Patent Application
20170314044
This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office via EFS-Web as an ASCII text file entitled “11005490101SequenceListing_ST25.txt” having a size of 98 kilobytes and created on May 2, 2017. Due to the electronic filing of the Sequence Listing, the electronically submitted Sequence Listing serves as both the paper copy required by 37 CFR §1.821(c) and the CRF required by §1.821(e). The information contained in the Sequence Listing is incorporated by reference herein and does not go beyond the disclosure in the International Application as filed.
This disclosure describes oncolytic adenovirus (OAd) constructs and methods on using the constructs. Generally, the oncolytic constructs include at least a partial deletion of the adenoviral region E3, a heterologous polynucleotide encoding a heterologous polypeptide in place of the at least partial deletion of E3, and a cell-specific regulatory polynucleotide operationally linked to the heterologous polynucleotide.
In some embodiments, the heterologous polynucleotide encodes a therapeutic polypeptide effective for treating cancer. In some of these embodiments, the therapeutic polypeptide can include a polypeptide that kills tumor cells. In other embodiments, the therapeutic polypeptide can include a polypeptide that sensitizes tumor cells to a primary cancer therapy such as radiotherapy, chemotherapy, and/or immunotherapy.
In some embodiments, the heterologous polynucleotide encodes a diagnostic polypeptide. In some of these embodiments, the diagnostic polypeptide binds to a radioisotope.
In some embodiments, the cell-specific regulatory polynucleotide can include a tumor-specific promoter.
In some embodiments, the construct can further include a genetic modification that increases transduction efficiency of the adenovirus construct into target cells. I some of these embodiments, the genetic modification can include a polynucleotide that encodes a fiber knob polypeptide of adenovirus type 3 (Ad3).
In some embodiments, the construct can lack a functional adenovirus death protein (ADP). In other embodiments, the construct can include an ADP coding region.
The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
The patent or application file contains at least one drawing or photograph executed in color. Copies of this patent or patent application publication with color drawing(s) or photographs(s) will be provided by the Office upon request and payment of the necessary fee.
This disclosure describes a novel oncolytic virus-based technology platform that has therapeutic and diagnostic utility. This technology is based on a genetically modified oncolytic adenovirus that can produce a level of transgene expression in tumor sites that can allow imaging of cancer cells and/or cancer therapy. For example, the oncolytic adenovirus can facilitate immunotherapy by expressing an immunostimulatory cytokine such as, for example, interferons, (IFNs, e.g., IFN-α, IFN-β; interleukins (ILs, e.g., IL-2, IL-6, IL-7, IL-8, IL-12); granulocyte-macrophage colony stimulating factor (GM-CSF), and/or therapeutic antibodies (e.g., anti-CTLA4 and anti-PD1). Viral-based immunotherapies can initiate local tumor lysis and/or stimulate immune response against cancer cells. Alternatively, the oncolytic adenovirus can be designed to express a therapeutic and/or imaging gene—e.g., the sodium iodine symporter (NIS), which can induce uptake of radioactive iodine by tumor cells. NIS expression allows noninvasive imaging of cancer with different radioisotopes (e.g., 123I, 124I, 99mTcO4, 188ReO4) and facilitates therapy with radioiodine 131I. Another application of the technology is its ability to monitor tumor development, disease staging, and/or adenovirus biodistribution by expressing a reporter transgene such as, for example, the green fluorescent protein (GFP or EGFP), red fluorescent protein (RFP), fluorescent reporter mCherry, herpes simples virus type 1 thymidine kinase (HSV1-tk) or mutant HSV1-sr39tk, somatostatin receptor (SSTR), a radiolabeled antibody against carcinoembryonic antigen (CEA), or secreted embryonic alkaline phosphatase (SEAP).
In some embodiments, the oncolytic adenovirus lacks the adenovirus death protein (ADP), the deletion of which improves gene expression. Expression of the transgene is replication-dependent: a therapeutic transgene is expressed in tumor cells with each round of adenovirus replication. Moreover, the oncolytic adenovirus can be designed to replicate—and therefore express the transgene—only in cancer cells.
Tumor-specific promoters can be used to control adenovirus replication. One extensively used method to develop tumor-targeted oncolytic adenovirus is to control viral replication with a tumor-specific promoter. Several promoters possess a suitable profile for tumor-specific oncolytic adenovirus development such as, for example, cyclooxygenase-2 (Cox2), heparin-binding growth/differentiation factor midkine (MK) promoter, mucin 1 (Muc1) promoters for pancreatic cancer; probasin (PB) and prostate—specific antigen (PSA) promoters for prostate cancer; and other candidates promoters that share the characteristic of being overexpressed in cancer while possessing the “liver off” profile—e.g., human telomerase catalytic subunit (hTERT), gastrin releasing peptide (GRP), vascular endothelium growth factor (VEGF), secretory leukocyte protease inhibitor (SLPI), fms-like tyrosine kinase 1(FLT-1), and caveolin-1 promoters. These tumor-specific promoters can be active in primary and established cancer cells while exhibiting very low activity in normal tissues. In some cases, the tumor-specific promoter can exhibit low activity in normal liver tissue, which can be the organ of most concern involving ectopic adenoviral vector gene expression. Systemically-delivered oncolytic adenoviruses demonstrated a low level of toxicity and liver sequestration, indicating the feasibility of tumor-specific-promoter-based oncolytic adenoviruses for systemic cancer therapy.
In some embodiments, the oncolytic adenovirus can include a genetic modification that increases transduction efficiency of the oncolytic adenovirus in cancer cells. The genetic modification can include, for example, modification of the viral capsid. Many cancer cells are resistant to human adenovirus type 5 (Ad5) infection due to profoundly low expression of the primary adenovirus receptor (coxsackie-adenovirus receptor, CAR). Changing oncolytic adenovirus tropism via genetic modification of the viral capsid proteins enables CAR-independent entry. Thus, replacing the fiber knob of Ad type 5 with the fiber knob of Ad type 3 (Ad3) retargets the modified oncolytic adenovirus to the Ad3 receptor (identified as CD46 and human desmoglein-2 receptor), which is typically highly expressed in cancer cells. The Ad5/Ad3 modification of the Ad fiber increases the viral infectivity in many different cancer cells in vitro and in vivo (e.g. esophageal adenocarcinoma, pancreatic cancer, lung cancer, melanoma, colon cancer, breast cancer, and ovarian cancers).
In some embodiments, an increase in antitumor effects can be achieved by incorporating an anti-tumor transgene into the genome of an oncolytic adenovirus. This local transgene expression, coupled with strong vector spread, can significantly advance oncolytic virotherapy for cancer. One strategy for expressing a transgene upon Ad replication in tumor cells uses an antitumor therapeutic transgene—e.g., NIS—placed into the Ad E3 region of the oncolytic adenovirus. Some embodiments can overexpress Adenoviral Death Protein (ΔE3/+ADP): ADP facilitates viral release and leads to more efficient intratumoral spread of the virus. In some embodiments, the oncolytic adenovirus lacks the Adenovirus Death Protein (ADP), the deletion of which improves transgene expression. Transgene expression from the E3 region is controlled by the adenoviral major late promoter and is therefore consistent with the replication cycle, a property that can be further exploited to monitor the adenovirus.
A ΔE3-based oncolytic adenovirus may be designed to express any suitable transgenes from the adenoviral E3 region. Exemplary transgenes that can be expressed in this manner include, for example, therapeutic transgenes such as IFN-α, INF-β, IL-2, IL-6, IL-7, IL-8, IL-12, GM-C SF, anti-CTLA4 antibody, anti-PD1 antibody, or NIS; or imaging/reporter transgenes such Luc, RFP, GFP, EGFP, mCherry, SSTR, TKsr39, CEA, SEAP, or NIS.
In some embodiments, the oncolytic adenovirus can be used to express a therapeutic compound. One exemplary embodiment involves the use of an oncolytic adenovirus designed to deliver interferon-based therapy. In one embodiment of the ΔE3/+ADP oncolytic adenovirus system, the oncolytic adenovirus was designed to express human interferon-a gene (Ad-IFN). IFN-α can exhibit one or more properties—e.g., antitumor effects through immunomodulation, direct inhibition of tumor cell growth, antiangiogenesis, radio sensitization and/or chemosensitization—that can exploited in a cancer therapy regimen. IFN-α-expressing oncolytic adenovirus can stimulate immune effect against pancreatic cancer cells and can be used as a therapeutic agent for treating pancreatic cancer.
In addition, a multidrug analysis revealed that a combination of oncolytic Ad-IFN with chemotherapeutics (e.g., fluorouracil (5FU), gemcitabine, cisplatin) and radiotherapy (e.g., X-ray) killed human and hamster pancreatic cancer cells significantly better than either of the single treatments in vitro and in vivo. These results support the impact of oncolytic adenovirus-mediated IFN-α to sensitize chemotherapy and radiation for pancreatic cancer (
In some embodiments, the oncolytic adenovirus can provide both therapeutic and diagnostic utility. In one exemplary embodiment, the oncolytic adenovirus can be designed to express a transgene that encodes the sodium iodine symporter (NIS), a human protein that is involved in concentrating iodine in the thyroid. Natural expression of NIS in the thyroid has been exploited as a way to achieve radiotherapy of benign thyroid disease and thyroid cancer for 70 years. This therapy has proven to be safe and effective. NIS expression can be directly imaged at high sensitivity using noninvasive radioiodine scanning and routine nuclear medicine techniques. This imaging requires only a minimal diagnostic dose of radioisotope. Because the uptake of radioiodine in tissues can be quantitated, both the distribution and the quantity of NIS expression can be easily and safely monitored noninvasively. For example, combined SPECT/CT or PET/CT scanners allow the fusion of functional data with high-resolution anatomical (CT) data to provide accurate localization and quantitative estimate of radioactivity. NIS can promote cellular uptake of different radioisotopes, e.g. 123I, 124I, 99mTcO4, 188ReO4, and 131I. Moreover, NIS can induce uptake of radioactive iodine by tumor cells and enable radiotherapy with 131I, thus synergistically enhancing the effect of viral oncolysis.
The NIS-expressing oncolytic adenovirus was constructed based on a ΔE3-based adenovirus. Although adenoviral death protein (ADP) enhances oncolysis and viral release in solid tumors, oncolysis also can negatively affect gene expression from the adenovirus and reduce uptake of radiotracer. Two NIS-expressing adenovirus constructs, lacking ADP, were generated. One included a wild type replication vector (Wt-ΔE3/ΔADP-NIS) and the other was Cox2-controlled (Cox2-ΔE3/ΔADP-NIS) (
The ability of a NIS-expressing oncolytic adenovirus to kill cancer cells in vitro was evaluated using pancreatic cancer cell line models. The ADP+ construct was significantly more cytolytic than noADP counterpart at earlier time points (
Oncolytic adenovirus expression of NIS in pancreatic cancer cell lines was evaluated by Western Blot. All NIS-OAds efficiently produce glycosylated NIS multimers as early as two days post-infection. However, infection with no-ADP vectors resulted in a significantly greater radioiodine uptake (125I) compared to ADP+ viruses (
Similarly, flow cytometry revealed that infection with noADP vectors resulted in a higher percentage of NIS-positive cells when compared to ADP+ infections in vitro (p<0.05) (
Primary cell cultures from patient cancer tissues may not necessarily accurately reflect in vivo activity, however. For example, isolating the primary cells can result in the loss of tissue structure, a change of cell character, and/or dropout of some cell populations, each of which can alter the character of the cell compared to a comparable cell in a live tissue. Therefore, to analyze the oncolytic adenovirus functionality and safety with live human tissues, ex vivo tissue slicer technology was used to prepare live tissues from de-identified specimens of biopsy-proven human pancreatic ductal adenocarcinoma and adjacent normal pancreas. Data provided in
The Cox2-ΔE3/ΔADP-NIS construct also showed increased radiotracer uptake than its ADP+ counterpart and ADCMV-NIS positive control in various in vivo models (
Similarly, evaluation of NIS-expressing adenovirus constructs using a pre-clinical human pancreatic adenocarcinoma patient-derived xenograft (PDX) model, demonstrated a significantly greater imaging with the noADP construct. The Cox2-ΔE3/ΔADP-NIS vector sustained a significantly earlier, higher intensity, and longer lasting radiotracer [99mTcO4]− uptake compared to other NIS-expressing vectors (
To understand how the noADP construct provides improved and sustained radioisotope uptake in vivo, immunocytochemistry analyses of NIS protein in tumor tissues of virus-treated animals were performed. The Cox2-ΔE3/ΔADP-NIS construct produced more NIS-positive regions in tumors and cell foci than its ADP+ counterpart (
Further clinical evaluation of NIS-expressing adenovirus constructs also demonstrated the ability of oncolytic adenovirus to facilitate therapy with a radioactive iodine [131I]. The combination of [131I] with Cox2-ΔE3/ΔADP-NIS significantly reduced tumor progression in a mouse model of human pancreatic cancer (
Together, these data demonstrate that deleting the Adenovirus Death Protein from the ΔE3-based oncolytic adenovirus improves expression of OAd-delivered transgene at tumor sites. Constructs lacking ADP (noADP) exhibited increased transgene protein expression and radiotracer uptake compare to otherwise identical ADP-positive counterparts. Gene expression from the ΔE3/ΔADP-based vectors was more sustainable and resulted in higher uptake of radioisotope in vitro and in in vivo cancer models, including the clinically relevant patient derived xenograft (PDX) model. In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
The Ad type 5 vectors containing the therapeutic and/or imaging gene in the E3 region are constructed using the “ΔE3/ΔADP” cloning strategy. Briefly, adenoviral death protein (ADP) and all other nonessential E3 genes (12.5 K, 6.7K, gp19K, RID-α and RID-β, and 14.7K) are deleted (
1. Transgene Inserted into the Adenoviral E3 Region
Deleting the adenoviral E3 genes, as described immediately above, liberates ˜3-5 kb of cloning capacity to accommodate the gene of interest. Expressing the gene of interest from the adenovirus E3 region follows a late profile due to control by the major late promoter and is therefore consistent with the replication cycle. This property is exploited to monitor adenovirus replication and to visualize the target tumors. A ΔE3-based oncolytic adenovirus may be designed to express any suitable transgenes from the adenoviral E3 region. Exemplary transgenes that can be expressed in this manner include, for example, therapeutic genes such as IFNα, INFβ, IL-2, IL-6, IL-7, IL-8, IL-12, GM-CSF, anti-CTLA4 and anti-PD1 antibodies, NIS, or imaging/reporter transgenes such Luc, RFP, GFP, EGFP, mCherry, SSTR, TKsr39, CEA, SEAP, or NIS.
The “ΔE3/ΔADP OAds” are controlled with a Tumor Specific Promoter. Vector replication is made tumor-specific by virtue of the tumor-specific promoter control of the adenoviral E1A region. As a consequence, gene expression is restricted to permissive tumors. The Cox-2 promoter is used as a model tumor-specific promoter, providing “tumor on, liver off” profile in pancreatic cancer. However, the promoter may be selected to provide selective expression in appropriate cells for a given disease. Examples include MK, Muc1, PB, PSA, hTERT, GRP, VEGF, SLPI, FLT-1, and caveolin-1 promoters.
For tumors deficient in the primary adenovirus receptor (coxsackie-adenovirus receptor, CAR—e.g. pancreatic cancer, prostate cancer), the vector can include the Ad5/Ad3-modified fiber. In this fiber, the adenovirus type 5 knob is replaced with the knob taken from Adenovirus type 3, which binds to CD46 and human desmoglein-2 receptor. For tumors that are CAR-positive (i.e., lung cancer), one can use the wild type Ad5 fiber.
Pancreatic cancer cells were grown in 6 well plates (5×105) until 80% confluent. Confluent cells were left untreated or treated with 1 viral particle per cell for three hours in approximately 500 μl of 5%FBS+ 1%P/S +DMEM cell growth media in a humidified 37° C. chamber. After three hours, media was removed and cells sub-cultured in fresh growth media for another 2-6 days. At 2 days, 4 days, and 6 days post-infection, experimental cells were collected via scraping into tubes on ice. Cells were washed 1× in cold 1× phosphate buffered saline (PBS). Washed cells were fixed in a 4% paraformaldehyde solution, careful to maintain a single cell suspension and washed again in 1× PBS before permeabilization with 0.05% Triton-X100 in PBS solution. Fixed and permeabilized cells were incubated in a 5% Bovine serum albumin (BSA) in 1× PBS solution containing a (1:500) dilution of anti-NIS primary antibody (FPSA, Santa Cruz Biotechnology, Inc., Dallas, Tex.) for approximately one hour on ice. Cells were washed in 1× PBS and re-suspended in 5% BSA containing secondary antibody conjugated to Phycoerythrin (PE) (Red) and incubated for 30 minutes at room temperature. Antibody stained cells were washed and re-suspended in 1% paraformaldehyde solution for one hour. Cells were washed and re-suspended in 1× PBS and analyzed immediately using a FacsCanto (BDBioSciences, San Jose, Calif.).
The in vitro cytocidal effect was analyzed via crystal violet staining. One day after 2×105 cells/well were grown in a 12-well plate, viruses were added at strengths of 1 vp/cell. After 6 days, 7 days, or 9 days of cultivation, the cells were fixed with 10% buffered formalin for 10 minutes and stained with 1% crystal violet in 70% ethanol for 20 minutes. Thereafter, the plates were washed three times with tap water and allowed to air dry.
3,000 cells/well were cultured in 96-well plates and subsequently infected with adenoviral vectors at strengths of 100 vp/cell, 1000 vp/cell, or 10,000 vp/cell. The number of surviving cells was analyzed by a colorimetric method using the Cell Titer Aqueous One Solution Cell Proliferation Assay (Promega, Madison, Wis.) as described by the manufacturer. Absorbance was measured at a wavelength of 490 nm in a FLUOstar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany). The proportion of living cells at each time point was normalized to the number of living uninfected cells.
Pancreatic cancer cells were grown in 10 cm2 dishes until 80% confluent. Confluent cells were left untreated or infected with 1 viral particle per cell for three hours in 5 ml of normal cell growth media in a humidified 37° C. chamber. After three hours, media was removed and cells sub-cultured in fresh growth media for another two days. At two days post-infection, experimental cells were collected via scraping into tubes on ice. Cells were washed 1× in cold 1× phosphate buffered saline (PBS) and transferred to fresh microfuge tubes. Washed cell pellets were lysed in 100 l of RIPA lysis solution containing fresh PMSF and proteases (Thermo Fisher Scientific, Inc., Waltham, Mass.) on ice. Lysates were fractionated at 14,000× g for 15 minutes in cold, supernatants were collected and placed into fresh chilled microfuge tubes for storage at −70° C. until further use.
Protein concentrations were determined using the DC Protein Estimation Assay and a BSA protein standard curve. Approximately 100 μg of diluted protein samples were diluted 1:4 with sample buffer and heated at 100° C./7 minutes prior to separation using a 7-9% Bis-Tris SDS-PAGE under standard conditions. Proteins were transferred under standard conditions onto PVDF membranes. Membranes were blocked using a 5% non-fat milk in a Tris buffered saline solution containing Tween (TBST) for one hour at room temperature. Blocked membranes were incubated with a (1:500) dilution with anti-NIS primary antibody or (1:200) or anti-actin (FPSA or actin, Santa Cruz Biotechnology, Inc., Dallas, Tex.) overnight with shaking in cold. After incubation, blots were washed in TBST three times for 10 minutes followed by incubation with a secondary antibody conjugated to infrared for 45 minutes shaking at room temperature. Membranes were then imaged using an imaging system (ODYSSEY, Li-Cor Biotechnology, Lincoln, Nebr.).
Cells were plated in 96-well plates (Corning, Inc., Corning, N.Y.) in the density of 8×103 cells/well. Next day, cells were infected with 100 μl of DMEN 5% FBS and 1% penicillin/streptomycin with 1 vp/cell of each virus. Infection was carried out for four hours and media was replaced with appropriate media. DMEM 5% FBS and 1% penicillin/streptomycin with 10 μM or 20 μM 5-FU. Plates were incubated at 37° C. in CO2 humidified incubator. CellTiter 96 Aqueous One Solution Cell Proliferation Assay MTS (Promega, Madison, Wis.) reagent was added to each well, and plates were incubated for one hour at 37° C. in 5% CO2 humidified incubator. Plates were read at 490 nm using a plate reader.
Crystal Violet assays
A total of 2×105 cells were plated in a 12-well cell culture plate (Corning, Inc., Corning, N.Y.). Next day, plates were infected with 50 vp/cell or 100 vp/cells of RGDΔE3ADPIFN. After 10 days of cultivation, the cells were fixed with 10% buffered formalin for 10 minutes and stained with 1% crystal violet in 70% ethanol for 20 minutes, followed by washing three times with tap water and air drying.
Immunofluorescence of NIS Pancreatic cancer cells were grown on two 8-well chamber slides (5×104 per well) until 80% confluent. Confluent cells were left untreated or treated with 1 viral particle per cell for three hours in 100 μl of cell growth media in a humidified 37° C. chamber. After three hours, media was removed and cells sub-cultured in fresh growth media for another 3-5 days. At 3 and 5 days post-infection, cells were fixed in chamber slides by first washing with cold 1× PBS. Washed cells were fixed in a 4% paraformaldehyde solution for 20 minutes on ice and then permeabilization with 0.05% Triton-X100 in PBS. Fixed and permeabilized cells were incubated in a 5% Bovine serum albumin (BSA) in 1× PBS solution at a (1:500) dilution with anti-NIS primary antibody or anti-actin (FPSA, Santa Cruz Biotechnology, Inc., Dallas, Tex.) for approximately one hour on ice. Cells were washed in 1× PBS and re-suspended in 5% BSA containing secondary antibody conjugated to phycoerythrin (PE) (Red) and incubated for 30 minutes at room temperature. Antibody stained cells were cover-slipped with DAPI/nuclear stain just prior to image capturing using a fluorescent microscope (40×).
Tumor growth assay was conducted by injecting Panc-1 cells subcutaneous in flanks of nude mice. Tumors were injected with either vehicle or 1×109viral particle per tumor.
Pancreatic tumors were embedded at the time of removal into paraffin and later sectioned onto glass slides. Sections were dewaxed in xylenes, washed with ethanol (70%, 50%, 90%, 100%) and rehydrated using distilled water. An additional antigen retrieval step was taken using a universal antigen retrieval solution 1× as instructed (R&D Systems, Inc., Minneapolis, Minn.). Hydrophobic pens were used to draw an outline around tissues. Tissues were permeabilized using a 0.05% Triton-X100 in PBS for five minutes on ice. Sections were washed and incubated in a 5% bovine serum albumin (BSA) in 1× PBS solution for one hour at room temperature to reduce background. Sections were then incubated in a 5% BSA in 1× PBS solution at a (1:500) dilution with anti-NIS primary antibody or anti-hexon-FITC (green) (FPSA, Santa Cruz Biotechnology, Inc., Dallas, Tex.) in cold temperature overnight. Sections were then washed in 1× PBS and incubated in a 5% BSA containing secondary antibody conjugated to phycoerythrin (PE) (Red) and incubated for one hour at room temperature. Antibody stained sections were cover-slipped with DAPI/nuclear stain just prior to image capturing using a fluorescent microscope (40×).
Human freshly isolated pancreatic cancer tissues or pancreatic cancer cell line Panc-1 was used to establish subcutaneous tumor models in immunodefficient mice. After tumor nodules reached 8-10 mm, a single injection of each virus was given.
At different time points after adenovirus administration, animals received intraperitopneal injections of 0.5 mCi of sodium pertechnetate ([99mTcO4]−). To measure radionuclide uptake induced by viral replication, a high-resolution micro-SPECT/CT system was used for planar and fusion images. Planar, real-time images were taken at each time point one hour after isotope injection, with five replicates per viral construct. Tumor size in the region-of-interest was determined by the number of counts present in the tumor, adjusted for background and isotope decay. Tumor growth was measured over the 21 days or up until just prior to its removal.
Three days after adenovirus administration, animals bearing human pancreatic cancer xenografts received intraperitopneal injections of I−131 (3 mCi). Tumor growth was measured over the 27 days for evaluation of the tumor size. Eight days later I−131 treated mice tumors and leg muscle were harvested and relative I−131 uptake was determined using a gamma counter.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims priority to U.S. Provisional Patent Application No. 62/330,467, filed May 2, 2016, which is incorporated herein by reference.
This invention was made with government support under 5R01-CA174861 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62330467 | May 2016 | US |
