CD40-targeted adenoviral GM-CSF gene transfer enhances and prolongs the maturation of human CML-derived dendritic cells upon cytokine deprivation.

Vaccination with autologous leukaemia-derived dendritic cells (DC) presents an adjuvant treatment option for chronic myeloid leukaemia (CML). Here, we show that high-efficiency CD40-targeted adenoviral gene transfer of GM-CSF to CML-derived DC induces long-lived maturation in the absence of exogenous cytokines and may thus ensure protracted stimulation of CML-specific T cells upon vaccination.

Adenoviral vectors targeted to CD40 enhance the efficacy of dendritic cell-based vaccination against human papillomavirus 16-induced tumor cells in a murine model.

Dendritic cells (DCs) represent a unique junction from which to initiate antigen-specific immunity. One of the most challenging obstacles for DC-based immunotherapy has been the means by which to convey tumor antigen-encoding genes to DCs. In this study, we show that adenoviral (or adenovirus, Ad) vectors targeted to CD40 by means of bispecific antibodies can enhance gene transfer to murine DCs. Moreover, we illustrate that this vector initiates phenotypic changes characteristic of DC maturation. To explore the in vivo potential of this strategy, we coupled this targeting approach with an Ad vector carrying the gene for a tumor antigen. In particular, the human papillomavirus (HPV) E7 antigen represents an attractive target for antigen-specific immunity of cervical cancer. Relative to DCs infected by untargeted Ad, DCs infected by AdE7 targeted to the receptor CD40 enhanced protection against HPV-16-induced tumor cells in a murine model. We have further established that this protection was both antigen specific and CD8+ T-cell dependent. Illustrating that Ad-modified DCs may be used in repeated vaccination, we report that preimmunization of animals with Ad infected DCs prior to E7 vaccination only moderately reduced vaccine efficacy. Finally, we have observed that CD40-targeted AdE7 can initiate partial therapeutic immunity in mice bearing established tumors. These findings suggest that gene-based vaccination of DCs with tumor antigens can elicit productive antitumoral immunity and that enhancements in gene transfer efficacy and/or DC maturation may facilitate this process.

Tumor-specific gene transfer via an adenoviral vector targeted to the pan-carcinoma antigen EpCAM.

The utility of adenoviral vectors for cancer therapy is limited due to their lack of specificity for tumor cells. In order to target adenovirus to tumor, the natural tropism of the adenovirus should be ablated and replaced by a tumor-specific binding domain. To this end, a neutralizing anti-fiber antibody conjugated to an anti-EpCAM antibody was created that targets the adenovirus to the EpCAM antigen present on tumor cells. The EpCAM antigen was chosen as the target because this antigen is highly expressed on a variety of adenocarcinomas of different origin such as breast, ovary, colon and lung, whereas EpCAM expression is limited in normal tissues. In these studies, the EpCAM-targeted adenovirus was shown to infect specifically cancer cell lines of different origin expressing EpCAM such as ovary, colon and head and neck. Gene transfer was blocked by excess anti-EpCAM antibody and dramatically reduced in EpCAM negative cell lines, thus showing the specificity of the EpCAM-targeted adenovirus. Importantly, infection with targeted adenovirus was independent of CAR, which is the natural receptor for adenovirus binding, since blocking of CAR with recombinant fiber knob did not affect infection with targeted adenovirus. Apart from the cancer cell lines, the efficacy of targeted viral infection was studied in freshly isolated primary human colon cancer cells. As colon cancer predominantly metastasizes to liver, and adenovirus has a high tropism for hepatocytes, we also sought to determine if the EpCAM-targeted adenovirus showed reduced infectivity of human liver cells. The bispecific antibody could successfully mediate gene transfer to primary human colon cancer cells, whereas it almost completely abolished infection of liver cells. This work thus demonstrates that EpCAM-targeted adenoviral vectors can be specifically directed to a wide variety of adenocarcinomas. This approach may prove to be useful for selective gene therapy of cancer.

Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.

Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies.