Anti-D monoclonal antibodies from 23 human and rodent cell lines display diverse IgG Fc-glycosylation profiles that determine their clinical efficacy

Belinda M Kumpel,Natalia I Olovnikova,Pauline M Rudd,Radka Saldova,Rick Kapur,Jodie L Abrahams,Gestur Vidarsson,Agnes Hipgrave Ederveen,Manfred Wuhrer,Carolien A M Koeleman,Kathryn L Armour

doi:10.1038/s41598-019-57393-9

Abstract

Anti-D immunoglobulin (Anti-D Ig) prophylaxis prevents haemolytic disease of the fetus and newborn. Monoclonal IgG anti-Ds (mAb-Ds) would enable unlimited supplies but have differed in efficacy in FcγRIIIa-mediated ADCC assays and clinical trials. Structural variations of the oligosaccharide chains of mAb-Ds are hypothesised to be responsible. Quantitative data on 12 Fc-glycosylation features of 23 mAb-Ds (12 clones, 5 produced from multiple cell lines) and one blood donor-derived anti-D Ig were obtained by HPLC and mass spectrometry using 3 methods. Glycosylation of mAb-Ds from human B-lymphoblastoid cell lines (B) was similar to anti-D Ig although fucosylation varied, affecting ADCC activity. In vivo, two B mAb-Ds with 77–81% fucosylation cleared red cells and prevented D-immunisation but less effectively than anti-D Ig. High fucosylation (>89%) of mouse-human heterohybridoma (HH) and Chinese hamster ovary (CHO) mAb-Ds blocked ADCC and clearance. Rat YB2/0 mAb-Ds with <50% fucosylation mediated more efficient ADCC and clearance than anti-D Ig. Galactosylation of B mAb-Ds was 57–83% but 15–58% for rodent mAb-Ds. HH mAb-Ds had non-human sugars. These data reveal high galactosylation like anti-D Ig (>60%) together with lower fucosylation (<60%) as safe features of mAb-Ds for mediating rapid red cell clearance at low doses, to enable effective, inexpensive prophylaxis.

Highlights

Anti-D immunoglobulin is a very safe and effective prophylactic therapy to prevent haemolytic disease of the fetus and newborn (HDFN)
Glycosylation of intravenous immunoglobulin (IVIG) was similar to other products[26] and that of the IgG anti-D purified from Rhophylac 300 anti-D Ig (Rhophylac) agreed with an earlier report[25]
Glycosylation profiles of the monoclonal antibodies (mAb-Ds) depended on the producer cell lines

Summary

Introduction

Anti-D immunoglobulin (anti-D Ig, RhIG) is a very safe and effective prophylactic therapy to prevent haemolytic disease of the fetus and newborn (HDFN). Anti-D Ig preparations consist of IgG fractionated from pooled plasma of hyperimmunised D-negative donors. Prophylactic anti-D accelerates the clearance of fetal D-positive red blood cells (RBC) from the maternal circulation[14], preventing D-immunisation which may otherwise result in HDFN. Anti-D monoclonal antibodies (mAb-Ds) would be safe, inexpensive, standardised products potentially capable of replacing anti-D Ig. Several groups have made mAb-Ds and tested them in relevant biological assays in vitro and in human studies of RBC clearance and prevention of D-immunisation. Human IgG has a highly conserved branched glycan chain covalently attached to Asn[297] of each Cγ2 domain (Fig. 1a). This glycan contains variable amounts of fucose, galactose, sialic acid, and bisecting N-acetylglucosamine (GlcNAc). We have found that alloimmune IgG1 responses against platelets and RBC antigens, including anti-D, are characterised by low fucosylation and increased galactosylation in most sera[22,23,24] as well as in the anti-D component of anti-D Ig preparations[25]

Methods

Results

Conclusion