Patent Application
20120100109
The present invention is directed to methods and compositions to significantly increase the yield and dissemination of oncolytic Herpes simplex viruses (HSVs) in semipermissive or resistant tumor cells. The present invention also relates to the combined administration of PI3K/AKT/mTOR pathway inhibitors (e.g., rapamycin and LY294002) and a HSV-derived oncolytic virus (a virus that can selectively kill tumor cells) to either block or reverse the growth of tumors that are otherwise resistant to the therapeutic effect of either agent alone. This invention has important applications in potentiating the activity of oncolytic HSVs against difficult-to-treat human tumors and/or in preventing the emergence of resistant tumor cells during virotherapy.
Virotherapy has shown substantial promise as a new treatment modality for a broad range of human tumors (Russell, et al., Viruses as anticancer drugs, Trends Pharmacol. Sci. 2007; 28:326-33; Liu, et al., Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress, Nat. Clin. Pract. Oncol. 2007; 4:101-17). Oncolytic herpes simplex virus (HSV) is currently in phase III clinical trials for development as a novel therapeutic agent against a broad range of human tumors. Although results have been promising, clinical outcome is likely to be compromised by intrinsic and acquired resistance to HSV replication, leading us to test agents that may overcome this obstacle. The antitumor activity of an oncolytic virus derives mainly from its ability to replicate after it infects a tumor cell, with subsequent spread of the progeny virus to the nearby tumor cells. The resultant extent of tumor destruction often exceeds that achieved with many other types of cancer biotherapeutic agents (Thorne, et al., Oncolytic virotherapy: approaches to tumor targeting and enhancing antitumor effects, Semin. Oncol. 2005; 32:537-48). Consequently, the ability of an oncolytic virus to replicate robustly in tumor cells is a key factor in securing a favorable outcome from virotherapy (Everts, et al., Replication-selective oncolytic viruses in the treatment of cancer, Cancer Gene Ther. 2005; 12:141-61).
Herpes simplex virus (HSV) has a broad cell tropism, and oncolytic viruses derived from parental HSV strains can lyse tumor cells of many different tissue origins (Rabkin, et al, Replication-Competent Viruses for Cancer Therapy, Basel: Karger, 2001:1-45). Nonetheless, tumor cells that are resistant to HSV oncolysis are encountered from time to time and pose significant barriers to therapeutic outcomes. Several strategies have been proposed to overcome the resistance of tumor cells to HSV. It has been reported, for example, that serial passage of an oncolytic HSV (a γ34.5-deleted mutant) in resistant glioma cells can select for viral progeny that replicate more efficiently in the tumor cells and then show an enhanced antitumor effect against these resistant gliomas in vivo (Shah, et al., Serial passage through human glioma xenografts selects for a Deltagamma134.5 herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice with experimental brain tumors, J. Virol. 2006; 80:7308-15). Prior art shows that although cyclophosphamide did not improve oncolytic HSV replication in the resistant Lewis lung carcinoma cells, its in vivo administration still enhanced the antitumor effect of the virotherapy (Li, et al., Coadministration of a herpes simplex virus-2 based oncolytic virus and cyclophosphamide produces a synergistic antitumor effect and enhances tumor-specific immune responses, Cancer Res. 2007; 67:7850-5).
Two groups have recently reported that rapamycin, an inhibitor of the mTOR (mammalian target of rapamycin) pathway, can increase the permissiveness of some resistant tumor cells to oncolytic myxoma virus or vesicular stomatitis virus (VSV). Stanford et al, for example, used rapamycin to pretreat human tumor cell lines that normally restrict myxoma virus replication and observed a striking increase in viral tropism and spread (Stanford, et al., Oncolytic virotherapy synergism with signaling inhibitors: Rapamycin increases myxoma virus tropism for human tumor cells, J. Virol. 2007; 81:1251-60). The enhanced replication of the myxoma virus in cells with a silenced mTOR pathway appeared to be linked to an increase in Akt kinase, suggesting that rapamycin could be used to improve the efficacy of oncolytic poxviruses in cancer treatment. Alain et al reported that rapamycin could significantly increase the replicative capability of an interferon (IFN)-sensitive VSV mutant (DeltaM51) in malignant glioma cells (Alain, et al., Vesicular stomatitis virus oncolysis is potentiated by impairing mTORC1-dependent type I IFN production, Proc. Natl. Acad. Sci. USA 2010; 107:1576-81). This enhancing effect apparently derived from the reduced inhibitory effect of type I IFNs on VSV (DeltaM51) replication once mTORC1 signaling is interrupted by rapamycin. More recently, rapamycin has been reported to enhance the therapeutic effect of a recombinant adenovirus carrying the gene encoding eukaryotic initiation factor 4E binding protein-1 (Mishra, et al., Adenovirus-mediated eukaryotic initiation factor 4E binding protein-1 in combination with rapamycin inhibits tumor growth of pancreatic ductal adenocarcinoma in vivo, Int. J. Oncol. 2009; 34:1231-40) and a newly developed mTOR inhibitor, Torin1, has been shown to enhance human cytomegalovirus replication (Moorman, et al., Rapamycin-resistant mTORC1 kinase activity is required for herpesvirus replication, J. Virol. 2010; 84:5260-9). However, none of these researchers have specifically investigated the effect of rapamycin on resistant tumor cells, nor did they look at oncolytic HSVs.
Previous studies failed to find any effective enhancement effect of rapamycin on replication of oncolytic HSVs in permissive tumor cells. For example, Some tumor cell lines are known to be fully permissive to the replication of oncolytic HSVs (Fu, et al., Expression of a fusogenic membrane glycoprotein by an oncolytic herpes simplex virus provides potent synergistic anti-tumor effect, Mol. Ther. 2003; 7:748-54; Fu, et al., A Mutant Type 2 Herpes Simplex Virus Deleted for the Protein Kinase Domain of the ICP10 Gene Is a Potent Oncolytic Virus, Mol. Ther. 2006; 13:882-90; Fu, et al., Potent systemic antitumor activity from an oncolytic herpes simplex virus of syncytial phenotype, Cancer Res. 2002; 62:2306-12). Baco-1 replicates to a high titer in these permissive tumor cells, reaching more than 1×107 plaque-forming-units (pfu) per milliliter as illustrated in
Oncolytic viruses have the potential to improve clinical outcome for a spectrum of human tumors, but this promise is compromised by the existence or emergence of treatment-resistant tumor cells. Tumor cells become resistant to virotherapy for several reasons. They may lack receptors for a particular oncolytic virus, as reported for adenovirus-based oncolytic viruses (Van Beusechem, et al., Conditionally replicative adenovirus expressing a targeting adapter molecule exhibits enhanced oncolytic potency on CAR-deficient tumors, Gene Ther. 2003; 10:1982-91), but this mechanism is unlikely to apply to oncolytic HSVs, which rely on more ubiquitous cellular receptors for entry (Spear, Herpes simplex virus: receptors and ligands for cell entry, Cell Microbiol. 2004; 6:401-10). Instead, tumor cell resistance to HSV-based oncolytic viruses probably reflects failure of virus replication once the cell has been infected. The present invention overcomes this known limitation by using drugs such as rapamycin or LY294002 to release the restriction placed on oncolytic HSV replication in resistant tumor cells, therefore increasing the virus yield as well as the spread to nearby cells. The current invention clearly demonstrates that the combination of one or more than one drugs and an oncolytic HSV can potentiate viral replication in highly resistant tumor cells, leading to a significantly enhanced antitumor effect.
The present invention addresses one of the core issues in the cancer treatment field. Therapy-resistant tumor formation is one of the main causes for reducing treatment effectiveness in the clinic. Methods and/or strategies to sensitize resistant tumors to a particular therapeutic modality can be extremely beneficial to the prognosis of cancer patients. The present invention discloses a method to sensitize resistant tumors to the treatment of herpes simplex virus (HSV)-based oncolytic virotherapy. This invention also emphasizes that HSV-derived oncolytic treatment of these semipermissive or resistant tumor cells with PI3K/AKT/mTOR pathway inhibitors, including but not limited to rapamycin or LY294002, can efficiently sensitize the cells to HSV-derived oncolytic viruses, i.e. increase tumor cell permissiveness to HSV-derived viruses. Without the drug treatment, HSV derived oncolytic viruses replicate and spread poorly in these tumor cells. When PI3K/AKT/mTOR pathway inhibitors are administered in an HSV-derived virotherapy, the replication and spread of the viruses are dramatically enhanced. For example, in animal models, administration of an inhibitor in an HSV-based virotherapy can efficiently shrink or even eradicate these tumors, while these components show little therapeutic effect if they are used individually. This discovery has a clear clinical value in forming a combinatorial treatment regimen for the treatment of resistant tumors.
P<0.05 vs. all other groups; ★P<0.01 vs. all other groups. Two-tailed t-test.
P<0.01 vs. Baco-1 treatment alone.
The present invention relates to the design, construction, characterization and use of a novel method to significantly increase the yield and dissemination of oncolytic Herpes simplex viruses (HSVs) in resistant tumor cells. The present invention also relates to the combined administration of PI3K/AKT/mTOR pathway inhibitors (e.g., rapamycin and LY294002) and a HSV-derived oncolytic virus to either block or reverse the growth of tumors that are otherwise resistant to the therapeutic effect of either agent alone. This invention has important applications in potentiating the activity of oncolytic HSVs against difficult-to-treat human tumors and/or in preventing the emergence of resistant tumor cells during virotherapy.
In a preferred embodiment of the present invention the inhibition of PI3K/AKT/mTOR signaling pathway improves the replication potential of oncolytic HSVs in cells that are known to be highly resistant to oncolytic HSV replication (Fu, et al., Virotherapy induces massive infiltration of neutrophils in a subset of tumors defined by a strong endogenous interferon response activity. Cancer Gene Ther. 2011 August 26. doi: 10.1038/cgt.2011.46). The presence of rapamycin or LY294002 in the medium increases the replication of Baco-1 (an oncolytic HSV) in EC9706 cells (known to be highly resistant to oncolytic HSV replication) more than 6-fold as illustrated in
In another preferred embodiment of the present invention the use of rapamycin or LY294002 promotes the spread of oncolytic HSV in highly resistant, but not fully permissive tumor cells. Because Baco-1 contains the Green Fluorescent Protein (GFP) gene, it is possible to visualize GFP expression during virus infection and thus monitor the spread of virus among tumor cells.
1Relative permissiveness was estimated by checking for GFP positive cells 24 h after Baco-1 infection at 0.1 pfu/cell.
2Fold of change in virus yield was calculated by dividing the virus yield from the well with rapamycin with that from the well without the drug.
In another preferred embodiment of the present invention the combined administration of one or more than one drug, such as rapamycin and LY294002, a P13 kinase inhibitor, when combined with HSV-based virotherapy, enhances the therapeutic effect—while use of either agent alone produces only transient inhibitory effect.
In another preferred embodiment of the present invention the potentiating effect of rapamycin on virus replication in highly resistant tumor cells is applied to other oncolytic HSVs, including strain 17 (17+), a wild-type HSV-1, FusOn-H2, which is constructed from an HSV-2 by mutating the N-terminal region of the ICP 10 gene (Fu, et al., A Mutant Type 2 Herpes Simplex Virus Deleted for the Protein Kinase Domain of the ICP10 Gene Is a Potent Oncolytic Virus, Mol. Ther. 2006; 13:882-90), and ApE-Mir-3, an HSV-1-based oncolytic virus in which the glycoprotein H (gH) gene is controlled by tissue-specific microRNAs (miRNAs), including let-7 and mir-122. To that end, EC9706 cells are infected with these viruses at 0.1 pfu/cell for 1 h and then cultured the cells in media with or without rapamycin at a concentration of 100 nM. The results in
In yet another preferred embodiment of the present invention the coadministration of rapamycin significantly increases the antitumor effect of Baco-1 in vivo. To test the beneficial effect of rapamycin on the oncolytic activity of Baco-1 in vivo, tumors from the highly resistant EC9706 line of human esophageal carcinoma cells are established by implanting tumor cells into the right flank of immune-deficient mice. When tumors reach the approximate size of 5 mm in diameter, the mice are divided randomly into four groups and treated by: (i) intratumoral injection of PBS only; (ii) intratumoral injection of Baco-1; (iii) intraperitoneal administration of rapamycin; and (iv) intratumoral injection of Baco-1 and intraperitoneal administration of rapamycin. Rapamycin is given daily at the dose of 50 μg/kg body weight, a dose that had been shown to only marginally affect EC9706 tumor growth when the drug is given alone (Hou, et al., mTOR inhibitor rapamycin alone or combined with cisplatin inhibits growth of esophageal squamous cell carcinoma in nude mice, Cancer Lett. 2010; 290:248-54). Antitumor effects are assessed over 3 weeks. While Baco-1 and rapamycin given alone slowed the growth of EC9706 tumors in mice, the combined administration of these agents (Baco-1-Rapa) blocked the growth entirely as illustrated in
Rapamycin enhances oncolytic HSV replication in tumor cells that do not fully support the virus growth. A. EC9706 cells were either preincubated with rapamycin overnight (Pre-Inf) or incubated with the drug during the virus infection (During-Inf). They were then infected with Baco-1 at 0.1 pfu/cell and for 72 h. Fold increase in virus yield was calculated by dividing the yield in the control well with that in the rapamycin treated well. Rapamycin was found to increase the yield of an oncolytic HSV by almost 6-fold. B. MCF-7 and HeLa cells were infected with Baco-1 at 0.01 pfu/cell for 1 h. Then the cells were cultured in medium without (control) or with rapamycin at a concentration of 100 nM for 72 h before harvesting for virus titration. Rapamycin was found to increase the yield of an oncolytic HSV by 3-5 fold. C. without the drug treatment, the yield of oncolytic virus in these resistant tumor cells was quite low (around 1×105 plaque forming units (pfu). In the presence of rapamycin, the virus yield was increased by more than a log, to almost 5×106 pfu.
Rapamycin promotes the spread of oncolytic HSV in semipermissive tumor cells and tumor cells that do not fully support the virus growth. Three permissive tumor cells (MDA-MB-231, Huh-7 and Hep-G2 cells) are infected with Baco-1 at 0.1 pfu/cell and incubated without or with rapamycin (100 nM). Micrographs taken at 48 h postinfection did not show any effect on the spread of Baco-1 in these permissive tumor cells. In the same experiment, three highly resistant tumor cells (EC9706, MCF-7 and Hela cells) are infected with Baco-1 at 0.01 pfu/cell and incubated without or with rapamycin at a concentration of 100 nM, or LY294002 at 50 μM concentration. Both rapamycin and LY294002 were found to dramatically enhance the spread of the oncolytic HSV (Baco-1) in all the three resistant tumor cells.
Rapamycin enhances the replication of several other types of oncolytic HSVs in tumor cells that do not fully support the virus growth. EC9706 cells were infected with three other types of oncolytic HSVs, including FusOn-H2 (an oncolytic HSV derived from HSV-2, while Baco-1 was derived from HSV-1) and Ape-Mir3 (an oncolytic HSV specifically targets to hepatocellular carcinoma), at 0.1 pfu/cell and then incubated with medium without or with rapamycin (100 nM) for 72 h before harvesting for virus titration. Rapamycin was found to increase the yield of these viruses by 3-10 folds in this highly resistant tumor cells.
Rapamycin potentiates the antitumor effect of oncolytic HSV against resistant tumor xenografts growing in immunodeficient animals. Mice bearing implanted EC9706 tumors on the right flank were mocked treated (PBS), treated with either Baco-1 (1×106 intratumorally) or rapamycin (50 μg/kg intraperitoneally), or treated with the combination of these agents (n=8 mice per group). Tumor size was periodically monitored after treatment. The results show that the combinatorial treatment resulted in a therapeutic effect that was significantly better than either treatment alone, indicating that rapamycin can greatly potentiate the antitumor effect of the HSV-virotherapy.
Rapamycin increases oncolytic HSV spreading within tumor xenografts. Mice bearing implanted EC9706 tumors were mocked treated (PBS), treated with either Baco-1 (1×106 intratumorally) or rapamycin (50 μg/kg intraperitoneally), or treated with the combination of these agents. Tumor samples were collected 3 days after treatment. Because of possible interference of nonspecific autofluorescence from the tumor tissues, tumor sections were immunohistochemically stained for GFP, as a means to assess the distribution of Baco-1 within tumor masses. The first antibody is rabbit anti-GFP polyclonal antibody and the second antibody is Texas red-conjugated goat anti-rabbit IgG antibody. Co-administration of rapamycin with oncolytic HSV (Baco-1) to tumor-bearing animals can greatly increase the spread of the virus within tumor tissues, as judged by visualization of tumor sections after immunohistochemical staining for GFP and by counting GFP-positive tumor cells isolated from the treated tumor tissues.
While the invention described herein specifically focuses on use of a novel method to significantly increase the yield and dissemination of oncolytic HSVs in semipermissive tumor cells, one of ordinary skills in the art, with the benefit of this disclosure, would recognize the extension of the approach to other combinatorial treatment regimens for the treatment of resistant tumors in a clinical setting.
The present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. The particular embodiments disclosed above are illustrative only, as the present invention may be modified and practiced in different but equivalent manners apparent to those skilled in the art having the benefit of the teachings herein. Furthermore, no embodiments disclosed herein are intended to limit the scope of the claims of this invention. It is therefore evident that the particular illustrative embodiments disclosed above may be altered or modified and all such variations are considered within the scope and spirit of the present invention.
This application claims priority to provisional application No. 61/406,951 filed on Oct. 26, 2010, which is herein incorporated by reference in its entirety.
The U.S. Government has a paid-up license in this invention and the rights in limited circumstances to require the patent owners to license others on reasonable terms as provided for by the terms of grant Nos. 7R01CA132792-03 and 7R01CA106671-07 awarded by the National Institute of Health.
| Number | Date | Country | |
|---|---|---|---|
| 61406951 | Oct 2010 | US |
