Glycoengineering Approach to Half-Life Extension of Recombinant Biotherapeutics

Abstract

The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.

Glycosaminoglycan Conjugation for Improving the Duration of Therapeutic Action of Glucagon-Like Peptide-1

“…24−28 In addition, we have recently shown that chondroitin (CH), a type of glycosaminoglycan (GAG) composed of disaccharide repeats of glucuronic acid (GlcA) and N -acetylgalactosamine (GalNAc), improves the PK of protein drugs. 29 CH-conjugated interferon fully retains its bioactivity, and its plasma activity lasts 100 h after intravenous (iv) injection.…”

Emerging therapies for haemophilia ‐ Global perspective

“…To date, fusion and PEGylation technologies have been successfully used to improve the pharmacokinetic properties of CFCs including half‐life extension. In the fusion approach, clotting factors FVIIa, FVIII and FIX have been linked to several long half‐life moieties including the fragment crystallisable (Fc), recombinant human albumin, carboxy terminal peptide (CTP) and polysialic acid (PSA) . The FC and albumin fusion with rFVIII and FIX have completed their phase 3 studies whilst the CTP and PSA approaches are currently ongoing in their phase 1 and phase 2 clinical developments.…”

Glycotherapy: New Advances Inspire a Reemergence of Glycans in Medicine

“…A new approach aiming to harness the beneficial effects of sialylation has been the incorporation of polysialic acid (PSA) onto therapeutic proteins produced in engineered HEK cells (Chen et al, 2012a). A polysialylated anti-HER2 single-chain variable fragment (scFv) showed an almost 30-fold increase in blood half-life but, unfortunately, PSA attachment also decreased receptor-mediated endocytosis.…”