A cathepsin-cleavage site between the adenovirus capsid protein IX and a tumor-targeting ligand improves targeted transduction.

Human adenoviruses have a great potential as anticancer agents. One strategy to improve their tumor-cell specificity and anti-tumor efficacy is to include tumor-specific targeting ligands in the viral capsid. This can be achieved by fusion of polypeptide-targeting ligands with the minor capsid protein IX. Previous research suggested that protein IX-mediated targeting is limited by inefficient release of protein IX-fused ligands from their cognate receptors in the endosome. This thwarts endosomal escape of the virus particles. Here we describe that the targeted transduction of tumor cells is augmented by a cathepsin-cleavage site between the protein IX anchor and the HER2/neu-binding ZH Affibody molecule as ligand. The cathepsin-cleavage site did not interfere with virus production and incorporation of the Affibody molecules in the virus capsid. Virus particles harboring the cleavable protein IX-ligand fusion in their capsid transduced the HER2/neu-positive SKOV-3 ovarian carcinoma cells with increased efficiency in monolayer cultures, three-dimensional spheroid cultures and in SKOV-3 tumors grown on the chorioallantoic membrane of embryonated chicken eggs. These data show that inclusion of a cathepsin-cleavage sequence between protein IX and a high-affinity targeting ligand enhances targeted transduction. This modification further augments the applicability of protein IX as an anchor for coupling tumor-targeting ligands.

The chicken chorioallantoic membrane tumor assay as model for qualitative testing of oncolytic adenoviruses.

Oncolytic adenoviruses are promising anticancer agents. To study and optimize their tumor-killing potency, genuine tumor models are required. Here we describe the use of the chicken chorioallantoic membrane (CAM) tumor model in studies on oncolytic adenoviral vectors. Suspensions of human melanoma, colorectal carcinoma and glioblastoma multiforme cell lines were grafted on the CAM of embryonated chicken eggs. All cell lines tested formed 5-10 mm size tumors, which recapitulated hallmarks of corresponding human specimens. Furthermore, melanoma tumors were injected with adenoviral vector-carrying gene encoding the fusion protein of parainfluenza virus type 5. This led to the induction of cell fusion and syncytia formation in the infected cells. At 6 days post-injection, histological and immunohistochemical analyses of tumor sections confirmed adenovirus replication and syncytia formation. These results demonstrate that the CAM model allows rapid assessment of oncolytic viruses in three-dimensional tumors. Hence, this model constitutes an easy and affordable system for preclinical characterization of viral oncolytic agents that may precede the mandatory process of animal testing. Application of this model will help reducing the use of human xenografts in mice for preclinical evaluation of oncolytic viruses and other anticancer agents.

Adenovirus targeting to HLA-A1/MAGE-A1-positive tumor cells by fusing a single-chain T-cell receptor with minor capsid protein IX.

Adenovirus vectors have great potential in cancer gene therapy. Targeting of cancer-testis (CT) antigens, which are specifically presented at the surface of tumor cells by human leukocyte antigen (HLA) class I molecules, is an attractive option. In this study, a single-chain T-cell receptor (scTCR) directed against the CT antigen melanoma-associated antigen (MAGE)-A1 in complex with the HLA class I molecule of haplotype HLA-A1 is fused with the C terminus of the adenovirus minor capsid protein IX. Propagation of a protein-IX (pIX)-gene-deleted human adenovirus 5 (HAdV-5) vector on cells that constitutively express the pIXscTCR fusion protein yielded viral particles with the pIXscTCR fusion protein incorporated in their capsid. Generated particles specifically transduced melanoma cell lines expressing the HLA-A1/MAGE-A1 target complex with at least 10-fold higher efficiency than control viruses. Whereas loading of HLA-A1-positive cells with MAGE-A1 peptides leads to enhanced transduction of the cells, the efficiency of virus transduction is strongly reduced if the HLA-A1 molecules are not accessible at the target cell. Taken together, these data provide proof of principle that pIXscTCR fusions can be used to target HAdV-5 vectors to tumor cells expressing intracellular CT antigens.

Bovine herpesvirus 1 interferes with TAP-dependent peptide transport and intracellular trafficking of MHC class I molecules in human cells.

Bovine herpesvirus 1 (BoHV-1), the cause of infectious bovine rhinotracheitis and infectious pustular vulvovaginitis in cattle, establishes a lifelong infection, despite the presence of antiviral immunity in the host. BoHV-1 has been shown to elude the host immune system, but the viral gene products responsible for this interference have not yet been identified. Studies aiming at the identification of BoHV-1-encoded immune evasion genes have been hampered by the lack of bovine-specific immunological reagents. Some of the immune evasion molecules identified for other herpesviruses are host species specific; others can act across the species barrier. In this study, experiments were performed to investigate whether BoHV-1 can infect human cells and interfere with antigen processing and presentation in these cells. A human melanoma cell line, Mel JuSo, appeared to be permissive for BoHV-1 infection. BoHV-1 induced expression of major viral glycoproteins at the surface of these cells and produced progeny virus up to 10(5) plaque forming units per ml. BoHV-1 infection resulted in impaired intracellular transport of human MHC class I molecules and inhibition of human TAP. These data indicate that the BoHV-1-encoded molecule(s) that block antigen presentation in bovine cells are able to interact with homologous components of the human MHC class I presentation pathway. The fact that immune evasion by BoHV-1 can be studied in human cells will facilitate the identification of the BoHV-1 gene products involved in this process. Moreover, the data presented here suggest that the BoHV-1 encoded inhibitors of antigen presentation represent potential immune suppressive agents for use in humans.