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, 10 (1), 1464

Anti-D Monoclonal Antibodies From 23 Human and Rodent Cell Lines Display Diverse IgG Fc-glycosylation Profiles That Determine Their Clinical Efficacy

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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 et al. Sci Rep.

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.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Glycosylation of anti-D IgG-Fc. (a) Cartoon of the branched oligosaccharide chain covalently attached to Asn297 of each Fc in the Cγ2 domain of IgG. The sugar linkages are shown. Dotted lines indicate the structures that may or may not be present on different glycans. (b) Summary of glycosylation of anti-Ds. Bar chart showing the average glycosylation of IgG1 mAb-Ds produced from human B, mouse HH + NS0, hamster CHO and rat YB2/0 cell lines, compared to Rhophylac anti-D. The percentage of glycans with fucose, galactose, agalactose (G0), monogalactose (G1), digalactose (G2), sialic acid and bisecting GlcNAc of the total samples of each cell line group is illustrated.
Figure 2
Figure 2
Functional activities of anti-D in ADCC assay. (a) The anti-D dependent lysis of red cells by NK cells is shown throughout the dilution range of antibody activity. Rhophylac anti-D is compared to mAb-Ds from human B cell lines, mouse cell lines, and hamster CHO and rat YB2/0 cell lines. Error bars indicate standard deviation (±s.d.). (b) Correlation between ADCC activities of anti-Ds and their glycosylation. Comparison is made of the percentage fucosylation, sialylation, galactosylation and bisecting GlcNAc with the percentage lysis of the anti-Ds (mAb-Ds and Rhophylac anti-D) shown at 25 ng/ml (left column) and 750 ng/ml (right column). The Pearson correlation coefficients for ADCC lysis and fucosylation are shown on the graphs, both were significant to p < 0.0001. There was no correlation between ADCC activity and percentage sialylation, galactosylation or bisection.

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