Affilin-based retargeting of adenoviral vectors to the epidermal growth factor receptor.

INTRODUCTION: Treatment of head and neck squamous cell carcinomas (HNSCC) by oncolytic adenoviral vectors holds promise as an efficient anti-cancer therapy. The epidermal growth factor receptor (EGFR) represents an attractive target receptor since it is frequently overexpressed in many types of HNSCC. METHODS: To achieve EGFR-specific targeting by human adenovirus type 5 (HAdV-5) based vectors, the EGFR affinity ligand Affilin was covalently attached in a position specific manner either to the fiber or the hexon protein of the vector capsid. In vitro and in vivo studies investigated EGFR-specific cancer cell transduction, susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of Affilin-decorated vectors. RESULTS: Affilin-decorated vectors showed strongly enhanced and EGFR-specific cancer cell transduction in vitro and less susceptibility to known sequestration mechanisms of HAdV-5 particles. However, in vivo neither systemic nor intratumoral vector administration resulted in an improved transduction of EGFR-positive tumors. Comprehensive analyses indicated hampered EGFR-targeting by Affilin-decorated vectors was caused by rapid vector particle consumption due to binding to the murine EGFR, insufficient tumor vascularization and poor target accessibility for Affilin in the solid tumor caused by a pronounced tumor stroma. CONCLUSION: In vitro studies yielded proof-of-concept results demonstrating that covalent attachment of a receptor-specific Affilin to the adenoviral capsid provides an effective and versatile tool to address cancer-specific target receptors by adenoviral vectors. Regarding EGFR as the vector target, off-target tissue transduction and low receptor accessibility within the tumor tissue prevented efficient tumor transduction by Affilin-decorated vectors, rendering EGFR a difficult-to-target receptor for adenoviral vectors.

Process- and product-related impurities in the ChAdOx1 nCov-19 vaccine.

ChAdOx1 nCov-19 and Ad26.COV2.S are approved vaccines inducing protective immunity against SARS-CoV-2 infection in humans by expressing the Spike protein of SARS-CoV-2. We analyzed protein content and protein composition of ChAdOx1 nCov-19 and Ad26.COV2.S by biochemical methods and by mass spectrometry. Four out of four tested lots of ChAdOx1 nCoV-19 contained significantly higher than expected levels of host cell proteins (HCPs) and of free viral proteins. The most abundant contaminating HCPs belonged to the heat-shock protein and cytoskeletal protein families. The HCP content exceeded the 400 ng specification limit per vaccine dose, as set by the European Medicines Agency (EMA) for this vaccine, by at least 25-fold and the manufacturer's batch-release data in some of the lots by several hundred-fold. In contrast, three tested lots of the Ad26.COV2.S vaccine contained only very low amounts of HCPs. As shown for Ad26.COV2.S production of clinical grade adenovirus vaccines of high purity is feasible at an industrial scale. Correspondingly, purification procedures of the ChAdOx1 nCov-19 vaccine should be modified to remove protein impurities as good as possible. Our data also indicate that standard quality assays, as they are used in the manufacturing of proteins, have to be adapted for vectored vaccines.

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids.

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

Chorioallantoic Membrane Tumor Model for Evaluating Oncolytic Viruses.

Oncolytic viruses are promising anticancer agents; however, regarding their clinical efficacy, there is still significant scope for improvement. Preclinical in vivo evaluation of oncolytic viruses is mainly based on syngeneic or xenograft tumor models in mice, which is labor-intensive and time-consuming. Currently, a large proportion of developmental work in the research field of oncolytic viruses is directed toward overcoming cellular and noncellular barriers to achieve improved virus delivery to primary tumors and metastases. To evaluate the large number of genetically or chemically modified viruses regarding tumor delivery and biodistribution patterns, it would be valuable to have an in vivo model available that would allow easy screening experiments, that is of higher complexity than monoclonal cell lines, and that could be used as a platform method before confirmatory studies in small and large animals. Based on our data, we believe that the chicken chorioallantoic membrane (CAM) assay is a quick and low-cost high-throughput tumor model system for the in vivo analysis of oncolytic viruses. Here we describe the establishment, careful characterization, and optimization of the CAM model as an in vivo model for the evaluation of oncolytic viruses. We have used human adenovirus type 5 (HAdV-5) as an example for validation but are confident that the model can be used as a test system for replicating viruses of many different virus families. We show that the CAM tumor model enables intratumoral and intravenous virus administration and is a feasible and conclusive model for the analysis of relevant virus-host interactions, biodistribution patterns, and tumor-targeting profiles.