Methodology

In vitro phenotypic screening of novel anti-cancer therapeutics is often performed using 2-D, monolayer immortalised cell line cultures. This study reports the use of primary tumour cells as the source of the antigen which steers hit selection towards more relevant in vivo phenotypes that could be more predictive of clinical outcomes.

ScFv antibodies and designed ankyrin repeat proteins (DARPins) were isolated by phage display selection against primary cells from non-small cell lung cancer patients. Cells were grown in multiple formats, including 3-D cultures, and were monitored to detect the anti-proliferative and pro-apoptotic activity of each molecule.

Multiple antibodies demonstrated a desired activity. The specific antigens that these phenotypic antibodies target were then identified using the Retrogenix cell microarray technology. Briefly, 2505 expression vectors, each encoding a full-length human cell surface protein, were arrayed across multiple microarray slides. HEK293 cells were grown over the vector array, leading to reverse transfection at each array location. After fixing the cells, the interaction between antibodies and the cells presenting the receptor was detected using a fluorescently-conjugated goat anti-human antibody. Each receptor ‘hit’ was confirmed by respotting the vector and repeating the binding experiment along with appropriate positive and negative controls.