[show abstract][hide abstract] ABSTRACT: Species C human adenovirus serotype 5 (HAdV-C5) is widely used as a vector for cancer gene therapy, because it efficiently transduces target cells. A variety of HAdV-C5 vectors have been developed and tested in vitro and in vivo for cancer gene therapy. While clinical trials with HAdV-C5 vectors resulted in effective responses in many cancer patients, administration of HAdV-C5 vectors to solid tumors showed responses in a limited area. A biological barrier in tumor mass is considered to hinder viral spread of HAdV-C5 vectors from infected cells. Therefore, efficient virus-spread from an infected tumor cell to surrounding tumor cells is required for successful cancer gene therapy. In this study, we compared HAdV-C5 to sixteen other HAdV serotypes selected from species A to G for virus-spread ability in vitro. HAdV-D9 showed better virus-spread ability than other serotypes, and its viral progeny were efficiently released from infected cells during viral replication. Although the HAdV-D9 fiber protein contains a binding site for coxsackie B virus and adenovirus receptor (CAR), HAdV-D9 showed expanded tropism for infection due to human CAR (hCAR)-independent attachment to target cells. HAdV-D9 infection effectively killed hCAR-negative cancer cells as well as hCAR-positive cancer cells. These results suggest that HADV-D9, with its better virus-spread ability, could have improved therapeutic efficacy in solid tumors compared to HAdV-C5.
PLoS ONE 01/2014; 9(2):e87342. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.
[show abstract][hide abstract] ABSTRACT: Postoperative abdominal/pelvic peritoneal adhesions are a major source of morbidity (bowel obstruction, infertility, ectopic gestation as well as chronic pelvic pain) in women. In this study, we screened various transduction and transcription modifications of adenovirus (Ad) to identify those that support maximal Ad-mediated gene delivery to human adhesion fibroblasts, which in turn would enhance the efficacy of this novel treatment/preventative strategy for postoperative adhesions. We transduced primary cultures of human peritoneal adhesion fibroblasts with fiber-modified Ad vectors Ad5-RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc as well as transcriptional targeting viruses Ad5-survivin-luc, Ad5-heparanase-luc, Ad5-mesothelin (MSLN)-CRAd-luc and Ad5-secretory leukoprotease inhibitor (SLPI)-luc, and compared their activity to wild-type Ad5-luc. At 48 h, luciferase activity was measured and normalized to the total protein content in the cells. Among the fiber-modified Ad vectors, Ad5-Sigma-luc and among the transcriptional targeting modified Ad vectors, Ad5-MSLN-CRAd-luc showed significantly increased expression levels of luciferase activity at 5, 10 and 50 plaque forming units/cell in adhesion fibroblast cells compared with wild-type Ad5-luc (p < 0.05). Specific modifications of Ad improve their gene delivery efficiency towards human peritoneal adhesion fibroblasts. Developing a safe localized method to prevent/treat postoperative adhesion formation would have a major impact on women health.
Gynecologic and Obstetric Investigation 08/2013; · 1.10 Impact Factor
[show abstract][hide abstract] ABSTRACT: Is targeted adenovirus vector, Ad-SSTR-RGD-TK (Adenovirus -human somatostatin receptor subtype 2- arginine, glycine and aspartate-thymidine kinase), given in combination with ganciclovir (GCV) against immortalized human leiomyoma cells (HuLM) a potential therapy for uterine fibroids?
Ad-SSTR-RGD-TK/GCV, a targeted adenovirus, effectively reduces cell growth in HuLM cells and to a significantly greater extent than in human uterine smooth muscle cells (UtSM).
Uterine fibroids (leiomyomas), a major cause of morbidity and the most common indication for hysterectomy in premenopausal women, are well-defined tumors, making gene therapy a suitable and potentially effective non-surgical approach for treatment. Transduction of uterine fibroid cells with adenoviral vectors such as Ad-TK/GCV (herpes simplex virus thymidine kinase gene) decreases cell proliferation.
An in vitro cell culture method was set up to compare and test the efficacy of a modified adenovirus vector with different multiplicities of infection in two human immortalized cell lines for 5 days.
Immortalized human leiomyoma cells and human uterine smooth muscle cells were infected with different multiplicities of infection (MOI) (5-100 plaque-forming units (pfu)/cell) of a modified Ad-SSTR-RGD-TK vector and subsequently treated with GCV. For comparison, HuLM and UtSM cells were transfected with Ad-TK/GCV and Ad-LacZ/GCV. Cell proliferation was measured using the CyQuant assay in both cell types. Additionally, western blotting was used to assess the expression of proteins responsible for regulating proliferation and apoptosis in the cells.
Transduction of HuLM cells with Ad-SSTR-RGD-TK/GCV at 5, 10, 50 and 100 pfu/cell decreased cell proliferation by 28, 33, 45, and 84%, respectively (P < 0.05) compared with untransfected cells, whereas cell proliferation in UtSM cells transfected with the same four MOIs of Ad-SSTR-RGD-TK/GCV compared with that of untransfected cells was decreased only by 8, 23, 25, and 28%, respectively (P < 0.01). Western blot analysis showed that, in comparison with the untargeted vector Ad-TK, Ad-SSTR-RGD-TK/GCV more effectively reduced expression of proteins that regulate the cell cycle (Cyclin D1) and proliferation (PCNA, Proliferating Cell Nuclear Antigen), and it induced expression of the apoptotic protein BAX, in HuLM cells.
Results from this study need to be replicated in an appropriate animal model before testing this adenoviral vector in a human trial.
Effective targeting of gene therapy to leiomyoma cells enhances its potential as a non-invasive treatment of uterine fibroids.
This work was supported by a grant from the National Institute of Child Health and Human Development, National Institutes of Health [R01 HD046228]. None of the authors has any conflict of interest to declare.
[show abstract][hide abstract] ABSTRACT: OBJECTIVE: The conditionally replicative adenovirus Ad5/3-Δ24 has a type-3 knob incorporated into the type-5 fiber that facilitates enhanced ovarian cancer infectivity. Preclinical studies have shown that Ad5/3-Δ24 achieves significant oncolysis and anti-tumor activity in ovarian cancer models. The purpose of this study was to evaluate in a Phase I trial the feasibility and safety of intraperitoneal (IP) Ad5/3-Δ24 in recurrent ovarian cancer patients. METHODS: Eligible patients were treated with IP Ad5/3-Δ24 for 3 consecutive days in one of three dose cohorts ranging 1 x 10(10)-1 x 10(12) vp. Toxicity was assessed utilizing CTC grading and efficacy with RECIST. Ascites, serum, and other samples were obtained to evaluate gene transfer, generation of wildtype virus, viral shedding, and antibody response. RESULTS: Nine of 10 patients completed treatment per protocol. A total of 15 vector-related adverse events were experienced in 5 patients. These events included fever or chills, nausea, fatigue, and myalgia. All were grade 1-2 in nature, transient, and medically managed. Of the 8 treated patients evaluable for response, six patients had stable disease and 2 patients had progressive disease. Three patients had decreased CA-125 from pretreatment levels one month after treatment. Ancillary biologic studies indicated Ad5/3-Δ24 replication in patients in the higher dose cohorts. All patients experienced an anti-adenoviral neutralizing antibody effect. CONCLUSIONS: This study suggests the feasibility and safety of a serotype chimeric infectivity-enhanced CRAd, Ad5/3-Δ24, as a potential therapeutic option for recurrent ovarian cancer patients.
[show abstract][hide abstract] ABSTRACT: We have previously designed a conditionally replicative oncolytic adenovirus (CRAd) named Ad-F512 that can target both the stromal and the malignant melanoma cell compartments. The replication capacity of this CRAd is driven by a 0.5 Kb SPARC promoter fragment (named F512). To improve CRA\[dacute]s efficacy we cloned into F512, motives responsive to hypoxia (HRE) and inflammation (NF-κB) to obtain a chimeric promoter named κBF512HRE. Using luciferase as a reporter gene we observed 10-15 fold increased activity under hypoxia and 10-80-fold induction upon TNF-α addition. We next constructed a CRAd (Ad-κBF512HRE) where E1A activity was under κBF512HRE regulation. Treatment of nude mice harboring established tumors made of a mix of SB2 melanoma cells and WI-38 fibroblasts with Ad-κBF512HRE led to the complete elimination of tumors in 100% of mice (8/8). Moreover, Ad-5/3-κBF512HRE, a viral variant pseudotyped with a chimeric 5/3 fiber exerted a strong lytic effect on CAR-negative melanoma cells and was highly effective in vivo on established tumors made of melanoma cells and WI-38 fibroblasts leading to the complete elimination of 4/5 tumors. These results indicate that this improved stroma-targeted oncolytic adenovirus can override the resistance of melanoma tumors and might become of significant importance for melanoma therapeutics.Journal of Investigative Dermatology accepted article preview online, 19 April 2013; doi:10.1038/jid.2013.191.
Journal of Investigative Dermatology 04/2013; · 6.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is a highly fatal disease mandating development of novel, targeted therapies to elicit prolonged survival benefit to the patients. Insulin-like growth factor-binding protein-7 (IGFBP7), a secreted protein belonging to the IGFBP family, functions as a potential tumor suppressor for HCC. In the present study, we evaluated the therapeutic efficacy of a replication-incompetent adenovirus expressing IGFBP7 (Ad.IGFBP7) in human HCC. Ad.IGFBP7 profoundly inhibited viability and induced apoptosis in multiple human HCC cell lines by inducing reactive oxygen species (ROS) and activating a DNA damage response (DDR) and p38 MAPK. In orthotopic xenograft models of human HCC in athymic nude mice, intravenous administration of Ad.IGFBP7 profoundly inhibited primary tumor growth and intrahepatic metastasis. In a nude mice subcutaneous model, xenografts from human HCC cells were established in both flanks and only left-sided tumors received intratumoral injection of Ad.IGFBP7. Growth of both left-sided injected tumors and right-sided uninjected tumors were markedly inhibited by Ad.IGFBP7 with profound suppression of angiogenesis. These findings indicate that Ad.IGFBP7 might be a potent therapeutic eradicating both primary HCC and distant metastasis and might be an effective treatment option for terminal HCC patients.Molecular Therapy (2013); doi:10.1038/mt.2012.282.
[show abstract][hide abstract] ABSTRACT: CONTRIBUTING REVIEWERS: The editors of Journal of Ovarian Research would like to thank all of our reviewers who have contributed to the journal in volume 5 (2012).
Journal of Ovarian Research 01/2013; 6(1):16. · 2.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: Vectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.
As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.
These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.
PLoS ONE 01/2013; 8(2):e55533. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: New approaches targeting metastatic neovasculature are needed. Payload capacity, cellular transduction efficiency, and first-pass cellular uptake following systemic vector administration, motivates persistent interest in tumor vascular endothelial cell (EC) adenoviral (Ad) vector targeting. While EC transductional and transcriptional targeting has been accomplished, vector administration approaches of limited clinical utility, lack of tumor-wide EC expression quantification, and failure to address avid liver sequestration, challenged prior work. Here, we intravenously injected an Ad vector containing 3 kb of the human roundabout4 (ROBO4) enhancer/promoter transcriptionally regulating an enhanced green fluorescent protein (EGFP) reporter into immunodeficient mice bearing 786-O renal cell carcinoma subcutaneous (SC) xenografts and kidney orthotopic (KO) tumors. Initial experiments performed in human coxsackie virus and adenovirus receptor (hCAR) transgenic:Rag2 knockout mice revealed multiple ECs with high-level Ad5ROBO4-EGFP expression throughout KO and SC tumors. In contrast, Ad5CMV-EGFP was sporadically expressed in a few tumor vascular ECs and stromal cells. As the hCAR transgene also facilitated Ad5ROBO4 and control Ad5CMV vector EC expression in multiple host organs, follow-on experiments engaged warfarin-mediated liver vector detargeting in hCAR non-transgenic mice. Ad5ROBO4-mediated EC expression was undetectable in most host organs, while the frequencies of vector expressing intratumoral vessels and whole tumor EGFP protein levels remained elevated. In contrast, AdCMV vector expression was only detectable in one or two stromal cells throughout the whole tumor. The Ad5ROBO4 vector, in conjunction with liver detargeting, provides tractable genetic access for in-vivo EC genetic engineering in malignancies.
PLoS ONE 01/2013; 8(12):e83933. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165 000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.Gene Therapy advance online publication, 22 November 2012; doi:10.1038/gt.2012.91.
[show abstract][hide abstract] ABSTRACT: Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 10(10) v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15-40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression.Molecular Therapy (2012); doi:10.1038/mt.2012.147.
[show abstract][hide abstract] ABSTRACT: Pancreatic adenocarcinoma is an aggressive malignancy. Oncolytic adenoviruses (Ads) are modified genetically to target tumor cells while sparing normal cells. We modified the knob domain of the Ad serotype 5 with a serotype 3 knob domain and incorporated the CXCR4 promoter to regulate Ad E1A gene expression (Ad5/3-CXCR4-E1A). These modifications were made to efficiently infect and lyse pancreatic tumors.
Human pancreatic cancer lines CFPAC-1, PANC-1, AsPC-1, and BxPC-3 were obtained from the American Type Culture Collection. Efficiency of Ad infection in the cells was determined by the use of an Ad construct expressing the green fluorescence protein (GFP) marker in place of the E1A gene (Ad5/3-CXCR4-GFP) and quantified by flow cytometry. Oncolytic activity in the pancreatic cancer cells was determined with the Ad5/3-CXCR4-E1A oncolytic Ad by a crystal violet staining method. To determine the oncolytic effect in vivo, pancreatic cancer cells were implanted on the flanks of 40 SCID mice (4 groups). Tumors were injected intratumorally for 3 days with Ad5/3-CXCR4-E1A, Ad5 wild-type (a positive control), or phosphate-buffered saline (a no virus control). Tumor size, overall survival, and body condition scale score were recorded. Statistical analyses included the Kaplan-Meier survival curve, the log-rank test, and one-way analysis of variance.
The serotype 3 fiber-modified Ad with the CXCR4 promoter (Ad5/3-CXCR4-E1A) was most efficient in infecting and lysing pancreatic cancer cells compared with an Ad containing an unmodified fiber knob (Ad5-CXCR4-E1A). Treatment of pancreatic tumor xenografts in vivo with Ad5/3-CXCR4-E1A group resulted in smaller tumors (P = .001), greater body condition scale score (P = .01), and greater survival time (P = .04) than the other treatment groups.
Ad5/3-CXCR4-E1A treatment significantly prolonged survival in SCID mice pancreatic tumor xenografts. This novel construct represents a potential new therapy against pancreatic cancer.
Surgery 07/2012; 152(3):441-8. · 3.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adenoviral infection induces nucleoplasmic redistribution of a nucleolar nucleophosmin 1/NPM1/B23.1. NPM1 is preferentially localized in the nucleoli of normal cells, whereas it is also present at the nuclear matrix in cancer cells. However, the biological roles of NPM1 during infection are unknown. Here, by analyzing a pV-deletion mutant, Ad5-dV/TSB, we demonstrate that pV promotes the NPM1 translocation from the nucleoli to the nucleoplasm in normal cells, and the NPM1 translocation is correlated with adenoviral replication. Lack of pV causes a dramatic reduction of adenoviral replication in normal cells, but not cancer cells, and Ad5-dV/TSB was defective in viral assembly in normal cells. NPM1 knockdown inhibits adenoviral replication, suggesting an involvement of NPM1 in adenoviral biology. Further, we show that NPM1 interacts with empty adenovirus particles which are an intermediate during virion maturation by immunoelectron microscopy. Collectively, these data implicate that pV participates in a process of viral assembly through NPM1.
[show abstract][hide abstract] ABSTRACT: Ad5.SSTR/TK.RGD is an infectivity-enhanced adenovirus expressing a therapeutic thymidine kinase suicide gene and a somatostatin receptor (SSTR) that allows for noninvasive gene transfer imaging. The purpose of this study was to identify the maximum tolerated dose (MTD), toxicities, clinical efficacy, and biologic effects of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer.
Eligible patients were treated intraperitoneally for 3 days with 1 × 10(9) to 1 × 10(12) vp/dose of Ad5.SSTR/TK.RGD followed by intravenous ganciclovir for 14 days. Toxicity and clinical efficacy were assessed using Common Toxicity Criteria (CTC) Adverse Events grading and Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Imaging using In-111 pentetreotide was obtained before and after treatment. Tissue samples were obtained to evaluate for gene transfer, generation of wild-type virus, viral shedding, and antibody response.
Twelve patients were treated in three cohorts. The most common vector-related clinical toxicities were grade I/II constitutional or pain symptoms, experienced most often in patients treated at the highest dose. MTD was not identified. Five patients showed stable disease; all others experienced progressive disease. One patient with stable disease experienced complete resolution of disease and normalization of CA125 on further follow-up. Imaging detected increased In-111 pentetreotide retention in patients treated at the highest dose. Ancillary studies showed presence of Ad5.SSTR/TK.RGD virus and HSV1-tk expression in ascites samples collected at various time points in most patients treated within the higher dose cohorts.
This study shows the safety, potential efficacy, and possible gene transfer imaging capacity of Ad5.SSTR/TK.RGD in patients with recurrent gynecologic cancer. Further development of this novel gene therapeutic appears to be warranted.
Clinical Cancer Research 04/2012; 18(12):3440-51. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: The ability of dendritic cells (DCs) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DCs for clinical administration, their loading with tumor associated antigens (TAAs) and their activation, is laborious and expensive, and, as a result of inter-individual variability in the personalized vaccines, remains poorly standardized. An attractive alternative approach is to load resident DCs in vivo by targeted delivery of TAAs, using viral vectors and activating them simultaneously. To this end, we have constructed genetically-modified adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAAs to the CD40 receptor on DCs. Pre-clinical human and murine studies conducted so far have clearly demonstrated the suitability of a 'two-component' (i.e. Ad and adaptor molecule) configuration for targeted modification of DCs in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in the clinical translation of this approach.
The Journal of Gene Medicine 01/2012; 14(6):416-27. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cancer gene therapy approaches have benefited greatly from the utilization of molecular-based therapeutics. Of these, adenovirus-based interventions hold much promise as a platform for targeted therapeutic delivery to tumors. However, a barrier to this progression is the lack of native adenovirus receptor expression on a variety of cancer types. As such, any adenovirus-based cancer therapy must take into consideration retargeting the vector to nonnative cellular surface receptors. Predicated upon the knowledge gained in native adenovirus biology, several strategies to transductionally retarget adenovirus have emerged. Herein, we describe the biological hurdles as well as strategies utilized in adenovirus transductional targeting, covering the progress of both adapter-based and genetic manipulation-based targeting. Additionally, we discuss recent translation of these targeting strategies into a clinical setting.
Advances in Cancer Research 01/2012; 115:39-67. · 6.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.
PLoS ONE 01/2012; 7(10):e46981. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Adenoviral (Ad) vectors show promise as platforms for vaccine applications against infectious diseases including HIV. However, the requirements for eliciting protective neutralizing antibody and cellular immune responses against HIV remain a major challenge. In a novel approach to generate 2F5- and 4E10-like antibodies, we engineered an Ad vector with the HIV membrane proximal ectodomain region (MPER) epitope displayed on the hypervariable region 2 (HVR2) of the viral hexon capsid, instead of expressed as a transgene. The structure and flexibility of MPER epitopes, and the structural context of these epitopes within viral vectors, play important roles in the induced host immune responses. In this regard, understanding the critical factors for epitope presentation would facilitate optimization strategies for developing viral vaccine vectors. Therefore we undertook a cryoEM structural study of this Ad vector, which was previously shown to elicit MPER-specific humoral immune responses. A subnanometer resolution cryoEM structure was analyzed with guided molecular dynamics simulations. Due to the arrangement of hexons within the Ad capsid, there are twelve unique environments for the inserted peptide that lead to a variety of conformations for MPER, including individual α-helices, interacting α-helices, and partially extended forms. This finding is consistent with the known conformational flexibility of MPER. The presence of an extended form, or an induced extended form, is supported by interaction of this vector with the human HIV monoclonal antibody 2F5, which recognizes 14 extended amino acids within MPER. These results demonstrate that the Ad capsid influences epitope structure, flexibility and accessibility, all of which affect the host immune response. In summary, this cryoEM structural study provided a means to visualize an epitope presented on an engineered viral vector and suggested modifications for the next generation of Ad vectors with capsid-incorporated HIV epitopes.
PLoS ONE 01/2012; 7(11):e49607. · 3.73 Impact Factor