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A generally applicable, easy-to-use method of focusing a patient's immune system to eradicate or prevent cancer has been elusive. We are attempting to develop a targeted virus to accomplish these aims. We previously created a recombinant replicating vesicular stomatitis virus (VSV) that preferentially infected Her2/neu expressing breast cancer cells and showed therapeutic efficacy in an implanted Balb/c mouse tumor model. The current work shows that this therapy generated therapeutic anti-tumor CD4 T cells against multiple tumor antigens. CD4 T cells transferred directly from cured donor mice could eradicate established tumors in host mice. T cells were transferred directly from donor mice and were not stimulated ex vivo. Both tumors that expressed Her2/neu and those that did not were cured by transferred T cells. Analysis of cytokines secreted by anti-tumor memory CD4 T cells displayed a multifunctional pattern with high levels of interferon-γ, interleukin (IL)-4 and IL-17. Anti-tumor memory CD4 T cells traveled to the mesenteric lymph nodes and were activated there. Treatment with targeted recombinant replicating VSV is a potent immune adjuvant that generates therapeutic, multifunctional anti-tumor memory CD4 T cells that recognize multiple tumor antigens. Immunity elicited by viral therapy is independent of host major histocompatibility complex or knowledge of tumor antigens. Virus-induced tumor immunity could have great benefit in the prevention and treatment of tumor metastases. [ABSTRACT FROM AUTHOR]

Interleukin-32 (IL-32) is a pro-inflammatory cytokine conditionally produced by T cells, natural killer (NK) cells, monocytes, epithelial cells and keratinocytes, which has an important role in host resistance against infectious disease. Interestingly, elevated levels of IL-32 transcripts in fine needle aspirates of tumor tissue have also been correlated with objective clinical responses in cancer patients receiving immunotherapy. To evaluate the antitumor impact of IL-32 gene therapy, we treated BALB/c mice bearing established subcutaneous CMS4 sarcomas with intratumoral (i.t.) injections of syngenic dendritic cells (DCs) engineered to express human IL-32β complementary DNA (that is, DC.IL32). Although ectopic expression of IL-32β by DC resulted in only modest phenotypic changes in these antigen-presenting cells, DC.IL32 produced higher levels of IL-12p70 than control DC. DC.IL32 were more potent activators of type-1 T-cell responses in vitro and in vivo, with i.t. administration of DC.IL32 leading to the CD8+ T-cell-dependent (but CD4+ T-cell- and NK cell-independent) suppression of tumor growth. Effective DC.IL32-based therapy promoted infiltration of tumors by type-1 (that is, CXCR3+VLA-4+GrB+) CD8+ T cells and CD11b+CD11c+ host myeloid DC, but led to reductions in the prevalence of CD11b+Gr1+ myeloid-derived suppressor cells and CD31+ blood vessels. [ABSTRACT FROM AUTHOR]

Targeted oncolytic poxviruses hold promise for the treatment of cancer. Arming these agents with immunostimulatory cytokines (for example, granulocyte-monocyte colony-stimulating factor; GM-CSF) can potentially increase their efficacy and/or alter their safety. However, due to species-specific differences in both human GM-CSF (hGM-CSF) activity and poxviruses immune avoidance proteins, the impact of hGM-CSF expression from an oncolytic poxvirus cannot be adequately assessed in murine or rat tumor models. We developed a rabbit tumor model to assess toxicology, pharmacodynamics, oncolytic efficacy and tumor-specific immunity of hGM-CSF expressed from a targeted oncolytic poxvirus JX-963. Recombinant purified hGM-CSF protein stimulated a leukocyte response in this model that paralleled effects of the protein in humans. JX-963 replication and targeting was highly tumor-selective after i.v. administration, and intratumoral replication led to recurrent, delayed systemic viremia. Likewise, hGM-CSF was expressed and released into the blood during JX-963 replication in tumors, but not in tumor-free animals. hGM-CSF expression from JX-963 was associated with significant increases in neutrophil, monocyte and basophil concentrations in the peripheral blood. Finally, tumor-specific cytotoxic T lymphocytes (CTL) were induced by the oncolytic poxvirus, and expression of hGM-CSF from the virus enhanced both tumor-specific CTL and antitumoral efficacy. JX-963 had significant efficacy against both the primary liver tumor as well as metastases; no significant organ toxicity was noted. This model holds promise for the evaluation of immunostimulatory transgene-armed oncolytic poxviruses, and potentially other viral species. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV), an avian paramyxovirus, has a potential oncolytic effect that may be of significance in the treatment of a variety of cancer diseases. An attenuated lentogenic isolate of NDV (HUJ) demonstrated a selective cytopathic effect upon a panel of human and mouse lung tumor cells, as compared to human nontumorigenic lung cells. The virus-selective oncolytic effect is apoptosis dependent, and related to higher levels of viral transcription, translation and progeny virus formation. Furthermore, NDV-HUJ oncolytic activity is directed in-cis and not through induction of cytokines, that may act in-trans on neighboring cells. Development of primary lung tumors and of the consequent metastasis in mice inoculated with mouse lung tumor cells 3LL-D122 was decreased following treatment with NDV-HUJ. The preferential killing of the tumor cells is not due to a deficiency in the interferon (IFN) system, as expression of the IFN-β gene, in the infected cells, is properly induced. Moreover, pretreatment with IFN effectively protected the tumor cells from the virus oncolytic effect. We conclude therefore, that NDV-HUJ should have a significant benefit in the treatment of lung cancer as well as other malignancies.Cancer Gene Therapy (2008) 15, 795–807; doi:10.1038/cgt.2008.31; published online 6 June 2008 [ABSTRACT FROM AUTHOR]

Previous studies have shown that intravenously applied bacteria can accumulate in tumors and lead to sporadic tumor regression. Recently, systemic administration of attenuated Salmonella typhimurium was demonstrated to generate no significant side effects in humans, but also no antitumor responses. We report the enhanced antitumor activity in preclinical mouse cancer models of nonvirulent S. typhimurium engineered to synthesize the cytokine Interleukin-18 (IL-18). IL-18-producing bacteria (but not control bacteria) inhibit the growth of primary subcutaneous tumors as well as pulmonary metastases in immunocompetent mice challenged with syngeneic multidrug-resistant clones of murine carcinoma cell lines, without overt toxicity to normal tissues. Antitumor activity was associated with increased accumulation of T-lymphocytes and NK cells in tumors, and massive infiltration of granulocytes, as well as increased intra-tumoral production of several cytokines. In summary, these findings provide evidence of promising preclinical antitumor activity of IL-18-expressing, attenuated S. typhimurium, suggesting a novel strategy for cancer immunotherapy.Cancer Gene Therapy (2008) 15, 787–794; doi:10.1038/cgt.2008.48; published online 25 July 2008 [ABSTRACT FROM AUTHOR]

Adoptive transfer of ex vivo expanded tumor-specific T cells is a promising therapeutic modality for promoting or augmenting antitumor immunity. Several groups, including ours, are developing antigen receptor gene transfer strategies as a means of generating effector cells for adoptive therapy. Chimeric antigen receptors (CARs) have been described that use single-chain antibodies or cytokine ligands as tumor targeting domains. Here, we describe the capacity of a tumor-binding peptide identified by phage display combinatorial library screening to serve as a CAR targeting domain. A phage library-selected high-affinity 12-mer peptide (Bpep) specific for alpha(v) beta(6) integrin (αvβ6) was chosen for these studies. Primary human T cells were genetically modified to express the Bpep-CAR consisting of an αvβ6-specific peptide and human IgG4 hinge-Fc extracellular domain fused to the cytoplasmic tail of CD3-ζ. T cell expression of the Bpep-CAR was assessed by Western blot analysis, and trafficking of the Bpep-CAR to the cell surface was demonstrated by flow cytometry. Functionally, Bpep-CAR redirected cytotoxic T lymphocytes specifically kill integrin αvβ6+ ovarian tumor targets, and are activated for interferon gamma secretion. Our data suggest that large new repertoires of tumor-specific T cell antigen receptor transgenes might be available through merging combinatorial peptide libraries with CAR construct design.Cancer Gene Therapy (2007) 14, 91–97. doi:10.1038/sj.cgt.7700993; published online 6 October 2006 [ABSTRACT FROM AUTHOR]

Replication-competent adenoviruses could provide an efficient method for delivering therapeutic genes to tumors. The most promising strategies among adenovirus-based oncolytic systems are designed to exploit free E2F-1 activity in cancer cells, which in the absence of pRb activates transcription and regulates the expression of genes involved in differentiation, proliferation, and apoptosis. We previously developed?24, an E1A-mutant, conditionally replicative oncolytic adenovirus. Here, we examine the ability of a second-generation?24 (?24-hyCD) engineered to express a humanized form of the Saccharomyces cerevisiae cytosine deaminase gene (hyCD). Real-time quantitative PCR, Western blotting, thin-layer chromatography, and radioisotope quantitative enzymatic assays confirmed the production of a catalytically active hyCD enzyme in the setting of an oncolytic infection in vitro; other experiments assessing local production of 5-fluorouracil and a concomitant bystander effect showed improved cytotoxicity. The IC50 dose of 5-fluorocytosine (5-FC) required for a complete cytopathic effect by the?24-hyCD virus was fivefold lower than with?24 alone in U251MG and U87MG malignant glioma (MG) cell lines. Intratumoral treatment of mice bearing intracranial U87MG xenografts with?24-hyCD+5-FC significantly improved survival, confirming that?24-hyCD with 5-FC is a more efficient anticancer tool than?24 alone. Histopathologically,?24-hyCD replication was accompanied by progressively augmented oncolysis and drug-induced necrosis. These findings demonstrate that?24-hyCD with concomitant systemic 5-FC is a significant improvement over the earlier?24 oncolytic tumor-selective strategy for therapy of experimental gliomas.Cancer Gene Therapy (2005) 12, 284-294. doi:10.1038/sj.cgt.7700750 Published online 14 January 2005 [ABSTRACT FROM AUTHOR]

lmmunotherapy in combination with suicide gene therapy for breast cancer was tested using a metastatic animal model. Subcutaneous injection of the nonimmunogenic breast cancer cell line 4T1 in BALB/C mice gave rise to tumors in 100% of mice with both micrometastases and macrometastases in the lung. We used the herpes simplex virus thymidine kinase (HSV-TK) gene along with the cytokine genes granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) to determine their effect on tumor regression and inhibition of lung metastasis. Adenoviral (AV) vectors carrying these transgenes, in separate constructs, were used in this study. lntratumoral administration of AV-TK followed by 10 days of ganciclovir treatment resulted in a delay in tumor growth and, in some cases, in a Iow to moderate reduction in tumor volume. Inclusion of either GM-CSF or IL-2 gene with HSV-TK resulted in a slightly greater reduction in tumor volume, although these data were not significantly different from those obtained for TK treatment alone. However, when both cytokine genes were combined with TK, a substantial reduction in tumor growth was observed compared with HSV-TK alone (P < .02). Tumor weight data also exhibited superior efficacy of TK/GM-CSF/IL-2 treatment when compared with animals treated with TK gene only (P < .01). More importantly, TK/GM-CSF/IL-2 combination gene therapy induced a significant reduction in lung metastasis compared with any other treatment groups in the 4T1 model (P < .001 between TK GM-CSF/IL-2 versus TK only). Surgical excision of primary tumors after TK/GM-CSF/IL-2 plus ganciclovir treatment resulted in anti-metastatic activity that was similar to that observed for animals in which no surgery was performed. Survival analysis showed a significant difference between animals treated with AV-TK/GM-CSF/IL-2 and animals treated with TK only at 35 days after virus injection (P < .01). lmmunophenotypic data suggest infiltration of lymphocytes... [ABSTRACT FROM AUTHOR]