U.S. patent number RE43,586 [Application Number 12/283,636] was granted by the patent office on 2012-08-14 for antibodies to prostate-specific membrane antigen.
This patent grant is currently assigned to Sloan-Kettering Institute for Cancer Research. Invention is credited to Maryann Fair, legal representative, William R. Fair, Warren D. W. Heston, Ron S. Israeli.
United States Patent |
RE43,586 |
Israeli , et al. |
August 14, 2012 |
Antibodies to prostate-specific membrane antigen
Abstract
This invention provides purified antibodies to the outer
membrane domain of prostate-specific membrane (PSM) antigen,
compositions of matter comprising PSM antigen antibodies conjugated
to a radioisotope or a toxin, and a method of imaging prostate
cancer by using PSM antigen antibodies.
Inventors: |
Israeli; Ron S. (Staten Island,
NY), Heston; Warren D. W. (Cleveland, OH), Fair; William
R. (New York, NY), Fair, legal representative; Maryann
(Longboat, FL) |
Assignee: |
Sloan-Kettering Institute for
Cancer Research (New York, NY)
|
Family
ID: |
36780204 |
Appl.
No.: |
12/283,636 |
Filed: |
September 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08403803 |
Mar 17, 1995 |
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PCT/US93/10624 |
Nov 5, 1993 |
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07973337 |
Nov 5, 1992 |
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Reissue of: |
08470735 |
Jun 6, 1995 |
7105159 |
Sep 12, 2006 |
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Current U.S.
Class: |
530/388.1;
530/388.8 |
Current CPC
Class: |
C07K
14/705 (20130101); C07K 14/4748 (20130101); C07K
16/3069 (20130101); G01N 2800/342 (20130101) |
Current International
Class: |
C07K
16/00 (20060101) |
References Cited
[Referenced By]
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0 173 951 |
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Dec 1986 |
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EP |
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0173951 |
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EP |
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May 2003 |
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EP |
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Oct 2006 |
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EP |
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WO 93/17715 |
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Sep 1993 |
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WO |
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WO 94/09820 |
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May 1994 |
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WO |
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WO 95/04548 |
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Feb 1995 |
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WO |
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WO 96/08570 |
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WO |
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WO 96/26272 |
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WO |
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WO |
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WO |
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WO |
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WO |
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Jul 2000 |
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WO |
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WO 01/009192 |
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WO |
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WO |
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WO |
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WO 02/096460 |
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WO |
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WO 03/064606 |
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WO |
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WO 2004/098535 |
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Nov 2004 |
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WO |
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WO 2006/125481 |
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Nov 2006 |
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WO |
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|
Primary Examiner: Yu; Misook
Assistant Examiner: Halvorson; Mark
Attorney, Agent or Firm: White; John P. Cooper & Dunham
LLP
Government Interests
This invention disclosed herein was made in part with Government
support under NIH Grants No. DK47650 and CA58192 from the
Department of Health and Human Services. Accordingly, the U.S.
Government has certain rights in this invention.
Parent Case Text
This application is a continuation application of U.S. Ser. No.
08/403,803, filed (Mar. 17, 1995, .Iadd.now abandoned .Iaddend.and
a continuation of PCT International Application No. PCT/US93/10624,
filed Nov. 5, 1993; which is a continuation-in-part of U.S. Ser.
No. 07/973,337, filed Nov. 5, 1992, now abandoned the contents of
which are hereby incorporated by reference.
Claims
What is claimed is:
1. A purified monoclonal antibody which binds to a fragment of an
outer membrane domain of prostate specific membrane antigen, which
fragment has within its structure the consecutive amino acid
sequence Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35).
2. A purified monoclonal antibody which binds to a fragment of an
outer membrane domain of prostate specific membrane antigen, which
fragment has within its structure the consecutive amino acid
sequence Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO: 36).
3. A purified monoclonal antibody which binds to a fragment of an
outer membrane domain of prostate specific membrane antigen, which
fragment has within its structure the consecutive amino acid
sequence Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37).
.[.4. A purified monoclonal antibody which binds to a fragment of
an outer membrane domain of prostate specific membrane antigen,
which fragment has within its structure each of the following amino
acid sequences: (a) Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35); (b)
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO: 36); (c)
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37); and (d)
Ala-Gly-Ala-Leu-Val-Leu-Ala-Gly-Gly-Phe-Phe-Leu-Leu-Gly-Phe-Leu-Phe
(SEQ ID 80:38)..].
5. A purified monoclonal antibody which binds to a fragment of an
outer membrane domain of prostate specific membrane antigen, which
fragment has within its structure each of the following amino acid
sequences: (a) Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35); (b)
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO: 36); and (c)
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37).
6. A purified monoclonal antibody which binds to a fragment of
prostate specific membrane antigen, which fragment corresponds to a
hydrophilic region of an outer membrane domain of prostate specific
membrane antigen, the amino acid sequence of which antigen is set
forth in SEQ ID NO:2.
7. A purified monoclonal antibody which binds to a hydrophilic
region of an outer membrane domain of prostate specific membrane
antigen, which hydrophilic region has within its structure the
consecutive amino acid sequence Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO:
35).
8. A purified monoclonal antibody which binds to a hydrophilic
region of an outer membrane domain of prostate specific membrane
antigen, which hydrophilic region has within its structure the
consecutive amino acid sequence Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO:
36).
9. A purified monoclonal antibody which binds to a hydrophilic
region of an outer membrane domain of prostate specific membrane
antigen, which hydrophilic region has within its structure the
consecutive amino acid sequence Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO:
37).
10. A purified monoclonal antibody which binds to an outer membrane
domain of prostate specific membrane antigen, the amino acid
sequence of which antigen is set forth in SEQ ID NO:2.
11. A purified monoclonal antibody which binds to a hydrophilic
region of an outer membrane domain of prostate specific membrane
antigen, the amino acid sequence of which antigen is set forth in
SEQ ID NO:2.
.[.12. The purified antibody of any one of claims 1-11, wherein the
antibody is a monoclonal antibody..].
13. A composition of matter comprising .[.the.]. .Iadd.a
.Iaddend.monoclonal antibody .[.of any one of 1-11.]. and an agent
conjugated .[.to the monoclonal antibody.]. .Iadd.thereto, wherein
the monoclonal antibody binds to an outer membrane domain of
prostate-specific membrane antigen, the amino acid sequence of
which antigen is set forth in SEQ ID NO:2.Iaddend..
14. The composition of matter of claim 13, wherein the agent is a
radioisotope or toxin.
15. A composition comprising a carrier and the composition of
matter of claim 13.
16. A method of imaging prostate cancer in a subject which
comprises administering to the subject the composition of matter of
claim 13, wherein the agent is an imaging agent under conditions
permitting formation of a complex between the composition of matter
and prostate specific membrane antigen, and obtaining an image of
any complex so formed.
17. A monoclonal antibody having an antigen-binding region
-specific for the extracellular domain of prostate specific
membrane antigen, the amino acid sequence of which antigen is set
forth in SEQ ID NO:2.
.Iadd.18. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a fragment of an outer membrane domain of
prostate-specific membrane antigen, which fragment has within its
structure the consecutive amino acid sequence
Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO:35)..Iaddend.
.Iadd.19. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a fragment of an outer membrane domain of
prostate-specific membrane antigen, which fragment has within its
structure the consecutive amino acid sequence
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO:36)..Iaddend.
.Iadd.20. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a fragment of an outer membrane domain of
prostate-specific membrane antigen, which fragment has within its
structure the consecutive amino acid sequence
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO:37)..Iaddend.
.Iadd.21. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a fragment of an outer membrane domain of
prostate-specific membrane antigen, which fragment has within its
structure each of the following amino acid sequences: (a)
Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35); (b)
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO: 36); and (c)
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37)..Iaddend.
.Iadd.22. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a fragment of prostate-specific membrane antigen, which
fragment corresponds to a hydrophilic region of an outer membrane
domain of prostate-specific membrane antigen, the amino acid
sequence of which antigen is set forth in SEQ ID NO:2..Iaddend.
.Iadd.23. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a hydrophilic region of an outer membrane domain of
prostate-specific membrane antigen, which hydrophilic region has
within its structure the consecutive amino acid sequence
Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO:35)..Iaddend.
.Iadd.24. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a hydrophilic region of an outer membrane domain of
prostate-specific membrane antigen, which hydrophilic region has
within its structure the consecutive amino acid sequence
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO:36)..Iaddend.
.Iadd.25. A composition of comprising a monoclonal antibody and an
agent conjugated thereto, wherein the monoclonal antibody binds to
a hydrophilic region of an outer membrane domain of
prostate-specific membrane antigen, which hydrophilic region has
within its structure the consecutive amino acid sequence
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO:37)..Iaddend.
.Iadd.26. A composition of matter comprising a monoclonal antibody
and an agent conjugated thereto, wherein the monoclonal antibody
binds to a hydrophilic region of an outer membrane domain of
prostate specific membrane antigen, the amino acid sequence of
which antigen is set forth in SEQ ID NO:2..Iaddend.
.Iadd.27. The composition of matter of any of claim 18-20, or
21-26, wherein the agent is a radioisotope or toxin..Iaddend.
.Iadd.28. A composition comprising a carrier and the composition of
matter of any of claim 18-20 or 21-26..Iaddend.
.Iadd.29. A method of imaging prostate cancer in a subject which
comprises administering to the subject the composition of matter of
any of claim 18-20 or 21-26, wherein the agent is an imaging agent,
under conditions permitting formation of a complex between the
composition of matter and prostate-specific membrane antigen, and
obtaining an image of any complex so formed..Iaddend.
.Iadd.30. A composition comprising a carrier and the composition of
matter of claim 27..Iaddend.
.Iadd.31. The composition of claim 30, wherein the agent is a
radioisotope..Iaddend.
.Iadd.32. The composition of claim 30, wherein the agent is a
toxin..Iaddend.
.Iadd.33. The method of claim 29, wherein the imaging agent is a
radioisotope..Iaddend.
Description
BACKGROUND OF THE INVENTION
Throughout this application various references are referred to
within parentheses. Disclosures of these publications in their
entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains. Full bibliographic citation for these
references may be found at the end of of each series of
experiments.
Prostate cancer is among the most significant medical problems in
the United States, as the disease is now the most common malignancy
diagnosed in American males. In 1992 there were over 132,000 new
cases of prostate cancer detected with over 36,000 deaths
attributable to the disease, representing a 17.3% increase over 4
years (2). Five year survival rates for patients with prostate
cancer range from 88% for those with localized disease to 29% for
those with metastatic disease. The rapid increase in the number of
cases appears to result in part from an increase in disease
awareness as well as the widespread use of clinical markers such as
the secreted proteins prostate-specific antigen (PSA) and prostatic
acid phosphatase (PAP) (37).
The prostate gland is a site of significant pathology affected by
conditions such as benign growth (BPH), neoplasia (prostatic
cancer) and infection (prostatitis). Prostate cancer represents the
second leading cause of death from cancer in man (1). However
prostatic cancer is the leading site for cancer development in men.
The difference between these two facts relates to prostatic cancer
occurring with increasing frequency as men age, especially in the
ages beyond 60 at a time when death from other factors often
intervenes. Also, the spectrum of biologic aggressiveness of
prostatic cancer is great, so that in some men following detection
the tumor remains a latent histologic tumor and does not become
clinically significant, whereas in other it progresses rapidly,
metastasizes and kills the man in a relatively short 2-5 year
period (1, 3).
In prostate cancer cells, two specific proteins that are made in
very high concentrations are prostatic acid phosphatase (PAP) and
prostate specific antigen (PSA) (4, 5, 6). These proteins have been
characterized and have been used to follow response to therapy.
With the development of cancer, the normal architecture of the
gland becomes altered, including loss of the normal duct structure
for the removal of secretions and thus the secretions reach the
serum. Indeed measurement of serum PSA is suggested as a potential
screening method for prostatic cancer. Indeed, the relative amount
of PSA and/or PAP in the cancer reduces as compared to normal or
benign tissue.
PAP was one of the earliest serum markers for detecting metastatic
spread (4). PAP hydrolyses tyrosine phosphate and has a broad
substrate specificity. Tyrosine phosphorylation is often increased
with oncogenic transformation. It has been hypothesized that during
neoplastic transformation there is less phosphatase activity
available to inactivate proteins that are activated by
phosphorylation on tyrosine residues. In some instances, insertion
of phosphatases that have tyrosine phosphatase activity has
reversed the malignant phenotype.
PSA is a protease and it is not readily appreciated how loss of its
activity correlates with cancer development (5, 6). The proteolytic
activity of PSA is inhibited by zinc. Zinc concentrations are high
in the normal prostate and reduced in prostatic cancer. Possibly
the loss of zinc allows for increased proteolytic activity by PSA.
As proteases are involved in metastasis and some proteases
stimulate mitotic activity, the potentially increased activity of
PSA could be hypothesized to play a role in the tumors metastases
and spread (7).
Both PSA and PAP are found in prostatic secretions. Both appear to
be dependent on the presence of androgens for their production and
are substantially reduced following androgen deprivation.
Prostate-specific membrane antigen (PSM) which appears to be
localized to the prostatic membrane has been identified. This
antigen was identified as the result of generating monoclonal
antibodies to a prostatic cancer cell, LNCaP (8).
Dr. Horoszewicz established a cell line designated LNCaP from the
lymph node of a hormone refractory, heavily pretreated patient (9).
This line was found to have an aneuploid human male karyotype. It
maintained prostatic differentiation functionality in that it
produced both PSA and PAP. It possessed an androgen receptor of
high affinity and specificity. Mice were immunized with LNCaP cells
and hybridomas were derived from sensitized animals. A monoclonal
antibody was derived and was designated 7E11-C5 (8). The antibody
staining was consistent with a membrane location and isolated
fractions of LNCaP cell membranes exhibited a strongly positive
reaction with immunoblotting and ELISA techniques. This antibody
did not inhibit or enhance the growth of LNCaP cells in vitro or in
vivo. The antibody to this antigen was remarkably specific to
prostatic epithelial cells, as no reactivity was observed in any
other component. Immunohistochemical staining of cancerous
epithelial cells was more intense than that of normal or benign
epithelial cells.
Dr. Horoszewicz also reported detection of immunoreactive material
using 7E11-C5 in serum of prostatic cancer patients (8). The
immunoreactivity was detectable in nearly 60% of patients with
stage D-2 disease and in a slightly lower percentage of patients
with earlier stage disease, but the numbers of patients in the
latter group are small. Patients with benign prostatic hyperplasia
(BPH) were negative. Patients with no apparent disease were
negative, but 50-60% of patients in remission yet with active
stable disease or with progression demonstrated positive serum
reactivity. Patients with non prostatic tumors did not show
immunoreactivity with 7E11-C5.
The 7E11-CS monoclonal antibody is currently in clinical trials.
The aldehyde groups of the antibody were oxidized and the
linker-chelator glycol-tyrosyl- (n,
.epsilon.-diethylenetriamine-pentacetic acid)-lysine (GYK-DTPA) was
coupled to the reactive aldehydes of the heavy chain (10). The
resulting antibody was designated CYT-356. Immunohistochemical
staining patterns were similar except that the CYT-356 modified
antibody stained skeletal muscle. The comparison of CYT-356 with
7E11-CS monoclonal antibody suggested both had binding to type 2
muscle fibers. The reason for the discrepancy with the earlier
study, which reported skeletal muscle to be negative, was suggested
to be due to differences in tissue fixation techniques. Still, the
most intense and definite reaction was observed with prostatic
epithelial cells, especially cancerous cells. Reactivity with mouse
skeletal muscle was detected with immunohistochemistry but not in
imaging studies. The Indium.sup.111-labeled antibody localized to
LNCaP tumors grown in nude mice with an uptake of nearly 30% of the
injected dose per gram tumor at four days. In-vivo, no selective
retention of the antibody was observed in antigen negative tumors
such as PC-3 and DU-145, or by skeletal muscle.
Very little was known about the PSM antigen. An effort at
purification and characterization has been described at meetings by
Dr. George Wright and colleagues (11, 12). These investigators have
shown that following electrophoresis on acrylamide gels and Western
blotting, the PSM antigen maintains a molecular weight of 100
kilodaltons (kd). Chemical and enzymatic treatment showed that both
the peptide and carbohydrate moieties of the PSM antigen are
required for recognition by the 7E11-C5 monoclonal antibody.
Competitive binding studies with specific lectins suggested that
galNAc is the dominant carbohydrate of the antigenic epitope.
The 100 kd glycoprotein unique to prostate cells and tissues was
purified and characterized. The protein was digested
proteolytically with trypsin and nine peptide fragments were
sequenced. Using the technique of degenerate PCR (polymerase chain
reaction), the full-length 2.65 kilobase (kb) cDNA coding for this
antigen was cloned. Preliminary results have revealed that this
antigen is highly expressed in prostate cancer tissues, including
bone and lymph node metastases (13). The entire DNA sequence for
the cDNA as well as the predicted amino acid sequence for the
antigen was determined. Further characterization of the PSM antigen
is presently underway in the applicants' laboratory including:
analysis of PSM gene expression in a wide variety of tissues,
transfection of the PSM gene into cells not expressing the antigen,
chromosome localization of the PSM gene, cloning of the genomic PSM
gene with analysis of the PSM promoter and generation of polyclonal
and monoclonal antibodies against highly antigenic peptide domains
of the PSM antigen, and identification of any endogenous PSM
binding molecules (ligands).
Currently, LNCaP cells provide the best in-vitro model system to
study human prostate cancer, since they produce all three prostatic
bio-markers; PSA, PAP and PSM. The cells possess an aneuploid male
karyotype with a Y chromosome, express a high affinity androgen
receptor, and are hormonally responsive to both testosterone and
DHT. Because PSM appears to be a transmembrane glycoprotein, it is
considered an attractive target for both antibody-directed imaging
and targeting of prostatic tumor deposits (38). We have
demonstrated expression of PSM protein in LNCAP cell membranes and
in PC-3 cells transfected with PSM cDNA and also the
characterization of PSM mRNA expression in human tissues, and in
response to steroid hormones.
BRIEF DESCRIPTION OF FIGURES
FIG. 1: Signal in lane 2 represent the 100 kD PSM antigen. The EGFr
was used as the positive control and is shown in lane 1. Incubation
with rabbit antimouse (RAM) antibody alone served as negative
control and is shown in lane 3.
FIG. 2 A-D: Upper two photos show LNCaP cytospins staining
positively for PSM antigen. Lower left in DU-145 and lower right is
PC-3 cytospin, both negative for PSM antigen expression.
FIG. 3 A-D: Upper two panels are human prostate sections (BPH)
staining positively for PSM antigen. The lower two panels show
invasive prostate carcinoma human sections staining positively for
expression of the PSM antigen.
FIG. 4: 100 kD PSM antigen following immunoprecipitation of
.sup.35S-Methionine labelled LNCaP cells with Cyt-356 antibody.
FIG. 5: 3% agarose gels stained with Ethidium bromide revealing PCR
products obtained using the degenerate PSM antigen primers. The
arrow points to sample IN-20, which is a 1.1 kb PCR product which
we later confirmed to be a partial cDNA coding for the PSM
gene.
FIG. 6 A-B: 2% agarose gels of plasmid DNA resulting from TA
cloning of PCR products. Inserts are excised from the PCR II vector
(Invitrogen Corp.) by digestion with EcoRI. 1.1 kb PSM gene partial
cDNA product is shown in lane 3 of gel 1.
FIG. 7: Autoradiogram showing size of cDNA represented in
applicants' LNCaP library using M-MLV reverse transcriptase.
FIG. 8: Restriction analysis of full-length clones of PSM gene
obtained after screening cDNA library. Samples have been cut with
Not I and Sal I restriction enzymes to liberate the insert.
FIG. 9: Plasmid Southern autoradiogram of full length PSM gene
clones. Size is approximately 2.7 kb.
FIG. 10: Northern blot revealing PSM expression limited to LNCaP
prostate cancer line and H26 Ras-transfected LNCaP cell line. PC-3,
DU-145, T-24, SKRC-27, HELA, MCF-7, HL-60, and others were are all
negative.
FIG. 11: Autoradiogram of Northern analysis revealing expression of
2.8 kb PSM message unique to the LNCaP cell line (lane 1), and
absent from the DU-145 (lane 2) and PC-3 cell lines (lane 3). RNA
size ladder is shown on the left (kb), and 28S and 18S ribosomal
RNA bands are indicated on the right.
FIG. 12 A-B: Results of PCR of human prostate tissues using PSM
gene primers. Lanes are numbered from left to right. Lane 1, LNCaP;
Lane 2, H26; Lane 3, DU-145; Lane 4, Normal Prostate; Lane 5, BPH;
Lane 6, Prostate Cancer; Lane 7, BPH; Lane 8, Normal; Lane 9, BPH;
Lane 10, BPH; Lane 11, BPH; Lane 12, Normal; Lane 13, Normal; Lane
14, Cancer; Lane 15, Cancer; Lane 16, Cancer, Lane 17, Normal; Lane
13, Cancer; Lane 19, IN-20 Control; Lane 20, PSM cDNA
FIG. 13: Isoelectric point of PSM antigen (non-glycosylated)
FIG. 14; 1-8 Secondary structure of antigen (panels 14-4 to 14-H:
SEQ ID NO:2)
FIG. 15:A-B A. Hydrophilicity plot of PSM antigen B. Prediction of
membrane spanning segments (SEQ ID NOS: 35-37).
FIG. 16:1-11 Homology of PSMA antigen (SEQ ID NO:1) with chicken
(SEQ ID NO:27), rat (SEQ ID NO:28) and human (SEQ ID NO:29)
transferrin receptor sequence.
FIG. 17A-C: Immunohistochemical detection of PSM antigen expression
in prostate cell lines. Top panel reveals uniformly high level of
expression in LNCaP cells; middle panel and lower panel are DU-145
and PC-3 cells respectively, both negative.
FIG. 18: Autoradiogram of protein gel revealing products of PSM
coupled in-vitro transcription/translation. Non-glycosylated PSM
polypeptide is seen at 84 kDa (lane 1) and PSM glycoprotein
synthesized following the addition of microsomes is seen at 100 kDa
(lane 2).
FIG. 19: Western Blot analysis detecting PSM expression in
transfected non-PSM expressing PC-3 cells. 100 kDa PSM glycoprotein
species is clearly seen in LNCaP membranes (lane 1), LNCaP crude
lysate (lane 2), and PSM-transfected PC-3 cells (lane 4), but is
undetectable in native PC-3 cells (lane 3).
FIG. 20: Autoradiogram of ribonuclease protection gel assaying for
PSM mRNA expression in normal human tissues. Radiolabeled 1 kb DNA
ladder (Gibco-BRL) is shown in lane 1. Undigested probe is 400
nucleotides (lane 2), expected protected PSM band-is 350
nucleotides, and tRNA control is shown (lane 3). A strong signal is
seen in human prostate (lane 11), with very faint, but detectable
signals seen in human brain (lane 4) and human salivary gland (lane
12).
FIG. 21: Autoradiogram of ribonuclease protection gel assaying for
PSM mRNA expression in LNCaP tumors grown in nude mice, and in
human prostatic tissues. .sup.32P-labeled 1 kb DNA ladder is shown
in lane 1. 298 nucleotide undigested probe is shown (lane 2), and
tRNA control is shown (lane 3). PSM mRNA expression is clearly
detectable in LNCaP cells (lane 4), orthotopically grown LNCaP
tumors in nude mice with and without matrigel (lanes 5 and 6), and
subcutaneously implanted and grown LNCaP tumors in nude mice (lane
7). PSM mRNA expression is also seen in normal human prostate (lane
8), and in a moderately differentiated human prostatic
adenocarcinoma (lane 10). Very faint expression is seen in a sample
of human prostate tissue with benign hyperplasia (lane 9).
FIG. 22: Ribonuclease protection assay for PSM expression in LNCaP
cells treated with physiologic doses of various steroids for 24
hours. .sup.32-Plabeled DNA ladder is shown in lane 1. 298
nucleotide undigested probe is shown (lane 2), and tRNA control is
shown (lane 3). PSM mRNA expression is highest in untreated LNCaP
cells in charcoal-stripped media (lane 4). Applicant see
significantly diminished PSM expression in LNCaP cells treated with
DHT (lane 5) Testosterone (lane 6), Estradiol (lane 7), and
Progesterone (lane 8), with little response to Dexamethasone (lane
9).
FIG. 23: Data illustrating results of PSM DNA and RNA presence in
transfect Dunning cell lines employing Southern and Northern
blotting techniques
FIG. 24:A-B Figure A indicates the power of cytokine transfected
cells to teach unmodified cells. Administration was directed to the
parental flank or prostate cells. The results indicate the
microenvironment considerations. Figure B indicates actual potency
at a particular site. The tumor was implanted in prostate cells and
treated with immune cells at two different sites.
FIG. 25:A-B Relates potency of cytokines in inhibiting growth of
primary tumors. Animals administered un-modified parental tumor
cells and administered as a vaccine transfected cells. Following
prostatectomy of rodent tumor results in survival increase.
FIG. 26: PCR amplification with nested primers improved our level
of detection of prostatic cells from approximately one prostatic
cell per 10,000 MCF-7 cells to better than one cell per million
MCF-7 cells, using either PSA.
FIG. 27: PCR amplification with nested primers improved our level
of detection of prostatic cells from approximately one prostatic
cell per 10,000 MCF-7 cells to better than one cell per million
MCF-7 cells, using PSM-derived primers.
FIG. 28: A representative ethidium stained gel photograph for
PSM-PCR. Samples run in lane A represent PCR products generated
from the outer primers and samples in lanes labeled B are products
of inner primer pairs.
FIG. 29: PSM Southern blot autoradiograph. The sensitivity of the
Southern blot analysis exceeded that of ethidium staining, as can
be seen in several samples where the outer product is not visible
on FIG. 3 A-D, but is detectable by Southern blotting as shown in
FIG. 4.
FIG. 30: Characteristics of the 16 patients analyzed with respect
to their clinical stage, treatment, serum PSA and PAP values, and
results of assay.
SUMMARY OF THE INVENTION
This invention provides an isolated mammalian nucleic acid molecule
encoding a mammalian prostate-specific membrane (PSM) antigen. The
isolated mammalian nucleic acid may be DNA, cDNA or RNA.
This invention also provides nucleic acid molecule comprising a
nucleic acid molecule of at least 15 nucleotides capable of
specifically hybridizing with a sequence included within the
sequence of a nucleic acid molecule encoding the PSM antigen. The
nucleic acid molecule may-either be DNA or RNA.
This invention provides nucleic acid molecule of at least 15
nucleotides capable of specifically hybridizing with a sequence of
a nucleic acid molecule which is complementary to the nucleic acid
molecule encoding a mammalian prostate-specific membrane
antigen.
This invention further provides a method of detecting expression of
the PSM antigen which comprises obtaining total mRNA from the cell
and contacting the mRNA so obtained with a labelled PSM antigen
specific nucleic acid molecule under hybridizing conditions,
determining the presence of mRNA hybridized to the probe, and
thereby detecting the expression of the PSM antigen by the cell.
The PSM antigen in tissue sections may be similarly detected.
This invention provides isolated nucleic acid molecule of PSM
antigen operatively linked to a promoter of RNA transcription. This
invention further provides a vector which comprises an isolated
mammalian nucleic acid molecule of PSM antigen.
This invention further provides a host vector system for the
production of a polypeptide having the biological activity of a
mammalian PSM antigen which comprises the vector comprising the
mammalian nucleic acid molecule encoding a mammalian PSM antigen
and a suitable host. The suitable host for the expression of PSM
antigen may be a bacterial cell, insect cell, or mammalian
cell.
This invention also provides a method of producing a polypeptide
having the biological activity of a mammalian PSM antigen which
comprises growing the host cell of vector system having a vector
comprising the isolated mammalian nucleic acid molecule encoding a
mammalian PSM antigen and a suitable host under suitable conditions
permitting production of the polypeptide and recovery of the
polypeptide so produced.
This invention provides a method for determining whether a ligand
can bind to a mammalian PSM antigen which comprises contacting a
mammalian cell having an isolated mammalian DNA molecule encoding a
mammalian PSM antigen with the ligand under conditions permitting
binding of ligands to the mammalian PSM antigen, and determining
whether the ligand binds to a mammalian PSM antigen. This invention
further provides ligands which bind to PSM antigen.
This invention provides purified mammalian PSM antigen. This
invention also provides a polypeptide encoded by the isolated
mammalian nucleic acid molecule encoding a mammalian PSM antigen.
This invention further provides a method to identify and purify
ligands of mammalian PSM antigen.
This invention further provides a method to produce both polyclonal
and monoclonal antibody using purified PSM antigens or polypeptides
encoded by an isolated mammalian nucleic acid molecule encoding a
mammalian PSM antigen.
This invention provides polyclonal and monoclonal antibody most
likely but not limited to directed either to peptide
Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No. 35), or Asn-Glu-Asp-Gly-Asn-Glu
(SEQ ID No. 36) or Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID No. 37) of the
PSM antigen.
This invention provides a therapeutic agent comprising an antibody
directed against a mammalian PSM antigen and a cytotoxic agent
conjugated thereto.
This invention also provides a method of imaging prostate cancer in
human patients which comprises administering to the patient at
least one antibody directed against PSM antigen, capable of binding
to the cell surface of the prostate cancer cell and labeled with an
imaging agent under conditions so as to form a complex between the
monoclonal antibody and the cell surface PSM antigen. This
invention further provides a composition comprising an effective
imaging amount of the antibody directed against PSM antigen and a
pharmaceutically acceptable carrier.
This invention further provides a method of imaging prostate cancer
in human patients which comprises administering to the patient
multiple antibodies directed towards different PSM epitopes.
The invention also provides a method of imaging prostate cancer in
human patients which comprises administering to the patient at
least one ligand, capable of binding to the cell surface of the
prostate cancer cell and labelled with an imaging agent under
conditions so as to form a complex between the ligand and the cell
surface PSM antigen. This invention further provides a composition
comprising an effective imaging amount of PSM antigen and a
pharmaceutically acceptable carrier.
This invention provides an immunoassay for measuring the amount of
the PSM antigen in a biological sample, e.g. serum, comprising
steps of a) contacting the biological sample with at least one PSM
antibody to form a complex with said antibody and the PSM antigen,
and b) measuring the amount of PSM antigen in said biological
sample by measuring the amount of said complex.
This invention also provides an immunoassay for measuring the
amount of the PSM antigen in a biological sample comprising steps
of a) contacting the biological sample with at least one PSM ligand
to form a complex with said ligand and the PSM antigen, and b)
measuring the amount of the PSM antigen in said biological sample
by measuring the amount of said complex.
This invention provides a method to purify mammalian PSM antigen
comprising steps of a) coupling the antibody directed against PSM
antigen to a solid matrix; b) incubating the coupled antibody of a)
with a cell lysate containing PSM antigen under the condition
permitting binding of the antibody and PSM antigen c) washing the
coupled solid matrix to eliminate impurities and d) eluting the PSM
antigen from the bound antibody.
This invention further provides transgenic nonhuman mammals which
comprises an isolated nucleic acid molecule of PSM antigen. This
invention also provides a transgenic nonhuman mammal whose genome
comprises antisense DNA complementary to DNA encoding a mammalian
PSM antigen so placed as to be transcribed into antisense mRNA
complementary to mRNA encoding the PSM antigen and which hybridizes
to mRNA encoding the PSM antigen thereby reducing its
translation.
This invention provides a method of suppressing or modulating
metastatic ability of prostate tumor cells, prostate tumor growth
or elimination of prostate tumor cells comprising introducing a DNA
molecule encoding a prostate specific membrane antigen operatively
linked to a 5' regulatory element into a tumor cell of a subject,
in a way that expression of the prostate specific membrane antigen
is under the control of the regulatory element, thereby suppressing
or modulating metastatic ability of prostate tumor cells, prostate
tumor growth or elimination of prostate tumor cells.
This invention provides a method of suppressing or modulating
metastatic ability of prostate tumor cells, prostate tumor growth
or elimination of prostate tumor cells, comprising introducing a
DNA molecule encoding a prostate specific membrane antigen
operatively linked to a 5' regulatory element coupled with a
therapeutic DNA into a tumor cell of a subject, thereby suppressing
or modulating metastatic ability of prostate tumor cells, prostate
tumor growth or elimination of prostate tumor cells.
This invention provides a therapeutic vaccine for preventing human
prostate tumor growth or stimulation of prostate tumor cells in a
subject, comprising administering an effective amount to the
prostate cell, and a pharmaceutical acceptable carrier, thereby
preventing the tumor growth or stimulation of tumor cells in the
subject.
This invention provides a method of detecting hematogenous
micrometastic tumor cells of a subject, comprising (A) performing
nested polymerase chain reaction (PCR) on blood, bone marrow, or
lymph node samples of the subject using the prostate specific
membrane antigen primers, and (B) verifying micrometastases by DNA
sequencing and Southern analysis, thereby detecting hematogenous
micrometastic tumor cells of the subject.
This invention provides a method of abrogating the mitogenic
response due to transferrin, comprising introducing a DNA molecule
encoding prostate specific membrane antigen operatively linked to a
5' regulatory element into a tumor cell, the expression of which
gene is directly associated with a defined pathological effect
within a multicellular organism, thereby abrogating mitogen
response due to transferrin.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this application, references to specific nucleotides are
to nucleotides present on the coding strand of the nucleic acid.
The following standard abbreviations are used throughout the
specification to indicate specific nucleotides: C=cytosine
A=adenosine T=thymidine G=guanosine A "gene" means a nucleic acid
molecule, the sequence of which includes all the information
required for the normal regulated production of a particular
protein, including the structural coding sequence, promoters and
enhancers.
This invention provides an isolated mammalian nucleic acid encoding
a mammalian prostate-specific membrane (PSM) antigen.
This invention further provides an isolated mammalian DNA molecule
of an isolated mammalian nucleic acid molecule encoding a mammalian
prostate-specific membrane antigen. This invention also provides an
isolated mammalian cDNA molecule encoding a mammalian
prostate-specific membrane antigen. This invention provides an
isolated mammalian RNA molecule encoding a mammalian
prostate-specific membrane antigen.
In the preferred embodiment of this invention, the isolated nucleic
sequence is cDNA from human as shown in sequence ID number 1. This
human sequence was submitted to-GenBank (Los Alamos National
Laboratory, Los Alamos, N.M.) with Accession Number, M99487 and the
description as PSM, Homo sapiens, 2653 base-pairs.
This invention also encompasses DNAs and cDNAs which encode amino
acid sequences which differ from those of PSM antigen, but which
should not produce phenotypic changes. Alternatively, this
invention also encompasses DNAs and cDNAs which hybridize to the
DNA and cDNA of the subject invention. Hybridization methods are
well known to those of skill in the art.
The DNA molecules of the subject invention also include DNA
molecules coding for polypeptide analogs, fragments or derivatives
of antigenic polypeptides which differ from naturally-occurring
forms in terms of the identity or location of one or more amino
acid residues (deletion analogs containing less than all of the
residues specified for the protein, substitution analogs wherein
one or more residues specified are replaced by other residues and
addition analogs where in one or more amino acid residues is added
to a terminal or medial portion of the polypeptides) and which
share some or all properties of naturally-occurring forms. These
molecules include: the incorporation of codons "preferred" for
expression by selected non-mammalian hosts; the provision of sites
for cleavage by restriction endonuclease enzymes; and the provision
of additional initial, terminal or intermediate DNA sequences that
facilitate construction of readily expressed vectors.
The DNA molecules described and claimed herein are useful for the
information which they provide concerning the amino acid sequence
of the polypeptide and as products for the large scale synthesis of
the polypeptide by a variety of recombinant techniques. The
molecule is useful for generating new cloning and expression
vectors, transformed and transfected prokaryotic and eukaryotic
host cells, and new and useful methods for cultured growth of such
host cells capable of expression of the polypeptide and related
products.
Moreover, the isolated mammalian nucleic acid molecules encoding a
mammalian prostate-specific membrane antigen are useful for the
development of probes to study the tumorigenesis of prostate
cancer.
This invention also provides nucleic acid molecules of at least 15
nucleotides capable of specifically hybridizing with a sequence of
a nucleic acid molecule encoding the prostate-specific membrane
antigen.
This nucleic acid molecule produced can either be DNA or RNA. As
used herein, the phrase "specifically hybridizing" means the
ability of a nucleic acid molecule to recognize a nucleic acid
sequence complementary to its own and to form double-helical
segments through hydrogen bonding between complementary base
pairs.
This nucleic acid molecule of at least 15 nucleotides capable of
specifically hybridizing with a sequence of a nucleic acid molecule
encoding the prostate-specific membrane antigen can be used as a
probe. Nucleic acid probe technology is well known to those skilled
in the art who will readily appreciate that such probes may vary
greatly in length and may be labeled with a detectable label, such
as a radioisotope or fluorescent dye, to facilitate detection of
the probe. DNA probe molecules may be produced by insertion of a
DNA molecule which encodes PSM antigen into suitable vectors, such
as plasmids or bacteriophages, followed by transforming into
suitable bacterial host cells, replication in the transformed
bacterial host cells and harvesting of the DNA probes, using
methods well known in the art. Alternatively, probes may be
generated chemically from DNA synthesizers.
RNA probes may be generated by inserting the PSM antigen molecule
downstream of a bacteriophage promoter such as T3, T7 or SP6. Large
amounts of RNA probe may be produced by incubating the labeled
nucleotides with the linearized PSM antigen fragment where it
contains an upstream promoter in the presence of the appropriate
RNA polymerase.
This invention also provides a nucleic acid molecule of at least 15
nucleotides capable of specifically hybridizing with a sequence of
a nucleic acid molecule which is complementary to the mammalian
nucleic acid molecule encoding a mammalian prostate-specific
membrane antigen. This molecule may either be a DNA or RNA
molecule.
The current invention further provides a method of detecting the
expression of a mammalian PSM antigen expression in a cell which
comprises obtaining total mRNA from the cell, contacting the mRNA
so obtained with a labelled nucleic acid molecule of at least 15
nucleotides capable of specifically hybridizing with a sequence of
the nucleic acid molecule encoding a mammalian PSM antigen under
hybridizing conditions, determining the presence of mRNA hybridized
to the molecule and thereby detecting the expression of the
mammalian prostate-specific membrane antigen in the cell. The
nucleic acid molecules synthesized above may be used to detect
expression of a PSM antigen by detecting the presence of mRNA
coding for the PSM antigen. Total mRNA from the cell may be
isolated by many procedures well known to a person of ordinary
skill in the art. The hybridizing conditions of the labelled
nucleic acid molecules may be determined by routine experimentation
well known in the art. The presence of mRNA hybridized to the probe
may be determined by gel electrophoresis or other methods known in
the art. By measuring the amount of the hybrid made, the expression
of the PSM antigen by the cell can be determined. The labelling may
be radioactive. For an example, one or more radioactive nucleotides
can be incorporated in the nucleic acid when it is made.
In one embodiment of this invention, nucleic acids are extracted by
precipitation from lysed cells and the mRNA is isolated from the
extract using an oligo-dT column which binds the poly-A tails of
the mRNA molecules (13). The mRNA is then exposed to radioactively
labelled probe on a nitrocellulose membrane, and the probe
hybridizes to and thereby labels complementary mRNA sequences.
Binding may be detected by luminescence autoradiography or
scintillation counting. However, other methods for performing these
steps are well known to those skilled in the art, and the
discussion above is merely an example.
This invention further provides another method to detect expression
of a PSM antigen in tissue sections which comprises contacting the
tissue sections with a labelled nucleic acid molecule of at least
15 nucleotides capable of specifically hybridizing with a sequence
of nucleic acid molecules encoding a mammalian PSM antigen under
hybridizing conditions, determining the presence of mRNA hybridized
to the molecule and thereby detecting the expression of the
mammalian PSM antigen in tissue sections. The probes are also
useful for in-situ hybridization or in order to locate tissues
which express this gene, or for other hybridization assays for the
presence of this gene or its mRNA in various biological tissues.
The in-situ hybridization using a labelled nucleic acid molecule is
well known in the art. Essentially, tissue sections are incubated
with the labelled nucleic acid molecule to allow the hybridization
to occur. The molecule will carry a marker for the detection
because it is "labelled", the amount of the hybrid will be
determined based on the detection of the amount of the marker and
so will the expression of PSM antigen.
This invention further provides isolated PSM antigen nucleic acid
molecule operatively linked to a promoter of RNA transcription. The
isolated PSM antigen sequence can be linked to vector systems.
Various vectors including plasmid vectors, cosmid vectors,
bacteriophage vectors and other viruses are well known to ordinary
skilled practitioners. This invention further provides a vector
which comprises the isolated nucleic acid molecule encoding for the
PSM antigen.
As an example to obtain these vectors, insert and vector DNA can
both be exposed to a restriction enzyme to create complementary
ends on both molecules which base pair with each other and are then
ligated together with DNA ligase. Alternatively, linkers can be
ligated to the insert DNA which correspond to a restriction site in
the vector DNA, which is then digested with the restriction enzyme
which cuts at that site. Other means are also available and known
to an ordinary skilled practitioner.
In an embodiment, the PSM sequence is cloned in the Not I/Sal I
site of pSPORT/vector (Gibco.RTM.--BRL). This plasmid, p55A-PSM,
was deposited on Aug. 14, 1992 with the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852,
U.S.A. under the provisions of the Budapest Treaty for the
International Recognition of the Deposit of Microorganism for the
Purposes of Patent Procedure. Plasmid, p55A-PSM, was accorded ATCC
Accession Number 75294.
This invention further provides a host vector system for the
production of a polypeptide having the biological activity of the
prostate-specific membrane antigen. These vectors may be
transformed into a suitable host cell to form a host cell vector
system for the production of a polypeptide having the biological
activity of PSM antigen.
Regulatory elements required for expression include is promoter
sequences to bind RNA polymerase and transcription initiation
sequences for ribosome binding. For example, a bacterial expression
vector includes a promoter such as the lac promoter and for
transcription initiation the Shine-Dalgarno sequence and the start
codon AUG (14). Similarly, a eukaryotic expression vector includes
a heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors may
be obtained commercially or assembled from the sequences described
by methods well known in the art, for example the methods described
above for constructing vectors in general. Expression vectors are
useful to produce cells that express the PSM antigen.
This invention further provides an isolated DNA or cDNA molecule
described hereinabove wherein the host cell is selected from the
group consisting of bacterial cells (such as E. coli), yeast cells,
fungal cells, insect cells and animal cells. Suitable animal cells
include, but are not limited to Vero cells, HeLa cells, Cos cells,
CV1 cells and various primary mammalian cells.
This invention further provides a method of producing a polypeptide
having the biological activity of the prostate-specific membrane
antigen which comprising growing host cells of a vector system
containing the PSM antigen sequence under suitable conditions
permitting production of the polypeptide and recovering the
polypeptide so produced.
This invention provides a mammalian cell comprising a DNA molecule
encoding a mammalian PSM antigen, such as a mammalian cell
comprising a plasmid adapted for expression in a mammalian cell,
which comprises a DNA molecule encoding a mammalian PSM antigen and
the regulatory elements necessary for expression of the DNA in the
mammalian cell so located relative to the DNA encoding the
mammalian PSM antigen as to permit expression thereof.
Numerous mammalian cells may be used as hosts, including, but not
limited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa
cells, Ltk cells, Cos cells, etc. Expression plasmids such as that
described supra may be used to transfect mammalian cells by methods
well known in the art such as calcium phosphate precipitation,
electroporation or DNA encoding the mammalian PSM antigen may be
otherwise introduced into mammalian cells, e.g., by microinjection,
to obtain mammalian cells which comprise DNA, e.g., cDNA or a
plasmid, encoding a mammalian PSM antigen.
This invention provides a method for determining whether a ligand
can bind to a mammalian prostate-specific membrane antigen which
comprises contacting a mammalian cell comprising an isolated DNA
molecule encoding a mammalian prostate-specific membrane antigen
with the ligand under conditions permitting binding of ligands to
the mammalian prostate-specific membrane antigen, and thereby
determining whether the ligand binds to a mammalian
prostate-specific membrane antigen.
This invention further provides ligands bound to the mammalian PSM
antigen.
This invention also provides a therapeutic agent comprising a
ligand identified by the above-described method and a cytotoxic
agent conjugated thereto. The cytotoxic agent may either be a
radioisotope or a toxin. Examples of radioisotopes or toxins are
well known to one of ordinary skill in the art.
This invention also provides a method of imaging prostate cancer in
human patients which comprises administering to the patients at
least one ligand identified by the above-described method, capable
of binding to the cell surface of the prostate cancer cell and
labelled with an imaging agent under conditions permitting
formation of a complex between the ligand and the cell surface PSM
antigen. This invention further provides a composition comprising
an effective imaging agent of the PSM antigen ligand and a
pharmaceutically acceptable carrier. Pharmaceutically acceptable
carriers are well known to one of ordinary skill in the art. For an
example, such a pharmaceutically acceptable carrier can be
physiological saline.
Also provided by this invention is a purified mammalian PSM
antigen. As used herein, the term "purified prostate-specific
membrane antigen" shall mean isolated naturally-occurring
prostate-specific membrane antigen or protein (purified from nature
or manufactured such that the primary, secondary and tertiary
conformation, and posttranslational modifications are identical to
naturally-occurring material) as well as non-naturally occurring
polypeptides having a primary structural conformation (i.e.
continuous sequence of amino acid residues). Such polypeptides
include derivatives and analogs.
This invention further provides a polypeptide encoded by the
isolated mammalian nucleic acid sequence of PSM antigen.
It is believed that there may be natural ligand interacting with
the PSM antigen. This invention provides a method to identify such
natural ligand or other ligand which can bind to the PSM antigen. A
method to identify the ligand comprises a) coupling the purified
mammalian PSM antigen to a solid matrix, b) incubating the coupled
purified mammalian PSM protein with the potential ligands under the
conditions permitting binding of ligands and the purified PSM
antigen; c) washing the ligand and coupled purified mammalian PSM
antigen complex formed in b) to eliminate the nonspecific binding
and impurities and finally d) eluting the ligand from the bound
purified mammalian PSM antigen. The techniques of coupling proteins
to a solid matrix are well known in the art. Potential ligands may
either be deduced from the structure of mammalian PSM or by other
empirical experiments known by ordinary skilled practitioners. The
conditions for binding may also easily be determined and protocols
for carrying such experimentation have long been well documented
(15). The ligand-PSM antigen complex will be washed. Finally, the
bound ligand will be eluted and characterized. Standard ligands
characterization techniques are well known in the art.
The above method may also be used to purify ligands from any
biological source. For purification of natural ligands in the cell,
cell lysates, serum or other biological samples will be used to
incubate with the mammalian PSM antigen bound on a matrix. Specific
natural ligand will then be identified and purified as above
described.
With the protein sequence information, antigenic areas may be
identified and antibodies directed against these areas may be
generated and targeted to the prostate cancer for imaging the
cancer or therapies.
This invention provides an antibody directed against the amino acid
sequence of a mammalian PSM antigen.
This invention provides a method to select specific regions on the
PSM antigen to generate antibodies. The protein sequence may be
determined from the PSM DNA sequence. Amino acid sequences may be
analyzed by methods well known to those skilled in the art to
determine whether they produce hydrophobic or hydrophilic regions
in the proteins which they build. In the case of cell membrane
proteins, hydrophobic regions are well known to form the part of
the protein that is inserted into the lipid bilayer of the cell
membrane, while hydrophilic regions are located on the cell
surface, in an aqueous environment. Usually, the hydrophilic
regions will be more immunogenic than the hydrophobic regions.
Therefore the hydrophilic amino acid sequences may be selected and
used to generate antibodies specific to mammalian PSM antigen. For
an example, hydrophilic sequences of the human PSM antigen shown in
hydrophilicity plot of FIG. 16 may be easily selected. The selected
peptides may be prepared using commercially available machines. As
an alternative, DNA, such as a cDNA or a fragment thereof, may be
cloned and expressed and the resulting polypeptide recovered and
used as an immunogen.
Polyclonal antibodies against these peptides may be produced by
immunizing animals using the selected peptides. Monoclonal
antibodies are prepared using hybridoma technology by fusing
antibody producing B cells from immunized animals with myeloma
cells and selecting the resulting hybridoma cell line producing the
desired antibody. Alternatively, monoclonal antibodies may be
produced by in vitro techniques known to a person of ordinary skill
in the art. These antibodies are useful to detect the expression of
mammalian PSM antigen in living animals, in humans, or in
biological tissues or fluids isolated from animals or humans.
In one embodiment, peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No.
35), Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 36) and
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID No. 37) of human PSM antigen are
selected.
This invention further provides polyclonal and monoclonal
antibody(ies) against peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No.
35), Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 36) and
Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID No. 37).
This invention provides a therapeutic agent comprising antibodies
or ligand(s) directed against PSM antigen and a cytotoxic agent
conjugated thereto or antibodies linked enzymes which activate
prodrug to kill the tumor. The cytotoxic agent may either be a
radioisotope or toxin.
This invention provides a method of imaging prostate cancer in
human patients which comprises administering to the patient the
monoclonal antibody directed against the peptide of the mammalian
PSM antigen capable of binding to the cell surface of the prostate
cancer cell and labeled with an imaging agent under conditions
permitting formation of a complex between the monoclonal antibody
and the cell surface prostate-specific membrane antigen. The
imaging agent is a radioisotope such as Indium.sup.111.
This invention further provides a prostate cancer specific imaging
agent comprising the antibody directed against PSM antigen and a
radioisotope conjugated thereto.
This invention also provides a composition comprising an effective
imaging amount of the antibody directed against the PSM antigen and
a pharmaceutically acceptable carrier. The methods to determine
effective imaging amounts are well known to a skilled practitioner.
One method is by titration using different amounts of the
antibody.
This invention further provides an immunoassay for measuring the
amount of the prostate-specific membrane antigen in a biological
sample comprising steps of a) contacting the biological sample with
at least one antibody directed against the PSM antigen to form a
complex with said antibody and the prostate-specific membrane
antigen, and b) measuring the amount of the prostate-specific
membrane antigen in said biological sample by measuring the amount
of said complex. One example of the biological sample is a serum
sample.
This invention provides a method to purify mammalian
prostate-specific membrane antigen comprising steps of a) coupling
the antibody directed against the PSM antigen to a solid matrix; b)
incubating the coupled antibody of a) with lysate containing
prostate-specific membrane antigen under the condition which the
antibody and prostate membrane specific can bind; c) washing the
solid matrix to eliminate impurities and d) eluting the
prostate-specific membrane antigen from the coupled antibody.
This invention also provides a transgenic nonhuman mammal which
comprises the isolated nucleic acid molecule encoding a mammalian
PSM antigen. This invention further provides a transgenic nonhuman
mammal whose genome comprises antisense DNA complementary to DNA
encoding a mammalian prostate-specific membrane antigen so placed
as to be transcribed into antisense mRNA complementary to mRNA
encoding the prostate-specific membrane antigen and which
hybridizes to mRNA encoding the prostate specific antigen thereby
reducing its translation.
Animal model systems which elucidate the physiological and
behavioral roles of mammalian PSM antigen are produced by creating
transgenic animals in which the expression of the PSM antigen is
either increased or decreased, or the amino acid sequence of the
expressed PSM antigen is altered, by a variety of techniques.
Examples of these techniques include, but are not limited to: 1)
Insertion of normal or mutant versions of DNA encoding a mammalian
PSM antigen, by microinjection, electroporation, retroviral
transfection or other means well known to those skilled in the art,
into appropriate fertilized embryos in order to produce a
transgenic animal (16) or 2) Homologous recombination (17) of
mutant or normal, human or animal versions of these genes with the
native gene locus in transgenic animals to alter the regulation of
expression or the structure of these PSM antigen sequences. The
technique of homologous recombination is well known in the art. It
replaces the native gene with the inserted gene and so is useful
for producing an animal that cannot express native PSM antigen but
does express, for example, an inserted mutant PSM antigen, which
has replaced the native PSM antigen in the animal's genome by
recombination, resulting in underexpression of the transporter.
Microinjection adds genes to the genome, but does not remove them,
and so is useful for producing an animal which expresses its own
and added PSM antigens, resulting in overexpression of the PSM
antigens.
One means available for producing a transgenic animal, with a mouse
as an example, is as follows: Female mice are mated, and the
resulting fertilized eggs are dissected out of their oviducts. The
eggs are stored in an appropriate medium such as M2 medium (16).
DNA or cDNA encoding a mammalian PSM antigen is purified from a
vector by methods well known in the art. Inducible promoters may be
fused with the coding region of the DNA to provide an experimental
means to regulate expression of the trans-gene. Alternatively or in
addition, tissue specific regulatory elements may be fused with the
coding region to permit tissue-specific expression of the
trans-gene. The DNA, in an appropriately buffered solution, is put
into a microinjection needle (which may be made from capillary
tubing using a pipet puller) and the egg to be injected is put in a
depression slide. The needle is inserted into the pronucleus of the
egg, and the DNA solution is injected. The injected egg is then
transferred into the oviduct of a pseudopregnant mouse (a mouse
stimulated by the appropriate hormones to maintain pregnancy but
which is not actually pregnant), where it proceeds to the uterus,
implants, and develops to term. As noted above, microinjection is
not the only method for inserting DNA into the egg cell, and is
used here only for exemplary purposes.
Another use of the PSM antigen sequence is to isolate homologous
gene or genes in different mammals. The gene or genes can be
isolated by low stringency screening of either cDNA or genomic
libraries of different mammals using probes from PSM sequence. The
positive clones identified will be further analyzed by DNA
sequencing techniques which are well known to an ordinary person
skilled in the art. For example, the detection of members of the
protein serine kinase family by homology probing (18).
This invention provides a method of suppressing or modulating
metastatic ability of prostate tumor cells, prostate tumor growth
or elimination of prostate tumor cells comprising introducing a DNA
molecule encoding a prostate specific membrane antigen operatively
linked to a 5' regulatory element into a tumor cell of a subject,
in a way that expression of the prostate specific membrane antigen
is under the control of the regulatory element, thereby suppressing
or modulating metastatic ability of prostate tumor cells, prostate
tumor growth or elimination of prostate tumor cells. The subject
may be a mammal or more specifically a human.
In one embodiment, the DNA molecule encoding prostate specific
membrane antigen operatively linked to a 5' regulatory element
forms part of a transfer vector which is inserted into a cell or
organism. In addition the vector is capable or replication and
expression of prostate specific membrane antigen. The DNA molecule
encoding prostate specific membrane antigen can be integrated into
a genome of a eukaryotic or prokaryotic cell or in a host cell
containing and/or expressing a prostate specific membrane
antigen.
Further, the DNA molecule encoding prostate specific membrane
antigen may be introduced by a bacterial, viral, fungal, animal, or
liposomal delivery vehicle. Other means are also available and
known to an ordinary skilled practitioner.
Further, the DNA molecule encoding a prostate specific membrane
antigen operatively linked to a promoter or enhancer. A number of
viral vectors have been described including those made from various
promoters and other regulatory elements derived from virus sources.
Promoters consist of short arrays of nucleic acid sequences that
interact specifically with cellular proteins involved in
transcription. The combination of different recognition sequences
and the cellular concentration of the cognate transcription factors
determines the efficiency with which a gene is transcribed in a
particular cell type.
Examples of suitable promoters include a viral promoter. Viral
promoters include: adenovirus promoter, an simian virus 40 (SV40)
promoter, a cytomegalovirus CCMV) promoter, a mouse mammary tumor
virus (MMTV) promoter, a Malony murine leukemia virus promoter, a
murine sarcoma virus promoter, and a Rous sarcoma virus
promoter.
Further, another suitable promoter is a heat shock promoter.
Additionally, a suitable promoter is a bacteriophage promoter.
Examples of suitable bacteriophage promoters include but not
limited to, a T7 promoter, a T3 promoter, an SP6 promoter, a lambda
promoter, a baculovirus promoter.
Also suitable as a promoter is an animal cell promoter such as an
interferon promoter, a metallothionein promoter, an immunoglobulin
promoter. A fungal promoter is also a suitable promoter. Examples
of fungal promoters include but are not limited to, an ADC1
promoter, an ARG promoter, an ADH promoter, a CYC1 promoter, a CUP
promoter, an ENO1 promoter, a GAL promoter, a PRO promoter, a PGK
promoter, a GAPDH promoter, a mating type factor promoter. Further,
plant cell promoters and insect cell promoters are also suitable
for the methods described herein.
This invention provides a method of suppressing or modulating
metastatic ability of prostate tumor cells, prostate tumor growth
or elimination of prostate tumor cells, comprising introducing a
DNA molecule encoding a prostate specific membrane antigen
operatively linked to a 5' regulatory element coupled with a
therapeutic DNA into a tumor cell of a subject, thereby suppressing
or modulating metastatic ability of prostate tumor cells, prostate
tumor growth or elimination of prostate tumor cells. The subject
may be a mammal or more specifically a human.
Further, the therapeutic DNA which is coupled to the DNA molecule
encoding a prostate specific membrane antigen operatively linked to
a 5' regulatory element into a tumor cell may code for a cytokine,
viral antigen, or a pro-drug activating enzyme. Other means are
also available and known to an ordinary skilled practitioner.
The cytokine used may be interleukin-2, interleukin-12, interferon
alpha, beta or gamma, granulocytic macrophage--colony stimulating
factor, or other immunity factors.
In addition, this invention provides a prostate tumor cell,
comprising a DNA molecule isolated from mammalian nucleic acid
encoding a mammalian prostate-specific membrane antigen under the
control of a prostate specific membrane antigen operatively linked
to a 5' regulatory element.
As used herein, DNA molecules include complementary DNA (cDNA),
synthetic DNA, and genomic DNA.
This invention provides a therapeutic vaccine for preventing human
prostate tumor growth or stimulation of prostate tumor cells in a
subject, comprising administering an effective amount to the
prostate cell, and a pharmaceutical acceptable carrier, thereby
preventing the tumor growth or stimulation of tumor cells in the
subject. Other means are also available and known to an ordinary
skilled practitioner.
This invention provides a method of detecting hematogenous
micrometastic tumor cells of a subject, comprising (A) performing
nested polymerase chain reaction (PCR) on blood, bone marrow or
lymph node samples of the subject using the prostate specific
membrane antigen primers, and (B) verifying micrometastases by DNA
sequencing and Southern analysis, thereby detecting hematogenous
micrometastic tumor cells of the subject. The subject may be a
mammal or more specifically a human.
The micrometastatic tumor cell may be a prostatic cancer and the
DNA primers may be derived from prostate specific antigen. Further,
the subject may be administered with simultaneously an effective
amount of hormones, so as to increase expression of prostate
specific membrane antigen.
This invention provides a method of abrogating the mitogenic
response due to transferrin, comprising introducing a DNA molecule
encoding prostate specific membrane antigen operatively linked to a
5' regulatory element into a tumor cell, the expression of which
gene is directly associated with a defined pathological effect
within a multicellular organism, thereby abrogating mitogen
response due to transferrin. The tumor cell may be a prostate
cell.
This invention will be better understood from the Experimental
Details which follow. However, one skilled in the art will readily
appreciate that the specific methods and results discussed are
merely illustrative of the invention as described more fully in the
claims which follow thereafter.
EXPERIMENTAL DETAILS
First Series of Experiments
Materials and Methods
The approach for cloning the gene involved purification of the
antigen in large quantities by immunoprecipitation, and
microsequencing of several internal peptides for use in
synthesizing degenerate oligonucleotide primers for subsequent use
in the polymerase chain reaction (19, 20). A partial cDNA was
amplified as a PCR product and this was used as a homologous probe
to clone the full-length cDNA molecule from a LNCaP (Lymph Node
Carcinoma of Prostate) cell line cDNA plasmid library (8). Early
experiments revealed to us that the CYT-356 antibody (9) was not
capable of detecting the antigen produced in bacteria since the
epitope was the glycosylated portion of the PSM antigen, and this
necessitated our more difficult, yet elaborate approach.
Western Analysis of the PSM Antigen
Membrane proteins were isolated from cells by hypotonic lysis
followed by centrifugation over a sucrose density gradient (21).
10-20 .mu.g of LNCaP, DU-145, and PC-3 membrane proteins were
electrophoresed through a 10% SDS-PAGE resolving gel with a 4%
stacking gel at 9-10 milliamps for 16-18 hours. Proteins were
electroblotted onto PVDF membranes (Millipore.RTM. Corp.) in
transfer buffer (48 mM Tris base, 39 mM Glycine, 20% Methanol) at
25 volts overnight at 4.degree. C. Membranes were blocked in TSB
(0.15M NaCl, 0.01M Tris base, 5% BSA) for 30 minutes at room
temperature followed by incubation with 10-15 .mu.g/ml of CYT-356
monoclonal antibody (Cytogen Corp.) for 2 hours. Membranes were
then incubated with 10-15 .mu.g/ml of rabbit anti-mouse
immunoglobulin (Accurate Scientific) for 1 hour at room temperature
followed by incubation with .sup.125I-Protein A (Amersham.RTM.) at
1.times.10.sup.6 cpm/ml at room temperature. Membranes were then
washed and autoradiographed for 12-24 hours at -70.degree. C. (FIG.
1).
Immunohistochemical Analysis of PSM Antigen Expression
The avidin-biotin method of immunohistochemical detection was
employed to analyze both human tissue sections and cell lines for
PSM Antigen expression (22). Cryostat-cut prostate tissue sections
(4-6% thick) were fixed in methanol/acetone for 10 minutes. Cell
cytospins were made on glass slides using 50,000 cells/100
.mu.l/slide. Samples were treated with 1% hydrogen peroxide in PBS
for 10-15 minutes in order to remove any endogenous peroxidase
activity. Tissue sections were washed several times in PBS, and
then incubated with the appropriate suppressor serum for 20
minutes. The suppressor serum was drained off and the sections or
cells were then incubated with the diluted CYT-356 monoclonal
antibody for 1 hour. Samples were then washed with PBS and
sequentially incubated with secondary antibodies (horse or goat
immunoglobulins, 1:200 dilution for 30 minutes), and with
avidin-biotin complexes (1:25 dilution for 30 minutes). DAB was
used as a chromogen, followed by hematoxylin counterstaining and
mounting. Frozen sections of prostate samples and duplicate cell
cytospins were used as controls for each experiment. As a positive
control, the anti-cytokeratin monoclonal antibody CAM 5.2 was used
following the same procedure described above. Tissue sections are
considered by us to express the PSM antigen if at least 5% of the
cells demonstrate immunoreactivity. Our scoring system is as
follows: 1=<5%; 2=5-19%; 3=20-75%; and 4=>75% positive cells.
Homogeneity versus heterogeneity was accounted for by evaluating
positive and negative cells in 3-5 high power light microscopic
fields (400.times., recording the percentage of positive cells
among 100-500 cells. The intensity of immunostaining is graded on a
1+ to 4+ scale, where 1-represents mild, 2-3+ represents moderate,
and 4+ represents intense immunostaining as compared to positive
controls.
Immunoprecipitation of the PSM Antigen
80%-confluent LNCaP cells in 100 mm petri dishes were starved in
RPMI media without methionine for 2 hours, after which
.sup.33S-Methionine was added at 100 .mu.Ci/ml and the cells were
grown for another 16-18 hours. Cells were then washed and lysed by
the addition of 1 ml of lysis buffer (1% Triton X-100, 50 mM Hepes
pH 7.5, 10% glycerol, 150 MM MgCl.sub.2, 1 mM PMSF, and 1 mM EGTA)
with incubation for 20 minutes at 40.degree. C. Lysates were
pre-cleared by mixing with Pansorbin.RTM. cells (Calbiochem) for 90
minutes at 4.degree. C. Cell lysates were then mixed with Protein A
Sepharose.RTM. CL-4B beads (Pharmacia.RTM.) previously bound with
CYT-356 antibody (Cytogen Corp.) and RAM antibody (Accurate
Scientific) for 3-4 hours at 4.degree. C. 12 .mu.g of antibody was
used per 3 mg of beads per petri dish. Beads were then washed with
HNTG buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.1% Triton X-100,
10% glycerol, and 2 mM Sodium Orthovanadate), resuspended in sample
loading buffer containing mercaptoethanol, denatured at 95.degree.
C. for 5-10 minutes and run on a 10% SDS-PAGE gel with a 4.degree.
stacking gel at 10 milliamps overnight. Gels were stained with
Coomassie Blue, destained with acetic acid/methanol, and dried down
in a vacuum dryer at 60.degree. C. Gels were then autoradiographed
for 16-24 hours at -70.degree. C. (FIG. 2 A-D).
Large-Scale Immunoprecigitation and Peptide Sequencing
The procedure described above for immunoprecipitation was repeated
with 8 confluent petri dishes containing approximately
6.times.10.sup.7 LNCaP cells. The immunoprecipitation product was
pooled and loaded into two lanes of a 10% SDS-PAGE gel and
electrophoresed at 9-10 milliamps for 16 hours. Proteins were
electroblotted onto Nitrocellulose BA-85 membranes (Schleicher and
Schuell.RTM.) for 2 hours at 75 volts at 4.degree. C. in transfer
buffer. Membranes were stained with Ponceau Red to visualize the
proteins and the 100 KD protein band was excised, solubilized, and
digested proteolytically with trypsin. HPLC was then performed on
the digested sample on an Applied Biosystems Model 171C and clear
dominant peptide peaks were selected and sequenced by modified
Edman degradation on a modified post liquid Applied Biosystems
Model 477A Protein/Peptide Microsequencer (23). Sequencing data on
all of the peptides is included within this document. We attempted
to sequence the amino-terminus of the PSM antigen by a similar
method which involved purifying the antigen by immunoprecipitation
and transfer via electro-blotting to a PVDF membrane
(Millipore.RTM.). Protein was analyzed on an Applied Biosystems
Model 477A Protein/Peptide Sequencer and the amino terminus was
found to be blocked, and therefore no sequence data could be
obtained by this technique.
TABLE-US-00001 PSM Antigen Peptide Sequences: 2T17 SLYES(W)TK (SEQ
ID No. 3) #5 2T22 (S)YPDGXNLPGG(g)VQR (SEQ ID No. 4) #9 2T26
FYDPMFK (SEQ ID No. 5) #3 2T27 IYNVIGTL(K) (SEQ ID No. 6) #4 2T34
FLYXXTQIPHLAGTEQNFQLAK (SEQ ID NO. 7) #6 2T35 G/PVILYSDPADYFAPD/GVK
(SEQ ID No. 8, 9) #2 2T38 AFIDPLGLPDRPFYR (SEQ ID No. 10) #1 2146
YAGESFPGIYDALFDIESK (SEQ ID No. 11) #8 2T47
TILFAS(W)DAEEFGXX(q)STE(e)A(E) . . . (SEQ ID No. 12) #7
Notes: X means that no residue could be identified at this
position. Capital denotes identification but with a lower degree of
confidence. (lower case) means residue present but at very low
levels. . . . indicates sequence continues but has dropped below
detection limit.
All of these peptide sequences were verified to be unique after a
complete homology search of the translated Genbank computer
database.
Degenerate PCR
Sense and anti-sense 5'-unphosphorylated degenerate oligonucleotide
primers 17 to 20 nucleotides in length corresponding to portions of
the above peptides were synthesized on an Applied Biosystems Model
394A DNA Synthesizer. These primers have degeneracies from 32 to
144. The primers used are shown below. The underlined amino acids
in the peptides represent the residues used in primer design.
Peptide 3: (SEQ ID No. 5)
PSM Primer "A" TT(C or T)--TA(C or T)--GA(C or T)--CCX--ATG--TT
(SEQ ID No.13)
PSM Primer "B" AAC--ATX--GG(A or G)--TC(A or G)--TA(A or G)--AA
(SEQ ID No. 14)
Primer A is sense primer and B is anti-sense. Degeneracy is
32-fold.
Peptide 4: IYNVIGTL(K) (SEQ ID No. 6)
PSM Primer "C" AT(T or C or A)--TA(T or C)--AA(T or C)--GTX--AT(T
or C or A)--GG (SEQ ID No. 15)
PSM Primer "D" CC(A or T or G)--ATX&13 AC(G or A)--TT(A or
G)--TA(A or G or T)--AT (SEQ ID No. 16)
Primer C is sense primer and D is anti-sense. Degeneracy is
144-fold.
Peptide 2: G/PVILYSDPADYFAPD/GVK (SEQ ID No. 8,9)
PSM Primer "E" CCX--GCX--GA(T or C)--TA(T or C)--TT(T or C)--CC
(SEQ ID No. 17)
PSM Primer "F" GC(G or A)--AA(A or G)--TA(A or G)--TXC--GCX--GG
(SEQ ID No. 16)
Primer E is sense primer and F is antisense primer. Degeneracy is
128-fold.
Peptide 6. FLYXXTQIPHLAGTEONFQLAK (SEQ ID No. 7)
PSM Primer "I" ACX--GA(A or G)--CA(A or G)--AA(T or C)--TT(T or
C)--CA(A or G)--CT (SEQ ID No. 19)
PSM Primer "J" AG--(T or C)TG--(A or G)AA--(A or G)TT--(T or
C)TG--(T or C)TC--XGT (SEQ ID No. 20)
PSM Primer "K" GA(A or G)--CA(A or G)--AA(T or C)--TT(T or C) CA(A
or G)--CT (SEQ ID No. 21)
PSM Primer "L" AG--(T or C)TG--(A or G)AA--(A or G)TT--(T or
C)TG--(T or C)TC (SEQ ID No. 22)
Primers I and K are sense printers and J and L are anti-sense. I
and J have degeneracies of 128-fold and K and L have 32-fold
degeneracy.
Peptide 7: TILFAS(W)DAEEPGXX(q)STE(e)A(E) . . . (SEQ ID No. 12)
PSM Primer "M" TGG--GA(T or C)--GCX--GA(A or G)--GA(A or G)--TT(C
or T)--GG (SEQ ID No. 23)
PSM Primer "N" CC--(G or A)AA--(T or C)TC--(T or C)TC--XGC--(A or
G)TC--CCA (SEQ ID No. 24)
PSM Primer row TGG--GA(T or C)--GCX--GA(A or G)--GA(A or G)--TT
(SEQ ID No. 25)
PSM Primer "p" AA--(T or C)TC--(T or C)TC--XGC--(A or G)TC--CCA
(SEQ ID No. 26)
Primers M and O are sense primers and N and P are anti-sense. M and
N have degeneracy of 64-fold and O and P are 32- fold
degenerate.
Degenerate PCR was performed using a Perkin-Elmer Model 480 DNA
thermal cycler. cDNA template for the PCR was prepared from LNCaP
mRNA which had been isolated by standard methods of oligo dT
chromatography (Collaborative Research). The cDNA synthesis was
carried out as follows:
4.5 .mu.l LNCaP poly A+RNA (2 .mu.g)
1.0 .mu.l Oligo dT primers (0.5 .mu.g)
4.5 .mu.l dH.sub.2O
10 .mu.l
Incubate at 68.degree. C..times.10 minutes.
Quick chill on ice .times.5 minutes.
Add:
4 .mu.l 5 .times. RT Buffer
2 .mu.l 0.1M DTT
1 .mu.l 10 mM dNTPs
0.5 .mu.l RNasin (Promega)
1.5 .mu.l dH.sub.2O
19 .mu.l
Incubate for 2 minutes at 37.degree. C.
Add 1 .mu.l Superscript.RTM. Reverse Transcriptase (Gibco.RTM.-BRL)
Incubate for 1 hour at 37.degree. C.
Add 30 .mu.l dH.sub.2.
Use 2 .mu.l per PCR reaction.
Degenerate PCR reactions were optimized by varying the annealing
temperatures, Mg++ concentrations, primer concentrations, buffer
composition, extension times and number of cycles. Our optimal
thermal cycler profile was: Denaturation at 94.degree. C..times.30
seconds, Annealing at 45-55.degree. C. for 1 minute (depending on
the mean T.sub.m of the primers used), and Extension at 72.degree.
C. for 2 minutes.
5 .mu.l 10 .times. PCR Buffer*
5 .mu.l 2.5 mM dNTP Mix
5 .mu.l Primer Mix (containing 0.5-1.0 g each of sense d anti-sense
primers)
5 .mu.l 100 mM .beta.-mercaptoethanol
2 .mu.l LNCaP cDNA template
5 .mu.l 25 mM MgCl.sub.2 (2.5 mM final)
21 .mu.l dH.sub.2O
20 .mu.l diluted Tag Polymerase (0.5U/.mu.l)
50 .mu.l total volume
Tubes were overlaid with 60 .mu.l of light mineral oil and
amplified for 30 cycles. PCR products were analyzed by
electrophoresing 5 .mu.l of each sample on a 2-3% agarose gel
followed by staining with Ethidium bromide and photography.
*10.times. PCR Buffer
166 mM NH.sub.4SO.sub.4
670 mM Tris, pH 8.8
2 mg/ml BSA
Representative photographs displaying PCR products are shown in
FIG. 5.
Cloning of PCR Products
In order to further analyze these PCR products, these products-were
cloned into a suitable plasmid vector using "TA Cloning"
(Invitrogen.RTM. Corp.). The cloning strategy employed here is to
directly ligate PCR products into a plasmid vector possessing
overhanging T residues at the insertion site, exploiting the fact
that Tag polymerase leaves overhanging A residues at the ends of
the PCR products. The ligation mixes are transformed into competent
E. coli cells and resulting colonies are grown up, plasmid DNA is
isolated by the alkaline lysis method (24), and screened by
restriction analysis (FIG. 6 A-B).
DNA Sequencing of PCR Products
TA Clones of PCR products were then sequenced by the dideoxy method
(25) using Sequenase (U.S. Biochemical). 3-4 .mu.g of each plasmid
DNA was denatured with NaOH and ethanol precipitated. Labeling
reactions were carried out as per the manufacturers recommendations
using .sup.35S-ATP, and the reactions were terminated as per the
same protocol. Sequencing products were then analyzed on 6%
polyacrylamide/7M Urea gels using an IBI sequencing apparatus. Gels
were run at 120 watts for 2 hours. Following electrophoresis, the
gels were fixed for 15-20 minutes in 10% methanol/10% acetic acid,
transferred onto Whatman 3MM paper and dried down in a Biorad.RTM.
vacuum dryer at 80.degree. C. for 2 hours. Gels were then
autoradiographed at room temperature for 16-24 hours. In order to
determine whether the PCR products were the correct clones, we
analyzed the sequences obtained at the 5' and 3' ends of the
molecules looking for the correct primer sequences, as well as
adjacent sequences which corresponded to portions of the peptides
not used in the design of the primers.
IN-20 was confirmed to be correct and represent a partial cDNA for
the PSM gene. In this PCR reaction, I and N primers were used. The
DNA sequence we obtained when reading from the I primer was:
TABLE-US-00002 (SEQ ID No. 30) ACG GAG CAA AJLC TTT CAG CTT GCA AAG
(SEQ ID No. 31) T E O N P O L A X
The underlined amino acids were the portion of peptide 6 that was
used to design this sense primer and the remaining amino acids
which agree with those present within our peptide confirm that this
end of the molecule represents the correct protein (PSM
antigen).
When we analyzed the other end of the molecule by reading from the
N primer the sequence was:
TABLE-US-00003 (SEQ ID No. 32) CTC TTC GGC ATC CCA GGT TGC ALAA CAA
ATT TGT TCT
Since this represents the anti-sense DNA sequence, we need to show
the complementary sense sequence in order to find our peptide.
Sense Sequence:
TABLE-US-00004 (SEQ ID No. 33) AGA ACA ATT TTG TTT GCK AGC TGG GAT
GCC AAG GAG (SEQ ID No. 34) R T I L P A S W D A E B
The underlined amino acids here represent the portion of peptide 7
used to create primer N. All of the amino acids upstream of this
primer are correct in the IN-20 clone, agreeing with the amino
acids found in peptide 7. Further DNA sequencing has enabled us to
identify the presence of our other PSM peptides within the DNA
sequence of our positive clone.
The DNA sequence of this partial cDKA was found to be unique when
screened on the Genbank computer database.
cDNA Library Construction and Cloning of Full--Length PSM cDNA
A cDNA library from LNCaP mRNA was constructed using the
Superscript.RTM. plasmid system (BRL.RTM.-Gibco). The library was
transformed using competent DH5-.alpha. cells and plated onto 100
mm plates containing LB plus 100 .mu.g/ml of Carbenicillin. Plates
were grown overnight at 37.degree. C. and colonies were transferred
to nitrocellulose filters. Filters were processed and-screened as
per Grunstein and Hogness (26), using our 1.1 kb partial cDNA
homologous probe which was radiolabelled with .sup.32P-dCTP by
random priming (27). We obtained eight positive colonies which upon
DNA restriction and sequencing analysis proved to represent
full-length cDNA molecules coding for the PSM antigen. Shown in
FIG. 7 is an autoradiogram showing the size of the cDNA molecules
represented in our library and in FIG. 8 restriction analysis of
several full-length clones is shown. FIG. 9 is a plasmid Southern
analysis of the samples in FIG. 6, showing that they all hybridize
to the 1.1 kb partial cDNA probe.
Both the cDNA as well as the antigen have been screened through the
Genbank Computer database (Human Genome Project) and have been
found to be unique.
Northern Analysis of PSM Gene Expression
Northern analysis (28) of the PSM gene has revealed that expression
is limited to the prostate and to prostate carcinoma.
RNA samples (either 10 .mu.g of total RNA or 2 .mu.g of poly A+RNA)
were denatured and electrophoresed through 1.1%
agarose/formaldehyde gels at 60 milliamps for 6-8 hours. RNA was
then transferred to Nytran.RTM. nylon membranes (Schleicher and
Schuell.RTM.) by pressure blotting in 10.times. SSC with a
Posi-blotter (Stratagene.RTM.). RNA was cross-linked to the
membranes using a Stratalinker (Stratagene.RTM.) and subsequently
baked in a vacuum oven at 80.degree. C. for 2 hours. Blots were
pre-hybridized at 65.degree. C. for 2 hours in prehybridization
solution (BRL.RTM.) and subsequently hybridized for 16 hours in
hybridization buffer (BRL.RTM.) containing 1-2.times.10.sup.6
cpm/ml of .sup.32P-labelled random-primed cDNA probe. Membranes
were washed twice in 1.times. SSPE/1% SDS and twice in 0.1 .times.
SSPE/1% SDS at 42.degree. C. Membranes were then air-dried and
autoradiographed for 12-36 hours at -70.degree. C.
PCR Analysis of PSM Gene Expression in Human Prostate Tissues
PCR was performed on 15 human prostate samples to determine PSM
gene expression. Five samples each from normal prostate tissue,
benign prostatic hyperplasia, and prostate cancer were used
(histology confirmed by MSKCC Pathology Department).
10 .mu.g of total RNA from each sample was reverse transcribed to
made cDNA template as previously described in section IV. The
primers used corresponded to the 5 and 3' ends of our 1.1 kb
partial cDRA, IN-20, and therefore the expected size of the
amplified band is 1.1 kb. Since the T.sub.m of our primers is
64.degree. C. we annealed the primers in our PCR at 60.degree. C.
We carried out the PCR for 35 cycles using the same conditions
previously described in section IV.
LNCaP and H26--Ras transfected LNCaP (29) were included as a
positive control and DU-145 as a negative control. 14/15 samples
clearly amplified the 1.1 kb band and therefore express the
gene.
Experimental Results
The gene which encodes the 100 kD PSM antigen has been identified.
The complete cDNA sequence is shown in Sequence ID #1. Underneath
that nucleic acid sequence is the predicted translated amino acid
sequence. The total number of the amino acids is 750, ID.#2. The
hydrophilicity of the predicted protein sequence is shown in FIG.
16. Shown in FIG. 17 are three peptides with the highest point of
hydrophilicity. They are: Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No. 35);
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 36; and Lys-Ser-Pro-Asp-Glu-Gly
(SEQ ID No. 37).
By the method of Klein, Kanehisa and DeLisi, a specific
membrane-spanning domain is identified. The sequence is from the
amino acid #19 to amino acid #44:
Ala-Gly-Ala-Leu-Val-Leu-Aal-Gly-Gly-Phe-Phe-Leu-Leu-Gly-Phe-Leu-
-Phe (SEQ ID No. 38).
This predicted membrane-spanning domain was computed on PC Gene
(computer software program). This data enables prediction of inner
and outer membrane domains of the PSM antigen which aids in
designing antibodies for uses in targeting and imaging prostate
cancer.
When the PSM antigen sequence with other known sequences of the
GeneBank were compared, homology between the PSM antigen sequence
and the transferrin receptor sequence were found. The data are
shown in FIG. 18.
EXPERIMENTAL DISCUSSIONS
Potential Uses for PSM Antigen: 1. Tumor detection:
Microscopic:
Unambiguous tumor designation can be accomplished by use of probes
for different antigens. For prostatic cancer, the PSM antigen probe
may prove beneficial. Thus PSM could be used for diagnostic
purposes and this could be accomplished at the microscopic level
using in-situ hybridization using sense (control) and antisense
probes derived from the coding region of the cDNA cloned by the
applicants. This could be used in assessment of local
extraprostatic extension, involvement of lymph node, bone or other
metastatic sites. As bone metastasis presents a major problem in
prostatic cancer, early detection of metastatic spread is required
especially for staging. In some tumors detection of tumor cells in
bone marrow portends a grim prognosis and suggests that
interventions aimed at metastasis be tried. Detection of PSM
antigen expression in bone marrow aspirates or sections may provide
such early information. PCR amplification or in-situ hybridization
may be used. This could be developed for any possible metastatic
region. 2. Antigenic site identification
The knowledge of the cDNA for the antigen also provides for the
identification of areas that would serve as good antigens for the
development of antibodies for use against specific amino acid
sequences of the antigen. Such sequences may be at different
regions such as outside, membrane or inside of the PSM antigen. The
development of these specific antibodies would provide for
immunohistochemical identification of the antigen. These derived
antibodies could then be developed for use, especially ones that
work in paraffin fixed sections as well as frozen section as they
have the greatest utility for immunodiagnosis. 3. Restriction
fragment length polymorphism and genomic DNA
Restriction fragment length polymorphisms (RFLPS) have proven to be
useful in documenting the progression of genetic damage that occurs
during tumor initiation and promotion. It may be that RFLP analysis
will demonstrate that changes in PSM sequence restriction mapping
may provide evidence of predisposition to risk or malignant
potential or progression of the prostatic tumor.
Depending on the chromosomal location of the PSM antigen, the PSM
antigen gene may serve as a useful chromosome location marker for
chromosome analysis. 4. Serum
With the development of antigen specific antibodies, if the antigen
or selected antigen fragments appear in the serum they may provide
for a serum marker for the presence of metastatic disease and be
useful individually or in combination with other prostate specific
markers. 5. Imaging
As the cDNA sequence implies that the antigen has the
characteristics of a membrane spanning protein with the majority of
the protein on the exofacial surface, antibodies, especially
monoclonal antibodies to the peptide fragments exposed and specific
to the tumor may provide for tumor imaging local extension of
metastatic tumor or residual tumor following prostatectomy or
irradiation. The knowledge of the coding region permits the
generation of monoclonal antibodies and these can be used in
combination to provide for maximal imaging purposes. Because the
antigen shares a similarity with the transferrin receptor based on
cDNA analysis (approximately 54%), it may be that there is a
specific normal ligand for this antigen and that identification of
the ligand(s) would provide another means of imaging. 6. Isolation
of ligands
The PSM antigen can be used to isolate the normal ligand(s) that
bind to it. These ligand(s) depending on specificity may be used
for targeting, or their serum levels may be predictive of disease
status. If it is found that the normal ligand for PSM is a carrier
molecule then it may be that PSM could be used to bind to that
ligand for therapy purposes (like an iron chelating substance) to
help remove the ligand from the circulation. If the ligand promotes
tumor growth or metastasis then providing soluble PSM antigen would
remove the ligand from binding the prostate. Knowledge of PSM
antigen structure could lend to generation of small fragment that
binds ligand which could serve the same purpose. 7. Therapeutic
uses
a) Ligands. The knowledge that the cDNA structure of PSM antigen
shares structural homology with the transferrin receptor (54% on
the nucleic acid level) implies that there may be an endogenous
ligand for the receptor that may or may not be transferrin-like.
Transferrin is thought to be a ligand that transports iron into the
cell after binding to the transferrin receptor. However,
apotransferrin is being reported to be a growth factor for some
cells which express the transferrin receptor (30). Whether
transferrin is a ligand for this antigen or some other ligand binds
to this ligand remains to be determined. If a ligand is identified
it may carry a specific substance such as a metal ion (iron or zinc
or other) into the tumor and thus serve as a means to deliver toxic
substances (radioactive or cytotoxic chemical i.e. toxin like ricin
or cytotoxic alkylating agent or cytotoxic prodrug) to the
tumor.
The main metastatic site for prostatic tumor is the bone. The bone
and bone stroma are rich in transferrin. Recent studies suggest
that this microenvironment is what provides the right "soil" for
prostatic metastasis in the bone (31). It may be that this also
promotes attachment as well, these factors which reduce this
ability may diminish prostatic metastasis to the bone and prostatic
metastatic growth in the bone.
It was found that the ligand for the new antigen (thought to be an
oncogene and marker of malignant phenotype in breast carcinoma)
served to induce differentiation of breast cancer cells and thus
could serve as a treatment for rather than promotor of the disease.
It may be that ligand binding to the right region of PSM whether
with natural ligand or with an antibody may serve a similar
function.
Antibodies against PSM antigen coupled with a cytotoxic agent will
be useful to eliminate prostate cancer cells. Transferrin receptor
antibodies with toxin conjugates are cytotoxic to a number of tumor
cells as tumor cells tend to express increased levels of
transferrin receptor (32). Transferrin receptors take up molecules
into the cell by endocytosis. Antibody drug combinations can be
toxic. Transferrin linked toxin can be toxic.
b) Antibodies against PSM antigen coupled with a cytotoxic agent
will be useful to eliminate prostate cancer cells. The cytotoxic
agent may be a radioisotope or toxin as known in ordinary skill of
the art. The linkage of the antibody and the toxin or radioisotope
can be chemical. Examples of direct linked toxins are doxorubicin,
chlorambucil, ricin, pseudomonas exotoxin etc., or a hybrid toxin
can be generated 1/2 with specificity for PSM and the other 1/2
with specificity for the toxin. Such a bivalent molecule can serve
to bind to the tumor and the other 1/2 to deliver a cytotoxic to
the tumor or to bind to and activate a cytotoxic lymphocyte such as
binding to the T.sub.1-T.sub.3 receptor complex. Antibodies of
required specificity can also be cloned into T cells and by
replacing the immunoglobulin domain of the T cell receptor (TcR);
cloning in the desired MAb heavy and light chains; splicing the
U.sub.b and U.sub.L gene segments with the constant regions of the
.alpha. and .beta. TCR chains and transfecting these chimeric
Ab/TcR genes in the patients' T cells, propagating these hybrid
cells and infusing them into the patient (33). Specific knowledge
of tissue specific antigens for targets and generation of MAb's
specific for such targets will help make this a usable approach.
Because the PSM antigen coding region provides knowledge of the
entire coding region, it is possible to generate a number of
antibodies which could then be used in combination to achieve an
additive or synergistic anti-tumor action. The antibodies can be
linked to enzymes which can activate non-toxic prodrugs at its site
of the tumor such as Ab-carboxypeptidase and 4-(bis(2
chloroethyl)amino) benzoyl-.alpha.-glutamic acid and its active
parent drug in mice (34).
It is possible to produce a toxic genetic chimera such as TP-40 a
genetic recombinant that possesses the cDNA from TGF-alpha and the
toxic portion of pseudomonas exotoxin so the TGF and portion of the
hybrid binds the epidermal growth factor receptor (EGFR), and the
pseudomonas portion gets taken up into the cell enzymatically and
inactivates the ribosomes ability to perform protein synthesis
resulting in cell death. When we know the ligand for the PSM
antigen we can do the same.
In addition, once the ligand for the PSM antigen is identified,
toxin can be chemically conjugated to the ligands. Such conjugated
ligands can be therapeutically useful. Examples of the toxins are
daunomycin, chlorambucil, ricin, pseudomonas exotoxin, etc.
Alternatively, chimeric construct can be created linking the cDNA
of the ligand with the cDNA of the toxin. An example of such toxin
is TGF.alpha. and pseudomonas exotoxin (35). 8. Others
The PSM antigen may have other uses. It is well known that the
prostate is rich in zinc, if the antigen provides function relative
to this or other biologic function the PSM antigen may provide for
utility in the treatment of other prostatic pathologies such as
benign hyperplastic growth and/or prostatitis.
Because purified PSM antigen can be generated, the purified PSM
antigen can be linked to beads and use it like a standard
"affinity" purification. Serum, urine or other biological samples
can be used to incubate with the PSM antigen bound onto beads. The
beads may be washed thoroughly and then eluted with salt or pH
gradient. The eluted material is SDS gel purified and used as a
sample for microsequencing. The sequences will be compared with
other known proteins and if unique, the technique of degenerated
PCR can be employed for obtaining the ligand. Once known, the
affinity of the ligand will be determined by standard protocols
(15).
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Isolation of plasma membranes from human skin fibroblasts. J.
Membrane Biology, 36:191-211. 24. Hsu, S. M., et al. (1981)
Comparative study of the immunoperoxidase, anti-peroxidase, and
avidin-biotin complex method for studying polypeptide hormones with
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using standard phenylisothiocyanate reagent and subpicomole high
performance liquid chromatography analysis. Analytical Biochem.
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chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA,
74:5463-5467. 28. Grunstein, M., et al. (1975) Colony hybridization
as a method for the isolation of cloned DNAs that contain a
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A. P., et al. (1983) A technique for radiolabeling DNA restriction
endonuclease fragments to high specific activity. Anal. Biochem,
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mRNAs transferred to diazobenzylomethyl paper from formaldehyde
gels. Nucleic Acids Research, 6:3559. 31. Voeller, H. J., et al.
(1991) v-rasH expression confers hormone-independent in-vitro
growth to LNCaP prostate carcinoma cells. Molec. Endocrinology.
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(1992) Selective stimulation of prostatic carcinoma cell
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E., Purnell, G., and Harwood, S. J. Monoclonal antibodies and
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SECOND SERIES OF EXPERIMENTS
Expression of the Prostate-Specific Membrane Antigen
Applicant's have recently cloned a 2.65 kb complementary DNA
encoding PSM, the prostate-specific membrane antigen recognized by
the 7E11-C5.3 anti-prostate monoclonal antibody.
Immunohistochemical analysis of the LNCaP, DU-145, and PC-3
prostate cancer cell lines for PSM expression using the 7E11-C5.3
antibody reveals intense staining in the LNCaP cells, with no
detectable expression in both the DU-145 and PC-3 cells. Coupled
in-vitro transcription/translation of the 2.65 kb full-length PSM
cDNA yields an 84 kDa protein corresponding to the predicted
polypeptide molecular weight of PSM. Post-translational
modification of this protein with pancreatic canine microsames
yields the expected 100 kDa PSM antigen. Following transfection of
PC-3 cells with the full-length PSM cDNA in a eukaryotic expression
vector applicant's detect expression of the PSM glycoprotein by
Western analysis using the 7E11-C5.3 monoclonal antibody.
Ribonuclease protection analysis demonstrates that the expression
of PSM mRNA is almost entirely prostate-specific in human tissues.
PSM expression appears to be highest in hormone-deprived states and
is hormonally modulated by steroids, with DHT downregulating PSM
expression in the human prostate cancer cell line LNCaP by 8-10
fold, testosterone downregulating PSM by 3-4 fold, and
corticosteroids showing no significant effect. Normal and malignant
prostatic tissues consistently show high PSM expression, whereas we
have noted heterogeneous, and at times absent, expression of PSM in
benign prostatic hyperplasia. LNCaP tumors implanted and grown both
orthotopically and subcutaneously in nude mice, abundantly express
PSM providing an excellent in-vivo model system to study the
regulation and modulation of PSM expression.
EXPERIMENTAL DETAILS
Materials and Methods
Cells and Reagents:
The LNCaP, DU-145, and PC-3 cell lines were obtained from the
American Type Culture Collection. Details regarding the
establishment and characteristics of these cell lines have been
previously published (5A,7A,8A). Unless specified otherwise, LNCaP
cells were grown in RPMI 1640 media supplemented with L-glutamine,
nonessential amino acids, and 5% fetal calf serum (Gibco-BRL,
Gaithersburg, Md.) in a CO.sub.2 incubator at 37C DU-145 and PC-3
cells were grown in minimal essential medium supplemented with 10%
fetal calf serum. All cell media were obtained from the MSKCC Media
Preparation Facility. Restriction and modifying enzymes were
purchased from Gibco-BRL unless otherwise specified.
Immunohistochemical Detection of PSM
We employed the avidin-biotin method of detection to analyze
prostate cancer cell lines for PSM antigen expression (9A). Cell
cytospins were made on glass slides using 5.times.10.sup.4
cells/100 ul per slide. Slides were washed twice with PBS and then
incubated with the appropriate suppressor serum for 20 minutes. The
suppressor serum was drained off and the cells were incubated with
diluted 7E11-CS.3 (5 g/ml) monoclonal antibody for 1 hour. Samples
were then washed with PBS and sequentially incubated with secondary
antibodies for 30 minutes and with avidin-biotin complexes for 30
minutes. Diaminobenzidine served as our chromogen and color
development followed by hematoxylin counter-staining and mounting.
Duplicate cell cytospins were used as controls for each experiment.
As a positive control, the anti-cytokeratin monoclonal antibody CAM
5.2 was used following the same procedure described above. Human EJ
bladder carcinoma cells served as a negative control.
In-Vitro Transcription/Translation of PSM Antigen
Plasmid 55A containing the full length 2.65 kb PSM cDNA in the
plasmid pSPORT 1 (Gibco-BRL) was transcribed in-vitro using the
Promega TNT system (Promega Corp. Madison, Wis.). T7 RNA polymerase
was added to the cDNA in a reaction mixture containing rabbit
reticulocyte lysate, an amino acid mixture lacking methionine,
buffer, and .sup.35S-Methionine (Amersham) and incubated at 30C for
90 minutes. Post-translational modification of the resulting
protein was accomplished by the addition of pancreatic canine
microsomes into the reaction mixture (Promega Corp. Madison, Wis.).
Protein products were analyzed by electrophoresis on 10% SDS-PAGE
gels which were subsequently treated with Amplify autoradiography
enhancer (Amersham, Arlington Heights, Ill.) according to the
manufacturers instructions and dried at 80C in a vacuum dryer. Gels
were autoradiographed overnight at -70C using Hyperfilm MP
(Amersham).
Transfection of PSM into PC-3 Cells
The full length PSM cDNA was subcloned into the pREP7 eukaryotic
expression vector (Invitrogen, San Diego, Calif.). Plasmid DNA was
purified from transformed DH5-alpha bacteria (Gibco-BRL) using
Qiagen maxi-prep plasmid isolation columns (Qiagen Inc.,
Chatsworth, Calif.). Purified plasmid DNA (6.10 g) was diluted with
900 ul of Optimem media (Gibco-BRL) and mixed with 30 ul of
Lipofectin reagent (Gibco-BRL) which had been previously diluted
with 9001 of Optimem media. This mixture was added to T-75 flasks
of 40-50% confluent PC-3 cells in Optimem media. After 24-36 hours,
cells were trypsinized and split into 100 mm dishes containing RPMI
1640 media supplemented with 10% fetal calf serum and 1 mg/ml of
Hygromycin B (Calbiochem, La Jolla, Calif.). The dose of Hygromycin
B used was previously determined by a time course/dose response
cytotoxicity assay. Cells were maintained in this media for 2-3
weeks with changes of media and Hygromycin B every 4-5 days until
discrete colonies appeared. Colonies were isolated using 6 mm
cloning cylinders and expanded in the same media. As a control,
PC-3 cells were also transfected with the pREP7 plasmid alone. RNA
was isolated from the transfected cells and PSM mRNA expression was
detected by both RNase Protection analysis (described later) and by
Northern analysis.
Western Blot Detection of PSM Expression
Crude protein lysates, were isolated from LNCaP, PC-3, and
PSM-transfected PC-3 cells as previously described (10A). LNCaP
cell membranes were also isolated according to published methods
(10A). Protein concentrations were quantitated by the Bradford
method using the BioRad protein reagent kit (BioRad, Richmond,
Calif.). Following denaturation, 20 g of protein was
electrophoresed on a 10% SDS-PAGE gel at 25 mA for 4 hours. Gels
were electroblotted onto Immobilon P membranes (Millipore, Bedford,
Mass.) overnight at 4C. Membranes were blocked in 0.15M NaCl/0.01M
Tris-HCl (TS) plus 5% BSA followed by a 1 hour incubation with
7E11-C5.3 monoclonal antibody (10 g/ml). Blots were washed 4 times
with 0.15M NaCl/0.01M Tris-HCl/0.05% Triton-X 100 (TS-X) and
incubated for 1 hour with rabbit anti-mouse IgG (Accurate
Scientific, Westbury, N.Y.) at a concentration of 10 g/ml.
Blots were then washed 4 times with TS-X and labeled with
.sup.125I-Protein A (Amersham, Arlington Heights, Ill.) at a
concentration of 1 million cpm/ml. Blots were then washed 4 times
with TS-X and dried on Whatman 3MM paper, followed by overnight
autoradiography at -70C using Hyperfilm MP (Amersham).
Orthotopic and Subcutaneous LNCaP Tumor Growth in Nude Mice
LNCaP cells were harvested from sub-confluent cultures by a one
minute exposure to a solution of 0.25% trypsin and 0.02% EDTA.
Cells were resuspended in RPMI 1640 media with 5% fetal bovine
scrum, washed and diluted in either Matrigel (Collaborative
Biomedical Products, Bedford, Mass.) or calcium and magnesium-free
Hank's balanced salt solution (HBSS). Only single cell suspensions
with greater than 90% viability by trypan blue exclusion were used
for in vivo injection. Male athymic Swiss (nu/nu) nude mice 4-6
weeks of age were obtained from the Memorial Sloan-Kettering Cancer
Center Animal Facility. For subcutaneous tumor cell injection one
million LNCaP cells resuspended in 0.2 mils. of Matrigel were
injected into the hindlimb of each mouse using a disposable syringe
fitted with a 28 gauge needle. For orthotopic injection, mice were
first anesthetized with an intraperitoneal injection of
Pentobarbital and placed in the supine position. The abdomen was
cleansed with Betadine and the prostate was exposed through a
midline incision. 2.5 million LNCaP tumor cells in 0.1 ml. were
injected directly into either posterior lobe using a 1 ml
disposable syringe and a 28 gauge needle. LNCaP cells with and
without Matrigel were injected. Abdominal closure was achieved in
one layer using Autoclip wound clips (Clay Adams, Parsippany,
N.J.). Tumors were harvested in 6-8 weeks, confirmed histologically
by faculty of the Memorial Sloan-Kettering Cancer Center Pathology
Department, and frozen in liquid nitrogen for subsequent RNA
isolation.
RNA Isolation
Total cellular RNA was isolated from cells and tissues by standard
techniques (11,12) as well as by using RNAzol B (Cinna/Biotecx,
Houston, Tex.). RNA concentrations and quality were assessed by UV
spectroscopy on a Beckman DU 640 spectrophotometer and by gel
analysis. Human tissue total RNA samples were purchased from
Clontech Laboratories, Inc., Palo Alto, Calif.
Ribonuclease Protection Assays
A portion of the PSM cDNA was subcloned into the plasmid vector
pSPORT 1 (Gibco-BRL) and the orientation of the cDNA insert
relative to the flanking T7 and SP6 RNA polymerase promoters was
verified by restriction analysis. Linearization of this plasmid
upstream of the PSM insert followed by transcription with SP6 RNA
polymerase yields a 400 nucleotide antisense RNA probe, of which
350 nucleotides should be protected from RNase digestion by PSM
RNA. This probe was used in FIG. 20. Plasmid IN-20, containing a 1
kb partial PSM cDNA in the plasmid pCR II (Invitrogen) was also
used for riboprobe synthesis. IN-20 linearized with Xmn I
(Gibco-BRL) yields a 298 nucleotide anti-sense RNA probe when
transcribed using SP6 RNA polymerase, of which 260 nucleotides
should be protected from RNase digestion by PSM mRNA. This probe
was used in FIGS. 21 and 22. Probes were synthesized using SP6 RNA
polymerase (Gibco-BRL), rNTPs (Gibco-BRL) , RNAsin (Promega), and
.sup.32P-rCTP (NEN, Wilmington, Del.) according to published
protocols (13). Probes were purified over NENSORB 20 purification
columns (NEN) and approximately 1 million cpm of purified,
radiolabeled PSM probe was mixed with 10 g of each RNA and
hybridized overnight at 45C using buffers and reagents from the RPA
II kit (Ambion, Austin, Tex.). Samples were processed as per
manufacturer's instructions and analyzed on 5% polyacrilamide/7M
urea denaturing gels using Seq ACRYL reagents (ISS, Natick, Mass.).
Gels were pre-heated to 55C and run for approximately 1-2 hours at
25 watts. Gels were then fixed for 30 minutes in 10% methanol/10%
acetic acid, dried onto Whatman 3MM paper at 80C in a BioRad vacuum
dryer and autoradiographed overnight with Hyperfilm MP (Amersham)).
Quantitation of PSM expression was determined by using a scanning
laser densitometer (LKB, Piscataway, N.J.).
Steroid Modulation Experiment
LNCaP cells (2 million) were plated onto T-75 flasks in RPMI 1640
media supplemented with 5% fetal calf serum and grown 24 hours
until approximately 30-40% confluent. Flasks were then washed
several times with phophate-buffered saline and RPMI medium
supplemented with 5T charcoal-extracted scrum was added. Cells were
then grown for another 24 hours, at which time dihydrotesterone,
testosterone, estradiol, progesterone, and dexamethasone
(Steraloids Inc., Wilton, N.H.) were added at a final concentration
of 2 nM. Cells were grown for another 24 hours and RNA was then
harvested as previously described and PSM expression analyzed by
ribonuclease protection analysis.
Experimental Results
Immunohistochemical Detection of PSM:
Using the 7E11-C5.3 anti-PSM monoclonal antibody, PSM expression is
clearly detectable in the LNCaP prostate cancer cell line, but not
in the PC-3 and DU-145 cell lines (FIG. 17) in agreement with
previously published results (4A). All normal and malignant
prostatic tissues analyzed stained positively for PSM expression
(unpublished data).
In-Vitro Transcription/Translation of PSM Antigen:
As shown in FIG. 18, coupled in-vitro transcription/translation of
the 2.65 kb full-length PSM cDNA yields an 84 kDa protein species
in agreement with the expected protein product from the 750 amino
acid PSM open reading frame. Following post-translational
modification using pancreatic canine microsomes we obtained a 100
kDa glycosylated protein species consistent with the mature, native
PSM antigen.
Detection of PSM Antigen in LNCaP Cell Membranes and Transfected
PC-3 Cells:
PC-3 cells transfected with the full length PSM cDNA in the pREP7
expression vector were assayed for expression of SM mRNA by
Northern analysis (data not shown). A clone with high PSM mRNA
expression was selected for PSM antigen analysis by Western
blotting using the 7E11-C5.3 antibody. In FIG. 19, the 100 kDa PSM
antigen is well expressed in LNCaP cell lysate and membrane
fractions, as well as in PSM-transfected PC-3 cells but not in
native PC-3 cells. This detectable expression in the transfected
PC-3 cells proves that the previously cloned 2.65 kb PSM cDNA
encodes the antigen recognized by the 7E11-C5.3 anti-prostate
monoclonal antibody and that the antigen is being appropriately
glycosylated in the PC-3 cells, since the antibody recognizes a
carbohydrate-containing epitope on PSM.
PSH mRNA Expressions
Expression of PSM mRNA in normal human tissues was analyzed using
ribonuclease protection assays. Tissue expression of PSM appears
predominantly within the prostate, with very low levels of
expression detectable in human brain and salivary gland (FIG. 20).
No detectable PSM mRNA expression was evident in non-prostatic
human tissues when analyzed by Northern analysis (data not shown).
We have also noted on occasion detectable PSM expression in normal
human small intestine tissue, however this mRNA expression is
variable depending upon the specific riboprobe used (data not
shown). All samples of normal human prostate and human prostatic
adenocarcinoma assayed have revealed clearly detectable PSM
expression, whereas we have noted generally decreased or absent
expression of PSM in tissues exhibiting benign hyperplasia (FIG.
21). In human LNCaP tumors grown both orthotopically and
subcutaneously in nude mice we detected abundant PSM expression
with or without the use of matrigel, which is required for the
growth of subcutaneously implanted LNCaP cells (FIG. 21). PSM mRNA
expression is distinctly modulated by the presence of steroids in
physiologic doses (FIG. 22). DHT downregulated expression by 8-10
fold after 24 hours and testosterone diminished PSM expression by
3-4 fold. Estradiol and progesterone also downregulated PSM
expression in LNCaP cells, perhaps as a result of binding to the
mutated androgen receptor known to exist in the LNCaP cell.
Overall, PSM expression is highest in the untreated LNCaP cells
grown in steroid-depleted media, a situation that we propose
simulates the hormone-deprived (castrate) state in-vivo. This
experiment was repeated at steroid dosages ranging from 2-200 nM
and at time points from 6 hours to 7-days with similar results;
maximal downregulation of PSM mRNA was seen with DHT at 24 hours at
doses of 2-20 nM.
Experimental Discussion
In order to better understand the biology of the human prostate in
both normal and neoplastic states, we need to enhance our knowledge
by studying the various proteins and other features that are unique
to this important gland. Previous research has provided two
valuable prostatic biomarkers, PAP and PSA, both of which have had
a significant impact on the diagnosis, treatment, and management of
prostate malignancies. Our present work describing the preliminary
characterization of the prostate-specific membrane antigen (PSM)
reveals it to be a gene with many interesting features. PSM is
almost entirely prostate-specific as are PAP and PSA, and as such
may enable further delineation of the unique functions and behavior
of the prostate. The predicted sequence of the PSM protein (3) and
its presence in the LNCaP cell membrane as determined by Western
blotting and immunohistochemistry, indicate that it is an integral
membrane protein. Thus, PSM provides an attractive cell surface
epitope for antibody-directed diagnostic imaging and cytotoxic
targeting modalities (14). The ability to synthesize the PSM
antigen in-vitro and to produce tumor xenografts maintaining high
levels of PSM expression provides us with a convenient and
attractive model system to further study and characterize the
regulation and modulation of PSM expression. Also, the high level
of PSM expression in the LNCaP cells provides an excellent in-vitro
model system. Since PSM expression is hormonally-responsive to
steroids and may be highly expressed in hormone-refractory disease
(15), it is imperative to elucidate the potential role of PSM in
the evolution of androgen-independent prostate cancer. The
detection of PSM mRNA expression in minute quantities in brain,
salivary gland, and small intestine warrants further investigation,
although these tissues were negative for expression of PSM antigen
by immunohistochemistry using the 7E11-C5.3 antibody (16). In all
of these tissues, particularly small-intestine, we detected mRNA
expression using a probe corresponding to a region of the PSM cDNA
near the 3' end, whereas we were unable to detect expression when
using a 5' end PSM probe. These results may indicate that the PSM
mRNA transcript undergoes alternative splicing in different
tissues. Previous protein studies have suggested that the 7E11-C5.3
antibody may actually detect two other slightly larger protein
species in addition to the 100 kDa PSM antigen (17). These other
protein species can be seen in the LNCaP lysate and membrane
samples in FIG. 19. Possible origins of these proteins include
alternatively spliced PSM mRNA, other genes distinct from but
closely related to PSM, or different post-translational
modifications of the PSM protein. We are currently investigating
these possibilities.
Applicnat's approach is based on prostate tissue specific
promotor:enzyme or cytokine chimeras. We will examine promotor
specific activation of prodrugs such as non toxic gancyclovir which
is converted to a toxic metabolite by herpes simplex thymidine
kinase or the prodrug 4-(bis (2chloroethyl)amino)benzoyl-1-glutamic
acid to the benzoic acid mustard alkylating agent by the
pseudomonas carboxy peptidase G2. As these drugs are activated by
the enzyme (chimera) specifically in the tumor the active drug is
released only locally in the tumor environment, destroying the
surrounding tumor cells. We will also examine the promotor specific
activation of cytokines such as IL-12, IL-2 or GM-CSF for
activation and specific antitumor vaccination. Lastly the tissue
specific promotor activation of cellular death genes may also prove
to be useful in this area.
Gene Therapy Chimeras
The establishment of "chimeric DNA" for gene therapy requires the
joining of different segments of DNA together to make a new DNA
that has characteristics of both precursor DNA species involved in
the linkage. In this proposal the two pieces being linked involve
different functional aspects of DNA, the promotor region which
allows for the reading of the DNA for the formation of mRNA will
provide specificity and the DNA sequence coding for the mRNA will
provide for therapeutic functional DNA.
DNA-Specified Enzyme or Cytokine mRNA:
When effective, antitumor drugs can cause the regression of very
large amounts of tumor. The main requirements for antitumor drug
activity is the requirement to achieve both a long enough time (t)
and high enough concentration (c) (c.times.t) of exposure of the
tumor to the toxic drug to assure sufficient cell damage for cell
death to occur. The drug also must be "active" and the toxicity for
the tumor greater than for the hosts normal cells (22). The
availability of the drug to the tumor depends on tumor blood flow
and the drugs diffusion ability. Blood flow to the tumor does not
provide for selectivity as blood flow to many normal tissues is
often as great or greater than that to the tumor. The majority of
chemotherapeutic cytotoxic drugs are often as toxic to normal
tissue as to tumor tissue. Dividing cells are often more sensitive
than non-dividing normal cells, but in many slow growing solid
tumors such as prostatic cancer this does not provide for antitumor
specificity (22).
Previously a means to increase tumor specificity of antitumor drugs
was to utilize tumor associated enzymes to activate nontoxic
prodrugs to cytotoxic agents. (19). A problem with this approach
was that most of the enzymes found in tumors were not totally
specific in their activity and similar substrate active enzymes or
the same enzyme at only slightly lower amounts was found in other
tissue and thus normal tissues were still at risk for damage.
To provide absolute specificity and unique activity, viral,
bacterial and fungal enzymes which have unique specificity for
selected prodrugs were found which were not present in human or
other animal cells. Attempts to utilize enzymes such as herpes
simplex thymidine kinase, bacterial cytosine deaminase and
carboxypeptidase G-2 were linked to antibody targeting systems with
modest success (19). Unfortunately, antibody targeted enzymes limit
the number of enzymes available per cell. Also, most antibodies do
not have a high tumor target to normal tissue ratio thus normal
tissues are still exposed reducing the specificity of these unique
enzymes. Antibodies are large molecules that have poor diffusion
properties and the addition of the enzymes molecular weight further
reduces the antibodies diffusion.
Gene therapy could produce the best desired result if it could
achieve the specific expression of a protein in the tumor and not
normal tissue in order that a high local concentration of the
enzyme be available for the production in the tumor environment of
active drug (21).
Cytokines:
Applicant's research group has demonstrated that Applicant's can
specifically and non-toxically "cure" an animal of an established
tumor, in models of bladder or prostate cancer. The prostate cancer
was the more difficult to cure especially if it was grown
orthotopically in the prostate.
Our work demonstrated that tumors such as the bladder and prostate
were not immunogenic, that is the administration of irradiated
tumor cells to the animal prior to subsequent administration of
non-irradiated tumor cells did not result in a reduction of either
the number of tumor cells to produce a tumor nor did it reduce the
growth rate of the tumor. But if the tumor was transfected with a
retrovirus and secreted large concentrations of cytokines such as
Il-2 then this could act as an antitumor vaccine and could also
reduce the growth potential of an already established and growing
tumor. IL-2 was the best, GM-CSF also had activity whereas a number
of other cytokines were much less active. In clinical studies just
using IL-2 for immunostimulation, very large concentrations had to
be given which proved to be toxic. The key to the success of the
cytokine gene modified tumor cell is that the cytokine is produced
at the tumor site locally and is not toxic and that it stimulates
immune recognition of the tumor and allows specific and non toxic
recognition and destruction of the tumor. The exact mechanisms of
how IL-2 production by the tumor cell activates immune recognition
is not fully understood, but one explanation is that it bypasses
the need for cytokine production by helper T cells and directly
stimulates tumor antigen activated cytotoxic CD8 cells. Activation
of antigen presenting cells may also occur.
Tissue Promotor-Specific Chimera DNA Activation
Non-Prostatic Tumor Systems:
It has been observed in non-prostatic tumors that the use of
promotor specific activation can selectively lead to tissue
specific gene expression of the transfected gene. In melanoma the
use of the tyrosinase promotor which codes for the enzyme
responsible for melanin expression produced over a 50 fold greater
expression of the promotor driven reporter gene expression in
melanoma cells and not non melanoma cells. Similar specific
activation was seen in the melanoma cells transfected when they
were growing in mice. In that experiment no non-melanoma or
melanocyte cell expressed the tyrosinase drive reporter gene
product. The research group at Welcome Laboratories have cloned and
sequenced the promoter region of the gene -coding for
carcinoembryonic antigen (CEA). CEA is expressed on colon and colon
carcinoma cells but specifically on metastatic cytosine deaminase
which converts 5 flurorocytosine into 5 fluorouracil and observed a
large increase in the ability to selectively kill CEA promotor
driven colon tumor cells but non dividing not dividing normal liver
cells. In vivo they observed that bystander tumor cells which were
not transfected with the cytosine deaminase gene were also killed,
and that there was no toxicity to the host animal as the large
tumors were regressing following treatment. Herpes simplex virus,
(HSV), thymidine kinase similarly activates the prodrug gancyclovir
to be toxic towards dividing cancer cells and HSV thymidine kinase
has been shown to be specifically activatable by tissue specific
promoters.
Prostatic Tumor Systems:
The therapeutic key to effective cancer therapy is to achieve
specificity and spare the patient toxicity. Gene therapy may
provide a key part to specificity in that non-essential tissues
such as the prostate and prostatic tumors produce tissue specific
proteins, such as acid phosphatase (PAP), prostate specific antigen
(PSA), and a gene which we cloned, prostate-specific membrane
antigen (PSM). Tissues such as the prostate contain selected tissue
specific transcription factors which are responsible for binding to
the promoter region of the DNA of these tissue specific mRNA. The
promoter for PSA has been cloned and we are investigating its use
as a prostate specific promotor for prostatic tumor cells. Usually
patients who are being treated for metastatic prostatic cancer have
been put on androgen deprivation therapy which dramatically reduces
the expression of mRNA for PSA. PSM on the other hand increases in
expression with hormone deprivation which-means it would be even
more intensely expressed on patients being treated with hormone
therapy. Preliminary work in collaboration with Dr. John Isaacs'
Laboratory demonstrates that PSM is expressed when the human
chromosome region containing the human PSM gene is transferred to
the rat tumor AT-6. AT-6 is a metastatic androgen independent
tumor. The same chromosome transferred into non prostate derived
tissues or tumors is not expressed and thus these cells could be
used as an animal model for these experiments. PSA, PSM positive
Huan LNCaP cells will be used for testing in nude mice.
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Abdel-Nabi, H., Wright, G. L., Gulfo, J. V., Petrylak, D. P., Neal,
C. E., Texter, J. E., Begun, F. P., Tyson, I., Heal, A., Mitchell,
E., Purnell, G.. and Harwood, S. J. Monoclonal antibodies and
radioimmunoconjugates in the diagnosis and treatment of prostate
cancer. Semin. Urol., 10: 45-54, 1992. 7. Stone, K. R., Mickey, D.
D., Wunderli, H.. Mickey, G. H., and Paulson, D. F. Isolation of a
human prostate carcinoma cell line (DU-145). Int. J. Cancer, 22:
274-281, 1978. 8. Kaign, M. E.. Narayan, K. S., Ohnuki, Y., and
Lechner, J. F. Establishment and characterization of a human
prostatic carcinoma cell line (PC-3). Invest. Urol., 17: 16-23,
1979. 9. Hsu, S. M., Raine, L., and Panger, H. Review of present
methods of immunohistochemical detection. Am. J. Clin. Path. 75:
734-738, 1981. 10. Harlow, E., and Lane, D. Antibodies: A
Laboratory Manual. New York: Cold Spring Harbor Laboratory, p. 449.
1968. 11. Glisin, V, Crkvenjakov, R., and Byus, C. Ribonucleic acid
isolated by cesium chloride centrifugation. Biochemistry, 13:
2633-2637, 1974. 12. Aviv, H., and Leder, P. Purification of
biologically active globin messenger RNA by chromotography on
oligothymidylic acid cellulose. Proc. Natl. Acad. Sci. USA, 69:
1408-1412, 1972. 13. Melton, D. A., Krieg, P. A., Rebagliati, M.
R., Maniatis, T. A., Zinn, K., and Careen, M. R. Efficient in-vitro
synthesis of biologically active RNA and RNA hybridization probes
from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids.
Res. 12: 7035-7056, 1984. 14. Personal Communication from Cytogen
Corporation, Princeton, N.J. 15. Axelrod, H. R., Gilman, S. C.,
D'Aleo, C. J., Petrylak, D., Reuter, V., Gulfo, J. V., Saad, A.,
Cordon-Cardo, C., and Scher, H. I. Preclinical results and human
immunohistochemical studies with .sup.90Y-CYT-356; a new prostatic
cancer therapeutic agent. AUA Proceedings, Abstract 596, 1992. 16.
Lopes, A. D., Davis, W. L., Rosenstraus, M. J., Uveges, A. J., and
Gilman, S. C. Immunohistochemical and pharmacokinetic
characterization of the site-specific immunoconjugate CYT-356
derived from antiprostate monoclonal antibody 7E11-C5. Cancer Res.,
50: 6423-6429, 1990. 17. Troyer, J. K.. Qi, F., Beckett, M. L.,
Morningstar, M. M., and Wright, G. L. Molecular characterization of
the 7E11-CS prostate tumor-associated antigen. AUA Proceedings.
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strategies for human gene therapy. FEBS. 223:212-225. 19. Antonie,
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established murine melanomas following direct intratumoral
injection of DNA. Cancer Res. 53:3860-3864, 1993.
THIRD SERIES OF EXPERIMENTS
Sensitive Detection of Prostatic Hematogenous Micrometastases Using
PSA and PSM-Derived Primers in the Polymerase Chain Reaction
We have developed a PCR-based assay enabling sensitive detection of
hematogenous micrometastases in patients with prostate cancer. We
performed "nested PCR", amplifying mRNA sequences unique to
prostate-specific antigen and to the prostate-specific membrane
antigen, and have compared their respective results.
Micrametastases were detected in 2/30 patients (6.7%) by PCR with
PSA-derived primers, while PSM-derived primers detected tumor cells
in 19/16 patients (63.3%). All 8 negative controls were negative
with both PSA and PSM PCR. Assays were repeated to confirm results,
and PCR products were verified by DNA sequencing and Southern
analysis. Patients harboring circulating prostatic tumor cells as
detected by PSM, and not by PSA-PCR included 4 patients previously
treated with radical prostatectomy and with non-measurable serum
PSA levels at the time of this assay. The significance of these
findings with respect to future disease recurrence and progression
will be investigated.
Improvement in the overall survival of patients with prostate
cancer will depend upon earlier diagnosis. Localized disease,
without evidence of extra-prostatic spread, is successfully treated
with either radical prostatectomy or external beam radiation, with
excellent long-term results (2,3). The major problem is that
approximately two-thirds of men diagnosed with prostate cancer
already have evidence of advanced extra-prostatic spread at the
time of diagnosis, for which there is at present no cure (4). The
use of clinical serum markers such as prostate-specific antigen
(PSA) and prostatic acid phosphatase (PAP) have enabled clinicians
to detect prostatic carcinomas earlier and provide useful
parameters to follow responses to therapy (5). Yet, despite the
advent of sensitive serum PSA assays, radionuclide bone scans, CT
scans and other imaging modalities, we are still unable to detect
the presence of micrometastatic cells prior to their establishment
of solid metastases. Previous work has been done utilizing the
polymerase chain reaction to amplify mRNA sequences unique to
breast, leukemia, and other malignant cells in the circulation and
enable early detection of micrometastases (6,7). Recently, a
PCR-based approach utilizing primers derived from the PSA DNA
sequence was published (8). In this study 3/12 patients with
advanced, stage D prostate cancer had detectable hematogenous
micrometastases.
We have recently identified and cloned a 2.65 kb cDNA encoding the
100 kDa prostate-specific membrane antigen (PSM) recognized by the
anti-prostate monoclonal antibody 7E11-CS.3 (9). PSM appears to be
an integral membrane glycoprotein which is very highly expressed in
prostatic tumors and metastases and is almost entirely
prostate-specific (10). Many anaplastic tumors and bone metastases
have variable and at times no detectable expression of PSA, whereas
these lesions appear to consistently express high levels of PSM.
Prostatic tumor cells that escape from the prostate gland and enter
the circulation are likely to have the potential to form metastases
and are possibly the more aggressive and possibly anaplastic cells,
a population of cells that may not express high levels of PSA, but
may retain high expression of PSM. We therefore chose to utilize
DNA primers derived from the sequences of both PSA and PSM in a PCR
assay to detect micrometastatic cells in the peripheral
circulation. -Despite the high level of amplification and
sensitivity of conventional RNA PCR, we have utilized a "nested"
PCR approach in which we first amplify a target sequence, and
subsequently use this PCR product as the template for another round
of PCR amplification with a new set of primers totally contained
within the sequence of the previous product. This approach has
enabled us to increase our level of detection from one prostatic
tumor cell per 10,000 cells to better than one cell per ten million
cells.
EXPERIMENTAL DETAILS
Materials and Methods
Cells and Reagents:
LNCaP and MCF-7 cells were obtained from the American Type Culture
Collection (Rockville, Md.). Details regarding the establishment
and characteristics of these cell lines have been previously
published (11,12). Cells were grown in RPMI 1640 media supplemented
with L-glutamine, nonessential amino acids, obtained from the MSKCC
Media Preparation Facility, and 5% fetal calf serum (Gibco-BRL,
Gaithersburg, Md.) in a CO.sub.2 incubator at 37C. All cell media
was obtained from the MSKCC Media Preparation Facility. Routine
chemical reagents were of the highest grade possible and were
obtained from Sigma Chemical Company, St. Louis, Mo.
Patient Blood Specimens
All blood specimens used in this study were from patients seen in
the outpatient offices of urologists on staff at MSKCC. Two
anti-coagulated (purple top) tubes per patient were obtained at the
time of their regularly scheduled blood draws. Specimen procurement
was conducted as per the approval of the MSKCC Institutional Review
Board. Samples were promptly brought to the laboratory for
immediate processing. Serum PSA and PAP determinations were
performed by standard techniques by the MSKCC Clinical Chemistry
Laboratory. PSA determinations were performed using the Tandem PSA
assay (Hybritech, San Diego, Calif.). The eight blood specimens
used as negative controls were from 2 males with normal scrum PSA
values and biopsy-proven BPH, one healthy female, 3 healthy males,
one patient with bladder cancer, and one patient with acute
promyelocytic leukemia.
Blood Sample Processing/RNA Extraction
4 ml of whole anticoagulated venous blood was mixed with 3 ml of
ice cold phosphate buffered saline and then carefully layered atop
8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in a 15-ml polystyrene
tube. Tubes were centrifuged at 200 .times. g for 30 min. at 4C.
Using a sterile pasteur pipette, the buffy coat layer (approx. 1
ml.) was carefully removed and rediluted up to 50 ml with ice cold
phosphate buffered saline in a 50 ml polypropylene tube. This tube
was then centrifuged at 2000 .times. g for 30 min at 4C. The
supernatant was carefully decanted and the pellet was allowed to
drip dry. One ml of RNazol B was then added to the pellet and total
RNA was isolated as per manufacturers directions (Cinna/Biotecx,
Houston, Tex.). RNA concentrations and purity were determined by UV
spectroscopy on a Beckman DU 640 spectrophotometer and by
gelanalysis.
Determination of PCR Sensitivity
RNA was isolated from LNCaP cells and from mixtures of LNCaP and
MCF-7 cells at fixed ratios (i.e. 1:100, 1:1000, etc.) using RNAzol
B. Nested PCR was then performed as described below with both PSA
and PSM primers in order to determine the limit of detection for
the assay. LNCaP:MCF-7 (1:100,000) cDNA was diluted with distilled
water to obtain concentrations of 1:1.000,000 and 1:10,000,000.
MCF-7 cells were chosen because they have been previously tested
and shown not to express PSM by PCR.
Polymerase Chain Reaction
The PSA outer primers used span portions of exons 4 and 5 to yield
a 486 bp PCR product and enable differentiation between cDNA and
possible contaminating genomic DNA amplification. The upstream
primer sequence beginning at nucleotide 494 in PSA cDNA sequence is
5'-TACCCACTGCATCAGGAACA-3' (SEQ. ID. No. 39) and the downstream
primer at nucleotide 960 is 5'-CCTTGAAGCACACCATTACA-3' (SEQ. ID.
No. 40). The PSA inner upstream primer (beginning at nucleotide
559) 5'-ACACAGGCCAGGTATTTCAG-3' (SEQ. ID. No. 41) and the
downstream primer (at nucleotide 894) 5'-GTCCAGCGTCCAGCACACAG-3'
(SEQ. ID. No. 42) yield a 355 bp PCR product. All primers were
synthesized by the MSKCC Microchemistry Core Facility. 5 g of total
RNA was reverse-transcribed into cDNA in a total volume of 201
using Superscript reverse transcriptase (Gibco-BRL) according to
the manufacturers recommendations. 11 of this cDNA served as the
starting template for the outer primer PCR reaction. The 201 PCR
mix included: 0.5U Taq polymerase (Promega Corp., Madison, Wis.),
Promega reaction buffer, 1.5MM MgCl.sub.2, 200M dNTPs, and 1.0M of
each primer. This mix was then transferred to a Perkin Elmer 9600
DNA thermal cycler and incubated for 25 cycles. The PCR profile was
as follows: 94C.times.15 sec., 60C.times.15 sec., and 72C for 45
sec. After 25 cycles, samples were placed on ice, and 11 of this
reaction mix served as the template for another round of PCR using
the inner primers. The first set of tubes were returned to the
thermal cycler for 25 additional cycles. PSM-PCR required the
selection of primer pairs that also spanned an intron in order to
be certain that cDNA and not genomic DNA were being amplified.
Since the genomic DNA sequence of PSM has not yet been determined,
this involved trying different primer pairs until a pair was found
that produced the expected size PCR product when cDNA was
amplified, but with no band produced from a genomic DNA template,
indicating the presence of a large intron. The PSM outer primers
yield a 946 bp product and the inner primers a 434 bp product. The
PSM outer upstream primer used was 5'-ATGGTGTTTGTGGTATTACC-3' (SEQ.
ID. No. 43) (beginning at nucleotide 1401) and the downstream
primer (at nucleotide 2348) was 5'-TGCTTGGAGCATAGATGACATGC-3' (SEQ.
ID. No. 44) The PSM inner upstream primer (at nucleotide 1581) was
5'-ACTCCTTCAAGAGCGTGGCG-3' (SEQ. ID. No. 45) and the downstream
primer (at nucleotide 2015) was 5'-AACACCATCCCTCCTCGAACC-3' (SEQ.
ID. No. 46). cDNA used was the same as for the PSA assay. The 501
PCR mix included: 1U Tag Polymerase (Promega), 250M dNTPs, 10mM
-mercaptoethanol, 2 mM MgCl.sub.2, and 51 of a 10.times. buffer mix
containing: 166mM NH.sub.4SO.sub.4, 670mM Tris pH 8.8, and 2 mg/ml
of acetylated BSA. PCR was carried out in a Perkin Elmer 480 DNA
thermal cycler with the following parameters: 94C.times.4 minutes
for 1 cycle, 94C.times.30 sec., 58C.times.1 minute, and 72C.times.1
minute for 25 cycles, followed-by 72C.times.10 minutes. Samples
were then iced and 21 of this reaction mix was used as the template
for another 25 cycles with a new reaction mix containing the inner
PSM primers. cDNA quality was verified by performing control
reactions using primers derived from -actin yielding a 446 bp PCR
product. The upstream primer used was 5'-AGGCCAACCGCGAGAAGATGA-3'
(SEQ. ID. No. 47) (exon 3) and the downstream primer was
5'-ATGTCCACTGGGGAAGC-3' (SEQ. ID. No. 48) (exon 4). The entire PSA
mix and 101 of each PSM reaction mix were run on 1.5-2% agarose
gels, stained with ethidium bromide and photographed in an Eagle
Eye Video Imaging System (Stratagene, Torrey Pines, Calif.). Assays
were repeated at least 3 times to verify results.
Cloning and Sequencing of PCR Products
PCR products were cloned into the pCR II plasmid vector using the
TA cloning system (Invitrogen). These plasmids were transformed
into competent E. coli cells using standard methods (13) and
plasmid DNA was isolated using Magic Minipreps (Promega) and
screened by restriction analysis. TA clones were then sequenced by
the dideoxy method (14) using Sequenase (U.S. Biochemical). 3-4g of
each plasmid was denatured with NaOH and ethanol precipitated.
Labeling reactions were carried out according to the manufacturers
recommendations using .sup.35S-dATP (NEN), and the reactions were
terminated as discussed in the same protocol. Sequencing products
were then analyzed on 6% polyacrilamide/7M urea gels run at 120
watts for 2 hours. Gels were fixed for 20 minutes in 10%
methanol/10% acetic acid, transferred to Whatman 3MM paper and
dried down in a vacuum dryer for 2 hours at 80C. Gels were then
autoradiographed at room temperature for 18 hours.
Southern Analysis
Ethidium-stained agarose gels of PCR products were soaked for 15
minutes in 0.2N HCl, followed by 30 minutes each in 0.5N NaOH/1.5M
NaCl and 0.1M Tris pH 7.5/1.5M NaCl. Gels were then equilibrated
for 10 minutes in 10.times. SSC (1.5M NaCl/0.15M Sodium Citrate.
DNA was transferred onto Nytran nylon membranes (Schleicher and
Schuell) by pressure blotting in 10.times. SSC with a Posi-blotter
(Stratagene). DNA was cross-linked to the membrane using a UV
Stratalinker (Stratagene). Blots were pre-hybridized at 65C for 2
hours and subsequently hybridized with denatured .sup.32P-labeled,
random-primed cDNA probes (either PSM or PSA) (9,15). Blots were
washed twice in 1.times. SSPE/0.5% SDS at 42C and twice in
0.1.times. SSPE/0.5% SDS at SOC for 20 minutes each. Membranes were
air-dried and autoradiographed for 30 minutes to 1 hour at -70C
with Kodak X-Omat film.
Experimental Results
Our technique of PCR amplification with nested primers improved our
level of detection of prostatic cells from approximately one
prostatic cell per 10,000 MCF-7 cells to better than one cell per
million MCF-7 cells, using either PSA or PSM-derived primers (FIGS.
26 and 27). This represents a substantial improvement in our
ability to detect minimal disease. Characteristics of the 16
patients analyzed with respect to their clinical stage, treatment,
serum PSA and PAP values, and results of our assay are shown in
table I. In total, PSA-PCR detected tumor cells in 2/30 patients
(6.7%), whereas PSM-PCR detected cells in 19/30 patients (63.3%).
There were no patients positive for tumor cells by PSA and not by
PSM, while PSM provided 8 positive patients not detected by PSA.
Patients 10 and 11 in table 1, both with very advanced
hormone-refractory disease were detected by both PSA and PSM. Both
of these patients have died since the time these samples were
obtained. Patients 4, 7, and 12, all of whom were treated with
radical prostatectomies for clinically localized disease, and all
of whom have non-measurable serum PSA values 1-2 years
postoperatively were positive for circulating prostatic tumor cells
by PSM-PCR, but negative by PSA-PCR. A representative ethidium
stained gel photograph for PSM-PCR is shown in FIG. 28. Samples run
in lane A represent PCR products generated from the outer primers
and samples in lanes labeled B are products of inner primer pairs.
The corresponding PSM Southern blot autoradiograph is shown in FIG.
29. The sensitivity of the Southern blot analysis exceeded that of
ethidium staining, as can be seen in several samples where the
outer product is not visible on FIG. 28, but is detectable by
Southern blotting as shown in FIG. 29. In addition, sample 3 on
FIGS. 28 and 29 (patient 6 in FIG. 30) appears to contain both
outer and inner bands that are smaller than the corresponding bands
in the other patients. DNA sequencing has confirmed that the
nucleotide sequence of these bands matches that of PSM, with the
exception of a small deletion. This may represent either an
artifact of PCR, alternative splicing of PSM mRNA in this patient,
or a PSM mutation. We have noted similar findings with other
samples on several occasions (unpublished data). All samples
sequenced and analyzed by Southern analysis have been confirmed as
true positives for PSA and PSM.
Experimental Details
The ability to accurately stage patients with prostate cancer at
the time of diagnosis is clearly of paramount importance in
selecting appropriate therapy and in predicting long-term response
to treatment, and potential cure. Pre-surgical staging presently
consists of physical examination, serum PSA and PAP determinations,
and numerous imaging modalities including transrectal
ultrasonography, CT scanning, radionuclide bone scans, and even MRI
scanning. No present modality, however, addresses the issue of
hematogenous micrometastatic disease and the potential negative
impact on prognosis that this may produce. Previous work has shown
that only a fractional percentage of circulating tumor cells will
inevitably go on to form a solid metastasis (16), however, the
detection of and potential quantification of circulating tumor cell
burden may prove valuable in more accurately staging disease. The
long-term impact of hematogenous micrometastatic disease must be
studied by comparing the clinical courses of patients found to have
these cells in their circulation with patients of similar stage and
treatment who test negatively.
The significantly higher level of detection of tumor cells with PSM
as compared to PSA is not surprising to us, since we have noted
more consistent expression of PSM in prostate carcinomas of all
stages and grades as compared to variable expression of PSA in more
poorly differentiated and anaplastic prostate cancers. We were
surprised to detect tumor cells in the three patients that had
undergone radical prostatectomies with subsequent undetectable
amounts of serum PSA. These patients would be considered to be
surgical "cures" by standard criteria, yet they apparently continue
to harbor prostatic tumor cells. It will be interesting to follow
the clinical course of these patients as compared to others without
PCR evidence of residual disease. We are presently analyzing larger
numbers of patient samples in order to verify these findings and
perhaps identify patients at risk for metastatic disease.
REFERENCES
1. Boring, C. C., Squires, T. S., and Tong, T.: Cancer Statistics,
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P. C.: Long-term results of radical prostatectomy in clinically
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S., and Ray, G. R.: Status of radiation treatment of prostate
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Thompson, I. M., Rounder, J. B., Teague, J. L., et al.: Impact of
routine screening for adenocarcinoma of the prostate on stage
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National Cancer Institute roundtable on prostate cancer,
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A., Ben-Ezra, J., and Colombero, A.: Detection of micrometastasis
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E.: The polymerase chain reaction: A new tool for the detection of
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R., Monne, M., Vihko, P., Mulholland, S. G., and Gomella, L. G.:
Detection of hematogenous micrometastasis in patients with prostate
cancer. Cancer Res., 52:6110-6112, 1992. 9. Israeli, R. S., Powell,
C. T., Fair, W. R., and Heston, W. D. W.: Molecular cloning of a
complementary DNA encoding a prostate-specific membrane antigen.
Cancer Res., 53:227-230, 1993. 10. Israeli, R. S., Powell, C. T.,
Corr, J. G., Fair, W. R., and Heston, W. D. W.: Expression of the
prostate-specific membrane antigen (PSM).: Submitted to Cancer
Research. 11. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr,
J. P., Rosenthal, H., Chu. T. M., Mirand, E. A., and Murphy, G. R:
LNCaP model of human prostatic carcinoma. Cancer Res.,
43:1809-1818, 1983. 12. Soule, H. D., Vazquez, J., Long, A.,
Albert, S., and Brennan, M.: A human cell line from a pleural
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FOURTH SERIES OF EXPERIMENTS
EXPRESSION OF THE PROSTATE SPECIFIC MEMBRANE ANTIGEN (PSM)
DIMINISHES THE MITOGENIC STIMULATION OF AGGRESSIVE HUMAN PROSTATIC
CARCINOMA CELLS BY TRANSFERRIN
An association between transferrin and human prostate cancer has
been suggested by several investigators. It has been shown that the
expressed prostatic secretions of patients with prostate cancer are
enriched with respect to their content of transferrin and that
prostate cancer cells are rich in transferrin receptors (J. Urol.
143, 381, 1990). Transferrin derived from bone marrow has been
shown to selectively stimulate the growth of aggressive prostate
cancer cells (PNAS 89, 6197, 1992). We have previously reported the
cloning of the cDNA encoding the 100 kDa PSM antigen (Cancer Res.
53, 208, 1993). DNA sequence analysis has revealed that a portion
of the coding region, from nucleotide 1250 to 1700 possesses a 54%
homology to the human transferrin receptor. PC-3 cells do not
express PSM mRNA or protein and exhibit increased cell growth in
response to transferrin, whereas, LNCaP prostate cancer cells which
highly express PSM have a very weak response to transferrin. To
determine whether PSM expression by prostatic cancer cells impacts
upon their mitogenic response to transferrin we stably transfected
the full-length PSM cDNA into the PC-3 prostate cancer cells.
Clones highly expressing PSM mRNA were identified by Northern
analysis and expression of PSM protein was verified by Western
analysis using the anti-PSM monoclonal antibody 7E11-C5.3.
We plated 2.times.10.sup.4 PC-3 or PSM-transfected PC-3 cells per
well in RPMI medium supplemented with 10% fetal bovine serum and at
24 hrs. added 1 .mu.g per ml. of holotransferrin to the cells.
Cells were counted at 1 day to be highly mitogenic to the PC-3
cells. Cells were counted at 1 day to determine plating efficiency
and at 5 days to determine the effect of the transferrin.
Experiments were repeated to verify the results.
We found that the PC-3 cells experienced an average increase of
275% over controls, whereas the LNCaP cells were only stimulated
43%. Growth kinetics revealed that the PSM-transfected PC-3 cells
grew 30% slower than native PC-3 cells. This data suggests that PSM
expression in aggressive, metastatic human prostate cancer cells
significantly abrogates their mitogenic response to
transferrin.
The use of therapeutic vaccines consisting of cytokine-secreting
tumor cell preparations for the treatment cf established prostate
cancer was investigated in the Dunning R3327-MatLyLu rat prostatic
adenocarcinoma model. Only IL-2 secreting, irradiated tumor cell
preparations were capable of curing animals from subcutaneously
established tumors, and engendered immunological memory that
protected the animals from another tumor challenge. Immunotherapy
was less effective when tumors were induced orthotopically, but
nevertheless led to improved outcome, significantly delaying, and
occasionally preventing recurrence of tumors after resection of the
cancerous prostate. Induction of a potent immune response in tumor
bearing animals against the nonimmunogenic MatLyLu tumor supports
the view that active immunotherapy of prostate cancer may have
therapeutic benefits.
SEQUENCE LISTINGS
1
3812653DNAHuman 1ctcaaaaggg gccggatttc cttctcctgg aggcagatgt
tgcctctctc tctcgctcgg 60attggttcag tgcactctag aaacactgct gtggtggaga
aactggaccc caggtctgga 120gcgaattcca gcctgcaggg ctgataagcg
aggcattagt gagattgaga gagactttac 180cccgccgtgg tggttggagg
gcgcgcagta gagcagcagc acaggcgcgg gtcccgggag 240cccggctctg
ctcgcgccga gatgtggaat ctccttcacg aaaccgactc ggctgtggcc
300accgcgcgcc gcccgcgctg gctgtgcgct ggggcgctgg tgctggcggg
tggcttcttt 360ctcctcggct tcctcttcgg gtggtttata aaatcctcca
atgaagctac taacattact 420ccaaagcata atatgaaagc atttttggat
gaattgaaag ctgagaacat caagaagttc 480ttatataatt ttacacagat
accacattta gcaggaacag aacaaaactt tcagcttgca 540aagcaaattc
aatcccagtg gaaagaattt ggcctggatt ctgttgagct agcacattat
600gatgtcctgt tgtcctaccc aaataagact catcccaact acatctcaat
aattaatgaa 660gatggaaatg agattttcaa cacatcatta tttgaaccac
ctcctccagg atatgaaaat 720gtttcggata ttgtaccacc tttcagtgct
ttctctcctc aaggaatgcc agagggcgat 780ctagtgtatg ttaactatgc
acgaactgaa gacttcttta aattggaacg ggacatgaaa 840atcaattgct
ctgggaaaat tgtaattgcc agatatggga aagttttcag aggaaataag
900gttaaaaatg cccagctggc aggggccaaa ggagtcattc tctactccga
ccctgctgac 960tactttgctc ctggggtgaa gtcctatcca gatggttgga
atcttcctgg aggtggtgtc 1020cagcgtggaa atatcctaaa tctgaatggt
gcaggagacc ctctcacacc aggttaccca 1080gcaaatgaat atgcttatag
gcgtggaatt gcagaggctg ttggtcttcc aagtattcct 1140gttcatccaa
ttggatacta tgatgcacag aagctcctag aaaaaatggg tggctcagca
1200ccaccagata gcagctggag aggaagtctc aaagtgccct acaatgttgg
acctggcttt 1260actggaaact tttctacaca aaaagtcaag atgcacatcc
actctaccaa tgaagtgaca 1320agaatttaca atgtgatagg tactctcaga
ggagcagtgg aaccagacag atatgtcatt 1380ctgggaggtc accgggactc
atgggtgttt ggtggtattg accctcagag tggagcagct 1440gttgttcatg
aaattgtgag gagctttgga acactgaaaa aggaagggtg gagacctaga
1500agaacaattt tgtttgcaag ctgggatgca gaagaatttg gtcttcttgg
ttctactgag 1560tgggcagagg agaattcaag actccttcaa gagcgtggcg
tggcttatat taatgctgac 1620tcatctatag aaggaaacta cactctgaga
gttgattgta caccgctgat gtacagcttg 1680gtacacaacc taacaaaaga
gctgaaaagc cctgatgaag gctttgaagg caaatctctt 1740tatgaaagtt
ggactaaaaa aagtccttcc ccagagttca gtggcatgcc caggataagc
1800aaattgggat ctggaaatga ttttgaggtg ttcttccaac gacttggaat
tgcttcaggc 1860agagcacggt atactaaaaa ttgggaaaca aacaaattca
gcggctatcc actgtatcac 1920agtgtctatg aaacatatga gttggtggaa
aagttttatg atccaatgtt taaatatcac 1980ctcactgtgg cccaggttcg
aggagggatg gtgtttgagc tagccaattc catagtgctc 2040ccttttgatt
gtcgagatta tgctgtagtt ttaagaaagt atgctgacaa aatctacagt
2100atttctatga aacatccaca ggaaatgaag acatacagtg tatcatttga
ttcacttttt 2160tctgcagtaa agaattttac agaaattgct tccaagttca
gtgagagact ccaggacttt 2220gacaaaagca acccaatagt attaagaatg
atgaatgatc aactcatgtt tctggaaaga 2280gcatttattg atccattagg
gttaccagac aggccttttt ataggcatgt catctatgct 2340ccaagcagcc
acaacaagta tgcaggggag tcattcccag gaatttatga tgctctgttt
2400gatattgaaa gcaaagtgga cccttccaag gcctggggag aagtgaagag
acagatttat 2460gttgcagcct tcacagtgca ggcagctgca gagactttga
gtgaagtagc ctaagaggat 2520tctttagaga atccgtattg aatttgtgtg
gtatgtcact cagaaagaat cgtaatgggt 2580atattgataa attttaaaat
tggtatattt gaaataaagt tgaatattat atataaaaaa 2640aaaaaaaaaa aaa
26532750PRTHuman 2Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala Val
Ala Thr Ala Arg1 5 10 15Arg Pro Arg Trp Leu Cys Ala Gly Ala Leu Val
Leu Ala Gly Gly Phe 20 25 30Phe Leu Leu Gly Phe Leu Phe Gly Trp Phe
Ile Lys Ser Ser Asn Glu 35 40 45Ala Thr Asn Ile Thr Pro Lys His Asn
Met Lys Ala Phe Leu Asp Glu 50 55 60Leu Lys Ala Glu Asn Ile Lys Lys
Phe Leu Tyr Asn Phe Thr Gln Ile65 70 75 80Pro His Leu Ala Gly Thr
Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile 85 90 95Gln Ser Gln Trp Lys
Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His 100 105 110Tyr Asp Val
Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn Tyr Ile 115 120 125Ser
Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn Thr Ser Leu Phe 130 135
140Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val Ser Asp Ile Val Pro
Pro145 150 155 160Phe Ser Ala Phe Ser Pro Gln Gly Met Pro Glu Gly
Asp Leu Val Tyr 165 170 175Val Asn Tyr Ala Arg Thr Glu Asp Phe Phe
Lys Leu Glu Arg Asp Met 180 185 190Lys Ile Asn Cys Ser Gly Lys Ile
Val Ile Ala Arg Tyr Gly Lys Val 195 200 205Phe Arg Gly Asn Lys Val
Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly 210 215 220Val Ile Leu Tyr
Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys225 230 235 240Ser
Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val Gln Arg Gly 245 250
255Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro Leu Thr Pro Gly Tyr
260 265 270Pro Ala Asn Glu Tyr Ala Tyr Arg Arg Gly Ile Ala Glu Ala
Val Gly 275 280 285Leu Pro Ser Ile Pro Val His Pro Ile Gly Tyr Tyr
Asp Ala Gln Lys 290 295 300Leu Leu Glu Lys Met Gly Gly Ser Ala Pro
Pro Asp Ser Ser Trp Arg305 310 315 320Gly Ser Leu Lys Val Pro Tyr
Asn Val Gly Pro Gly Phe Thr Gly Asn 325 330 335Phe Ser Thr Gln Lys
Val Lys Met His Ile His Ser Thr Asn Glu Val 340 345 350Thr Arg Ile
Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu Pro 355 360 365Asp
Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp Val Phe Gly 370 375
380Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val His Glu Ile Val
Arg385 390 395 400Ser Phe Gly Thr Leu Lys Lys Glu Gly Trp Arg Pro
Arg Arg Thr Ile 405 410 415Leu Phe Ala Ser Trp Asp Ala Glu Glu Phe
Gly Leu Leu Gly Ser Thr 420 425 430Glu Trp Ala Glu Glu Asn Ser Arg
Leu Leu Gln Glu Arg Gly Val Ala 435 440 445Tyr Ile Asn Ala Asp Ser
Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 450 455 460Asp Cys Thr Pro
Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu465 470 475 480Leu
Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr Glu Ser 485 490
495Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly Met Pro Arg Ile
500 505 510Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu Val Phe Phe Gln
Arg Leu 515 520 525Lys Ile Ala Ser Gly Arg Ala Arg Tyr Thr Lys Asn
Trp Glu Thr Asn 530 535 540Lys Phe Ser Gly Tyr Pro Leu Tyr His Ser
Val Tyr Glu Thr Tyr Glu545 550 555 560Leu Val Glu Lys Phe Tyr Asp
Pro Met Phe Lys Tyr His Leu Thr Val 565 570 575Ala Gln Val Arg Gly
Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val 580 585 590Leu Pro Phe
Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala 595 600 605Asp
Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met Lys Thr 610 615
620Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val Lys Asn Phe
Thr625 630 635 640Glu Ile Ala Ser Lys Phe Ser Glu Arg Leu Gln Asp
Phe Asp Lys Ser 645 650 655Asn Pro Ile Val Leu Arg Met Met Asn Asp
Gln Leu Met Phe Leu Glu 660 665 670Arg Ala Phe Ile Asp Pro Leu Gly
Leu Pro Asp Arg Pro Phe Tyr Arg 675 680 685His Val Ile Tyr Ala Pro
Ser Ser His Asn Lys Tyr Ala Gly Glu Ser 690 695 700Phe Pro Gly Ile
Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp705 710 715 720Pro
Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala Ala 725 730
735Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val Ala 740 745
75038PRTHuman 3Ser Leu Tyr Glu Ser Trp Thr Lys1
5415PRTHumanMISC_FEATURE(6)..(7)Xaa=unknown 4Ser Tyr Pro Asp Gly
Xaa Xaa Leu Pro Gly Gly Gly Val Gln Arg1 5 10 1557PRTHuman 5Phe Tyr
Asp Pro Met Phe Lys1 569PRTHuman 6Ile Tyr Asn Val Ile Gly Thr Leu
Lys1 5722PRTHumanMISC_FEATURE(4)..(5)Xaa=unknown 7Phe Leu Tyr Xaa
Xaa Thr Gln Ile Pro His Leu Ala Gly Thr Glu Gln1 5 10 15Asn Phe Gln
Leu Ala Lys 20817PRTHuman 8Gly Val Ile Leu Tyr Ser Asp Pro Ala Asp
Tyr Phe Ala Pro Asp Val1 5 10 15Lys917PRTHuman 9Pro Val Ile Leu Tyr
Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val1 5 10 15Lys1015PRTHuman
10Ala Phe Ile Asp Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg1 5 10
151119PRTHuman 11Tyr Ala Gly Glu Ser Phe Pro Gly Ile Tyr Asp Ala
Leu Phe Asp Ile1 5 10 15Glu Ser
Lys1222PRTHumanMISC_FEATURE(14)..(15)Xaa=unknown 12Thr Ile Leu Phe
Ala Ser Trp Asp Ala Glu Glu Phe Gly Xaa Xaa Gly1 5 10 15Ser Thr Glu
Glu Ala Glu 201317DNAartificial sequenceprimer 13ttytaygayc cnatgtt
171417DNAartificial sequenceprimer 14aacatnggrt crtaraa
171517DNAartificial sequenceprimer 15athtayaayg tnathgg
171617DNAartificial sequenceprimer 16ccdatnacrt trtadat
171717DNAartificial sequenceprimer 17ccngcngayt ayttygc
171817DNAartificial sequenceprimer 18gcraartart ncgcngg
171920DNAartificial sequenceprimer 19acngarcara ayttycarct
202020DNAartificial sequenceprimer 20agytgraart tytgytcngt
202117DNAartificial sequenceprimer 21garcaraayt tycarct
172217DNAartificial sequenceprimer 22agytgraart tytgytc
172320DNAartificial sequenceprimer 23tgggaygcng argarttygg
202420DNAartificial sequenceprimer 24ccraaytcyt cngcrtccca
202517DNAartificial sequenceprimer 25tgggaygcng argartt
172617DNAartificial sequenceprimer 26aaytcytcng crtccca
1727780DNAchickenmisc_feature(82)..(84)n=any nucleotide
27tacacttatc ccattcggac atgcccacct tggaactgga gacccttaca ccccaggctt
60cccttcgttc aaccacaccc annngtttcc accagttgaa tcttcaggac taccccacat
120tgctgttcag accatctcta gcagtgcagc agccaggctg ttcagcaaaa
tggatggaga 180cacatgctct ganagnngtt ggaaaggtgc gatccannnt
tcctgtaagg tnngacnnaa 240caaagcagga gannnngcca gantaatggt
gaaactagat gtgaacaatt ccatgaaaga 300caggaagatt ctgaacatct
tcggtgctat ccagggattt gaagaacctg atcggtatgt 360tgtgattgga
gcccagagag actcctgggg cccaggagtg gctaaagctg gcactggaac
420tgctatattg ttggaacttg cccgtgtgat ctcagacata gtgaaaaacg
agggctacaa 480accgaggcga agcatcatct ttgctagctg gagtgcagga
gactacggag ctgtgggtgc 540tactgaatgg ctggaggggt actctgccat
gctgcatgcc aaagctttca cttacatcan 600ngcttggatg ctccagtcct
gggagcaagc catgtcaaga tttctgccag ccccttgctg 660tatatgctgc
tggggagtat tatgaagggg gtgaagaatc cagcagcagt ctcagagagc
720nnnnctctat aacagacttg gcccagactg ggtaaaagca gttgttcctc
ttggcctgga 78028660DNAratmisc_feature(284)..(284)n=any nucleotide
28tgcagaaaag ctattcaaaa acatggaagg aaactgtcct cctagttgga atatagattc
60ctcatgtaag ctggaacttt cacagaatca aaatgtgaag ctcactgtga acaatgtact
120gaaagaaaca agaatactta acatctttgg cgttattaaa ggctatgagg
aaccagaccg 180ctacattgta gtaggagccc agagagacgc ttggggccct
ggtngttgcg aagtccagtg 240tgggaacagg tcttnctgtt gaaacttgcc
caagtattct cagatatgat ttcaaaagat 300ggatttagac ccagcaggag
tattatcttt gccagctgga ctgcaggaga ctatggagct 360gttggtccga
ctgagtggct ggaggggtac ctttcatctt tgcatctaaa gnnngctttc
420acttacatta atnctggata aagtcgtcct gggtactagc aacttcaagg
tttctgccag 480ccccctatta tatacactta tggggaagat aatgcaggan
ncgtaaagca tccgannnnn 540nnnttgatgg aaaatatcta tatcgaaaca
gtaattggat tagcaaaatt gaggaacttt 600ccttggacaa tgctgcattc
ccttttcttg catattcagg aatcccagca gtttctttct
66029540DNAhumanmisc_feature(214)..(214)n=any nucleotide
29tatggaagga gactgtccct ctgactggaa aacagactct acatgtagga tggtaacctc
60agaaagcaag aatgtgaagc tcactgtgag caatgtgctg aaagagataa aaattcttaa
120catctttgga gttattaaag gctttgtaga accagatcac tatgttgtag
ttggggccca 180gagagatgca tggggccctg gagctgcaaa atcncggtgt
aggcacagct ctcctattga 240aacttgccca gatgttctca gatatggtct
taaaagatgg gtttcagccc agcagaagca 300ttatctttgc cagttggagt
gctggagact ttggatcggt tggtgccact gaatggctag 360agggatacct
ttcgtcncct gcatttaaag gctttcactt atattaatct ggataaagcg
420gttcttggta ccagcaactt caaggtttct gccagcccac tgttgtatac
gcttattgag 480aaaacaatgc aaaatgtgaa gcatccggtt actgggcaat
ttctatatca ggacagcaac 5403027DNAartificial sequenceprimer
30acggagcaaa actttcagct tgcaaag 27319PRTartificial sequenceprimer
31Thr Glu Gln Asn Phe Gln Leu Ala Lys1 53236DNAartificial
sequenceprimer 32ctcttcggca tcccagcttg caaacaaaat tgttct
363336DNAartificial sequenceprimer 33agaacaattt tgtttgcaag
ctgggatgcc aaggag 363412PRTartificial sequenceprimer 34Arg Thr Ile
Leu Phe Ala Ser Trp Asp Ala Glu Glu1 5 10356PRThuman 35Asp Glu Leu
Lys Ala Glu1 5366PRThuman 36Asn Glu Asp Gly Asn Glu1 5376PRThuman
37Lys Ser Pro Asp Glu Gly1 53817PRThuman 38Ala Gly Ala Leu Val Leu
Ala Gly Gly Phe Phe Leu Leu Gly Phe Leu1 5 10 15Phe
* * * * *
References