Upon local delivery, adenovirus (Ad) serotype 5 viruses use the coxsackie and Ad receptor (CAR) for cell binding and αv integrins for internalization. When administered systemically, however, their ...role in liver tropism is limited because CAR-permissive and mutated viruses show similar biodistribution, a finding recently attributed to blood coagulation factor (F) IX or complement protein C4BP binding to the adenovirus fiber and “bridging” to either low-density lipoprotein receptor-related protein or heparan sulfate proteoglycans. Here, we show that hepatocyte transduction in vitro can be enhanced by the vitamin K-dependent factors FX, protein C, and FVII in addition to FIX but not by prothrombin (FII), FXI, and FXII. This phenomenon was not dependent on proteolytic activation or cell signaling activity and for FX was mediated by direct virus-factor binding. Human FX substantially enhanced hepatocyte transduction by CAR-permissive and mutated viruses in an ex vivo liver perfusion model. In vivo, global down-regulation of vitamin K-dependent zymogens by warfarin significantly diminished liver uptake of CAR-deleted Ads; however, this phenomenon was fully rescued by acute infusion of human FX. Our results indicate a common and pivotal role for distinct vitamin K-dependent coagulation factors in mediating hepatocyte transduction by adenoviruses in vitro and in vivo.
Viral-mediated oncolysis is a novel cancer therapeutic approach with the potential to be more effective and less toxic than current therapies due to the agents selective growth and amplification in ...tumor cells. To date, these agents have been highly safe in patients but have generally fallen short of their expected therapeutic value as monotherapies. Consequently, new approaches to generating highly potent oncolytic viruses are needed. To address this need, we developed a new method that we term "Directed Evolution" for creating highly potent oncolytic viruses.
Taking the "Directed Evolution" approach, viral diversity was increased by pooling an array of serotypes, then passaging the pools under conditions that invite recombination between serotypes. These highly diverse viral pools were then placed under stringent directed selection to generate and identify highly potent agents. ColoAd1, a complex Ad3/Ad11p chimeric virus, was the initial oncolytic virus derived by this novel methodology. ColoAd1, the first described non-Ad5-based oncolytic Ad, is 2-3 logs more potent and selective than the parent serotypes or the most clinically advanced oncolytic Ad, ONYX-015, in vitro. ColoAd1's efficacy was further tested in vivo in a colon cancer liver metastasis xenograft model following intravenous injection and its ex vivo selectivity was demonstrated on surgically-derived human colorectal tumor tissues. Lastly, we demonstrated the ability to arm ColoAd1 with an exogenous gene establishing the potential to impact the treatment of cancer on multiple levels from a single agent.
Using the "Directed Evolution" methodology, we have generated ColoAd1, a novel chimeric oncolytic virus. In vitro, this virus demonstrated a >2 log increase in both potency and selectivity when compared to ONYX-015 on colon cancer cells. These results were further supported by in vivo and ex vivo studies. Furthermore, these results have validated this methodology as a new general approach for deriving clinically-relevant, highly potent anti-cancer virotherapies.
Baculovirus vectors are able to transduce a large variety of mammalian cell types and express transgenes placed under the control of heterologous promoters. In this study, we evaluated the potential ...of baculovirus vectors for malaria vaccination. To induce efficient CD4(+) and CD8(+) T-cell responses, we produced a series of vectors that display the Plasmodium falciparum circumsporozoite (CS) protein in the virion envelope and/or allow for CS expression upon transduction of mammalian cells. We found that baculovirus vectors can transduce professional antigen-presenting cells and trigger their maturation, which is a prerequisite for efficient antigen presentation. Upon intramuscular injection into mice, the vector that both displayed and expressed CS induced higher anti-CS antibody titers (of the immunoglobulin (IgG)1 and IgG2a type) and a higher frequency of interferon-γ-producing T cells specific to CS, than the vectors which either only displayed or only expressed CS. The baculovirus CS display/expression vector was also superior in inducing CS-specific CD4(+) and CD8(+) T-cell responses in vitro using human peripheral blood mononuclear cells from naive donors. This, together with the absence of pre-existing immunity to baculoviruses in humans, the absence of viral gene expression in mammalian cells, and the relative low immunogenicity of baculovirus virions, makes these vectors promising tools for vaccination. Furthermore, the ability to produce large amounts in serum-free medium at a low cost adds a further advantage to this vector system.
Accumulating data indicate that tumor-infiltrating regulatory T cells (Treg) are present in human tumors and locally suppress antitumor immune cells. In this study, we found an increased Treg/CD8 ...ratio in human breast and cervical cancers. A similar intratumoral lymphocyte pattern was observed in a mouse model for cervical cancer (TC-1 cells). In this model, systemic Treg depletion was inefficient in controlling tumor growth. Furthermore, systemic CTL-associated antigen-4 (CTLA-4) blockade, an approach that can induce tumor immunity in other tumor models, did not result in TC-1 tumor regression but led to spontaneous development of autoimmune hepatitis. We hypothesized that continuous expression of an anti-CTLA-4 antibody localized to the tumor site could overcome Treg-mediated immunosuppression and locally activate tumor-reactive CD8+ cells, without induction of autoimmunity. To test this hypothesis, we created TC-1 cells that secrete a functional anti-CTLA-4 antibody (TC-1/alphaCTLA-4-gamma1 cells). When injected into immunocompetent mice, the growth of TC-1/alphaCTLA-4-gamma1 tumors was delayed compared with control TC-1 cells and accompanied by a reversion of the intratumoral Treg/CD8 ratio due to an increase in tumor-infiltrating IFNgamma-producing CD8+ cells. When local anti-CTLA-4 antibody production was combined with Treg inhibition, permanent TC-1 tumor regression and immunity was induced. Importantly, no signs of autoimmunity were detected in mice that received local CTLA-4 blockade alone or in combination with Treg depletion.
Heat shock proteins such as gp96 have the ability to chaperone peptides and activate antigen-presenting cells. In this study, we tested whether adenovirus-mediated overexpression of secreted or ...membrane-associated forms of gp96 in tumor cells would stimulate an antitumor immune response. Studies were carried out in C57Bl/6 mice bearing aggressively growing s.c. tumors derived from syngeneic TC-1 cells, a cell line that expresses HPV16 E6 and E7 proteins. We found that secreted gp96 can induce protective and therapeutic antitumor immune responses. Our data also indicate that the antitumor effect of sgp96 expression seems to be limited by the induction of suppressive regulatory T cells (Treg). TC-1 tumor transplantation increased the number of splenic and tumor-infiltrating Tregs. Importantly, treatment of mice with low-dose cyclophosphamide decreased the number of Tregs and enhanced the immunostimulatory effect of sgp96 expression. We also tested whether an oncolytic vector (Ad.IR-E1A/TRAIL), that is able to induce tumor cell apoptosis and, potentially, release cryptic tumor epitopes in immunogenic form, could stimulate antitumor immune responses. Although tumor cells infected ex vivo with Ad.IR-E1A/TRAIL had no antitumor effect when used as a vaccine alone, the additional treatment with low-dose cyclophosphamide resulted in the elimination of pre-established tumors. This study gives a rationale for testing approaches that suppress Tregs in combination with oncolytic or immunostimulatory vectors.
Vectors containing group B adenovirus (Ad) fibers are able to efficiently transduce gene therapy targets that are refractory to infection with standard Ad serotype 5 (Ad5) vectors, including ...malignant tumor cells, hematopoietic stem cells, and dendritic cells. Preliminary studies in mice indicate that, after intravenous injection, B-group fiber-containing Ads do not efficiently transduce most organs and cause less acute toxicity than Ad5 vectors. However, biodistribution and safety studies in mice are of limited value because the mouse analog of the B-group Ad receptor, CD46, is expressed only in the testis, whereas in humans, CD46 is expressed on all nucleated cells. Unlike mice, baboons have CD46 expression patterns and levels that closely mimic those in humans. We conducted a biodistribution and toxicity study of group B Ad fiber-containing vectors in baboons. Animals received phosphate-buffered saline, Ad5-bGal (a first-generation Ad5 vector), or B-group fiber-containing Ads (Ad5/35-bGal and Ad5/11-bGal) at a dose of 2 x 10(12) VP/kg, and vector biodistribution and safety was analyzed over 3 days. The amount of Ad5/35-bGal and Ad5/11-bGal vector genomes was in most tissues one to three orders of magnitude below that of Ad5. Significant Ad5/35- and Ad5/11-mediated transgene (beta-galactosidase) expression was seen only in the marginal zone of splenic follicles. Compared with the animal that received Ad5-bGal, all animals injected with B-group fiber-containing Ad vectors had lower elevations in serum proinflammatory cytokine levels. Gross and histopathology were normal in animals that received B-group Ad fiber-containing Ads, in contrast to the Ad5-infused animal, which showed widespread endothelial damage and inflammation. In a further study, a chimeric Ad5/35 vector carrying proapoptotic TRAIL and Ad E1A genes under tumor-specific regulation was well tolerated in a 30-day toxicity study. No major clinical, serologic, or pathologic abnormalities were noticed in this animal.
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