Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions

Julie Gaston,Nicolas Maestrali,Guilhem Lalle,Tarik Dabdoubi,Bruno Dumas,Marie Gagnaire,Katarina Radošević,Pierre-François Berne,Alessandro Masiero

doi:10.1038/s41598-019-55091-0

Abstract

Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. However, a large number of diseases involve cytosolic targets and designing antibodies able to efficiently reach intracellular compartments would expand the antibody-tractable conditions. Here, we genetically fused cell penetrating peptides (CPPs) at various positions to an antibody targeting cancer cells, evaluated the developability features of the resulting antibody-peptide fusions and the ability of selected constructs to reach the cytosol. We first determined positions in the IgG structure that were permissive to CPP incorporation without destabilizing the antibody. Fusing CPPs to the C-terminus of the light chain and either before or after the hinge had the least effect on antibody developability features. These constructs were further evaluated for cell penetration efficiency. Two out of five tested CPPs significantly enhanced antibody penetration into the cytosol, in particular when fused before or after the hinge. Finally, we demonstrate that specific antibody binding to the cell surface target is necessary for efficient cell penetration of the CPP-antibody fusions. This study provides a solid basis for further exploration of therapeutic antibodies for intracellular targets.

Highlights

Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane
As the hinge is a flexible domain and this position has been described as allowing introduction of a scFv23, we evaluated the impact of inserting a cell penetrating peptides (CPPs) directly before the hinge domain (“before hinge”) as well as directly after the hinge domain (“after hinge”)
Pep-1 was chosen as the first amphipathic CPP to be tested as it has previously been chemically bound to anti-LAMP1 and anti-beta-actin and demonstrated to facilitate antibody penetration into cells[26]

Summary

Introduction

Because of their favorable properties as macromolecular drugs, antibodies are a very successful therapeutic modality for interfering with disease-relevant targets in the extracellular space or at the cell membrane. Since this cytosol-penetrating sequence is within a CDR, a complex engineering approach may be necessary in order to facilitate antibody penetration while not impairing the affinity and specificity of the parental antibody In this approach, fusion of an integrin-binding peptide to the antibody was necessary to enable target cell specificity and enhance efficacy of uptake, further complicating the design of potential therapeutic antibody. Another approach to get large molecules into the cells is using cell-penetrating peptides (CPPs) as a cross-membrane transport vehicle. This category of small peptides (ranging from 5 to 30 amino acids), has become an interesting tool for intracellular delivery, as they have demonstrated their ability to cross cellular membranes[17,18,19]

Methods

Results

Conclusion