Oncolytic adenovirus based on serotype 3.

Oncolytic adenoviruses have been safe in clinical trials but the efficacy has been mostly limited. All published trials have been performed with serotype 5 based viruses. The expression level of the Ad5 receptor CAR may be variable in advanced tumors. In contrast, the Ad3 receptor remains unclear, but is known to be abundantly expressed in most tumors. Therefore, we hypothesized that a fully serotype 3 oncolytic adenovirus might be useful for treating cancer. Patients exposed to adenoviruses develop high titers of serotype-specific neutralizing antibodies, which might compromise re-administration. Thus, having different serotype oncolytic viruses available might facilitate repeated dosing in humans. Ad3-hTERT-E1A is a fully serotype 3 oncolytic adenovirus controlled by the promoter of the catalytic domain of human telomerase. It was effective in vitro on cell lines representing seven major cancer types, although low toxicity was seen in non-malignant cells. In vivo, the virus had anti-tumor efficacy in three different animal models. Although in vitro oncolysis mediated by Ad3-hTERT-E1A and wild-type Ad3 occurred more slowly than with Ad5 or Ad5/3 (Ad3 fiber knob in Ad5) based viruses, in vivo the virus was at least as potent as controls. Anti-tumor efficacy was retained in presence of neutralizing anti-Ad5 antibodies whereas Ad5 based controls were blocked. In summary, we report generation of a non-Ad5 based oncolytic adenovirus, which might be useful for testing in cancer patients, especially in the context of high anti-Ad5 neutralizing antibodies.

Prolonged systemic circulation of chimeric oncolytic adenovirus Ad5/3-Cox2L-D24 in patients with metastatic and refractory solid tumors.

Eighteen patients with refractory and progressive solid tumors were treated with a single round of triple modified oncolytic adenovirus (Ad5/3-Cox2L-D24). Ad5/3-Cox2L-D24 is the first non-Coxsackie-adenovirus receptor-binding oncolytic adenovirus used in humans. Grades 1-2 flu-like symptoms, fever, and fatigue were seen in most patients, whereas transaminitis or thrombocytopenia were seen in some. Non-hematological grades 3-5 side effects were seen in one patient with grade 3 ileus. Treatment resulted in high neutralizing antibody titers within 3 weeks. Virus appeared in serum 2-4 days after treatment in 83% of patients and persisted for up to 5 weeks. One out of five radiologically evaluable patients had partial response (PR), one had minor response (MR), and three had progressive disease (PD). Two patients scored as PD had a decrease in tumor density. Tumor reductions not measurable with Response Evaluation Criteria In Solid Tumors (RECIST) were seen in a further four patients. PR, MR, stable disease, and PD were seen in 12, 23.5, 35, and 29.5% of tumor markers analyzed, respectively (N=17). Ad5/3-Cox2L-D24 appears safe for treatment of cancer in humans and extended virus circulation results from a single treatment. Objective evidence of anti-tumor activity was seen in 11/18 (61%) of patients. Clinical trials are needed to extend these findings.

Utility of TK/GCV in the context of highly effective oncolysis mediated by a serotype 3 receptor targeted oncolytic adenovirus.

Arming oncolytic adenoviruses with therapeutic transgenes and enhancing transduction of tumor cells are useful strategies for eradication of advanced tumor masses. Herpes simplex virus thymidine kinase (TK) together with ganciclovir (GCV) has been promising when coupled with viruses featuring low oncolytic potential, but their utility is unknown in the context of highly effective infectivity-enhanced viruses. We constructed Ad5/3-Delta24-TK-GFP, a serotype 3 receptor-targeted, Rb/p16 pathway-selective oncolytic adenovirus, where a fusion gene encoding TK and green fluorescent protein (GFP) was inserted into 6.7K/gp19K-deleted E3 region. Ad5/3-Delta24-TK-GFP killed ovarian cancer cells effectively, which correlated with GFP expression. Delivery of GCV immediately after infection abrogated viral replication, which might have utility as a safety switch. Due to the bystander effect, killing of some cell lines in vitro was enhanced by GCV regardless of timing. In murine models of metastatic ovarian cancer, Ad5/3-Delta24-TK-GFP improved antitumor efficacy over the respective replication-deficient virus with GCV. However, GCV did not further enhance efficacy of Ad5/3-Delta24-TK-GFP in vivo. Simultaneous detection of tumor load and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis. In summary, TK/GCV may not add antitumor activity in the context of highly potent oncolysis.

Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

Employment of liver tissue slice analysis to assay hepatotoxicity linked to replicative and nonreplicative adenoviral agents.

Whereas virotherapy has emerged as a novel and promising approach for neoplastic diseases, appropriate model systems have hampered preclinical evaluation of candidate conditionally replicative adenovirus agents (CRAds) with respect to liver toxicity. This is due to the inability of human viral agents to cross species. We have recently shown the human liver tissue slice model to be a facile means to validate adenoviral replication. On this basis, we sought to determine whether our ex vivo liver tissue slice model could be used to assess CRAd-mediated liver toxicity. We analyzed and compared the toxicity of a conditionally replicative adenovirus (AdDelta24) to that of a replication incompetent adenovirus (Adnull [E1-]) in mouse and human liver tissue slices. To accomplish this, we examined the hepatic apoptosis expression profile by DNA microarray analyses, and compared these results to extracellular release of aminotransferase enzymes, along with direct evidence of apoptosis by caspase-3 immunhistochemical staining and TUNEL assays. Human and mouse liver tissue slices demonstrated a marked increase in extracellular release of aminotransferase enzymes on infection with AdDelta24 compared to Adnull. AdDelta24-mediated liver toxicity was further demonstrated by apoptosis induction, as detected by caspase-3 immunohistochemical staining, TUNEL assay and microarray analysis. In conclusion, concordance of CRAd-mediated apoptosis in both the human and the mouse liver tissue slice models was demonstrated, despite the limited replication ability of CRAds in mouse liver slices. The results of this study, defining the CRAd-mediated apoptosis gene expression profiles in human and mouse liver, may lay a foundation for preclinical liver toxicity analysis of CRAd agents.

A human adenoviral vector with a chimeric fiber from canine adenovirus type 1 results in novel expanded tropism for cancer gene therapy.

The development of novel therapeutic strategies is imperative for the treatment of advanced cancers like ovarian cancer and glioma, which are resistant to most traditional treatment modalities. In this regard, adenoviral (Ad) cancer gene therapy is a promising approach. However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in cancer gene therapy clinical trials to date has been limited, mainly due to the paucity of the primary Ad5 receptor, the coxsackie and adenovirus receptor (CAR), on human cancer cells. To circumvent CAR deficiency, Ad5 vectors have been retargeted by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes. Recently, more radical modifications based on 'xenotype' knob switching with non-human adenovirus have been exploited. Herein, we present the characterization of a novel vector derived from a recombinant Ad5 vector containing the canine adenovirus serotype 1 (CAV-1) knob (Ad5Luc1-CK1), the tropism of which has not been previously described. We compared the function of this vector with our other chimeric viruses displaying the CAV-2 knob (Ad5Luc1-CK2) and Ad3 knob (Ad5/3Luc1). Our data demonstrate that the CAV-1 knob can alter Ad5 tropism through the use of a CAR-independent entry pathway distinct from that of both Ad5Luc1-CK2 and Ad5/3-Luc1. In fact, the gene transfer efficiency of this novel vector in ovarian cancer cell lines, and more importantly in patient ovarian cancer primary tissue slice samples, was superior relative to all other vectors applied in this study. Thus, CAV-1 knob xenotype gene transfer represents a viable means to achieve enhanced transduction of low-CAR tumors.

Transductional and transcriptional targeting of adenovirus for clinical applications.

Adenovirus (Ad) targeting is a novel approach for the design and administration of therapeutic agents wherein the agent is rationally designed to localize and restrict transgene expression to the site of disease in a self-directed manner, usually via exploitation of unique biophysical and genetic properties specific to the diseased tissue. The ablation of promiscuous native Ad tropism coupled with active targeting modalities has demonstrated that innate gene delivery efficiency may be retained while circumventing Ad dependence on its primary cellular receptor, the coxsackie and adenovirus receptor (CAR), to achieve CAR-independent vector tropism. Herein, we describe advances in Ad targeting that are predicated not only on fundamental understanding of vector/cell interplay, but also on the specific transcriptional profiles of target tissues. Further, targeting is discussed in the context of improving the safety and efficacy of clinical approaches utilizing adenoviral vectors and replication competent oncolytic agents. In summary, existing results suggest a critical linkage between targeted agents and increases in therapeutic utility.

A cyclooxygenase-2 promoter-based conditionally replicating adenovirus with enhanced infectivity for treatment of ovarian adenocarcinoma.

Conditionally replicating adenoviruses (CRADs) take advantage of tumor-specific characteristics for preferential replication and subsequent oncolysis of cancer cells. The antitumor effect is determined by the capability to infect tumor cells. Here, we used RGDCRADcox-2R, which features the cyclooxygenase-2 promoter for replication control and an integrin binding RGD-4C motif for enhanced infectivity of ovarian cancer cells. RGDCRADcox-2R replicated in and killed human ovarian cancer cells effectively, while the replication in nonmalignant cells was low. Importantly, the therapeutic efficacy, as evaluated in an orthotopic model of peritoneally disseminated ovarian cancer, was significantly improved and toxicity was lower than with a wild-type virus. Thus, this CRAD could be tested for treatment of ovarian cancer in humans.

[Risk of uterine rupture after cesarean section--analysis of 1,086 births]. / Das Risiko der Uterusruptur nach vorausgegangenem Kaiserschnitt--eine Analyse von 1,086 Geburten.

OBJECTIVE: In the presented paper, obstetrical management after previous caesarian section was studied in a large patient collective at the University Department of Gynaecology and Obstetrics in Cologne from 1979 to 1995. Particular attention was given to the feared complication of rupture of the uterus. PATIENTS: From a total of 15,166 deliveries, 1,086 of the births had been preceded by one or more caesarian section. These 1,086 births formed the patient collective for the present study. RESULTS: Vaginal delivery was attempted in 44.5% of patients and was successful in 86% of those cases. If there had been a previous caesarian section, the percentage shifted in favour of vaginal delivery. All patients with more than two previous caesarian sections were delivered by a primary caesarian section. The feared complication of rupture of the uterus occurred in four cases, for which case reports are presented. In view of such cases, signs of imminent uterus rupture often constitute an indication for primary (11.5%) or secondary resectioning (31.9%). No relationship was found between fetal outcome and mode of delivery. CONCLUSION: This retrospective study confirms the general recommendation of vaginal delivery following previous caesarian section as long as risks are minimized by a readiness to proceed with resectioning when signs of imminent rupture of the uterus arise.

The risk of rupture of the uterus: an analysis of 1086 births after previous caesarean section.

In the work presented here, obstetric management after a previous caesarean section was studied in a large patient group at the University Department of Gynecology and Obstetrics in Cologne from 1979 to 1995. Particular attention was given to the feared complication of uterine rupture. From a total of 15 166 deliveries, 1086 of the births had been preceded by one or more caesarean sections. These 1086 births formed the basis for the present study. Vaginal delivery was attempted in 44.5% of patients and was successful in 86% of those cases. Where there had been only one previous caesarean section, the percentage shifted in favor of vaginal delivery. All patients with more than two previous caesarean sections were delivered by elective caesarean section. The feared complication of rupture of the uterus occurred in four cases, for which case reports are presented. In view of such cases, signs of imminent uterus rupture often constitute an indication for elective (11.5%) or emergency resectioning (31.9%). No relationship was found between fetal outcome and mode of delivery. This retrospective study confirms the general recommendation and safety of vaginal delivery after a previous caesarean section as long as risks are minimised by a readiness to proceed with a repeat caesarean when signs of imminent rupture of the uterus arise.