Glycan Structure Determinants for Cation-Independent Mannose 6-Phosphate Receptor Binding and Cellular Uptake of a Recombinant Protein

Abstract

The cation-independent mannose 6-phosphate receptor (CI-MPR) plays a critical role in intracellular transport of lysosomal enzymes as well as the uptake of recombinant proteins. To define the minimal glycan structure determinants necessary for receptor binding and cellular uptake, we synthesized a series of glycans containing mono-, di-, tri-, tetra-, and hexamannoses terminated with either one or two phosphates for conjugating to a model protein, recombinant human acid α-glucosidase. A high affinity interaction with the CI-MPR can be achieved for the enzyme conjugated to a dimannose glycan with a single phosphate. However, tightest binding to a CI-MPR affinity column was observed with a hexamannose structure containing two phosphates. Moreover, maximal cellular uptake and a 5-fold improvement in in vivo potency were achieved when the bisphosphorylated hexamannose glycan is conjugated to the protein by a β linker. Nevertheless, even a monophosphorylated dimannose glycan conjugate showed stronger binding to the receptor affinity column, higher cellular uptake, and significantly greater in vivo efficacy compared to the unconjugated protein which contains a low level of high affinity glycan structure. These results demonstrate that the phosphorylated dimannose moiety appears to be the minimal structure determinant for enhanced CI-MPR binding and that the orientation of the glycan is critical for maximum receptor interaction. In summary, we have improved the understanding of the mechanism of CI-MPR binding and developed a simple alternative for CI-MPR targeting.

Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies

“…Design of a manufacturing process allowing for precise regulation of protein N -glycosylation is highly advantageous and urgently needed, as N -glycosylation often profoundly affects biological efficacy and pharmacokinetic properties of the manufactured drug substance [ 4 , 5 , 6 , 7 ]. Indeed, numerous preclinical studies have supported the strong relationship between the glycosylation structure decorating therapeutic proteins and their clinical properties, including protein stability, solubility, circulatory half-life, and efficacy [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. A well-characterized example of an N -glycosylation pattern affecting therapeutic efficacy was shown with the removal of the α-1,6-fucose residue attached to the innermost N -acetylglucosamine (GlcNAc) of the N -glycosylation core present on some anticancer antibodies such as rituximab and trastuzumab.…”

Chemoenzymatic Synthesis and Receptor Binding of Mannose-6-Phosphate (M6P)-Containing Glycoprotein Ligands Reveal Unusual Structural Requirements for M6P Receptor Recognition

“…7–14 For example, conjugation of synthetic M6P oligosaccharides to the recombinant human acid α -glucosidase (rhGAA) through selectively oxidized sialic acid or galactose residues in the N -glycans of the enzyme led to M6P-modified rhGAA that showed an enhanced uptake and demonstrated up to 5-fold greater potency than the unmodified rhGAA in a Pompe disease mouse model. 9–11 Despite these promising studies, chemical conjugation suffers from heterogeneity in linkage sites, potential instability of the conjugates, and introduction of unnatural linkers that may be immunogenic in humans. Moreover, as to the molecular recognition between CI-MPR receptor and M6P-glycoproteins, it is still not fully understood how the location of the M6P moiety in the oligosaccharide, the valency of the M6P ligands, and/or the oligosaccharide context affect the binding and affinity for the M6P receptor CI-MPR.…”

Glyco-engineering strategies for the development of therapeutic enzymes with improved efficacy for the treatment of lysosomal storage diseases

“…In contrast to the unsuccessful targeting of HP-GAA, a series of approaches employing chemical conjugation of M-6-P glycans have been shown to improve the clearance of accumulated glycogen in the skeletal muscles of Pompe mice as well as MPR binding and subsequent targeting to lysosomes (28 - 32) . In a proof-of-concept study, Genzyme researchers isolated M-6-P glycans from recombinant GLA (Agalsidase beta) and, after derivatization to glycosylhdrazines, attached these M-6-P glycan to periodate-oxidized sialic acids of GAA using carbonyl chemistry (28) .…”

“…Clinical trials examining the safety and efficacy of this GAA are currently underway (33) . Recently, glycan structures for efficient CI-MPR binding were determined using the chemical conjugation of various glycans containing phosphate groups (32) . Zhou et al , reported that the tightest binding to CI-MPR was achieved with a hexamnnose structure containing two phosphates, while the phosphorylated dimannose moiety appears to be the minimal structure for binding.…”