| Immune effector cells pre-infected with oncolytic virus -> Monitor Keywords |
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Immune effector cells pre-infected with oncolytic virusRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Immune effector cells pre-infected with oncolytic virus description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070077231, Immune effector cells pre-infected with oncolytic virus. Brief Patent Description - Full Patent Description - Patent Application Claims
BACKGROUND OF THE INVENTION [0002] Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites it will likely result in death of the individual. [0003] The desired goal of cancer therapy is to kill cancer cells preferentially, without having a deleterious effect on normal cells. Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy, and chemotherapy. [0004] Local treatments, such as radiation therapy and surgery, offer a way of reducing the tumor mass in regions of the body that are accessible through surgical techniques or high doses of radiation therapy. However, more effective local therapies with fewer side effects are needed. Moreover, these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients. To combat the spread of tumor cells, systemic therapies are used. [0005] The primary weapon against cancer is chemotherapy. However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. This failure is in part due to the intrinsic or acquired drug resistance of many tumor cells. Another drawback to the use of chemotherapeutic agents is their severe side effects. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. [0006] Proposed alternative therapies include the administration of oncolytic viruses, and the use of viral vectors to deliver a transgene whose expression product activates a chemotherapeutic agent. The genetic engineering of viruses for use as oncolytic agents has initially focused on the use of replication-incompetent viruses. This strategy was hoped to prevent damage to non-tumor cells by the viruses. A major limitation of this approach is that these replication-incompetent viruses require a helper virus to be able to integrate and/or replicate in a host cell. These viruses are limited in their effectiveness, because each replication-defective retrovirus particle can enter only a single cell and cannot productively infect others thereafter. Therefore, they cannot spread far from the producer cell, and are unable to completely penetrate many tumors in vivo. More recently, genetic engineering of oncolytic viruses has focused on the generation of "replication-conditional" viruses in an attempt to avoid systemic infection, while allowing the virus to spread to other tumor cells. Replication-conditional viruses are designed to preferentially replicate in actively dividing cells, such as tumor cells. Thus, these viruses should target tumor cells for oncolysis, and replicate in these cells so that the virus can spread to other tumor cells. [0007] However, while the virus-based approach has provided evidence of significant therapeutic effects in animal models of tumors, the method is limited by the efficiency of viral infection; the requirement of a helper virus or producer cell line for some viral vectors; tumor cell heterogeneity for the cellular factor(s) complementing viral mutant growth for other viral vectors; and antiviral immune responses. [0008] A variety of immune cell-based cancer therapies have also been proposed, many of which rely on the identification of tumor-associated antigens that are often weak or expressed on only a subset of tumor cells. Cytokine induced killer (CIK) cells are a population of cells derived from human PBMC's following ex vivo expansion with .gamma.IFN, anti-CD3 antibody and IL-2. They bear phenotypic markers of NK and T cells, express NKG2D and have been found to mediate killing of tumor cells through recognition of a class of stress-associated ligands expressed on the tumor cell surface (NKG2D ligands). CIK cells therefore do not rely on specific antigens and they have also been shown to target a variety of tumors and exert their cytotoxic effects following systemic delivery. Previous pre-clinical imaging studies found that at 72 hours (h) after intravenous delivery signals from CIK cells were found primarily at the tumor site. However, tumor cell killing required effector to target ratios of five to ten CIK cells per tumor cell in vitro, and a dependence on over expression of NKG2D ligands on the tumor targets. [0009] Targeted biological therapies hold tremendous potential for the treatment of cancers, yet their effective use has been limited by constraints on delivery and effective tumor targeting. There exists a need for a local therapy that provides for effective killing of tumor cells. The present invention addresses this need. Relevant Literature [0010] Leemhuis et al. (2005) Biol Blood Marrow Transplant 11, 181-7 (2005); Lu & Negrin (1994) J Immunol 153, 1687-96; Kim et al. (2001) Nat Med 7, 781-7 (2001). Thorne & Kim (2004) Expert Opin Biol Ther 4, 1307-21. Puhlmann et al. (2000) Cancer Gene Ther 7, 66-73 (2000). Hamerman et al. (2005) Curr Opin Immunol 17, 29-35. SUMMARY OF THE INVENTION [0011] Methods are provided for the treatment of cancer, through administration to a patient of an effector cell population that is pre-infected with an oncolytic virus. The effector cells are preferably T cells, which may be autologous or allogeneic. In some embodiments, the cells are cytokine induced killer cells, which do not rely on recognition through the T cell antigen receptor for cytoxicity. In other embodiments, the cells are tumor infiltrating T lymphocytes. The effector cells are infected with an oncolytic virus, preferably a replication competent virus, e.g. vaccinia, adenovirus, etc. Oncolytic viruses of interest are replication-selective or tropism modified viruses that are either only capable of entering into, or of completing a successful infection cycle within, transformed cells. [0012] Pre-infection of effector cells with oncolytic virus resulted in a prolonged eclipse period where the virus remained within the cells until interaction with, and infiltration into, the tumor. The infected cells are preferably administered to the patient during the eclipse phase. In the combined therapeutic, the effector cells were shown to retain their ability to traffic to tumors. At the tumor site the oncolytic virus was released deep in the tumor rather than merely at the surface; thus the cell mediated delivery of the virus led to enhanced biodistribution within the tumor. In addition, the cytotoxic effects of the effector cells may be increased by viral replication in the tumor target. This combined therapeutic has been demonstrated to be safe, with minimal viral infection of normal tissues, and highly effective, producing an enhanced anti-tumor effect compared to either therapy alone. The methods of the invention thus provide for a synergistic effect based on the combined biotherapeutics. [0013] Other objects and advantages of the present invention will become evident from the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [0014] These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the detailed description of the invention when considered in connection with the accompanying drawings which show as follows: [0015] FIG. 1A-1C. Vaccinia virus displays unusual replication kinetics in CIK cells. (a) Viral replication was followed for different vaccinia strains in CIK cells after infection at a multiplicity of infection (MOI) of 1.0 plaque forming units (PFU)/cell (*p=0.034, T12 test, SEM). (b) Viral replication of the same vaccinia strains in the B-cell lymphoma cell line OCI-ly8 (MOI of 1.0 PFU/cell). (c) Kinetics of replication of vvDD in CIK cells following infection at an MOI of 1.0 PFU/cell. Viral infectious units (PFU/ml) were titered separately in the cell and media fractions of the infected plate at times indicated. Cell-associated and cell-free viral titers are plotted to reveal viral replication kinetics. CIK cell numbers within infected or uninfected plates were counted at the same time points. [0016] FIG. 2A-2C. CIK cells retain their ability to kill target cells despite pre-infection with vvDD, and infection of CIK-resistant target cells with vvDD increases their susceptibility to CIK-mediated killing due to NKG2D ligand expression. (a) UCI-101 target cells expressing luciferase were mixed with different numbers of CIK effector cells (ratios of 100:1, 50:1, 20:1 and 10:1 or target only). CIK effector cells had been pre-infected with vvDD for 24-h, 48-h or 72-h (MOI 1.0 PFU/cell). Luciferase output, as an indication of cytotoxicity, was measured using an IVIS50 system (Xenogen Corp.) after 4 h. (b) SKOV-3 target cells expressing luciferase, alone or pre-infected with vvDD (MOI 1.0 PFU/cell) for 24-h, were mixed with CIK cells at effector:target ratios of 20:1. Luciferase signal was measured after 4 h (p=0.0076, T-test, SEM). (c) UCI-101 or SKOV-3 cells alone or pre-infected with vvDD (MOI 1.0 PFU/ml for 8 h) were stained with anti-MICA/MICB antibody conjugated to PE and the numbers of cells expressing these cell surface markers were determined by FACS analysis. Isotype controls produced less than 1% positive staining. [0017] FIG. 3A-3D. Biodistribution of vvDD expressing luciferase delivered to mice bearing subcutaneous UCI-101 tumors either alone or within CIK cells and immunofluorescence microscopy of tumor sections (UCI-101). (a) Mice received 1.times.10.sub.7 CIK cells pre-infected with vvDD-luc (MOI 1.0 PFU/cell) via tail vein injection (left) or 1.times.10.sub.7 PFU vvDD-luc via tail vein injection (right). Injections occurred on day 0. Animals were imaged at the times indicated (in days post therapy) using an IVIS200 system (Xenogen Corp.). Alternatively, vvDD-GFP was used (lower panel day 3) in an equivalent experiment, and animals were imaged using a Maestro (CRI, Boston Mass.) imaging system. (b) Quantification of light output per tumor is plotted relative to time 13 as an indication of viral replication and distribution. Values are averages for 3 animals per group, error bars are SEM; p=0.0079 at day 35 (T-test). (c) Tumors from animals receiving vvDD-GFP (1.times.10.sub.7 PFU) or 1.times.10.sub.7 CIK pre-infected with vvDD-GFP (MOI 1.0 PFU/cell) via tail vein injection. Sections (48 h post-injection) were stained with anti-CD31 (endothelial cell marker) (magnification 400.times.) (d) Tumor from animals receiving 1.times.10.sub.7 CIK cells conjugated to Cy5.5 (red) and pre-infected with vvDD-GFP (MOI 1.0 PFU/cell). Sections were stained with Sytox blue (DNA binding)(blue) and anti-GFP antibody (green) (tumors taken 72 h post-injection). Arrows indicate area of overlapping green and red, indicating infected CIK cells within the tumor (scale bar 100 m). [0018] FIG. 4A-4B. Survival and tumor burden of animals bearing UCI-101 or SKOV-3 tumors. (a) Kaplan-Meier survival curves of animals carrying UCI-101 or SKOV-3 intraperitoneal tumors (cell lines expressed luciferase and tumor burden was measured using bioluminescence imaging). Each animal received a single tail vein injection of either PBS; 1.times.10.sub.7 CIK cells; 1.times.10.sub.7 PFU vvDD; or 1.times.10.sub.7 CIK cells preinfected with vvDD (MOI 1.0 PFU/cell), n=8 animals/group. Combination therapy significantly increased survival compared to any other treatment (Logrank test; p=<0.05). (e.g. CIK+vvDD compared to vvDD alone; p=0.0072 (UCI-101) or 0.0379 (SKOV-3)). (b) The tumor burden (measured by bioluminescence imaging) for each individual animal was plotted against time. A single intravenous treatment was delivered on day 3 (arrows) of PBS (black) or combination therapy (green) (top) or CIK (red) or vvDD (blue) (bottom). Grey shaded area indicates range of tumor burden for the PBS treated group. [0019] FIG. 5A-5B. Fluoresence and bioluminescence imaging of trafficking of uninfected and vvDD infected CIK cells. (a) Mice carrying subcutaneous SKOV-3 (top panel) or UCI-101 (bottom panel) tumors were treated (day 0) with a single intravenous injection of either 1.times.10.sub.7 CIK cells labeled with Cy5.5 (left animal) or 1.times.10.sub.7 Cy5.5 labeled CIK cells infected with vvDD-GFP (MOI 1.0) (right animal). Cy5.5 fluorescence was imaged using an IVIS200 system (Xenogen Corp.). A PBS control mouse was included 14 for comparison on day 2. (b) In an equivalent experiment, a mouse bearing an UCI-101 tumor was treated with 1.times.10.sub.7 CIK cells transfected with retrovirus to express luciferase and infected with vvDD-GFP (MOI 1.0) on day 0. Bioluminescence was imaged using an IVIS200. Arrows indicate locations of tumors. [0020] FIG. 6A-6E. Immunofluorescence microscopy of MICA/B expression in a treated SKOV-3 tumor. A mouse bearing a subcutaneous SKOV-3 tumor was treated with a tail vein injection of 1.times.10.sub.7 CIK cells labeled with Cy5.5 and infected with vvDD-GFP (MOI 1.0). Tumors were recovered 48 hours after treatment and frozen sections were stained with (a) a nuclear dye (Sytox Blue, Molecular Probes); (b) anti-GFP antibody: or (c) anti-MICA/B. Fluorescence was imaged, along with Cy5.5 fluorescence (d) using a Leica confocal microscope. An overlay image is also shown (e) [0021] FIG. 7. Immunofluorescence microscopy of CIK delivery of vvDD in a UCI-101 tumor. A mouse bearing a subcutaneous UCI-101 tumor was treated with a tail vein injection of 1.times.10.sub.7 CIK cells labeled with Cy5.5 and infected with vvDD-GFP (MOI 1.0). Tumors were recovered 24 hours after treatment and frozen sections were stained with a nuclear dye (Sytox Blue, Molecular Probes) and anti-GFP antibody (green). Fluorescence was imaged, along with Cy5.5 fluorescence (red) using a Leica confocal microscope. An overalay image is shown>Style tag for figure legends. Continue reading about Immune effector cells pre-infected with oncolytic virus... Full patent description for Immune effector cells pre-infected with oncolytic virus Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Immune effector cells pre-infected with oncolytic virus patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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