AB1056F Sigma-AldrichAnti-Adenovirus Antibody, FITC-conjugated

The efficacy of cancer immunotherapy is limited because of central and peripheral immune tolerance towards tumor-antigens. We propose a novel approach based on the fact that the immune system has not evolved tolerance towards adenoviruses (Ads) and that Ads have not evolved efficient mechanisms for immune-escape. The host-response to intratumoral Ad-vector injection in mice that were immunologically tolerant to neu-positive syngeneic mammary-cancer (MMC) was investigated. Intratumoral injection with replication-deficient, transgene-devoid Ad induced immune responses at two different anatomical sites: the tumor-draining lymph nodes and the tumor microenvironment. The lymph nodes supported the generation of both neu- and Ad-specific T effector cells, while inside the tumor microenvironment only Ad-specific T cells expanded. Importantly, Ad-specific T cells were anti-tumor-reactive despite the presence of active regulatory T cell-mediated immune tolerance inside MMC tumors and anti-tumor efficacy of Ad was increased by pre-immunization against Ad despite the production of Ad-neutralizing antibodies.

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Recent attempts to circumvent the limitations of adenovirus (Ad) vectors derived from species C serotype Ad5 have focused on the use of alternative human serotypes. These new serotypes have multiple benefits including a low prevalence of neutralizing antibodies in humans and alternate tropisms. To investigate the characteristics of alternatives to Ad5 vectors, we compared the biodistribution and safety of Ads from species B (Ad3, 11p, 35), C (Ad5), E (Ad4), and F (Ad41), or chimeric Ad5 viruses containing the Ad11 or Ad35 fibers (Ad5/11 and Ad5/35), after intravenous (IV) delivery into hCD46 transgenic mice. Our data suggest that (i) mechanisms of cell and tissue sequestration differ; (ii) levels of sequestration to lung, liver, or spleen do not correlate with toxicity; (iii) delivery of all serotypes causes activation of coagulation, possibly through platelet interaction; (iv) despite binding to the same receptor in vitro, Ad serotypes act differently in vivo; and (v) platelet depletion affects blood clearance, organ sequestration and chemokine/cytokine release of some, but not all Ad serotypes. Overall, our data indicate that Ad5-based vectors are relatively safe as compared to other serotypes. This data should be taken into consideration in future studies about the clinical use of Ad vectors.

We have developed a new class of adenovirus vectors that selectively replicate in tumor cells. The vector design is based on our recent observation that a variety of human tumor cell lines support DNA replication of adenovirus vectors with deletions of the E1A and E1B genes, whereas primary human cells or mouse liver cells in vivo do not. On the basis of this tumor-selective replication, we developed an adenovirus system that utilizes homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome resulting in replication-dependent activation of transgene expression in tumors (Ad.IR vectors). Here, we used this system to achieve tumor-specific expression of adenoviral wild-type E1A in order to enhance viral DNA replication and spread within tumor metastases. In vitro DNA replication and cytotoxicity studies demonstrated that the mechanism of E1A-enhanced replication of Ad.IR-E1A vectors is efficiently and specifically activated in tumor cells, but not in nontransformed human cells. Systemic application of the Ad.IR-E1A vector into animals with liver metastases achieved transgene expression exclusively in tumors. The number of transgene-expressing tumor cells within metastases increased over time, indicating viral spread. Furthermore, the Ad.IR-E1A vector demonstrated antitumor efficacy in subcutaneous and metastatic models. These new Ad.IR-E1A vectors combine elements that allow for tumor-specific transgene expression, efficient viral replication, and spread in liver metastases after systemic vector application.

Adenoviruses (Ads) that selectively replicate in tumor cells have shown promising preliminary results in clinical trials, especially in combination with chemotherapy. Here, we describe a system that combines the antitumor synergy of Ads and chemotherapeutic agents with the benefits of enzyme-activated prodrug therapy. In this system, a functional transgene expression cassette is created by homologous recombination during adenoviral DNA replication. Transgene expression is strictly dependent on viral DNA replication, which in turn is tumor specific. We constructed replication-activated Ad vectors to express a secreted form of beta-glucuronidase and a cytosine deaminase/uracil phosphoribosyltransferase, which activate the prodrugs 9-aminocamptothecin glucuronide to 9-aminocamptothecin and 5-fluorocytosine to 5-fluorouracil (5-FU) and further to 5-fluoro-UMP, respectively. We demonstrated replication-dependent transgene expression, prodrug activation, and induction of tumor cell toxicity by secreted beta-glucuronidase and cytosine deaminase/uracil phosphoribosyltransferase. Furthermore, exposure of cells to activated prodrug or drug at subtoxic concentrations enhanced viral DNA replication. Characteristically, these agents induced changes in the cell cycle status of exposed cells (G(2) arrest), which closely resembled the effect of wild-type Ad infection, and are thought to be favorable for viral replication. We tested a number of cytostatic drugs (camptothecin, etoposide, daunorubicin, cisplatin, 5-fluorouracil, hydroxyurea, Taxol, and actinomycin D) for their effect on viral DNA replication and found considerable differences between individual agents. Finally, we show that the combination of viral and prodrug therapy enhances viral replication and spread in liver metastases derived from human colon carcinoma or cervical carcinoma in a mouse model. Our data indicate that specific vector/drug combinations tailored to be synergistic may have the potential to improve the potency of either therapeutic approach. These data also provide a new rationale for expressing prodrug-activating enzymes from conditionally replicating Ads.

Preclinical studies were designed to investigate the safety of recombinant adenoviruses infused into the portal vein of adult rhesus monkeys, as well as the safety and efficacy of readministration of these agents. The vectors used were recombinant adenoviruses, the E1 region of which was replaced with a marker gene expression cassette. Four 3- to 5-kg rhesus monkeys underwent portal vein cannulation, and infusion of escalating doses of recombinant first-generation vector. Serial sequential liver biopsies were performed, and necropsies were performed out to 14 months. X-Gal histochemical analysis of the liver showed evidence of dose-dependent increased gene transfer throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was also present in transduced livers, and occurred in a dose-dependent manner. Severe toxicity, including mortality, was noted at the highest dose of vector. Readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response. The data suggest that intraportal administration and readministration of recombinant adenoviral E1-deleted vectors are feasible and safe. Vector administration at the highest dose (1 x 10(13) particles/kg) was associated with severe clinical and biochemical toxicity, and significant gene expression was associated with transaminitis. Readministration of vector is safe, but gene transfer is limited by the presence of neutralizing antibody.

We have recently shown that the overexpression of a heat shock protein 70 (hsp 70) in a rat myogenic cell line confers protection against simulated ischemia. We also developed and demonstrated that overexpression of this protein, in the hearts of transgenic mice, protects against ischemia/reperfusion injury. We have now inserted the hsp70 gene in an adenoviral vector and show that we are able to transfer and achieve overexpression of this protein in neonatal cardiomyocytes and in the rat myogenic cell line H9c2. We find that cells infected with the adenoviral-hsp70i construct are rendered tolerant to simulated ischemia as compared to cells infected with a control recombinant adenoviral construct. In conclusion, our results demonstrate the feasibility of using adenoviral vectors to overexpress the hsp70 in myogenic cells, specially in cardiomyocytes, and the efficiency of this approach for providing protection against myocardial ischemia.