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WO2006078892A2 - Use of a novel prostate specific membrane antigen for cancer diagnosis and therapy - Google Patents

Use of a novel prostate specific membrane antigen for cancer diagnosis and therapy
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WO2006078892A2
WO2006078892A2PCT/US2006/002020US2006002020WWO2006078892A2WO 2006078892 A2WO2006078892 A2WO 2006078892A2US 2006002020 WUS2006002020 WUS 2006002020WWO 2006078892 A2WO2006078892 A2WO 2006078892A2
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psma
cancer
cell surface
fragment
extracellular domain
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PCT/US2006/002020
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French (fr)
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WO2006078892A3 (en
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Ayyappan K. Rajasekaran
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The Regents Of The University Of California
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Abstract

The present invention provides, for the first time, the finding that a soluble extracellular domain of PSMA or a peptide fragment thereof specifically binds to PSMA-overexpressing, without any of the delivery problems or toxic side-effects associated with the use of such antibodies. In particular, the PSMA polypeptides and peptides described herein bind to PSMA on the surface of cells such as cancer cells and therefore have clinical significance for diagnosing, providing a prognosis for, imaging, and/or treating a cancer that overexpresses cell surface PSMA such as prostate cancer as well as endothelial cells of solid tumors. Compositions and kits for carrying out the methods of the present invention are also provided.

Description

USE OF A NOVEL PROSTATE SPECIFIC MEMBRANE ANTIGEN
FOR CANCERDIAGNOSIS AND THERAPY CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/645,930, filed January 21, 2005, the content of which is hereby incorporated herein by reference in its entirety for all purposes.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant No. DAMD 17-02- 1-0661, awarded by the U.S. Department of Defense. The U.S. Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
[0003] Cancer is the second leading cause of death behind heart disease. In fact, cancer incidence and death figures account for about 10% of the U.S. population in certain areas of the United States (National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) database and Bureau of the Census statistics; see, Harrison's Principles of Internal Medicine, Kasper et ah, 16th ed., 2005, Chapter 66). The five leading causes of cancer deaths among men are lung cancer, prostate cancer, colon and rectum cancer, pancreatic cancer, and leukemia. The five leading causes of cancer deaths among women are lung cancer, breast cancer, colon cancer, ovarian cancer, and pancreatic cancer. When detected at locally advanced or metastatic stages, no consistently curative treatment regimen exists. Treatment for metastatic cancer includes immunotherapy, hormonal ablation, radiation therapy, chemotherapy, hormonal therapy, and combination therapies. Unfortunately, for prostate cancer and hormone dependent tumors, there is frequent relapse of an aggressive androgen independent disease that is insensitive to further hormonal manipulation or to treatment with conventional chemotherapy (Ghosh et al., Proc. Natl. Acad. Sci. USA, 95:13182-13187 (1998)).
[0004] Prostate cancer is the most common non-skin cancer in the United States and accounts for over 30,000 deaths and over 230,000 new cases each year. Prostate specific membrane antigen (PSMA), which is highly expressed in prostate cancer cells versus normal prostate tissue, has been an attractive marker for the development of PSMA-targeted prostate cancer therapeutics and diagnostics. Monoclonal antibodies against full-length PSMA have shown high affinity and specificity for prostate cancer cells in vitro and in mouse models. However, the cellular localization of PSMA indicates that immunotherapy using anti-PSMA antibodies has limited therapeutic value for the treatment of prostate cancer. In particular, PSMA is localized to the apical plasma membrane, which is not accessible to conventional circulating therapeutic reagents in the blood such as antibodies. Tight junctions present at the boundary between apical and basolateral plasma membrane domains prevent antibodies from passing through and reaching the apical plasma membrane. In fact, high grade and metastatic prostate cancer tissues maintain glandular architecture with distinct apical and basolateral polarity. As a result, current approaches using anti-PSMA antibodies are inadequate for the treatment of prostate cancer due to problems encountered with antibody delivery. Thus, there is a need in the art for alternative approaches for the diagnosis and treatment of prostate cancer. The present invention satisfies this and other needs.
BRIEF SUMMARY OF THE INVENTION [0005] The present invention provides, for the first time, the finding that a soluble extracellular domain of PSMA or peptide fragments thereof specifically bind to PSMA- overexpressing cells, without any of the delivery problems or toxic side-effects associated with the use of such antibodies. In particular, the PSMA polypeptides and peptides described herein advantageously bind to PSMA on the surface of cells such as cancer cells and therefore have clinical significance for diagnosing, providing a prognosis for, imaging, and/or treating a cancer that overexpresses cell surface PSMA such as prostate cancer as well as endothelial cells of solid tumors. Suitable fragments of the PSMA extracellular domain for use in the present invention can be determined using simple binding assays to cellular PSMA. The PSMA-specific polypeptide or peptide can be utilized as a targeting domain to deliver reporter molecules for use in cancer diagnosis, e.g., in vitro or in vivo applications such as in vivo imaging. Additionally, toxins or radionuclides can be conjugated to the PSMA-specific polypeptide or peptide to generate highly-specific anti-cancer therapeutics. Compositions and kits for carrying out the diagnostic, prognostic, imaging, and therapeutic methods of the present invention are also provided.
[0006] In one aspect, the present invention provides a composition comprising a soluble extracellular domain of prostate specific membrane antigen (PSMA) or a fragment thereof that binds to cell surface PSMA and a physiologically acceptable carrier. [0007] In one embodiment, the soluble extracellular domain of PSMA or fragment thereof is linked to a detectable moiety {i.e., imaging agent) such as a radionuclide, a nanoparticle, a fluorescent dye, a fluorescent marker, or an enzyme. In another embodiment, the soluble extracellular domain of PSMA or fragment thereof is linked to an anti-cancer therapeutic such as a radionuclide, a toxin, a cytotoxic agent, an apoptotic agent, or any other chemotherapeutic, radiotherapeutic, or hormonal therapeutic agent known in the art. In certain instances, the soluble extracellular domain of PSMA or fragment thereof is expressed on the surface of a retrovirus such as a lentivirus, and the retrovirus is packaged with an imaging agent and/or an anti-cancer therapeutic.
[0008] In another aspect, the present invention provides a method of diagnosing a cancer that overexpresses cell surface PSMA, the method comprising the steps of:
(a) contacting a tissue sample with a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and
(b) determining whether or not PSMA protein is overexpressed in the sample, thereby diagnosing the cancer that overexpresses cell surface PSMA.
[0009] In yet another aspect, the present invention provides a method of providing a prognosis for a cancer that overexpresses cell surface PSMA, the method comprising the steps of:
(a) contacting a tissue sample with a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and
(b) determining whether or not PSMA protein is overexpressed in the sample, thereby providing a prognosis for the cancer that overexpresses cell surface PSMA.
[0010] The present invention also provides a method of imaging a cancer that overexpresses cell surface PSMA in a subject, the method comprising the steps of: (a) administering to the subject a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and
(b) determining where the soluble extracellular domain of PSMA or fragment thereof is concentrated in the subject, thereby imaging the cancer that overexpresses cell surface PSMA.
[0011] Generally, the methods find particular use in diagnosing, providing a prognosis for, or in vivo imaging cancers such as prostate cancer, renal cancer, bladder cancer, lung cancer, ovarian cancer, breast cancer, colon cancer, leukemias, B-cell lymphomas {e.g., non- Hodgkin's lymphomas, including Burkitt's, Small Cell, and Large Cell lymphomas), hepatocarcinoma, or multiple myeloma. Preferably, the cancer that overexpresses cell surface PSMA is prostate cancer.
[0012] In another aspect, the present invention provides a method of treating or inhibiting a cancer that overexpresses cell surface PSMA in a subject comprising administering to the subject a therapeutically effective amount of a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA.
[0013] The soluble extracellular domain of PSMA or fragment thereof can be administered alone or co-administered (e.g., concurrently or sequentially) in combination therapy with conventionally used chemotherapy, radiation therapy, hormonal therapy, and/or immunotherapy. The methods find particular use in treating prostate cancer, renal cancer, bladder cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, leukemias, B-cell lymphomas (e.g., non-Hodgkin's lymphomas, including Burkitt's, Small Cell, and Large Cell lymphomas), hepatocarcinoma, multiple myeloma, or other cancers that overexpress PSMA on the cell surface.
[0014] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 shows a schematic diagram of the structure of PSMA. PSMA is a type II transmembrane protein with a short N-terminal cytoplasmic domain (CD), a hydrophobic transmembrane region (TM), and a large extracellular domain (ED). The CD contains an endocytic targeting motif and filamin A binding site (A). The large ED is highly glycosylated with 9 predicted N-glycosylation sites (Y). The ED contains two domains of unknown function that span amino acids 44-150 (B) and 151-274 (D), proline- and glycine- rich regions that span amino acids 145-172 and 249-273, respectively (C and E), a catalytic domain that spans amino acids 274-587 (F), and a final domain of unknown function (amino acids 587-750) to which a helical dimerization domain (amino acids 601-750) is located (G).
[0016] Figure 2 shows an exemplary amino acid sequence of full-length PSMA (amino acids 1-750) and the PSMA extracellular domain (amino acids 44-750).
[0017] Figure 3 shows the purification of the soluble extracellular domain of PSMA using a nickel charged HiTrap™ Chelating HP Column. Figure 3 A shows a silver stained gel of 15 μl aliquots of starting conditioned media, column flowthrough, a 10 mM imidazole wash, and elution fractions of increasing imidazole concentrations. Figure 3B shows a Coomassie Blue stained gel of 15 μl of the resultant concentrated pool fractions of soluble PSMA. Arrows indicate bands corresponding to the expected molecular weight of soluble PSMA.
[0018] Figure 4 shows the binding of the soluble extracellular domain of PSMA to human prostate cancer cells that overexpress PSMA on the cell surface. Figure 4A shows that soluble PSMA specifically binds to the prostate cancer cells. Figure 4B shows that control cells do not display any detectable binding.
DETAILED DESCRIPTION OF THE INVENTION I. Introduction
[0019] Prostate specific membrane antigen (PSMA) is a metallopeptidase transmembrane protein localized to the apical plasma membrane in the secretory epithelial cells of the prostate gland. Although the expression of this protein is undetectable to very low in normal prostate epithelial cells, its level increases several fold in high grade prostate cancers and metastatic disease as well as in other cancers. In addition to prostate cancer cells, PSMA is expressed in the neovasculature of solid tumors but not in normal endothelial cells, indicating a role for PSMA in angiogenesis.
[0020] The expression pattern of PSMA in prostate cancer and in other cancers make it an attractive molecule for various diagnostic and therapeutic strategies. Currently, several antibodies against PSMA are being used in the diagnosis and therapy of prostate cancer. However, the cellular localization of PSMA strongly indicates that using anti-PSMA antibodies has limited value for the diagnosis or treatment of prostate cancer. In particular, PSMA is localized to the apical plasma membrane, which is not accessible to conventional circulating antibody-based reagents in the blood. Tight junctions present at the boundary between apical and basolateral plasma membrane domains prevent antibodies from passing through and reaching the apical plasma membrane. In fact, high grade and metastatic prostate cancer tissues maintain glandular architecture with distinct apical and basolateral polarity. As a result, current approaches using anti-PSMA antibodies are inadequate for the diagnosis or treatment of prostate cancer due to problems encountered with antibody delivery. Current approaches using anti-PSMA antibodies are also inadequate due to the frequent occurrence of toxic side-effects such as fever, chills and shivering, rash, headache, wheezing, drop in blood pressure, nausea and vomiting, breathing difficulties, and even heart failure in patients receiving anti-PSMA antibodies.
[0021] The present invention is based, in part, on the surprising discovery that a soluble extracellular domain of PSMA or peptide fragments thereof specifically bind to PSMA on the surface of cells such as cancer cells, in some case with affinity and specificity comparable to that of anti-PSMA antibodies, without any of the delivery problems or toxic side-effects associated with the use of such antibodies. In particular, the PSMA polypeptides and peptides described herein advantageously bind to cells that overexpress PSMA on the cell surface. In certain instances, the PSMA peptide fragment is a short peptide of the extracellular domain of PSMA that associates with PSMA-overexpressing cells. The peptide crosses the tight junction barrier, thereby reaching PSMA expressed at the apical plasma membrane. Most importantly, this peptide has a tremendous impact on the accessibility of PSMA expressed in the endothelial cells of tumor vasculature since small peptides can pass through various biological impediments presented by the solid tumors.
[0022] The present invention therefore provides PSMA polypeptides or peptides derived from the extracellular domain that can associate with PSMA expressed on the surface of cells such as prostate cancer cells as well as endothelial cells of solid tumors. As described below, the PSMA polypeptide or peptide can be linked (e.g., conjugated) to detectable labels or therapeutic reagents for diagnosis, prognosis, imaging, and/or therapy of prostate cancer and other cancers. Soluble forms of shorter fragments of epitope tagged extracellular domain of PSMA (i.e., PSMA peptide) bind to live PSMA-overexpressing prostate cancer and endothelial cells in culture. An ELISA-based assay can be used to determine the binding of PSMA peptide to target cells. Tight junction permeability of the peptide can be monitored and compared to the permeability of anti-PSMA antibody in reaching PSMA expressed at the apical plasma membrane in polarized epithelial cells. Also, the internalization of PSMA peptide can be monitored in cultured cells. In certain instances, additional PSMA peptide sequences can be added to improve internalization. The ability of this peptide to reach prostate tumors can be tested using in vivo xenografts or transgenic mice expressing PSMA in their prostate. Accessibility of this peptide to tumor endothelial cells can also be monitored using an in vivo xenograft model. Non-invasive imaging techniques such as positron emission tomography or luciferase-mediated visualization of PSMA peptide binding to tumors can be utilized to monitor the ability of this peptide to target PSMA expressed on prostate cancer or tumor endothelial cells. [0023] Accordingly, in a first aspect, the present invention provides a composition comprising a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA and a physiologically acceptable carrier.
[0024] In one embodiment, the soluble extracellular domain of PSMA is a polypeptide comprising the entire extracellular domain of full-length PSMA (amino acids 44-750). In another embodiment, a fragment of the soluble extracellular domain of PSMA is a polypeptide comprising 1, 2, 3, 4, 5, or more domains within the extracellular domain of full- length PSMA. Preferably, the fragment comprises the dimerization domain of full-length PSMA (amino acids 601-750), alone or in combination with neighboring domains or portions thereof. In yet another embodiment, a fragment of the soluble extracellular domain of PSMA is a peptide of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Preferably, the fragment comprises at least about 10 consecutive amino acids within the dimerization domain of full-length PSMA. The PSMA polypeptides and peptides of the present invention bind to cell surface PSMA, that is, they are capable of binding to cells such as cancerous cells that overexpress PSMA on the cell surface. Methods for synthesizing, expressing, and/or purifying polypeptides and peptides are well known in the art and are also described below.
[0025] In certain instances, the PSMA polypeptide or peptide of the present invention is linked to a detectable moiety such as a radionuclide, a nanoparticle, a fluorescent dye, a fluorescent marker, or an enzyme. In certain other instances, the PSMA polypeptide or peptide is linked to an anti-cancer therapeutic such as a radionuclide, a toxin, a cytotoxic agent, an apoptotic agent, or any other chemotherapeutic, radiotherapeutic, or hormonal therapeutic agent known in the art. hi some embodiments, the PSMA polypeptide or peptide is expressed on the surface of a retrovirus such as a lentivirus, and the retrovirus is packaged with one or more imaging agents and/or anti-cancer therapeutics. Methods for expressing heterologous proteins on the surface of retroviruses and for packaging retroviruses are known in the art and are also described below.
[0026] m another embodiment, the physiologically acceptable carrier comprises any liquid, solid, or semi-solid substance that is used as a diluent or vehicle for the PSMA polypeptide or peptide and selected in accordance with the route of administration and standard pharmaceutical practice for a particular dosage form. Examples of liquid carriers include, but are not limited to, physiological saline, phosphate buffer, normal buffered saline (135-150 niM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Non-limiting examples of solid or semi-solid carriers include mannitol, sorbitol, xylitol, maltodextrin, lactose, dextrose, sucrose, glucose, inositol, powdered sugar, molasses, starch, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, acacia gum, guar gum, tragacanth gum, alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, Veegum®, larch arabogalactan, gelatin, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyacrylic acid (e.g., Carbopol), calcium silicate, calcium phosphate, dicalcium phosphate, calcium sulfate, kaolin, sodium chloride, polyethylene glycol, and combinations thereof.
[0027] The present invention also provides a method of diagnosing a cancer that overexpresses cell surface PSMA, the method comprising the steps of:
(a) contacting a tissue sample with a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and (b) determining whether or not PSMA protein is overexpressed in the sample, thereby diagnosing the cancer that overexpresses cell surface PSMA.
[0028] The present invention further provides a method of providing a prognosis for a cancer that overexpresses cell surface PSMA, the method comprising the steps of:
(a) contacting a tissue sample with a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and
(b) determining whether or not PSMA protein is overexpressed in the sample, thereby providing a prognosis for the cancer that overexpresses cell surface PSMA.
[0029] The diagnostic and prognostic methods of the present invention can be carried out by determining the extent of binding between a soluble extracellular domain of PSMA or fragment thereof and cell surface PSMA protein from a subject, wherein increased binding relative to a healthy subject indicates a cancerous phenotype. In certain instances, the methods of diagnosis or prognosis are carried out by determining the extent by which the soluble extracellular domain of PSMA or fragment thereof binds to test tissue compared to normal tissue, for example, by employing an in vitro binding assay.
[0030] Generally, the methods of the present invention find particular use in diagnosing or providing a prognosis for prostate cancer, renal cancer, bladder cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, leukemias, B-cell lymphomas (e.g., non-Hodgkin's lymphomas, including Burkitt's, Small Cell, and Large Cell lymphomas), hepatocarcinoma, or multiple myeloma. Preferably, the methods of the present invention are used in diagnosing or providing a prognosis for prostate cancer or a subtype thereof. In carrying out the diagnostic or prognostic methods described herein, the determination of whether or not PSMA is overexpressed can be made, e.g., by comparing a test biological sample to a control autologous biological sample from normal tissue.
[0031] In carrying out the diagnostic or prognostic methods of the present invention, the tissue sample can be taken from a tissue of a primary tumor or a metastatic tumor. A tissue sample can be taken, for example, by an excisional biopsy, an incisional biopsy, a needle biopsy, a surgical biopsy, a bone marrow biopsy, or any other biopsy technique known in the art. In some embodiments, the tissue sample is microlaser microdissected cells from a needle biopsy. In other embodiments, the tissue sample is a metastatic cancer tissue sample. In yet other embodiments, the tissue sample is fixed, e.g., with paraformaldehyde, and embedded, e.g., in paraffin. Suitable tissue samples can be obtained from cancers such as prostate, kidney, bladder, ovary, lung, colon, breast, etc., as well as from the blood, serum, saliva, urine, bone, lymph node, liver, or tissue.
[0032] Preferably, the PSMA polypeptide or peptide of the present invention is linked to a detectable moiety such as a radionuclide, a nanoparticle, a fluorescent dye, a fluorescent marker, or an enzyme, e.g., to determine the extent of binding between the PSMA polypeptide or peptide and the test or normal tissue sample using an in vitro binding assay.
[0033] The present invention also provides a method of imaging a cancer that overexpresses cell surface PSMA in a subject, the method comprising the steps of:
(a) administering to the subject a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and (b) determining where the soluble extracellular domain of PSMA or fragment thereof is concentrated in the subject, thereby imaging the cancer that overexpresses cell surface PSMA.
[0034] The in vivo imaging methods described herein find particular use in detecting prostate cancer, renal cancer, bladder cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, leukemias, B-cell lymphomas {e.g., non-Hodgkin's lymphomas, including Burkitt's, Small Cell, and Large Cell lymphomas), hepatocarcinoma, multiple myeloma, or other cancers that overexpress PSMA on the cell surface. [0035] Preferably, the PSMA polypeptide or peptide of the present invention is linked to a detectable moiety such as a radionuclide, a nanoparticle, a fluorescent dye, a fluorescent marker, or an enzyme. In certain instances, the PSMA polypeptide or peptide is expressed on the surface of a retrovirus such as a lentivirus, and the retrovirus is packaged with one or more imaging agents.
[0036] Various devices and means for determining where the soluble extracellular domain of PSMA or fragment thereof is concentrated in the subject are described below and include, for example, Single Photon Emission Computerized Tomography (SPECT), Positron Emission Tomography (PET), Magnetic Resonance Imaging (MRI), as well as any in vivo optical imaging technique known to one of skill in the art.
[0037] In another aspect, the present invention provides a method of treating or inhibiting a cancer that overexpresses cell surface PSMA in a subject comprising administering to the subject a therapeutically effective amount of a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA.
[0038] In carrying out the methods of treatment, the one or more PSMA polypeptides or peptides that bind to cancerous cells overexpressing PSMA on the cell surface can be administered simultaneously or sequentially with conventional therapies, for example, currently used chemotherapy, radiation therapy, hormonal therapy, or immunotherapy treatments. In one embodiment, one or more PSMA polypeptides or peptides are administered prior to administering another therapeutic agent. In other embodiments, one or more PSMA polypeptides or peptides are administered concurrently with another therapeutic agent or after administering another therapeutic agent. Alternatively, one or more PSMA polypeptides or peptides are administered without any additional therapeutic agents.
[0039] As a non-limiting example, the PSMA polypeptide or peptide of the present invention can be co-administered with conventional chemotherapeutic agents including alkylating agents (e.g., cisplatin, cyclophosphamide, carboplatin, ifosfamide, chlorambucil, busulfan, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g., 5-fluorouracil, azathioprine, methotrexate, fludarabine, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, plicamycin, etc.), and the like. [0040] The PSMA polypeptide or peptide of the present invention can also be coadministered with conventional hormonal therapaeutic agents including, but not limited to, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, tamoxifen, and gonadotropin-releasing hormone agonists (GnRH) such as goserelin.
[0041] Additionally, the PSMA polypeptide or peptide of the present invention can be coadministered with conventional immunotherapeutic agents including, but not limited to, immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alpha- interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA- DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to111In,90Y, or131I, etc.).
[0042] In a further embodiment, the PSMA polypeptide or peptide of the present invention can be co-administered with conventional radiotherapeutic agents including, but not limited to, radionuclides such as47Sc,64Cu,67Cu,89Sr,86Y,87Y,90Y,105Rh,111Ag,111In,117mSn,
149Pm,153Sm,166Ho,177Lu,186Re,188Re,211At, and212Bi, optionally conjugated to antibodies directed against tumor antigens.
[0043] The therapeutic methods described herein find particular use in treating prostate cancer, renal cancer, bladder cancer, ovarian cancer, lung cancer, breast cancer, colon cancer, leukemias, B-cell lymphomas (e.g., non-Hodgkin's lymphomas, including Burkitt's, Small Cell, and Large Cell lymphomas), hepatocarcinoma, multiple myeloma, or other cancers that overexpress PSMA on the cell surface.
[0044] Preferably, the PSMA polypeptide or peptide of the present invention is linked to an anti-cancer therapeutic such as a radionuclide, a toxin, a cytotoxic agent, an apoptotic agent, or any other chemotherapeutic, radiotherapeutic, or hormonal therapeutic agent known in the art. In certain instances, the PSMA polypeptide or peptide is expressed on the surface of a retrovirus such as a lentivirus, and the retrovirus is packaged with one or more anti-cancer therapeutics.
II. Definitions [0045] As used herein, the following terms have the meanings ascribed to them unless specified otherwise. [0046] "Prostate specific membrane antigen" or "PSMA" refers to nucleic acids {e.g., gene, pre-mRNA, rnRNA), polypeptides, polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, preferably about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein, including the extracellular domain of PSMA, which corresponds to amino acids 44- 750 of full-length wild-type PSMA as shown in Figure 2; (2) specifically bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising a referenced amino acid sequence, immunogenic fragments thereof, and conservatively modified variants thereof, including the extracellular domain of PSMA as described above; (3) specifically hybridize under stringent hybridization conditions to a nucleic acid encoding a referenced amino acid sequence, and conservatively modified variants thereof, including a nucleic acid encoding the extracellular domain of PSMA as described above; and/or (4) have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to a reference nucleic acid sequence, including a reference nucleic acid encoding the extracellular domain of PSMA as described above. A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate (e.g., human), rodent (e.g., rat, mouse, hamster), cow, pig, horse, sheep, or any mammal. The nucleic acids and proteins of the present invention include both naturally- occurring and recombinant molecules. The full-length sequence of PSMA (protein and nucleic acid) is provided in U.S. Patent Nos. 6,569,432 and 5,935,818. An exemplary human nucleic acid encoding PSMA is provided by Accession No. NM_ 004476; exemplary protein sequences are provided by Accession Nos. NP_004467, A56881, and AAM34479. Truncated, alternatively spliced, precursor, and mature forms of PSMA are also included in the foregoing definition.
[0047] Topologically, PSMA has an "extracellular domain," a "transmembrane domain," and a "cytoplasmic domain." These domains can be structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and hydrophilic domains (see, e.g., Stryer, Biochemistry (3rd ed. 1988) and any of a number of Internet based sequence analysis programs, such as those found at http://dot.imgen.bcm.tmc.edu). "Extracellular domain" therefore refers to the domains of PSMA that are exposed to the extracellular face of the cell. The extracellular domain of PSMA corresponds to amino acids 44-750 of full-length wild-type PSMA, which is typically 750 amino acids in length.
[0048] As used herein, a "fragment" of PSMA refers to any portion of the extracellular domain of PSMA that corresponds to amino acids 44-750 of full-length wild-type PSMA. The term "fragment" encompasses peptides or polypeptides as small as 5 amino acids, preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acids, up to the full extracellular domain of PSMA, and also includes subdomains within the extracellular domain, such as the dimerization domain of PSMA (amino acids 601- 750). In certain instances, the extracellular domain of PSMA or a fragment thereof can be optionally conjugated to a heterologous molecule, e.g., a heterologous peptide, to assist in crossing tight junctions and cellular membranes. Examples of such molecules include, but are not limited to, hydrophobic and amphipathic peptides such as an 11 amino acid peptide of the tat protein of HIV, a 20 residue peptide sequence which corresponds to amino acids 84- 103 of the pl6 protein {see, e.g., Fahraeus et ah, Current Biology, 6:84 (1996)), the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et ah, J. Biol. Chem., 269:10444 (1994)), the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et ah, J. Biol. Chem., 270:1 4255-14258 (1995)), and the VP22 translocation domain from HSV (Elliot et ah, Cell, 88 :223-233 (1997)).
[0049] The term "cancer" refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, solid and lymphoid cancers, etc. Examples of different types of cancer include, but are not limited to, prostate cancer, renal cancer {i.e., renal cell carcinoma), bladder cancer, lung cancer, breast cancer, thyroid cancer, liver cancer {i.e., hepatocarcinoma), pleural cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, anal cancer, pancreatic cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gall bladder cancer, rectal cancer, appendix cancer, small intestine cancer, stomach (gastric) cancer, cancer of the central nervous system, skin cancer, choriocarcinoma; head and neck cancer, blood cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, B-cell lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Cell lymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, and multiple myeloma. In preferred embodiments, the compositions and methods of the present invention are useful for diagnosing, imaging, proving a prognosis for, and treating prostate cancer or a subtype thereof.
[0050] The terms "overexpress," "overexpression," or "overexpressed" interchangeably refer to a gene that is transcribed or translated at a detectably greater level, usually in a cancer cell, in comparison to a normal cell. Overexpression therefore refers to both overexpression of PSMA protein and RNA, as well as local overexpression due to altered protein trafficking patterns and/or augmented functional activity. Overexpression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.) or rnRNA (e.g., RT-PCR, PCR, hybridization, etc.). One skilled in the art will know of other techniques suitable for detecting overexpression of PSMA protein or mRNA. Cancerous cells, e.g., cancerous prostate cells, can overexpress PSMA on the cell surface at a level of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% in comparison to normal, non-cancerous cells such as prostate cells. Cancerous cells can also have at least about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or 7-fold higher level of PSMA transcription or translation in comparison to normal, non-cancerous cells. In certain instances, the cancer cell sample is autologous. In some cells, PSMA expression is very low or undetectable. As such, expression includes no expression, i.e., expression that is undetectable or insignificant.
[0051] "Therapy resistant" cancers, tumor cells, and tumors refer to cancers that have become resistant to both apoptosis-mediated (e.g., through death receptor cell signaling, for example, Fas ligand receptor, TRAIL receptors, TNF-Rl, chemotherapeutic drugs, radiation, etc.) and non-apoptosis mediated (e.g., toxic drugs, chemicals, etc.) cancer therapies including, but not limited to, chemotherapy, hormonal therapy, radiotherapy, immunotherapy, and combinations thereof.
[0052] "Therapeutic treatment" and "cancer therapies" refers to apoptosis-mediated and non-apoptosis mediated cancer therapies including, without limitation, chemotherapy, hormonal therapy, radiotherapy, immunotherapy, and combinations thereof.
[0053] By "therapeutically effective amount or dose" or "sufficient amount or dose" herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).
[0054] The term "biological sample" includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include blood and blood fractions or products {e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells {e.g., primary cultures, explants, and transformed cells), stool, urine, other biological fluids {e.g., prostatic fluid, gastric fluid, intestinal fluid, renal fluid, lung fluid, cerebrospinal fluid, and the like), etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
[0055] A "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated {e.g., prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, etc.), the size and type of the tumor {e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. A diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core- needle biopsy" of the tumor mass, or a "fine-needle aspiration biopsy" which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, for example, in Harrison 's Principles of Internal Medicine, Kasper, et al, eds., 16th ed., 2005, Chapter 70, and throughout Part V.
[0056] The terms "cancer-associated antigen," "tumor-specific marker," or "tumor marker" interchangeably refers to a molecule (typically protein, carbohydrate, or lipid) that is preferentially expressed in a cancer cell in comparison to a normal cell, and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. A marker or antigen can be expressed on the cell surface or intracellularly. Oftentimes, a cancer- associated antigen is a molecule that is overexpressed or stabilized with minimal degradation in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression, or more in comparison to a normal cell. Oftentimes, a cancer-associated antigen is a molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions, or mutations in comparison to the molecule expressed on a normal cell. Oftentimes, a cancer-associated antigen will be expressed exclusively in a cancer cell and not synthesized or expressed in a normal cell. Exemplified cell surface tumor markers include the proteins c-erbB-2 and human epidermal growth factor receptor (HER) for breast cancer, PSMA for prostate cancer, and carbohydrate mucins in numerous cancers, including breast, ovarian, and colorectal. Exemplified intracellular tumor markers include, for example, mutated tumor suppressor or cell cycle proteins, including p53.
[0057] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (e.g., about 60% identity, preferably about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like). Such sequences are then said to be "substantially identical." This definition also refers to, or may be applied to, the complement of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or over a region that is about 50-100 amino acids or nucleotides in length.
[0058] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0059] A "comparison window," as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from about 20 to about 600, usually from about 50 to about 200, more usually from about 100 to about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoI Biol, 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat 'I. Acad. ScL USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al, eds. 1987-2005, Wiley Interscience)).
[0060] A preferred example of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al, J. MoI. Biol, 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the present invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff, Proc. Natl. Acad. ScL USA, 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
[0061] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally-occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
[0062] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et ah, J.
Biol. Ghent, 260:2605-2608 (1985); Rossolini et al, MoI. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
[0063] A particular nucleic acid sequence also implicitly encompasses "splice variants" and nucleic acid sequences encoding truncated forms of PSMA, e.g., nucleic acid sequences encoding the extracellular domain of PSMA. Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by a splice variant or truncated form of that nucleic acid. "Splice variants," as the name suggests, are products of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. Nucleic acids can be truncated at the 5'-end or at the 3 '-end. Polypeptides can be truncated at the N- terminal end or the C-terminal end. Truncated versions of nucleic acid or polypeptide sequences can be naturally-occurring or recombinantly created.
[0064] The term "peptide" refers to a compound made up of a single chain of D- or L- amino acids or a mixture of D- and L-amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length. Preferably, the PSMA peptides of the present invention that bind to cell surface PSMA (i.e., PSMA peptides that bind to cells overexpressing PSMA on the cell surface) are about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. Methods for synthesizing, expressing, and/or purifying peptides are well known in the art and are described below.
[0065] The term "protein" refers to a compound that is composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation. "Protein" encompasses "polypeptide," although polypeptides need not have a well-defined conformation. Proteins and polypeptides, as opposed to peptides, generally consist of chains of about 50 or more amino acids, e.g., about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or more amino acids in length. Typically, polypeptides comprise one or more domains or regions of a full- length protein. For example, the PSMA polypeptides of the present invention can comprise the entire extracellular domain of PSMA (amino acids 44-750) or 1, 2, 3, 4, 5, or more domains within the extracellular domain, such the dimerization domain (amino acids 601- 750). Methods for synthesizing, expressing, and/or purifying proteins and polypeptides are well known in the art and are described below.
[0066] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ- carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid.
[0067] Amino acids may be referred to. herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0068] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids that encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences. [0069] As to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the present invention.
[0070] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) {see, e.g., Creighton, Proteins (1984)).
[0071] A "label," "detectable moiety," or "imaging agent" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. A detectable moiety can be coupled either directly or indirectly to the PSMA polypeptide or peptide fragment described herein using methods well known in the art. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes {e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers {e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes {e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, electron-dense reagents, biotin, digoxigenin, haptens, and the like.
[0072] The term "radionuclide" refers to a nuclide that exhibits radioactivity. A "nuclide" refers to a type of atom specified by its atomic number, atomic mass, and energy state, such as carbon 14 (14C). "Radioactivity" refers to the radiation, including alpha particles, beta particles, nucleons, electrons, positrons, neutrinos, and gamma rays, emitted by a radioactive substance. Radionuclides suitable for use in the present invention include, but are not limited to, fluorine 18 (18F), phosphorus 32 (32P), scandium 47 (47Sc), cobalt 55 (55Co), copper 60 (60Cu), copper 61 (61Cu)3 copper 62 (62Cu), copper 64 (64Cu), gallium 66 (66Ga), copper 67 C'Cu), gallium 67 (0Oa), gallium 68 (MGa), rubidium 82 (82Rb), yttrium 86 (86Y), yttrium 87 (87Y), strontium 89 (89Sr), yttrium 90 (90Y), rhodium 105 (105Rh), silver 111 (111Ag), indium 111 (111In), iodine 124 (124T), iodine 125 (125I), iodine 131 (131I), tin 117m (117mSn), technetium 99m (99mTc), promethium 149 (149Pm), samarium 153 (153Sm), holmium 166 (166Ho), lutetium 177 (177Lu), rhenium 186 (186Re), rhenium 188 (188Re), thallium 201 (201Tl), astatine 211 (211At), and bismuth 212 (212Bi). As used herein, the V in117mSn and99mTc stands for meta state. Additionally, naturally-occurring radioactive elements such as uranium, radium, and thorium, which typically represent mixtures of radioisotopes, are suitable examples of radionuclides.
[0073] As described herein, compositions comprising a radionuclide coupled to a polypeptide corresponding to the extracellular domain of PSMA or a peptide fragment thereof that binds to cell surface PSMA are particularly useful for therapeutic, imaging, diagnostic, or prognostic purposes in a subject. The radionuclide can be directly coupled to the PSMA polypeptide or peptide fragment, directly coupled to a linking group (e.g., a peptide linking group), or bound to a chelating agent. Methods for coupling radionuclides to proteins or linking groups or binding radionuclides to chelating agents are known to one of skill in the art. In certain instances, the therapeutic compositions of the present invention comprise PSMA polypeptides or peptides conjugated to a bifunctional chelating agent that contains a radionuclide such as47Sc,64Cu,67Cu,89Sr,86Y,87Y,90Y,105Rh,111Ag,111In,117mSn,149Pm,153Sm,166Ho,177Lu,186Re,188Re,211At, and/or212Bi bound thereto.
Alternatively, the therapeutic compositions of the present invention comprise PSMA polypeptides or peptides or linking groups conjugated thereto that are radiolabeled with a radionuclide such as F, I, I, and/or I. In certain other instances, the imaging compositions of the present invention comprise PSMA polypeptides or peptides conjugated to a bifunctional chelating agent that contains a radionuclide such as55Co,60Cu,61Cu,62Cu,64Cu,66Ga,67Cu,67Ga,68Ga,82Rb,86Y,87Y,90Y,111In,99mTc, and/or201Tl bound thereto. Alternatively, the imaging compositions of the present invention comprise PSMA polypeptides or peptides or linking groups conjugated thereto that are radiolabeled with a radionuclide such as18F and/or131I.
[0074] A "chelating agent" refers to a compound which binds to a metal ion, such as a radionuclide, with considerable affinity and stability. In addition, the chelating agents of the present invention are bifunctional, having a metal ion chelating group at one end and a reactive functional group capable of binding to peptides, polypeptides, or proteins at the other end. Methods for conjugating bifunctional chelating agents to peptides, polypeptides, or proteins are well known in the art. Suitable bifunctional chelating agents include, but are not limited to, 1 ,4,7, 10-tetraazacyclododecane-N,N',NII,Nl"-tetraacetic acid (DOTA), a bromoacetamidobenzyl derivative of DOTA (BAD), 1,4,8,11-tetraazacyclotetradecane- N,N',N",N'"-tetraacetic acid (TETA), diethylenetriaminepentaacetic acid (DTPA), the dicyclic dianhydride of diethylenetriaminepentaacetic acid (ca-DTPA), 2-(p- isothiocyanatobenzyl) diethylenetriaminepentaacetic acid (SCNBzDTPA), and 2-(p- isothiocyanatobenzyl)-5(6)-methyl-diethylenetriaminepentaacetic acid (MxDTPA) {see, e.g., Ruegg et al, Cancer Res., 50:4221-4226 (1990); DeNardo et al, Clin. Cancer Res., 4:2483- 2490 (1998)). Other chelating agents include EDTA, NTA, HDTA and their phosphonate analogs such as EDTP, HDTP, and NTP (see, e.g., Pitt et al, INORGANIC CHEMISTRY IN BIOLOGY AND MEDICINE, Martell, Ed., American Chemical Society, Washington, D.C., 1980, pp. 279-312; Lindoy, THE CHEMISTRY OF MACROCYCLIC LIGAND COMPLEXES, Cambridge University Press, Cambridge, 1989; Dugas, BIOORGANIC CHEMISTRY, Springer-Verlag, New York, 1989).
[0075] The term "nanoparticle" refers to a microscopic particle whose size is measured in nanometers, e.g., a particle with at least one dimension less than about 100 nm. Nanoparticles are particularly useful as detectable moieties because they are small enough to scatter visible light rather than absorb it. For example, gold nanoparticles possess significant visible light extinction properties and appear deep red to black in solution. As a result, compositions comprising PSMA polypeptides and peptides conjugated to nanoparticles can be used for the in vivo imaging of tumors or cancerous cells in a subject. Methods for attaching polypeptides or peptides nanoparticles are well known in the art and are described in, e.g., Liu et al., Biomacromolecules, 2:362-368 (2001); Tomlinson et al, Methods MoI. Biol, 303:51-60 (2005); and Tkachenko et al, Methods MoI Biol, 303:85-99 (2005). At the small end of the size range, nanoparticles are often referred to as clusters. Metal, dielectric, and semiconductor nanoparticles have been formed, as well as hybrid structures (e.g., core- shell nanoparticles). Nanospheres, nanorods, and nanocups are just a few of the shapes that have been grown. Semiconductor quantum dots and nanocrystals are examples of additional types of nanoparticles. Such nanoscale particles, when conjugated to a PSMA polypeptide or peptide of the present invention, can be used as imaging agents for the in vivo detection of tumor tissue such as prostate cancer tissue. Alternatively, nanoparticles can be used in therapeutic applications as drug carriers that, when conjugated to a PSMA polypeptide or peptide of the present invention, deliver chemotherapeutic agents, hormonal therapaeutic agents, radiotherapeutic agents, toxins, or any other cytotoxic or anti-cancer agent known in the art to cancerous cells that overexpress PSMA on the cell surface.
[0076] The term "recombinant," when used with reference, e.g., to a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.
[0077] The term "heterologous," when used with reference to portions of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). Examples of heterologous proteins suitable for use in the present invention include, but are not limited to, fusion proteins containing a subsequence that binds to the extracellular domain of PSMA fused to a reporter subsequence (e.g., green fluorescent protein, luciferase, horseradish peroxidase, alkaline phosphatase, etc.), a cytotoxic subsequence (e.g., bacterial toxins, etc.), or other sequences (e.g., Fc, HA, GST, etc.). Methods for constructing and expressing such heterologous proteins are well known to those skilled in the art.
[0078] As used herein, the term "administering" means oral administration, administration as a suppository, topical contact, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal or subcutaneous administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra- arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc. By "co-administer" it is meant that a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA is administered at the same time, just prior to, or just after the administration of one or more additional cancer therapies such as chemotherapy, hormonal therapy, radiotherapy, or immunotherapy.
III. Prostate Specific Membrane Antigen
[0079] Prostate specific membrane antigen (PSMA) is a metallopeptidase predominantly expressed in prostate cancer cells. PSMA was originally identified by the monoclonal antibody 7El 1 derived from immunization with a partially purified membrane preparation from the LNCaP prostatic adenocarcinoma cell line (Horoszewiez et al, Anticancer Res., 7:927-935 (1987)). A 2.65 kb cDNA fragment encoding the PSMA protein was cloned and subsequently mapped to chromosome 1 IpI 1.2 (Israeli et al., Cancer Res., 53:227-230 (1993); O'Keefe et al, Biochim. Biophys. Acta, 1443:113-127 (1998)).
[0080] Initial analysis of PSMA demonstrated the widespread expression within the cells of the prostatic secretory epithelium. Immunohistochemical staining demonstrated that PSMA was absent to moderate in hyperplastic and benign tissues, while malignant tissues stained with the greatest degree of intensity (Horoszewiez et al, supra). Subsequent investigations have recapitulated these results and evinced PSMA expression as a universal feature in practically every prostatic tissue examined to date. These reports further demonstrate that expression of PSMA increases precipitously in a manner proportional to tumor aggressiveness (Burger et al, Int. J. Cancer, 100:228-237 (2002); Chang et al, Cancer Res., 59:3192-3198 (1999); Chang et al, Urology, 57:1179-1183 (2001); Kawakami et al, Cancer Res., 57:2321-2324 (1997); Liu et al, Cancer Res., 57:3629-3634 (1997); Ross et al, CHn. Cancer Res., 9:6357-6362 (2003); Silver et al, Clin. Cancer Res., 3:81-85 (1997); Sweat s al, Urology, 52:637-640 (1998); Troyer et al, Int. J. Cancer, 62:552-558 (1995); Wright et al, Urology, 48:326-334 (1996)).
[0081] Consistent with the correlation between PSMA expression and tumor stage, increased levels of PSMA are associated with androgen-independent prostate cancer. Analysis of tissue samples from prostate cancer patients demonstrated elevated PSMA levels following physical castration or androgen-deprivation therapy. Unlike expression of PSA, which is downregulated following androgen ablation, PSMA expression is significantly increased in both primary and metastatic tumor specimens (Kawakami et al, supra; Wright et al, supra). Consistent with the elevated expression in androgen-independent tumors, PSMA transcription is also known to be downregulated by steroids, and administration of testosterone mediates a dramatic reduction in PSMA protein and rnRNA levels (Israeli et al, Cancer Res., 54:1807-1811 (1994); Wright et al, supra). PSMA is also highly expressed in secondary prostatic tumors and occult metastatic disease. Immunohistochemical analysis revealed relatively intense and homogenous expression of PSMA within metastatic lesions localized to lymph nodes, bone, soft tissue, and lungs, as compared to benign prostatic tissue (Chang et al, Urology, 57:1179-1183 (2001); Murphy et al, Cancer, 78:809-818 (1996); Sweat et al, Urology, 52:637-640 (1998)).
[0082] Some reports have also indicated PSMA expression in extraprostatic tissues, including a subset of renal proximal tubules, cells of the intestinal brush border, and cells in the colonic crypt (Chang et al, Cancer Res., 59:3192-3198 (1999); Horoszewiez et al, supra; Israeli et al, Cancer Res., 54:1807-1811 (1994); Lopes et al, Cancer Res., 50:6423- 6429 (1990); Troyer et al, Int. J. Cancer, 62:552-558 (1995)). PSMA is also expressed in the tumor-associated neo vasculature of most solid cancers, but is absent in the normal vascular endothelium (Chang et al, Cancer Res., 59:3192-3198 (1999); Liu et al, supra; Silver et al, supra). Although the significance of PSMA expression within the vasculature is unknown, the specificity of tumor-associated endothelium expression of PSMA makes it an important target for the treatment of many forms of malignancies.
[0083] The expression of PSMA and its upregulation in advanced carcinoma and metastatic disease indicate a promising role for PSMA as a clinical biomarker for the diagnosis, detection, and treatment of cancers such as prostate cancer. For example, immunoscintographic scanning can be performed using an111In labeled form of the 7El 1 monoclonal antibody for the detection of PSMA-expressing tumor cells. However, positive signals detected with the 7El 1 monoclonal antibody are likely ascribed to immunoreactivity with dead or dying cells within a tumor mass because this antibody recognizes an intracellular epitope and is incapable of binding to viable cells. In addition to imaging strategies, PSMA-specific antibodies are also being used for therapeutic purposes, e.g., by conjugating them to radionuclides or cytotoxic drugs. However, current immunotherapeutic approaches using anti-PSMA antibodies are inadequate for the treatment of cancers such as prostate cancer due to antibody delivery problems and toxic side-effects. A. Structure of PSMA
[0084] The PSMA gene consists of 19 exons that span approximately 60 kb of genomic DNA. As shown in Figure 1, this gene encodes a type II transmembrane protein with a short, N-terminal cytoplasmic tail (amino acids 1-19), a single hydrophobic transmembrane domain (amno acids 20-43), and a large extracellular domain (amino acids 44-750) at the C-terminus (Israeli et al, Cancer Res., 53:227-230 (1993); O'Keefe et al, Biochim. Biophys. Acta, 1443:113-127 (1998)). The extracellular domain contains two domains of unknown function that span amino acids 44-150 and 151-274; proline- and glycine-rich regions that span amino acids 145-172 and 249-273, respectively; a catalytic domain that spans amino acids 274-587; and a final domain of unknown function (amino acids 587-750) containing a helical dimerization domain that spans amino acids 601-750.
[0085] The extracellular domain of PSMA is highly glycosylated, with N-lmked oligosaccharides accounting for up to 25% of the molecular weight of the native protein (Holmes et al, Prostate Suppl, 7:25-29 (1996)). Regions within this domain share modest degrees of homology with the transferrin receptor (TfR) (Israeli et al. , Cancer Res. , 53 :227- 230 (1993)) and with members of the M28 family of co-catalytic aminopeptidases (Rawlings et al, Biochim. Biophys. Acta, 1339:247-252 (1997)). Although TfR has only a vestigial catalytic site, PSMA is known to possess both N-acetylated alpha-linked acidic dipeptidase (NAALADase) and folate hydrolase activities (Carter et al, Proc. Natl. Acad. ScL USA, 93:749-753 (1996); Pinto et al, Clin. Cancer Res., 2:1445-1451 (1996)). These two related hydrolase activities hydrolyze gamma-peptide bonds between N-acetylaspartate and glutamate in the abundant neuropeptide N-acetylaspartylglutamate (NAAG) and the gamma- glutamyl linkages in pteroylpolyglutamate, respectively. Thus, this enzyme has been alternatively referred to as both glutamate carboxypeptidase II (GCPII) and folate hydrolase I (FOLHl). The enzymatic activity of PSMA is largely inhibited by phosphate, even at millimolar concentrations (Slusher et al, Nat. Med., 5:1396-1402 (1999)), and is dependent upon glycosylation and dimerization for proper function (Ghosh et al, Prostate, 57:140-151 (2003); Schulke et al, Proc. Natl. Acad. Sd. USA, 100:12590-12595 (2003)). In spite of being only 19 amino acids in length, the cytoplasmic domain of PSMA interacts with a number of proteins and has a major impact on the localization and molecular properties of PSMA (Anilkumar et al, Cancer Res., 63:2645-2648 (2003); Rajasekaran et al, MoI Biol. Cell, 14:4835-4845 (2003)). [0086] Evidence using RT-PCR indicates the existence of alternative PSMA isoforms, including PSM1, PSM-B, and PSM-C. In contrast to the integral transmembrane orientation of full-length PSMA, these variants are believed to exist within the cytosol and are thought to be the consequence of alternative splicing of the PSMA gene (Schmittgen et al, Int. J. Cancer, 107:323-329 (2003); Su et al, Cancer Res., 55:1441-1443 (1995)).
B. Dimerization of PSMA
[0087] Homodimerization is a fundamental feature of many transmembrane receptors. Homodimer formation is often induced by ligand binding, which is in turn necessary for mediating the cellular response of the receptor (Schlessinger, Cell, 110:669-672 (2002)). The TfR is an archetypal example of one such receptor. This type II transmembrane protein is involved in regulating cellular iron homeostasis through binding and internalization of iron- laden transferrin (Aisen, Int. J. Biochem. Cell Biol, 36:2137-2143 (2004)).
[0088] PSMA shares homology with TfR, both at the level of amino acid identity and at the level of domain organization (Mahadevan et al, Protein ScI, 8:2546-2549 (1999)). Like TfR, PSMA is expressed as a non-covalently linked homodimer on the cell surface
(Lawrence et al, Science, 286:779-782 (1999); Schulke et al, Proc. Natl. Acad. Sd. USA, 100:12590-12595 (2003)). This dimerization is mediated by epitopes within the large extracellular domain, as truncated versions of PSMA lacking the cytoplasmic and transmembrane domains are still capable of interacting {see, Example 1 below). PSMA dimerization is critical to maintain the conformation and enzymatic activity of PSMA
(Schulke et al, supra). Although the possibility has yet to be fully addressed, the similarity between PSMA and TfR at the amino acid and structural level, combined with the common dimerization requirement, indicate that these proteins share similar receptor and ligand transport functions.
C. Resemblance of PSMA Cellular Trafficking with Membrane Receptors
[0089] A variety of transmembrane receptors and membrane components are internalized from the plasma membrane and trafficked through the endocytic system. This endocytic trafficking allows cells to maintain homeostasis and internalize vital nutrients, lipids, and proteins. For example, binding of iron-bound transferrin to TfR results in an induction of receptor internalization and iron transport into the cell (Klausner et al, Proc. Natl. Acad. Sd. USA, 81:3005-3009 (1984)). Additionally, endocytosis of membrane receptors is also an established mechanism to downregulate signal transduction cascades. One classical example is the regulation of epidermal growth factor receptor (EGFR) signaling. Binding of epidermal growth factor (EGF) induces EGFR endocytosis and signal attenuation (Chang et a!., J. Biol. Chem., 268:19312-19320 (1993)).
[0090] Like the transferrin and EGF receptors, PSMA undergoes endocytosis from the plasma membrane. This endocytosis occurs through clathrin-coated pits and involves the first five N-terminal amino acids of the cytoplasmic tail. This motif of MWNLL appears to constitute a novel endocytic-targeting signal and likely interacts with the AP-2 adaptor protein complex (Rajasekaran et al, supra). Although PSMA is constitutively internalized from the cell surface, binding of antibodies or related antibody fragments to the extracellular domain increases the rate of PSMA internalization (Liu et al, Cancer Res., 58:4055-4060 (1998)). These antibodies may be acting like a natural ligand, thus indicating that, like TfR, PSMA may have a receptor function involved in endocytosis of a putative unknown ligand.
[0091] Interestingly, the NAALADase activity of PSMA is inhibited by the millimolar concentration of phosphate present in culture media (Tiffany et al, Eur. J. Pharmacol., 427:91-96 (2001)). Since internalization assays are performed under normal culture conditions, NAALADase activity is not required for the internalization function of PSMA. In addition, NAAG, a well-known substrate of PSMA, does not increase the rate of PSMA internalization in prostate cancer cells.
[0092] Following endocytosis, a number of receptors are recycled back to the plasma membrane surface. While some proteins are recycled directly from early endosomes, other receptors are first targeted to a tubulovesicular membrane structure proximal to the centrosomes referred to as the recycling endosomal compartment (REC) (Mukherjee et al, Physiol. Rev., 77:759-803 (1997); Ren et al, Proc. Natl. Acad. Sd. USA, 95:6187-6192 (1998)). TfR is one of the best-studied markers for the REC. Upon internalization, PSMA is targeted to the REC with similar kinetics to TfR (Anilkumar et al, supra; Rajasekaran et al, supra). As such, the antibody induced, clathrin-mediated internalization of PSMA and the accumulation in the REC supports the hypothesis that PSMA might function as a receptor internalizing a putative ligand.
D. Association of PSMA with Filamin A [0093] Filamin A (FLNa) is a dimeric actin cross-linking phosphoprotein that plays a vital role in the stabilization of many receptors at the plasma membrane (Stossel et al, Nat. Rev. MoI. Cell. Biol, 2: 138-145 (2001)). It is known that many membrane receptors like the metabotropic glutamate receptor, dopamine receptor, calcitonin receptor, tumor necrosis factor receptor, and insulin receptor interact with FLNa. The interaction between FLNa and these receptors plays a crucial role in modulating receptor function (Enz, FEBS Lett., 514:184-188 (2002); He et ah, J. Biol. Chem., 278:27096-27104 (2003); Seek et ah, J. Biol. Chem., 278:10408-10416 (2003)).
[0094] Using the N-terminal 19 amino acids as bait, the cytoplasmic domain was shown to interact with the 23rd to 24th repeat of FLNa in a yeast two-hybrid assay. When expressed in a filamin-negative cell line, PSMA was rapidly internalized from the cell surface. However, ectopic expression of FLNa in these cells resulted in a 50% reduction in the rate of PSMA internalization. These data suggest that FLNa may stabilize PSMA at the cell surface by tethering it to the actin cytoskeleton, likely preventing AP-2 from binding. Interestingly, expression of FLNa also reduced the NAALADase activity of PSMA at the cell surface, perhaps by inducing a conformational change in the extracellular domain (Anilkumar et al., Cancer Res., 63:2645-2648 (2003)). These data suggest that competitive binding of AP-2 and FLNa to the PSMA cytoplasmic tail regulates endocytosis, recycling, and the enzymatic activities of PSMA. Furthermore, the fact that the glutamate receptor, a protein that transports glutamate, and PSMA, an enzyme that releases glutamate, both bind to FLNa raises the intriguing possibility that PSMA and the glutamate receptor exist as a multiprotein complex on the plasma membrane. This interaction would facilitate the generation and transport of glutamate into the cell.
[0095] FLNa is also known to play a role in cell adhesion and motility. Calderwood and colleagues have shown that β integrin interacts with FLNa and this interaction is inhibitory to cell migration (Calderwood et al., Nat. Cell Biol., 3:1060-1068 (2001)). Another binding partner for FLNa is RaIA, a small GTP binding protein known to play a role in filopodia formation (Ohta et al., Proc. Natl. Acad. Sd. USA, 96:2122-2128 (1999)). Although an accumulation of PSMA in filopodial structures has been observed, it is not known whether PSMA plays a role in cell migration.
E. Resemblance of PSMA to Multifunctional Peptidases
[0096] Numerous examples indicate a role for enzymatic peptidases in mediating cell migration by affecting signaling cascades. For example, the interaction of the neutral endopeptidase (NEP) cytoplasmic tail with Lyn kinases blocks the activation of PI3 -kinase, thereby preventing FAK phosphorylation-mediated cell migration (Sumitomo et ah, J. Clin. Invest, 106:1399-4407 (2000)). NEP is also known to inhibit the proliferation of prostate epithelial cells by its direct association with PTEN (Sumitomo et al, Cancer Cell, 5:67-78 (2004)). PTEN is a lipid and protein phosphatase, which inhibits PI3-kinase mediated activation of Akt, a kinase involved in cell survival. A catalytic mutant of NEP could also block the cell proliferation and migration, suggesting that the enzymatic activity was not required. Mutational analysis of the cytoplasmic tail of NEP identified a basic amino acid rich motif containing five lysine and arginine residues proximal to the transmembrane domain that mediates the interaction between NEP and PTEN (Sumitomo et al, supra). The cytoplasmic tail of PSMA also has a stretch of three basic arginine residues proximal to the transmembrane domain, thus raising a possibility that PSMA may also interact with PTEN.
[0097] CD26 is another interesting example of a multifunctional type II cell surface glycoprotein with important roles in cell signaling. This molecule is expressed on a wide variety of cells and possesses dipeptidyl peptidase IV (DPIV) activity (Buhling et al, Immunol. Lett, 45:47-51 (1995); Buhling et al, Nat. Immun., 13:270-279 (1994); Gorrell et al, Cell Immunol, 134:205-215 (1991)). CD26 has been shown to regulate cell migration and proliferation independent of its enzymatic activity (Gorrell et al, Scand. J. Immunol, 54:249-264 (2001)). CD26 is known to bind adenosine deaminase, an enzyme involved in irreversible deamination of adenosine, and this association has been shown to be essential for the promotion of cell proliferation and cytokine production (Kameoka et al, Science, 261 :466-469 (1993)). CD26 has also been shown to affect the migratory behavior of T cells through interactions with extracellular matrix proteins such as collagen and fibronectin (Hanski et al, Biol Chem. Hoppe Seyler, 366:1169-1176 (1985); Piazza et al, Biochem. J., 262:327-334 (1989)). These examples indicate that although PSMA is a peptidase, it can have multiple roles, not only as an enzyme but also as a protein with cell survival and migratory functions.
F. Potential Role of PSMA Enzyme Activity in Prostate Cancer
[0098] Increased PSMA enzymatic peptidase activity is associated with metastatic prostate cancer (Lapidus et al, Prostate, 45:350-354 (2000)).
[0099] The prostate gland is mainly composed of stromal, epithelial, and neuroendocrine cells. The cellular and tissue homeostasis in general is maintained by the dynamic balance of cell proliferation, differentiation, and apoptosis. This balance is generated by the continuous cross-talk among these cell populations (Sung et al, Differentiation, 70:506-521 (2002)). For this purpose, epithelial and stromal cells secrete various types of growth factors, chemokines, and neuropeptides (Wong et al, Int. Rev. Cytol, 199:65-116 (2000)). Deregulation in this paracrine communication can result in derangement of the prostate gland such as benign prostate hyperplasia and prostate carcinoma (Dawson et al, Br. J. Cancer, 90:1577-1582 (2004)). For example, the peptidase NEP normally acts to inhibit the migratory properties of prostate epithelial cells. NEP achieves the inhibition of prostatic epithelial cell migration by cleaving critical neuropeptides such as bombesin and endothelin, thereby preventing the relay of signal transduction mediated by G-protein coupled receptors (Sumitomo et al., J. Clin. Invest, 106:1399-4407 (2000)). CD26 is also involved in the regulation of paracrine signaling by promoting the cleavage of growth factors, chemokines, neuropeptides, and hormones, thus contributing to the regulation of T cell and monocyte migration (Gorrell et al, Scand. J. Immunol., 54:249-264 (2001)).
[0100] Like NEP and CD26, PSMA is also a type II transmembrane glycoprotein with co- catalytic metallopeptidase activity. The increased expression of PSMA in prostatic adenocarcinoma may indicate a role in the cleavage of signaling molecules involved in maintaining prostate gland architecture and function. As such, the overexpression of PSMA could disturb the growth balance of the prostate gland.
IV. Synthesis of PSMA Peptides and Polypeptides
[0101] Any of a variety of methods and devices known in the art can be used to synthesize, express, and/or purify the PSMA polypeptides and peptides of the present invention, e.g., a polypeptide comprising the extracellular domain of PSMA (amino acids 44-750) or the dimerization domain of PSMA (amino acids 601-750), or a peptide of at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids that corresponds to a portion of the PSMA dimerization domain and is capable of binding to PSMA on the cell surface.
[0102] The PSMA polypeptides and peptides described herein can be prepared via a wide variety of well-known chemical synthesis techniques. Polypeptides and peptides are typically synthesized in solution or on a solid support in accordance with conventional techniques (see, e.g., Merrifield, Am. Chem. Soc, 85:2149 2154 (1963)). Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols (see, e.g., Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., 1984). For example, solid phase synthesis of the PSMA polypeptides and peptides of the present invention can be performed by the sequential addition of the remaining amino acids in a sequence after the C- terminal amino acid of the sequence is attached to an insoluble support. Using solid phase synthesis methods, one or more D-amino acids can be inserted, instead of L-amino acids, into a PSMA peptide or polypeptide at any desired location or locations, e.g., to increase its stability. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid- Phase Peptide Synthesis, pp. 3-284, in The Peptides: Analysis, Synthesis, Biology, Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al, J. Am. Chem. Soc, 85:2149- 2156 (1963); and Stewart et al, Solid Phase Peptide Synthesis, 2nd ed., 1984.
[0103] After chemical synthesis, the PSMA polypeptide or peptide may possess a conformation substantially different than the native conformation of the constituent polypeptide. In this case, it is helpful to denature and reduce the polypeptide or peptide and then to cause it to refold into the preferred conformation. Methods of reducing and denaturing polypeptides or peptides and inducing refolding are well known to those of skill in the art {see, e.g., Debinski et al, J. Biol. Chem., 268:14065-14070 (1993); Kreitman et al, Bioconjug. Chem., 4:581-585 (1993); Buchner et al, Anal Biochem., 205:263-270 (1992)). For example, Debinski et al describes the denaturation and reduction of inclusion body polypeptides in guanidine-DTE. The polypeptide is then refolded in a redox buffer containing oxidized glutathione and L-arginine.
[0104] The present invention also relies on routine techniques in the field of recombinant expression of proteins in prokaryotic and eukaryotic organisms. Basic texts disclosing the general methods of use in the present invention include, e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, 2nd ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Current Protocols in Molecular Biology, Ausubel et al, eds., 1994.
[0105] To obtain high level expression of nucleic acids encoding the PSMA polypeptides or peptides described herein, the nucleic acids are typically subcloned into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al and Ausubel et al, supra. Bacterial expression systems for expressing PSMA polypeptides and peptides are available in, e.g., E. coli, Bacillus sp., and Salmonella (see, e.g., Palva et al, Gene, 22:229- 235 (1983); Mosbach et al, Nature, 302:543-545 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. In one preferred embodiment, retroviral expression systems are used to synthesize and/or deliver the PSMA polypeptides and peptides of the present invention.
[0106] Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application. The promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function, hi addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the PSMA polypeptide- or peptide- encoding nucleic acid in host cells. A typical expression cassette thus contains a promoter operably linked to the nucleic acid sequence encoding PSMA polypeptides or peptides and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites. In addition to a promoter sequence, the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
[0107] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc, HA, 6xHis, etc. Sequence tags may be included in an expression cassette for nucleic acid rescue. Markers such as fluorescent proteins, green or red fluorescent protein, β-gal, CAT, luciferase, and the like can be included in the vectors as markers for vector transduction. [0108] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral vectors, and vectors derived from the Epstein-Barr virus. Other exemplary eukaxyotic vectors include pMSG, pAV009/A+, pMTO10/A+, ρMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, S V40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
[0109] Expression of proteins from eukaryotic vectors can also be regulated using inducible promoters. With inducible promoters, expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.
[0110] In one embodiment, the vectors of the present invention for expressing PSMA polypeptides or peptides that bind to cell surface PSMA have a regulatable promoter, e.g., tet- regulated systems and the RU-486 system (see, e.g., Gossen et al, Proc. Natl. Acad. ScL USA, 89:5547 (1992); Oligino et al, Gene Ther., 5:491-496 (1998); Wang et al, Gene Ther., 4:432-441 (1997); Neering et al, Blood, 88:1147-1155 (1996); Rendahl et al, Nat. Biotechnol, 16:757-761 (1998)). Some expression systems have markers that provide gene amplification such as thymidine kinase or dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a PSMA polypeptide- or peptide-encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoter.
[0111] The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences. The particular antibiotic resistance gene chosen is not critical, and any of the many resistance genes known in the art are suitable. The prokaryotic sequences are preferably chosen such that they do not interfere with the replication of the DNA in eukaryotic cells, if necessary.
[0112] Standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large quantities of PSMA polypeptides or peptides, which are then purified using standard techniques (see, e.g., Colley et al, J. Biol. Chem., 264:17619- 17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182, Deutscher, ed., 1990). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J Bact, 132:349-351 (1977); Clark-Curtiss et al, Methods in Enzymology, 101:347-362 (1983)).
[0113] Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include, but are not limited to, the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, biolistics, liposomes, microinjection, plasma vectors, viral vectors, and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into a host cell (see, e.g., Sambrook et al, supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one nucleic acid into the host cell capable of expressing PSMA polypeptides or peptides.
[0114] After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of PSMA polypeptides or peptides, which is recovered from the culture using standard techniques identified below.
[0115] The recombinant PSMA polypeptides and peptides described herein can be purified from any suitable expression system. Similarly, chemically synthesized PSMA polypeptides and peptides can also be purified.
[0116] The PSMA polypeptides and peptides of the present invention may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, immunopurification methods, and others (see, e.g., Scopes, Protein Purification: Principles and Practice, 1982; U.S. Patent No. 4,673,641; Ausubel et al, supra; Sambrook et al, supra). A number of procedures can be employed when a recombinant PSMA polypeptide or peptide is being purified. For example, proteins having established molecular adhesion properties can be reversibly fused to the PSMA polypeptide or peptide. With the appropriate ligand, PSMA polypeptides and peptides can be selectively adsorbed to a purification column and then freed from the column in a relatively pure form. The fused protein is then removed by enzymatic activity. Finally, PSMA polypeptides or peptides can be purified using immunoaffinity columns.
[0117] Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive. Promoter induction with IPTG is one example of an inducible promoter system. Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein. Proteins expressed in bacteria may form insoluble aggregates (inclusion bodies). Several protocols are suitable for purification of PSMA polypeptides or peptides from inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation, and/or purification of inclusion bodies by disruption of bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL pH 7.5, 50 mM NaCl, 5 mM MgC12, 1 mM DTT, 0.1 mM ATP, and 1 mM PMSF. The cell suspension can be lysed using 2-3 passages through a French Press, homogenized using a Polytron (Brinkman Instruments), or sonicated on ice. Alternate methods of lysing bacteria are apparent to those of skill in the art (see, e.g., Sambrook et al, supra; Ausubel et ah, supra).
[0118] If necessary, the inclusion bodies are solubilized, and the lysed cell suspension is typically centrifuged to remove unwanted insoluble matter. Proteins that formed the inclusion bodies may be renatured by dilution or dialysis with a compatible buffer. Suitable solvents include, but are not limited to, urea (e.g., from about 4 M to about 8 M), formamide (e.g., at least about 80% on a volume/volume basis), and guanidine hydrochloride (e.g., from about 4 M to about 8 M). Some solvents which are capable of solubilizing aggregate-forming proteins such as sodium dodecyl sulfate (SDS) and 70% formic acid are inappropriate for use in this procedure due to the possibility of irreversible denaturation of the proteins, accompanied by a lack of binding activity. Although guanidine hydrochloride and similar agents are denaturants, this denaturation is not irreversible and renaturation may occur upon removal (by dialysis, for example) or dilution of the denaturant, allowing reformation of biologically active protein. Other suitable buffers are known to those skilled in the art. PSMA polypeptides or peptides are separated from other bacterial proteins by standard separation techniques, e.g., with Ni-NTA agarose resin, glutathione agarose resin, etc.
[0119] Alternatively, it is possible to purify PSMA polypeptides and peptides from the bacterial periplasm. After lysis of the bacteria, when the PSMA polypeptide or peptide is exported into the periplasm of the bacteria, the periplasmic fraction of the bacteria can be isolated by cold osmotic shock in addition to other methods known to skill in the art. To isolate recombinant proteins from the periplasm, the bacterial cells are centrifuged to form a pellet. The pellet is resuspended in a buffer containing 20% sucrose. To lyse the cells, the bacteria are centrifuged and the pellet is resuspended in ice-cold 5 mM MgSO4 and kept in an ice bath for approximately 10 minutes. The cell suspension is centrifuged and the supernatant is decanted and saved. The recombinant proteins present in the supernatant can be separated from the host proteins by standard separation techniques well known to those of skill in the art.
[0120] Often as an initial step, particularly if the protein mixture is complex, an initial salt fractionation can separate many of the unwanted host cell proteins (or proteins derived from the cell culture media) from the recombinant protein of interest {e.g., a PSMA polypeptide or peptide of the present invention). In certain instances, the salt used for solubility fractionation is ammonium sulfate. Ammonium sulfate precipitates proteins by effectively reducing the amount of water in the protein mixture. Proteins then precipitate on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations. A typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration will precipitate the most hydrophobic of proteins. The precipitate is then discarded (unless the protein of interest is hydrophobic) and ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed, if necessary, either through dialysis or filtration. Other methods that rely on the solubility of proteins, such as cold ethanol precipitation, are well known to those of skill in the art and can also be used to fractionate complex protein mixtures.
[0121] The molecular weight of the PSMA polypeptides and peptides described herein can be used to isolate them from proteins of greater and lesser size using size differential ultrafiltration through membranes of different pore size {e.g., Amicon or Millipore membranes). As a first step, the protein mixture is ultrafiltered through a membrane with a pore size that has a lower molecular weight cut-off than the molecular weight of the protein of interest. The retentate of the ultrafiltration is then ultrafiltered against a membrane with a molecular cut off greater than the molecular weight of the protein of interest. The recombinant protein will pass through the membrane into the filtrate. The filtrate can then be chromatographed as described below.
[0122] The PSMA polypeptides and peptides of the present invention can also be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands. In addition, antibodies raised against PSMA polypeptides and peptides can be conjugated to column matrices and the proteins immunopurified. AU of these methods are well known in the art. It will be apparent to one of skill that chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech).
V. Diagnostic and Prognostic Methods
[0123] In certain aspects, the present invention provides methods of diagnosing or providing a prognosis for cancer, e.g., a cancer that overexpresses PSMA such as prostate cancer. As used herein, the term "providing a prognosis" refers to providing a prediction of the probable course and outcome of a cancer or the likelihood of recovery from the cancer. In certain instances, cancer patients with negative or low PSMA expression have a longer disease-specific survival as compared to those with high PSMA expression. As such, the level of PSMA expression can be used as a prognostic indicator, with negative or low expression as an indication of a good prognosis, e.g., a longer disease-specific survival.
[0124] The methods of the present invention can also be useful for diagnosing the severity of a cancer, e.g., a cancer that overexpresses PSMA. As a non-limiting example, the level of PSMA expression can be used to determine the stage or grade of a cancer such as prostate cancer, e.g., according to the Tumor/Nodes/Metastases (TNM) system of classification (International Union Against Cancer, 6th edition, 2002) or the Whitmore-Jewett staging system (American Urological Association). Typically, cancers are staged using a combination of physical examination, blood tests, and medical imaging. If tumor tissue is obtained via biopsy or surgery, examination of the tissue under a microscope can also provide pathologic staging. In certain instances, cancer patients with high PSMA expression have a more severe stage or grade of that type of cancer. As such, the level of PSMA expression can be used as a diagnostic indicator of the severity of a cancer or of the risk of developing a more severe stage or grade of the cancer. In certain other instances, the stage or grade of a cancer assists a practitioner in determining the prognosis for the cancer and in selecting the appropriate cancer therapy.
[0125] The diagnostic and prognostic methods of the present invention advantageously utilize novel PSMA polypeptides that correspond to the extracellular domain of PSMA or peptide fragments thereof that bind to cell surface PSMA. Such polypeptides and peptide fragments can be used to determine a level of PSMA expression in tumor tissue or cancerous cells and then compared to a baseline value or range. Typically, the baseline value is representative of PSMA expression levels in a healthy person not suffering from cancer. Variation of PSMA levels from the baseline range (i.e., either up or down) indicates that the subject has a cancer or is at risk of developing a cancer. In some embodiments, the level of PSMA expression is measured by taking a blood, urine, or tumor tissue sample from a subject and measuring the amount of PSMA in the sample using any number of detection methods known in the art. For example, a pull-down assay can be performed on samples such as serum or prostatic fluid using the PSMA polypeptides and peptide fragments described herein coupled to magnetic beads (e.g., Dynabeads®; Invitrogen Corp., Carlsbad, CA) to determine the level of PSMA expression.
[0126] The present invention is based, in part, on the surprising discovery that polypeptides corresponding to the extracellular domain of PSMA or peptide fragments thereof bind to PSMA on the surface of cancer cells with affinity and specificity comparable to that of antibodies directed against cell surface PSMA, without any of the delivery problems or toxic side-effects associated with immunotherapy. As a result, any antibody-based technique for determining a level of expression of a protein of interest can be used to measure the level of PSMA expression in tumor tissue or cancerous cells. For example, immunoassays such as ELISA assays, immunoprecipitation assays, and immunohistochemical assays can be modified by replacing the use of antibodies with the use of PSMA polypeptides or peptides of the present invention coupled to a detectable moiety. One skilled in the art will know of additional antibody-based techniques that can be modified for determining a level of PSMA expression according to the methods of the present invention.
[0127] In some embodiments, the expression of PSMA in a cancerous or potentially cancerous tissue may be evaluated by visualizing the presence and/or localization of PSMA in the subject. Any technique known in the art for visualizing tumors, tissues, or organs in live subjects can be used in the imaging methods of the present invention. Preferably, the in vivo imaging of cancerous or potentially cancerous tissue is performed using a polypeptide corresponding to the extracellular domain of PSMA or a peptide fragment thereof that binds to the surface of cells expressing PSMA, wherein the PSMA polypeptide or peptide fragment is linked to an imaging agent such as a detectable moiety (i.e., a contrast agent). A detectable moiety can be coupled either directly or indirectly to the PSMA polypeptide or peptide fragment described herein using methods well known in the art. A wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the PSMA polypeptide or peptide component, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides as described above, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers {e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes {e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.
[0128] The detectable moiety can be visualized in a subject using any device or method known in the art. For example, methods such as Single Photon Emission Computerized Tomography (SPECT), which detects the radiation from a single photon gamma-emitting radionuclide using a rotating gamma camera, and radionuclide scintigraphy, which obtains an image or series of sequential images of the distribution of a radionuclide in tissues, organs, or body systems using a scintillation gamma camera, may be used for detecting the radiation emitted from a detectable moiety linked to a PSMA peptide or polypeptide of the present invention. Positron Emission Tomography (PET) is another suitable technique for detecting radiation in a subject to visualize tumors in living patients according to the methods of the present invention. Furthermore, U.S. Patent No. 5,429,133 describes a laparoscopic probe for detecting radiation concentrated in solid tissue tumors. Miniature and flexible radiation detectors intended for medical use are produced by Intra-Medical LLC, Santa Monica, California. Magnetic Resonance Imaging (MRI) or any other imaging technique known to one of skill in the art {e.g., radiography {i.e., X-rays), computed tomography (CT), fluoroscopy, etc.) is also suitable for detecting the radioactive emissions of radionuclides.
[0129] Various in vivo optical imaging techniques that are suitable for the visualization of fluorescent and/or enzymatic labels or markers include, but are not limited to, fluorescence microendoscopy {see, e.g., Flusberg et ah, Optics Lett., 10:2212-221 A (2005)), fiber-optic fluorescence imaging {see, e.g., Flusberg et ah, Nature Methods, 2:941-950 (2005)), fluorescence imaging using a flying-spot scanner {see, e.g., Ramanujam et ah, IEEE Trans. Biomed. Eng, 48:1034-1041 (2001)), catheter-based imaging systems {see, e.g., Funovics et ah, Radiology, 231:659-666 (2004)), near-infrared imaging systems {see, e.g., Mahmood et ah, Radiology, 213:866-870 (1999)), fluorescence molecular tomography {see, e.g., Gurfinkel et ah, Dis. Markers, 19:107-121 (2004)), and bioluminescent imaging {see, e.g., Dikmen et ah, Turk. J. Med. ScL, 35:65-70 (2005)).
[0130] The PSMA polypeptides or peptide fragments of the present invention, when conjugated to any of the above-described detectable moieties, can be administered in doses effective to achieve the desired image of tumor tissue or cancerous cells in a subject. Such doses may vary widely, depending upon the particular detectable label employed, the type of tumor tissue or cancerous cells subjected to the imaging procedure, the imaging equipment being used, and the like. However, regardless of the detectable moiety or imaging technique used, such detection is aimed at determining where the PSMA polypeptide or peptide fragment is concentrated in a subject, with such concentration being an indicator of the location of a tumor or tumor cells. Alternatively, such detection is aimed at determining the extent of tumor regression in a subject, with the size of the tumor being an indicator of the efficacy of cancer therapy.
VI. Methods of Administration and Pharmaceutical Compositions
[0131] As described herein, polypeptides corresponding to the extracellular domain of PSMA or peptide fragments thereof that bind to PSMA on the surface of cells such as cancer cells are particularly useful in treating, imaging, diagnosing, and/or providing a prognosis for cancers such as prostate cancer. For therapeutic applications, the PSMA polypeptides and peptide fragments of the present invention can be administered alone or co-administered in combination with conventional chemotherapy, radiotherapy, hormonal therapy, and/or immunotherapy.
[0132] As a non-limiting example, PSMA polypeptides and peptide fragments can be coadministered with conventional chemotherapeutic agents including alkylating agents (e.g., cisplatin, cyclophosphamide, carboplatin, ifosfamide, chlorambucil, busulfan, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g., 5-fluorouracil, azathioprine, methotrexate, fludarabine, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), topoisomerase inhibitors (e.g., amsacrine, etoposide (VP 16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, plicamycin, etc.), and the like.
[0133] PSMA polypeptides and peptide fragments can also be co-administered with conventional hormonal therapaeutic agents including, but not limited to, steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, tamoxifen, and gonadotropin-releasing hormone agonists (GnRH) such as goserelin.
[0134] Additionally, PSMA polypeptides and peptide fragments can be co-administered with conventional immunotherapeutic agents including, but not limited to, immunostimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti- VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody- calicheamicin conjugate, anti-CD22 monoclonal antibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy (e.g., anti-CD20 monoclonal antibody conjugated to111In,90Y, or131I, etc.).
[0135] In a further embodiment, PSMA polypeptides and peptide fragments can be coadministered with conventional radiotherapeutic agents including, but not limited to, radionuclides such as47Sc,64Cu,67Cu,89Sr,86Y,87Y,90Y,105Rh,111Ag,111In,117mSn,149Pm, '53Sm, '66Ho,177Lu, '86Re, '88Re,211 At, and212Bi, optionally conjugated to antibodies directed against tumor antigens.
[0136] In some embodiments, the compositions of the present invention comprise the extracellular domain of PSMA or a fragment thereof and a physiologically (i.e., pharmaceutically) acceptable carrier. As used herein, the term "carrier" refers to a typically inert substance used as a diluent or vehicle for a drug such as a therapeutic agent. The term also encompasses a typically inert substance that imparts cohesive qualities to the composition. Typically, the physiologically acceptable carriers are present in liquid, solid, or semi-solid form. Examples of liquid carriers include physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), and the like. Examples of solid or semi-solid carriers include mannitol, sorbitol, xylitol, maltodextrin, lactose, dextrose, sucrose, glucose, inositol, powdered sugar, molasses, starch, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, acacia gum, guar gum, tragacanth gum, alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, Veegum®, larch arabogalactan, gelatin, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, polyacrylic acid (e.g., Carbopol), calcium silicate, calcium phosphate, dicalcium phosphate, calcium sulfate, kaolin, sodium chloride, polyethylene glycol, and combinations thereof. Since physiologically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989). [0137] The pharmaceutical compositions of the present invention may be sterilized by conventional, well-known sterilization techniques or may be produced under sterile conditions. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions can contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.
[0138] Formulations suitable for oral administration can comprise: (a) liquid solutions, such as an effective amount of a packaged PSMA polypeptide or peptide fragment suspended in diluents, e.g., water, saline, or PEG 400; (b) capsules, sachets, or tablets, each containing a predetermined amount of a PSMA polypeptide or peptide fragment, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise a PSMA polypeptide or peptide fragment in a flavor, e.g., sucrose, as well as pastilles comprising the polypeptide or peptide fragment in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like, containing, in addition to the polypeptide or peptide, carriers known in the art.
[0139] The PSMA polypeptide or peptide fragment of choice, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be
"nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
[0140] Suitable formulations for rectal administration include, for example, suppositories, which comprises an effective amount of a packaged PSMA polypeptide or peptide fragment with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons, hi addition, it is also possible to use gelatin rectal capsules which contain a combination of the PSMA polypeptide or peptide fragment of choice with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons.
[0141] Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intratumoral, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Injection solutions and suspensions can also be prepared from sterile powders, granules, and tablets. In the practice of the present invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally. Parenteral administration, oral administration, and intravenous administration are the preferred methods of administration. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
[0142] The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component, e.g., a PSMA polypeptide or peptide fragment. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form. The composition can, if desired, also contain other compatible therapeutic agents.
[0143] In therapeutic use for the treatment of cancer, the PSMA polypeptides or peptide fragments utilized in the pharmaceutical compositions of the present invention are administered at the initial dosage of about 0.001 mg/kg to about 1000 mg/kg daily. A daily dose range of about 0.01 mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, or about 1 mg/kg to about 100 mg/kg, or about 10 mg/kg to about 50 mg/kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the PSMA polypeptide or peptide fragment being employed. For example, dosages can be empirically determined considering the type and stage of cancer diagnosed in a particular patient. The dose administered to a patient, in the context of the present invention, should be sufficient to affect a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular PSMA polypeptide or peptide fragment in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the PSMA polypeptide or peptide fragment. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
[0144] One factor in the administration of the PSMA polypeptide and peptide compounds of the present invention is ensuring that the polypeptide or peptide fragment has the ability to traverse the plasma membrane of a cell, or a tight junction, which maintains a barrier that prevents diffusion of molecules across the epithelial cell layer, or the membrane of an intracellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides across a cell membrane.
[0145] For example, "membrane translocation polypeptides" have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology, 6:629-634 (1996)). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem., 270:1 4255-14258 (1995)).
[0146] Examples of peptide sequences that can be linked to a PSMA polypeptide or peptide of the present invention, e.g., for facilitating the uptake of proteins and peptides into cells, include, but are not limited to, an 11 amino acid peptide of the tat protein of HIV, a 20 residue peptide sequence which corresponds to amino acids 84-103 of the pi 6 protein (see, e.g., Fahraeus et al, Current Biology, 6:84 (1996)), the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al, J. Biol. Chem., 269:10444 (1994)), the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al, supra), and the VP22 translocation domain from HSV (Elliot et al, Cell, 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to the PSMA polypeptides or peptides described herein.
[0147] Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules are composed of at least two parts (called "binary toxins"): a translocation or binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, other clostridial toxins, diphtheria toxin, Pseudomonas exotoxin A, pertussis toxin, botulinum toxin, tetanus toxin, Bacillus anthracis toxin, and pertussis adenylate cyclase, can be used to deliver peptides or polypeptides to the cell cytosol as internal or amino-terminal fusions {see, e.g., Arora et al, J. Biol Chem., 268:3334-3341 (1993); Perelle et al, Infect. Immun., 61:5147-5156 (1993); Stenmark et al, J. Cell Biol, 113:1025-1032 (1991); Donnelly et al, Proc. Natl Acad. ScL U.S.A., 90:3530-3534 (1993); Carbonetti et al, Abstr. Annu. Meet. Am. Soc. Microbiol, 95:295 (1995); Sebo et al, Infect. Immun., 63:3851-3857 (1995); Klimpel et al, Proc. Natl. Acad. Sd. U.S.A., 89:10277-10281 (1992); and Novak et al, J. Biol Chem., 267:17186-17193 (1992)).
[0148] Such toxin translocation domains can be used to translocate the PSMA polypeptides and peptides of the present invention across a cell membrane, tight junction, or organelle membrane. The PSMA polypeptides and peptides described herein can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the PSMA polypeptide or peptide and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
[0149] The toxin domain from any of the bacterial toxins described above can be used as a cytotoxic agent for the treatment of cancers such as prostate cancer when conjugated to the PSMA polypeptides and peptides of the present invention. The PSMA polypeptides and peptides can be conveniently fused to or derivatized with such sequences. Typically, the toxin sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the PSMA polypeptide or peptide and the toxin sequence. Any suitable linker can be used, e.g., a peptide linker.
[0150] In a preferred embodiment, the PSMA polypeptides and peptides of the present invention (e.g., soluble polypeptides and peptides that bind to PSMA located on the surface of cells such as cancer cells) are delivered using retroviral vectors (i.e., retroviruses) as described in Morizono et al, Cell Cycle, 4:854-856 (2005). Briefly, oncoretroiviral or lentiviral vectors are pseudotyped with a chimeric Sindbis virus envelope protein in which an Fc binding region has been inserted into the receptor binding region of the envelope protein. A heterologous protein containing a fusion between the Fc region and a PSMA polypeptide or peptide of the present invention is then expressed on (e.g., bound to) the surface of the retroviral vector through the interaction of the Fc region with the Fc binding region of the chimeric Sindbis virus envelope protein. Alternatively, nucleic acid encoding the PSMA polypeptide or peptide can be introduced into the retroviral vector and expressed on the surface of the retrovirus. When the resulting retroviral vector is delivered to a subject, e.g., by intravenous injection, it will be specifically targeted to cells such as cancer cells that express PSMA on the cell surface. For the in vivo detection of tumors or cancerous cells, the retroviral vector can contain one or more of the imaging agents described above. Alternatively, the retroviral vector can be packaged with one or more of the chemotherapeutic agents, hormonal therapaeutic agents, radiotherapeutic agents, or toxins described above or any other cytotoxic or anti-cancer agent known in the art for the treatment of cancers such as prostate cancer.
[0151] The pharmaceutical preparations are typically delivered to a mammal, including humans and non-human mammals. Non-human mammals treated or imaged using the present compositions include domesticated animals (e.g., canine, feline, murine, rodentia, lagomorpha, etc.) and agricultural animals (bovine, equine, ovine, porcine, etc).
VIL Kits
[0152] The present invention also provides kits for carrying out the therapeutic, diagnostic, prognostic, and imaging assays described herein. The kits will typically be comprised of one or more containers containing PSMA polypeptides or peptide fragments thereof that bind to cell surface PSMA, e.g., in dehydrated form, with instructions for their rehydration and administration. For example, one container of a kit may hold the dehydrated PSMA polypeptides or peptides and another container may hold a buffer suitable for rehydrating the polypeptides or peptides. Kits can include any of the compositions noted above, and optionally further include additional components such as instructions to practice the desired method, control polypeptides or peptides such as scrambled PSMA sequences, a robotic armature for mixing kit components, and the like.
VIII. Screening Methods
[0153] The present invention also provides methods of identifying compounds that inhibit cancer growth or progression, for example, by potentiating the binding of a soluble extracellular domain of PSMA or a fragment thereof to PSMA located on the surface of cells such as cancer cells. As a result, the identified compounds find use in inhibiting the growth of and promoting the regression of a tumor by promoting the interaction between the PSMA polypeptides and peptides of the present invention and cell surface PSMA. The identified compounds can inhibit cancer growth or progression alone, or when used in combination with other cancer therapies, including chemotherapies, radiation therapies, hormonal therapies, immunotherapies, and combinations thereof.
[0154] Using the assays described herein, one can identify lead compounds that are suitable for further testing to identify those that are therapeutically effective modulating agents by screening a variety of compounds and mixtures of compounds for their ability to potentiate the binding of the PSMA polypeptides and peptides described herein to cells overexpressing PSMA on the cell surface. Compounds of interest can be either synthetic or naturally- occurring.
[0155] Screening assays can be carried out in vitro or in vivo. Typically, initial screening assays are carried out in vitro, and can be confirmed in vivo using cell based assays or animal models. For instance, compounds that potentiate the binding of the PSMA polypeptides and peptides of the present invention to PSMA located on the surface of cells such as cancer cells can promote cellular apoptosis resulting from the increased binding interaction in comparison to cells unexposed to the test compound.
[0156] The screening methods are designed to screen large chemical or polymer {e.g., peptides, small organic molecules, etc.) libraries by automating the assay steps and providing compounds from any convenient source to the assays, which are typically run in parallel {e.g., in microtiter formats on microtiter plates in robotic assays). [0157] The present invention also provides in vitro assays in a high throughput format. For each of the assay formats described, "no modulator" control reactions, which do not include a modulator, provide, e.g., a background level of the binding interaction of a soluble extracellular domain of PSMA or a fragment thereof to cell surface PSMA. In the high throughput assays of the present invention, it is possible to screen up to several thousand different modulators in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different compounds. It is possible to assay many different plates per day; assay screens for up to about 6,000-20,000, and even up to about 100,000-1,000,000 different compounds is possible using the integrated systems of the present invention. The steps of labeling, addition of reagents, fluid changes, and detection are compatible with full automation, for instance, using programmable robotic systems or "integrated systems" commercially available, for example, through BioTX Automation, Conroe, TX; Qiagen, Valencia, CA; Beckman Coulter, Fullerton, CA; and Caliper Life Sciences, Hopkinton, MA.
[0158] Essentially, any chemical compound can be tested as a potential modulator of the binding between a soluble extracellular domain of PSMA or a fragment thereof and cells overexpressing PSMA on the cell surface. Most preferred are generally compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions. It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland), as well as providers of small organic molecule and peptide libraries ready for screening, including Chembridge Corp. (San Diego, CA), Discovery Partners International (San Diego, CA), Triad Therapeutics (San Diego, CA), Nanosyn (Menlo Park, CA), Affymax (Palo Alto, CA), ComGenex (South San Francisco, CA), and Tripos, Inc. (St. Louis, MO).
[0159] In one preferred embodiment, modulators of PSMA polypeptide or peptide binding to cell surface PSMA are identified by screening a combinatorial library containing a large number of potential therapeutic compounds (potential modulator compounds). Such "combinatorial chemical or peptide libraries" can be screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
[0160] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length {i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
[0161] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art {see, for example, Beeler et ah, Curr Opin Chem Biol. 9:277 (2005) and Shang and Tan, Curr Opin Chem Biol. 9:248 (2005). Libraries of use in the present invention can be composed of amino acid compounds, carbohydrates, or small organic compounds. Carbohydrate libraries have been described in, for example, Liang et ah, Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853.
[0162] Representative amino acid compound libraries include, but are not limited to, peptide libraries {see, e.g., U.S. Patent Nos. 5,010,175, 6,828,422, and 6,844,161; Furka, /nt. J. Pept. Prot. Res., 37:487-493 (1991); Houghton et ah, Nature, 354:84-88 (1991); and Eichler, Comb Chem High Throughput Screen., 8:135 (2005)), peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio- oligomers (PCT Publication No. WO 92/00091), vinylogous polypeptides (Hagihara et ah, J. Amer. Chem. Soc, 114:6568 (1992)), nonpeptidal peptidomimetics with β-D-glucose scaffolding (Hirschrnann et ah, J. Amer. Chem. Soc, 114:9217-9218 (1992)), peptide nucleic acid libraries {see, e.g., U.S. Patent 5,539,083), antibody libraries {see, e.g., U.S. Patent Nos. 6,635,424 and 6,555,310; PCT/US96/10287; and Vaughn et ah, Nature Biotechnology, 14(3):309-314 (1996)), and peptidyl phosphonates (Campbell et ah, J. Org. Chem., 59:658 (1994)).
[0163] Representative small organic molecule libraries include, but are not limited to, diversomers such as hydantoins, benzodiazepines, and dipeptides (Hobbs et ah, Proc. Nat. Acad. ScL USA, 90:6909-6913 (1993)); analogous organic syntheses of small compound libraries (Chen et ah, J. Amer. Chem. Soc, 116:2661 (1994)); oligocarbamates (Cho et ah, Science, 261:1303 (1993)); benzodiazepines {e.g., U.S. Patent No. 5,288,514; and Baum, C&EN, Jan 18, page 33 (1993)); isoprenoids (e.g., U.S. Patent No. 5,569,588); thiazolidinones and metathiazanones (e.g., U.S. Patent No. 5,549,974); pyrrolidines (e.g., U.S. Patent Nos. 5,525,735 and 5,519,134); morpholino compounds (e.g., U.S. Patent No. 5,506,337); tetracyclic benzimidazoles (e.g., U.S. Patent No. 6,515,122); dihydrobenzpyrans (e.g., U.S. Patent No. 6,790,965); amines (e.g., U.S. Patent No. 6,750,344); phenyl compounds (e.g., U.S. Patent No. 6,740,712); azoles (e.g., U.S. Patent No. 6,683,191); pyridine carboxamides or sulfonamides (e.g., U.S. Patent No. 6,677,452); 2- aminobenzoxazoles (e.g., U.S. Patent No. 6,660,858); isoindoles, isooxyindoles, or isooxyquinolines (e.g., U.S. Patent No. 6,667,406); oxazolidinones (e.g., U.S. Patent No. 6,562,844); and hydroxylamines (e.g., U.S. Patent No. 6,541,276).
[0164] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem. Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).
IX. Examples
[0165] The following examples are offered to illustrate, but not to limit, the claimed invention.
Example 1. Soluble PSMA Binds to Cancer Cells that Overexpress PSMA [0166] The extracellular domain of PSMA ("soluble PSMA") was cloned into the pSecTag2 expression vector (rnvitrogen; Carlsbad, CA) in frame with the secretion signal from the mouse Ig Kappa-chain. Cloning into the pSecTag2 expression vector also introduced a C-terminal polyhistidine tag (6xHis), which can be used for purification, and a c-myc epitope useful for localization and characterization studies. 6xHis-tagged soluble PSMA was purified using a nickel charged HiTrap™ Chelating HP Column (GE Healthcare; Buckinghamshire, England). As shown in Figure 3 A, 15 μl aliquots of starting conditioned media, column flowthrough, a 10 mM imidazole wash, and elution fractions of increasing imidazole concentrations were analyzed by SDS-PAGE followed by silver staining to determine soluble PSMA recovery and purity. Fractions containing soluble PSMA eluted between 20-100 mM imidazole and were pooled and concentrated using Centricon filters (Millipore; Billerica, MA). Figure 3B shows a Coomassie Blue stained gel of 15 μl of the resultant concentrated pool fractions of soluble PSMA. [0167] To determine whether soluble PSMA binds to cancer cells that overexpress PSMA on the cell surface, LNCaP cells were used. LNCaP cells are human prostate cancer cells that overexpress PSMA endogenously. LNCaP cells were seeded onto glass coverslips and incubated with 10 μg/ml soluble PSMA. The cells were then fixed and incubated with anti- myc antibody. Cells were washed, incubated with FITC-conjugated secondary antibody, and visualized by fluorescent microscopy. Control cells did not receive anti-myc antibody. As shown in Figure 4, soluble PSMA specifically bound to LNCaP cells (Figure 4A), whereas control cells did not display any detectable fluorescence (Figure 4B).
Example 2. Preparation of Retroviral Vectors Conjugated with Soluble PSMA [0168] The soluble PSMA polypeptides and peptides of the present invention can be delivered to target cells that overexpress PSMA such as cancerous cells using retroviral vectors as described in, e.g., Morizono et al, Cell Cycle, 4:854-856 (2005). Briefly, oncoretroiviral or lentiviral vectors are pseudotyped with a chimeric Sindbis virus envelope protein in which an Fc binding region has been inserted into the receptor binding region of the envelope protein. A heterologous protein containing a fusion between soluble PSMA and an Fc region is constructed, expressed, and purified as described above. The soluble PSMA- Fc fusion is then conjugated to the surface of the retroviral vector through the interaction of the Fc region with the Fc binding region of the chimeric Sindbis virus envelope protein. The resulting retroviral vector can be packaged with one or more of the imaging agents and/or anti-cancer agents using techniques known in the art.
[0169] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. AU publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:
L A composition comprising a soluble extracellular domain of prostate specific membrane antigen (PSMA) or a fragment thereof that binds to cell surface PSMA and a physiologically acceptable carrier.
2. The composition of claim 1, wherein the soluble extracellular domain of PSMA comprises amino acids 44-750 of full-length PSMA.
3. The composition of claim 1 , wherein the fragment thereof comprises amino acids 601 -750 of full-length PSMA.
4. The composition of claim 1, wherein the fragment thereof comprises at least about 10 consecutive amino acids within amino acids 601-750 of full-length PSMA.
5. The composition of claim 1, wherein the soluble extracellular domain of PSMA or fragment thereof is linked to a detectable moiety.
6. The composition of claim 5, wherein the detectable moiety is selected from the group consisting of a radionuclide, a nanoparticle, a fluorescent dye, a fluorescent marker, and an enzyme.
7. The composition of claim 1, wherein the soluble extracellular domain of PSMA or fragment thereof is linked to an anti-cancer agent.
8. The composition of claim 7, wherein the anti-cancer agent is selected from the group consisting of a radionuclide, a toxin, a chemotherapeutic agent, a hormonal therapaeutic agent, and a radiotherapeutic agent.
9. A method of diagnosing a cancer that overexpresses cell surface PSMA, the method comprising the steps of: (a) contacting a tissue sample with a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and (b) determining whether or not PSMA protein is overexpressed in the sample, thereby diagnosing the cancer that overexpresses cell surface PSMA.
10. The method of claim 9, wherein the cancer that overexpresses cell surface PSMA is selected from the group consisting of prostate cancer, renal cancer, bladder cancer, ovarian cancer, breast cancer, colon cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma, and hepatocarcinoma.
11. The method of claim 10, wherein the cancer that overexpresses cell surface PSMA is prostate cancer.
12. The method of claim 9, wherein the tissue sample is a biopsy.
13. The method of claim 9, wherein the tissue sample is from prostate, kidney, bladder, ovary, bone, colon, lung, lymph node, or liver.
14. The method of claim 9, wherein the soluble extracellular domain of PSMA or fragment thereof is linked to a detectable moiety.
15. A method of providing a prognosis for a cancer that overexpresses cell surface PSMA, the method comprising the steps of: (a) contacting a tissue sample with a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and (b) determining whether or not PSMA protein is overexpressed in the sample, thereby providing a prognosis for the cancer that overexpresses cell surface PSMA.
16. The method of claim 15, wherein the cancer that overexpresses cell surface PSMA is selected from the group consisting of prostate cancer, renal cancer, bladder cancer, ovarian cancer, breast cancer, colon cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma, and hepatocarcinoma.
17. The method of claim 16, wherein the cancer that overexpresses cell surface PSMA is prostate cancer.
18. The method of claim 15, wherein the tissue sample is a biopsy.
19. The method of claim 15, wherein the tissue sample is from prostate, kidney, bladder, ovary, bone, colon, lung, lymph node, or liver.
20. The method of claim 15, wherein the soluble extracellular domain of PSMA or fragment thereof is linked to a detectable moiety.
21. A method of imaging a cancer that overexpresses cell surface PSMA in a subject, the method comprising the steps of: (a) administering to the subject a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA; and (b) determining where the soluble extracellular domain of PSMA or fragment thereof is concentrated in the subject, thereby imaging the cancer that overexpresses cell surface PSMA.
22. The method of claim 21 , wherein the cancer that overexpresses cell surface PSMA is selected from the group consisting of prostate cancer, renal cancer, bladder cancer, ovarian cancer, breast cancer, colon cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma, and hepatocarcinoma.
23. The method of claim 22, wherein the cancer that overexpresses cell surface PSMA is prostate cancer.
24. The method of claim 21, wherein the soluble extracellular domain of PSMA or fragment thereof is linked to a detectable moiety.
25. The method of claim 24, wherein the detectable moiety is selected from the group consisting of a radionuclide, a nanoparticle, a fluorescent dye, a fluorescent marker, and an enzyme.
26. The method of claim 21 , wherein the soluble extracellular domain of PSMA or fragment thereof is expressed on the surface of a retrovirus.
27. The method of claim 26, wherein the retrovirus is a lentivirus.
28. The method of claim 26, wherein the retrovirus is packaged with a detectable moiety.
29. A method of treating or inhibiting a cancer that overexpresses cell surface PSMA in a subject comprising administering to the subject a therapeutically effective amount of a soluble extracellular domain of PSMA or a fragment thereof that binds to cell surface PSMA.
30. The method of claim 29, wherein the cancer that overexpresses cell surface PSMA is selected from the group consisting of prostate cancer, renal cancer, bladder cancer, ovarian cancer, breast cancer, colon cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, multiple myeloma, and hepatocarcinoma.
31. The method of claim 30, wherein the cancer that overexpresses cell surface PSMA is prostate cancer.
32. The method of claim 29, wherein the soluble extracellular domain of PSMA or fragment thereof is co-administered with an additional cancer therapy.
33. The method of claim 32, wherein the additional cancer therapy is selected from the group consisting of chemotherapy, immunotherapy, hormonal therapy, and radiotherapy.
34. The method of claim 29, wherein the soluble extracellular domain of PSMA or fragment thereof is administered without an additional cancer therapy.
35. The method of claim 29, wherein the soluble extracellular domain of PSMA or fragment thereof is linked to an anti-cancer agent.
36. The method of claim 35, wherein the anti-cancer agent is selected from the group consisting of a radionuclide, a toxin, a chemotherapeutic agent, a hormonal therapaeutic agent, and a radiotherapeutic agent.
37. The method of claim 29, wherein the soluble extracellular domain of PSMA or fragment thereof is expressed on the surface of a retrovirus.
38. The method of claim 37, wherein the retrovirus is a lentivirus.
39. The method of claim 37, wherein the retrovirus is packaged with an anti-cancer agent.
PCT/US2006/0020202005-01-212006-01-19Use of a novel prostate specific membrane antigen for cancer diagnosis and therapyWO2006078892A2 (en)

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WO2018187791A1 (en)2017-04-072018-10-11Juno Therapeutics, IncEngineered cells expressing prostate-specific membrane antigen (psma) or a modified form thereof and related methods

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* Cited by examiner, † Cited by third party
Publication numberPriority datePublication dateAssigneeTitle
WO2018134691A2 (en)2017-01-202018-07-26Juno Therapeutics GmbhCell surface conjugates and related cell compositions and methods
US11517627B2 (en)2017-01-202022-12-06Juno Therapeutics GmbhCell surface conjugates and related cell compositions and methods
WO2018187791A1 (en)2017-04-072018-10-11Juno Therapeutics, IncEngineered cells expressing prostate-specific membrane antigen (psma) or a modified form thereof and related methods

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