OC 15.3 - Terminal sialylation of commercial rVWF concentrate is responsible for attenuated macrophage-mediated clearance via the MGL receptor.

Sunday, June 23, 2024

10:00 – 10:15 ICT

Room: 110 A-C

Presenting Author(s)

Ciara Byrne, PhD

Postdoc
RCSI Dublin
Dublin, Dublin, Ireland

Co-Author(s)

SW

Soracha Ward

Post-Doctoral Fellow
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.
Dublin, Dublin, Ireland
AM

Alan B. Moran

Scientist
Ludger Ltd
Oxfordshire, England, United Kingdom
DS

Daniel Spencer

Scientist
Ludger Ltd
Oxfordshire, England, United Kingdom
RG

Richard Gardner

Scientist
Ludger Ltd
Oxfordshire, England, United Kingdom
MM

Marco Monopoli

Senior Lecturer, Dept of Chemistry
RCSI Dublin
Dublin, Dublin, Ireland
PL

Patricia Lopes

PhD student
RCSI Dublin
Dublin, Dublin, Ireland
Alain Chion, PhD

Post-Doctoral Fellow
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.
Dublin, Dublin, Ireland
Jamie O'Sullivan

Lecturer/StAR Investigator in Vascular Biology
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland.
Dublin, Dublin, Ireland
Ross Baker, MB BS BMedSc FRACP, FRCPA

Professor
Western Australia Centre for Thrombosis and Haemostasis, Perth Blood Institute, Murdoch University, Perth, Australia
Perth, Western Australia, Australia
Peter L. Turecek, PhD, PharmD

Scientific Expert, PDT Global Medical Affairs
Baxalta Innovations GmbH, a Takeda company, Vienna, Austria
Vienna, Austria
JO

James S. O'Donnell

Professor of Vascular Biology
Irish Centre for Vascular Biology, School of Pharmacy and Biomolecular Sciences, RCSI University of Medicine and Health Sciences, Dublin, Ireland.
Dublin, Dublin, Ireland

Background: Phase 1 and 3 clinical trials have shown that the half-life of recombinant human VWF (rVWF) expressed in CHO cells (Takeda) is significantly longer than plasma-derived (pd-)VWF concentrates.

Aims: We hypothesized that differences in terminal sialylation mediate the attenuated clearance of rVWF.

Methods: Clearance of r- and pd-VWF were compared in VWF-/- mice in the presence or absence of clodronate-induced macrophage depletion. Binding to macrophages and HepG2 cells was assessed by flow cytometry. VWF interaction with purified macrophage galactose-type lectin (MGL) and asialoglycoprotein 1 (ASGPR1) receptors was studied in plate-binding assays. N- and O-glycomes of pd-VWF and rVWF were analysed using lectin-binding arrays and mass spectrometry (Ludger Glycosciences).

Results: Consistent with human data, rVWF clearance was significantly reduced in VWF-/- mice compared to pd-VWF (MRT 55.3 versus 41.8mins; p< 0.05) (Fig.1A). rVWF clearance was attenuated following clodronate-induced macrophage depletion (Fig.1B). Clearance rates for asialo-pdVWF and asialo-rVWF were identical (Fig.1C). Consistent with the in vivo prolongation in rVWF half-life, rVWF binding to murine macrophages was significantly (p=0.012) reduced compared to pd-VWF (Figs.1D-E). Furthermore, significantly reduced MGL-and ASGPR binding was seen for rVWF compared to pd-VWF (Fig.1F). SNA lectin (binds α2-6 linked sialic acid) binding to rVWF was markedly reduced compared to pd-VWF (p=0.0005) (Fig.2A). Conversely, Maackia agglutinin (binds α2-3 linked sialic acid) binding to rVWF was significantly increased (p=0.033) (Fig.2B). MS analysis confirmed that α2-6 linked sialylation represented the majority of terminal sialylation on pd-VWF (Fig.2C). Conversely, rVWF synthesized in CHO cells did not express any α2-6 linked sialic acid. Instead, α2-3 linked sialylation was significantly increased on rVWF, which also had less exposed β-Gal residues.

Conclusion(s): Our findings demonstrate that altered sialylation of rVWF is responsible for attenuated macrophage-mediated clearance. This effect, mediated in large part via the MGL receptor, might explain the extended plasma half-life of rVWF.