Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy.

Marie Godar,Virginia Morello,Christophe Blanchetot,Valérie Hanssens,Cristina Basilico,Paolo Michieli,Hans De Haard,Michael Saunders,Anna Hultberg,Ava Sadi,Mohamed El Khattabi,Natalie De Jonge,Bart N Lambrecht

doi:10.1038/srep31621

Abstract

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

Highlights

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes
The physical and thermal stability, or better, the capacity of VHHs to refold completely after denaturation enables the apparent infinite reusability of the columns[15]. These characteristics explained that these binding domains have already been successfully applied as affinity ligands in chromatography processes covering a variety of biotherapeutics[16,17,18,19], can serve as target-specific capture reagents[20], and can replace the conventional and expensive protein A purification step[21,22]. To validate this dual anti-idiotypic purification process, we established a proof-of-principle study using two previously generated antagonistic monospecific antibodies (mAbs) that compete with HGF for binding to MET, targeting two unique, non-overlapping epitopes on the extracellular region of the MET receptor (Fig. 2a,b)[23]
We showed that cooperation between these two mAbs was reproducibly observed in vitro in a variety of biochemical and biological assays, the WT46 and WT52 did not always show a sound synergistic effect in mice[23]

Summary

Introduction

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. We present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind to the correct heavy chain/light chain pairings of each arm. Bispecific antibodies (BsAb) represent new emerging antibody (Ab) -based therapeutics They are generally built with different antigen (Ag)-binding sites (Fab or single-chain Fv), each one used to provide a different target-binding specificity, albeit within the same molecule[1]. The high immunogenicity triggered in humans with the mouse and rat components limit such approach clinically, which can only be used in very specific indications for a short period of time[11]

Methods

Results

Conclusion