FAP-retargeted Ad5 enables in vivo gene delivery to stromal cells in the tumor microenvironment.

Fibroblast activation protein (FAP) is a cell surface serine protease that is highly expressed on reactive stromal fibroblasts, such as cancer-associated fibroblasts (CAFs), and generally absent in healthy adult tissues. FAP expression in the tumor stroma has been detected in more than 90% of all carcinomas, rendering CAFs excellent target cells for a tumor site-specific adenoviral delivery of cancer therapeutics. Here, we present a tropism-modified human adenovirus 5 (Ad5) vector that targets FAP through trivalent, designed ankyrin repeat protein-based retargeting adapters.

CRISPR-clear imaging of melanin-rich B16-derived solid tumors.

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging.

A structural model of a Ras-Raf signalosome.

The protein K-Ras functions as a molecular switch in signaling pathways regulating cell growth. In the human mitogen-activated protein kinase (MAPK) pathway, which is implicated in many cancers, multiple K-Ras proteins are thought to assemble at the cell membrane with Ras effector proteins from the Raf family. Here we propose an atomistic structural model for such an assembly.

Engineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action.

The receptor tyrosine kinase HER2 acts as oncogenic driver in numerous cancers. Usually, the gene is amplified, resulting in receptor overexpression, massively increased signaling and unchecked proliferation. However, tumors become frequently addicted to oncogenes and hence are druggable by targeted interventions.

Intermolecular biparatopic trapping of ErbB2 prevents compensatory activation of PI3K/AKT via RAS-p110 crosstalk.

Compensatory mechanisms, such as relief of AKT-ErbB3-negative feedback, are known to desensitize ErbB2-dependent tumours to targeted therapy. Here we describe an adaptation mechanism leading to reactivation of the PI3K/AKT pathway during trastuzumab treatment, which occurs independently of ErbB3 re-phosphorylation. This signalling bypass of phospho-ErbB3 operates in ErbB2-overexpressing cells via RAS-PI3K crosstalk and is attributable to active ErbB2 homodimers.

Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.

Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express.

Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging.

Purpose: Human epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert.

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters.

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus' original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice.

Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer.

Designed ankyrin repeat proteins are a novel class of specific binding molecules, which display increased thermodynamic stability, smaller size and at least equal target affinity compared to immunoglobulins, making them potentially powerful tools in diagnostic pathology and therapeutic oncology. Here, we investigated whether designed ankyrin repeat proteins can reliably identify the amplification status of the epidermal growth factor receptor 2 in breast cancer. Designed ankyrin repeat proteins specific for epidermal growth factor receptor 2 were tested in paraffin-embedded tissue sections.

Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size.

Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome.