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

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

The PSI domain of the MET oncogene encodes a functional disulfide isomerase essential for the maturation of the receptor precursor

The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of alpha and beta chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand-hepatocyte growth factor (HGF)-such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein

Dative constructions in Romance and beyond

This book offers a comprehensive account of dative structures across languages –with an important, though not exclusive, focus on the Romance family. As is well-known, datives play a central role in a variety of structures, ranging from ditransitive constructions to cliticization of indirect objects and differentially marked direct objects, and including also psychological predicates, possessor or causative constructions, among many others. As interest in all these topics has increased significantly over the past three decades, this volume provides an overdue update on the state of the art. Accordingly, the chapters in this volume account for both widely discussed patterns of dative constructions as well as those that are relatively unknown

Dative constructions in Romance and beyond

This book offers a comprehensive account of dative structures across languages –with an important, though not exclusive, focus on the Romance family. As is well-known, datives play a central role in a variety of structures, ranging from ditransitive constructions to cliticization of indirect objects and differentially marked direct objects, and including also psychological predicates, possessor or causative constructions, among many others. As interest in all these topics has increased significantly over the past three decades, this volume provides an overdue update on the state of the art. Accordingly, the chapters in this volume account for both widely discussed patterns of dative constructions as well as those that are relatively unknown

Dative constructions in Romance and beyond

This book offers a comprehensive account of dative structures across languages –with an important, though not exclusive, focus on the Romance family. As is well-known, datives play a central role in a variety of structures, ranging from ditransitive constructions to cliticization of indirect objects and differentially marked direct objects, and including also psychological predicates, possessor or causative constructions, among many others. As interest in all these topics has increased significantly over the past three decades, this volume provides an overdue update on the state of the art. Accordingly, the chapters in this volume account for both widely discussed patterns of dative constructions as well as those that are relatively unknown

Dative constructions in Romance and beyond

This book offers a comprehensive account of dative structures across languages –with an important, though not exclusive, focus on the Romance family. As is well-known, datives play a central role in a variety of structures, ranging from ditransitive constructions to cliticization of indirect objects and differentially marked direct objects, and including also psychological predicates, possessor or causative constructions, among many others. As interest in all these topics has increased significantly over the past three decades, this volume provides an overdue update on the state of the art. Accordingly, the chapters in this volume account for both widely discussed patterns of dative constructions as well as those that are relatively unknown