Intratumoral administration of an adenovirus expressing a kinase dead form of ErbB-2 inhibits tumor growth.

ErbB-2 is amplified or overexpressed in a number of different cancers including breast, ovarian, lung, prostate and stomach. This overexpression leads to enhanced receptor dimer formation and stabilization allowing the receptor to remain in an active state. The clinical consequences of ErbB-2 overexpression include increased tumor aggressiveness, poor prognosis, decreased patient survival and resistance to chemotherapy. As a result, a variety of different strategies are being examined to inhibit its function or expression. In this study, we explored the efficacy of a type 5 recombinant adenovirus encoding a kinase dead form of ErbB-2, AderbB-2 Delta tk, as a potential therapeutic agent for breast cancer using a murine breast model expressing constitutively active ErbB-2. Co-expression in tumor cells of the kinase dead form of ErbB-2 inhibits receptor activity and induces the death of cells expressing constitutively active ErbB-2. In addition, AderbB-2 Delta tk exhibits antitumor activity in both immune-competent and immune-deficient animals with increased antitumor activity in the immune-competent animals. The results suggest both immune and non-immune mechanisms contribute to the antitumor efficacy of this vector.

Combined CXC chemokine and interleukin-12 gene transfer enhances antitumor immunity.

It has been shown that intratumor administration of an adenovirus vector expressing IL-12 produces a potent T cell-mediated response that leads to significant tumor regression in a murine breast cancer model. IP-10 and MIG are CXC chemokines that recruit mononuclear cells in vivo. In addition to their chemotactic roles, IP-10 and MIG inhibit angiogenesis. We tested whether the addition of IP-10 or MIG may both enhance the antitumor immune response of IL-12 through T cell recruitment and inhibit tumor growth through angiostasis. Adenovirus vectors expressing IP-10 or MIG and/or IL-12 were administered intratumorally in a murine model of mammary adenocarcinoma and fibrosarcoma. Administration of IP-10 or MIG in combination with IL-12 resulted in considerable tumor regression and increased survival time of tumor-bearing animals as compared with IP-10, MIG, IL-12 alone or control-treated animals, with the IP-10 IL-12 combination being most effective. These results suggest augmenting the antitumor immune response and inhibiting tumor angiogenesis with adenoviral vectors expressing IP-10 in combination with IL-12 is a novel way to enhance tumor regression.

Induction of ErbB-2/neu-specific protective and therapeutic antitumor immunity using genetically modified dendritic cells: enhanced efficacy by cotransduction of gene encoding IL-12.

Overexpression of ErbB-2/neu occurs in 20-30% of patients with breast cancer and indicates a poor prognosis. The presence of a detectable immune response to ErbB-2/neu in some patients suggests that this oncogene may be a useful target for vaccine therapy. We evaluated whether genetic immunization using dendritic cells (DC) transduced ex vivo with an adenovirus expressing the ErbB-2/neu gene (AdNeuTK) could induce protective and therapeutic immunity against a breast tumor cell line overexpressing ErbB-2/neu. Subcutaneous (s.c.) immunization with the DC vaccine elicited protective immunity in an average of 60% of animals. CTL analysis demonstrated specific cytotoxic activity against breast tumor cells, as well as syngeneic fibroblasts transduced with AdNeuTK. In vivo depletion studies demonstrated both CD4+ and CD8+ T cells were required. In a therapeutic setting, immunization with the DC vaccines could cure mice with pre-established tumors and efficacy was further enhanced by cotransducing DCs with a vector expressing murine IL-12 (AdmIL-12). These studies support DC vaccines as a therapeutic strategy for human breast cancer, while emphasizing the importance of optimizing an immune response by combining tumor antigen presentation with immunostimulatory cytokines.

Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila.

The Su(var)2-5 locus, an essential gene in Drosophila, encodes the heterochromatin-associated protein HP1. Here, we show that the Su(var)2-5 lethal period is late third instar. Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein. We demonstrate that heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in heterochromatin-mediated transcriptional regulation in these larvae. However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations. These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes.

Adenoviral vectors expressing lymphotactin and interleukin 2 or lymphotactin and interleukin 12 synergize to facilitate tumor regression in murine breast cancer models.

We have previously demonstrated that intratumoral injection with Ad vectors expressing IL-2 or IL-12 can induce regression in a murine breast cancer model. These IL-2- or IL-12-induced antitumor responses were mainly mediated by Ag-specific T cells. Lymphotactin is a novel lymphocyte chemokine that can cause local accumulation of CD4+, CD8+, and NK cells. We hypothesized that addition of lymphotactin may enhance the antitumor immune responses induced by locally produced IL-2 and IL-12 as we have previously shown. To this end we constructed two double-recombinant adenoviral vectors expressing lymphotactin along with either IL-2 (Ad5 Lym/IL-2) or IL-12 (Ad5 Lym/IL-12). Subcutaneous injection of polyoma middle T (PyMT) or Neu (8142) transgenically derived breast adenocarcinoma cells, in the hind flank of FVB/n mice, results in the formation of tumor nodules in 14-21 days. We show that these constructs elicit potent antitumor responses when administered intratumorally. The antitumor responses are long lasting as determined by rechallenge experiments and hence demonstrate a protective immunity. These observations indicate that by augmenting the antitumor response with adenoviral vectors expressing lymphotactin in combination with IL-2 or IL-12 is a novel way to enhance immunotherapeutic approaches.

Murine dendritic cells transduced with an adenoviral vector expressing a defined tumor antigen can overcome anti-adenovirus neutralizing immunity and induce effective tumor regression.

In this study transduced dendritic cells (DCs) were used to enhance immunogenecity of a specific tumor antigen. Using a polyoma middle T (PyMT) transgenic mammary carcinoma model we found that injections of DCs transduced with an adenoviral (Ad) vector expressing PyMT (DCAd-PymT) led to potent specific anti-tumor immunity. Efficacy was not affected by neutralizing Abs (high or low titers) and naive animals did not produce detectable anti-Ad Abs following two injections of transduced DCs. Repeated injections of transduced DCs significantly improved therapeutic efficacy in mice with established lung metastases. These data emphasize the ability of Ad-infected DCs to: i) minimize anti-Ad Ab production, ii) overcome pre-existing anti-Ad humoral immunity, and iii) improve vaccination efficacy when injected more than once.

Transient gene transfer of non-ELR chemokines to rodent lung induces mononuclear cell accumulation and activation.

The in vivo function of the CXC chemokines interferon-inducible protein-10 (IP-10) and monokine induced by gamma (MIG) was examined using replication-deficient adenoviral vectors expressing human IP-10 (AdIP-10) or murine MIG (AdMIG). Intratracheal and intranasal administration of AdIP-10 or AdMIG into rats and mice produced transient chemokine overexpression from the bronchial epithelium. IP-10 concentrations in the bronchoalveolar lavage fluid (BAL) of AdIP-10-treated animals showed peak expression (>2 ng/ml) 24-48 h after AdIP-10 administration. Dramatic transient increases in BAL cellularity (macrophages, monocytes, lymphocytes, and neutrophils) were observed in AdIP-10-treated and AdMIG-treated animals, and histologic examination of AdIP-10-treated lungs revealed transient infiltrations of mononuclear cells primarily localized around the bronchus and extending throughout the lung parenchyma. However, in immunocomprised SCID mice, only increases in natural killer cell populations were detected in BAL following AdIP-10 intranasal administration, indicating that monocyte/macrophage and neutrophil accumulation was likely the result of factors released from activated lymphocytes.

Enhanced immune response to the melanoma antigen gp100 using recombinant adenovirus-transduced dendritic cells.

Glycoprotein 100 (gp100) is one of a series of well-characterized human melanoma-associated antigens expressed by most melanoma cells. Immunization of C57BL/6 mice with an adenovirus (Ad) vector encoding human gp100 (Adhgp100) has been shown to induce limited protective immunity against challenge with murine melanoma B16 cells. In the current study we determined whether gp100-specific immunity can be enhanced using bone-marrow-derived dendritic cells (DCs) transduced with Adhgp100 ex vivo. Subcutaneous injection of Adhgp100-infected DCs resulted in potent T-cell-mediated protective immunity and a greater than 80% reduction of established tumors when administered to B16 tumor-bearing hosts. Compared to direct injection of Adhgp100 vector alone, immunization with Adhgp100-infected DCs induced markedly greater antitumor activity. In vitro CTL analysis demonstrated that DC-Adhgp100 immunization activated both CD4(+) and CD8(+) CTLs, while no lytic activity was generated by vaccination with Adhgp100 alone. In vivo depletion of CD4(+) T cells, but not CD8(+) T cells, completely abrogated CTL activity, suggesting that Adhgp100-transduced DCs result in activation of both CD4(+) and CD8(+) CTLs via a CD4(+)-dependent mechanism. We speculate that this improved efficacy of Adhgp100-transduced DCs compared to direct immunization with Adhgp100 may be the result of direct DC-mediated CD4(+) T cell activation. These results emphasize the importance of CD4(+) T cells in the development of therapeutic antigen-specific cancer vaccines.

Enhanced interleukin-2 gene transfer immunotherapy of breast cancer by coexpression of B7-1 and B7-2.

The capability of B7-1 to augment the antitumor activity of some cytokines has been shown primarily for such cytokines as interleukin-12 (IL-12), IL-7, and to a lesser extent IL-2. In this study, we investigate the ability of B7-1 and B7-2 to augment the antitumor activity of IL-2. Considering the affinity of both molecules for CD28 (T cell receptor for B7-1 and B7-2), we postulated that their potential to augment IL-2 antitumor activity would be similar. Two murine transgenic adenocarcinoma models were chosen to investigate the activity of adenoviral vectors constructed to express either B7-1 and IL-2 or B7-2 and IL-2. Before administering the vector intratumorally to tumor-bearing mice, we determined the expression of B7-1, B7-2, MHC I, and MHC II on these tumor cells and demonstrated positive expression of only MHC I. Intratumoral injection of the vector expressing B7-1 and IL-2 resulted in complete regression of all tumors treated. In contrast, the vector expressing B7-2 and IL-2 was significantly less effective at regressing PyMT tumors, whereas both double recombinant vectors demonstrated similar levels of complete regression in the Neu (NDL 8142) model. Regressed mice were all protected for rechallenge in both models and demonstrated antigen-specific cytotoxic T lymphocytes (CTL) in the PyMT model. These findings indicate that the combination of IL-2 with B7-1 or B7-2 significantly enhances the antitumor activity of IL-2.

A double recombinant adenovirus expressing the costimulatory molecule B7-1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model.

Tumors that express tumor-specific antigens can maintain growth in an immunocompetent organism. Current hypotheses tend toward T cell anergy as a key component for the inhibition of immunoreactivity against such tumors. Anergy is thought to occur from hyperactive stimulation of the TCR in the absence of costimulation (costimulation leads to proliferation via IL-2 production). Subcutaneous injection of transgenic polyoma middle T transformed breast adenocarcinoma tumor cells (PyMT) in the hind flank of FVB/n mice results in the formation of tumor nodules at this site. We determined the MHC class I and class II, B7-1, and B7-2 expression in the tumor cells by flow cytometry and showed positive staining for only MHC class I. We show that a single E1-deleted adenovirus constructed to express both the costimulatory molecule B7-1 (murine) and human IL-2 genes (Ad5E1 mB7-1/human IL-2) elicits a very potent antitumor response when administered intratumorally. Ad5E1 mB7-1/human IL-2 induced rapid and complete regression (100%) of all tumors compared with Ad5 E1 mB7-1 (38%), Ad CAIL-2 (42%), and Ad5E1 dl70-3 (control vector) (0%). All mice that exhibited complete tumor regression were fully protected in tumor cell challenge experiments. The systemic immunity generated by intratumoral administration of the Ad vectors was associated with a strong anti-PyMT CTL response. These observations indicate that augmenting the immunogenicity of the tumor with coincident expression of B7-1 in combination with IL-2 may prove beneficial in direct tumor immunotherapy.

Interleukin 12 and B7-1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression.

Stimulation of antitumor immune mechanisms is the primary goal of cancer immunotherapy, and accumulating evidence suggests that effective alteration of the host-tumor relationship involves immunomodulating cytokines and also the presence of costimulatory molecules. To examine the antitumor effect of direct in vivo gene transfer of murine interleukin 12 (IL-12) and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1, that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1 gene in E3 under control of the murine cytomegalovirus promoter. This vector expressed high levels of IL-12 and B7-1 in infected murine and human cell lines and in primary murine tumor cells. In mice bearing tumors derived from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete regression in 70% of treated animals. By contrast, administration of a similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted in only a delay in tumor growth. Interestingly, coinjection of two different viruses expressing either IL-12 or B7-1 induced complete tumor regression in only 30% of animals treated at this dose. Significantly, cured animals remained tumor free after rechallenge with fresh tumor cells, suggesting that protective immunity had been induced by treatment with AdIL12-B7-1. These results support the use of Ad vectors as a highly efficient delivery system for synergistically acting molecules and show that the combination of IL-12 and B7-1 within a single Ad vector might be a promising approach for in vivo cancer therapy.

The manganese superoxide dismutase gene of Drosophila: structure, expression, and evidence for regulation by MAP kinase.

The gene encoding manganese superoxide dismutase (MnSOD) from Drosophila melanogaster has been isolated and its expression has been studied. In contrast to several mammalian MnSOD genes, the Drosophila gene contains a single intron and is transcribed into a single 0.8-kb transcript. Whole-mount in situ hybridization reveals extensive transcript accumulation in ovarian nurse cells and a heavy maternal contribution to the early embryo. Larval imaginal discs are enriched with MnSOD transcripts relative to other larval tissues, further suggesting a possible relationship between high MnSOD expression and mitotic activity. The 5'-upstream region contains several well-known regulatory elements including metal response, antioxidant response, and xenobiotic response elements (MRE, ARE, and XRE, respectively), sites for activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), adenosine 3',5'-cyclic monophosphate regulator binding element factor (CREB), as well as classic TATA and CAAT boxes. That MnSOD expression in Drosophila is regulated in part by the transcription factor AP-1 via the MAP kinase signal transduction pathway is suggested by experiments which show that a hypomorphic mutation of the MAP kinase-encoding rolled gene substantially reduces levels of MnSOD transcripts and correlates with reduced resistance to oxidative stress in rolled mutants.