Potent and long-term antiangiogenic efficacy mediated by FP3-expressing oncolytic adenovirus.

Various ways to inhibit vascular endothelial growth factor (VEGF), a key facilitator in tumor angiogenesis, are being developed to treat cancer. The soluble VEGF decoy receptor (FP3), due to its high affinity to VEGF, is a highly effective and promising strategy to disrupt VEGF signaling pathway. Despite potential advantage and potent therapeutic efficacy, its employment has been limited by very poor in vivo pharmacokinetic properties. To address this challenge, we designed a novel oncolytic adenovirus (Ad) expressing FP3 (RdB/FP3). To demonstrate the VEGF-specific nature of RdB/FP3, replication-incompetent Ad expressing FP3 (dE1/FP3) was also generated. dE1/FP3 was highly effective in reducing VEGF expression and functionally elicited an antiangiogeneic effect. Furthermore, RdB/FP3 exhibited a potent antitumor effect compared with RdB or recombinant FP3. Consistent with these data, RdB/FP3 was shown to greatly decrease VEGF expression level and vessel density and increase apoptosis in both tumor endothelial and tumor cells, verifying potent suppressive effects of RdB/FP3 on VEGF-mediated tumor angiogenesis in vivo. Importantly, the therapeutic mechanism of antitumor effect mediated by RdB/FP3 is associated with prolonged VEGF silencing efficacy and enhanced oncolysis via cancer cell-specific replication of oncolytic Ad. Taken together, RdB/FP3 provides a new promising therapeutic approach in the treatment of cancer and angiogenesis-related diseases.

Soluble vascular endothelial growth factor decoy receptor FP3 exerts potent antiangiogenic effects.

The binding of vascular endothelial growth factor (VEGF) to its receptors stimulates tumor growth; therefore, modulation of VEGF would be a viable approach for antiangiogenic therapy. We constructed a series of soluble decoy receptors containing different VEGF receptor 1 (FLT1) and VEGF receptor 2 (KDR) extracellular domains fused with the Fc region of human immunoglobulin (Ig) and evaluated their antiangiogenic effects and antitumor effects. Results of in vitro binding and cell proliferation assays revealed that decoy receptor FP3 had the highest affinity to VEGF-A and -B. Compared with bevacizumab, FP3 more effectively inhibited human umbilical vein endothelial cell (HUVEC) migration and vessel sprouting from rat aortic rings. FP3 significantly reduced phosphorylation of AKT and ERK1/2, critical proteins in the VEGF-mediated survival pathway in endothelial cells. Moreover, FP3 inhibited tumor growth in human hepatocellular carcinoma (HepG2), breast cancer (MCF-7), and colorectal cancer (LoVo) tumor models, and reduced microvessel density in tumor tissues. The FP3-mediated inhibition of tumor growth was significantly higher than that of bevacizumab at the same dose. FP3 also demonstrated synergistic antitumor effects when combined with 5-fluorouracil (5-FU). Taken together, FP3 shows a high affinity for VEGF and produced antiangiogenic effects, suggesting its potential for treating angiogenesis-related diseases such as cancer.

A phase 1 study of KH902, a vascular endothelial growth factor receptor decoy, for exudative age-related macular degeneration.

PURPOSE: To determine the safety, tolerability, and bioactivity of KH902, a fully human fusion protein containing key domains from vascular endothelial growth factor receptors 1 and 2 with human immunoglobulin Fc. DESIGN: Prospective, single-center, open-label, dose-escalating, interventional case series. PARTICIPANTS: Twenty-eight patients with choroidal neovascularization (CNV) resulting from exudative age-related macular degeneration (AMD) with lesion size of 12 disc areas or less and best-corrected visual acuity (VA) of 55 letters or worse. METHODS: A single intravitreal injection of KH902 at 1 of 6 escalating doses if no dose-limiting toxicity (DLT) occurred through postinjection day 14 of the previous dose level. Follow-up examinations were performed on postinjection days 1, 3, 5, 7, 14, 28, and 42. The primary end point was at 42 days, and patients were monitored for an additional 6 weeks (12 weeks total). MAIN OUTCOME MEASURES: The primary safety measures were changes from baseline in VA, intraocular pressure (IOP), intraocular inflammation, and production of anti-KH902 antibody. Dose-limiting toxicity was defined as intraocular inflammation, elevated IOP, significantly reduced vision, or retinal hemorrhage within 42 days after injection. Bioactivity measures included mean change from baseline in VA, central retinal thickness, and total macular volume on optical coherence tomography and CNV changes on fluorescein angiography. RESULTS: All patients completed the study with no DLT and no serious or drug-related adverse events. Ocular adverse events were mild to moderate in severity, including transient IOP elevation and injection-site subconjunctival hemorrhage after KH902 injections. No serum anti-KH902 antibodies were detected. On day 42 after injection, the mean change in VA from baseline was +19.6 letters with no subjects losing 1 letter or more and 57% of patients gaining 15 letters or more from baseline. The mean change in center point thickness from baseline was -77.2 µm and the mean decrease in CNV area was 12.6%. CONCLUSIONS: No safety concerns were detected after a single, intravitreal injection of KH902 up to 3.0 mg in this phase 1 study. Bioactivity of KH902 was suggested with improvements in VA, reduction in central retinal thickness, and a decrease in CVN area in patients with CNV resulting from exudative AMD, indicating that further study is warranted.

Combined use of chemotherapeutics and oncolytic adenovirus in treatment of AFP-expressing hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) which is always refractory to most chemotherapeutic agents may result in poor survival of patients with advanced HCC. Oncolytic adenovirus is a new form for cancer gene therapy via its ability to replicate and kill tumor cells in a tumor-specific manner. In order to eradicate tumors effectively, the combination of chemotherapeutic agents and oncolytic adenovirus has been considered. This study aimed to systematically analyze the possibility of synergistic cytotoxicity of oncolytic adenoviruses in combination with chemotherapeutic agents. METHODS: Several types of human HCC cell lines were used to determine the specificity and cytotoxicity of oncolytic adenovirus Ad5-HC and Ad5-AFP (IRES) by measuring cell viability in vitro and antitumor efficiency in vivo. The cytotoxicity of Ad5-HC and Ad5-AFP (IRES) combined with chemotherapeutic agents were also assessed by the methyl thiazolyl tetrazolium assay. RESULTS: Both Ad5-HC and Ad5-AFP (IRES) were significantly cytotoxic to HCC cells with great specificity in vitro and in vivo. The combination of oncolytic adenovirus with 5-FU, doxorubicin, and paclitaxel was synergistically effective for the killing of HCC cells. CONCLUSIONS: These data suggest that oncolytic adenovirus sensitize tumors to chemotherapy and the combination therapy of chemotherapeutic agents and oncolytic adenovirus has an enhanced antitumor effect on HCC cells.

Enhanced gene transfer and oncolysis of head and neck cancer and melanoma cells by fiber chimeric oncolytic adenoviruses.

PURPOSE: The purpose of this study was to evaluate a fiber knob replacement strategy to improve infectivity and efficacy of Ad5 fiber chimeric oncolytic viruses for treatment of melanoma and head and neck cancers (HNC). EXPERIMENTAL DESIGN: Adenoviral receptors and transduction levels were used to determine the level of infectivity of fiber-modified, green fluorescent protein-expressing, replication-deficient viruses in a panel of melanoma and HNC cell lines in vitro. Virus yield and cytotoxicity assays were used to determine the tumor specificity and virus replication-mediated cytotoxicity of the fiber-modified oncolytic viruses in the same panel of melanoma and HNC in vitro. Xenograft tumor models were used to assess the antitumor activity of those fiber-modified chimeric viruses compared with the parental virus. RESULTS: Marker gene expression following gene transfer of the fiber chimeric vectors in melanoma and HNC cell lines was approximately 10-fold higher than that obtained with parental Ad5 vector. The fiber chimeric oncolytic variants mediated killing of melanoma and HNC cells that was 2- to 576-fold better than with the parental virus. In addition, fiber chimeric variants produced 2- to 7-fold more progeny virus in tumor cells than the parental virus. Moreover, a high multiplicity of infection was needed for the fiber chimeric viruses to produce cytotoxicity in normal cells. A significantly stronger antitumor response and survival advantage were shown in the tested melanoma and HNC xenograft models following i.t. injections. CONCLUSIONS: In vitro and in vivo studies showed the improved transduction, replication, cytotoxicity, antitumor efficacy, and survival advantage in melanoma and HNC tumor models, suggesting a potential use of these oncolytic agents for the treatment of melanoma and HNCs.

CG0070, a conditionally replicating granulocyte macrophage colony-stimulating factor--armed oncolytic adenovirus for the treatment of bladder cancer.

PURPOSE: The purpose of this study was to examine the tumor specificity, cytotoxicity, and granulocyte macrophage colony-stimulating factor expression of CG0070, a conditionally replicating oncolytic adenovirus, in human bladder transitional cell carcinoma (TCC) cell lines and determine its antitumor efficacy in bladder TCC tumor models. EXPERIMENTAL DESIGN: Virus yield and cytotoxicity assays were used to determine tumor specificity and virus replication-mediated cytotoxicity of CG0070 in a panel of human bladder TCC cell lines and primary cells in vitro. Two s.c. and one orthotopic bladder TCC xenograft tumor models were used to assess antitumor activity of CG0070. RESULTS: In a matched isogenic pair of cell lines with differing retinoblastoma (Rb) pathway status, CG0070 showed selective E1a and granulocyte macrophage colony-stimulating factor (GM-CSF) expression in Rb pathway-defective cells. CG0070 replicated in Rb-defective bladder TCC cell lines as efficiently as wild-type adenovirus but produced 100-fold less virus in normal human cells. CG0070 was up to 1,000-fold more cytotoxic in Rb pathway-defective bladder TCC cells in comparison with normal human cells. Antitumor activity of CG0070 was shown in two bladder TCC s.c. xenograft tumor models following intratumoral injections and intravesical treatment in an orthotopic xenograft tumor model when compared with PBS treatment. CONCLUSIONS: In vitro and in vivo studies showed the selective replication, cytotoxicity, GM-CSF production, and antitumor efficacy of CG0070 in several bladder TCC models, suggesting a potential utility of this oncolytic agent for the treatment of bladder cancer. Further studies are warranted to show the role of human GM-CSF in the antitumor efficacy of CG0070.

Dual promoter-controlled oncolytic adenovirus CG5757 has strong tumor selectivity and significant antitumor efficacy in preclinical models.

PURPOSE: Transcriptionally controlled oncolytic adenovirus CG5757 is engineered with two tumor-specific promoters from E2F-1 and human telomerase reverse transcriptase genes. This virus has broad anticancer spectrum and higher specificity. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of E2F-1 and human telomerase reverse transcriptase promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and cytotoxicity assays followed by a newly developed ex vivo tumor culture assay. Subsequently, in vivo antitumor efficacy and toxicology studies were carried out to assess the therapeutic potential of this oncolytic agent. RESULTS: In a broad panel of cells, E2F-1 and human telomerase reverse transcriptase promoters were activated in a tumor-selective manner. Under the control of these promoters, expression of E1a and E1b genes appears only in tumor cells. This specificity is extended to viral replication and hence the cytotoxicity in a broad range of cancer cells. Furthermore, CG5757 only replicates in cancer tissues but not in normal tissues that are derived from clinical biopsies. The safety profile was further confirmed in in vivo toxicology studies, and strong efficacy was documented in several tumor xenograft models after CG5757 was given via different routes and regimens. CONCLUSIONS: CG5757 has strong antitumor selectivity and potency. It has low toxicity and has great potential as a therapeutic agent for different types of cancers.

Development of transcriptionally regulated oncolytic adenoviruses.

Changes initiated at the cellular and systemic levels as a result of viral infection or neoplastic transformation share significant overlap. Therefore, the use of replicating viruses to treat tumors has long been postulated as a promising avenue for oncolytic therapy. Over the last 10 years, transcriptionally regulated adenoviruses have become a popular platform for the development of such oncolytic viruses. Placement of heterologous promoters in front of key adenoviral transcription units to achieve tumor- or tissue-specific viral replication is well documented. Various derivatives of this general strategy have led to considerable insight into its limitations, pitfalls, and potential. Although a general process can be described by which to develop transcriptionally regulated adenoviruses, it is apparent that few set rules can yet be defined as to what constitutes a safe, stable, and therapeutically effective vector. Clinical experiences to date suggest the short-term potential for this class of therapeutics lies in combination therapy regimens. Such lessons from the clinic suggest the next generation of transcriptionally regulated oncolytic adenoviruses take advantage of the ability of the platform to carry transgenes in order to deliver a multimodal therapy from a single agent. Beyond this 'arming' of the vectors lies the detargeting, retargeting, and coating of adenoviruses to improve the delivery of the agent to the treatment site(s). As a therapeutic platform, transcriptionally regulated adenoviruses are at an early stage of development with considerable opportunities for advancement.

Oncolytic adenovirus CG7870 in combination with radiation demonstrates synergistic enhancements of antitumor efficacy without loss of specificity.

Conditionally replicating adenoviruses that selectively replicate in tumor cells, but not in normal cells, are being explored as virotherapeutic agents for cancer. A prostate-specific oncolytic adenovirus, CG7870 is currently being evaluated in phase 1/2 clinical trials for the treatment of prostate cancer. To decrease the effective dose and further increase the therapeutic efficacy of CG7870, the combination of virotherapy with radiation therapy was explored in this study. CG7870 is an oncolytic adenovirus in which tumor-specific promoters are driving the expression of E1A and E1B proteins. The effects of combined treatment with CG7870 and radiation on cultured cells were determined in cytotoxicity and virus yield assays. The antitumor efficacy of CG7870 (1 x 10(7) particles/mm3 of tumor), 10 Gy of local radiation or both was evaluated in established subcutaneous LNCaP xenografts in nude mice. In vitro, the dual agent treatment resulted in synergistically enhanced potency at suboptimal doses of radiation and virus. Virus yield in irradiated cells increased relative to yield in nonirradiated cells without compromising the specificity of the vector for its target cell types. In vivo, CG7870 treatment alone suppressed tumor growth and extended tumor nonprogression time. The average tumor-volume of the groups treated with CG7870 only and radiation only was 121 and 126% of baseline, respectively, 39 days after treatment. The average tumor-volume of the combination group was 34% of baseline 39 days after a single dose of treatment. No significant body weight loss was observed in any treatment group. There was a significant drop in serum level of prostate-specific antigen (PSA) in the combination group compared to the group treated with either agent alone. In mice treated with CG7870 only or radiation only, serum PSA levels changed to 26 and 383% of baseline, respectively, by study day 46. In contrast, PSA levels in mice treated with CG7870 plus radiation decreased to less than 11% of baseline by study day 46. Histological analysis of tumor sections collected from the combination group revealed enhanced necrosis and more apoptotic cells. Combination of CG7870 with radiotherapy significantly increased antitumor efficacy compared to either agent alone. These results suggest that CG7870 in combination with radiation has improved antitumor efficacy at lower doses and with no additional side effects.

Complex mosaicism is a novel approach to infectivity enhancement of adenovirus type 5-based vectors.

The use of adenovirus type 5 (Ad5) for cancer therapy is limited by deficiency of its primary cell attachment receptor, coxsackie and adenovirus receptor (CAR), on cancer cells. Ad5 retargeting to alternate receptors through fiber genetic modification can be used to circumvent CAR dependence of its tropism, and thereby achieve infectivity enhancement. Here we propose and test a novel "complex mosaicism" approach for fiber modification, which combines serotype chimerism with peptide ligand(s) incorporation in a single-fiber molecule. We incorporated integrin-binding peptide RGD-4C in the HI-loop, at the carboxy (C)-terminus, or both locales of the Ad3 knob, in the context of Ad5/3 chimera fiber in order to retarget simultaneously the Ad vector to integrins and Ad3 receptors. The infectivity enhancement of the fiber modifications was assessed in various cancer cell lines as cancer-targeting models. Replication-defective complex mosaic Ad-luc vectors bearing chimeric fiber (F.5/3), with or without C-terminal RGD-modification of Ad3 knob, demonstrated up to 55-fold gene transfer increase in bladder cancer cell lines. Although this augmentation was primarily due to Ad3 receptor targeting, some contribution of RGD-mediated integrin-targeting was also observed, suggesting that complex mosaic modification can function in a dual-receptor targeting via a single Ad3 fiber knob.

Identification of pretreatment agents to enhance adenovirus infection of bladder epithelium.

Adenovirus has been used widely as a gene transfer vector in the laboratory and clinic for the purpose of gene therapy. Conditionally replication-competent oncolytic adenoviruses are capable of multiplying up to a thousand old in target cells, a property that might prove to be of tremendous potential in the area of cancer therapy. Intravesicular therapy of refractory superficial bladder cancer employing an oncolytic adenovirus would allow for local administration and efficient delivery of virus to bladder tumor. The glycosaminoglycan layer on the surface of the bladder urothelium acts as a nonspecific antiadherence barrier and may be a significant roadblock to efficient infection of the urothelium by adenoviruses. Several laboratories have investigated the potential utility of bladder pretreatment with chemical agents to enhance the adenovirus infection of bladder urothelium but with limited success. A class of compounds has been identified that is effective for pretreatment of urothelium, permitting efficient adenoviral infection. In a murine model, pretreatment of the bladder with 0.1% dodecyl-beta-D-maltoside (DDM) or sodium dodecyl sulfate (SDS) for 5 min resulted in >90% transduction of the urothelial layer within 15 min after exposure to a replication-defective adenovirus compared to

Carcinoembryonic antigen-producing cell-specific oncolytic adenovirus, OV798, for colorectal cancer therapy.

Human carcinoembryonic antigen (CEA) is overexpressed in most colorectal cancers and has been widely used as a clinical marker for the management of colon cancer patients. The transcriptional regulatory elements (TREs) of CEA include two enhancer elements and a promoter in the 5'-flanking region of the CEA gene. By using these elements in different combinations to control reporter gene expression and the replication of adenovirus variants in various tumor cells, we have identified an optimal CEA regulatory cassette that tightly controls gene expression and viral replication in CEA-producing colon cancer cells. One of these variants, OV798, in which this regulatory cassette controls E1A expression, was further characterized. OV798 preferentially replicates in and kills CEA-producing colorectal cancer cell lines such as LoVo and SW1463, but its replication is attenuated by 1000-fold in the CEA-negative cell lines Colo-320DM (colon cancer), PA-1 (ovarian cancer), G361 (melanoma), U118 MG (glioma), and HBL-100 (human breast epithelial cell). The antitumor activity of OV798 was further examined in BALB/c nu/nu mice carrying s.c. human colon tumor xenografts. A single intratumoral administration of OV798 resulted in growth inhibition of human LoVo colon cancer xenografts. Six weeks after treatment, relative tumor volume decreased to 90% of baseline for the OV798 treatment group, compared to an increase to 1200% of baseline at 4 weeks for the vehicle-treated group. In vitro and in vivo characterization indicate that OV798 could be used as a therapy for human colon cancer.

Selectively replicating oncolytic adenoviruses as cancer therapeutics.

The resurgence of interest in virotherapy over the course of the last decade has led to several promising modalities for the treatment of human cancers. Among these are transcriptionally regulated adenovirus variants that preferentially replicate in and destroy target tumor cells. To date, these oncolytic adenoviruses, such as CG-7060 (Cell Genesys Inc) and CG-7870 (Cell Genesys Inc), have demonstrated good safety profiles and significant antitumor activity. Additionally, synergistic effects have been seen when oncolytic viruses have been used in combination with traditional chemotherapy or radiotherapy. Future clinical trials are needed to further test the utility of the first generation of oncolytic adenoviruses. Much of the upcoming development in this field will revolve around improving the antitumor efficacy of the viruses, modulating host immune responses and maximizing the synergistic effects of oncolytic viruses administered with traditional chemotherapy or radiotherapy.

Identification of human uroplakin II promoter and its use in the construction of CG8840, a urothelium-specific adenovirus variant that eliminates established bladder tumors in combination with docetaxel.

Uroplakins (UPs) are a group of integral membrane proteins that are synthesized as the major differentiation products of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but very little is known about its transcription response elements. To identify the promoter elements, a DNA fragment of 2239 bp upstream of the UPII gene was amplified by PCR and linked to a promoterless firefly luciferase reporter gene. Transient transfection experiments showed that the DNA segment located between -1809 and +1 bp resulted in preferential expression in bladder carcinoma cells with negligible expression in nonurothelial cells. This promoter was engineered into adenovirus (Ad) type 5 to drive the expression of the E1A and E1B genes and to create an attenuated replication-competent Ad variant, termed CG8840. Viral replication and the cytopathic effect of CG8840 were evaluated by virus yield and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in bladder transitional cell carcinoma (TCC) cell lines RT4 and SW780; nonbladder cancer cell lines G361 (melanoma), LNCaP (prostate cancer), PA-1 (ovarian cancer), and U118 (brain cancer); and human primary cells including lung fibroblasts, bladder smooth muscle cells, and mammary epithelial cells. CG8840 replicated in and eliminated bladder TCC efficiently with high specificity (10,000:1) in comparison with nonbladder cells. The antitumor activity of CG8840 was examined in BALB/c nu/nu mice carrying s.c. human TCC xenografts. Intratumoral and i.v. administration of CG8840 in RT4 human bladder cancer xenografts caused significant (P < 0.01) inhibition of tumor growth. Synergistic antitumor efficacy was observed when CG8840 was combined with docetaxel, resulting in significant regression of RT4 bladder cancer xenograft tumors within 6 weeks after i.v. administration of CG8840 (3.33 x 10(9) plaque-forming units/animal on day 1) and docetaxel (20 mg/kg on days 2, 6, and 9). These results demonstrate the utility of the UPII promoter in the generation of urothelium-specific adenoviral vectors and provide a potential foundation for the development of bladder tumor-specific oncolytic viral therapies.