Résumé
BACKGROUND: Monoclonal antibodies (mAbs) are an important part of targeted therapies in many fields and particularly in cancerology by reaching their targets overexpressed on tumour cells (“on target - on tumour”). However, most of these targets are also expressed at basal level by healthy tissues generating some toxicities (“on target - off tumour”). An affinity antibody property for their targets is a key component of this targeting efficiency (1-3). Moreover, the diffusion of antibodies in the tumor is a poorly studied subject and closely related to the targeting efficiency of the mAb and the affinity (4, 5). The aim of this project is to study the role of antibody affinity/avidity in tumour binding, diffusion and in the decrease of “on target - off tumour”.METHOD: We developed a range of human antibodies by phage display processing on human EGFR target with different affinity levels. We studied these affinity levels by enzyme-linked immunosorbent assay (ELISA) and BIACORE assay by Surface Plasmon Resonance (SPR). We added this affinity feature by variable pH dependency properties. We confirmed this in vitro validation by cytometry assays (FACS) on different cell lines characterized by a gradient of EGFR level (from 103 to 106 receptors/cells). RESULTS: We generated six human antibodies specific against human EGFR. Using different in vitro assays, we ranked these antibodies as low, medium and high affinity against EGFR compared with the high affinity Cetuximab and Panitumumab positive controls. We also observed pH dependency properties. Cytometry experiments on different cell lines expressing EGFR showed that only medium affinity level antibodies could bind overexpressed EGFR cell lines but not cell lines with medium and low expression of EGFR (that could mimic healthy tissues) at physiological pH. However, at acidic pH, these medium affinity antibodies could recover their binding abilities on cell line expressing medium level of EGFR without any binding on cell line expressing low level of this target. These medium affinity antibodies should be as effective as high affinity antibodies on tumors while being much less toxic on healthy tissues expressing low levels of the target.PERSPECTIVES: Our hypothesis needs to be confirmed in vivo. We plan to use antibodies radiolabelled with Zirconium-89 to perform non-invasive quantitative biodistribution study by Positron Emission Tomography Computed Tomography (PET-CT) on mice xenografted with both tumor and “healthy tissue”. Binding and diffusion of antibodies will be quantified.