U.S. patent application number 11/480319 was filed with the patent office on 2007-06-28 for prostate-specific membrane antigen.
This patent application is currently assigned to Sloan Kettering Institute for Cancer Research. Invention is credited to William R. Fair, Warren D. W. Heston, Ron S. Israeli.
Application Number | 20070148662 11/480319 |
Document ID | / |
Family ID | 36613669 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148662 |
Kind Code |
A1 |
Israeli; Ron S. ; et
al. |
June 28, 2007 |
Prostate-specific membrane antigen
Abstract
This invention provides an isolated mammalian nucleic acid
molecule encoding a mammalian prostate-specific membrane antigen.
This invention also provides prostate-specific membrane nucleic
acid of at least 15 nucleotides capable of specifically hybridizing
with a sequence of a nucleic acid molecule encoding a mammalian
prostate-specific membrane antigen. This invention further provides
vector and vector host expression system for the prostate-specific
membrane antigen. This invention also provides methods to identify
ligands which bind to the prostate-specific membrane antigen, to
generate antibody against a complete prostate-specific membrane
antigen or a portion of the antigen. This invention further
provides purified prostate-specific membrane antigen. This
invention provides a therapeutic agent comprising an antibody
directed against to prostate-specific membrane antigen and a
cytotoxic agent conjugated thereto. This invention also provides a
method of imaging prostate cancer and an immunoassay for measuring
the amount of the prostate-specific membrane antigen in a
biological simple. This invention further provides transgenic
nonhuman mammal which comprises the isolated nucleic acid molecule
encoding a mammalian prostate-specific membrane antigen.
Inventors: |
Israeli; Ron S.; (Forest
Hills, NY) ; Heston; Warren D. W.; (New York, NY)
; Fair; William R.; (New York, NY) |
Correspondence
Address: |
Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Assignee: |
Sloan Kettering Institute for
Cancer Research
|
Family ID: |
36613669 |
Appl. No.: |
11/480319 |
Filed: |
June 30, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08481916 |
Jun 7, 1995 |
7070782 |
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11480319 |
Jun 30, 2006 |
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08325553 |
Oct 18, 1994 |
5538866 |
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08481916 |
Jun 7, 1995 |
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07973337 |
Nov 5, 1992 |
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08325553 |
Oct 18, 1994 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 435/7.23; 530/350; 536/23.5 |
Current CPC
Class: |
C12Q 1/6883 20130101;
G01N 2800/342 20130101; C07K 14/705 20130101; C07K 14/4748
20130101; A61K 39/00 20130101; C12Q 2600/158 20130101 |
Class at
Publication: |
435/006 ;
435/007.23; 435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/705 20060101
C07K014/705 |
Claims
1. An isolated mammalian nucleic acid molecule encoding a mammalian
prostate-specific membrane antigen.
2-21. (canceled)
22. A method for determining whether a ligand can bind to a
mammalian prostate-specific membrane antigen which comprises
contacting a mammalian cell having 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 determining
whether the ligand binds to a mammalian prostate-specific membrane
antigen.
23. (canceled)
24. Purified mammalian prostate-specific membrane antigen.
25-33. (canceled)
34. An antibody directed against the amino acid sequence of a
mammalian prostate-specific membrane antigen.
35. An antibody directed either to peptide Asp-Glu-Leu-Lys-Ala-Glu
(SEQ ID No. 39), or Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 39), or
Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 40) or Lys-Ser-Pro-Asp-Glu-Gly
(SEQ ID No. 41) of the prostate-specific membrane antigen.
36-47. (canceled)
48. 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 mammalian
prostate-specific membrane antigen thereby reducing its
translation.
Description
BACKGROUND OF THE INVENTION
[0001] 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 this application, preceding
the sequence listing and the claims.
[0002] 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, 2).
[0003] In prostate cancer cells, two specific proteins that are
made in very high concentrations are prostatic acid phosphatase
(PAP) and prostate specific antigen (PSA) (3, 4, 5). 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.
[0004] PAP was one of the earliest serum markers for detecting
metastatic spread (3). 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.
[0005] PSA is a protease and it is not readily appreciated how loss
of its activity correlates with cancer development (4, 5). 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 (6).
[0006] 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.
[0007] 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 (7).
[0008] Dr. Horoszewicz established a cell line designated LNCaP
from the lymph node of a hormone refractory, heavily pretreated
patient (8). 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 (7). 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.
[0009] Dr. Horoszewicz also reported detection of immunoreactive
material using 7E11-C5 in serum of prostatic cancer patients (7).
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.
[0010] The 7E11-C5 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 (9). 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-C5 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.
[0011] 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 (10, 11). These investigators have
shown that following electrophoresis on acrylamide gels and Western
blotting, the PSM antigen appears to have 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.
[0012] A 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 (12). 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).
BRIEF DESCRIPTION OF FIGURES
[0013] 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.
[0014] FIG. 2 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.
[0015] FIG. 3 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.
[0016] FIG. 4 100 kD PSM antigen following immunoprecipitation of
.sup.35S-Methionine labelled LNCaP cells with Cyt-356 antibody.
[0017] 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.
[0018] FIG. 6 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.
[0019] FIG. 7 Autoradiogram showing size of cDNA represented in
applicants' LNCaP library.
[0020] 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.
[0021] FIG. 9 Plasmid Southern autoradiogram of full length PSM
gene clones. Size is approximately 2.7 kb.
[0022] 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.
[0023] 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.
[0024] FIG. 12 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
18, Cancer; Lane 19, IN-20 Control; Lane 20, PSM cDNA
[0025] FIG. 13 Isoelectric point of PSM antigen
(non-glycosylated)
[0026] FIG. 14 Secondary structure of PSM antigen
[0027] FIG. 15 A. Hydrophilicity plot of PSM antigen B. Prediction
of membrane spanning segments
[0028] FIG. 16 Homology with chicken, rat and human transferrin
receptor sequence
SUMMARY OF THE INVENTION
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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.
[0033] This invention provides isolated nucleic acid sequence 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] This invention provides a therapeutic agent comprising an
antibody directed against a mammalian PSM antigen and a cytotoxic
agent conjugated thereto.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] This invention provides a method to purify mammalian PSM
antigen comprising steps of: [0047] 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.
[0048] 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.
DETAILED DESCRIPTION OF THE INVENTION
[0049] 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:
[0050] C=cytosine A=adenosine [0051] T=thymidine G=guanosine
[0052] This invention provides an isolated mammalian nucleic acid
encoding a mammalian prostate-specific membrane (PSM) antigen.
[0053] 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 mammalina
prostate-specific membrane antigen.
[0054] 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. Mex.) with Accession Number,
M99487 and the description as PSM, Homo sapiens, 2653
base-pairs.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] Regulatory elements required for expression include 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] Numerous mammalian cells may be used as hosts, including,
but not limited to, the mouse fibroblast cell NIH3T3, CHO cells,
HeLa cells, Ltk.sup.- 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.
[0076] 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.
[0077] This invention further provides ligands bound to the
mammalian PSM antigen.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] This invention further provides a polypeptide encoded by the
isolated mammalian nucleic acid sequence of PSM antigen.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] This invention provides an antibody directed against the
amino acid sequence of a mammalian PSM antigen.
[0086] 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. 15A 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.
[0087] 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.
[0088] 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.
[0089] 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).
[0090] 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.
[0091] 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.
[0092] This invention further provides a prostate cancer specific
imaging agent comprising the antibody directed against PSM antigen
and a radioisotope conjugated thereto.
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] 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.
[0099] 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).
[0100] 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
Materials and Methods
[0101] 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. [0102] I.
Western Analysis of the PSM Antigen
[0103] 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 (Millipores 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). [0104]
II. Immunohistochemical Analysis of PSM Antigen Expression
[0105] 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 .mu.m 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. [0106] III. Immunoprecipitation of the PSM Antigen
[0107] 80%-confluent LNCaP cells in 100 mm petri dishes were
starved in RPMI media without methionine for 2 hours, after which
.sup.35S-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 4.degree. C. Lysates were
pre-cleared by mixing with Pansorbin.RTM. cells (Calbiochem.RTM.)
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
.beta.-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). [0108] IV. Large-Scale
Immunoprecipitation and Peptide Sequencing
[0109] 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.
[0110] PSM Antigen Peptide Sequences: TABLE-US-00001 2T17 #5
SLYES(W)TK (SEQ ID No. 3) 2T22 #9 (S)YPDGXNLPGG(g)VQR (SEQ ID No.
4) 2T26 #3 FYDPMFK (SEQ ID No. 5) 2T27 #4 IYNVIGTL(K) (SEQ ID No.
6) 2T34 #6 FLYXXTQIPHLAGTEQNFQLAK (SEQ ID No. 7) 2T35 #2
G/PVILYSDPADYFAPD/GVK (SEQ ID No. 8, 9) 2T38 #1 AFIDPLGLPDRPFYR
(SEQ ID No. 10) 2T46 #8 YAGESFPGIYDALFDIESK (SEQ ID No. 11) 2T47 #7
TILFAS(W)DAEEFGXX(q)STE(e)A (SEQ ID No. 12) (E) . . .
[0111] 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.
[0112] All of these peptide sequences were verified to be unique
after a complete homology search of the translated Genbank computer
database. [0113] IV. Degenerate PCR
[0114] 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.
[0115] Peptide 3: FYDPMFK (SEQ ID No. 5) TABLE-US-00002 PSM Primer
"A" TT(C or T) - TA(C or T) - GA (SEQ ID No. 13) (C or T) - CCX -
ATG - TT PSM Primer "B" AAC - ATX - GG(A or G) - TC (SEQ ID No. 14)
(A or G) - TA(A or G) - AA
[0116] Primer A is sense primer and B is anti-sense. Degeneracy is
32-fold.
[0117] Peptide 4: IYNVIGTL(K) (SEQ ID No. 6) TABLE-US-00003 PSM
Primer "C" AT(T or C or A) - TA(T or C) - AA (SEQ ID No. 15) (T or
C) - GTX - AT(T or C or A) - GG PSM Primer "D" CC(A or T or G) -
ATX - AC(G or (SEQ ID No. 16) A) - TT(A or G) - TA(A or G or T) -
AT
[0118] Primer C is sense-primer and D is anti-sense. Degeneracy is
144-fold.
[0119] Peptide 2: .alpha./PVILYSDPADYFAPD/GVK (SEQ ID No. 8, 9)
TABLE-US-00004 PSM Primer "E" CCX - GCX - GA(T or C) - TA(T or (SEQ
ID No. 17) C) - TT(T or C) - GC PSM Primer "F" GC(G or A) - AA(A or
G) - TA(A or (SEQ ID No. 18) G) - TXC - GCX - GG
[0120] Primer E is sense primer and F is antisense primer.
Degeneracy is 128-fold.
[0121] Peptide 6: FLYXXTQIPBLAGTEONFOLAK (SEQ ID No. 7)
TABLE-US-00005 PSM Primer "I" ACX - GA(A or G) - CA(A or G) - AA
(SEQ ID No. 19) (T or C) - TT(T or C) - CA(A or G) - CT PSM Primer
"J" AG - (T or C)TG - (A or G)AA - (A (SEQ ID No. 20) or G)TT - (T
or C)TG - (T or C) TC - XGT PSM Primer "K" GA(A or G) - CA(A or G)
- AA(T or (SEQ ID No. 21) C) - TT(T or C) CA(A or G) - CT PSM
Primer "L" AG - (T or C)TG - (A or G)AA - (A (SEQ ID No. 22) or
G)TT - (T or C)TG - (T or C)TC
Primers I and K are sense primers and J and L are anti-sense. I and
J have degeneracies of 128-fold and K and L have 32-fold
degeneracy.
[0122] Peptide 7: TILFAS(W)DAEEFGXX(q)STE(e)A(E) . . . (SEQ ID No.
12) TABLE-US-00006 PSM Primer "M" TGG - GA(T or C) - GCX - GA(A or
(SEQ ID No. 23) G) - GA(A or G) - TT(C or T) - GG PSM Primer "N" CC
- (G or A)AA - (T or C)TC - (T (SEQ ID No. 24) or C)TC - XGC - (A
or G)TC - CCA PSM Primer "O" TGG - GA(T or C) - GCX - GA(A or (SEQ
ID No. 25) G) - GA(A or G) - TT PSM Primer "P" AA - (T or C)TC - (T
or C)TC - (SEQ ID No. 26) XGC - (A or G)TC - CCA
[0123] 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.
[0124] 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: TABLE-US-00007 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
[0125] Incubate at 68.degree. C..times.10 minutes.
[0126] Quick chill on ice.times.5 minutes.
[0127] Add: TABLE-US-00008 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
[0128] Incubate for 2 minutes at 37.degree. C.
[0129] Add 1 .mu.g Superscript.RTM. Reverse Transcriptase
(Gibco.RTM.-BRL)
[0130] Incubate for 1 hour at 37.degree. C.
[0131] Add 30 .mu.l dH.sub.2O.
[0132] Use 2 .mu.l per PCR reaction.
[0133] 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. TABLE-US-00009 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 .mu.g each of sense and 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 2 .mu.l diluted Taq
Polymerase (0.5 U/.mu.l) 50 .mu.l total volume
[0134] 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.
[0135] *10.times.PCR Buffer [0136] 166 mM NH.sub.4SO.sub.4 [0137]
670 mM Tris, pH 8.8 [0138] 2 mg/ml BSA
[0139] Representative photographs displaying PCR products are shown
in FIG. 5. [0140] V. Cloning of PCR Products
[0141] 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 Taq 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). [0142] VI. DNA
Sequencing of PCR Products
[0143] 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.
[0144] 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-00010 ACG GAG CAA AAC TTT CAG CTT GCA (SEQ ID No. 30)
AAG T E Q N F Q L A (SEQ ID No. 31) K
[0145] 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).
[0146] When we analyzed the other end of the molecule by reading
from the N primer the sequence was: TABLE-US-00011 CTC TTC GGC ATC
CCA GCT TGC AAA (SEQ ID No. 32) CAA AAT TGT TCT
[0147] Since this represents the anti-sense DNA sequence, we need
to show the complementary sense sequence in order to find our
peptide.
[0148] Sense Sequence: TABLE-US-00012 AGA ACA ATT TTG TTT GCA AGC
TGG (SEQ ID No. 33) GAT GCC AAG GAG R T I L F A S W (SEQ ID No. 34)
D A E E
[0149] 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.
[0150] The DNA sequence of this partial cDNA was found to be unique
when screened on the Genbank computer database. [0151] VII. cDNA
Library Construction and Cloning of Full-Length PSM cDNA
[0152] A cDNA library from LNCaP mRNA was constructed using the
Superscripts 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. 8, showing that they all hybridize
to the 1.1 kb partial cDNA probe.
[0153] 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. [0154] VIII. Northern Analysis of PSM
Gene Expression
[0155] Northern analysis (28) of the PSM gene has revealed that
expression is limited to the prostate and to prostate
carcinoma.
[0156] 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.
[0157] IX. PCR Analysis of PSM Gene Expression in Human Prostate
Tissues
[0158] 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).
[0159] 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 cDNA, 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.
[0160] 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
[0161] 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. 15A. Shown in FIG. 15B 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).
[0162] 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).
[0163] 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.
[0164] 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. 16.
Experimental Discussions
[0165] Potential Uses for PSM Antigen:
[0166] 1. Tumor Detection:
[0167] Microscopic:
[0168] 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.
[0169] 2. Antigenic Site Identification
[0170] 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.
[0171] 3. Restriction Fragment Length Polymorphism and Genomic
DNA
[0172] 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.
[0173] Depending on the chromosomal location of the PSM antigen,
the PSM antigen gene may serve as a useful chromosome location
marker for chromosome analysis.
[0174] 4. Serum
[0175] 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.
[0176] 5. Imaging
[0177] 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.
[0178] 6. Isolation of Ligands
[0179] 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.
[0180] 7. Therapeutic Uses
[0181] 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.
[0182] 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.
[0183] It was found that the ligand for the neu 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.
[0184] 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.
[0185] 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.h 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).
[0186] 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.
[0187] 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).
[0188] 8. Others
[0189] 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.
[0190] 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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Sequence CWU 1
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