American Association for Cancer Research
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Figure S4 from A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2–Bispecific Antibody for Combination Cancer Therapy

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journal contribution
posted on 2023-03-28, 15:00 authored by Amr El Zawily, Frederick S. Vizeacoumar, Renuka Dahiya, Sara L. Banerjee, Kalpana K. Bhanumathy, Hussain Elhasasna, Glinton Hanover, Jessica C. Sharpe, Malkon G. Sanchez, Paul Greidanus, R. Greg Stacey, Kyung-Mee Moon, Ilya Alexandrov, Juha P. Himanen, Dimitar B. Nikolov, Humphrey Fonge, Aaron P. White, Leonard J. Foster, Bingcheng Wang, Behzad M. Toosi, Nicolas Bisson, Tajib A. Mirzabekov, Franco J. Vizeacoumar, Andrew Freywald

Fig. S4. A. Heatmap of the shRNA screen hits that are enriched in the top 15 GO processesgenerated using GSEA software. B. Gene Set Enrichment Analysis plots of the top 15 GOprocess for the shRNA screen data produced by GSEA software used to generate the heatmap inA.



Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment.

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