In Silico Analysis of Therapeutic Antibody Aggregation and the Influence of Glycosylation.

The aggregation of therapeutic antibodies is a major issue for the pharmaceutical industry leading to loss of drug quality, increased dosage, and unwanted immune responses such as the production of anti-drug antibodies (ADA). As aggregation can occur at various stages of development and storage, much work has been performed to reduce or eliminate it. In this report we analyzed four antibodies available in the PDB (1IGT, 1IGY, 1HZH, and 5DK3) using the online software UCSF Chimera to study the structural features of the proteins and the associated N-linked glycans in the CH2 domains of the Fc region. To study antibody aggregation in silico we used the online software TANGO and AGGRESCAN to identify aggregation prone regions (APR) in the antibodies and the influence of the Fc glycans on hydrophobic and aromatic residues present in the APRs. In the 3D structures of 1IGT and 1IGY the glycan chains are in close enough proximity to influence and protect these hydrophobic regions. However, in the 3D structures of 1HZH and 5DK3 the glycans do not appear to influence the likely APRs of the antibodies. Therefore, in these structures we modified the Fc glycan regions by adjusting the glycosylated asparagine side chains and glycosidic bonds. We successfully adjusted the glycan chains of 1HZH and 5DK3 and reduced the distance between them and the APRs to show potential influence on aggregation. However, similar to 5DK3, the influence of glycosylation on the APRs of the antibody was limited due to the size of the glycans present in the 3D structure. This report is based on in silico studies to show how antibody glycans can influence aggregation.

Metabolic Labeling of Primary Neurons Using Carbohydrate Click Chemistry.

Glycans play an important role in many neuronal processes, such as neurotransmitter release and reuptake, cell-cell communication and adhesion, modulation of ion channel activity, and immune function. Carbohydrate click chemistry is a powerful technique for studying glycan function and dynamics in vitro, in vivo, and ex vivo. Here, we use commercially available synthetic tetraacetylated azido sugars, copper and copper-free click chemistry to metabolically label and analyze primary rat cortical neurons. In addition, we use high resolution confocal and STED microscopy to image and analyze different forms of glycosylation in ultrahigh resolution. We observe different patterns of GlcNAz, GalNAz, and ManNAz distribution at different stages of neuronal development. We also observe highly sialylated structures on the neuronal plasma membrane, which warrant further investigation.

Nanobodies: The Future of Antibody-Based Immune Therapeutics.

Targeted therapy is a fast evolving treatment strategy to reduce unwanted damage to healthy tissues, while increasing efficacy and specificity. Driven by state-of-the-art technology, this therapeutic approach is especially true of cancer. Antibodies with their remarkable specificity have revolutionized therapeutic strategies for autoimmune conditions and cancer, particularly blood-borne cancers, but have severe limitations in treating solid tumors. This is mainly due to their large molecular size, low stability, tumor-tissue penetration difficulties, and pharmacokinetic properties. The tumor microenvironment, rich in immune-suppressing molecules is also a major barrier in targeting solid tumors by antibody-based drugs. Nanobodies have recently emerged as an alternative therapeutic agent to overcome some of the drawbacks of traditional antibody treatment. Nanobodies are the VHH domains found on the heavy-chain only antibodies of camelids and are the smallest naturally available antibody fragments with excellent antigen-binding specificity and affinity, equivalent to conventional antibodies but with molecular weights as low as 15 kDa. The compact size, high stability, enhanced hydrophilicity, particularly in framework regions, excellent epitope interactions with protruding CDR3 regions, and improved tissue penetration make nanobodies the next-generation therapeutics (Nano-BioDrugs). In this review, the authors discuss the interesting properties of nanobodies and their advantages over their conventional counterparts and provide insight into how nanobodies are being utilized as agonists and antagonists, bispecific constructs, and drug and enzyme-conjugates to combat the tumor microenvironment and treat disease.

Dichloroacetate Stabilizes Mitochondrial Fusion Dynamics in Models of Neurodegeneration.

Mitochondrial dysfunction is a recognized hallmark of neurodegenerative diseases and abnormal mitochondrial fusion-fission dynamics have been implicated in the pathogenesis of neurodegenerative disorders. This study characterizes the effects of metabolic flux inhibitors and activators on mitochondrial fusion dynamics in the neuronal cell culture model of differentiated PC12 cells. Using a real time confocal microscopy assay, it was found that the carnitine palmitoyltransferase I (CPTI) inhibitor, etomoxir, reduced mitochondrial fusion dynamics in a time-dependent manner. Etomoxir also decreased JO2, ΔΨm and reactive oxygen species (ROS) production rates. The mitochondrial pyruvate carrier (MPC) inhibitor, UK5099, reduced fusion dynamics and in combination with etomoxir these inhibitory effects were amplified. Use of the pyruvate dehydrogenase (PDH) kinase inhibitor dichloroacetate, which is known to increase metabolic flux through PDH, reversed the etomoxir-induced effects on fusion dynamics, JO2, ΔΨm but not ROS production rates. Dichloroacetate also partially reversed inhibition of mitochondrial fusion dynamics caused by the parkinsonian-inducing neurotoxin, MPP+. These results suggest that dichloroacetate-induced activation of metabolic flux in the mitochondrion may be a mechanism to restore normal mitochondrial fusion-fission dynamics in metabolically challenged cells.

Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIaArg131/His131 (CD32a), RIIb (CD32b) and RIIIaPhe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIaPhe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIaPhe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a stabilizing role of FcγR glycans in the antibody binding interaction.

Fc gamma receptors: glycobiology and therapeutic prospects.

Therapeutic antibodies hold great promise for the treatment of cancer and autoimmune diseases, and developments in antibody-drug conjugates and bispecific antibodies continue to enhance treatment options for patients. Immunoglobulin (Ig) G antibodies are proteins with complex modifications, which have a significant impact on their function. The most important of these modifications is glycosylation, the addition of conserved glycans to the antibody Fc region, which is critical for its interaction with the immune system and induction of effector activities such as antibody-dependent cell cytotoxicity, complement activation and phagocytosis. Communication of IgG antibodies with the immune system is controlled and mediated by Fc gamma receptors (FcγRs), membrane-bound proteins, which relay the information sensed and gathered by antibodies to the immune system. These receptors are also glycoproteins and provide a link between the innate and adaptive immune systems. Recent information suggests that this receptor glycan modification is also important for the interaction with antibodies and downstream immune response. In this study, the current knowledge on FcγR glycosylation is discussed, and some insight into its role and influence on the interaction properties with IgG, particularly in the context of biotherapeutics, is provided. For the purpose of this study, other Fc receptors such as FcαR, FcεR or FcRn are not discussed extensively, as IgG-based antibodies are currently the only therapeutic antibody-based products on the market. In addition, FcγRs as therapeutics and therapeutic targets are discussed, and insight into and comment on the therapeutic aspects of receptor glycosylation are provided.

Metabolic flux control in glycosylation.

Glycosylation is a common post-translational protein modification, in which glycans are built onto proteins through the sequential addition of monosaccharide units, in reactions catalysed by glycosyltransferases. Glycosylation influences the physicochemical and biological properties of proteins, with subsequent effects on subcellular and extracellular protein trafficking, cell-cell recognition, and ligand-receptor interactions. Glycan structures can be complex, as is the regulation of their biosynthesis, and it is only recently that the systems biology of metabolic flux control and glycosyltransferase networks has become a study in its own right. We review various models of glycosylation that have been proposed to date, based on current knowledge of Golgi structure and function, and consider how metabolic flux through glycosyltransferase networks regulates glycosylation events in the cell.

Galactosyltransferase 4 is a major control point for glycan branching in N-linked glycosylation.

Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein.

Fc gamma receptor glycosylation modulates the binding of IgG glycoforms: a requirement for stable antibody interactions.

FcγRs play a critical role in the immune response following recognition of invading particles and tumor associated antigens by circulating antibodies. In the present study we investigated the role of FcγR glycosylation in the IgG interaction and observed a stabilizing role for receptor N-glycans. We performed a complete glycan analysis of the recombinant FcγRs (FcγRIa, FcγRIIa, FcγRIIb, FcγRIIIa(Phe158/Val158), and FcγRIIIb) expressed in human cells and demonstrate that receptor glycosylation is complex and varied between receptors. We used surface plasmon resonance to establish binding patterns between rituximab and all receptors. Complex binding was observed for FcγRIa and FcγRIIIa. The IgG-FcγR interaction was further investigated using a combination of kinetic experiments and enzymatically deglycosylated FcγRIa and FcγRIIIa(Phe158/Val158) receptors in an attempt to determine the underlying binding mechanism. We observed that antibody binding levels decreased for deglycosylated receptors, and at the same time, binding kinetics were altered and showed a more rapid approach to steady state, followed by an increase in the antibody dissociation rate. Binding of rituximab to deglycosylated FcγRIIIa(Phe158) was now consistent with a 1:1 binding mechanism, while binding of rituximab to FcγRIIIa(Val158) remained heterogeneous. Kinetic data support a complex binding mechanism, involving heterogeneity in both antibody and receptor, where fucosylated and afucosylated antibody forms compete in receptor binding and in receptor molecules where heterogeneity in receptor glycosylation plays an important role. The exact nature of receptor glycans involved in IgG binding remains unclear and determination of rate and affinity constants are challenging. Here, the use of more extended competition experiments appear promising and suggest that it may be possible to determine dissociation rate constants for high affinity afucosylated antibodies without the need to purify or express such variants. The data described provide further insight into the complexity of the IgG-FcγR interaction and the influence of FcγR glycosylation.

Glycosylation and Fc receptors.

Immunoglobulins and Fc receptors are critical glycoprotein components of the immune system. Fc receptors bind the Fc (effector) region of antibody molecules and communicate information within the innate and adaptive immune systems. Glycosylation of antibodies, particularly in the Fc region of IgG, has been extensively studied in health and disease. The N-glycans in the identical heavy chains have been shown to be critical for maintaining structural integrity, communication with the Fc receptor and the downstream immunological response. Less is known about glycosylation of the Fc receptor in either healthy or disease states, however, recent studies have implicated an active role for receptor associated oligosaccharides in the antibody-receptor interaction. Research into Fc receptor glycosylation is increasing rapidly, where Fc receptors are routinely used to analyze the binding of therapeutic monoclonal antibodies and where glycosylation of receptors expressed by cells of the immune system could potentially be used to mediate and control the differential binding of immunoglobulins. Here we discuss the glycosylation of immunoglobulin antibodies (IgA, IgE, IgG) and the Fc receptors (FcαR, FcεR, FcγR, FcRn) that bind them, the function of carbohydrates in the immune response and recent advances in our understanding of these critical glycoproteins.

N-linked glycan structures of the human Fcγ receptors produced in NS0 cells.

Immune recognition of nonself is coordinated through complex mechanisms involving both innate and adaptive responses. Circulating antibodies communicate with effector cells of the innate immune system through surface receptors known as Fcγ receptors (FcγRs). The FcγRs are single-pass transmembrane glycoproteins responsible for regulating innate effector responses toward antigenic material. Although immunoglobulin G (IgG) antibodies bind to a range of receptors, including complement receptors and C-type lectins, we have focused on the Fcγ receptors. A total of five functional FcγRs are broadly classified into three families (FcγRI, FcγRII, and FcγRIII) and together aid in controlling both inflammatory and anti-inflammatory responses of the innate immune system. Due to the continued success of monoclonal antibodies in treating cancer and autoimmune disorders, research is typically directed toward improving the interaction of antibodies with the FcγRs through manipulation of IgG properties such as N-linked glycosylation. Biochemical studies using recombinant forms of the FcγRs are often used to quantitate changes in binding affinity, a key indicator of a likely biological outcome. However, analysis of the FcγRs themselves is imperative as recombinant FcγRs differ greatly from those observed in humans. In particular, the N-linked glycan composition is significantly important due to its function in the IgG-FcγR interaction. Here, we present data for the N-linked glycans present on FcγRs produced in NS0 cells, namely, FcγRIa, FcγRIIa, FcγRIIB, FcγRIIIa, and FcγRIIIb. Importantly, these FcγRs demonstrate typical murine glycosylation in the form of α-galactose epitopes, N-glycolylneuraminic acid, and other key glycan properties that are generally expressed in murine cell lines and therefore are not typically observed in humans.

The effect of pH on the initial rate kinetics of the dimeric biliverdin-IXalpha reductase from the cyanobacterium Synechocystis PCC6803.

Biliverdin-IXalpha reductase from Synechocystis PCC6803 (sBVR-A) is a stable dimer and this behaviour is observed under a range of conditions. This is in contrast to all other forms of BVR-A, which have been reported to behave as monomers, and places sBVR-A in the dihydrodiol dehydrogenase/N-terminally truncated glucose-fructose oxidoreductase structural family of dimers. The cyanobacterial enzyme obeys an ordered steady-state kinetic mechanism at pH 5, with NADPH being the first to bind and NADP(+) the last to dissociate. An analysis of the effect of pH on k(cat) with NADPH as cofactor reveals a pK of 5.4 that must be protonated for effective catalysis. Analysis of the effect of pH on k(cat)/K(m)(NADPH) identifies pK values of 5.1 and 6.1 in the free enzyme. Similar pK values are identified for biliverdin binding to the enzyme-NADPH complex. The lower pK values in the free enzyme (pK 5.1) and enzyme-NADPH complex (pK 4.9) are not evident when NADH is the cofactor, suggesting that this ionizable group may interact with the 2'-phosphate of NADPH. His84 is implicated as a crucial residue for sBVR-A activity because the H84A mutant has less than 1% of the activity of the wild-type and exhibits small but significant changes in the protein CD spectrum. Binding of biliverdin to sBVR-A is conveniently monitored by following the induced CD spectrum for biliverdin. Binding of biliverdin to wild-type sBVR-A induces a P-type spectrum. The H84A mutant shows evidence for weak binding of biliverdin and appears to bind a variant of the P-configuration. Intriguingly, the Y102A mutant, which is catalytically active, binds biliverdin in the M-configuration.