Gutless Helper-Dependent and First-Generation HAdV5 Vectors Have Similar Mechanical Properties and Common Transduction Mechanisms.

Delivering vectorized information into cells with the help of viruses has been of high interest to fundamental and applied science, and bears significant therapeutic promise. Human adenoviruses (HAdVs) have been at the forefront of gene delivery for many years, and the subject of intensive development resulting in several generations of agents, including replication-competent, -defective or retargeted vectors, and recently also helper-dependent (HD), so-called gutless vectors lacking any viral protein coding information. While it is possible to produce HD-AdVs in significant amounts, physical properties of these virus-like particles and their efficiency of transduction have not been addressed. Here, we used single-cell and single virus particle assays to probe the effect of genome length on HAdV-C5 vector transduction. Our results demonstrate that first-generation C5 vectors lacking the E1/E3 regions of the viral genome as well as HD-AdV-C5 particles with a wild type (wt) ∼36 kbp or an undersized double-strand DNA genome are similar to human adenovirus C5 (HAdV-C5) wt regarding attachment to human lung epithelial cells, endocytic uptake, endosome penetration and dependency on the E3 RING ubiquitin ligase Mind Bomb 1 for DNA uncoating at the nuclear pore complex. Atomic force microscopy measurements of single virus particles indicated that small changes in the genome length from 94% to 103% of HAdV-C5 have no major impact on physical and mechanical features of AdV vectors. In contrast, an HD-AdV-C5 with ∼30 kbp genome was slightly stiffer and less heat-resistant than the other particles, despite comparable entry and transduction efficiencies in tissue culture cell lines, including murine alveolar macrophage-like Max-Planck-Institute (MPI)-2 cells. Together, our in vitro studies reinforce the use of HD-AdV vectors for effective single round gene delivery. The results illustrate how physical properties and cell entry behavior of single virus particles can provide functional information for anticipated therapeutic vector applications.

DARPin-fused T cell engager for adenovirus-mediated cancer therapy.

Bispecific T cell engagers are a promising class of therapeutic proteins for cancer therapy. Their potency and small size often come with systemic toxicity and short half-life, making intravenous administration cumbersome. These limitations can be overcome by tumor-specific in situ expression, allowing high local accumulation while reducing systemic concentrations. However, encoding T cell engagers in viral or non-viral vectors and expressing them in situ ablates all forms of quality control performed during recombinant protein production. It is therefore vital to design constructs that feature minimal domain mispairing, and increased homogeneity of the therapeutic product. Here, we report a T cell engager architecture specifically designed for vector-mediated immunotherapy. It is based on a fusion of a designed ankyrin repeat protein (DARPin) to a CD3-targeting single-chain antibody fragment, termed DATE (DARPin-fused T cell Engager). The DATE induces potent T cell-mediated killing of HER2+ cancer cells, both as recombinantly produced therapeutic protein and as in situ expressed payload from a HER2+-retargeted high-capacity adenoviral vector (HC-AdV). We report remarkable tumor remission, DATE accumulation, and T cell infiltration through in situ expression mediated by a HER2+-retargeted HC-AdV in vivo. Our results support further investigations and developments of DATEs as payloads for vector-mediated immunotherapy.