Chemoenzymatic Glycoengineering Research Articles

Evaluation of Two Chemoenzymatic Glycan Remodeling Approaches to Generate Site-Specific Antibody-Drug Conjugates.

Fc-glycosite-specific antibody-drug conjugation represents a promising direction for the preparation of site-specific antibody-drug conjugates (ADCs). In the present research, we conducted a systemic evaluation of two endoglycosidase-catalyzed chemoenzymatic glycoengineering technologies to prepare glycosite-specific ADCs. In the first two-step approach, the antibody was deglycosylated and then reglycosylated with a modified intact N-glycan oxazoline. In the second one-pot approach, antibodies were deglycosylated and simultaneously glycosylated with a functionalized disaccharide oxazoline. For the comprehensive evaluation, we first optimized and scaled-up the preparation of azido glycan oxazolines. Afterwards, we proved that the one-pot glycan-remodeling approach was efficient for all IgG subclasses. Subsequently, we assembled respective ADCS using two technology routes, with two different linker-payloads combinations, and performed systemic in vitro and in vivo evaluations. All the prepared ADCs achieved high homogeneity and illustrated excellent stability in buffers with minimum aggregates, and exceptional stability in rat serum. All ADCs displayed a potent killing of BT-474 breast cancer cells. Moving to the mouse study, the ADCs prepared from two technology routes displayed potent and similar efficacy in a BT-474 xenograft model, which was comparable to an FDA-approved ADC generated from random conjugation. These ADCs also demonstrated excellent safety and did not cause body weight loss at the tested dosages.

Homogeneity of antibody-drug conjugates critically impacts the therapeutic efficacy in brain tumors.

SUMMARYGlioblastoma multiforme (GBM) is the most aggressive and fatal disease of all brain tumor types. Most therapies rarely provide clinically meaningful outcomes in the treatment of GBM. Although antibody-drug conjugates (ADCs) are promising anticancer drugs, no ADCs have been clinically successful for GBM, primarily because of poor blood-brain barrier (BBB) penetration. Here, we report that ADC homogeneity and payload loading rate are critical parameters contributing to this discrepancy. Although both homogeneous and heterogeneous conjugates exhibit comparable in vitro potency and pharmacokinetic profiles, the former shows enhanced payload delivery to brain tumors. Our homogeneous ADCs provide improved antitumor effects and survival benefits in orthotopic brain tumor models. We also demonstrate that overly drug-loaded species in heterogeneous conjugates are particularly poor at crossing the BBB, leading to deteriorated overall brain tumor targeting. Our findings indicate the importance of homogeneous conjugation with optimal payload loading in generating effective ADCs for intractable brain tumors.

Challenges and Opportunities for the Large-Scale Chemoenzymatic Glycoengineering of Therapeutic N-Glycosylated Monoclonal Antibodies

Variability in the glycosylation profile of therapeutic monoclonal antibodies (mAbs), due to recombinant production technologies, leads to inconsistencies in effector functions and pharmacokinetic properties, both batch-to-batch and within single batches. It also poses regulatory concerns over the effectiveness of commercially available formulations. In vitro chemoenzymatic glycoengineering of variants displaying a homogeneous glycan profile is a trending strategy for ensuring consistent, controlled, and enhanced therapeutic performance, but reported successes are largely limited to small-scale applications. The major challenges for the industrial-scale introduction of the technique stem from the need for activated sugar donors, which can participate in undesired side reactions, and from the economic cost of the additional enzymatic steps and purification stages. While recent developments within the area address some of these obstacles, it appears that more effort is required in order to access the untapped potential of biocatalysis to enable the robust production of therapeutically superior constructs.

Enhanced Immunomodulatory Effect of Intravenous Immunoglobulin by Fc Galactosylation and Nonfucosylation.

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in the treatment of various autoimmune/inflammatory diseases although its mechanism of action remains elusive. Recently, nonfucosylated IgG has been shown to be preferentially bound to Fcγ receptor IIIa (FcγRIIIa) on circulating natural killer cells; therefore, we hypothesized that nonfucosylated IVIG may modulate immune responses through FcγRIIIa blockade. Here, homogeneous fucosylated or nonfucosylated glycoforms of normal polyclonal IgG bearing sialylated, galactosylated or nongalactosylated Fc oligosaccharides were generated by chemoenzymatic glycoengineering to investigate whether the IgG glycoforms can inhibit antibody-dependent cellular cytotoxicity (ADCC). Among the six IgG glycoforms, galactosylated, nonfucosylated IgG [(G2)2] had the highest affinity to FcγRIIIa and 20 times higher potency to inhibit ADCC than native IgG. A pilot study of IVIG treatment in mice with collagen antibody-induced arthritis highlighted the low-dose (G2)2 glycoform of IVIG (0.1 g/kg) as an effective immunomodulatory agent as the 10-fold higher dose of native IVIG. These preliminary results suggest that the anti-inflammatory activity of IVIG is in part mediated via activating FcγR blockade by galactosylated, nonfucosylated IgG and that such nonfucosylated IgG glycoforms bound to FcγRs on immune cells play immunomodulatory roles in health and disease. This study provides insights into improved therapeutic strategies for autoimmune/inflammatory diseases using glycoengineered IVIG and recombinant Fc.

Enhanced transglycosylation activity of an Endo-F3 mutant by ligand-directed localization.

At present, numerous studies have been reported to remodel the N-glycans of therapeutic antibodies for the gain of functions. Among the ways of remodeling antibody N-glycans, the chemoenzymatic glycoengineering approach by endoglycosidase (ENGase) has been deeply investigated and provided a significant tool for IgG glycoengineering. Among these cases, the transglycosylation activity of Endo-F3, compared to Endo-S and S2, is insufficient and limits its power in remodeling IgG glycosylation. Herein, we chemically conjugated the Endo-F3 mutant D165A with an Fc binding peptide (FcBP), aiming to improve the affinity of Endo-F3 D165A to IgGs, and therefore enhance the transglycosylation activity of D165A. In this report, we investigated the conjugation site of FcBP to D165A and the linkers between them and found that the conjugation indeed significantly increases the transglycosylation activity of D165A. Meanwhile, we optimized the FcBP-D165A catalyzed transglycosylation process, including the enzyme quantity, oxazoline concentration, and so on. Finally, by this method, we remodeled the N-glycans of rituximab and trastuzumab into homogeneous S2G2F, G2F, GN2M3, and M3 types with decreased enzyme quantity, oxazoline ratio, and catalyzing time. This method not only provides an enhanced ENGase for IgG glycoengineering but also suggests that ligand-directed localization of enzymes is a potential strategy to enhance the activity of enzymes towards the targeted substrate.

Homogeneous Antibody-Drug Conjugates via Glycoengineering.

Conventional antibody-drug conjugates (ADCs) randomly assemble small-molecule drugs onto Lys or Cys residues of a tumor-targeting antibody, featured with heterogeneity in payload numbers and conjugation positions. Glycosite-specific ADCs (gsADCs) link payload drugs onto IgG Fc N-glycans with high homogeneity that facilitates structural optimization and quality control for ADC drug development. In this protocol, we report two strategies for preparation of homogeneous ADCs via chemoenzymatic glycoengineering. First, an azido-tagged unnatural N-glycan substrate is transferred onto Fc glycosites of a therapeutic antibody through Endo-S-catalyzed glycoremodeling, followed by click reaction with an alkyne-tagged payload drug to give a well-defined gsADC. In an alternative way, glycoengineering of antibody with a natural sialylated N-glycan and successive selective oxidation of sialic acid moieties using sodium periodate provided an aldehyde handle on the glycans for conjugation with an aminooxy-assembled payload. These two strategies both enable gsADCs with high homogeneity in their conjugation sites, payload numbers, and glycoforms, which are characterized of a single mass under mass-spectral detection.

Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody

The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, posttranslational site-selective modifications of glycans in antibodies and other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial α1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the FcγIIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

Glycoengineered antibodies: towards the next-generation of immunotherapeutics.

Monoclonal antibodies (mAbs) are currently the largest and fastest growing class of biopharmaceuticals, and they address unmet medical needs, e.g., in oncology and in auto-immune diseases. Their clinical efficacy and safety is significantly affected by the structure and composition of their glycosylation profile which is commonly heterogeneous, heavily dependent on the manufacturing process, and thus susceptible to variations in the cell culture conditions. Glycosylation is therefore considered a critical quality attribute for mAbs. Commonly, in currently marketed therapeutic mAbs, the glycosylation profile is suboptimal in terms of biological properties such as antibody-dependent cell-mediated cytotoxicity or may give rise to safety concerns due to the presence of non-human glycans. This article will review recent innovative developments in chemo-enzymatic glycoengineering, which allow generating mAbs carrying single, well-defined, uniform Fc glycoforms, which confers the desired biological properties for the target application. This approach offers significant benefits such as enhanced Fc effector functions, improved safety profiles, higher batch-to-batch consistency, decreased risks related to immunogenicity and manufacturing process changes, and the possibility to manufacture mAbs, in an economical manner, in non-mammalian expression systems. Overall, this approach could facilitate and reduce mAb manufacturing costs which in turn would translate into tangible benefits for both patients and manufacturers. The first glycoengineered mAbs are about to enter clinical trials and it is expected that, once glycoengineering reagents are available at affordable costs, and in-line with regulatory requirements, that targeted remodeling of antibody Fc glycosylation will become an integral part in manufacturing the next-generation of immunotherapeutics.

Pharmacokinetic and Pharmacodynamic Profiles of Glyco-Modified Atrial Natriuretic Peptide Derivatives Synthesized Using Chemo-enzymatic Synthesis Approaches.

Atrial natriuretic peptide (ANP) exerts beneficial pharmacological effects in the treatment of various cardiovascular disorders, such as acute congestive heart failure (ADHF). However, the clinical use of ANP is limited to the continuous intravenous infusion owing to its short half-life (2.4 ± 0.7 min). In the present study, we conjugated the glyco-modified ANP with a monoclonal antibody (mAb) or an Fc via chemo-enzymatic glyco-engineering using EndoS D233Q/Q303L. The most potent derivative SG-ANP-Fc conjugate extended the half-life to 14.9 d and the duration of blood pressure lowering effect to over 28 d. This new biologic modality provides an opportunity to develop outpatient therapy after ADHF.

Crystallizable Fragment Glycoengineering for Therapeutic Antibodies Development.

Monoclonal antibody (mAb)-based therapeutics are the fastest growing class of human pharmaceuticals. They are typically IgG1 molecules with N-glycans attached to the N297 residue on crystallizable fragment (Fc). Different Fc glycoforms impact their effector function, pharmacokinetics, stability, aggregation, safety, and immunogenicity. Fc glycoforms affect mAbs effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by modulating the Fc–FcγRs and Fc–C1q interactions. While the terminal galactose enhances CDC activity, the fucose significantly decreases ADCC. Defucosylated immunoglobulin Gs (IgGs) are thus highly pursued as next-generation therapeutic mAbs with potent ADCC at reduced doses. A plethora of cell glycoengineering and chemoenzymatic glycoengineering strategies is emerging to produce IgGs with homogenous glycoforms especially without core fucose. The chemoenzymatic glycosylation remodeling also offers useful avenues for site-specific conjugations of small molecule drugs onto mAbs. Herein, we review the current progress of IgG-Fc glycoengineering. We begin with the discussion of the structures of IgG N-glycans and biosynthesis followed by reviewing the impact of IgG glycoforms on antibody effector functions and the current Fc glycoengineering strategies with emphasis on Fc defucosylation. Furthermore, we briefly discuss two novel therapeutic mAbs formats: aglycosylated mAbs and Fc glycan specific antibody–drug conjugates (ADCs). The advances in the understanding of Fc glycobiology and development of novel glycoengineering technologies have facilitated the generation of therapeutic mAbs with homogenous glycoforms and improved therapeutic efficacy.

Glycoengineering and glycosite-specific labeling of serum IgGs from various species

Chemoenzymatic glycoengineering of immunoglobulin G (IgG) catalyzed by Endo-S is a powerful approach to remodel the heterogeneous N-glycoforms of Fc domain with a homogeneous synthetic glycan structure for enhanced Fc receptor-mediated effector functions. The previous researches on the method development mainly focused on human or humanized IgGs with therapeutic potentials. Here, for the first time we report the extended application of this method on glycan-remodeling of serum IgGs from other species including rabbit, mouse, and goat. Harnessing an azido-tagged non-natural N-glycan substrate and successive click reaction, glycosite-specific fluorescent labeling of IgGs was enabled. This study provided a new avenue for glycoengineering and Fc-specific labeling of IgGs with minimized influence on antigen-binding domains, and this method was adaptive to thousands of commercial antibody reagents from various species with great application potentials.

Labeling glycans on living cells by a chemoenzymatic glycoengineering approach.

ABSTRACTStructural glycobiology has traditionally been a challenging field due to a limited set of tools available to investigate the diverse and complex glycan molecules. However, we cannot ignore that glycans play critical roles in health as well as in disease, and are present in more than 50% of all proteins and on over 80% of all surface proteins. Chemoenzymatic glycoengineering (CGE) methods are a powerful set of tools to synthesize complex glycans, but the full potential of these methods have not been explored in cell biology yet. Herein, we report the labeling of live Chinese hamster ovary (CHO) cells by employing three highly specific glycosyltransferases: a sialyltransferase, a galactosyltransferase, and an N-acetyl-glucosaminyl transferase. We verified our results by bio-orthogonal blots and further rationalized them by computational modeling. We expect CGE applications in cell biology to rise and their implementation will assist in structural-functional discoveries in glycobiology. This research will contribute to this effort.

Chemoenzymatic Glyco-engineering of Monoclonal Antibodies.

Monoclonal antibodies (mAbs) are an important class of therapeutic glycoproteins widely used for the treatment of cancer, inflammation, and infectious diseases. Compelling data have shown that the presence and fine structures of the conserved N-glycans at the Fc domain can profoundly affect the effector functions of antibodies. However, mAbs are usually produced as mixtures of Fc glycoforms and the control of glycosylation to a favorable, homogeneous status in various host expression systems is still a challenging task. In this chapter, we describe a detailed procedure of chemoenzymatic glyco-engineering of monoclonal antibodies, using rituximab (a therapeutic monoclonal antibody) as a model system. The protocol includes the deglycosylation of a mAb by an endoglycosidase (such as wild type EndoS) to remove the heterogeneous Fc N-glycans, leaving only the innermost GlcNAc or the core-fucosylated GlcNAc at the glycosylation site. Then the deglycosylated IgG serves as an acceptor for an endoglycosidase-catalyzed transglycosylation to add a desired N-glycan to the GlcNAc acceptor to reconstitute a defined, homogeneous natural glycoform of IgG, using a glycosynthase mutant as the enzyme and activated glycan oxazoline as the donor substrate. A semi-synthesis of sialylated and asialylated biantennary N-glycan oxazolines is also described. This detailed procedure can be used for the Fc glycosylation remodeling of other mAbs to provide homogeneous Fc glycoforms for various applications.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities.

Backgroundα-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This “capping” of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins.ResultsHuman α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates.ConclusionsFunctional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 – 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0138-8) contains supplementary material, which is available to authorized users.

Structural Characterization of Anti-Inflammatory Immunoglobulin G Fc Proteins

Immunoglobulin G (IgG) is a central mediator of host defense due to its ability to recognize and eliminate pathogens. The recognition and effector responses are encoded on distinct regions of IgGs. The diversity of the antigen recognition Fab domains accounts for IgG's ability to bind with high specificity to essentially any antigen. Recent studies have indicated that the Fc effector domain also displays considerable heterogeneity, accounting for its complex effector functions of inflammation, modulation, and immune suppression. Therapeutic anti-tumor antibodies, for example, require the pro-inflammatory properties of the IgG Fc to eliminate tumor cells, while the anti-inflammatory activity of intravenous IgG requires specific Fc glycans for activity. In particular, the anti-inflammatory activity of intravenous IgG is ascribed to a small population of IgGs in which the Asn297-linked complex N-glycans attached to each Fc CH2 domain include terminal α2,6-linked sialic acids. We used chemoenzymatic glycoengineering to prepare fully disialylated IgG Fc and solved its crystal structure. Comparison of the structures of asialylated Fc, sialylated Fc, and F241A Fc, a mutant that displays increased glycan sialylation, suggests that increased conformational flexibility of the CH2 domain is associated with the switch from pro-inflammatory to anti-inflammatory activity of the Fc.

Chemoenzymatic Glycoengineering of Intact IgG Antibodies for Gain of Functions

The fine structures of Fc N-glycans can modulate the effector functions of IgG antibodies. It has been demonstrated that lack of the core fucose on the Fc N-glycans leads to drastic enhancement of antibody-dependent cellular cytotoxicity (ADCC), while terminal α2,6-sialylation of Fc glycan plays a critical role for the anti-inflammatory activity of human intravenous immunoglobulin (IVIG). We describe in this paper a highly efficient chemoenzymatic method for site-selective Fc glycoengineering of intact monoclonal antibody and IVIG. Two new glycosynthase mutants (EndoS-D233A and D233Q) were generated by site-directed mutagenesis of EndoS (an endoglycosidase from Streptococcus pyogenes ) and were found to be capable of efficiently transferring predefined N-glycans from corresponding glycan oxazolines to the Fc-deglycosylated intact IgGs without product hydrolysis. As a model study, rituximab (a therapeutic monoclonal antibody) was successfully transformed from mixtures of G0F, G1F, and G2F glycoforms to well-defined homogeneous glycoforms, including a fully sialylated (S2G2F) glycoform that may gain anti-inflammatory activity, a nonfucosylated G2 glycoform that showed significantly enhanced FcγIIIa receptor-binding activity, and an azido-tagged glycoform that can be further transformed into other glycoforms. We also found that EndoS could selectively remove the Fc N-glycans in the presence of FAB glycosylation. This finding, coupled with the remarkable transglycosylation activity of the EndoS glycosynthase mutants, permitted a highly selective glycoengineering of the IVIG's Fc glycans into a fully sialylated Fc glycoform, which may possess significantly enhanced anti-inflammatory activity. The glycoengineering approach described here provides a general platform to modulate the effector functions of IgG antibodies, enabling the optimization of therapeutic efficacy and gain of new functions of monoclonal antibodies and IVIG.