Identified as the key receptor for lysosomal delivery, the ubiquitous cation-independent mannose 6-phosphate membrane receptor (CI-M6PR, hereafter referred to as M6PR) has the ability to bind M6P-bearing ligands and release them in the endo-lysosomal network [[1], [2], [3]]. The M6P signal is essential to lysosomal proteins to reach the lysosome through the M6PR pathway. Several strategies have been developed to address recombinant proteins to the lysosome for the treatment of rare lysosomal storage disorders [[4], [5], [6], [7]]. The efficient technology based on the chemical grafting of phosphonate derivatives (M6Pn), the Analogues of Mannose 6-phosphate Functionalized on Anomeric position (AMFA), was demonstrated to improve the delivery of the enzyme replacement therapy via M6PR and to achieve a better therapeutic response in a mouse model of glycogen storage disease type II (also called Pompe disease) even at a late stage [8,9].

Here, we study a novel application of these M6Pn derivatives, the AMFA, adapted to therapeutic monoclonal antibodies (mAb) that combines high target specificity for antigens with an acquired affinity for M6PR. Antibodies engineered with AMFA (mAb-AMFA) are expected to efficiently transport the antigen into cells and reach the endo-lysosomal compartments where the antigen can be degraded. C. Bertozzi and coworkers [10] reported that the binding of a large polyglyco-peptide bearing 20 to 90 M6Pn to mAbs, called lysosomal targeting chimaera (LYTAC), increased antibody uptake and antigen degradation in cells.

Our engineering approach consists in the conjugation of AMFA to the glycosylated chains located on the Fc domain of mAb. N-glycan modifications of the Fc domain are known to be critical for the antibody integrity and its ability to interact with Fc receptors [11]. Based on our previous results with the acid alpha-glucosidase (GAA)-AMFA [8,9], it was predictable that the antibody-AMFA would also be addressed into the lysosome but it was uncertain if the antibody-AMFA would be degraded. To provide a therapeutic advantage, antibody engineering must maintain unaltered all the parameters essential to its efficacy, including antigen binding, antibody stability and interactions with Fc-gamma receptors (FcγR), essential for antibody effector functions, or FcRn, involved in antibody half-life and biodistribution. FcγR are expressed on the hematopoietic cells and are involved in the immune response such as antibody dependent cell cytotoxicity or phagocytosis. In contrast, FcRn is expressed in many cell types such as endothelial, epithelial, or hematopoietic cells [[12], [13], [14]]. It is the main receptor for immunoglobulin G (IgG) recycling and transcytosis thus ensuring the prolonged half-life of mAb [12,15,16]. It binds IgG at acidic pH in the endosomes and recycles them back to extracellular medium at physiological pH [17,18]. In this context, the binding of antibody-AMFA to FcRn would ensure antibody recycling to the cell surface and thus avoid lysosomal proteolysis.

In this paper, two anti-TNFα mAbs, infliximab (IFX) and adalimumab (ADA), were grafted with AMFA on the N-glycans of their heavy chains and used to investigate the impact of AMFA engineering on their recycling. We provided in vitro evidences with different molecular tools that the conjugation of AMFA onto mAb does not alter FcRn recognition and antibody recycling, and increases the drug delivery in cells via M6PR. Indeed, a greater fluorescence is observed in cells treated with IFX-3AMFA comparing to IFX. In addition, the excess of AMFA reverses IFX-3AMFA uptake. Functional FcRn binding was supported by in vivo experiments, showing that IFX-AMFA and ADA-AMFA have a similar pharmacokinetic profile to their unmodified counterpart.