Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14 ؉ subpopulation of PBMCs but OECs developed exclusively from the CD14 ؊ fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new celltherapeutic approaches to ischemic cardiovascular disease.

Relatively little attention has been paid to the role of virotherapy in promoting antitumor immune responses. Here, we show that CD8+ T cells are critical for the efficacy of intratumoral vesicular stomatitis virus virotherapy and are induced against both virally encoded and tumor-associated immunodominant epitopes. We tested three separate immune interventions to increase the frequency/activity of activated antitumoral T cells. Depletion of Treg had a negative therapeutic effect because it relieved suppression of the antiviral immune response, leading to early viral clearance. In contrast, increasing the circulating levels of tumor antigenspecific T cells using adoptive T cell transfer therapy, in combination with intratumoral virotherapy, generated significantly improved therapy over either adoptive therapy or virotherapy alone. Moreover, the incorporation of a tumorassociated antigen within the oncolytic vesicular stomatitis virus increased the levels of activation of naïve T cells against the antigen, which translated into increased antitumor therapy. Therefore, our results show that strategies which enhance immune activation against tumor-associated antigens can also be used to enhance the efficacy of virotherapy.

In situ killing of tumor cells using suicide gene transfer to generate death by a non-apoptotic pathway was associated with high immunogenicity and induction of heat shock protein (hsp) expression. In contrast, a syngeneic colorectal tumor line, CMT93, killed predominantly by apoptosis, showed low levels of hsp expression and less immunogenicity. When apoptosis was inhibited in CMT93 cells by overexpression of bcl-2, hsp was also induced. Furthermore, when cDNA encoding hsp70 was stably transfected into B16 and CMT93 cells, its expression significantly enhanced the immunogenicity of both tumors. Increased levels of hsp, induced by non-apoptotic cell killing, may provide an immunostimulatory signal in vivo which helps break tolerance to tumor antigens. These findings have important implications for the development of novel anti-cancer therapies aimed at promoting patients' immune responses to their own tumors.

Introduction:

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.

Purpose: To determine the safety and feasibility of daily i.v. administration of wild-type oncolytic reovirus (type 3 Dearing) to patients with advanced cancer, assess viral excretion kinetics and antiviral immune responses, identify tumor localization and replication, and describe antitumor activity. Experimental Design: Patients received escalating doses of reovirus up to 3 Â 10 10 TCID 50 for 5 consecutive days every 4 weeks.Viral excretion was assessed by reverse transcription-PCR and antibody response by cytotoxicity neutralization assay. Pretreatment and post-treatment tumor biopsies were obtained to measure viral uptake and replication. Results: Thirty-three patients received 76 courses of reovirus from 1 Â 10 8 for 1 day up to 3 Â 10 10 TCID 50 for 5 days, repeated every four weeks. Dose-limiting toxicity was not seen. Common grade 1to 2 toxicities included fever, fatigue, and headache, which were dose and cycle independent.Viral excretion at day 15 was not detected by reverse transcription-PCR at 25 cycles and only in 5 patients at 35 cycles. Neutralizing antibodies were detected in all patients and peaked at 4 weeks. Viral localization and replication in tumor biopsies were confirmed in 3 patients. Antitumor activity was seen by radiologic and tumor marker (carcinoembryonic antigen, CA19.9, and prostate-specific antigen) evaluation. Conclusions: Oncolytic reovirus can be safely and repeatedly administered by i.v. injection at doses up to 3 Â 10 10 TCID 50 for 5 days every 4 weeks without evidence of severe toxicities. Productive reoviral infection of metastatic tumor deposits was confirmed. Reovirus is a safe agent that warrants further evaluation in phase II studies.

IntroductionStandard gene-therapy approaches to cancer treatment, such as transfer of suicide genes that confer sensitivity to prodrugs, have limitations as cytoreductive strategies owing to insufficient bystander effects of the therapeutic gene combined with suboptimal transduction efficiency of currently available gene delivery vectors. A more compelling approach in this situation is the use of a vector or virus that is able to replicate within the tumor tissue, resulting in direct cell death through cytolysis or toxicity of viral proteins. Ideally, such an agent should also be capable of stimulating a potent immune response to the tumor within which it can replicate.Studies throughout the twentieth century have documented the lytic effects of various viruses on many types of human cancer, 1 and systematic study of candidate oncolytic viruses is intensifying. Viruses under investigation as oncolytic agents include human adenoviruses, ONYX-015, 2,3 reovirus, 4 herpes viruses 5,6 and vesicular stomatitis virus. 7 All of these viruses have shown promise in preclinical studies, and clinical studies of some of the agents are now in progress. 8 Viruses of the Paramyxoviridae family are also oncolytic. Almost 30 years ago, the human paramyxovirus, mumps, was administered to 90 patients with advanced malignancy, 9 resulting in significant (although mostly short-lived) responses. Toxicity was minimal. More recently, Newcastle disease virus, an avian paramyxovirus, has also shown promising results in preclinical studies, [10][11][12] and clinical trials in human subjects have begun.In this study, we have investigated another human paramyxovirus, measles, as a potential antitumor agent for lymphoid malignancies. Measles virus (MV) may be particularly promising as an oncolytic virus for the treatment of lymphoid malignancy for a number of reasons. First, a nonpathogenic strain of MV is available, well characterized, and safe. Live attenuated MV vaccines, derived from the Edmonston-B strain (MV-Ed), 13 have been used worldwide for more than 30 years, and in excess of 160 million doses have been administered in the United States alone with an excellent safety record. Second, although many human cell types are permissive for MV infection in vitro, in the presence of an intact immune system, virus replication after natural infection is limited to a few cell types in vivo. Lymphoid organs are prominent sites of MV replication; indeed, multinucleated giant cells develop during infection in lymph nodes as a result of gross cell-cell fusion. 14 Third, we have recently shown that expression of virally derived fusogenic membrane glycoproteins in tumor cells, including MV fusion (F) and hemagglutinin (H) glycoproteins, 15-17 results in a potent cytopathic effect mediated by massive cell-cell fusion. The considerable local bystander effect implies that transduction of all tumor cells would not be necessary to achieve significant tumor cell kill. However, the use of MV as a replicating vector with which to deliver the F and H glycopro...

Oncolytic viruses can exert their antitumor activity via direct oncolysis or activation of antitumor immunity. Although reovirus is currently under clinical investigation for the treatment of localized or disseminated cancer, any potential immune contribution to its efficacy has not been addressed. This is the first study to investigate the ability of reovirus to activate human dendritic cells (DC), key regulators of both innate and adaptive immune responses. Reovirus induced DC maturation and stimulated the production of the proinflammatory cytokines IFN-α, TNF-α, IL-12p70, and IL-6. Activation of DC by reovirus was not dependent on viral replication, while cytokine production (but not phenotypic maturation) was inhibited by blockade of PKR and NF-κB signaling. Upon coculture with autologous NK cells, reovirus-activated DC up-regulated IFN-γ production and increased NK cytolytic activity. Moreover, short-term coculture of reovirus-activated DC with autologous T cells also enhanced T cell cytokine secretion (IL-2 and IFN-γ) and induced non-Ag restricted tumor cell killing. These data demonstrate for the first time that reovirus directly activates human DC and that reovirus-activated DC stimulate innate killing by not only NK cells, but also T cells, suggesting a novel potential role for T cells in oncolytic virus-induced local tumor cell death. Hence reovirus recognition by DC may trigger innate effector mechanisms to complement the virus’s direct cytotoxicity, potentially enhancing the efficacy of reovirus as a therapeutic agent.