Microorganisms for imaging and/or treatment of tumors

Benefit of priority is claimed under 35 U.S.C. §119(e) to U.S. Provisional Application Ser. No. 60/934,768, to Nanhai Chen, Yuman Fong, Aladar A. Szalay, Yong A. Yu and Qian Zhang, filed on Jun. 15, 2007, entitled “MICROORGANISMS FOR IMAGING AND/OR TREATMENT OF TUMORS.” The subject matter of this application is incorporated by reference in its entirety.

This application is related to International Application No. (Attorney Dkt. No. 0119356-00132/112PC) to Nanhai Chen, Yuman Fong, Aladar A. Szalay, Yong A. Yu and Qian Zhang, filed on Jun. 13, 2008, entitled “MICROORGANISMS FOR IMAGING AND/OR TREATMENT OF TUMORS,” which also claims priority to U.S. Provisional Application Ser. No. 60/934,768. The subject matter of this application is incorporated by reference in its entirety.

An electronic version on compact disc (CD-R) of the Sequence Listing is filed herewith in duplicate (labeled Copy # 1 and Copy # 2), the contents of which are incorporated by reference in their entirety. The computer-readable file on each of the aforementioned compact discs, created on Jun. 13, 2008, is identical, 992 kilobytes in size, and entitled 112SEQ.001.txt.

Modified recombinant viruses for diagnosis and therapy are provided. Diagnostic and therapeutic methods using the modified recombinant viruses also are provided.

Cancers, such as pancreatic cancer and malignant pleural mesothelioma, are highly aggressive diseases. The annual incidence in the United States was estimated to be ˜40,000 cases for pancreatic cancer and ˜4,000 cases for malignant mesothelioma in the year 2004, with increasing incidence worldwide for mesothelioma, especially in industrialized nations due to the etiology of this disease from asbestos exposure (Bianchi and Bianchi (2007) Ind Health 45: 379-87). Both of these tumors are highly resistant to standard therapies, with 5-year survival rates of only 5% for pancreatic cancer and 9% for mesothelioma. Even with combined surgery, chemotherapy and radiation, only a small minority of patients are rendered disease-free for a prolonged period of time (Adusumilli et al. (2006) J Gene Med 8:603-15.

Oncolytic viral therapy has been studied and tested over the past century, and many viral types, including adenovirus, herpes simplex virus, Newcastle disease virus, myxoma virus, vaccinia virus and vesicular stomatitis virus, are being investigated as novel agents for the treatment of human cancer (Woo et al. (2006) Curr Opin Investig Drugs 7:549-59). Accordingly, effective tumor diagnostic and therapeutic viral agents that are highly selective for tumors are needed. In addition, there exists a need to provide reagents and methods for tracking and monitoring viral distribution, tumor targeting, proliferation and persistence in oncolytic viral therapies by noninvasive imaging, which provide important safety, efficacy and toxicity data. Such real-time monitoring also would provide useful viral-dose and administration schedule information for optimization of therapy and would obviate the need for multiple and repeated tissue biopsies.

Provided are recombinant viruses, particularly, vaccinia virus, such as LIVP, that accumulates in tumors or other immunoprivileged tissues, such as wounds and inflamed tissues, and not accumulate to toxic levels in other tissues. These viruses encoded a protein that enhances uptake or retention of a compound that emits a signal that permits detection, such as by non-optical imaging. Proteins that enhance uptake or retention include transporter proteins. These viruses also can be used for treatment of tumors, wounded tissues and inflammations within a subject. The compound that is taken up or retained can be a therapeutic compound or can be modified, such as by conjugate to a therapeutic compound, to have therapeutic activity. The viruses can be used for detection, detection and treatment, detection and monitoring of treatment. Methods for detection, detection and treatment, detection and monitoring of treatment are provided as are uses of recombinant viruses, such as vaccinia viruses for detection, detection and treatment, detection and monitoring of treatment.

Provided herein are recombinant vaccinia viruses that encode a sodium-dependent transporter protein. Sodium-dependent transporter proteins include those from the solute carrier 5 and solute carrier 6 transporter protein families, such as a norepinephrine transporter (NET) and a sodium-iodide symporter (NIS), including a human norepinephrine transporter (hNET) and a human sodium-iodide symporter (hNIS) as well as allelic and species variants thereof and other variants, including any having at least about or at least 60, 65, 70, 75, 80, 85, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 percent or more sequence identity with those disclosed herein. These include modified forms that retain transporter activity sufficient for the methods provided herein.

Recombinant vaccinia virus include of any of claims 1-5 that is a Lister strain viruses, such as the LIVP strain. Nucleic acid encoding transporter protein can be inserted anywhere in the virus such that the virus expresses it and replicates in a subject. In exemplary embodiments, the nucleic acid encoding the transporter protein is inserted into a nonessential locus or gene, such as the hemagglutinin (HA), thymidine kinase (TK) or F14.5 gene or locus. Exemplary of such viruses are those provided herein that include GLV-1h99, GLV-1h100, GLV-1h101, GLV-1h139, GLV-1h146, GLV-1h150, GLV-1h151, GLV-1h152 and GLV-1h153. These viruses can be further modified to encode a therapeutic protein. Generally the encoding nucleic acid is inserted into a different locus from the nucleic acid that encodes the transporter protein. Exemplary therapeutic agents include, but are not limited to an anti-cancer agents and anti-angiogenic agents. A therapeutic agent, includes, but is not limited to, a cytokine, a chemokine, an immunomodulatory molecule, an antigen, an antibody or fragment thereof, antisense RNA, prodrug converting enzyme, siRNA, angiogenesis inhibitor, a toxin, an antitumor oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anti-cancer polypeptide antibiotic, and tissue factor, such as single chain antibody (scFv), including an anti-VEGF single chain antibody, a plasminogen K5 domain, a human tissue factor-αvβ3-integrin RGD fusion protein, interleukin-24 or an IL-6-IL-6 receptor fusion protein and fusion proteins of substrates for the transporter protein and a therapeutic agent, such as a chemotherapeutic compound or a toxin. The viruses can encoded a plurality of therapeutic agents and/or transporter proteins.

Also provided are combination that contain one or more of the recombinant viruses, particularly vaccinia viruses, provided herein, and a substrate transported into a cell that expresses the transporter; and/or an anti-cancer compound. Substrates can be detectable or can induce a detectable signal or can modified to be detectable or to induce a detectable signal, such as electromagnetic radiation. The substrate can be radiolabeled; it can be conjugated to a cytotoxic agent, such as a cytokine, a chemokine, a growth factor, a photosensitizing agent, a toxin, an anti-cancer antibiotic, a chemotherapeutic compound, a radionuclide, an angiogenesis inhibitor, a signaling modulator, an anti-metabolite, an anti-cancer vaccine, an anti-cancer oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anti-cancer antibody, an anti-cancer antibiotic, an immunotherapeutic agent, a bacterium and combinations thereof.

Conjugation can be chemical or, where the substrate and cytotoxic agent are proteins can be a fusion protein. Conjugate can be direct or via a linker. Exemplary cytotoxic agents, include, but are not limited to, radiolabels, a cytotoxins and chemotherapeutic drugs and cytotoxic drugs. Exemplary cytotoxic agents, include, but are not limited to, double-chain ricin, ricin A chain, abrin, abrin A chain, saporin, modeccin, modeccin A chain, Pseudomonas aeruginosa exotoxin, Cholera toxin, Shigella toxin, E. coli heat labile toxin and Diphtheria toxin. doxorubicin, daunomycin, 5-fluorouracil, methotrexate, taxol, ricin A, colchicine, cytochasins, monensin, ouabain, mitoxanthrone, vindesine, vinblastine, vincristine, enterotoxin, cisplatin, carboplatin, gemcitabine, irinotecan, an anti-EGFR antibody and an anti-VEGF antibody. In addition to conjugation the substrate and cytotoxic agent can be separate and can be separately administered. Proteinaceous cytotoxic agents and conjugates also can be expressed by the virus. In the combinations, the substrate and virus can be formulated as a single composition or separately in two compositions. Also, provided are kits that contain the combinations and optionally reagents and other components for use of the combinations and instructions for use thereof.

Also provided are pharmaceutical composition containing the recombinant viruses provided herein in a pharmaceutically acceptable carrier. They can be formulated for any type of administration include local or systemic administration.

The recombinant viruses provided herein also can be vaccines, including smallpox vaccines.

Also provided are methods of imaging or detecting a tumor, an inflammation or a wound within a subject practiced by administering any virus provided herein to a subject suspected of a having a tumor and/or internal wound or inflammation. In practicing the method, the virus is administered with or sequentially or intermittently with a substrate that is transported into a cell that expresses the transporter encoded by the virus. The substrate or its accumulation can be detected, thereby detecting or imaging a tumor, wound and/or inflammation. Detection and imaging can be effected by fluorescence imaging, magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), positron emission tomography (PET), scintigraphy, gamma camera, a β+ detector, a γ detector and combinations thereof.

Methods of treatment are provided. The methods are effected by administering a any virus provided herein to a subject to effect treatment. Treatment can be for any disease or disorder for which administration of a virus, particularly a vaccinia virus, is effective. Such diseases and disorders include, tumors and cancers and/or metastasis. The method can further include administering a substrate that is transported into a cell that expresses the transporter encoded by the virus. The substrate itself can be therapeutic or it can be conjugated to a therapeutic agent, whereby treatment is effected. The substrate can be administered before or after or simultaneously with the virus or it can be encoded by nucleic acid that is administered, such as another virus or other vector that encodes it. The substrate can be conjugated to a cytotoxic agent as described above.

For the methods of treatment, the virus can be administered by any suitable route, including systemically, intravenously, intraarterially, intratumorally, endoscopically, intralesionally, intramuscularly, intradermally, intraperitoneally, intravesicularly, intraarticularly, intrapleurally, percutaneously, subcutaneously, orally, parenterally, mucosally, intranasally, intratracheally, by inhalation, intracranially, intraprostaticaly, intravitreally, topically, ocularly, vaginally and rectally. The virus or virus and substrate can be administered with an anticancer agent or treatment. The anticancer agent or treatment can be administered before or after or simultaneously or intermittently with the virus or the virus and substrate. Anticancer agents include any noted above, including, but not limited to, a cytokine, a chemokine, a growth factor, a photosensitizing agent, a toxin, an anti-cancer antibiotic, a chemotherapeutic compound, a radionuclide, an angiogenesis inhibitor, a signaling modulator, an anti-metabolite, an anti-cancer vaccine, an anti-cancer oligopeptide, a mitosis inhibitor protein, an antimitotic oligopeptide, an anti-cancer antibody, an anti-cancer antibiotic, an immunotherapeutic agent, hyperthermia or hyperthermia therapy, a bacterium, radiation therapy and any combination thereof.