Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF DIAGNOSING,
MONITORING, STAGING, IMAGING AND TREATING PROSTATE CANCER
FIELD OF THE INVENTION
This invention relates, in part, to newly developed
assays for detecting, diagnosing, monitoring, staging,
prognosticating, imaging and treating cancers, particularly
prostate cancer.
BACKGROUND OF THE INVENTION
Cancer of the prostate is the most prevalent malignancy
IO in adult males, excluding skin cancer, and is an increasingly
prevalent health problem in the United States. In 1996, it
was estimated that 91,400 deaths would result from this
disease in the United States alone, indicating that prostate
cancer is second only to lung cancer as the most common cause
of death in the same population. If diagnosed and treated
early, when the cancer is still confined to the prostate, the
chances of cure is significantly higher.
Treatment decisions for an individual are linked to the
stage of prostate cancer present in that individual. A common
classification of the spread of prostate cancer was developed
by the American Urological Association (AUA). The AUA system
divides prostate tumors into four stages, A to D. Stage A,
microscopic cancer within prostate, is further subdivided into
stages A1 and A2. Sub-stage A1 is a well-differentiated
cancer confined to one site within the prostate. Treatment
is generally observation, radical prostatectomy, or radiation.
Sub-stage A2 is a moderately to poorly differentiated cancer
at multiple sites within the prostate. Treatment is radical
prostatectomy or radiation. Stage B, palpable lump within the
prostate, is also further subdivided into sub-stages B1 and
B2. In sub-stage B1, the cancer forms a small nodule in one
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lobe of the prostate. In sub-stage B2, the cancer forms large
or multiple nodules, or occurs in both lobes of tl~e prostate.
Treatment for sub-stages B1 and B2 is either radical
prostatectomy or radiation. Stage C is a large cancer mass
involving most or all of the prostate and is also further
subdivided into two sub-stages. In sub-stage C1, the cancer
forms a continuous mass that may have extended beyond the
prostate. In sub-stage C2, the cancer forms a continuous mass
that invades the surrounding tissue. Treatment for both these
sub-stages is radiation with or without drugs to address the
cancer. The fourth stage, Stage D is metastatic cancer and
is also subdivided into two sub-stages. In sub-stage D1, the
cancer appears in the lymph nodes of the pelvis. In sub-stage
D2, the cancer involves tissues beyond lymph nodes. Treatment
for both of these sub-stages is systemic. drugs to address the
cancer as well as pain.
However, current prostate cancer staging methods are
limited. As many as 50% of prostate cancers initially staged
as A2, 8, or C are actually stage D, metastatic. Discovery
of metastasis is significant because patients with metastatic
cancers have a poorer prognosis and require significantly
different therapy than those with localized cancers. The five
year survival rates for patients with localized and metastatic
prostate cancers are 93% and 29%, respectively.
Accordingly, there is a great need for more sensitive
and accurate methods for the staging of a cancer in a human
to determine whether or not such cancer has metastasized and
for monitoring the progress of a cancer in a human which has
not metastasized for the onset of metastasis.
It has now been found that a number of proteins in the
public domain are useful as diagnostic markers for prostate
cancer. These diagnostic markers are referred to herein as
cancer specific genes or CSGs and include, but are not limited
to: Pro109 which is a human zinc-a 2-glycoprotein (Freje et
al. Genomics 1993 18(3):575-587); Proll2 which is a human
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cysteine-rich protein with a zinc-finger motif (Liebhaber et
al. Nucleic Acid Research 1990 18(13):3871-3879; fi9lkJ9514772 and
W09895436); Pro111 which is a prostate-specific
transglutaminase (Dubbink et al. Genomics 1998 51(3):434-944);
Pro115 which is a novel serine protease with transmembrane,
LDLR, and SRCR domains and maps to 21q22.3 (Paoloni-Giacobino
et al. Genomics 1997 44(3):309-320; W09837418 and W0987093);
Pro110 which is a human breast carcinoma fatty acid synthase
(U.S. Patent 5,665,874 and W09403599); Pro113 which is a
homeobox gene, HOXB13 (Steinicki et al. J. Invest. Dermatol.
1998 111:57-63); Pro114 which is a human tetraspan NET-1
(W09839446); and Pro118 which is a human JM27 protein
(W09845435). ESTs for these CSGs are set forth in SEQ ID NO:
1, 3, 5, 7, 9, 11, 13 and 15 while the full length contigs for
these CSGs are set forth in SEQ ID N0:2, 4, 6, 8, 10, 12, 14
and I6, respectively. Additional CSGs for use in the present
invention are depicted herein in SEQ ID N0: 17, 18, 19 and 20.
In the present invention, methods are provided for
detecting, diagnosing, monitoring, staging, prognosticating,
imaging and treating prostate cancer via the cancer specific
genes referred to herein as CSGs. For purposes of the present
invention, CSG refers, among other things, to native protein
expressed by the gene comprising a polynucleotide sequence of
SEQ ID NO:l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
I6, 17, 18, 19 or 20. By "CSG" it is also meant herein
polynucleotides which, due to degeneracy in genetic coding,
comprise variations i.n nucleotide sequence as compared to SEQ
ID NO: 1-20, but which still encode the same protein. In the
alternative, what is meant by CSG as used herein, means the
native mRNA encoded by the gene comprising the polynucleotide
sequence of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20, levels of the gene
comprising the polynucleotide sequence of SEQ ID NO:1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20, or levels of a polynucleotide which is capable of
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hybridizing under stringent conditions to the antisense
sequence of SEQ ID N0:1, 2, 3, 4, 5, 6, 7, 8, 9, ~-0, 11, 12,
13, 14, 15, 16, 17, 18, 19 or 20.
Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in
the art from the following description. It should be
understood, however, that the following description and the
specific examples, while indicating preferred embodiments of
the invention are given by way of illustration only. Various
changes and modifications within the spirit and scope of the
disclosed invention will become readily apparent to those
skilled in the art from reading the following description and
from reading the other parts of the present disclosure.
SUN~ARY OF THE INVENTION
Toward these ends, and others, it is an object of the
present invention to provide a method for diagnosing the
presence of prostate cancer by analyzing for changes in levels
of CSG in cells, tissues or bodily fluids compared with levels
of CSG in preferably i:he same cells, tissues, or bodily fluid
type of a normal human control, wherein a change in levels of
CSG in the patient versus the normal human control is
associated with prostate cancer.
Further provided is a method of diagnosing metastatic
prostate cancer in a patient having prostate cancer which is
not,known to have metastasized by identifying a human patient
suspected of having prostate cancer that has metastasized;
analyzing a sample of cells, tissues, or bodily fluid from
such patient for CSG; comparing the CSG levels in such cells,
tissues, or bodily fluid with levels of CSG in preferably the
same cells, tissues, or bodily fluid type of a normal human
control, wherein an increase in CSG levels in the patient
versus the normal human control is associated with prostate
cancer which has metastasized.
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Also provided by the invention is a method of staging
prostate cancer in a human which has such ~ cancer by
identifying a human patient having such cancer; analyzing a
sample of cells, tissues, or bodily fluid from such patient
for CSG; comparing CSG levels in such cells, tissues, or
bodily fluid with levels of CSG in preferably the same cells,
tissues, or bodily fluid type of a normal human control
sample, wherein an increase in CSG levels in the patient
versus the normal human control is associated with a cancer
which is progressing and a decrease in the levels of CSG is
associated with a cancer which is regressing or in remission.
Further provided is a method of monitoring prostate
cancer in a human having such cancer for the onset of
metastasis. The method comprises identifying a human patient
having such cancer that is not known to have metastasized;
periodically analyzing a sample of cells, tissues, or bodily
fluid from such patient for CSG; comparing the CSG levels in
such cells, tissue, or bodily fluid with levels of CSG in
preferably the same cells, tissues, or bodily fluid type of
a normal human control sample, wherein an increase in CSG
levels in the patient versus the normal human control is
associated with a cancer which has metastasized.
Further provided is a method of monitoring the change
in stage of prostate cancer in a human having such cancer by
looking at levels of CSG in a human having such cancer. The
method comprises identifying a human patient having such
cancer; periodically analyzing a sample of cells, tissues, or
bodily fluid from such patient for CSG; comparing the CSG
levels in such cells, tissue, or bodily fluid with levels of
CSG in preferably the same cells, tissues, or bodily fluid
type of a normal human control sample, wherein an increase in
CSG levels in the patient versus the normal human control is
associated with a cancer which is progressing and a decrease
in the levels of CSG is associated with a cancer which is
regressing or in remission.
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Further provided are methods of designing new
therapeutic agents targeted to a CSG for use in 4imaging and
treating prostate cancer. For example, in one embodiment,
therapeutic agents such as antibodies targeted against CSG or
fragments of such antibodies can be used to detect or image
localization of CSG in a patient for the purpose of detecting
or diagnosing a disease or condition. Such antibodies can be
polyclonal, monoclonal, or omniclonal or prepared by molecular
biology techniques. The term "antibody", as used herein and
throughout the instant specification is also meant to include
aptamers and single-stranded oligonucleotides such as those
derived from an in vitro evolution protocol referred to as
SELEX and well known to those skilled in the art. Antibodies
can be labeled with a variety of detectable labels including,
but not limited to, radioisotopes and paramagnetic metals.
Therapeutics agents such as antibodies or fragments thereof
can also be used in the treatment of diseases characterized
by expression of CSG. In these applications, the antibody can
be used without or with derivatization to a cytotoxic agent
such as a radioisotope, enzyme, toxin, drug or a prodrug.
Other objects, features, advantages and aspects of the
present invention will become apparent to those of skill in
the art from the following description. It should be
understood, however, that the following description and the
specific examples, while indicating preferred embodiments of
the invention, are given by way of illustration only. Various
changes and modifications within the spirit and scope of the
disclosed invention will become readily apparent to those
skilled in the art from reading the following description and
from reading the other parts of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to diagnostic assays and
methods, both quantitative and qualitative for detecting,
diagnosing, monitoring, staging and prognosticating cancers
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by comparing levels of CSG in a human patient with those of
CSG in a normal human control. For purposes of~the present
invention, what is meant be CSG levels is, among other things,
native protein expressed by the gene comprising a
polynucleotide sequence of SEQ ID N0:1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 1?, 18, 19 or 20. By "CSG" it
is also meant herein polynucleotides which, due to degeneracy
in genetic coding, comprise variations in nucleotide sequence
as compared to SEQ ID NO: 1-20, but which still encode the
same protein. The native protein being detected, may be
whole, a breakdown product, a complex of molecules or
chemically modified. In the alternative, what is meant by
CSG as used herein, means the native mRNA encoded by the gene
comprising the polynucleotide sequence of SEQ ID NO:l, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, I9 or
20, levels of the gene comprising the polynucleotide sequence
of SEQ ID NO:1, 2, 3, 4, 5, 5, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20, or levels of a polynucleotide which
is capable of hybridizing under stringent conditions to the
antisense sequence of SEQ ID NO:l, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Such levels are
preferably determined in at least one of, cells, tissues
and/or bodily fluids, including determination of normal and
abnormal levels. Thus, for instance, a diagnostic assay in
accordance with the invention for diagnosing overexpression
of CSG protein compared to normal control bodily fluids,
cells, or tissue samples may be used to diagnose the presence
of prostate cancer.
All the methods of the present invention may optionally
include determining the levels of other cancer markers as well
as CSG. Other cancer markers, in addition to CSG, useful in
the present invention will depend on the cancer being tested
and are known to those of skill in the art.
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Diagnostic Assays
The present invention provides methods for di~'dgnosing the
presence of prostate cancer by analyzing for changes in levels
of CSG in cells, tissues or bodily fluids compared with levels
of CSG in cells, tissues or bodily fluids of preferably the
same type from a normal human control, wherein an increase in
levels of CSG in the patient versus the normal human control
is associated with the presence of prostate cancer.
Without limiting the instant invention, typically, for
a quantitative diagnostic assay a positive result indicating
the patient being tested has cancer is one in which cells,
tissues or bodily fluid levels of the cancer marker, such as
CSG, are at least two times higher, and most preferably are
at least five times higher, than in preferably the same cells,
tissues or bodily fluid of a normal human control.
The present invention also provides a method of
diagnosing metastatic prostate cancer in a patient having
prostate cancer which has not yet metastasized for the onset
of metastasis. In the method of the present invention, a
human cancer patient suspected of having prostate cancer which
may have metastasized (but which was not previously known to
have metastasized) is identified. This is accomplished by a
variety of means known to those of skill in the art.
In the present invention, determining the presence of CSG
levels in cells, tissues or bodily fluid, is particularly
useful for discriminating between prostate cancer which has
not metastasized and prostate cancer which has metastasized.
Existing techniques have difficulty discriminating between
prostate cancer which has metastasized and prostate cancer
-30 which has not metastasized and proper treatment selection is
often dependent upon such knowledge.
In the present invention, the cancer marker levels
measured in such cel:Ls, tissues or bodily fluid is CSG, and
are compared with levels of CSG in preferably the same cells,
tissue or bodily fluid type of a normal human control. That
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is, if the cancer marker being observed is just CSG in serum,
this level is preferably compared with the leveik of CSG in
serum of a normal human control. An increase in the CSG in
the patient versus the normal human control is associated with
prostate cancer which has metastasized.
Without limiting the instant invention, typically, for
a quantitative diagnostic assay a positive result indicating
the cancer in the patient being tested or monitored has
metastasized is one in which cells, tissues or bodily fluid
levels of the cancer marker, such as CSG, are at least two
times higher, and most preferably are at least five times
higher, than in preferably the same cells, tissues or bodily
fluid of a normal patient.
Normal human control as used herein includes a human
patient without cancer and/or non cancerous samples from the
patient; in the methods for diagnosing or monitoring for
metastasis, normal human control may preferably also include
samples from a human patient that is determined by reliable
methods to have prostate cancer which has not metastasized.
2 0 Staging
The invention also provides a method of staging prostate
cancer in a human patient. The method comprises identifying
a human patient having such cancer and analyzing cells,
tissues or bodily fluid from such human patient for CSG. The
CSG levels determined in the patient are then compared with
levels of CSG in preferably the same cells, tissues or bodily
fluid type of a normal human control, wherein an increase in
CSG levels in the human patient versus the normal human
control is associated with a cancer which is progressing and
~0 a decrease in the levels of CSG (but still increased over true
normal levels) is associated with a cancer which is regressing
or in remission.
Moai toririg
Further provided is a method of monitoring prostate
cancer in a human patient having such cancer for the onset of
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metastasis. The method comprises identifying a human patient
having such cancer that is not known to have mcs'tastasized;
periodically analyzing cells, tissues or bodily fluid from
such human patient for CSG; and comparing the CSG levels
determined in the human patient with levels of CSG in
preferably the same cells, tissues or bodily fluid type of a
normal human control, wherein an increase in CSG levels in the
human patient versus the normal human control is associated
with a cancer which has metastasized. :Cn this method, normal
human control samples may also include prior patient samples.
Further provided by this invention is a method of
monitoring the change in stage of prostate cancer in a human
patient having such cancer. The method comprises identifying
a human patient having such cancer; periodically analyzing
cells, tissues or bodily fluid from such human patient for
CSG; and comparing the CSG levels determined in the human
patient with levels of CSG in preferably the same cells,
tissues or bodily fluid type of a normal human control,
wherein an increase in CSG levels in the human patient versus
the normal human control is associated with a cancer which is
progressing in stage and a decrease in the levels of CSG is
associated with a cancer which is regressing in stage or in
remission. In this method, normal human control samples may
also include prior patient samples.
Monitoring a patient for onset of metastasis is periodic
and,preferably done on a quarterly basis. However, this may
be more or less frequent depending on the cancer, the
particular patient, and the stage of the cancer.
Assay Techniques
Assay techniques that can be used to determine levels of
gene expression (including protein levels), such as CSG of the
present invention, in a sample derived from a patient are well
known to those of skill in the art. Such assay methods
include, without limitation, radioimmunoassays, reverse
transcriptase PCR (RT-PCR) assays, immunohistochemistry
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assays, in situ hybridization assays, competitive-binding
assays, Western Blot analyses, ELISA assays a~?'ti proteomic
approaches: two-dimensional gel electrophoresis (2D
electrophoresis) and non-gel based approaches such as mass
spectrometry or protein interaction profiling. Among these,
ELISAs are frequently preferred to diagnose a gene's expressed
protein in biological fluids.
An ELISA assay initially comprises preparing an antibody,
if not readily available from a commercial source, specific
to CSG, preferably a monoclonal antibody. In addition a
reporter antibody generally is prepared which binds
specifically to CSG. The reporter antibody is attached to a
detectable reagent such as radioactive, fluorescent or
enzymatic reagent, for example horseradish peroxidase enzyme
or alkaline phosphatase.
To carry out the ELISA, antibody specific to CSG is
incubated on a solid support, e.g. a polystyrene dish, that
binds the antibody. Any free protein binding sites on the
dish are then covered by incubating with a non-specific
protein such as bovine serum albumin. Next, the sample to be
analyzed is incubated in the dish, during which time CSG binds
to the specific antibody attached to the polystyrene dish.
Unbound sample is washed out with buffer. A reporter antibody
specifically directed to CSG and linked to a detectable
reagent such as horseradish peroxidase is placed in the dish
resulting in binding of the reporter antibody to any
monoclonal antibody bound to CSG. Unattached reporter
antibody is then washed out. Reagents for peroxidase
activity, including a colorimetric substrate are then added
to the dish. Immobilized peroxidase, linked to CSG
antibodies, produces a colored reaction product. The amount
of color developed in a given time period is proportional to
the amount of CSG protein present in the sample. Quantitative
results typically ar_e obtained by reference to a standard
curve,
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A competition assay can also be employed wherein
antibodies specific to CSG are attached to a solid~support and
labeled CSG and a sample derived from the host are passed over
the solid support. The amount of label detected which is
attached to the solid support can be correlated to a quantity
of CSG in the sample.
Nucleic acid methods can also be used to detect CSG mRNA
as a marker for prostate cancer. Polymerase chain reaction
(PCR) and other nucleic acid methods, such as ligase chain
reaction (LCR) and nucleic acid sequence based amplification
(NASABA), can be used to detect malignant cells for diagnosis
and monitoring of various malignancies. For example, reverse-
transcriptase PCR (RT-PCR) is a powerful technique which can
be used to detect the presence of a specific mRNA population
in a complex mixture of thousands of other mRNA species. In
RT-PCR, an mRNA species is first reverse transcribed to
complementary DNA (cDNA) with use of the enzyme reverse
transcriptase: the cDNA is then amplified as in a standard PCR
reaction. RT-PCR can thus reveal by amplification the
presence of a single species of mRNA. Accordingly, if the
mRNA is highly specific for the cell that produces it, RT-PCR
can be used to identify the presence of a specific type of
cell.
Hybridization to clones or oligonucleotides arrayed on
a solid support (i.e. gridding) can be used to both detect the
expression of and quantitate the level of expression of that
gene. In this approach, a cDNA encoding the CSG gene is fixed
to a substrate. The substrate may be of any suitable type
including but not limited to glass, nitrocellulose, nylon or
plastic. At least a portion of the DNA encoding the CSG gene
is attached to the substrate and then incubated with the
analyte, which may be RNA or a complementary DNA (cDNA) copy
of the RNA, isolated from the tissue of interest.
Hybridization between the substrate bound DNA and the analyte
can be detected and quantitated by several means including but
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not limited to radioactive labeling or fluorescence labeling
of the analyte or a secondary molecule designed tt~ detect the
hybrid, Quantitation of the level of gene expression can be
done by comparison of the intensity of the signal from the
analyte compared with that determined from known standards.
The standards can be obtained by in vitro transcription of the
target gene, quantitating the yield, and then using that
material to generate a standard curve.
Of the proteomic approaches, 2D electrophoresis is a
technique well known to those in the art. Isolation of
individual proteins from a sample such as serum is
accomplished using sequential separation of proteins by
different characteristics usually on polyacrylamide gels.
First, proteins are separated by size using an electric
current. The current acts uniformly on all proteins, so
smaller proteins move farther on the gel than larger proteins.
The second dimension applies a current perpendicular to the
first and separates proteins not on the basis of size but on
the specific electric charge carried by each protein. Since
no two proteins with different sequences are identical on the
basis of both size and charge, the result of a 2D separation
is a square gel in which each protein occupies a unique spot.
Analysis of the spots with chemical or antibody probes, or
subsequent protein microsequencing can reveal the relative
abundance of a given protein and the identity of the proteins
in the sample.
The above tests can be carried out on samples derived
from a variety of cells, bodily fluids and/or tissue extracts
such as homogenates or solubilized tissue obtained from a
.30 patient. Tissue extracts are obtained routinely from tissue
biopsy and autopsy material. Bodily fluids useful in the
present invention include blood, urine, saliva or any other
bodily secretion or derivative thereof. By blood it is meant
to include whole blood, plasma, serum or any derivative of
blood.
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In Vivo Targeting of CSGs
Identification of these CSGs is also us~ul in the
rational design of new therapeutics for imaging and treating
cancers, and in particular prostate cancer. For example, in
one embodiment, antibodies which specifically bind to CSG can
be raised and used in vivo in patients suspected of suffering
from prostate cancer. Antibodies which specifically bind a
CSG can be injected into a patient suspected of having
prostate cancer for diagnostic and/or therapeutic purposes.
The preparation and use of antibodies for in vivo diagnosis
is well known in the art. For example, antibody-chelators
labeled with Indium-111 have been described for use in the
radioimmunoscintographic imaging of carcinoembryonic antigen
expressing tumors (Sumerdon et al. Nucl. Med. Biol. 1990
17:297-254). In particular, these antibody-chelators have
been used in detecting tumors in patients suspected of having
recurrent colorectal cancer (Griffin et al. J. Clin. Onc. 1991
9:631-640). Antibodies with paramagnetic ions as labels for
use in magnetic resonance imaging have also been described
(Lauffer, R.B. Magnetic Resonance in Medicine 1991 22:339-
342). Antibodies directed against CSG can be used in a
similar manner. Labeled antibodies which specifically bind
CSG can be injected into patients suspected of having prostate
cancer for the purpose of diagnosing or staging of the disease
status of the patient. The label used will be selected in
accordance with the imaging modality to be used. For example,
radioactive labels such as Indium-117., Technetium-99m or
Iodine-131 can be used for planar scans or single photon
emission computed tomog~raphy (SPECT). Positron emitting
labels such as Fluorine-19 can be used in positron emission
tomography. Paramagnetic ions such as Gadlinium (III) or
Manganese (II) can be used in magnetic resonance imaging
(MRI). Localization of the label permits determination of the
spread of the cancer. The amount of label within an organ or
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tissue also allows determination of the presence or absence
of cancer in that organ or tissue.
For patients diagnosed with prostate cancer, injection
of an antibody which specifically binds CSG can also have a
therapeutic benefit. The antibody may exert its therapeutic
effect alone. Alternatively, the antibody can be conjugated
to a cytotoxic agent such as a drug, toxin or radionuclide to
enhance its therapeutic effect. Drug monoclonal antibodies
have been described in the art for example by Garnett and
Baldwin, Cancer Research 1986 96:2407-2412. The use of toxins
conjugated to monoclonal antibodies for the therapy of various
cancers has also been described by Pastan et al. Cell 1986
47:641-648. Yttrium-90 labeled monoclonal antibodies have
been described for maximization of dose delivered to the tumor
while limiting toxicity to normal tissues (Goodwin and Meares
Cancer Supplement 1997 80:2675-2680). Other cytotoxic
radionuclides including, but not limited to Copper-67, Iodine-
131 and Rhenium-186 can also be used for labeling of
antibodies against CSG.
Antibodies which can be used in these in vivo methods
include polyclonal, monoclonal and omni.clonal antibodies and
antibodies prepared via molecular biology techniques.
Antibody fragments and aptamers and single-stranded
oligonucleotides such as those derived from an in vitro
evolution protocol referred to as SELEX and well known to
those skilled in the art can also be used.
Small molecules predicted via computer imaging to
specifically bind to regions of CSGs can also be designed and
synthesized and tested for use in the imaging and treatment
of prostate cancer. Further, libraries of molecules can be
screened for potential anticancer agents by assessing the
ability of the molecule to bind to CSGs identified herein.
Molecules identified in the library as being capable of
binding to CSG are key candidates for further evaluation for
use in the treatment of prostate cancer.
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EXAMPLES
The present invention is further described by the
following examples. These examples are provided solely to
illustrate the invention by reference to specific embodiments.
These exemplifications, while illustrating certain aspects of
the invention, do not portray the limitations or circumscribe
the scope of the disclosed invention.
All examples outlined here were carried out using
standard techniques, which are well known and routine to those
of skill in the art, except where otherwise described in
detail. Routine molecular biology techniques of the following
example can be carried out as described in standard laboratory
manuals, such as Sambrook et al., MOLECULAR CLONING: A
LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989).
Example l: Identification of CSGs
Identification of CSGs were carried out by a systematic
analysis of data in the LIFESEQ database available from Incyte
Pharmaceuticals, Palo Alto, CA, using the data mining Cancer
Leads Automatic Search Package (CLASP) developed by diaDexus
LLC, Santa Clara, CA.
The CLASP performs the following steps: selection of
highly expressed organ specific genes based on the abundance
level of the corresponding EST in the targeted organ versus
all the other organs; analysis of the expression level of each
highly expressed organ specific genes in normal, tumor tissue,
disease tissue and tissue libraries associated with tumor or
disease; selection of the candidates demonstrating component
ESTs were exclusively or more frequently found in tumor
libraries. The CLASP allows the identification of highly
expressed organ and cancer specific genes. A final manual in
depth evaluation is then performed to finalize the CSGs
selection.
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Clones depicted in the following Table 1 are CSGs useful
in diagnosing, monitoring, staging, imaging aid treating
prostate cancer.
Table l: CSGs
Clone ID Pro # SEQ ID NO:
3924528H1 Pro109 1,2
578349H1 Pro112 3,4
1794013H1 Prolll 5,6
2189835H1 Pro115 7,8
3277219H1 Pro110 9,10
1857415 Pro113 11,12
1810463H1 Pro119 13,14
zr65G11 Pro118 15,16
2626135H1 17
zd46d08 18
1712252H1 19
789583H1 20
Example 2: Relative Quantitation of Gene Expression
Real-Time quantitative PCR with fluorescent Taqman probes
is a quantitation detection system utilizing the 5'- 3'
nuclease activity of Taq DNA polymerase. The method uses an
internal fluorescent oligonucleotide probe (Taqman) labeled
with a 5' reporter dye and a downstream, 3' quencher dye.
During PCR, the 5'-3' nuclease activity of Taq DNA polymerase
releases the reporter, whose fluorescence can then be detected
by the laser detector of the Model 7700 Sequence Detection
System (PE Applied Biosystems, Foster City, CA, USA).
Amplification of an endogenous control is used to
standardize the amount: of sample RNA added to the reaction and
normalize for Reverse Transcriptase (RT) efficiency. Either
cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
ATPase, or 18S ribosomal RNA (rRNA) is used as this endogenous
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control. To calculate relative quantitation between all the
samples studied, the target RNA levels for one ' ample were
used as the basis for comparative results (calibrator).
Quantitation relative to the "calibrator" can be obtained
using the standard curve method or the comparative method
(User Bulletin #2: ABI PRISM 7700 Sequence Detection System).
The tissue distribution and the level of the target gene
were evaluated for every sample in normal and cancer tissues.
Total RNA was extracted from normal tissues, cancer tissues,
and from cancers and the corresponding matched adjacent
tissues. Subsequently, first strand cDNA was prepared with
reverse transcriptase and the polymerase chain reaction was
done using primers and Taqman probes specific to each target
gene. The results were analyzed using the ABI PRISM 7700
Sequence Detector. The absolute numbers are relative levels
of expression of the target gene in a particular tissue
compared to the calibrator tissue.
Expression of Clone ID 3424528H1 (Pro109):
For the CSG Pro109, real-time quantitative PCR was
performed using the following primers:
Forward Primer:
5'- ATCAGAACAAAGAGGCTGTGTC - 3' (SEQ ID N0:21)
Reverse Primer:
5'- ATCTCTAAAGCCCCAACCTTC - 3' (SEQ ID N0:22)
The absolute numbers depicted in Table 2 are relative levels
of expression of the CSG referred to as Pro109 in 12 normal
different tissues. All the values are compared to normal
stomach (calibrator). These RNA samples are commercially
available pools, originated by pooling samples of a particular
tissue from different individuals.
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Table 2: Relative Levels of CSG Pro109 Expression in Pooled
Samples w
Tis sue NORMnT.
Colon 0.02
Endometrium 0.01
Kidney 0.48
Liver 14.83
Ovary 0.08
Pancreas 4.38
Prostate 11.24
Small Intestine 0.42
Spleen p
Stomach 1
Testis 0.62
Uterus 0.02
The relative levels of expression in Table 2 show that with
the exception of liver (14.83), Prol09 mRNA expression is
higher (11.24) in prostate compared with all other normal
tissues analyzed. Pancreas, with a relative expression level
of 4.38, is the only other tissue expressing considerable mRNA
for Pro109.
The absolute numbers in Table 2 were obtained analyzing
pools of samples of a particular tissue from different
individuals. They cannot be compared to the absolute numbers
originated from RNA obtained from tissue samples of a single
individual in Table 3.
The absolute numbers depicted in Table 3 are relative
levels of expression of Pro109 in 28 pairs of matching samples
and 4 unmatched samples. All the values are compared to
normal stomach (cali.brator). A matching pair is formed by
mRNA from the cancer sample for a particular tissue and mRNA
from the normal adjacent sample for that same tissue from the
same individual.
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Table 3: Relative Levels of CSG Pro109 Expression in
Individual Samples '"
Sample ID Tissue Cancer Matcliing
Normal
Adjacent
Pro34B Prostate 1 5.98 6.06
Pro65XB Prostate 2 16.68 3.85
Pro69XB Prostate 3 20.46 6.82
Pro78XB Prostate 4 1.39 1.4
ProlOIXB Prostate 5 24.8 9.8
Prol2B Prostate 6 9.1 0.2
Prol3XB Prostate 7 0.5 9.7
Pro20XB Prostate 8 13 12.5
Pro23B Prostate 9 16.8 3
Ovr100050 Ova ry 1 0.4
Ovr1028 Ovary 2 1.9
Ovrl8GA Ovary 3 0.1
Ovr206I Ovary 4 0.1
Maml2X Mammary Gland 1 13.5 1.4
Mam47XP Mammary Gland 2 0.7 0.2
Lng47XQ Lung 1 2.36 0.03
Lng60XL Lung 2 7.39 0.2
Lng75XC Lung 3 0.77 0.27
StoAC44 Stomach 1 0.05 1.19
StoAC93 Stomach 2 0.55 0.8
StoAC99 Stomach 3 0.12 3.04
ColAS43 Colon 1 7.6.11 0.07
ColAS45 Colon 2 0.11 0.08
ColAS46 Colon 3 9.99 0.4
Livl5XA Liver 1 8.43 10.97
Liv42X Liver 2 1.57 20.82
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Liv99XA Liver 3 2.98 9.19
Pan77X Pancreas 1 36 32
Pan82XP Pancreas 2 0.09 7.09
Pan92X Pancreas 3 0.7 0
Pan7lXL Pancreas 9 2.48 0.73
Pan10343 Pancreas 5 46 5.5
U = Negative
In the analysis of matching samples, the higher levels
of expression were in prostate, showing a high degree of
tissue specificity for prostate tissue. Of all the samples
different than prostate analyzed, only 4 cancer samples (the
cancer sample mammary 1 with 13.5, colon 1 with 16.11, liver
1 with 8.43, and lung 2 with 7,39) showed an expression
comparable to the mRNA expression in prostate. These results
confirmed some degree of tissue specificity as obtained with
the panel of normal poaled samples (Table 2).
Furthermore, the level of mRNA expression was compared
in cancer samples and the isogenic normal adjacent tissue from
the same individual. This comparison provides an indication
of specificity for the cancer (e. g. higher levels of mRNA
expression in the cancer sample compared to the normal
adjacent). Table 3 shows overexpression of Pro109 in 6 out
of 9 primary prostate cancer tissues compared with their
respective normal adjacents. Thus, overexpression in the
cancer tissue was observed in 66.66% of the prostate matching
samples tested (total of 9 prostate matching samples).
Altogether, the degree of tissue specificity, plus the
mRNA overexpression in 66.66% of the primary prostate matching
samples tested is indicative of Pro109 being a diagnostic
marker far prostate cancer.
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Expression of Clone ID 578349H1 (Pro112):
For the CSG Pro112, real-time quantitative PCR was
performed using the following primers:
Forward Primer
5'- TGCCGAAGAGGTTCAGTGC - 3' (SEQ ID N0:23)
Reverse Primer
5'- GCCACAGTGGTACTGTCCAGAT - 3' (SEQ ID N0:24)
The absolute numbers depicted in Table 4 are relative
levels of expression of the CSG Pro112 in 12 normal different
tissues. All the values are compared to normal thymus
(calibrator). These RNA samples are commercially available
pools, originated by pooling samples of a particular tissue
from different individuals.
Table 4: Relative Levels of CSG Pro112 Expression in Pooled
Samples
Tissue NORMAL
Brain _ 2.9
Heart 0.1
Kidney 0.2
Liver 0.2
Lung 7.7
Mammary 9 _ 2
Muscle 0.1
Prostate 5.5
Small Intestine 1.8
Testis 1
Thymus 1
Uterus 21
The relative levels of expression in Table 4 show that
Pro112 mRNA expression is the 3rd most highly expressed gene
(after uterus and mammary) in the pool of normal prostate
tissue compared to a total of 12 tissues analyzed. The
absolute numbers in Table 4 were obtained analyzing pools of
samples of a particular tissue from different individuals.
These results demonstrate that Proll2 mRNA expression is
specific for prostate thus indicating Pro112 to be a
diagnostic marker for prostate disease especially cancer.
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Expression of Clone ID 1794013H1 (Prolll):
For the CSG Prolll, real-time quantitative PCR was
performed using the following primers:
Forward Primer
5'- GCTGCAAGTTCTCCACATTGA - 3' (SEQ ID N0:25)
Reverse Primer
5'- CAGCCGCAGGTGAAACAC - 3' (SEQ ID N0:26)
The absolute numbers depicted in Table 5 are relative levels
of expression of the CSG Prolll in 12 normal different
tissues. All the values are compared to normal testis
(calibrator). These RNA samples are commercially available
pools, originated by pooling samples of a particular tissue
from different individuals.
Table 5: Relative Levels of CSG Prolll Expression in Pooled
Samples
Tissue NORMAL
Brain 0.04
Heart p
Kidney 0
Liver 0
Lung 0.05
Mamma ry 0 . 19
Muscle 5166.6
Prostate 1483.72
Small Intestine 0.33
Testis 1
Thymus 0.49
Uterus 0.07
The relative levels of expression in Table 5 show that Prolll
mRNA expression is extraordinarily high in the pool of normal
prostate (1483.72) compared to all the other tissues analyzed
with the exception of muscle (5166.6). These results
demonstrate that Prolll mRNA expression shows specificity for
prostate and muscle.
The absolute numbers in Table 5 were obtained analyzing
pools of samples of a particular tissue from different
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individuals. They cannot be compared to the absolute numbers
originated from RNA obtained from tissue samples ~'f a single
individual in Table 6.
The absolute numbers depicted in Table 6 are relative
levels of expression of Prolll in 48 pairs of matching and 18
unmatched samples. All the values are compared to normal
testis (calibrator). A matching pair is formed by mRNA from
the cancer sample for a particular tissue and mRNA from the
normal adjacent sample for that same tissue from the same
individual.
Table 6: Relative Levels of CSG Prolll Expression in
Individual Samples
Sample ID Tissue Cancer Matching
Normal
Adjacent
Pro101XB Prostate 1 8.3 21.8
Prol2B Prostate 2 2336 133
Prol3XB Prostate 3 3.4 23
Pro20XB Prostate 9 21.6 121.5
Pro23B Prostate 5 19.4 3.7
Pro34B Prostate 6 15 39
Pro65XB Prostate 7 8 867
Pro69XB Prostate 8 56 94
Pro78XB Prostate 9 24 1515
Pro.84XB Prostate 20 119 15.35
Pro90XB Prostate 11 8.08 112.2
Pro9lXB Prostate 12 0.88 51.8
ProC215 Prostate 13 0.3
ProC234 Prostate 14 0.35
ProC280 Prostate 15 436.5
Pro109XB Prostate 16 3.43 265
Pro110 Prostate 17 18.2 8.73
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Pro125XB Prostate 18 0.34 186
Pro326 Prostate 19 1392 110
ProlOR Prostate 20 0.5
(prostatitis)
Pro20R Prostate 21 24.1
(prostatitis)
Pro258 Prostate 22 (BPH) 9610
Pro263C Prostate 23 (BPH) 0
Pro267A Prostate 24 (BPH) 1.46
Pro271A Prostate 25 (BPH) 0
Pro460Z Prostate 26 (BPH) 1.47
ProC032 Prostate 27 (BPH) 14.9
Tst39X Testis 1 0 0
B1d32XK Bladder 1 0.49 0.41
B1d96XK Bladder 2 0 0
B1d66X Bladder 3 0 0
BldTRl4 Bladder 4 0 0
Kid106XD Kidney 1 0 0
Kid107XD Kidney 2 0 0
Kid109XD Kidney 3 0 0
Pan10343 Pancreas 1 0 0
Pan7lXL Pancreas 2 0 0
Pan77X Pancreas 3 0 0
Livl5XA Liver 1 0 0
Liv42X Liver 2 0 0
ClnAS43 Colon 1 0 0
ClnAS45 Colon 2 0 0
ClnAS46 Colon 3 0 0
ClnAS67 Colon 4 0 0
ClnACl9 Colon 5 0 0
ClnASl2 Colon 6 0 0
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SmI2IXA Small Intestine 1 0 0
SmIH89 Sma:l1 Intestine 0 0
2
Lng47XQ Lung 1 0.7 0
Lng60XL Lung 2 0 0
Lng75XC Lung 3 0 p
Lng90X Lung 9 0 0
Maml2X Mammary Gland 1 0 1.4
Mam59X Mammary Gland 2 0.2 0
MamA06X Mammary Gland 3 0 0
MamS127 Mammary Gland 4 0 p
Mam162X Mammary Gland 5 0 0
Mam42DN Mammary Gland 6 0 0
Ovr103X Ovary 1 0.14 0
Ovr10050 Ovary 2 0.2
Ovr1028 Ovary 3 0
Ovr10400 Ovary 4 0.2
OvrlBGA Ovary 5 p
Ovr206I Ovary 6 0
Ovr20GA Ovary 7 0.2
Ovr25GA Ovary 8 0
u= megamve
In the analysis of matching samples, the higher levels
of .expression were in prostate showing a high degree of tissue
specificity for prostate. These results confirm the tissue
specificity results obtained with normal pooled samples (Table
5) .
Furthermore, the level of mRNA expression in cancer
samples and the isogenic normal adjacent tissue from the same
individual were compared. This comparison provides an
indication of specificity for cancer (e.g. higher levels of
mRNA expression in the cancer sample compared to the normal
adjacent). Table 6 shows overexpression of Prolll in 5 out
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of 15 primary prostate cancer samples compared with their
respective normal adjacent (prostate samples 2, 5 ~''10, 17, and
19). Similar expression levels were observed in 3 unmatched
prostate cancers (prostate samples 13, 14, 15), 2 prostatitis
(prostate samples 20, 21), and 6 benign prostatic hyperplasia
samples (prostate samples 22 through 27). Thus, there is
overexpression in the cancer tissue of 31.250 of the prostate
matching samples tested (total of 16 prostate matching
samples).
Altogether, the high level of tissue specificity, plus
the mRNA overexpression in 31.25% of the prostate matching
samples tested are indicative of Prolll being a diagnostic
marker for prostate cancer.
Expression of Clone ID 2189835H1 (Pro115):
For the CSG Pro115, real-time quantitative PCR was
performed using the following primers:
Forward Primer
5'- TGGCTTTGAACTCAGGGTCA - 3' (SEQ ID N0:27)
Reverse Primer
5'- CGGATGCACCTCGTAGACAG - 3' (SEQ ID N0:28)
The absolute numbers depicted in Table 7 are relative levels
of expression of the CSG Pro115 in 12 normal different
tissues. All the values are compared to normal thymus
(calibrator). These RNA samples are commercially available
pools, originated by pooling samples of a particular tissue
from different individuals.
Table 7: Relative Levels of CSG Pro115 Expression in Pooled
Samples
Tissue _ NORMAL
Brain 0.016
Heart ~ 0.002
Kidney g,Og
Liver 2.20
Lun 112.99
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Mammary 29.45
Muscle _ 0.05
Prostate 337.79
Small Intestine 7.54
Testis 1.48
Thymus 1
Uterus 1.4
The relative levels of expression in Table 7 show that
Proll5 mRNA expression is higher (337.79) in prostate compared
with all the other normal tissues analyzed. Lung, with a
relative expression level of 112.99, and mammary (29.446) are
the other tissues expressing moderate levels of mRNA for
Pro115. These results establish Pro115 mRNA expression to be
highly specific for prostate.
The absolute numbers in Table 7 were obtained analyzing
pools of samples of a particular tissue from different
individuals. They cannot be compared to the absolute numbers
originated from RNA obtained from tissue samples of a single
individual in Table 8.
The absolute numbers depicted in Table 8 are relative
levels of expression of Pro115 in 17 pairs of matching and 21
unmatched samples. All the values are compared to normal
thymus (calibrator). A matching pair is formed by mRNA from
the cancer sample for a particular tissue and mRNA from the
normal adjacent sample for that same tissue from the same
individual.
Table 8: Relative bevels of CSG ProllS Expression in
Individual Samples
Sample ID Tissue Cancer Matching
Normal
Adjacent
Prol2B Prostate 1 1475.9 190.3
ProC234 Prostate 2 169.61
Pro109XB Prostate 3 639.53
ProlOIXB Prostate 4 1985.2 2882.9
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Prol3XB Prostate 5 34.9 13.9
Pro215 Prostate 6 525.59
Pro125XB Prostate 7 556.05
Pro23B Prostate 8 1891.4 1118.6
ProC280 Prostate 9 454.3
Pro20XB Prostate 10 1332.6
Pro34B Prostate 11 362.91
Pro65XB Prostate 12 135.06
Pro69XB Prostate 13 179.67
ProIOR Prostate 14 143.82
(prostatitis)
Pro20R Prostate 15 397.79
(prostatitis)
Pro258 Prostate 16 (BPH) 216.6
Pro263C Prostate 17 (BPH) 601.25
Pro267A Prostate 18 (BPH) 200.28
Pro271A Prostate 19 (BPH) 111.43
Pro460Z Prostate 20 (BPH) 53.84
ProC032 Prostate 21 (BPH) _'i6.94
SmI2IXA Small Intestine 28.8 29.9
1
SmIH89 Small Intestine 70.8 348.5
2
ClnACl9 Colon 1 22.73 446.47
ClnASl2 Colon 2 116.97 493.18
Kid106XD Kidney 1 86.13 41.14
Kid107XD Kidney 2 0.26 35.14
Lng47XQ Lung 1 5.13 20.98
Lng60XL Lung 2 13.93 114.78
Lng75XC Lung 3 16.47 53.79
Maml2X Mammary Gland 1 6.25 10.75
Mam162X Mammary Gland 2 1.84 2.54
Mam42DN Mammary Gland 3 23.08 35.51
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Ovr10050 Ovary 1 0.9
.",.
Ovr1028 Ovary 2 261.4
Ovr103X Ovary 3 7 0.1
Ovr20GA Ovary 4 - 0
Ovr25GA Ovary 5 0
0 = Negative
Higher levels of expression were seen in prostate,
showing a high degree of tissue specificity for prostate
tissue. Of all the analyzed samples different from prostate,
only two cancer samples (colon 2 with 116.97 and ovary 2 with
261.4 ), and 5 normal- adjacent tissue samples (small intestine
2, colon 1, colon 2, kidney 1, and lung 2), showed an
expression comparable to the mRNA expression in prostate.
These results confirmed the tissue specificity results
obtained with the panel of normal pooled samples (Table 7).
Furthermore, the levels of mRNA expression in cancer
samples and the isogenic normal adjacent tissue from the same
individual were compared. This comparison provides an
indication of specificity for the cancer (e. g. higher levels
of mRNA expression in the cancer sample compared to the normal
adjacent). Table 8 shows higher expression of Proll5 in 3 out
of 9 matched prostate cancer tissues (prostate samples l, 5
& 8) .
Altogether, the high level of tissue specificity, plus
the. higher expression in 750 of the prostate matching samples
tested, are indicative of Pro115 being a diagnostic marker for
prostate cancer.
Expression of Clone ID 3277219H1 (Pro110):
For the CSG Pro110, real-time quantitative PCR was
performed using the following primers:
Forward Primer
5'- CGGCAACCTGGTAGTGAGTG - 3' (SEQ ID N0:29)
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Reverse Primer
5'- CGCAGCTCCTTGTAAACTTCAG - 3' (SEQ I~ N0:30)
The absolute numbers depicted in Table 9 are relative levels
of expression of the CSG Pro110 in 12 normal different
tissues. All the values are compared to normal small
intestine (calibrator). These RNA samples are commercially
available pools, originated by pooling samples of a particular
tissue from different individuals.
Table 9: Relative Levels of CSG ProllO Expression in Pooled
Samples
Tissue NORMAL
Brain 6.61
Heart. 0 . 7
Kidney 0.74
Live r 7 . 94
Lung 11.88
Mammary 22 . 7 8
Muscle 6.77
Prostate 3.01
Small Intestine 1
Testis 2.58
Thymus 13.79
Uterus 2.61
The relative levels of expression in Table 9 show that Pro110
mRNA expression is not as high in normal prostate (3.01)
compared with all the other normal tissues analyzed.
The absolute numbers in Table 9 were obtained analyzing
pools of samples of a particular tissue from different
individuals. They cannot be compared to the absolute numbers
originated from RNA obtained from tissue samples of a single
individual in Table 10.
The absolute numbers depicted in Table 10 are relative
levels of expression of Pro110 in 33 pairs of matching
samples. All the values are compared to normal small
intestine (calibrator). A matching pair is formed by mRNA
from the cancer sample for a particular tissue and mRNA from
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the normal adjacent sample for that same tissue from the same
individual.
Table 10: Relative Levels of CSG Pro110 Expression in
Individual Samples
Sample ID Tissue Cancer Matching
Normal
Adjacent
Prol2B Prostate 1 11.8 0.3
Pro78XB Prostate 2 14.3 6.3
ProlOIXB Prostate 3 33.2 10.7
Prol3XB Prostate 9 0.3 0.4
Pro23XB Prostate 5 25.5 14.4
Pro20XB Prostate 6 43.3 4
Pro34XB Prostate 7 31.8 18.7
Pro65XB Prostate 8 26.9 3.4
Pro69XB Prostate 9 12.5 7
Lng75XC Lung 1 1.9 3
Lng90X Lung 2 5.5 0.5
LngACll Lung 3 9.3 9.7
LngAC32 Lung 4 11.2 2.2
Lng47XQ Lung 5 11.3 0.3
Lng60XL Lung 6 29.1 6.8
Maml2B Mammary Gland 1 19.8 0
Mam603X Mammary Gland 2 13.7 0
Mam82XI Mammary Gland 3 73.5 0
MamA04 Mammary Gland 4 0 24.6
MamBOIIX Mammary Gland 5 17.4 2
MamC012 Mammary Gland 6 0 12.8
MamC034 Mammary Gland 7 0 61
Maml2X Mammary Gland 8 14 2.2
Mam59X Mammary Gland 9 33 2.2
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MamA06X Mammary Gland 10 16.4 0.8
LivlSXA Liver 1 4.7 0.6
Liv42X Liver 2 7.5 2.6
Liv94XA Liver 3 0.4 1.4
ClnAS93 Colon 1 52.9 1.4
ClnAS45 Colon 2 2.1 0.8
ClnAS46 Colon 3 39.8 3.7
SmI2lX Small Intestine 1 0.9 0.1
SmIH89 Small. Intestine 5.8 0.9
2
0 = Negative
The levels of mRNA expression in cancer samples and the
isogenic normal adjacent tissue from the same individual were
compared. This comparison provides an indication of
specificity for the cancer (e. g. higher levels of mRNA
expression in the cancer sample compared to the normal
adjacent). Table 10 shows overexpression of Pro110 in 8 of
the 9 primary prostate cancer tissues compared with their
respective normal adjacent (except prostate 4). Thus, there
was overexpression in 88.88 of the cancer prostate tissue
as compared to the prostate matching samples tested (total of
9 prostate matching samples).
Although not tissue specific, Pro110 mRNA expression is
upregulated in prostate cancer tissues. The mRNA
overexpression in 88.880 of the primary prostate matching
cancer samples tested is indicative of Pro110 being a
diagnostic marker for prostate cancer. Pro110 also showed
overexpression in several other cancers tested including small
intestine, colon, liver, mammary and lung (see Table 10).
Accordingly Pro110 may be a diagnostic marker for other types
of cancer as well.
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Expression of Clone ID 1857415; Gene ID 346880 (Proll3):
For the CSG Pro113, real-time quantitati~~e PCR was
performed using the following primers:
Forward Primer
5'- CGGGAACCTACCAGCCTATG - 3' (SEQ TD N0:31)
Reverse Primer
5'- CAGGCAACAGGGAGTCATGT - 3' (SEQ ID N0:32)
The absolute numbers depicted in Table 11 are relative levels
of expression of the CSG Proll3 in 12 normal different
tissues. All the values are compared to normal thymus
(calibrator). These RNA samples are commercially available
pools, originated by pooling samples of a particular tissue
from different individuals.
Table 11: Relative Levels of CSG Pro113 Expression in
Pooled Samples
Tissue NORMAL
Brain 0.03
Heart 0
Kidney 0.01
Liver 0
Lung 0
Mammary Gland 0
Muscle 0.04
Prostate 489.44
Small Intestine 0.02
Testis 0.35
Thymus 1
Uterus 0.13
The relative levels of expression in Table 11 show that Pro113
mRNA expression is higher (489.44) in prostate compared with
all the other normal tissues analyzed. Testis, with a
relative expression level of 0.35, uterus (0.13), thymus
(1.0), kidney (0.01) and brain (0.03) were among the other
tissues expressing lower mRNA levels for Pro113. These
results establish that Pro113 mRNA expression is highly
specific for prostate.
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The absolute numbers in Table 11 were obtained analyzing
pools of samples of a particular tissue from- different
individuals. They cannot be compared to the absolute numbers
originated from RNA obtained from tissue samples of a single
individual in Table 12.
The absolute numbers depicted in Table 12 are relative
levels of expression of Pro113 in 78 pairs of matching and 25
unmatched tissue samples. All the values are compared to
normal thymus (calibrator). A matching pair is formed by mRNA
from the cancer sample for a particular_ tissue and mRNA from
the normal adjacent sample for that same tissue from the same
individual. In cancers (for example, ovary) where it was not
possible to obtain normal adjacent samples from the same
individual, samples from a different normal individual were
analyzed.
Table 12: Relative Levels of CSG Proll3 Expression in
Individual Samples
Sample ID Tissue Cancer Matched or
Unmatched
Normal
Adjacent
Pro780B/781B Prostate 1 375.58 446.29
Pro1291B/1292B Prostate 2 1060 31
Pro139B96/140B96 Prostate 3 91 32
Pro209B96/210B96 Prostate 9 505 255
Pro1256B/1257B Prostate 5 165.79 141.63
Pro1293B/1294B Prostate 6 1613.7 874.61
Pro694B/695B Prostate 7 458.6 142.21
Pro1012B/1013B Prostate 8 1520 864
Pro1222B/1223B Prostate 9 939 530
Pro845B/896B Prostate 10 1552.4 374.6
Pro1094B/1095B Prostate 11 278.37 135.89
Pro650B/651B Prostate 12 532.81 640.85
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Pro902B/9038 Prostate 13 609.05 415.86
Pro916B/917B Prostate 14 699.42 401.24
Pro9821110A/110B Prostate 15 156 487.8
ProS9821326A/26B Prostate 16 794.4 472.8
Pro9407c215 Prostate 17 1389.2
Pro9907c234 Prostate 18 305.5
Pro9407c280A Prostate 19 894.5
Pro9409COlOR Prostate 20 269.7
(prostatitis)
Pro9404C120R Prostate 21 299.2
(prostatitis)
Pro1000258 Prostate 22 199.6
{BPH)
Pro4001263C Prostate 23 576
(BPH)
Pro9001267A Prostate 29 132.1
(BPH)
Pro9411C032 Prostate 25 118.2
(BPH)
Pro4001960Z Prostate 26 276.3
{BPH)
Pro4001271A Prostate 27 58.7
(BPH)
Kid1064D/65D Kidney 1 0 0.1
Kid1079D/1080D Kidney 2 0.3 0.02
Kid1097D/1098D Kidney 3 35.14 0.32
Kid1029D/1025D Kidney 9 1.31 0
Kid1183D/1184D Kidney 5 24.79 0
Kid1242D/1243D Kidney 6 0 0
B1d469K Bladder 1 2.88
B1d467K/468K Bladder 2 2.65
B1d327K/328K Bladder 3 0 9.05
B1d470K Bladder 4 1.64
B1d665T/669T Bladder S 0.21 1.99
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B1d1496K/1497K Bladder 6 13.55 1.14
B1d1721K/1722K Bladder 7 120.16 ~ 1.34
Tst239X/240X Testis 1 31.5 0.73
TstS9820647A/97B Testis 2 15.7 0
TstS9820663A/663B Testis 3 72 1.4
SknS9821248A/298B Skin 1 1.8 0,5
SknS99448A/448B Skin 2 251.6 0
Skn99816A/816B Skin 3 33 0.7
Sto4004864A9/B4 Stomach 1 14.12 0
Sto4004509A3/B1 Stomach 2 40.74 39
SmI9807A212A/213A Small 0.1 0
Intestine 1
SmI9802H008/H009 Small 5.8 0.1
Intestine 2
C1n9608B012/BO11 Colon 1 4.5 0
C1n9709c079ra/073ra Colon 2 65.8 3.1
C1n4004709A1/709B1 Colon 3 1.1 0.9
C1n9405C199/C200 Colon 4 34.76 0.73
C1n9707c004gb/006ga Colon 5 90.26 0.96
C1n96-09-B004/B003 Colon 6 17.9 20.64
C1n9612B006/B005 Colon 7 17.56 0.3
C1n9705F002D/FOO1C Colon 8 21.39 0
CInCXGA Colon 9 929.14 142.69
Pan10343a Pancreas 1 0 0
Pan776P/777P Pancreas 2 0 0.15
Pan9210/9220 Pancreas 3 7.36 0
Pan714L/715L Pancreas 4 13.57 0.11
Pan829P/825P Pancreas 5 0 0
Lng476Q/477Q Lung 1 0 0
Lng605L/606L Lung 2 0 0.1
Lng11145B/11195C Lung 3 85.9 0
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Lng0008632A/32B Lung 4 23.85 0
Lng750C/751C Lung 5 0.32 0.25
Lng8890A/8890B Lung 6 10.63 0
Lng8926A/8926B Lung 7 15.37 0
Lng0010239A/39B Lung 8 26.17 0
Lng9502C109R/110R Lung 9 0.68 0
LngS9821944a/44b Lung 10 0 0
Mam00042D01/42N01 Mammary Gland 8.5 0
1
Mam59XC Mammary Gland 61.07 0
2
Mam9706A066G/67C Mammary Gland 9.84 0
3
Mam14153a1C Mammary Gland 9.72 6.99
9
Mam1620F/1621F Mammary Gland 0.91 0
5
Mam00014D05 Mammary Gland 2.45 0
6
End10479B/D Endometrium 1 133.43 1.12
End9705A125A/126A Endometrium 2 0 0.39
End9704C281A/282A Endometrium 3 23.5 1.56
End680o97/681097 Endometrium 4 88.89 79.02
Utr13590/13580 Uterus 1 0.2 0
Utr850U/851U Uterus 2 0 0
Utr14170/14180 Uterus 3 14 0.9
Utr233U96/234U96 Uterus 4 8.65 4.64
CvxVNM00052D01/52N01 Cervix 1 0.82 77.15
CvxVNM00083D01/83N01 Cervix 2 0.78 221.48
CvxND00023D01/23N01 Cervix 3 3.25 15.22
Ovr10370/10380 Ovary 1 0.1 0
Ovr10050 Ovary 2 18.96
Ovr1028 Ovary 3 0
Ovr14638A1C Ovary 4 3.2
Ovr14603A1D Ovary 5 882.3
Ovr7730 Ovary 6 0
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Ovr9702C018GA Ovary 7 0.15
Ovr206I Ovary 8 p
Ovr9702C020GA Ovary 9 0
Ovr9702C025GA Ovary 10 p
Ovr9701C035GA Ovary 11 0.07
Ovr9701C050GB Ovary 12 0.58
a = wegarive
In the analysis of matching samples, the higher levels
of expression were in prostate, showing a high degree of
tissue specificity for prostate tissue. In addition to the
higher expression levels in prostate cancer samples, Pro113
expression was found to be either induced (where not expressed
in normal adjacent tissues) or somewhat upregulated in several
other cancers. However, the relative expression and the fold
increase in prostate cancer samples far exceeds that in other
cancer tissues and is highly significant.
Furthermore, the levels of mRNA expression in cancer
samples and the isogenic normal adjacent tissue from the same
individual were compared. This comparison provides an
indication of specificity for the cancer (e. g. higher levels
of mRNA expression in the cancer sample compared to the normal
adjacent). Table 12 shows overexpression of Pro113 in 13 out
of 16 primary prostate cancer tissues compared with their
respective normal adjacent (prostate samples 2, 3, 4, 5, 6 7,
8, 9, 10, 11, 13, 14, 16). Thus, there was overexpression in
the cancer tissue for 81.250 of the prostate matching samples
tested. The median for the level of expression in prostate
cancer tissue samples is 509, whereas the median for all other
cancers is only 7.93, with the exception of one colon sample,
colon 9, whose expression was similar to that found in
prostate cancer tissues.
Altogether, the high level of tissue specificity, plus
the mRNA overexpression in 81.250 of the primary prostate
matching samples tested are indicative of Pro113 being a
CA 02347081 2001-04-18
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- 40 -
diagnostic marker for prostate cancer. Expression was also
found to be higher in other cancer tissues compareca~-with their
respective normal adjacent tissues (kidney, bladder, testis,
skin, stomach, small intestine, colon, pancreas, lung,
mammary, endometrium, uterus, and ovary) thus indicating
Pro113 to be a pan cancer marker.
Expression of Clone ID 1810463ii1 (Pro114):
For the CSG Pro114, real-time quantitative PCR was
performed using the following primers:
Forward Primer
5'- TGGGCATCTGGGTGTCAA - 3' (SEQ ID N0:33)
Reverse Primer
5'- CGGCTGCGATGAGGAAGTA - 3' (SEQ ID N0:34)
The absolute numbers depicted in Table 13 are relative
levels of expression of the CSG Pro114 in 12 normal different
tissues. All the values are compared to normal muscle
(calibrator). These RNA samples are commercially available
pools, originated by pooling samples of a particular tissue
from different indiv_Lduals.
Table 13: Relative Levels of CSG Proll4 Expression in
Pooled Samples
Tissue - NORMAL
Brain g,7
Heart 0.7
Kidney 414.4
Liver 4
Lung 882.2
Mammary 4 4
Muscle 1
Prostate 1951
Small Intestine 22
Testis 367.1
Thymus 25.8
Uterus 139.6
The relative levels of expression in Table 13 show that Proll9
mRNA expression is higher (1951) in prostate compared with all
the other normal tissues analyzed. Lung, with a relative
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WO 00/23111 PCT/US99/24331
- 41 -
expression level of 882.2, kidney 914.4, testis 367.1 and
uterus 139.x, are the other tissues expressing higher levels
of mRNA~ for Proll4. These results establish Proll4 mRNA
expression to be more specific for prostate than other tissues
examined.
The high level of tissue specificity is indicative of
Proll4 being a diagnostic marker for diseases of the prostate,
especially cancer.
Expression of Clone ID zr65g11 (Pro118):
For the CSG Pro118, real-time guantitative PCR was
performed using the following primers:
Forward Primer
5'- GCCCATCTCCTGCTTCTTTAGT - 3' (SEQ ID N0:35)
Reverse Primer
5'- CGTGGAGATGGCTCTGATGTA - 3' (SEQ ID N0:36)
The absolute numbers depicted in Table 19 are relative
levels of expression of the CSG Pro118 in 12 normal different
tissues. All the values are compared to normal kidney
(calibrator). These RNA samples are commercially available
pools, originated by pooling samples of a particular tissue
from different individuals.
Table 14: Relairive Levels of CSG Pro118 Expression in
Pooled Samples
Tissue ~ NORMAL
Colon O.g7
Endometrium 19282
Kidney 1
Liver 0
Ovary 86.22'
Pancreas 0
Prostate 962.1
Small Intestine0
Spleen 0.75
Stomach 0.54
Testis 343.7
Uterus 1064
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The relative levels of expression in Table l~4 show that
ProllB mRNA expression is the 3'° highest in prostate (962.1)
next to endometrium (19282) and uterus (1064), which are
female-specific tissues. Testis, with a relative expression
level of 343.7 is the only other male tissue expressing
moderate levels of mRNA for Pro118. These results establish
Pro118 mRNA expression to be highly specific for reproductive
tissues including the prostate.
The absolute numbers in Table 14 were obtained analyzing
pools of samples of a particular tissue from different
individuals. They cannot be compared to the absolute numbers
originated from RNA obtained from tissue samples of a single
individual in Table 15.
The absolute numbers depicted in Table 15 are relative
levels of expression of Pro118 in 59 pairs of matching and 21
unmatched samples. All the values are compared to normal
kidney (calibrator). A matching pair is formed by mRNA from
the cancer sample for a particular tissue and mRNA from the
normal adjacent sample for that same tissue from the same
individual.
Table 15: Relative Levels of CSG Proll8 Expression in
Individual Samples
Sample ID Tissue Cancer Matching
Normal
Adjacent
Prol2B Prostate 1 41700.7 22242.83
ProC234 Prostate 2 40087
Pro78XB Prostate 3 4075.6 7066.7
Pro109XB Prostate 4 334.4 777.2
Pro84XB Prostate 5 11684 58290
Pro101XB Prostate 6 21474.13 100720.8
Pro9lX Prostate 7 14849 33717
Prol3XB Prost=ate 8 202.57 146.91
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ProC215 Prostate 9 73243
Pro125XB Prostate 10 629.6 521.4
Pro23B Prostate 11 157532.6 110654.4
Pro90XB Prostate 12 2317 64134
ProC280 Prostate 13 42020
Pro20XB Prostate 14 2909.31
Pro34B Prostate 15 29610 23264
Pro110 Prostate 16 13354 30991
Pro65XB Prostate 17 10126 11270
Pro69XB Prostate 18 2671.42
Pro326 Prostate 19 9962.3 19231
ProlOR Prostate 20 27355
(prostatitis)
Pro20R Prostate 21 21081
(prostatitis)
Pro258 Prostate 22 (BPH) '79916.32
Pro263C Prostate 23 (BPH) 108924.5
Pro267A Prostate 24 (BPH) 92910.22
Pro271A Prostate 25 (BPH) 57004.9
Pro460Z Prostate 26 (BPH) 57949.23
ProC032 Prostate 27 (BPH) 95781.94
Kid106XD Kidney 1 3.08 217.36
Kid107XD Kidney 2 0 38.36
Kid109XD Kidney 3 0 123.5
KidlOXD Kidney 4 17.69 67.8
KidllXD Kidney 5 16.79 360.8
Kid124D Kidney 6 0 167.4
B1d32XK Bladder 1 0 0
B1d47K Bladder 2 36.38
B1d66X Bladder 3 0 4.52
BldTRl4 Bladder 4 0 12.17
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BldTRl7 Bladder 5 0 0
B1d46XK Bladder 6 16.5 ~ 0
Tst39X Testis 1 116.6 24.35
Tst647T Testis 2 856.16 43.5
StoAC49 Stomach 1 0 0
StoAC93 Stomach 2 0 0
SmI2IXA Small Intestine i 68.45 0
SmIH89 Small Intestine 2 0 0
ClnACl9 Colon 1 149 21.33
ClnASl2 Colon 2 0 0
ClnB34 Colon 3 0 0
ClnB56 Colon 9 13.04 5.22
ClnAS43 Colon 5 0 0
Lng47XQ Lung 1 0 0
Lng60XL Lung 2 0 0
Lng75XC Lung 3 0 3.38
Lng90X Lung 4 0 0
LngBR26 Lung 5 0 26.82
Pan10343 Pancreas 1 50.47 0
Pan77X Pancreas 2 281.1 0
Pan92X Pancreas 3 18.41 0
Pan7lXL Pancreas 9 0 0
Pan82XP Pancreas 5 0 0
PanC044 Pancreas 6 0 0
Maml2X Mammary Gland 1 0 0
Mam162X Mammary Gland 2 0 0
Mam42DN Mammary Gland 3 0 0
MamS127 Mammary Gland 4 12.58 0
Maml4DN Mammary Gland 5 0 0
End28XA Endometrium 1 331.9 1824
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End3AX Endometrium 2 27825 65839
End4XA Endometrium 3 10.3 ~ 15935
Utr1410 Uterus 1 18885 18116
Utr23XU Uterus 2 3358 7674
CvxKS52 Cervix 1 0 0
CvxKS83 Cervix 2 0 0
Ovr10050 Ovary 1 72.86
Ovr1028 Ovary 2 0
Ovr638A Ovary 3 0
Ovr63A Ovary 4 90.88
Ovr7730 Ovary 5 1.21
Ovr10400 Ovary 6 5.08
Ovr1050 Ovary 7 0
Ovr1118 Ovary 8 7.41
Ovr103X Ovary 9 32.78
Ovr20GA Ovary 10 0
Ovr25GA Ovary 11 1173.83
Ovr35GA Ovary 12 313.4
Ovr50GB Ovary 13 823.1
Ovrl8GA Ovary 19 40.6
Ovr206I Ovary 15 1264
Ovr230A Ovary 16 1285
a = wegaza.ve
Tn the analysis of matching samples, the higher levels of
expression were in prostate, endometrium, testis, and ovary
showing a high degree of tissue specificity for reproductive
tissues. These results confirmed the tissue specificity
results obtained with the panel of normal pooled samples
(Table 14).
Furthermore, the levels of mRNA expression in cancer
samples and the isogenic normal adjacent tissue from the same
individual were compared. This comparison provides an
CA 02347081 2001-04-18
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- 46 -
indication of specificity for the cancer (e. g. higher levels
of mRNA expression in the cancer sample compared tg.the normal
adjacentj. Table 15 shows overexpression of Pro118 in 5 out
of 14 primary prostate cancer tissues (prostate samples 1, 8,
10, 11, 15) compared with their respective normal adjacent.
Thus, there was overexpression in the cancer tissue for 35.710
of the prostate matching samples tested (total of 14 prostate
matching samples). Expression of Proll8 was similarly higher
in 3 unmatched cancer tissues (prostat.e samples 9, 13, 14),
2 prostatitis (prostate samples 20, 21), and 6 benign
hyperplasia tissues (prostate samples 22 through 27).
Altogether, the high level of tissue specificity, plus
the mRNA overexpression in 35.710 of the primary prostate
matching samples tested are indicative of Pro118 being a
diagnostic marker for prostate cancer.
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
SEQUENCE LISTING
<110> Salceda, Susana
Recipon, Herve
Cafferkey, Robert
diaDexus, LLC
<120> Method of Diagnosing, Monitoring, Staging, Imaging and
Treating Prostate Cancer
<130> DEX-0052
<140>
<141>
<150> 60/104,737
<151> 1998-10-19
<160> 36
<170> PatentIn Ver. 2.0
<210> 1
<211> 188
<212> DNA
<213> Homo sapiens
<400> 1
ggtaaacacc tgcttttatc atcagaacaa agaggctgtg tcccctgccc tatgaggtcc 60
atttctgaga gttgtggcta atgggcaaga aggttggggc tttagagatt tgggataaag 120
atatcaaaca ccagaaaggt agaaagaagt gatcagatta gggttactta ggtgatgata 180
tgaactct 188
<210> 2
<211> 9819
<212> DNA
<213> Homo sapiens
<400> 2
cagctggggt ctacccaggt ccatgtcttg gacatgttga gagtttttct ggaaggcagg 60
gatacagtgt ggtccaaaaa cacacaaatg cccctactgg cccaggggtt gtcacaatag 120
actggaaggg tgacacatcc caggcgcttg ccacccatca cacgcacctc ctacccactg 180
gcatccttcc accccaggca cacacaaagc ctcagtccag agatcaactc tggactcagc 240
tctgaatttg catatcctgt gtgtagattc attcttcata acctctgccc agcctagctt 300
gtgtatcatt tttttttctc tattagggga ggagcccgtc ctggcactcc cattggcctg 360
tagattcacc tcccctgggc agggccccag gacccaggat aatatctgtg cctcctgccc 420
agaaccctcc aagcagacac aatggtaaga atggtgcctg tcctgctgtc tctgctgctg 480
cttctgggtc ctgctgtccc ccaggagaac caagatggtg agtggggaaa gcaagggatg 540
1
SUBSTITUTE SHEEP (RULE 26~
CA 02347081 2001-04-18
WO 00/23111 PCTNS99/24331
ggtgctggag aggactggaa ggaggtgagg aacaggacat gtggctggga gacaggctgg 600
atgcagctgg gataccctgg catacggcag gaatgggtgc ccaaggctgt caactccctc 660
agctcacaca cttccaggag cattcaggga gcctctgcgc tggcccgaaa taagaccttc 720
aggaatctga atctaaaacc cctagtttac agtgaaaaca aagactccaa agaccaagcg 780
acctgcttgg ggtagacagt caggacggag taggaaccat atgcctggag ct~cttctgc 840
tcctgttcct tccctccttc cgatggctgg gtacacctgc ctgacgctga ggaaaagaga 900
gagcagcccc aaggggaaag tgggaaggca ggttggctgg agggatggtg ctagaaggaa 960
acccgtgccc aaatcccaca ctcagacacc actgcagtgg gtctggaagg cgagtggctg 1020
gaagagaaga gagtgggagc tccgggagat caagagtcac tcctaggata agggaaggag 1080
gctgtttgtg gcatgagaat gtgcaggata aagacatgga agcgaatggc ttctcagttg 1140
tgtgagttta aaattcatga catttacaaa ttgtcagaaa aggtgttata tgtttgttat 1200
ataacaatca ctttggaatg ttaatctgat tctgtgccaa aatctgaatt actcagggtt 1260
ctccagagaa acagaactaa taggtggtac acatatacat atatatgtac gtacacatac 1320
atacatacac tgtatacaca tggatacaca cacacatagg aagagattta catatatgta 1380
tacaaaagag agagagagta gagatttatt ttaagaaatt gactcacact attgggagga 1440
gtaacaagtc ctaaatcttc agagccggcc agcaggctgg agacccaggg aagagttgat 1500
gtcttagtct tgattccaag ggcagactgt aggcagaatt ctttcctctt taggggacat 1560
ctgaggcttt ttctcttaag gccttcaact gattggatga agcccaccac tatggagagt 1620
aatccacttt actcaaggtc tactgatttt tttgtaaatt aaaaaaaaaa ctgtgggtgc 1680
atagtatgtg tatatattta tggggtacat gagaggtttt gattcaggca tgcaatgtga 1740
aataatcaca tcatcaaaaa tgaggtatcc atcccttcaa gcttttatcg tttgtgttac 1800
agacaatcca attatacttt tttggttatt ttagttttta aaagtatttg attatttatt 1860
tatttattta tttttgagac agagtctcac tctgtcaccc aggcaggagt gcagtggcat 1920
gatctcggct cactgcaacc tccgcctccc aggttcaagc aattttcctg cctcagtctc 1980
ctgagtagct aggactacag gcacctgcca ccacacctgg ct:aatttttt tgtattttta 2040
gtagagacgg tttcatcatg ttggccaggc tagtcttgat atcctgacct cgtgatctgc 2100
ccgccttggt ctcccaaagt gccgggatta caggtgtcag caactgcgcc tggcctctct 2160
tttggttatt taaaagtgta caattaaatt atgattatta ttattatttt tgagatggat 2220
tcttgttctg tcacccaggc tggagtgcag tggcgtgatc ttggcttact gcaaacctcc 2280
gcctgttggg ttcaagcaat tatcttgcct cgggtgtaca ctgccacaca cggctaactt 2340
atgtattttt aatagagata gggLttcacc atgttggcta gactggtctt gacctcttga 2400
cctcaagtga tccactcact tcagcctccc agagtgctgg aattacaggc acgagccacc 2460
acacctggcc ccagttaaat tattattgac tatagtcacc ctgttgtgct atcaaatagt 2520
aggtcttatt cattcttctt tttttttttt tttttgtgac agagttgccc aggctggaat 2580
gcagtggtgc aatcttggct cactgcaacc tctgcctccc gggcttaagc gattctcctg 2640
cctcagcctt ctgagtcgct gggactacag gtgtgtgcca ccacgcccgg ctaatttatg 2700
tatttttagt agagatgggg tttcaccatg ttggccaggc tggtttcgaa ctcctgacct 2760
caagtgaccc acctgcctca gcttcccaaa gtgttggaat tacaggcatg agccaccaca 2820
cctggcccca gttaaattat tattcactgg agtcactttg ttgtgctatc aaatagtttt 2880
ctaactattt tttttgtacc cattaaccac cctcccaatt tccccccaac cctgccacta 2940
cccttcccag cctttggtaa ccatccttct actctctatg tccatgaatt caattgtagg 3000
gtctactgat ttaaaggcta atcacattta gacactcagg agcaagaata attttagtaa 3060
ttgaactagg attctgccat atgacctcca acatcattag cacctgtgta aattgtatca 3120
taaaataatt atggaactat tatggaaatg tccctctctc ccagatccca ccttgtacca 3180
aaatgcaagg tacaaccccg ggaattctga gctccatcct agtcttaccc tgtgctaatt 3240
cagtctgggt catttcttga attttctggt aaattctcct ttcaaccctt tctaactata 3300
tgtatttgtc aggttaagct agaagtgtta attttttttt tttttgagat ggagccttgc 3360
tttgtcacct aggctgaagt gcagtggcat gatctcagct cactgcaagc tccgcctccc 3420
2
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCTNS99/24331
gggttcatgc cattctcctg cctcagcctc ctgagtagct gggactacag gcacccgcca 3480
ccatgcttgg ctaatttttt gaattcttag tagagacggg gtttcaccat gttagccagg 3540
atggtctcga tctcctgacc tcgtgatcca cccgcctcgg ccccctaaag tgctgggatt 3600
acaggcgtga gccactgagc ccggacgaaa tgttaatttg ttttttttga gacggagtct 3660
cactctgtca tccaagctgg agtgcagtgg catgatcttg gcttgttgca a~tctgcct 3720
ctctggttca agtgattttc ctgcctcagc ctccagcatg actgggatta caggcccgca 3780
ccaccatgcc cagctaattt ttgtattttt taatagagat ggggtttcac catgttggcc 3840
aggctggtct tcaactcctg atctcaagta atctgcctgc cttggcctcc caaagtcctg 3900
ggattacagg catgagccac ggagcccagc ctagaaatgt taatttctaa cgcatgtcag 3960
attccatgca cactgggcaa ggttccattc ctccatgggg tgactcaggg atccaggcca 4020
attgcatatt gagactcttt catattatcc tgtggccttc aaagtcgtca cctctaggga 4080
tgagaaacaa aagggaaagc cagctggtag ggtcttggac aagaagaaag acatcacttc 4140
tgctcacatt ctcttttgac aaaactcagt cacatggtcc caatatatct tcgaggtggc 4200
tgagtaatgt tatcttccta tgtgtcaagc agaggaaata atgtagtgaa gacacaggat 4260
ggtctctgaa atatcatctc aggcatgaaa gtagagcata Ctcacttgag tgagcctcca 4320
gtggtgtgaa gttgatggca ggagaaagag ctggggaaga aaaggccagt ggcaggtctc 4380
ccctcctagc cctatgcagc cccacagtgg gacccttgca tggacctcaa ccatcagaat 4440
cttttctttt gcaggtcgtt actctctgac ctatatctac actgggctgt ccaagcatgt 4500
tgaagacgtc cccgcgtttc aggcccttgg ctcactcaat gacctccagt tctttagata 4560
caacagtaaa gacaggaagt ctcagcccat gggactctgg agacaggtgg aaggaatgga 4620
ggattggaag caggacagcc aacttcagaa ggccagggag gacatcttta tggagaccct 4680
gaaagacatt gtggagtatt acaacgacag taacggtcag tgaataacag accacagggg 4740
tggaaggtct aacccaagag gcagcccccc cagtgtgagt ggcaagggat cagcaggatg 4800
gaaatagtcc caatcccagg ggaagaacag gagacacagc agaaacacag acatgtccgc 4860
atcccaccca ccccacagca caggtgctcc ccgcttcccc atcaattgcc ccatcctcat 4920
cccaggcctc aggtcacaca ggaagtgatg gcagagtcac ttcctatcca ggcacctatg 4980
acctctcacc tccacacccc acccatcgga ggctgatacc cccgtgagaa ggcatcagac 5040
tcacccctgt ccagggaggt tgcctggaga gtgagccact ctcaaagtca ctcagacctg 5100
ggctcacctg gtggttctgc cagtcctagc tgttgacagt gaaacgttcc caaaatatct 5160
ggttgaaatc tgcaaacatt ggagcactga gacctacctc caaacaagtc tgtaatattt 5220
aactatgtct gttctatgaa ggatgtcaca gtctgtcctg atctcccttg cagctccatc 5280
acctagcaca gggtacagcc aatattggct caattgaaat ttgtggaatc cacagagaaa 5340
agcacccggc acacaccgta gcccatgctg ggggctcagg aagtgctgga ttcaaaactg 5400
tgggctgtta gagttccttg gagccctaaa gttcctcctt accatacgat gcagacccag 5460
gaagggccac ctgcgctatg gtcagaggag ctggtggcag agcccgtgca gagatggtcc 5520
ctgtgccccc ggcccagtgc tctttctcct aaaccacact gccagcccca aggcagccaa 5580
cctcaggtct ggtgaactgc tggtgttaaa ttatcataga gtgggtgtca aaagatgggc 5640
tactaagtac aaaaatgccc aaggtgctac atgggatctg aagattttca aaaggaggca 5700
agaaagagat aggcagatgt ttcaaggatg tggggtgggg gaggtcttgg taaggaaaat 5760
ggcccaggct gtgtgtcagc aataggagag gagggggcac aggtgatcag aaaagacact 5820
gggggaagca ttgatggaca ggaatagaaa tggcaaagtg gataattaag aggaaggagg 5880
atgaggagat gaacacaggg tattagaaaa taatagaagg cagggcttgg tggctcactc 5940
ttgtaatccc agcactttgg gaggctgagg caggcagatc acctaaggtc aggagttcga 6000
gaccagcccg gccaacatgg tgaaaccctg tctctactaa taatacaaaa atagcctggc 6060
atggtggcac acgtctgtgg tcccagctac tcaggaggct gaggcaggag aattgcttga 6120
acccaggagg cagaggttac agtggccaaa atcctaccat tgcactacag cctgggtgac 6180
aagagtgaaa cgttgtctaa aaacaaaaaa caaaaaacaa aaaaaggaaa taatagtagc 6240
tgacatttac tgagcactta ctttgtgcca ggcccatcta tgagcatata taatgctcag 6300
3
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/2311 I PCT/US99/24331
aatagccccc taaaacagtg ctcttggcat tgccatttca gaggtgagga aatagaggca 6360
cagggagttg agtggctcca gttcaggcaa cacaccaggt gggggtgggg ggctggggag 6420
agacctggga cgtgagccca gacagcttga gagctttcag agtctatgcc aacagcacca 6480
accagtgctg ggtaaacacc tgcttttatc atcagaacaa agaggctgtg tcccctgccc 6540
tatgaggtcc atttctgaga gttgtggcta atgggcaaga aggttggggc ttt~gagatt 6600
tgggataaag atatcaaaca ccagaaaggt agaaagaagt gatcagatta gggttactta 6660
ggtgatgata tgaactcttc ctagaactga gagaaaaaga gagccttcct ttactcatat 6720
gaaatcacaa ataatttcta tccaatttgg aagtacactt tggtgtagtt gtgacagctt 6780
cctcaggact cagcataaat tcaaacaaat aattgtcctt agaagagatg ctatagaaga 6840
gatagaaata tattcatatt ctgtagcttt tttttttttg agatggagtt ttgctcttgt 6900
cacccaagct ggagtgcagt gatgcaatct cagctcactg caaactttgc ctcctgggtt 6960
caagggattc tcctgcctca gcctcccgat aactgggact acaggctaca ggcatgtgtc 7020
actactcctg gttaattttt tttttttttt tttaagactg agtcttgctc tgtctttcag 7080
gctgatgtac aatggctcca tctcggctca ctacaacttc tgtcccccag gttcaagcga 7140
ttctcctgcc tcagcctcat gagtagctgg gattacaggc atgtgccagc acacccagca 7200
aatttttgta tttttagtag agatgaggtc ttaccatgtt ggccaggctg gtctcaaact 7260
cctgacctca ggtgatcctt tggcctcagc ctccctaact gctgggatta caggcatgag 7320
ccactgcgtc cagcctaatt ttatattttt ggtagagatg gggtttcacc atattggcca 7380
ggctggtctc gaactcatga cctaaggtga tccatcctcc tcagcctctc aaagtgctgg 7440
gattacaagt gtgagccact gggcctggtg cttttttttt tttttttttt tttttttttt 7500
tgagataggg tctcactctg tcacccaggc tgaaatgcag tagtgtgatt ttggctcatt 7560
gcagccttga cttcccaggc tgaagtgatc ctcccacctc agcctcctga gtagctgggg 7620
ctacaggcat gcaccaccat gctgcgctaa tttttatatt ttttgtagtg gtgggatttc 7680
gccatatcac cctggctggt ctggaacccc tgggctcaag cgatccactc gcttcagctt 7740
ctcaaagtgc tgggattaca ggcatgagcc acagcgccca ggctgtagct ctcttaagga 7800
ggaacatatc tcatctgaga caaacctgaa atgccaaacc aaactgagtt agcccctctc 7860
tgtctgttgt atatattgga gtaataacct atttgtcttg ataaagggat tgcatgcttg 7920
aattgcaaaa acctttattt cttttgggtt gcccaatgtg caagactaag agttattttg 7980
ataaatttct caccaggctg actgtctctc tgtggggtcg ggggagtttt cagggtctca 8040
cgtattgcag ggaaggtttg gttgtgagat cgagaataac agaagcagcg gagcattctg 8100
gaaatattac tatgatggaa aggactacat tgaattcaac aaagaaatcc cagcctgggt 8160
ccccttcgac ccagcagccc agataaccaa gcagaagtgg gaggcagaac cagtctacgt 8220
gcagcgggcc aaggcttacc tggaggagga gtgccctgcg actctgcgga aatacctgaa 8280
atacagcaaa aatatcctgg accggcaagg tactcactgc ttcctgctcc ccagtactga 8340
gcccagaata aaagacgatc tcaggctagg agctcaggca acatcttagt ccggtctcat 8400
ctgttcctgg atgtccctca gacccccagc tttcatcttt taggatttat tccttccctg 8460
ggataatata atttgtggtc caaaaagaac atcatcaaaa tttcaggcag aatgggccag 8520
gaaggccatt ctttcttgat gagtgtcccc aaatcatctc caattaacag acaaggagct 8580
tgaggttagg gaggtgaggg taacactgtc tgtaagaggc agagctggga ctcaaattcc 8640
agatttcaga ttccaaatcc catcgttttt tatctctaca atgatgcctc ccatctgggt 8700
ggtggagaga agggaggcgt gtaaaagtca gccccagaag gacaagagca agccagtgtg 8760
agcggaattg atggctgcaa gctgagactt ggattggaga cgtagtgaga ctcaggattg 8820
tgcagtgctg cagggaagtg gttgctggat agaggcatgg gctgaaccaa gcagctggac 8880
tgagactggg ggacagaact ccaaagccca ctgagatgtg ggaaaacatg gagaagcaca 8940
cggagcattc acaacttatt gccgtcagag tcaatacatg ggtgaggtgg ggattgggca 9000
agagggaaag cgtcagcctt ccctgatatt ctggaaagtc tcccggggct gggggtgggc 9060
aggtacagag cttcgagctc tgctgatcgc tgacatccag gggtgggggt aggaagagac 9120
ctgggccggg agaagtccac ctcaagcctg cagtgtcaca ctctatccct ccacagatcc 9180
4
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCTNS99/24331
tccctctgtg gtggtcacca gccaccaggc cccaggagaa aagaagaaac tgaagtgcct 9240
ggcctacgac ttctacccag ggaaaattga tgtgcactgg actcgggccg gcgaggtgca 9300
ggagcctgag ttacggggag atgttcttca caatggaaat ggcacttacc agtcctgggt 9360
ggtggtggca gtgcccccgc aggacacagc cccctactcc tgccacgtgc agcacagcag 9420
cctggcccag cccctcgtgg tgccctggga ggccagctag gaagcaaggg ttggaggcaa 9480
tgtgggatct cagacccagt agctgccctt cctgcctgat gtgggagctg aaccacagaa 9540
atcacagtca atggatccac aaggcctgag gagcagtgtg gggggacaga caggaggtgg 9600
atttggagac cgaagactgg gatgcctgtc ttgagtagac ttggacccaa aaaatcatct 9660
caccttgagc ccacccccac cccattgtct aatctgtaga agctaataaa taatcatccc 9720
tccttgccta gcataacaga gaatcctttt tttaacggtg atgcgctgta gaaatgtgac 9780
tagattttct cattggttct gccctcaagc actgaattc 9819
<210> 3
<211> 250
<212> DNA
<213> Homo Sapiens
<400> 3
cgcccctgcg ccgccgagcc agctgccaga atgccgaact ggggaggagg caagaaatgt 60
ggggtgtgtc agaagacggt ttactttgcc gaagaggttc agtgcgaagg caacagcttc 120
cataaatcct gcttcctgtg catggtctgc aagaagaatc tggacagtac cactgtggcc 180
gtgcatggtg aggagattta ctgcaagtcc tgctacggca agaagtatgg gcccaaaggc 240
tatggctacg 250
<210> 4
<211> 1900
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (16)
<220>
<221> unsure
<222> (18)
<220>
<221> unsure
<222> (20)
<220>
<221> unsure
<222> (1887)
<220>
<221> unsure
<222> (1894)
5
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
<400> 4
acgccttccg cggagnanan caaaacggcg cgcaggccgg gcgcacccag ccgccacttc 60
cgagagcgcc tgccgcccct ggcgccgccg agccagctgc cagaatgccg aactggggag 120
gaggcaagaa atgtggggtg tgtcaagaag acggtttact ttgccgaaga ggt~cagtgc 180
gaaggcaaca gcttccataa atcctgcttc ctgtgcatgg tctgcaagaa gaatctggac 240
agtaccactg tgggccgtgc atggtgagga gatttactgg caagtccctg ctacggcaag 300
aagtatgggc ccaaaggcta tggctacggg ccagggcgca ggcaccctca gcactgacaa 360
gggggagtcg ctgggtatca agcacgagga agcccctggg ccacaggccc accaccaacc 420
ccaatggcat ccaaatttgc ccagaagatt ggtggctccg agcgctgccc ccgatgcagc 480
caggcagtct atgctgcgga gaaggtgatt ggtgctggga agtcctggca taaggcctgc 540
tttcgatgtg ccaagtgtgg caaaggcctt gagtcaacca ccctgggcag acaaggatgg 600
cgagatttac tgcaaaggat gttatgctaa aaacttcggg cccaagggct ttggttttgg 660
gcaaggagct ggggccttgg tccactctga gtgaggccac catcacccac cacaccctgc 720
ccactcctgc gcttttcatc gccattccat tcccagcagc tttggagacc tccaggatta 780
tttctctgtc agccctgcca catatcacta atgacttgaa cttgggcatc tggctccctt 840
tggtttgggg gtctgcctga ggtcccaccc cactaaaggg ctccccaggc ctgggatctg 900
acaccatcac cagtaggaga cctcagtgtt ttgggtctag gtgagagcag gcccctctcc 960
ccacacctcg ccccacagag ctctgttctt agcctcctgt gctgcgtgtc catcatcagc 1020
tgaccaagac acctgaggac acatcttggc acccagagga gcagcagcaa caggctggag 1080
ggagagggaa gcaagaccaa gatgaggagg ggggaaggct gggttttttg gatctcagag 1140
attctcctct gtgggaaaga ggttgagctt cctggtgtcc ctcagagtaa gcctgaggag 1200
tcccagctta gggagttcac tattggaggc agagaggcat gcaggcaggg tcctaggagc 1260
ccctgcttct ccaggcctct tgcctttgag tctttgtgga atggatagcc tcccactagg 1320
actgggagga gaataaccca ggtcttaagg accccaaagt caggatgttg tttgatcttc 1380
tcaaacatct agttccctgc ttgatgggag gatcctaatg aaatacctga aacatatatt 1440
ggcatttatc aatggctcaa atcttcattt atctctggcc ttaaccctgg ctcctgaggc 1500
tgcggccagc agagcccagg ccagggctct gttcttgcca cacctgcttg atcctcagat 1560
gtggagggag gtaggcactg cctcagtctt catccaaaca cctttccctt tgccctgaga 1620
cctcagaatc ttccctttaa cccaagaccc tgcctcttcc actccaccct tctccaggga 1680
cccttagatc acatcactcc acccctgcca ggccccaggt taggaatagt ggtgggagga 1740
aggggaaagg gctgggcctc accgctccca gcaactgaaa ggacaacact atctggagcc 1800
acccactgaa agggctgcag gcatgggctg tacccaagct gatttctcat ctggtcaata 1860
aagctgttta gaccagaaaa aaaaaanaaa aaanaaaagg 1900
<210> 5
<211> 273
<212> DNA
<213s Homo Sapiens
<400> 5
gatgcatcaa aagagctgca agttctccac attgacttct tgaatcagga caacgccgtt 60
tctcaccaca catgggagtt ccaaacgagc agtcctgtgt tccggcgagg acaggtgttt 120
cacctgcggc tggtgctgaa ccagccccta caatcctacc accaactgaa actggaattc 180
agcacagggc cgaatcctag catcgccaaa cacaccctgg tggtgctcga cccgaggacg 240
ccctcagacc actacaactg gcaggcaacc ctt 273
<210> 6
6
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCTNS99/24331
<211> 3021
<212> DNA
<213> Homo Sapiens
<400> 6
tgtggaagca ccaggcatca gagatagagt cttccctggc attgcaggag agaatctgaa 60
gggatgatgg atgcatcaaa agagctgcaa gttctccaca ttgacttctt gaatcaggac 120
aacgccgttt ctcaccacac atgggagttc caaacgagca gtcctgtgtt ccggcgagga 180
caggtgtttc acctgcggct ggtgctgaac cagcccctac aatcctacca ccaactgaaa 240
ctggaattca gcacagggcc gaatcctagc atcgccaaac acaccctggt ggtgctcgac 300
ccgaggacgc cctcagacca ctacaactgg caggcaaccc ttcaaaatga gtctggcaaa 360
gaggtcacag tggctgtcac cagttccccc aatgccatcc tgggcaagta ccaactaaac 420
gtgaaaactg gaaaccacat ccttaagtct gaagaaaaca tcctatacct tctcttcaac 480
ccatggtgta aagaggacat ggttttcatg cctgatgagg acgagcgcaa agagtacatc 540
ctcaatgaca cgggctgcca ttacgtgggg gctgccagaa gtatcaaatg caaaccctgg 600
aactttggtc agtttgagaa aaatgtcctg gactgctgca tttccctgct gactgagagc 660
tccctcaagc ccacagatag gagggacccc gtgctggtgt gcagggccat gtgtgctatg 720
atgagctttg agaaaggcca gggcgtgctc attgggaatt ggactgggga ctatgaaggt 780
ggcacagccc catacaagtg gacaggcagt gccccgatcc tgcagcagta ctacaacacg 840
aagcaggctg tgtgctttgg ccagtgctgg gtgtttgctg ggatcctgac tacagtgctg 900
agagcgttgg gcatcccagc acgcagtgtg acaggcttcg attcagctca cgacacagaa 960
aggaacctca cggtggacac ctatgtgaat gagaatggca agaaaatcac cagtatgacc 1020
cacgactctg tctggaattt ccatgtgtgg acggatgcct ggatgaagcg accggatctg 1080
cccaagggct acgacggctg gcaggctgtg gacgcaacgc cgcaggagcg aagccagggt 1140
gtcttctgct gtgggccatc accactgacc gccatccgca aaggtgacat ctttattgtc 1200
tatgacacca gattcgtctt ctcagaagtg aatggtgaca ggctcatctg gttggtgaag 1260
atggtgaatg ggcaggagga gttacacgta atttcaatgg agaccacaag catcgggaaa 1320
aacatcagca ccaaggcagt gggccaagac aggcggagag atatcaccta tgagtacaag 1380
tatccagaag gctcctctga ggagaggcag gttcatggat catgccttcc tccttctcag 1440
ttctgagagg gagcacagac gacctgtaaa agagaacttt cttcacatgt cggtacaatc 1500
agatgatgtg ctgctgggaa actctgttaa tttcaccgtg attcttaaaa ggaagaccgc 1560
tgccctacag aatgtcaaca tcttgggctc ctttgaacta cagttgtaca ctggcaagaa 1620
gatggcaaaa ctgtgtgacc tcaataagac ctcgcagatc caaggtcaag tatcagaagt 1680
gactctgacc ttggactcca agacctacat caacagcctg gctatattag atgatgagcc 1740
agttatcaga ggtttcatca ttgcggaaat tgtggagtct aaggaaatca tggcctctga 1800
agtattcacg tctttccagt accctgagtt ctctatagag ttgcctaaca caggcagaat 1860
tggccagcta cttgtctgca attgtatctt caagaatacc ctggccatcc ccttgactga 1920
cgtcaagttc tctttggaaa gcctgggcat ctcctcacta cagacctctg accatgggtg 1980
agtctgcctg aggacggtgc agcctggtga gaccatccaa tcccaaataa aatgcacccc 2040
aataaaaatg gacccaagaa atttatcgtc aagttaagtt ccaaacaagt gaaagagatt 2100
aatgctcaga agattgttct catcaccaag tagccttgtc tgatgctgtg gagccttagt 2160
tgagatttca gcatttccta ccttgtggct tagctttcag attatggatg attaaatttg 2220
atgacttata tgagggcaga ttcaagagcc agcaggtcaa aaaggccaac acaaccataa 2280
gcagccagac ccacaaggcc aggtcctgtg ctatcacagg gtcaccttct tttacagtta 2340
gaaacaccag ccgaggccac agaatcccat ccctttcctg agtcatggcc tcaaaaatca 2400
gggccaccat tgtctcaatt caaatccata gatttcgaag ccacagattc tctccctgga 2460
gcaagcatga ctatgggcag cccagtgctg ccacctgctg acgacccttg agaagctgcc 2520
atatcttcag gccatgggtt caccagccct gaaggcacct gtcaactgga gtgctctctc 2580
7
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
agcactggga tgggcctgat agaagtgcat tctcctccta ttgcctccat tctcctctct 2640
ctatccctga aatccaggaa gtccctctcc tggtgctcca agcagtttga agcccaatct 2700
gcaaggacat ttctcaaggg ccatgtggtt ttgcagacaa ccctgtcctc aggcctgaac 2760
tcaccataga gacccatgtc agcaaacggt gaccagcaaa tcctcttccc ttattctaaa 2820
gctgcccctt gggagactcc agggagaagg cattgcttcc tccctggtgt gaa tctttc 2880
tttggtattc catccactat cctggcaact caaggctgct tctgttaact gaagcctgct 2940
ccttcttgtt ctgccctcca gagatttgct caaatgatca ataagcttta aattaaactc 3000
tacttcaaga aaaaaaaacc g 3021
<210> 7
<211> 267
<212> DNA
<213> Homo sapiens
<400> 7
gaacattcca gatacctatc attactcgat gctgttgata acagcaagat ggctttgaac 60
tcagggtcac caccagctat tggaccttac tatgaaaacc atggatacca accggaaaac 120
ccctatcccg cacagcccac tgtggtcccc actgtctacg aggtgcatcc ggctcagtac 180
tacccgtccc ccgtgcccca gtacgccccg agggtcctga cgcaggcttc caaccccgtc 240
gtctgcacgc agcccaaatc cccatcc 267
<210> 8
<211> 3443
<212> DNA
<213> Homo sapiens
<400> B
gggcgggccg ggccgagtag gcgcgagcta agcaggaggc ggaggcggag gcggagggcg 60
aggggcgggg agcgccgcct ggagcgcggc aggtcatatt gaacattcca gatacctatc 120
attactcgat gctgttgata acagcaagat ggctttgaac tcagggtcac caccagctat 180
tggaccttac tatgaaaacc atggatacca accggaaaac ccctatcccg cacagcccac 240
tgtggtcccc actgtctacg aggtgcatcc ggctcagtac tacccgtccc ccgtgcccca 300
gtacgccccg agggtcctga cgcaggcttc caaccccgtc gtctgcacgc agcccaaatc 360
cccatccggg acagtgtgca cctcaaagac taagaaagca ctgtgcatca ccttgaccct 420
ggggaccttc ctcgtgggag ctgcgctggc cgctggccta ctctggaagt tcatgggcag 480
caagtgctcc aactctggga tagagtgcga ctcctcaggt acctgcatca acccctctaa 540
ctggtgtgat ggcgtgtcac actgccccgg cggggaggac gagaatcggt gtgttcgcct 600
ctacggacca aacttcatcc ttcaggtgta ctcatctcag aggaagtcct ggcaccctgt 660
gtgccaagac gactggaacg agaactacgg gcgggcggcc tgcagggaca tgggctataa 720
gaataatttt tactctagcc aaggaatagt ggatgacagc ggatccacca gctttatgaa 780
actgaacaca agtgccggca atgtcgatat ctataaaaaa ctgtaccaca gtgatgcctg 840
ttcttcaaaa gcagtggttt ctttacgctg tatagcctgc ggggtcaact tgaactcaag 900
ccgccagagc aggatcgtgg gcggcgagag cgcgctcccg ggggcctggc cctgggcagg 960
tcagcctgca cgtccagaac gtccacgtgt gcggaggctc catcatcacc cccgagtgga 1020
tcgtgacagc cgcccactgc gtggaaaaac ctcttaacaa tccatggcat tggacggcat 1080
ttgcggggat tttgagacaa tctttcatgt tctatggagc cggataccaa gtagaaaaag 1140
tgatttctca tccaaattat gactccaaga ccaagaacaa tgacattgcg ctgatgaagc 1200
tgcagaagcc tctgactttc aacgacctag tgaaaccagt gtgtctgccc aacccaggca 1260
8
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCTNS99/24331
tgatgctgca gccagaacag ctctgctgga tttccgggtg gggggccacc gaggagaaag 1320
ggaagacctc agaagtgctg aacgctgcca aggtgcttct cattgagaca cagagatgca 1380 _
acagcagata tgtctatgac aacctgatca caccagccat gatctgtgcc ggcttcctgc 1440
aggggaacgt cgattcttgc cagggtgaca gtggagggcc tctggtcact tc ~aagaaca 1500
atatctggtg gctgataggg gatacaagct ggggttctgg ctgtgccaaa gcttacagac 1560
caggagtgta cgggaatgtg atggtattca cggactggat ttatcgacaa atgagggcag 1620
acggctaatc cacatggtct tcgtccttga cgtcgtttta caagaaaaca atggggctgg 1680
ttttgcttcc ccgtgcatga tttactctta gagatgattc agaggtcact tcatttttat 1740
taaacagtga acttgtctgg ctttggcact ctctgccatt ctgtgcaggc tgcagtggct 1800
cccctgccca gcctgctctc cctaacccct tgtccgcaag gggtgatggc cggctggttg 1860
tgggcactgg cggtcaagtg tggaggagag gggtggaggc tgccccattg agatcttcct 1920
gctgagtcct ttccaggggc caattttgga tgagcatgga gctgtcacct ctcagctgct 1980
ggatgacttg agatgaaaaa ggagagacat ggaaagggag acagccaggt ggcacctgca 2040
gcggctgcct ctggggccac ttggtagtgt ccccagccta cctctccaca aggggatttt 2100
gctgatgggt tcttagagcc ttagcagccc tggatggtgg ccagaaataa agggaccagc 2160
ccttcatggg tggtgacgtg gtagtcacct tgtaagggga acagaaacat ttttgttctt 2220
atggggtgag aatatagaca gtgcccttgg gtgcgaggga agcaattgaa aaggaacttg 2280
ccctgagcac tcctggtgca ggtctccacc tgcacattgg gtggggctcc tgggagggag 2340
actcagcctt cctcctcatc ctccctgacc ctgctcctag caccctggag agtgcacatg 2400
ccccttggtc ctgggcaggg gcgccaagtc tggcaccatg ttggcctctt caggcctgct 2460
agtcactgga aattgaggtc catgggggaa atcaaggatg ctcagtttaa ggtacactgt 2520
ttccatgtta tgtttctaca cattgctacc tcagtgctcc tggaaactta gcttttgatg 2580
tctccaagta gtccaccttc atttaactct ttgaaactgt atcatctttg ccaagtaaga 2640
gtggtggcct atttcagctg ctttgacaaa atgactggct cctgacttaa cgttctataa 2700
atgaatgtgc tgaagcaaag tgcccatggt ggcggcgaag aagagaaaga tgtgttttgt 2760
tttggactct ctgtggtccc ttccaatgct gtgggtttcc aaccagggga agggtccctt 2820
ttgcattgcc aagtgccata accatgagca ctactctacc atggttctgc ctcctggcca 2880
agcaggctgg tttgcaagaa tgaaatgaat gattctacag ctaggactta accttgaaat 2940
ggaaagtctt gcaatcccat ttgcaggatc cgtctgtgca catgcctctg tagagagcag 3000
cattcccagg gaccttggaa acagttggca ctgtaaggtg cttgctcccc aagacacatc 3060
ctaaaaggtg ttgtaatggt gaaaacgtct tccttcttta ttgccccttc ttatttatgt 3120
gaacaactgt ttgtcttttt ttgtatcttt tttaaactgt aaagttcaat tgtgaaaatg 3180
aatatcatgc aaataaatta tgcgattttt ttttcaaagt aaccactgca tctttgaagt 3240
tctgcctggt gagtaggacc agcctccatt tccttataag ggggtgatgt tgaggctgct 3300
ggtcagagga ccaaaggtga ggcaaggcca gacttggtgc tcctgtggtt ggtgccctca 3360
gttcctgcag cctgtcctgt tggagaggtc cctcaaatga ctccttctta ttattctatt 3420
agtctgtttc catgggcgtg ata 3443
<210> 9
<211> 254
<212> DNA
<213> Homo sapiens
<400> 9
gtgctgcacc aggccaccat cctgcccaag actgggacag tgtccctgga ggtacggctc 60
ctggaggcct cccgtgcctt cgaggtgtca gagaacggca acctggtagt gagtgggaag 120
gtgtaccagt gggatgaccc tgaccccagg ctcttcgacc acccggaaag ccccaccccc 180
aaccccacgg agcccctctt cctggcccag gctgaagttt acaaggagct gcgtctgcgt 240
9
SUBSTITUTE SHEET (RULE 26~
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
ggctacgact acgg
254
<210> 10
<211> 8470
<212> DNA
<213> Homo sapiens
<220>
<221> unsure
<222> (4131)
<220>
<221> unsure
<222> (5117)
<220>
<221> unsure
<222> (5552)
<400> 10
cggccgtcga cacggcagcg gccccggcct ccctctccgc cgcgcttcag cctcccgctc 60
cgccgcgctc cagcctcgct ctccgccgcc cgcaccgccg cccgcgccct caccagagca 120
gccatggagg aggtggtgat tgccggcatg tccgggaagc tgccagagtc ggagaacttg 180
caggagttct gggacaacct catcggcggt gtggacatgg tcacggacga tgaccgtcgc 240
tggaaggcgg ggctctacgg cctgccccgg cggtccggca agctgaagga cctgtctagg 300
tttgatgcct ccttcttcgg agtccacccc aagcaggcac acacgatgga ccctcagctg 360
cggctgctgc tggaagtcac ctatgaagcc atcgtggacg gaggcatcaa cccagattca 420
ctccgaggaa cacacactgg cgtctgggtg ggcgtgagcg gctctgagac ctcggaggcc 480
ctgagccgag accccgagac actcgtgggc tacagcatgg tgggctgcca gcgagcgatg 540
atggccaacc ggctctcctt cttcttcgac ttcagagggc ccagcatcgc actggacaca 600
gcctgctcct ccagcctgat ggccctgcag aacgcctacc aggccatcca cagcgggcag 660
tgccctgccg ccatcgtggg gggcatcaat gtcctgctga agcccaacac ctccgtgcag 720
ttcttgaggc tggggatgct cagccccgag ggcacctgca aggccttcga cacagcgggg 780
aatgggtact gccgctcgga gggtgtggtg gccgtcctgc tgaccaagaa gtccctggcc 840
cggcgggtgt acgccaccat cctgaacgcc ggcaccaata cagatggctt caaggagcaa 900
ggcgtgacct tcccctcagg ggatatccag gagcagctca tccgctcgtt gtaccagtcg 960
gccggagtgg cccctgagtc atttgaatac atcgaagccc acggcacagg caccaaggtg 102C
ggcgaccccc aggagctgaa tggcatcacc cgagccctgt gcgccacccg ccaggagccg 1080
ctgctcatcg gctccaccaa gtccaacatg gggcacccgg agccagcctc ggggctggca 1140
gccctggcca aggtgctgct gtccctggag cacgggctct gggcccccaa cctgcacttc 1200
catagcccca accctgagat cccagcgctg ttggatgggc ggctgcaggt ggtggaccag 1260
cccctgcccg tccgtggcgg caacgtgggc atcaactcct ttggcttcgg gggctccaaa 1320
cgtgcacatc atcctgaggc ccaacacgca gccgcccccc gcacccggcc cacatgccac 1380
cctgccccgt ctgctgcggg ccagcggacg cacccctgag gccgtgcaga agctgctgga 1440
gcagggcctc cggcacagcc agggcctggc tttcctgagc atgtgaacga catcgcggct 1500
gtccccgacc accgccatgc ccttccgtgg ctacgctgtg ctgggtggtg agacgcggtg 1560
gcccagaggt gcagcaggtg cccgctggcg agcgcccgct ctggttcatc tgctctggga 1620
tgggcacaca gtggcgcggg atggggctga gcctcatgcg cctggaccgc ttccgagatt 1680
10
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
ccatcctacg ctccgatgag gctgtgaacc gattcggcct gaaggtgtca cagctgctgc 1740
tgagcacaga cgagagcacc tttgatgaca tcgtccattc gtttgtgagc ctgactgcca 1800
tccagatagg cctcatagac ctgctgagct gcatggggct gaggccagat ggcatcgtcg 1860
gccactccct gggggaggtg gcctgtggct acgccgacgg ctgcctgtcc caggaggagg 1920
ccgtcctcgc tgcctactgg aggggacagt gcatcaaaga agcccatctc ccg~cgggcg 1980
ccatggcagc cgtgggcttg tcctgggagg agtgtaaaca gcgctgcccc ccggcggtgg 2040
tgcccgccgc cacaactcca aggacacagt caccatctcg ggacctcagg ccccggtgtt 2100
tgagttcgtg gagcagctga ggaaggaggg tgtgtttgcc aaggaggtgc ggaccggcgg 2160
tatggccttc cactcctact tcatggaggc catcgcaccc ccactgctgc aggagctcaa 2220
gaaggtgatc cgggagccga agccacgttc agcccgctgg ctcagcacct ctatccccga 2280
ggcccagtgg cacagcagcc tggcacgcac gtcctccgcc gagtacaatg tcaacaacct 2340
ggtgagccct gtgctgttcc aggaggccct gtggcacgtg cctgagcacg cggtggtgct 2400
ggagatcgcg ccccacgccc tgctgcaggc tgtcctgaag cgtggcctga agccgagctg 2460
caccatcatc cccctgatga agaaggatca cagggacaac ctggagttct tcctggccgg 2520
catcggcagg ctgcacctct caggcatcga cgccaacccc aatgccttgt tcccacctgt 2580
ggagtcccca gctccccgag gaactcccct catctcccca ctcatcaagt gggaccacag 2640
cctggcctgg gacgcgccgg crgccgagga cttccccaac ggttcaggtt ccccctcagc 2700
caccatctac acatgcacac caagctccga gtctcctgac cgctacctgg tggaccacac 2760
catcgacggt cgcgtcctct tccccgccac tggctacctg agcatagtgt ggaagacgct 2820
ggcccgaccc ctgggcctgg gcgtcgagca gctgcctgtg gtgtttgagg atgtggtgct 2880
gcaccaggcc accatcctgc ccaagactgg gacagtgtcc ctggaggtac ggctcctgga 2940
ggcctcccgt gccttcgagg tgtcagagaa cggcaacctg gtagtgagtg ggaaggtgta 3000
ccagtgggat gaccctgacc ccaggctctt cgaccacccg gaaagcccca cccccaaccc 3060
cacggagccc ctcttcctgg cccaggctga agtttacaag gagctgcgtc tgcgtggcta 3120
cgactacggc cctcatttcc agggcatcct ggaggccagc ctggaaggtg actcggggag 3180
gctgctgtgg aaggataatg ggtgagttca tggacaccat gctgcagatg tccatcctgg 3240
gtcggccaag cacggcctgt acctgcccac ccgtgtcacc gccatccaca tcgaccctgc 3300
cacccacagg cagaagctgt acacactgca ggacaaggcc caagtggctg acgtggtggt 3360
gagcaggtgg ctgagggtra cagtggccgg aggcgtccac atctccgggc tccacactga 3420
gtcggccccg cggcggcagc aggagcagca ggtgcccatc ctggagaagt tttgcttcac 3480
tccccacacg gaggaggggt gcctgtctga gcacgctgcc ctcgaggagg agctgcaact 3540
gtgcaagggg ctggtcgagg cactcgagac caaggtgacc cagcaggggc tgaagatggt 3600
ggtgcccgga ctggatgggg cccagatccc cccgggaccc ctcacagcag gaactgcccc 3660
ggctgttgtc ggctgcctgr aggcttcagc tcaacgggaa cctgcagctg gagctggcgc 3720
aggtgctggc ccaggagagg cccaagctgc cagaggaccc tctgctcagc ggcctcctgg 3780
actccccggc actcaaggcc tgcctggaca ctgccgtgga gaacatgccc agcctgaaga 3840
tgaaggtggt ggaggtgctg gccggccacg gtcacctgta ttcccgcatc ccaggcctgc 3900
tcagccccca tcccctgctg cagctgagct acacggccac cgaccgccac ccccaggccc 3960
tggazggctgc ccaggccgag ctgcagcagc acgacgttgc ccagggccag tgggatcccg 4020
cagaccctgc ccccagcgcc ctgggcagcg cggacctcct ggtgtgcaac tgtgctgtgg 4080
ctgccctcgg ggacccgcct cagctctcag caacatggtg gctgccctga nagaaggggg 4140
ctttctgctc ctgcacacac tgctccgggg gcaccccctc ggggacatcg tggccttcct 4200
cacctccact gagccgcagt atggccaggg catcctgagc caggacgcgt gggagagcct 4260
cttctccagg gtgtcgctgc gcctggtggg cctgaagaag tccttctacg gctccacgct 4320
cttcctgtgc cgccggccca ccccgcagga cagccccatc ttcctgccgg tggacgatac 4380
cagcttccgc tgggtggagt ctctgaaggg catcctggct gacgaagact ctttcccggc 4440
ctgtgtggct gaaggccatc aactgttcca cctcgggcgt ggtgggcttg gtgaactgtc 4500
tccgccgaga gcccggcgga acgctccggt gtgtgctgct ctccaacctc agcagcacct 4560
11
SUBStITUTE SHEET (RULE 26~
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
cccacgtccc ggaggtggac ccgggctccg cagaactgca gaaggtgttg cagggagacc 4620
tggtgatgaa cgtctaccgc gacggggcct ggggggcttt ccgccacttc ctgctggagg 4680
aggacaagcc tgaggagccg acggcacatg cctttgtgag caccctcacc cggggggacc 4740
tgtccctcca tccgctgggt ctgctcctcg ctgcgccatg cccagcccac ctgccctggc 4800
gcccagctct gcacggtcta ctacgcctcc ctcaacttcc gcgacatcat gc ggccact 4860
ggcaagctgt cccctgatgc catcccaggg aagtggacct cccaggacag cctgctaggt 4920
atggagttct cgggccgaga cgccagcggc aagcgtgtga tgggactggt gcctgccaag 4980
ggcctggcca cctctgtcct gctgtcaccg gacttcctct gggatgtgcc ttccaactgg 5040
acgctggagg aggcggcctc ggtgcctgtc gtctacagca cggcctacta cgcgctggtg 5100
gtgcgtgggc gggtgcnccc cggggagacg ctgctcatcc actcgggctc gggcggcgtg 5160
ggccaggccg ccatcgccat cgccctcagt ctgggctgcc gcgtcttcac caccgtgggg 5220
tcggctgaga agcgggcgta cctccaggcc aggttccccc agctcgacag caccagcttc 5280
gccaactccc gggacacatc cttcgagcag catgtgctgt ggcacacggg cgggaagggc 5340
gttgacctgg tcttgaactc cttggcggaa gagaagctgc aggccagcgt gaggtgcttg 5400
gctacgcacg gtcgcttcct ggaaattggc aaattcgacc tttctcagaa ccacccgctc 5460
ggcatggcta tcttcctgaa gaacgtgaca ttccacgggg tcctactgga tgcgttcttc 5520
aacgagagca gtgctgactg gcgggaggtg tnggcgcttg tgcaggccgg catccgggat 5580
ggggtggtac ggcccctcaa gtgcacggtg ttccatgggg cccaggtgga ggacgccttc 5640
cgctacatgg cccaagggaa gcacattggc aaagtcgtcg tgcaggtgct tgcggaggag 5700
ccggaggcag tggctgaagg gggccaaacc caagctgatg tcggccatct ccaagacctt 5760
ctgcccggcc cacaagagct acatcatcgc tggtggtctg ggtggcttcg gcctggagtt 5820
ggcgcagtgg ctgatacagc gtggggtgca gaagctcgtg ttgacttctc gctccgggat 5880
ccggacaggc taccaggcca agcaggtccg ccggtggagg cgccagggcg tacaggtgca 5940
ggtgtccacc agcaacatca gctcactgga gggggcccgg ggcctcattg ccgaggcggc 6000
gcagcttgag gcccgtgggc ggcgtcttca acctggccgt ggtcttgaga gatggcttgc 6060
tggagaacca gaccccagag ttcttccagg acgtctgcaa gcccaagtac agcggcaccc 6120
tgaacctgga cagggtgacc cgagggcgtg ccctgagctg gactactttg tggtcttctc 6180
ctctgtgagc tgcgggcgtg gcaatgcggg acagagcaac tacggctttg ccaatttccg 6240
ccatggagcg tatctgtgag aaacgccggc acgaaggcct cccaggcctg gccgtgcagt 6300
ggggcgccat cggcgacgtg ggcattttgg tggagacgat gagcaccaac gacacgatcg 6360
tcagtggcac gctgccccag cgcatggcgt cctgcctgga ggtgctggac ctcttcctga 6420
accagcccca catggtcctg agcagctttg tgctggctga gaaggctgcg gcctataggg 6480
acagggacag ccagcgggac ctggtggagg ccgtggcaca catcctgggc atccgcgact 6540
tggctgctgt caacctggac agctcactgg cggacctggg cctggactcg ctcatgagcg 6600
tggaggtgcg ccagacgctg gagcgtgagc tcaacctggt gctgtccgtg cgcgaggtgc 6660
ggcaactcac gctccggaaa ctgcaggagc tgtcctcaaa ggcggatgag gccagcgagc 6720
tgggcatgcc ccacgcccaa ggaggatggt ctggcccagc agcagactca gctgaacctg 6780
cgctccctgc tggtgaaccc ggagggcccc accctgatgc ggctcaactg ccgtgcagag 6840
ctcggagcgg cccctgttcc tggtgcaccc aattcgaggg ctccaccacc gtgttccaca 6900
gcctggcctc ccggctcagc atccccacct atggcctgca gtgcacccga gctgcgcccc 6960
ttgacagcat ccacagcctg gctgcctact acatcgactg catcaggcag gtgcagcccg 7020
agggccccta ccgcgtggcc ggctactcct acggggcctg cgtggccttt gaaatgtgct 7080
cccagctgca ggcccagcag agcccagccc ccacccacaa cagcctcttc ctgttcgacg 7140
gctcgcccac ctacgtactg gcctacaccc agagctaccg ggcaaagctg accccaggct 7200
gtgaggctga ggctgagacg gaggccatat gcttcttcgt gcagcagttc acggacatgg 7260
agcacaacag ggtgctggag gcgctgctgc cgctgaaggg cctagaggag cgtgtggcag 7320
ccgccgtgga cctgatcatc aagagccacc agggcctgga ccgccaggag ctgagctttg 7380
cggcccggtc cttctactac aagctgcgtg ccgctgagca gtacacaccc aaggccaagt 7440
12
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
accatggcaa cgtgatgcta ctgcgcgcca agacgggtgg cgcctacggc gaggacctgg 7500
gcgcggacta caacctctcc caggtatgcg acgggaaagt atccgtccac gtcatcgagg 7560
gtgaccaccg cacgctgctg gagggcagcg gcctggagtc catcatcagc atcatccaca 7620
gctccctggc tgagccacgc gtgagcgtgc gggagggcta ggcccgtgcc cccgcctgcc 7680
accggaggtc actccaccat ccccacccca tcccacccca cccccgccat gc acgggat 7740
tgaagggtcc tgccggtggg accctgtccg gcccagtgcc actgcccccc gaggctagct 7800
agacgtaggt gttaggcatg tcccacccac ccgccgcctc ccacggcacc tcggggacac 7860
cagagctgcc gacttggaga ctcctggtct gtgaagagcc ggtggtgccc gtgcccgcag 7920
gaactggggc tgggcctcgt gcgcccgtgg ggtctgcgct tggtctttct gtgcttggat 7980
ttgcatattt attgcattgc tggtagagac ccccaggcct gtccaccctg ccaagactcc 8040
tcaggcagcg tgtgggtccc gcactctgcc cccatttccc cgatgtcccc tgcgggcgcg 8100
ggcagccacc caagcctgct ggctgcggcc ccctctcggc caggcattgg ctcagcccgc 8160
tgagtggggg gtcgtgggcc agtccccgag gactgggccc ctgcacaggc acacagggcc 8220
cggccacacc cagcggcccc ccgcacagcc acccgtgggg tgctgccctt atgcccggcg 8280
ccgggcacca actccatgtt tggtgtttgt ctgtgtttgt ttttcaagaa atgattcaaa 8340
ttgctgcttg gattttgaaa tttactgtaa ctgtcagtgt acacgtctgg accccgtttc 8400
atttttacac caatttggta aaaatgctgc tctcagcctc ccacaattaa accgcatgtg 8460
atctccaaaa 8470
<210> 11
<211> 812
<212> DNA
<213> Homo sapiens
<400> 11
gccgcagcca atcagcgcgc gtgcccgggc ccctgcgtct cttgcgtcaa gacggccgtg 60
ctgagcgaat gcaggcgact tgcgagctgg gagcgattta aaacgctttg gattcccccg 120
gcctgggtgg ggagagcgag ctgggtgccc cctagattcc ccgcccccgc acctcatgag 180
ccgaccctcg gctccatgga gcccggcaat tatgccacct tggatggagc caaggatatc 240
gaaggcttgc tgggagcggg aggggggcgg aatctggtcg cccactcccc tctgaccagc 300
cacccagcgg cgcctacgct gatgcctgct gtcaactatg cccccttgga tctgccaggc 360
tcggcggagc gccaaagcaa tgccacccat gccctggggt gccccagggg acgtccccag 420
ctcccgtgcc ttatggttac tttggaggcg ggtactactc ctgccgagtg tcccggagct 480
cgctgaaacc ctgtgcccag gcagccaccc tggccgcgta ccccgcggag actcccacgg 540
ccggggaaga gtaccccagc cgccccactg agtttgcctt ctatccggga tatccgggaa 600
cctaccagcc tatggccagt tacctggacg tgtctgtggt gcagactctg ggtgctcctg 660
gagaaccgcg acatgactcc ctgttgcctg tggacagtta ccagtcttgg gctctcgctg 720
gtggctggaa cagccagatg tgttgccagg gagaacagaa cccaccaggt cccttttgga 780
aggcagcatt tgcagactcc agcgggcagc ac 812
<210> 12
<211> 2385
<212> DNA
<213> Homo sapiens
<400> 12
ataagctggg gtaaagtatt ttcgcagttt ctgcctttag gattttatta gcttctctcc 60
cccaggccgc agccaatcag cgcgcgtgcc cgggcccctg cgtctcttgc gtcaagacgg 120
13
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
ccgtgctgag cgaatgcagg cgacttgcga gctgggagcg atttaaaacg ctttggattc 180
ccccggcctg ggtggggaga gcgagctggg tgccccctag attccccgcc cccgcacctc 240 _
atgagccgac cctcggctcc atggagcccg gcaattatgc caccttggat ggagccaagg 300
atatcgaagg cttgctggga gcgggagggg ggcggaatct ggtcgcccac tcccctctga 360
ccagccaccc agcggcgcct acgctgatgc ctgctgtcaa ctatgccccc ttggatctgc 420
caggctcggc ggagccgcca aagcaatgcc acccatgccc tggggtgccc caggggacgt 480
ccccagctcc cgtgccttat ggttactttg gaggcgggta ctactcctgc cgagtgtccc 540
ggagctcgct gaaaccctgt gcccaggcag ccaccctggc cgcgtacccc gcggagactc 600
ccacggccgg ggaagagtac cccagccgcc ccactgagtt tgccttctat ccgggatatc 660
cgggaaccta ccagcctatg gccagttacc tggacgtgtc tgtggtgcag actctgggtg 720
ctcctggaga accgcgacat gactccctgt tgcctgtgga cagttaccag tcttgggctc 780
tcgctggtgg ctggaacagc cagatgtgtt gccagggaga acagaaccca ccaggtccct 840
tttggaaggc agcatttgca gactccagcg ggcagcaccc tcctgacgcc tgcgcctttc 900
gtcgcggccg caagaaacgc attccgtaca gcaaggggca gttgcgggag ctggagcggg 960
agtatgcggc taacaagttc atcaccaagg acaagaggcg caagatctcg gcagccacca 1020
gcctctcgga gcgccagatt accatctggt ttcagaaccg ccgggtcaaa gagaagaagg 1080
ttctcgccaa ggtgaagaac agcgctaccc cttaagagat ctccttgcct gggtgggagg 1140
agcgaaagtg ggggtgtcct ggggagacca ggaacctgcc aagcccaggc tggggccaag 1200
gactctgctg agaggcccct agagacaaca cccttcccag gccactggct gctggactgt 1260
tcctcaggag cggcctgggt acccagtatg tgcagggaga cggaacccca tgtgacagcc 1320
cactccacca gggttcccaa agaacctggc ccagtcataa tcattcatcc tgacagtggc 1380
aataatcacg ataaccagta ctagctgcca tgatcgttag cctcatattt tctatctaga 1440
gctctgtaga gcactttaga aaccgctttc atgaattgag ctaattatga ataaatttgg 1500
aaggcgatcc ctttgcaggg aagctttctc tcagaccccc ttccattaca cctctcaccc 1560
tggtaacagc aggaagactg aggagagggg aacgggcaga ttcgttgtgt ggctgtgatg 1620
tccgtttagc atttttctca gctgacagct gggtaggtgg acaattgtag aggctgtctc 1680
ttcctccctc cttgtccacc ccatagggtg tacccactgg tcttggaagc acccatcctt 1740
aatacgatga tttttctgtc gtgtgaaaat gaagccagca ggctgcccct agtcagtcct 1800
tccttccaga gaaaaagaga tttgagaaag tgcctgggta attcaccatt aatttcctcc 1860
cccaaactct ctgagtcttc ccttaatatt tctggtggtt ctgaccaaag caggtcatgg 1920
tttgttgagc atttgggatc ccagtgaagt agatgtttgt agccttgcat acttagccct 1980
tcccaggcac aaacggagtg gcagagtggt gccaaccctg ttttcccagt ccacgtagac 2040
agattcacgt gcggaattct ggaagctgga gacagacggg ctctttgcag agccgggact 2100
ctgagaggga catgagggcc tctgcctctg tgttcattct ctgatgtcct gtacctgggc 2160
tcagtgcccg gtgggactca tctcctggcc gcgcagcaaa gccagcgggt tcgtgctggt 2220
ccttcctgca ccttaggctg ggggtggggg gcctgccggc gcattctcca cgattgagcg 2280
cacaggcctg aagtctggac aacccgcaga accgaagctc cgagcagcgg gtcggtggcg 2340
agtagtgggg tcggtggcga gcagttggtg gtgggccgcg gccgc 2385
<210> 13
<211> 221
<212> DNA
<213> Homo Sapiens
<400> 13
dsdnrstatc tttctgtgtg gtgcagccct gttggcagtg ggcatctggg tgtcaatcga 60
tggggcatcc tttctgaaga tcttcgggcc actgtcgtcc agtgccatgc agtttgtcaa 120
cgtgggctac ttcctcatcg cagccggcgt tgtggtcttt gctcttggtt tcctgggctg 180
14
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/2311 I PCT/US99/24331
ctatggtgct aagactgaga gcaagtgtgc cctcgtgacg t 221
<210> 14
<211> 1533
<212> DNA
<213> Homo sapiens
<400> 14
gggcacgcag acattctggg aagccacttg ccccacccct gggctgcttc ttcttgagat 60
caggaggggc gttgcccagg gctggtgttg ccaggtggag gcctgctgag gcagtggttg 120
tggggatcgg tctccaggca gcagggggca gcagggtcaa ggagaggcta actggccacg 180
ggtggggcca gcaggcgggc agaaggaggc tttaaagcgc ctaccctgcc tgcaggtgag 240
cagtggtgtg tgagagccag gccgtccctc tgcctgccca ctcagtggca acacccggga 300
gctgttttgt cctttgtgga gcctcagcag ttccctgctt tcagaactca ctgccaagag 360
ccctgaacag gagccaccat ggcagtgctt cagcttcatt aagaccatga tgatcctctt 420
caatttgctc atctttctgt gtggtgcagc cctgttggca gtgggcatct gggtgtcaat 480
cgatggggca tcctttctga agatcttcgg gccactgtcg tccagtgcca tgcagtttgt 540
caacgtgggc tacttcctca tcgcagccgg cgttgtggtc tttgctcttg gtttcctggg 600
ctgctatggt gctaagactg agagcaagtg tgccctcgtg acgttcttct tcatcctcct 660
cctcatcttc attgctgagg ttgcagctgc tgtggtcgcc ttggtgtaca ccacaatggc 720
tgagcacttc ctgacgttgc tggtagtgcc tgccatcaag aaagattatg gttcccagga 780
agacttcact caagtgtgga acaccaccat gaaagggctc aagtgctgtg gcttcaccaa 840
ctatacggat tttgaggact caccctactt caaagagaac agtgcctttc ccccattctg 900
ttgcaatgac aacgtcacca acacagccaa tgaaacctgc accaagcaaa aggctcacga 960
ccaaaaagta gagggttgct tcaatcagct tttgtatgac atccgaacta atgcagtcac 1020
cgtgggtggt gtggcagctg gaattggggg cctcgagctg gctgccatga ttgtgtccat 1080
gtatctgtac tgcaatctac aataagtcca cttctgcctc tgccactact gctgccacat 1140
gggaactgtg aagaggcacc ctggcaagca gcagtgattg ggggagggga caggatctaa 1200
caatgtcact tgggccagaa tggacctgcc ctttctgctc cagacttggg gctagatagg 1260
gaccactcct tttaggcgat gcctgacttt ccttccattg gtgggtggat gggtgggggg 1320
cattccagag cctctaaggt agccagttct gttgcccatt cccccagtct attaaaccct 1380
tgatatgccc cctaggccta gtggtgatcc cagtgctcta ctgggggatg agagaaaggc 1440
attttatagc ctgggcataa gtgaaatcag cagagcctct gggtggatgt gtagaaggca 1500
cttcaaaatg cataaacctg ttacaatgtt gcc 1533
<210> 15
<211> 472
<212> DNA
<213> Homo sapiens
<400> 15
tcagagaaaa ctcaaacttt attgagagaa ttttcaaatt ttcagtcaca ttttcaatgt 60
gacatcagcc atgtgtgtag cttcagcttg tcttcttttt aacttatggc tgcccatctc 120
ctgcttcttt agtcttagca tgcttaggat taggtggagt cttctctttt acatcagagc 180
catctccacg ctcactccga gtcttttcca gatccatttc ctggcaatca ccttctactt 240
tacgttcttc gatcggaggt gttccttctc tctcttgtcc aggttcaata tcctgattgt 300
cagttggtgg ttcctcttgc tgagattcac cgggagccac gaatgcaacc acatcgggag 360
cctcctgacc atctcctctt cctctggatc ttgatctcac tcgtgcactc atcgctgcaa 420
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
ctagaagatc gtgaactgaa gaacttgagt cagcagagag cctggcgaag as 472
<210> 16
<211> 478
<212> DNA
<213> Homo sapiens
<400> 16
cttcattctt cgccaggctc tctgctgact caagttcttc agttcacgat cttctagttg 60
cagcgatgag tgcacgagtg agatcaagat ccagaggaag aggagatggt caggaggctc 120
ccgatgtggt tgcattcgtg gctcccggtg aatctcagca agaggaacca ccaactgaca 180
atcaggatat tgaacctgga caagagagag aaggaacacc tccgatcgaa gaacgtaaag 240
tagaaggtga ttgccaggaa atggatctgg aaaagactcg gagtgagcgt ggagatggct 300
ctgatgtaaa agagaagact ccacctaatc ctaagcatgc taagactaaa gaagcaggag 360
atgggcagcc ataagttaaa aagaagacaa gctgaagcta cacacatggc tgatgtcaca 420
ttgaaaatgt gactgaaaat ttgaaaattc tctcaataaa gtttgagttt tctctgaa 478
<210> 17
<211> 198
<212> DNA
<213> Homo Sapiens
<220>
<223> unsure
<222> (191)
<400> 17
cccgctgtac caccccagca tgttctgcgc cggcggaggg caagaccaga aggactcctg 60
caacggtgac tctggggggc ccctgatctg caacgggtac ttgcagggcc ttgtgtcttt 120
cggaaaagcc ccgtgtggcc aagttggcgt gccaggtgtc tacaccaacc tctgcaaatt 180
cactgagtgg nattaagg
198
<210> 18
<211> 465
<212> DNA
<213> Homo Sapiens
<400> 18
tggagatgga gtatgtattt attttacaaa aataaatcac catcttcgga ccatttgtag 60
actggaacat ttcgagcaat gagtgcgcca cacggacgag tgccctggtg actccctgat 120
gttcgcgtca cccccagggc caccttggcg cccgcatgag cctcgcttcc cactcccggc 180
ctccaactcc cttccctcgc agccgccatt caccttctgc tgtttatttg tctgcagagc 240
gcctggacac cggaaaaggc gattccctga gcgcctggag ttggagacaa ttcctggttc 300
agaatttaaa catctttcta aggtaagcgc tgctccaaaa ctcttcgccg cgtggggact 360
ttgcaccagg ggcggttggg aaggaagttg gccctccacg ggttcctggg caaccgcggc 420
ctgttgaaaa aaggttctgg gtcaaataat ttaacttcgg aggag 465
<210> 19
16
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCTNS99/24331
<211> 204
<212> DNA
<213> Homo Sapiens
<400> 19
ggcgggaaca ggcggcgctg gacctgtacc cctacgacgc cgggacggac agcggcttca 60
ccttctcctc ccccaacttc gccaccatcc cgcaggacac ggtgaccgag ataacgtcct 120
cctctcccag ccacccggcc aactccttct actacccgcg gctgaaggcc ctgcctccca 180
tcgccagggt gacactggtg cggc 204
<210>20
<211>294
<212>DNA
<213>Homo Sapiens
<220>
<221> unsure
<222> (287)
<400> 20
gagatttctc ttcaatggct tcctgtgagc tagagtttga aaatatctta aaatcttgag 60
ctagagatgg aagtagcttg gacgattttc attatcatgt aaatcgggtc actcaagggg 120
ccaaccacag ctgggagcca ctgctcaggg gaaggttcat atgggacttt ctactgccca 180
aggttctata caggatataa aggtgcctca cagtatagat ctggtagcaa agtaagaaga 240
aacaaacact gatctctttc tgccacccct ctgacccttt ggaactnctc tgac 294
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 21
atcagaacaa agaggctgtg tc 22
<210> 22
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 22
atctctaaag ccccaacctt c 21
17
SUBSTITUTE SHEET (RUSE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
<210> 23
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 23
tgccgaagag gttcagtgc lg
<2i0> 24
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 24
gccacagtgg tactgtccag at 22
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 25
gctgcaagtt ctccacattg a 21
<210> 26
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 26
cagccgcagg tgaaacac
18
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence
18
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 27
tggctttgaa ctcagggtca 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 28
cggatgcacc tcgtagacag 20
<210> 29
<211> 20
<2T2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 29
cggcaacctg gtagtgagtg 20
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 30
cgcagctcct tgtaaacttc ag 22
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
19
SUBSTITUTE SHEET (RULE 26~
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
<400> 31
cgggaaccta ccagcctatg 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 32
caggcaacag ggagtcatgt 20
<210> 33
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: synthetic
<400> 33
tgggcatctg ggtgtcaa 18
<210> 34
<211> I9
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 34
cggctgcgat gaggaagta 19
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 35
gcccatctcc tgcttcttta gt 22
<210> 36
SUBSTITUTE SHEET (RULE 26)
CA 02347081 2001-04-18
WO 00/23111 PCT/US99/24331
<211> 21
<212> DNA
<213> Artificial Sequence -
<220>
<223> Description of Artificial Sequence: Synthetic
<400> 36
cgtggagatg gctctgatgt a
21
21
SUBSTITUTE SHEET (RULE 26)
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...~_~..~.....
