Stephen R. Indelicato

Characterization of Replication-Competent Adenovirus Isolates from Large-Scale Production of a Recombinant Adenoviral Vector

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.

The Use of Laser Scanning Cytometry to Assess Depth of Penetration of Adenovirus p53 Gene Therapy in Human Xenograft Biopsies

SCH58500 is an agent for gene therapy of cancer, consisting of a replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5/beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.

Use of high-performance liquid chromatographic and microbiological analyses for evaluating the presence or absence of active metabolites of the antifungal posaconazole in human plasma.

Posaconazole (SCH 56592) is a novel broad spectrum triazole antifungal agent that is currently in phase III clinical trials for the treatment of systemic fungal infections. This study was initiated to determine if orally administered posaconazole to humans would result in the formation of active metabolite(s). Plasma samples from a multiple-rising dose study in healthy volunteers were analyzed by validated HPLC and microbiological methods. The HPLC analysis involved extraction with a mixture of organic solvent (methylene chloride-hexane) followed by separation on a C18 column and quantification by UV absorbance at 262 nm. The microbiological assay was performed utilizing an agar diffusion method using Candida pseudorropicalis ATCC 46764 as the test organism. Potency was determined by comparing the growth inhibition zones produced by the test sample to those produced by standard concentrations prepared in plasma. Individual and mean plasma concentration-time profiles were similar for both HPLC and microbiological assays. The area under the plasma concentration-time curves of the microbiological and HPLC results were similar with a mean (RSD) ratio of 105.5% 15.3%), indicating that there was no relevant biologically active metabolite of posaconazole in human plasma.

Noncompetitive Antibody Neutralization of IL-10 Revealed by Protein Engineering and X-Ray Crystallography

IL-10 is a dimeric cytokine that must engage its high-affinity cell surface receptor, IL-10R1, to induce multiple cellular activities. Here we report the 1.9 A crystal structure of an engineered IL-10 monomer (IL-10M1) in complex with a neutralizing Fab fragment (9D7Fab). 9D7Fab and IL-10R1 bind distinct nonoverlapping surfaces on IL-10M1. Antagonism of the IL-10M1/IL-10R1 interaction is the result of 9D7Fab-induced conformational changes in the CD loop of IL-10M1 that indirectly alter the structure of the IL-10R1 binding site. A single mutation (Ile87Ala) in the same CD loop region of the Epstein-Barr virus IL-10 (ebvIL-10) also reduces IL-10R1 binding affinity, suggesting that ebvIL-10 and 9D7Fab use similar allosteric mechanisms to modulate IL-10R1 affinity and biological activity.

Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models.

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a “lowered threshold” for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3–11–fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDA-MB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies. Cancer Gene Therapy (2000) 7, 1469–1480