Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2001-05-08
2002-11-05
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S198200, C435S007100, C435S007230, C530S388800
Reexamination Certificate
active
06475389
ABSTRACT:
BACKGROUND OF THE INVENTION
It is becoming increasingly evident that the monitoring of prostate-specific membrane antigens (PSMA) is desirable for the detection and management of prostate cancer.
Prostate cancer is the most common cancer in males in the United States. The efficiency of early detection of prostate cancer has increased with a serum test for the prostate-specific antigen (PSA). However, PSA is neither tissue-specific nor disease-specific. Many other conditions of the prostate gland, some of them being benign, can also result in abnormal elevation of PSA level in the serum (Polascik, et al., Prostate-specific antigen: A decade of discovery—what we have learned and where we are going, J. Urol, vol 162, pp 293-306, 1999).
Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein with both intra and extracellular domains, and is highly specific for the prostate tissue (Israeli, et al., Molecular cloning of a complimentary DNA encoding of prostate-specific membrane antigen, Cancer Res, vol 53, pp 227-230, 1993). PSMA is expressed in benign and malignant prostatic epithelium and can be detected immunohistochemically (Horoszewicz, et al. Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients, Anticancer Res, vol 7, pp 927-936, 1987; Silver, et al., Prostate-specific membrane antigen expression in normal and malignant human tissues, Clin Cancer Res, vol 3, pp 81-85, 1997; Wright, et al., Expression of prostate-specific membrane antigen (PSMA) in normal, benign, and malignant prostate tissues, Urol Oncol, vol 1, pp 18-28, 1995; Bostwick, et al., Prostate-specific membrane antigen expression in prostatic intraepithelial neoplasia and adenocarcinoma: A study of 184 cases, Cancer, vol 82, pp 2256-2261, 1998; and Sweat, et al., Prostate-specific membrane antigen expression is greatest in prostate adenocarcinoma and lymph node metastasis, Urology, vol 52, pp 637-640, 1998). PSMA serum levels have been proposed to be of prognostic significance in prostate cancer patients with advanced disease, see e.g. Grasso, et al., Combined nested RT-PCR assay for prostate-specific antigen and prostate-specific membrane antigen in prostate cancer patients: correlation with pathological stage, Cancer Research vol 58, pp 1456-1459, 1998.
Attempts to reliably detect PSMA in serum have not been successful and currently, there is no routine methodology available to monitor PSMA levels in the sera of prostate cancer patients.
PSMA expression in human prostate tissue was documented for the first time by immunohistochemical staining using 7E11.C5-antibody (Horoszewicz, et al, supra). The 7E11.C5 monoclonal antibody that was raised against human prostate cancer cells (LNCaP) is known to bind to intracellular epitope of PSMA near the amino terminus. An isotope conjugated form of 7E11.C5, designated CYT-356, has been utilized for the identification of local and distant prostate cancer metastasis and recurrence. 7E11.C5 antibody is deposited with the American Type Culture Collection at Rockville, Md., USA having ATCC access number HB10494. The PSMA immunoreactivity was found to be greater in high-grade prostate tumors as compared to benign prostatic hyperplasia (BPH) and normal cells (Horoszewicz, et al. supra; Silver, et al., supra; Wright, et al, supra; Bostwick, et al, supra; and Sweat, et al., supra). However, among 33 cell lines tested by Horoszewicz, et al., supra, PSMA immunoreactivity was detected only in lymph node carcinoma of prostate (LNCaP) cells. In 1997, the US Food and Drug Administration approved the use of radioimmuno-conjugate form of 7E11.C5 (CYT 356) for the detection of localized prostate cancer.
Attempts made to measure PSMA in the serum of patients having prostate cancer by Western-blot analysis have not been conclusive, (Troyer, et al., Detection and characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids, Int. J. Cancer, vol 62, pp 552-558, 1995; Rochon, et al., Western-blot assay for prostate-specific membrane antigen in serum of prostate cancer patients, Prostate, vol 25, pp 219-223, 1994; and Beckett, et al., Prostate-specific membrane antigen levels in sera from healthy men and patients with benign prostate hyperplasia or prostate cancer, Clin Cancer Res, vol 5, pp 4034-4040, 1999). It has been reported that Western-blot analysis can show an increase in serum PSMA levels in patients with later stage C
1
, D
1
and D
2
prostate cancer as compared to normal (Rochon, et al. supra). On the other hand, Troyer, et al. supra reported that they were unable to detect PSMA in the serum of prostate cancer patients. Beckett, et al. supra reported that they were able to detect PSMA in the serum, but they were unable to distinguish between early and late stage of the disease.
Prior to the present invention, attempts to use the simpler ELISA to detect prostate cancer have not been successful. In “competitive-inhibition” ELISA, PSMA was previously detected in 46% of sera from prostate cancer patients and none in BPH or normal individuals (Horoszewicz, et al. supra). This is clearly insufficient reliability for a determinative test.
In our prior studies, it has been shown that PSA has strong affinity for Thiophilic gels (1S, 2S and 3S) as described in co-pending U.S. patent application Ser. No. 09/624,692, filed Jul. 24, 2000. Recently, we have made an observation that PSMA also has a strong affinity for T-gels. Briefly, a T-gel slurry is packed in a column (0.5×5 cm) and equilibrated with 25 mM Hepes buffer, containing 1M sodium sulfate, pH 7.0. A source of PSMA, e.g. solubilized cell extract from LNCaP cells (5×10
7
cells/ml) or human serum (400 &mgr;l) is reconstituted with column equilibrating buffer and applied to the column. The column is then washed with approximately 10 void volumes of column equilibrating buffer and the bound proteins are eluted with 25 mM Hepes buffer, pH 7.0. The presence of PSMA in various column fractions is then detected by SDS-PAGE and Western-blot analysis using a monoclonal anti PSMA antibody, such as 7E11.C5.
As an example, electrophoresis of fractions was carried out under non-reducing conditions in 4-15% gradient polyacrylamide gel. Samples were loaded in equal volumes containing comparable protein levels and ran for 40 min at 200 volts. After electrophoresis, the proteins in the gel were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. The transfer buffer contained 25 mM Tris, 192 mM glycine and 20% methanol at pH 8.3. The transfer was performed at 100 volts for one hour. After the transfer, the membrane was blocked for one hour in a broad function protein based blocker with gentle agitation. Such blockers include albumin, casein or dry milk solution. Such a blocker is commercially available as NAP-Sure blocker™ (Geno-Technology, Inc., St. Louis, Mo.). Immunostaining was carried out using 7E11.C5 antibody or monoclonal antibody to &agr;
1
-antichymotrypsin at 5 &mgr;g/ml concentration diluted in NAP-Sure blocker™ (Geno-Technology, Inc., St. Louis, Mo.), for 1 hr. Secondary antibody, horse radish peroxidase conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories, Inc., West Grove, Pa.) at 1:5,000 dilution in 1% albumin/PBS/0.1% Tween 20, was incubated with the membrane for 45 min. The proteins were detected with enhanced luminol (ECL) reagent for horse radish peroxidase. Such a reagent is available under the trademark NEL 102, (NEN, Boston, Ma.). The complexed protein was then exposed to X-ray film to visualize the positive bands.
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Chadha Kailash C.
Kawinski Elzbieta
Dunn Michael L.
Health Research , Inc.
Therkorn Ernest G.
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