Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies

Yu, Xiaojie; Baruah, Kavitha; Harvey, David J.; Vasiljević, Snežana; Alonzi, Dominic S.; Song, Byeong Doo; Higgins, Matthew K.; Bowden, Thomas A.; Scanlan, Christopher N.; Crispin, Max

doi:10.1021/ja4014375

Cited by 82 publications

(126 citation statements)

References 67 publications

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“…This switch in receptor specificity coincides with a switch in effector function in vivo, as sialylated IgG suppresses inflammation in mouse models of autoimmunity. Recent structural data support an earlier computational model that sialylation increases the conformational flexibility of the C H 2 domain, sampling conformations compatible with type II FcR binding (8,20,36).…”

Section: Discussionsupporting

confidence: 62%

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

Fiebiger

Maamary

Pincetic

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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The antiinflammatory activity of intravenous immunoglobulin (IVIG) is dependent on the presence of sialic acid in the core IgG fragment crystallizable domain (Fc) glycan, resulting in increased conformational flexibility of the C H 2 domain with corresponding modulation of Fc receptor (FcR) binding specificity from type I to type II receptors. Sialylated IgG Fc (sFc) increases the activation threshold of innate effector cells to immune complexes by stimulating the upregulation of the inhibitory receptor FcγRIIB. We have found that the structural alterations induced by sialylation can be mimicked by specific amino acid modifications to the C H 2 domain. An IgG Fc variant with a point mutation at position 241 (F→A) exhibits antiinflammatory activity even in the absence of sialylation. F241A and sFc protect mice from arthritis in the K/BxN-induced model and, in the T cell-mediated experimental autoimmune encephalomyelitis (EAE) mouse model, suppress disease by specifically activating regulatory T cells (T reg cells). Protection by these antiinflammatory Fcs in both antibody-and T cell-mediated autoimmune diseases required type II FcRs and the induction of IL-33. These results further clarify the mechanism of action of IVIG in both antibody-and T cellmediated inflammatory diseases and demonstrate that Fc variants that mimic the structural alterations induced by sialylation, such as F241A, can be promising therapeutic candidates for the treatment of various autoimmune disorders.IgG Fc sialylation | conformational change | antiinflammatory | T reg cells

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Section: Discussionsupporting

confidence: 62%

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

Fiebiger

Maamary

Pincetic

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…In this case, we identified GnT1 activity as being modulated, showing that not only trimming, but also the initial part of the elongation is affected by surface glycan interaction, in line with other reports that suggested the effect of N-glycan-carbohydrate interaction on glycan processing and protein structure (44,45,50). For example, studies performed on antibodies showed the role of a phenylalanine on the IgG glycan distribution by modifying the protein stability (37,38).…”

Section: Discussionmentioning

confidence: 99%

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Losfeld¹,

Scibona²,

Lin³

et al. 2017

FASEB j.

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To study how the interaction between N-linked glycans and the surrounding amino acids influences oligosaccharide processing, we used protein disulfide isomerase (PDI), a glycoprotein bearing 5 N-glycosylation sites, as a model system and expressed it transiently in a Chinese hamster ovary (CHO)-S cell line. PDI was produced as both secreted Sec-PDI and endoplasmic reticulum-retained glycoprotein (ER)-PDI, to study glycan processing by ER and Golgi resident enzymes. Quantitative site-specific glycosylation profiles were obtained, and flux analysis enabled modeling site-specific glycan processing. By altering the primary sequence of PDI, we changed the glycan/ protein interaction and thus the site-specific glycoprofile because of the improved enzymatic fluxes at enzymatic bottlenecks. Our results highlight the importance of direct interactions between N-glycans and surface-exposed amino acids of glycoproteins on processing in the ER and the Golgi and the possibility of changing a site-specific N-glycan profile by modulating such interactions and thus the associated enzymatic fluxes. Altering the primary protein sequence can therefore be used to glycoengineer recombinant proteins.-Losfeld, M.-E., Scibona, E., Lin, C.-W., Villiger, T. K., Gauss, R., Morbidelli, M., Aebi, M. Influence of protein/glycan interaction on site-specific glycan heterogeneity. FASEB J. 31, 4623-4635 (2017). www.fasebj.org KEY WORDS: glycoengineering • glycobiology • glycoprotein maturation • Golgi processing • enzymatic flux N-glycosylation is a major posttranslational modification of proteins that modulates folding, maturation, trafficking, and secretion, as well as specific protein functions (1-3). Contrary to many other posttranslational modifications, N-glycans are composed of many monosaccharide units, creating a large diversity of glycan structures (4, 5). In eukaryotic cells, the N-glycosylation occurs in the endoplasmic reticulum (ER) after a conserved pathway. The oligosaccharide GlcNAc 2 Man 9 Glc 3 is assembled on the lipid dolicholpyrophosphate at the membrane of the ER and then is covalently linked to the side-chain amide of the asparagine residue in the sequon N-X-S/T by oligosaccharyltransferase (OST) (1, 6, 7). Multiple sites on one glycoprotein can be modified by the OST complex and with the increasing complexity of multicellular organism, an increasing number of N-glycosylation sites in the glycoproteome are observed. It is estimated that up to 6000 different sites are modified by OST in mammalian cells (8). In the second phase of the N-glycosylation pathway, the protein-linked glycan is trimmed by glucosidases and mannosidases (1, 9) and is subsequently modified by different glycosyltransferases spatially distributed along the secretory pathway. N-glycan processing enzymes have defined substrate specificities and locations in the secretory pathway (9, 10), and their expression level can be regulated upon internal and external signals.Glycan maturation in the secretory pathway is not a template-driven process. The...

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“…6,19,20 The impact of the presence of terminal sialic acid residues is also uncertain. [21][22][23][24][25][26][27][28][29][30] Indeed, the discrepancies in the methods used for evaluating biological activity, the variability in the type and level of sialylation, as well as the lack of a systematic in-depth glycan characterization, all contribute to the ambiguity of its role in IgG functions. In humans, sialic acids can be attached to the Fc-glycans either on the C3-or the C6-hydroxyl group of the terminal galactose, through the action of a2,3-sialyltransferases (ST3) or the a2,6-sialyltransferase-1 (ST6).…”

Section: Introductionmentioning

confidence: 99%

“…22 Contradictory results were also reported on sialylated Fcs' ability to bind FcgRIIIa, but using dissimilar IgG preparations and affinity measurement protocols. 21,28 In other studies assessing the anti-inflammatory properties of a2,6-sialylated IgGs, 21,24,26,30 blood-derived or recombinant antibodies were enriched by affinity chromatography using Sambucus Nigra agglutinin-resins that were shown to catch mainly IgGs bearing Fab-sialylated glycans to the detriment of Fc-sialylated species. 24,32,37 While the specific involvement of Fc's a2,6-sialylation in anti-inflammatory activity was shown by Anthony et al in their model, 35 which receptor the sialylated Fcs are targeting remains controversial.…”

Section: Introductionmentioning

confidence: 99%

“…41,42 The very high number of potent mutations and the possibility that the mutations themselves rather than the resulting altered glycan structure may affect antibody function render the understanding of Fc glycan structure/function a complex task. 28 Most cell engineering efforts directed toward altering glycosylation have focused on the manipulation of Chinese hamster ovary (CHO) cell lines because they are widely used for the manufacturing of therapeutic mAbs. 43 Their glycosylation machinery is very similar to that of humans, but they lack a functional ST6, limiting their sialylation capability to the addition of a2,3SA residues.…”

Section: Introductionmentioning

confidence: 99%

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Production of α2,6-sialylated IgG1 in CHO cells

Raymond

Robotham

Spearman

et al. 2015

mAbs

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The presence of a2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized a2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan a2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human a2,6-sialyltransferase 1 (ST6) and b1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by a2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The a2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl-and sialyl-transferases substrate specificities.

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Engineering Hydrophobic Protein–Carbohydrate Interactions to Fine-Tune Monoclonal Antibodies

Cited by 82 publications

References 67 publications

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

Protection in antibody- and T cell-mediated autoimmune diseases by antiinflammatory IgG Fcs requires type II FcRs

Influence of protein/glycan interaction on site‐specific glycan heterogeneity

Production of α2,6-sialylated IgG1 in CHO cells

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