Discovering anti-cancer biologics using phenotypic screening and Retrogenix’s target deconvolution technology

A recent project involving scientists from MedImmune, OcellO and Retrogenix has been successful in using 3-D phenotypic screening to select novel antibodies that are functional against primary tumour cells. The group then went on to identify the specific cell surface antigen targets of the most promising phenotypic molecules. The results were published in the journal ‘Molecular Cancer’.

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.

 

Results

Distinct phenotypic effects were observed for a subset of antibodies that all bind to CUB-domain containing protein 1 (CDCP1) – a cell surface transmembrane protein that is upregulated in many tumour cells and associated with invasive and metastatic phenotypes. These strong functional effects led the group to test an anti-CDCP1 IgG antibody from their panel for the potential to inhibit tumour growth in vivo using a mouse xenograft model. Although the antibody treatment alone was not efficacious, significant enhancement of tumour growth inhibition and increased survival times were observed when the antibody was co-administered with cisplatin, compared to cisplatin alone at the same dose. This identifies CDCP1 as a potential target for cisplatin combination therapy.

 

Impact

Target deconvolution has traditionally been a well-recognised bottleneck in phenotypic discovery. This study demonstrates both the utility of 3-D phenotypic screening in identifying functional molecules as well as the power of the Retrogenix target deconvolution platform in pinpointing specific antigen targets. Retrogenix’s success in this area is providing researchers with the critical information that allows promising phenotypic molecules to progress and can also uncover novel receptor targets that can provide a real competitive advantage.

Authored by Alan M. Sandercock et al., the paper is entitled ‘Identification of anti-tumour biologics using primary tumour models, 3-D phenotypic screening and image-based multi-parametric profiling’. To view the full, open-access article please click here.

 

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