A three-dimensional assay for measurement of viral-induced oncolysis.

Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Delta24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Delta24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells.

A promising approach to immunotherapy involves the loading of dendritic cells (DCs) with genetic material to facilitate sustained expression of a relevant antigen in this population of potent antigen presenting cells (APC). Viral vectors such as adenovirus (Ad) have been used for this purpose. Existing methods for DC infection are limited by lack of specificity and a requirement for DC exposure to high viral doses. Targeting of Ad to DCs with bispecific antibodies has significantly augmented levels of transgene expression. Genetic fusion of the extracellular portion of coxsackievirus-adenovirus receptor (CAR) to cell-specific ligands has also proved successful in targeting Ad to cells of interest. We report here the production and primary characterization of a new fusion protein comprising the ecto-domain of CAR connected to a single chain antibody (scFv) G28-5 against human CD40 present on the surface of DCs. We demonstrate that the fusion protein (CAR/G28) specifically interacts with both recombinant Ad fiber knob and the ecto-domain of human CD40 in a binding assay (ELISA). Finally, we show that the CAR/G28 fusion protein promotes highly efficient transduction of DCs of both rhesus monkey and human origin.

Heat shock and heat shock protein 70i enhance the oncolytic effect of replicative adenovirus.

Replication-competent viruses are currently being evaluated for their cancer cell-killing properties. These vectors are designed to induce tumor regression after selective viral propagation within the tumor. However, replication-competent viruses have not resulted heretofore in complete tumor eradication in the clinical setting. Recently, heat shock has been reported to partially alleviate replication restriction on an avian adenovirus (Ad) in a human lung cancer cell line. Therefore, we hypothesized that heat shock and overexpression of heat shock protein (hsp) would support the oncolytic effect of a replication-competent human Ad. To this end, we tested the oncolytic and burst kinetics of a replication-competent Ad after exposure to heat shock or to inducible hsp 70 overexpression by a replication-deficient Ad (Adhsp 70i). Heat-shock resulted in augmentation of Ad burst and oncolysis while decreasing total intracellular Ad DNA. Overexpression of hsp 70i also enhanced Ad-mediated oncolysis but did not decrease intracellular Ad DNA levels. We conclude that heat shock and Adhsp 70i enhance the Ad cell-killing potential via distinct mechanisms. A potential therapeutic implication would be the use of local hyperthermia to augment oncolysis by increasing the burst of replication-competent Ad. The role of hsp in Ad-mediated oncolysis should be additionally explored.

Improved gene transfer efficiency to primary and established human pancreatic carcinoma target cells via epidermal growth factor receptor and integrin-targeted adenoviral vectors.

In this study we analyzed two ways of retargeting of Ad-vectors to human pancreatic carcinoma with the aim of enhancing the gene transfer efficiency. First, we analyzed the expression of the epidermal growth factor receptor (EGFR) on primary, as well as established pancreatic carcinoma cells by flow cytometry which revealed high expression levels of EGFR on the surface of these cells. We showed that EGFR-retargeted entry pathway using a bispecific fusion protein formed by a recombinant soluble form of truncated Coxsackie and Adenovirus Receptor (sCAR) genetically fused with human EGF (sCAR-EGF) redirects them to the EGFR leading to an enhanced gene transfer efficiency to pancreatic carcinoma cells. Since flow cytometry revealed absence of CAR expression, but the presence of at least one of both alphav integrins on the pancreatic carcinoma cells, a second way of targeting was investigated using a genetically modified Ad vector which has an RGD (Arg-Gly-Asp)-containing peptide inserted into the HI-loop of the fiber knob. This RGD targeted Ad (AdlucRGD) revealed efficient CAR-independent infection by allowing binding to cellular integrins resulting in a dramatic enhancement of gene transfer. These findings have direct relevance for Ad-vector based gene therapy strategies for pancreatic carcinoma.

Human immunodeficiency virus type 1-mediated syncytium formation is compatible with adenovirus replication and facilitates efficient dispersion of viral gene products and de novo-synthesized virus particles.

Conditionally replicative adenovirus (CRAd) vectors are designed for specific oncolytic replication in tumor tissues with concomitant sparing of normal cells. As such, CRAds offer an unprecedented level of anticancer potential for malignancies that have been refractory to previous cancer gene therapy interventions. CRAd efficacy may, however, be compromised by inefficient dispersion of the replicating vector within the tumor tissue. To address this issue, we evaluated the utility of a fusogenic membrane glycoprotein (FMG), which induces the fusion of neighboring cellular membranes to form multinucleated syncytia. We hypothesized that the FMG-mediated syncytia would facilitate dispersion of the adenovirus (Ad) gene products and viral progeny. To test this, human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins, which induce syncytia in the presence of CD4+ target cells, were expressed by an Ad (Ad5HIVenv) in permissive (CD4-positive) and nonpermissive (CD4-negative) cell lines. After validating this Ad-FMG model, the efficiency of Ad replication in the presence or absence of syncytia was evaluated. The results demonstrated that syncytium formation was compatible with Ad replication and dramatically increased the dispersion of virus gene products within the cytoplasm of the syncytia as well as viral particles in the nuclei of the syncytial mass. Moreover, progeny virions were released more efficiently from syncytia compared with nonsyncytial cells. These data demonstrate the utility of FMGs as a dispersion agent and suggest that FMGs can improve the efficacy of CRAd gene therapy.

Midkine and cyclooxygenase-2 promoters are promising for adenoviral vector gene delivery of pancreatic carcinoma.

Midkine (MK), a heparin binding growth factor, and cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandin, are both up-regulated at the mRNA or protein level in many human malignant tumors. Here, we investigated the tumor specificity of both MK and COX-2 promoters in human pancreatic cancer, with the aim to improve the selectivity of therapeutic gene expression. We constructed recombinant adenoviral (Ad) vectors containing either the luciferase (Luc) reporter gene under the control of the COX-2 or MK promoter or the herpes simplex virus thymidine kinase (HSV Tk) gene under the control of the COX-2 promoter and compared the expression with the cytomegalovirus (CMV) promoter. AdMKLuc achieved moderate to relatively high activity upon infection to both primary and established pancreatic carcinoma cells. Of the two COX-2 promoter regions (COX-2M and COX-2L), both revealed a high activity in primary pancreatic carcinoma cells, whereas in the established pancreatic carcinoma cell lines, COX-2L has an approximately equal high activity compared to CMV. In addition, both AdCOX-2M Tk and AdCOX-2L Tk induced marked cell death in response to ganciclovir (GCV) in three of four established pancreatic carcinoma cell lines. From these results, and because it has been reported that AdMKTk and AdCOX-2L Tk in combination with GCV did not reveal significant liver toxicity, we conclude that the MK as well as the COX-2 promoters are promising tumor-specific promoters for Ad vector-based gene therapy of pancreatic cancer.

Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid.

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.

Selective gene delivery to head and neck cancer cells via an integrin targeted adenoviral vector.

In vivo cancer gene therapy approaches for squamous cell carcinoma of the head and neck (SCCHN) based on adenoviral vector-mediated gene delivery have been limited by the suboptimal efficacy of gene transfer to tumor cells. We hypothesized that this issue was due to deficiency of the primary adenoviral receptor, the coxsackie-adenovirus receptor (CAR), on the tumor targets. Studies of CAR levels on SCCHN cell lines confirmed that their relative refractoriness to the adenoviral vector was based on this deficiency. To circumvent this deficiency, we applied an adenoviral vector targeted to a tumor cell marker characteristic of SCCHN. In this regard, integrins of the alpha2beta1 and alpha3beta1 class are frequently overexpressed in SCCHN. Furthermore, these integrins recognize the RGD peptide motif. On this basis, we applied an adenoviral vector genetically modified to contain such a peptide within the HI loop of the fiber protein as a means to alter viral tropism. Studies confirmed that the CAR-independent gene delivery achieved via this strategy allowed enhanced gene transfer efficiencies to SCCHN tumor cells. Importantly, this strategy could achieve preferential augmentation of gene transfer in tumor cells compared with normal cells. The ability to achieve enhanced and specific gene transfer to tumor cells via adenoviral vectors has important implications for gene therapy strategies for SCCHN and for other neoplasms in general.

Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma.

BACKGROUND: Adenovirus-mediated gene therapy has been used for squamous cell carcinoma of the head and neck (SCCHN), but the in vivo efficacy has been limited by a lack of tissue specificity and low infection efficiency. We are interested in improving cancer gene therapy strategies using targeted adenovirus vectors. OBJECTIVE: To determine if the infection efficiency of adenovirus-mediated gene transfer to SCCHN cells could be enhanced by retargeting to the epidermal growth factor receptor (EGFR), which is known to be overexpressed in these tumors. DESIGN: Epidermal growth factor receptor retargeting in SCCHN cells was accomplished with a bispecific antibody that recognized the knob domain of adenovirus as well as EGFR. Using this retargeting schema, we compared the infection efficiency and specificity of unmodified and EGFR-retargeted adenovirus. RESULTS: Squamous cell carcinoma of the head and neck cell lines were shown to be infected by adenovirus with low efficiency, which is likely because of the low level of adenovirus receptor expressed in the SCCHN cells. Epidermal growth factor receptor retargeting markedly enhanced transduction in both SCCHN cell lines and primary tumor tissue, as indicated by the elevated levels of reporter gene expression. Furthermore, retargeting enhanced infection of tumor tissue compared with normal tissue from the same patient. CONCLUSIONS: Epidermal growth factor receptor retargeting enhanced adenovirus infection of SCCHN cells and, in doing so, augments the potency of the vector. This modification makes the vector potentially more valuable in the clinical setting.

Translation elongation factor-1 alpha interacts with the 3' stem-loop region of West Nile virus genomic RNA.

The conserved 3'-terminal stem-loop (3' SL) of the West Nile virus (WNV) genomic RNA was previously used to probe for cellular proteins that may be involved in flavivirus replication and three cellular proteins were detected that specifically interact with the WNV 3' SL RNA (J. L. Blackwell and M. A. Brinton, J. Virol. 69:5650-5658, 1995). In this study, one of these cellular proteins was purified to apparent homogeneity by ammonium sulfate precipitation and liquid chromatography. Amino acid sequence Western blotting, and supershift analyses identified the cellular protein as translation elongation factor-1 alpha (EF-1 alpha). Competition gel mobility shift assays demonstrated that the interaction between EF-1 alpha and WNV 3' SL RNA was specific. Dephosphorylation of EF-1 alpha by calf intestinal alkaline phosphatase inhibited its binding to WNV 3' SL RNA. The apparent equilibrium dissociation constant for the interaction between EF-1 alpha and WNV 3' SL RNA was calculated to be 1.1 x 10(-9) M. Calculation of the stoichiometry of the interaction indicated that one molecule of EF-1 alpha binds to each molecule of WNV 3' SL RNA. Using RNase footprinting and nitrocellulose filter binding assays, we detected a high-activity binding site on the main stem of the WNV 3' SL RNA. Interaction with EF-1 alpha at the high-activity binding site was sequence specific, since nucleotide substitution in this region reduced the binding activity of the WNV 3' SL RNA for EF-1 alpha by approximately 60%. Two low-activity binding sites were also detected, and each accounted for approximately 15 to 20% of the binding activity. Intracellular association between the host protein and the viral RNA was suggested by coimmunoprecipitation of WNV genomic RNA and EF-1 alpha, using an anti-EF-1 alpha antibody.

BHK cell proteins that bind to the 3' stem-loop structure of the West Nile virus genome RNA.

The first 83 3' nucleotides of the genome RNA of the flavivirus West Nile encephalitis virus (WNV) form a stable stem-loop (SL) structure which is followed in the genome by a smaller SL. These 3' structures are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and as such might specifically bind to cellular or viral proteins. Cellular proteins from uninfected and WNV-infected BHK-21 S100 cytoplasmic extracts formed three distinct complexes with the WNV plus-strand 3' SL [(+)3'SL] RNA in a gel mobility shift assay. Subsequent competitor gel shift analyses showed that two of these RNA-protein complexes, complexes 1 and 2, contained cell proteins that specifically bound to the WNV (+)3'SL RNA. UV-induced cross-linking and Northwestern blotting analyses detected WNV (+)3'SL RNA-binding proteins of 56, 84, and 105 kDa. When the S100 cytoplasmic extracts were partially purified by ion-exchange chromatography, a complex that comigrated with complex 1 was detected in fraction 19, while a complex that comigrated with complex 2 was detected in fraction 17. UV-induced cross-linking experiments indicated that an 84-kDa cell protein in fraction 17 and a 105-kDa protein in fraction 19 bound specifically to the WNV (+)3'SL RNA. In addition to binding to the (+)3'SL RNA, the 105-kDa protein bound to the SL structure located at the 3' end of the WNV minus-strand RNA. Initial mapping studies indicated that the 84- and 105-kDa proteins bind to different regions of the (+)3'SL RNA. The 3'-terminal SL RNA of another flavivirus, dengue virus type 3, specifically competed with the WNV (+)3'SL RNA in gel shift assays, suggesting that the host proteins identified in this study are flavivirus specific.

Outcome of treated pregnancies in women with antiphospholipid syndrome: an update of the Utah experience.

OBJECTIVE: To determine the outcome of treated pregnancies in women with well-characterized antiphospholipid syndrome. METHODS: We reviewed 82 consecutive pregnancies in 54 women with antiphospholipid syndrome who were treated during pregnancy with the following: 1) prednisone and low-dose aspirin; 2) heparin and low-dose aspirin; 3) prednisone, heparin, and low-dose aspirin; or 4) other combinations of these medications or immunoglobulin. RESULTS: The overall neonatal survival rate was 73%, excluding spontaneous abortions, but treatment failures (fetal and neonatal deaths) occurred in all treatment groups. Patients with successful treated pregnancies had fewer previous fetal deaths than those with unsuccessful treated pregnancies. There were no significant differences in outcome among the four treatment groups. Preeclampsia and fetal distress occurred in half of all pregnancies, and fetal growth impairment occurred in nearly one-third. Preterm delivery due to maternal or fetal indications was required in 37% of the pregnancies. Four pregnancies were also complicated by postpartum thrombosis during treatment. CONCLUSIONS: Pregnancy in women with antiphospholipid syndrome appears to be improved by treatment, but fetal loss may occur despite treatment. Preeclampsia, fetal distress, fetal growth impairment, and premature delivery are common. Because of the clinically significant risk of thrombotic episodes, thrombosis prophylaxis should be considered in these patients.