Duane E. Johnson

Re-engineering adenovirus regulatory pathways to enhance oncolytic specificity and efficacy.

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.

Impact of Human Neutralizing Antibodies on Antitumor Efficacy of an Oncolytic Adenovirus in a Murine Model

Purpose: The purpose of this study was to assess the impact of anti-adenovirus neutralizing antibodies (AdNAbs) on the distribution, tolerability, and efficacy of intravenously administered oncolytic adenovirus. A translational model was developed to evaluate the impact of humoral immunity on intravenous administration of oncolytic adenovirus in humans. Experimental Design: Initially, severe combined immunodeficient (SCID)/beige mice were passively immunized with various amounts of human sera to establish a condition of preexisting humoral immunity similar to humans. A replication-deficient adenovirus encoding β-galactosidase (rAd-βgal) was injected intravenously into these mice. An AdNAb titer that mitigated galactosidase transgene expression was determined. A xenograft tumor-bearing nude mouse model was developed to assess how a similar in vivo titer would impact the activity of 01/PEME, an oncolytic adenovirus, after intravenous administration. Results: In SCID/beige mice, there was a dose dependence between AdNAbs and galactosidase transgene expression; 90% of transgene expression was inhibited when the titer was 80. A similar titer reconstituted in the nude mice with human serum, as was done in the SCID/beige mice, did not abrogate the antitumor efficacy of the replicating adenovirus after intravenous administration. Viral DNA increased in tumors over time. Conclusions: In intravenous administration, preexisting AdNAb titer of 80 significantly attenuated the activity of a 2.5 × 1012 particles per kilogram dose of nonreplicating adenovirus; the same titer had no affect on the activity of an equivalent dose of replicating adenovirus. Our results suggest that a majority of patients with preexisting adenovirus immunity would be candidates for intravenous administration of oncolytic adenovirus.

Intratumoral Spread and Increased Efficacy of a p53-VP22 Fusion Protein Expressed by a Recombinant Adenovirus

ABSTRACT In vitro experiments have demonstrated intercellular trafficking of the VP22 tegument protein of herpes simplex virus type 1 from infected cells to neighboring cells, which internalize VP22 and transport it to the nucleus. VP22 also can mediate intercellular transport of fusion proteins, providing a strategy for increasing the distribution of therapeutic proteins in gene therapy. Intercellular trafficking of the p53 tumor suppressor protein was demonstrated in vitro using a plasmid expressing full-length p53 fused in-frame to full-length VP22. The p53-VP22 chimeric protein induced apoptosis both in transfected tumor cells and in neighboring cells, resulting in a widespread cytotoxic effect. To evaluate the anti-tumor activity of p53-VP22 in vivo, we constructed recombinant adenoviruses expressing either wild-type p53 (FTCB) or a p53-VP22 fusion protein (FVCB) and compared their effects in p53-resistant tumor cells. In vitro, treatment of tumor cells with FVCB resulted in enhanced p53-specific apoptosis compared to treatment with equivalent doses of FTCB. However, in normal cells there was no difference in the dose-related cytotoxicity of FVCB compared to that of FTCB. In vivo, treatment of established tumors with FVCB was more effective than equivalent doses of FTCB. The dose-response curve to FVCB was flatter than that to FTCB; maximal antitumor responses could be achieved using FVCB at doses 1 log lower than those obtained with FTCB. Increased antitumor efficacy was correlated with increased distribution of p53 protein in FVCB-treated tumors. This study is the first demonstration that VP22 can enhance the in vivo distribution of therapeutic proteins and improve efficacy in gene therapy.

Selective Expression of Nonsecreted Interferon by an Adenoviral Vector Confers Antiproliferative and Antiviral Properties and Causes Reduction of Tumor Growth in Nude Mice

Replication-deficient adenoviruses expressing human interferon-alpha2b (HuIFN-alpha2b) or the hybrid IFN-alpha2alpha1 or those with the secretory signal deleted, whose express is driven by the alpha-fetoprotein (AFP) promoter, were constructed and characterized. Synthesis of IFN protein and secretion or intracellular retention were tested by Western blotting and immunoassay. Expression of IFN by the recombinant adenoviruses was restricted to cells that constitutively express AFP. In these cells, expression of both secreted and nonsecreted recombinant IFN resulted in inhibition of cell proliferation, resistance to viral infection, induction of major histocompatibility complex (MHC) class I expression, increased apoptosis, and activation of an IFN-stimulated response element (ISRE)-containing promoter. Also, the induction of protein kinase R (PKR), increased phosphorylation of Stat1, and accumulation of hypophosphorylated pRb were observed for both the secreted and nonsecreted IFN, suggesting that the nonsecreted IFN may act through a similar pathway. Hep3B cells, an AFP-positive line derived from a patient with hepatocellular carcinoma (HCC), were injected subcutaneously (s.c.) into athymic nude mice to generate established tumors. Intratumoral injection of recombinant adenoviruses expressing secreted as well as the nonsecreted IFN caused suppression of tumor growth. As the AFP promoter is activated in many HCC cells but is silent in normal cells, these constructs may be useful in restricting IFN effects to the tumor cells while reducing toxicity to the neighboring tissues.

Interferon α2b gene delivery using adenoviral vector causes inhibition of tumor growth in xenograft models from a variety of cancers

A recombinant adenovirus expressing human interferon α2b driven by the cytomegalovirus promoter, IACB, was shown to produce and secrete biologically active protein in vitro and in vivo. Intravenous administration of IACB in Buffalo rats resulted in circulating levels of biologically active human interferon at 70,000 international units/mL for up to 15 days. Distribution of interferon protein after IACB administration was different from that seen with the subcutaneous delivery of interferon protein. Higher levels of interferon protein were observed in liver and spleen after IACB delivery compared to protein delivery. The antitumor efficacy of IACB, as measured by suppression of tumor growth, was tested in athymic nude mice bearing established human tumor xenografts from different types of human cancer. Subcutaneous tumors most responsive to the intratumoral administration of IACB ranked as U87MG (glioblastoma) and K562 (chronic myelogenous leukemia), followed by Hep 3B (hepatocellular carcinoma) and LN229 cells (glioblastoma). Intravenous administration of IACB in animals bearing U87MG or Hep 3B xenografts was also effective in suppressing tumor growth, although to a lesser extent than the intratumoral administration. IACB was also tested in a metastatic model in beige/SCID mice generated with H69 (small cell lung carcinoma) cells and was found to prolong survival in tumor-bearing animals. This suggested that interferon gene delivery can be effective in suppressing tumor growth in a wide variety of cells. Cancer Gene Therapy (2001) 8, 788–795