Production,characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

The effector functions of therapeutic antibodies are strongly affected by the specific glycans added to the Fc domain during post-translational processing. Antibodies bearing high levels of N-linked mannose-5 glycan (Man5) have been reported to exhibit enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) compared with antibodies with fucosylated complex or hybrid glycans. To better understand the relationship between antibodies with high levels of Man5 and their biological activity in vivo, we developed an approach to generate substantially homogeneous antibodies bearing the Man5 glycoform. A mannosidase inhibitor, kifunensine, was first incorporated in the cell culture process to generate antibodies with a distribution of high mannose glycoforms. Antibodies were then purified and treated with a mannosidase for trimming to Man5 in vitro. This 2-step approach can consistently generate antibodies with > 99% Man5 glycan. Antibodies bearing varying levels of Man5 were studied to compare ADCC and Fcγ receptor binding, and they showed enhanced ADCC activity and increased binding affinity to the FcγRIIIA. In addition, the clearance rate of antibodies bearing Man8/9 and Man5 glycans was determined in a pharmacokinetics study in mice. When compared with historical data, the antibodies bearing the high mannose glycoform exhibited faster clearance rate compared with antibodies bearing the fucosylated complex glycoform, while the pharmacokinetic properties of antibodies with Man8/9 and Man5 glycoforms appeared similar. In addition, we identified the presence of a mannosidase in mouse serum that converted most Man8/9 to Man6 after 24 h.     

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC₅₀ values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.     

Glycoform-resolved FcɣRIIIa affinity chromatography–mass spectrometry

ABSTRACT

Determination of the impact of individual antibody glycoforms on Fc?RIIIa affinity, and consequently antibody-dependent cell-mediated cytotoxicity (ADCC) previously required high purity glycoengineering. We hyphenated Fc?RIIIa affinity chromatography to mass spectrometry, which allowed direct affinity comparison of glycoforms of intact monoclonal antibodies. The approach enabled reproduction and refinement of known glycosylation effects, and insights on afucosylation pairing as well as on low-abundant, unstudied glycoforms. Our method greatly improves the understanding of individual glycoform structure–function relationships. Thus, it is highly relevant for assessing Fc-glycosylation critical quality attributes related to ADCC.     

Production of an endo-beta-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein

Enterococcus faecalis is associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man(5)- to Man(9)-GlcNAc(2)), to support growth of E. faecalis was investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linked N-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action of Streptomyces plicatus endo-beta-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only one N-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity in E. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans by E. faecalis may play a role in the survival and persistence of this nosocomial pathogen in vivo.     

Isolation of a mutant Arabidopsis plant that lacks N-acetyl glucosaminyl transferase I and is unable to synthesize Golgi-modified complex N-linked glycans.

The complex asparagine-linked glycans of plant glycoproteins, characterized by the presence of beta 1-->2 xylose and alpha 1-->3 fucose residues, are derived from typical mannose9(N-acetylglucosamine)2 (Man9GlcNAc2) N-linked glycans through the activity of a series of glycosidases and glycosyl transferases in the Golgi apparatus. By screening leaf extracts with an antiserum against complex glycans, we isolated a mutant of Arabidopsis thaliana that is blocked in the conversion of high-manne to complex glycans. In callus tissues derived from the mutant plants, all glycans bind to concanavalin A. These glycans can be released by treatment with endoglycosidase H, and the majority has the same size as Man5GlcNAc1 glycans. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, the mutant cells synthesize Man9GlcNAc2 and Man8GlcNAc2 glycans, suggesting that the biochemical lesion in the mutant is not in the biosynthesis of high-mannose glycans in the endoplasmic reticulum but in their modification in the Golgi. Direct enzyme assays of cell extracts show that the mutant cells lack N-acetyl glucosaminyl transferase I, the first enzyme in the pathway of complex glycan biosynthesis. The mutant plants are able to complete their development normally under several environmental conditions, suggesting that complex glycans are not essential for normal developmental processes. By crossing the complex-glycan-deficient strain of A. thaliana with a transgenic strain that expresses the glycoprotein phytohemagglutinin, we obtained a unique strain that synthesizes phytohemagglutinin with two high-mannose glycans, instead of one high-mannose and one complex glycan.     

Fc glycans terminated with N-acetylglucosamine residues increase antibody resistance to papain

Glycosylation in the CH2 domain of Fc is required for immunoglobulins G (IgGs) to exhibit immune effector functions including complement-dependent cytotoxicity (CDC) and antibody-dependent (Ab-dependent) cellular cytotoxicity (ADCC). We recently established that glycosylated Abs are more resistant to papain digestion than non-glycosylated IgGs (Biochem. Biophys. Res. Commun. 2006, 341, 797-803). To test whether specific Fc glycan structures affect Ab resistance to papain, we used in vitro glycoengineering methods to prepare homogeneous Ab glycoforms terminated with either sialic acid (G2S2), beta-galactose (G2), or N-acetylglucosamine (G0) and subjected them to papain digestions. Analyses of aliquots taken at different times during the digestions by matrix-assisted laser desorption-time-of-flight-mass spectroscopy (MALDI-TOF-MS) and high-performance liquid chromatography (HPLC) methods showed that the G0 glycoform was at least two times more resistant to papain digestion than the G2 and G2S2 glycoforms. The increased resistance of the G0 glycoform over the G2 and G2S2 glycoforms was independent of the specific Ab analyzed. A mouse/human chimeric version of Ab1, a fully human version of Ab2, and a humanized version of Ab3 exhibited a similar pattern of glycoform-dependent resistance. These data suggest that terminal sugars of Fc glycans may play important roles in Ab stability and affect resistance to proteases in addition to impacting Ab effector functions.     

Carrier protein-modulated presentation and recognition of an N-glycan: observations on the interactions of Man(8) glycoform of ribonuclease B with conglutinin

Conglutinin is a serum lectin of the innate immune system, which binds high mannose N-glycans when these are appropriately presented on proteins. Here we use the conglutinin-ribonuclease B (RNaseB)-recognition system as a model to investigate the structural basis of selective recognition of protein-bound oligosaccharides by this carbohydrate-binding receptor. Conglutinin shows little binding to the isolated RNaseB-Man(8 )glycoform, and no binding to Man(5-6) glycoforms. In contrast, when the protein moiety is reduced and denatured we observe that conglutinin binds strongly to the isolated RNaseB-Man(8) glycoform and weakly to the Man(5-6) glycoforms. These results are in accord with observations on the binding to the N-glycans in the absence of carrier protein. NMR analyses of native RNaseB-Man(8) and -Man(5-6) glycoforms reveal that the three-dimensional structure of the protein moiety is essentially identical to that of non-glycosylated RNase (RNaseA). Thus there are no perceptible differences between the RNase protein forms that could account for differential availability of the N-glycan for conglutinin-binding. After reduction and denaturation, the NMR spectrum became typical of a non-structured polypeptide, although the conformational preferences of the N-glycosidic linkage were unchanged, and most importantly, the Man(8 )oligosaccharide retained the average conformational behavior of the free oligosaccharide irrespective of the carrier protein fold. This conformational freedom is clearly not translated into full availability of the oligosaccharide for the carbohydrate-recognition protein. We propose, therefore, that the differing bioactivity of the N-glycan is a reflection of the existence of different geometries of presentation of the carbohydrate determinant in relation to the protein surface within the glycan:carrier protein ensemble.     

In vitro galactosylation of human IgG at 1 kg scale using recombinant galactosyltransferase

The Fc effector functions of immunoglobulin G (IgG) antibodies are in part determined by structural features of carbohydrates linked to each of the paired gamma heavy chains in the antibody constant domain (C(H)2). One glycoform that has been shown to be advantageous is G2, where both arms of complex bi-antennary N-glycans terminate in galactose. In vitro treatment with glycosyltransferases can remodel heterogeneous IgG glycoforms, enabling preparation of IgG molecules with homogeneous glycan chains. Here we describe optimization of conditions for use of a soluble recombinant galactosyltransferase in vitro to remodel glycans of human serum IgG, and we demonstrate a scaled-up reaction in which >98% of neutral glycans attached to 1 kg IgG are converted to the G2 glycoform. Removal of glycosylation reagents from the product is achieved in one step by affinity chromatography on immobilized Protein A.     

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety,functionality and efficacy

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as nextgeneration therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.     

Development of a simple and rapid method for producing non-fucosylated oligomannose containing antibodies with increased effector function

Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.     

Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway

A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation.     

The effect of Fc glycan forms on human IgG2 antibody clearance in humans

Several studies using a variety of approaches have investigated the impact of the Fc glycan structure on IgG clearance rates. Most, but not all, of these studies have concluded that glycan structural differences do not affect clearance. Here we investigated the impact of glycan on the clearance of a human antibody in humans. To monitor glycan-dependent changes, a human IgG2 was affinity purified in a single step from serum samples from a human pharmacokinetic study. The glycan profile from the purified antibody samples was determined by RP-HPLC/MS analysis of the 2-aminobenzamide-labeled glycans. Relative levels of high-mannose species (M6-M9) decreased over circulation time. Differences in the individual high-mannose structural isoform clearance rates were measured from extracted ion current profiles. Similar changes to the glycan profile could be achieved through incubation of the antibody in serum in vitro, suggesting that the changes observed in vivo were the result of glycan cleavage, not differential antibody clearance. These results confirm that antibody clearance is not significantly affected by the Fc glycan structure and provide evidence for the presence of circulating mannosidase activity in humans.     

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.Key words: antibodies, IgGs, glycans, oligosaccharides, sialic acid, sialidase, ADCC, CDC, effector functions, cells, Fc receptors, proteases     

Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcγ receptor binding and antibody-dependent cell-mediated cytotoxicity activities

The presence or absence of core fucose in the Fc region N-linked glycans of antibodies affects their binding affinity toward FcγRIIIa as well as their antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, the quantitative nature of this structure-function relationship remains unclear. In this study, the in vitro biological activity of an afucosylated anti-CD20 antibody was fully characterized. Further, the effect of fucose reduction on Fc effector functions was quantitatively evaluated using the afucosylated antibody, its “regular” fucosylated counterpart and a series of mixtures containing varying proportions of “regular” and afucosylated materials. Compared with the “regular” fucosylated antibody, the afucosylated antibody demonstrated similar binding interactions with the target antigen (CD20), C1q and FcγRIa, moderate increases in binding to FcγRIIa and IIb, and substantially increased binding to FcγRIIIa. The afucosylated antibodies also showed comparable complement-dependent cytotoxicity activity but markedly increased ADCC activity. Based on EC₅₀ values derived from dose-response curves, our results indicate that the amount of afucosylated glycan in antibody samples correlate with both FcγRIIIa binding activity and ADCC activity in a linear fashion. Furthermore, the extent of ADCC enhancement due to fucose depletion was not affected by the FcγRIIIa genotype of the effector cells.     

Comparative characterization of the glycosylation profiles of an influenza hemagglutinin produced in plant and insect hosts

The main objective of this study was to characterize the N-linked glycosylation profiles of recombinant hemagglutinin (HA) proteins expressed in either insect or plant hosts, and to develop a mass spectrometry based workflow that can be used in quality control to assess batch-to-batch reproducibility for recombinant HA glycosylation. HA is a surface glycoprotein of the influenza virus that plays a key role in viral infectivity and pathogenesis. Characterization of the glycans for plant recombinant HA from the viral strain A/California/04/09 (H1N1) has not yet been reported. In this study, N-linked glycosylation patterns of the recombinant HAs from both insect and plant hosts were characterized by precursor ion scan-driven data-dependent analysis followed by high-resolution MS/MS analysis of the deglycosylated tryptic peptides. Five glycosylation sites (N11, N23, N276, N287, and N481) were identified containing high mannose type glycans in plant-expressed HAs, and complex type glycoforms for the insect-expressed HA. More than 95% site occupancy was observed for all glycosylation sites except N11, which was 60% occupied. Multiple-reaction monitoring based quantitation analysis was developed for each glycopeptide isoform and the quantitative results indicate that the Man(8) GlcNAc(2) is the dominant glycan for all sites in plant-expressed HAs. The relative abundance of the glycoforms at each specific glycosylation site and the relative quantitation for each glycoform among three HAs were determined. Few differences in the glycosylation profiles were detected between the two batches of plant HAs studied, but there were significant differences between the glycosylation patterns in the HAs generated in plant and insect expression hosts.     

Glycoengineering of the methylotrophic yeast Hansenula polymorpha for the production of glycoproteins with trimannosyl core N-glycan by blocking core oligosaccharide assembly

The initial lipid-linked oligosaccharide Glc(3)Man(9)GlcNAc(2)-dolichyl pyrophosphate (Dol-PP) for N-glycan is synthesized and assembled at the membrane of the endoplasmic reticulum (ER) and subsequently transferred to a nascent polypeptide by the oligosaccharide transferase complex. We have identified an ALG3 homolog (HpALG3) coding for a dolichyl-phosphate-mannose dependent alpha-1,3-mannosyltransferase in the methylotrophic yeast Hansenula polymorpha. The detailed analysis of glycan structure by linkage-specific mannosidase digestion showed that HpALG3 is responsible for the conversion of Man5GlcNAc(2)-Dol-PP to Man(6)GlcNAc(2)-Dol-PP, the first step to attach a mannose to the lipid-linked oligosaccharide in the ER. The N-glycosylation pathway of H. polymorpha has been remodeled by deleting the HpALG3 gene in the Hpoch1 null mutant strain blocked in the yeast-specific outer mannose chain synthesis and by introducing an ER-targeted Aspergillus saitoi alpha-1,2-mannosidase gene. This glycoengineered H. polymorpha strain produced glycoproteins mainly containing trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core backbone of various human-type N-glycans. The results demonstrate the high potential of H. polymorpha to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.     

Simultaneous estimation of the association constants of glycoprotein glycoforms to a common protein by capillary electrophoresis

The efficacy of our capillary electrophoresis method for simultaneous estimation of the association constants of glycoprotein glycoforms to a common target protein was demonstrated using ribonuclease and ovalbumin glycoforms as glycoform models and Lens culinaris agglutinin (LCA) as a protein model. The ribonuclease glycoforms were fairly well separated in the absence of LCA at pH 5.8, but the peaks were retarded without any change of separation profile in the presence of LCA, the retardation becoming greater as LCA concentration increased. The estimated values of apparent association constant (K(a)) were at the 10(6)M(-1) level for all the ribonuclease glycoforms, and there was no significant difference among glycoforms. The high-mannose-type N-glycans released from a mixture of ribonuclease glycoforms gave lower values of K(a) at the 10(4)-10(5)M(-1) level to the same protein, and the glycans having a larger number of the mannose residue gave larger K(a) values. These results imply that the glycan moiety in this glycoprotein might contribute to its binding to the protein, but the polypeptide core played the major role. In contrast, ovalbumin glycoforms gave poorly resolved peaks in the absence of LCA, but they were separated into several peaks in the presence of LCA, which were tentatively assigned based on the knowledge of affinity to this lectin, and K(a) values were estimated simultaneously. The estimated K(a) values were smaller than those of the ribonuclease glycoforms, suggesting the major role of the N-glycan moiety. Thus, capillary electrophoresis allowed simultaneous estimation of K(a) values under common conditions using small amounts of glycoform mixtures and proteins without prior isolation and purification. Comparison of the obtained values will provide useful information on the glycan structure-affinity correlation.     

核心岩藻糖基化控制在治疗性抗体研究中的应用

对于人类许多疾病,抗体已成为了一种很重要的治疗制剂。如何进一步提高抗体的效能,是目前研究的主要热点之一。在抗体治疗过程中,抗体依赖性细胞介导的细胞毒作用(antibody dependent cellular cytotoxicity,ADCC)被认为是治疗性抗体临床疗效中一个重要的治疗功能。抗体恒定区Fc片段(crystalline fragment)的糖基化对ADCC起着至关重要的作用,尤其是抗体恒定区的核心岩藻糖基化。近些年来,很多研究报告表明,去除或降低岩藻糖基化的治疗性抗体不论在体内还是在体外都表现出更高的效能。这主要是由于去除或降低岩藻糖基化的治疗性抗体相对于岩藻糖基化的抗体,可以在更低浓度下通过与FcγRIIIa的高亲和力而表现出较强的ADCC作用。因此,去除或降低岩藻糖基化抗体的应用有望成为提高下一代治疗性抗体效能的有效手段。在此综述中,我们主要讨论了控制治疗性抗体恒定区岩藻糖基化的重要性及当前去除或降低岩藻糖基化治疗性抗体的生产控制方法。     

Engineering host cell lines to reduce terminal sialylation of secreted antibodies

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.     

Glycan engineering reveals interrelated effects of terminal galactose and core fucose on antibody-dependent cell-mediated cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been identified as one of the potentially critical effector functions underlying the clinical efficacy of some therapeutic immunoglobin G1 (IgG1) antibodies. It has been well established that higher levels of afucosylated N-linked glycan structures on the Fc region enhance the IgG binding affinity to the FcγIIIa receptor and lead to increased ADCC activity. However, whether terminal galactosylation of an IgG1 impacts its ADCC activity is less understood. Here, we used a new strategy for glycan enrichment and remodeling to study the impact of terminal galactose on ADCC activity for therapeutic IgG1s. Our results indicate that the degree of influence of terminal galactose on in vitro ADCC activity depends on the presence or absence of the core fucose, which is typically linked to the first N-acetyl glucosamine residue of an N-linked glycosylation core structure. Specifically, terminal galactose on afucosylated IgG1 mAbs enhanced ADCC activity with impact coefficients (ADCC%/Gal%) more than 20, but had minimal influence on ADCC activity on fucosylated structures with impact coefficient in the range of 0.1–0.2. Knowledge gained here can be used to guide product and process development activities for biotherapeutic antibodies that require effector function for efficacy, and also highlight the complexity in modulating the immune response through N-linked glycosylation of antibodies.