Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t 1/2 < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t 1/2 = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA. Mol Cancer Ther; 9(11); 3052-64. ©2010 AACR.

The expanding use of adenoviral vectors for gene therapy has brought about the need for new analytical tools. We have developed an anion-exchange high-performance liquid chromatography method to analyze recombinant adenovirus serotype 5 samples. Before this assay, available analytical methods consisted of either long-term biological assays or required highly purified test articles. These methods were inadequate for optimizing adenovirus production and purification. This assay can quantitate viral particles in either crude lysates or highly pure samples. It can be used to assess particles in both dilute and concentrated samples over a wide dynamic range. Moreover, the population of viral particles eluted in the peak contains most of the infectious virions. This assay is a sensitive technique that overcomes the limitations of previous methods. It provides an essential tool to accomplish process optimization.

We have investigated the use of column chromatography for the purification of ACN53, a recombinant adenovirus type 5 encoding the human p53 tumor suppressor protein. Anion exchange, size exclusion, hydrophobic interaction, and metal chelating resins were tested; each was found to have distinct advantages and disadvantages. Based on these data, a rapid method was devised for the purification of ACN53. The resultant product was characterized and compared to cesium chloride density-gradient purified virus by SDS-PAGE, Western blot analysis, absorbance spectrum, total particle-to-infectious particle ratio, expression of p53 gene product in Saos-2 cells, growth inhibition of Saos-2 cells, and contamination by ATCC-293 host cell proteins. The results show that column chromatography offers an alternative to ultracentrifugation for the purification of recombinant adenoviruses for use in human gene therapy trials and other research applications.

Replicating adenoviruses may prove to be effective anticancer agents if they can be engineered to selectively destroy tumor cells. We have constructed a virus (01/PEME) containing a novel regulatory circuit in which p53-dependent expression of an antagonist of the E2F transcription factor inhibits viral replication in normal cells. In tumor cells, however, the combination of p53 pathway defects and deregulated E2F allows replication of 01/PEME at near wild-type levels. The re-engineered virus also showed significantly enhanced efficacy compared with extensively studied E1b-deleted viruses such as dl1520 in human xenograft tumor models.

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