Peter Krajcsi

Tissue-Specific, Tumor-Selective, Replication-Competent Adenovirus Vector for Cancer Gene Therapy

ABSTRACT We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a “vector spread” assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

An Oncolytic Adenovirus Vector Combining Enhanced Cell-to-Cell Spreading, Mediated by the ADP Cytolytic Protein, with Selective Replication in Cancer Cells with Deregulated Wnt Signaling

We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3–11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear β-catenin.

A 14,500 MW protein is coded by region E3 of group C human adenoviruses

There is an ORF in the early region E3 transcription unit of human adenovirus 5 (Ad5) which could encode a protein of 14,500 MW (14.5K). This ORF is conserved in Ad5 and Ad2, both group C adenoviruses, and also in Ad3 and Ad7, both group B adenoviruses. To address whether the 14.5K protein is synthesized, we prepared antisera against synthetic peptides corresponding to residues 19-34 or 118-132 in the Ad5 version of 14.5K, and also against a TrpE-14.5K fusion protein expressed in Escherichia coli. These antisera immunoprecipitated the [35S]Met-labeled 14.5K protein from KB cells infected with rec700 (an Ad5-Ad2-Ad5 recombinant), Ad2, and a variety of E3 mutants. Mutants in the 14.5K ORF did not produce the 14.5K protein. The 14.5K is coded in large part, although probably not exclusively, by E3 mRNA f, as indicated by immunoprecipitation of 14.5K from cells infected with mutants that overproduce or underproduce mRNA f. The 14.5K migrated as five to six bands on SDS-PAGE after immunoprecipitation or Western blot, suggesting that it undergoes post-translational modification. Two bands of 14.5K were obtained by cell-free translation of 14.5K from mRNA purified by hybridization from infected cells.

Adenovirus RIDβ Subunit Contains a Tyrosine Residue That Is Critical for RID-Mediated Receptor Internalization and Inhibition of Fas- and TRAIL-Induced Apoptosis

ABSTRACT The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDα and RIDβ subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these “death” receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDβ that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDβ were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDβ C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxφ). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDβ but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RIDs ability to down-regulate receptors.

Adenovirus replication-competent vectors (KD1, KD3) complement the cytotoxicity and transgene expression from replication-defective vectors (Ad-GFP, Ad-Luc).

The successful clinical application of adenovirus (Ad) in cancer control has been of limited success because of the current inability to infect the majority of cancer cells with a large amount of vector. In this study, we show that when human lung tumors growing in immunodeficient nude mice were coinfected with a replication-defective (RD) Ad vector expressing green fluorescent protein and a replication-competent (RC) Ad vector named KD3, KD3 enhanced the expression of green fluorescent protein throughout the tumor. Also, KD3 and another RC vector named KD1 complemented the expression of luciferase from a RD vector in a human liver tumor xenotransplant in nude mice. Altogether, these results suggest that the combination of a RD vector with a RC vector might be a more effective treatment for cancer than either vector alone due to more widespread dissemination of the virus.

A 6700 MW membrane protein is encoded by region E3 of adenovirus type 2.

There is an open reading frame between ATG1022 and TGA1205 in the E3 transcription unit of adenovirus 2 that could encode a protein of MW 6700 (6.7K) (61 amino acids). To address whether this protein is expressed, we prepared an antiserum against a synthetic peptide corresponding to residues 47-61 in the 6.7K protein. This antiserum immunoprecipitated two series of protein bands, a 7K-8K doublet and a 15K-16K doublet or triplet, as observed by electrophoresis on 10-18% gradient SDS-polyacrylamide gels. These bands were not obtained from cells infected with mutants that lack the 6.7K gene. Most, if not all, of the 7K-8K and 15K-16K bands were detected by immunoblot, indicating that they are modified versions of the 6.7K protein. Only an 8K band was observed after cell-free translation of hybridization-purified mRNA, suggesting that this may be the primary translation product. As judged by DNA sequence, the 6.7K protein has a hydrophobic domain of at least 22 residues (residues 16-37), suggesting that 6.7K may be a membrane protein. Consistent with this, the 7K-8K and 15K-16K bands were observed in the crude membrane but not the cytosol or nuclear fractions of biochemically fractionated cells. The 6.7K protein was underproduced by mutants which underproduce E3 mRNAs a and c, indicating that 6.7K is translated from these mRNAs. Since the E3-gp 19K protein is also translated from mRNAs a and c, these mRNAs are bicistronic. The 6.7K protein is well-conserved in Ad5 (Ad2 and Ad5 are group C adenoviruses), and appears to be marginally conserved in Ad3 (group B).

The E3-10.4K protein of adenovirus is an integral membrane protein that is partially cleaved between Ala22 and Ala23 and has a Ccyt orientation

The Ad2 E3-10.4K protein is required together with the E3-14.5K protein to down-regulate the epidermal growth factor receptor in adenovirus-infected cells. Both proteins are also required to prevent tumor necrosis factor cytolysis under certain conditions. 10.4K is a 91 amino acid membrane-associated protein that migrates as two bands, upper and lower, on SDS-PAGE. We show here that the upper band is the primary translation product which initiates at AUG2173 in the E3 transcription unit of Ad2. The upper band is processed slowly (greater than 4 hr to complete) into the lower band by proteolytic cleavage between residues Ala22 and Ala23 by a microsome-associated protease. The upper and lower bands become equal in abundance, after which they are very stable. The N-terminus of the in vivo-derived upper band is not blocked to sequencing and it retains its initiating Met. 10.4K has a hydrophobic domain (H1) near its N-terminus that is probably a signal sequence for membrane insertion; cleavage of this signal is atypical because it was not cotranslational in vivo and it was not complete. 10.4K has a second hydrophobic domain (H2) located within residues 35-60. H2 appears to be a transmembrane (stop transfer) domain because both the upper and the lower 10.4K bands remained associated with membranes after extraction at pH 11.5, because both bands were extracted into the detergent phase with Triton X-114, and because both bands were only partially reduced in size when 10.4K-containing microsomes were digested with proteinase K. These proteinase K-digested bands were immunoprecipitated with an antipeptide antiserum against residues 19-34 but not with an antiserum against residues 68-80 or 77-91, indicating that both 10.4K bands are orientated in the membrane with the C-terminus in the cytoplasm. We conclude that the lower band of 10.4K is a type I bitopic membrane protein and suggest that the upper band is a polytopic membrane protein with both the H1 and the H2 hydrophobic domains spanning the membrane.

The adenovirus E3-14.5K protein which is required for prevention of TNF cytolysis and for down-regulation of the EGF receptor contains phosphoserine

The E3-14.5K and E3-10.4K proteins form a complex and function to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. Both 14.5K and 10.4K are cytoplasmic membrane proteins with a Ccyt orientation in the membrane. We show here that 14.5K is phosphorylated on serine residues in cells infected by adenoviruses that synthesize both 14.5K and 10.4K. 14.5K is phosphorylated on both serine and threonine in cells infected by a mutant that does not synthesize 10.4K; thus, the presence or absence of 10.4K affects the phosphorylation of 14.5K. Phosphotyrosine was not detected. 14.5K is also phosphorylated when translated in vitro in a rabbit reticulocyte extract. Both in vivo and in vitro, at least one of the phosphorylation sites is near the C-terminus, in the cytoplasmic domain of 14.5K. This C-terminal region of 14.5K is the most conserved among Ad5, Ad2, Ad3, and Ad7, and it is essential for 14.5K to prevent tumor necrosis factor cytolysis.

A real-time PCR method to rapidly titer adenovirus stocks.

A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the length of time required to perform the assay (2-4 wk). A major drawback of particle titration methods is the lack of consistent correlation between the resultant titer and the infectious titer. To overcome these problems, a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay was developed that detects encapsidated full-length genomes. Importantly, there is a linear correlation between the titer determined by the realtime PCR assay and the infectious titer determined by a plaque assay. This chapter provides step-by-step guidance for preparing viral DNA, conducting the real-time PCR assay, and using the resultant data to calculate a viral titer.