US20100041126A1 - Methods for diagnosing and treating prostate cancer - Google Patents

Methods for diagnosing and treating prostate cancer Download PDF

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US20100041126A1
US20100041126A1 US12/541,824 US54182409A US2010041126A1 US 20100041126 A1 US20100041126 A1 US 20100041126A1 US 54182409 A US54182409 A US 54182409A US 2010041126 A1 US2010041126 A1 US 2010041126A1
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gpc3
prostate cancer
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cancer
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Bruce Wang
Matthias Wabl
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate

Definitions

  • This disclosure relates generally to methods for diagnosing and treating prostate cancer.
  • Prostate cancer is the most common malignant cancer in North American men. It is estimated that approximately 200,000 new cases and 31,500 prostate cancer-related deaths will occur in the United States annually. Prostate cancer is now the second leading cause of cancer death in men, exceeded only by lung cancer. It accounts for 29% of all male cancers and 11% of male cancer-related deaths.
  • PSA prostate-specific antigen
  • serum PSA prostate-specific antigen
  • PSA is produced by both normal and cancerous glands.
  • the serum levels can be elevated with both localized and advanced or disseminated disease.
  • PSA levels are generally proportional to the volume of the cancer. Because there is a significant overlap between PSA levels found in cancer and benign prostatic hyperplasia, it is important to obtain sequential levels in low or borderline elevated values.
  • fPSA free PSA
  • Prostate cancer like other forms of cancer, is caused by genetic aberrations, i.e., mutations.
  • mutations In the mutant cells the normal balance between the factors that promote and restrain growth is disrupted, and as a result, these mutant cells proliferate continuously—the hallmark of tumor cells.
  • Mutations can arise spontaneously or by external factors such as chemical mutagens, radiation, or viral integration, which inserts extra-genomic DNA that may or may not contain an oncogene.
  • a cellular gene can be modified by point mutation, insertion and frame shift (including truncation), (functional) deletion (including silencing), or translocation, which sometimes can result in gene fusion.
  • protooncogenes can become oncogenes, which promote proliferation, and tumor suppressor genes can become inactivated, also inducing tumor growth.
  • Any combination of the above-mentioned changes in DNA can contribute to tumor formation. The consequences of these changes may or may not be held in check by the immune system (immune surveillance).
  • GPC3 a heparin sulfate proteoglycan anchored to the cell membrane via glycosylphosphatidylinositol (1).
  • the protein has a molecular weight of 65.6 kDa and the polypeptide chain has 580 amino acid residues.
  • the heparin sulfate chain of the proteoglycans interacts with heparin-binding growth factors and thus serves as a co-receptor in cell signaling (2), although GPC3 might bind also in a different way (3).
  • GPC3 modulates BMP and EGF-mediated effects during renal branching morphogenesis (4).
  • GPC3 is not expressed in systemic organs (6). Because its expression is decreased in lung adenocarcinoma (7, 8), human gastric cancer (9), ovarian cancer cell lines (10, 11), mesotheliomas (10,11), and breast tumors (12), GPC3 may function as a tumor suppressor. But because its mRNA and protein expression is increased in hepatocellular carcinomas (13-15), colorectal malignancies (16), and embryonal tumors (17) as compared to normal tissue, GPC3 is also considered an onco(fetal) protein.
  • GPC3 is a promising diagnostic marker (6). It is also known that in these cancers, GPC3 modulates FGF2 and BMP-7 signaling (18), and promotes growth by stimulating canonical Wnt signaling (3).
  • GPC3 mRNA and protein are expressed (19).
  • GPC3 protein is found in sera from 40% of melanoma patients but not in sera from subjects with large congenital melanocytic nevus and from healthy donors. GPC3 expression disappeared in sera from one third of the patients after surgical removal of the melanoma (19).
  • GPC3 levels increase significantly in prostate cancer cells, and (ii) this increase can be measured the ductile fluid and blood-fluid sample of patients.
  • the present disclosure includes, in one aspect, a method of screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject.
  • the method includes assaying a body-fluid sample from the subject for the level of GPC3 protein in the sample, and determining from the assayed level, whether the subject has an elevated GPC3 level associated with prostate cancer, as an indicator of prostate ca ncer.
  • the assayed level of GPC3 indicative or prostate cancer is preferably in a range between 1 and 10 ng/ml.
  • the assayed level of GPC3 indicative or prostate cancer is preferably in a range between 1 and 10 ng/ml.
  • the assaying step may involve applying the body fluid to a solid-phase immunoassay device, the level of GPC3 in the sample may be indicated qualitatively by a calorimetric or fluorometric indicator, and the determining may include comparing the indicator with a known standard.
  • the level of assayed GPC3 is indicative of the stage of the cancer in the subject.
  • a diagnostic device for use in for screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject.
  • the device includes a (i) structure for receiving a body-fluid sample from the subject, (ii) an anti-GPC-3 binding antibody associated with the structure and capable of reacting with body-fluid received in the structure, to produce, in combination with other reagents associated with the structure, a detectable reaction indicative of the level of GPC3 in the sample, and (iii) a known-standard indicator against which the level of detectable reaction produced can be assessed as an elevated level associated with prostate cancer.
  • the known-standard indicator includes two or more indicators that represent different levels of GPC3 associated with different stages of prostate cancer.
  • the structure in the device includes a porous pad or strip having the anti-GPC3 binding protein embedded therein, for reaction with the fluid sample when the sample is applied to the pad, the detectable reaction is indicated by a colorimetric or fluorimetric indicator, and the known standard indicator includes an indicia that represents a level of GPC3 corresponding to that associated with prostate cancer.
  • the device includes an instrument for generating a signal related to the level of GPC3 produced, a microprocessor for comparing the signal with a know-standard signal value associated with prostate cancer, and a display for displaying an output of the microprocessor.
  • a histological method for examining human prostate tissue for the presence and extent of prostate cancer.
  • the method includes staining the prostate tissue with an anti-GPC-3 binding protein labeled with a detectable marker, to attach the marker to the surface of tissue cells, and determining, based on an elevated distribution and extent of detectable marker with respect to the distribution and extent of marker in normal prostate cells, the presence and extent of prostate cancer in the tissue.
  • the distribution and extent of detectable marker in the tissue may be used to determine the stage of cancer in the tissue, as a method of staging the patient for anti-cancer treatment.
  • method is practiced by (a) extracting genomic DNA from human patients having prostate cancer, (b) for each extracted DNA, comparing the sequence of the DNA in a selected region 0-200 kbases upstream of the GPC3 gene on chromosome interval Xq26, with a homologous region of DNA from one or more wildtype individuals, and (c), by the comparing, identifying one or more mutations in the region associated with an increased risk prostate cancer.
  • this method further includes (d) for each mutation identified in step (c) producing a gene fragment capable binding selectively to genomic DNA fragments carrying that mutation, but not to corresponding wildtype DNA fragments, and (e) attaching the gene fragments constructed in step (d) at known positions on a chip-chip substrate.
  • a method of reducing tumor burden in a subject with prostate cancer by exposing subject antigen-presenting cells to human GPC3 polypeptide or antigenic fragment(s) thereof, and by the exposing, stimulating and causing clonal expansion of memory CD4 helper cells, CD8 Tc cytotoxic lymphocytes and CD8 non-cytotoxic T-suppressor lymphocytes thereby causing expansion of GPC3 antigen-specific memory CD4 helper cells, GPC3 antigen-specific CD8 Tc cytotoxic lymphocytes and GPC3 antigen-specific CD8 non-cytotoxic T-suppressor lymphocytes in the subject.
  • the treatment method may be practiced by exposing the subject's antigen-presenting cells ex vivo to the human GPC3 polypeptide or antigenic fragment(s) thereof, under conditions effective to activate the cells, and injecting the activated cells into the subject.
  • the method may be practiced by injecting the subject with the human GPC3 polypeptide or fragments thereof, carried in a suitable adjuvant.
  • a patient's body-fluid sample is assayed for the level of GPC3 protein in the sample, to determine from the assayed level, whether the subject has an elevated GPC3 level associated with prostate cancer. If the patient has such an elevated GPC3 level, the patient is treated by administering a therapeutically effective amount of a GPC3 binding agent effective, when bound to GPC3 on the surface of prostate cancer cells, to inhibit growth or viability of the cells.
  • the GPC3 binding agent administered may be human or humanized anti-GPC3 antibody, effective, when bound to GPC3 on the surface of prostate cancer cells, to promote antibody-dependent cell cytotoxicity.
  • FIG. 1 shows the genomic organization of the mouse Gpc3 locus, as view by a customized screen print of the UCSC genome web site browser (March 2005 version of the mm6 gene assembly). Top, base position on chromosome X. Vertical handle bars in red, green and blue represent the retroviral insertions into the locus in 82 independent tumors.
  • FIGS. 2A and 2B shows immunohistochemical stains of human prostate tumor ( 2 A) and matched normal tissue from the same patient ( 2 B).
  • the polyclonal rabbit antibody serum used reacts with 2 peptide epitopes: DLFIDKKVLKVAHVEHEET (SEQ ID NO:1; amino acid residues 365 to 383, encoded by exon 4) and LAYDLDVDDAPGNSQQ (SEQ ID NO: 2; amino acid residues 526 to 541, encoded by exon 8);
  • FIGS. 3A and 3B show a solid-phase diagnostic device for determining GPC3 levels in a human patient, at initial ( 3 A) and final stages ( 3 B) of the assay;
  • FIG. 4 shows a portion of a gene chip useful for diagnosing genetic predisposition to prostate cancer, constructed in accordance with the present disclosure.
  • “Screening” for prostate cancer means testing individuals for a level of GPC3 that is indicative of prostate cancer or an elevated risk of prostate cancer.
  • “Staging” treatment of prostate cancer involves determining the stage of prostate cancer in an individual, based on the level of GPC3 detected, and tailoring the treatment to that stage.
  • stages of prostate cancer which are defined by the degree of localization of cancer cells.
  • prostate cancer may be defined as early stage at which the cancer is responsive to a number of hormonal-based therapies, and a later, more serious androgen-independent stage.
  • mice A number of cancer genes (oncogenes and tumor suppressor genes) associated with prostate cancer were identified in a high throughput manner using proviral tagging in mice.
  • proviral tagging mice are infected with a retrovirus that does not contain an oncogene (e.g., murine leukemia virus, MLV or murine mammary tumor virus, MMTV)(20-24).
  • an oncogene e.g., murine leukemia virus, MLV or murine mammary tumor virus, MMTV
  • MLV murine leukemia virus
  • MMTV murine mammary tumor virus
  • the virus During retroviral infection, the virus integrates into the cellular genome and inserts its DNA near or within genes, which leads to various outcomes: (i) The insertion site is too far away from a protooncogene and thus does not activate it. In this case, there will be no selection for that cell. (ii) The provirus inserts within 200 kb of a protooncogene, but not within the gene (type 1). Here, either the viral promoter or the viral enhancer increases the expression level of the protooncogene. (iii) The provirus inserts within a gene, destroying or altering its function (type 2).
  • a tumor suppressor may be scored if a retrovirus lands within a gene and truncates or destroys it. In these cases, the suppressor may be haplo-insufficient, or alternatively, the mutation on the other allele is provided spontaneously by the mouse.
  • the integration event may also lead to more complex consequences, such as a dominant negative effect of the truncated gene product or the transcription of anti-sense or microRNA.
  • GPC3 protein was assayed in situ using an anti-GPC3 antibody, prepared as described above. Briefly, tissue samples from the ductile epithelium in normal and cancerous human tissue were exposed to unlabeled rabbit anti-GPC3 antibody under standard in situ labeling conditions. After washing the cells to remove unbound antibody, the tissue was stained with a horseradish peroxidase polymer-conjugated anti-rabbit secondary antibody (Zymed Laboratories), and viewed by photomicroscopy, with the results shown in FIGS. 2A (cancerous tissue) and 2 B (normal tissue).
  • the disclosure provides a histological method for examining human prostate tissue for the presence and extent of prostate cancer.
  • prostate tissue is stained with a labeled anti-GPC-3 binding protein, e.g., fluorescence-labeled anti-GPC3 antibody (see Section 11 below), to attach the marker to the surface of tissue cells.
  • a labeled anti-GPC-3 binding protein e.g., fluorescence-labeled anti-GPC3 antibody (see Section 11 below
  • elevated levels of GPC3 antigen as an indicator of prostate cancer, increased risk of prostate cancer, or the stage of prostate cancer, can be assayed directly from a body-fluid sample, e.g., seminal fluid or a blood sample.
  • the anti-GPC3 antibody used in the present disclosure can be obtained by any a variety of conventional methods as a monoclonal, polyclonal, or recombinant antibody.
  • One preferred antibody, particularly for diagnostic use, is a mouse monoclonal antibody, prepared according to well-known hybridoma methodology. Briefly, human GPC3 may be first obtained, for example, by expressing the GPC3 (MXR7) gene as disclosed by Lü, H. et al (Gene 188 (1997), 151-156). The purified GPC3 protein is as an immunogen. Alternatively, a partial peptide of GPC3 can be used as a sensitization antigen.
  • the partial peptide can be obtained by chemical synthesis from the amino acid sequence of human GPC3.
  • Two GPC3 sequences that may be employed are: DLFIDKKVLKVAHVEHEET (SEQ ID NO: 1; amino acid residues 365 to 383, encoded by exon 4) and LAYDLDVDDAPGNSQQ (SEQ ID NO: 2; amino acid residues 526 to 541, encoded by exon 8).
  • Anti-GPC3 antibodies useful in diagnostic applications may be labeled with a variety of detectable labels, including detectable reporters, such as enzymes for enzyme-linked immunosorbent assays (ELISA), detectable particles, such as gold particles and reporter-carrying liposomes, colorimetric or fluorescent reporters, radiolabels, and labels such as a biotin label by which secondary detectable labels, such as a reporter-labeled streptavidin label can be attached.
  • detectable reporters such as enzymes for enzyme-linked immunosorbent assays (ELISA)
  • detectable particles such as gold particles and reporter-carrying liposomes
  • colorimetric or fluorescent reporters such as a biotin label by which secondary detectable labels, such as a reporter-labeled streptavidin label can be attached.
  • an unlabeled anti-GPC3 antibody for example, a mouse IgG antibody, is detected by reaction with a labeled antibody, e.g., a labeled anti-mouse IgG antibody.
  • human monoclonal antibodies having binding activity to glypican 3 can be produced by sensitizing in vitro human lymphocytes with GPC3, and causing the sensitized lymphocytes to fuse with the human-derived myeloma cells having a permanent division potential.
  • GPC3 as an antigen can be administered to a transgenic animal having all the repertories of a human antibody gene to obtain anti-glypican 3 antibody-producing cells, and then human antibodies for GPC3 may be obtained from the immortalized anti-GPC3 antibody-producing cells (see International Patent Publication Nos. WO 94/25585, WO 93/12227, WO 92/03918 and WO 94/02602).
  • human or humanized antibodies specific against GPC3 antigen can be prepared by recombinant technique, such as have been reported (see, for example, U.S. Pat. Nos. 6,090,382 and 6,258,562).
  • the present disclosure includes a method of screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject, by assaying a body-fluid sample from the subject for the level of GPC3 protein in the sample, and determining from the assayed level, whether the subject has an elevated GPC3 level associated with prostate cancer, as an indicator of prostate cancer.
  • Preferred body-fluid samples are (a) seminal fluid and (b) blood.
  • seminal fluid is assayed
  • the assayed level of GPC3 indicative or prostate cancer is in a range between 1 and 10 ng/ml.
  • blood is assayed
  • the assayed level of GPC3 indicative or prostate cancer is in a range between 1 and 10 ng/ml.
  • the assay may be carried out by any of a variety of assay methods used for detecting body-fluid antigens, including ELISA techniques, homogeneous assays, for example, involving fluorescence quenching, and a variety of solid-phase sandwich assays in which the GPC3 antigen is captured on by an anti-GPC3 antibody carried on a solid support, and the immobilized antigen-antibody complex is labeled with a second anti-GPC3 antibody, e.g., a second antibody carrying a calorimetric or gold-particle reporter.
  • assay methods used for detecting body-fluid antigens, including ELISA techniques, homogeneous assays, for example, involving fluorescence quenching, and a variety of solid-phase sandwich assays in which the GPC3 antigen is captured on by an anti-GPC3 antibody carried on a solid support, and the immobilized antigen-antibody complex is labeled with a second anti-GPC3 antibody, e
  • FIGS. 3A and 3B illustrate a solid-phase assay strip constructed in accordance with an embodiment of the present disclosure, suitable for carrying out a sandwich immunoassay of the type just mentioned, and shown in initial and final assay states, respectively.
  • the strip indicated generally at 10 , includes a porous support or pad 12 having a sample-application zone 14 in an upstream region of the support and a sample-detection zone 16 in a downstream region.
  • the sample-application zone includes a detectable anti-GPC3 antibody reagent, e.g., anti-GPC3 antibodies labeled with gold particles, and carried in the zone in an unbound, i.e., non-immobilized form.
  • This reagent is indicated by solid circles, such as at 18 .
  • Anti-GPC3 antibodies which may be the same or different from those in the labeled antibody reagent, are immobilized to the solid support within the detection zone, and are indicated by the “Y” shapes, such as at 20 .
  • zone 22 which is located adjacent the detection zone and has one or more colored or shaded regions corresponding to different assay levels of GPC3 in a body-fluid sample.
  • zone 22 includes three regions 22 a , 22 b , and 22 c , corresponding to an assayed level of GPC3 (a) below that associated with prostate cancer, (b) corresponding to a lower threshold level associated with prostate cancer, and (c) a level that is substantially higher, e.g., 2-3 times, higher than the threshold layer in region 22 b , respectively.
  • These three regions provide a known standard indicator against which the level of detectable reaction produced can be assessed as a level associated with prostate cancer.
  • the assay strip and reference zone constitute an assay device for use in screening for prostate cancer in a human subject, or for staging treatment of prostate in a human subject.
  • a known volume of a body-fluid sample to be tested is added to the sample-application zone of the strip, where it diffuses into the zone, allowing the antibody reagent to react with GPC3 antigen in the sample to form an antigen-antibody complex.
  • This complex and unbound antibody reagent then migrate downstream by capillarity toward the detection zone, where the antigen-antibody complex is captured by the immobilize antibody and the unbound reagent is carried to the end of the support, as indicated at 24 .
  • the higher the concentration of antigen in the body fluid the higher the density of captured reagent in the detection zone and the greater the color or intensity in this zone.
  • This color or intensity produced in the detection zone is compared with the standards in the reference zone to determine a qualitative level of GPC3 associated with the presence or absence of prostate cancer. If a sub-threshold level of GPC3 is observed in the assay, the subject can be classified in a low-probability category for the presence of cancer. If a threshold or higher level of antigen is observed, the subject may be recommended for additional testing and/or more frequent testing.
  • the assay device includes an assay strip like that described above, but where the known-reference indicator is provided by a strip-reader instrument reader having (i) a reader slot for receiving the assay strip, (ii) a light source and an optical detection, e.g., a spectrophotometric detector, for detecting an assay-related optical condition at the detection zone of the assay strip, (iii) an electronics or processor unit which records and processes a signal from the optical detector, and converts the signal to an assayed level of GPC3, and (iv) a user display screen or window.
  • a strip-reader instrument reader having (i) a reader slot for receiving the assay strip, (ii) a light source and an optical detection, e.g., a spectrophotometric detector, for detecting an assay-related optical condition at the detection zone of the assay strip, (iii) an electronics or processor unit which records and processes a signal from the optical detector, and converts the signal to an assa
  • the instrument may report the actual GPC3 body-fluid sample detected, allowing the operator to compare the displayed value with known standard indicator levels provided with the assay strip or instrument, to assess whether the subject has an elevated GPC3 level associated with prostate cancer, or to assess the possible stage of the cancer, for purposes of treatment design.
  • the instrument itself may contain stored known-standard indicator levels which can be compared internally with an assayed level to generate an output that indicates whether an elevated GPC3 level associated with prostate cancer has been detected, or to indicate the stage of the cancer.
  • the present disclosure provides a method for identifying mutations associated with increased risk of prostate cancer in a human subject.
  • the method is based on the finding discussed in Section IIA above that many virally induced mutations introduced into the region 0-200 kbases upstream of the GPC3 gene of chromosomal interval region Xq26 was4e associated with prostate cancer.
  • genomic DNA is extracted from human patients having prostate cancer, preferably including patients from men representing different racial and age groups, and in particular, patients that are known to have a family history of the disease, eig., fathers or other close male relatives with prostate cancer.
  • the Xq26 from the long arm of the X chromosome is isolated according to known methods, and these fragments are sequenced by standard sequencing techniques that may involve, in one known methods (i) cutting the fragments with selected endonucleases, (ii) cloning the cut fragments in a suitable cloning vector, e.g., single-stranded sequencing vector, and sequencing the resulting clones.
  • Mutations are one or more sites along the region are identified by comparing each of the 200 kbase upstream sequences with sequences from the same region derived from normal (wildtype) males with no known family predisposition to prostate cancer. Preferably sequences from a number of wildtype men are determined to ensure a true wildtype sequence. For each extracted DNA, the patient and wildtype sequences are compared to identify mutations in the patient sequences, and thus mutations that are likely associated with increased risk of prostate cancer.
  • the device includes a gene chip, such as shown at 30 in FIG. 4 , having an array of regions, such as regions 34 , 36 , each containing bound known-sequence fragments, such as fragment 37 in region 34 .
  • the fragments or probes are preferably 25-70 bases in lengths, and each includes one of the above-identified mutations upstream of the GPC3 gene that is associated with prostate cancer.
  • the array sequences are designed in length and sequence to bind to those Xq26 mutations identified above, but not to associated fragments. Gene-chip construction, and detection of mutant sequences with such chips, are well known.
  • a typical genetic-screening procedure patient cells are obtained, genomic DNA is extracted, and Xq26 sequence regions of interest are amplified by standard PCR, employing fluoresceinated probes.
  • the amplified material is then reacted with the chip-array sequences, under suitable hybridization conditions, and the array surface is washed to remove unbound material, then scanned with a suitable chip reader to identify any mutated sequences associated with prostate cancer.
  • the figure shows binding of a labeled genomic DNA fragment, indicated at 42 , to an array region 38 having bound probe molecules 40 . Detection of a fluorescent signal in this array region is diagnostic of a known genetic mutation in the critical upstream GPC3 region of Xq26 interval and may be diagnostic of a genetic predisposition to prostate cancer.
  • the mutated identified as above are used to construct a set of molecular inversion probes (MIPs) capable of identifying the presence of genomic mutations by the steps of:
  • MIPs molecular inversion probes
  • the present disclosure also includes methods for treating, e.g., reducing the tumor burden in a human subject with prostate cancer.
  • a GPC3 antigen e.g., full length GPC3 or an antigenic peptide, such as one of the GPC3 peptides disclosed above, is used to activate immune cells that participate in inducing cytotoxic T cells specific against prostate tumor cells. This may be done, in one embodiment, by exposing antigen-presenting cells obtained from the patient ex vivo with the GPC3 antigen, under conditions effective to activate the cells, e.g., in the presence of GM-CSF.
  • the cells are reintroduced into the patient, where the activated cells are effective in stimulating clonal expansion of cytotoxic T cells against the tumor.
  • This immunotherapy approach is described, for example, in U.S. Pat. No. 6,080,409 and pertinent references cited therein.
  • the GPC3 antigen may be administered to the patient as a vaccine, typically present in a suitable adjuvant, such as one containing GM-CSF.
  • the peptide vaccine is effective to stimulate and causing clonal expansion of memory CD4 helper cells, CD8 Tc cytotoxic lymphocytes and CD8 non-cytotoxic T-suppressor lymphocytes, causing causing expansion of GPC3 antigen-specific memory CD4 helper cells, GPC3 antigen-specific CD8 Tc cytotoxic lymphocytes and GPC3 antigen-specific CD8 non-cytotoxic T-suppressor lymphocytes in the subject.
  • antigen-containing compositions suitable for injection, and suitable antigen doses for immuno-stimulation of cytotoxic T cells have been described in a number of patents and literature publications on T-cell induction by immunotherapy. Those methods are applicable in the present method involving GPC3 antigen for the treatment of prostate cancer. Following treatment, the patient is monitored for change in status of the cancer, typically by a combination of a tumor-visualization procedure, such as MRI or CAT scan, and levels of prostate-related antigens, including GPC3 itself.
  • a tumor-visualization procedure such as MRI or CAT scan
  • levels of prostate-related antigens including GPC3 itself.
  • a patient diagnosed with prostate cancer is first confirmed as having elevated levels of GPC3, according to assay methods described above. If the subject tests positive in this assay, he is treated by administration of anti-GPC3 antibody.
  • the antibody is a human or humanized antibody, prepared as described above, and is administered by IV or subcutaneous injection in a suitable physiological carrier.
  • the antibody dose is preferably 1 to 10 mg/injection, and the patient is treated at intervals of every 14 days or so.
  • the patient is monitored for change in status of the cancer, typically by a combination of a tumor-visualization procedure and levels of prostate-related antigens, as above.
  • the treatment may be carried out in combination with other prostate-cancer treatments, including drug or radio-isotope therapy, and may be continued until a desired diminution in tumor size is observed.
  • SEQ ID NO:1 SEQ ID NO:1; DLFIDKKVLKVAHVEHEET (amino acid residues 365 to 383 of human GPC3 antigen, encoded by exon 4 of the human GPC3 gene).

Abstract

Devices and methods for diagnosing and treating prostate cancer are disclosed. In practicing the method, a subject's body-fluid sample is assayed for GPC3 protein, and the GPC3 level observed is used in determining whether the subject has an elevated GPC3 level associated with prostate cancer. Patients with such elevated levels may be treated, in accordance with the present disclosure, with a variety of GPC3-related immunotherapy agents.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional application of currently pending U.S. application Ser. No. 11/325,847, filed 4 Jan. 2006, the contents of which are expressly incorporated herein by reference.
    • INTRODUCTION
  • This disclosure relates generally to methods for diagnosing and treating prostate cancer.
    • REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
  • A “Sequence Listing” is submitted with this application in the form of a text file, created 14 Aug. 2009, and named “598498001US00SeqList.txt” (1,233 bytes), the contents of which are incorporated herein by reference in their entirety.
    • REFERENCES
  • The following references are cited below in support of the present disclosure or methods employed in its practice.
    • 1 Filmus, J., and S. B. Selleck. 2001. Glypicans: proteoglycans with a surprise. J Clin Invest 108:497.
    • 2. Zhang, Z., C. Coomans, and G. David. 2001. Membrane heparan sulfate proteoglycan-supported FGF2-FGFR1 signaling: evidence in support of the “cooperative end structures” model, J Biol Chem 276:41921.
    • 3. Capurro, M. I., Y. Y. Xiang, C. Lobe, and J. Filmus. 2005. Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling. Cancer Res 65:6245.
    • 4. Grisaru, S., D. Cano-Gauci, J. Tee, J. Filmus, and N. D. Rosenblum. 2001. Glypican-3 modulates BMP- and FGF-mediated effects during renal branching morphogenesis. Dev Biol 231:31.
    • 5. Paine-Saunders, S., B. L. Viviano, J. Zupicich, W. C. Skarnes, and S. Saunders, 2000. Glypican-3 controls cellular responses to Bmp4 in limb patterning and skeletal development. Dev Biol 225:179.
    • 6. Yamauchi, N., A. Watanabe, M. Hishinuma, K. I. Ohashi, Y. Midorikawa, Y. Morishita, T. Niki, J. Shibahara, M. Mori, M. Makuuchi, Y. Hippo, T. Kodama, H. Iwanari, H. Aburatani, and M. Fukayama. 2005. The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol.
    • 7. Powell, C. A., G. Xu, J. Filmus, S. Busch, J. S. Brody, and P. B. Rothman. 2002. Oligonucleotide microarray analysis of lung adenocarcinoma in smokers and nonsmokers identifies GPC3 as a potential lung tumor suppressor. Chest 121:6 S.
    • 8. Kim, H., G. L. Xu, A. C. Borczuk, S. Busch, J. Filmus, M, Capurro, J. S. Brody, J. Lange, J, M. D'Armiento, P. B. Rothman, and C. A. Powell. 2003. The heparan sulfate proteoglycan GPC3 is a potential lung tumor suppressor. Am J Respir Cell Mol Biol 29:694.
    • 9. Zhu, Z., H. Friess, J. Kleeff, L. Wang, M. Wirtz, A. Zimmermann, M. Korc, and M. W. Buchler. 2002. Glypican-3 expression is markedly decreased in human gastric cancer but not in esophageal cancer. Am J Surg 184:78.
    • 10. Lin, H., R. Huber, D. Schlessinger, and P. J. Morin. 1999. Frequent silencing of the GPC3 gene in ovarian cancer cell lines. Cancer Res 59:807.
    • 11. Murthy, S. S., T. Shen, A. De Rienzo, W. C. Lee, P. C. Ferriola, S. C. Jhanwar, B. T. Mossman, J. Filmus, and J. R. Testa. 2000. Expression of GPC3, an X-linked recessive overgrowth gene, is silenced in malignant mesothelioma. Oncogene 19:410.
    • 12. Xiang, Y. Y., V. Ladeda, and J. Filmus. 2001. Glypican-3 expression is silenced in human breast cancer. Oncogene 20:7408.
    • 13. Sung, Y. K., S. Y. Hwang, M. K. Park, M. Farooq, I. S. Han, H. I. Bae, J. C. Kim, and M. Kim. 2003. Glypican-3 is overexpressed in human hepatocellular carcinoma. Cancer Sci 94:259.
    • 14. Hsu, H. C., W. Cheng, and P. L. Lai. 1997. Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution. Cancer Res 57:5179.
    • 15. Zhu, Z. W., H. Friess, L. Wang, M. Abou-Shady, A. Zimmermann, A. D. Lander, M. Korc, J. Kleeff, and M. W. Buchler. 2001. Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders. Gut 48:558.
    • 16. Lage, H., M. Dietel, G. Froschle, and A, Reymann. 1998. Expression of the novel mitoxantrone resistance associated gene MXR7 in colorectal malignancies, Int J Clin Pharmacol Ther 36:58.
    • 17. Saikali, Z., and D. Sinnett. 2000. Expression of glypican 3 (GPC3) in embryonal tumors. Int J Cancer 89:418.
    • 18. Midorikawa, Y., S. Ishikawa, H. Iwanari, T. Imamura, H, Sakamoto, K. Miyazono, T. Kodama, M. Makuuchi, and H, Aburatani. 2003. Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling. Int J Cancer 103:455.
    • 19. Nakatsura, T., T. Kageshita, S. Ito, K. Wakamatsu, M. Monji, Y. Ikuta, S. Senju, T. Ono, and Y. Nishimura. 2004. Identification of glypican-3 as a novel tumor marker for melanoma. Clin Cancer Res 10:6612.
    • 20. Nusse, R., A, van Ooyen, D. Cox, Y. K. Fung, and H. Varmus. 1984, Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307:131.
    • 21. Nusse, R., and H. E. Varmus. 1982. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell 31:99.
    • 22. Sorensen, A. B., M. Duch, H. W. Amtoft, P. Jorgensen, and F. S. Pedersen. 1996. Sequence tags of provirus integration sites in DNAs of tumors induced by the murine retrovirus SL3-3. J Virol 70:4063.
    • 23. Lund, A. H., G. Turner, A. Trubetskoy, E. Verhoeven, E. Wientjens, D. Hulsman, R. Russell, R. A. DePinho, J. Lenz, and M. van Lohuizen. 2002. Genome-wide retroviral insertional tagging of genes involved in cancer in Cdkn2a-deficient mice. Nat Genet 32:160.
    • 24. Mikkers, H., J. Allen, P. Knipscheer, L. Romeijn, A. Hart, E. Vink, A. Berns, and L. Romeyn. 2002. Highthroughput retroviral tagging to identify components of specific signaling pathways in cancer, Nat Genet 32:153.
    • 25. Collier, L. S., C. M. Carlson, S. Ravimohan, A. J. Dupuy, and D. A. Largaespada. 2005. Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse, Nature 436:272.
    • 26. Dupuy, A. J., K. Akagi, D. A. Largaespada, N. G. Copeland, and N. A. Jenkins. 2005. Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436:221.
    BACKGROUND
  • Prostate cancer is the most common malignant cancer in North American men. It is estimated that approximately 200,000 new cases and 31,500 prostate cancer-related deaths will occur in the United States annually. Prostate cancer is now the second leading cause of cancer death in men, exceeded only by lung cancer. It accounts for 29% of all male cancers and 11% of male cancer-related deaths.
  • Currently, the FDA has approved serum PSA (prostate-specific antigen) for use as a prostate cancer screening laboratory test. Like many serum tumor markers, PSA is produced by both normal and cancerous glands. In men with prostate cancer, the serum levels can be elevated with both localized and advanced or disseminated disease. PSA levels are generally proportional to the volume of the cancer. Because there is a significant overlap between PSA levels found in cancer and benign prostatic hyperplasia, it is important to obtain sequential levels in low or borderline elevated values.
  • The introduction of free PSA (fPSA) testing has introduced a greater level of specificity in identifying early prostate cancer. In 1998, the FDA approved fPSA testing as a diagnostic aid for men with total PSA values between 4.0-10.0 ng/mL. This has often been the diagnostic gray zone for total PSA testing and fPSA may aid in the stratification. In general, at any free PSA level, the more enlarged the prostate, the more likely the prostate may be cancerous. However, these tests remain qualitative at best, and more reliable types of detection, and means for staging the cancer treatment, are needed.
  • Prostate cancer, like other forms of cancer, is caused by genetic aberrations, i.e., mutations. In the mutant cells the normal balance between the factors that promote and restrain growth is disrupted, and as a result, these mutant cells proliferate continuously—the hallmark of tumor cells. Mutations can arise spontaneously or by external factors such as chemical mutagens, radiation, or viral integration, which inserts extra-genomic DNA that may or may not contain an oncogene. A cellular gene can be modified by point mutation, insertion and frame shift (including truncation), (functional) deletion (including silencing), or translocation, which sometimes can result in gene fusion. In this way protooncogenes can become oncogenes, which promote proliferation, and tumor suppressor genes can become inactivated, also inducing tumor growth. Any combination of the above-mentioned changes in DNA can contribute to tumor formation. The consequences of these changes may or may not be held in check by the immune system (immune surveillance).
  • One protein whose expression has been implicated in certain cancers is the Glypican 3 protein, or GPC3, a heparin sulfate proteoglycan anchored to the cell membrane via glycosylphosphatidylinositol (1). The protein has a molecular weight of 65.6 kDa and the polypeptide chain has 580 amino acid residues. The heparin sulfate chain of the proteoglycans interacts with heparin-binding growth factors and thus serves as a co-receptor in cell signaling (2), although GPC3 might bind also in a different way (3). In embryonic development, GPC3 modulates BMP and EGF-mediated effects during renal branching morphogenesis (4). It also controls cellular responses to BMP4 in limb patterning and skeletal development (5). Except for weak expression in bronchiolar epithelial cells, GPC3 is not expressed in systemic organs (6). Because its expression is decreased in lung adenocarcinoma (7, 8), human gastric cancer (9), ovarian cancer cell lines (10, 11), mesotheliomas (10,11), and breast tumors (12), GPC3 may function as a tumor suppressor. But because its mRNA and protein expression is increased in hepatocellular carcinomas (13-15), colorectal malignancies (16), and embryonal tumors (17) as compared to normal tissue, GPC3 is also considered an onco(fetal) protein. For hepatocarcinoma, GPC3 is a promising diagnostic marker (6). It is also known that in these cancers, GPC3 modulates FGF2 and BMP-7 signaling (18), and promotes growth by stimulating canonical Wnt signaling (3).
  • In more than 80% of melanoma and melanocytic nevus, both GPC3 mRNA and protein are expressed (19). Interestingly, GPC3 protein is found in sera from 40% of melanoma patients but not in sera from subjects with large congenital melanocytic nevus and from healthy donors. GPC3 expression disappeared in sera from one third of the patients after surgical removal of the melanoma (19).
  • Heretofore, there has been no demonstrated link between changes in GPC3 levels and prostate cancer. Such a link could have a number of important diagnostic and therapeutic applications. In accordance with the present disclosure, it has now been discovered that (i) GPC3 levels increase significantly in prostate cancer cells, and (ii) this increase can be measured the ductile fluid and blood-fluid sample of patients.
    • SUMMARY
  • The present disclosure includes, in one aspect, a method of screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject. The method includes assaying a body-fluid sample from the subject for the level of GPC3 protein in the sample, and determining from the assayed level, whether the subject has an elevated GPC3 level associated with prostate cancer, as an indicator of prostate ca ncer.
  • Where the body-fluid sample is seminal fluid, the assayed level of GPC3 indicative or prostate cancer is preferably in a range between 1 and 10 ng/ml. Where the body-fluid sample is blood or serum, the assayed level of GPC3 indicative or prostate cancer is preferably in a range between 1 and 10 ng/ml.
  • In one embodiment of the method, the assaying step may involve applying the body fluid to a solid-phase immunoassay device, the level of GPC3 in the sample may be indicated qualitatively by a calorimetric or fluorometric indicator, and the determining may include comparing the indicator with a known standard.
  • For use in staging the treatment of prostate cancer in a human, the level of assayed GPC3 is indicative of the stage of the cancer in the subject.
  • In a related aspect, a diagnostic device is provided for use in for screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject. The device includes a (i) structure for receiving a body-fluid sample from the subject, (ii) an anti-GPC-3 binding antibody associated with the structure and capable of reacting with body-fluid received in the structure, to produce, in combination with other reagents associated with the structure, a detectable reaction indicative of the level of GPC3 in the sample, and (iii) a known-standard indicator against which the level of detectable reaction produced can be assessed as an elevated level associated with prostate cancer. For use in staging the treatment of prostate cancer in a human, the known-standard indicator includes two or more indicators that represent different levels of GPC3 associated with different stages of prostate cancer.
  • In one embodiment, the structure in the device includes a porous pad or strip having the anti-GPC3 binding protein embedded therein, for reaction with the fluid sample when the sample is applied to the pad, the detectable reaction is indicated by a colorimetric or fluorimetric indicator, and the known standard indicator includes an indicia that represents a level of GPC3 corresponding to that associated with prostate cancer.
  • In another embodiment, the device includes an instrument for generating a signal related to the level of GPC3 produced, a microprocessor for comparing the signal with a know-standard signal value associated with prostate cancer, and a display for displaying an output of the microprocessor.
  • In still another aspect, a histological method is provided for examining human prostate tissue for the presence and extent of prostate cancer. The method includes staining the prostate tissue with an anti-GPC-3 binding protein labeled with a detectable marker, to attach the marker to the surface of tissue cells, and determining, based on an elevated distribution and extent of detectable marker with respect to the distribution and extent of marker in normal prostate cells, the presence and extent of prostate cancer in the tissue. The distribution and extent of detectable marker in the tissue may be used to determine the stage of cancer in the tissue, as a method of staging the patient for anti-cancer treatment.
  • Also disclosed is a method for identifying genetic mutations associated with an increased risk of prostate cancer. Here method is practiced by (a) extracting genomic DNA from human patients having prostate cancer, (b) for each extracted DNA, comparing the sequence of the DNA in a selected region 0-200 kbases upstream of the GPC3 gene on chromosome interval Xq26, with a homologous region of DNA from one or more wildtype individuals, and (c), by the comparing, identifying one or more mutations in the region associated with an increased risk prostate cancer.
  • For use in constructing a gene chip designed for genetic screening for risk of prostate cancer, this method further includes (d) for each mutation identified in step (c) producing a gene fragment capable binding selectively to genomic DNA fragments carrying that mutation, but not to corresponding wildtype DNA fragments, and (e) attaching the gene fragments constructed in step (d) at known positions on a chip-chip substrate.
  • In still another aspect, provided is a method of reducing tumor burden in a subject with prostate cancer, by exposing subject antigen-presenting cells to human GPC3 polypeptide or antigenic fragment(s) thereof, and by the exposing, stimulating and causing clonal expansion of memory CD4 helper cells, CD8 Tc cytotoxic lymphocytes and CD8 non-cytotoxic T-suppressor lymphocytes thereby causing expansion of GPC3 antigen-specific memory CD4 helper cells, GPC3 antigen-specific CD8 Tc cytotoxic lymphocytes and GPC3 antigen-specific CD8 non-cytotoxic T-suppressor lymphocytes in the subject.
  • The treatment method may be practiced by exposing the subject's antigen-presenting cells ex vivo to the human GPC3 polypeptide or antigenic fragment(s) thereof, under conditions effective to activate the cells, and injecting the activated cells into the subject. Alternatively, the method may be practiced by injecting the subject with the human GPC3 polypeptide or fragments thereof, carried in a suitable adjuvant.
  • In another immunotherapy treatment method, in accordance with the present disclosure, a patient's body-fluid sample is assayed for the level of GPC3 protein in the sample, to determine from the assayed level, whether the subject has an elevated GPC3 level associated with prostate cancer. If the patient has such an elevated GPC3 level, the patient is treated by administering a therapeutically effective amount of a GPC3 binding agent effective, when bound to GPC3 on the surface of prostate cancer cells, to inhibit growth or viability of the cells. The GPC3 binding agent administered may be human or humanized anti-GPC3 antibody, effective, when bound to GPC3 on the surface of prostate cancer cells, to promote antibody-dependent cell cytotoxicity.
  • These and other aspects, objects, advantages, and features of the present disclosure will become apparent to those persons skilled in the art upon reading the details of the disclosure as more fully described below.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 shows the genomic organization of the mouse Gpc3 locus, as view by a customized screen print of the UCSC genome web site browser (March 2005 version of the mm6 gene assembly). Top, base position on chromosome X. Vertical handle bars in red, green and blue represent the retroviral insertions into the locus in 82 independent tumors.
  • FIGS. 2A and 2B shows immunohistochemical stains of human prostate tumor (2A) and matched normal tissue from the same patient (2B). The polyclonal rabbit antibody serum used reacts with 2 peptide epitopes: DLFIDKKVLKVAHVEHEET (SEQ ID NO:1; amino acid residues 365 to 383, encoded by exon 4) and LAYDLDVDDAPGNSQQ (SEQ ID NO: 2; amino acid residues 526 to 541, encoded by exon 8);
  • FIGS. 3A and 3B show a solid-phase diagnostic device for determining GPC3 levels in a human patient, at initial (3A) and final stages (3B) of the assay; and
  • FIG. 4 shows a portion of a gene chip useful for diagnosing genetic predisposition to prostate cancer, constructed in accordance with the present disclosure.
    •  DETAILED DESCRIPTION I. Definitions
  • The following terms have the definitions given below, unless otherwise indicated in the specification.
  • “Screening” for prostate cancer, in accordance with the present disclosure, means testing individuals for a level of GPC3 that is indicative of prostate cancer or an elevated risk of prostate cancer.
  • “Staging” treatment of prostate cancer, in accordance with the present disclosure, involves determining the stage of prostate cancer in an individual, based on the level of GPC3 detected, and tailoring the treatment to that stage. There are four recognized stages of prostate cancer, which are defined by the degree of localization of cancer cells. In addition, prostate cancer may be defined as early stage at which the cancer is responsive to a number of hormonal-based therapies, and a later, more serious androgen-independent stage.
    • II. Prostate Cancer and GPC3 Expression A. Identification of GPC3 as an Oncogene in a Retroviral Mutagenesis Screen.
  • A number of cancer genes (oncogenes and tumor suppressor genes) associated with prostate cancer were identified in a high throughput manner using proviral tagging in mice. In proviral tagging, mice are infected with a retrovirus that does not contain an oncogene (e.g., murine leukemia virus, MLV or murine mammary tumor virus, MMTV)(20-24). Recently, the host range of this approach has been broadened by the use of a transposon (25, 26).
  • During retroviral infection, the virus integrates into the cellular genome and inserts its DNA near or within genes, which leads to various outcomes: (i) The insertion site is too far away from a protooncogene and thus does not activate it. In this case, there will be no selection for that cell. (ii) The provirus inserts within 200 kb of a protooncogene, but not within the gene (type 1). Here, either the viral promoter or the viral enhancer increases the expression level of the protooncogene. (iii) The provirus inserts within a gene, destroying or altering its function (type 2). There will be no selection for a cell that contains either type 1 and type 2 insertion events in a gene that is not a protooncogene or tumor suppressor gene. If integration results in the formation of a tumor, genes adjacent to the integration site can be identified, and classified as either protooncogenes or tumor suppressor genes (20). A tumor suppressor may be scored if a retrovirus lands within a gene and truncates or destroys it. In these cases, the suppressor may be haplo-insufficient, or alternatively, the mutation on the other allele is provided spontaneously by the mouse. The integration event may also lead to more complex consequences, such as a dominant negative effect of the truncated gene product or the transcription of anti-sense or microRNA.
  • In a screen with T lymphotropic virus SL3-3, 81 independent tumors were recovered that contained proviral integrations distributed over a range from 25 to 152 kb from the 5′ end of the Gpc3 gene (FIG. 1, “Picobella_SL3” and “FSP_SL3”; green and blue bars), Similarly, in a screen with B lymphotropic virus Akv1-99, one tumor was recovered that contained a proviral integration within the same region (FIG. 1, “PicoAKV2”, red bar). These integrations cause the overexpression of the Gpc3 gene. These studies demonstrate that prostate cancer is associated with GPC3 overexpression, indicating that the gene functions as an oncogene in prostate cancer.
    • B. Expression of GPC3 in Human Prostate Tumors and Normal Tissue
  • The mutations that were identified as causal in mouse tumor formation increased the steady-state level of protein expression. To confirm that overexpression of the GPC3 gene product is characteristic of prostate cancer, GPC3 protein was assayed in situ using an anti-GPC3 antibody, prepared as described above. Briefly, tissue samples from the ductile epithelium in normal and cancerous human tissue were exposed to unlabeled rabbit anti-GPC3 antibody under standard in situ labeling conditions. After washing the cells to remove unbound antibody, the tissue was stained with a horseradish peroxidase polymer-conjugated anti-rabbit secondary antibody (Zymed Laboratories), and viewed by photomicroscopy, with the results shown in FIGS. 2A (cancerous tissue) and 2B (normal tissue). As seen, GPC3 overexpression in prostate cancer leads to a higher concentration of GPC3 in the lumen of the ducts (FIG. 2A). More generally, the disclosure provides a histological method for examining human prostate tissue for the presence and extent of prostate cancer. In the method, prostate tissue is stained with a labeled anti-GPC-3 binding protein, e.g., fluorescence-labeled anti-GPC3 antibody (see Section 11 below), to attach the marker to the surface of tissue cells. The presence and extent of prostate cancer in the tissue is then determined based on an elevated distribution and extent of detectable marker with respect to the distribution and extent of marker in normal prostate cells.
  • As will be seen in Section lV below, elevated levels of GPC3 antigen, as an indicator of prostate cancer, increased risk of prostate cancer, or the stage of prostate cancer, can be assayed directly from a body-fluid sample, e.g., seminal fluid or a blood sample.
    • III. Preparation of Anti-GPC3 Antibody
  • This section describes production of anti-GPC3 antibodies useful for diagnostic and therapeutic purposes, as described further in the sections below. The anti-GPC3 antibody used in the present disclosure can be obtained by any a variety of conventional methods as a monoclonal, polyclonal, or recombinant antibody. One preferred antibody, particularly for diagnostic use, is a mouse monoclonal antibody, prepared according to well-known hybridoma methodology. Briefly, human GPC3 may be first obtained, for example, by expressing the GPC3 (MXR7) gene as disclosed by Lage, H. et al (Gene 188 (1997), 151-156). The purified GPC3 protein is as an immunogen. Alternatively, a partial peptide of GPC3 can be used as a sensitization antigen. The partial peptide can be obtained by chemical synthesis from the amino acid sequence of human GPC3. Two GPC3 sequences that may be employed are: DLFIDKKVLKVAHVEHEET (SEQ ID NO: 1; amino acid residues 365 to 383, encoded by exon 4) and LAYDLDVDDAPGNSQQ (SEQ ID NO: 2; amino acid residues 526 to 541, encoded by exon 8).
  • Anti-GPC3 antibodies useful in diagnostic applications may be labeled with a variety of detectable labels, including detectable reporters, such as enzymes for enzyme-linked immunosorbent assays (ELISA), detectable particles, such as gold particles and reporter-carrying liposomes, colorimetric or fluorescent reporters, radiolabels, and labels such as a biotin label by which secondary detectable labels, such as a reporter-labeled streptavidin label can be attached. In some assay formats, an unlabeled anti-GPC3 antibody, for example, a mouse IgG antibody, is detected by reaction with a labeled antibody, e.g., a labeled anti-mouse IgG antibody.
  • For therapeutic uses, human monoclonal antibodies having binding activity to glypican 3, (see Japanese Patent Publication (Kokoku) No. 1-59878 B (1989)) can be produced by sensitizing in vitro human lymphocytes with GPC3, and causing the sensitized lymphocytes to fuse with the human-derived myeloma cells having a permanent division potential. Alternatively, GPC3 as an antigen can be administered to a transgenic animal having all the repertories of a human antibody gene to obtain anti-glypican 3 antibody-producing cells, and then human antibodies for GPC3 may be obtained from the immortalized anti-GPC3 antibody-producing cells (see International Patent Publication Nos. WO 94/25585, WO 93/12227, WO 92/03918 and WO 94/02602).
  • In still other methods, human or humanized antibodies specific against GPC3 antigen can be prepared by recombinant technique, such as have been reported (see, for example, U.S. Pat. Nos. 6,090,382 and 6,258,562).
    • IV. Diagnostic Methods and Reagents
  • In one aspect, the present disclosure includes a method of screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject, by assaying a body-fluid sample from the subject for the level of GPC3 protein in the sample, and determining from the assayed level, whether the subject has an elevated GPC3 level associated with prostate cancer, as an indicator of prostate cancer.
  • Preferred body-fluid samples are (a) seminal fluid and (b) blood. Where seminal fluid is assayed, the assayed level of GPC3 indicative or prostate cancer is in a range between 1 and 10 ng/ml. Where blood is assayed, the assayed level of GPC3 indicative or prostate cancer is in a range between 1 and 10 ng/ml.
  • The assay may be carried out by any of a variety of assay methods used for detecting body-fluid antigens, including ELISA techniques, homogeneous assays, for example, involving fluorescence quenching, and a variety of solid-phase sandwich assays in which the GPC3 antigen is captured on by an anti-GPC3 antibody carried on a solid support, and the immobilized antigen-antibody complex is labeled with a second anti-GPC3 antibody, e.g., a second antibody carrying a calorimetric or gold-particle reporter.
  • FIGS. 3A and 3B illustrate a solid-phase assay strip constructed in accordance with an embodiment of the present disclosure, suitable for carrying out a sandwich immunoassay of the type just mentioned, and shown in initial and final assay states, respectively. The strip, indicated generally at 10, includes a porous support or pad 12 having a sample-application zone 14 in an upstream region of the support and a sample-detection zone 16 in a downstream region. The sample-application zone includes a detectable anti-GPC3 antibody reagent, e.g., anti-GPC3 antibodies labeled with gold particles, and carried in the zone in an unbound, i.e., non-immobilized form. This reagent is indicated by solid circles, such as at 18. Anti-GPC3 antibodies, which may be the same or different from those in the labeled antibody reagent, are immobilized to the solid support within the detection zone, and are indicated by the “Y” shapes, such as at 20.
  • Also shown is a reference zone 22 which is located adjacent the detection zone and has one or more colored or shaded regions corresponding to different assay levels of GPC3 in a body-fluid sample. In the embodiment shown, zone 22 includes three regions 22 a, 22 b, and 22 c, corresponding to an assayed level of GPC3 (a) below that associated with prostate cancer, (b) corresponding to a lower threshold level associated with prostate cancer, and (c) a level that is substantially higher, e.g., 2-3 times, higher than the threshold layer in region 22 b, respectively. These three regions provide a known standard indicator against which the level of detectable reaction produced can be assessed as a level associated with prostate cancer. Together, the assay strip and reference zone constitute an assay device for use in screening for prostate cancer in a human subject, or for staging treatment of prostate in a human subject.
  • In operation, a known volume of a body-fluid sample to be tested is added to the sample-application zone of the strip, where it diffuses into the zone, allowing the antibody reagent to react with GPC3 antigen in the sample to form an antigen-antibody complex. This complex and unbound antibody reagent then migrate downstream by capillarity toward the detection zone, where the antigen-antibody complex is captured by the immobilize antibody and the unbound reagent is carried to the end of the support, as indicated at 24. As can be appreciated, the higher the concentration of antigen in the body fluid, the higher the density of captured reagent in the detection zone and the greater the color or intensity in this zone. This color or intensity produced in the detection zone is compared with the standards in the reference zone to determine a qualitative level of GPC3 associated with the presence or absence of prostate cancer. If a sub-threshold level of GPC3 is observed in the assay, the subject can be classified in a low-probability category for the presence of cancer. If a threshold or higher level of antigen is observed, the subject may be recommended for additional testing and/or more frequent testing.
  • In another embodiment, the assay device includes an assay strip like that described above, but where the known-reference indicator is provided by a strip-reader instrument reader having (i) a reader slot for receiving the assay strip, (ii) a light source and an optical detection, e.g., a spectrophotometric detector, for detecting an assay-related optical condition at the detection zone of the assay strip, (iii) an electronics or processor unit which records and processes a signal from the optical detector, and converts the signal to an assayed level of GPC3, and (iv) a user display screen or window. The instrument may report the actual GPC3 body-fluid sample detected, allowing the operator to compare the displayed value with known standard indicator levels provided with the assay strip or instrument, to assess whether the subject has an elevated GPC3 level associated with prostate cancer, or to assess the possible stage of the cancer, for purposes of treatment design. Alternatively, the instrument itself may contain stored known-standard indicator levels which can be compared internally with an assayed level to generate an output that indicates whether an elevated GPC3 level associated with prostate cancer has been detected, or to indicate the stage of the cancer.
    • V. Identifying Genetic Mutation Associated with Prostate Cancer
  • In another aspect, the present disclosure provides a method for identifying mutations associated with increased risk of prostate cancer in a human subject. The method is based on the finding discussed in Section IIA above that many virally induced mutations introduced into the region 0-200 kbases upstream of the GPC3 gene of chromosomal interval region Xq26 wer4e associated with prostate cancer.
  • In practicing the method, genomic DNA is extracted from human patients having prostate cancer, preferably including patients from men representing different racial and age groups, and in particular, patients that are known to have a family history of the disease, eig., fathers or other close male relatives with prostate cancer. The Xq26 from the long arm of the X chromosome is isolated according to known methods, and these fragments are sequenced by standard sequencing techniques that may involve, in one known methods (i) cutting the fragments with selected endonucleases, (ii) cloning the cut fragments in a suitable cloning vector, e.g., single-stranded sequencing vector, and sequencing the resulting clones.
  • Mutations are one or more sites along the region are identified by comparing each of the 200 kbase upstream sequences with sequences from the same region derived from normal (wildtype) males with no known family predisposition to prostate cancer. Preferably sequences from a number of wildtype men are determined to ensure a true wildtype sequence. For each extracted DNA, the patient and wildtype sequences are compared to identify mutations in the patient sequences, and thus mutations that are likely associated with increased risk of prostate cancer.
  • Once a large number of these mutations are identified, edg., at least 50-200 or more, they may be used in constructing a genetic screening device, e.g., a gene chip, useful for screening individuals for genetic predisposition to prostate cancer. In one embodiment, the device includes a gene chip, such as shown at 30 in FIG. 4, having an array of regions, such as regions 34, 36, each containing bound known-sequence fragments, such as fragment 37 in region 34. The fragments or probes are preferably 25-70 bases in lengths, and each includes one of the above-identified mutations upstream of the GPC3 gene that is associated with prostate cancer. In particular, the array sequences are designed in length and sequence to bind to those Xq26 mutations identified above, but not to associated fragments. Gene-chip construction, and detection of mutant sequences with such chips, are well known.
  • In a typical genetic-screening procedure, patient cells are obtained, genomic DNA is extracted, and Xq26 sequence regions of interest are amplified by standard PCR, employing fluoresceinated probes. The amplified material is then reacted with the chip-array sequences, under suitable hybridization conditions, and the array surface is washed to remove unbound material, then scanned with a suitable chip reader to identify any mutated sequences associated with prostate cancer. The figure shows binding of a labeled genomic DNA fragment, indicated at 42, to an array region 38 having bound probe molecules 40. Detection of a fluorescent signal in this array region is diagnostic of a known genetic mutation in the critical upstream GPC3 region of Xq26 interval and may be diagnostic of a genetic predisposition to prostate cancer.
  • In an alternative embodiment, the mutated identified as above are used to construct a set of molecular inversion probes (MIPs) capable of identifying the presence of genomic mutations by the steps of: The construction and use of MIPs for identifying genetic mutations have been described (see, for example, Wang, et al, Nucleic acids research (England) 2005, Vol. 33, p. 21).
    • VI. Treatment Methods and Pharmaceutical Preparations
  • The present disclosure also includes methods for treating, e.g., reducing the tumor burden in a human subject with prostate cancer. In one approach, a GPC3 antigen, e.g., full length GPC3 or an antigenic peptide, such as one of the GPC3 peptides disclosed above, is used to activate immune cells that participate in inducing cytotoxic T cells specific against prostate tumor cells. This may be done, in one embodiment, by exposing antigen-presenting cells obtained from the patient ex vivo with the GPC3 antigen, under conditions effective to activate the cells, e.g., in the presence of GM-CSF. Once activated ex Vivo, the cells are reintroduced into the patient, where the activated cells are effective in stimulating clonal expansion of cytotoxic T cells against the tumor. This immunotherapy approach is described, for example, in U.S. Pat. No. 6,080,409 and pertinent references cited therein.
  • Alternatively, the GPC3 antigen may be administered to the patient as a vaccine, typically present in a suitable adjuvant, such as one containing GM-CSF. The peptide vaccine is effective to stimulate and causing clonal expansion of memory CD4 helper cells, CD8 Tc cytotoxic lymphocytes and CD8 non-cytotoxic T-suppressor lymphocytes, causing causing expansion of GPC3 antigen-specific memory CD4 helper cells, GPC3 antigen-specific CD8 Tc cytotoxic lymphocytes and GPC3 antigen-specific CD8 non-cytotoxic T-suppressor lymphocytes in the subject.
  • Preparation of antigen-containing compositions suitable for injection, and suitable antigen doses for immuno-stimulation of cytotoxic T cells have been described in a number of patents and literature publications on T-cell induction by immunotherapy. Those methods are applicable in the present method involving GPC3 antigen for the treatment of prostate cancer. Following treatment, the patient is monitored for change in status of the cancer, typically by a combination of a tumor-visualization procedure, such as MRI or CAT scan, and levels of prostate-related antigens, including GPC3 itself.
  • In a second general immunotherapy approach, a patient diagnosed with prostate cancer is first confirmed as having elevated levels of GPC3, according to assay methods described above. If the subject tests positive in this assay, he is treated by administration of anti-GPC3 antibody. Preferably the antibody is a human or humanized antibody, prepared as described above, and is administered by IV or subcutaneous injection in a suitable physiological carrier. The antibody dose is preferably 1 to 10 mg/injection, and the patient is treated at intervals of every 14 days or so. During treatment, the patient is monitored for change in status of the cancer, typically by a combination of a tumor-visualization procedure and levels of prostate-related antigens, as above. The treatment may be carried out in combination with other prostate-cancer treatments, including drug or radio-isotope therapy, and may be continued until a desired diminution in tumor size is observed.
  • While the present disclosure describes particular embodiments and applications, it will be appreciated that various changes and modification may be made without departing from the present disclosure as claimed.
    • Sequences:
  • SEQ ID NO:1; DLFIDKKVLKVAHVEHEET (amino acid residues 365 to 383 of human GPC3 antigen, encoded by exon 4 of the human GPC3 gene).
  • SEQ ID NO: 2; LAYDLDVDDAPGNSQQ (amino acid residues 526 to 541 of human GPC3 antigen, encoded by exon 8 of the human GPC3 gene).

Claims (4)

1. A diagnostic device for use in for screening for prostate cancer in a human subject, or staging treatment of prostate cancer in a subject, comprising
a structure for receiving a body-fluid sample from the subject,
an anti-GPC-3 binding antibody associated with said structure and capable of reacting with body-fluid received in said structure, to produce, in combination with other reagents associated with the structure, a detectable reaction indicative of the level of GPC3 in the sample, and
a known-standard indicator against which the level of detectable reaction produced can be assessed as an elevated level associated with prostate cancer.
2. The diagnostic device of claim 1, wherein said structure includes a porous pad having the anti-GPC3 binding protein embedded therein, for reaction with the fluid sample when the sample is applied to the pad, said detectable reaction is indicated by a calorimetric or fluorimetric indicator, and said known-standard indicator includes an index that represents a level of GPC3 corresponding to that associated with prostate cancer.
3. The diagnostic device of claim 1, which includes a spectrophotometic detector for generating a signal related to the level of GPC3 produced, a microprocessor for comparing said signal with a known-standard signal value associated with prostate cancer, and a display for displaying an output of the microprocessor.
4. The diagnostic device of claim 1, for use in staging the treatment of prostate cancer in a human, wherein said known-standard indicator includes two or more indicators that represent different levels of GPC3 associated with different stages of prostate cancer.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110577600A (en) * 2018-06-07 2019-12-17 中国科学院上海药物研究所 GPC 3-targeted antibody-drug conjugate, and preparation method and application thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9522940B2 (en) 2012-05-23 2016-12-20 Pieris Pharmaceuticals Gmbh Lipocalin muteins with binding-affinity for glypican-3 (GPC-3) and use of lipocalin muteins for target-specific delivery to cells expressing GPC-3
TWI693073B (en) 2012-12-21 2020-05-11 日商中外製藥股份有限公司 Therapeutic agent for GPC3 target is the effective therapeutic agent for GPC3 target administered to patients
EP3088890B1 (en) 2013-12-24 2020-09-23 Chugai Seiyaku Kabushiki Kaisha Method for measuring soluble gpc3 protein
JP6691060B2 (en) * 2014-01-17 2020-04-28 グリセリックス リミティド Cell surface prostate cancer antigen for diagnostics
EP2944652A1 (en) * 2014-05-13 2015-11-18 Technische Universität München Glypican-3-specific T-cell receptors and their uses for immunotherapy of hepatocellular carcinoma
MA40764A (en) 2014-09-26 2017-08-01 Chugai Pharmaceutical Co Ltd THERAPEUTIC AGENT INDUCING CYTOTOXICITY
CN105759051B (en) * 2016-02-25 2018-12-18 广州科方生物技术股份有限公司 A kind of quantification assay kit of Monophosphoinositideproteoglycans proteoglycans-3 and preparation method thereof
JPWO2022154119A1 (en) 2021-01-18 2022-07-21

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06138129A (en) * 1992-10-30 1994-05-20 Wako Pure Chem Ind Ltd Micro-plate reader
US6500673B1 (en) * 1998-07-28 2002-12-31 Jacques Toledano Electrostatic device and method for immunological detection
US20030105000A1 (en) * 2000-11-03 2003-06-05 Pero Stephanie C. Methods and compositions for inhibiting GRB7
US20040236080A1 (en) * 2001-06-22 2004-11-25 Hiroyuki Aburatani Cell proliferation inhibitors containing anti-glypican 3 antibody

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004022597A1 (en) 2002-09-04 2004-03-18 Chugai Seiyaku Kabushiki Kaisha Antibody against blood-solubilized n-terminal peptide in gpc3
WO2004023145A1 (en) * 2002-09-04 2004-03-18 Perseus Proteomics Inc. Method of diagnosing cancer by detecting gpc3

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06138129A (en) * 1992-10-30 1994-05-20 Wako Pure Chem Ind Ltd Micro-plate reader
US6500673B1 (en) * 1998-07-28 2002-12-31 Jacques Toledano Electrostatic device and method for immunological detection
US20030105000A1 (en) * 2000-11-03 2003-06-05 Pero Stephanie C. Methods and compositions for inhibiting GRB7
US20040236080A1 (en) * 2001-06-22 2004-11-25 Hiroyuki Aburatani Cell proliferation inhibitors containing anti-glypican 3 antibody

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Abaza et al. (Journal of Protein Chemistry, Vol. 11, No. 5, 1992, pages 433-444) *
American Cancer Society (http://www.cancer.org/Treatment/UnderstandingYourDiagnosis/staging, 5/31/2011) *
Burgess et al (J of Cell Bio. 111:2129-2138, 1990) *
Coleman et al. (Research in Immunology, 1994; 145(1): 33-36) *
Corona Electric (Absorbance Microplate Reader MTP-310/MTP-310LAB, 11/3/2011) *
Rudikoff et al. (PNAS, USA, 1982, 79: 1979-1983) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110577600A (en) * 2018-06-07 2019-12-17 中国科学院上海药物研究所 GPC 3-targeted antibody-drug conjugate, and preparation method and application thereof

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