Selective targeting of adenovirus to alphavbeta3 integrins, VEGFR2 and Tie2 endothelial receptors by angio-adenobodies.

Tumor angiogenesis is a prominent mechanism, driving the development and progression of solid tumors and the formation of cancer cell metastasis. Newly formed tumor vessels represent an elective target for the activity and the delivery of cancer therapeutics. We targeted adenovirus (Ad5) to endothelial receptors which are up-regulated during the formation of new blood vessels, to enhance the efficiency of anticancer gene therapy applications. Bifunctional angio-adenobodies were constructed by the fusion of a single chain antibody directed against the adenoviral fiber knob, to different peptides recognizing the alpha(v)beta(3) integrins, VEGFR2 and Tie2 receptors on endothelial cells. The angio-adenobodies were coupled to the adenoviral vector, containing luciferase and GFP as reporter genes. In vitro data showed selective targeting of the Ad5 to the endothelial receptors both in mouse (H5V) and human cell lines (HUVEC). H5V cells, refractory to Ad5 infection, showed high level of luciferase expression when cells were infected with targeted virus. Viral transgene expression increased in HUVEC cells when cells were infected with Ad5 conjugated with angio-adenobody thereby demonstrating the affinity of the peptides for human endothelial cells also. In vivo data obtained from mice bearing a C26 colon carcinoma subcutaneously show viral transgene expression only in tumors infected with angio-adenobodies retargeted adenovirus. The results of the present study demonstrate that endothelial targeted angio-adenobodies represent a versatile tool to direct adenovirus from its native receptors to the integrins alpha(v)beta(3), VEGFR2 and Tie2 receptors that are fundamental in many angiogenesis related diseases such as cancer.

Potent systemic anticancer activity of adenovirally expressed EGFR-selective TRAIL fusion protein.

Previously, we demonstrated potent tumor cell-selective pro-apoptotic activity of scFv425:sTRAIL, a recombinant fusion protein comprised of EGFR-directed antibody fragment (scFv425) genetically fused to human soluble TNF-related apoptosis-inducing ligand (sTRAIL). Here, we report on the promising therapeutic systemic tumoricidal activity of scFv425:sTRAIL when produced by the replication-deficient adenovirus Ad-scFv425:sTRAIL. In vitro treatment of EGFR-positive tumor cells with Ad-scFv425:sTRAIL resulted in the potent induction of apoptosis of not only infected tumor cells, but importantly also of up to 60% of noninfected EGFR-positive tumor cells. A single intraocular injection of Ad-scFv425:sTRAIL in tumor-free nu/nu mice resulted in predominant liver infection and concomitant high blood plasma levels of scFv425:sTRAIL. These mice showed no sign of Ad-scFv425:sTRAIL-related liver toxicity. Identical treatment of mice with established intraperitoneal renal cell carcinoma xenografts resulted in rapid and massive tumor load reduction and subsequent long-term survival. Taken together, adenoviral-mediated in vivo production of scFv425:sTRAIL may be exploitable for systemic treatment of EGFR-positive cancer.

Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

Differences in heat sensitivity between normal and acute myeloid leukemic stem cells: feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts.

OBJECTIVES: In autologous stem cell transplantation contamination of the graft with malignant cells is frequently noticed and necessitates the use of in vivo or in vitro purging modalities. The hematopoietic recovery after transplantation depends on the number of stem and progenitor cells in the transplant. Therefore, in the present study the effects of hyperthermic treatment on the human normal and acute myeloid leukemic (AML) stem cell compartment were investigated. METHODS: Normal bone marrow and AML blasts were heat treated up to 120 minutes at 43 degrees C. The surviving fractions of the different stem cell subsets were determined using in vitro methylcellulose and cobblestone area-forming cell (CAFC) clonogenic assays, as well as the in vivo NOD/SCID repopulating assay. The leukemic nature of the colonies from AML cells was confirmed by RT-PCR analysis. In order to increase the therapeutic index of the hyperthermic purging modality, the heat treatment was preceded by a 3-hour incubation at 37 degrees C with the ether lipid ET-18-OCH(3) (25 microg/mL). RESULTS: It could be demonstrated that normal progenitor cells are far more resistant to hyperthermia than leukemic progenitor cells (56%+/-7% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). Furthermore, normal hematopoietic stem cells appear to be extremely resistant to the heat treatment (94%+/-9% survival after 60 minutes at 43 degrees C). In contrast, in the leukemic stem cell compartment no significant differences in heat sensitivity between the stem cells and progenitor subsets could be observed (12.3%+/-2.9% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). The combined treatment resulted in a survival for normal progenitor and stem cells of 32%+/-6% and 85%+/-15% after 60 minutes at 43 degrees C, respectively. Under these conditions the number of leukemic stem cells was reduced to 1%+/-0.3%. After 120 minutes at 43 degrees C, no AML-colonies could be detected anymore. CONCLUSIONS: Our data demonstrate that leukemic stem cells have an increased hyperthermic sensitivity compared to their normal counterparts and that this difference can be further increased in combination with ET-18-OCH(3). These striking differences in heat sensitivity warrant the use of hyperthermia as a clinically applicable purging modality in autologous stem cell transplantation.

Peripheral blood stem cells differ from bone marrow stem cells in cell cycle status, repopulating potential, and sensitivity toward hyperthermic purging in mice mobilized with cyclophosphamide and granulocyte colony-stimulating factor.

Peripheral blood stem cells (PBSCs) are increasingly used in autologous stem cell transplantations. We investigated the mobilizing effect of a combined cyclophosphamide (CTX) and granulocyte colony-stimulating factor (G-CSF) treatment on progenitor cells (STRA) and primitive stem cells (LTRA) in normal and splenectomized CBA/H mice. This combined treatment not only resulted in mobilization but also in expansion of hematopoietic stem cell subsets. The latter phenomenon was somewhat suppressed in splenectomized animals, but in these mice an enhanced mobilization of STRA and LTRA cells into the peripheral blood was observed. Furthermore, we studied the engraftment potential of mobilized PBSCs. Mice transplanted with PBSCs engrafted significantly better compared to mice transplanted with bone marrow stem cells from control and mobilized mice. The repopulation curve was characterized by a less-deep nadir indicating that the differences occur during the initial phase after transplantation. Contamination of autologous PBSC transplants with malignant cells is noticed frequently and is the basis for urging the use of purging modalities. Here we used hyperthermia and found that the mobilized progenitor cells in peripheral blood are more resistant to hyperthermia than those in the bone marrow (i.e., a survival of 11 +/- 5% after 90 min at 43 degrees C for peripheral blood progenitors, compared to 0.5 +/- 0.4% in bone marrow of mobilized animals and 1.6 +/- 0.5% in normal animals, respectively). Hyperthermic purging does not eliminate the superior repopulating features of a PBSC graft, as is demonstrated by an increased median survival time of lethally irradiated mice transplanted with purged PBSCs. In conclusion, our data demonstrate that CTX + G-CSF-mobilized PBSCs have an enhanced engraftment potential concomitantly with a decreased cycling activity and hence a decreased hyperthermic sensitivity. These findings support the use of these mobilized PBSCs for autologous stem cell transplantation and strengthen the basis for using hyperthermia as a purging modality.