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The IgG-specific endoglycosidases EndoS and EndoS2 from Streptococcus pyogenes can remove conserved N-linked glycans present on the Fc region of host antibodies to inhibit Fc-mediated effector functions. These enzymes are therefore being investigated as therapeutics for suppressing unwanted immune activation, and have additional application as tools for antibody glycan remodeling. EndoS and EndoS2 differ in Fc glycan substrate specificity due to structural differences within their catalytic glycosyl hydrolase domains. However, a chimeric EndoS enzyme with a substituted glycosyl hydrolase from EndoS2 loses catalytic activity, despite high structural homology between the two enzymes, indicating either mechanistic divergence of EndoS and EndoS2, or improperly-formed domain interfaces in the chimeric enzyme. Here, we present the crystal structure of the EndoS2-IgG1 Fc complex determined to 3.0 Å resolution. Comparison of complexed and unliganded EndoS2 reveals relative reorientation of the glycosyl hydrolase, leucine-rich repeat and hybrid immunoglobulin domains. The conformation of the complexed EndoS2 enzyme is also different when compared to the earlier EndoS-IgG1 Fc complex, and results in distinct contact surfaces between the two enzymes and their Fc substrate. These findings indicate mechanistic divergence of EndoS2 and EndoS. It will be important to consider these differences in the design of IgG-specific enzymes, developed to enable customizable antibody glycosylation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding via the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked N-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.

Antibodies can mediate immune recruitment or clearance of immune complexes through the interaction of their Fc domain with cellular Fc receptors. Clustering of antibodies is a key step in generating sufficient avidity for efficacious receptor recognition. However, Fc receptors may be saturated with prevailing, endogenous serum immunoglobulin and this raises the threshold by which cellular receptors can be productively engaged. Here, we review the factors controlling serum IgG levels in both healthy and disease states, and discuss how the presence of endogenous IgG is encoded into the functional activation thresholds for low- and high-affinity Fc receptors. We discuss the circumstances where antibody engineering can help overcome these physiological limitations of therapeutic antibodies. Finally, we discuss how the pharmacological control of Fc receptor saturation by endogenous IgG is emerging as a feasible mechanism for the enhancement of antibody therapeutics., Competing Interests: Declaration of Competing Interest S.A.B. has acted as a consultant for a number of biotechs and receives institutional support for grants and patents from BioInvent. M.C. has acted as a consultant for a number of biotechs and legal firms and is a named inventor on a patent application describing “Combined therapeutic use of antibodies and endoglycosidases” (WO2013110946A1) which was acquired by Hansa Biopharma., (Copyright © 2023. Published by Elsevier B.V.)

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Tumor vasculature is structurally abnormal, with anatomical deformities, reduced pericyte coverage and low tissue perfusion. As a result of this vascular dysfunction, tumors are often hypoxic, which is associated with an aggressive tumor phenotype, and reduced delivery of therapeutic compounds to the tumor. We have previously shown that a peptide containing the thrombospondin-1 type I repeats (3TSR) specifically targets tumor vessels and induces vascular normalization in a mouse model of epithelial ovarian cancer (EOC). However, due to its small size, 3TSR is rapidly cleared from circulation. We now introduce a novel construct with the 3TSR peptide fused to the C-terminus of each of the two heavy chains of the Fc region of human IgG1 (Fc3TSR). We hypothesize that Fc3TSR will have greater anti-tumor activity in vitro and in vivo compared to the native compound., Methods: Fc3TSR was evaluated in vitro using proliferation and apoptosis assays to investigate differences in efficacy compared to native 3TSR. In light of the multivalency of Fc3TSR, we also investigate whether it induces greater clustering of its functional receptor, CD36. We also compare the compounds in vivo using an orthotopic, syngeneic mouse model of advanced stage EOC. The impact of the two compounds on changes to tumor vasculature morphology was also investigated., Results: Fc3TSR significantly decreased the viability and proliferative potential of EOC cells and endothelial cells in vitro compared to native 3TSR. High-resolution imaging followed by image correlation spectroscopy demonstrated enhanced clustering of the CD36 receptor in cells treated with Fc3TSR. This was associated with enhanced downstream signaling and greater in vitro and in vivo cellular responses. Fc3TSR induced greater vascular normalization and disease regression compared to native 3TSR in an orthotopic, syngeneic mouse model of advanced stage ovarian cancer., Conclusion: The development of Fc3TSR which is greater in size, stable in circulation and enhances receptor activation compared to 3TSR, facilitates its translational potential as a therapy in the treatment of metastatic advanced stage ovarian cancer., Competing Interests: Declaration of Competing Interest J.P. and J.L. are co-inventors on US patent US20140271641A1 for the treatment of ovarian cancer with 3TSR., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of the complement system in HIV-1 immunity and pathogenesis is multifaceted, and an improved understanding of complement activities mediated by HIV-1-specific antibodies has the potential to inform and advance clinical development efforts. A seminal nonhuman primate challenge experiment suggested that complement was dispensable for the protective effect of the early broadly neutralizing antibody (bnAb) b12, but recent experiments have raised questions about the breadth of circumstances under which this conclusion may hold. Here, we reassess the original observation using Fc variants of IgG1 b12 that enhance complement activity and report that complement fixation on recombinant antigen, virions, and cells and complement-dependent viral and cellular lysis in vitro vary among bnAbs. Specifically, while the clinically significant V3 glycan-specific bnAb 10-1074 demonstrates activity, we found that b12 does not meaningfully activate the classical complement cascade. Consistent with avid engagement by C1q and its complex system of regulatory factors, these results suggest that complement-mediated antibody activities demonstrate a high degree of context dependence and motivate revisiting the role of complement in antibody-mediated prevention of HIV-1 infection by next-generation bnAbs in new translational studies in animal models. IMPORTANCE Given the suboptimal outcome of VRC01 antibody-mediated prevention of HIV-1 infection in its first field trial, means to improve diverse antiviral activities in vivo have renewed importance. This work revisits a loss-of-function experiment that investigated the mechanism of action of b12, a similar antibody, and finds that the reason why complement-mediated antiviral activities were not observed to contribute to protection may be the inherent lack of activity of wild-type b12, raising the prospect that this mechanism may contribute in the context of other HIV-specific antibodies.

Small-angle neutron scattering (SANS) and small- angle X-ray scattering (SAXS) are powerful techniques for the structural characterization of biomolecular complexes. In particular, SANS enables a selective observation of specific components in complexes by selective deuteration with contrast-matching techniques. In most cases, however, biomolecular interaction systems with heterogeneous oligomers often contain unfavorable aggregates and unbound species, hampering data interpretation. To overcome these problems, SAXS has been recently combined with size exclusion chromatography (SEC), which enables the isolation of the target complex in a multi-component system. By contrast, SEC-SANS is only at a preliminary stage. Hence, we herein perform a feasibility study of this method based on our newly developed inverse contrast-matching (iCM) SANS technique using antibody interactions as model systems. Immunoglobulin G (IgG) or its Fc fragment was mixed with 75% deuterated Fc-binding proteins, i.e. a mutated form of IgG-degrading enzyme of Streptococcus pyogenes and a soluble form of Fcγ receptor IIIb, and subjected to SEC-SANS as well as SEC-SAXS as reference. We successfully observe SANS from the non-deuterated IgG or Fc formed in complex with these binding partners, which were unobservable in terms of SANS in D2O, hence demonstrating the potential utility of the SEC-iCM-SANS approach., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The Fc portion of immunoglobulin G (IgG) promotes defensive effector functions in the immune system by interacting with Fcγ receptors and complement component C1q. These interactions critically depend on N-glycosylation at Asn297 of each C H 2 domain, where biantennary complex-type oligosaccharides contain microheterogeneities resulting primarily from the presence or absence of non-reducing terminal galactose residues. Crystal structures of Fc have shown that a pair of N-glycans is located between the two C H 2 domains. Here we applied our metabolic isotope labeling technique using mammalian cells for in-solution structural characterization of mouse IgG2b-Fc glycoforms with a molecular mass of 54 kDa. Based on spectral assignments of the N-glycans as well as polypeptide backbones of Fc, we probed conformational perturbations of Fc induced by N-glycan trimming, especially enzymatic degalactosylation. The results indicated that degalactosylation structurally perturbed the Fc region through rearrangement of glycan-protein interactions. The spectral assignments of IgG2b-Fc glycoprotein will provide the basis for NMR investigation of its dynamic conformations and interactions with effector molecules in solution.

Pathogens frequently use multivalent binding to sialic acid to infect cells or to modulate immunity through interactions with human sialic acid-binding immunoglobulin-type lectins (Siglecs). Molecules that interfere with these interactions could be of interest as diagnostics, anti-infectives or as immune modulators. This review describes the development of molecular scaffolds based on the crystallizable fragment (Fc) region of immunoglobulin (Ig) G that deliver high-avidity binding to innate immune receptors, including sialic acid-dependent receptors. The ways in which the sialylated Fc may be engineered as immune modulators that mimic the anti-inflammatory properties of intravenous polyclonal Ig or as blockers of sialic-acid-dependent infectivity by viruses are also discussed.

Background: Outbreaks of porcine epidemic diarrhea virus (PEDV) infection have re-emerged and spread rapidly worldwide, resulting in significant economic losses. Vaccination is the best way to prevent PEDV infection in young piglets. Objective: To enhance the efficacy of an inactivated vaccine against PEDV, we evaluated the adjuvant properties of Fc domain of IgG. Methods: Fifteen crossbred gilts (180 ∼ 210 days old) were used. Five pigs in group 1 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 10 6 TCID 50 of inactivated PEDV. Five pigs in group 2 were intramuscularly vaccinated twice at 4 weeks and 2 weeks prior to farrowing with 10 6 TCID 50 of inactivated PEDV-sFc. Five pigs in group 3 were not vaccinated and served as negative controls. Serum samples were collected at farrowing and subjected to ELISA, a serum neutralizing (SN) test, and a cytokine assay. Statistical analysis was performed by a two-tailed unpaired t-test. Results: Vero cells expressing swine IgG Fc on its surface was established. When PEDV was propagated in the cells expressing the swine Fc, PEDV virion incorporated the Fc. Immunization of pigs with inactivated PEDV harbouring Fc induced significantly higher antibody production against PEDV, comparing to the immunization with normal inactivated PEDV. In addition, we observed significantly increased IFN-γ levels in sera. Conclusion: Our results indicate that Fc molecule facilitate immune responses and PEDV harbouring Fc molecule could be a possible vaccine candidate. However, a challenge experiment would be needed to investigate the protective efficacy of PEDV harbouring Fc.

Complement-dependent cytotoxicity (CDC) is a potent effector mechanism, engaging both innate and adaptive immunity. Although strategies to improve the CDC activity of antibody therapeutics have primarily focused on enhancing the interaction between the antibody crystallizable fragment (Fc) and the first subcomponent of the C1 complement complex (C1q), the relative importance of intrinsic affinity and binding valency of an antibody to the target antigen is poorly understood. Here we show that antibody binding affinity to a cell surface target antigen evidently affects the extent and efficacy of antibody-mediated complement activation. We further report the fundamental role of antibody binding valency in the capacity to recruit C1q and regulate CDC. More specifically, an array of affinity-modulated variants and functionally monovalent bispecific derivatives of high-affinity anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth factor receptor 2 (HER2) therapeutic immunoglobulin Gs (IgGs), previously reported to be deficient in mediating complement activation, were tested for their ability to bind C1q by biolayer interferometry using antigen-loaded biosensors and to exert CDC against a panel of EGFR and HER2 tumor cells of various histological origins. Significantly, affinity-reduced variants or monovalent derivatives, but not their high-affinity bivalent IgG counterparts, induced near-complete cell cytotoxicity in tumor cell lines that had formerly been shown to be resistant to complement-mediated attack. Our findings suggest that monovalent target engagement may contribute to an optimal geometrical positioning of the antibody Fc to engage C1q and deploy the complement pathway.

A series of well-defined N-glycosylated IgG4-Fc variants were utilized to investigate the effect of glycan structure on their physicochemical properties (conformational stability and photostability) and interactions with an Fc γ receptor IIIA (FcγRIIIA). High mannose (HM, GlcNAc 2 Man (8+n) [n = 0-4]), Man 5 (GlcNAc 2 Man 5 ), GlcNAc 1 , and N297Q IgG4-Fc were prepared in good quality. The physical stability of these IgG4-Fc variants was examined with differential scanning calorimetry and intrinsic fluorescence spectroscopy. Photostability was assessed after photoirradiation between 295 and 340 nm (λ max  = 305 nm), and HPLC-MS/MS analysis of specific products was performed. The size of glycans at Asn 297 affects the yields of light-induced Tyr side-chain fragmentation products, where the yields decreased in the following order: N297Q > GlcNAc 1 > Man 5 > HM. These yields correlate with the thermal stability of the glycoforms. The HM and Man 5 glycoforms display increased affinity for FcγRIIIA by at least 14.7-fold compared with GlcNAc 1 IgG4-Fc. The affinities measured for the HM and Man 5 IgG4-Fc (0.39-0.52 μM) are similar to those measured for fucosylated IgG1. Dependent on the mechanisms of action of IgG4 therapeutics, such glycoforms may need to be carefully monitored. The nonglycosylated N297Q IgG4-Fc did not present measurable affinity to FcγRIIIA., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Guillain-Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the C H 2 and C H 3 domains. These data indicate that the IgG assembly is mediated through Fc-Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain-Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

Despite being the least abundant immunoglobulin G in human plasma, IgG4 are used therapeutically when weak effector functions are needed. The increase in engineered IgG4-based antibodies on the market led us to study the patent landscape of IgG4 Fc engineering, i.e ., patents claiming modifications in the heavy chain. Thirty-seven relevant patent families were identified, comprising hundreds of IgG4 Fc variants focusing on removal of residual effector functions (since IgG4s bind to FcγRI and weakly to other FcγRs), half-life enhancement and IgG4 stability. Given the number of expired or soon to expire major patents in those 3 areas, companies developing blocking antibodies now have, or will in the near future, access to free tools to design silenced, half-life extended and stable IgG4 antibodies.

Forced degradation experiments of monoclonal antibodies (mAbs) aid in the identification of critical quality attributes (CQAs) by studying the impact of post-translational modifications (PTMs), such as oxidation, deamidation, glycation, and isomerization, on biological functions. Structure-function characterization of mAbs can be used to identify the PTM CQAs and develop appropriate analytical and process controls. However, the interpretation of forced degradation results can be complicated because samples may contain mixtures of asymmetrically and symmetrically modified mAbs with one or two modified chains. We present a process to selectively create symmetrically and asymmetrically modified antibodies for structure-function characterization using the bispecific DuoBody® platform. Parental molecules mAb1 and mAb2 were first stressed with peracetic acid to induce methionine oxidation. Bispecific antibodies were then prepared from a mixture of oxidized or unoxidized parental mAbs by a controlled Fab-arm exchange process. This process was used to systematically prepare four bispecific mAb products: symmetrically unoxidized, symmetrically oxidized, and both combinations of asymmetrically oxidized bispecific mAbs. Results of this study demonstrated chain-independent, 1:2 stoichiometric binding of the mAb Fc region to both FcRn receptor and to Protein A. The approach was also applied to create asymmetrically deamidated mAbs at the asparagine 330 residue. Results of this study support the proposed 1:1 stoichiometric binding relationship between the FcγRIIIa receptor and the mAb Fc. This approach should be generally applicable to study the potential impact of any modification on biological function.

As the link between antigen binding and immune activation, the antibody Fc region has received extensive structural study. In this report, the structure of the Fc fragment of the NIST IgG1 mAb (reference material 8671) is described at 2.1 Å resolution in space group P2 1 2 1 2 1 , with approximate unit-cell parameters a = 50, b = 80, c = 138 Å. Prior Fc structures with a wide variety of modifications are also surveyed, focusing on those in the same crystal form. To facilitate the analysis of conformations, a reference frame and a two-parameter metric are proposed, considering the CH2 domains as mobile with respect to a fixed dimeric CH3 core. Over several human Fc structures, a significant variation in Fc elbow conformations is observed, which may serve to facilitate the regulation of Fc effector signaling., (open access.)

To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract, we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17 to 36 (like mouse MAb RG1), and JWW3, a mouse IgG2b derivative of Mab24 specific for L2 residues 58 to 64. By Western blotting, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1 and 25/34 for JWW3. WW1 IgG and F(ab') 2 bound HPV16 pseudovirions similarly; however, whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild-type and neonatal Fc receptor (FcRn)-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption, required for genital infection and induced by either brushing or nonoxynol-9 treatment, released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3, and RG1 was reduced in Fc receptor knockout compared to wild-type mice. However, levels of in vitro neutralization by WW1 IgG were similar in TRIM21 knockout and wild-type cells, indicating that Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellular pseudovirions for phagocytes in protecting mice from intravaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without the need for FcRn-mediated transcytosis. IMPORTANCE At least 15 alpha HPV types are causative agents for 5% of all cancers worldwide, and beta types have been implicated in nonmelanoma skin cancer, whereas others produce benign papillomas, such as genital warts, associated with considerable morbidity and health systems costs. Vaccines targeting the minor capsid protein L2 have the potential to provide broad-spectrum immunity against medically relevant HPVs of divergent genera via the induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reaches the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest that this also permits the local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes for humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays., (Copyright © 2018 American Society for Microbiology.)

The therapeutic efficacy of an antibody drug depends on the variable domains and on the constant crystallizable fragment (Fc). IgG variable domains have been the targets of extensive molecular engineering in search of more specific binders with higher affinities for their targets. Similarly, Fc engineering approaches have led to modulating both the immune effector responses and serum half-lives of therapeutic antibodies. A high-affinity interaction between the IgG Fc and neonatal Fc receptor (FcRn) at a slightly acidic pH can protect IgG molecules from undergoing lysosomal or serum proteinase-induced degradation. Here we describe an optimized protocol for the development of a tailored, synthetic human Fc repertoire to select Fc mutants which show highly pH-restricted FcRn binding with high affinity.

Multimeric fragment crystallizable (Fc) regions and Fc-fusion proteins are actively being explored as biomimetic replacements for IVIG therapy, which is deployed to manage many diseases and conditions but is expensive and not always efficient. The Fc region of human IgG1 (IgG1-Fc) can be engineered into multimeric structures (hexa-Fcs) that bind their cognate receptors with high avidity. The critical influence of the unique N -linked glycan attached at Asn-297 on the structure and function of IgG1-Fc is well documented; however, whether the N -linked glycan has a similarly critical role in multimeric, avidly binding Fcs, is unknown. Hexa-Fc contains two N -linked sites at Asn-77 (equivalent to Asn-297 in the Fc of IgG1) and Asn-236 (equivalent to Asn-563 in the tail piece of IgM). We report here that glycosylation at Asn-297 is critical for interactions with Fc receptors and complement and that glycosylation at Asn-563 is essential for controlling multimerization. We also found that introduction of an additional fully occupied N -linked glycosylation site at the N terminus at position 1 (equivalent to Asp-221 in the Fc of IgG1) dramatically enhances overall sialic acid content of the Fc multimers. Furthermore, replacement of Cys-575 in the IgM tail piece of multimers resulted in monomers with enhanced sialic acid content and differential receptor-binding profiles. Thus insertion of additional N -linked glycans into either the hinge or tail piece of monomers or multimers leads to molecules with enhanced sialylation that may be suitable for managing inflammation or blocking pathogen invasion., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.

As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue)., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Biosimilarity assessments are performed to decide whether 2 preparations of complex biomolecules can be considered "highly similar." In this work, a machine learning approach is demonstrated as a mathematical tool for such assessments using a variety of analytical data sets. As proof-of-principle, physical stability data sets from 8 samples, 4 well-defined immunoglobulin G1-Fragment crystallizable glycoforms in 2 different formulations, were examined (see More et al., companion article in this issue). The data sets included triplicate measurements from 3 analytical methods across different pH and temperature conditions (2066 data features). Established machine learning techniques were used to determine whether the data sets contain sufficient discriminative power in this application. The support vector machine classifier identified the 8 distinct samples with high accuracy. For these data sets, there exists a minimum threshold in terms of information quality and volume to grant enough discriminative power. Generally, data from multiple analytical techniques, multiple pH conditions, and at least 200 representative features were required to achieve the highest discriminative accuracy. In addition to classification accuracy tests, various methods such as sample space visualization, similarity analysis based on Euclidean distance, and feature ranking by mutual information scores are demonstrated to display their effectiveness as modeling tools for biosimilarity assessments., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The Fc portion of immunoglobulin G (IgG) recruits complements and its cognate receptors, thereby promoting defensive mechanisms in the humoral immune system. These effector functions critically depend on N-glycosylation at the Fc region, which is therefore regarded as a crucial factor in the design and production of therapeutic antibodies. NMR spectroscopy plays a unique role in the characterization of conformational dynamics and intermolecular interactions of IgG-Fc in solutions. Here, we report NMR assignments of the glycosylated Fc fragment (Mr 53 kDa), cleaved from a chimeric antibody with human IgG1 constant regions, which was produced in Chinese hamster ovary cells with uniform (13)C- and (15)N-labeling.

The structural integrity and conformational stability of various IgG1-Fc proteins produced from the yeast Pichia pastoris with different glycosylation site occupancy (di-, mono-, and nonglycosylated) were determined. In addition, the physical stability profiles of three different forms of nonglycosylated Fc molecules (varying amino-acid residues at site 297 in the CH 2 domain due to the point mutations and enzymatic digestion of the Fc glycoforms) were also examined. The physical stability of these IgG1-Fc glycoproteins was examined as a function of pH and temperature by high-throughput biophysical analysis using multiple techniques combined with data visualization tools (three index empirical phase diagrams and radar charts). Across the pH range of 4.0-6.0, the di- and monoglycosylated forms of the IgG1-Fc showed the highest and lowest levels of physical stability, respectively, with the nonglycosylated forms showing intermediate stability depending on solution pH. In the aglycosylated Fc proteins, the introduction of Asp (D) residues at site 297 (QQ vs. DN vs. DD forms) resulted in more subtle changes in structural integrity and physical stability depending on solution pH. The utility of evaluating the conformational stability profile differences between the various IgG1-Fc glycoproteins is discussed in the context of analytical comparability studies., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Blood-based anti-amyloid-β (Aβ) immunoglobulins (IgGs) and peripheral inflammation are factors correlating with development of Alzheimer's disease (AD). IgG functionality can drastically change from anti- to pro-inflammatory via alterations in the IgG-Fc N-glycan structure. Herein, we tested if IgG-Fc glycosylation in plasma is indeed altered during the development of AD. Samples from age-matched subjects of 23 controls, 58 patients with stable mild cognitive impairment (SMCI), 34 patients with progressive (P)MCI, and 31 patients with AD were investigated. Label-free shotgun proteomics was applied without glycoprotein enrichment. Glycans on peptides EEQYNSTYR (IgG1) and EEQFNSTFR (IgG2) were quantified, and their abundances were normalized to total IgGn glycoform abundance. Univariate and multivariate statistics were employed to investigate the correlations between the patients groups and the abundances of the IgG glycoforms as well as those of inflammatory mediating proteins. Significant differences (p ≤ 0.05) were found, with a lower abundance of complex galactosylated and sialylated forms in AD. For females, a decline in glycoform complexity correlated with disease progress but an inverse change was found in males prior to the onset of AD. Principal component analysis (PCA; Males: R(2)X(cum) = 0.65, Q(2)(cum) = 0.34; Females: R(2)X(cum) = 0.62, Q(2)(cum) = 0.36), confirmed the gender similarities (for controls, SMCI and AD) as well as differences (for PMCI), and showed a close correlation between pro-inflammatory protein markers, AD, female PMCI, and truncated IgG-Fc glycans. The differences observed between genders prior to the onset of AD may indicate a lower ability in females to suppress peripheral inflammation, which may lead to exacerbated disease progression.

A key element for the successful development of novel therapeutic antibodies is to fully understand their pharmacokinetic and pharmacodynamic behavior before performing clinical trials. While many in vitro modeling approaches exist, these simply cannot substitute for data obtained from appropriate animal models. It was established quite early that the unusual long serum half-life of immunoglobulin G's (IgGs) and Fc domains are due to their rescue and recycling by the neonatal Fc receptor (FcRn). The diverse roles of FcRn became apparent after isolation and cloning. Interesting are the significant species differences between rodent and human FcRn reactivity, rendering wild type rodents an inadequate model for studying IgG serum half-life. With the advance of genetic engineering mouse models have been established expressing human FcRn, and lacking mouse FcRn protein. These models have become highly relevant tools for serum half-life analysis of Fc-containing compounds., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The liver isoform of the enzyme alkaline phosphatase (AP) has been used classically as a serum biomarker for hepatic disease states such as hepatitis, steatosis, cirrhosis, drug-induced liver injury, and hepatocellular carcinoma. Recent studies have demonstrated a more general anti-inflammatory role for AP, as it is capable of dephosphorylating potentially deleterious molecules such as nucleotide phosphates, the pathogenic endotoxin lipopolysaccharide (LPS), and the contact clotting pathway activator polyphosphate (polyP), thereby reducing inflammation and coagulopathy systemically. Yet the mechanism underlying the observed increase in liver AP levels in circulation during inflammatory insults is largely unknown. This paper hypothesizes an immunological role for AP in the liver and the potential of this system for damping generalized inflammation along with a wide range of ancillary pathologies. Based on the provided framework, a mechanism is proposed in which AP undergoes transcytosis in hepatocytes from the canalicular membrane to the sinusoidal membrane during inflammation and the enzyme's expression is upregulated as a result. Through a tightly controlled, nucleotide-stimulated negative feedback process, AP is transported in this model as an immune complex with immunoglobulin G by the asialoglycoprotein receptor through the cell and secreted into the serum, likely using the receptor's State 1 pathway. The subsequent dephosphorylation of inflammatory stimuli by AP and uptake of the circulating immune complex by endothelial cells and macrophages may lead to decreased inflammation and coagulopathy while providing an early upstream signal for the induction of a number of anti-inflammatory gene products, including AP itself., (© 2013.)

Immunoglobulin G (IgG) antibodies coordinate immune effector responses by interacting with effector cells via fragment crystallizable γ (Fcγ) receptors. The IgG Fc domain directs effector responses through subclass and glycosylation variation. Although each Fc variant has been extensively characterized in isolation, during immune responses, IgG is almost always produced in Fc mixtures. How this influences effector responses has not been examined. Here, we measure Fcγ receptor binding to mixed Fc immune complexes. Binding of these mixtures falls along a continuum between pure cases and quantitatively matches a mechanistic model, except for several low-affinity interactions mostly involving IgG2. We find that the binding model provides refined estimates of their affinities. Finally, we demonstrate that the model predicts effector cell-elicited platelet depletion in humanized mice. Contrary to previous views, IgG2 exhibits appreciable binding through avidity, though it is insufficient to induce effector responses. Overall, this work demonstrates a quantitative framework for modeling mixed IgG Fc-effector cell regulation.

The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered Fc domains that confer a longer circulation half-life by virtue of more favorable pH-dependent binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically to recapitulate all the key processes relevant to human antibody persistence in circulation, namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which are important for the clearance of target pathogenic cells and also has favorable developability.

HIV-specific broadly neutralizing antibodies (bNAbs) confer protection after passive immunization, but the immunological mechanisms that drive their development are poorly understood. Structural features of bNAbs indicate that they originate from extensive germinal center (GC) selection, which relies on persistent GC activity. However, why a fraction of infected individuals are able to successfully drive more effective affinity maturation is unclear. Delivery of antigens in the form of antibody-immune complexes (ICs), which bind to complement receptors (CRs) or Fc receptors (FcRs) on follicular dendritic cells, represents an effective mechanism for antigen delivery to the GC. We sought to define whether IC-FcR or CR interactions differ among individuals who develop bNAb responses to HIV. Enhanced Fc effector functions and FcR/CR interactions, via altered Fc glycosylation profiles, were observed among individuals with neutralizing antibody responses to HIV compared with those without neutralizing antibody activity. Moreover, both polyclonal neutralizer ICs and monoclonal IC mimics of neutralizer antibodies induced higher antibody titers, higher-avidity antibodies, and expanded GC B cell reactions after immunization of mice via accelerated antigen deposition within B cell follicles in a complement-dependent manner. Thus, these data point to a direct role for altered Fc profile/complement interactions in shaping the maturation of the humoral immune response, providing insights into how GC activity may be enhanced to drive affinity maturation in next-generation vaccine approaches.

Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy.

The importance of therapeutic recombinant proteins in medicine has led to a variety of tactics to increase their circulation time or to enable routes of administration other than injection. One clinically successful tactic to improve both protein circulation and delivery is to fuse the Fc domain of IgG to therapeutic proteins so that the resulting fusion proteins interact with the human neonatal Fc receptor (FcRn). As an alternative to grafting the high molecular weight Fc domain to therapeutic proteins, we have modified their N and/or C termini with a short peptide sequence that interacts with FcRn. Our strategy was motivated by results [Mezo AR, et al. (2008) Proc Natl Acad Sci USA 105:2337-2342] that identified peptides that compete with human IgG for FcRn. The small size and simple structure of the FcRn-binding peptide (FcBP) allows for expression of FcBP fusion proteins in Escherichia coli and results in their pH-dependent binding to FcRn with an affinity comparable to that of IgG. The FcBP fusion proteins are internalized, recycled, and transcytosed across cell monolayers that express FcRn. This strategy has the potential to improve protein transport across epithelial barriers, which could lead to noninvasive administration and also enable longer half-lives of therapeutic proteins.

Abundant evidence points to a critical role for antibodies in protection and pathology across infectious diseases. While the antibody variable domain facilitates antibody binding and the blockade of infection, the constant domain (Fc) mediates cross talk with the innate immune system. The biological activity of the Fc region is controlled genetically via class switch recombination, resulting in the selection of distinct antibody isotypes and subclasses. However, a second modification is made to all antibodies, via post-translational changes in antibody glycosylation. Studies from autoimmunity and oncology have established the role of immunoglobulin G (IgG) Fc glycosylation as a key regulator of humoral immune activity. However, a growing body of literature, exploring IgG Fc glycosylation through the lens of infectious diseases, points to the role of inflammation in shaping Fc-glycan profiles, the remarkable immune plasticity in antibody glycosylation across pathogen-exposed populations, the canonical and noncanonical functions of glycans and the existence of antigen-specific control over antibody Fc glycosylation. Ultimately, this work provides critical new insights into the functional roles for antibody glycosylation as well as lays the foundation for leveraging antibody glycosylation to drive prevention or control across diseases. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies, engineered antibodies, and antibody fragments have become important biological therapeutic platforms. The IgG format with bivalent binding sites has a modular structure with different biological roles, i.e., effector and binding functions, in different domains. We demonstrated the reconstruction of an IgG-like domain structure in vitro by protein ligation using protein trans-splicing. We produced various binding domains to replace the binding domain of IgG from Escherichia coli and the Fc domain of human IgG from Brevibacillus choshinensis as split-intein fusions. We showed that in vitro protein ligation could produce various Fc-fusions at the N-terminus in vitro from the independently produced domains from different organisms. We thus propose an off-the-shelf approach for the combinatorial production of Fc fusions in vitro with several distinct binding domains, particularly from naturally occurring binding domains. Antiviral lectins from algae are known to inhibit virus entry of HIV and SARS coronavirus. We demonstrated that a lectin could be fused with the Fc-domain in vitro by protein ligation, producing an IgG-like molecule as a “lectibody”. Such an Fc-fusion could be produced in vitro by this approach, which could be an attractive method for developing potential therapeutic agents against rapidly emerging infectious diseases like SARS coronavirus without any genetic fusion and expression optimization.

Guillain−Barré syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the CH2 and CH3 domains. These data indicate that the IgG assembly is mediated through Fc−Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain−Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

A family of chimeric immunoglobulins (Igs) bearing the murine variable region directed against the hapten dansyl linked to human IgG1, -2, -3, and -4 has been characterized with respect to binding to the human high affinity Fc gamma receptor, Fc gamma RI. Chimeric IgG1 and -3 have the highest affinity association (Ka = 10(9) M-1), IgG4 is 10-fold reduced from this level, and IgG2 displays no detectable binding. A series of genetic manipulations was undertaken in which domains from the strongly binding subclass IgG3 were exchanged with domains from the nonbinding subclass IgG2. The subclass of the CH2 domain was found to be critical for determining IgG receptor affinity. In addition, the hinge region was found to modulate the affinity of the IgG for Fc gamma RI, possibly by determining accessibility of Fc gamma RI to the binding site on Fc. A series of amino acid substitutions were engineered into the CH2 domain of IgG3 and IgG4 at sites considered potentially important to Fc receptor binding based on homology comparisons of binding and nonbinding IgG subclasses. Characterization of these mutants has revealed the importance for Fc gamma RI association of two regions of the genetic CH2 domain separated in primary structure by nearly 100 residues. The first of these is the hinge-link or lower hinge regions, in which two residues, Leu (234) and Leu(235) in IgG1 and -3, are critical to high affinity binding. Substitution at either of these sites reduces the IgG association constant by 10-100-fold. The second region that appears to contribute to receptor binding is in a hinge-proximal bend between two beta strands within the CH2 domain, specifically, Pro(331) in IgG1 and -3. As a result of beta sheet formation within this domain, this residue lies within 11 A of the hinge-link region. Substitution at this site reduces the Fc receptor association constant by 10-fold.

The majority of human NK cells express low affinity IgG Fc receptors (CD16+), whereas a minor subset of NK cells lack Fc receptor expression (CD16-). In contrast to CD16+ NK cells that express only p75 IL-2 receptors, CD16- NK cells constitutively co-express both p75 and p55 IL-2 receptors in vivo and preferentially respond to low concentrations of IL-2 with increased cytolytic activation and proliferation. Scatchard analysis demonstrated the presence of approximately 1,200 high affinity (approximately 25 pM kD) and approximately 9,600 intermediate affinity (approximately 2 nM kD) IL-2 receptors on CD16- NK cells. CD16+ NK cells expressed only a single intermediate affinity IL-2 receptor of approximately 1.9 nM kD (approximately 9,000 sites per cell). The IL-2 binding data thus substantiated the phenotypic and functional studies and definitively show that the differential responsiveness of CD16- and CD16+ NK cells to IL-2 is manifested through different affinity IL-2 receptors.

The Fc portion of immunoglobulin G (IgG) is a horseshoe-shaped homodimer, which interacts with various effector proteins, including Fcγ receptors (FcγRs). These interactions are critically dependent on the pair of N-glycans packed between the two CH2 domains. Fucosylation of these N-glycans negatively affects human IgG1-FcγRIIIa interaction. The IgG1-Fc crystal structures mostly exhibit asymmetric quaternary conformations with divergent orientations of CH2 with respect to CH3. We aimed to provide dynamic views of IgG1-Fc by performing long-timescale molecular dynamics (MD) simulations, which were experimentally validated by small-angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our simulation results indicated that the dynamic conformational ensembles of Fc encompass most of the previously reported crystal structures determined in both free and complex forms, although the major Fc conformers in solution exhibited almost symmetric, stouter quaternary structures, unlike the crystal structures. Furthermore, the MD simulations suggested that the N-glycans restrict the motional freedom of CH2 and endow quaternary-structure plasticity through multiple intramolecular interaction networks. Moreover, the fucosylation of these N-glycans restricts the conformational freedom of the proximal tyrosine residue of functional importance, thereby precluding its interaction with FcγRIIIa. The dynamic views of Fc will provide opportunities to control the IgG interactions for developing therapeutic antibodies.

Although antibody functions are executed in heterogeneous blood streams characterized by molecular crowding and promiscuous intermolecular interaction, detailed structural characterizations of antibody interactions have thus far been performed under homogeneous in vitro conditions. NMR spectroscopy potentially has the ability to study protein structures in heterogeneous environments, assuming that the target protein can be labeled with NMR-active isotopes. Based on our successful development of isotope labeling of antibody glycoproteins, here we apply NMR spectroscopy to characterize antibody interactions in heterogeneous extracellular environments using mouse IgG-Fc as a test molecule. In human serum, many of the HSQC peaks originating from the Fc backbone exhibited attenuation in intensity of various magnitudes. Similar spectral changes were induced by the Fab fragment of polyclonal IgG isolated from the serum, but not by serum albumin, indicating that a subset of antibodies reactive with mouse IgG-Fc exists in human serum without preimmunization. The metaepitopes recognized by serum polyclonal IgG cover the entire molecular surface of Fc, including the binding sites to Fc receptors and C1q. In-serum NMR observation will offer useful tools for the detailed characterization of biopharamaceuticals, including therapeutic antibodies in physiologically relevant heterogeneous environments, also giving deeper insight into molecular recognition by polyclonal antibodies in the immune system. [ABSTRACT FROM AUTHOR]

Résumé Les immunoglobulines polyvalentes IgG sont utilisées en thérapeutique humaine depuis plus de 60 ans à partir de la description des premiers déficits humoraux. D’autres indications dans les maladies auto-immunes ou inflammatoires sont apparues dans les années 80. Après un rappel historique, nous présentons les indications reconnues de l’utilisation de ces IgG, leurs mécanismes d’actions et les modalités d’utilisation actuelle par voie intraveineuse ou sous-cutanée. Summary Polyvalent human immunoglobulines IgG are used in human diseases since fifties for primitive immunodeficiency. New indications in autoimmune and inflammatory diseases appeared in eighties. After a brief historical review, we discuss clinical Indications of IgG, their mechanisms of action, and the intravenous or subcutaneous routes of clinical use.of IgG [ABSTRACT FROM AUTHOR]

Biosimilarity assessments are performed to decide whether 2 preparations of complex biomolecules can be considered "highly similar." In this work, a machine learning approach is demonstrated as a mathematical tool for such assessments using a variety of analytical data sets. As proof-of-principle, physical stability data sets from 8 samples, 4 well-defined immunoglobulin G1-Fragment crystallizable glycoforms in 2 different formulations, were examined (see More et al., companion article in this issue). The data sets included triplicate measurements from 3 analytical methods across different pH and temperature conditions (2066 data features). Established machine learning techniques were used to determine whether the data sets contain sufficient discriminative power in this application. The support vector machine classifier identified the 8 distinct samples with high accuracy. For these data sets, there exists a minimum threshold in terms of information quality and volume to grant enough discriminative power. Generally, data from multiple analytical techniques, multiple pH conditions, and at least 200 representative features were required to achieve the highest discriminative accuracy. In addition to classification accuracy tests, various methods such as sample space visualization, similarity analysis based on Euclidean distance, and feature ranking by mutual information scores are demonstrated to display their effectiveness as modeling tools for biosimilarity assessments. [ABSTRACT FROM AUTHOR]

The Fc portion of immunoglobulin G (IgG) recruits complements and its cognate receptors, thereby promoting defensive mechanisms in the humoral immune system. These effector functions critically depend on N-glycosylation at the Fc region, which is therefore regarded as a crucial factor in the design and production of therapeutic antibodies. NMR spectroscopy plays a unique role in the characterization of conformational dynamics and intermolecular interactions of IgG-Fc in solutions. Here, we report NMR assignments of the glycosylated Fc fragment ( Mr 53 kDa), cleaved from a chimeric antibody with human IgG1 constant regions, which was produced in Chinese hamster ovary cells with uniform C- and N-labeling. [ABSTRACT FROM AUTHOR]

Key message: We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis. Abstract: A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a N-enriched plant growth medium. The Fc fragment derived from the N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells. [ABSTRACT FROM AUTHOR]