Abstract 5612: A streamlined workflow for preclinical assessment of monoclonal antibody therapies: A case study
Authors: Lopez David Cobeta , Klingner Kerstin , Carkill Marie , Nunan Robert , Avrutskaya Anya , de Marval Paula Miliani , Blackwell Amber , Dawson Sarah , Barnes Donna , Freeth Jim , Bruce Deborah , Bazin Richard , McLaughlin René , Schueler Julia , Moiset Gemma , Vlaming Maria L. 
Published: June 2022

Abstract
Monoclonal antibody (mAb) therapies have become the dominant product class within the biopharmaceutical industry mainly due to their intrinsic capacity to bind endogenous immune receptors and targeted antigens. In fact, this kind of therapeutic agent accounts for one fifth of the FDA’s new drug approvals each year. In addition, their stability and specificity make them the ideal scaffold to develop more complex and efficacious drug modalities such as bispecific antibodies and antibody-drug conjugates. However, in order to advance mAb therapies to the clinic, there are a number of parameters that need to be considered during early-stage development. The purpose of this study was to showcase important preclinical characterization and efficacy experiments aimed at assessing the biological activity, binding profile, mechanism of action and in vivo potency of cetuximab, a mAb therapy. Cetuximab targets EGFR, a well characterized receptor present in the epithelial cell membrane that is overexpressed in several cancer types, such as non-small cell lung cancer, breast cancer and colorectal cancer. In normal tissues EGFR activation initiates several intracellular signaling events involved in development and homeostasis. However, when overexpressed, it stimulates the growth, metastasis and invasion of tumors. For this reason, EGFR has been considered an important target for the development of new drugs. Here we measured the binding affinity of cetuximab to two EGFR expressing cancer cell lines (A-431 and A-549) and its off-target binding to a broad range of full-length human proteins employing Retrogenix Cell Microarray Technology. Using the AlphaLISA system, we observed that cetuximab significantly inhibits EGF binding to EGFR. The consequences of cetuximab treatment on EGF binding and the initiation of the signaling cascade were investigated by looking at the phosphorylation status of EGFR via intracellular staining and flow cytometry. Moreover, we tested the ability of cetuximab to induce Antibody Dependent Cellular Cytotoxicity (ADCC) where the target cell lines were co-cultured with freshly isolated NK cells. ADCC was assessed via both flow cytometry and live cell imaging. Lastly, we studied the efficacy of cetuximab in vivo. A tumor progression mouse model generated from A-431 cells and several Patient Derived Xenografts (PDX) mouse models representing a wide variety of cancers were treated with cetuximab and a significant reduction in tumor growth was observed for most of these cancers. The in vivo efficacy correlated directly with the EGFR expression level determined by IHC. With this case study we have generated a complete and valuable preclinical data package that could be used to advance this mAb therapy into the clinic. Moreover, this study serves as the basis for a streamlined workflow for mAb lead optimization and development as well as comparability studies for biosimilars.
Citation Format: David Cobeta Lopez, Kerstin Klingner, Marie Carkill, Robert Nunan, Anya Avrutskaya, Paula Miliani de Marval, Amber Blackwell, Sarah Dawson, Donna Barnes, Jim Freeth, Deborah Bruce, Richard Bazin, René McLaughlin, Julia Schueler, Gemma Moiset, Maria L. Vlaming. A streamlined workflow for preclinical assessment of monoclonal antibody therapies: A case study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5612.