Note: Descriptions are shown in the official language in which they were submitted.
CA 02262624 1999-01-29
W 09810~349 PCT~US97/13~50
A NOVEL ~IETHOD OF DETECTING AND TREATING CANCER
RELATED APPLICATION:
This application is a continuation-in-part application of USSN 08/714.744,
filed September 16, 1996, which is also continuation-in-part of USSN 08/691,479,filed August 2, 1996. The above mentioned patent applications are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
This invention relates, in part, to newly developed assay for diagnosing
cancers, particularly prostate cancer, and benign prostate hyperplasia (BPH), and
methods for identifying agents which modulate PLA2 activity and therapeutic a_ents
that modulate PLA2 activity for treating cancers and BPH.
BACKGROUND OF THE INVENTION
Cancer of the prostate is the most prevalent m:~lign~ncy in adult males,
excluding skin cancer, and is an increasingly prevalent health problem in the United
States. In 1994, it was estimated that in the US, 38,000 deaths resulted from this
disease, in~lic~ting 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.
Accordingly, there is a great need for sensitive methods for the detection of organ-
confined prostate cancer.
Extracellular Phospholipase A2 (PLA2) enzymes appear to mediate a variety
of responses including cellular proliferation, chemotaxis and infl~mm~ion. Thereare two major groups of extracellular PLA2 enzymes: pancreatic or group I and
rheumatoid arthritis synovial fluid (RASF) or group II. The group I enzyme
functions in digestion and also in modulating proliferation and chemotaxis.
Currently, RASF-PLA~ is predominantly thought to play a role in inflammatory
responses including arthritis, septic shock and lung injury. The level of RASF-
PLA~ is regulated at the mRNA level by a variety agents including interleukin-6,interleukin- I and tumor necrosis factor, all of which are involved in inflammatory
-1-
. . .
CA 02262624 1999-01-29
W 098/05349 PCTrUS97/13550
responses. While elevated leveis of PLA2 enzyme activity have been reported in ~prostate cancer tissue in rats (F.H. Faas et al., The Journal of Urolo,v. Vol 156, 2~3-
248, 1996), there do not appear to be any reports of alterations of RASF-PLA~
mRNA or polypeptide level in prostate cancer or benign prostate hyperplasia in
5 humans.
Northern blot analysis was done on equivalent amounts of mRNA isolated
from prostate cancer (PC), benign prostatic hyperplasia (BPH) and norrnal prostate
(NP) according to methods published in Maniatis (MOLECULAR CLONING
Maniatis, et. al., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).10 Probe was synthesized from the full length cDNA encoding RASF-PLA2. Results
indicated the following ratios of RASF-PLA~ mRNA: 10: 0.25: 1 for PC, BPH and
NP, respectively. Loading differences were normalized using actin mRNA levels.
The result showed that RASF-PLA2 appears to be upregulated in prostate cancer and
potentially downregulated in BPH.
As used hereinbelow "PLA2" refers to group II or RASF-PLA~.
SUMl~IARY OF THE INVENTION
Toward these ends, and others, it is an object of the present invention to
provide a new method for diagnosing, treating, and monitoring progression. remission
20 or recurrence of various forms of abnormal cell growth, such as cancers, particulary
prostate cancer, and benign prostate hyperplasia (BPH). Further provided are methods
to screen for therapeutic agents and pharmaceutical compositions for treating
abnormal celi growth, such as cancers, particularly prostate cancer, and BPH. Further
provided is the utilization of such agents or compositions for the for the treatement of
25 cancer. patricularly prostate cancer, and BPH.
Thus, in accordance with one aspect of the present inven~ion there are provided
methods of screening for compounds which bind to and inhibit activation of the PLA~.
In accordance with another aspect of the present invention there is provided a
method of using such inhibiting compounds for treating conditions associated with
30 over-expression of the PLA n
In accordance with yet another aspect of the present invention, there are
provided PLA~ antaoonists (inhibitors). Among the preferred antagonists are those
CA 02262624 l999-0l-29
W O 98/05349 PCTAUS97/13550
which mimic PLA~ so as to bind to PLA2 binding molecules but not elicit a PLA2-
induced response or more than one PLA~-induced response. Also among the preferred
anta~onists are molecules that bind to or interact with PLA2 so as to inhibit an effec
of PLA2 or more th;m one effect of PLA2 or which prevent expression of PLA~.
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.
DESCRIPI'ION OF THE INVENTION
The present invention relates to diagnostic assays, both quantitative and
qualitative for detecting levels of PLA2 protein or PLA2 mRNA in cells, tissues and
bodily fluids, including determination of normal and abnorrnal levels. Thus, forinct~nCe. a diagnostic assay in accordance with the invention for detecting over-
expression of PLA2 protein compared to normal control bodily fluids or tissue
sarnples may be used to detect the presence of cancers, including prostate cancer.
Further. the present method of quantifying protein PLA~ protein level is particularly
useful for discrimin~ting between BPH and prostate cancer, since the existing methods
such as prostatic specific antigen (PSA), digital examination, and transurethralultrasound tests have difficulty discrimin~ting between prostate cancer and BPH. For
example, the existing PSA diagnostic tests detect 20-28% of BPH patients and 62-81% of prostate cancer patients with PSA blood levels above approximately 99% ofthe norrnal population. Assay techniques that can be used to determine levels of gene
expression, such as PLA ~ of the present invention, in a sample derived from a host are
well-known to those of skill in the art. Such assay methods include
radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays,
immunohistochemistry assays, in situ hybridization assays, competitive-binding
assays. Western Blot analyses and ELISA assays. Among these, ELISAs are
-3 -
CA 02262624 1999-01-29
WO 98/05349 PCTtUS97/13550
frequently preferred to detect a gene's expressed protein in biological iluids. An
ELISA assay initially comprises preparing an antibody, if not readily available from a
commercial source, specific to PLA2, preferably a monoclonal antibody. In addition
a reporter antibody generally is prepared which binds specifically to PLA~. The
S 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 PLA2 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 PLA2 binds to the specific antibody attached to the polystyrene dish. Unbound
sample is washed out with buffer. A reporter antibody specifically directed to PLA2
and linked to horseradish peroxidase is placed in the dish resulting in binding of the
reporter antibody to any monoclonal antibody bound to PLA2. Unattached reporter
antibody is then washed out. Reagents for peroxidase activity, including a colorimetric
substrate are then added to the dish. Imrnobilized peroxidase, linked to PLA2
antibodies, produces a colored reaction product. The amount of color developed in a
given time period is proportional to the amount of PLA2 protein present in the
sample. Quantitative results typically are obtained by reference to a standard curve.
Without lirniting the instant invention, typically, for a quantitative diagnostic assay a
positive result indicating the disease is one in which blood levels are higher than
three standard deviations above the mean blood level for a normal healthy
population of individuals (99.86% of the population).
A competition assay may be employed wherein antibodies specific to PLA2
attached to a solid support and labeled PLA2 and a sample derived from the host are
passed over the solid support and the amount of label detected attached to the solid
support can be correlated to a quantity of PLA2 in the sarnple.
Nucleic acid methods may be used to detect PLA2 mRNA as a marker for
BPH and cancer, particularly prostate cancer. Polymerase chain reaction (PCR) and
other nucleic acid methods, such as ligase chain reaction (LCR) and nucleic acidsequence based amplification (NASABA), can be used to detect malignant cells for
-4-
CA 02262624 1999-01-29
WO 98105349 PCT/US97/13550
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 rnRNA population in a complex mixture of thousands of other rrlRNA species.
In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA
S (cDN~) with use of the enzyme reverse transcriptase; the cDNA is then arnplified as in
a standard PCR reaction. RT-PCR can thus reveal by arnplification 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 arrayed on a grid (i.e. griddin~,) can be used to both
10 detect the expression of and qu~nti~:lte the level of expression of that gene. In this
approach, a cDNA encoding the PLA2 gene is fixed to a substrate. The substrate
may be of any suitable type including but not lirnited to glass, nitrocellulose, nylon
or plastic. DNA encoding the PLA2 clone is attached to the substrate and then
incubated with the analyte, which may be RNA or a complementray DNA (cDNA)
15 copy of the RNA, isolated from the tissue of interest. Hybridization between the
substrate bound clone and the analyte can be detected and quantitated by severalmeans including but not limited to radioactive labeling or fluorescence labeling of
the analyte or a secondary molecule designed to detect the hybrid. Quantitation of
the level of gene expression can be done by comparison of the intensity of the signal
20 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.
The above tests can be carried out on samples derived from patients' bodily
fluids and tissue extracts (homogenates or solubilized tissue) such as from blood.
25 urine. s31iva, tissue biopsy and autopsy material.
Antibodies
The PLA~ polypetide. its fr~gm~rl~c or other derivatives, or analogs thereof. orcells e.l~pressin~ them can be used as an immunogen to produce antibodies thereto.
30 These antibodies can be, for example, polyclonal or monoclonal antibodies. The
present invention also includes chimeric, single chain, and hl~m~ni7ed antibodies. as
CA 02262624 1999-01-29
W 098/05349 PCT~US97/13550
well as Fab fr~gm~rltc or the product of a Fab expression library. Various procedures
known in the ar~ may be used for the production of such antibodies and fragments.
Antibodies generated against PLA~ can be obtained by direct injection of the
polypeptide into an animal or by l~minicr~ring the polypeptide to an animal,
preferably a nonh~ n. The antibody so obtained will then bind the polypeptide itself.
In this manner. even a sequence encoding only a fragment of the polypeptide can be
used to generate antibodies binding the whole native polypeptide.
For ~lcp~tion of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used. Examples include
the hybridoma technique (Kohler, G. and Milstein, C., Nature 256:495-497 (1975)),
the trioma technique, the human B-cell hybridoma technique (Kozbor et al.,
Immunology To~ay 4:72 (1983)) and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND
CANCE~ THERAPY, Alan R. Liss, Inc. (1985)).
Techniques described for the production of single chain antibodies (U.S. Patent
No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic
polypeptide products of this invention. Also, transgenic mice, or other organisms such
as other m~lnm:llc, may be used to express hllm~ni7~d antibodies to irnmunogenic
PLA2.
Thus, among others. antibodies against PLA2 may be employed to treat/inhibit
various forms of cancer, including prostate cancer, and BPH.
PLA2 binding molec~ s and assays
PLA2 could be used to isolate proteins which interact with it and this
interaction could be a target for interference. Inhibitors of protein-protein
interactions between PLA2 and other factors could lead to the development of
pharmaceutical agents for the modulation of PLA~ activity. As used herein, the term
"modulate" refer to affecting the PLA2 function.
Thus, this invention also provides a method for identification of binding
molecules to PLA~. Genes encoding proteins for binding molecules to PLA2 can be
identified by numerous methods known to those of skill in the art, for example, ligand
panning and FACS sorting. Such methods are described in many laboratory manuals
-6 -
CA 02262624 1999-01-29
W O 98105349 PCT~US97/13550
such as, for instance, Coligan et al., Current Protocols in Immunology I (Rivelt. A.J.
Biochem. J. 291:1-10 (1993)): Chapter 5 (1991).
For example, the yeast two-hybrid system provides methods for detecting the
interaction between a first test protein and a second test protein, in vivo, using
. 5 reconstitution of the activity of a transcriptional activator. The method is disclosed
in U.S. Patent No. 5,283,173; reagents are available from Clontech and Stratagene.
Briefly, PLA2 cDNA is fused to a Gal4 transcription factor DNA binding domain
and expressed in yeast cells. cDNA library members obtained from cells of interest
are fused to a transactivation domain of Gal4. cDNA clones which express proteins
which can interact with PLA2 will lead to reconstitution of Gal4 activity and
transactivation of expression of a reporter gene such as Gall-lacZ.
An alternative method is screening of ~gtl l, ~ZAP (Stratagene) or
equivalent cDNA expression libraries with recombinant PLA2. Recombinant PLA~
protein or fragments thereof are fused to small peptide tags such as FLAG, HSV or
GST. The peptide tags can possess convenient phosphorylation sites for a kinase
such as heart muscle creatine kinase or they can be biotinylated. Recombinant
PLAa can be phosphorylated with 32[p] or used unlabeled and detected with
streptavidin or antibodies against the tags. ~gtl lcDNA expression libraries aremade from cells of interest and are incubated with the recombinant PLA2, washed
and cDNA clones isolated which interact with PLA2. See, e.g., T. Maniatis et al,
supra.
Another method is the screening of a m~mm~ n expression library in which
the cDNAs are cloned into a vector between a m~mm~ n promoter and
polyadenylation site and transiently transfected in COS or 293 cells followed bydetection of the binding protein 48 hours later by incubation of fixed and washed
cells with a labelled PLA2, preferably iodinated, and detection of bound PLA2 byautoradiography. See Sims et aL, Science 241:585-589 ( 1988) and McMahan et al.,EMBO J. 10:2821-2832 (1991). In this manner, pools of cDNAs containing the
cDNA encoding the binding protein of interest can be selected and the cDNA of
interest can be isolated by further subdivision of each pool followed by cycles of
transient transfection, binding and autoradiography. Alternatively, the cD~A of
interest can be isolated by transfecting the entire cDNA library into m.~mm~lian cells
-7 -
. . ~ ~ . . .
CA 02262624 1999-01-29
W 098/05349 PCTAUS97/13550
and panning the cells on a dish containing PLA~ bound to the plate. Cells which
attach after washing are Iysed and the plasmid DNA isolated, amplified in bacteria,
and the cycle of transfection and panning repeated until a single cDNA clone is
obtained. See Seed et al, Proc. Natl. Acad. Sci. USA 84:3365 ( 1987) and Aruffo et
al., EMBO J. 6:3313 (1987~. If the binding protein is secreted, its cDNA can be
obtained by a similar pooling strategy once a binding or neutralizing assay has been
established for assaying supernatants from transiently transfected cells. General
methods for screening supematants are disclosed in Wong et al., Science 228:810-815 (1985).
Another alternative method is isolation of proteins interacting with PLA2
directly from cells. Fusion proteins of PLA ~ with GST or small peptide tags aremade and immobilized on beads. Biosynthetically labeled or unlabeled protein
extracts from the cells of interest are prepared, incubated with the beads and washed
with buffer. Proteins interacting with PLA2 are eluted specifically from the beads
and analyzed by SDS-PAGE. Binding partner primary amino acid sequence data are
obtained by microsequencing. Optionally, the cells can be treated with agents that
induce a functional response such as tyrosine phosphorylation of cellular proteins.
An example of such an agent would be a growth factor or cytokine such as
interleukin-2.
Another alternative method is immunoaffinity purification. Recombinant
PLA2 is incubated with labeled or unlabeled cell extracts and imrnunoprecipitated
with anti- PLA2 antibodies. The imrnunoprecipitate is recovered with protein A-
Sepharose and analyzed by SDS-PAGE. Unlabelled proteins are labeled by
biotinylation and detected on SDS gels with streptavidin. Binding partner proteins
are analyzed by microsequencing. Further, standard biochemical purification steps
known to those skilled in the art may be used prior to microsequencing.
Yet another alternative method is screening of peptide libraries for binding
partners. Recombinant tagged or labeled PLA2 is used to select peptides from a
peptide or phosphopeptide library which interact with PLA2. Sequencing of the
peptides leads to identification of consensus peptide sequences which might be
found in interacting proteins.
CA 02262624 1999-01-29
W 098/05349 PCT~US97/13550
PLA~ binding partners identified by any of these methods or other methods
which would be known to those of ordinary skill in the art as well as those putative
binding partners discussed above can be used in the assay method of the invention.
Assaying for the presence of PLA2/binding partner complex are accomplished by,
for example, the yeast two-hybrid system, ELISA or immunoassays using antibodiesspecific for the complex. In the presence of test substances which interrupt or inhibit
forrnation of PLA2/binding partner interaction, a decreased amount of complex will
be determined relative to a control lacking the test substance.
Assays for free PLA2 or binding partner are accomplished by, for example,
ELISA or immunoassay using specific antibodies or by incubation of radiolabeled
PLA2 with cells or cell membranes followed by centrifugation or filter separation
steps. In the presence of test substances which interrupt or inhibit formation of
PLA~/binding partner interaction, an increased amount of free PLA2 or free binding
partner will be determined relative to a control lacking the test substance.
Polypeptides of the invention also can be used to assess PLA2 binding capacity
of PLA~ binding molecules in cells or in cell-free preparations.
Agonists and antagonists - assays and molE,~ s
The PLA2 may be employed in a process for screening for compounds which
either inhibit, promote or modulate the enzymatic activity of PLA2. One standardassay for PLA2 uses ~linoleoyl-1-14C] labeled L~ acyl-2-
linoleoylphosphatidylethanolamine as a substrate and follows the release of 14C
labeled free fatty acid. This assay or others could be used to identify either agonists or
antagoniStS of PLA2
Examples of potential PLA~ antagonists are small molecules such as organic
molecules or peptides, antibodies, or in some cases an oligonucleotide, which binds to
PLA~ and prevents enzymatic activity.
Potential antagonists also include small molecules or proteins which are
closely related to the binding molecules of the PLA~, e.g. a fragment of the binding
molecules. which have lost biological function, and when bind to the PLA2
polypeptide inhibit its activity. ("Binding molecules" as used herein refer to molecules
that specifcally bind to or interact with PLA~ polypeptide of the present invention.
CA 02262624 1999-01-29
W 098105349 PCT~US97/13550
Included in the definition of binding molecules are other factors, co-factors, units or
subunits which enhance PLA2 activity or diminish it. Such binding molecules are a
part of the present invention. Binding molecules also may be non-naturally occurring,
such as antibodies and antibody-derived reagents that bind specifically to PLA2.)
A potential antagonist also includes an antisense construct prepared through
the use of antisense technology. Anticen.ce technology can be used to control gene
expression throu~ah triple-helix formation or antisense DNA or RNA, both of which
methods are based on binding of a polynucleotide to DNA or RNA. For exarnple, the
5' coding portion of the polynucleotide sequence, which encodes for the mature PLA2,
is used to design an :~nti~en.ce RNA oligonucleotide of from about 10 to 40 base pairs
in length. A DNA oligonucleotide is decign.od to be complementary to a region of the
gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073
(1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251:1360
(1991)), thereby preventing transcription and the production of PLA2 polypeptide.
The ~ntisence RNA oligonucleotide hybridizes to the mRNA in ~ivo and blocks
translation of the mRNA molecule into the PLA2 polypeptide (~nticellce - Okano, J.
Neuroc~tem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene
Expression, CRC Press, Boca Raton, E:L (1988)). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of the PLA2 polypeptide. Included in this
delivery is by gene therapy.
Another potential antagonist is a small molecule which binds to the PLA2
making it in~rceccible to binding molecules (e.g. substrates) such that normal
biological activity is prevented. Examples of small molecules include, but are not
limited to, small peptides or peptide-like molecules and organic compounds.
PLA2 are ubiquitous in the animal host and are responsible for many
biological functions, including many pathologies. Accordingly, it is desirous to find
compounds and drugs which can inhibit the function of a PLA2.
This invention additionally provides a method of treating an abnormal
condition related to an excess of PLA2 activity, such as BPH and various forrns of
cancer. including prostate cancer, which comprises ~lminict~ring to a subject the
inhibitor compounds (antagonists) as hereinabove described along with a
-10-
CA 02262624 1999-01-29
W 098/05349 PCTrUS97/13550
pharnl:-reutic~lly acceptable carrier in an amount effective to inhibit PLA2 activity
directly or by blocking binding of binding molecules to PLA ~ polypeptide.
Cor.ro~ilions and Kits
The compounds which inhibit such PLA2, may be employed in combination
with a suitable pharm~eutic ~l carrier. Such compositions comprise a therapeutically
effective amount of the polypeptide or compound, and a ph~rm~reutil~lly acceptable
carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should
suit the mode of ~flminictration
The invention further relates to pharrn~l~eutic~l packs and kits compricing one
or more containers filled with one or more of the ingredients of the aforementioned
compositions of the invention.
A-lminictration
Polypeptides and other compounds of the present invention may be employed
alone or in conjunction with other compounds, such as therapeutic compounds.
The pharm~eutic~l compositions may be administered in any effective,
convenient marmer including, for inct~nce administration by topical, oral, anal,vaginal, intravenous, intraperitoneal, intr~mnccul~r, subcut~neous, intranasal or
intradermal routes, among others.
The pharmaceutical compositions generally are administered in an amount
effective for treatment or prophylaxis of a specific indication or indications. In
general, the compositions are administered in an arnount of at least about 10 ,ug/kg
body weight. In most cases they will be ~rlminict~red in an amount not in excess of
about 8 mg/kg body weight per day. Preferably, in most cases, dose is from about 10
,ug/kg to about I mg/kg body weight, daily. It will be appreciated that op~
dosage will be determined by standard methods for each treatment modality and
indication, taking into account the indication, its severity, route of ~rlminictration,
complicating conditions and the like.
Vaccine
CA 02262624 l999-0l-29
W 098/05349 PCTAUS97/13550
Another aspec~ of the invention relates to a method for inducing an
immunological response in an animal, particularly in a m~mm~l, which comprises
inoculating the animal with PLA2, or a fragment or variant thereof, adequate to
produce antibody to protect said animal from BPH or various forms of cancer,
5 including prostate cancer. Yet another aspect of the invention relates to a method of
inducing immunological response in an animal which comprises, through gene
therapy, delivering gene encoding PLA2, or a fragment or a variant thereof, for
expressing PLA2, or a fragment or a variant thereof in vivo in order to induce an
immunological response to produce antibody to protect said animal from disease.
Further aspect of the invention relates to an immunological composition
which, when introduced into an animal, particularly m~mm~ n host, induces an
immunological response in that animal to a given PLA2 gene or protein coded
therefrom, wherein the composition comprises a recombinant PLA2 gene or protein
coded therefrom comprising DNA which codes for and expresses an antigen of said
PLA2 gene or protein coded therefrom.
The PLA2 or a fragment thereof may be fused with co-protein which may not
by itself produce antibodies, but is capable of stabilizing the first protein and
producing a fused protein which will have immunogenic and protective properties.Thus fused recombinant protein, preferably further comprises an antigenic co-
protein. such as Glutathione-S-transferase (GST) or beta-galactosidase, relatively
large co-proteins which solubilize the protein and facilitate production and
purification thereof. Moreover, the co-protein may act as an adjuvant in the sense of
providing a generalized stimulation of the immune system. The co-protein may be
attached to either the amino or carboxy terminus of the first protein.
The present invention also includes a vaccine formulation which comprises
the immunogenic recombinant protein together with a suitable carrier. Since the
protein may be broken down in the stomach, it is preferably administered
parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc.
injection). Formulations suitable for parenteral administration include aqueous and
non-aqueous sterile injection solutions which may contain anti-oxidants, buffers,
bacteriostats and solutes which render the formulation instonic with the blood of the
recipient: and aqueous and non-aqueous sterile suspensions which may include
-12-
CA 02262624 1999-01-29
W O 98/05349 PCT~US97/13550
suspending agents or thickening agents. The formulations may be presented in unit-
dose or multi-dose containers, for example, sealed ampoules and vials and may bestored in a freeze-dried condition requiring only the addition of the sterile liquid
carrier immediately prior to use. The vaccine formulation may also include adjuvant
systems for enhancing the immunogenicity of the forrnulation, such as oil-in water
systems and other systems known in the art. The dosage will depend on the specific
activity of the vaccine and can be readily determined by routine experimentation.
Whilst the invention has been described with reference to PLA2, it is to be
understood that this covers fragments of the naturally occurring protein and similar
.proteins (for example, having sequence homologies of 50% or greater) with
additions, deletions or substitutions which do not substantially affect the
immunogenic properties of the recombinant protein.
The present invention also provides a method for the production of
transgenic animals with altered PLA2 levels for the productions of animals bearing
PLA2 induced diseases. Transgenic, non-human, animals may be obtained by
transfecting appropriate fertilized eggs or embryos of a host with nucleic acidsencoding the PLA 7 disclosed herein, see for example U.S.Patents 4,736,866;
5,175,385; 5,175,384 and 5,175,386. The resultant transgenic animal may be used
as a model for the study of altered PLA2 levels. Particularly, useful transgenicanimals are those which display a detectable phenotype associated with the altered
expression of the PLA2 polypeptide. Drugs may then be screened for their ability to
reverse or exacerbate the relevant phenotype.
EXA~IPLES
The present invention is further described by the following ex:-mpl~. The
examples are provided solely to illustrate the invention by reference to specific
embodiments. These exemplification's, while illustrating certain specific aspects of
the invention, do not portray the limitations or circumscribe the scope of the disclosed
invention.
All examples are 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 examples can be carried out as
-13-
, .. .~ . . . .. .
CA 02262624 1999-01-29
W 098/05349 PCT~US97113550
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), herein referred to as "Sambrook."
All parts or amounts set out in the following examples are by weight, unless
otherwise specified.
Unless otherwise stated size separation of fragments in the exarnples below is
carried out using standard techniques of agarose and polyacrylamide gel
electrophoresis ("PAGE") in Sambrook and numerous other references such as, for
inct~nre, by Goeddel et al., Nucleic Acids Res. 8:4057 ( 1980).
F,Y~nlrle 1
mRNA is prepared from normal and cancerous prostate tissue, size fractionated on an
agarose gel and then transferred to a nylon membrane (northem blot). This blot is
then hybridized to a PLA~ specific cDNA probe that has been labeled with radioactive
lS [32P]-dCTP. Following incubation, the blot is then washed under stringent conditions
(described in Sambrook) and then exposed to film. Changes in mRNA levels as a
function of disease will be seen as a change in intensity of the signal seen in diseased
versus normal tissue.
F~Y~n1PIe 2
Below are the results from the PLA2 levels in serum as found by an ELISA assay.
A. Normal healthy men without prostate disease ( 17 individuals): 4.85
ng/ml + 3.29
B. Normal healthy women (20 individuals): 5.31 ng/ml + 3.44
C. Combined normal healthy men and women (37 individuals): 5.11 ng/ml
30 + 3.33
D. Patients with progressive prostate cancer (10 individuals): 32.65 ng/ml
+ 18.7
Eight (80%) of ten patients with progressive prostate cancer had PLA~
-14-
CA 02262624 1999-01-29
W 098/05349 PCTAUS97/13550
blood levels at least three standard deviations above the combined mean
of normal healthy men and women.
E. Patients with prostate cancer in remission ( 10 individuals) 7.77 ng/ml
5 + 8.67
Only one of nine (I 1.1%) patients with prostate cancer in remission had
PLA ~ blood levels at least three standard deviations above the
combined
mean of normal healthy men and women.
F. Patients with stabilized prostate cancer (S individuals): 8.74 ng/ml +
8.12
Only one of five patients (20%) with stable prostate cancer had PLA2
blood levels at least three standard deviations above the combined
mean of normal healthy men and women.
G. Patients with benign prostate hypertrophy (13 individuals): 4.59 ng/ml
+ 3.29
No patients with benign prostate hypertrophy had PLA2 blood levels at
least three standard deviations above the combined mean of norrnal
healthy men and women.
H. Patients with prostatitis (7 individuals): 8.47 ng/ml + 9.73
Only one of seven ( 14.3%) patients with prostatitis had PLA2 blood levels at
least three standard deviations above the combined mean of normal healthy
men and women.
...... . . .... .. ... . . . . . .
