Graphical Abstract Highlights d NK cell-activating antibodies are selectively transferred across the placenta d Digalactosylated Fc glycans are preferentially transferred across the placenta d Digalactosylated antibodies bind more effectively to FcRn and FCGR3A d Although immature, neonatal NK cells are highly responsive to immune complexes SUMMARY Despite the worldwide success of vaccination, newborns remain vulnerable to infections. While neonatal vaccination has been hampered by maternal antibody-mediated dampening of immune responses, enhanced regulatory and tolerogenic mechanisms, and immune system immaturity, maternal pre-natal immunization aims to boost neonatal immunity via antibody transfer to the fetus. However, emerging data suggest that antibodies are not transferred equally across the placenta. To understand this, we used systems serology to define Fc features associated with antibody transfer. The Fc-profile of neonatal and maternal antibodies differed, skewed toward natural killer (NK) cell-activating antibodies.This selective transfer was linked to digalactosylated Fc-glycans that selectively bind FcRn and FCGR3A, resulting in transfer of antibodies able to efficiently leverage innate immune cells present at birth. Given emerging data that vaccination may direct antibody glycosylation, our study provides insights for the development of next-generation maternal vaccines designed to elicit antibodies that will most effectively aid neonates. Antibodies against pertussis derived filamentous hemagglutinin (FHA), pertactin (PTN), fimbriae (FIM), and pertussis toxin (PTX) antigens were compared in 14 mother:cord pairs. (A) The flow cytometric plots depict the gating strategy for antibody dependent cellular phagocytosis (ADCP). (B) The connected dot-plot shows the phagocytic activity across mother:cord pairs. (C) The box-and-whisker plot shows the transfer ratio of ADCP. The dotted line indicates a 100% transfer efficiency (equivalent levels across both compartments). (D) The flow plots highlight the gating strategy for antibody dependent neutrophil phagocytosis (ADNP). (E) The dot-plot shows the relationship between ADNP activity across mother:cord pairs for each antigen-specificity. (F) The whisker plots show the transfer ratio for ADNP. (G) The flow plots highlighting the gating strategy for the NK cell activation assay. (H-J) The dot-line plots show NK-dependent degranulation plotted as the percentage of NK cells positive for CD107a (H), IFNg (I), and MIP-1b (J). (K-M) The whisker plots depict the transfer ratio across the three NK cell activation markers, CD107a (K), IFNg (L), and MIP-1b (M).

No abstract

SummaryN-linked glycosylation affects the potency, safety, immunogenicity, and pharmacokinetic clearance of several therapeutic proteins including monoclonal antibodies. A robust control strategy is needed to dial in appropriate glycosylation profile during the course of cell culture processes accurately. However, N-glycosylation dynamics remains insufficiently understood owing to the lack of integrative analyses of factors that influence the dynamics, including sugar nucleotide donors, glycosyltransferases, and glycosidases. Here, an integrative approach involving multi-dimensional omics analyses was employed to dissect the temporal dynamics of glycoforms produced during fed-batch cultures of CHO cells. Several pathways including glycolysis, tricarboxylic citric acid cycle, and nucleotide biosynthesis exhibited temporal dynamics over the cell culture period. The steps involving galactose and sialic acid addition were determined as temporal bottlenecks. Our results show that galactose, and not manganese, is able to mitigate the temporal bottleneck, despite both being known effectors of galactosylation. Furthermore, sialylation is limited by the galactosylated precursors and autoregulation of cytidine monophosphate-sialic acid biosynthesis.

Small-molecule control of antibody Nglycosylation in engineered mammalian cells The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Chang, Michelle M. et al. "Small-molecule control of antibody Nglycosylation in engineered mammalian cells." Nature Chemical Biology 15, 7 (May 2019