U.S. patent application number 13/157691 was filed with the patent office on 2011-11-24 for novel, prostate-specific gene for diagnosis, prognosis and management of prostate cancer.
Invention is credited to Gang An, Robert Veltri.
Application Number | 20110286917 13/157691 |
Document ID | / |
Family ID | 22933947 |
Filed Date | 2011-11-24 |
United States Patent
Application |
20110286917 |
Kind Code |
A1 |
An; Gang ; et al. |
November 24, 2011 |
Novel, Prostate-Specific Gene for Diagnosis, Prognosis and
Management of Prostate Cancer
Abstract
Disclosed are nucleic acid and amino acid sequences encoded by a
novel, prostate specific gene (UC41) and diagnostic techniques for
the detection of human prostate cancer utilizing such nucleic acid
and amino acid sequences. Genetic probes and methods useful in
monitoring the progression and diagnosis of prostate cancer are
described. Methods of treatment for prostate cancer utilizing
antisense constructs or antibodies specific for UC41 gene products
are also described.
Inventors: |
An; Gang; (Oklahoma City,
OK) ; Veltri; Robert; (Oklahoma City, OK) |
Family ID: |
22933947 |
Appl. No.: |
13/157691 |
Filed: |
June 10, 2011 |
Related U.S. Patent Documents
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11706417 |
Feb 12, 2007 |
7993830 |
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13157691 |
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09962902 |
Sep 25, 2001 |
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11706417 |
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09579236 |
May 24, 2000 |
6369195 |
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09962902 |
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09247188 |
Feb 9, 1999 |
6156515 |
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09579236 |
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Current U.S.
Class: |
424/1.49 ;
424/135.1; 424/139.1; 424/9.1; 435/6.11; 435/6.12; 435/6.13;
435/7.1; 435/7.92; 436/501; 506/9; 514/44R |
Current CPC
Class: |
C12Q 2600/106 20130101;
C12Q 2600/158 20130101; Y10S 530/866 20130101; C12Q 2600/136
20130101; C12Q 1/6886 20130101; Y10S 530/85 20130101; C07K 14/4748
20130101; G01N 33/57434 20130101; A61P 13/08 20180101; A61P 43/00
20180101; A61K 39/0011 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/1.49 ;
436/501; 435/7.92; 435/7.1; 435/6.13; 435/6.12; 435/6.11; 506/9;
424/139.1; 514/44.R; 424/9.1; 424/135.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/577 20060101 G01N033/577; G01N 21/64 20060101
G01N021/64; A61K 51/00 20060101 A61K051/00; C40B 30/04 20060101
C40B030/04; A61K 31/7088 20060101 A61K031/7088; A61P 35/00 20060101
A61P035/00; A61K 49/00 20060101 A61K049/00; G01N 33/574 20060101
G01N033/574; C12Q 1/68 20060101 C12Q001/68 |
Claims
1-23. (canceled)
23. A method of treating a subject with prostate cancer, comprising
the steps of: (a) obtaining a biological sample from a subject with
prostate cancer; (b) screening said sample for the expression of a
prostate cancer marker using an antibody that binds immunologically
to a polypeptide comprising SEQ ID NO:2 or SEQ ID NO:5; and (c)
administering an effective amount of said antibody to said
subject.
24. A method of treating a subject with prostate cancer, comprising
the steps of: (a) obtaining a biological sample from a subject with
prostate cancer; (b) screening said sample for the expression of a
prostate cancer marker; (c) providing an antisense DNA molecule
that encodes an RNA molecule that binds to a polynucleotide
comprising SEQ ID NOs: 1, wherein said antisense DNA molecule
comprises a vector containing human regulatory elements for the
production of said RNA molecule; and (d) administering an effective
amount of said vector to said subject.
25-27. (canceled)
28. A method for detecting prostate cancer cells in a biological
sample, comprising the following steps: (a) obtaining a biological
sample from a subject; (b) providing an antibody that binds
immunologically to a polypeptide comprising SEQ ID NO:2 or SEQ ID
NO:5; (c) contacting the biological sample with the antibody; and
(d) detecting the antibody bound to the biological sample, wherein
detection of the antibody to the biological sample indicates the
presence of prostate cancer cells in the biological sample.
29-32. (canceled)
33. The method of claim 24, wherein said antisense DNA molecule
encodes a full length complementary sequence to SEQ ID NO:1 or a
portion of SEQ ID NO:1 comprising at least 8 contiguous base pairs
in length.
34. The method of claim 23, wherein said antibody is in the form of
a single chain antibody.
35. The method of claim 34, wherein said administering step
comprises providing an expression vector that encodes said
single-chain antibody.
36. The method of claim 28, wherein the antibody comprises a
polyclonal antibody or a monoclonal antibody.
37. The method of claim 28, wherein the antibody comprises an
antibody fragment.
38. The method of claim 28, wherein the antibody is a monoclonal
antibody and is conjugated to a detectable moiety.
39. The method of claim 38, wherein the detectable moiety comprises
a radioactive, fluorescent, biological or enzymatic moiety.
40. The method of claim 39, wherein the radioactive moiety is a
radionuclide.
41. The method of claim 28, further comprising separating the
antibody bound to the biological sample of step (c) from unbound
antibody.
42. The method of claim 28, wherein the antibody bound to the
biological sample is detected by an enzyme-linked immunosorbent
assay (ELISA), a radioimmunoassay (RIA), a Western blot assay, a
dot blotting assay, a slot blot assay, a fluorescence activated
cell sorting (FACS) assay or an immunohistochemistry assay.
43. The method of claim 28, wherein the biological sample comprises
a sample that is suspected of containing a polypeptide comprising
SEQ ID NO:2 or SEQ ID NO:5.
44. The method of claim 28, wherein the biological sample comprises
normal prostate cells, primary prostate cancer cells, metastatic
prostate cancer cells or a combination thereof
45. The method of claim 28, wherein the biological sample comprises
a sample that is suspected of containing prostate cancer cells.
46. The method of claim 45, wherein the biological sample comprises
prostate tissue , lymph node tissue , spleen tissue, skin tissue,
organ tissue, bone marrow tissue, or isolated cells thereof.
47. The method of claim 28, wherein the biological sample comprises
prostate tissue, lymph node tissue or a biological fluid that comes
into contact with prostate tissue or lymph node tissue.
48. The method of claim 28, wherein the biological sample comprises
a fresh-frozen paraffin-embedded tissue blocks or a formalin-fixed
paraffin-embedded tissue block.
49. The method of claim 28, further comprising the steps of: (e)
quantifying the amount of a prostate cancer marker in the
biological sample using the antibody that binds immunologically to
a polypeptide comprising SEQ ID NO:2 or SEQ ID NO:5; and (f)
comparing the amount of the prostate cancer marker in the
biological sample of step (e) to the amount of the prostate cancer
marker in a normal reference sample, wherein an increased amount of
the prostate cancer marker in the biological sample of step (e)
relative to the amount of the prostate cancer marker in the normal
reference sample is indicative of prostate cancer cells in the
biological sample of step (e).
50. The method of claim 28, further comprising determining the
prognosis of a subject having prostate cancer, wherein the
biological sample is obtained from said subject, by quantifying the
amount of the prostate cancer marker in the biological sample bound
by the antibody that binds immunologically to a polypeptide
comprising SEQ ID NO:2 or SEQ ID NO:5, wherein an amount of the
prostate cancer marker above a cut-off amount corresponds to a
relatively worse prognosis for the subject as compared to the
prognosis corresponding to prostate marker levels below the cut-off
amount.
51. The method of claim 28, further comprising determining the
diagnosis of a subject suspected of having prostate cancer, wherein
the biological sample is obtained from said subject, by quantifying
the amount of the prostate cancer marker in the biological sample
bound by the antibody that binds immunologically to a polypeptide
comprising SEQ ID NO:2 or SEQ ID NO:5, wherein an increased amount
of the prostate cancer marker relative to a cut-off amount
corresponds to a diagnosis of prostate cancer in the subject.
52. A method for detecting prostate cancer cells in a subject
comprising the following steps: (a) administering an
imaging-effective amount of a detectably-labeled antibody that
binds immunologically to a polypeptide comprising SEQ ID NO:2 or
SEQ ID NO:5 and a pharmaceutically effective carrier to a subject;
(b) detecting the antibody that is bound to prostate cancer cells
in the subject.
53. The method of claim 52, wherein the antibody bound to the
prostate cancer cells in the subject is detected by a radio-imaging
assay.
54. The method of claim 52, wherein the detectably-labeled antibody
comprises an antibody conjugated to a radioactive moiety.
55. The method of claim 54, wherein the radioactive moiety is a
radionuclide.
56. A method of determining the ability of a candidate substance to
inhibit expression of the UC41 gene, the method comprising the
steps of: (a) providing at least one UC41-expressing prostate cell;
(b) contacting the at least one UC41-expressing prostate cell with
a candidate substance; (c) measuring the level of UC41 expression
in the at least one UC41-expressing prostate cell; and (d)
comparing the level of UC41 expression in the at least one
UC41-expressing prostate cell of step (c) with the level of UC41
expression measured in at least one UC41-expressing prostate cell
in the absence of the candidate substance, wherein a candidate
substance inhibits expression of the UC41 gene when the level of
UC41 expression in the at least one UC41-expressing prostate cell
of step (c) is less than the level of UC41 expression measured in
the at least one UC41-expressing prostate cell in the absence of
the candidate substance.
57. The method of claim 56, wherein UC41 expression is measured by
detection of expression of a polynucleotide that hybridizes to a
polynucleotide comprising the sequence set forth in SEQ ID NOs. 1,
3 or 4.
58. The method of any one of claims 56, wherein the at least one
UC41 expressing prostate cell is provided as an adherent cell on a
culture dish, as part of an alginate biomatrix, or in a suspension
culture.
59. The method of claim 56, wherein the level of UC41 expression is
measured by a Northern blot assay or a slot blot assay.
60. The method of claim 57, wherein the level of UC41 expression is
measured by an enzyme-linked immunosorbent assay (ELISA), a
radioimmunoassay (RIA), a Western blot assay, a dot blotting assay,
a fluorescence activated cell sorting (FACS) assay or an
immunohistochemistry assay.
61. The method of claim 56, wherein the at least one prostate
cancer cell comprises a biological sample from a subject with
prostate cancer.
62. The method of claim 56, wherein the at least one
UC41-expressing prostate cancer cell is in a subject with prostate
cancer.
63. The method of claim 62, wherein the level of UC41 expression is
measured by a radio-imaging assay.
64. The method of claim 63, wherein the radio-imaging assay
comprises the steps of: (a) administering to the subject an
imaging-effective amount of a detectably-labeled antibody that
binds immunologically to a polypeptide comprising SEQ ID NO:2 or
SEQ ID NO:5 and a pharmaceutically effective carrier; (b) detecting
the antibody bound to prostate cancer cells in the subject.
65. The method of claim 64, wherein the detectably-labeled antibody
comprises an antibody conjugated to a radioactive moiety.
66. The method of claim 65, wherein the radioactive moiety is a
radionuclide.
67. The use of a prostate cell which expresses a UC41 gene encoding
the polynucleotide sequence set forth in SEQ NOs. 1, 3 or 4 to
screen for modulators of UC41 expression.
68. The use of claim 67, wherein the UC41-expressing prostate cell
is provided as an adherent cell on a culture dish, as part of an
alginate biomatrix, or in a suspension culture.
Description
1.0 BACKGROUND OF THE INVENTION
[0001] 1.1 Field of the Invention
[0002] The present invention relates generally to novel nucleic
acid sequences, polypeptides encoded by the novel nucleic acid
sequences and antibodies specific for such polypeptides, useful as
probes or primers for the diagnosis, prognosis and management of
prostate cancer and methods relating thereto. More particularly,
the present invention concerns probes, primers and methods useful
in diagnosing, identifying and monitoring the progression of
prostate cancer through measurements of gene products. The present
invention also concerns a novel prostate specific gene and methods
of treatment for prostate cancer, based upon the disclosed nucleic
acid and polypeptide sequences.
[0003] 1.2 Description of the Related Art
[0004] Genetic detection of human disease states is a rapidly
developing field (Taparowsky et al., 1982; Slamon et al., 1989;
Sidransky et al., 1992; Mild et al., 1994; Dong et al., 1995;
Morahan et al., 1996; Lifton, 1996; Barinaga, 1996). However, some
problems exist with this approach. A number of known genetic
lesions merely predispose to development of specific disease
states. Individuals carrying the genetic lesion may not develop the
disease state, while other individuals may develop the disease
state without possessing a particular genetic lesion. In human
cancers, genetic defects may potentially occur in a large number of
known tumor suppresser genes and proto-oncogenes.
[0005] The genetic detection of cancer has a long history. One of
the earliest genetic lesions shown to predispose to cancer was
transforming point mutations in the ras oncogenes (Taparowsky et
al., 1982). Transforming ras point mutations may be detected in the
stool of individuals with benign and malignant colorectal tumors
(Sidransky et al., 1992). However, only 50% of such tumors
contained a ras mutation (Sidransky et al., 1992). Similar results
have been obtained with amplification of HER-2/neu in breast and
ovarian cancer (Slamon et al., 1989), deletion and mutation of p53
in bladder cancer (Sidransky et al., 1991), deletion of DCC in
colorectal cancer (Fearon et al., 1990) and mutation of BRCA1 in
breast and ovarian cancer (Miki et al., 1994).
[0006] None of these genetic lesions are capable of predicting a
majority of individuals with cancer and most require direct
sampling of a suspected tumor, making screening difficult.
[0007] Further, none of the markers described above are capable of
distinguishing between metastatic and non-metastatic forms of
cancer. In effective management of cancer patients, identification
of those individuals whose tumors have already Metastasized or are
likely to metastasize is critical. Because metastatic cancer kills
560,000 people in the US each year (ACS home page), identification
of markers for metastatic prostate cancer would be an important
advance.
[0008] A particular problem in cancer detection and diagnosis
occurs with prostate cancer. Carcinoma of the prostate (PCA) is the
most frequently diagnosed cancer among men in the United States
(Veltri et al., 1996). Prostate cancer was diagnosed in
approximately 189,500 men in 1998 and about 40,000 men succumbed to
the malignancy (Landis et al, 1998). Although relatively few
prostate tumors progress to clinical significance during the
lifetime of the patient, those which are progressive in nature are
likely to have metastasized by the time of detection. Survival
rates for individuals with metastatic prostate cancer are quite
low. Between these extremes are patients with prostate tumors that
will metastasize but have not yet done so, for whom surgical
prostate removal is curative. Determination of which group a
patient falls within is critical in determining optimal treatment
and patient survival.
[0009] The FDA approval of the serum prostate specific antigen
(PSA) test in 1984 changed the way that prostate disease was
managed (Allhoff et al., 1989; Cooner et al., 1990; Jacobson et
al., 1995; Orozco et al., 1998). PSA is widely used as a serum
biomarker to detect and monitor therapeutic response in prostate
cancer patients (Badalament et al., 1996; O'Dowd et al., 1997).
Several modifications in PSA assays (Partin and Oesterling, 1994;
Babian et al., 1996; Zlotta et al., 1997) have resulted in earlier
diagnoses and improved treatment.
[0010] Although PSA has been widely used as a clinical marker of
prostate cancer since 1988 (Partin and Oesterling, 1994), screening
programs utilizing PSA alone or in combination with digital rectal
examination (DRE) have not been successful in improving the
survival rate for men with prostate cancer (Partin and Oesterling,
1994). Although PSA is specific to prostate tissue, it is produced
by normal and benign as well as malignant prostatic epithelium,
resulting in a high false-positive rate for prostate cancer
detection (Partin and Oesterling, 1994).
[0011] While an effective indicator of prostate cancer when serum
levels are relatively high, PSA serum levels are more ambiguous
indicators of prostate cancer when only modestly elevated, for
example when levels are between 2-10 ng/ml. At these modest
elevations, serum PSA may have originated from non-cancerous
disease states such as BPH (benign prostatic hyperplasia),
prostatitis or physical trauma (McCormack et al., 1995). Although
application of the lower 2.0 ng/ml cancer detection cutoff
concentration of serum PSA has increased the diagnosis of prostate
cancer, especially in younger men with non-palpable early stage
tumors (Stage Tlc) (Soh et al., 1997; Carter and Coffey, 1997;
Harris et al., 1997; Orozco et al., 1998), the specificity of the
PSA assay for prostate cancer detection at low serum PSA levels
remains a problem.
[0012] Several investigators have sought to improve upon the
specificity of serologic detection of prostate cancer by examining
a variety of other biomarkers besides serum PSA concentration
(Ralph and Veltri, 1997). One of the most heavily investigated of
these other biomarkers is the ratio of free versus total PSA (f/t
PSA) in a patient's blood. Most PSA in serum is in a molecular form
that is bound to other proteins such as .alpha.1-antichymotrypsin
(ACT) or .alpha.2-macroglobulin (Christensson et al., 1993; Stenman
et al., 1991; Lilja et al., 1991). Free PSA is not bound to other
proteins. The ratio of free to total PSA (f/tPSA) is usually
significantly higher in patients with BPH compared to those with
organ confined prostate cancer (Marley et al., 1996; Oesterling et
al., 1995; Pettersson et al., 1995). When an appropriate cutoff is
determined for the f/tPSA assay, the f/tPSA assay can help
distinguish patients with BPH from those with prostate cancer in
cases in which serum PSA levels are only modestly elevated (Marley
et al., 1996; Partin and Oesterling, 1996). Unfortunately, while
f/tPSA may improve on the detection of prostate cancer, information
in the f/tPSA ratio is insufficient to improve the sensitivity and
specificity of serologic detection of prostate cancer to desirable
levels.
[0013] Other markers that have been used for prostate cancer
detection include prostatic acid phosphatase (PAP) and prostate
secreted protein (PSP). PAP is secreted by prostate cells under
hormonal control (Brawn et al., 1996). It has less specificity and
sensitivity than does PSA. As a result, it is used much less now,
although PAP may still have some applications for monitoring
metastatic patients that have failed primary treatments. In
general, PSP is a more sensitive biomarker than PAP, but is not as
sensitive as PSA (Huang et al., 1993). Like PSA, PSP levels are
frequently elevated in patients with BPH as well as those with
prostate cancer.
[0014] Another serum marker associated with prostate disease is
prostate specific membrane antigen (PSMA) (Horoszewicz et al.,
1987; Carter and Coffey, 1996; Murphy et al., 1996). PSMA is a Type
II cell membrane protein and has been identified as Folic Acid
Hydrolase (FAH) (Carter and Coffey, 1996). Antibodies against PSMA
react with both normal prostate tissue and prostate cancer tissue
(Horoszewicz et al., 1987). Murphy et al. (1995) used ELISA to
detect serum PSMA in advanced prostate cancer. As a serum test,
PSMA levels are a relatively poor indicator of prostate cancer.
However, PSMA may have utility in certain circumstances. PSMA is
expressed in metastatic prostate tumor capillary beds (Silver et
al., 1997) and is reported to be more abundant in the blood of
metastatic cancer patients (Murphy et al., 1996). PSMA messenger
RNA (mRNA) is down-regulated 8-10 fold in the LNCaP prostate cancer
cell line after exposure to 5-.alpha.-dihydroxytestosterone (DHT)
(Israeli et al., 1994).
[0015] Two relatively new potential biomarkers for prostate cancer
are human kallekrein 2 (HK2) (Piironen et al., 1996) and prostate
specific transglutaminase (pTGase) (Dubbink et al., 1996). HK2 is a
member of the kallekrein family that is secreted by the prostate
gland (Piironen et al., 1996). Prostate specific transglutaminase
is a calcium-dependent enzyme expressed in prostate cells that
catalyzes post-translational cross-linking of proteins (Dubbink et
al., 1996). In theory, serum concentrations of HK2 or pTGase may be
of utility in prostate cancer detection or diagnosis, but the
usefulness of these markers is still being evaluated.
[0016] Interleukin 8 (IL-8) has also been reported as a marker for
prostate cancer. (Veltri et al, 1999). Serum IL-8 concentrations
were reported to be correlated with increasing stage of prostate
cancer and to be capable of differentiating BPH from malignant
prostate tumors. (Id.) The wide-scale applicability of this marker
for prostate cancer detection and diagnosis is still under
investigation.
[0017] In addition to these protein markers for prostate cancer,
several genetic changes have been reported to be associated with
prostate cancer, including: allelic loss (Bova, et al., 1993;
Macoska et al., 1994; Carter et al., 1990); DNA hypermethylation
(Isaacs et al., 1994); point mutations or deletions of the
retinoblastoma (Rb), p53 and KAI1 genes (Bookstein et al., 1990a;
Bookstein et al., 1990b; Isaacs et al, 1991; Dong et al., 1995);
and aneuploidy and aneusomy of chromosomes detected by fluorescence
in situ hybridization (FISH) (Macoska et al., 1994; Visakorpi et
al., 1994; Takahashi et al, 1994; Alcaraz et al., 1994). None of
these has been reported to exhibit sufficient sensitivity and
specificity to be useful as general screening tools for
asymptomatic prostate cancer.
[0018] A recent discovery was that differential expression of both
full-length and truncated forms of HER2/neu oncogene receptor was
correlated with prostate cancer. (An et al., 1998). Analysis by
RT-PCR.TM. indicated that overexpression of the HER2/neu gene is
associated with prostate cancer progression. (Id.)
[0019] In current clinical practice, the serum PSA assay and
digital rectal exam (DRE) is used to indicate which patients should
have a prostate biopsy (Lithrup et al., 1994; Orozco et al., 1998).
Histological examination of the biopsied tissue is used to make the
diagnosis of prostate cancer. Based upon the 189,500 cases of
diagnosed prostate cancer in 1998 (Landis, 1998) and a known cancer
detection rate of about 35% (Parker et al., 1996), it is estimated
that in 1998 over one-half million prostate biopsies were performed
in the United States (Orozco et al., 1998; Veltri et al., 1998).
Clearly, there would be much benefit derived from a serological
test that was sensitive enough to detect small and early stage
prostate tumors that also had sufficient specificity to exclude a
greater portion of patients with noncancerous or clinically
insignificant conditions.
[0020] There remain deficiencies in the prior art with respect to
the identification of the genes linked with the progression of
prostate cancer and the development of diagnostic methods to
monitor disease progression. Likewise, the identification of genes
which are differentially expressed in prostate cancer would be of
considerable importance in the development of a rapid, inexpensive
method to diagnose cancer. Although a few prostate specific genes
have been cloned (PSA, PSMA, HK2, pTGase, etc.), these are
typically not up-regulated in prostate cancer. The identification
of a novel, prostate specific gene that is differentially expressed
in prostate cancer, compared to non-malignant prostate tissue,
would represent a major, unexpected advance for the diagnosis,
prognosis and treatment of prostate cancer.
2.0 SUMMARY OF THE INVENTION
[0021] The present invention addresses deficiencies in the prior
art by identifying a novel, prostate specific gene that is
differentially expressed in human prostate cancer compared to
normal human prostate or benign prostatic hyperplasia (BPH). The
encoded mRNA species and the corresponding encoded protein species
have utility, for example, as markers of prostate cancer.
Antibodies against the encoded protein species, as well as
antisense constructs specific for the mRNA species, have utility
for methods of therapeutic treatment of prostate cancer. In
addition, the cDNA sequence can be used to design probes and
primers for identification of a full length genomic sequence, as
well as the promoter sequence for the gene, of use in the design of
prostate specific expression vectors of utility in the gene therapy
of prostate cancer.
[0022] The nucleic acid sequence of this novel, prostate specific
gene can be used to design specific oligonucleotide probes and
primers. When used in combination with nucleic acid hybridization
and amplification procedures, these probes and primers permit the
rapid analysis of prostate biopsy core specimens, serum samples,
etc. This will assist physicians in diagnosing prostate cancer and
in determining optimal treatment courses for individuals with
prostate tumors of varying malignancy. The same probes and primers
also may be used for in situ hybridization or in situ PCR.TM.
detection and diagnosis of prostate cancer.
[0023] The novel gene sequence also may be used to identify and
isolate a full length genomic DNA sequence and its associated
regulatory elements, including the promoter, from genomic human DNA
libraries. The cDNA sequence identified in the present invention is
first used to construct hybridization probes to screen genomic
human DNA libraries by standard techniques. Once partial genomic
clones have been identified, full-length genes are isolated by
"chromosomal walking" (also called "overlap hybridization"). See,
Chinault and Carbon, 1979. Nonrepetitive sequences at or near the
ends of the partial genomic clones are then used as hybridization
probes in further genomic library screening, ultimately allowing
the isolation of entire genomic sequence for the novel prostate
specific gene reported herein. Those experienced in the art will
realize that full length genes may be obtained using the cDNA
sequence described herein using technology currently available
(Sambrook et al., 1989; Chinault and Carbon, 1979).
[0024] In the practice of this method, the cDNA sequence identified
in the present disclosure is used as a hybridization probe to
screen human genomic DNA libraries by standard techniques. In a
preferred practice, a high quality human genomic DNA library is
obtained from commercial or other sources. The library is plated
on, for example, agarose plates containing nutrients, antibiotics
and other standard ingredients. Individual colonies are transferred
to nylon or nitrocellulose membranes and the cDNA probes are
hybridized to complementary sequences on the membranes.
Hybridization is detected by radioactive or enzyme-linked tags
associated with the hybridized probes. Positive colonies are grown
up and sequenced by, for example, dideoxy nucleotide sequencing or
similar methods well known in the art. Comparison of cloned
sequences with known human or animal cDNA or genomic sequences is
performed using computer programs and databases well known to the
skilled practitioner.
[0025] In one embodiment of the present invention, the isolated
nucleic acids of the present invention are incorporated into
expression vectors and expressed as the encoded proteins or
peptides. Such proteins or peptides may in certain embodiments be
used as antigens for induction of monoclonal or polyclonal antibody
production.
[0026] One aspect of the present invention is thus, oligonucleotide
hybridization probes and primers that hybridize selectively to
samples of prostate cancer. These probes and primers are selected
from those sequences designated herein as SEQ ID NO:1, SEQ ID NO:3
and SEQ ID NO:4. The availability of probes and primers specific
for such prostate specific nucleic acid sequences, that are
differentially expressed in prostate cancer, provides the basis for
diagnostic kits useful for distinguishing between BPH, prostate
organ confined cancer and metastatic prostate tumors.
Alternatively, the availability of probes and primers that
hybridize to one or more nucleic acids corresponding to SEQ ID
NO:1, SEQ ID NO:3 or SEQ ID NO:4 provide the basis for diagnostic
kits useful in the detection of prostate cancer.
[0027] In one broad aspect, the present invention encompasses kits
for use in detecting prostate cancer cells in a biological sample.
Such a kit may comprise one or more pairs of primers for amplifying
nucleic acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:4. The kit may further comprise samples of total mRNA derived
from tissue of various physiological states, such as normal, BPH,
confined tumor and metastatically progressive tumor, for example,
to be used as controls. The kit also may comprise buffers,
nucleotide bases, and other compositions to be used in
hybridization and/or amplification reactions. Each solution or
composition may be contained in a vial or bottle and all vials held
in close confinement in a box for commercial sale. Another
embodiment of the present invention encompasses a kit for use in
detecting prostate cancer cells in a biological sample comprising
oligonucleotide probes effective to bind with high affinity to
nucleic acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:4 in a Northern blot assay and containers for each of these
probes. In a further embodiment, the invention encompasses a kit
for use in detecting prostate cancer cells in a biological sample
comprising antibodies specific for proteins encoded by nucleic
acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4,
identified in the present invention.
[0028] In one broad aspect, the present invention encompasses
methods for treating prostate cancer patients by administration of
effective amounts of antibodies specific for the peptide products
of nucleic acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ
ID NO:4, or by administration of effective amounts of vectors
producing antisense messenger RNAs that bind to nucleic acids
corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, thereby
inhibiting expression of the protein products of a prostate
specific gene that is overexpressed in prostate cancer. Antisense
nucleic acid molecules also may be provided as RNAs, as some stable
forms of RNA with a long half-life that may be administered
directly without the use of a vector are now known in the art. In
addition, DNA constructs may be delivered to cells by liposomes,
receptor mediated transfection and other methods known in the art.
Delivery of the present agents, by any means known in the art would
be encompassed by the present claims.
[0029] One aspect of the present invention is novel isolated
nucleic acid segments that are useful as described herein as
hybridization probes and primers that specifically hybridize to
nucleic acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:4. These nucleic acids are described herein as species shown to
be differentially expressed in prostate cancer as compared to BPH
and normal prostate tissue. The invention further comprises an
isolated nucleic acid of between about 14 and about 100 bases in
length, either identical to or complementary to a portion of the
same length occurring within the disclosed sequences.
[0030] The present invention comprises proteins and peptides with
amino acid sequences encoded by the aforementioned isolated nucleic
acid segments.
[0031] The invention further comprises methods for detecting
prostate cancer cells in biological samples, using hybridization
primers and probes designed to specifically hybridize to nucleic
acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4.
This method further comprises measuring the amounts of nucleic acid
amplification products formed when primers selected from the
designated sequences are used.
[0032] The invention further comprises the prognosis and/or
diagnosis of prostate cancer by measuring the amounts of nucleic
acid amplification products formed as above. The invention
comprises methods of treating individuals with prostate cancer by
providing effective amounts of antibodies and/or antisense DNA
molecules which bind to the products of the above mentioned
isolated nucleic acids. The invention further comprises kits for
performing the above-mentioned procedures, containing antibodies,
amplification primers and/or hybridization probes.
[0033] The present invention further comprises production of
antibodies specific for proteins or peptides encoded by nucleic
acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, and
the use of those antibodies for diagnostic applications in
detecting prostate cancer. The invention further comprises
therapeutic treatment of prostate cancer by administration of
effective doses of inhibitors specific for the aforementioned
encoded proteins.
3.0 BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1. Identification of the UC41 cDNA clone and
confirmation of differential expression. Panel A. Results of an
agarose gel-based differential display study, comparing RNA from
normal prostate tissues (N) with RNA from prostate cancer tissues
(C). The position of the UC41 band is at the bottom of the panel,
just above the label "UC41". Panel B. Confirmation of differential
expression of UC4I by RT-PCR.TM. of normal prostate tissues (N) and
prostate cancer tissues (C). The position of the UC41 band in this
panel is adjacent to the label "UC41".
[0035] FIG. 2. UC41 expression in pair-matched normal and cancer
tissues. The pair-matched tissue was microdissected from OCT
embedded radical prostatectomies as described below in the section
MATERIAL AND METHODS. .beta..sub.2 microglobin RT-PCR.TM. was used
as a control. Matched samples were from normal prostate (N) and
adjacent prostate cancer tissues (C). The insert on the far right
side of the FIG. shows the results of RT-PCR.TM. for UC41 and
.beta..sub.2 microglobin in the LNCaP, PC-3 and DU 145 prostate
cancer cell lines and the Lu 23 and Lu 35 prostate cancer
xenografts.
[0036] FIG. 3. In situ hybridization performed with both sense and
antisense probes labeled with digoxigenin-dUTP as described below
in the section MATERIAL AND METHODS. UC41 was predominantly
expressed in the basal cells of normal prostate (far left). Its
expression was upregulated in prostate cancer (middle and far
right).
[0037] FIG. 4. In situ hybridization performed with both sense and
antisense probes labeled with digoxigenin-dUTP as described below
in the section MATERIAL AND METHODS. UC41 was predominantly
expressed in the basal cells of normal prostate (far left). Its
expression was upregulated in prostate cancer metastatic to lymph
nodes (middle and far right).
[0038] FIG. 5. In situ hybridization performed with both sense and
antisense probes labeled with digoxigenin-dUTP as described below
in the section MATERIAL AND METHODS. UC41 was predominantly
expressed in the basal cells of normal prostate (far left). Its
expression was upregulated in prostate cancer metastatic to bone
(middle and far right).
[0039] FIG. 6. Expression of UC41 in different normal tissues
analyzed by Northern blot hybridization. The human multiple tissue
northern blot (Clontech, 2 .mu.g of poly A.sup.+ RNA per lane) was
blotted as described below in the section MATERIAL AND METHODS.
Relative expression of UC41 is shown in human spleen (lane 1),
thymus (lane 2), prostate (lane 3), testis (lane 4), ovary (lane
5), small intestine (lane 6), colon (mucosal lining, lane 7) and
peripheral blood leukocyte tissues (lane 8). The UC41 band of
approximately 2.4 kb is expressed only in normal prostate
tissue.
[0040] FIG. 7. Confirmation of prostate specific expression in UC41
by slot blot analysis. A human RNA master filter (Clontech, 89-514
ng of poly A.sup.+ RNA per lane) was probed with a UC41 specific
probe as described below in the section MATERIAL AND METHODS.
Expression of UC41 was examined in RNA samples from (top row) whole
brain, amygdala, caudate nucleus, cerebellum, cerebral cortex,
frontal lobe, hippocampus, medulla oblongata, (second row)
occipital lobe, putamen, substantia nigra, temporal lobe, thalamus,
nucleus acumens, spinal cord, (third row) heart, aorta, skeletal
muscle, colon, bladder, uterus, prostate, stomach, (fourth row)
testis, ovary, pancreas, pituitary gland, adrenal gland, thyroid
gland, salivary gland, mammary gland, (fifth row) kidney, liver,
small intestine, spleen, thymus, peripheral leukocyte, lymph node,
bone marrow, (sixth row) appendix, lung, trachea, placenta,
(seventh row) fetal brain, fetal heart, fetal kidney, fetal liver,
fetal spleen, fetal thymus, fetal lung, (bottom row) yeast total
RNA, yeast tRNA, E. coli rRNA. E. coli DNA, Poly r(A), human Cot1
DNA, human DNA (100 ng) and human DNA (500 ng).
[0041] FIG. 8. Results of UC41 FISH mapping. Panel A shows the FISH
signals on a metaphase chromosome. Panel B shows the same mitotic
figure stained with DAPI to identify chromosome 3. Panel C shows a
diagram of the FISH mapping results, wherein each dot represents
the double FISH signals detected on human chromosome 3.
[0042] FIG. 9. Sequence analysis of UC41 cDNA. The UC41 cDNA
sequence and predicted amino acid sequence are shown. The predicted
leader sequence is indicated by the underlined box. The predicted
transmembrane region is underlined in bold. The prediction of
transmembrane region is based on statistical analysis of Tmbase, a
database of naturally occurring transmembrane proteins.
4.0 DETAILED DESCRIPTION OF THE INVENTION
[0043] Previous work by the present inventors and others resulted
in the identification of an expressed sequence tag (EST), referred
to as UC41, whose expression was increased in prostate cancer cells
compared to normal or benign prostate tissues. These and other
results were reported in U.S. patent application Ser. No.
08/692,787, the entire text of which is incorporated herein by
reference.
[0044] The present invention discloses the entire cDNA sequence of
the UC41 gene (SEQ ID NO:1), and identifies UC41 as a novel,
prostate specific gene whose expression is upregulated in prostate
cancer. As such, the UC41 gene is an indicator of malignant
transformation of prostate tissues. The skilled artisan will
recognize that such a differentially expressed, prostate specific
gene has utility in the early detection, diagnosis, prognosis and
treatment of prostate cancer, within the scope of the present
invention.
[0045] Those skilled in the art will realize that the nucleic acid
sequences disclosed herein will find utility in a variety of
applications in prostate cancer detection, diagnosis, prognosis and
treatment. Examples of such applications within the scope of the
present invention comprise amplification of one or more nucleic
acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4
using specific primers; detection of nucleic acids corresponding to
SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4 by hybridization with
oligonucleotide probes; incorporation of isolated nucleic acids
into vectors; expression of RNA, peptides or polypeptides from the
vectors; development of immunologic reagents corresponding to
proteins encoded by isolated nucleic acids corresponding to SEQ ID
NO:1, SEQ ID NO:3 or SEQ ID NO:4; and therapeutic treatments of
prostate cancer using antibodies, antisense nucleic acids, or other
inhibitors specific for the identified prostate specific gene
products.
4.1 Nucleic Acids
[0046] As described herein, an aspect of the present disclosure is
nucleic acids corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:4. These sequences, in turn, correspond to: the entire cDNA
sequence of UC41 (SEQ ID NO:1, FIG. 9); the cDNA sequence of UC41
that is 5' to the UC41 EST sequence disclosed in U.S. patent
application Ser. No. 08/692,787 (SEQ ID NO:3, FIG. 9, bases 1-1322
of cDNA sequence); and the cDNA sequence of UC41 that is 3' to the
UC41 EST sequence (SEQ ID NO:4, FIG. 9, bases 1501-1934 of cDNA
sequence).
[0047] In one embodiment, the nucleic acid sequences disclosed
herein will find utility as hybridization probes or amplification
primers. These nucleic acids may be used, for example, in
diagnostic evaluation of tissue samples or employed to clone full
length genomic clones, including promoter and other regulatory
sequences, corresponding thereto. In certain embodiments, these
probes and primers consist of oligonucleotide fragments. Such
fragments should be of sufficient length to provide specific
hybridization to an RNA or DNA tissue sample. The sequences
typically will be 10-20 nucleotides, but may be longer. Longer
sequences, e.g., 40, 50, 100, 500 and even up to full length, are
preferred for certain embodiments.
[0048] Nucleic acid molecules having contiguous stretches of about
10, 13, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 75,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,
1300, 1400, 1500, 1600, 1700, 1800 or 1900 nucleotides, up to the
full length of the disclosed sequences, from a sequence selected
from SEQ ID NO:3 and SEQ ID NO:4. are contemplated. Molecules that
are complementary to the above mentioned sequences and that bind to
these sequences under high stringency conditions also are
contemplated. These probes will be useful in a variety of
hybridization embodiments, such as Southern and Northern blotting.
In some cases, it is contemplated that probes may be used that
hybridize to multiple target sequences without compromising their
ability to effectively diagnose cancer.
[0049] Various probes and primers can be designed around the
disclosed nucleotide sequences. Primers may be of any length but,
typically, are 10-20 bases in length. By assigning numeric values
to a sequence, for example, the first residue is 1, the second
residue is 2, etc., an algorithm defining all primers can be
proposed:
n to n+y
[0050] where n is an integer from 1 to the last number of the
sequence and y is the length of the primer minus one (9 to 19),
where n+y does not exceed the last number of the sequence. Thus,
for a 10-mer, the probes correspond to bases 1 to 10, 2 to 11, 3 to
12 . . . and so on. For a 15-mer, the probes correspond to bases 1
to 15, 2 to 16, 3 to 17 . . . and so on. For a 20-mer, the probes
correspond to bases 1 to 20, 2 to 21, 3 to 22 . . . and so on.
[0051] The values of n in the algorithm above for each of the
nucleic acid sequences is: SEQ ID NO:3, n=1322; SEQ ID NO:4,
n=434.
[0052] The use of a hybridization probe of between 14 and 100
nucleotides in length allows the formation of a duplex molecule
that is both stable and selective. Molecules having complementary
sequences over stretches greater than 20 bases in length are
generally preferred, in order to increase stability and selectivity
of the hybrid, and thereby improve the quality and degree of
particular hybrid molecules obtained. One will generally prefer to
design nucleic acid molecules having stretches of 20 to 30
nucleotides, or even longer where desired. Such fragments may be
readily prepared by, for example, directly synthesizing the
fragment by chemical means or by introducing selected sequences
into recombinant vectors for recombinant production.
[0053] Accordingly, the nucleotide sequences of the invention may
be used for their ability to selectively form duplex molecules with
complementary stretches of genes or RNAs or to provide primers for
amplification of DNA or RNA from tissues. Depending on the
application envisioned, one will desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of probe towards target sequence.
[0054] For applications requiring high selectivity, one will
typically desire to employ relatively stringent conditions to form
the hybrids, e.g., one will select relatively low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.10 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. Such high stringency conditions tolerate little, if
any, mismatch between the probe and the template or target strand,
and would be particularly suitable for isolating specific genes or
detecting specific mRNA transcripts. It is generally appreciated
that conditions can be rendered more stringent by the addition of
increasing amounts of formamide.
[0055] For certain applications, for example, substitution of amino
acids by site-directed mutagenesis, it is appreciated that lower
stringency conditions are required. Under these conditions,
hybridization may occur even though the sequences of probe and
target strand are not perfectly complementary, but are mismatched
at one or more positions. Conditions may be rendered less stringent
by increasing salt concentration and decreasing temperature. For
example, a medium stringency condition could be provided by about
0.1 to 0.25 M NaCl at temperatures of about 37.degree. C. to about
55.degree. C., while a low stringency condition could be provided
by about 0.15 M to about 0.9 M salt, at temperatures ranging from
about 20.degree. C. to about 55.degree. C. Thus, hybridization
conditions can be readily manipulated, and thus will generally be a
method of choice depending on the desired results.
[0056] The following codon chart may be used, in a site-directed
mutagenic scheme, to produce nucleic acids encoding the same or
slightly different amino acid sequences of a given nucleic
acid:
TABLE-US-00001 TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0057] In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl.sub.2, 10 mM dithiothreitol, at temperatures between
approximately 20.degree. C. to about 37.degree. C. Other
hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 .mu.M MgCl.sub.2, at temperatures
ranging from approximately 40.degree. C. to about 72.degree. C.
[0058] In certain embodiments, it will be advantageous to employ
nucleic acid sequences of the present invention in combination with
an appropriate means, such as a label, for determining
hybridization. A wide variety of appropriate indicator means are
known in the art, including fluorescent, radioactive, enzymatic or
other ligands, such as avidin/biotin, which are capable of being
detected. In preferred embodiments, one may desire to employ a
fluorescent label or an enzyme tag such as urease, alkaline
phosphatase or peroxidase, instead of radioactive or other
environmentally undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known which can be employed
to provide a detection means visible to the human eye or
spectrophotometrically, to identify specific hybridization with
complementary nucleic acid-containing samples.
[0059] In general, it is envisioned that the hybridization probes
described herein will be useful both as reagents in solution
hybridization, as in PCR.TM., for detection of expression of
corresponding genes, as well as in embodiments employing a solid
phase. In embodiments involving a solid phase, the test DNA (or
RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to hybridization with selected probes under desired conditions. The
selected conditions will depend on the particular circumstances
based on the particular criteria required (depending, for example,
on the G+C content, type of target nucleic acid, source of nucleic
acid, size of hybridization probe, etc.). Following washing of the
hybridized surface to remove non-specifically bound probe
molecules, hybridization is detected, or even quantified, by means
of the label.
[0060] It will be understood that this invention is not limited to
the particular probes disclosed herein and particularly is intended
to encompass at least nucleic acid sequences that are hybridizable
to the disclosed sequences or are functional sequence analogs of
these sequences. For example, a partial sequence may be used to
identify a structurally-related gene or the full length genomic or
cDNA clone from which it is derived. Those of skill in the art are
well aware of the methods for generating cDNA and genomic libraries
which can be used as a target for the above-described probes
(Sambrook et al., 1989).
[0061] For applications in which the nucleic acid segments of the
present invention are incorporated into vectors, such as plasmids,
cosmids or viruses, these segments may be combined with other DNA
sequences, such as promoters, polyadenylation signals, restriction
enzyme sites, multiple cloning sites, other coding segments, and
the like, such that their overall length may vary considerably. It
is contemplated that a nucleic acid fragment of almost any length
may be employed, with the total length preferably being limited by
the ease of preparation and use in the intended recombinant DNA
protocol.
[0062] DNA segments encoding a specific gene may be introduced into
recombinant host cells and employed for expressing a specific
structural or regulatory protein. Alternatively, through the
application of genetic engineering techniques, subportions or
derivatives of selected genes may be employed. Upstream regions
containing regulatory regions such as promoter regions may be
isolated and subsequently employed for expression of the selected
gene.
[0063] Where an expression product is to be generated, it is
possible for the nucleic acid sequence to be varied while retaining
the ability to encode the same product. Reference to the codon
chart, provided above, will permit those of skill in the art to
design any nucleic acid encoding for the product of a given nucleic
acid.
4.2 Encoded Proteins
[0064] Once the entire coding sequence of the differentially
expressed, prostate specific gene has been determined, the gene can
be inserted into an appropriate expression system. The gene can be
expressed in any number of different recombinant DNA expression
systems to generate large amounts of the polypeptide product, which
can then be purified and used to vaccinate animals to generate
antisera with which further studies may be conducted.
[0065] Examples of expression systems known to the skilled
practitioner in the art include bacteria such as E. coli, yeast
such as Pichia pastoris, baculovirus, and mammalian expression
systems such as in COS or CHO cells. A complete gene can be
expressed or, alternatively, fragments of the gene encoding
portions of polypeptide can be produced.
[0066] In certain broad applications of the invention, the gene
sequence encoding the polypeptide is analyzed to detect putative
transmembrane sequences. Such sequences are typically very
hydrophobic and are readily detected by the use of standard
sequence analysis software, such as MacVector (IBI, New Haven,
Conn.). The presence of transmembrane sequences is often
deleterious when a recombinant protein is synthesized in many
expression systems, especially E. coli, as it leads to the
production of insoluble aggregates which are difficult to renature
into the native conformation of the protein. Deletion of
transmembrane sequences typically does not significantly alter the
conformation of the remaining protein structure.
[0067] Moreover, transmembrane sequences, being by definition
embedded within a membrane, are inaccessible. Antibodies to these
sequences may not, therefore, prove useful in in vivo or in situ
studies. Deletion of transmembrane-encoding sequences from the
genes used for expression can be achieved by standard techniques.
For example, fortuitously-placed restriction enzyme sites can be
used to excise the desired gene fragment, or PCR.TM.-type
amplification can be used to amplify only the desired part of the
gene.
[0068] Computer sequence analysis may be used to determine the
location of the predicted major antigenic determinant epitopes of
the polypeptide. Software capable of carrying out this analysis is
readily available commercially, for example MacVector (IBI, New
Haven, Conn.). The software typically uses standard algorithms such
as the Kyte/Doolittle or Hopp/Woods methods for locating
hydrophilic sequences may be found on the surface of proteins and
are, therefore, likely to act as antigenic determinants.
[0069] Once this analysis is made, polypeptides may be prepared
which contain at least the essential features of the antigenic
determinant and which may be employed in the generation of antisera
against the polypeptide. Minigenes or gene fusions encoding these
determinants may be constructed and inserted into expression
vectors by standard methods, for example, using PCR.TM. cloning
methodology.
[0070] The gene or gene fragment encoding a polypeptide may be
inserted into an expression vector by standard subcloning
techniques. An E. coli expression vector may be used which produces
the recombinant polypeptide as a fusion protein, allowing rapid
affinity purification of the protein. Examples of such fusion
protein expression systems are the glutathione S-transferase system
(Pharmacia, Piscataway, N.J.), the maltose binding protein system
(NEB, Beverley, Mass.), the FLAG system (IBI, New Haven, Conn.),
and the 6.times. His system (Qiagen, Chatsworth, Calif.).
[0071] Some of these systems produce recombinant polypeptides
bearing only a small number of additional amino acids, which are
unlikely to affect the antigenic ability of the recombinant
polypeptide. For example, both the FLAG system and the 6.times. His
system add only short sequences, both of which are known to be
poorly antigenic and which do not adversely affect folding of the
polypeptide to its native conformation. Other fusion systems are
designed to produce fusions wherein the fusion partner is easily
excised from the desired polypeptide. In one embodiment, the fusion
partner is linked to the recombinant polypeptide by a peptide
sequence containing a specific recognition sequence for a protease.
Examples of suitable sequences are those recognized by the Tobacco
Etch Virus protease (Life Technologies, Gaithersburg, Md.) or
Factor Xa (New England Biolabs, Beverley, Mass.).
[0072] The expression system used also may be one driven by the
baculovirus polyhedron promoter. The gene encoding the polypeptide
may be manipulated by standard techniques in order to facilitate
cloning into the baculovirus vector. One baculovirus vector is the
pBlueBac vector (Invitrogen, Sorrento, Calif.). The vector carrying
the gene for the polypeptide is transfected into Spodoptera
frugiperda (Sf9) cells by standard protocols, and the cells are
cultured and processed to produce the recombinant antigen. See
Summers et al., U.S. Pat. No. 4,215,051 (incorporated herein by
reference).
[0073] As an alternative to recombinant polypeptides, synthetic
peptides corresponding to the antigenic determinants may be
prepared. Such peptides are at least six amino acid residues long,
and may contain up to approximately 35 residues, which is the
approximate upper length limit of automated peptide synthesis
machines, such as those available from Applied Biosystems (Foster
City, Calif.). Use of such small peptides for vaccination typically
requires conjugation of the peptide to an immunogenic carrier
protein such as hepatitis B surface antigen, keyhole limpet
hemocyanin or bovine serum albumin. Methods for performing this
conjugation are well known in the art.
[0074] Amino acid sequence variants of the polypeptide also may be
prepared. These may, for instance, be minor sequence variants of
the polypeptide which arise due to natural variation within the
population or they may be homologues found in other species. They
also may be sequences which do not occur naturally but which are
sufficiently similar that they function similarly and/or elicit an
immune response that cross-reacts with natural forms of the
polypeptide. Sequence variants may be prepared by standard methods
of site-directed mutagenesis such as those described herein for
removing the transmembrane sequence.
[0075] Amino acid sequence variants of the polypeptide may be
substitutional, insertional or deletion variants. Deletion variants
lack one or more residues of the native protein which are not
essential for function or immunogenic activity, and are exemplified
by the variants lacking a transmembrane sequence. Another common
type of deletion variant is one lacking secretory signal sequences
or signal sequences directing a protein to bind to a particular
part of a cell. An example of the latter sequence is the SH2
domain, which induces protein binding to phosphotyrosine
residues.
[0076] Substitutional variants typically contain an alternative
amino acid at one or more sites within the protein, and may be
designed to modulate one or more properties of the polypeptide such
as stability against proteolytic cleavage. Substitutions preferably
are conservative, that is, one amino acid is replaced with one of
similar size and charge. Conservative substitutions are well known
in the art and include, for example, the changes of: alanine to
serine; arginine to lysine; asparagine to glutamine or histidine;
aspartate to glutamate; cysteine to serine; glutamine to
asparagine; glutamate to aspartate; glycine to proline; histidine
to asparagine or glutamine; isoleucine to leucine or valine;
leucine to valine or isoleucine; lysine to arginine, glutamine, or
glutamate; methionine to leucine or isoleucine; phenylalanine to
tyrosine, leucine or methionine; serine to threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine; and valine to isoleucine or leucine.
[0077] Insertional variants include fusion proteins such as those
used to allow rapid purification of the polypeptide and also may
include hybrid proteins containing sequences from other proteins
and polypeptides which are homologues of the polypeptide. For
example, an insertional variant may include portions of the amino
acid sequence of the polypeptide from one species, together with
portions of the homologous polypeptide from another species. Other
insertional variants may include those in which additional amino
acids are introduced within the coding sequence of the polypeptide.
These typically are smaller insertions than the fusion proteins
described above and are introduced, for example, to disrupt a
protease cleavage site.
[0078] Major antigenic determinants of the polypeptide may be
identified by an empirical approach in which portions of the gene
encoding the polypeptide are expressed in a recombinant host, and
the resulting proteins tested for their ability to elicit an immune
response. For example, PCR.TM. may be used to prepare a range of
peptides lacking successively longer fragments of the C-terminus of
the protein. The immunoprotective activity of each of these
peptides then identifies those fragments or domains of the
polypeptide which are essential for this activity. Further studies
in which only a small number of amino acids are removed at each
iteration then allows the location of the antigenic determinants of
the polypeptide.
[0079] Another method for the preparation of the polypeptides
according to the invention is the use of peptide mimetics. Mimetics
are peptide-containing molecules which mimic elements of protein
secondary structure. See, for example, Johnson et al. (1993). The
underlying rationale behind the use of peptide mimetics is that the
peptide backbone of proteins exists chiefly to orient amino acid
side chains in such a way as to facilitate molecular interactions,
such as those of antibody and antigen. A peptide mimetic is
expected to permit molecular interactions similar to the natural
molecule.
[0080] Successful applications of the peptide mimetic concept have
thus far focused on mimetics of .beta.-turns within proteins, which
are known to be highly antigenic. Likely .beta.-turn structure
within a polypeptide may be predicted by computer-based algorithms
as discussed herein. Once the component amino acids of the turn are
determined, peptide mimetics may be constructed to achieve a
similar spatial orientation of the essential elements of the amino
acid side chains.
4.3 Preparation of Antibodies Specific for Encoded Proteins
4.3.1 Expression of Proteins from Cloned cDNAs
[0081] The cDNA species specified in SEQ ID NO:1, SEQ ID NO:3 and
SEQ ID NO:4. may be expressed as encoded peptides or proteins. The
engineering of DNA segment(s) for expression in a prokaryotic or
eukaryotic system may be performed by techniques generally known to
those of skill in recombinant expression. It is believed that
virtually any expression system may be employed in the expression
of the claimed nucleic acid sequences.
[0082] Both cDNA and genomic sequences are suitable for eukaryotic
expression, as the host cell will generally process the genomic
transcripts to yield functional mRNA for translation into protein.
In addition, it is possible to use partial sequences for generation
of antibodies against discrete portions of a gene product, even
when the entire sequence of that gene product remains unknown.
Computer programs are available to aid in the selection of regions
which have potential immunologic significance. For example,
software capable of carrying out this analysis is readily available
commercially from MacVector (IBI, New Haven, Conn.). The software
typically uses standard algorithms such as the Kyte/Doolittle or
Hopp/Woods methods for locating hydrophilic sequences which are
characteristically found on the surface of proteins and are,
therefore, likely to act as antigenic determinants.
[0083] As used herein, the terms "engineered" and "recombinant"
cells are intended to refer to a cell into which an exogenous DNA
segment or gene, such as a cDNA or gene has been introduced through
the hand of man. Therefore, engineered cells are distinguishable
from naturally occurring cells which do not contain a recombinantly
introduced exogenous DNA segment or gene. Recombinant cells include
those having an introduced cDNA or genomic gene, and also include
genes positioned adjacent to a heterologous promoter not naturally
associated with the particular introduced gene.
[0084] To express a recombinant encoded protein or peptide, whether
mutant or wild-type, in accordance with the present invention one
would prepare an expression vector that comprises one of the
claimed isolated nucleic acids under the control of, or operatively
linked to, one or more promoters. To bring a coding sequence "under
the control of" a promoter, one positions the 5' end of the
transcription initiation site of the transcriptional reading frame
generally between about 1 and about 50 nucleotides "downstream"
(i.e., 3') of the chosen promoter. The "upstream" promoter
stimulates transcription of the DNA and promotes expression of the
encoded recombinant protein. This is the meaning of "recombinant
expression" in this context.
[0085] Many standard techniques are available to construct
expression vectors containing the appropriate nucleic acids and
transcriptional/translational control sequences in order to achieve
protein or peptide expression in a variety of host-expression
systems. Cell types available for expression include, but are not
limited to, bacteria, such as E. coli and B. subtilis transformed
with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA
expression vectors.
[0086] Certain examples of prokaryotic hosts are E. coli strain
RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as
well as E. coli W3110 (F-, lambda-, prototrophic, ATCC No. 273325);
bacilli such as Bacillus subtilis; and other enterobacteriaceae
such as Salmonella typhimurium, Serratia marcescens, and various
Pseudomonas species.
[0087] In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host
cell are used in connection with these hosts. The vector ordinarily
carries a replication site, as well as marking sequences which are
capable of providing phenotypic selection in transformed cells. For
example, E. coli is often transformed using pBR322, a plasmid
derived from an E. coli species. pBR322 contains genes for
ampicillin and tetracycline resistance and thus provides a simple
means for identifying transformed cells. The pBR plasmid, or other
microbial plasmid or phage must also contain, or be modified to
contain, promoters which may be used by the microbial organism for
expression of its own proteins.
[0088] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism may be
used as transforming vectors in connection with these hosts. For
example, the phage lambda GEM.TM.-11 may be utilized in making a
recombinant phage vector which may be used to transform host cells,
such as E. coli LE392.
[0089] Further useful vectors include pIN vectors (Inouye et al.,
1985); and pGEX vectors, for use in generating glutathione
S-transferase (GST) soluble fusion proteins for later purification
and separation or cleavage. Other suitable fusion proteins are
those with .beta.-galactosidase, ubiquitin, or the like.
[0090] Promoters that are most commonly used in recombinant DNA
construction include the .beta.-lactamase (penicillinase), lactose
and tryptophan (trp) promoter systems. While these are the most
commonly used, other microbial promoters have been discovered and
utilized, and details concerning their nucleotide sequences have
been published, enabling those of skill in the art to ligate them
functionally with plasmid vectors.
[0091] For expression in Saccharomyces, the plasmid YRp7, for
example, is commonly used (Stinchcomb et al., 1979; Kingsman et
al., 1979; Tschemper et al., 1980). This plasmid already contains
the trp1 gene which provides a selection marker for a mutant strain
of yeast lacking the ability to grow in tryptophan, for example
ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trp1
lesion as a characteristic of the yeast host cell genome then
provides an effective environment for detecting transformation by
growth in the absence of tryptophan.
[0092] Suitable promoting sequences in yeast vectors include the
promoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980) or
other glycolytic enzymes (Hess et al., 1968; Holland et al., 1978),
such as enolase, glyceraldehyde-3-phosphatedehydrogenase,
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate
kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plasmids, the
termination sequences associated with these genes are also ligated
into the expression vector 3' of the sequence desired to be
expressed to provide polyadenylation of the mRNA and
termination.
[0093] Other suitable promoters, which have the additional
advantage of transcription controlled by growth conditions, include
the promoter region for alcohol dehydrogenase 2, isocytochrome C,
acid phosphatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization.
[0094] In addition to micro-organisms, cultures of cells derived
from multicellular organisms also may be used as hosts. In
principle, any such cell culture is workable, whether from
vertebrate or invertebrate culture. In addition to mammalian cells,
these include insect cell systems infected with recombinant virus
expression vectors (e.g., baculovirus); and plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing one or more coding sequences.
[0095] In a useful insect system, Autographa californica nuclear
polyhidrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The isolated
nucleic acid coding sequences are cloned into non-essential regions
(for example the polyhedrin gene) of the virus and placed under
control of an AcNPV promoter (for example the polyhedrin promoter).
Successful insertion of the coding sequences results in the
inactivation of the polyhedrin gene and production of non-occluded
recombinant virus (i.e., virus lacking the proteinaceous coat coded
for by the polyhedrin gene). These recombinant viruses are then
used to infect Spodoptera frugiperda cells in which the inserted
gene is expressed (e.g., U.S. Pat. No. 4,215,051 (Smith)).
[0096] Examples of useful mammalian host cell lines are VERO and
HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK,
COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines. In addition, a
host cell strain may be chosen that modulates the expression of the
inserted sequences, or modifies and processes the gene product in
the specific fashion desired. Such modifications (e.g.,
glycosylation) and processing (e.g., cleavage) of protein products
may be important for the function of the encoded protein.
[0097] Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cells lines or host systems may be chosen
to ensure the correct modification and processing of the foreign
protein expressed. Expression vectors for use in mammalian cells
ordinarily include an origin of replication (as necessary), a
promoter located in front of the gene to be expressed, along with
any necessary ribosome binding sites, RNA splice sites,
polyadenylation site, and transcriptional terminator sequences. The
origin of replication may be provided either by construction of the
vector to include an exogenous origin, such as may be derived from
SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may
be provided by the host cell chromosomal replication mechanism. If
the vector is integrated into the host cell chromosome, the latter
is often sufficient.
[0098] The promoters may be derived from the genome of mammalian
cells (e.g., metallothionein promoter) or from mammalian viruses
(e.g., the adenovirus late promoter; the vaccinia virus 7.5K
promoter). Further, it is also possible, and may be desirable, to
utilize promoter or control sequences normally associated with the
desired gene sequence, provided such control sequences are
compatible with the host cell systems.
[0099] A number of viral based expression systems may be utilized,
for example, commonly used promoters are derived from polyoma,
Adenovirus 2, and most frequently Simian Virus 40 (SV40). The early
and late promoters of SV40 virus are particularly useful because
both are obtained easily from the virus as a fragment which also
contains the SV40 viral origin of replication. Smaller or larger
SV40 fragments also may be used, provided there is included the
approximately 250 bp sequence extending from the HindIII site
toward the BglI site located in the viral origin of
replication.
[0100] In cases where an adenovirus is used as an expression
vector, the coding sequences may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing proteins in infected
hosts.
[0101] Specific initiation signals also may be required for
efficient translation of the claimed isolated nucleic acid coding
sequences. These signals include the ATG initiation codon and
adjacent sequences. Exogenous translational control signals,
including the ATG initiation codon, may additionally need to be
provided. One of ordinary skill in the art would readily be capable
of determining this and providing the necessary signals. It is well
known that the initiation codon must be in-frame (or in-phase) with
the reading frame of the desired coding sequence to ensure
translation of the entire insert. These exogenous translational
control signals and initiation codons may be of a variety of
origins, both natural and synthetic. The efficiency of expression
may be enhanced by the inclusion of appropriate transcription
enhancer elements or transcription terminators (Bittner et al.,
1987).
[0102] In eukaryotic expression, one will also typically desire to
incorporate into the transcriptional unit an appropriate
polyadenylation site (e.g., 5'-AATAAA-3') if one was not contained
within the original cloned segment. Typically, the poly A addition
site is placed about 30 to 2000 nucleotides "downstream" of the
termination site of the protein at a position prior to
transcription termination.
[0103] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express constructs encoding proteins may be engineered.
Rather than using expression vectors that contain viral origins of
replication, host cells may be transformed with vectors controlled
by appropriate expression control elements (e.g., promoter,
enhancer, sequences, transcription terminators, polyadenylation
sites, etc.), and a selectable marker. Following the introduction
of foreign DNA, engineered cells may be allowed to grow for 1-2
days in an enriched media, and then are switched to a selective
media. The selectable marker in the recombinant plasmid confers
resistance to the selection and allows cells to stably integrate
the plasmid into their chromosomes and grow to form foci which in
turn may be cloned and expanded into cell lines.
[0104] A number of selection systems may be used, including but not
limited to, the herpes simplex virus thymidine kinase (Wigler et
al., 1977), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska et al., 1962) and adenine phosphoribosyltransferase
genes (Lowy et al., 1980), in tk-, hgprt- or aprt- cells,
respectively. Also, antimetabolite resistance may be used as the
basis of selection for dhfr, that confers resistance to
methotrexate (Wigler et al., 1980; O'Hare et al., 1981); gpt, that
confers resistance to mycophenolic acid (Mulligan et al., 1981);
neo, that confers resistance to the aminoglycoside G-418
(Colberre-Garapin et al., 1981); and hygro, that confers resistance
to hygromycin (Santerre et al., 1984).
[0105] It is contemplated that the isolated nucleic acids of the
invention may be "overexpressed," i.e., expressed in increased
levels relative to its natural expression in human prostate, cells,
or even relative to the expression of other proteins in the
recombinant host cell. Such overexpression may be assessed by a
variety of methods, including radio-labeling and/or protein
purification. However, simple and direct methods are preferred, for
example, those involving SDS/PAGE and protein staining or Western
blotting, followed by quantitative analyses, such as densitometric
scanning of the resultant gel or blot. A specific increase in the
level of the recombinant protein or peptide in comparison to the
level in natural human prostate, cells is indicative of
overexpression, as is a relative abundance of the specific protein
in relation to the other proteins produced by the host cell and,
e.g., visible on a gel.
4.3.2 Purification of Expressed Proteins
[0106] Further aspects of the present invention concern the
purification, and in particular embodiments, the substantial
purification, of an encoded protein or peptide. The term "purified
protein or peptide" as used herein, is intended to refer to a
composition, isolatable from other components, wherein the protein
or peptide is purified to any degree relative to its
naturally-obtainable state, i.e., in this case, relative to its
purity within a prostate, cell extract. A purified protein or
peptide therefore also refers to a protein or peptide, free from
the environment in which it may naturally occur.
[0107] Generally, "purified" will refer to a protein or peptide
composition which has been subjected to fractionation to remove
various other components, and which composition substantially
retains its expressed biological activity. Where the term
"substantially purified" is used, this will refer to a composition
in which the protein or peptide forms the major component of the
composition, such as constituting about 50% or more of the proteins
in the composition.
[0108] Various methods for quantifying the degree of purification
of the protein or peptide will be known to those of skill in the
art in light of the present disclosure. These include, for example,
determining the specific activity of an active fraction, or
assessing the number of polypeptides within a fraction by SDS/PAGE
analysis. A preferred method for assessing the purity of a fraction
is to calculate the specific activity of the fraction, to compare
it to the specific activity of the initial extract, and to thus
calculate the degree of purity, herein assessed by a "-fold
purification number". The actual units used to represent the amount
of activity will, of course, be dependent upon the particular assay
technique chosen to follow the purification and whether or not the
expressed protein or peptide exhibits a detectable activity.
[0109] Various techniques suitable for use in protein purification
will be well known to those of skill in the art. These include, for
example, precipitation with ammonium sulfate, PEG, antibodies and
the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse
phase, hydroxylapatite and affinity chromatography; isoelectric
focusing; gel electrophoresis; and combinations of such and other
techniques. As is generally known in the art, it is believed that
the order of conducting the various purification steps may be
changed, or that certain steps may be omitted,.and still result in
a suitable method for the preparation of a substantially purified
protein or peptide.
[0110] There is no general requirement that the protein or peptide
always be provided in the most purified state. Indeed, it is
contemplated that less substantially purified products will have
utility in certain embodiments. Partial purification may be
accomplished by using fewer purification steps in combination, or
by utilizing different forms of the same general purification
scheme. For example, it is appreciated that a cation-exchange
column chromatography performed utilizing an HPLC apparatus will
generally result in a greater fold purification than the same
technique utilizing a low pressure chromatography system. Methods
exhibiting a lower degree of relative purification may have
advantages in total recovery of protein product, or in maintaining
the activity of an expressed protein.
[0111] It is known that the migration of a polypeptide may vary,
sometimes significantly, with different conditions of SDS/PAGE
(Capaldi et al., 1977). It will therefore be appreciated that under
differing electrophoresis conditions, the apparent molecular
weights of purified or partially purified expression products may
vary.
4.3.3 Antibody Generation
[0112] For some embodiments, it will be desirable to produce
antibodies that bind with high specificity to the polypeptide
product(s) of an isolated nucleic acid selected from SEQ ID NO:1,
SEQ ID NO:3 and SEQ ID NO:4. Means for preparing and characterizing
antibodies are well known in the art (See, e.g., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, 1988;
incorporated herein by reference).
[0113] Methods for generating polyclonal antibodies are well known
in the art. Briefly, a polyclonal antibody is prepared by
immunizing an animal with an immunogenic composition and collecting
antisera from that immunized animal. A wide range of animal species
may be used for the production of antisera. Typically the animal
used for production of anti-antisera is a rabbit, a mouse, a rat, a
hamster, a guinea pig or a goat. Because of the relatively large
blood volume of rabbits, a rabbit is a preferred choice for
production of polyclonal antibodies.
[0114] As is well known in the art, a given composition may vary in
its immunogenicity. It is often necessary therefore to boost the
host immune system, as may be achieved by coupling a peptide or
polypeptide immunogen to a carrier. Exemplary and preferred
carriers are keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA). Other albumins such as ovalbumin, mouse serum
albumin or rabbit serum albumin also may be used as carriers. Means
for conjugating a polypeptide to a carrier protein are well known
in the art and include glutaraldehyde,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and
bis-biazotized benzidine.
[0115] As is also well known in the art, the immunogenicity of a
particular immunogen composition may be enhanced by the use of
non-specific stimulators of the immune response, known as
adjuvants. Exemplary and preferred adjuvants include complete
Freund's adjuvant (a non-specific stimulator of the immune response
containing killed Mycobacterium tuberculosis), incomplete Freund's
adjuvants and aluminum hydroxide adjuvant.
[0116] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes may
be used to administer the immunogen (subcutaneous, intramuscular,
intradermal, intravenous and intraperitoneal). The production of
polyclonal antibodies may be monitored by sampling blood of the
immunized animal at various points' following immunization. A
second, booster injection, also may be given. The process of
boosting and titering is repeated until a suitable titer is
achieved. When a desired level of immunogenicity is obtained, the
immunized animal may be bled and the serum isolated and stored,
and/or the animal may be used to generate MAbs. For production of
rabbit polyclonal antibodies, the animal may be bled through an ear
vein or alternatively by cardiac puncture. The removed blood is
allowed to coagulate and then centrifuged to separate serum
components from whole cells and blood clots. The serum may be used
as is for various applications or else the desired antibody
fraction may be purified by well-known methods, such as affinity
chromatography using another antibody or a peptide bound to a solid
matrix.
[0117] Monoclonal antibodies (MAbs) may be readily prepared through
use of well-known techniques, such as those exemplified in U.S.
Pat. No. 4,196,265, incorporated herein by reference. Typically,
this technique involves immunizing a suitable animal with a
selected immunogen composition, e.g., a purified or partially
purified expressed protein, polypeptide or peptide. The immunizing
composition is administered in a manner effective to stimulate
antibody producing cells.
[0118] The methods for generating monoclonal antibodies (MAbs)
generally begin along the same lines as those for preparing
polyclonal antibodies. Rodents such as mice and rats are preferred
animals, however, the use of rabbit, sheep or frog cells is also
possible. The use of rats may provide certain advantages (Goding,
1986, pp. 60-61), but mice are preferred, with the BALB/c mouse
being most preferred as this is most routinely used and generally
gives a higher percentage of stable fusions.
[0119] The animals are injected with antigen as described above.
The antigen may be coupled to carrier molecules such as keyhole
limpet hemocyanin if necessary. The antigen would typically be
mixed with adjuvant, such as Freund's complete or incomplete
adjuvant. Booster injections with the same antigen would occur at
approximately two-week intervals.
[0120] Following immunization, somatic cells with the potential for
producing antibodies, specifically B lymphocytes (B cells), are
selected for use in the MAb generating protocol. These cells may be
obtained from biopsied spleens, tonsils or lymph nodes, or from a
peripheral blood sample. Spleen cells and peripheral blood cells
are preferred, the former because they are a rich source of
antibody-producing cells that are in the dividing plasmablast
stage, and the latter because peripheral blood is easily
accessible. Often, a panel of animals will have been immunized and
the spleen of the animal with the highest antibody titer will be
removed and the spleen lymphocytes obtained by homogenizing the
spleen with a syringe. Typically, a spleen from an immunized mouse
contains approximately 5.times.10.sup.7 to 2.times.10.sup.8
lymphocytes.
[0121] The antibody-producing B lymphocytes from the immunized
animal are then fused with cells of an immortal myeloma cell,
generally one of the same species as the animal that was immunized.
Myeloma cell lines suited for use in hybridoma-producing fusion
procedures preferably are non-antibody-producing, have high fusion
efficiency, and enzyme deficiencies that render then incapable of
growing in certain selective media which support the growth of only
the desired fused cells (hybridomas).
[0122] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, pp. 65-66, 1986;
Campbell, pp. 75-83, 1984). For example, where the immunized animal
is a mouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1,
Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul;
for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and
U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in
connection with human cell fusions.
[0123] One preferred murine myeloma cell is the NS-1 myeloma cell
line (also termed P3-NS-1-Ag4-1), which is readily available from
the NIGMS Human Genetic Mutant Cell Repository by requesting cell
line repository number GM3573. Another mouse myeloma cell line that
may be used is the 8-azaguanine-resistant mouse murine myeloma
SP2/0 non-producer cell line.
[0124] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 2:1 proportion, though the
proportion may vary from about 20:1 to about 1:1, respectively, in
the presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Sendai
virus have been described by Kohler and Milstein (1975; 1976), and
those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by
Gefter et al. (1977). The use of electrically induced fusion
methods is also appropriate (Goding pp. 71-74, 1986).
[0125] Fusion procedures usually produce viable hybrids at low
frequencies, about 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are
differentiated from the parental, unfused cells (particularly the
unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0126] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in FLAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B cells may operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B cells.
[0127] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired reactivity. The assay should be sensitive, simple and
rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity
assays, plaque assays, dot immunobinding assays, and the like.
[0128] The selected hybridomas would then be serially diluted and
cloned into individual antibody-producing cell lines, which clones
may then be propagated indefinitely to provide MAbs. The cell lines
may be exploited for MAb production in two basic ways. A sample of
the hybridoma may be injected (often into the peritoneal cavity)
into a histocompatible animal of the type that was used to provide
the somatic and myeloma cells for the original fusion. The injected
animal develops tumors secreting the specific monoclonal antibody
produced by the fused cell hybrid. The body fluids of the animal,
such as serum or ascites fluid, may then be tapped to provide MAbs
in high concentration. The individual cell lines also may be
cultured in vitro, where the MAbs are naturally secreted into the
culture medium from which they may be readily obtained in high
concentrations. MAbs produced by either means may be further
purified, if desired, using filtration, centrifugation and various
chromatographic methods such as HPLC or affinity
chromatography.
[0129] Large amounts of the monoclonal antibodies of the present
invention also may be obtained by multiplying hybridoma cells in
vivo. Cell clones are injected into mammals which are
histocompatible with the parent cells, e.g., syngeneic mice, to
cause growth of antibody-producing tumors: Optionally, the animals
are primed with a hydrocarbon, especially oils such as pristane
(tetramethylpentadecane) prior to injection.
[0130] In accordance with the present invention, fragments of the
monoclonal antibody of the invention may be obtained from the
monoclonal antibody produced as described above, by methods which
include digestion with enzymes such as pepsin or papain and/or
cleavage of disulfide bonds by chemical reduction. Alternatively,
monoclonal antibody fragments encompassed by the present invention
may be synthesized using an automated peptide synthesizer.
[0131] The monoclonal conjugates of the present invention are
prepared by methods known in the art, e.g., by reacting a
monoclonal antibody prepared as described above with, for instance,
an enzyme in the presence of a coupling agent such as
glutaraldehyde or periodate. Conjugates with fluorescein markers
are prepared in the presence of these coupling agents or by
reaction with an isothiocyanate. Conjugates with metal chelates are
similarly produced. Other moieties to which antibodies may be
conjugated include radionuclides such as .sup.3H, .sup.125I,
.sup.131I .sup.32P, .sup.35S, .sup.14C, .sup.51Cr, .sup.36Cl,
.sup.57Co, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu, and
.sup.99mTc. Radioactively labeled monoclonal antibodies of the
present invention are produced according to well-known methods in
the art. For instance, monoclonal antibodies may be iodinated by
contact with sodium or potassium iodide and a chemical oxidizing
agent such as sodium hypochlorite, or an enzymatic oxidizing agent,
such as lactoperoxidase. Monoclonal antibodies according to the
invention may be labeled with technetium-.sup.99 by ligand exchange
process, for example, by reducing pertechnate with stannous
solution, chelating the reduced technetium onto a Sephadex column
and applying the antibody to this column or by direct labeling
techniques, e.g., by incubating pertechnate, a reducing agent such
as SNCl.sub.2, a buffer solution such as sodium-potassium phthalate
solution, and the antibody.
[0132] It will be appreciated by those of skill in the art that
monoclonal or polyclonal antibodies specific for proteins that are
preferentially expressed in metastatic or nonmetastatic human
prostate cancer will have utilities in several types of
applications. These may include the production of diagnostic kits
for use in detecting or diagnosing human prostate cancer. An
alternative use would be to link such antibodies to therapeutic
agents, such as chemotherapeutic agents, followed by administration
to individuals with prostate cancer, thereby selectively targeting
the prostate cancer cells for destruction. The skilled practitioner
will realize that such uses are within the scope of the present
invention.
4.4 Immunodetection Assays
4.4.1 Immunodetection Methods
[0133] In still further embodiments, the present invention concerns
immunodetection methods for binding, purifying, removing,
quantifying or otherwise generally detecting biological components.
The encoded proteins or peptides of the present invention may be
employed to detect antibodies having reactivity therewith, or,
alternatively, antibodies prepared in accordance with the present
invention, may be employed to detect the encoded proteins or
peptides. The steps of various useful immunodetection methods have
been described in the scientific literature, such as, e.g.,
Nakamura et al. (1987).
[0134] In general, the immunobinding methods include obtaining a
sample suspected of containing a protein, peptide or antibody, and
contacting the sample with an antibody or protein or peptide in
accordance with the present invention, as the case may be, under
conditions effective to allow the formation of immunocomplexes.
[0135] The immunobinding methods include methods for detecting or
quantifying the amount of a reactive component in a sample, which
methods require the detection or quantitation of any immune
complexes formed during the binding process. Here, one would obtain
a sample suspected of containing a prostate specific protein,
peptide or a corresponding antibody, and contact the sample with an
antibody or encoded protein or peptide, as the case may be, and
then detect or quantify the amount of immune complexes formed under
the specific conditions.
[0136] In terms of antigen detection, the biological sample
analyzed may be any sample that is suspected of containing a
prostate cancer-specific antigen, such as a prostate or lymph node
tissue section or specimen, a homogenized tissue extract, an
isolated cell, a cell membrane preparation, separated or purified
forms of any of the above protein-containing compositions, or even
any biological fluid that comes into contact with prostate tissues,
including blood or lymphatic fluid.
[0137] Contacting the chosen biological sample with the protein,
peptide or antibody under conditions effective and for a period of
time sufficient to allow the formation of immune complexes (primary
immune complexes) is generally a matter of simply adding the
composition to the sample and incubating the mixture for a period
of time long enough for the antibodies to form immune complexes
with, i.e., to bind to, any antigens present. After this time, the
sample-antibody composition, such as a tissue section, ELISA plate,
dot blot or Western blot, will generally be washed to remove any
non-specifically bound antibody species, allowing only those
antibodies specifically bound within the primary immune complexes
to be detected.
[0138] In general, the detection of immunocomplex formation is well
known in the art and may be achieved through the application of
numerous approaches. These methods are generally based upon the
detection of a label or marker, such as any radioactive,
fluorescent, biological or enzymatic tags or labels of standard use
in the art. U.S. Patents concerning the use of such labels include
U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149 and 4,366,241, each incorporated herein by
reference. Of course, one may find additional advantages through
the use of a secondary binding ligand such as a second antibody or
a biotin/avidin ligand binding arrangement, as is known in the
art.
[0139] The encoded protein, peptide or corresponding antibody
employed in the detection may itself be linked to a detectable
label, wherein one would then simply detect this label, thereby
allowing the amount of the primary immune complexes in the
composition to be determined.
[0140] Alternatively, the first added component that becomes bound
within the primary immune complexes may be detected by means of a
second binding ligand that has binding affinity for the encoded
protein, peptide or corresponding antibody. In these cases, the
second binding ligand may be linked to a detectable label. The
second binding ligand is itself often an antibody, which may thus
be termed a "secondary" antibody. The primary immune complexes are
contacted with the labeled, secondary binding ligand, or antibody,
under conditions effective and for a period of time sufficient to
allow the formation of secondary immune complexes. The secondary
immune complexes are then generally washed to remove any
non-specifically bound labeled secondary antibodies or ligands, and
the remaining label in the secondary immune complexes is then
detected.
[0141] Further methods include the detection of primary immune
complexes by a two step approach. A second binding ligand, such as
an antibody, that has binding affinity for the encoded protein,
peptide or corresponding antibody is used to form secondary immune
complexes, as described above. After washing, the secondary immune
complexes are contacted with a third binding ligand or antibody
that has binding affinity for the second antibody, again under
conditions effective and for a period of time sufficient to allow
the formation of immune complexes (tertiary immune complexes). The
third ligand or antibody is linked to a detectable label, allowing
detection of the tertiary immune complexes thus formed. This system
may provide for signal amplification if this is desired.
[0142] The immunodetection methods of the present invention have
evident utility in the diagnosis of conditions such as prostate
cancer. Here, a biological or clinical sample suspected of
containing either the encoded protein or peptide or corresponding
antibody is used. However, these embodiments also have applications
to non-clinical samples, such as in the titering of antigen or
antibody samples, in the selection of hybridomas, and the like.
[0143] In the clinical diagnosis or monitoring of patients with
prostate cancer, the detection of an antigen encoded by a nucleic
acid corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4, or
an increase in the levels of such an antigen, in comparison to the
levels in a corresponding biological sample from a normal subject
is indicative of a patient with prostate cancer. The basis for such
diagnostic methods lies, in part, with the finding that the novel
prostate specific gene identified in the present invention is
overexpressed in prostate cancer tissue samples (see Examples
below). By extension, it may be inferred that said gene produces
elevated levels of encoded protein(s), that may be used as prostate
cancer markers.
[0144] Those of skill in the art are very familiar with
differentiating between significant expression of a prostate
specific gene, which represents a positive identification, and low
level or background expression of the same gene. Indeed, background
expression levels are often used to form a "cut-off" above which
increased staining will be scored as significant or positive.
Significant expression may be represented by high levels of
antigens in tissues or within body fluids, or alternatively, by a
high proportion of cells from within a tissue that each give a
positive signal.
4.4.2 Immunohistochemistry
[0145] The antibodies of the present invention may be used in
conjunction with both fresh-frozen and formalin-fixed,
paraffin-embedded tissue blocks prepared by immunohistochemistry
(IHC). Any IHC method well known in the art may be used such as
those described in Diagnostic Immunopathology, 2nd edition. edited
by, Robert B. Colvin, Atul K. Bhan and Robert T. McCluskey. Raven
Press, New York., 1995, (incorporated herein by reference) and in
particular, Chapter 31 of that reference entitled Gynecological and
Genitourinary Tumors (pages 579-597), by Debra A. Bell, Robert H.
Young and Robert E. Scully and references therein.
4.4.3 ELISA
[0146] As noted, it is contemplated that the encoded proteins or
peptides of the invention will find utility as immunogens, e.g., in
connection with vaccine development, in immunohistochemistry and in
ELISA assays. One evident utility of the encoded antigens and
corresponding antibodies is in immunoassays for the detection of
prostate cancer specific proteins, as needed in diagnosis and
prognostic monitoring.
[0147] Immunoassays, in their most simple and direct sense, are
binding assays. Certain preferred immunoassays are the various
types of enzyme linked immunosorbent assays (ELISAs) and
radioimmunoassays (RIA) known in the art. Immunohistochemical
detection using tissue sections is also particularly useful.
However, it will be readily appreciated that detection is not
limited to such techniques, and Western blotting, dot blotting,
FACS analyses, and the like also may be used.
[0148] In one exemplary ELISA, antibodies binding to the encoded
proteins of the invention are immobilized onto a selected surface
exhibiting protein affinity, such as a well in a polystyrene
microtiter plate. Then, a test composition suspected of containing
the prostate cancer marker antigen, such as a clinical sample, is
added to the wells. After binding and washing to remove
non-specifically bound immunecomplexes, the bound antigen may be
detected. Detection is generally achieved by the addition of a
second antibody specific for the target protein, that is linked to
a detectable label. This type of ELISA is a simple "sandwich
ELISA." Detection also may be achieved by the addition of a second
antibody, followed by the addition of a third antibody that has
binding affinity for the second antibody, with the third antibody
being linked to a detectable label.
[0149] In another exemplary ELISA, the samples suspected of
containing the prostate cancer marker antigen are immobilized onto
the well surface and then contacted with the antibodies of the
invention. After binding and washing to remove non-specifically
bound immunecomplexes, the bound antigen is detected. Where the
initial antibodies are linked to a detectable label, the
immunecomplexes may be detected directly. Again, the
immunecomplexes may be detected using a second antibody that has
binding affinity for the first antibody, with the second antibody
being linked to a detectable label.
[0150] Another ELISA in which the proteins or peptides are
immobilized, involves the use of antibody competition in the
detection. In this ELISA, labeled antibodies are added to the
wells, allowed to bind to the prostate cancer marker protein, and
detected by means of their label. The amount of marker antigen in
an unknown sample is then determined by mixing the sample with the
labeled antibodies before or during incubation with coated wells.
The presence of marker antigen in the sample acts to reduce the
amount of antibody available for binding to the well and thus
reduces the ultimate signal. This is appropriate for detecting
antibodies in an unknown sample, where the unlabeled antibodies
bind to the antigen-coated wells and also reduces the amount of
antigen available to bind the labeled antibodies.
[0151] Irrespective of the format employed, ELISAs have certain
features in common, such as coating, incubating or binding, washing
to remove non-specifically bound species, and detecting the bound
immunecomplexes. These are described as follows:
[0152] In coating a plate with either antigen or antibody, one will
generally incubate the wells of the plate with a solution of the
antigen or antibody, either overnight or for a specified period of
hours. The wells of the plate will then be washed to remove
incompletely adsorbed material. Any remaining available surfaces of
the wells are then "coated" with a nonspecific protein that is
antigenically neutral with regard to the test antisera. These
include bovine serum albumin (BSA), casein and solutions of milk
powder. The coating allows for blocking of nonspecific adsorption
sites on the immobilizing surface and thus reduces the background
caused by nonspecific binding of antisera onto the surface.
[0153] In ELISAs, it is probably more customary to use a secondary
or tertiary detection means rather than a direct procedure. Thus,
after binding of a protein or antibody to the well, coating with a
non-reactive material to reduce background, and washing to remove
unbound material, the immobilizing surface is contacted with the
control human prostate, cancer and/or clinical or biological sample
to be tested under conditions effective to allow immunecomplex
(antigen/antibody) formation. Detection of the immunecomplex then
requires a labeled secondary binding ligand or antibody, or a
secondary binding ligand or antibody in conjunction with a labeled
tertiary antibody or third binding ligand.
[0154] "Under conditions effective to allow immunecomplex
(antigen/antibody) formation" means that the conditions preferably
include diluting the antigens and antibodies with solutions such as
BSA, bovine gamma globulin (BGG) and phosphate buffered saline
(PBS)/Tween. These added agents also tend to assist in the
reduction of nonspecific background.
[0155] The "suitable" conditions also mean that the incubation is
at a temperature and for a period of time sufficient to allow
effective binding. Incubation steps are typically from about 1 to 2
to 4 h, at temperatures preferably on the order of 25.degree. to
27.degree. C., or may be overnight at about 4.degree. C. or so.
[0156] Following all incubation steps in an ELISA, the contacted
surface is washed so as to remove non-complexed material. A
preferred washing procedure includes washing with a solution such
as PBS/Tween, or borate buffer. Following the formation of specific
immunecomplexes between the test sample and the originally bound
material, and subsequent washing, the occurrence of even minute
amounts of immunecomplexes may be determined.
[0157] To provide a detecting means, the second or third antibody
will have an associated label to allow detection. Preferably, this
will be an enzyme that will generate color development upon
incubating with an appropriate chromogenic substrate. Thus, for
example, one will desire to contact and incubate the first or
second immunecomplex with a urease, glucose oxidase, alkaline
phosphatase or hydrogen peroxidase-conjugated antibody for a period
of time and under conditions that favor the development of further
immunecomplex formation (e.g., incubation for 2 h at room
temperature in a PBS-containing solution such as PBS-Tween).
[0158] After incubation with the labeled antibody, and subsequent
to washing to remove unbound material, the amount of label is
quantified, e.g., by incubation with a chromogenic substrate such
as urea and bromocresol purple or
2,2'-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS] and
H.sub.2O.sub.2, in the case of peroxidase as the enzyme label.
Quantitation is then achieved by measuring the degree of color
generation, e.g., using a visible spectra spectrophotometer.
4.4.4 Use of Antibodies for Radioimaging
[0159] The antibodies of this invention will be used to quantify
and localize the expression of the encoded marker proteins. The
antibody, for example, will be labeled by any one of a variety of
methods and used to visualize the localized concentration of the
cells producing the encoded protein.
[0160] The invention also relates to an in vivo method of imaging a
pathological prostate, cancer condition using the above described
monoclonal antibodies. Specifically, this method involves
administering to a subject an imaging-effective amount of a
delectably-labeled prostate, cancer-specific monoclonal antibody or
fragment thereof and a pharmaceutically effective carrier and
detecting the binding of the labeled monoclonal antibody to the
diseased tissue. The term "in vivo imaging" refers to any method
which permits the detection of a labeled monoclonal antibody of the
present invention or fragment thereof that specifically binds to a
diseased tissue located in the subject's body. A "subject" is a
mammal, preferably a human. An "imaging effective amount" means
that the amount of the detectably-labeled monoclonal antibody, or
fragment thereof, administered is sufficient to enable detection of
binding of the monoclonal antibody or fragment thereof to the
diseased tissue.
[0161] A factor to consider in selecting a radionuclide for in vivo
diagnosis is that the half-life of a nuclide be long enough so that
it is still detectable at the time of maximum uptake by the target,
but short enough so that deleterious radiation upon the host, as
well as background, is minimized. Ideally, a radionuclide used for
in vivo imaging will lack a particulate emission, but produce a
large number of photons in a 140-2000 keV range, which may be
readily detected by conventional gamma cameras.
[0162] A radionuclide may be bound to an antibody either directly
or indirectly by using an intermediary functional group.
Intermediary functional groups which are often used to bind
radioisotopes which exist as metallic ions to antibody are
diethylenetriaminepentaacetic acid (DTPA) and ethylene
diaminetetracetic acid (EDTA). Examples of metallic ions suitable
for use in this invention are .sup.99mTc, .sup.123I, .sup.131I
.sup.111In, .sup.131I, .sup.97Ru, .sup.67Cu, .sup.67Ga, .sup.125I,
.sup.68Ga, .sup.72As, .sup.89Zr, and .sup.201Tl.
[0163] In accordance with this invention, the monoclonal antibody
or fragment thereof may be labeled by any of several techniques
known to the art. The methods of the present invention also may use
paramagnetic isotopes for purposes of in vivo detection. Elements
particularly useful in Magnetic Resonance Imaging ("MRI") include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Cr, and .sup.56Fe.
[0164] Administration of the labeled antibody may be local or
systemic and accomplished intravenously, intraarterially, via the
spinal fluid or the like. Administration also may be intradermal or
intracavitary, depending upon the body site under examination.
After a sufficient time has lapsed for the monoclonal antibody or
fragment thereof to bind with the diseased tissue, for example 30
min to 48 h, the area of the subject under investigation is
examined by routine imaging techniques such as MRI, SPECT, planar
scintillation imaging and emerging imaging techniques, as well. The
exact protocol will necessarily vary depending upon factors
specific to the patient, as noted above, and depending upon the
body site under examination, method of administration and type of
label used; the determination of specific procedures would be
routine to the skilled artisan. The distribution of the bound
radioactive isotope and its increase or decrease with time is then
monitored and recorded. By comparing the results with data obtained
from studies of clinically normal individuals, the presence and
extent of the diseased tissue may be determined.
[0165] It will be apparent to those of skill in the art that a
similar approach may be used to radio-image the production of the
encoded prostate cancer marker proteins in human patients. The
present invention provides methods for the in vivo diagnosis of
prostate, cancer in a patient. Such methods generally comprise
administering to a patient an effective amount of a prostate,
cancer specific antibody, which antibody is conjugated to a marker,
such as a radioactive isotope or a spin-labeled molecule, that is
detectable by non-invasive methods. The antibody-marker conjugate
is allowed sufficient time to come into contact with reactive
antigens that be present within the tissues of the patient, and the
patient is then exposed to a detection device to identify the
detectable marker.
4.4.5 Kits
[0166] In still further embodiments, the present invention concerns
immunodetection kits for use with the immunodetection methods
described above. As the encoded proteins or peptides may be
employed to detect antibodies and the corresponding antibodies may
be employed to detect encoded proteins or peptides, either or both
of such components may be provided in the kit. The immunodetection
kits will thus comprise, in suitable container means, an encoded
protein or peptide, or a first antibody that binds to an encoded
protein or peptide, and an immunodetection reagent.
[0167] In certain embodiments, the encoded protein or peptide, or
the first antibody that binds to the encoded protein or peptide,
may be bound to a solid support, such as a column matrix or well of
a microtiter plate.
[0168] The immunodetection reagents of the kit may take any one of
a variety of forms, including those detectable labels that are
associated with or linked to the given antibody or antigen, and
detectable labels that are associated with or attached to a
secondary binding ligand. Exemplary secondary ligands are those
secondary antibodies that have binding affinity for the first
antibody or antigen, and secondary antibodies that have binding
affinity for a human antibody.
[0169] Further suitable immunodetection reagents for use in the
present kits include the two-component reagent that comprises a
secondary antibody that has binding affinity for the first antibody
or antigen, along with a third antibody that has binding affinity
for the second antibody, the third antibody being linked to a
detectable label.
[0170] The kits may further comprise a suitably aliquoted
composition of the encoded protein or polypeptide antigen, whether
labeled or unlabeled, as may be used to prepare a standard curve
for a detection assay.
[0171] The kits may contain antibody-label conjugates either in
fully conjugated form, in the form of intermediates, or as separate
moieties to be conjugated by the user of the kit. The components of
the kits may be packaged either in aqueous media or in lyophilized
form.
[0172] The container means of the kits will generally include at
least one vial, test tube, flask, bottle, syringe or other
container means, into which the antibody or antigen may be placed,
and preferably, suitably aliquoted. Where a second or third binding
ligand or additional component is provided, the kit will also
generally contain a second, third or other additional container
into which this ligand or component may be placed. The kits of the
present invention will also typically include a means for
containing the antibody, antigen, and any other reagent containers
in close confinement for commercial sale. Such containers may
include injection or blow-molded plastic containers into which the
desired vials are retained.
4.5 Detection and Quantitation of RNA Species
[0173] One embodiment of the instant invention comprises a method
for identification of prostate cancer cells in a biological sample
by amplifying and detecting nucleic acids corresponding to the
novel prostate specific gene (UC41) reported herein. The biological
sample may be any tissue or fluid in which prostate cancer cells
might be present. Various embodiments include radical prostatectomy
specimens, pathological specimens, bone marrow aspirate, bone
marrow biopsy, lymph node aspirate, lymph node biopsy, spleen
tissue, fine needle aspirate, skin biopsy or organ tissue biopsy.
Other embodiments include samples where the body fluid is
peripheral blood, serum, plasma, lymph fluid, ascites, serous
fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal
fluid, stool, prostatic fluid or urine.
[0174] Nucleic acid used as a template for amplification is
isolated from cells contained in the biological sample, according
to standard methodologies. (Sambrook et al., 1989) The nucleic acid
may be genomic DNA or fractionated or whole cell RNA. Where RNA is
used, it may be desired to convert the RNA to a complementary cDNA.
In one embodiment, the RNA is whole cell RNA and is used directly
as the template for amplification.
[0175] Pairs of primers that selectively hybridize to nucleic acids
corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4 are
contacted with the isolated nucleic acid under conditions that
permit selective hybridization. Once hybridized, the nucleic
acid:primer complex is contacted with one or more enzymes that
facilitate template-dependent nucleic acid synthesis. Multiple
rounds of amplification, also referred to as "cycles," are
conducted until a sufficient amount of amplification product is
produced.
[0176] Next, the amplification product is detected. In certain
applications, the detection may be performed by visual means.
Alternatively, the detection may involve indirect identification of
the product via chemiluminescence, radioactive scintigraphy of
incorporated radiolabel or fluorescent label or even via a system
using electrical or thermal impulse signals (Affymax technology;
Bellus, 1994).
[0177] Following detection, one may compare the results seen in a
given patient with a statistically significant reference group of
normal patients and prostate, cancer patients. In this way, it is
possible to correlate the amount of nucleic acid detected with
various clinical states.
4.5.1 Primers
[0178] The term primer, as defined herein, is meant to encompass
any nucleic acid that is capable of priming the synthesis of a
nascent nucleic acid in a template-dependent process. Typically,
primers are oligonucleotides from ten to twenty base pairs in
length, but longer sequences may be employed. Primers may be
provided in double-stranded or single-stranded form, although the
single-stranded form is preferred.
4.5.2 Template Dependent Amplification Methods
[0179] A number of template dependent processes are available to
amplify the nucleic acid sequences present in a given template
sample. One of the best known amplification methods is the
polymerase chain reaction (referred to as PCR.TM.) which is
described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and
4,800,159, and in Innis et al., 1990, each of which is incorporated
herein by reference in its entirety.
[0180] Briefly, in PCR.TM., two primer sequences are prepared which
are complementary to regions on opposite complementary strands of
the target nucleic acid sequence. An excess of deoxynucleoside
triphosphates are added to a reaction mixture along with a DNA
polymerase, e.g., Taq polymerase. If the target nucleic acid
sequence is present in a sample, the primers will bind to the
target nucleic acid and the polymerase will cause the primers to be
extended along the target nucleic acid sequence by adding on
nucleotides. By raising and lowering the temperature of the
reaction mixture, the extended primers will dissociate from the
target nucleic acid to form reaction products, excess primers will
bind to the target nucleic acid and to the reaction products and
the process is repeated.
[0181] A reverse transcriptase PCR.TM. amplification procedure may
be performed in order to quantify the amount of mRNA amplified.
Methods of reverse transcribing RNA into cDNA are well known and
described in Sambrook et al., 1989. Alternative methods for reverse
transcription utilize thermostable DNA polymerases. These methods
are described in WO 90/07641 filed Dec. 21, 1990. Polymerase chain
reaction methodologies are well known in the art.
[0182] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in European Application No. 320 308,
incorporated herein by reference in its entirely. In LCR, two
complementary probe pairs are prepared, and in the presence of the
target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR.TM., bound ligated units dissociate
from the target and then serve as "target sequences" for ligation
of excess probe pairs. U.S. Pat. No. 4,883,750 describes a method
similar to LCR for binding probe pairs to a target sequence.
[0183] Qbeta Replicase, described in PCT Application No.
PCT/US87/00880, also may be used as still another amplification
method in the present invention. In this method, a replicative
sequence of RNA which has a region complementary to that of a
target is added to a sample in the presence of an RNA polymerase.
The polymerase will copy the replicative sequence which may then be
detected.
[0184] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'-[.alpha.-thio]-triphosphates in one strand of a restriction site
also may be useful in the amplification of nucleic acids in the
present invention. Walker et al. (1992), incorporated herein by
reference in its entirety.
[0185] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis,
i.e., nick translation. A similar method, called Repair Chain
Reaction (RCR), involves annealing several probes throughout a
region targeted for amplification, followed by a repair reaction in
which only two of the four bases are present. The other two bases
may be added as biotinylated derivatives for easy detection. A
similar approach is used in SDA. Target specific sequences also may
be detected using a cyclic probe reaction (CPR). In CPR, a probe
having 3' and 5' sequences of non-specific DNA and a middle
sequence of specific RNA is hybridized to DNA which is present in a
sample. Upon hybridization, the reaction is treated with RNase H,
and the products of the probe identified as distinctive products
which are released after digestion. The original template is
annealed to another cycling probe and the reaction is repeated.
[0186] Still other amplification methods described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, each of which is incorporated herein by reference
in its entirety, may be used in accordance with the present
invention. In the former application, "modified" primers are used
in a PCR.TM. like, template and enzyme dependent synthesis. The
primers may be modified by labeling with a capture moiety (e.g.,
biotin) and/or a detector moiety (e.g., enzyme). In the latter
application, an excess of labeled probes are added to a sample. In
the presence of the target sequence, the probe binds and is cleaved
catalytically. After cleavage, the target sequence is released
intact to be bound by excess probe. Cleavage of the labeled probe
signals the presence of the target sequence.
[0187] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR. Kwoh et al.
(1989); Gingeras et al., PCT Application WO 88/10315, incorporated
herein by reference in their entirety. In NASBA, the nucleic acids
may be prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a clinical sample, treatment with
lysis buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has target specific
sequences. Following polymerization, DNA/RNA hybrids are digested
with RNase H while double stranded DNA molecules are heat denatured
again. In either case the single stranded DNA is made fully double
stranded by addition of second target specific primer, followed by
polymerization. The double-stranded DNA molecules are then multiply
transcribed by a polymerase such as T7 or SP6. In an isothermal
cyclic reaction, the RNA's are reverse transcribed into double
stranded DNA, and transcribed once against with a polymerase such
as T7 or SP6. The resulting products, whether truncated or
complete, indicate target specific sequences.
[0188] Davey et al., European Application No. 329 822 (incorporated
herein by reference in its entirely) disclose a nucleic acid
amplification process involving cyclically synthesizing
single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA
(dsDNA), which may be used in accordance with the present
invention. The ssRNA is a first template for a first primer
oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from the
resulting DNA:RNA duplex by the action of ribonuclease H (RNase H,
an RNase specific for RNA in duplex with either DNA or RNA). The
resultant ssDNA is a second template for a second primer, which
also includes the sequences of an RNA polymerase promoter
(exemplified by T7 RNA polymerase) 5' to its homology to the
template. This primer is then extended by DNA polymerase
(exemplified by the large "Klenow" fragment of E. coli DNA
polymerase I), resulting in a double-stranded DNA ("dsDNA")
molecule, having a sequence identical to that of the original RNA
between the primers and having additionally, at one end, a promoter
sequence. This promoter sequence may be used by the appropriate RNA
polymerase to make many RNA copies of the DNA. These copies may
then re-enter the cycle leading to very swift amplification. With
proper choice of enzymes, this amplification may be done
isothermally without addition of enzymes at each cycle. Because of
the cyclical nature of this process, the starting sequence may be
chosen to be in the form of either DNA or RNA.
[0189] Miller et al., PCT Application WO 89/06700 (incorporated
herein by reference in its entirety) disclose a nucleic acid
sequence amplification scheme based on the hybridization of a
promoter/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. This
scheme is not cyclic, i.e., new templates are not produced from the
resultant RNA transcripts. Other amplification methods include
"race" and "one-sided PCR.TM.." Frohman (1990) and Ohara et al.
(1989), each herein incorporated by reference in their
entirety.
[0190] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, also may be used in the amplification step of
the present invention. Wu et al. (1989), incorporated herein by
reference in its entirety.
4.5.3 Separation Methods
[0191] Following amplification, it may be desirable to separate the
amplification product from the template and the excess primer for
the purpose of determining whether specific amplification has
occurred. In one embodiment, amplification products are separated
by agarose, agarose-acrylamide or polyacrylamide gel
electrophoresis using standard methods. See Sambrook et al.,
1989.
[0192] Alternatively, chromatographic techniques may be employed to
effect separation. There are many kinds of chromatography which may
be used in the present invention: adsorption, partition,
ion-exchange and molecular sieve, and many specialized techniques
for using them including column, paper, thin-layer and gas
chromatography (Freifelder, 1982).
4.5.4 Identification Methods
[0193] Amplification products must be visualized in order to
confirm amplification of the target nucleic acid sequences. One
typical visualization method involves staining of a gel with
ethidium bromide and visualization under UV light. Alternatively,
if the amplification products are integrally labeled with radio- or
fluorometrically-labeled nucleotides, the amplification products
may then be exposed to x-ray film or visualized under the
appropriate stimulating spectra, following separation.
[0194] In one embodiment, visualization is achieved indirectly.
Following separation of amplification products, a labeled, nucleic
acid probe is brought into contact with the amplified target
nucleic acid sequence. The probe preferably is conjugated to a
chromophore but may be radiolabeled. In another embodiment, the
probe is conjugated to a binding partner, such as an antibody or
biotin, where the other member of the binding pair carries a
detectable moiety.
[0195] In one embodiment, detection is by Southern blotting and
hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art and
may be found in many standard books on molecular protocols. See
Sambrook et al., 1989. Briefly, amplification products are
separated by gel electrophoresis. The gel is then contacted with a
membrane, such as nitrocellulose,permitting transfer of the nucleic
acid and non-covalent binding. Subsequently, the membrane is
incubated with a chromophore-conjugated probe that is capable of
hybridizing with a target amplification product. Detection is by
exposure of the membrane to x-ray film or ion-emitting detection
devices.
[0196] One example of the foregoing is described in U.S. Pat. No.
5,279,721, incorporated by reference herein, which discloses an
apparatus and method for the automated electrophoresis and transfer
of nucleic acids. The apparatus permits electrophoresis and
blotting without external manipulation of the gel and is ideally
suited to carrying out methods according to the present
invention.
4.5.5 Kit Components
[0197] All the essential materials and reagents required for
detecting UC41 nucleic acids in a biological sample may be
assembled together in a kit. The kit generally will comprise
preselected primer pairs for nucleic acids corresponding to SEQ ID
NO:1, SEQ ID NO:3 or SEQ ID NO:4. Also included may be enzymes
suitable for amplifying nucleic acids including various polymerases
(RT, Taq, etc.), deoxynucleotides and buffers to provide the
necessary reaction mixture for amplification. Preferred kits also
may comprise primers for the detection of a control,
non-differentially expressed RNA such as .beta.-actin, for
example.
[0198] The kits generally will comprise, in suitable means,
distinct containers for each individual reagent and enzyme as well
as for each primer pair. Preferred pairs of primers for amplifying
nucleic acids are selected to amplify the sequences designated
herein as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4.
[0199] In certain embodiments, kits will comprise hybridization
probes designed to hybridize to a sequence or a complement of a
sequence designated herein as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID
NO:4. Such kits generally will comprise, in suitable means for
close confinement, distinct containers for each individual reagent
and enzyme as well as for each hybridization probe.
4.6 Use of RNA Fingerprinting
[0200] RNA fingerprinting is a means by which RNAs isolated from
many different tissues, cell types or treatment groups may be
sampled simultaneously to identify RNAs whose relative abundances
vary. Two forms of this technology were developed simultaneously
and reported in 1992 as RNA fingerprinting by differential display
(Liang and Pardee, 1992; Welsh et al., 1992). (See also Liang and
Pardee, U.S. Pat. No. 5,262,311, incorporated herein by reference
in its entirety.) Both techniques were utilized in the studies
described below. Some of the studies described herein were
performed similarly to Donahue et al., 1994.
[0201] The basic technique of differential display has been
described in detail (Liang and Pardee, 1992). Total cell RNA is
primed for first strand reverse transcription with an anchoring
primer composed of oligo dT. The oligo dT primer is extended using
a reverse transcriptase, for example, Moloney Murine Leukemia Virus
(MMLV) reverse transcriptase. The synthesis of the second strand is
primed with an arbitrarily chosen oligonucleotide, using reduced
stringency conditions. Once the double-stranded cDNA has been
synthesized, amplification proceeds by standard PCR.TM. techniques,
utilizing the same primers. The resulting DNA fingerprint is
analyzed by gel electrophoresis and ethidium bromide staining or
autoradiography. A side by side comparison of fingerprints obtained
from different cell derived RNAs using the same oligonucleotide
primers identifies mRNAs that are differentially expressed.
[0202] RNA fingerprinting technology has been demonstrated as being
effective in identifying genes that are differentially expressed in
cancer (Liang et al., 1992; Wong et al., 1993; Sager et al., 1993;
Mok et al., 1994; Watson et al., 1994; Chen et al., 1995a; Chen et
al., 1995b; An et al., 1995). The present invention utilizes the
RNA fingerprinting technique to identify genes that are
differentially expressed in prostate, cancer. These studies
utilized RNAs isolated from tumor tissues and tumor-derived cell
lines that behave as tumors cells with different metastatic
potential.
[0203] The underlying concept of these studies was that genes that
are differentially expressed in cells with different metastatic
potentials may be used as indicators of metastatic potential. Since
metastasis is a prerequisite for prostate, cancer progression to
life threatening pathologies, indicators of metastatic potential
are likely to be indicators of pathological potential.
[0204] Reverse transcription (RT) of RNA to cDNA followed by
relative quantitative PCR.TM. (RT-PCR.TM.) may be used to determine
the relative concentrations of specific mRNA species in a series of
total cell RNAs isolated from normal, benign and cancerous
prostate, tissues. By determining that the concentration of a
specific mRNA species varies, it is shown that the gene encoding
the specific mRNA species is differentially expressed. This
technique may be used to confirm that mRNA transcripts shown to be
differentially regulated by RNA fingerprinting are differentially
expressed in prostate, cancer progression.
[0205] The problems inherent in clinical samples are that they are
of variable quantity (making normalization problematic), and that
they are of variable quality (necessitating the co-amplification of
a reliable internal control, preferably of larger size than the
target). Both of these problems are overcome if the RT-PCR.TM. is
performed as a relative quantitative RT-PCR.TM. with an internal
standard in which the internal standard is an amplifiable cDNA
fragment that is larger than the target cDNA fragment and in which
the abundance of the mRNA encoding the internal standard is roughly
5-100 fold higher than the mRNA encoding the target. This assay
measures relative abundance, not absolute abundance of the
respective mRNA species.
[0206] Other studies described below were performed using a more
conventional relative quantitative RT-PCR.TM. with an external
standard protocol. These assays sample the PCR.TM. products in the
linear portion of their amplification curves. The number of PCR.TM.
cycles that are optimal for sampling must be empirically determined
for each target cDNA fragment. In addition, the reverse
transcriptase products of each RNA population isolated from the
various tissue samples must be carefully normalized for equal
concentrations of amplifiable cDNAs. This is very important since
this assay measures absolute mRNA abundance. Absolute mRNA
abundance may be used as a measure of differential gene expression
only in normalized samples. While empirical determination of the
linear range of the amplification curve and normalization of cDNA
preparations are tedious and time consuming processes, the
resulting RT-PCR.TM. assays may be superior to those derived from
the relative quantitative RT-PCR.TM. with an internal standard.
[0207] One reason for this is that without the internal
standard/competitor, all of the reagents may be converted into a
single PCR.TM. product in the linear range of the amplification
curve, increasing the sensitivity of the assay. Another reason is
that with only one PCR.TM. product, display of the product on an
electrophoretic gel or some other display method becomes less
complex, has less background and is easier to interpret.
4.7 Diagnosis and Prognosis of Human Cancer
[0208] In certain embodiments, the present invention allows the
diagnosis and prognosis of human prostate cancer by screening for
prostate specific nucleic acids, particularly those that are
overexpressed in prostate cancer. The field of cancer diagnosis and
prognosis is still uncertain. Various markers have been proposed to
be correlated with metastasis and malignancy. They may be
classified generally as cytologic, protein or nucleic acid
markers.
[0209] Cytologic markers include such things as "nuclear
roundedness" (Diamond et al, 1982) and cell ploidy. Protein markers
include prostate specific antigen (PSA) and CA125. Nucleic acid
markers have included amplification of Her2/neu, point mutations in
the p53 or ras genes, and changes in the sizes of triplet repeat
segments of particular chromosomes.
[0210] All of these markers exhibit certain drawbacks, associated
with false positives and false negatives. A false positive result
occurs when an individual without malignant cancer exhibits the
presence of a "cancer marker". For example, elevated serum PSA has
been associated with prostate carcinoma. However, it also occurs in
some individuals with non-malignant, benign hyperplasia of the
prostate. A false negative result occurs when an individual
actually has cancer, but the test fails to show the presence of a
specific marker. The incidence of false negatives varies for each
marker, and frequently also by tissue type. For example, ras point
mutations have been reported to range from a high of 95 percent in
pancreatic cancer to a low of zero percent in some gynecologic
cancers.
[0211] Additional problems arise when a marker is present only
within the transformed cell itself. Ras point mutations may only be
detected within the mutant cell, and are apparently not present in,
for example, the blood serum or urine of individuals with
ras-activated carcinomas. This means that, in order to detect a
malignant tumor, one must take a sample of the tumor itself, or its
metastatic cells. Since the object of cancer detection is to
identify and treat tumors before they metastasize, essentially one
must first identify and sample a tumor before the presence of the
cancer marker can be detected.
[0212] Finally, specific problems occur with markers that are
present in normal cells but absent in cancer cells. Most tumor
samples will contain mixed populations of both normal and
transformed cells. If one is searching for a marker that is present
in normal cells, but occurs at reduced levels in transformed cells,
the "background" signal from the normal cells in the sample may
mask the presence of transformed cells.
[0213] The ideal cancer marker would be one that is present in
malignant cancers, and either missing or else expressed at
significantly lower levels in benign tumors and normal cells. The
present invention addresses this need for prostate cancer markers
by identifying a novel, prostate specific gene (UC Band #41) which
is expressed at much higher levels in malignant prostate carcinoma
than in benign or normal prostate. In particular, the results for
UC Band #41 (SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:4) discussed in
Examples 1-5 below, are quite promising in that this marker is
apparently only overexpressed in malignant tumors and is present at
very low levels in BPH or normal prostate. Further, this gene is
significantly elevated in a high percentage of human prostate
cancers examined to date.
[0214] It is expected that in clinical applications, human tissue
samples will be screened for the presence of the expression
products of UC41. Such samples could consist of needle biopsy
cores, surgical resection samples, lymph node tissue, or serum. In
certain embodiments, nucleic acids would be extracted from these
samples and amplified as described above. Some embodiments would
utilize kits containing pre-selected primer pairs or hybridization
probes. The amplified nucleic acids would be tested for UC41
expression products by, for example, gel electrophoresis and
ethidium bromide staining, or Southern blotting, or a solid-phase
detection means as described above. These methods are well known
within the art. The levels of expression product(s) detected would
be compared with statistically valid groups of metastatic,
non-metastatic malignant, benign or normal prostate samples. The
diagnosis and prognosis of the individual patient would be
determined by comparison with such groups.
[0215] Another embodiment of the present invention involves
application of RT-PCR.TM. techniques to detect circulating prostate
cancer cells (i.e., those that have already metastasized), using
probes and primers selected from sequences or their complements
designated herein as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4.
Similar techniques have been described in PCT Patent Application
No. WO 94/10343, incorporated herein by reference.
[0216] In this embodiment, metastatic prostate cancer cells are
detected in hematopoietic samples by amplification of prostate
cancer-specific nucleic acid sequences. Samples taken from blood or
lymph nodes are treated as described below to purify total cell
RNA. The isolated RNA is reverse transcribed using a reverse
transcriptase and primers selected to bind under high stringency
conditions to a nucleic acid sequence to the sequence of SEQ ID
NO:1, SEQ ID NO:3 or SEQ ID NO:4. Following reverse transcription,
the resulting cDNAs are amplified using standard PCR.TM. techniques
(described below) and a thermostable DNA polymerase.
[0217] The presence of amplification products corresponding to UC41
nucleic acids may be detected by several alternative means. In one
embodiment, the amplification product may be detected by gel
electrophoresis and ethidium bromide staining. Alternatively,
following the gel electrophoresis step the amplification product
may be detected by standard Southern blotting techniques, using an
hybridization probe selected to bind specifically to a nucleic acid
corresponding to SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4. Probe
hybridization may in turn be detected by a standard labeling means,
for example, by incorporation of [.sup.32P]-nucleotides followed by
autoradiography. The amplification products may alternatively be
detected using a solid phase detection system as described above,
utilizing a hybridization probe specific for SEQ ID NO:1, SEQ ID
NO:3 or SEQ ID NO:4 and an appropriate labeling means. The presence
of UC41 nucleic acids in blood or lymph node samples may be taken
as indicative of a patient with metastatic prostate cancer.
4.8 Targeted Inhibition of a Prostate Specific Gene
[0218] In principal, the novel prostate specific gene (UC41)
identified in the present invention may serve as a target for
therapeutic intervention in prostate cancer.
[0219] Inhibitors could potentially be designed for UC41. This is
complicated by the fact that no specific function has been
identified for this gene products, and no data is available on its
three-dimensional structures.
[0220] Identification of protein function may be extrapolated, in
some cases, from the primary sequence data, provided that sequence
homology exists between the unknown protein and a protein of
similar sequence and known function. Proteins tend to occur in
large families of relatively similar sequence and function. For
example, a number of the serine proteases, like trypsin and
chymotrypsin, have extensive sequence homologies and relatively
similar three-dimensional structures. Other general categories of
homologous proteins include different classes of transcriptional
factors, membrane receptor proteins, tyrosine kinases, GTP-binding
proteins, etc. The putative amino acid sequences encoded by the
prostate specific gene of the present invention may be
cross-checked for sequence homologies versus the protein sequence
database of the National Biomedical Research Fund. Homology
searches are standard techniques for the skilled practitioner.
[0221] Even three-dimensional structure may be inferred from the
primary sequence data of the encoded protein(s). Again, if
homologies exist between the encoded amino acid sequences and other
proteins of known structure, then a model for the structure of the
encoded protein may be designed, based upon the structure of the
known protein. An example of this type of approach was reported by
Ribas de Pouplana and Fothergill-Gilmore (1994). These authors
developed a detailed three-dimensional model for the structure of
Drosophila alcohol dehydrogenase, based in part upon sequence
homology with the known structure of 3-.alpha.,
20-.beta.-hydroxysteroiddehydrogenase. Once a three-dimensional
model is available, inhibitors may be designed by standard computer
modeling techniques. This area has been reviewed by Sun and Cohen
(1993), herein incorporated by reference.
4.8.1 Antisense Constructs
[0222] The term "antisense" is intended to refer to polynucleotide
molecules complementary to a portion of an RNA expression product
of UC41, as defined herein. "Complementary" polynucleotides are
those which are capable of base-pairing according to the standard
Watson-Crick complementarity rules. That is, the larger purines
will base pair with the smaller pyrimidines to form combinations of
guanine paired with cytosine (G:C) and adenine paired with either
thymine (A:T) in the case of DNA, or adenine paired with uracil
(A:U) in the case of RNA. Inclusion of less common bases such as
inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others
in hybridizing sequences does not interfere with pairing.
[0223] Antisense polynucleotides, when introduced into a target
cell, specifically bind to their target polynucleotide and
interfere with transcription, RNA processing, transport,
translation and/or stability. Antisense RNA constructs, or DNA
encoding such antisense RNA's, may be employed to inhibit gene
transcription or translation or both within a host cell, either in
vitro or in vivo, such as within a host animal, including a human
subject.
[0224] The intracellular concentration of monovalent cation is
approximately 160 mM (10 mM Na.sup.+; 150 mM K.sup.+). The
intracellular concentration of divalent cation is approximately 20
mM (18 mM Mg.sup.+; 2 mM Ca.sup.++). The intracellular protein
concentration, which would serve to decrease the volume of
hybridization and, therefore, increase the effective concentration
of nucleic acid species, is 150 mg/ml. Constructs can be tested in
vitro under conditions that mimic these in vivo conditions.
[0225] Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. It is contemplated that the most effective
antisense Constructs for the present invention will include regions
complementary to the mRNA start site, or to those sequences
identified herein as SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:4. One
can readily test such constructs simply by testing the constructs
in vitro to determine whether levels of the target protein are
affected. Similarly, detrimental non-specific inhibition of protein
synthesis also can be measured by determining target cell viability
in vitro.
[0226] As used herein, the terms "complementary" or "antisense"
mean polynucleotides that are substantially complementary over
their entire length and have very few base mismatches. For example,
sequences of fifteen bases in length may be termed complementary
when they have a complementary nucleotide at thirteen or fourteen
nucleotides out of fifteen. Naturally, sequences which are
"completely complementary" will be sequences which are entirely
complementary throughout their entire length and have no base
mismatches.
[0227] Other sequences with lower degrees of homology also are
contemplated. For example, an antisense construct which has limited
regions of high homology, but also contains a non-homologous region
(e.g., a ribozyme) could be designed. These molecules, though
having less than 50% homology, would bind to target sequences under
appropriate conditions.
[0228] As stated above, although the antisense sequences may be
full length cDNA copies, or large fragments thereof, they also may
be shorter fragments, or "oligonucleotides," defined herein as
polynucleotides of 50 or less bases. Although shorter oligomers
(8-20) are easier to make and increase in vivo accessibility,
numerous other factors are involved in determining the specificity
of base-pairing. For example, both binding affinity and sequence
specificity of an oligonucleotide to its complementary target
increase with increasing length. It is contemplated that
oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 25, 30, 35, 40, 45, 50 or 100 base pairs will be used. While
all or part of the gene sequence may be employed in the context of
antisense construction, statistically, any sequence of 14 bases
long should occur only once in the human genome and, therefore,
suffice to specify a unique target sequence.
[0229] In certain embodiments, one may wish to employ antisense
constructs which include other elements, for example, those which
include C-5 propyne pyrimidines. Oligonucleotides which contain C-5
propyne analogues of uridine and cytidine have been shown to bind
RNA with high affinity and to be potent antisense inhibitors of
gene expression (Wagner et al., 1993).
[0230] As an alternative to targeted antisense delivery, targeted
ribozymes may be used. The term "ribozyme" is refers to an
RNA-based enzyme capable of targeting and cleaving particular base
sequences in both DNA and RNA. Ribozymes have specific catalytic
domains that possess endonuclease activity (Kim and Cech, 1987;
Gerlach et al., 1987; Forster and Symons, 1987). For example, a
large number of ribozymes accelerate phosphoester transfer
reactions with a high degree of specificity, often cleaving only
one of several phosphoesters in an oligonucleotide substrate (Cech
et at, 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub,
1992). This specificity has been attributed to the requirement that
the substrate bind via specific base-pairing interactions to the
internal guide sequence ("IGS") of the ribozyme prior to chemical
reaction.
[0231] Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic
acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No.
5,354,855 reports that certain ribozymes can act as endonucleases
with a sequence specificity greater than that of known
ribonucleases and approaching that of the DNA restriction enzymes.
Thus, sequence-specific ribozyme-mediated inhibition of gene
expression may be particularly suited to therapeutic applications
(Scanlon et at, 1991; Sarver et al., 1990). Recently, it was
reported that ribozymes elicited genetic changes in some cell lines
to which they were applied; the altered genes included the
oncogenes H-ras, c-fos and genes of HIV. Most of this work involved
the modification of a target mRNA, based on a specific mutant codon
that is cleaved by a specific ribozyme.
[0232] Ribozymes can either be targeted directly to cells, in the
form of RNA oligonucleotides incorporating ribozyme sequences, or
introduced into the cell as an expression vector encoding the
desired ribozymal RNA. Ribozymes may be used and applied in much
the same way as described for antisense polynucleotide. Ribozyme
sequences also may be modified in much the same way as described
for antisense polynucleotide. For example, one could incorporate
non-Watson-Crick bases, or make mixed RNA/DNA oligonucleotides, or
modify the phosphodiester backbone, or modify the 2'-hydroxy in the
ribose sugar group of the RNA.
[0233] Alternatively, the antisense oligo- and polynucleotides
according to the present invention may be provided as RNA via
transcription from expression constructs that carry nucleic acids
encoding the oligo- or polynucleotides. Throughout this
application, the term "expression construct" is meant to include
any type of genetic construct containing a nucleic acid encoding an
antisense product in which part or all of the nucleic acid sequence
is capable of being transcribed. Typical expression vectors include
bacterial plasmids or phage, such as any of the pUC or
Bluescript.TM. plasmid series or, as discussed further below, viral
vectors adapted for use in eukaryotic cells.
[0234] In preferred embodiments, the nucleic acid encodes an
antisense oligo- or polynucleotide under transcriptional control of
a promoter. A "promoter" refers to a DNA sequence recognized by the
synthetic machinery of the cell, or introduced synthetic machinery,
required to initiate the specific transcription of a gene. The
phrase "under transcriptional control" means that the promoter is
in the correct location and orientation in relation to the nucleic
acid to control RNA polymerase initiation.
[0235] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the I-ISV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 by of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0236] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0237] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 by upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 by apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either co-operatively or independently to activate
transcription.
[0238] The particular promoter that is employed to control the
expression of a nucleic acid encoding the inhibitory peptide is not
believed to be important, so long as it is capable of expressing
the peptide in the targeted cell. Thus, where a human cell is
targeted, it is preferable to position the nucleic acid coding the
inhibitory peptide adjacent to and under the control of a promoter
that is active in the human cell. Generally speaking, such a
promoter might include either a human or viral promoter.
[0239] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter and the Rous
sarcoma virus long terminal repeat can be used to obtain high-level
expression of various proteins. The use of other viral or mammalian
cellular or bacterial phage promoters which are well-known in the
art to achieve expression of peptides according to the present
invention is contemplated as well, provided that the levels of
expression are sufficient for a given purpose.
[0240] By employing a promoter with well-known properties, the
level and pattern of expression of an antisense oligo- or
polynucleotide can be optimized. Further, selection of a promoter
that is regulated in response to specific physiologic signals can
permit inducible expression of an inhibitory protein. For example,
a nucleic acid under control of the human PAI-1 promoter results in
expression inducible by tumor necrosis factor. Additionally any
promoter/enhancer combination (as per the Eukaryotic Promoter Data
Base EPDB) also could be used to drive expression of a nucleic acid
according to the present invention. Use of a T3, T7 or SP6
cytoplasmic expression system is another possible embodiment.
[0241] Eukaryotic cells can support cytoplasmic transcription from
certain bacterial promoters if the appropriate bacterial polymerase
is provided, either as part of the delivery complex or as an
additional genetic expression construct.
[0242] Tables 2 and 3 list several elements/promoters which may be
employed, in the context of the present invention, to regulate the
expression of the gene of interest. This list is not intended to be
exhaustive of all the possible elements involved in the promotion
of gene expression but, merely, to be exemplary thereof.
[0243] Enhancers are genetic elements that increase transcription
from a promoter located at a distant position on the same molecule
of DNA. Enhancers are organized much like promoters. That is, they
are composed of many individual elements, each of which binds to
one or more transcriptional proteins.
[0244] The basic distinction between enhancers and promoters is
operational. An enhancer region as a whole must be able to
stimulate transcription at a distance; this need not be true of a
promoter region or its component elements. On the other hand, a
promoter must have one or more elements that direct initiation of
RNA synthesis at a particular site and in a particular orientation,
whereas enhancers lack these specificities. Promoters and enhancers
are often overlapping and contiguous, often seeming to have a very
similar modular organization.
[0245] Below is a list of viral promoters, cellular
promoters/enhancers, and inducible promoters/enhancers that could
be used in combination with the nucleic acid encoding a gene of
interest in an expression construct (Table 2 and Table 3).
Additionally, any promoter/enhancer combination (as per the
Eukaryotic Promoter Data Base EPDB) also could be used to drive
expression of the gene. Eukaryotic cells can support cytoplasmic
transcription from certain bacterial promoters if the appropriate
bacterial polymerase is provided, either as part of the delivery
complex or as an additional genetic expression construct.
TABLE-US-00002 TABLE 2 ENHANCER/PROMOTER Immunoglobulin Heavy Chain
Immunoglobulin Light Chain T-Cell Receptor HLA DQ .alpha. and DQ
.beta. .beta.-Interferon Interleukin-2 Interleukin-2 Receptor MHC
Class II 5 MHC Class II HLA-DR.alpha. .beta.-Actin Prealbumin
(Transthyretin) Muscle Creatine Kinase Elastase I Metallothionein
Collagenase Albumin Gene .alpha.-Fetoprotein .tau.-Globin
.beta.-Globin e-fos c-HA-ras Insulin Neural Cell Adhesion Molecule
(NCAM) .alpha.1-Antitrypsin H2B (TH2B) Histone Mouse or Type I
Collagen Glucose-Regulated Proteins (GRP94 and GRP78) Rat Growth
Hormone Human Serum Amyloid A (SAA) Troponin I (TN I)
Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40
Polyoma Retroviruses Papilloma Virus Hepatitis B Virus Human
Immunodeficiency Virus Cytomegalovirus
TABLE-US-00003 TABLE 3 Element Inducer MT II Phorbol Ester (TPA)
Heavy metals MMTV (mouse mammary Glucocorticoids tumor virus)
.beta.-Interferon poly(rl)X, poly(rc) Adenovirus 5 E2 Ela c-jun
Phorbol Ester (TPA), H.sub.2O.sub.2 Collagenase Phorbol Ester (TPA)
Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA)
Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene
A23187 .alpha.-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene
H-2kB Interferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol
Ester-TPA Tumor Necrosis Factor FMA Thyroid Stimulating Thyroid
Hormone Hormone .alpha. Gene Insulin E Box Glucose
[0246] Where a cDNA insert is employed, typically one will
typically include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed, such as human growth hormone and SV40 polyadenylation
signals. Also contemplated as an element of the expression
construct is a terminator. These elements can serve to enhance
message levels and to minimize read through from the construct into
other sequences.
[0247] In certain embodiments of the invention, the delivery of a
nucleic acid in a cell may be identified in vitro or in vivo by
including a marker in the expression construct. The marker would
result in an identifiable change to the transfected cell permitting
identification of expression. Enzymes such as herpes simplex virus
thymidine kinase (tk) (eukaryotic) or chloramphenicol
acetyltransferase(CAT) (prokaryotic) may be employed.
[0248] One also may include a polyadenylation signal to effect
proper polyadenylation of the transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed. For example, the SV40, .beta.-globin or adenovirus
polyadenylation signal may be employed. Also contemplated as an
element of the expression cassette is a terminator. These elements
can serve to enhance message levels and to minimize read through
from the cassette into other sequences.
4.8.2 Single-Chain Antibodies
[0249] In yet another embodiment, one gene may comprise a
single-chain antibody. Methods for the production of single-chain
antibodies are well known to those of skill in the art. The skilled
artisan is referred to U.S. Pat. No. 5,359,046, (incorporated
herein by reference) for such methods. A single chain antibody is
created by fusing together the variable domains of the heavy and
light chains using a short peptide linker, thereby reconstituting
an antigen binding site on a single molecule.
[0250] Single-chain antibody variable fragments (scFvs) in which
the C-terminus of one variable domain is tethered to the N-terminus
of the other via a 15 to 25 amino acid peptide or linker, have been
developed without significantly disrupting antigen binding or
specificity of the binding (Bedzyk et al., 1990; Chaudhary et al.,
1990). These Fvs lack the constant regions (Fc) present in the
heavy and light chains of the native antibody. Single-chain
antibodies to the protein products of the UC41 gene are
contemplated within the scope of the present invention.
[0251] Single-chain antibodies can be synthesized by a cell,
targeted to particular cellular compartments, and used to interfere
in a highly specific manner with cell growth and metabolism.
Recently, single-chain antibodies were utilized for the phenotypic
knockout of growth-factor receptors, the functional inactivation of
p21ras, and the inhibition of HIV-1 replication. Intracellular
antibodies offer a simple and effective alternative to other forms
of gene inactivation, as well as demonstrate a clear potential as
reagents for cancer therapy and for the control of infectious
diseases. Single-chain antigen-binding proteins also represent
potentially unique molecules for targeted delivery of drugs,
toxins, or radionuclides to a tumor site, and show increased
accessibility to tumor cells in vivo (Yokoda et al., 1992).
[0252] It is also contemplated by the present invention that
single-chain antibody therapy can be combined with chemotherapeutic
or radiotherapeutic intervention. The discussion S of combined
therapy with traditional chemotherapy or radiotherapy employed
herein is incorporated into this section by reference.
4.8.3 Liposomal Formulations
[0253] In certain broad embodiments of the invention, the antisense
oligo- or polynucleotides and/or expression vectors may be
entrapped in a liposome. Liposomes are vesicular structures
characterized by a phospholipid bilayer membrane and an inner
aqueous medium. Multilamellar liposomes have multiple lipid layers
separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation of
closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated
are cationic lipid-nucleic acid complexes, such as
lipofectamine-nucleic acid complexes.
[0254] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1)
(Kato et al, 1991). In yet further embodiments, the liposome may be
complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression vectors have been successfully employed in
transfer and expression of a polynucleotide in vitro and in vivo,
then they are applicable for the present invention. Where a
bacterial promoter is employed in the DNA construct, it also will
be desirable to include within the liposome an appropriate
bacterial polymerase.
[0255] "Liposome" is a generic term encompassing a variety of
single and multilamellar lipid vehicles formed by the generation of
enclosed lipid bilayers. Phospholipids are used for preparing the
liposomes according to the present invention and can carry a net
positive charge, a net negative charge or are neutral. Dicetyl
phosphate can be employed to confer a negative charge on the
liposomes, and stearylamine can be used to confer a positive charge
on the liposomes.
[0256] Lipids suitable for use according to the present invention
can be obtained from commercial sources. For example, dimyristyl
phosphatidylcholine ("DMPC") can be obtained from Sigma Chemical
Co., dicetyl phosphate ("DCP") is obtained from K & K
Laboratories (Plainview, N.Y.); cholesterol ("Chol") is obtained
from Calbiochem (La Jolla, Calif.); dimyristyl phosphatidylglycerol
("DMPG") and other lipids may be obtained from Avanti Polar Lipids,
Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform,
chloroform/methanol or t-butanol can be stored at about -20.degree.
C. Preferably, chloroform is used as the only solvent since it is
more readily evaporated than methanol.
[0257] Phospholipids from natural sources, such as egg or soybean
phosphatidylcholine, brain phosphatidic acid, brain or plant
phosphatidylinositol, heart cardiolipin and plant or bacterial
phosphatidylethanolamineare preferably not used as the primary
phosphatide, i.e., constituting 50% or more of the total
phosphatide composition, because of the instability and leakiness
of the resulting liposomes.
[0258] Liposomes used according to the present invention can be
made by different methods. The size of the liposomes varies
depending on the method of synthesis. A liposome suspended in an
aqueous solution is generally in the shape of a spherical vesicle,
having one or more concentric layers of lipid bilayer molecules.
Each layer consists of a parallel array of molecules represented by
the formula XY, wherein X is a hydrophilic moiety and Y is a
hydrophobic moiety. In aqueous suspension, the concentric layers
are arranged such that the hydrophilic moieties tend to remain in
contact with an aqueous phase and the hydrophobic regions tend to
self-associate. For example, when aqueous phases are present both
within and without the liposome, the lipid molecules will form a
bilayer, known as a lamella, of the arrangement XY-YX.
[0259] Liposomes within the scope of the present invention can be
prepared in accordance with known laboratory techniques. In one
preferred embodiment, liposomes are prepared by mixing liposomal
lipids, in a solvent in a container, e.g., a glass, pear-shaped
flask. The container should have a volume ten-times greater than
the volume of the expected suspension of liposomes. Using a rotary
evaporator, the solvent is removed at approximately 40.degree. C.
under negative pressure. The solvent normally is removed within
about 5 min to 2 h, depending on the desired volume of the
liposomes. The composition can be dried further in a desiccator
under vacuum. The dried lipids generally are discarded after about
1 wk because of a tendency to deteriorate with time.
[0260] Dried lipids can be hydrated at approximately 25-50 mM
phospholipid in sterile, pyrogen-free water by shaking until all
the lipid film is resuspended. The aqueous liposomes can be then
separated into aliquots, each placed in a vial, lyophilized and
sealed under vacuum.
[0261] In the alternative, liposomes can be prepared in accordance
with other known laboratory procedures: the method of Bangham et
al. (1965), the contents of which are incorporated herein by
reference; the method of Gregoriadis, as described in DRUG CARRIERS
IN BIOLOGY AND MEDICINE, G. Gregoriadis (1979), the contents of
which are incorporated herein by reference; the method of Deamer
and Uster (1983), the contents of which are incorporated by
reference; and the reverse-phase evaporation method as described by
Szoka and Papahadjopoulos (1978). The aforementioned methods differ
in their respective abilities to entrap aqueous material and their
respective aqueous space-to-lipid ratios.
[0262] The dried lipids or lyophilized liposomes prepared as
described above may be reconstituted in a solution of nucleic acid
and diluted to an appropriate concentration with an suitable
solvent, e.g., DPBS. The mixture is then vigorously shaken in a
vortex mixer. Unencapsulated nucleic acid is removed by
centrifugation at 29,000.times.g and the liposomal pellets washed.
The washed liposomes are resuspended at an appropriate total
phospholipid concentration, e.g., about 50-200 mM. The amount of
nucleic acid encapsulated can be determined in accordance with
standard methods. After determination of the amount of nucleic acid
encapsulated in the liposome preparation, the liposomes may be
diluted to appropriate concentration and stored at 4.degree. C.
until use.
[0263] In a preferred embodiment, the lipid
dioleoylphosphatidylcholine is employed. Nuclease-resistant
oligonucleotides were mixed with lipids in the presence of excess
t-butanol. The mixture was vortexed before being frozen in an
acetone/dry ice bath. The frozen mixture was lyophilized and
hydrated with Hepes-buffered saline (1 mM Hepes, 10 mM NaCl, pH
7.5) overnight, and then the liposomes were sonicated in a bath
type sonicator for 10 to 15 min. The size of the
liposomal-oligonucleotides typically ranged between 200-300 nm in
diameter as determined by the submicron particle sizer autodilute
model 370 (Nicomp, Santa Barbara, Calif.).
4.8.4 Viral Delivery Systems
[0264] There are a number of ways in which expression vectors may
introduced into cells. In certain embodiments of the invention, the
expression construct comprises a virus or engineered construct
derived from a viral genome. The ability of certain viruses to
enter cells via receptor-mediated endocytosis, to integrate into a
host cell genome, and express viral genes stably and efficiently
have made them attractive candidates for the transfer of foreign
genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein,
1988; Baichwal and Sugden, 1986; Temin, 1986). Preferred gene
therapy vectors are generally viral vectors.
[0265] Although some viruses that can accept foreign genetic
material are limited in the number of nucleotides they can
accommodate and in the range of cells they infect, these viruses
have been demonstrated to successfully effect gene expression.
However; adenoviruses do not integrate their genetic material into
the host genome and therefore do not require host replication for
gene expression making them ideally suited for rapid, efficient,
heterologous gene expression. Techniques for preparing replication
infective viruses are well I known in the art.
[0266] Of course in using viral delivery systems, one will desire
to purify the virion sufficiently to render it essentially free of
undesirable contaminants, such as defective interfering viral
particles or endotoxins and other pyrogens such that it will not
cause any untoward reactions in the cell, animal or individual
receiving the vector construct. A preferred means of purifying the
vector involves the use of buoyant density. gradients, such as
cesium chloride gradient centrifugation.
[0267] Viruses used as gene vectors such as DNA viruses may include
the papovaviruses (e.g., simian virus 40, bovine papilloma virus,
and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and
adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986).
[0268] One of the preferred methods for in vivo delivery involves
the use of an adenovirus expression vector. Although adenovirus
vectors are known to have a low capacity for integration into
genomic DNA, this feature is counterbalanced by the high efficiency
of gene transfer afforded by these vectors. "Adenovirus expression
vector" is meant to include those constructs containing adenovirus
sequences sufficient to (a) support packaging of the construct and
(b) to express an antisense polynucleotide that has been cloned
therein.
[0269] The expression vector comprises a genetically engineered
form of adenovirus.
[0270] Knowledge of the genetic organization of adenovirus, a 36
kb, linear, double-stranded DNA virus, allows substitution of large
pieces of adenoviral DNA with foreign sequences up to 7 kb
(Grunhaus and Horwitz, 1992). In contrast to retroviral infection,
the adenoviral infection of host cells does not result in
chromosomal integration because adenoviral DNA can replicate in an
episomal manner without potential genotoxicity. Also, adenoviruses
are structurally stable, and no genome rearrangement has been
detected after extensive amplification. Adenovirus can infect
virtually all epithelial cells regardless of their cell cycle
stage. So far, adenoviral infection appears to be linked only to
mild disease such as acute respiratory disease in humans.
[0271] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized genome, ease of
manipulation, high titer, wide target cell range and high
infectivity. Both ends of the viral genome contain 100-200 base
pair inverted repeats (ITRs); which are cis elements necessary for
viral DNA replication and packaging. The early (E) and late (L)
regions of the genome contain different transcription units that
are divided by the onset of viral DNA replication. The E1 region
(E1A and E1B) encodes proteins responsible for the regulation of
transcription of the viral genome and a few cellular genes. The
expression of the E2 region (E2A and E2B) results in the synthesis
of the proteins for viral DNA replication. These proteins are
involved in DNA replication, late gene expression and host cell
shut-off (Renan, 1990). The products of the late genes, including
the majority of the viral capsid proteins, are expressed only after
significant processing of a single primary transcript issued by the
major late promoter (MLP). The MLP, (located at 16.8 m.u.) is
particularly efficient during the late phase of infection, and all
the inRNAs issued from this promoter possess a 5'-tripartite leader
(TPL) sequence which makes them preferred mRNAs for
translation.
[0272] In currently used systems, recombinant adenovirus is
generated from homologous recombination between shuttle vector and
provirus vector. Due to the possible recombination between two
proviral vectors, wild-type adenovirus may be generated from this
process. Therefore, it is critical to isolate a single clone of
virus from an individual plaque and examine its genomic
structure.
[0273] Generation and propagation of adenovirus vectors which are
replication deficient depend on a unique helper cell line,
designated 293, which is transformed from human embryonic kidney
cells by Ad5 DNA fragments and constitutively expresses E1 proteins
(Graham et al., 1977). Since the E3 region is dispensable from the
adenovirus genome (Jones and Shenk, 1978), the current adenovirus
vectors, with the help of 293 cells, carry foreign DNA in either
the E1, the E3, or both regions (Graham and Prevec, 1991). In
nature, adenovirus can package approximately 105% of the wild-type
genome (Ghosh-Choudhury et al., 1987), providing capacity for about
2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA
that is replaceable in the E1 and E3 regions, the maximum capacity
of the current adenovirus vector is under 7.5 kb, or about 15% of
the total length of the vector. More than 80% of the adenovirus
viral genome remains in the vector backbone and is the source of
vector-borne cytotoxicity. Also; the replication deficiency of the
E1-deleted virus is incomplete. For example, leakage of viral gene
expression has been observed with the currently available vectors
at high multiplicities of infection (MOI) (Mulligan, 1993).
[0274] Helper cell lines may be derived from human cells such as
human embryonic kidney cells, muscle cells, hematopoietic cells or
other human embryonic mesenchymal or epithelial cells.
Alternatively, the helper cells, may be derived from the cells of
other mammalian species that are permissive for human adenovirus.
Such cells include, e.g., Vero cells or other monkey embryonic
mesenchymal or epithelial cells. As discussed, the preferred helper
cell line is 293.
[0275] Recently, Racher et al. (1995) disclosed improved methods
for culturing 293 cells and propagating adenovirus. In one format,
natural cell aggregates are grown by inoculating individual cells
into 1 liter siliconized spinner flasks (Techne, Cambridge, UK)
containing 100-200 ml of medium. Following stirring at 40 rpm, the
cell viability is estimated with trypan blue. In another format,
Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) are
employed as follows. A cell innoculum, resuspended in 5 ml of
medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer
flask and left stationary, with occasional agitation, for 1 to 4 h.
The medium is then replaced with 50 ml of fresh medium and shaking
is initiated. For virus production, cells are allowed to grow to
about 80% confluence, after which time the medium is replaced (to
25% of the final volume) and adenovirus added at an MOI of 0.05.
Cultures are left stationary overnight, following which the volume
is increased to 100% and shaking is commenced for another 72 h.
[0276] Other than the requirement that the adenovirus vector be
replication defective, or at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovinis may be of
any of the 42 different known serotypes or subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material
in order to obtain the conditional replication-defective adenovirus
vector for use in the present invention. This is because Adenovirus
type 5 is a human adenovirus about which a great deal of
biochemical and genetic information is known, and it has
historically been used for most constructions employing adenovirus
as a vector.
[0277] A typical vector applicable to practicing the present
invention is replication defective and will not have an adenovirus
E1 region. Thus, it will be most convenient to introduce the
polynucleotide encoding the UC41 gene at the position from which
the E1-coding sequences have been removed. However, the position of
insertion of the construct within the adenovirus sequences is not
critical. The polynucleotide encoding the UC41 gene also may be
inserted in lieu of the deleted E3 region in E3 replacement vectors
as described by Karlsson et al., (1986) or in the E4 region where a
helper cell line or helper virus complements the E4 defect.
[0278] Adenovirus is easy to grow and manipulate and exhibits broad
host range in vitro and in vivo. This group of viruses can be
obtained in high titers, e.g, 10.sup.9-10.sup.11 plaque-forming
units per ml, and they are highly infective. The life cycle of
adenovirus does not require integration into the host cell genome.
The foreign genes delivered by adenovirus vectors are episomal and,
therefore, have low genotoxicity to host cells. No side effects
have been reported in studies of vaccination with wild-type
adenovirus (Couch et al., 1963; Top et al., 1971), demonstrating
their safety and therapeutic potential as in vivo gene transfer
vectors.
[0279] Adenovirus vectors have been used in eukaryotic gene
expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and
vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec,
1991). Recently, animal studies suggested that recombinant
adenovirus could be used for gene therapy (Stratford-Perricaudet
and Perricaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et
al., 1993). Studies in administering recombinant adenovirus to
different tissues include trachea instillation (Rosenfeld et al.,
1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,
1993), peripheral intravenous injections (Herz and Gerard, 1993)
and stereotactic inoculation into the brain (Le Gal La Salle et
al., 1993).
[0280] Other gene transfer vectors may be constructed from
retroviruses. The retroviruses are a group of single-stranded RNA
viruses characterized by an ability to convert their RNA to
double-stranded DNA in infected cells by a process of
reverse-transcription (Coffin, 1990). The resulting DNA then stably
integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins. The integration results in the
retention of the viral gene sequences in the recipient cell and its
descendants. The retroviral genome contains three genes, gag, pol,
and env. that code for capsid proteins, polymerase enzyme, and
envelope components, respectively. A sequence found upstream from
the gag gene contains a signal for packaging of the genome into
virions. Two long terminal repeat (LTR) sequences are present at
the 5' and 3' ends of the viral genome. These contain strong
promoter and enhancer sequences, and also are required for
integration in the host cell genome (Coffin, 1990).
[0281] In order to construct a retroviral vector, a nucleic acid
encoding a UC41 gene is inserted into the viral genome in the place
of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol, and env genes, but without the
LTR and packaging components, is constructed (Mann et al., 1983).
When a recombinant plasmid containing a cDNA, together with the
retroviral LTR and packaging sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the
packaging sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then
secreted into the culture media (Nicolas and Rubenstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors arc
capable of infecting a broad variety of cell types. However,
integration and stable expression require the division of host
cells (Paskind et al., 1975).
[0282] A novel approach designed to allow specific targeting of
retrovirus vectors was recently developed based on the chemical
modification of a retrovinis by the chemical addition of lactose
residues to the viral envelope. This modification could permit the
specific infection of hepatocytes via sialoglycoprotein
receptors.
[0283] A different approach to targeting of recombinant
retroviruses has been designed in which biotinylated antibodies
against a retroviral envelope protein and against a specific cell
receptor were used. The antibodies were coupled via the biotin
components by using streptavidin (Roux et al., 1989). Using
antibodies against major histocompatibility complex class I and
class II antigens, the infection of a variety of human cells that
bear those surface antigens with an ecotropic virus in vitro was
demonstrated (Roux et al., 1989).
[0284] There are certain limitations to the use of retrovirus
vectors. For example, retrovirus vectors usually integrate into
random sites in the cell genome. This can lead to insertional
mutagenesis through the intemiption of host genes or through the
insertion of viral regulatory sequences that can interfere with the
function of flanking genes (Varmus et al., 1981). Another concern
with the use of defective retrovirus vectors is the potential
appearance of wild-type replication-competent virus in the
packaging cells. This may result from recombination events in which
the intact sequence from the recombinant virus inserts upstream
from the gag, pol, env sequence integrated in the host cell genome.
However, new packaging cell lines are now available that should
greatly decrease the likelihood of recombination (Markowitz et al.,
1988; Hersdorffer et al., 1990).
[0285] Other viral vectors may be employed as expression
constructs. Vectors derived from viruses such as vaccinia virus
(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),
adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden,
1986; Hermonat and Muzycska, 1984), and herpes viruses may be
employed. They offer several attractive features for various
mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and
Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0286] With the recent recognition of defective hepatitis B
viruses, new insight has been gained into the structure-function
relationship of different viral sequences. In vitro studies showed
that the virus could retain the ability for helper-dependent
packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990). This suggests that large
portions of the genome can be replaced with foreign genetic
material. The hepatotropism and persistence (integration) are
particularly attractive properties for liver-directed gene
transfer. Chang et al. (1991) recently introduced the
chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B
virus genome in the place of the polymerase, surface, and
pre-surface coding sequences. It was co-transfected with wild-type
virus into an avian hepatoma cell line. Culture media containing
high titers of the recombinant virus were used to infect primary
duckling hepatocytes. Stable CAT gene expression was detected for
at least 24 days after transfection (Chang et al., 1991).
[0287] To effect expression of sense or antisense gene constructs,
the expression construct must be delivered into a cell. This
delivery may be accomplished in vitro, as in laboratory procedures
for transforming cells lines, or in vivo or ex vivo, as in the
treatment of certain disease states. One mechanism for delivery is
via viral infection where the expression construct is encapsidated
in an infectious viral particle.
4.8.5 Non-Viral Methods
[0288] Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells also are contemplated by
the present invention. These include calcium phosphate
precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;
Rippe et al., 1990), DEAE-dextran (Gopal, 1985), electroporation
(Tur-Kaspa et al., 1986; Potter et al., 1984), direct
microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes
(Nicolau and Sene, 1982; Fraley et al., 1979) and lipofectamine-DNA
complexes, cell sonication (Fechheimer et al., 1987), gene
bombardment using high velocity microprojectiles (Yang et al.,
1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and
Wu, 1988). Some of these techniques may be successfully adapted for
in vivo or ex vivo use.
[0289] In one embodiment of the invention, the expression construct
may simply consist of naked recombinant vector. Transfer of the
construct may be performed by any of the methods mentioned above
which physically or chemically permeabilize the cell membrane. For
example, Dubensky et al. (1984) successfully injected polyomavirus
DNA in the form of CaPO.sub.4 precipitates into liver and spleen of
adult and newborn mice demonstrating active viral replication and
acute infection. Benvenisty and Neshif (1986) also demonstrated
that direct intraperitoneal injection of CaPO.sub.4 precipitated
plasmids results in expression of the transfected genes. It is
envisioned that DNA encoding an antisense UC41 construct also may
be transferred in a similar manner in vivo.
[0290] Once the expression construct has been delivered into the
cell the nucleic acid encoding the UC41 gene may be positioned and
expressed at different sites. In certain embodiments, the nucleic
acid encoding the gene may be stably integrated into the genome of
the cell. This integration may be in the cognate location and
orientation via homologous recombination (gene replacement) or it
may be integrated in a random, non-specific location (gene
augmentation). In yet further embodiments, the nucleic acid may be
stably maintained in the cell as a separate, episomal segment of
DNA. Such nucleic acid segments or "episomes" encode sequences
sufficient to permit maintenance and replication independent of or
in synchronization with the host cell cycle. How the expression
construct is delivered to a cell and where in the cell the nucleic
acid remains is dependent on the type of expression construct
employed.
4.8.6 Pharmaceutical Compositions and Routes of Administration
[0291] Where clinical application of liposomes containing antisense
oligo- or polynucleotides or expression vectors is undertaken, it
will be necessary to prepare the liposome complex as a
pharmaceutical composition appropriate for the intended
application. Generally, this will entail preparing a pharmaceutical
composition that is essentially free of pyrogens, as well as any
other impurities that could be harmful to humans or animals. One
also will generally desire to employ appropriate buffers to render
the complex stable and allow for uptake by target cells.
[0292] Aqueous compositions of the present invention comprise an
effective amount of the antisense expression vector encapsulated in
a liposome as discussed above, further dispersed in
pharmaceutically acceptable carrier or aqueous medium. Such
compositions also are referred to as innocula. The phrases
"pharmaceutically or pharmacologically acceptable" refer to
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or a human, as
appropriate.
[0293] As used herein, "phannaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0294] Solutions of therapeutic compositions can be prepared in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0295] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection also may
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per
milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the
like.
[0296] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according to
well known parameters.
[0297] An effective amount of the therapeutic composition is
determined based on the intended goal. The term "unit dose" or
"dosage" refers to physically discrete units suitable for use in a
subject, each unit containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired
responses, discussed above, in association with its administration,
i.e., the appropriate route and treatment regimen. The quantity to
be administered, both according to number of treatments and unit
dose, depends on the protection desired.
[0298] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting dose include physical and clinical
state of the patient, the route of administration and the potency,
stability and toxicity of the particular therapeutic substance. For
the instant application, it is envisioned that the amount of
therapeutic composition comprising a unit dose will range from
about 5-30 mg of polynucleotide.
4.9 Methods for Treating UC41 Related Malignancies
[0299] The present invention also contemplates, in another
embodiment, the treatment of prostate cancer. The types of cancer
that may be treated, according to the present invention, are
limited only by the involvement of UC41. By involvement is meant
that, it is not even a requirement that UC41 be mutated or
abnormal--the overexpression or underexpression of the protein(s)
encoded by this gene may be a primary factor in the development of
prostate cancer. Thus, it is contemplated that tumors may be
treated using antisense or expression therapy targeted to the UC41
gene product(s).
[0300] In many contexts, it is not necessary that the tumor cell be
killed or induced to undergo normal cell death or "apoptosis."
Rather, to accomplish a meaningful treatment, all that is required
is that the tumor growth be slowed to some degree. It may be that
the tumor growth is completely blocked, however, or that some tumor
regression is achieved. Clinical terminology such as "remission"
and "reduction of tumor" burden also are contemplated given their
normal usage.
4.9.1 Genetic Based Therapies
[0301] One of the therapeutic embodiments contemplated by the
present inventors is the intervention, at the molecular level, in
the events involved in the tumorigenesis of some cancers.
Specifically, the present inventors intend to provide, to a cancer
cell, an antisense construct capable of inhibiting expression of
UC41 in that cell. Particularly preferred expression vectors are
viral vectors such as adenovirus, adeno-associated virus, herpes
virus, vaccinia virus and retrovirus. Also preferred is
liposomally-encapsulated expression vector.
[0302] Those of skill in the art are well aware of how to apply
gene delivery to in vivo and ex vivo situations. For viral vectors,
one generally will prepare a viral vector stock. Depending on the
kind of virus and the titer attainable, one will deliver between
about 1.times.10.sup.4 and 1.times.10.sup.12 infectious particles
to the patient. Similar figures may be extrapolated for liposomal
or other non-viral formulations by comparing relative uptake
efficiencies. Formulation as a pharmaceutically acceptable
composition is discussed below.
[0303] Various routes are contemplated for various tumor types. The
section below on routes contains an extensive list of possible
routes. For practically any tumor, systemic delivery is
contemplated. This will prove especially important for attacking
microscopic or metastatic cancer. Where discrete tumor mass may be
identified, a variety of direct, local and regional approaches may
be taken. For example, the tumor may be injected directly with the
expression vector. A tumor bed may be treated prior to, during or
after resection. Following resection, one generally will deliver
the vector by a catheter left in place following surgery. One may
utilize the tumor vasculature to introduce the vector into the
tumor by injecting a supporting vein or artery. A more distal blood
supply route also may be utilized.
[0304] In a different embodiment, ex vivo gene therapy is
contemplated. This approach is particularly suited, although not
limited, to treatment of bone marrow associated cancers. In an ex
vivo embodiment, cells from the patient are removed and maintained
outside the body for at least some period of time. During this
period, a therapy is delivered, after which the cells are
reintroduced into the patient. Preferably, any tumor cells in the
sample have been killed.
4.9.2 Immunotherapies
[0305] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0306] According to the present invention, native or wild type UC41
may be a target for an immune effector. It is possible UC41 may be
targeted by immunotherapy, either using antibodies, antibody
conjugates, or immune effector cells.
[0307] Alternatively, immunotherapy could be used as part of a
combined therapy, in conjunction with UC41-targeted gene therapy.
The general approach for combined therapy is discussed below.
Generally, the tumor cell must bear some marker that is amenable to
targeting, i.e., is not present on the majority of other cells.
Many tumor markers exist and any of these may be suitable for
targeting in the context of the present invention. Common tumor
markers include carcinoembryonic antigen, prostate specific
antigen, urinary tumor associated antigen, fetal antigen,
tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA,
MucB, PLAP, estrogen receptor, laminin receptor, erb B and
p155.
4.93 Combined Therapy with Immunotherapy, Chemotherapy or
Radiotherapy
[0308] Tumor cell resistance to DNA damaging agents represents a
major problem in clinical oncology. One goal of current cancer
research is to find ways to improve the efficacy of chemo- and
radiotherapy. One way is by combining such traditional therapies
with gene therapy. For example, the herpes simplex-thymidine kinase
(HS-tk) gene, when delivered to brain tumors by a retroviral vector
system, successfully induced susceptibility to the antiviral agent
ganciclovir (Culver et al., 1992). In the context of the present
invention, it is contemplated that UC41 gene therapy could be used
similarly in conjunction with chemo- or radiotherapeutic
intervention.
[0309] To kill cells, inhibit cell growth, inhibit metastasis,
inhibit angiogenesis or otherwise reverse or reduce the malignant
phenotype of tumor cells, using the methods and compositions of the
present invention, one would generally contact a "target" cell with
an antisense construct of UC41 and at least one other agent. These
compositions would be provided in a combined amount effective to
kill or inhibit proliferation of the cell. This process may involve
contacting the cells with the antisense construct and the agent(s)
or factor(s) at the same time. This may be achieved by contacting
the cell with a single composition or pharmacological formulation
that includes both agents, or by contacting the cell with two
distinct compositions or formulations simultaneously, wherein one
composition includes the antisense or expression construct and the
other includes the agent.
[0310] Alternatively, the gene therapy treatment may precede or
follow the other agent treatment by intervals ranging from min to
wk. In embodiments where the other agent and expression construct
are applied separately to the cell, one would generally ensure that
a significant period of time did not expire between the time of
each delivery, such that the agent and expression construct would
still be able to exert an advantageously combined (e.g.,
synergistic) effect on the cell. In such instances, it is
contemplated that one would contact the cell with both modalities
within about 12-24 h of each other and, more preferably, within
about 6-12 h of each other, with a delay time of only about 12 h
being most preferred. In some situations, it may be desirable to
extend the duration of treatment with only the therapeutic agent
significantly, for example, where several days (2, 3, 4, 5, 6 or 7)
to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
[0311] It also is conceivable that more than one administration of
either UC41 antisense construct or the other agent will be desired.
Various combinations may be employed, where UC41 is "A" and the
other agent is "B", as exemplified below:
[0312] A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
[0313] A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
[0314] A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/BB B/A/B/B B/B/A/B
[0315] To achieve cell killing, both agents are delivered to a cell
in a combined amount effective to kill the cell.
[0316] Agents or factors suitable for use in a combined therapy are
any chemical compound or treatment method that induces DNA damage
when applied to a cell. Such agents and factors include radiation
and waves that induce DNA damage such as .gamma.-irradiation,
X-rays, UV-irradiation, microwaves, electronic emissions, and the
like. A variety of chemical compounds, also described as
"chemotherapeutic agents," function to induce DNA damage, all of
which are intended to be of use in the combined treatment methods
disclosed herein. Chemotherapeutic agents contemplated to be of use
include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16),
camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even
hydrogen peroxide. The invention also encompasses the use of a
combination of one or more DNA damaging agents, whether
radiation-based or actual compounds, such as the use of X-rays with
cisplatin or the use of cisplatin with etoposide.
[0317] In treating cancer according to the invention, one would
contact the tumor cells with an agent in addition to the antisense
construct. This may be achieved by irradiating the localized tumor
site with radiation such as X-rays, UV-light, .gamma.-rays or even
microwaves. Alternatively, the tumor cells may be contacted with
the agent by administering to the subject a therapeutically
effective amount of a pharmaceutical composition comprising a
compound such as, adriamycin, 5-fluorouracil, etoposide,
camptothecin, actinomycin-D, or mitomycin C. The agent may be
prepared and used as a combined therapeutic composition, or kit, by
combining it with a UC41 antisense construct, as described
above.
[0318] Agents that directly cross-link nucleic acids, specifically
DNA, are envisaged to facilitate DNA damage leading to a
synergistic, antineoplastic combination with UC41. Agents such as
cisplatin, and other DNA alkylating agents may be used.
[0319] Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in a clinical setting for the treatment of neoplasms,
these compounds are administered intravenously through bolus
injections at doses ranging from 25-75 mg/m.sup.2 at 21 day
intervals for adriamycin, to 35-50 mg/m.sup.2 for etoposide
intravenously or double the intravenous dose orally.
[0320] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors and subunits also lead to DNA damage. A number of
nucleic acid precursors have been developed for this purpose.
Particularly useful are agents that have undergone extensive
testing and are readily available, such as 5-fluorouracil (5-FU).
Although quite toxic, 5-FU is applicable in a wide range of
carriers, including topical. However intravenous administration
with doses ranging from 3 to 15 mg/kg/day is commonly used.
[0321] Other factors that cause DNA damage and have been used
extensively include .gamma.-rays, X-rays, and/or the directed
delivery of radioisotopes to tumor cells. Other forms of DNA
damaging factors also are contemplated such as microwaves and
UV-irradiation. It is most likely that all of these factors effect
a broad range of damage to DNA, on the precursors of DNA, the
replication and repair of DNA, and the assembly and maintenance of
chromosomes. Dosage ranges for X-rays range from daily doses of 50
to 200 roentgens for prolonged periods of time (3 to 4 wk), to
single doses of 2000 to 6000 roentgens. Dosage ranges for
radioisotopes vary widely, and depend on the half-life of the
isotope, the strength and type of radiation emitted, and the uptake
by the neoplastic cells.
[0322] The skilled artisan is directed to "Remington's
Pharmaceutical Sciences" 15th Edition, chapter 33, and in
particular to pages 624-652. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations should meet
sterility, pyrogenicity, and general safety and purity standards as
required by the FDA Office of Biologics standards.
[0323] The inventors propose that the regional delivery of UC41
antisense constructs to patients with prostate cancer will be a
very efficient method for delivering a therapeutically effective
gene to counteract the clinical disease. Similarly, chemo- or
radiotherapy may be directed to a particular, affected region of
the subject's body. Alternatively, systemic delivery of expression
construct and/or the agent may be appropriate in certain
circumstances, for example, where extensive metastasis has
occurred.
[0324] In addition to combining UC41-targeted therapies with chemo-
and radiotherapies, it also is contemplated that combination with
other gene therapies will be advantageous. For example,
simultaneous targeting of therapies directed toward UC41 and p53
may produce an improved anti-cancer treatment. Any other
tumor-related gene conceivably can be targeted in this manner, for
example, p21, Rb, APC, DCC, NF-1, NF-2, p16, FHIT, WT-1, MEN-I,
MEN-II, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fms, jun, trk,
ret, gsp, hst, bcl and abl.
4.9.4 Screening for Modulators of UC41
[0325] Cells exhibiting elevated UC4I expression may be screened to
identify effectors of UC41 expression. Therefore, within certain
embodiments of the invention, methods are provided for screening
for modulators of UC41 expression. UC41 expression, in turn, may be
examined by Northern blot or slot blot analysis of the RNA products
of the gene, Western blot analysis of the protein products of the
gene, or by other standard techniques as described herein.
Compounds which modulate UC41 expression may be detected by their
effect on the amounts of these RNA or protein gene products present
in a given cell line or tissue sample.
[0326] Screening methods may use the target cells as adherent cells
on a culture dish, as part of an alginate biomatrix, in suspension
culture or in any other form that permits the expression of the
polypeptide or nucleic acid to be monitored. These cells are then
used as reagents to screen small molecule and peptide libraries to
identify modulators of UC41 function.
[0327] Regulation of expression can occur at any phase in the
synthesis and release of a nucleotide or polypeptide, including
gene transcription; stability of the mRNA; translation;
post-translational modifications such as proteolytic processing,
formation of disulfide bonds, amidation, and glycosylation; and
subcellular locallization. Screening methods will monitor the
expression of these gene products in the absence of the candidate
substance and comparing such results to the assay performed. In the
presence of candidate substances.
[0328] It is contemplated that this screening technique will prove
useful in the general identification of a compound that will serve
the purpose of decreasing, inhibiting, or otherwise abrogating the
expression of UC41. Such compounds will be useful in the treatment
of prostate cancer. In these embodiments, the present invention is
directed to a method for determining the ability of a candidate
substance to inhibit UC41 expression. The method including
generally the steps of: [0329] (a) providing at least one UC41
expressing cell; [0330] (b) contacting said cell with said
candidate substance; [0331] (c) measuring the level of UC41
expression in said cell; and [0332] (d) comparing the UC41
expression of the cell in step (c) with the UC41 expression of the
cell of step (a).
[0333] To identify a candidate substance as being capable of
inhibiting UC41 expression in the assay above, one would measure or
determine levels of UC41 expression in an appropriate cell line in
the absence of the added candidate substance. One would then add
the candidate substance to the cell and determine the level of UC41
expression in the presence of the candidate substance. A candidate
substance which decrease UC41 expression relative to the expression
level in its absence, is indicative of a candidate regulatory
substance for UC41 expression.
[0334] As used herein the term "candidate substance" refers to any
molecule that is capable of modulating UC41 expression. The
candidate substance may be a protein or fragment thereof, a small
molecule inhibitor, or even a nucleic acid molecule. It may prove
to be the case that the most useful pharmacological compounds for
identification through application of the screening assay will be
compounds that are structurally related to other known modulators
of expression. The active compounds may include fragments or parts
of naturally-occurring compounds or may be only found as active
combinations of known compounds which are otherwise inactive.
However, prior to testing of such compounds in humans or animal
models, it will possibly be necessary to test a variety of
candidates to determine which have potential.
[0335] Accordingly, the active compounds may include fragments or
parts of naturally-occurring compounds or may be only found as
active combinations of known compounds which are otherwise
inactive. Accordingly, the present invention provides screening
assays to identify agents which stimulate or inhibit cellular UC41
expression, it is proposed that compounds isolated from natural
sources, such as animals, bacteria, fungi, plant sources, including
leaves and bark, and marine samples may be assayed as candidates
for the presence of potentially useful pharmaceutical agents. It
will be understood that the pharmaceutical agents to be screened
could also be derived or synthesized from chemical compositions or
man-made compounds. Thus, it is understood that the candidate
substance identified by the present invention may be polypeptide,
polynucleotide, small molecule inhibitors or any other compounds
that may be designed through rational drug design starting from
known regulators of gene expression, as well as known agents for
therapeutic treatment of prostate cancer.
[0336] The candidate screening assays are simple to set up and
perform. Thus, in assaying for a candidate UC41 regulatory
compound, after obtaining a UC41 expressing cell line, one will
admix a candidate substance with the cell, under conditions which
would allow measurable expression to occur.
[0337] "Effective amounts" in certain circumstances are those
amounts effective to reproducibly stimulate expression from the
cell in comparison to their normal levels. Compounds that achieve
significant appropriate changes in UC41 expression will be
used.
[0338] Significant changes in UC41 expression are represented by a
decrease in expression of at least about 30%-40%, and most
preferably, by decreases of at least about 50%, with higher values
of course being possible.
[0339] It will, of course, be understood that all the screening
methods of the present invention are useful in themselves
notwithstanding the fact that effective candidates may not be
found. The invention provides methods for screening for such
candidates, not solely methods of finding them.
5.0 EXAMPLES
[0340] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus may be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes may be made in the
particular embodiments which are disclosed and still obtain a like
or similar result without departing from the spirit and scope of
the invention.
5.1 Materials and Methods
5.1.1 Tissue Acquisition
[0341] Normal prostate, benign prostatic hyperplasia (BPH),
prostate cancer (CaP) and metastatic prostate cancer tissue were
from radical prostatectomies. Xenografts were from two cell lines,
Lu23 and Lu35, passaged in nude mice. All tissues were either
processed immediately for RNA isolation or fresh frozen in liquid
nitrogen and then stored at -80.degree. C. for further use. The
prostate cancer cell lines LNCaP, PC-3 and DU 1145 were maintained
in RPMI 1640 medium.
5.1.2 Differential Display
[0342] A modified Differential Display method (Liang and Pardee,
1992; An et al., 1995) was used to identify UC41 as a gene
up-regulated in prostate cancer. Total RNA was isolated from frozen
normal prostate and prostate cancer tissues according to
Chomczynski and Sacchi (1987). RNA (10 .mu.g) from each tissue was
treated with 5 units of RNase-free DNase I (GIBCO/BRL,
Gaithersburg, Md.) in the presence of 20 mM Tris-HCl, pH 8.4, 50 mM
KCl, 2 mM MgCl.sub.2 and 20 units of RNase inhibitor (Boehringer
Mannheim Biochemicals, Indianapolis, Ind.). After extraction with
phenol/chloroform and ethanol precipitation, the RNA was
redissolved in DEPC-treated H.sub.2O.
[0343] One .mu.g from each RNA sample was reverse transcribed into
cDNA using random hexamers and M-MLV reverse transcriptase
(GIBCO/BRL, Gaithersburg, Md.), following the manufacturer's
instructions. The reaction mixture contained 50 mM Tris-HCl, pH
8.3, 75 mM KCl, 3 mM MgCl.sub.2, 10 mM DTT, 500 .mu.M dNTP, 2 .mu.m
random hexamers and 400 U M-MLV reverse transcriptase. PCR.TM. was
performed with two arbitrary 10-mer primers.
[0344] Arbitrary primers used for identifying UC41:
TABLE-US-00004 5' CTAGTGATTA 3' SEQ ID NO: 6 5' GTCCTTATGA 3' SEQ
ID NO: 7
[0345] PCR.TM. was performed in 1.times. PCR.TM. buffer (GIBCO/BRL,
Gaithersburg, Md.), 50 dNTPs, 0.2 .mu.M arbitrary primer(s), 1/20
volume (1 .mu.l) of the cDNA and 1 U of Tag DNA polymerase
(GIBCO/BRL, Gaithersburg, Md.) in a final volume of 20 .mu.l. The
amplification parameters included 40 cycles of reaction with 30 sec
denaturing at 94.degree. C., 1 min 30 sec annealing at 38.degree.
C. and 1 min extension at 72.degree. C. A final extension at
72.degree. C. was performed for 15 min. The PCR.TM. products were
then separated on a 3% metaphor agarose gel (FMC BioProducts,
Rockland, Me.) with 0.5 .mu.g/ml ethidium bromide and positive
bands were identified under UV light. Positive bands were excised
with a razor blade, purified on Qiaex resin (Qiagen, Valencia,
Calif.) according to the manufacturer's instructions and cloned
directly into a plasmid using the TA cloning system (pGEM-T system,
Promega, Madison, Wis.). The differential expression of positive
bands was confirmed by relative quantitative RT-PCR.TM..
5.1.3 Prostate cDNA Library Screening and Sequence Analysis
[0346] An [.alpha.-.sup.32P]-dATP labeled probe was made by random
labeling (Gibco/BRL, Gaithersburg, Md.) from a 183 by EST fragment
of UC41 (identified as SEQ ID NO:23 in U.S. patent application Ser.
No. 08/692,787 the entire text of which is incorporated herein by
reference.) This fragment corresponds to bases 1323-1500 of SEQ ID
NO:1. A normal human prostate cDNA library in .lamda.gt11
(Clontech) was plated in duplicate and screened according to the
product manual. Hybridization of the plated library was performed
at 68.degree. C. overnight in ExpressHyb solution (Clontech, Palo
Alto, Calif.). Filters were washed twice in 2.times.SSC, 0.05% SDS
at 37.degree. C., then once in 0.1.times.SSC, 0.1% SDS at
50.degree. C. The labeled filter was exposed to XAR-5 film (Kodak,
Rochester, N.Y.) at -70.degree. C.
[0347] From about 3.times.10.sup.5 plaques, 2 independent positive
clones were isolated after three rounds of screening. The inserts
from the two clones were re-amplified and sub-cloned into
sequencing vector pCR2-TOPO (Invitrogen, Carlsbad, Calif.). The
sequence was determined by cycle sequencing with both M13 forward
and reverse primers and analyzed with Sequencher software (Gene
Codes). The complete cDNA sequence identified for UC41 is presented
in SEQ ID NO:1. The full length cDNA fragment (SEQ ID NO: 1)
inserted in vector pCR2-TOPO is identified herein as pCR TOPO-UC41.
The generated sequence was analyzed for the prediction of open
reading frame, translation initiation site, signaling peptide,
transmembrane region and potential modification domain.
5.1.4 Northern Blot and Dot Blot Analyses
[0348] The probe used to screen Northern blots and dot blots
consisted of a 697 by DNA fragment excised from pCR TOPO-UC41 by
Sph I and Pst I digestion and labeled with [.alpha..sup.32P]-dATP
using a random primer DNA labeling kit (Gibco/BRL, Gaithersburg,
Md.). The Sph I-Pst I fragment corresponds to bases 157 to 854 of
SEQ ID NO:1. The multiple tissue northern (including eight normal
adult tissues) and human RNA master (including 43 adult tissues and
7 fetal tissues) filters (Clontech, Palo Alto, Calif.) containing 2
.mu.g or 89-514 ng, respectively, of normalized poly A.sup.+ RNA
per lane or dot were hybridized with
[.alpha.-.sup.32P]-dATP-labeled probe as described above and
exposed to XAR-5 film (Kodak, Rochester, N.Y.) at -70.degree. C.
for 3 days.
5.1.5 In Situ Hybridization
[0349] To generate the probe for in situ hybridization studies, the
UC41 cDNA (SEQ ID NO:1) was amplified by PCR.TM. using the
following primers for 30 cycles at 94.degree. C. 30 sec, 58.degree.
C. 1 min, 72.degree. C. 1 min each and a final extension of 5 min
at 72.degree. C.
TABLE-US-00005 Forward primer (at position 1319 of SEQ ID NO: 1)
(SEQ ID NO: 8) 5'-AAAACGATATCATGCTTTCTCATCTCTCC-3' Reverse primer
(at position 1694 of SEQ ID NO: 1) (SEQ ID NO: 9)
5'-CAATTGCGGCCGCTCTATCAGCCTCTTTGGAG-3'
[0350] The PCR.TM. amplified product was then cloned into the
pGEM-T vector to create the plasmid pGEMT-UC41. Both sense and
antisense digoxigenin-dUTP labeled RNA probes were synthesized from
1 .mu.g of linearized plasmid pGEMT-UC41 (digested with Nco 1 or
Eco RV) in a standard in vitro transcription reaction, using the T7
and SP6 promoters and a DIG RNA labeling kit (Boehringer Mannheim,
Indianapolis, Ind.).
[0351] In situ hybridization was performed with a GenII automatic
in situ system (Ventana Medical Systems, Tucson, Ariz.). Sections
(5 .mu.m) from formalin-fixed, paraffin-embedded tissue were
mounted onto Proma plus slides. The slides were then dewaxed at
65.degree. C. for 2 h and rehydrated. Before hybridization,
sections were digested with proteinase I cocktail for 12 min at
37.degree. C. Then 10 ng of either antisense or sense probe in the
hybridization buffer, in a volume of 70 .mu.l, were applied to the
sections, denatured at 65.degree. C. and then incubated at
42.degree. C. for 360 min. After sequential washing with 2.times.,
1.times. and 0.1.times. SSC buffer, the specific signal was
detected using mouse anti-digoxigenin antibody, followed by biotin
conjugated anti-mouse antibody and streptavidin-horseradish
peroxidase and developed with DAB H.sub.2O.sub.2.
5.1.6 RT-PCR.TM.
[0352] Microdissections of normal prostate and prostate cancer
tissues, as guided by hematoxylin and eosin (H&E) staining,
were performed on OCT (Optimal Cutting Temperature compound, Miles,
Inc., Elkhart Ind.) embedded tissues taken following radical
prostatectomies. Total RNA was isolated from the corresponding
normal prostate and prostate cancer tissue, prostate cancer cell
lines and xenografts with a STAT 60 kit (Tel-Test, Friendswood,
Tex.) according to the manufacturer's instructions. One .mu.g of
total RNA from each sample was reverse-transcribed into cDNA with
hexamer random primers and a SuperTrancriptase II kit (GIBCO/BRL,
Gaithersburg, Md.). One microliter each of the resultant cDNA was
amplified by PCR.TM. for 28 cycles at 94.degree. C. for 30 sec,
58.degree. C. for 1 min, 72.degree. C. for 1 min each, using either
the UC41 specific or .beta..sub.2 microglobin primers identified
below.
TABLE-US-00006 UC41 forward primer (position 169 of SEQ ID NO: 1)
5'-CAAATAGCAAGCCCTGCCCACTCA-3'. (SEQ ID NO: 10) UC41 reverse primer
(position 503 of SEQ ID NO: 1) 5'-CTCCTAATCTCACCCCTTCCGCTAT-3' (SEQ
ID NO: 11) .beta..sub.2 microglobin forward primer
5'-CACGTCATCCAGCAGAGAATGGAAAGTC-3' (SEQ ID NO: 12) .beta..sub.2
microglobin reverse primer 5'-GAGAATAGGTTGTAGTTGTAGAACCAGT-3' (SEQ
ID NO: 13)
5.1.7 Chromosomal Locallization of the UC41 Gene by FISH
[0353] The chromosomal location of UC41 was identified by FISH
using the procedure of Heng et al. (1992). Lymphocytes isolated
from human blood were cultured in minimum essential medium (MEM)
supplemented with 10% fetal calf serum and phytohemagglutinin (PHA)
at 37.degree. C. for 68-72 h. The lymphocyte cultures were then
treated with bromodeoxyuridine (BrdU, 0.18 mg/ml, Sigma Chemical
Co., St. Louis, Mo.) to synchronize the cell population. The cells
were washed with serum-free medium to release the block in cell
cycle and recultured at 37.degree. C. for 6 h in MEM with thymidine
(2.5 .mu.g/ml, Sigma).
[0354] Cells were harvested and slides were made by standard
procedures, including hypotonic treatment and fixation followed by
air-drying. Slides were baked at 55.degree. C. for 1 h. After
treatment with RNase, the slides were denatured in 70% formamide in
2.times.SSC for 2 min at 70.degree. C., followed by ethanol
dehydration. A 0.9 kb UC41 cDNA probe was biotinylated with dATP
for 1 h at 15.degree. C. using a BioNick labeling kit (GIBCO/BRL,
Gaithersburg, Md.). The probe was prepared by Hpa I and Nco I
digestion and corresponds to bases 870 to 1840 of SEQ ID NO:1.
[0355] The labeled probe was denatured at 75.degree. C. for 5 min
in a hybridization solution containing 50% formamide, 10% dextran
sulfate and human Cot I DNA. The denatured probe was added to the
slides and allowed to hybridize with chromosomal DNA overnight.
Slides were then washed, detected and amplified by standard
procedures. FISH signals and DAPI banding pattern were recorded
separately by sequential photography of the same microscope field.
The assignment of the FISH mapping data with chromosomal bands was
achieved by superimposing FISH signals on the DAPI banded
chromosomes.
[0356] A Stanford G3 panel (Research Genetics, Huntsville, Ala.)
was used to determine the linkage of UC41 with known microsatellite
markers. The G3 panel contained 83 hamster-human radiation-reduced
cell hybrids with an estimated resolution of approximately 500 kb.
PCR.TM. was performed with the following UC41 specific primers.
TABLE-US-00007 Forward primer (at position 1191 of SEQ ID NO: 1)
5'-CTGCCAACTTCCTCTCTATGC-3' (SEQ ID NO: 14) Reverse primer (at
position 1707 of SEQ ID NO: 1) 5'-GTCCTCATCTATCAGCCTCTTTG-3' (SEQ
ID NO: 15)
[0357] Thermal cycling was performed for 35 cycles at 94.degree. C.
for 30 sec, 56.degree. C. for 1 min, 72.degree. C. for 1 min,
followed by extension at 72.degree. C. for 10 min. The PCR.TM.
product was resolved in 1% agarose gel and the G3 panel of UC41 was
ordered using the RII-MAP program on the RH server (University of
Washington).
5.2 Example 1
Identification of UC41 as an Overexpressed Gene in Prostate Cancer
by Differential Display
[0358] A modified agarose gel-based mRNA differential display
method was used to identify genes differentially expressed in
prostate cancer tissue. UC41 was identified as a gene that was very
significantly up-regulated in prostate cancer. As shown in FIG. 1
(panel A), a strong band (UC41) was seen in all three prostate
cancer tissues used for the differential display study, while UC41
band intensity was very low all four normal tissues tested.
[0359] The UC41 band was excised, purified and cloned directly into
the pGEM-T plasmid by TA cloning. The UC41 cDNA clone was sequenced
by standard techniques as described above. The resulting 183 by EST
fragment of UC41 (identified as SEQ ID NO:23 in U.S. patent
application Ser. No. 08/692,787 the entire text of which is
incorporated herein by reference) corresponds to bases 1323-1500 of
SEQ ID NO:1.
[0360] Using this sequence data, forward and reverse primers for
UC41 (SEQ ID NO:10 and SEQ ID NO:11) were designed and used in a
relative quantitative RT-PCR.TM. study, as described above, to
confirm the up-regulation of this gene in prostate cancer. As shown
in FIG. 1, panel B, the expression of UC41 was confirmed to be
significantly up-regulated in all six prostate cancer tissues
tested, while expression was very low in normal prostate tissues.
Separate studies confirmed that expression of UC41 is .
significantly elevated in prostate cancer compared with BPH. Thus,
overexpression of UC41 appears to be diagnostic for malignant
prostate tumors.
[0361] The expression of UC41 was compared in pair-matched samples
of normal prostate and prostate cancer tissues taken from the same
subjects, as determined by semi-quantitative RT-PCR.TM.. All eight
samples show increased expression of UC41 in prostate cancer
tissues, with different degrees of overexpression observed in
individual subjects (FIG. 2). UC41 expression was examined in three
prostate cancer cell lines, LNCaP, PC-3, DU145 and two CaP
xenografts, Lu 23 and Lu 35. Lu 23 expressed a high level of UC41,
while Lu 35 showed relatively low expression and the three prostate
cancer cell lines were almost negative for expression of UC41 (FIG.
2).
5.3 Example 2
Isolation of a Full Length cDNA Clone of UC41
[0362] The 183 by UC41 fragment identified in Example 1 was used as
a probe to screen a normal prostate tissue cDNA library. This
screen yielded six independent positive clones on duplicated
filters (containing over 3.times.10.sup.6 recombinant clones).
These six isolated clones were subjected to further screening. Two
of these clones gave strong positive signals on repetitive
hybridization. The inserts of these two clones were amplified with
flanking M13 primers and subcloned into the Top vector (Invitrogen,
Carlsbad, Calif.). DNA sequences were determined from the both ends
of the inserts and found to overlap with each other and with the
UC41 fragment identified by differential display.
[0363] The full-length cDNA for UC41 gene was cloned by a
combination of cDNA library screening and RACE (Rapid Cloning of
cDNA Ends) methods (Frohman, 1990 incorporated by reference). The
complete, 1934 by cDNA sequence identified is shown in SEQ ID NO:1
(FIG. 9).
[0364] Bioinformatic-generated translation analysis of the cDNA
indicated an ORF (open reading frame) of 125 amino acids (SEQ ID
NO:2), with an ATG initiation codon at bp 1320 of SEQ ID NO:1 and a
termination codon at by 1695 of SEQ ID NO:1. The 183 by EST
identified in Example 1 starts immediately after the initiation
codon and ends approximately halfway through the ORF.
[0365] The translated protein has a calculated molecular mass of
13.7 kDa and an isoelectric pH of 10.48. Two potential
myristylation sites are located at residues 59-64 (GQVSTR) and
80-85 (GISNSG) of SEQ ID NO:2. Three putative protein kinase C
phosphorylation sites are located at residues 27-29 (TLR), 62-64
(STR) and 84-86 (SGR) of SEQ ID NO:2. A hydropathy plot of SEQ ID
NO:2 yielded a lightly hydrophilic N-terminal region that may serve
as a signal peptide (residues 2-24 of SEQ ID NO:2) and a strongly
hydrophobic C-terminal area which has the characteristic of a
transmembrane domain (residues 99-121 of SEQ ID NO:2). Extensive
computer homology searches conducted in the GenBank, ECM, and Swiss
sequence banks did not find any known nucleotide/amino acid
sequence having homology with the UC41 cDNA or deduced amino acid
sequences.
5.4 Example 3
In Situ Hybridization
[0366] Studies were performed to investigate the expression of UC41
and to localize UC41 mRNA in formalin-fixed paraffin-embedded
prostate cancer and prostate cancer metastatic to lymph node and
bone, compared with normal prostate tissue. FIG. 3 shows a
comparison of UC41 staining in prostate cancer tissue compared to
normal prostate. A significant level of UC41 was localized in
prostate adenocarcinoma tissue, while only minimal levels of UC41
mRNA were detected in adjacent samples of normal and benign
prostatic epithelial cells (FIG. 3). UC41 expression in normal
prostate tissue appears to be locallized to luminal epithelial
cells, preferentially in luminal basal cells. (FIG. 3) Prostate
cancer cells metastatic to the lymph nodes showed very heavy
staining with a UC41 mRNA probe (FIG. 4), while normal lymph nodes
exhibited no detectable staining (FIG. 4). Similar results were
observed with metastatic prostate cancer cells in bone marrow,
compared to normal bone marrow (FIG. 5).
5.5 Example 4
Specific Expression of UC41 in Prostate Tissue
[0367] Tissue-specific expression of UC41 was examined by Northern
blot analysis with with mRNA from spleen, thymus, prostate, testis,
ovary, small intestine, colon and peripheral blood leukocytes (FIG.
6). A strong hybridization signal was observed specifically in
prostate tissue (FIG. 6). The results in prostate are consistent
with the existence of two different splicing variants of the UC41
gene, with a major band migrating at approximately 1.5 kb and a
minor band at approximately 2.1 kb (FIG. 6). These results
indicated that expression of the UC41 gene is specific for prostate
tissue.
[0368] The prostate specific expression of UC41 was confirmed by
dot blot hybridization, using an expanded panel of RNA samples from
fifty different adult human or fetal human tissues (FIG. 7). A
strong hybridization signal was again observed only in prostate RNA
(FIG. 7), with a very minor amount of hybridization detected in
bladder RNA (FIG. 7).
5.6 Example 5
Chromosomal Localization of UC41
[0369] The chromosomal locus of UC41 was identified by comparison
of UC41 FISH hybridization (FIG. 8, panel A) and DAPI staining
(FIG. 8, panel B). Based on this comparison, UC41 was assigned to
the long arm of chromosome 3. The detailed position of UC41 was
further determined based on a summary of data from 10 different
FISH/DAPI comparisons (FIG. 8, insert on far right), which mapped
the gene to 3q22-23. Both FISH mapping (FIG. 8) and human genomic
DNA Southern hybridization indicate that the UC41 gene exists as a
single copy.
[0370] UC41 was typed on the G3 radiation hybrid panel developed at
Stanford and analyzed with the RH-MAP program (University of
Washington), as described above. The two-point analyses ordered
UC41 with the microsatellite marker D3S1541, with a distance of 11
cR (about 250 kb).
[0371] Those experienced in the art will recognize that the gene
and gene products (RNAs and proteins) for UC41 are included within
the scope of the invention herein described. Those experienced in
the art will also recognize that the diagnosis and prognosis of
prostatic cancer by detection of the nucleic acid or protein
products of this gene are included within the scope of the present
invention.
[0372] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the composition, methods and in the
steps or in the sequence of steps of the method described herein
without departing from the concept, spirit and scope of the
invention.
[0373] More specifically, it will be apparent that certain agents
which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
1811934DNAHomo sapiensprostate specific UC41 cDNA, UC Band #41
1ataaatactc acactttcaa agtaaacgct cttcttttct atgtgctcca tatcgctttg
60ccctctttat ttgagcaacg taccctgtag tgtgtaaata agagatgaca gctcctataa
120tacgtgtggg tgaggaatgg gttcgataaa acagaagcat gcacaattca
aatagcaagc 180cctgtcccac tcagtttatg cacaaataac ttgcagattc
tctgattttc tgccagcaac 240tgcctcctct tcccctcccc actgccttcc
agaagtctct cagaatcata tccggcacgg 300tgtatagaga taggagtaat
agggataaga cttgtttttt ctgaatttta attattgtca 360tttagcattt
gctcagtgtt ttgtgatgaa aatcgttggg ttttacttat ttttactatg
420ggcaaattga gatgccttta atcataaagg cagccccaac ccaaggtacc
cttgcataat 480agcggaaggg gtgagattag gagtaagcct ttagagcacc
ggtgcaggac tcaggatatg 540gatttgtggc ttgttcttat atgccgtatt
tgtcttatgg gtaaatgctg tatatcgttt 600tgatttttcc tatcgtggaa
cacttttcag ttatatgtct ggtggaatca agtgtttcat 660gttactttta
aatgtacata ggtggcttaa ttttttttat ttacaattca gcacttcaga
720tgtgaagaga tatggctttt tctttttttt tttttttaca acagaatata
acttgcttct 780gagccctcat ttctgattgg tggtgatgga aaccttagct
tgcctgttgg aggtaactgt 840cacttccaat tcactgcagt cttgtccagg
ttaacacaat catgttggtg taaatagtca 900actgaggatt taaatagtca
attcaaaatg caaacttctt atctaagtga ttctcccctc 960tcaaggaatt
tccccttcct gtcctctttc cagtatgtta tctgggttca gagtgggtac
1020ttgtataact acacaacaca gaggaagaga gtccagttct catgcagtca
tctggatccc 1080tgctggccac ctctgaagta gatgtctata tccactgtgc
taagtggtct tggaaatgga 1140tccagttggt cattagtgga gctctagaac
ctgctgatga ttcatggtcc ctgccaactt 1200cctctctatg ccctcctcat
tcaaagtgaa ggtttctttc agcttattgg ccctttggcc 1260cattcccatt
ctttgtagaa acttctgcag accacattca gccactggaa aaccaaaata
1320tgctttctca tctctctcct ctccttcacg atgccattct gccatttctg
ttttgtggta 1380gacaggttgg cccaggcact ctaaggccca ggctggcaca
ggttggccca ggcacttcaa 1440gcctaagtcc atttacagtt tctattccat
ctattcctaa agaaggaggc agagggcagg 1500tctcaactcg tgtttcagca
ctgctgtttt acacacacac acacaccctc tctccaggca 1560tctctaactc
aggcagaact tttatttcct cccaatatgg cagaaaccac cttcaatttc
1620atatcaggat atttccttct ttattgttta tagtttactt tgcaactgag
gtgatctcca 1680aagaggctga tagatgagga ctaattgcta actgcactcc
cagctgcaac aggcatgaag 1740gaagatatgg gtggtccatc tccatgttca
ttacagtgat aggtcagctg tctccaacct 1800ttttggcatc agggactagt
tttgtggaag acaatttttc catggacatg gggtggggaa 1860ggaaggagat
gattttgggt tgaaactgtt ccacttcaga ctcagatcat caggcgttag
1920attatcataa ggac 19342125PRTHomo sapiensprostate specific UC41,
UC Band #41 2Met Leu Ser His Leu Ser Pro Leu Leu His Asp Ala Ile
Leu Pro Phe1 5 10 15Leu Phe Cys Gly Arg Gln Val Gly Pro Gly Thr Leu
Arg Pro Arg Leu 20 25 30Ala Gln Val Gly Pro Gly Thr Ser Ser Leu Ser
Pro Phe Thr Val Ser 35 40 45Ile Pro Ser Ile Pro Lys Glu Gly Gly Arg
Gly Gln Val Ser Thr Arg 50 55 60Val Ser Ala Leu Leu Phe Tyr Thr His
Thr His Thr Leu Ser Pro Gly65 70 75 80Ile Ser Asn Ser Gly Arg Thr
Phe Ile Ser Ser Gln Tyr Gly Arg Asn 85 90 95His Leu Gln Phe His Ile
Arg Ile Phe Pro Ser Leu Leu Phe Ile Val 100 105 110Tyr Phe Ala Thr
Glu Val Ile Ser Lys Glu Ala Asp Arg 115 120 12531322DNAArtificial
Sequencesynthetic portion of UC41 cDNA 5' to UC41 EST sequence,
UC41 cDNA residues 1-1322 3ataaatactc acactttcaa agtaaacgct
cttcttttct atgtgctcca tatcgctttg 60ccctctttat ttgagcaacg taccctgtag
tgtgtaaata agagatgaca gctcctataa 120tacgtgtggg tgaggaatgg
gttcgataaa acagaagcat gcacaattca aatagcaagc 180cctgtcccac
tcagtttatg cacaaataac ttgcagattc tctgattttc tgccagcaac
240tgcctcctct tcccctcccc actgccttcc agaagtctct cagaatcata
tccggcacgg 300tgtatagaga taggagtaat agggataaga cttgtttttt
ctgaatttta attattgtca 360tttagcattt gctcagtgtt ttgtgatgaa
aatcgttggg ttttacttat ttttactatg 420ggcaaattga gatgccttta
atcataaagg cagccccaac ccaaggtacc cttgcataat 480agcggaaggg
gtgagattag gagtaagcct ttagagcacc ggtgcaggac tcaggatatg
540gatttgtggc ttgttcttat atgccgtatt tgtcttatgg gtaaatgctg
tatatcgttt 600tgatttttcc tatcgtggaa cacttttcag ttatatgtct
ggtggaatca agtgtttcat 660gttactttta aatgtacata ggtggcttaa
ttttttttat ttacaattca gcacttcaga 720tgtgaagaga tatggctttt
tctttttttt tttttttaca acagaatata acttgcttct 780gagccctcat
ttctgattgg tggtgatgga aaccttagct tgcctgttgg aggtaactgt
840cacttccaat tcactgcagt cttgtccagg ttaacacaat catgttggtg
taaatagtca 900actgaggatt taaatagtca attcaaaatg caaacttctt
atctaagtga ttctcccctc 960tcaaggaatt tccccttcct gtcctctttc
cagtatgtta tctgggttca gagtgggtac 1020ttgtataact acacaacaca
gaggaagaga gtccagttct catgcagtca tctggatccc 1080tgctggccac
ctctgaagta gatgtctata tccactgtgc taagtggtct tggaaatgga
1140tccagttggt cattagtgga gctctagaac ctgctgatga ttcatggtcc
ctgccaactt 1200cctctctatg ccctcctcat tcaaagtgaa ggtttctttc
agcttattgg ccctttggcc 1260cattcccatt ctttgtagaa acttctgcag
accacattca gccactggaa aaccaaaata 1320tg 13224434DNAArtificial
Sequencesynthetic portion of UC41 cDNA 3' to UC41 EST sequence,
UC41 cDNA residues 1501-1934 4tctcaactcg tgtttcagca ctgctgtttt
acacacacac acacaccctc tctccaggca 60tctctaactc aggcagaact tttatttcct
cccaatatgg cagaaaccac cttcaatttc 120atatcaggat atttccttct
ttattgttta tagtttactt tgcaactgag gtgatctcca 180aagaggctga
tagatgagga ctaattgcta actgcactcc cagctgcaac aggcatgaag
240gaagatatgg gtggtccatc tccatgttca ttacagtgat aggtcagctg
tctccaacct 300ttttggcatc agggactagt tttgtggaag acaatttttc
catggacatg gggtggggaa 360ggaaggagat gattttgggt tgaaactgtt
ccacttcaga ctcagatcat caggcgttag 420attatcataa ggac
434565PRTArtificial Sequencesynthetic positions 61-125 of UC41,
antibody binding peptide 5Val Ser Thr Arg Val Ser Ala Leu Leu Phe
Tyr Thr His Thr His Thr1 5 10 15Leu Ser Pro Gly Ile Ser Asn Ser Gly
Arg Thr Phe Ile Ser Ser Gln 20 25 30Tyr Gly Arg Asn His Leu Gln Phe
His Ile Arg Ile Phe Pro Ser Leu 35 40 45Leu Phe Ile Val Tyr Phe Ala
Thr Glu Val Ile Ser Lys Glu Ala Asp 50 55 60Arg65610DNAArtificial
Sequencesynthetic arbitrary 10-mer PCR primer for identifying UC41
6ctagtgatta 10710DNAArtificial Sequencesynthetic arbitrary 10-mer
PCR primer for identifying UC41 7gtccttatga 10829DNAArtificial
Sequencesynthetic amplification PCR forward primer for generating
UC41 probe for in situ hybridization 8aaaacgatat catgctttct
catctctcc 29932DNAArtificial Sequencesynthetic amplification PCR
reverse primer for generating UC41 probe for in situ hybridization
9caattgcggc cgctctatca gcctctttgg ag 321024DNAArtificial
Sequencesynthetic UC41 specific semi-quantitative RT-PCR
amplification forward primer 10caaatagcaa gccctgccca ctca
241125DNAArtificial Sequencesynthetic UC41 specific
semi-quantitative RT-PCR amplification reverse primer 11ctcctaatct
caccccttcc gctat 251228DNAArtificial Sequencesynthetic beta2
microglobulin specific PCR amplification forward primer
12cacgtcatcc agcagagaat ggaaagtc 281328DNAArtificial
Sequencesynthetic beta2 microglobulin specific PCR amplification
reverse primer 13gagaataggt tgtagttgta gaaccagt 281421DNAArtificial
Sequencesynthetic UC41 specific PCR forward primer 14ctgccaactt
cctctctatg c 211523DNAArtificial Sequencesynthetic UC41 specific
PCR reverse primer 15gtcctcatct atcagcctct ttg 23166PRTArtificial
Sequencesynthetic 6XHis additional amino acids 16His His His His
His His1 5176PRTArtificial Sequencesynthetic UC41 potential
myristoylation site, residues 59-64 17Gly Gln Val Ser Thr Arg1
5186PRTArtificial Sequencesynthetic UC41 potential myristoylation
site, residues 80-85 18Gly Ile Ser Asn Ser Gly1 5
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