IgG antibodies contain a conserved -glycosylation site on the Fc domain to which a complex, biantennary glycan is attached. The fine structures of this glycan modulate antibody effector functions by affecting the binding affinity of the Fc to diverse Fc receptor family members. For example, core fucosylation significantly decreases antibody-dependent cellular cytotoxicity (ADCC), whereas terminal α2,6-sialylation plays a critical role in the anti-inflammatory activity of human i.v. immunoglobulin therapy. The effect of specific combinations of sugars in the glycan on ADCC remains to be further addressed, however. Therefore, we synthesized structurally well-defined homogeneous glycoforms of antibodies with different combinations of fucosylation and sialylation and performed side-by-side in vitro FcγR-binding analyses, cell-based ADCC assays, and in vivo IgG-mediated cellular depletion studies. We found that core fucosylation exerted a significant adverse effect on FcγRIIIA binding, in vitro ADCC, and in vivo IgG-mediated cellular depletion, regardless of sialylation status. In contrast, the effect of sialylation on ADCC was dependent on the status of core fucosylation. Sialylation in the context of core fucosylation significantly decreased ADCC in a cell-based assay and suppressed antibody-mediated cell killing in vivo. In contrast, in the absence of fucosylation, sialylation did not adversely impact ADCC.

Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).

Antibodies are immunoglobulins that play essential roles in immune systems. All antibodies are glycoproteins that carry at least one or more conserved N-linked oligosaccharides ( N-glycans) at the Fc domain. Many studies have demonstrated that both the presence and fine structures of the attached glycans can exert a profound impact on the biological functions and therapeutic efficacy of antibodies. However, antibodies usually exist as mixtures of heterogeneous glycoforms that are difficult to separate in pure glycoforms. Recent progress in glycoengineering has provided useful methods that enable production of glycan-defined and site-selectively modified antibodies for functional studies and for improved therapeutic efficacy. This review highlights major approaches in glycoengineering of antibodies with a focus on recent advances in three areas: glycoengineering through glycan biosynthetic pathway manipulation, glycoengineering through in vitro chemoenzymatic glycan remodeling, and glycoengineering of antibodies for site-specific antibody–drug conjugation.

OBJECTIVESThe objective of the present study was to evaluate short- and mid-term outcomes of the left subclavian artery (LSA) chimney stent implantation (LSACSI) during thoracic endovascular aortic repair (TEVAR), and to summarize our experience with this technique.METHODSFrom June 2010 to September 2012, 59 patients (49 men; mean age of 57.4 ± 13.3 years, range from 26 to 83 years) who underwent TEVAR and LSACSI were enrolled. Patients suffered from Stanford type B aortic dissection (n = 27), penetrating aortic ulcer (n = 18), aortic arch aneurysm (n = 9), pseudoaneurysm of the aortic arch (n = 4) and proximal type I endoleak after TEVAR of aortic dissection (n = 1). Elective settings were performed in 72% and emergent in 38% of all patients. Follow-up was performed at postoperative 3 months, 6 months and yearly thereafter.RESULTSThe technical success rate was 98.3% (58/59), and 69 thoracic stent grafts were used. Sixty-two chimney stents, including 55 uncovered and 7 covered stents, were implanted in 59 LSAs. The overall immediate endoleak rate was 15.3% (9/59); type I endoleak was observed in 5 patients and type II in 4 patients. The difference in the immediate endoleak rate related to the anatomy between the outer and the inner curvature was statistically significant (35 vs 4%, P = 0.018). Chimney stent compression was observed in 3 patients and another stent was deployed inside the first one. Perioperative complications included stroke (3.4%, 2/59) and left upper limb ischaemia (1.7%, 1/59). The median follow-up period was 16.5 (range 1–39 months). The mortality rate during follow-up was 5.4% (3/56). Complications during follow-up included endoleak [overall, n = 8 (14.3%, 8/56); type I, n = 5; type II, n = 3], retrograde type A aortic dissection (n = 1), collapse (n = 3, 5.4%) or occlusion (n = 2, 3.6%) of the chimney stent.CONCLUSIONSShort- and mid-term results showed that it is feasible to preserve the patency of the LSA in TEVAR with the chimney technique for thoracic aortic pathologies close to the LSA. However, TEVAR combined with LSACSI was not advocated for lesions located at the outer curve of the aortic arch due to a high possibility of endoleak.