length of amino acid sequence A is
not equal to the length of amino
acid sequence B, the % amino acid sequence identity of A to B will not equal
the % amino acid sequence identity
ofBtoA.
In addition, % amino acid sequence identity may also be determined using the
WU-BLAST-2 computer
S program (Altschul et aL, Methods in Enzvmolo~v, 266:460-480 (1996)). Most of
the WU-BLAST-2 search
parameters are set to the default values. Those not set to default values,
i.e., the adjustable parameters, are set with
the following values: overlap span = 1, overlap fraction = 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 poiypeptide
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.
"PR0187 variant polynucleotide" or "PR0187 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0187 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) a nucleic acid sequence which encodes
residues 1 or about 23 to 205 of the
PR0187 polypeptide shown in Figure 2 (SEQ ID N0:2), (b) a nucleic acid
sequence which encodes amino acids
X to 205 of the PRO 187 polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X
is any amino acid residue from
18 to 27 of Figure 2 (SEQ ID N0:2), or (c) a nucleic acid sequence which
encodes another specifically derived
fragment of the amino acid sequence shown in Figure 2 (SEQ ID N0:2).
Ordinarily, a PR0187 variant
polynucleotide will have at least about 80% nucleic acid sequence identity,
more preferably at least about 81%
nucleic acid sequence identity, more preferably at least about 82% nucleic
acid sequence identity, more preferably
at least about 83% nucleic acid sequence identity, more preferably at least
about 84% nucleic acid sequence identity,
more preferably at least about 85% nucleic acid sequence identity, more
preferably at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) a nucleic acid sequence which encodes residues 1 or about 23 to 205
of the PROI 87 polypeptide shown
in Figure 2 (SEQ ID N0:2), (b) a nucleic acid sequence which encodes amino
acids X to 205 of the PR0187
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polypeptide shown in Figure 2 (SEQ ID N0:2), wherein X is any amino acid
residue from 18 to 2? of Figure 2
(SEQ ID N0:2), or (c) a nucleic acid sequence which encodes another
specifically derived fragment of the amino
acid sequence shown in Figure 2 (SEQ ID N0:2). PR0187 polynucleotide variants
do not encompass the native
PR0187 nucleotide sequence.
S "PR0533 variant polynucleotide" or "PR0533 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PROS33 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 23 to 216 of the
PROS33 polypeptide shown in Figure
4 (SEQ ID N0:7), (b) X to 216 of the PR0533 polypeptide shown in Figure 4 (SEQ
ID N0:7), wherein X is any
amino acid residue from 18 to 27 of Figure 4 (SEQ ID N0:7) or (c) another
specifically derived fragment of the
l0 amino acid sequence shown in Figure 4 (SEQ ID N0:7). Ordinarily, a PR0533
variant polynucleotide will have
at least about 80% nucleic acid sequence identity, more preferably at least
about 81 % nucleic acid sequence identity,
more preferably at least about 82% nucleic acid sequence identity, more
preferably at least about 83% nucleic acid
sequence identity, more preferably at least about 84% nucleic acid sequence
identity, more preferably at least about
8S% nucleic acid sequence identity, more preferably at least about 86% nucleic
acid sequence identity, more
I S preferably at least about 87% nucleic acid sequence identity, more
preferably at least about 88% nucleic acid
sequence identity, more preferably at least about 89% nucleic acid sequence
identity, more preferably at least about
90% nucleic acid sequence identity, more preferably at least about 91% nucleic
acid sequence identity, more
preferably at least about 92% nucleic acid sequence identity, more preferably
at least about 93% nucleic acid
sequence identity, more preferably at least about 94% nucleic acid sequence
identity, more preferably at least about
20 95% nucleic acid sequence identity, more preferably at least about 96%
nucleic acid sequence identity, more
preferably at least about 97% nucleic acid sequence identity, more preferably
at least about 98% nucleic acid
sequence identity and yet more preferably at least about 99% nucleic acid
sequence identity with either (a) residues
1 or about 23 to 216 of the PROS33 polypeptide shown in Figure 4 (SEQ ID
N0:7), (b) X to 216 of the PR0533
polypeptide shown in Figure 4 (SEQ ID N0:7), wherein X is any amino acid
residue from 18 to 27 of Figure 4
25 (SEQ ID N0:7) or (c) another specifically derived fragment of the amino
acid sequence shown in Figure 4 (SEQ
tD N0:7). PR0533 polynucleotide variants do not encompass the native PR0533
nucleotide sequence.
"PR0214 variant polynucleotide" or "PR0214 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0214 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 30 to 420 of the
PR0214 polypeptide shown in Figure
30 6 (SEQ 1D N0:12), (b) X to 420 of the PR0214 polypeptide shown in Figure 6
(SEQ ID N0:12), wherein X is any
amino acid residue from 25 to 34 of Figure 6 (SEQ ID N0:12), (c) 1 or about 30
to X of Figure 6 (SEQ ID N0:12),
wherein X is any amino acid from amino acid 367 to amino acid 376 of Figure 6
(SEQ ID N0:12) or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 6
(SEQ ID N0:12). Ordinarily, a
PR0214 variant polynucleotide wil I have at least about 80% nucleic acid
sequence identity, more preferably at least
3S about $ I% nucleic acid sequence identity, more preferably at least about
82% nucleic acid sequence identity, more
preferably at least about 83% nucleic acid sequence identity, more preferably
at least about 84% nucleic acid
sequence identity, more preferably at least about 8S% nucleic acid sequence
identity, more preferably at least about
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86% nucleic acid sequence identity, more preferably at least about 87% nucleic
acid sequence identity, more
preferably at least about 88% nucleic acid sequence identity, more preferably
at least about 89% nucleic acid
sequence identity, more preferably at least about 90% nucleic acid sequence
identity, more preferably at least about
91% nucleic acid sequence identity, more preferably at least about 92% nucleic
acid sequence identity, more
preferably at least about 93% nucleic acid sequence identity, more preferably
at least about 94% nucleic acid
sequence identity, more preferably at least about 95% nucleic acid sequence
identity, more preferably at least about
96% nucleic acid sequence identity, more preferably at least about 97% nucleic
acid sequence identity, more
preferably at least about 98% nucleic acid sequence identity and yet more
preferably at least about 99% nucleic acid
sequence identity with either (a) residues 1 or about 30 to 420 of the PR0214
polypeptide shown in Figure 6 (SEQ
ID N0:12), (b) X to 420 of the PR0214 polypeptide shown in Figure 6 (SEQ ID
N0:12), wherein X is any amino
acid residue from 25 to 34 of Figure 6 (SEQ ID N0:12), (c) 1 or about 30 to X
of Figure 6 (SEQ ID N0:12),
wherein X is any amino acid from amino acid 367 to amino acid 376 of Figure 6
(SEQ ID N0:12) or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 6
(SEQ ID N0:12). PR0214
polynucleotide variants do not encompass the native PR0214 nucleotide
sequence.
"PR0240 variant polynucleotide" or "PR0240 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0240 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 31 to 229 of the
PR0240 polypeptide shown in Figure
8 (SEQ ID N0:17), (b) X to 229 of the PR0240 polypeptide shown in Figure 8
(SEQ ID N0:17), wherein X is any
amino acid residue from 26 to 35 of Figure 8 (SEQ ID N0:17), (c) 1 or about 31
to X of Figure 8 (SEQ ID N0:17),
wherein X is any amino acid from amino acid 193 to amino acid 202 of Figure 8
(SEQ ID N0:17) or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 8
(SEQ ID N0:17). Ordinarily, a
PR0240 variant polynucleotide will have at least about 80% nucleic acid
sequence identity, more preferably at least
about 81 % nucleic acid sequence identity, more preferably at least about 82%
nucleic acid sequence identity, more
preferably at least about 83% nucleic acid sequence identity, more preferably
at least about 84% nucleic acid
sequence identity, more preferably at least about 85% nucleic acid sequence
identity, more preferably at least about
86% nucleic acid sequence identity, more preferably at least about 87% nucleic
acid sequence identity, more
preferably at least about 88% nucleic acid sequence identity, more preferably
at least about 89% nucleic acid
sequence identity, more preferably at least about 90% nucleic acid sequence
identity, more preferably at least about
91% nucleic acid sequence identity, more preferably at least about 92% nucleic
acid sequence identity, more
preferably at least about 93% nucleic acid sequence identity, more preferably
at least about 94% nucleic acid
sequence identity, more preferably at least about 95% nucleic acid sequence
identity, more preferably at least about
96% nucleic acid sequence identity, more preferably at least about 97% nucleic
acid sequence identity, more
preferably at least about 98% nucleic acid sequence identity and yet more
preferably at least about 99% nucleic acid
sequence identity with either (a) residues 1 or about 31 to 229 of the PR0240
polypeptide shown in Figure 8 (SEQ
ID N0:17), (b) X to 229 of the PR0240 polypeptide shown in Figure 8 (SEQ ID
N0:17), wherein X is any amino
acid residue from 26 to 35 of Figure 8 (SEQ ID N0:17), (c) 1 or about 31 to X
of Figure 8 (SEQ ID N0:17),
wherein X is any amino acid from amino acid 193 to amino acid 202 of Figure 8
(SEQ ID N0:17) or (d) another
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specifically derived fragment of the amino acid sequence shown in Figure 8
(SEQ ID N0:17). PR0240
polynucleotide variants do not encompass the native PR0240 nucleotide
sequence.
"PR0211 variant polynucleotide" or "PR021 I variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0211 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 25 to 353 of the
PR0211 polypeptide shown in Figure
l0 (SEQ ID N0:22), (b) X to 353 of the PR021 I polypeptide shown in Figure l0
(SEQ ID N0:22), wherein X is
any amino acid residue from 20 to 29 of Figure 10 (SEQ ID N0:22) or (c)
another specifically derived fragment
of the amino acid sequence shown in Figure 10 (SEQ ID N0:22). Ordinarily, a
PR0211 variant polynucleotide
will have at least about 80% nucleic acid sequence identity, more preferably
at least about 81% nucleic acid
sequence identity, more preferably at least about 82% nucleic acid sequence
identity, more preferably at least about
83% nucleic acid sequence identity, more preferably at least about 84% nucleic
acid sequence identity, more
preferably at least about 85% nucleic acid sequence identity, more preferably
at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
IS preferably at least about 90% nucleic acid sequence identity, more
preferably at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) residues 1 or about 25 to 353 of the PR02 I 1 polypeptide shown in
Figure 10 (SEQ ID N0:22), (b) X to
353 of the PR0211 polypeptide shown in Figure 10 (SEQ ID N0:22), wherein X is
any amino acid residue from
20 to 29 of Figure 10 (SEQ ID N0:22) or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure 10 (SEQ ID N0:22). PR0211 polynucleotide variants do not
encompass the native PR0211
nucleotide sequence.
"PR0230 variant polynucleotide" or "PR0230 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0230 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 22 to 164 of the
PR0230 polypeptide shown in Figure
12 {SEQ ID N0:27), (b) X to I 64 of the PR0230 polypeptide shown in Figure 12
(SEQ ID N0:27), wherein X is
any amino acid residue from 17 to 26 of Figure 12 (SEQ ID N0:27) or {c)
another specifically derived fragment
of the amino acid sequence shown in Figure 12 (SEQ ID N0:27). Ordinarily, a
PR0230 variant polynucleotide
will have at least about 80% nucleic acid sequence identity, more preferably
at least about 81% nucleic acid
sequence identity, more preferably at least about 82% nucleic acid sequence
identity, more preferably at least about
83% nucleic acid sequence identity, more preferably at least about 84% nucleic
acid sequence identity, more
preferably at least about 85% nucleic acid sequence identity, more preferably
at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
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preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91 % nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 9S% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) residues I or about 22 to 164 of the PR0230 polypeptide shown in
Figure 12 (SEQ ID N0:27), (b) X to
164 of the PR0230 polypeptide shown in Figure 12 (SEQ ID N0:27), wherein X is
any amino acid residue from
17 to 26 of Figure 12 (SEQ ID N0:27) or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure 12 (SEQ ID N0:27). PR0230 polynucleotide variants do not
encompass the native PR0230
nucleotide sequence.
"PR0261 variant polynucleotide" or "PR0261 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0261 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 24 to 2S0 of the
PR0261 polypeptide shown in Figure
l S 14 (SEQ ID N0:32), (b) X to 2S0 of the PR0261 polypeptide shown in Figure
14 (SEQ ID N0:32), wherein X is
any amino acid residue from 19 to 28 of Figure 14 (SEQ ID N0:32) or (c)
another specifically derived fragment
of the amino acid sequence shown in Figure 14 (SEQ ID N0:32). Ordinarily, a
PR0261 variant polynucleotide
will have at least about 80% nucleic acid sequence identity, more preferably
at least about 81 % nucleic acid
sequence identity, more preferably at least about 82% nucleic acid sequence
identity, more preferably at least about
83% nucleic acid sequence identity, more preferably at least about 84% nucleic
acid sequence identity, more
preferabty at least about 85% nucleic acid sequence identity, more preferably
at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
2S sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
preferably at least about 9S% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) residues I or about 24 to 2S0 of the PR0261 polypeptide shown in
Figure 14 (SEQ ID N0:32), (b) X to
2S0 of the PR0261 polypeptide shown in Figure 14 (SEQ ID N0:32), wherein X is
any amino acid residue from
19 to 28 of Figure 14 (SEQ ID N0:32) or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure t4 (SEQ ID N0:32). PR0261 polynucleotide variants do not
encompass the native PR0261
nucleotide sequence.
3S "PR0246 variant polynucleotide" or "PR0246 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0246 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 30 to 390 of the
PR0246 polypeptide shown in Figure
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16 (SEQ ID N0:37), (b) X to 390 of the PR0246 polypeptide shown in Figure 16
(SEQ ID N0:37), wherein X is
any amino acid residue from 25 to 34 of Figure 16 (SEQ ID N0:37), (c) 1 or
about 30 to X of Figure 16 (SEQ ID
N0:37), wherein X is any amino acid from amino acid 242 to amino acid 25l of
Figure 16 (SEQ ID N0:37) or (d)
another specifically derived fragment ofthe amino acid sequence shown in
Figure 16 (SEQ ID N0:37). Ordinarily,
a PR0246 variant polynucleotide will have at least about 80% nucleic acid
sequence identity, more preferably at
least about 81% nucleic acid sequence identity, more preferably at least about
82% nucleic acid sequence identity,
more preferably at least about 83% nucleic acid sequence identity, more
preferably at least about 84% nucleic acid
sequence identity, more preferably at least about 85% nucleic acid sequence
identity, more preferably at least about
86% nucleic acid sequence identity, more preferably at least about 87% nucleic
acid sequence identity, more
preferably at least about 88% nucleic acid sequence identity, more preferably
at least about 89% nucleic acid
sequence identity, more preferably at least about 90% nucleic acid sequence
identity, more preferably at least about
91% nucleic acid sequence identity, more preferably at least about 92% nucleic
acid sequence identity, more
preferably at least about 93% nucleic acid sequence identity, more preferably
at least about 94% nucleic acid
sequence identity, more preferably at least about 95% nucleic acid sequence
identity, more preferably at least about
96% nucleic acid sequence identity, more preferably at least about 97% nucleic
acid sequence identity, more
preferably at least about 98% nucleic acid sequence identity and yet more
preferably at least about 99% nucleic acid
sequence identity with either (a) residues 1 or about 30 to 390 of the PR0246
polypeptide shown in Figure 16 (SEQ
ID N0:37), (b) X to 390 of the PR0246 polypeptide shown in Figure 16 (SEQ ID
N0:37), wherein X is any amino
acid residue from 25 to 34 of Figure 16 (SEQ ID N0:37), (c) 1 or about 30 to X
of Figure 16 (SEQ ID N0:37),
wherein X is any amino acid from amino acid 242 to amino acid 251 of Figure 16
(SEQ ID N0:37) or (d) another
specifically derived fragment of the amino acid sequence shown in Figure 16
(SEQ ID N0:37). PR0246
polynucleotide variants do not encompass the native PR0246 nucleotide
sequence.
"PR0317 variant polynucleotide" or "PR0317 variant nucleic acid sequence"
means a nucleic acid
molecule which encodes an active PR0317 polypeptide as defined below and which
has at least about 80% nucleic
acid sequence identity with either (a) residues 1 or about 19 to 366 of the
PR0317 polypeptide shown in Figure
18 (SEQ ID N0:42), (b) X to 366 of the PR0317 polypeptide shown in Figure 18
(SEQ ID N0:42), wherein X is
any amino acid residue from 14 to 23 of Figure 18 (SEQ ID N0:42) or (c)
another specifically derived fragment
of the amino acid sequence shown in Figure l8 (SEQ ID N0:42). Ordinarily, a
PR0317 variant polynucleotide
will have at least about 80% nucleic acid sequence identity, more preferably
at least about 81% nucleic acid
sequence identity, more preferably at least about 82% nucleic acid sequence
identity, more preferably at least about
83% nucleic acid sequence identity, more preferably at least about 84% nucleic
acid sequence identity, more
preferably at least about 85% nucleic acid sequence identity, more preferably
at least about 86% nucleic acid
sequence identity, more preferably at least about 87% nucleic acid sequence
identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about 89% nucleic
acid sequence identity, more
preferably at least about 90% nucleic acid sequence identity, more preferably
at least about 91% nucleic acid
sequence identity, more preferably at least about 92% nucleic acid sequence
identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about 94% nucleic
acid sequence identity, more
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preferably at least about 95% nucleic acid sequence identity, more preferably
at least about 96% nucleic acid
sequence identity, more preferably at least about 97% nucleic acid sequence
identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least about 99%
nucleic acid sequence identity with
either (a) residues l or about 19 to 366 of the PR0317 polypeptide shown in
Figure 18 (SEQ ID N0:42), (b) X to
366 of the PR0317 polypeptide shown in Figure 18 (SEQ ID N0:42), wherein X is
any amino acid residue from
14 to 23 of Figure 18 (SEQ ID N0:42) or (c) another specifically derived
fragment of the amino acid sequence
shown in Figure 18 (SEQ ID N0:42). PR0317 polynucleotide variants do not
encompass the native PR0317
nucleotide sequence.
Ordinarily, PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 and
PR0317
I 0 variant polynucleotides are at least about 30 nucleotides in length, often
at least about 60 nucleotides in length, more
often at least about 90 nucleotides in length, more often at least about 120
nucleotides in length, more often at least
about 150 nucleotides in length, more often at least about I 80 nucleotides in
length, more often at least about 210
nucleotides in length, more often at least about 240 nucleotides in length,
more often at least about 270 nucleotides
in length, more often at least about 300 nucleotides in length, more often at
least about 450 nucleotides in length,
more often at least about 600 nucleotides in length, more often at least about
900 nucleotides in length, or more.
"Percent (% ) nucleic acid sequence identity" with respect to the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 and PR0317 polypeptide-encodingnucieic acid
sequences identified herein
is defined as the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in a
PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317
polypeptide-encoding
nucleic acid sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum
percent sequence identity. Alignment for purposes of determining percent
nucleic acid sequence identity can be
achieved in various ways that are within the skill in the art, for instance,
using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)
software. Those skilled in
the art can determine appropriate parameters for measuring alignment,
including any algorithms needed to
achieve maximal alignment over the full-length of the sequences being
compared. For purposes herein, however,
~ nucleic acid sequence identity values are obtained as described below by
using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the ALIGN-2
program is provided in
Figures 20A-Q. The ALIGN-2 sequence comparison computer program was authored
by Genentech, Inc., and
the source code shown in Figures 20A-Q 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 Figures 20A-Q. The ALIGN-2 program
should be compiled for use
on a UNIX operating system, preferably digital UNIX V4.OD. Ail sequence
comparison parameters are set by
the ALIGN-2 program and do not vary.
For purposes herein, the % nucleic acid sequence identity of a given nucleic
acid sequence C to, with,
or against a given nucleic acid sequence D (which can alternatively be phrased
as a given nucleic acid sequence
C that has or comprises a certain % nucleic acid sequence identity to, with,
or against a given nucleic acid
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WO 00/15666 PCT/US99/20594
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, Figures 19C-D
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. 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
mufti-pass = 25, dropoff for final gapped alignment = 25 and scoring matrix =
BLOSUM62.
1n 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 cart
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 Enz:vmoloev. 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 % nucleic acid sequence identity
value is detetrnined by dividing
(a) the number of matching identical nucleotides between the nucleic acid
sequence of the PRO polypeptide-
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encoding nucleic acid molecule of interest having a sequence derived from the
native sequence PRO polypeptide-
encoding nucleic acid and the comparison nucleic acid molecule of interest
(i.e., the sequence against which the
PRO polypeptide-encoding nucleic acid molecule of interest is being compared
which may be a variant PRO
polynucleotide) as determined by WLJ-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 187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 and
PR0317 variant polynucleotides are nucleic acid molecules that encode an
active PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide and which are
capable of hybridizing,
preferably under stringent hybridization and wash conditions, to nucleotide
sequences encoding the full-length
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide shown in
Figure 2 (SEQ ID N0:2), Figure 4 (SEQ ID N0:7), Figure 6 (SEQ ID NO: l2),
Figure 8 (SEQ ID N0:17), Figure
10 (SEQ ID N0:22), Figure 12 (SEQ ID N0:27), Figure l4 (SEQ ID N0:32), Figure
16 (SEQ ID N0:37), or
Figure 18 (SEQ ID N0:42), respectively. PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261,
PR0246 or PR0317 variant polypeptides may be those that are encoded by a
PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 variant polynucleotide.
The term "positives", in the context of the amino acid sequence identity
comparisons performed as
described above, includes amino acid residues in the sequences compared that
are not only identical, but also
those that have similar properties. Amino acid residues that score a positive
value to an amino acid residue of
interest are those that are either identical to the amino acid residue of
interest or are a preferred substitution (as
defined in Table 1 below) of the amino acid residue of interest.
For purposes herein, the % value of positives of a given amino acid sequence A
to, with, or against a
given amino acid sequence B (which can alternatively be phrased as a given
amino acid sequence A that has or
comprises a certain % positives to, with, or against a given amino acid
sequence B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scoring a positive value as
defined above by the sequence
alignment program ALIGN-2 in that program's alignment of A and B, and where Y
is the total number of amino
acid residues in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the
length of amino acid sequence B, the % positives of A to B will not equal the
% positives of B to A.
"Isolated," when used to describe the various polypeptides disclosed herein,
means polypeptide that has
been identified and separated and/or recovered from a component of its natural
environment. Preferably, the
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
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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 I S
residues of N-terminal or internal amino acid sequence by use of a spinning
cup sequenator, or (2) to homogeneity
by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or,
preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within recombinant cells,
since at least one component of the
PRO 187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317
natural environment will
not be present. Ordinarily, however, isolated polypeptide will be prepared by
at least one purification step.
An "isolated" nucleic acid molecule encoding a PR0187, PR0533, PR0214, PR0240,
PR021 I, PR0230,
PR0261, PR0246 or PR0317 polypeptide or an "isolated" nucleic acid encoding an
anti-PR0187, anti-PR0533,
anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-PR0261, anti-PR0246
or anti-PR0317 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 PR0187-, PR0533-
, PR0214-, PR0240-, PR0211-,
PR0230-, PR0261-, PR0246- or PR0317-encoding nucleic acid or the anti-PR0187-,
anti-PR0533-, anti-
PR0214-, anti-PR0240-, anti-PR0211-, anti-PR0230-, anti-PR0261-, anti-PR0246-
or anti-PR0317-encoding
nucleic acid. Preferably, the isolated nucleic acid is free of association
with all components with which it is
naturally associated. An isolated PR0187-, PR0533-, PR0214-, PR0240-, PR0211-,
PR0230-, PR0261-,
PR0246- or PR0317-encoding nucleic acid molecule or an anti-PR0187-, anti-
PR0533-, anti-PR0214-, anti-
PR0240-, anti-PR0211-, anti-PR0230-, anti-PR0261-, anti-PR0246- or anti-PR0317-
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 PR0187-, PR0533-, PR02 i 4-, PR0240-, PR0211-,
PR0230-, PR0261-, PR0246- or
PR0317-encoding nucleic acid molecule or the anti-PR0187-, anti-PR0533-, anti-
PR0214-, anti-PR0240-, anti-
PR0211-, anti-PR0230-, anti-PR0261-,anti-PR0246-or anti-PR0317-encoding
nucleic acid molecule as it exists
in natural cells. However, an isolated nucleic acid molecule encoding a
PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 poiypeptide or an anti-PR0187, anti-
PROS33, anti-PR0214,
anti-PR0240,anti-PR0211,anti-PR0230,anti-PR0261,anti-PR0246oranti-
PR0317antibodyincludesPR0187-
PR0533-, PR0214-, PR0240-, PR0211-, PR0230-, PR0261-, PR0246-or PR0317-nucleic
acid molecules and
anti-PR0187-, anti-PR0533-, anti-PR0214-, anti-PR0240-, anti-PR0211-, anti-
PR0230-, anti-PR0261-, anti-
PR0246- or anti-PR0317-encoding nucleic acid molecules contained in cells that
ordinarily express PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptides
or express anti-
PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-
PR0261, anti-PR0246 or
anti-PR0317 antibodies where, for example, the nucleic acid molecule is in a
chromosomal location different from
that of natural cells.
The term "control sequences" refers to DNA sequences necessary for the
expression of an operably linked
coding sequence in a particular host organism. The control sequences that are
suitable for prokaryotes, for example,
include a promoter, optionally an operator sequence, and a ribosome binding
site. Eukaryotic cells are known to
utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional
relationship with another nucleic acid
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sequence. For example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide
if it is expressed as a preprotein that participates in the secretion of the
polypeptide; a promoter or enhancer is
operably linked to a coding sequence if it affects the transcription of the
sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, "operably linked"
means that the DNA sequences being linked are contiguous, and, in the case of
a secretory leader, contiguous and
in reading phase. However, enhancers do not have to be comiguous. Linking is
accomplished by ligation at
convenient restriction sites. If such sites do not exist, the synthetic
oligonucleotide adaptors or linkers are used in
accordance with conventional practice.
The term "antibody" is used in the broadest sense and specifically covers, for
example, single anti-
PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-PR0230, anti-
PR0261, anti-PR0246 or
anti-PR0317 monoclonal antibodies (including agonist, antagonist,and
neutralizingantibodies),anti-PROI 87, anti-
PR0533, anti-PR0214, anti-PR0240, anti-PR021 l, anti-PR0230, anti-PR0261, anti-
PR0246 or anti-PR0317
antibody compositions with polyepitopic specificity, single chain anti-PRO
187, anti-PR0533, anti-PR0214, anti-
PR0240, anti-PRO211, anti-PR0230, anti-PR026 I , anti-PR0246 or anti-PR0317
antibodies, and fragments of
anti-PR0187, anti-PR0533, anti-PR0214,anti-PR0240,anti-PR0211, anti-PR0230,
anti-PR0261, anti-PR0246
or anti-PR0317 antibodies (see below). The term "monoclonal antibody" as used
herein refers to an antibody
obtained from a population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the
population are identical except for possible naturally-occurring mutations
that may be present in minor amounts.
"Stringency" of hybridization reactions is readily determinable by one of
ordinary skill in the art, and
generally is an empirical calculation dependent upon probe length, washing
temperature, and salt concentration.
In general, longer probes require higher temperatures for proper annealing,
while shorter probes need lower
temperatures. Hybridization generally depends on the ability of denatured DNA
to reanneal when complementary
strands are present in an environment below their melting temperature. The
higher the degree of desired homology
between the probe and hybridizable sequence, the higher the relative
temperature which can be used. As a result,
it follows that higher relative temperatures would tend to make the reaction
conditions more stringent, while tower
temperatures less so. For additional details and explanation of stringency of
hybridization reactions, see Ausubel
et al., Current Protocols in Molecular Bioloev 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% FicolUO.l%
polyvinylpynolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium
chloride, 75 mM sodium
citrate at 42°C; of (3) employ 50% formamide, 5 x SSC (0.75 M NaCI,
0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), O.I% sodium pyrophosphate, 5 x Denhardt's solution,
sonicated salmon sperm DNA (50
~cg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at
42°C in 0.2 x SSC (sodium chloride/sodium
citrate) and 50% formamide at 55°C, followed by a high-stringency wash
consisting of 0.1 x SSC containing EDTA
at 55°C.
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"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 NaCI, 15 mM trisodium citrate), SO mM sodium
phosphate (pH 7.6), 5 x Denhardt's
solution, l0% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm
DNA, followed by washing the filters
in 1 x SSC at about 35-50°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 PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 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
I S 50 amino acid residues (preferably, between about 10 and 20 amino acid
residues).
"Active" or "activity" for the purposes herein refers to forms) of PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptides which retain a
biological and/or an immunological
activity/property of a native or naturally-occurring PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230,
PR0261, PR0246 or PR0317 poiypeptide, wherein "biological" activity refers to
a function (either inhibitory or
stimulatory) caused by a native or naturally-occurring PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230,
PR0261, PR0246 or PR0317 polypeptide other than the ability to induce the
production of an antibody against
an antigenic epitope possessed by a a native or naturally-occurring PRO l 87,
PR0533, PR0214, PR0240, PR02 I 1,
PR0230, PR0261, PR0246 or PR0317 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 PR0187,
PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317 polypeptide.
"Biological activity" in the context of an antibody or another antagonist
molecule 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 bind or complex with the polypeptides
encoded by the amplified genes identified
herein, or otherwise interfere with the interaction of the encoded
poiypeptides with other cellular proteins or
otherwise interfere with the transcription or translation of a PR0187, PR0533,
PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide. A preferred biological activity
is growth inhibition of a
target tumor cell. Another preferred biological activity is cytotoxic activity
resulting in the death of the target tumor
cell.
The term "biological activity" in the context of a PROi87, PR0533, PR0214,
PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide means the ability of a PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide to induce neoplastic cell
growth or uncontrolled cell
growth.
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The phrase ''immunological activity" means immunological cross-reactivity with
at least one epitope of
a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide.
"Immunological cross-reactivity" as used herein means that the candidate
polypeptide is capable of
competitively inhibiting the qualitative biological activity of a PR0187,
PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide having this activity with
polyclonal antisera raised against the
known active PR0187, PR0533. PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or
PR0317
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 conesponding PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261,
PR0246 or PR0317
polypeptide is significantly higher (preferably at least about 2-times, more
preferably at least about 4-times, even
more preferably at least about 8-times, most preferably at least about 10-
times higher) than the binding affinity of
that molecule to any other known native polypeptide.
The term "antagonist" is used in the broadest sense, and includes any molecule
that partially or fully
blocks, inhibits, or neutralizes a biological activity of a native PR0187,
PR0533, PR0214, PR0240, PR021 I,
PR0230, PR0261, PR0246 or PR03 I7 polypeptide disclosed herein or the
transcription or translation thereof.
Suitableantagonistmoleculesspecifically include antagonist antibodies or
antibody fragments, fragments, peptides,
small organic molecules, anti-sense nucleic acids, etc. Included are methods
for identifying antagonists of a
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
poiypeptide with a
candidate antagonist molecule and measuring a detectable change in one or more
biological activities normally
associated with the PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261,
PR0246 or PR0317
polypeptide.
A "small molecule" is defined herein to have a molecular weight below about
500 Daltons.
"Antibodies" (Abs) and "immunoglobulins" (lgs) are glycoproteins having the
same structural
characteristics. While antibodies exhibit binding specificity to a specific
antigen; immunoglobulins include both
antibodies and other antibody-like molecules which lack antigen specificity.
Polypeptides of the latter kind are,
for example, produced at low levels by the lymph system and at increased
levels by myelomas. The term
"antibody" is used in the broadest sense and specifically covers, without
limitation, intact monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies)
formed from at least two intact
antibodies, and antibody fragments so long as they exhibit the desired
biological activity.
"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
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disulfide bridges. Each heavy chain has at one end a variable domain (V")
followed by a number of constant
domains. Each light chain has a variable domain at one end (V,,) and a
constant domain at its other end; the
constant domain of the light chain is aligned with the first constant domain
of the heavy chain, and the light-chain
variable domain is aligned with the variable domain of the heavy chain.
Particular amino acid residues are believed
to form an interface between the light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions of the variable
domains differ extensively in
sequence among antibodies and are used in the binding and specificity of each
particular antibody for its particular
antigen. However, the variability is not evenly distributed throughout the
variable domains of antibodies. It is
concentrated in three segments called complementarily-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 (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 p-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 Pub(. No.91-3242. Vol. I, pages 647-669 (
1991 )). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit various
effector functions, such as participation
of the antibody in antibody-dependent cellular toxicity.
The term "hypervariable region" when used herein refers to the amino acid
residues of an antibody which
are responsible for antigen-binding. The hypervariable region comprises amino
acid residues from a
"complementarily determining region" to "CDR" (i.e., residues 24-34 (L 1 ), 50-
56 (L2) and 89-97 (L3) in the light
chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy
chain variable domain; Kabat et
al., Senuences of Proteins of Immunological Interest. 5th Ed. Public Health
Service, National Institute of Health,
Bethesda, MD. [ l 991 ]) and/or those residues from a "hypervariable loop" (i.
e., residues 26-32 (L 1 ), 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'),, and Fv fragments;
diabodies; linearantibodies(Zapata etal., Protein Ene. , 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, whose
name reflects its ability to crystallize
readily. Pepsin treatment yields an F(ab')Z fragment that has two antigen-
combining sites and is still capable of
cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen-
recognition and -binding site.
This region consists ofa dimer ofone heavy- and one light-chain variable
domain in tight, non-covalent association.
It is in this configuration that the three CDRs of each variable domain
interact to define an antigen-binding site on
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the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-
binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising only three
CDRs specific for an antigen) has
the ability to recognize and bind antigen, although at a lower affinity than
the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH 1 )
S of the heavy chain. Fab fragments differ from Fab' fragments by the addition
of a few residues at the carboxy
terminus ofthe heavy chain CH I domain including one or more cysteines from
the antibody hinge region. Fab'-SH
is the designation herein for Fab' in which the cysteine residues) of the
constant domains bear a free thiol group.
F(ab'), antibody fragments originally were produced as pairs of Fab' fragments
which have hinge cysteines between
them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be assigned to one
of two clearly distinct types, called kappa (K) and lambda (.1), based on the
amino acid sequences of their constant
domains.
Depending'on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins
can be assigned to different classes. There are five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and
IgM, and several ofthese may be further divided into subclasses (isotypes),
e.g., IgG 1, IgG2, IgG3, IgG4, IgA, and
IgA2. The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called
a, 8, e, y, and p, respectively. The subunit structures and three-dimensional
configurations of different classes of
immunoglobulins are well known.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of
substantially homogeneous antibodies,. i.e., the individual antibodies
comprising the population are identical except
for possible naturally occurring mutations that may be present in minor
amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site. Furthermore, in
contrast to conventional (polyclonal)
antibodypreparationswhichtypicallyincludedifferent 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 et al., Nature. 256:495 [ 1975],
or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567). The "monoclonal antibodies"
may also be isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature.
352:624-628 ( 1991 ] and Marks et al., J. Mol. Biol.. 222:581-597 ( 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
chains) is identical with or homologous to corresponding sequences in
antibodies derived from another species
or belonging to another antibody class or subclass, as well as fragments of
such antibodies, so long as they exhibit
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the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-
6855 [ 1984]).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab'), or
other antigen-binding subsequences
of antibodies) which contain minimal sequence derived from non-human
immunoglobulin. For the most part,
humanized antibodies are human immunoglobulins (recipient antibody) in which
residues from a CDR of the
recipient are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit
having the desired specificity, affinity, and capacity. In some instances, Fv
FR residues of the human
immunoglobulin are replaced by corresponding non-human residues. Furthermore,
humanized antibodies may
comprise residues which are found neither in the recipient antibody nor in the
imported CDR or framework
sequences. These modifications are made to further refine and maximize
antibody performance. In general, the
humanized antibody will comprise substantially all of at least one, and
typically two, variable domains, in which
all or substantially all of the CDR regions correspond to those of a non-human
immunoglobulin and all or
substantially all of the FR regions are those of a human immunoglobulin
sequence. The humanized antibody
t 5 optimally also wilt 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. Oo. Struct. Biol., 2:593-596 (1992). The
humanized antibody includes a
PRIMATIZEDT"' antibody wherein the antigen-bindingregion ofthe antibody is
derived from an antibody produced
by immunizing macaque monkeys with the antigen of interest.
"Single-chain Fv" or "sFv" antibody fragments comprise the VH and V~ domains
of antibody, wherein
these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a
polypeptide linker between the VH and 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. 1 l3, Rosenburg
and Moore eds., Springer-Verlag, New York, pp. 269-315 ( 1994).
The term "diabodies" refers to small antibody fragments with two antigen-
binding sites, which fragments
comprise a heavy-chain variable domain (V") connected to a light-chain
variable domain (V~) in the same
polypeptide chain (VH - V~). By using a linker that is too short to allow
pairing between the two domains on the
same chain, the domains are forced to pair with the complementary domains of
another chain and create two
antigen-binding sites. Diabodies are described more fully in, for example, EP
404,097; WO 93/11161; and
Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 ( 1993).
An "isolated" antibody is one which has been identified and separated 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
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blue or, preferably, silver stain. Isolated antibody includes the antibody in
situ within recombinant cells since at
least one component of the antibody's natural environment will not be present.
Ordinarily, however, isolated
antibody will be prepared by at least one purification step.
The word "label" when used herein refers to a detectable compound or
composition which is conjugated
directly or indirectly to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself
(e.g., radioisotope labels or fluorescent labels) or, in the case of an
enrymatic label, may catalyze chemical
alteration of a substrate compound or composition which is detectable.
Radionuclides that can serve as detectable
labels include, for example, I-131,1-123, I-125, Y-90, Re-188, Re-186, At-211,
Cu-67, Bi-212, and Pd-109.
By "solid phase" is meant a non-aqueous matrix to which the antibody of the
present invention can adhere.
Examples of solid phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled
pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene,
polyvinyl alcohol and silicones. In
certain embodiments, depending on the context, the solid phase can comprise
the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography column). This
term also includes a discontinuous solid
phase of discrete particles, such as those described in U.S. Patent No.
4,275,149.
I S 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 PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261,
PR0246 or PR0317 polypeptide or antibody thereto and, optionally, a
chemotherapeutic agent) to a mammal. The
components of the liposome are commonly arranged in a bilayer formation,
similar to the lipid arrangement of
biological membranes.
As used herein, the term "immunoadhesin" designates antibody-like molecules
which combine the binding
specificity of a heterologous protein (an "adhesin") with the effector
functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of an amino acid
sequence with the desired binding
specificity which is other than the antigen recognition and binding site of an
antibody (i.e., is "heterologous"), and
an immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a
contiguous amino acid sequence comprising at least the binding site of a
receptor or a ligand. The immunoglobulin
constant domain sequence in the immunoadhesin may be obtained from any
immunoglobuiin, such as IgG-1, IgG-2,
IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), lgE, IgD or IgM.
II. Compositions and Methods of the Invention
A. Full-length PRO 187. PR0533. PR0214. PR0240. PR021 t . PR0230. PR0261,
PR0246 and PR0317
polvoeptides
The present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides
referred to in the present application as PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261,
PR0246 and PR0317. In particular, cDNA encoding PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230,
PR0261, PR0246 and PR0317 polypeptides has been identified and isolated, as
disclosed in further detail in the
Examples below. It is noted that proteins produced in separate expression
rounds may be given different PRO
numbers but the UNQ number is unique for any given DNA and the encoded
protein, and will not be changed.
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acid substitutions can be the result of replacing one amino acid with another
amino acid having similar structural
and/or chemical properties, such as the replacement of a leucine with a
serine, i.e., conservative amino acid
replacements. Insertions or deletions may optionally be in the range of about
1 to S amino acids. The variation
allowed may be determined by systematically making insertions, deletions or
substitutions of amino acids in the
sequence and testing the resulting variants for activity exhibited by the full-
length or mature native sequence.
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 and PR0317
polypeptide
fragments are provided herein. Such fragments may be truncated at the N-
terminus or C-terminus, or may lack
internal residues, for example, when compared with a full length native
protein. Certain fragments lack amino acid
residues that are not essential for a desired biological activity of the
PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide.
PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317
fragments may
be prepared by any of a number of conventional techniques. Desired peptide
fragments may be chemically
synthesized. An alternative approach involves generating PR0187, PR0533,
PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 fragments by enzymatic digestion, e.g., by
treating the protein with an
enzyme known to cleave proteins at sites defined by particular amino acid
residues, or by digesting the DNA with
suitable restriction enzymes and isolating the desired fragment. Yet another
suitable technique involves isolating
and amplifying a DNA fragment encoding a desired polypeptide fragment, by
polymerise chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA fragment are
employed at the 5' and 3' primers in the
PCR. Preferably, PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
polypeptide fragments share at least one biological and/or immunological
activity with the native PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide.
In particular embodiments, conservative substitutions of interest are shown in
Tabie t 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 l, or as further
described below in reference to amino acid
classes, are introduced and the products screened.
Table 1
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
Ite (I) leu; val; met; ala;
phe;
norleucine leu
Leu (L) norleucine; ile; val;
met; ala; phe ile
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Lys (K) arg; gln; asn arg
Met (M) leu; phe; ile leu
Phe (F) leu; val; ile; ala; leu
tyr
Pro (P) ala ala
Ser(S) thr thr
Thr(T) ser ser
Trp (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
polypeptide are accomplished by
selecting substitutions that differ significantly in their effect on
maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain. Naturally occurring residues are
divided into groups based on common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile;
(2) neutral hydrophilic: cys, ser, thr;
(3) acidic: asp, glu;
(4) basic: asn, gln, his, lys, arg;
(5) residues that influence chain orientation: gly, pro; and
(6) aromatic: trp, tyr, phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another class.
Such substituted residues also may be introduced into the conservative
substitution sites or, more preferably, into
the remaining (non-conserved) sites.
The variations can be made using methods known in the art such as
oligonucleotide-mediated (site-
directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed
mutagenesis [Carter et al., Nucl.
Acids Res.. 13:4331 ( 1986); Zoller et al., Nucl. Acids Res.. 10:6487 (
1987)], cassette mutagenesis [Wells et al.,
Gene. 34:315 (1985)], restriction selection mutagenesis [Wells etal., Philos.
Trans. R. Soc. London SerA 317:415
(1986)] or other known techniques can be performed on the cloned DNA to
produce the PR0187, PR0533,
PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 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, gfycine, serine, and cysteine. Alanine is
typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the beta-carbon
and is less likely to alter the main-
chain conformation of the variant [Cunningham and Wells, Science. 244: 1081-
1085 (1989)]. Alanine is also
typically preferred because it is the most common amino acid. Further, it is
frequently found in both buried and
exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N.Y.);
Chothia, J. Mol. Biol.. 150:1 (1976)].
If alanine substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
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C. Modifications of PR0187, PR0533. PR0214, PR0240. PR021 I PR0230 PR0261
PR0246 and
PR0317
Covalent modifications of PROl87, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246
and PR0317 are included within the scope of this invention. One type of
covalent modification includes reacting
targeted amino acid residues ofa PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or
PR0317 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 187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or
PR0317. Derivatization with bifunctional agents is useful, for instance, for
crosslinking PROl87, PR0533,
PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 to a water-insoluble
support matrix or surface
l0 for use in the method for purifying anti-PR0187, anti-PR0533, anti-PR0214,
anti-PR0240, anti-PR0211, anti-
PR0230, anti-PR0261, anti-PR0246 or anti-PR0317 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 ~spartyl residues, respectively, hydroxylation of proline and
lysine, phosphorylation of hydroxyl
groups ofseryl orthreonyl residues, methylation ofthe a-amino groups of
lysine, arginine, and histidine side chains
[T.E. Creighton, Proteins: Structure and Molecular Properties W.H. Freeman &
Co., San Francisco, pp. 79-86
(1983)], acetylation of the N-terminal amine, and amidation of any C-terminal
carboxyl group.
Another type of covalent modification of the PR0212, PR0290, PR0341, PR0535,
PR0619, PR0717,
PR0809, PR0830, PR0848, PR0943, PRO1005, PR01009, PR01025, PROi030, PR01097,
PR01107,
PRO111 (, PR01153, PR01182, PROI 184, PR01187, PRO 1281,
PR023,PR039,PR0834,PR01317,PR01710,
PR02094, PR02145 or PR02198 polypeptide included within the scope of this
invention comprises altering the
native glycosylation pattern ofthe polypeptide. "Altering the native
glycosylation pattern" is intended for purposes
herein to mean deleting one or more carbohydrate moieties found in native
sequence PRO 187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 (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 187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317. In addition, the phrase includes qualitative changes in the
glycosylation ofthe native proteins,
involving a change in the nature and proportions of the various carbohydrate
moieties present.
Addition ofglycosylation sites to the PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261,
PR0246 or PR0317 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 PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or
PR0317 (for O-linked
glycosylation sites). The PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 orPR0317
amino acid sequence may optionally be altered through changes at the DNA
level, particularly by mutating the
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DNA encoding the PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
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 PR0187,
PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide is by chemical or
enrymatic 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 PROl87, PR0533, PR0214,
PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide may be accomplished chemically or
enrymatically or by
mutational substitution ofcodons 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.
Biophvs., 259:52 ( 1987) and by Edge etal., Anal. Biochem.. l 18:131 { 1981 ).
Enrymatic cleavage ofcarbohydrate
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 PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230,
PR0261, PR0246 or PR0317 comprises linking the PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230,
PR0261, PR0246 or PR0317 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 PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
of the
present invention may also be modified in a way to form a chimeric molecule
comprising PR0187, PR0533,
PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 fused to another,
heterologous potypeptide
or amino acid sequence.
In one embodiment, such a chimeric molecule comprises a fusion of the PR0187,
PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 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 PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317. The
presence ofsuch epitope-tagged forms ofthe PR0187, PR0533, PR0214, PR0240,
PR021 I, PR0230, PR0261,
PR0246 or PR0317 can be detected using an antibody against the tag
polypeptide. Also, provision of the epitope
tag enables the PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317 to be
readily purified by affinity purification using an anti-tag antibody or
another type of affinity matrix that binds to
the epitope tag. Various tag polypeptides and their respective antibodies are
well known in the art. Examples
include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its
antibody 12CA5 [Field et al., Mol. Cell. Biol.. 8:2159-2165 (1988)]; the c-myc
tag and the 8F9, 3C7, 6E(0, G4,
B7 and 9E 10 antibodies thereto [Evan et al., Molecular and Cellular Biolosy.
5:3610-3616 ( 1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al.,
Protein Eneineerin7, 3(6):547-553 ( 1990)].
Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnoloey.
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. Bioi.
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Chem.. 266:15163-15 I 66 ( I 991 )]; 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 PR0187, PR0533,
PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 with an
immunoglobulin or a particular
S 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 fvsions preferably include
the substitution of a soluble (transmembrane domain deleted or inactivated)
form of a PR0187, PR0533, PR0214,
PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317 polypeptide in place of at
least one variable region
within an Ig molecule. In a particularly preferred embodiment, the
immunoglobulin fusion includes the hinge, CH2
and CH3, or the hinge, CH 1, CH2 and CH3 regions of an IgG 1 molecule. For the
production of immunoglobulin
fusions see also, US Patent No. 5,428,130 issued June 27, 1995.
D. Preparation of PR0187. PR0533, PR0214 PR0240 PR0211 PR0230 PR0261 PR0246
and
PR0317 Polvoeptides
The description below relates primarily to production of PR0187, PR0533,
PR0214, PR0240, PR021 l,
PR0230, PR0261, PR0246 or PR0317 by culturing cells transformed or transfected
with a vector containing
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
nucleic acid. It is, of
course, contemplated that alternative methods, which are well known in the
art, may be employed to prepare
PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317. For
instance, the
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
sequence, or portions
thereof, may be produced by direct peptide synthesis using solid-phase
techniques [see, e.g., Stewart et al., Solid-
Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA ( 1969);
Merrifield, J. Am. Chem. Soc.. 85:2149-
2154 ( 1963)). In virro 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 PR0187, PR0533,
PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 may be chemically synthesized separately and
combined using chemical
or enrymatic methods to produce the full-length PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230,
PR0261, PR0246 or PR0317.
a. Isolation of DNA Encodine a PR0187 PR0533 PR0214 PR0240 PR0211 PR0230
PR0261. PR0246 or PR0317 Polvpeptide
DNA encoding PRO I 87, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317
may be obtained from a cDNA library prepared from tissue believed to possess
the PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 mRNA and to express it at a
detectable level.
Accordingly, human PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
DNA can be conveniently obtained from a cDNA library prepared from human
tissue, such as described in the
Examples. PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or
PR0317 encoding
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gene may also be obtained from a genomic library or by oiigonucleotide
synthesis.
Libraries can be screened with probes (such as antibodies to the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide, or oligonucleotides of
at least about 20-80 bases)
designed to identify the gene of interest orthe protein encoded by it.
Screening the cDNA or genomic library with
S the selected probe may be conducted using standard procedures, such as
described in Sambrook et al., Molecular
Clonine: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1989). An alternative means
to isolate the gene encoding PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or
PR0317 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
oiigonucleotide sequences
selected as probes should be of sufficient length and sufficiently unambiguous
that false positives are minimized.
The oligonucleotide is preferably labeled such that it can be detected upon
hybridization to DNA in the library being
screened. Methods of labeling are well known in the art, and include the use
of radiolabels like''-P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions, including moderate
stringency and high stringency,
are provided in Sambrook et al., supra.
Sequences identified in such library screening methods can be compared and
aligned to other known
sequences deposited and available in public databases such as GenBank or other
private sequence databases.
Sequence identity (at either the amino acid or nucleotide level) within
defined regions of the molecule or across the
full-length sequence can be determined using methods known in the art and as
described herein.
Nucleic acid having protein coding sequence may be obtained by screening
selected cDNA or genomic
libraries using the deduced amino acid sequence disclosed herein for the first
time, and, if necessary, using
conventional primer extension procedures as described in Sambrook et al.,
supra, to detect precursors and
processing intermediates of mRNA that may not have been reverse-transcribed
into cDNA.
b. Selection and Transformation of Host Cells
Host cel is are transfected or transformed with expression or cloning vectors
described herein for PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 production
and cultured in
conventional nutrient media modified as appropriate for inducing promoters,
selecting transformants, oramplifying
the genes encoding the desired sequences. The culture conditions, such as
media, temperature, pH and the like, can
be selected by the skilled artisan without undue experimentation. In general,
principles, protocols, and practical
techniques for maximizing the productivity of cell cultures can be found in
Mammalian Cell Biotechnoloev: a
Practical Approach. M. Butler, ed. (IRL Press, 1991 ) and Sambrook et al.,
supra.
Methods of eukaryotic cell transfection and prokaryotic cell transformation
are known to the ordinarily
skilled artisan, for example, CaCI:, CaPO,, liposome-mediated and
electroporation. Depending on the host cell
used, transformation is performed using standard techniques appropriate to
such cells. The calcium treatment
employing calcium chloride, as described in Sambrook et al., supra, or
electroporation is generally used for
prokaryotes. Infection with Agrobacterium mmefaciens is used for
transformation of certain plant cells, as
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described by Shaw et al., Gene. 23:315 ( 1983) and WO 89/05859 published 29
June 1989. For mammalian cells
without such cell walls, the calcium phosphate precipitation method of Graham
and van der Eb, ViroloQV, 52:456-
457 ( 1978) can be employed. General aspects of mammalian cell host system
transfections have been described
in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried
out according to the method of Van
Solingen et al., J. Bact., 130:946 ( 1977) and Hsiao et al., Proc. Natl. Acad.
Sci. (USA), 76:3829 (1979). However,
other methods for introducing DNA into cells, such as by nuclear
microinjection, electroporation, bacterial
protoplast fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various
techniques fortransforming mammalian cells, see, Keown et al., Methods in
Enzvmoloey, 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. toll.
Various E. toll strains are publicly
available, such as E. toll K12 strain MM294 (ATCC 31,446); E. toll X1776 (ATCC
31,537); E. toll strain W3110
(ATCC 27,325) and E. Coli strain KS 772 (ATCC 53,635). Other suitable
prokaryotic host cells include
Enterobacteriaceae such as Escherichia, e.g., E. toll, Enterobacter, Erwinia,
Klebsiella, Proteus, Salmonella, e.g.,
Salmonella tvphimurium, Serratia, e.g., Serratia marcescans, and Shigella, as
wel I as Bacilli such as B. subtilis and
B. licheniformis (e.g., B. licheniformis 4 l P disclosed in DD 266,710
published 12 April 1989), Pseudomonas such
as P. aeruginosa, and Streptomyces. These examples are illustrative rather
than limiting. Strain W3110 is one
particularly preferred host or parent host because it is a common host strain
for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts of
proteolytic enrymes. For example, strain
W3110 may be modified to effect a genetic mutation in the genes encoding
proteins endogenous to the host, with
examples of such hosts including E. toll W3110 strain I A2, which has the
complete genotype tonA ; E. toll W3110
strain 9E4, which has the complete genotype tonA ptr3; E. toll W3110 strain
27C7 (ATCC 55,244), which has the
complete genotype tonA ptr3 phoA EIS (argF lac)169 degP ompTkan'; E. toll
W3110 strain 37D6, which has
the complete genotype tonA ptr3 phoA El.i (argh'-lac) 169 degP ompT rbs7 ilvG
kan ; E. toll W3110 strain 4OB4,
which is strain 37D6 with a non-kanamycin resistant degP deletion mutation;
and an E. toll strain having mutant
periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August
1990. Alternatively, in vitro methods
of cloning, e.g., PCR or other nucleic acid polymerase reactions, are
suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable cloning or
expression hosts for PR0187-, PR0533-, PR0214-, PR0240-, PR0211-, PR0230-,
PR0261-, PR0246- or
PR0317-encoding vectors. Saccharomyces cerevisiae is a commonly used lower
eukaryotic host microorganism.
Others include Schizosaccharomyces pombe (Beach and Nurse, Nature. 290: 140 [
1981 ]; EP 139,383 published
2 May 1985); Kltryveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al.,
Bio/Technoloey, 9: 968-975 ( 1991 ))
such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.
Bacteriol.. 737 [1983]), K. Jragilis
(ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.
waltii (ATCC 56,500), K.
drosophilarum (ATCC 36,906; Vanden Berg et al., Bio/Technoloey, 8:135 (1990)),
K. thermotolerans, and K.
marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol.. 28:265-
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278 [ I 988]); 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, Penicillium,
Tolypocladium (WO 91/00357
published 10 January 1991 ), and Aspergillus hosts such as A. nidulans
(Ballance et al., Biochem. Bioohys. Res.
S Commun.. 112:284-289 [1983]; Tilburn et al., Gene. 26:205-221 [ 1983];
Yelton et al., Proc. Natl. Acad. Sci. USA
81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479
[1985]). Methylotropic yeasts are
suitable herein and include, but are not limited to, yeast capable of growth
on methanol selected from the genera
consisting of Harrsenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula. A list of
specific species that are exemplary of this class of yeasts may be found in C.
Anthony, The Biochemistry of
Methvlotrophs, 269 (1982).
Suitable host cells for the expression of glycosyiated PR0187, PR0533, PR0214,
PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 are derived from multicellular organisms.
Examples of invertebrate cells
include insect cells such as Drosophiia S2 and Spodoptera Sf9, as well as
plant cells. Examples of useful
mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells.
More specific examples include
monkey kidney CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human
embryonic kidney line (293
or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen
Virol., 36:59 (1977)); Chinese
hamster ovary cel Is/-DHFR (CHO), Urlaub and Chasin, Proc. Natl. Acad. Sci.
USA. 77:4216 ( 1980)); mouse sertoli
cells (TM4, Mather, Biol. R~rod., 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.
Selection and Use of a Reolicable Vector
The nucleic acid (e.g., cDNA or genom is DNA) encoding PRO 187, PR0533, PR02 (
4, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 may be inserted into a replicable vector for
cloning (amplification of the
DNA) or for expression. Various vectors are publicly available. The vector
may, for example, be in the form of
a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid
sequence may be inserted into the vector
by a variety of procedures. In general, DNA is inserted into an appropriate
restriction endonuclease sites) using
techniques known in the art. Vector components generally include, but are not
limited to, one or more of a signal
sequence, an origin of replication, one or more marker genes, an enhancer
element, a promoter, and a transcription
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 PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317
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 187-, PR0533-, PR0214-, PR0240-, PR02 I I -, PR0230-, PR0261-, PR0246-
or PR0317-encodingDNA
that is inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected, for example,
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from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable
enterotoxin II leaders. For yeast
secretion the signal sequence may be, e.g., the yeast invertase leader, alpha
factor leader (including Saccharomyces
and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No.
5,010,182), or acid phosphatase leader,
the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or
the signal described in WO 90113646
S 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~c plasmid origin
is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus,
VSV or BPV) are useful for cloning
vectors in mammalian cells.
Expression and cloning vectors will typically contain a selection gene, also
termed a selectable marker.
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, or (c) supply critical nutrients
not available from complex media, e.g., the gene encoding D-alanine racemase
for Bacilli.
An example of suitable selectable markers for mammalian cells are those that
enable the identification of
cells competent to take up the PR0187-, PR0533-, PR0214-, PR0240-, PR0211-,
PR0230-, PR0261-, PR0246-
or PR0317-encoding nucleic acid, such as DHFR or thymidine kinase. An
appropriate host cell when wild-type
DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and
propagated as described by
Urlaub et al., Proc. Natl. Acad. Sci. USA. 77:4216 ( 1980). A suitable
selection gene for use in yeast is the trp 1
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 trp 1 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 PR0187-, PR0533-,
PR0214-, PR0240-, PR0211-, PR0230-, PR0261-, PR024b-orPR0317-
encodingnucleicacidsequencetodirect
mRNA synthesis. Promoters recognized by a variety of potential host cells are
well known. Promoters suitable
for use with prokaryotic hosts include the (i-lactamase and lactose promoter
systems [Chang et al., Nature. 275:615
( 1978); Goeddel et al., Nature, 281:544 ( 1979)],alkalinephosphatase, 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 PR0187, PR0533, PR0214,
PR0240, PR021 l, PR0230,
PR0261, PR0246 or PR0317.
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 Res.. 7:149 ( I 968); Holland, Biochemistry. 17:4900 (
1978)], such as enolase, glyceraldehyde-
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3-phosphate dehydrogenase. hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphogiycerate 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.
PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317
transcription
from vectors in mammalian host cells is controlled, for example, by promoters
obtained from the genomes of
viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July
1989), adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin
promoter, and from heat-shock promoters, provided such promoters are
compatible with the host cell systems.
Transcription ofa DNA encodingthePR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261,
PR0246 or PR0317 by higher eukaryotes may be increased by inserting an
enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp,
that act on a promoter to increase its
transcription. Many enhancer sequences are now known from mammalian genes
(globin, elastase, albumin, a-
fetoprotein, and insulin). Typically, however, one will use an enhancer from a
eukaryotic cell virus. Examples
include the SV40 enhancer on the late side of the replication origin (bp 100-
270), the cytomegalovirus early
promoter enhancer, the polyoma enhancer on the late side ofthe replication
origin, and adenovirus enhancers. The
enhancer may be spliced into the vector at a position 5' or 3'to the PR0187,
PR0533, PR0214, PR0240, PR021 l,
PR0230, PR0261, PR0246 or PR0317 coding sequence, but is preferably located at
a site 5' from the promoter.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated
cells from other multicellular organisms) will also contain sequences
necessary for the termination oftranscription
and for stabilizing the mRNA. Such sequences are commonly available from the
5' and, occasionally 3',
untranslated regionsofeukaryotic orviral DNAs or cDNAs. These regions contain
nucleotide segments transcribed
as polyadenylated fragments in the untranslated portion of the mRNA encoding
PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317.
Still other methods, vectors, and host cells suitable for adaptation to the
synthesis of PR0187, PR0533,
PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 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.
d. Detectins Gene Amplification/Ex~ression
Gene amplification and/or expression may be measured in a sample directly, for
example, by conventional
Southern blotting, Northern blotting to quantitate the transcription of mRNA
[Thomas, Proc. Natl. Acad. Sci. USA.
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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
immunohistochemical staining and/or assay of
sample fluids may be either monoclonal or polyclonal, and may be prepared in
any mammal. Conveniently, the
antibodies may be prepared against a native sequence PROl87, PR0533, PR0214,
PR0240, PR0211, PR0230,
PR0261, PR0246 or PR0317 polypeptide or against a synthetic peptide based on
the DNA sequences provided
herein or against an exogenous sequence fused to PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230,
PR0261, PR0246 or PR0317 DNA and encoding a specific antibody epitope.
e. Purification of Polvpeptide
Forms of PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or
PR0317 may
be recovered from culture medium or from host cell lysates. If membrane-bound,
it can be released from the
membrane using a suitable detergent solution (e.g., Triton-X 100) or by
enzymatic cleavage. Cells employed in
expression of PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317 can be
disrupted by various physical or chemical means, such as freeze-thaw cycling,
sonication, mechanical disruption,
or cell lysing agents.
It may be desired to purify PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230,
PR0261, PR0246
or PR0317 from recombinant cell proteins or polypeptides. The following
procedures are exemplary of suitable
purification procedures: by fractionation on an ion-exchange column; ethanol
precipitation; reverse phase HPLC;
chromatography on silica or on a canon-exchange resin such as DEAF;
chromatofocusing; SDS-PAGE; ammonium
sulfate precipitation; gel filtration using, for example, Sephadex G-75;
protein A Sepharose columns to remove
contaminants such as lgG; and metal chelating columns to bind epitope-tagged
forms of the PR0187, PR0533,
PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317. Various methods of
protein purification
may be employed and such methods are known in the art and described for
example in Deutscher, Methods in
Enzymoloey, I 82 (1990); Scopes, Protein Purification: Princieles and
Practice, Springer-VerIag,New York ( 1982).
The purification steps) selected will depend, for example, on the nature of
the production process used and the
particular PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or
PR0317 produced.
E. Amplification ofGenes Encodine the PROI 87. PR0533 PR0214 PR0240 PR0211
PR0230
PR0261, PR0246 or PR0317 Polypeptides in Tumor Tissues and Cell Lines
The present invention is based on the identification and characterization of
genes that are amplified in
certain cancer cells.
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The genome of prokaryotic and eukaryotic organisms is subjected to two
seemingly conflicting
requirements. One is the preservation and propagation of DNA as the genetic
information in its original form, to
guarantee stable inheritance through multiple generations. On the other hand,
cells or organisms must be able to
adapt to lasting environmental changes. The adaptive mechanisms can include
qualitative or quantitative
modifications of the genetic material. Qualitative modifications include DNA
mutations, in which coding
sequences are altered resulting in a structurally and/or functionally
different protein. Gene amplification is a
quantitative modification, whereby the actual number ofcomplete coding
sequence, i.e., a gene, increases, leading
to an increased number of available templates for transcription, an increased
number oftranslatable transcripts, and,
ultimately, to an increased abundance of the protein encoded by the amplified
gene.
The phenomenon of gene amplification and its underlying mechanisms have been
investigated in vitro in
several prokaryotic and eukaryotic culture systems. The best-characterized
example of gene amplification involves
the culture of eukaryotic cells in medium containing variable concentrations
of the cytotoxic drug methotrexate
(MTX). MTX is a folic acid analogue and interferes with DNA synthesis by
blocking the enzyme dihydrofolate
reductase (DHFR). During the initial exposure to low concentrations of MTX
most cells (>99.9%) will die. A
I 5 small number of cells survive, and are capable of growing in increasing
concentrations of MTX by producing large
amounts of DHFR-RNA and protein. The basis of this overproduction is the
amplification of the single DHFR
gene. The additional copies of the gene are found as extrachromosomal copies
in the foam of small, supernumerary
chromosomes (double minutes) or as integrated chromosomal copies.
Gene amplification is most commonly encountered in the development of
resistance to cytotoxic drugs
(antibiotics for bacteria and chemotherapeutic agents for eukaryotic cells)
and neoplastic transformation.
Transformation of a eukaryotic cell as a spontaneous event or due to a viral
or chemicaUenvironmental insult is
typically associated with changes in the genetic material of that cell. One of
the most common genetic changes
observed in human malignancies are mutations of the p53 protein. p53 controls
the transition of cells from the
stationary (G 1 ) to the replicative (S) phase and prevents this transition in
the presence of DNA damage. In other
words, one of the main consequences of disabling p53 mutations is the
accumulation and propagation of DNA
damage, i.e., genetic changes. Common types of genetic changes in neoplastic
cells are, in addition to point
mutations, amplifications and gross, structural alterations, such as
translocations.
The amplification of DNA sequences may indicate specific functional
requirement as illustrated in the
DHFR experimental system. Therefore, the amplification of certain oncogenes in
malignancies points toward a
causative role of these genes in the process of malignant transformation and
maintenance of the transformed
phenotype. This hypothesis has gained support in recent studies. For example,
the bcl-2 protein was found to be
amplified in certain types ofnon-Hodgkin's lymphoma. This protein inhibits
apoptosis and leads to the progressive
accumulation of neoplastic cells. Members of the gene family of growth factor
receptors have been found to be
amplified in various types of cancers suggesting that overexpression of these
receptors may make neoplastic cells
less susceptible to limiting amounts of available growth factor. Examples
include the amplification ofthe androgen
receptor in recurrent prostate cancer during androgen deprivation therapy and
the amplification of the growth factor
receptor homologue ERB2 in breast cancer. Lastly, genes involved in
intracellular signaling and control of cell
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cycle progression can undergo amplification during malignant transformation.
This is illustrated by the
amplification of the bcl-I and ras genes in various epithelial and lymphoid
neoplasms.
These earlier studies illustrate the feasibility of identifying amplified DNA
sequences in neoplasms,
because this approach can identify genes important for malignant
transformation. The case of ERB2 also
demonstrates the feasibility from a therapeutic standpoint, since transforming
proteins may represent novel and
specific targets for tumor therapy.
Several different techniques can be used to demonstrate amplified genomic
sequences. Classical
cytogenetic analysis of chromosome spreads prepared from cancer cells is
adequate to identify gross structural
alterations, such as translocations, deletions and inversions. Amplified
genomic regions can only be visualized,
if they involve large regions with high copy numbers or are present as
extrachromosomal material. While
cytogenetics was the first techniqueto demonstrate the consistent association
of specific chromosomal changes with
particular neopiasms, it is inadequate for the identification and isolation of
manageable DNA sequences. The more
recently developed technique of comparative genomic hybridization (CGH) has
illustrated the widespread
phenomenon ofgenomic amplification in neoplasms. Tumor and normal DNA are
hybridized simultaneously onto
metaphases of normal cells and the entire genome can be screened by image
analysis for DNA sequences that are
present in the tumor at an increased frequency. ( WO 93/ 18,186; Gray et al.,
Radiation Res.. 137:275-289 [ 1994]).
As a screening method, this type of analysis has revealed a large number of
recurring amplicons (a stretch of
amplified DNA) in a variety of human neoplasms. Although CGH is more sensitive
than classical cytogenetic
analysis in identifying amplified stretches of DNA, it does not allow a rapid
identification and isolation of coding
sequences within the amplicon by standard molecular genetic techniques.
The most sensitive methods to detect gene amplification are polymerase chain
reaction (PCR)-based
assays. These assays utilize very small amount of tumor DNA as starting
material, are exquisitely sensitive, provide
DNA that is amenable to further analysis, such as sequencing and are suitable
for high-volume throughput analysis.
The above-mentioned assays are not mutually exclusive, but are frequently used
in combination to identify
amplifications in neoplasms. While cytogenetic analysis and CGH represent
screening methods to survey the entire
genome for amplified regions, PCR-based assays are most suitable for the final
identification of coding sequences,
i.e., genes in amplified regions.
According to the present invention, such genes have been identified by
quantitative PCR (S. Gelmini et
al., Clin. Chem., 43:752 [ 1997]), by comparing DNA from a variety of primary
tumors, including breast, lung,
colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis,
ovary, uterus, etc. tumor, or tumor cell lines,
with pooled DNA from healthy donors. Quantitative PCR was performed using a
TaqMan instrument (ABI).
Gene-specific primers and fluorogenic probes were designed based upon the
coding sequences of the DNAs.
HumanlungcarcinomacelllinesincludeA549(SRCC768),Calu-1 (SRCC769),Calu-
6(SRCC770),H157
(SRCC771), H441 (SRCC772), H460 (SRCC773), H522 (SRCC832), H810 (SRCC833),
SKMES-1 (SRCC774)
and SW900 (SRCC775), all available from ATCC. Primary human lung tumor cells
usually derive from
adenocarcinomas, squamous cell carcinomas, large cell carcinomas, non-small
cell carcinomas, small cell
carcinomas, and broncho alveolar carcinomas, and include, for example, SRCC724
(adenocarcinoma, abbreviated
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as "AdenoCa")(LT1), SRCC725 (squamous cell carcinoma, abbreviated as
"SqCCa)(LTIa), SRCC726
(adenocarcinoma)(LT2), SRCC727 (adenocarcinoma)(LT3), SRCC728
(adenocarcinoma)(LT4), SRCC729
(squamous cell carcinoma)(LT6), SRCC730 (adeno/squamous cell carcinoma)(LT7),
SRCC825
(adenocarcinoma)(LT8), SRCC731 (adenocarcinoma)(LT9), SRCC732 (squamous cell
carcinoma)(LT10),
SRCC733 (squamous cell carcinoma){LT11), SRCC734 (adenocarcinoma)(LT12),
SRCC735 (adeno/squamous
cell carcinoma)(LT13), SRCC736 (squamous cell carcinoma)(LT
15),SRCC737(squamouscellcarcinoma)(LT16),
SRCC738 (squamous cell carcinoma)(LTl7), SRCC739 (squamous cell carcinoma)(LTl
8), SRCC740 (squamous
cell carcinoma)(LT19), SRCC741 (lung cell carcinoma, abbreviated as
"LCCa")(LT21), SRCC811
(adenocarcinomaxLT22), SRCC887 (squamous cell carcinoma) (LT26), SRCC888
(adeno-BAC carcinoma)
(LT27), SRCC889 (squamous cell carcinoma) (LT28), SRCC890 (squamous cell
carcinoma) (LT29), SRCC891
(adenocarcinoma) (LT30), SRCC892 (squamous cell carcinoma) (LT31), SRCC894
(adenocarcinoma) (LT33).
Also included are human lung tumors designated SRCC 1125 [HF-000631 ], SRCC
1129 [HF-000643], SRCC 1133
[HF-000840] and SRCC1135 [HF-000842].
Colon cancer cell lines include, for example, ATCC cell lines SW480
(adenocarcinoma, SRCC776),
I S SW620 (lymph node metastasis of colon adenocarcinoma, SRCC777), Co1o320
(carcinoma, SRCC778), Co1o205
(carcinoma, SRCC828), HCC2998 (carcinoma, SRCC830), HT29 (adenocarcinoma,
SRCC779), HM7 (carcinoma,
SRCC780),KM 12 (carcinoma,SRCC831 ),CaWiDr (adenocarcinoma, SRCC781 ), HCTI 5
(carcinoma,SRCC829),
HCTI 16 (carcinoma, SRCC782), SKCO1 (adenocarcinoma, SRCC783), SW403
(adenocarcinoma, SRCC784),
LS 174T (carcinoma, SRCC785), and HM7 (a high mucin producing variant of ATCC
colon adenocarcinoma cell
line LS 174T, SRCC780, obtained from Dr. Robert Warren, UCSF). Primary colon
tumors include colon
adenocarcinomas designated CT1 (SRCC751), CT2 (SRCC742), CT3 (SRCC743), CT4
(SRCC752), CTS
(SRCC753), CT6 (SRCC754), CT7 {SRCC755), CT8 (SRCC744), CT9 (SRCC756), CT10
(SRCC745), CTI 1
(SRCC757), CT12 (SRCC746), CT 14 (SRCC747), CT 15 (SRCC748),CT 16 (SRCC749),CT
17 (SRCC750), CT 18
(SRCC758), CT25 (adenocarcinoma, SRCC912), CT28 (adenocarcinoma, SRCC915) CT35
(adenocarcinoma,
SRCC921 ). Also included are human colon tumor centers designated SRCC 1051
[HF-000499], SRCC 1052 [HF-
000539], SRCC 1053 [HF-000575], SRCC 1054 [HF-000698], SRCC 1060 [HF-000756],
SRCC 1144 [HF-000789]
and SRCCI 14$[HF-000811] and human colon tumor margin designated SRCC1059 [HF-
000755].
Human breast carcinoma cell lines include, for example, HBL100 (SRCC759),
MB435s (SRCC760),
T47D (SRCC761), MB468(SRCC762), MB175 (SRCC763), MB361 (SRCC764), BT20
(SRCC765), MCF7
(SRCC766), and SKBR3 (SRCC767), and human breast tumor center designated SRCC
1057 [HF-000545]. Also
included are human breast tumors designated SRCC 1094, SRCC 1095, SRCC 1096,
SRCC 1097, SRCC 1098,
SRCC 1099, SRCC 1100 and SRCC I 1 O I .
Human kidney tumor centers include SRCC989 [HF-000611 ] and SRCC 1014 [HF-
000613]. Lymph node
tumor includes SRCC1004 [HF-000854]. Rectal tumor margin includes SRCC82 [HF-
000551]. Testis tumor
center includes SRCCI001 [HF-000733] and testis tumor margin SRCC999 [HF-
000716].
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F. Tissue Distribution
The results of the gene amplification assays herein can be verified by further
studies, such as, by
determining mRNA expression in various human tissues.
As noted before, gene amplification andlor gene expression in various tissues
may be measured by
conventional Southern blotting, Northern blotting to quantitate the
transcription of mRNA (Thomas, Proc. Natl.
Acad. Sci. USA. 77:5201-5205 [ 1980]), dot blotting (DNA analysis), or insitu
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.
Gene expression in various tissues, alternatively, may be measured by
immunological methods, such as
immunohistochemical staining of tissue sections and assay of cell culture or
body fluids, to quantitate directly the
expression of gene product. Antibodies useful for immunohistochemical staining
and/or assay of sample fluids may
be either monoclonal or polyclonal, and may be prepared in any mammal.
Conveniently, the antibodies may be
prepared against a native sequence PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246
or PR0317 polypeptide or against a synthetic peptide based on the DNA
sequences provided herein or against
exogenous sequence fused to sequence PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261,
PR0246 or PR0317 DNA and encoding a specific antibody epitope. General
techniques for generating antibodies,
and special protocols for Northern blotting and in situ hybridization are
provided hereinbelow.
G. Chromosome Manpine
If the amplification of a given gene is functionally relevant, then that gene
should be amplified more than
neighboring genomic regions which are not important for tumor survival. To
test this, the gene can be mapped to
a particular chromosome, e.g., by radiation-hybrid analysis. The amplification
level is then detet~rnined at the
location identif ed, and at neighboring genomicregion. Selective or
preferential amplif canon at the genomic region
to which the gene has been mapped is consistent with the possibility that the
gene amplification observed promotes
tumor growth or survival. Chromosome mapping includes both framework and
epicenter mapping. For further
details see e.g., Stewart et al., Genome Research. 7:422-433 ( 1997).
H. Antibody Binding_Studies
The results of the gene amplification study can be further verified by
antibody binding studies, in which
the ability ofanti-PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211,
anti-PR0230, anti-PR0261,
anti-PR0246 or anti-PR0317 antibodies to inhibit the expression of the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptides on tumor (cancer) cells
is tested. Exemplary
antibodies include polyclonal, monoclonal, humanized, bispecific, and
heteroconjugate antibodies, the preparation
of which will be described hereinbelow.
Antibody binding studies may be can led out in any known assay method, such as
competitive binding
assays, direct and indirect sandwich assays, and immunoprecipitation assays.
Zola, Monoclonal Antibodies: A
Manual of Techniaues, pp.147-158 (CRC Press, Inc., 1987).
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Competitive binding assays rely on the ability of a labeled standard to
compete with the test sample
analyte for binding with a limited amount of antibody. The amount of target
protein (encoded by a gene amplified
in a tumor cell) in the test sample is inversely proportional to the amount of
standard that becomes bound to the
antibodies. To facilitate determining the amount of standard that becomes
bound, the antibodies preferably are
insolubilized before or after the competition, so that the standard and
analyte that are bound to the antibodies may
conveniently be separated from the standard and analyte which remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to
a different immunogenic
portion, or epitope, of the protein to be detected. In a sandwich assay, the
test sample analyze is bound by a first
antibody which is immobilized on a solid support, and thereafter a second
antibody binds to the analyte, thus
forming an insoluble three-part complex. See, e.g., U.S. Patent No. 4,376,110.
The second antibody may itself be
labeled with a detectable moiety (direct sandwich assays) or may be measured
using an anti-immunoglobulin
antibody that is labeled with a detectable moiety (indirect sandwich assay).
For example, one type of sandwich
assay is an ELISA assay, in which case the detectable moiety is an enzyme.
For immunohistochemistry, the tumor sample may be fresh or frozen or may be
embedded in paraffin and
fixed with a preservative such as formalin, for example.
Cell-Based Tumor Assavs
Cell-based assays and animal models for tumors (e.g., cancers) can be used to
verify the findings of the
gene amplification assay, and further understand the relationship between the
genes identified herein and the
development and pathogenesis of neoplastic cell growth. The role of gene
products identified herein in the
development and pathology of tumor or cancer can be tested by using primary
tumor cells or cells lines that have
been identified to amplify the genes herein. Such cells include, for example,
the breast, colon and lung cancer cells
and cell tines listed above.
In a different approach, cells of a cell type known to be involved in a
particular tumor are transfected with
the cDNAs herein, and the ability of these cDNAs to induce excessive growth is
analyzed. Suitable cells include,
for example, stable tumor cells lines such as, the B 104-1-1 cell line (stable
NIH-3T3 cell line transfected with the
neu protooncogene) and ras-transfected NIH-3T3 cells, which can be transfected
with the desired gene, and
monitored for tumorogenic growth. Such transfected cell lines can then be used
to test the ability of poly- or
monoclonal antibodies or antibody compositions to inhibit tumorogenic cell
growth by exerting cytostatic or
cytotoxic activity on the growth of the transformed cells, or by mediating
antibody-dependent cellular cytotoxicity
(ADCC). Cells transfected with the coding sequences of the genes identified
herein can further be used to identify
drug candidates for the treatment of cancer.
In addition, 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]).
Animal Models
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A variety of well known animal models can be used to further understand the
role of the genes identified
herein in the development and pathogenesis of tumors, and to test the efficacy
of candidate therapeutic agents,
including antibodies, and other antagonists of the native polypeptides,
including small molecule antagonists. The
in vivo nature of such models makes them particularly predictive of responses
in human patients. Animal models
of tumors and cancers (e.g., breast cancer, colon cancer, prostate cancer,
lung cancer, etc.) include both non-
recombinant and recombinant (transgenic) animals. Non-recombinant animal
models include, for example, rodent,
e.g., murine models. Such models can be generated by introducing tumor cells
into syngeneic mice using standard
techniques, e.g., subcutaneous injection, tail vein injection, spleen
implantation, intraperitoneal implantation,
implantation under the renal capsule, or orthopin implantation, e.g., colon
cancer cells implanted in colonic tissue.
(See, e.g., PCT publication No. WO 97/33551, published September 18, 1997).
Probably the most often used animal species in oncological studies are
immunodeficient mice and, in
particular, nude mice. The observation that the nude mouse with hypo/aplasia
could successfully act as a host for
human tumor xenografts has lead to its widespread use for this purpose. The
autosomal recessive 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, RII1 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 Oncology Research. E. Boven and B. Winograd, eds., CRC
Press, Inc., 1991.
The cells introduced into such animals can be derived from known tumor/cancer
cell lines, such as, any
of the above-listed tumor cell lines, and, for example, the B104-1-I cell line
(stable NIH-3T3 cell line transfected
with the neu protooncogene); ras-transfected NIH-3T3 cells; Caco-2 (ATCC HTB-
37); a moderately well-
differentiated grade II human colon adenocarcinoma cell line, HT-29 (ATCC HTB-
38), or from tumors and cancers.
Samples of tumor or cancer cells can be obtained from patients undergoing
surgery, using standard 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
subdetmal implants. In this location, the inoculum is deposited between the
lower part of the dermal connective
tissue and the s.c. tissue. Boven and Winograd ( 1991 ), supra.
Animal models of breast cancer can be generated, for example, by implanting
rat neuroblastoma cells
(from which the neu oncogen was initially isolated), or neu-transformed NIH-
3T3 cells into nude mice, essentially
as described by Drebin et al., PNAS 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
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Too et al., Cancer Research. 55:681-684 ( 1995). This model is based on the so-
called "METAMOUSE" sold by
Anticancer, Inc. (San Diego, California).
Tumors that arise in animals can be removed and cultured in vitro. Cells from
the in vitro cultures can then
be passaged to animals. Such tumors can serve as targets for further testing
or drug screening. Alternatively, the
tumors resulting from the passage can be isolated and RNA from pre-passage
cells and cells isolated after one or
more rounds of passage analyzed for differential expression of genes of
interest. Such passaging techniques can
be perfotlned with any known tumor or cancer cell fines.
For example, Meth A, CMS4, CMSS, CMS21, and WEHI-164 are chemically induced
fibrosarcomas of
BALB/c female mice (DeLeo et al., J. Exn. Med., 146:720 [1977)), which provide
a highly controllable model
system for studying the anti-tumor activities of various agents (Palladino et
al., J. Immunol., 138:4023-4032
[ 19$7)). 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 10x10°
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 ofthe lung
(SCCL). This tumor can be introduced in normal mice upon injection of tumor
fragments from an affected mouse
or of cells maintained in culture (Zupi et al., Br. J. Cancer. 41 auppl. 4:309
[ 1980]), and evidence indicates that
tumors can be started from injection of even a single cell and that a very
high proportion of infected tumor cells
survive. For further information about this tumor model see, Zacharski,
Haemostasis. 16:300-320 ( 1986)).
One way of evaluating the efficacy of a test compound in an animal model is
implanted tumor is to
measure the size of the tumor before and after treatment. Traditionally, the
size of implanted tumors has been
measured with a slide caliper in two or three dimensions. The measure limited
to two dimensions does not
accurately reflect the size of the tumor, therefore, it is usually converted
into the corresponding volume by using
a mathematical formula. However, the measurement of tumor size is very
inaccurate. The therapeutic effects of
a drug candidate can be better described as treatment-induced growth delay and
specific growth delay. Another
important variable in the description of tumor growth is the tumor volume
doubling time. Computer programs for
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 ofthe genes
identified herein into the genome of animals of interest, using standard
techniques for producing transgenic animals.
Animals that can serve as a target fortransgenic manipulation include, without
limitation, mice, rats, rabbits, guinea
pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees
and monkeys. Techniques known
in the art to introduce a transgene into such animals include pronucleic
microinjection (Hoppe and Wanger, U.S.
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Patent No. 4,873,191); retrovirus-mediated gene transfer into germ lines
(e.g., Van der Putten et al., Proc. Natl.
_Acad. Sci. USA. $2:6148-615 [ 1985]); gene targeting in embryonic stem cells
(Thompson et al., Cell, 56:313-321
[1989]); electroporation of embryos (Lo, Mol. Cell Biol.. 3:1803-1814 [1983]);
sperm-mediated gene transfer
(Lavitrano et al., C~ 57:717-73 [1989]). For review, see, for example, U.S.
Patent No. 4,736,866.
For the purpose of the present invention, transgenic animals include those
that carry the transgene only
in part of their cells ("mosaic animals"). The transgene can be integrated
either as a single transgene, or in
concatamers, e.g., head-to-head or head-to-tail tandems. Selective
introduction of a transgene into a particular cell
type is also possible by following, for example, the technique of Lasko et
al., Proc. Natl. Acad. Sci. USA. 89:6232-
636 ( 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.
Alternatively, "knock out" animals can be constructed which have a defective
or altered gene encoding
a PRO 187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide identified
herein, as a result of homologous recombination between the endogenous gene
encoding the polypeptide and altered
genomic DNA encoding the same polypeptide introduced into an embryonic cell of
the animal. For example,
cDNA encoding a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317
polypeptide can be used to clone genomic DNA encoding that polypeptide in
accordance with established
techniques. A portion ofthe genomic DNA encoding a particular PR0187, PR0533,
PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide can be deleted or replaced with
another gene, such as a gene
encoding a selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered
flanking DNA (both at the 5' and 3' ends) are included in the vector [see
e.g., Thomas and Capecchi, Cell. 51:503
( 1987) for a description of homologous recombination vectors]. The vector is
introduced into an embryonic stem
cell line (e.g., by electroporation) and cells in which the introduced DNA has
homologously recombined with the
endogenous DNA are selected [see e.g., Li et al., C~ 69:915 ( 1992)]. The
selected cells are then injected into a
biastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras
[see e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach E. J. Robertson, ed. (IRL,
Oxford, 1987), pp. 113-152]. A
chimeric embryo can then be implanted into a suitable pseudopregnant female
foster animal and the embryo brought
to term to create a "knock out" animal. Progeny harboring the homologously
recombined DNA in their germ cells
can be identified by standard techniques and used to breed animals in which
all cells of the animal contain the
homologously recombined DNA. Knockout animals can be characterized for
instance, by their ability to defend
against certain pathological conditions and by their development of
pathological conditions due to absence of the
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
polypeptide.
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
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the feline oral squamous cell carcinoma (SCC). Feline oral SCC is a highly
invasive, malignant tumor that is the
most common oral malignancy of cats, accounting for over 60% of the oral
tumors reported in this species. It rarely
metastasizes to distant sites, although this low incidence of metastasis may
merely be a reflection of the short
survival times for cats with this tumor. These tumors are usually not amenable
to surgery, primarily because of the
anatomy of the feline oral cavity. At present, there is no effective treatment
for this tumor. Prior to entry into the
study, each cat undergoes complete clinical examination, biopsy, and is
scanned by computed tomography (CT).
Cats diagnosed with sublingual oral squamous cell tumors are excluded from the
study. The tongue can become
paralyzed as a result of such tumor, and even if the treatment kills the
tumor, the animals may not be able to feed
themselves. Each cat is treated repeatedly, over a longer period of time.
Photographs of the tumors will be taken
daily during the treatment period, and at each subsequent recheck. After
treatment, each cat undergoes another CT
scan. CT scans and thoracic radiograms are evaluated every 8 weeks thereafter.
The data are evaluated for
differences in survival, response and toxicity as compared to control groups.
Positive response may require
evidence of tumor regression, preferably with improvement of quality of life
and/or increased life span.
In addition, other spontaneous animal tumors, such as fibrosarcoma,
adenocarcinoma, lymphoma,
chrondroma, leiomyosarcoma of dogs, cats, and baboons can also be tested. Of
these mammary adenocarcinonia
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.
K. Screenine Assavs for Drue Candidates
Screening assays for drug candidates are designed to identify compounds that
bind or complex with the
polypeptides encoded by the genes identified herein, or otherwise interfere
with the interaction of the encoded
polypeptides with other cellular proteins. Such screening assays will include
assays amenable to high-throughput
screening of chemical libraries, making them particularly suitable for
identifying small molecule drug candidates.
Small molecules contemplated include synthetic organic or inorganic compounds,
including peptides, preferably
solublepeptides,(poly)peptide-immunoglobulinfusions, 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
ft~agments. The assays can be performed in a variety of formats, including
protein-protein binding assays,
biochemical screening assays, immunoassays and cell based assays, which are
well characterized in the art.
All assays are common in that they call for contacting the drug candidate with
a polypeptide encoded by
a nucleic acid identified herein under conditions and for a time sufficient to
allow these two components to interact.
In binding assays, the interaction is binding and the complex formed can be
isolated or detected in the
reaction mixture. In a particular embodiment, the 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
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component, which may be labeled by a detectable label, to the immobilized
component, e.g., the coated surface
containing the anchored component. When the reaction is complete, the non-
reacted components are removed, e.g.,
by washing, and complexes anchored on the solid surface are detected. When the
originally non-immobilized
component carries a detectable label, the detection of label immobilized on
the surface indicates that complexing
occurred. Where the originally non-immobilized component does not carry a
label, complexing can be detected,
for example, by using a labeled antibody specifically binding the immobilized
complex.
If the candidate compound interacts with but does not bind to a particular
PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide encoded by a gene
identified herein, its
interaction with that polypeptide can be assayed by methods well known for
detecting protein-protein interactions.
Such assays include traditional approaches, such as, cross-linking, co-
immunoprecipitation, and co-purification
through gradients or chromatographic columns. In addition, protein-protein
interactions can be monitored by using
a yeast-based genetic system described by Fields and co-workers [Fields and
Song, Nature, 340:245-246 ( 1989);
Chien et al., Proc. Natl. Acad. Sci. USA. 88: 9578-9582 (1991)] as disclosed
by Chevray and Nathans, Proc. Natl.
Acad. Sci. USA. 89:5789-5793 (1991)]. Many transcriptional activators, such as
yeast GAL4, consist of two
physically discrete modular domains, one acting as the DNA-binding domain,
while the other one functioning as
the transcription activation domain. The yeast expression system described in
the foregoing publications (generally
referred to as the "two-hybrid system") takes advantage of this property, and
employs two hybrid proteins, one in
which the target protein is fused to the DNA-binding domain of GAL4, and
another, in which candidate activating
proteins are fused to the activation domain. The expression of a GAL1-lacZ
reporter gene under control of a
GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-
protein interaction. Colonies
containing interacting polypeptides are detected with a chromogenic substrate
for [i-galactosidase. A complete kit
(MATCHMAKERT"') for identifying protein-protein interactions between two
specific proteins using the two
hybrid technique is commercially available from Clontech. This system can also
be extended to map protein
domains involved in specific protein interactions as well as to pinpoint amino
acid residues that are crucial for these
interactions.
Compounds that interfere with the interaction of a PR018?-, PR0533-, PR0214-,
PR0240-, PR0211-,
PR0230-, PR0261-, PR0246- or PR0317-encoding gene identified herein and other
infra- or extracelluiar
components can be tested as follows: usually a reaction mixture is prepared
containing the product of the amplified
gene and the infra- or extracellular component under conditions and for a time
allowing for the interaction and
binding of the two products. To test the ability of a test compound to inhibit
binding, the reaction is run in the
absence and in the presence of the test compound. In addition, a placebo may
be added to a third reaction mixture,
to serve as positive control. The binding (complex formation) between the test
compound and the infra- or
extracellular component present in the mixture is monitored as described
hereinabove. The formation of a complex
in the control reactions) but not in the reaction mixture containing the test
compound indicates that the test
compound interferes with the interaction of the test compound and its reaction
partner.
To assay for antagonists, the PRO 187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246
or PR0317 polypeptide may be added to a cell along with the compound to be
screened for a particular activity and
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the ability of the compound to inhibit the activity of interest in the
presence of the PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide indicates that
the compound is an
antagonist to the PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261,
PR0246 or PR0317
polypeptide. Alternatively, antagonists may be detected by combining the PROI
87, PR0533, PR0214, PR0240,
PR021 I, PR0230, PR0261, PR0246 or PR0317 polypeptide and a potential
antagonist with membrane-bound
PR0187, PR0533, PR0214, PR0240, PR021 !, PR0230, PR0261, PR0246 or PR0317
polypeptide receptors
or recombinant receptors under appropriate conditions for a competitive
inhibition assay. The PRO 187, PR0533,
PR0214; PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide can be
labeled, such as by
radioactivity, such that the number of PROI 87, PR0533, PR0214, PR0240, PR021
l, PR0230, PR0261, PR0246
or PR0317 polypeptide molecules bound to the receptor can be used to determine
the effectiveness ofthe potential
antagonist. The gene encoding the receptor can be identified by numerous
methods known to those ofskill in the
art, for example, ligand panning and FACS sorting. Coligan et a/., Current
Protocols in Immun.. 1~2~: Chapter 5
(1991). Preferably, expression cloning is employed wherein polyadenylated RNA
is prepared from a cell
responsive to the PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
polypeptide and a cDNA library created from this RNA is divided into pools and
used to transfect COS cells or
other cells that are not responsive to the PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261,
PR0246 or PR0317 polypeptide. Transfected cells that are grown on glass slides
are exposed to labeled PR0187,
PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317 polypeptide.
The PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide
can be labeled by a
variety of means including iodination or inclusion of a recognition site for a
site-specific protein kinase. Following
fixation and incubation, the slides are subjected to autoradiographic
analysis. Positive pools are identified and sub-
pools are prepared and re-transfected using an interactive sub-pooling and re-
screening process, eventually yielding
a single clone that encodes the putative receptor.
As an alternative approach for receptor identification, labeled PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 poiypeptide can be photoaffinity-
linked with cell membrane or
extract preparations that express the receptor molecule. Cross-linked material
is resolved by PAGE and exposed
to X-ray film. The labeled complex containing the receptor can be excised,
resolved into peptide fragments, and
subjected to protein micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used
to design a set of degenerate oligonucleotide probes to screen a cDNA library
to identify the gene encoding the
putative receptor.
In another assay for antagonists, mammalian cells or a membrane preparation
expressing the receptor
would be incubated with labeled PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or
PR0317 polypeptide in the presence of the candidate compound. The ability of
the compound to enhance or block
this interaction could then be measured.
More specific examples of potential antagonists include an oligonucleotide
that binds to the fusions of
immunoglobulin with PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
polypeptide, and, in particular, antibodies including, without limitation,
poly- and monoclonal antibodies and
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antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and
chimeric or humanized versions of such
antibodies or fragments, as well as human antibodies and antibody fragments.
Alternatively, a potential antagonist
may be a closely related protein, for example, a mutated form of the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide that recognizes the
receptor but imparts no effect,
thereby competitively inhibiting the action of the PR0187, PR0533, PR0214,
PR0240, PR021 l, PR0230,
PR0261, PR0246 or PR0317 polypeptide.
Another potential PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or
PR0317 polypeptide antagonistisan antisenseRNA or DNA construct prepared using
antisense technology, where,
e.g., an antisense RNA or DNA molecule acts to block directly the translation
of mRNA by hybridizing to targeted
mRNA and preventing protein translation. Antisense technology can be used to
control gene expression through
triple-helix formation or antisense DNA or RNA, both of which methods are
based on binding of a polynucleotide
to DNA or RNA. For example, the 5' coding portion of the polynucleotide
sequence, which encodes the mature
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
poiypeptides herein, is
used to design an antisense RNA oligonucleotide of from about 10 to 40 base
pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the gene
involved in transcription (triple helix - see
Lee et al., Nucl. Acids Res.. 6:3073 (1979); Cooney et al., Science. 241: 456
(1988); Dervan et al., Science.
251:1360 ( 1991 )), thereby preventingtranscriptionand the production ofthe
PRO 187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide. The antisense RNA
oligonucleotide hybridizes to
the mRNA in vivo and blocks translation of the mRNA molecule into the PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide (antisense - Okano,
Neurochem.. 56:560 (1991 );
Ol~odeoxvnucleotides as Antisense Inhibitors of Gene Exaression (CRC Press:
Boca Raton, FL, 1988). The
oligonucleotides described above can also be delivered to cells such that the
antisense RNA or DNA may be
expressed in vivo to inhibit production of the PR0187, PR0533, PR0214, PR0240,
PR021 l, PR0230, PR0261,
PR0246 or PR0317 polypeptide. When antisense DNA is used,
oligodeoxyribonucleotides derived from the
translation-initiation site, e.g., between about -10 and +10 positions of the
target gene nucleotide sequence, are
preferred.
Antisense RNA or DNA molecules are generally at least about 5 bases in length,
about I 0 bases in length,
about 15 bases in length, about 20 bases in length, about 25 bases in length,
about 30 bases in length, about 35 bases
in length, about 40 bases in length, about 45 bases in length, about 50 bases
in length, about 55 bases in length,
about 60 bases in length, about 65 bases in length, about 70 bases in length,
about 75 bases in length, about 80 bases
in length, about 85 bases in length, about 90 bases in length, about 95 bases
in length, about 100 bases in length,
or more.
Potential antagonists include small molecules that bind to the active site,
the receptor binding site, or
growth factor or other relevant binding site of the PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230,
PR0261, PR0246 or PR0317 polypeptide, thereby blocking the normal biological
activity of the PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 polypeptide.
Examples of small
molecules include, but are not limited to, small peptides or peptide-like
molecules, preferably soluble peptides, and
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synthetic non-peptidyl organic or inorganic compounds.
Ribozymes are enzymatic RNA moleculescapable ofcatalyzing the specific
cleavage of RNA. Riborymes
act by sequence-specific hybridization to the complementary target RNA,
followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within a potential RNA target can be
identified by known techniques. For further
details see, e.g., Rossi, Current Biolosy, 4:469-471 (1994), and PCT
publication No. WO 97/33551 (published
September 18, 1997).
Nucleic acid molecules in triple-helix formation used to inhibit transcription
should be single-stranded and
composed of deoxynucleotides. The base composition of these otigonucleotides
is designed such that it promotes
triple-helix formation via Hoogsteen base-pairing rules, which generally
require sizeable stretches of purines or
pyrimidines on one strand of a duplex. For further details see, e.g., PCT
publication No. WO 97/33551, supra.
These small molecules can be identified by any one or more of the screening
assays discussed hereinabove
and/or by any other screening techniques well known for those skilled in the
art.
L. Compositions and Methods for the Treatment of Tumors
The compositions useful in the
treatmentoftumorsassociatedwiththeamplificationofthegenesidentified
herein include, without limitation, antibodies, small organic and inorganic
molecules, peptides, phosphopeptides,
antisense and riboryme molecules, triple helix molecules, etc. that inhibit
the expression and/or activity ofthe target
gene product.
For example, antisense RNA and RNA molecule act to directly block the
translation of mRNA by
hybridizing to targeted mRNA and preventing protein translation. When
antisense DNA is used,
oligodeoxyribonucleotides derived from the translation initiation site, e.g.,
between about -10 and +10 positions
of the target gene nucleotide sequence, are preferred.
RibozymesareenzymaticRNAmoleculescapableofcatalyzingthespecificcleavageofRNA.
Ribozymes
act by sequence-specific hybridization to the complementary target RNA,
followed by endonucleolytic cleavage.
Specific ribozyme cleavage sites within a potential RNA target can be
identified by known techniques. For further
details see, e.g., Rossi, Current Bioloev. 4:469-471 (1994), and PCT
publication No. WO 97/33551 (published
September 18, 1997).
Nucleic acid molecules in triple helix formation used to inhibit transcription
should be single-stranded and
composed of deoxynucleotides. The base composition of these oligonucleotides
is designed such that it promotes
triple helix formation via Hoogsteen base pairing rules, which generally
require sizeable stretches of purines or
pyrimidines on one strand of a duplex. For further details see, e.g., PCT
publication No. WO 97/33551, supra.
These molecules can be identified by any or any combination of the screening
assays discussed
hereinabove and/or by any other screening techniques well known for those
skilled in the art.
M. Antibodies
Some of the most promising drug candidates according to the present invention
are antibodies and antibody
fragments which may inhibit the production or the gene product of the
amplified genes identified herein and/or
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reduce the activity of the gene products.
Polyclonal Antibodies
Methods of preparing polyclonal antibodies are known to the skilled artisan.
Polyclonal antibodies can
be raised in a mammal, for example, by one or more injections of an immunizing
agent and, if desired, an adjuvant.
Typically, the immunizing agent and/or adjuvant will be injected in the mammal
by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the PR0187,
PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR0317 polypeptide or a fusion protein thereof. It
may be useful to conjugate the
immunizing agent to a protein known to be immunogenic in the mammal being
immunized. Examples of such
l0 immunogenic proteins include but are not limited to keyhole limpet
hemocyanin, serum albumin, bovine
thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may
be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
synthetictrehalose dicorynomycolate). The
immunization protocol may be selected by one skilled in the art without undue
experimentation.
2. Monoclonal Antibodies
The anti-PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-
PR0230, anti-PR0261,
anti-PR0246 oranti-PR0317 antibodies may, alternatively, be monoclonal
antibodies. Monoclonal antibodies may
be prepared using hybridoma methods, such as those described by Kohler and
Milstein, Nature, 256:495 (1975).
In a hybridoma method, a mouse, hamster, or other appropriate host animal, is
typically immunized with an
immunizing agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically
bind to the immunizing agent. Alternatively, the lymphocytes may be immunized
in vitro.
The immunizingagentwill typically include the PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230,
PR0261, PR0246 or PR0317 polypeptide, including fragments, or a fusion protein
of such protein or a fragment
thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if
cells of human origin are desired, or
spleen cells or lymph node cells are used if non-human mammalian sources are
desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma
cell [coding, Monoclonal Antibodies: Principles and Practice Academic Press, (
1986) pp. 59-103]. Immortalized
cell lines are usually transformed mammalian cells, particularly myeloma cells
of rodent, bovine and human origin.
Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may
be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit the growth
or survival of the unfused,
immortalized cells. For example, if the parental cells lack the enryme
hypoxanthine guanine phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support
stable high level expression of
antibody by the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More
preferred immortalized cell lines are murine myeloma lines, which can be
obtained, for instance, from the Salk
Institute Cell Distribution Center, San Diego, California and the American
Type Culture Collection (ATCC),
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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 Techniaues and Aaplications, 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 PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261,
PR0246 or PR0317. Preferably, the binding specificity ofmonoclonal antibodies
produced by the hybridoma cells
is determined by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme
linked immunoabsorbent assay (ELISA). Such techniques and assays are known in
the art. The binding affinity
of the monoclonal antibody can, for example, be determined by the Scatchard
analysis of Munson and Pollard,
Anal. Biochem.. 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned
by limiting dilution
procedures and grown by standard methods [coding, supra). Suitable culture
media for this purpose include, for
example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium.
Alternatively, the hybridoma cells may
be grown in vivo as ascites in a mammal.
I 5 The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture medium
orascites fiuidbyconventionalimmunoglobulin 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, 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
immunoglobulin coding sequence all
or part of the coding sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide
can be substituted for the constant domains of an antibody of the invention,
or can be substituted for the variable
domains of one antigen-combining site of an antibody of the invention to
create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent
antibodies are well
known in the art. For example, one method involves recombinant expression of
immunoglobulin light chain and
modified heavy chain. The heavy chain is truncated generally at any point in
the Fc region so as to prevent heavy
chain crosslinking. Alternatively, the relevant cysteine residues are
substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce
fragments thereof, particularly, Fab fragments, can be accomplished using
routine techniques known in the art.
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3. Human and Humanized Antibodies
The anti-PRO 187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR021 1, anti-
PR0230, anti-PR0261,
anti-PR0246 or anti-PR0317 antibodies may further comprise humanized
antibodies or human antibodies.
Humanized forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains
or fragments thereof (such as Fv, Fab, Fab', F(ab'), or other antigen-binding
subsequences of antibodies) which
contain minimal sequence derived from non-human immunoglobulin. Humanized
antibodies include human
immunoglobulins (recipient antibody) in which residues from a complementary
determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit
having the desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human
immunoglobulin are replaced by corresponding non-human residues. Humanized
antibodies may also comprise
residues which are found neither in the recipient antibody nor in the imported
CDR or framework sequences. In
general, the humanized antibody will comprise substantially all of at least
one, and typically two, variable domains,
in which all or substantially all of the CDR regions con espond 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
I S 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. Op. Struct. Biol.. 2_:593-596 ( 1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized
antibody has one or more amino acid residues introduced into it from a source
which is non-human. These non-
human amino acid residues are often referred to as "import" residues, which
are typically taken from an "import"
variable domain. Humanization can be essentially performed following the
method of Winter and co-workers
[Jones et al., Nature. 321:522-525 ( 1986); Riechmann et al., Nature. 332:323-
327 ( 1988); Verhoeyen et al.,
Science, 239:1534-1536 ( 1988)], by substituting rodent CDRs or CDR sequences
for the corresponding sequences
of a human antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (U.S. Patent No.
4,816,567), wherein substantially less than an intact human variable domain
has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues are
substituted by residues from analogous
sites in rodent antibodies.
Human antibodies can also be produced using various techniques known in the
art, including phage display
libraries [Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991); Marks er al.,
J. Mol. Biol.. 222:581 (1991)].
The techniques of Cole et al., and Boerner et al., are also available for the
preparation of human monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Theranv, Alan R.
Liss, p. 77 (1985) and Boetner et al.,
J. Immunol.. 147(1 ):86-95 ( 1991 )]. Similarly, human antibodies can be made
by introducing of human
immunoglobulin loci into transgenic animals, e.g., mice in which the
endogenous immunoglobulin genes have been
partially or completely inactivated. Upon challenge, human antibody production
is observed, which closely
resembles that seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807;
5,545,806; 5,569,825; 5,625,126;
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WO 00/15666 PCT/US99/20594
5,633,425; 5,661,016, and in the following scientific publications: Marks et
al., Bio/Technolo~y, 10:779-783
( 1992); Lonberg et al., Nature. 368:856-859 ( 1994); Morrison, Nature.
368:812-13 ( 1994); Fishwild et al., Nature
Biotechnoloev. 14:845-51 ( 1996); Neuberger, Nature Biotechnoloey, 14:826 (
1996); Lonberg and Huszar, Intern.
Rev. Immunol.. 13:65-93 (1995).
4. Antibody Dependent Enzyme Mediated Prodrue Therapy (ADEPT)
The antibodies of the present invention may also be used in ADEPT by
conjugating the antibody to a
prodrug-activating enzyme which converts a prodrug (e.g., a peptidyl
chemotherapeutic agent, see WO 81 /0l 145)
to an active anti-cancer drug. See, for example, WO 88/07378 and U. S. Patent
No. 4,975,278.
The enzyme component ofthe immunoconjugate useful for ADEPT includes any
enzyme capable ofacting
on a prodrug in such as way so as to convert it into its more active,
cytotoxic form.
Enzymes that are useful in the method ofthis invention include, but are not
limited to, glycosidase, glucose
oxidase, human lysosyme, human glucuronidase, alkaline phosphatase useful for
converting phosphate-containing
prodrugs into free drugs; arylsulfatase useful for converting sulfate-
containing prodrugs into free drugs; cytosine
deaminaseusefulforconvertingnon-toxic5-fluorocytosineintotheanti-cancerdrug5-
fluorouracil;proteases,such
as serratia protease, thermolysin, subtilisin, carboxypeptidases (e.g.,
carboxypeptidase G2 and carboxypeptidase
A) and cathepsins (such as cathepsins B and L), that are useful for converting
peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs that contain
D-amino acid substituents;
carbohydrate-cleaving enzymes such as (i-galactosidase and neuraminidase
useful for converting glycosylated
prodrugs into free drugs; ~3-lactamase useful for converting drugs derivatized
with ~3-lactams into free drugs; and
penicillin amidases, such as penicillin Vamidase or penicillin G amidase,
useful for converting drugs derivatized
at their amine nitrogens with phenoxyacetyl or phenyiacetyl groups,
respectively, into free drugs. Alternatively,
antibodies with enzymatic activity, also known in the art as "abrymes" can be
used to convert the prodrugs of the
invention into free active drugs (see, e.g., Massey, Nature. 328:457-458 (
1987)). Antibody-abzyme conjugates can
be prepared as described herein for delivery of the abzyme to a tumor cell
population.
The enzymes of this invention can be covaiently bound to the anti-PR0187, anti-
PR0533, anti-PR0214,
anti-PR0240, anti-PR0211, anti-PR0230, anti-PR0261, anti-PR0246 or anti-PR0317
antibodies by techniques
well known in the art such as the use ofthe heterobifunctional cross-linking
agents discussed above. Alternatively,
fusion proteins comprising at least the antigen binding region of the antibody
of the invention linked to at least a
functionally active portion of an enryme of the invention can be constructed
using recombinant DNA techniques
well known in the art (see, e.g., Neuberger et al., Nature. 312:604-608
(1984)).
Bis~,ecific 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
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 the
other one is for any
other antigen, and preferably for a cell-surface protein or receptor or
receptor subunit.
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Methods for making bispecific antibodies are known in the art. Traditionally,
the recombinant production
of bispecific antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where
the two heavy chains have different specificities (Milstein and Cuello,
Nature, 305:537-539 [1983]). Because of
the random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a
potential mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The
purification of the correct molecule is usually accomplished by affinity
chromatography steps. Similar procedures
are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al.,
EMBO J.. 10:3b55-3659 ( 1991 ).
Antibody variable domains with the desired binding specificities (antibody-
antigen combining sites) can
be fused to immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy
chain constant domain, comprising at least part of the hinge, CH2, and CH3
regions. It is preferred to have the first
heavy-chain constant region (CH 1 ) containing the site necessary for light-
chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if
desired, the immunoglobulin light
chain, are inserted into separate expression vectors, and are co-transfected
into a suitable host organism. For further
details of generating bispecific antibodies see, for example, Suresh et al.,
Methods in EnzymoloQV, 1311:210 ( 1986).
IS According to another approach described in WO 96/27011, the interface
between a pair of antibody
molecules can be engineered to maximize the percentage of heterodimers which
are recovered from recombinant
cell culture. The preferred interface comprises at least a part of the CH3
region of an antibody constant domain.
In this method, one or more small amino acid side chains from the interface of
the first antibody molecule are
replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory
"cavities" of identical or similar size
to the large side chains) are created on the interface of the second antibody
molecule by replacing large amino acid
side chains with smaller ones (e.g., alanine or threonine). This provides a
mechanism for increasing the yield of
the heterodimer over other unwanted end-products such as homodimers.
Bispecific antibodies can be prepared as full length antibodies or antibody
fragments (e.g., F(ab')2
bispecific antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been
described in the literature. For example, bispecific antibodies can be
prepared using chemical linkage. Brennan
et al., Science. 229:81 ( 1985) describe a procedure wherein intact antibodies
are proteolytically cleaved to generate
F(ab'), fragments. These fragments are reduced in the presence of the dithiol
complexing agent sodium arsenite
to stabilize vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then
converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-
thiol by reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB
derivative to form the bispecific antibody. The bispecific antibodies produced
can be used as agents for the
selective immobilization of enrymes.
Fab' fragments may be directly recovered from E. coli and chemically coupled
to form bispecific
antibodies. Shalaby et al., J. Exn. Med., 175:217-225 (1992) describe the
production of a fully humanized
bispecific antibody F(ab')Z molecule. Each Fab' fragment was separately
secreted from E. coli and subjected to
directed chemical coupling in vitro to form the bispecific antibody. The
bispecific antibody thus fotmted was able
to bind to cells overexpressing the ErbB2 receptor and normal human T cells,
as well as trigger the lytic activity
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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): I 547-1553 ( 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 V" and V~ domains of one fragment are forced to pair with the
complementary V~ and V" domains
of another fragment, thereby forming two antigen-binding sites. Another
strategy for making bispecific antibody
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
polypeptide herein.
Alternatively, an anti-poiypeptide 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 (CD16) so as to focus cellular
defense mechanisms to the cell
expressing the particular polypeptide. Bispecific antibodies may also be used
to localize cytotoxic agents to cells
which express a particular polypeptide. These antibodies possess a polypeptide-
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 polypeptide and further binds tissue factor
(TF).
6. Heteroconjueate Antibodies
Heteroconjugate antibodies are composed of two covatently 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.
7. Effector function en~ineerine
It may be desirable to modify the antibody of the invention with respect to
effector function, so as to
enhance the effectiveness of the antibody in treating cancer, for example. For
example, cysteine residues) may
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be introduced in 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-I 195 ( 1992) and Shopes, J. Immunol.. 148:2918-2922 ( 1992).
Homodimeric antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional cross-
linkers as described in Wolff et al., Cancer
Research. 53:2560-2565 ( 1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may
thereby have enhanced complement lysis and ADCC capabilities. See, Stevenson
et al., Anti-Cancer Drue Desien,
3:219-230 (1989).
8. Immunoconiueates
The invention also pertains to immunoconjugates comprising an antibody
conjugated to a cytotoxic agent
such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin
of bacterial, fungal, plant or animal
origin, or fragments thereof, or a small molecule toxin), or a radioactive
isotope (i.e., a radioconjugate).
Chemotherapeutic agents useful in the generation of such immunoconjugates have
been described above.
Enzymatically active protein toxins and fragments thereof which can be used
include diphtheria A chain,
nonbinding active fragments of diphtheria toxin, cholera toxin, botulinus
toxin, exotoxin A chain (from
Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-
sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolacaamericana proteins(PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, saporin, mitogellin,
restrictocin, phenomycin, enomycin and the
tricothecenes. Small molecule toxins include, for example, caticheamicins,
maytansinoids, palytoxin and CC 1065.
A variety of radionuclides are available for the production of radioconjugated
antibodies. Examples include z'=Bi,
~~~i, ~i~In, 9°Y and ~~Re.
Conjugates of the antibody and cytotoxic agent are made using a variety of
bifunctional protein coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT),bifunctionalderivatives
of imidoesters (such as dimethyl adipimidate HCL), active esters (such as
disuccinimidylsuberate), aldehydes (such
as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives
(such as bis-(p-diazoniumbenzoyøethylenediamine), 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-
labeled 1-isothiocyanatobenzyl-3-
methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating
agent for conjugation of
radionucleotide to the antibody. See, W094/11026.
In another embodiment, the antibody may be conjugated to a "receptor" (such as
streptavidin) for
utilization in tumor pretargeting wherein the antibody-receptor conjugate is
administered to the patient, followed
by removal of unbound conjugate from the circulation using a clearing agent
and then administration of a "ligand"
(e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a
radionucleotide).
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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. Patent Nos. 4,485,045 and
4,544,545. Liposomes with enhanced circulation time are disclosed in U.S.
Patent No. 5,013,556.
Particularly useful liposomes can be generated by the reverse phase
evaporation method with a lipid
composition comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-
PE). Liposomes are extruded through filters of defined pore size to yield
liposomes with the desired diameter. Fab'
fragments ofthe antibody ofthe present invention can be conjugated to the
liposomes as described in Martin etal.,
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).
N. Pharmaceutical Compositions
Antibodies specifically binding the product of an amplified gene identified
herein, as well as other
molecules identified by the screening assays disclosed hereinbefore, can be
administered for the treatment of
tumors, including cancers, in the form of pharmaceutical compositions.
If the protein encoded by the amplified gene is intracellular and whole
antibodies are used as inhibitors,
internalizing antibodies are preferred. However, lipofections or liposomes can
also be used to deliver the antibody,
or an antibody fragment, into cells. 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]).
Therapeutic formulations of the antibody are prepared for storage by mixing
the antibody having the
desired degree of purity with optional pharmaceutically acceptable carriers,
excipients or stabilizers (Remineton's
Pharmaceutical Sciences. 16th edition, Osol, A. ed. [1980]), in the form of
lyophilized formulations or aqueous
solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic
acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium
chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or
benryl alcohol; alkyl parabens such as
methylorpropyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-
cresol); low molecularweight(less
than about 10 residues) polypeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine, giutamine,
asparagine, histidine, arginine, or
lysine; monosaccharides, disaccharides, andothercarbohydrates 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 TWEEN7"',
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PLURONICST" or polyethylene glycol (PEG).
Non-antibody compounds identified by the screening assays of the present
invention can be formulated
in an analogous manner, using standard techniques well known in the art.
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
macroemuisions. Such techniques
are disclosed in Remineton's Pharmaceutical Sciences. Ibth 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.
Sustained-release preparations may be prepared. Suitable examples of sustained-
release preparations
include semipermeable matrices of solid hydrophobic polymers containing the
antibody, which matrices are in the
form of shaped articles, e.g., films or microcapsules. Examples of sustained-
release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No.
3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-
degradable ethylene-vinyl acetate,
degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT T""
(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-glycoiic acid
enable release of molecules for over
100 days, certain hydrogels release proteins for shorter time periods. When
encapsulated antibodies remain in the
body for a long time, they may denature or aggregate as a result of exposure
to moisture at 37°C, resulting in a loss
of biological activity and possible changes in immunogenicity. Rational
strategies can be devised for stabilization
depending on the mechanism involved. For example, if the aggregation mechanism
is discovered to be
intermolecular S-S bond formation through thin-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.
O. Methods of Treatment
It is contemplated that the antibodies and other anti-tumor compounds ofthe
present invention may be used
to treat various conditions, including those characterized by overexpression
and/or activation ofthe amplified genes
identified herein. Exemplary conditions or disorders to be treated with such
antibodies and other compounds,
including, but not limited to, small organic and inorganic molecules,
peptides, antisense molecules, etc., 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);
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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, e.g., antibodies, 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, by intramuscular, intraperitoneal, intracerobrospinal,
subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes, Intravenous
administration of the antibody is preferred.
Other therapeutic regimens may be combined with the administration of the anti-
cancer agents, e.g.,
antibodies of the instant invention. For example, the patient to be treated
with such anti-cancer agents may also
receive radiation therapy. Alternatively, or in addition, a chemotherapeutic
agent may be administered to the
patient. Preparation and dosing schedules for such chemotherapeutic agents may
be used according to
manufacturers' instructions or as determined empirically by the skilled
practitioner. Preparation and dosing
schedules for such chemotherapy are also described in Chemotherapy Service
Ed., M.C. Perry, Williams& Wilkins,
Baltimore, MD ( 1992). The chemotherapeutic agent may precede, or follow
administration of the anti-tumor agent,
e.g., antibody, or may be given simultaneously therewith. The antibody 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 other tumor
associated antigens, such as
antibodies which bind to the ErbB2, EGFR, ErbB3, ErbB4, or vascular
endothelial factor (VEGF). Alternatively,
or in addition, two or more antibodies binding the same or two or more
different antigens disclosed herein 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 antibodies herein are co-administered
with a growth inhibitory agent. For
example, the growth inhibitory agent may be administered first, followed by an
antibody of the present invention.
However, simultaneous administration or administration of the antibody 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, e. g., an antibody
herein will depend on the type of disease to be treated, as defined above, the
severity and course of the disease,
whether the agent is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical
history and response to the agent, and the discretion of the attending
physician. The agent is suitably administered
to the patient at one time or over a series of treatments.
For example, depending on the type and severity of the disease, about 1 ~g/kg
to I S mg/kg (e.g., 0.1-20
mg/kg) of antibody 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 pg/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
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conventional techniques and assays.
P. Articles of Manufacture
In another embodiment of the invention, an article of manufacture containing
materials useful for the
diagnosis or treatment of the disorders described above is provided. The
article of manufacture comprises a
container and a label. Suitable containers include, for example, bottles,
vials, syringes, and test tubes. The
containers may be formed from a variety of materials such as glass or plastic.
The container holds a composition
which is effective for diagnosing or treating the condition and may have a
sterile access port (for example the
container may be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection
needle). The active agent in the composition is usually an anti-tumor agent
capable of interfering with the activity
of a gene product identified herein, e.g., an antibody. 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
I S user standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions
for use.
Q. Diagnosis and Prognosis of Tumors
While cell surface proteins, such as growth receptors overexpressed in certain
tumors are excellent targets
for drug candidates or tumor (e.g., cancer) treatment, the same proteins along
with secreted proteins encoded by
the genes amplified in tumor cells find additional use in the diagnosis and
prognosis of tumors. For example,
antibodies directed against the protein products of genes amplified in tumor
cells can be used as tumor diagnostics
or prognostics.
Forexample, antibodies, including antibody fragments, can be used to
qualitatively or quantitatively detect
the expression of proteins encoded by the amplified genes ("marker gene
products"). The antibody preferably is
equipped with a detectable, e.g., fluorescent label, and binding can be
monitored by light microscopy, flow
cytometry, fluorimetry, or other techniques known in the art. These techniques
are particularly suitable, if the
amplified gene encodes a cell surface protein, e.g., a growth factor. Such
binding assays are performed essentially
as described in section 5 above.
In situ detection of antibody binding to the marker gene products can be
performed, for example, by
immunofluorescence or immunoelectron microscopy. For this purpose, a
histological specimen is removed from
the patient, and a labeled antibody is applied to it, preferably by overlaying
the antibody on a biological sample.
This procedure also allows for determining the distribution of the marker gene
product in the tissue examined. It
will be apparent for those skilled in the art that a wide variety of
histological methods are readily available for in
situ detection.
The following examples are offered for illustrative purposes only, and are not
intended to limit the scope
of the present invention in any way.
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All patent and literature references cited in the present specification are
hereby incorporated by reference
in their entirety.
EXAMPLES
Commercially available reagents referred to in the examples were used
according to manufacturer's
instructions unless otherwise indicated. The source of those cells identified
in the following examples, and
throughout the specification, by ATCC accession numbers is the American Type
Culture Collection, 10801
University Blvd., Manassas, VA 20110-2209. All original deposits referred to
in the present application were made
under the provisions of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for
the Purpose of Patent Procedure and the Regulations thereunder (Budapest
Treaty). This assures maintenance of
a viable culture of the deposit for 30 years from the date of deposit. The
deposit will be made available by ATCC
under the terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc., and ATCC, which
assures permanent and unrestricted availability of the progeny of the culture
of the deposit to the public upon
issuance of the pertinent U.S. patent or upon laying open to the public of any
U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to one
determined by the U.S. Commissioner of
Patents and Trademarks to be entitled thereto according to 35 USC ~ 122 and
the Commissioner's rules pursuant
thereto (including 37 CFR ~ 1.14 with particular reference to 886 OG 638).
Unless otherwise noted, the present invention uses standard procedures of
recombinant DNA technology,
such as those described hereinabove and in the following textbooks: Sambrook
et al., Molecular Clonine: A
Laboratory Manual, Cold Spring Harbor Press N.Y., 1989; Ausubel et al.,
Current Protocols in Molecular BioloQV,
Green Publishing Associates and Wiley Interscience, N.Y., 1989; Innis et al.,
PCR Protocols: A Guide to Methods
and Avolications, Academic Press, Inc., N.Y., 1990; Harlow et al., Antibodies:
A Laboratory Manual. Cold Shrine
Harbor Press. Cold Spring Harbor, 1988; Gait, Oligonucleotide Synthesis. IRL
Press, Oxford, 1984; R.I. Freshney,
Animal Cell Culture 1987; Coligan et al., Current Protocols in Immunoloev.
1991.
EXAMPLE 1
Isolation of eDNA clones Encodine a Human PRO 187
An expressed sequence tag (EST) DNA database ( LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA)
was searched and an EST [#843193] was identified which showed homology to
fibroblast growth factor (FGF-8)
also known as androgen-induced growth factor.
RNA for construction of cDNA libraries was then isolated from human fetal lung
tissue. The cDNA
libraries used to isolate the eDNA clones encoding human PR0187 were
constructed by standard methods using
commercially available reagents such as those from Invitrogen, San Diego, CA.
The cDNA was primed with oligo
dT containing a NotI site, linked with blunt to Sall hemikinased adaptors,
cleaved with Notl, sized appropriately
by get electrophoresis, and cloned in a defined orientation into a suitable
cloning vector (such as pRKB or pRKD;
pRKSB is a precursor of pRKSD that does not contain the Sfil site; see, Holmes
et al., Science, 253:1278-1280
( l 991 )) in the unique XhoI and Notl.
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Oligoziucleotide probes based upon the above described EST sequence were then
synthesized: 1 ) to
identify by PCR a cDNA library that contained the sequence of interest, and 2)
for use as probes to isolate a clone
of the full-length coding sequence for PR0187. Forward and reverse PCR primers
generally range from 20 to 30
nucleotides and are often designed to give a PCR product of about 100-1000 by
in length. The probe sequences
are typically 40-55 by in length. In order to screen several libraries for a
full-length clone, DNA from the libraries
was screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular BioloQV, szzpra, with the
PCR primer pair. A positive library was then used to isolate clones encoding
the gene of interest using the probe
oligonucleotide and one of the primer pairs.
The oligonucleotide probes employed were as follows:
forward PCR primer:
5'-CAGTACGTGAGGGACCAGGGCGCCATGA-3' (SEQ ID N0:3)
reverse PCR primer:
5'-CCGGTGACCTGCACGTGCTTGCCA-3' (SEQ ID N0:4)
hybridization probe:
5'-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3' (SEQ ID NO:S)
A full length clone was identified that contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 26-28 and a stop signal at nucleotide
positions 641-643 (Figure 1, SEQ ID
NO:1). The predicted polypeptide precursor is 205 amino acids long and is
shown in Figure 2 (SEQ ID N0:2)
Analysis of the full-length PR0187 sequence shown in Figure 2 (SEQ ID N0:2)
evidences the presence of an
important polypeptide domain as shown in Figure 2, wherein the location given
for that important polypeptide
domain is approximate as described above. Analysis of the full-length PR0187
sequence evidenced a signal
peptide from about amino acid 1 to about amino acid 22. Clone DNA27864-l 155
has been deposited with ATCC
on October 16, 1997 and is assigned ATCC deposit no. 209375.
An analysis ofthe Dayhoff database (version 35.45 SwissProt 35), usingthe
ALIGN-2 sequence alignment
analysis of the full-length sequence shown in Figure 2 (SEQ ID N0:2),
evidenced 74% sequence identity between
the PR0187 amino acid sequence and human fibroblast growth factor-8 (androgen-
induced growth factor).
EXAMPLE 2
Isolation of cDNA Clones Encodine a Human PR0533
The EST sequence accession number AF007268, a murine fibroblast growth factor
(FGF-15) was used
to search various public EST databases (e.g., GenBank, Dayhoff, etc.). The
search was performed using the
computer program BLAST or BLAST2 [Altschul et al., Methods in Enzvmoloev,
266:460-480 (1996)] as a
comparison of the ECD protein sequences to a 6 frame translation of the EST
sequences. The search resulted in
a hit with GenBank EST AA220994, which has been identified as stratagene NT2
neuronal precursor 937230.
RNA for construction of cDNA libraries was then isolated from human fetal
retina. The cDNA libraries
used to isolate the cDNA clones encoding human PR0533 were constructed by
standard methods using
commercially available reagents such as those from Invitrogen, San Diego, CA.
The cDNA was primed with oligo
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dT containing a NotI site, linked with blunt to Sall hemikinased adaptors,
cleaved with Notl, sized appropriately
by gel electrophoresis, and cloned in a defined orientation into a suitable
cloning vector (such as pRKB or pRKD;
pRKSB is a precursor of pRKSD that does not contain the SfiI site; see, Holmes
et al., Science. 253:1278-1280
( 1991 )) in the unique Xhol and Notl.
Oligonucleotide probes based upon the above described EST sequence were then
synthesized: I ) to
identify by PCR a cDNA library that contained the sequence of interest, and 2)
for use as probes to isolate a clone
of the full-length coding sequence for PR0533. Forward and reverse PCR primers
generally range from 20 to 30
nucleotides and are ofren designed to give a PCR product of about I00-1000 by
in length. The probe sequences
are typically 40-55 by in length. In order to screen several libraries for a
full-length clone, DNA from the Libraries
was screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular Biolosv, supra, with the
PCR primer pair. A positive library was then used to isolate clones encoding
the gene of interest using the probe
oiigonucleotide and one of the primer pairs.
The oligonucleotide probes employed were as follows:
FGFlS.f (forward PCR primer):
5'-ATCCGCCCAGATGGCTACAATGTGTA-3' (SEQ ID N0:8)
FGF 15.r (reverse PCR primer):
5'-CCAGTCCGGTGACAAGCCCAAA-3' (SEQ ID N0:9)
FGFlS.p (hybridization probe):
5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3' (SEQ ID NO:10)
A full length clone was identified that contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 464-466 and a stop signal at
nucleotide positions 11 l2-1114 (Figure 3, SEQ
ID N0:6). The predicted polypeptide precursor is 2l6 amino acids long and is
shown in Figure 4 (SEQ 1D N0:7)
Analysis of the full-length PR0533 sequence shown in Figure 4 (SEQ ID N0:7)
evidences the presence of an
important polypeptide domain as shown in Figure 4, wherein the location given
for that important polypeptide
domain is approximate as described above. Analysis ofthe full-length PR0533
sequence evidences a signal peptide
from about amino acid I to about amino acid 22. Clone DNA49435-1219 has been
deposited with ATCC on
November 21, 1997 and is assigned ATCC deposit no. 209480.
An analysis ofthe Dayhoffdatabase (version 35.45 SwissProt 35), usingthe ALIGN-
2 sequence alignment
analysis of the full-length sequence shown in Figure 4 (SEQ ID N0:7),
evidenced 53% sequence identity between
the PR0533 amino acid sequence and fibroblast growth factor.
EXAMPLE 3
Isolation of cDNA Clones Encoding a Human PR0214
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public EST databases (e.g., GenBank) and a proprietary EST
database (LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST2
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[Altschul et al., Methods in Enzvmolot=v, 266:460-480 ( 1996)] as a comparison
of the ~CD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
cases, 90) or greater that did not encode known proteins were clustered and
assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA28744. In some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences~discussed
above.
Based on the DNA28744 consensus sequence, oligonucleotides were synthesized: l
) 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 PR0214. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about 1-1.5 kbp. In order to screen several libraries for a full-length
clone, DNA from the libraries was
screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular Biolow, 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'-ATTCTGCGTGAACACTGAGGGC-3' (SEQ 1D N0:13)
reverse PCR primer:
5'-ATCTGCTTGTAGCCCTCGGCAC-3' (SEQ ID N0:14)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28744
sequence which had the following nucleotide sequence:
hybridization probe:
S'-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTCTCGATGTGGATGAGTGTGA-3' (SEQ ID
NO:15)
RNA for construction of the cDNA libraries was isolated from human fetal lung
tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a NotI
site, linked with blunt to Sall hemikinased adaptors, cleaved with Notl, sized
appropriately by gel electrophoresis,
and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
of pRKSD that does not contain the Sfil site; see, Holmes et al., Science.
253:1278-1280 ( 1991 )) in the unique Xhol
and NotI sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for a
full-length PR0214 polypeptide (designatedhereinas DNA32286-1191 [Figure 5,
SEQ ID NO: l l ]) and the derived
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protein sequence for that PR0214 polypeptide.
'The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 103-105 and a stop signal at
nucleotide positions 1363-1365 (Figure 5, SEQ
ID NO:11 ). The predicted polypeptide precursor is 420 amino acids long and is
shown in Figure 6 (SEQ ID
N0:12). Analysis of the full-length PR0214 sequence shown in Figure 6 (SEQ ID
N0:12) evidences the presence
of a variety of important polypeptide domains as shown in Figure 6, wherein
the locations given for those important
polypeptide domains are approximate as described above. Analysis of the full-
length PR0214 sequence evidences
the presence of the following: a signal peptide from about amino acid 1 to
about amino acid 29 and a
transmembrane domain from about amino acid 372 to about amino acid 392. Clone
DNA32286-1191 has been
deposited with ATCC on October 16, 1997 and is assigned ATCC deposit no.
209385.
An analysis ofthe Dayhoffdatabase (version35.45 SwissProt 35), using the ALIGN-
2 sequence alignment
analysis of the full-length sequence shown in Figure 6 (SEQ ID N0:12),
evidenced sequence identity between the
PR0214 amino acid sequence and HT protein and/or Fibulin (49% and 38%,
respectively).
EXAMPLE 4
Isolation of cDNA Clones Encoding a Human PR0240 Polvpeptide
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public EST databases (e.g., GenBank) and a proprietary EST
database (L1FESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST2
[Altschul et al., Methods in Enzymoloev, 266:460-480 ( 1996)] as a comparison
of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
cases, 90) or greater that did not encode known proteins were clustered and
assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA30873. In some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed
above.
Based on the DNA30873 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 PR0240. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about I-1.5 kbp. In order to screen several libraries for a full-length
clone, DNA from the libraries was
screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular Bioloev, supra, with the PCR
primer pair. A positive library was then used to isolate clones encoding the
gene of interest using the probe
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oligonucleotide and one of the primer pairs.
PCR primers (forward and reverse) were synthesized:
forward PCR primer:
5'-TCAGCTCCAGACTCTGATACTGCC-3' {SEQ ID N0:18)
reverse PCR primer:
5'-TGCCTTTCTAGGAGGCAGAGCTCC-3' (SEQ ID N0:19)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA30873
sequence which had the following nucleotide sequence:
hybridization probe:
5'-GGACCCAGAAATGTGTCCTGAGAATGGATCTTGTGTACCTGATGGTCCAG-3' (SEQ ID
N0:20)
RNA for construction of the cDNA libraries was isolated from human fetal liver
tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a NotI
site, linked with blunt to SaII hemikinased adaptors, cleaved with Notl, sized
appropriately by gel electrophoresis,
and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
of pRKSD that does not contain the Sfil site; see, Holmes et al., Science.
253:1278-1280 ( 1991 )) in the unique XhoI
and Notl sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for a
full-IengthPR0240polypeptide(designatedhereinasDNA34387-1138[Figure7,SEQ
IDN0:16])and the derived
protein sequence for that PR0240 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 12-l4 and a stop signal at nucleotide
positions 699-701 (Figure 7, SEQ ID
N0:16). The predicted polypeptide precursor is 229 amino acids long and is
shown in Figure 8 (SEQ ID N0:17).
Analysis of the full-length PR0240 sequence shown in Figure 8 (SEQ ID N0:17)
evidences the presence of a
variety of important polypeptide domains as shown in Figure 8, wherein the
locations given for those important
polypeptide domains are approximate as described above. Analysis of the full-
length PR0240 sequence evidences
the presence of the following: a signal peptide from about amino acid 1 to
about amino acid 30 and a
transmembrane domain from about amino acid 198 to about amino acid 212. Clone
DNA34387-1138 has been
deposited with ATCC on September 16, 1997 and is assigned ATCC deposit no.
209260.
An analysisofthe Dayhoffdatabase (version 35.45 SwissProt 35), using the ALIGN-
2 sequence alignment
analysis of the full-length sequence shown in Figure 8 (SEQ ID N0:17),
evidenced sequence identity between the
PR0240 amino acid sequence and the serrate precursor protein from Drosophilia
melanogaster and the C-serrate-1
protein from Gallus gallus (30% and 35%, respectively).
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EXAMPLE 5
Isolation of cDNA Clones Encodine a Human PR0211
The extracelluiar domain (ECD) sequences {including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public EST databases (e.g., Dayhoff, GenBank) and a
proprietary EST database (LIFESEQ~,
Incyte Pharmaceuticals, Palo Alto, CA). The search was performed using the
computer program BLAST or
BLAST2 [Altschul et al., Methods in Enzvmology. 266:460-480 (1996)] as a
comparison of the ECD protein
sequences to a 6 frame translation of the EST sequences. Those comparisons
resulting in a BLAST score of 70 (or
in some cases, 90) or greater that did not encode known proteins were
clustered and assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA28730. In some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed
above.
Based on the DNA28730 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 PR021 I. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about I-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 Bioloav. 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'-AGAGTGTATCTCTGGCTACGC-3' (SEQ ID N0:23)
reverse PCR primer:
5'-TAAGTCCGGCACATTACAGGTC-3' (SEQ ID N0:24)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28730
sequence which had the following nucleotide sequence:
hybridization probe:
5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3' (SEQ ID N0:25)
RNA for construction of the cDNA libraries was isolated from human fetal lung
tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a NotI
site, linked with blunt to SaII hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis,
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and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
of pRKSD that does not contain the Sfil site; see, Hoimes et al., Science,
253:1278-1280 ( 1991 )) in the unique Xhol
and Nott sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for a
full-length PR0211 polypeptide (designated hereinas DNA32292-1131 [Figure 9,
SEQ ID N0:21 ]) and the derived
protein sequence for that PR021 I polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 65-67and a stop signal at nucleotide
positions 1124-1126 (Figure 9, SEQ ID
N0:21 ). The predicted polypeptide precursor is 353 amino acids long with a
molecular weight of approximately
38,190 daltons [Figure 10; (SEQ ID N0:22)]. Analysis of the full-length PR0211
sequence shown in Figure 10
(SEQ ID N0:22) evidences the presence of an important polypeptide domain as
shown in Figure 10, wherein the
location given forthat important polypeptide domain is approximate as
described above. Analysis ofthe full-length
PR0211 sequence evidences the presence of a signal peptide from about amino
acid 1 to about amino acid 24.
Clone DNA32292-113 I has been deposited with ATCC on September 16, 1997 and is
assigned ATCC deposit no.
I S 209258.
An analysis ofthe Dayhoffdatabase (version 35.45 SwissProt35), using the ALIGN-
2sequencealignment
analysis of the full-length sequence shown in Figure I 0 (SEQ ID N0:22),
evidenced sequence identity between the
PR0211 amino acid sequence and EGF.
EXAMPLE 6
Isolation of cDNA Clones Encoding a Human PR0230
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public EST databases (e.g., GenBank) and a proprietary EST
database (LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST2
[Altschul et al., Methods in Enzvmoloev 266:460-480 ( 1996)] as a comparison
of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
cases, 90) or greater that did not encode known proteins were clustered and
assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA30857. An EST
proprietary to Genentech was employed
in the consensus assembly and is herein designated DNA20088. In some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed
above.
Based on the DNA30857 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
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coding sequence for PR0230. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about 1-1.5 kbp. In order to screen several libraries for a full-length
clone, DNA from the libraries was
screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular Bioloey, supra, with the PCR
primer pair. A positive library was then used to isolate clones encoding the
gene of interest using the probe
oligonucleotide and one of the primer pairs.
PCR primers (forward and reverse) were synthesized:
forward PCR primer:
l0 5'-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID N0:28)
reverse PCR primer:
5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID N0:29)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA30857
sequence which had the following nucleotide sequence:
I S hybridization probe:
5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCTGGG-3' (SEQ ID
N0:30)
RNA for construction of the cDNA libraries was isolated from human fetal lung
tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
20 reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a NotI
site, linked with blunt to SaII hemikinased adaptors, cleaved with Notl, sized
appropriately by gel electrophoresis,
and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
of pRKSD that does not contain the SfiI site; see, Holmes et al., Science,
253:1278-1280 ( 1991 )) in the unique Xhol
and NotI sites.
25 DNA sequencing of the clones isolated as described above gave the full-
length DNA sequence for a
full-length PR0230 polypeptide (designated herein as DNA33223-1136 [Figure 11,
SEQ ID N0:26]) and the
derived protein sequence for that PR0230 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 100-102 and a stop signal at
nucleotide positions 592-594 (Figure 11, SEQ
30 ID N0:26). The predicted polypeptide precursor is 164 amino acids long
(Figure 12; (SEQ ID N0:27)]. Analysis
of the full-length PR0230 sequence shown in Figure l2 (SEQ ID N0:27) evidences
the presence of an important
polypeptide domain as shown in Figure 12, wherein the location given for that
important polypeptide domain is
approximate as described above. Analysis of the full-length PR0230 sequence
evidences the presence of a signal
peptide from about amino acid 1 to about amino acid 21. Clone DNA33223-1136
has been deposited with ATCC
35 on September 16, 1997 and is assigned ATCC deposit no. 209264.
An analysis ofthe Dayhoff database (version 35.45 SwissProt35), using the
ALIGN-2 sequence alignment
analysis of the full-length sequence shown in Figure 12 (SEQ ID N0:27),
evidenced sequence identity between the
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PR0230 amino acid sequence and a rabbit tubulointerstitial nephritis antigen
precursor.
EXAMPLE 7
Isolation of cDNA Clones Encoding a Human PR0261
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public EST databases (e.g., GenBank) and a proprietary EST
database (LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST2
[Altschul er al., Methods in Enzvmoloey. 266:460-480 ( 1996)] as a comparison
of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
cases, 90) or greater that did not encode known proteins were clustered and
assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA30843. In some cases,
the consensus sequence derives
t 5 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 DNA30843 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 PR0261. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about I-l.5 kbp. In order to screen several libraries for a full-length
clone, DNA from the libraries was
screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular Bioloev. 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'-AAAGGTGCGTACCCAGCTGTGCC-3' (SEQ ID N0:33)
reverse PCR primer:
5'-TCCAGTCGGCAGAAGCGGTTCTGG-3' (SEQ !D N0:34)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA30843
sequence which had the following nucleotide sequence:
hybridization probe:
5'-CCTGGTGCTGGATGGCTGTGGCTGCTGCCGGGTATGTGCACGGCGGCTGGG-3' (SEQ ID
N0:35)
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RNA for construction of the cDNA libraries was isolated from human fetal lung
tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a Notl
site, linked with blunt to SaII hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis,
and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
ofpRKSD that does not contain the Sfil site; see, Holmes et al., Science.
253:1278-1280 ( I 991 )) in the unique XhoI
and Notl sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for a
full-length PR0261 polypeptide (designated herein as DNA33473-1176 [Figure 13,
SEQ ID N0:31)) and the
derived protein sequence for that PR0261 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 10-12 and a stop signal at nucleotide
positions 760-762 (Figure 13, SEQ ID
N0:3 I ). The predicted polypeptide precursor is 250 amino acids long [Figure
14; (SEQ ID N0:32)]. Analysis of
the full-length PR0261 sequence shown in Figure 14 (SEQ ID N0:32) evidences
the presence of an important
polypeptide domain as shown in Figure 14, wherein the location given for that
important polypeptide domain is
approximate as described above. Analysis of the full-length PR0261 sequence
evidences the presence of a signal
peptide from about amino acid 1 to about amino acid 23. Clone DNA33473-1176
has been deposited with ATCC
on October 17, 1997 and is assigned ATCC deposit no. 209391.
An analysis ofthe Dayhoffdatabase(version 35.45 SwissProt 35), usingthe ALIGN-
2 sequence alignment
analysis ofthe full-length sequence shown in Figure 14 (SEQ IDN0:32),
evidenced sequence identity between the
PR0261 amino acid sequence and CTCF, thereby indicating that PR0261 is a novel
growth factor.
EXAMPLE 8
Isolation of cDNA Clones Encoding a Human PR0246
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from
about 950 known secreted proteins from the Swiss-Prot public database were
used to search EST databases. The
EST databases included public EST databases (e.g., GenBank) and a proprietary
EST database (LIFESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST2
[Altschul et al., Methods in Enzrmoloev, 266:460-480 ( 1996)) as a comparison
of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
cases, 90) or greater that did not encode known proteins were clustered and
assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA30955. In some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed
above.
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Based on the DNA30955 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 PR0246. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about 1-1.5 kbp. In order to screen several libraries for a full-length
clone, DNA from the libraries was
screened by PCR amplification, as per Ausubel et al., Current Protocols in
Molecular BioloQV, 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'-AGGGTCTCCAGGAGAAAGACTG-3' (SEQ ID N0:38)
reverse PCR primer.
5'-ATTGTGGGCCTTGCAGACATAGAC-3' (SEQ ID N0:39)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA30955
sequence which had the following nucleotide sequence:
hybridization probe:
5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCAGCTCC-3' (SEQ ID
N0:40)
RNA for construction of the cDNA libraries was isolated from human fetal liver
tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a Notl
site, linked with blunt to SaII hemikinased adaptors, cleaved with Notl, sized
appropriately by gel electrophoresis,
and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
of pRKSD that does not contain the Sfil site; see, Holmes et al., Science.
253:1278-1280 ( 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 PR0246 polypeptide (designated herein as DNA35639-1172 [Figure 15,
SEQ ID N0:36]) and the
derived protein sequence for that PR0246 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparent translational
initiation site at nucleotide positions 126-128 and a stop signal at
nucleotide positions 1296-1298 (Figure 15, SEQ
ID N0:36). The predicted polypeptide precursor is 390 amino acids long [Figure
16; (SEQ ID N0:37)]. Analysis
of the full-length PR0246 sequence shown in Figure 16 (SEQ ID N0:37) evidences
the presence of a variety of
important polypeptide domains as shown in Figure 16, wherein the locations
given for those important polypeptide
domains are approximate as described above. Analysis ofthe full-length PR0246
sequence evidences the presence
of the following: a signal peptide from about amino acid I to about amino acid
29 and a transmembrane domain
from about amino acid 247 to about amino acid 266. Clone DNA35639-1 l76 has
been deposited with ATCC on
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October 17, 1997 and is assigned ATCC deposit no. 209396.
An analysisofthe Dayhoffdatabase (version 35.45 SwissProt 3S), using the ALIGN-
2 sequence alignment
analysis of the full-length sequence shown in Figure 16 (SEQ ID N0:37),
evidenced sequence identity between the
PR0246 amino acid sequence and the human cell surface protein HCAR, thereby
indicating that PR0246 may be
a novel cell surface virus receptor.
EXAMPLE 9
Isolation of cDNA Clones Encoding, a Human PR0317
The extracellular domain (ECD) sequences (including the secretion signal
sequence, if any) from about
950 known secreted proteins from the Swiss-Prot public database were used to
search EST databases. The EST
databases included public EST databases (e.g., GenBank)and a proprietary EST
database (L1FESEQ~, Incyte
Pharmaceuticals, Palo Alto, CA). The search was performed using the computer
program BLAST or BLAST2
[Altschul et al., Methods in Enzymoloey, 266:460-480 (1996)] as a comparison
of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons resulting in a
BLAST score of 70 (or in some
1 S cases, 90) or greater that did not encode known proteins were clustered
and assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of Washington,
Seattle, Washington).
A consensus DNA sequence was assembled relative to other EST sequences using
phrap as described
above. This consensus sequence is herein designated DNA28722. In some cases,
the consensus sequence derives
from an intermediate consensus DNA sequence which was extended using repeated
cycles of BLAST and phrap
to extend that intermediate consensus sequence as far as possible using the
sources of EST sequences discussed
above.
Based on the DNA28722 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 PR0317. Forward and reverse PCR primers generally range
from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 by in length. The
probe sequences are typically 40-55
by in length. In some cases, additional oligonucleotides are synthesized when
the consensus sequence is greater
than about 1-1.5 kbp. In order to screen several libraries for a full-length
clone, DNA from the libraries was
screened by PCR amplification, as per Ausubel er al., Current Protocols in
Molecular Bioloev, supra, with the PCR
primer pair. A positive library was then used to isolate clones encoding the
gene of interest using the probe
oligonucleotide and one of the primer pairs.
PCR primers (forward and reverse) were synthesized:
forward PCR primer:
5'-AGGACTGCCATAACTTGCCTG-3' (SEQ ID N0:43)
reverse PCR primer:
5'-ATAGGAGTTGAAGCAGCGCTGC-3' (SEQ ID N0:44)
Additionally, a synthetic oligonucleotide hybridization probe was constructed
from the consensus DNA28722
sequence which had the following nucleotide sequence:
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hybridization probe:
5'-TGTGTGGACATAGACGAGTGCCGCTACCGCTACTGCCAGCACCGC-3' (SEQ ID N0:45)
RNA for construction of the cDNA libraries was isolated from human fetal
kidney tissue. The cDNA
libraries used to isolate the cDNA clones were constructed by standard methods
using commercially available
reagents such as those from Invitrogen, San Diego, CA. The cDNA was primed
with oligo dT containing a Notl
site, linked with blunt to Salt hemikinased adaptors, cleaved with Notl, sized
appropriately by gel electrophoresis,
and cloned in a defined orientation into a suitable cloning vector (such as
pRKB or pRKD; pRKSB is a precursor
of pRKSD that does not contain the Sfi1 site; see, Holmes et al., Science.
253:1278-1280 ( 1991 )) in the unique XhoI
and Notl sites.
DNA sequencing of the clones isolated as described above gave the full-length
DNA sequence for a
full-length PR0317 polypeptide (designated herein as DNA33461-1199 [Figure 17,
SEQ ID N0:41]) and the
derived protein sequence for that PR0317 polypeptide.
The full length clone identified above contained a single open reading frame
with an apparenttranslational
initiation site at nucleotide positions 68-70 and a stop signal at nucleotide
positions l 166-1 l68 (Figure 17, SEQ
ID N0:41 ). The predicted polypeptide precursor is 366 amino acids long
[Figure 18; (SEQ ID N0:42)]. Analysis
of the full-length PR0317 sequence shown in Figure 18 (SEQ ID N0:42) evidences
the presence of important
polypeptide domains as shown in Figure l8, wherein the locations given for
those important polypeptide domains
are approximate as described above. Analysis of the full-length PR031?
sequence (Figure 18, SEQ ID N0:42)
evidences the following: a signal peptide from about amino acid 1 to about
amino acid 18, and an N-linked
glycosylation site at amino acid 160. Clone DNA33461-1199 has been deposited
with ATCC on October 1 S, 1997
and is assigned ATCC deposit no. 209367.
An analysis ofthe Dayhoffdatabase (version 35.45 SwissProt 35), usingthe ALIGN-
2 sequence alignment
analysis of the full-length sequence shown in Figure 18 (SEQ ID N0:42),
evidenced 92% sequence identity
between the PR0317 amino acid sequence and human EBAF-1. A significant
homology also exists between the
PR0317 amino acid sequence and human EBAF-2 and mouse LEFTY protein. PR0317
shows amino acid
sequence alignment with other members of the TGF-[3 superfamily. The C-
terminal end of the protein contains
many conserved sequences and the pattern expected of the TGF-(3 superfamily.
EXAMPLE 10
Gene Amplification
This example shows that the PR0187-, PR0533-, PR0214-, PR0240-, PR0211-,
PR0230-, PR0261-,
PR0246- or PR0317-encoding genes are amplified in the genome of certain human
lung, colon and/or breast
cancers and/or cell lines. Amplification is associated with overexpression of
the gene product, indicating that the
polypeptides are useful targets for therapeutic intervention in certain
cancers such as colon, lung, breast and other
cancers. Therapeutic agents may take the form of antagonists of PRO 187,
PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or PR031? polypeptide, for example, murine-human
chimeric, humanized or human
antibodies against a PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
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polypeptide.
The starting material for the screen was genomic DNA isolated from a variety
cancers. The DNA is
quantitated precisely, e.g., fluorometrically. As a negative conuol, DNA was
isolated from the cells of ten normal
healthy individuals which was pooled and used as assay controls for the gene
copy in healthy individuals (not
shown). The S' nuclease assay (for example, TaqManr") and real-time
quantitative PCR (for example, ABI Prizm
7700 Sequence Detection SystemT"' (Perkin Elmer, Applied Biosystems Division,
Foster City, CA)), were used
to find genes potentially amplified in certain cancers. The results were used
to determine whether the DNA
encoding PROl87, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or
PR0317 is
over-represented in any of the primary lung or colon cancers or cancer cell
lines or breast cancer cell lines that were
screened. The primary lung cancers were obtained from individuals with tumors
of the type and stage as indicated
in Table 2. An explanation of the abbreviations used for the designation of
the primary tumors listed in Table 2
and the primary tumors and cell lines referred to throughout this example has
been given hereinbefore.
The results of the TaqManT"' are reported in delta (0) Ct units. One unit
corresponds to 1 PCR cycle or
approximately a 2-fold amplification relative to normal, two units corresponds
to 4-fold, 3 units to 8-fold
l5 amplification and so on. Quantitation was obtained using primers and a
TaqManrM fluorescent probe derived from
the PRO 187-, PR0533-, PR02 I 4-, PR0240-, PR0211-, PR023 0-, PR0261-, PR0246-
or PR0317-encodinggene.
Regions of PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or
PR0317 which are
most likely to contain unique nucleic acid sequences and which are least
likely to have spliced out introns are
preferred for the primer and probe derivation, e.g., 3'-untranslated regions.
The sequences for the primers and
probes (forward, reverse and probe) used for the PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230,
PR0261, PR0246 or PR0317 gene amplification analysis were as follows:
PRO I 87 lDNA27864- I 155
27864.tm.p:
5'-GCAGATTTTGAGGACAGCCACCTCCA-3' (SEQ ID N0:46)
27864.tm.f
5'-GGCCTTGCAGACAACCGT-3' (SEQ ID N0:47)
27864.tm.r:
5'-CAGACTGAGGGAGATCCGAGA-3' (SEQ ID N0:48)
27864.tm.p2:
5'-CAGCTGCCCTTCCCCAACCA-3' (SEQ ID N0:49)
27864.tm.f2:
5'-CATCAAGCGCCTCTACCA-3' {SEQ ID NO:50)
27864.tm.r2:
5'-CACAAACTCGAACTGCTTCTG-3'
(SEQ ID NO:51)
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PROS33 (DNA4943S-12191:
4943S.tm.f:
S'-GGGACGTGCTTCTACAAGAACAG-3' (SEQ ID NO:S2)
4943S.tm.r:
S'-CAGGCTTACAATGTTATGATCAGACA-3' (SEQ ID N0:53)
49435.tm.p:
S'-TATTCAGAGTTTTCCATTGGCAGTGCCAGTT-3' (SEQ ID NO:S4)
PR0214 (DNA32286-1191 ):
32286.3utr-5:
S'-GGGCCATCACAGCTCCCT-3' (SEQ ID NO:SS)
32286.3utr-3b:
S'-GGGATGTGGTGAACACAGAACA-3' (SEQ ID NO:S6)
32286.3utr-probe:
1S S'-TGCCAGCTGCATGCTGCCAGTT-3'
(SEQ ID N0:57)
PR0240 (DNA34387-1138):
34387.tm.p 1:
S'-CAGCGCCGAAAAGCCAAGACTTCAT-3' (SEQ ID NO:SB)
34387.tm.fl:
' S'-GATTCTGGGAGCCACCACTCTAT-3' (SEQ ID NO:S9)
34387.tm.r 1:
S'-AGCTCCCTGACTGGGCTAAGATA-3' (SEQ ID N0:60)
34387.3utr-S:
S'-GTCAGGGAGCTCTGCTTCCTAG-3' (SEQ ID N0:61
)
34387.3utr-3:
S'-AATGGCGGCCTCAACCTT-3' (SEQ ID N0:62)
34387.3utr-probe.rc:
S'-CGAATCCACTGGCGAAAGATGCCTT-3' (SEQ ID N0:63)
PR0211 (DNA32292-1131):
32292.3utr-S:
S'-CAGAAGGATGTCCCGTGGAA-3'
(SEQ ID N0:64)
32292.3utr-3:
S'-GCCGCTGTCCACTGCAG-3' (SEQ ID N0:6S)
32292.3utr-probe.rc:
S'-GACGGCATCCTCAGGGCCACA-3'
(SEQ ID N0:66)
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PR0230 lDNA33223-1136):
33223.tm.p3:
5'-ATGTCCTCCATGCCCACGCG-3' (SEQ ID N0:67)
33223.tm.f3:
5'-GAGTGCGACATCGAGAGCTT-3' (SEQ ID N0:68)
33223.tm.r3:
5'-CCGCAGCCTCAGTGATGA-3' (SEQ ID N0:69)
33223.3utr-5:
5'-GAAGAGCACAGCTGCAGATCC-3' (SEQ ID N0:70)
33223.3utr-3:
5'-GAGGTGTCCTGGCTTTGGTAGT-3' (SEQ ID N0:71
)
33223.3 utr-probe:
5'-CCTCTGGCGCCCCCACTCAA-3' (SEQ ID N0:72)
PR0261 (DNA33473-11761:
33473.3utr-5:
5'-TCTAGCCCACTCCCTGCCT-3' (SEQ ID N0:73)
33473.3utr-3:
5'-GAAGTCGGAGAGAAAGCTCGC-3' (SEQ ID N0:74)
33473.3utr-probe:
5'-CACACACAGCCTATATCAAACATGCACACG-3' (SEQ ID N0:75)
PR0246 lDNA35639-1172):
35639.3utr-5:
5'-GGCAGAGACTTCCAGTCACTGA-3' (SEQ ID N0:76)
35639.3utr-3:
5'-GCCAAGGGTGGTGTTAGATAGG-3' (SEQ ID N0:77)
35639.3utr-probe:
S'-CAGGCCCCCTTGATCTGTACCCCA-3' (SEQ ID N0:78)
PR0317 (DNA33461-1199):
33461.tm.f:
5'-CCAGGAGAGCTGGCGATG-3' (SEQ ID N0:79)
33461.tm.r:
5'-GCAAATTCAGGGCTCACTAGAGA-3' (SEQ ID N0:80)
33461.tm.p:
5'-CACAGAGCATTTGTCCATCAGCAGTTCAG-3' (SEQ ID N0:81)
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The 5' nuclease assay reaction is a fluorescent PCR-based technique which
makes use of the 5' exonuclease
activity of Taq DNA polymerise enryme to monitor amplification in real time.
Two oligonucleotide primers are
used to generate an amplicon typical of a PCR reaction. A third
oligonucleotide, or probe, is designed to detect
nucleotide sequence located between the two PCR primers. The probe is non-
extendible by Taq DNA polymerise
enzyme, and is labeled with a reporter fluorescent dye and a quencher
fluorescent dye. Any laser-induced emission
from the reporter dye is quenched by the quenching dye when the two dyes are
located close together as they are
on the probe. During the amplification reaction, the Taq DNA polymerise enzyme
cleaves the probe in a
template-dependent manner. The resultant probe fragments disassociate in
solution, and signal from the released
reporter dye is free from the quenching effect of the second fluorophore. One
molecule of reporter dye is liberated
for each new molecule synthesized, and detection ofthe unquenched reporter dye
provides the basis for quantitative
interpretation of the data.
The 5' nuclease procedure is run on a real-time quantitative PCR device such
as the ABI Prism 7700TM
Sequence Detection. The system consists of a thermocycler, laser, charge-
coupled device (CCD) camera and
computer. The system amplifies samples in a 96-well format on a thermocycler.
During amplification,
1 S laser-induced fluorescent signal is collected in real-time through fiber
optics cables for all 96 wells, and detected
at the CCD. The system includes software for running the instrument and for
analyzing the data.
5' Nuclease assay data are initially expressed as Ct, or the threshold cycle.
This is defined as the cycle at
which the reporter signal accumulates above the background level of
fluorescence. The OCt values are used as
quantitative measurement of the relative number of starting copies of a
particular target sequence in a nucleic acid
sample when comparing cancer DNA results to normal human DNA results.
Table 2 describes the stage, T stage and N stage of various primary tumors
which were used to screen the
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
compounds of the
invention.
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Table 2
Primary Lung and Colon Tumor Profiles
Primary Tumor StaeeOther Stale Dukes
Stage T S_ tape
N Stage
Human lung tumor AdenoCa (SRCC724)IIA T1 Nl
[LT1]
Human lung tumor SqCCa (SRCC725)IIB T3 NO
[LTIa)
Human lung tumor AdenoCa (SRCC726)IB T2 NO
[LT2]
Human lung tumor AdenoCa (SRCC727)IIIAT1 N2
[LT3]
Human lung tumor AdenoCa (SRCC728)IB T2 NO
[LT4]
10Human lung tumor SqCCa (SRCC729)IB T2 NO
[LT6]
Human lung tumor Aden/SqCCa IA TI NO
(SRCC730) [LT7]
Human lung tumor AdenoCa (SRCC731IB T2 NO
) [LT9]
Human lung tumor SqCCa (SRCC732)IIB T2 NI
[LT10)
Human lung tumor SqCCa (SRCC733)IIA T1 N 1
[LT11 ]
I Human lung tumor AdenoCa (SRCC734)IV T2 NO
S [LT12]
Human lung tumor AdenoSqCCa T2 NO
(SRCC735)[LT13] IB
Human lung tumor SqCCa (SRCC736)IB T2 NO
[LT15]
Human lung tumor SqCCa (SRCC737)IB T2 NO
[LT16]
Human lung tumor SqCCa (SRCC738)IIB T2 N 1
[LT17]
20Human lung tumor SqCCa (SRCC739)1B T2 NO
[LT18]
Human lung tumor SqCCa (SRCC740)IB T2 NO
[LT19]
Human lung tumor LCCa (SRCC741IIB T3 N 1
) [LT21 ]
Human lung AdenoCa (SRCC811) IA T1 NO
[LT22]
Human colon AdenoCa (SRCC742) M 1 D pT4 NO
[CT2]
25Human colon AdenoCa (SRCC743) B pT3 NO
[CT3]
Human colon AdenoCa (SRCC B . T3 NO
744) [CT8]
Human colon AdenoCa (SRCC745) A pT2 NO
[CT10]
Human colon AdenoCa (SRCC746) MO, RI B T3 NO
[CT12]
Human colon AdenoCa (SRCC747) pMO, RO B pT3 pN0
[CT14]
30Human colon AdenoCa (SRCC748) M1, R2 D T4 N2
[CTIS]
Human colon AdenoCa (SRCC749) pM0 B pT3 pN0
[CT16)
Human colon AdenoCa (SRCC750) C i pT3 pN
[CT 17] 1
Human colon AdenoCa (SRCC751) MO, R1 B pT3 NO
[CTI]
Human colon AdenoCa (SRCC752) B pT3 MO
[CT4]
35Human colon AdenoCa (SRCC753) G2 C1 pT3 pN0
[CTS]
Human colon AdenoCa (SRCC754) pMO, RO B pT3 pN0
[CT6]
Human colon AdenoCa (SRCC755) G 1 A pT2 pN0
jCT?]
Human colon AdenoCa (SRCC756) G3 D pT4 pN2
[CT9)
Human colon AdenoCa (SRCC757) B T3 NO
[CTl 1 }
40Human colon AdenoCa (SRCC758) MO, RO B pT3 pN0
[CTI8]
DNA Preparation:
DNA was prepared from cultured cell lines, primary tumors, normal human blood.
The isolation was
45 performed using purification kit, buffer set and protease and all from
Quiagen, according to the manufacturer's
instructions and the description below.
Cell culture lysis:
Cells were washed and trypsinized at a concentration of 7.5 x 108 per tip and
pelleted by centrifuging at
1000 rpm for 5 minutes at 4°C, followed by washing again with I/2
volume of PBS recentrifugation. The pellets
50 were washed a third time, the suspended cells collected and washed 2x with
PBS. The cells were then suspended
into 10 mi PBS. Buffer C 1 was equilibrated at 4°C. Qiagen protease
#19155 was diluted into 6.25 mi cold ddHzO
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to a final concentration of 20 mg/ml and equilibrated at 4°C. 10 ml of
G2 Buffer was prepared by diluting Qiagen
RNAse A stock ( 100 mg/ml) to a final concentration of 200 ~cg/ml.
Buffer C I ( 10 ml, 4°C) and ddH20 (40 ml, 4°C) were then added
to the 10 ml of cell suspension, mixed
by inverting and incubated on ice for 10 minutes. The cell nuclei were
pelleted by centrifuging in a Beckman
swinging bucket rotor at 2500 rpm at 4°C for IS minutes. The
supernatant was discarded and the nuclei were
suspended with a vortex into 2 ml Buffer C 1 (at 4°C) and 6 ml ddH=O,
followed by a second 4°C centrifugation at
2500 rpm for I 5 minutes. The nuclei were then resuspended into the residual
buffer using 200 ul per tip. G2 buffer
( 10 m I) was added to the suspended nuclei while gentle vortexing was
applied. Upon completion of buffer addition,
vigorous vortexing was applied for 30 seconds. Quiagen protease (200 ~cl,
prepared as indicated above) was added
and incubated at 50°C for 60 minutes. The incubation and centrifugation
was repeated until the lysates were clear
(e.g., incubating additional 30-60 minutes, pelleting at 3000 x g for 10 min.,
4°C).
Solid human tumor sample preparation and lysis:
Tumor samples were weighed and placed into 50 ml conical tubes and held on
ice. Processing was limited
to no more than 250 mg tissue per preparation ( 1 tip/preparation). The
protease solution was freshly prepared by
I S diluting into 6.25 ml cold ddH=O to a final concentration of 20 mg/ml and
stored at 4°C. G2 buffer (20 ml) was
prepared by diluting DNAse A to a final concentration of 200 mg/ml (from 100
mglml stock). The tumor tissue
was homogenated in 19 ml G2 buffer for 60 seconds using the large tip of the
polytron in a laminar-flow TC hood
in order to avoid inhalation of aerosols, and held at room temperature.
Between samples, the polytron was cleaned
by spinning at 2 x 30 seconds each in 2L ddH:O, followed by G2 buffer (50 ml).
if tissue was still present on the
generator tip, the apparatus was disassembled and cleaned.
Quiagen protease (prepared as indicated above, 1.0 ml) was added, followed by
vortexing and incubation
at 50°C for 3 hours. The incubation and centrifugation was repeated
until the lysates were clear (e.g., incubating
additional 30-60 minutes, pelleting at 3000 x g for 10 min., 4°C).
Human blood preparation and lysis:
Blood was drawn from healthy volunteers using standard infectious agent
protocols and citrated into 10
ml samples per tip. Quiagen protease was freshly prepared by dilution into
6.25 ml cold ddH=O to a final
concentration of 20 mg/ml and stored at 4°C. G2 buffer was prepared by
diluting RNAse A to a final concentration
of200 ~g/ml from l00 mg/ml stock. The blood (10 ml) was placed into a 50 ml
conical tube and 10 ml C1 buffer
and 30 ml ddN,O (both previously equilibrated to 4°C) were added, and
the components mixed by inverting and
held on ice for 10 minutes. The nuclei were pelleted with a Beckman swinging
bucket rotor at 2500 rpm, 4°C for
15 minutes and the supernatant discarded. With a vortex, the nuclei were
suspended info 2 m( C 1 buffer (4°C) and
6 ml ddH20 (4°C). Vortexing was repeated until the pellet was white.
The nuclei were then suspended into the
residual buffer using a 200 ~cl tip. G2 buffer ( 10 ml) were added to the
suspended nuclei while gently vortexing,
followed by vigorous vortexing for 30 seconds. Quiagen protease was added (200
ul) and incubated at 50°C for
60 minutes. The incubation and centrifugation was repeated until the lysates
were clear (e.g., incubating additional
30-60 minutes, pelieting at 3000 x g for 10 min., 4°C).
Purifrcation of cleared lysates:
-91-
CA 02341304 2001-03-02
WO 00/15666 PCT/US99/20594
(1) Isolation of eenomic DNA:
Genomic DNA was equilibrated ( 1 sample per maxi tip preparation) with 10 ml
QBT buffer. QF elution
buffer was equilibrated at 50°C. The samples were vortexed for 30
seconds, then loaded onto equilibrated tips and
drained by gravity. The tips were washed with 2 x 15 ml QC buffer. The DNA was
eluted into 30 ml silanized,
autoclaved 30 ml Corex tubes with I S ml QF buffer (50°C). Isopropanoi
( 10.5 ml) was added to each sample, the
tubes covered with paraffin and mixed by repeated inversion until the DNA
precipitated. Samples were pelleted by
centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at 4°C.
The pellet location was marked, the
supernatant discarded, and 10 ml 70% ethanol (4°G) was added. Samples
were pelleted again by centrifugation on
the SS-34 rotor at 10,000 rpm for 10 minutes at 4°C. The pellet
location was marked and the supernatant discarded.
The tubes were then placed on their side in a drying rack and dried 10 minutes
at 37°C, taking care not to overdry
the samples.
After drying, the pellets were dissolved into 1.0 ml TE (pH 8.5) and placed at
50°C for 1-2 hours. Samples
were held overnight at 4°C as dissolution continued. The DNA solution
was then transferred to 1.5 ml tubes with
a 26 gauge needle on a tuberculin syringe. The transfer was repeated Sx in
order to shear the DNA. Samples were
then placed at 50°C for 1-2 hours.
(2) Ouantitation of eenomic DNA and preparation for gene amplification assay:
The DNA levels in each tube were quantifed by standard A,,~, AZg°
spectrophotometry on a 1:20 dilution
(5 ~cl DNA + 95 ~I ddH20) using the 0.1 ml quartz cuvetts in the Beckman DU640
spectrophotometer. A=~/AZ$o
ratios were in the range of I .8-1.9. Each DNA samples was then diluted
further to approximately 200 ng/ml in TE
(pH 8.5). If the original material was highly concentrated (about 700 nglul),
the material was placed at 50°C for
several hours until resuspended.
Fluorometric DNA quantitation was then performed on the diluted material (20-
600 ng/ml) using the
manufacturer's guidelines as modified below. This was accomplished by allowing
a Hoeffer DyNA Quant 200
fluorometer to warm-up for about 15 minutes. The Hoechst dye working solution
(#H33258, 10 ul, prepared within
12 hours of use) was diluted into l00 ml 1 x THE buffer. A 2 ml cuvette was
filled with the fluorometer solution,
placed into the machine, and the machine was zeroed. pGEM 3Zf(+) (2 ~cl, lot
#360851026) was added to 2 ml of
fluorometer solution and calibrated at 200 units. An additional 2 ~1 of pGEM
3Zf(+) DNA was then tested and the
reading confirmed at 400 +/- 10 units. Each sample was then read at least in
triplicate. When 3 samples were found
to be within 10% of each other, their average was taken and this value was
used as the quantification value.
The fluorometricly determined concentration was then used to dilute each
sample to 10 ng/~1 in ddH=O.
This was done simultaneously on all template samples for a single TaqMan plate
assay, and with enough material
to run 500-1000 assays. The samples were tested in triplicate with TaqmanT"
primers and probe both B-actin and
GAPDH on a single plate with normal human DNA and no-template controls. The
diluted samples were used
provided that the CT value of normal human DNA subtracted from test DNA was +/-
1 Ct. The diluted, lot-
qualified genomic DNA was stored in 1.0 mi aliquots at -80°C. Aliquots
which were subsequently to be used in
the gene amplification assay were stored at 4°C. Each 1 ml aliquot is
enough for 8-9 plates or 64 tests.
-92-
CA 02341304 2001-03-02
WO 00/15666 PCT/US99/20594
Gene amplification assay:
The PRO1$7, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246or PR0317
compounds
of the invention were screened in the following primary tumors and the
resulting ~Ct values are reported in Table
15
25
35
-93-
CA 02341304 2001-03-02
WO 00/15666 PCTNS99i20594
N ~ ~ h ~h
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CA 02341304 2001-03-02
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PR0187:
PR0187 (DNA27864-1155) was reexamined along with selected tumors from the
above initial screen
with framework mapping. Table 4 describes the framework markers that were
employed in association with
PR0187 (DNA27864-I 155). The framework markers are located approximately every
20 megabases along
Chromosome 8 (Figure 21 ), and are used to control aneuploidy. The OCt values
for the described framework
markers along Chromosome 8 relative to PR0187 (DNA27864) are indicated for
selected tumors in Table 6.
PR0187 (DNA27864- I I 55) was also reexamined along with selected tumors from
the above initial screen
with epicenter mapping. Table 5 describes the epicenter markers that were
employed in association with PR0187
(DNA27864-1 I55). These markers are located in close proximity to DNA27864 and
are used to assess the
amplification status of the region of Chromosome 8 in which DNA27864 is
located. The distance between markers
is measured in centirays (cR), which is a radiation breakage unit
approximately equal to a 1 % chance of a breakage
between two markers. One cR is very roughly equivalent to 20 kilobases. The
marker SHGC-9963 is the marker
found to be the closest to the location on Chromosome 8 where DNA27864-1 I55
closely maps.
Table 7 indicates the ~Ct values for results of epicenter mapping relative to
DNA27864, indicating the
relative amplification in the region more immediate to the actual location of
DNA27864 along Chromosome 8
(Figure 21 ).
Table 4
Framework Markers Used for DNA27864
Map Position on Chromosome 8 Stanford Genome Center Marker
Nsme
H9 EST00040
H59 WI-961
H 121 SHGC-11323
H200 SHGC-7433
H256 AFMa 183zf 1
Table 5
Epicenter Markers Along Chromosome 8 Used for DNA27864
Map Position on ChromosomeStanford Human GenomeDistance to Next
8 Center Marker Name Marker
(cR)
H60 SHGC-32556 9
H61 SHGC-15994 5
H62 SHGC-9963 5
H63 SHGC-33989 10
H64 UT5371 33
H65 SHGC-9507 -
-115-
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Table 6
Amplification of framework markers relative to DNA27864 (OCt)
Tumor H9 DNA27864 H59 H 121 H200 H256
CT1 0.00 0.50 0.07 -0.07 -O.13 0.58
S CT4 -0.42 0.20 -0.18 -0.49 -0.08 0.27
CTS -0.07 2.03 -0.54 -0.34 0.14 0.43
CT6 -0.08 0.13 0.04 -0.02 -0.09 -0.13
CT7 0.03 0.86 0.02 -0.06 -0.10 0.07
CT9 -0.39 -1.39 -0.14 -0.24 -0.29 0.08
CT11 -0.08 1.90 0.04 -0.05 -0.12 0.12
CTl8 0.12 0.11 0.02 -0.06 0.09 0.09
CT2 -0.25 2.92 0.23 0.16 0.14 0.34
CT3 -1.47 -0.05 0.09 0.07 -0.22 -0.01
CT8 -0.80 0.84 0.04 0.08 -0.11 0.29
IS CT10 -0.41 l.51 -0.48 0.03 0.05 0.25
CT12 -1.69 -0.57 -0.60 -0.07 0.43 0.83
CT14 -1.17 1.62 0.24 0.14 0.21 1.14
CTlS -1.05 0.30 -0.65 0.32 0.02 -15.63
CT16 -1.24 -0.09 0.13 0.16 0.07 0.11
CT17 -0.12 0.26 -0.03 0.15 0.19 0.16
LT11.1 -0.11 -0.30 -0.17 0.10 -0.11 0.06
LT12.1 0.26 2.26 0.59 -0.37 0.40 0.48
LT13.1 0.24 2.58 0.45 0.14 0.08 0.28
LT15.1 0.21 3.54 0.39 -0.16 -0.09 0.30
LT 16.2 0.03 2.07 -0.14 -0.66 -0.34 0.99
LT17.2 0.64 -0.10 0.39 0.03 -0.01 0.38
LT18.2 0.82 -0.73 0.27 0.09 -0.10 0.19
LT22.i 0.07 -1.57 -0.27 -0.22 0.43 0.45
LTI.I 0.05 -1.94 -0.62 -0.12 0.25 0.25
LTla.l -0.15 -0.71 -0.65 0.00 0.29 0.56
LT2.2 0.13 -0.56 0.22 0.21 0.25 0.43
LT3.1 -0.19 0.26 -0.10 0.12 0.20 0.13
-116-
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Tumor H9 DNA27864 H59 H 121 H200 H256
LT4.2 O.17 -1.35 -0.19 0.36 .1 0.8i 0.58
LT6.1 -0.38 -0.81 -0.82 0.25 0.15 0.36
LT7. -0.94 -1.12 -0.89 -0.50 0.35 0.73
i
LT9.1 0.11 -0.27 -0.10 -0.01 0.26 -0.01
LT10.1 0.10 0.25 0.00 0.10 -0.10 -0.04
Table 7
Amplification of Epicenter Markers Relative to DNA27864 (ACt)
Tumors H60 H61 H62 DNA H63 H64 H65
27864
LT1.1 -0.32 -0.56 0.00 -2.38 -0.77 -3.07 -0.22
LT 1 -0.09 -0.32 0.00 -0.66 -0.29 -1. I -0.09
a. 4
l
LT2.2 0.16 -0.31 0.00 -0.55 -0.52 1.14 -0.09
LT3.1 -0.47 -0.63 0.00 -0.45 -0.?S 0.29 -0.13
LT4.2 -0.32 -0.29 0.00 - I .61 -0.09 -3.46 -0.05
LT6.1 -0.52 -0.69 0.00 -0.99 -0.20 -0.65 -0.30
LT7.1 -1.16 -1.51 0.00 -1.54 -1.19 -2.79 -1.18
LT9.1 0.16 -0.15 0.00 -1.13 -0.61 -0.88 -0.05
LT I -0.27 -0.46 0.00 -0.41 -0.66 -1.25 -0.28
0.
I
LT11.1 1.87 0.20 0.00 -0.53 0.55 0.92 0.51
LT 12.10.48 0.02 0.00 1.96 -0.21 -0.54 0. I
1
LT 13.10.23 -0.19 0.00 2.32 0.11 1.74 0.10
LTlS.I 0.23 -0.27 0.00 3.43 -0.41 1.61 -0.03
LT16.2 -0.16 -0.72 0.00 1.88 -0.59 1.05 -0.32
LT17.2 2.44 0.20 0.00 0.14 0.71 0.63 0.01
LT18.2 0.17 -0.29 0.00 -0.60 -0.27 0.01 -0.05
LT22.1 0.38 -0.40 0.00 -1.42 -0.35 -9.53 -0.20
CT2 0.19 -0.10 0.00 3.25 0.13 3.15 0.05
CT3 0.15 -0.18 0.00 -0.02 0.15 2.34 0.09
CT8 0.33 0.18 0.00 1.05 -8.10 0.34 0.05
CT10 0.34 0.02 0.00 1.81 0.32 3.52 0.05
-117-
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Tumors H60 H61 H62 DNA H63 H64 H65
27864
CT12 -0.14 -0.27 0.00 -0.44 -0.46 0.71 0.29
CT14 0.90 0.24 0.00 1.86 0.45 0.34 0.02
CT16 0.28 0.09 0.00 0.27 0.33 1.88 -0.07
CT17 0.13 -0.22 0.00 0.32 0.40 0.29 -0.15
CT1 -0.05 -0.08 0.00 0.76 -0.25 -0.66 0.16
CT4 -0.47 -0.24 0.00 0.28 -0.47 -O.15 -0.36
CTS -0.35 -0.15 0.00 2.19 -0.47 0.00 0.06
CT6 -0.17 0.11 0.00 0.41 -0.06 -1.50 0.31
CT7 -0.29 -0.22 0.00 0.98 -0.25 0.81 0.07
10CT9 -0.33 -0.36 0.00 -1.89 0.06 -1.74 0.09
CTII -0.23 -0.30 0.00 1.89 0.33 0.36 0.38
CT18 -0.15 -0.12 0.00 -0.05 O.16 -1.36 0.31
PR0240:
l5 PR0240 (DNA34387-1138) was reexamined along with selected tumors from the
above initial screen
with framework mapping. Table 8 describes the framework markers that were
employed in association with
PR0240 (DNA34387-1138). The framework markers are located approximately every
20 megabases along
Chromosome 2 (Figure 22), and are used to control aneuploidy. The OCt values
for the described framework
markers along Chromosome 2 relative to PR0240 (DNA34387) are indicated for
selected tumors in Table I0.
20 PR0240 (DNA34387-I 138) was also reexamined along with selected tumors from
the above initial screen
with epicenter mapping. Table 9 describes the epicenter markers that were
employed in association with PR0240
(DNA34387-1138). These markers are located in close proximity to DNA34387 and
are used to assess the
amplification status of the region of Chromosome 2 (Figure 22) in which
DNA34387 is located. The distance
between individual markers is measured in centirays, which is a radiation
breakage unit approximately equal to a
25 1 % chance of a breakage between two markers. One cR is very roughly
equivalent to 20 kilobases. The marker
SHGC-14626 is the marker along Chromosome 2 which most closely maps to
DNA34387; however, the TaqManT"'
primers and probes for SHGC-14626 failed in our assay, due to technical
difficulties related to PCR. DNA34387
was also found to be contained within a BAC (Bacterial Artifical Chromosome).
The full BAC was about I OOKb.
A BAC containing DNA34387 was identified by screening a BAC library with the
TaqManTM primers and probe
30 for DNA34387. The ends of a DNA34387-positive clone were sequenced, and two
sets of TaqManT"' primers and
probes were made for the BAC end sequence (Tables 9 & I 1 ). This confirms the
validity of our original epicenter
mapping results. Table I I indicates the ACt values for results of epicenter
mapping relative to DNA34387,
indicating the relative amplification in the immediate chromosomal region
along Chromosome 2 (Figure 22).
-118-
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Table 8
Framework Markers Used for DNA3438?
Map Position on Chromosome Stanford Human Genome Center
2 Marker Name
B3 SHGC-30098
B45 SHGC-33615
B90 WI-7936
B 125 WI-469
B 169 SHGC-31624
B213 SHGC-10074
B251 SHGC-30979
B290 SHGC-9794
B33 I AFM21 Oxb2
B386 SHGC-13676
B44 I AFM 199yt2
B494 SHGC-33448
Table 9
Epicenter Markers Along Chromosome 2 Used for DNA34387
Map Position on ChromosomeStanford Human GenomeDistance to next marker
2 Center Marker Name (cR)
B60 AFMal36wh9 65 (gap)
B63 AFM254vc9 6
B64 SHGC-14574 28
B65 SHGC-14626 7
B66 SHGC-11736 ?
B67 SHGC-35430 17
B68 AFM234YA9 32
B71 CHLC.GATA8F07.440
189D13FOltl Marker from forward -
end of
BAC sequence
I 89D131tEV 1 Marker from forward
end of
BAC sequence
-119-
CA 02341304 2001-03-02
WO 00/15666 PCT/US99/Z0594
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M ' ~ ~ O N ~ O O N '~ V1 et M M O
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O O , ~ O O O O ~ - ~ ~. ~t
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v1 O O O O O O O O O O
~D O O O O O O O O O O
O O O O O O O O O O
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,co _. o ,... M 00 ~ h M OsO ~O ch ~ O M O
O O O O O O O O O O O O O O ~ O
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N p M O o0 ~ ~ M N O O p p ~ ~ O lV
O ~ .--~ O ~ ~ N N O O
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122
CA 02341304 2001-03-02
WO 00/1566b PCT/~JS99/20594
N
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'-'
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0
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123
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PR0230:
PR0230 (DNA33223-1136) was reexamined along with selected tumors from the
above initial screen
with framework mapping. Table 12 describes the framework markers that were
employed in association with
PR0230 (DNA33223-1136). The framework markers are located approximately every
20 megabases along
Chromosome 1 (Figure 23), and are used to control aneuploidy. The ACt values
for the described framework
markers along Chromosome I relative to PR0230 (DNA33223) are indicated for
selected tumors in Table 14.
PR0230 (DNA33223-1136) was also reexamined with epicenter mapping. A Bacterial
Artificial
Chromosome (BAC) containing DNA33223 was identified by screening a BAC
library. The ends of a DNA33223-
positive clone were sequenced, and two sets of TaqManT" primers and probes
were made for the BAC end
sequence. TaqManT"' primers and probes to the BAC ends were thus used as
markers to assess the amplification
status of the region of Chromosome 1 in which DNA33223 is located. BAC clones
are typically 100 to I 50 Kb.
DNA40625 (a novel gene) is also located on the BAC containing DNA33223. Table
13 describes the epicenter
markers that were employed in association with PR0230 (DNA33223). These
markers are located in close
proximity to DNA33223 and are used to assess the amplification status of the
region of Chromosome l in which
DNA33223 is located. The distance between markers is measured in centirays
(cR), which is a radiation breakage
unit approximately equal to a l% chance of a breakage between two markers. One
cR is very roughly equivalent
to 20 kilobases. The marker SHGC-35321 is the marker found to be the closest
to the location on Chromosome
1 where DNA33223-1136 maps (Figure 23).
Table 15 indicates the ACt values for results of epicenter mapping to
DNA33223, indicating the relative
amplification in the region more immediate to the actual location of DNA33223
along Chromosome 1 (Figure 23).
Table 12
Framework Markers Used for DNA33223
25Map Position Stanford Human Genome Center Marker Name
A I SHGC-33169
A40 SHGC-390 l
A84 AFM234tb6
A 129 ACTI B03
30A 180 SHGC-34001
A220 AFM338wb5
A263 EST00691
A312 SHGC-7599
A355 SHGC-32839
35A398 SHGC-37552
-l24-
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Map Position Stanford Human Genome Center Marker Name
A443 SHGC-11921
A485 SHGC-9327
A520 SHGC-30345
A553 SHGC-3055
Table 13
Epicenter Markers Along Chromosome I Used for DNA33223
Map Position Stanford Human GenomeDistance to Next Marker
on Center (cR)
Chromosome 1 Marker Name
10A83 SHGC-37693 97 (gap)
A84 AFM234tb6 9
A85 AFM240za9 46
A8fi AFM 199zd2 10
A87 SHGC-35321 1 I
15A88 SHGC-3252 12
A89 SHGC-11204 173
A I02 SHGC-11219 -
189D 13FOR 1 Marker from forward -
end of BAC
sequence
189D13FOR2 Marker from forward -
end of BAC
sequence
20DNA33223 - -
DNA40625 - -
189D13REV1 Marker from forward -
end of BAC
sequence
189D13REV2 Marker from forward -
end of BAC
sequence
-125-
CA 02341304 2001-03-02
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CA 02341304 2001-03-02
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CA 02341304 2001-03-02
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CA 02341304 2001-03-02
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131
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DISCUSSION AND CONCLUSION:
PR0187 fDNA27864-1155):
The ACt values for DNA27864-I 155 in a variety of tumors are reported in Table
3. A OCt of >1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA27864-1155
encoding PR0187 occurred (1) in
primary lung tumors: LTl2, LT13, LT15, LT16, LT19 and LT30; and (2) in primary
colon tumors: CT2, CT8,
CTIO, CT14, CTI, CTS, CT6, CT7, CT9 and CTI I. Amplification has been
confirmed by framework mapping
(Table 6) for DNA27864: in primary lung tumors: LTl2, LT13, LTIS and LT16; and
in primary colon tumors:
CT2, CTS, CT10, CTI1 and CTl4. The framework markers analysis reports the
relative amplification of
particular regions of Chromosome 8 in the indicated tumors, while the
epicenter markers analysis gives a more
precise reading of the relative amplification in the region immediately in the
vicinity of the gene of interest. The
amplification of the closest known framework markers (Table 6) does not occur
to a greater extent than that of
DNA27864.
Amplification has been confirmed by epicenter mapping (Table 7) for DNA27864-I
155 and resulted in
significant amplification: in primary colon tumors: CT2, CTS, CTB, CT10, CTI
land CT14; and in primary lung
tumors: LT12, LT 13, LT15, and LT16. In contrast, the amplification ofthe
closest known epicenter markers (with
the exception of H60 and H64) does not occur to a greater extent than that of
DNA27864 (Table 7). This strongly
suggests that DNA27864 is the gene responsible for the amplification of the
particular region on Chromosome 8.
Because amplification of DNA27864 occurs in various lung and colon tumors, it
is highly probable to play a
significant role in tumor formation or growth. As a result, antagonists (e.g.,
antibodies) directed against the protein
encoded by DNA27864 (PR0187) would be expected to have utility in cancer
therapy.
PR0533 lDNA49435-1219):
The OCt values for DNA49435-1219 in a variety of lung tumors are reported in
Table 3. A ~Ct of > 1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA49435-1219
encoding PR0533 occurred in primary
lung tumors: LTIa, LT7, LTI 1, LTt6, LT17, and LT19. Because amplification of
DNA49435-1219 occurs in
various lung tumors, it is likely associated with tumor formation and/or
growth. As a result, antagonists (e.g.,
antibodies) directed against the protein encoded by DNA49435 (PR0533) would be
expected to be useful in cancer
therapy.
PR0214 (DNA32286-1191 ):
The ACt values for DNA32286-1191 in a variety of tumors are reported in Table
3. A ACt of >1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA32286-
1191encoding PR0214 occurred: (1) in
primary lung tumors: LT3, LTI 1, LTI2, LT13, LT15, LT17 and LTI9; and (2) in
primary colon tumors: CT2,
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CT3, CTB, CT10, CT l2, CT14, CT15, CT16, CT17, CTI, CT4, CTS, CT6, CT9 and CTl
I. Because amplification
of DNA32286-1191 occurs in various tumors, it is likely associated with tumor
formation and/or growth. As a
result, antagonists (e.g., antibodies) directed against the protein encoded by
DNA32286 (PR0214) would be
expected to be useful in cancer therapy.
PR0240 (DNA34387-1138:
The ACt values for DNA34387-1138 in a variety of tumors are reported in Table
3. A OCt of >I was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA34387-1138
encoding PR0240 occurred: (1) in
primary lung tumors: LTIa, LT3, LT6, LT10, LT11, LT12, LT13, LTIS, LT16, LT17,
LT19, LT2I, LT26, LT28,
LT30 and in HF000840; (2) in primary colon tumors: CT2, CT3, GTB, CT10, CT12,
CT14, CT15, CT16, CT17,
CT1, CT4, CTS, CT6, CT7, CT11 and colon tumor centers HF000539, HF000575 and
HF000698; (3) in breast
tumors SRCC 1097, SRCC 1098, SRCC 1100, SRCC 11 O I and breast tumor center
HF000575; (4) in kidney tumor
center HF00061 I ; (5) in lymph node HF000854; and (6) in testis tumor center
HF000733 and testis tumor margin
HF000716.
Amplification has been confirmed by framework mapping (Table 10) for DNA34387-
1138: in primary
lung tumors: LTIa, LT3, LT11, LT12, LT13, LTIS, LT16, LT17 and LT19. In
contrast, the amplification ofthe
closest known epicenter markers (with the exception of markers B3 and B90)
does not occur to a greater extent than
that of DNA34387 (Table 10). The framework markers analysis reports the
relative amplification of particular
regions ofChromosome 2 in the indicated tumors, while the epicenter markers
analysis gives a more precise reading
of the relative amplification in the region immediately in the vicinity of the
gene of interest.
Amplification has also been confirmed by epicenter mapping (Table 11 ) for
DNA34387- I I 38 and resulted
in significant amplification: in primary lung tumors: LTIa, LT2, LT3, LT4,
LT6, LT7, LT9, LTlO, LT11, LTl2,
LT13, LT15, LT16, LT17, LT19 and LT21. It appears that DNA34387 is very close
to the BAC marker
2081021 Forl as the marker shows amplification in a similar pattern of tumors
as DNA34387, and the degree of
amplification is similar (Table 11 ).
The amplification shown by framework and epicenter mapping strongly suggests
that DNA34387 is the
gene responsible for the amplification of the particular region on Chromosome
2. Because amplification of
DNA34387 occurs in various tumors, it is highly probable to play a significant
role in tumor formation or growth.
As a result, antagonists (e.g., antibodies) directed against the protein
encoded by DNA34387 (PR0240) would be
expected to have utility in cancer therapy.
PR0211 (DNA32292-1131):
The ACt values for DNA32292- l 13 I in a variety of tumors are reported in
Table 3. A OCt of > 1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA32292-1131
encoding PR0211 occurred: (1) in
primary lung tumors: LTIa, LT3, LT4, LT9, LT10, LT1 l, LT12, LT13, LT15, LT16,
LT17, LT19 and LT21; (2)
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in primary colon tumors: CT2, CT1, CT4, CTS, CT6 and CTI 1; and (3) in lung
tumor cell line SW900. Because
amplification of DNA32292-1131 occurs in various tumors, it is likely
associated with tumor formation and/or
growth. As a result, antagonists (e.g., antibodies) directed against the
protein encoded by DNA32292 (PR0211 )
would be expected to be useful in cancer therapy.
PR0230 (DNA33223-l 136):
The OCt values for DNA33223-1136 in a variety of tumors are reported in Table
3. A OCt of > 1 was
typically used as the threshold;value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA33223-1136
encoding PR0230 occurred: ( 1 ) in
primary lung tumors: LTl 1, LT12, LT13, LT15, LT16, LTI7, LT19, LT21, LT30 and
HF000840; (2) in primary
colon tumors: CT3, CT12, CT16, CT 17, CT 1, CT4, CTS, CT7, CTl 1 and colon
tumor center HF000539; (3) in lung
tumor cell line Calu-1; (4) in colon tumor cell line SW620; (5) in kidney
tumor center HF000611; and (6) in testis
tumor center and tumor margin HF000733 and HF000716, respectively.
Amplification has been confirmed by framework mapping (Table 14) for DNA33223:
in primary lung
IS tumors: LTI 1, LT12, LT13, LT15, LT17 and LT19. Epicenter mapping (Table
15) for DNA33223 resulted in
significant amplification: in primary lung tumors: LTIa, LT3, LT6, LT9, LT10,
LTI 1, LT12, LTI3, LT15, LT16,
LT17and LT l9; and in primary colon tumors: CT2, CTS, CT10 and CT 11. The
framework markers analysis reports
the relative amplification of particular regions of Chromosome l in the
indicated tumors, while the epicenter
markers analysis gives a more precise reading of the relative amplification in
the region immediately in the vicinity
of the gene of interest. The amplification of the closest known framework
markers (Table 14) does not occur to
a greater extent than that of DNA33223. This strongly suggests that DNA33223
is the gene responsible for the
amplification of the particular region on Chromosome 1. Because amplification
of DNA33223 occurs in various
tumors and cell lines (especially lung), it is highly probable to play a
significant role in tumor formation or growth.
As a result, antagonists (e.g., antibodies) directed against the protein
encoded by DNA33223 (PR0230) would be
expected to have utility in cancer therapy.
PR0261 (DNA33473-1176):
The ACt values for DNA33473-1176 in a variety of tumors are reported in Table
3. A ACt of >1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA33473-1176
encoding PR0261occurred: (1) in
primary lung tumors: LTIa, LT10, LT11, LT12, LT13, LT15, LT16, LT17, LT18,
LT19 and LT21; (2) in primary
colon tumors: CT2, CT3, CT14, and CTS; {3) in colon tumor cell lines: SW480,
SW620, Co1o320, HT29, WiDr,
HCTI 16, SKCOI, SW403 and LS174T; and (4) in breast tumor cell lines HBL100,
MB435s, BT20, and SKBR3.
Because amplification of DNA33473-1176 occurs in various tumors, it is likely
associated with tumor formation
and/or growth. As a result, antagonists (e.g., antibodies) directed against
the protein encoded by DNA33473
(PR0261 ) would be expected to be useful in cancer therapy.
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PR0246 (DNA35639-1172):
The ACt values for DNA35639-1172 in a variety of tumors are reported in Table
3. A ACt of >1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA35639-1172
encoding PR0246 occurred: (1) in
primary lung tumors: LT3, LT10, LTl l, LT12, LT13, LTIS, LT17, LT19 and LT21;
and (2) in primary colon
tumors: CT4, CTS, CT9 and CT11. Because amplification of DNA35639-1172 occurs
in various tumors, it is
likely associated with tumor formation and/or growth. As a result, antagonists
(e.g., antibodies) directed against
the protein encoded by DNA35639 (PR0246) would be expected to be useful in
cancer therapy.
PR0317 (DNA33461-1199):
The ACt values for DNA33461-1199 in a variety of tumors are reported in Table
3. A ACt of >1 was
typically used as the threshold value for amplification scoring, as this
represents a doubling of gene copy. Table
3 indicates that significant amplification of nucleic acid DNA33461-1199
encoding PR0317 occurred: (1) in
primary lung tumors: LTIa, LT3, LT4, LT6, LT7, LT9, LT10, LTI 1, LT12, LT13,
LT15, LT16, LT17 and LT19;
and (2) in primary colon tumors: CT2, CT10, CT14, CT15, CT4 and CT9. Because
amplification of DNA33461-
1199 occurs in various tumors, it is likely associated with tumor formation
and/or growth. As a result, antagonists
(e.g., antibodies) directed against the protein encoded by DNA33461 (PR0317)
would be expected to be useful in
cancertherapy.
EXAMPLE 1 I
!n si~rr Hybridization
!n situ hybridization is a powerful and versatile technique for the detection
and localization of nucleic acid
sequences within cell or tissue preparations. It may be useful, for example,
to identify sites of gene expression,
analyze the tissue distribution of transcription, identify and localize viral
infection, follow changes in specific
mRNA synthesis and aid in chromosome mapping.
In situ hybridization was performed following an optimized version of the
protocol by Lu and Gillett, Cell
Vision, 1:169-176(1994), usingPCR-generated"P-labeledriboprobes.
Briefly,formalin-fixed, paraffin-embedded
human tissues were sectioned, deparaffinized, deproteinated in proteinase K
(20 g/ml) for 15 minutes at 37°C , and
further processed for in situ hybridization as described by Lu and Gillett,
supra. A ["-P] UTP-Labeled antisense
riboprobe was generated from a PCR product and hybridized at 55°C
overnight. The slides were dipped in Kodak
NTB2 nuclear track emulsion and exposed for 4 weeks.
"P-Ribonrobe synthesis
6.0 ~cl (125 mCi) of"P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) were speed vac
dried. To each
tube containing dried "P-UTP, the following ingredients were added:
2.0 girl Sx transcription buffer
1.0 icl DTT (100 mM)
2.0 ul NTP mix (2.5 mM : 10 ul; each of 10 mM GTP, CTP & ATP + 10 ~1 H20)
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1.0 ul UTP (50 ~M)
l.0 ~I Rnasin
1.0 ~cl DNA template (l~cg)
1.0 ~I H,O
l.0 ~d RNA polymerase (for PCR products T3 = AS, T7 = S, usually)
The tubes were incubated at 37°C for one hour. 1.0 ~l RQ1 DNase were
added, followed by incubation
at 37°C for 15 minutes. 90 ul TE (10 mM Tris pH 7.6/1mM EDTA pH 8.0)
were added, and the mixture was
pipetted onto DE81 paper. The remaining solution was loaded in a Microcon-50
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, 100 ~1 TE were added. 1 ul of the final product
was pipetted on DE81 paper and
counted in 6 ml of Biofluor Il.
The probe was run on a TBE/urea gel. I-3 ul of the probe or 5 ul of RNA Mrk
III were added to 3 ul of
loading buffer. After heating on a 37°C heat block for three minutes,
the gel was immediately placed on ice. The
wells of gel were flushed, the sample loaded, and run at I80-250 volts for 45
minutes. The gel was wrapped in
saran wrap and exposed to XAR film with an intensifying screen in -70°C
freezer one hour to overnight.
"P-Hybridization
Pretreatment of frozen sections: The slides were removed from the freezer,
placed on aluminium 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% parafotmaldehyde on
ice in the fume hood, and washed
in 0.5 x SSC for 5 minutes, at room temperature (25 ml 20 x SSC + 975 ml SQ
H:O). After deproteination in 0.5
~g/ml proteinase K for 10 minutes at 37°C (12.5 ~cl of 10 mg/ml stock
in 250 ml prewarmed RNase-free RNAse
buffer), the sections were washed in 0.5 x SSC for 10 minutes at room
temperature. The sections were dehydrated
in 70%, 95%, 100% ethanol, 2 minutes each.
Pretreatmentofparaffin-embedded sections: The slides weredeparaffinized,placed
in SQ HBO, and rinsed
twice in 2 x SSC at room temperature, for 5 minutes each time. The sections
were deproteinated in 20 ~g/ml
proteinase K (500 ul of 10 mg/ml in 250 ml RNase-free RNase buffer;
37°C, 15 minutes ) - human embryo, or 8
x proteinase K ( 100 ul in 250 ml Rnase buffer, 37°C, 30 minutes) -
formalin tissues. Subsequent rinsing in 0.5 x
SSC and dehydration were performed as described above.
Prehvbridization: The slides were laid out in a plastic box lined with Box
buffer (4 x SSC, 50%
formamide) - saturated filter paper. The tissue was covered with 50 ~1 of
hybridization buffer (3.75g Dextran
Sulfate + 6 ml SQ H20), vortexed and heated in the microwave for 2 minutes
with the cap loosened. After cooling
on ice, 18.75 ml formamide, 3.75 ml 20 x SSC and 9 ml SQ H=O were added, the
tissue was vortexed well, and
incubated at 42°C for t-4 hours.
Hybridization: 1.0 x 106 cpm probe and 1.0 ~cl tRNA (50 mg/ml stock) per slide
were heated at 95°C
for 3 minutes. The slides were cooled on ice, and 48 ul hybridization buffer
were added per slide. After vortexing,
50 ~,l "P mix were added to 50 ~cl prehybridization on slide. The slides were
incubated overnight at 55°C.
Washes: Washing was done 2x10 minutes with 2xSSC, EDTA at room temperature
(400 ml 20 x SSC
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+ 16 ml 0.25M EDTA, Vr=4L), followed by RNaseA treatment at 37°C for 30
minutes (500 ~cl of 10 mg/ml in 250
ml Rnase buffer = 20 ug/ml), The slides were washed 2x 10 minutes with 2 x
SSC, EDTA at room temperature.
The stringency wash conditions were as follows: 2 hours at 55°C, 0.1 x
SSC, EDTA (20 ml 20 x SSC + 16 ml
EDTA, V,~4L).
DNA49435-1219 (FGF homolog. FGF receptor 3 lieand)
Oligo A-2526 46 mer:
5'-GGATTCTAATACGACTCACTATAGGGCGGATCCTGGCCGGCCTCGG-3' (SEQ ID N0:82)
Oligo A-25 l H 48 mer:
5'-CTATGAAATTAACCCTCACTAAAGGGAGCCCGGGCATGGTCTCAGTTA-3' (SEQ ID N0:83)
Moderate expression was observed over cortical neurons in the fetal brain.
Expression was observed over
the inner aspect of the fetal retina, and possibly in the developing lens.
Expression was seen over fetal skin,
cartilage, small intestine, placental villi and umbilical cord. In adult
tissues, there was an extremely high level of
expression over the gall bladder epithelium. Moderate expression was seen over
the adult kidney, gastric and
colonic epithelia. These data are consistent with the potential role of this
molecule in cartilage and bone growth.
DNA32286-1191 (EGF-like homologZ:
Oligo B-1380 47mer:
5'-GGATTCTAATACGACTCACTATAGGGCCCCTCCTGCCTTCCCTGTCC-3' (SEQ ID N0:84)
Oligo A-1348 48mer:
5'-CTATGAAATTAACCCTCACTAAAGGGAGTGGTGGCCGCGATTATCTGC-3' (SEQ ID N0:85)
in fetal tissues, low level expression was observed throughout the mesenchyme.
Moderate expression was
seen in placental stromal cells in membraneous tissues, and in thyroid. Low
level expression was seen in cortical
neurons.
DNA34387-1138 (Jag~~ed/EGF homoloel:
Oligo B-231 W 48mer:
5'-GGATTCTAATACGACTCACTATAGGGCCCGAGATATGCACCCAATGTC-3' (SEQ ID N0:86)
Oligo B-231-X 47mer:
5'-CTATGAAATTAACCCTCACTAAAGGGATCCCAGAATCCCGAAGAACA-3' (SEQ 1D N0:87)
Expression pattern in human adult and fetal tissues
Elevated signal was observed at the following sites:
Fetal tissues: thyroid epithelium, small intestinal epithelium, gonad,
pancreatic epithelium, hepatocytes in liver
and renal tubules; expression was also seen in vascular tissue in developing
bones.
Adult tissues: moderate signal in placental cytotrophoblast, renal tubular
epithelium, bladder epithelium,
parathyroid and epithelial tumors.
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Expression in lung adenocarcinoma and sguanrous carcinoma
Expression was observed in all eight squamous carcinomas and in six out of
eight adenocarcinomas.
Expression was seen in in-situ and infiltrating components. Expression levels
were low to moderate in the
adenocarcinomas. In general, expression was higher in the squamous carcinomas
and in two the expression was
strong. No expression was seen in the tumor stroma, alveoli or normal
respiratory epithelium. There was possible
low level expression in lymph nodes.
DNA33223-1136 ftubulointerstitial nephritis antieen homoloe):
DNA33223 p 1:
S'-GGATTCTAATACGACTCACTATAGGGCGGCGATGTCCACTGGGGCTAC-3' (SEQ ID N0:88)
DNA33223p2:
S'-CTATGAAATTAACCCTCACTAAAGGGACGAGGAAGATGGGCGGATGGT-3' (SEQ ID N0:89)
Expression in human and fetal tissues
Tissue sections showed an intense signal associated with arterial and venous
vessels in the fetus. In
1 S arteries, the signal appeared to be confined to smooth muscle/pericytic
cells. The signal was also seen in capillary
vessels and in glomeruli. Expression was also observed in epithelium cells in
the fetal lens. Strong expression was
also seen in cells within placental trophoblastic villi. These cells lie
between the trophoblast and the fibroblast-like
cells that express HGF, and have an uncertain histogenesis.
In the adult, there was no evidence of expression in the wall of the aorta and
most vessels appeared to be
negative. However, expression was seen over vascular channels in the normal
prostate and in the epithelium lining
the gallbladder. Expression was seen in the vessels of the soft-tissue sarcoma
and the renal cell carcinoma. In
summary, this molecule showed relatively specific vascular expression in the
fetus as well as in some adult organs.
Expression was also observed in the fetal lens and the adult gallbladder.
Expression using breast and lung tumor tissues
2S Vascular expression similar to the above results was seen in fetal blocks.
Expression was in vascular
smooth muscle, rather than epithelium. Expression was also seen in smooth
muscle of the developing esophagus,
hence this molecule is not vascular specific. Expression was examined in 4
lung and 4 breast carcinomas.
Substantial expression was seen in vascular smooth muscle of at least 3 out of
4 lung cancers and 2 out of 4 breast
cancers. In addition, in one breast carcinoma (IF97-06551 3E), expression was
observed in peritumoral stromal
cells of uncertain histogenesis (possibly myofibroblasts).
Expression in lung adenocarcinoma and squamous carcinoma
One of sixteen ( 1/16) tumors showed a strong hybridization signal, and 2 out
of 16 showed positive signals
of weak to moderate intensity. All three tumors were classified as poorly
differentiated squamous cell carcinomas.
The remaining 6 squamous carcinomas and all 7 adenocarcinomas were negative.
As seen in previous studies
described above, expression was present in endothelial and smooth muscle ce!!s
of small and medium-sized
vascular channels and one case showed weak, focal expression in benign
glandular cells.
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DNA33473-1176 (CTGF homoloa):
Oligo D-1708 45 mer:
5'-GGATTCTAATACGACTCACTATAGGGCGCGAGGACGGCGGCTTCA-3' (SEQ ID N0:90)
Oligo D-170V 48 mer:
5'-CTATGAAATTAACCCTCACTAAAGGGAAGAGTCGCGGCCGCCCTTTTT (SEQ ID N0:91)
Strong expression was observed in dermal fibroblasts in normal adult skin.
Strong expression was also
seen in two cirrhotic livers, at sites of active hepatic fibrosis. Moderate
expression was found over fasiculata cells
of the adrenal cortex. This localization supports a role for this molecule in
extracellular matrix formation or
turnover.
DNA35639-t 172 (HCAR homoloe):
DNA3 5639p l
5'-GGATTCTAATACGACTCACTATAGGGCTTGCTGCGGTTTTTGTTCCTG-3' (SEQ ID N0:92)
DNA35639p2:
IS 5'-CTATGAAATTAACCCTCACTAAAGGGAGCTGCCGATCCCACTGGTATT-3' (SEQ ID N0:93)
This molecule was strongly expressed in fetal vascular endothelium, including
tissues of the CNS. Lower
level of expression was observed in adult vasculature, including CNS. It was
not obviously expressed at higher
levels in tumor vascular endothelium. Signal was also seen over bone matrix
and adult spleen, however, this signal
was obviously cell associated.
EXAMPLE 12
Use of PROI 87, PR0533. PR0214. PR0240 PR021 1. PR0230 PR026I PR0246 or PR0317
as a
hybridization probe
The following method describes use of a nucleotide sequence encoding a PR0187,
PR0533, PR0214,
PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317 polypeptide as a
hybridization probe.
DNA comprising the coding sequence of a full-length or mature "PRO"
polypeptide as disclosed herein
and/or fragments thereof may be employed as a probe to screen for homologous
DNAs (such as those encoding
naturally-occurring variants of PR0187, PR0533, PR0214, PR0240, PR021 I,
PR0230, PR0261, PR0246 or
PR0317 in human tissue cDNA libraries or human tissue genomic libraries.
Hybridization and washingoffilterscontainingeitherlibraryDNAs is
performedunderthefollowinghigh
stringency conditions. Hybridization of radiolabeled PR0187-, PR0533-, PR0214-
, PR0240-, PR0211-,
PR0230-, PR0261-, PR0246- or PR0317-derived probe to the filters is performed
in a solution of 50%
formamide, Sx SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium
phosphate, pH 6.8, 2x Denhardt's
solution, and 10% dextran sulfate at 42°C for 20 hours. Washing of the
filters is performed in an aqueous solution
of O.lx SSC and 0.1% SDS at 42°C.
DNAs having a desired sequence identity with the DNA encoding full-length
native sequence PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 can then be
identified using
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standard techniques known in the art.
EXAMPLE 13
Expression of "PRO" Polvneptides in E. toll.
This example illustrates preparation of an unglycosylated form of PR0187,
PR0533, PR0214, PR0240,
PR021 I, PR0230, PR0261, PR0246 or PR0317 by recombinant expression in E.
toll.
The DNA sequence encoding the PRO polypeptide of interest is initially
amplified using selected PCR
primers. The primers should contain restriction enryme 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. toll; see Bolivar et al., Gene. 2:95 (1977)) which
contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction enzyme and
dephosphorylated. The PCR amplified
sequences are then ligated into the vector. The vector will preferably include
sequences which encode for an
antibiotic resistance gene, atrp promoter, a poly-His leader (including the
first six STII codons, poly-His sequence,
and enterokinase cleavage site), the PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246
or PR0317 coding region, lambda transcriptional terminator, and an argU gene.
The ligation mixture is then used to transform a selected E. toll strain using
the methods described in
Sambrook et al., supra. Transformants are identified by their ability to grow
on LB plates and antibiotic resistant
colonies are then selected. Plasmid DNA can be isolated and confirmed by
restriction analysis and DNA
sequencing.
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
PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317
protein can then be
purified using a metal chelating column under conditions that allow tight
binding of the protein.
PRO 187, PR0533, PR0240 and PR0317 were successfully expressed in E. toll in a
poly-His tagged form
using the following procedure. The DNA encoding PRO l 87, PR0533, PR0240 or
PR0317 was initially amplified
using selected PCR primers. The primers contained restriction enzyme sites
which correspond to the restriction
enzyme sites on the selected expression vector, and other useful sequences
providing for efficient and reliable
translation initiation, rapid purification on a metal chelation column, and
proteolytic removal with enterokinase.
The PCR-amplified, poly-His tagged sequences were then ligated into an
expression vector, which was used to
transform an E. toll host based on strain 52 (W3110 fuhA(tonA) Ion galE
rpoHts(htpRts) clpP(laclq).
Transformants were first grown in LB containing 50 mg/ml carbenicillin at
30°C with shaking until an O.D.~ of
3-5 was reached. Cultures were then diluted 50-100 fold into CRAP media
(prepared by mixing 3.57 g (NH4)ZS04,
0.71 g sodium citrate~2H20, 1.07 g KCI, 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 MgSO,) and grown
for approximately 20-30
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hours at 30°C with shaking. Samples were removed to verify expression
by SDS-PAGE analysis, and the bulk
culture is centrifuged to pellet the cells. Cell pellets were frozen until
purification and refolding.
E. coli paste from 0.5 to 1 L fetrnentations (6-10 g pellets) was resuspended
in i0 volumes (w/v) in 7 M
guanidine, 20 mM Tris, pH 8 buffer. Solid sodium sulfite and sodium
tetrathionate is added to make final
concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred
overnight at 4 °C. This step results
in a denatured protein with all cysteine residues blocked by sulfitolization.
The solution was centrifuged at 40,000
rpm in a Beckman Ultracentifuge for 30 min. The supernatant was 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 was loaded onto a 5 ml Qiagen Ni'-'-NTA metal chelate column
equilibrated in the metal chelate column
buffer. The column was washed with additional buffer containing 50 mM
imidazole (Calbiochem, Utrol grade),
pH 7.4. The protein was eluted with buffer containing 250 mM imidazole.
Fractions containing the desired protein
were pooled and stored at 4°C. Protein concentration was estimated by
its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid sequence.
The proteins were refolded by diluting sample slowly into freshly prepared
refolding buffer consisting of
20 mM Tris, pH 8.6, 0.3 M NaCI, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1
mM EDTA. Refolding
volumes were chosen so that the final protein concentration was between 50 to
100 micrograms/ml. The refolding
solution was stirred gently at 4 °C for 12-36 hours. The refolding
reaction was 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
was filtered through a 0.22 micron filter and acetonitrile was added to 2-10%
final concentration. The refolded
protein was 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 Aze° absorbance were analyzed
on SDS polyacrylamide gels and fractions containing homogeneous refolded
protein were 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 higheracetonitrile concentrations. In
addition to resolving misfoided forms
of proteins from the desired form, the reversed phase step also removes
endotoxin from the samples.
Fractions containing the desired folded PR0187, PR0533, PR0240 and PR0317
proteins, respectively,
were pooled and the acetonitrile removed using a gentle stream of nitrogen
directed at the solution. Proteins were
formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4%
mannitol by dialysis or by gel filtration
using G25 Superfine (Phannacia) resins equilibrated in the formulation buffer
and sterile filtered.
EXAMPLE 14
Expression of PR0187, PR0533, PR0214, PR0240 PR0211 PR0230 PR0261 PR0246 or
PR0317 in
mammalian cells
This example illustrates preparation of a potentially glycosylated form of
PR0187, PR0533, PR0214,
PR0240, PR021 I, PR0230, PR0261, PR0246 or PR0317 by recombinant expression in
mammalian cells.
The vector, ARKS (see EP 307,247, published March 15, 1989), is employed as
the expression vector.
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Optionally, the PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261,
PR0246 or PR0317 DNA
is ligated into pRKS with selected restriction enrymes to allow insertion of
the PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR03 i 7 DNA using ligation methods
such as described in
Sambrook et al., supra. The resulting vector is called pRKS-PRO 187, pRKS-
PR0533, pRKS-PR0214, pRKS-
PR0240, pRKS-PR0211, pRKS-PR0230, pRKS-PR0261, pRKS-PR0246 or pRKS-PR0317.
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 I 0 ~g pRKS-PRO 187,
pRKS-PR0533, pRKS-PR0214,
pRKS-PR0240,pRK5-PR021 I,pRKS-PR0230,pRK5-PR0261,pRK5-PR0246orpRKS-PR0317 DNA
ismixed
with about 1 ug DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543
( 1982)] and dissolved in 500
ul of 1 mM Tris-HCI, 0.1 mM EDTA, 0.227 M CaCI,. To this mixture is added,
dropwise, 500 ~1 of 50 mM
HEPES (pH 7.35), 280 mM NaCI, I .5 mM NaPO,, and a precipitate is allowed to
form for 10 minutes at 25°C. The
precipitate is suspended and added to the 293 cells and allowed to settle for
about four hours at 37°C. The culture
medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30
seconds. The 293 cells are then washed
with serum free medium, fresh medium is added and the cells are incubated for
about 5 days.
Approximately 24 hours after the transfections, the culture medium is removed
and replaced with culture
medium (alone) or culture medium containing 200 ~Ci/ml'SS-cysteine and 200
E.cCi/ml'sS-methionine. After a
l2 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
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
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, PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246
or PR0317 DNA may be introduced into 293 cells transiently using the dextran
sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci.. 12:7575 ( 1981 ). 293 cells are
grown to maximal density in a spinner flask
and 700 ~g pRKS-PR0187, pRKS-PR0533, pRKS-PR0214, ARKS-PR0240, pRKS-PR021 I,
pRKS-PR0230,
pRKS-PR0261, pRKS-PR0246 or pRKS-PR0317 DNA is added. The cel Is 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 ug/ml
bovine insulin and 0.1 ~g/ml bovine
transferrin. After about four days, the conditioned media is centrifuged and
filtered to remove cells and debris.
The sample containing expressed PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246
or PR0317 can then be concentrated and purified by any selected method, such
as dialysis and/or column
chromatography.
In another embodiment PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or
PR0317 can be expressed in CHO cells. The pRKS-PR0187, ARKS-PR0533, pRKS-
PR0214, pRKS-PR0240,
pRKS-PR0211, pRKS-PR0230, pRKS-PR0261, pRKS-PR0246 or pRKS-PR0317 vector can
be transfected into
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CHO cells using known reagents such as CaP04 or DEAF-dextran. As described
above, the cell cultures can be
incubated, and the medium replaced with culture medium (alone) or medium
containing a radiolabel such as'sS_
methionine. Afterdeterminingthepresence ofPR0187, PR0533, PR0214, PR0240,
PR021 l, PR0230, PR0261,
PR0246 or PR0317 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 PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317 can then
be concentrated and purified by any selected method.
Epitope-tagged PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317
may also be expressed in host CHO cells. The PRO187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261,
PR0246 or PR0317 may be subcloned out of the pRKS vector. The subclone insert
can undergo PCR to fuse in
frame with a selected epitope tag such as a poly-His tag into a Baculovirus
expression vector. The poly-His tagged
PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317
insert can then be
subcloned into a SV40 driven vector containing a selection marker such as DHFR
for selection of stable clones.
Finally, the CHO cells can be transfected (as described above) with the SV40
driven vector. Labeling may be
1 S performed, as described above, to verify expression. The culture medium
containing the expressed poly-His tagged
PRO 187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 can
then be concentrated
and purified by any selected method, such as by Ni2'-chelate affinity
chromatography. Expression in CHO and/or
COS cells may also be accomplised by a transient expression procedure.
PR02I4, PR0240, PR0211, PR0230 and PR0261 were expressed in CHO cells by both
a transient and
a stable expression procedure. In addition, PR0246 was transiently expressed
in CHO cells.
Stable expression in CHO cells was performed using the following procedure.
The proteins were
expressed as an IgG construct (immunoadhesin), in which the coding sequences
for the soluble forms (e.g.,
extracellular domains) of the respective proteins were fused to an IgG I
constant region sequence containing the
hinge, CH2 and CH2 domains and/or is a poly-His tagged form.
Following PCR amplification, the respective DNAs were subcloned in a CHO
expression vector using
standard techniques as described in Ausubel et al., Current Protocols of
Molecular Bioloev. 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 for
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 dihydrofoiate reductase
(DHFR). DHFR expression permits
selection for stable maintenance of the plasmid following transfection.
Twelve micrograms ofthe desired plasmidDNA were introduced into approximately
i 0 million CHO cells
using commercially available transfection reagents Superfect~ (Quiagen),
Dosperm or Fugene~ (Boehringer
Mannheim). The cells were grown as described in Lucas et al., supra.
Approximately 3 x 10'' cells are froien in
an ampuie for further growth and production as described below.
The ampules containing the plasmid DNA were thawed by placement into water
bath and mixed by
vortexing. The contents were pipetted into a centrifuge tube containing 10 mls
of media and centrifuged at 1000
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rpm for 5 minutes. The supernatant was aspirated and the cells were
resuspended in 10 ml of selective media (0.2
~m filtered PS20 with 5% 0.2 um diafiltered fetal bovine serum). The cells
were then aliquoted into a 100 ml
spinner containing 90 ml of selective media. After I-2 days, the cells were
transferred into a 250 ml spinner filled
with 150 ml selective growth medium and incubated at 37°C. After
another 2-3 days, 250 ml, 500 ml and 2000 ml
spinners were seeded with 3 x l05 cells/ml. The cell media was exchanged with
fresh media by centrifugation and
resuspension in production medium. Although any suitable CHO media may be
employed, a production medium
described in US Patent No. 5,122,469, issued June 16, 1992 was actually used.
3L production spinner is seeded
at 1.2 x 10° cells/ml. On day 0, the cell number and pH were
determined. On day 1, the spinner was sampled and
sparging with filtered air was commenced. On day 2, the spinner was sampled,
the temperature shifted to 33°C,
and 30 ml of 500 g/L glucose and 0.6 ml of 10% antifoam (e.g., 35%
polydimethylsiloxane emulsion, Dow Corning
365 Medical Grade Emulsion). Throughout the production, pH was adjusted as
necessary to keep at around 7.2.
After 10 days, or until viability dropped below 70%, the cell culture was
harvested by centrifugation and filtered
through a 0.22 ~m filter. The filtrate was either stored at 4°C or
immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins were purified using a Ni ='-
NTA column (Qiagen). Before
( 5 purification, imidazole was added to the conditioned media to a
concentration of 5 mM. The conditioned media
was pumped onto a 6 ml Ni z'-NTA column equilibrated in 20 mM Hepes, pH 7.4,
buffer containing 0.3 M NaCI
and 5 mM imidazole at a flow rate of4-5 mUmin. at 4°C. After loading,
the column was washed with additional
equilibration buffer and the protein eluted with equilibration buffer
containing 0.25 M imidazole. The highly
purified protein was subsequently desalted into a storage buffer containing 10
mM Hepes, 0.14 M NaCI and 4%
mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column and stored at -
80°C.
Immunoadhesin (Fc containing) constructs were purified from the conditioned
media as follows. The
conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which
had been equilibrated in 20 mM
Na phosphate buffer, pH 6.8. After loading, the column was washed extensively
with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately
neutralized by collecting I ml
fractions into tubes containing 275 ul of 1 M Tris buffer, pH 9. The highly
purified protein was subsequently
desalted into storage buffer as described above for the poly-His tagged
proteins. The homogeneity was assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman
degradation.
EXAMPLE 15
Expression of PR0187. PR0533. PR0214, PR0240. PR0211, PR0230 PR0261 PR0246 or
PR0317 in
Yeast
The following method describes recombinant expression of PR0187, PR0533,
PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 in yeast.
First, yeast expression vectors are constructed for intracellular production
or secretion of PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 from the
ADH2/GAPDH
promoter. DNA encoding PR0187, PR0533, PR0214, PR0240, PR021 I, PR0230,
PR0261, PR0246 or
PR0317 and the promoter is inserted into suitable restriction enryme sites in
the selected plasmid to direct
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intracellular expression of PR0187, PR0533, PR0214. PR0240, PR021 I, PR0230,
PR0261, PR0246 or
PR0317. For secretion, DNA encoding PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261,
PR0246 or PR0317 can be cloned into the selected plasmid, together with DNA
encoding the ADH2/GAPDH
promoter, a native PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317
signal peptide or other mammalian signal peptide, or, for example, a yeast
alpha-factor or invertase secretory
signaUleader sequence, and linker sequences (if needed) for expression of
PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317.
Yeast cells, such as yeast strain AB I 10, can then be transformed with the
expression plasmids
described above and cultured in selected fermentation media. The transformed
yeast supernatants can be
analyzed by precipitation with 10% trichloroacetic acid and separation by SDS-
PAGE, followed by staining of
the gels with Coomassie Blue stain.
Recombinant PR0187, PR0533, PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or
PR0317 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
IS containing PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246
or PR0317 may
further be purified using selected column chromatography resins.
EXAMPLE 16
Expression of PROI 87. PR0533. PR0214. PR0240. PR021 I PR0230 PR0261 PR0246 or
PR0317 in
Baculovirus-infected Insect Cells
The following method describes recombinant expression in Baculovirus-infected
insect cells.
The sequence coding for PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246
or PR0317 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 piasmids may be employed,
including plasmids derived from commercially available plasmids such as
pVL1393 (Novagen). Briefly, the
sequence encoding PR0187, PR0533, PR0214, PR0240, PR0211, PR0230, PR0261,
PR0246 or PR0317 or
the desired portion of the coding sequence of PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261,
PR0246 or PR0317 (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 plasm id and
BaculoGoldT"'virus DNA
(Pharmingen) into Spodopterafrugiperda ("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 187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246
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or PR0317 can then be purified, for example, by Ni='-chelate affinity
chromatography as follows. Extracts are
prepared from recombinant virus-infected Std cells as described by Rupert et
al., Nature, 362:175-179 (1993).
Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 ml Hepes,
pH 7.9; 12.5 mM MgCI,; 0.1 mM
EDTA; 10% glycerol; O.l% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds
on ice. The sonicates are
cleared by centrifugation, and the supernatant is diluted 50-fold in loading
buffer (50 mM phosphate, 300 mM
NaCI, (0% glycerol, pH 7.8) and filtered through a 0.45 tcm 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 A~~ with loading buffer, at which point fraction collection
is started. Next, the column is
washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCI, 10%
glycerol, pH 6.0), which elutes
nonspecifically bound protein. After reaching A,8° baseline again, the
column is developed with a 0 to 500 mM
imidazole gradient in the secondary wash buffer. One ml fractions are
collected and analyzed by SDS-PAGE and
silver staining or Western blot with Ni=+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing
the eluted His,°-tagged PR0187, PR0533, PR0214, PR0240, PR0211, PR0230,
PR0261, PR0246 or PR0317,
I S respectively, are pooled and dialyzed against loading buffer.
Alternatively, purification of the IgG tagged (or Fc tagged) PR0187, PR0533,
PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317 can be performed using known
chromatography techniques,
including for instance, Protein A or protein G column chromatography.
PROl87, PR0533, PR0214, PR0240, PR0211, PR0230, PR0246 and PR0317 were
expressed in
baculovirus infected Sf9 insect cells. While expression was actually performed
in a 0.5-2 L scale, it can be readily
scaled up for larger (e.g., 8 L) preparations. The proteins were expressed as
an IgG construct (immunoadhesin),
in which the protein extracellular region was fused to an 1gG 1 constant
region sequence containing the hinge, CH2
and CH3 domains and/or in poly-His tagged forms.
Following PCR amplification, the respective coding sequences were subcloned
into a baculovirus
expression vector (pb.PH.IgG for 1gG fusions and pb.PH.His.c for poly-His
tagged proteins), and the vector and
Baculogold~ baculovirus DNA (Pharmingen) were co-transfected into 105
Spodoptera frugiperda ("Sf9") cells
(ATCC CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His are
modifications of the
commercially available baculovirus expression vector pVL 1393 (Pharmingen),
with modified polylinker regions
to include the His or Fc tag sequences. The cells were grown in Hink's TNM-FH
medium supplemented with 10%
FBS (Hyclone). Cells were incubated for 5 days at 28 °C. The
supernatant was harvested and subsequently used
for the first viral amplification by infecting Sf9 cells in Hink's TNM-FH
medium supplemented with 10% FBS at
an approximate multiplicity of infection (MOI) of l0. Cells were incubated for
3 days at 28°C. The supernatant
was harvested and the expression of the constructs in the baculovirus
expression vector was determined by batch
binding of 1 ml of supernatant to 25 ml of Ni Z'-NTA beads (QIAGEN) for
histidine tagged proteins or Protein-A
Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE
analysis comparing to a
known concentration of protein standard by Coomassie blue staining.
The first viral amplification supernatant was used to infect a spinner culture
(500 ml) of Sf9 cells grown
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in ESF-921 medium (Expression Systems LLC) at an approximate MOI of 0.1. Cells
were incubated for 3 days
at 28°C. The supernatant was harvested and filtered. Batch binding and
SDS-PAGE analysis was repeated, as
necessary, until expression of the spinner culture was confirmed.
The conditioned medium from the transfected cells (0.5 to 3 L) was harvested
by centrifugation to remove
the cells and filtered through 0.22 micron filters. For the poly-His tagged
constructs, the protein construct were
purified using a Ni Z+-NTA column (Qiagen). Before purification, imidazole was
added to the conditioned media
to a concentration of 5 mM. The conditioned media were pumped onto a 6 ml Ni
Z+-NTA column equilibrated in
20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a flow
rate of 4-5 mUmin. at 4°C.
After loading, the column was washed with additional equilibration buffer and
the protein eluted with equilibration
buffer containing 0.25 M imidazole. The highly purified protein was
subsequently desalted into a storage buffer
containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25 ml G25
Superfine (Pharmacia) column
and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the
conditioned media as
follows. The conditioned media were pumped onto a S ml Protein A column
(Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column
was washed extensively with
equilibration buffer before elution with 100 mM citric acid, pH 3.5. The
eluted protein was immediately neutralized
by collecting I ml fractions into tubes containing 275 ml of 1 M Tris buffer,
pH 9. The highly purified protein was
subsequently desalted into storage buffer as described above for the poly-His
tagged proteins. The homogeneity
of the proteins was verified by SDS poiyacrylamide gei (PEG) electrophoresis
and N-terminal amino acid
sequencing by Edman degradation.
Alternatively, a modified baculovirus procedure may be used incorporating high
5 cells. In this procedure,
the DNA encoding the desired sequence was amplified with suitable systems,
such as Pfu (Stratagene), or fused
upstream (S'-of) of an epitope tag contained with a baculovirus expression
vector. Such epitope tags include poly-
His tags and immunoglobulin tags (like Fc regions of IgG). A variety of
plasmids may be employed, including
plasmids derived from commercially available plasmids such as pIE 1-1
(Novagen). The pIE I -1 and pIE I-2 vectors
are designed for constitutive expression of recombinant proteins from the
baculovirus ie1 promoter in stably-
transformed insect cells. The plasmids differ only in the orientation of the
multiple cloning sites and contain all
promoter sequences known to be important for iel-mediated gene expression in
uninfected insect cells as well as
the hr5 enhancer element. pIE 1-1 and pIE1-2 include the translation
initiation site and can be used to produce fusion
proteins. Briefly, the desired sequence or the desired portion of the sequence
(such as the sequence encoding the
extracellular domain of a transmembrane protein) is amplified by PCR with
primers complementary to the 5' and
3' regions. The 5' primer may incorporate flanking (selected) restriction
enryme sites. The product was then
digested with those selected restriction enzymes and subcloned into the
expression vector. Forexample, derivatives
ofpIE 1-1 can include the Fc region ofhuman IgG (pb.PH.IgG) or an 8 histidine
(pb.PH.His) tag downstream {3'-of)
the desired sequence. Preferably, the vector construct is sequenced for
confirmation.
High 5 cells are grown to a confluency of 50% under the conditions of,
27°C, no CO2, NO penlstrep. For
each 150 mm plate, 30 ug of pIE based vector containing the sequence was mixed
with 1 ml Ex-Cell medium
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(Media: Ex-Cell 401 + 1/100 L-Glu JRH Biosciences #14401-78P (note: this media
is light sensitive)), and in a
separate tube, 100 ~cl of CellFectin (CeIIFECTIN (GibcoBRL #10362-010)
(vortexed to mix)) was mixed with 1
ml of Ex-Cell medium. The two solutions were combined and allowed to incubate
at room temperature for 15
minutes. 8 ml of Ex-Cell media was added to the 2 ml of DNA/CeIIFECTIN mix and
this is layered on High 5
cells that have been washed once with Ex-Cell media. The plate is then
incubated in darkness for l hour at room
temperature. The DNA/CelIFECTIN mix is then aspirated, and the cells are
washed once with Ex-Cell to remove
excess CelIFECTIN, 30 ml of fresh Ex-Cell media was added and the cells are
incubated for 3 days at 28°C. The
supernatant was harvested and the expression of the sequence in the
baculovirus expression vector was determined
by batch binding of I ml of supernatant to 25 ml ofNi z'-NTA beads (QIAGEN)
for histidine tagged proteins or
Protein-A Sepharose CL-4B beads (Pharmacia) for IgG tagged proteins followed
by SDS-PAGE analysis
comparing to a known concentration of protein standard by Coomassie blue
staining.
The conditioned media from the transfected cells (0.5 to 3 L) was harvested by
centrifugation to remove
the cells and filtered through 0.22 micron filters. For the poly-His tagged
constructs, the protein comprising the
sequence is purified using a Ni'-'-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned
media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml
Ni '-'-NTA column equilibrated
in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCI and 5 mM imidazole at a
flow rate of 4-5 ml/min. at 48°C.
After loading, the column was washed with additional equilibration buffer and
the protein eluted with equilibration
buffer containing 0.25 M imidazole. The highly purified protein was then
subsequently desalted into a storage
buffer containing 10 mM Hepes, 0.14 M NaCI and 4% mannitol, pH 6.8, with a 25
ml G25 Superfine (Pharmacia)
column and stored at -80°C.
Immunoadhesin (Fc containing) constructs of proteins were purified from the
conditioned media as
follows. The conditioned media was pumped onto a 5 ml Protein A column
(Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading, the column
was washed extensively with
equilibration buffer before elution with 100 mM citric acid, pH 3.5. The
eluted protein was immediately neutralized
by collecting 1 ml fractions into tubes containing 275 ml of 1 M Tris buffer,
pH 9. The highly purified protein was
subsequently desalted into storage buffer as described above for the poly-His
tagged proteins. The homogeneity
of the sequence was assessed by SDS polyacrylamide gels and by N-terminal
amino acid sequencing by Edman
degradation and other analytical procedures as desired or necessary.
PR0187, PR0533, PR0214, PR0240, PR0211 and PR0246 were successfully expressed
by the above
modified baculovirus procedure incorporating high S cells.
EXAMPLE 17
Demonstration of binding of PR0533 to FGF Recptor 3
PR0533 was expressed in baculovirus in a C-terminal His8 epitope tagged form
as described in Example
16, as was a control C-terminal His8 epitope protein. The extracellular
domains of FGF receptors 1-4 and TIE1
receptor were expressed as Fc fusion proteins. Proteins were allowed to
interact in binding buffer (DMEM media
+ lOmM Hepes pH 7.4 + 0.1% albumin + 200 ng/ml heparin) at room temperature
for one hour. Protein A
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Sepharose (Pharmacia) was added (0.01 ml) and binding continued for 30
minutes. Protein A Sepharose beads
were collected and washed twice in binding buffer. Samples were then resolved
by SDS PAGE under reducing
conditions. Western blot analysis was conducted with anti-His antibody
(Qiagen) as recommended by the
manufacturer. The results demonstrated a high specificity binding to FGF
Receptor 3 (FGFR3-Fc). This is very
significant, since most FGF ligands bind more than one FGF receptor.
EXAMPLE 18
Preparation of Antibodies that Bind PR0187. PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261~
PR0246 or PR0317
This example illustrates preparation of monoclonal antibodies which can
specifically bind PR0187,
PR0533, PR0214, PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317.
Techniques for producing the monoclonal antibodies are known in the art and
are described, for instance,
in Coding, supra. Immunogens that may be employed include purified PR0187,
PR0533, PR0214, PR0240,
PR0211, PR0230, PR0261, PR0246 or PR0317, fusion proteins containing PR0187,
PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317 and cells expressing
recombinant PR0187, PR0533,
PR0214, PR0240, PR021 l, PR0230, PR0261, PR0246 or PR0317 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 PR0187, PR0533, PR0214, PR0240,
PR0211, PR0230,
PR0261, PR0246 or PR0317 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
todetectanti-PR0187, anti-PR0533, anti-PR0214, anti-PR0240, anti-PR0211, anti-
PR0230, anti-PR0261, anti-
PR0246 or anti-PR0317 antibodies.
After a suitable antibody titer has been detected, the animals "positive" for
antibodies can be injected with
a final intravenous injection of PR0187, PR0533, PR0214, PR0240, PR0211,
PR0230, PR0261, PR0246 or
PR0317. 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 myeioma cell
line such as P3X63AgU. l, available
from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then
be plated in 96 well tissue
culture plates containing HAT (hypoxanthine, aminopterin, and thymidine)
medium to inhibit proliferation of non-
fused cells, myeloma hybrids, and spleen cell hybrids.
The hybridoma cells will be screened in an EL1SA for reactivity against
PR0187, PR0533, PR0214,
PR0240, PR0211, PR0230, PR0261, PR0246 or PR0317. Determinationof"positive"
hybridoma cells secreting
the desired monoclonal antibodies against PR0187, PR0533, PR0214, PR0240,
PR021 !, PR0230, PR0261,
PR0246 or PR0317 is within the skill in the art.
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The positive hybridoma cells can be injected intraperitoneally into syngeneic
Balb/c mice to produce
ascites containing the anti-PR0187, anti-PR0533, anti-PR0214, anti-PR0240,
anti-PR021 I, anti-PR0230, anti-
PR0261, anti-PR0246 or anti-PR0317 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.
Deposit of Material:
The following the American Type Culture
materials have Collection, 10801
been deposited
with
University
Blvd., Manassas,
VA 20110-2209,
USA (ATCC):
Material ATCC Deposit No.: Deposit Date
DNA27864-1155 209375 10/16/97
DNA49435-1219 209480 11/21/97
DNA32286-1191 209385 10/ 16/97
l5 DNA34387-1138209260 9/16/97
DNA32292- l 209258 9/ 16/97
131
DNA33223-1136 209264 9/16/97
DNA33473-1176 209391 10/17/97
DNA35639-1172 209396 10/17/97
DNA33461-1199209367 10/15/97
These deposits were made under the provisions of the Budapest Treaty on the
International Recognition
of the Deposit of Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest
Treaty). This assures the maintenance of a viable culture of the deposit for
30 years from the date of deposit. The
deposit will be made available by the ATCC under the terms of the Budapest
Treaty, and subject to an agreement
between Genentech, Inc., and the 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 C.F.R. ~ 1.14
with particular reference to 886 OG
638).
The assignee of the present application has agreed that if a culture of the
materials on deposit should die
or be lost or destroyed when cultivated under suitable conditions, the
materials will be promptly replaced on
notification with another of the same. Availability of the deposited material
is not to be construed as a license to
practice the invention in contravention of the rights granted under the
authority of any government in accordance
with its patent laws.
The foregoing written specification is considered to be sufficient to enable
one skilled in the art to practice
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WO 00/15666 PCT/US99/20594
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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