The coxsackievirus and adenovirus receptor acts as a tumour suppressor in malignant glioma cells.

The coxsackievirus and adenovirus receptor (CAR) is a membrane glycoprotein with a cytoplasmic domain, a transmembrane domain and an extracellular region consisting of two immunoglobulin-like domains, an amino-terminal immunoglobulin variable (IgV)-related domain (D1), which is distal to the cell surface, and a proximal IgC2 domain (D2). The coxsackievirus and adenovirus receptor has been shown to exhibit tumour suppression activity in human bladder and prostate cancer cells. In the current paper, we demonstrate that CAR is a tumour suppressor in glioma cells and that the extracellular D2 domain is not required for this inhibitory effect. This finding provides a biological basis for the observation that expression of CAR is downregulated in malignant glioma cells. This suggests that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realise the full potential of adenoviral vectors for cancer gene therapy.

The therapeutic efficacy of adenoviral vectors for cancer gene therapy is limited by a low level of primary adenovirus receptors on tumour cells.

Replication-defective adenoviral vectors are currently being employed as gene delivery vehicles for cancer gene therapy. To address the hypothesis that the therapeutic efficacy of adenoviral vectors is restricted by their inability to infect tumour cells expressing low levels of the primary cellular receptor for adenoviruses, the coxsackievirus and adenovirus receptor (CAR), we have employed a pair of ovarian cancer cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the direct evaluation of the relationship between the efficacy of an adenoviral vector and the primary receptor levels of the host cancer cell, without the confounding influence of other variable cellular factors. We demonstrate that a deficiency of the primary cellular receptor on the tumour cells restricts the efficacy of adenoviral vectors in two distinct cancer gene therapy approaches, TP53 gene replacement therapy and herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy. Moreover, we show that a deficiency of the primary receptor on the tumour cells limits the efficiency of adenovirus-mediated gene transfer in vivo. Since a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect adenoviruses to achieve CAR-independent infection will be necessary to realize the full potential of adenoviral vectors in the clinical setting.

Efficient oncolysis by a replicating adenovirus (ad) in vivo is critically dependent on tumor expression of primary ad receptors.

Replicating adenoviruses (Ads) are designed to replicate in and destroy cancer cells, generating viral progeny that spread within the tumor. To address the importance of the primary cellular receptor for Ads, the coxsackievirus and Ad receptor (CAR), in permitting intratumoral spread of a replicating Ad, we have used a pair of tumor cell lines differing only in the expression of a primary receptor for Ad5. This novel system thus allowed the first direct evaluation of the relationship between the efficacy of a replicating Ad and the primary receptor levels of the host cell without the confounding influence of other variable cellular factors. We demonstrate that the absence of the primary cellular receptor on the tumor cells restricts the oncolytic potency of a replicating Ad both in vitro and in vivo. Based on these findings, it is apparent that the potential therapeutic advantages afforded by viral replication would be negated by poor intratumoral spread of the viral progeny due to the failure to infect neighboring tumor cells. Because a number of studies have reported that primary cancer cells express only low levels of CAR, our results suggest that strategies to redirect Ads to achieve CAR-independent infection will be necessary to realize the full potential of replicating Ads in the clinical setting.

A Sindbis virus mRNA polynucleotide vector achieves prolonged and high level heterologous gene expression in vivo.

The direct intramuscular delivery of naked plasmid DNA has been demonstrated to allow expression of encoded heterologous genes in the target myocytes. The method has been employed to elicit immunization based upon delivery of antigen encoding plasmid DNA. For application in the context of achieving anti-tumor immunization against antigenic transforming oncoproteins, delivery of plasmid DNAs encoding these molecules would create significant potential safety hazards. As an alternative to DNA polynucleotide vectors, we explored the utility of mRNA vehicles for inducing foreign gene expression in muscle cells in vivo. Synthetic reporter-gene encoding mRNA transcripts were derived for this analysis. The Sindbis virus vector was also used to derive luciferase mRNA transcripts which possessed self-replication capacity. In these studies, it could be shown that the replicative vector was capable of directing significantly elevated levels of reporter gene expression in myocytes compared to a non-replicative mRNA species. In addition, the replicative species was capable of achieving significantly prolonged levels of in vivo gene expression compared to non-replicative mRNA. Both of these characteristics will make replicative mRNA vectors of utility for polynucleotide-based immunization protocols.

A carcinoembryonic antigen polynucleotide vaccine for human clinical use.

We have constructed a plasmid DNA encoding the full-length complementary DNA for human carcinoembryonic antigen (CEA) under transcriptional regulatory control of the cytomegalovirus early promoter/enhancer (pCEA) and demonstrated that this plasmid can function as a polynucleotide vaccine to elicit a CEA-specific immune response. This immune response protects against tumor challenge with syngeneic CEA-transduced colon carcinoma cells in mice. In the present work, the pCEA construct and purification method were modified to eliminate nonessential viral sequences, the ampicillin selectable marker, mutagens, and endotoxin to produce a reagent suitable for human clinical trials. The human use plasmid (pGT37) directs CEA expression at levels comparable with the original pCEA plasmid and can be propagated to yield large quantities of plasmid DNA based on kanamycin selection. A simple extraction technique greatly reduces contamination by endotoxin. Six weekly intramuscular injections of pGT37 elicited CEA-specific lymphoblastic transformation and antibody response in five of five mice and fully protected 10 of 10 mice against tumor challenge with syngeneic CEA-expressing colon cancer cells 42 days from the first plasmid injection. Thus, pGT37 encoding a tumor-associated antigen (CEA) has been shown to elicit cellular and humoral immune responses and mediate antitumor effects in vivo. This plasmid is suitable for human use and can be easily propagated in the laboratory.

A carcinoembryonic antigen polynucleotide vaccine has in vivo antitumor activity.

We have constructed a plasmid DNA encoding the full-length cDNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer and demonstrated that this plasmid can function as a polynucleotide vaccine. The immune response elicited by the CEA polynucleotide vaccine is dose and schedule dependent. There appears to be a threshold dose of 50 micrograms capable of inducing CEA-specific lymphoblastic transformation, lymphokine release, and antibody response. Doses of 10 micrograms were significantly less effective. When 50-micrograms doses are employed, thrice weekly or weekly vaccination schedules more reliably elicit CEA-specific immune responses by day 43 than does an every-3-weeks schedule. Furthermore the CEA polynucleotide vaccine can immunoprotect against challenge with syngeneic CEA-transduced colon carcinoma cells as early as 3 weeks after the first vaccination. Studies are ongoing to demonstrate the ability of CEA polynucleotide vaccination to treat pre-existing syngeneic mouse colon and breast carcinomas expressing human CEA.

Immune response to a carcinoembryonic antigen polynucleotide vaccine.

We have constructed a DNA plasmid encoding the full length complementary DNA for human carcinoembryonic antigen (CEA) driven by the cytomegalovirus early promoter/enhancer (plasmid DNA encoding human CEA) and demonstrated that this plasmid can function as a polynucleotide vaccine. This polynucleotide vaccine induced humoral and/or cellular immune responses specific for human CEA in all 5 immunized mice. Lymphoblastic transformation data with the use of enriched T-cell populations detected the presence of CEA-specific memory T-cells in 3 of 5 mice. Lymphocytes from 2 of 5 mice had interleukin 2/interleukin 4 release in response to CEA. CEA specificity was confirmed by the absence of reactivity to a control antigen and lack of CEA reactivity among mice vaccinated with a control plasmid encoding chloramphenicol acetyltransferase. Four of 5 mice vaccinated with plasmid DNA encoding human CEA demonstrated anti-CEA antibody responses. This immune response compared favorably with a positive control group of mice immunized with vaccinia-CEA by a dose and schedule previously shown to induce immunoprotection and therapy against a human CEA expressing syngeneic murine colon carcinoma model. Studies are ongoing to establish the construct, dose, and schedule to elicit optimal CEA-specific immune response as well as immunoprotection and therapy against human CEA expressing syngeneic murine adenocarcinoma models.

An animal model to predict the immunogenicity of murine V regions in humans.

Clinical trials with genetically engineered chimeric mouse/human monoclonal antibodies have demonstrated that mouse variable regions differ dramatically in their degree of immunogenicity. These observations led us to search for an animal model that could predict mouse variable region (V region) immunogenicity prior to human clinical trials. We selected monoclonal antibodies 17-1A and B72.3 for study because human trials have demonstrated the very low immunogenicity of the mouse 17-1A V region and the high immunogenicity of the mouse B72.3 V region. Random-bred New Zealand white rabbits were injected intravenously with mouse 17-1A, B72.3, or both using a dose and schedule comparable to human trials. After initial injection only two of ten rabbits developed an antibody response to mouse 17-1A, while all five animals receiving a second injection developed antibody that was entirely mouse constant region-specific. On the other hand, nine of ten rabbits demonstrated an antibody response to initial infusion of mouse B72.3 that was greater than 90% specific for the complementarity-determining region components of the V regions. Competitive inhibition with isolated heavy and light chains demonstrated specificity for heavy or light chains (approximately 60%) or a requirement for both (40%). Thus, as in humans, the V region of 17-1A elicited little or no immune response in rabbits, while the V region of B72.3 was highly immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)