Wt Ad5 Research Articles

Nonclinical characterization of ICVB-1042 as a selective oncolytic adenovirus for solid tumor treatment

ICVB-1042 is an oncolytic adenovirus containing modifications to enhance replication, lysis, and viral spreading in tumor cells. The anti-tumor activity, immune activation, tropism, selectivity, and mechanism of action were evaluated in preparation for a first-in-human study. ICVB-1042 was at least 100-fold more cytotoxic in A549 cells than in normal primary cells tested, demonstrating its high tumor selectivity and a low likelihood of targeting primary tissues. ICVB-1042 administered to mice intravenously or intratumorally was effective in reducing tumor burden. Its intravenous administration also inhibited tumor growth in orthotopic models. ICVB-1042 was well tolerated in mice compared to HAdV-C5 (Wt Ad5), with reduced liver sequestration, supporting safety of the drug for systemic delivery. These preclinical data demonstrating the safety and potency of ICVB-1042 for treatment of various solid tumors support the ongoing clinical investigation (NCT05904236).

Abstract B62: Development of infectivity-selective oncolytic adenovirus for systemic cancer therapy

Abstract Adenovirus (OAd) has high potential for systemic cancer therapy. However, its efficacy upon systemic application has been quite limited to date. Unlike locoregional therapy, systemic application of cancer therapy mandates better tumor distribution and transduction. When adenoviruses are injected intravenously, most of the virus goes to the liver due to high expression of wild-type Ad receptor (coxsackie-adenovirus receptor: CAR) and capture by Kuppfer cells. The large sequestration by liver reduces tumor transduction rate and the in vivo efficacy upon systemic therapy. Therefore, the improvement of cancer-selective transduction and reduction of liver distribution would overcome the obstacles for systemic delivery and enable efficient systemic treatment of cancer with OAd. To improve the tumor transduction, we have developed a new technology and generated the large diversity Ad-fiber library in OAd format. By screening with Mesothelin (MSLN) overexpressing cell line, we obtained an infectivity-selective OAd for pancreatic cancer. MSLN-targeted OAd showed selective and powerful antitumor effect against MSLN-positive subcutaneous tumor model in both intratumoral (i.t.) and intravenous (i.v.) injection. Importantly, when we assessed viral distribution after i.v. injection, the liver sequestration of MSLN-targeted OAd was lower than untargeted OAd (Ad5 WT virus). Also, the viral copy number of MSLN-targeted OAd in the tumor was significantly higher than Ad5 WT virus. Most importantly, when antitumor effect of MSLN-targeted OAd was assessed in patient-derived xenograft (PDX) model, only the MSLN-targeted OAd showed significant antitumor effect upon intravenous administration. To further reduce liver sequestration and increase antitumor efficacy, we developed an improved vector with second modification in hexon. The MSLN-targeted OAd was additionally modified by substitution of Ad5 hexon-hypervariable region 7 (HVR7) with Ad26 hexon-HVR7. We assessed liver distribution of the double-modified vector after i.v. injection. The virus copy number after double-modified OAd was significantly lower in the liver. These data suggest that hexon modification allowed to escape from the liver. The detailed mechanism of this finding is currently being investigated and focuses on the viral uptake by hepatocytes and Kupffer cells. In summary, MSLN-targeted OAd showed remarkable antitumor effect in both systemic and local injections, and hexon modification further reduced liver sequestration. We believe that the fiber and hexon double-modified vector might be a promising vector to reduce hepatotoxicity and enhance antitumor effect for systemic treatment of pancreatic cancer. Citation Format: Mizuho Sato-Dahlman, Masato Yamamoto. Development of infectivity-selective oncolytic adenovirus for systemic cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B62.

Abstract 5921: Systemic treatment with mesothelin-targeted oncolytic adenovirus shows efficacy patient-derived xenograft of pancreatic cancer

Abstract Pancreatic cancer is an aggressive malignant disease. Despite extensive efforts, systemic therapies have provided only limited efficacy for patients with this disease. Oncolytic Adenovirus (OAd) is a promising therapeutics, and it is also known for its efficient in vivo gene delivery. However, when adenovirus vectors are injected intravenously into mice, most of the virus goes to the liver and can lead to liver toxicity at high dosage. One of the reason for liver tropism is that hepatocytes express high levels of the primary adenovirus receptor, and non-parenchymal liver cells, such as Kupffer cell and epithelial cell, also capture the viral particle. As a consequence of large sequestration of adenovirus by liver, the tumor transduction rate is low and the in vivo efficacy is limited. Therefore, the improvement of cancer selective transduction and vector distribution to avoid liver sequestration would overcome the obstacles for systemic delivery required for efficient systemic treatment of spread and/or metastatic lesions of pancreatic cancer with OAd. To improve the tumor transduction, we have generated the pancreatic cancer-targeted OAd by high-throughput screening of Ad-fiber library in mesothelin (MSLN) expressing cells. The pancreatic cancer-targeted OAd binds to MSLN protein, which is overexpressed on the surface of pancreatic cancer. MSLN-targeted OAd showed selective and powerful anti-tumor effect against Panc-1 xenograft tumor model in both intratumoral (i.t.) and intravenous (i.v.) injection. Importantly, when we assessed viral distribution after i.v. injection, the liver sequestration of MSLN-targeted OAd was lower than untargeted OAd (Ad5 WT virus) at 48 hrs after injection. By day 7, the viral copy number of MSLN-targeted OAd in the tumor was significantly higher than Ad5 WT virus. These results suggest that systemic injection of the tumor targeted-OAd showed significantly lower liver sequestration and better tumor accumulation. Additionally, we performed multiple time point injection of MSLN-targeted OAd against regrown tumors. Four out of six tumors were controlled with repeated injection. Next, antitumor effect of MSLN-targeted OAd was assessed in patient-derived xenograft (PDX) model. After intravenous administration, only the MSLN-targeted OAd showed significant antitumor effect compared to the untreated group (p<0.05), while the growth of Ad5 WT virus injected group was same as untreated group. In this study, systemic injection of MSLN-targeted OAd showed remarkable anti-tumor effect in both systemic and intratumoral injections at low dose. Our results indicated that tumor targeted-OAd can embody efficient systemic treatment for pancreatic cancer which are mostly found with spread or metastatic lesions. Citation Format: Mizuho Sato-Dahlman, Yoshiaki Miura, Praveensingh Hajeri, Hideki Yoshida, Kari Jacobsen, Chikako Yanagiba, Masato Yamamoto. Systemic treatment with mesothelin-targeted oncolytic adenovirus shows efficacy patient-derived xenograft of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5921.

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922-947 (E1A CR2-deleted), dl1520 (E1B-55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel-resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non-CAR receptors to infect cells, are also significantly more effective in paclitaxel-resistant ovarian cell models. Inhibition of CDK4/6 using PD-0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms – if validated further, this information could have future clinical utility to aid patient selection for clinical trials.

The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3′ end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3′ end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5′ AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

Tu2022 Optimizing Outcomes of Complex Ventral Hernia Repairs Using a Sandwich Reconstruction Technique With Novel Hybrid-VAC Closure

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFN's properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinoma's low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.

Tu2021 Interferon Expressing Oncolytic Adenovirus for Esophageal Cancer

Introduction: Esophageal cancer remains a highly lethal malignancy with a low overall survival rate. In recent years, Western countries have seen a rapid increase in the incidence of esophageal adenocarcinoma. We have already demonstrated the effectiveness of cyclooxygenase-2 controlled conditionally replicative adenoviruses (CRAd) in esophageal adenocarcinoma cell lines in both in vitro and in vivo models. We now hypothesize that CRAds designed to express interferon alpha (IFN) could provide for increased cell killing capabilities. Additionally, when used as part of combination therapy with chemotherapy and radiation we hope to exploit IFN's properties as a chemoradiotherapy sensitizer to overcome barriers of conventional regimens for esophageal adenocarcinoma. Methods: An infectivity enhanced IFN-expressing CRAd was designed and generated (5/3 Cox2 CRAd ΔE3 ADP IFN). Crystal violet assays were used to determine the in vitro cytocidal effects of the virus across multiple esophageal cancer cell lines (OE19, OE33, and TE7) when compared to three control vectors. Furthermore, the IFN-expressing CRAd was used in conjunction with varying doses of cisplatin and radiation as part of a combination regimen. Results: Genetic modification of the virus capsid (Ad5/Ad3) was used to overcome esophageal adenocarcinoma's low expression of the primary adenovirus receptor and improve infectivity. The virus was also armed with the adenoviral death protein (ADP) to maximize adenoviral spread. At low viral titers, the IFN-expressing CRAd had a much improved cytocidal effect compared to its otherwise identical counterpart that expressed luciferase instead of IFN. Furthermore, the IFN-expressing CRAd had an equivalent cytocidal effect as a non-selective IFN-expressing virus at later time points. The IFN-expressing CRAd also greatly outperformed the standard control adenovirus (Ad5 Wt). The combination of the IFN-expressing CRAd with chemoradiation outperformed all monotherapy treatments (virus alone, chemotherapy alone, radiation alone) across all cell lines tested. Of note, when chemotherapy and radiation were tested in the OE19 cell line, increasing concentrations of cisplatin (up to 9 μM) and doses of radiation (up to 16 Gy) were unable to yield a complete cytocidal effect. Importantly, it was not until the addition of the IFN-expressing CRAd to the chemoradiotherapy regimen that total cancer cell death was achieved. Conclusion: We have demonstrated that an IFN-expressing CRAd has a potent cytocidal effect across multiple esophageal cancer cell lines. When used as part of a combination regimen with chemotherapy and radiation, the IFN-expressing CRAd affords greatly increased cancer cell death. Given these promising results, we are currently testing the IFN-expressing CRAd in an in vivo setting.

Targeted expression of herpes simplex virus thymidine kinase in adenovirus‐infected cells reduces virus titers upon treatment with ganciclovir in vitro

Adenoviruses are a frequent cause of life-threatening infections in immunocompromised patients. Available therapeutics still cannot completely prevent fatal outcomes. By contrast, herpes viruses are well treatable with prodrugs such as ganciclovir (GCV), which are selectively activated in virus-infected cells by virus-encoded thymidine kinases. This effective group of prodrugs is not applicable to adenoviruses and other DNA viruses because they lack those kinases. To render adenoviruses amenable to GCV treatment, we generated an adenoviral vector-based delivery system for targeted expression of herpes simplex virus thymidine kinase (HSV-TK) in wild-type adenovirus 5 (wt Ad5)-infected cells. HSV-TK expression was largely restricted to wt virus-infected cells by transcription of the gene from the Ad5 E4 promoter. Its activity is dependent on the adenoviral E1A gene product which is not produced by the vector but is only provided in cells infected with wt adenovirus. The anti-adenoviral effect of HSV-TK expression and concomitant treatment with GCV was assessed in vitro in four different cell lines or primary cells. E4 promoter-mediated HSV-TK background expression was sufficiently low to prevent cytotoxicity in the presence of low-levels GCV in cells not infected with wt Ad5. However, expression was several-fold increased in wt Ad5-infected cells and treatment with low levels of GCV efficiently inhibited wt Ad5 DNA replication. Genome copy numbers and output of infectious particles were reduced by up to > 99.99% and cell viability was greatly increased. We extended the concept of enzyme/prodrug therapy to adenovirus infections by selectively sensitizing adenovirus-infected cells to treatment with GCV.

HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production.

Adeno-associated virus type 2 (AAV) is a popular vector for human gene therapy, because of its safety record and ability to express genes long term. Yet large scale recombinant (r)AAV production remains problematic due to low particle yield. The adenovirus (Ad) and herpes (simplex) virus (HSV) helper genes for AAV have been widely used and studied, but the helper genes of human papillomavirus (HPV) have not. HPV-16 E1, E2 and E6 help wild type (wt) AAV productive infection in differentiating keratinocytes, however HEK293 cells are the standard cell line used for generating rAAV. Here we demonstrate that the three HPV genes were unable to stimulate significant rAAV replication in HEK293 cells when used alone. However, when used in conjunction (complementation) with the standard Ad5 helper gene set, E1, E2 and E6 were each capable of significantly boosting rAAV DNA replication and virus particle yield. Moreover, wt AAV DNA replication and virion yield were also significantly boosted by each HPV gene along with wt Ad5 virus co-infection. Mild to moderate changes in rep- and cap–encoded protein levels were evident in the presence of the E1, E2 and E6 genes. Higher wt AAV DNA replication was not matched by similar increases in the levels of rep-encoded protein. Moreover, while rep mRNA was up-regulated, cap mRNA was up-regulated more. Higher virus yields did correlate most consistently with increased Rep52, VP3 and VP-related 21/31 kDa species. The observed boost in wt and rAAV production by HPV genes was not unexpected, as the Ad and HPV helper gene sets do not seem to recapitulate each other. These results raise the possibility of generating improved helper gene sets derived from both the Ad and HPV helper gene sets.

A Novel Cancer-Targeting Adenovirus Expressing Interferon Alpha For Pancreatic Cancer Therapy

Introduction: More effective systemic therapy is clearly needed for pancreatic adenocarcinoma. Interferon alpha (IFNα) is promising in multimodality therapy, but has a short half-life and strong side effects. We have generated a novel oncolytic adenovirus using several strategies to enhance efficacy while reducing toxicity. The COX2 promoter is used to drive viral replication due to known overexpression in pancreatic tumor cells, while avoiding replication toxicity to normal organs, e.g., liver. Our design uses the Ad5/Ad3 fiber-knob chimera for optimal infectivity, and adenoviral death protein for enhanced apoptosis and viral spread. Moreover, our oncolytic adenovirus system allows massive transgene expression in target cells. We hypothesize that localized IFNα expression from oncolytic adenovirus will enhance anti-tumor effect with minimal toxicity. Methods: Qualitative assessment of effect was done with crystal violet staining at serial time points. Quantitative cell viability was assessed colorimetrically using a commercially-available kit. COX2 expression in cell lines was assessed by RT-PCR, and promoter function by a luciferase gene reporter assay with known COX2-positive and negative controls. IFNα expression levels were quantitiated by ELISA from supernatant. In vivo, nude mice were injected on each flank with 1×10^6 S2O13 tumor cells, then intratumorally with virus at 1×10^10 vp/50μl or PBS when tumors were 5-8mm in size. Results: in vitro a robust effect on highly aggressive metastatic pancreatic cancer S2 cell lines is seen. When compared to Ad5 wt, the standard control virus without cancer specificity, the new virus has a similarly potent effect on S2VP10, and is more powerful than Ad5 wt when used on S2O13. Our novel virus is also more powerful than the conventional non-replicating IFNα virus. Analysis of COX2 expression in pancreatic cancer cell lines shows that both S2 cell lines are COX2-positive. Our novel virus shows no toxicity to COX2-negative BT474 cells to day 8 post infection, indicating cancer specificity. IFNα expression is dependent on viral replication and reaches a much higher concentration using the novel virus than from the non-replicating IFNα virus. In vivo experiments show a strong suppression of tumor growth from the new virus (Fig 1). Conclusions: A new cancer-targeting oncolytic adenovirus was successfully designed to selectively replicate in cancer cells with a powerful antitumor effect. The effect of the targeted vector was as strong as the viruses without replication selectivity. By expressing IFNα locally in the tumor environment, toxicity from systemic delivery seen in current therapy should be avoided. We are actively working in an in vivo syngeneic model using immunocompetent hamsters.

Inhibition of the Inflammatory Cytokine TNF-α Increases Adenovirus Activity in Ovarian Cancer via Modulation of cIAP1/2 Expression

Oncolytic adenoviruses show promise as a cancer treatment. However, they generate acute inflammatory responses with production of cytokines, including tumor necrosis factor-α (TNF-α). We investigated whether inhibition of TNF-α augments efficacy of the E1A CR2-deleted adenovirus dl922-947 in ovarian cancer. dl922-947 induced transcription of TNF-α and its downstream signaling targets interleukin-6 and -8 (IL-6 and IL-8) in ovarian cancer cells. In vitro, RNAi-mediated knockdown of TNF-α reduced production of multiple inflammatory cytokines after infection and increased ovarian cancer cell sensitivity to virus cytotoxicity, as did treatment with the anti-TNF-α antibody infliximab. In vivo, stable knockdown of TNF-α in IGROV-1 xenografts increased the anticancer activity of dl922-947. In addition, inhibition of TNF-α using monoclonal antibodies also improved dl922-947 efficacy. This increased efficacy resulted from suppression of cellular inhibitor of apoptosis-1 and -2 (cIAP1 and cIAP2) transcription in malignant cells and a consequent increase in caspase-mediated apoptosis. These findings suggest that TNF-α acts as a survival factor in adenovirus-infected cells. Combining TNF-α inhibition with oncolytic adenoviruses could improve antitumor activity in clinical trials.

A 49-kilodalton isoform of the adenovirus type 5 early region 1B 55-kilodalton protein is sufficient to support virus replication.

The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48- to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection.

Replication-competent Adenovirus-mediated Suicide Gene Therapy with Radiation in a Preclinical Model of Pancreatic Cancer

In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer.

A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 × 10 10 vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA.

Activity of the Adenoviral E1A Deletion Mutant dl922-947 in Ovarian Cancer: Comparison with E1A Wild-type Viruses, Bioluminescence Monitoring, and Intraperitoneal Delivery in Icodextrin

The adenoviral mutant dl922-947 has potent activity in a variety of tumors. We investigated the efficacy of dl922-947 in ovarian carcinoma; compared its activity to wild-type adenovirus, dl309, and dl1520; and investigated the use of icodextrin to enhance activity in vivo. We also assessed the utility of luciferase bioluminescence imaging to quantify the response of human ovarian carcinoma xenografts to dl922-947. Ovarian carcinoma cell lines were transfected in vitro with dl922-947, adenovirus 5 wild-type (Ad5 WT), dl309, and dl1520 and monitored for S-phase induction, viral protein expression, replication, and overall survival. In vivo, the efficacy of dl922-947 when delivered in PBS or icodextrin to female nude mice bearing IGROV1 xenografts was determined. In vitro, dl922-947 induced lysis with greater efficacy than Ad5 WT, dl309, or dl1520 in all ovarian carcinoma cell lines tested, which was associated with earlier expression of viral proteins and S-phase induction. The lytic effect in immortalized ovarian surface epithelial cells confirmed that cellular retinoblastoma pathway status is a strong determinant of dl922-947 activity. In vivo, i.p. delivery of dl922-947 (5 x 10(9) particles daily x 5) increased median survival from 20 to 96 days (P < 0.0001) and delivery in icodextrin-enhanced survival further. However, delayed hepatic toxicity was evident in some dl922-947-treated mice, which was not dependent upon viral replication within tumor cells or the liver. dl922-947 has potency in ovarian carcinoma and i.p. delivery in icodextrin may enhance this activity. Immunocompetent models of ovarian carcinoma are required for further evaluation of hepatotoxicity.

499. Improving the Stability of Ad-AAV Hybrid Vectors

The Ad/AAV hybrid vector is an E1 gene-deleted adenovirus containing a transgene cassette flanked on its 5' and 3' ends by AAV ITR vector sequences. After coinfection of Ad/AAV and wt Ad5 of cells containing the rep-cap genes, the AAV ITR-containing transgene cassette is excised from the Ad/AAV, amplified, and then packaged into AAV particles.

311. Oncolytic Adenovirus CG5757 Preferentially Infects Primary Human Tumor Tissues and Shows Strong Anti-Tumor Activity in Tumor Models Following Systemic Administration

Top of pageAbstract A human adenovirus serotype 5 based oncolytic virus CG5757 was designed to preferentially replicate in and kill cancer cells that have a defective retinoblastoma (Rb) pathway and overexpress telomerase. Tumor selectivity of CG5757 was examined using a newly developed ex vivo primary tumor tissue culture model. Primary human colorectal tumors were obtained after surgical resection and placed in Millicell insert with media containing hormones, which can keep tissue viable for about 5 days. The paired tumor or normal tissues from same tissue sample were used in a viral infection experiment to determine the tumor selectivity of CG5757. Cultured tissues were infected with CG5757 or wild type (wt) Ad5 for five days, and progeny viral production was determined by TCID50 assay. Results of the paired tissue cultures from five colon cancer patients indicate that wt Ad5 has similar viral productivity in tumor and normal tissues, whereas CG5757 has higher productivity in tumor tissues. In general, CG5757 produces 100- to 1,000-fold more virus in tumor tissue compared to normal tissue. Immunohistochemical staining for adenoviral E1A shows that CG5757 has positive infection only in the cancer tissue (colon), whereas no signal is detected in normal tissues of colon, pancreas or spleen. Similarly in vitro viral specificity characterization using a panel of tumor cell lines also suggest a strong tumor selectivity of CG5757. Anti-tumor efficacy of CG5757 following intravenous injection was examined in a subcutaneous tumor model. In this study, pre-established prostate cancer LNCaP xenografts in nude mice were treated with two intravenous injections of 4|[times]|1010 viral particles of CG5757 given at a 3-day interval. Significant anti-tumor efficacy was observed in CG5757-treated animals. Eight weeks after treatment, CG5757-treated animals exhibited approximately 72% inhibition of tumor growth. These studies demonstrate the potential therapeutic efficacy of such dual promoter-controlled oncolytic adenoviruses in cancers that are Rb-defective and telomerase-positive.

640. E1 Bp-Specific CD8+ T Lymphocytes Are Highly Induced in the Liver and Spleen Eight Days after Adenoviral Infection

Top of pageAbstract The use of Adenovirus (Ad) as a gene delivery vector is limited by transient gene expression due to the host cytotoxic T lymphocyte (CTL) response and innate responses. We and others have shown that the cytotoxic response to Ad is only partially inhibited by administration of Ad to mutant mice deficient in producing a cytotoxic immune response, or by treatment with TNF inhibitors. However, analysis of the specific CTL response to Ad has not been possible due to the lack of reagents that recognize the rearranged T cell receptor expressed on Ad-specific cytotoxic T cells. We therefore developed a tetramer that recognizes the immunodominant epitope of E1B (192 VNIRNCCYI), referred to as E1Bp, in the context of class Ib MHC. The tetramer consists of H2-Db, β2-microglobulin and the peptide epitope, and is referred to as Db-E1Bp. First, to test the specificity of this peptide-MHC complex, C57BL/6 (H-2b; B6) mice were injected with 8×108i.u. of wild type (wt) Ad5. As a control, wt Ad5 injected DBA/2 (H-2d; D2) were analyzed. Lymphocytes (105 )isolated 8 days later from spleen or liver were restimulated with E1Bp in vitro and analyzed for IFN-γ production by an ELISPOT assay. There was a significant increase in spots (6 and 63 spots, respectively) compared to cells stimulated with human influenza virus peptide M1(58 GILGFVFTL) (0 spot, p 98%) of the Db-E1Bp+CD8+ T cells are CD44+. No Db-E1Bp+CD8+ T cells were found in immunized D2 or naive B6 mice. To determine peptide-specific lysis of target cells in vivo, we labeled target cells from syngeneic mice with either high (2μM; CFSEHi) or low (0.2μM; CFSELo) concentration of CFSE and then pulsed CFSEHi cells with E1Bp and CFSELo cells remained unpulsed. Target cells were adoptively transferred into B6 or D2 mice that were challenged 8 days earlier with wt Ad5, and the percentage of specific killing was determined 6 hours later. As a peptide specificity control, CFSEHi cells pulsed with M1 peptide and unpulsed CFSELo cells were transferred to identically immunized B6 mice. E1Bp-specific lysis was significantly increased in B6 mice compared to control mice or immunized D2 mice (p<0.001). There was a significant correlation between E1Bp-specific lysis and the frequency of Db-E1Bp+ CD8+ T cells in B6 mice (p<0.01). We next used these methods to measure the decreased Ad-specific CTL response in aged mice. Compared with 2-mo-old mice, there was a 30% decrease in killing efficiency in 18-mo-old mice and a 58% decrease in Db-E1Bp+CD8+ T cells. Our results indicate that this tetramer is novel, sensitive and specific tool to detect and follow the E1Bp-specific CTL response against Ad infection.

130. Targeting of Adenovirus Vectors to Mesothelin Via a Bispecific Antibody Reveals Enhanced Gene Transfer to Human Pancreatic Cancer Cells

Pancreatic cancer is the 5th leading cause of cancer death in the Western world with no effective treatment yet. Adenoviral (Ad) 5 vectors have been studied for gene delivery into human pancreatic cancer, showing that wild type Ad5 has a limited transduction efficiency. Since the extracellular mesothelin protein is upregulated on primary human pancreatic cancer cells (Argani et al. (2001) Clin Canc Res 7, 3862), while no or limited expression is detected on normal pancreatic cells, we chose to target Ad5 to this surface antigen by using the bispecific antibody approach. We cloned the cDNA encoding for the single-chain and bispecific antibody that binds to mesothelin into an expression vector, and produced the bispecific antibody that binds both to adenovirus fiber knob and to mesothelin. Ad5 could indeed be targeted to mesothelin expressing OVCAR-3 control cells as shown by improved transduction with Ad5CMVGFP complexed with bispecific antibody compared to wt Ad5CMVGFP without bispecific antibody. Subsequently, two near primary and four established pancreatic cancer cells were infected with the AdCMVGFP-bispecific antibody complex, in the presence or in the absence of Ad5 fiber protein and single-chain antibody. It was shown that adenovirus complexed with bispecific antibody revealed a significant enhanced gene transfer to one established and two near primary CAR negative and mesothelin positive pancreatic carcinoma cells, compared to wt Ad5. This effect could be prevented by competitive pre-incubation of the cells with the single-chain antibody. We conclude that adenovirus vectors complexed with bispecific antibody directed to mesothelin can enter mesothelin expressing pancreatic tumor cells with a high efficiency and efficacy compared to wt Ad5, suggesting that mesothelin might be a promising antigen for targeted adenoviral gene therapy of pancreatic cancer.

Caspase-Mediated Cleavage of Adenovirus Early Region 1A Proteins

Adenovirus 2 and 12 early region 1A (Ad2 and Ad12 E1A) proteins were cleaved during cisplatin-induced apoptosis of Ad-transformed rat and human cells. Cleavage was inhibited in the presence of caspase inhibitors such as Z-VAD-FMK. In Ad12 transformants both 13S and 12S E1A proteins were cleaved at a similar rate. In Ad2 transformants the E1A 13S component was appreciably less stable than the 12S component. In in vitro studies Ad2 and Ad12 E1A 13S and Ad2 12S proteins were rapidly cleaved by caspase 3 whereas Ad12 12S E1A and Ad12 13S E1A were rapidly degraded by caspase 7. Cleavage sites in Ad12 13S proteins for caspase 3 have been determined. Initial cleavage occurred at D24 and D150; this was followed by cleavage at D204 and D242. Caspase-3-mediated cleavage of Ad12 13S E1A destroyed its ability to bind to CBP and TBP but interaction between C terminal E1A polypeptides and CtBP was observed. During viral infection Ad5 and Ad12 E1A 12S proteins were markedly more stable than 13S proteins but no difference was observed in Ad E1A levels in the absence or presence of the caspase inhibitors Z-VAD-FMK or Z-D(OMe)-E(OMe)-V-D(OMe)-CH 2F. Limited caspase 3 and 10 activation occurred during infection with the E1B 19K − virus Ad2 pm1722 but little or no activation of caspase 3 was observed during wt virus infection. Examination of protein cleavage during viral infection of A549 cells showed proteolysis of lamin B and PARP in response to Ad5 wt and Ad2 pm1722. Protein degradation in response to both viruses was partially inhibited by Z-VAD-FMK. Following infection of human skin fibroblasts lamin B was degraded, although only limited changes in PARP levels were observed. We have concluded that Ad E1A is cleaved by caspases during apoptosis but not during viral infection. However, some of the processes commonly associated with apoptosis occur during viral infection, particularly with E1B 19K − mutants, although apoptosis per se is not evident.