Jason G. Fewell

Treatment of disseminated ovarian cancer using nonviral interleukin‐12 gene therapy delivered intraperitoneally

The poor prognosis associated with ovarian cancer is primarily the result of delayed diagnosis and the lack of an effective treatment for advanced disease. Use of novel immunotherapy strategies are being evaluated that work to enhance local and systemic immune responses against cancer cells and can possibly work together with traditional cytotoxic chemotherapy regimens to produce more effective treatment options.

Phase I trial of a formulated IL-12 plasmid in combination with carboplatin and docetaxel chemotherapy in the treatment of platinum-sensitive recurrent ovarian cancer

OBJECTIVES The primary objective of this study was to evaluate the safety and tolerability of a formulated IL-12 plasmid administered intraperitoneally (IP) in conjunction with intravenous (IV) carboplatin/docetaxel in platinum-sensitive ovarian cancer patients. METHODS Escalating doses of IL-12 plasmid (phIL-12) formulated with the lipopolymer PEG-PEI-Cholesterol (PPC) were administered IP every 10-11 days for a total of four treatments and the highest dose was expanded to eight treatments. Patients also received IV carboplatin (AUC 5) and docetaxel (75 mg/m(2)) every 21 days. Patients were followed for safety, biological activity and antitumor activity after phIL-12/PPC treatment. RESULTS All 13 patients enrolled in the study received both phIL-12/PPC and chemotherapy treatment. There were 49 plasmid-associated adverse events (AEs). The most common AEs were abdominal pain, transient hypotension, low grade fever, catheter site pain, chills, dysgeusia, infusion-related reaction, and nausea. These AEs appeared to be plasmid dose related. Grade 3 AEs included manageable abdominal pain and cytokine release syndrome. There were no dose limiting toxicities and the plasmid treatment did not augment the chemotherapy-associated AEs. The best overall antitumor response (17% CR, 33% PR, 42% SD and 8% PD) was typical of the patient population enrolled for the study. Translational studies showed rise in IFN-γ and TNF-α concentrations in a dose dependent manner. CONCLUSIONS The escalating doses and cycles of intraperitoneal phIL-12/PPC when combined with carboplatin/docetaxel chemotherapy in recurrent ovarian cancer patients were well tolerated and did not appear to exacerbate the side effects or attenuate the efficacy of the chemotherapy treatment.

Versatile cationic lipids for siRNA delivery.

Exploitation of the RNA interference (RNAi) pathway offers the promise of new and effective therapies for a wide variety of diseases. Clinical development of new drugs based on this platform technology is still limited, however, by a lack of safe and efficient delivery systems. Here we report the development of a class of structurally versatile cationic lipopolyamines designed specifically for delivery of siRNA which show high levels of target transcript knockdown in a range of cell types in vitro. A primary benefit of these lipids is the ease with which they may be covalently modified by the addition of functional molecules. For in vivo applications one of the core lipids (Staramine) was modified with methoxypolyethylene glycols (mPEGs) of varying lengths. Upon systemic administration, PEGylated Staramine nanoparticles containing siRNA targeting the caveolin-1 (Cav-1) transcript caused a reduction of the Cav-1 transcript of up to 60%, depending on the mPEG length, specifically in lung tissue after 48h compared to treatment with non-silencing siRNA. In addition, modification with mPEG reduced toxicity associated with intravenous administration. The ability to produce a high level of target gene knockdown in the lung with minimal toxicity demonstrates the potential of these lipopolyamines for use in developing RNAi therapeutics for pulmonary disease.

Regulated production of proteins from muscle using gene transfer: potential therapeutic applications

The ability to produce high-level transgene expression following the introduction of genetic material into a host cell has been well documented. Various vectors and methods for in vivo gene delivery have been shown to provide long-term expression from many different tissue types in rodents and large animals. However, many potential therapeutic targets for gene therapy involve the production of proteins that are toxic or lead to undesirable effects if overexpressed. Thus, the ability to achieve regulated gene expression following treatment will be required to ensure the safety of long-acting gene therapy products. Skeletal muscle, in particular, has been widely used as a target for gene therapy protocols, due to the ease of accessibility and ability to produce and secrete some proteins at very high levels. This review focuses on regulated gene therapy systems that are being evaluated for use in muscle, and discusses two classes of system: those dependent on exogenously administered drugs and those dependent on endogenously produced metabolites.

GEN-1 immunotherapy for the treatment of ovarian cancer

GEN-1 is a gene-based immunotherapy, comprising a human IL-12 gene expression plasmid and a synthetic plasmid delivery system, delivered intraperitoneally (ip.) to produce local and persistent levels of a pleiotropic immunocytokine, IL-12, at the tumor site in patients with advanced ovarian cancer. The goal of local and persistent IL-12 delivery is to remodel the highly immunosuppressive tumor microenvironment to favor immune stimulation while avoiding serious systemic toxicities, a major limitation of recombinant IL-12 therapy. Safe and sustained local production of IL-12 and related immunocytokines at the tumor site could produce potentially more favorable immunological changes in the tumor microenvironment and antitumor responses than a bolus systemic delivery of recombinant IL-12. Treatment safety, clinical benefits and biological activity of GEN-1 ip. in patients with ovarian cancer and in representative animal models are described.

Abstract 2078: Interleukin-12 gene therapy in combination with bevacizumab and pegylated liposomal doxorubicin for treatment of disseminated ovarian cancer

Despite recent improvements in treatment options for ovarian cancer patients, notably, the approval of using bevacizumab in combination with chemotherapies including pegylated liposomal doxorubicin (PLD), this disease is still the most deadly of all gynecological malignancies requiring new and novel therapeutics. Interleukin-12 (IL-12) is a highly active cytokine that can induce a potent anti-cancer immunity mediated through activation of cytotoxic T-lymphocytes, natural killer cell proliferation, and secretion of interferon-γ. We are developing an IL-12 based gene therapy for the treatment of gynecological malignancies that have spread into the peritoneal cavity. Our approach utilizes IL-12 plasmid (pIL-12) formulated with the PPC delivery system, which is comprised of a low molecular weight polyethylenimine covalently linked to polyethyleneglycol and cholesterol. Previously we have shown in a mouse model of disseminated ovarian cancer efficacy of a treatment regimen of pIL-12/PPC used in combination with paclitaxel and carboplatin. The combination treatment significantly improved survival compared to either pIL-12/PPC alone or chemotherapy alone and demonstrated the feasibility of using an IL-12 immunotherapy in combination with cytotoxic chemotherapies to achieve additive therapeutic effects. Results from a Phase I clinical trial in platinum resistant patients have shown that intraperitoneal delivery of pIL-12/PPC in combination with PLD produced an overall clinical benefit of 57.1% (PR = 21.4%; SD = 35.7%) in patients with measurable disease. The highest percentage of PRs were found at the highest dose level (28.6%) along with highest percentage of patients achieving SD (57.1%). Here we describe studies evaluating the combination of pIL-12/PPC with bevacizumab and PLD. For these studies 5,000,000 human SKOV3 cells were implanted into the peritoneal cavity of immunocompromised Hsd:Athymic Nude-Foxn1nu mice. Treatment with pIL-12/PPC alone and bevacizumab alone resulted in a 50% and a 39% reduction of animals with visible tumors at the end of the study. Combining pIL-12/PPC + bevacizumab improved the response to 78% of animals with no visible tumors. Further, combining pIL-12/PPC + bevacizumab + PLD resulted in a >98% decrease in tumor burden in animals compared to controls and ∼92% decrease in tumor burden compared to animals treated only with bevacizumab + PLD. All treatments were well tolerated and analysis of serum chemistries and hematology showed normal ranges of all parameters examined for all groups. There were no significant differences in animal weights between groups during the experiment. Together these results suggest synergistic efficacies can be achieved by combining a novel pIL-12/PPC immunotherapy with anti-angiogenesis therapies and cytotoxic chemotherapies in disseminated ovarian cancer. Citation Format: Jason G. Fewell, Majed M. Matar, Jennifer Rice, Diane McClure, Elaine Brunhoeber, Jeff Sparks, Stefanie Greenleaf, Kelley Smith, Khursheed Anwer. Interleukin-12 gene therapy in combination with bevacizumab and pegylated liposomal doxorubicin for treatment of disseminated ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2078.

Abstract B17: Inhibition of lung tumor progression in a metastatic mouse model following intravenous delivery of siRNA nanocomplexes

Abstract Exploiting the RNAi pathway offers the potential to advance the treatment of many diseases through highly specific and efficient silencing of gene products. Unfortunately, the requisite safe and efficient delivery of nucleic acids to target cells remains a fundamental problem for the development of RNA and DNA based therapeutics. We have developed a versatile lipopolyamine based delivery system that has been optimized for in vivo delivery by incorporating functional groups onto the core cationic lipopolyamine structure. The direct modification of the core structure (named Staramine) allows for the generation of nanoparticle formulations with a wide range of physicochemical properties and does not require the co-formulation of commercial helper or pegylated lipids. We have previously reported safe, efficient and persistent siRNA mediated transcript knockdown specifically in the lung endothelium using the Starmaine delivery system. Here we extend those observations by administering a siRNA that targets vascular endothelial growth factor receptor 2 (VEGFR-2; KDR/Flk-1) in an animal model of metastatic lung cancer. VEGFR-2, a receptor tyrosine kinase, has been shown to be mainly expressed on endothelial cells and plays a critical role in cell proliferation and differentiation and consequently is also important in angiogenesis associated tumor growth and metastasis. Repeated intravenous administration of nanocomplexes comprised of Staramine formulated VEGFR-2 siRNA (~2 mg/kg/dose) resulted in a significant (40%) reduction of VEGFR-2 transcript levels in isolated tumors from the lungs of mice compared to control injected animals. Similar levels of transcript knockdown were not achieved in tumors when using siRNAs against targets not having the high level of endothelial cell restriction that is seen with VEGFR-2. Immunohistopathology of the lungs of tumor bearing mice administered VEGFR-2 siRNA indicated a significant decrease in vascular density in lung tumors and was consistent with an overall reduction in tumor burden in the mouse lungs. These results support the continued evaluation of using Staramine to deliver siRNAs and other similar molecules as potential therapies for diseases of the lung where the vascular endothelium may be involved. Citation Format: Jason G. Fewell, Khursheed Anwer, Kevin Polach, Majed Matar, Jennifer Rice, Angela Rea-Ramsey, Jeff Sparks, Diane McClure, Casey Pence, Elaine Brunhoeber, Leslie Wilkinson. Inhibition of lung tumor progression in a metastatic mouse model following intravenous delivery of siRNA nanocomplexes [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr B17.

Abstract 5535: Delivery of siRNA via functionalized lipopolyamine systems

The efficient delivery of siRNA molecules to target cells is critical for the application of RNAi to the treatment of human cancers. To this end, we have developed novel cationic lipopolyamime structures that can be used to complex or encapsulate siRNA into nanoparticles for efficient in vitro or in vivo delivery. Unlike conventional cationic delivery systems, these lipopolyamines are chemically flexible which allow for covalent attachment of functional moieties which can modulate their biological activity. For example, modification of these systems with serum stabilizing agents allows for modulation of physical parameters important for biological properties such as toxicity and biodistribution. Intravenous (iv) injection of these functionalized nanoparticles showed preferential distribution to the lungs and corresponding depletion of targeted mRNA transcripts from lung tissue. We have investigated these systems for use in delivering siRNA targeting VEGF in mice with advanced disseminated ovarian cancer through intraperitoneal (ip) injection of siRNA-lipopolyamine nanoparticles. This model was established by injecting animals (ip) with 2.5×10 6 ID8 cells (malignant transformed ovarian surface epithelial cells that have been further modified to overexpress VEGF protein). Delivery of serum stabilized complexes to the peritoneal cavity of mice with disseminated ovarian cancer resulted in significant target gene knockdown in cells obtained from the malignant ascites fluid of these animals with modest knockdown in the peritoneal tumors themselves. A further modification of the core lipopolyamine structure incorporated attachment of a targeting peptide ligand through a linker molecule. Physico-chemical characterization of this targeted system and in vitro assays with ID8 cancer cells demonstrated specific delivery of siRNAs dependent on the presence of the targeting ligand on the liposome and the targeted receptor on the cell surface. These results have prompted in vivo testing using the targeted delivery systems to assess siRNA distribution as a function of delivered dose. Additional studies are underway that will establish the optimized delivery conditions for use in tumor bearing animals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5535.

279. A Novel Functionalized Polymeric Formulation for use in Delivering Therapeutic Genes for the Treatment of Solid Tumors

We have described the synthesis, physico-chemical properties and gene therapy application of a novel water soluble gene delivery system PEG-PEI-Chol (PPC). PPC is composed of a low molecular weight branched polyethyleneimine, cholesterol and polyethylene glycol and designed to promote DNA condensation into nanoparticles, enhance membrane uptake and improve biocompatibility and pharmacokinetics, respectively. The chemical composition and molar ratios between the individual components of the polymer were verified by NMR. Formulation of PPC with DNA produced particles of |[sim]|100nm mean diameter and zeta potential of |[sim]|20mV. In vitro optimization studies using a luciferase reporter gene were performed in order to evaluate the effects of varying amounts of PEG and increasing nitrogen to phosphorus ratio (N:P). Use of the PPC delivery system with a plasmid encoding for interleukin-12 (IL-12) for treatment of solid tumors was examined in mice. Significant inhibition in tumor growth rate and/or increased incidence of complete tumor rejection was seen in murine models of breast cancer and head and neck cancer following intratumoral injections. A combination therapy approach was evaluated where the IL-12/PPC treatment was combined with an investigative polymeric formulation of paclitaxel against 4T1 mammary tumors and it was shown that combined therapy led to a synergistic inhibition of tumor growth. This form of paclitaxel had little effect on SCCVII head and neck tumors however, cyclophosphamide strongly inhibited SCCVII tumor growth. Further, when IL-12/PPC intratumoral injections were combined with cyclophosphamide administration, there was a slight additional inhibition in tumor growth over the cyclophosphamide treatment alone and there was a significant increase in the rate of complete tumor rejection. Future studies will be aimed at optimizing dose and dosing regimens of combination therapies for the treatment of solid tumors.