Inflammation, apoptosis, and necrosis induced by neoadjuvant fas ligand gene therapy improves survival of dogs with spontaneous bone cancer.

Fas ligand (FasL) gene therapy for cancer has shown promise in rodents; however, its efficacy in higher mammals remains unknown. Here, we used intratumoral FasL gene therapy delivered in an adenovirus vector (Ad-FasL) as neoadjuvant to standard of care in 56 dogs with osteosarcoma. Tumors from treated dogs had greater inflammation, necrosis, apoptosis, and fibrosis at day 10 (amputation) compared to pretreatment biopsies or to tumors from dogs that did not receive Ad-FasL. Survival improvement was apparent in dogs with inflammation or lymphocyte-infiltration scores >1 (in a 3-point scale), as well as in dogs that had apoptosis scores in the top 50th percentile (determined by cleaved caspase-3). Survival was no different than that expected from standard of care alone in dogs with inflammation scores ≤1 or apoptosis scores in the bottom 50th percentile. Reduced Fas expression by tumor cells was associated with prognostically advantageous inflammation, and this was seen only in dogs that received Ad-FasL. Together, the data suggest that Ad-FasL gene therapy improves survival in a subset of large animals with naturally occurring tumors, and that at least in some tumor types like osteosarcoma, it is most effective when tumor cells fail to express Fas.

Mutation-selective tumor remission with Ras-targeted, whole yeast-based immunotherapy.

Activating mutations in Ras oncoproteins represent attractive targets for cancer immunotherapy, but few vectors capable of generating immune responses required for tumor killing without vector neutralization have been described. Whole recombinant yeast heterologously expressing mammalian mutant Ras proteins were used to immunize mice in a carcinogen-induced lung tumor model. Therapeutic immunization with the whole recombinant yeast caused complete regression of established Ras mutation-bearing lung tumors in a dose-dependent, antigen-specific manner. In combination with the genomic sequencing of tumors in patients, the yeast-based immunotherapeutic approach could be applied to treat Ras mutation-bearing human cancers.

Regulation of Fas-mediated apoptosis by N-ras in melanoma.

Oncogenic ras has been shown to downregulate Fas receptor expression and increase Fas ligand expression and thus contribute to resistance to Fas-mediated cell death in several cell types. The effects of ras on Fas-mediated apoptosis have not been studied in melanoma. We studied the effects of activated N-ras by measuring Fas, Fas ligand, and FLIP expression as well as susceptibility to Fas-ligand-induced cell death in transfectants of WM35, a radial growth phase human melanoma cell line. Based on quantitative polymerase chain reaction and fluorescence-activated cell sorter analysis, we found that the ras transfectants expressed less Fas mRNA and surface Fas receptor. Cr51 release cytotoxicity assays demonstrated less susceptibility to Fas-mediated apoptosis in ras transfectants, correlating with the Fas mRNA and protein expression results. Ras inhibition with the specific inhibitor FTI-277 showed that downregulation of Fas in the ras transfectants could be reversed. This correlates with cytotoxicity experiments showing that ras inhibition increases susceptibility to Fas-mediated apoptosis. The control transfectants expressed FLIP but ras did not affect FLIP expression. The control and ras transfectants did not express Fas ligand as demonstrated by reverse transcriptase polymerase chain reaction and fluorescence-activated cell sorter analysis. Cytotoxicity assays further confirmed that these melanoma ras transfectants do not express functional Fas ligand. These results suggest that ras contributes to tumor progression by decreasing susceptibility to Fas-mediated cell death at least in part through downregulation of Fas receptor at the transcriptional level.

Enhancing antimelanoma immune responses through apoptosis.

We examined the feasibility of using tumor apoptosis at accessible sites to enhance antimelanoma immune responses in a model of spontaneous canine melanoma. We show that priming peripheral blood mononuclear cells with apoptotic melanoma cells significantly enhanced autologous and allogeneic lymphokine-activated killing of tumor cells. Since various pathways required for intrinsic apoptosis are often inactivated in melanoma, we used Fas ligand (FasL) overexpression to promote extrinsic apoptosis. FasL induced apoptosis in five of six cell lines. Each of the susceptible lines, but not the resistant one, expressed Fas mRNA. In addition, direct intratumoral administration of FasL DNA to tumor-bearing dogs was safe, with no adverse events reported over 7 days of observation. A reduction of tumor burden was seen in three of five dogs treated. The reduction of tumor volume was correlated with Fas expression by the tumors, although one dog with a Fas-negative tumor survived for 82 weeks after treatment. Our data show that overexpression of FasL is suitable to promote apoptosis of Fas(+) melanomas, and support the notion that priming immune responder cells with apoptotic tumor cells may enhance antitumor responses. The results also suggest that intratumoral administration of FasL offers a safe route for therapeutic gene delivery.

Methods of analyzing chromatin changes accompanying apoptosis of target cells in killer cell assays.

Natural killer (NK) cells are a crucial component of the immune system. For example, the NK cell-mediated killing of tumor cells is a first line of defense against the development of cancer. Detection and quantification of apoptosis induced in target cells by NK cells is a useful tool for studies of the mechanisms of both immunological protection and pathogenesis. This chapter describes how to do this by using techniques with a broad application to both NK cells and cytotoxic T cells.

Whole recombinant yeast-based immunotherapy induces potent T cell responses targeting HCV NS3 and Core proteins.

Control of primary infection with hepatitis C virus (HCV) is associated with robust and broad T cell immunity. In contrast, chronic infection is characterized by weak T cell responses suggesting that an approach that boosts these responses could be a therapeutic advance. Saccharomyces cerevisiae is an effective inducer of innate and adaptive cellular immunity and we have generated recombinant yeast cells (GI-5005) that produce an HCV NS3-Core fusion protein. Pre-clinical studies in mice showed that GI-5005 induced potent antigen-specific proliferative and cytotoxic T cell responses that were associated with Th1-type cytokine secretion. In studies in which GI-5005 was administered up to 13 times, no detectable vector neutralization or induction of tolerance was observed. Prophylactic as well as therapeutic administration of GI-5005 in mice led to eradication of tumor cells expressing HCV NS3 protein. Immunotherapy with GI-5005 is being evaluated in chronic HCV infected individuals in a Phase 1 clinical trial.

Yeasts encoding tumour antigens in cancer immunotherapy.

Immunotherapy for cancer represents an attractive therapeutic target because of its specificity and lack of toxicity, but products investigated so far have been limited by neutralisation, complexity of manufacturing and requirement for patient-specific products. Recombinant yeast cells are capable of stimulating the immune system to produce highly specific and potent cellular responses against target protein antigens with little toxicity. Data from animal models suggest that Tarmogens (yeast-based immunotherapeutics) can elicit protective immunity against xenografted and chemically induced tumours. This concept is now being tested in a Phase I trial in patients with colorectal, pancreatic and non-small cell lung cancers.