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Multifunctional anti-HIV Fc-fusion proteins aim to tackle HIV efficiently through multiple modes of action. Although results have been promising, these recombinant proteins are hard to produce. This study explored the production and characterization of anti-HIV Fc-fusion proteins in plant-based systems, specifically Nicotiana benthamiana plants and tobacco BY-2 cell suspension. Fc-fusion protein expression in plants was optimized by incorporating codon optimization, ER retention signals, and hydrophobin fusion elements. Successful transient protein expression was achieved in N. benthamiana, with notable improvements in expression levels achieved through N-terminal hydrophobin fusion and ER retention signals. Stable expression in tobacco BY-2 resulted in varying accumulation levels being at highest 2.2.mg/g DW. The inclusion of hydrophobin significantly enhanced accumulation, providing potential benefits for downstream processing. Mass spectrometry analysis confirmed the presence of the ER retention signal and of N-glycans. Functional characterization revealed strong binding to CD64 and CD16a receptors, the latter being important for antibody-dependent cellular cytotoxicity (ADCC). Interaction with HIV antigens indicated potential neutralization capabilities. In conclusion, this research highlights the potential of plant-based systems for producing functional anti-HIV Fc-fusion proteins, offering a promising avenue for the development of these novel HIV therapies., Competing Interests: Declaration of Competing Interest JC and JB were founders of AlbaJuna Therapeutics. AB, JB, JC and FC were employees of AlbaJuna therapeutics. Other authors, no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Enteroviruses contain multiple serotypes and can cause severe neurological complications. The intricate life cycle of enteroviruses involving dynamic virus-receptor interaction hampers the development of broad therapeutics and vaccines. Here, using function-based screening, we identify a broadly therapeutic antibody h1A6.2 that potently protects mice in lethal models of infection with both enterovirus A71 and coxsackievirus A16 through multiple mechanisms, including inhibition of the virion-SCARB2 interactions and monocyte/macrophage-dependent Fc effector functions. h1A6.2 mitigates inflammation and improves intramuscular mechanics, which are associated with diminished innate immune signalling and preserved tissue repair. Moreover, cryogenic electron microscopy structures delineate an adaptive binding of h1A6.2 to the flexible and dynamic nature of the VP2 EF loop with a binding angle mimicking the SCARB2 receptor. The coordinated binding mode results in efficient binding of h1A6.2 to all viral particle types and facilitates broad neutralization of enterovirus, therefore informing a promising target for the structure-guided design of pan-enterovirus vaccine., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Antibody Fc regions harbour the binding sites for receptors that mediate effector functions following antigen engagement by the Fab regions. An extended "hinge" region in IgG allows flexibility between Fab and Fc, but in both the most primitive antibody, IgM, and in the evolutionarily more recent IgE, the hinge is replaced by an additional domain pair in the homodimeric six-domain Fc region. This permits additional flexibility within the Fc region, which has been exploited by nature to modulate antibody effector functions. Thus, in pentameric or hexameric IgM, the Fc regions appear to adopt a planar conformation in solution until antigen binding causes a conformational change and exposes the complement binding sites. In contrast, IgE-Fc principally adopts an acutely bent conformation in solution, but the binding of different receptors is controlled by the degree of bending, and there is allosteric communication between receptor binding sites., Methods: We sought to trace the evolution of Fc conformational diversity from IgM to IgE via the intermediate avian IgY by studying the solution conformations of their Fc regions by small-angle X-ray scattering. We compared four extant proteins: human IgM-Fc homodimer, chicken IgY-Fc, platypus IgE-Fc, and human IgE-Fc. These are examples of proteins that first appeared in the jawed fish [425 million years ago (mya)], tetrapod (310 mya), monotreme (166 mya), and hominid (2.5 mya) clades, respectively., Results and Discussion: We analysed the scattering curves in terms of contributions from a pool of variously bent models chosen by a non-negative linear least-squares algorithm and found that the four proteins form a series in which the proportion of acutely bent material increases: IgM-Fc < IgY-Fc < plIgE-Fc < huIgE-Fc. This follows their order of appearance in evolution. For the huIgM-Fc homodimer, although none are acutely bent, and a significant fraction of the protein is sufficiently bent to expose the C1q-binding site, it predominantly adopts a fully extended conformation. In contrast, huIgE-Fc is found principally to be acutely bent, as expected from earlier studies. IgY-Fc, in this first structural analysis of the complete Fc region, exhibits an ensemble of conformations from acutely bent to fully extended, reflecting IgY's position as an evolutionary intermediate between IgM and IgE., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Calvert, Nyamboya, Beavil and Sutton.)

Current cancer immunotherapy predominately focuses on eliciting type 1 immune responses fighting cancer; however, long-term complete remission remains uncommon 1,2 . A pivotal question arises as to whether type 2 immunity can be orchestrated alongside type 1-centric immunotherapy to achieve enduring response against cancer 3,4 . Here we show that an interleukin-4 fusion protein (Fc-IL-4), a typical type 2 cytokine, directly acts on CD8 + T cells and enriches functional terminally exhausted CD8 + T (CD8 + T TE ) cells in the tumour. Consequently, Fc-IL-4 enhances antitumour efficacy of type 1 immunity-centric adoptive T cell transfer or immune checkpoint blockade therapies and induces durable remission across several syngeneic and xenograft tumour models. Mechanistically, we discovered that Fc-IL-4 signals through both signal transducer and activator of transcription 6 (STAT6) and mammalian target of rapamycin (mTOR) pathways, augmenting the glycolytic metabolism and the nicotinamide adenine dinucleotide (NAD) concentration of CD8 + T TE cells in a lactate dehydrogenase A-dependent manner. The metabolic modulation mediated by Fc-IL-4 is indispensable for reinvigorating intratumoural CD8 + T TE cells. These findings underscore Fc-IL-4 as a potent type 2 cytokine-based immunotherapy that synergizes effectively with type 1 immunity to elicit long-lasting responses against cancer. Our study not only sheds light on the synergy between these two types of immune responses, but also unveils an innovative strategy for advancing next-generation cancer immunotherapy by integrating type 2 immune factors., (© 2024. The Author(s).)

Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

HLA-DQ molecules drive unwanted alloimmune responses after solid-organ transplants and several autoimmune diseases, including type 1 diabetes and celiac disease. Biologics with HLA molecules as part of the design are emerging therapeutic options for these allo- and autoimmune conditions. However, the soluble α and β chains of class II HLA molecules do not dimerize efficiently without their transmembrane domains, which hinders their production. In this study, we examined the feasibility of interchain disulfide engineering by introducing paired cysteines to juxtaposed positions in the α and β chains of HLA-DQ7, encoded by HLA-DQA1∗05:01 and HLA-DQB1∗03:01 respectively. We identified three variant peptide-HLA-DQ7-Fc fusion proteins (DQ7Fc) with increased expression and production yield, namely Y19C-D6C (YCDC), A83C-E5C (ACEC), and A84C-N33C (ACNC). The mutated residues were conserved across all HLA-DQ proteins and had limited solvent exposure. Further characterizations of the YCDC variant showed that the expression of the fusion protein is peptide-dependent; inclusion of a higher-affinity peptide correlated with increased protein expression. However, high-affinity peptide alone was insufficient for stabilizing the DQ7 complex without the engineered disulfide bond. Multiple DQ7Fc variants demonstrated expected binding characteristics with commercial anti-DQ antibodies in two immunoassays and by a cell-based assay. Lastly, DQ7Fc variants demonstrated dose-dependent killing of DQ7-specific B cell hybridomas in a flow cytometric, complement-dependent cytotoxicity assay. These data support inter-chain disulfide engineering as a novel approach to efficiently producing functional HLA-DQ molecules and potentially other class II HLA molecules as candidate therapeutic agents., Competing Interests: Conflict of interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. X.A., C.L., and H.G. have financial interests in Antiger Therapeutics Inc. A patent application on Class II HLA with Inter-Chain Disulfide Bond has been filed in the US patent office (Inventor: C.L. and H.G.; Antiger Therapeutics Inc; US Provisional Application No. 63/613880)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mutational changes that affect the binding of the C2 fragment of Streptococcal protein G (GB1) to the Fc domain of human IgG (IgG-Fc) have been extensively studied using deep mutational scanning (DMS), and the binding affinity of all single mutations has been measured experimentally in the literature. To investigate the underlying molecular basis, we perform in silico mutational scanning for all possible single mutations, along with 2 μs-long molecular dynamics (WT-MD) of the wild-type (WT) GB1 in both unbound and IgG-Fc bound forms. We compute the hydrogen bonds between GB1 and IgG-Fc in WT-MD to identify the dominant hydrogen bonds for binding, which we then assess in conformations produced by Mutation and Minimization (MuMi) to explain the fitness landscape of GB1 and IgG-Fc binding. Furthermore, we analyze MuMi and WT-MD to investigate the dynamics of binding, focusing on the relative solvent accessibility of residues and the probability of residues being located at the binding interface. With these analyses, we explain the interactions between GB1 and IgG-Fc and display the structural features of binding. In sum, our findings highlight the potential of MuMi as a reliable and computationally efficient tool for predicting protein fitness landscapes, offering significant advantages over traditional methods. The methodologies and results presented in this study pave the way for improved predictive accuracy in protein stability and interaction studies, which are crucial for advancements in drug design and synthetic biology.

Antigen-presenting cells (APCs) play an important role in virus infection control by bridging innate and adaptive immune responses. Macrophages and dendritic cells (DCs) possess various surface receptors to recognize/internalize antigens, and antibody binding can enhance pathogen-opsonizing uptake by these APCs via interaction of antibody fragment crystallizable (Fc) domains with Fc receptors, evoking profound pathogen control in certain settings. Here, we examined phagocytosis-enhancing potential of Fc domains directly oriented on a retroviral virion/virus-like particle (VLP) surface. We generated an expression vector coding a murine Fc fragment fused to the transmembrane region (TM) of a retroviral envelope protein, deriving expression of the Fc-TM fusion protein on the transfected cell surface and production of virions incorporating the chimeric Fc upon co-transfection. Incubation of Fc-displaying simian immunodeficiency virus (SIV) with murine J774 macrophages and bone marrow-derived DCs derived Fc receptor-dependent enhanced uptake, being visualized by imaging cytometry. Alternative preparation of a murine leukemia virus (MLV) backbone-based Fc-displaying VLP loading an influenza virus hemagglutinin (HA) antigen resulted in enhanced HA internalization by macrophages, stating antigen compatibility of the design. Results show that the Fc-TM fusion molecule can be displayed on certain viruses/VLPs and may be utilized as a molecular adjuvant to facilitate APC antigen uptake., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The effective attachment of antibodies to the immune sensing interface is a crucial factor that determines the detection performance of immunosensors. Therefore, this study aims to investigate a novel antibody immobilization material with low molecular weight, high stability, and excellent directional immobilization effect. In this study, we employed molecular docking technology based on the ZDOCK algorithm to virtually screen DNA functional ligands (DNAFL) for the Fc segment of antibodies. Through a comprehensive analysis of the key binding sites and contact propensities at the interface between DNAFL and IgG antibody, we have gained valuable insights into the affinity relationship, as well as the principles governing amino acid and nucleotide interactions at this interface. Furthermore, molecular affinity experiments and competitive binding experiments were conducted to validate both the binding ability of DNAFL to IgG antibody and its actual binding site. Through affinity experiments using multi-base sequences, we identified bases that significantly influence antibody-DNAFL binding and successfully obtained DNAFL with an enhanced affinity towards the IgG Fc segment. These findings provide a theoretical foundation for the targeted design of higher-affinity DNAFLs while also presenting a new technical approach for immunosensor preparation with potential applications in biodetection.

α1,6-Fucosyltransferase (Fut8) is the enzyme responsible for catalyzing core fucosylation. Exogenous L-fucose upregulates fucosylation levels through the GDP-fucose salvage pathway. This study investigated the relationship between core fucosylation and immunoglobulin G (IgG) amounts in serum utilizing WT (Fut8 +/+ ), Fut8 heterozygous knockout (Fut8 +/- ), and Fut8 knockout (Fut8 -/- ) mice. The IgG levels in serum were lower in Fut8 +/- and Fut8 -/- mice compared with Fut8 +/+ mice. Exogenous L-fucose increased IgG levels in Fut8 +/- mice, while the ratios of core fucosylated IgG versus total IgG showed no significant difference among Fut8 +/+ , Fut8 +/- , and Fut8 +/- mice treated with L-fucose. These ratios were determined by Western blot, lectin blot, and mass spectrometry analysis. Real-time PCR results demonstrated that mRNA levels of IgG Fc and neonatal Fc receptor, responsible for protecting IgG turnover, were similar among Fut8 +/+ , Fut8 +/- , and Fut8 +/- mice treated with L-fucose. In contrast, the expression levels of Fc-gamma receptor Ⅳ (FcγRⅣ), mainly expressed on macrophages and neutrophils, were increased in Fut8 +/- mice compared to Fut8 +/+ mice. The effect was reversed by administrating L-fucose, suggesting that core fucosylation primarily regulates the IgG levels through the Fc-FcγRⅣ degradation pathway. Consistently, IgG internalization and transcytosis were suppressed in FcγRⅣ-knockout cells while enhanced in Fut8-knockout cells. Furthermore, we assessed the expression levels of specific antibodies against ovalbumin and found they were downregulated in Fut8 +/- mice, with potential recovery observed with L-fucose administration. These findings confirm that core fucosylation plays a vital role in regulating IgG levels in serum, which may provide insights into a novel mechanism in adaptive immune regulation., Competing Interests: Conflicts 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.)

TRIM21 is a pivotal effector in the immune system, orchestrating antibody-mediated responses and modulating immune signaling. In this comprehensive study, we focus on the interaction of TRIM21 with Fc engineered antibodies and subsequent implications for viral neutralization. Through a series of analytical techniques, including biosensor assays, mass photometry, and electron microscopy, along with structure predictions, we unravel the intricate mechanisms governing the interplay between TRIM21 and antibodies. Our investigations reveal that the TRIM21 capacity to recognize, bind, and facilitate the proteasomal degradation of antibody-coated viruses is critically dependent on the affinity and avidity interplay of its interactions with antibody Fc regions. We suggest a novel binding mechanism, where TRIM21 binding to one Fc site results in the detachment of PRYSPRY from the coiled-coil domain, enhancing mobility due to its flexible linker, thereby facilitating the engagement of the second site, resulting in avidity due to bivalent engagement. These findings shed light on the dual role of TRIM21 in antiviral immunity, both in recognizing and directing viruses for intracellular degradation, and demonstrate its potential for therapeutic exploitation. The study advances our understanding of intracellular immune responses and opens new avenues for the development of antiviral strategies and innovation in tailored effector functions designed to leverage TRIM21s unique binding mode., Competing Interests: CK declares employment, patents and stock ownership with Roche. Authors JR, JT, JB, TJ, TS, and LL were employed by the company Roche Diagnostics GmbH. Authors LF and PR were employed by the company F.Hoffmann-La Roche Ltd. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Reusch, Franken, Then, Ringler, Butzer, Juroschek, Klein, Schlothauer and Larivière.)

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation modulates effector functions such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Consequently, assessing IgG Fc glycosylation is important for understanding the role of antibodies in infectious, alloimmune and autoimmune diseases. GlYcoLISA determines the Fc glycosylation of antigen-specific IgG by an immunosorbent assay with a liquid chromatography-mass spectrometry (LC-MS) readout. Detection of antigen-specific IgG glycosylation in a subclass- and site-specific manner is realized by LC-MS-based glycopeptide analysis after proteolytic cleavage. GlYcoLISA addresses challenges related to the low abundance of specific IgG and the high background of total IgG by using well-established immunosorbent assays for purifying antibodies of the desired specificity using immobilized antigen. Alternative methods with sufficient glycan resolution lack these important specificities. GlYcoLISA is performed in a 96-well plate format, and the analysis of 160 samples takes ~5 d, with 1 d for sample preparation, 2 d of LC-MS measurement and 2 d for partially automated data processing. GlYcoLISA requires expertise in LC-MS operation and data processing., (© 2024. Springer Nature Limited.)

Sialic acids as terminal sugar residues on cell surface or secreted proteins have many functional roles. In particular, the presence or absence of α2,6-linked sialic acid residues at the immunoglobulin G (IgG) Fc fragment can switch IgG effector functions from pro- to anti-inflammatory activity. IgG glycosylation is considered to take place inside the plasma blast/plasma cell while the molecule travels through the endoplasmic reticulum and Golgi apparatus before being secreted. However, more recent studies have suggested that IgG sialylation may occur predominantly post-antibody secretion. To what extent this extracellular IgG sialylation process contributes to overall IgG sialylation remains unclear, however. By generating bone marrow chimeric mice with a B cell-specific deletion of ST6Gal1, the key enzyme required for IgG sialylation, we now show that sialylation of the IgG Fc fragment exclusively occurs within B cells pre-IgG secretion. We further demonstrate that B cells expressing ST6Gal1 have a developmental advantage over B cells lacking ST6Gal1 expression and thus dominate the plasma cell pool and the resulting serum IgG population in mouse models in which both ST6Gal1-sufficient and -deficient B cells are present., Competing Interests: Authors MH, OZ, and GL were employed by Genos Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Werner, Hanić, Zaitseva, Lauc, Lux, Nitschke and Nimmerjahn.)

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.)

Antibodies can initiate lung injury in a variety of disease states such as autoimmunity, in reactions to transfusions, or after organ transplantation, but the key factors determining in vivo pathogenicity of injury-inducing antibodies are unclear. Harmful antibodies often activate the complement cascade. A model for how IgG antibodies trigger complement activation involves interactions between IgG Fc domains driving the assembly of IgG hexamer structures that activate C1 complexes. The importance of IgG hexamers in initiating injury responses was not clear, so we tested their relevance in a mouse model of alloantibody- and complement-mediated acute lung injury. We used 3 approaches to block alloantibody hexamerization (antibody carbamylation, the K439E Fc mutation, or treatment with domain B from staphylococcal protein A), all of which reduced acute lung injury. Conversely, Fc mutations promoting spontaneous hexamerization made a harmful alloantibody into a more potent inducer of acute lung injury and rendered an innocuous alloantibody pathogenic. Treatment with a recombinant Fc hexamer "decoy" therapeutic protected mice from lung injury, including in a model with transgenic human FCGR2A expression that exacerbated pathology. These results indicate an in vivo role of IgG hexamerization in initiating acute lung injury and the potential for therapeutics that inhibit or mimic hexamerization to treat antibody-mediated diseases.

The allosteric modulation of the homodimeric H10-03-6 protein to glycan ligands L1 and L2, and the STAB19 protein to glycan ligands L3 and L4, respectively, has been studied by molecular dynamics simulations and free energy calculations. The results revealed that the STAB19 protein has a significantly higher affinity for L3 (-11.38 ± 2.32 kcal/mol) than that for L4 (-5.51 ± 1.92 kcal/mol). However, the combination of the H10-03-6 protein with glycan L2 (1.23 ± 6.19 kcal/mol) is energetically unfavorable compared with that of L1 (-13.96 ± 0.35 kcal/mol). Further, the binding of glycan ligands L3 and L4 to STAB19 would result in the significant closure of the two CH2 domains of the STAB19 conformation with the decrease of the centroid distances between the two CH2 domains compared with the H10-03-6/L1/L2 complex. The CH2 domain closure of STAB19 relates directly to the formation of new hydrogen bonds and hydrophobic interactions between the residues Ser239, Val240, Asp265, Glu293, Asn297, Thr299, Ser337, Asp376, Thr393, Pro395, and Pro396 in STAB19 and glycan ligands L3 and L4, which suggests that these key residues would contribute to the specific regulation of STAB19 to L3 and L4. In addition, the distance analysis revealed that the EF loop in the H10-03-6/L1/L2 model presents a high flexibility and partial disorder compared with the stabilized STAB19/L3/L4 complex. These results will be helpful in understanding the specific regulation through the asymmetric structural characteristics in the CH2 and CH3 domains of the H10-03-6 and STAB19 proteins.

Development of novel therapeutic proteins and biosimilars requires a thorough understanding of the relationship between their structure and function. Particularly, how IgG glycosylation affects its effector functions is a point increasingly underscored in guidelines by the World Health Organization and regulatory agencies. Our results show that just a 1% decrease in Fc fucosylation can lead to a more than 25% increase in antibody-dependent cell-mediated cytotoxicity. The intercorrelated nature of glycan patterns, combined with the low variability and lack of well-defined glycan patterns in process development and manufacture samples, makes studying the effects of individual glycan structures challenging. The conventional approach to structure-function studies often relies on a suboptimal set of tools, such as the one-factor-at-a-time method for experimental planning and univariate data analysis. Here, we introduce a systematic approach to understanding and prediction of the impact of Fc glycans on effector functions, using a combination of the design of experiment, multivariate data analysis, and in-vitro glycoengineering. This approach adheres to quality-by-design principles and aligns with regulatory agency guidelines. A variety of analytical assays, including binding and cell-based assays, were applied to investigate the effect of individual glycans of the IgG1 molecule. The regression models developed here provide a quantitative explanation and prediction of the impact of individual glycan features on the binding to FcγRs and bioactivity of the therapeutic protein. To the best of our knowledge, this is the first report of a systematic approach to quantitatively understand the multivariate impact of glycosylation on the effector functionality of therapeutic monoclonal antibodies, providing valuable tools for advancing therapeutic protein development.

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been used in the clinic, but they have not been systematically compared. We have now produced a matched set of anti-CD20 antibodies with different Fc subclasses and variants and compared their activity for binding to C1q, Fc-gamma receptors and in cell-based assays. Most of the variants still have significant levels of activity in one or more of these assays and many of them have impaired temperature stability compared with the corresponding wild-type antibody.

The crystallizable fragment (Fc) domain of immunoglobulin subclass IgG1 antibodies is engineered for a wide variety of pharmaceutical applications. Two important structural variables in Fc constructs are the hinge region connecting the Fc to the antigen binding fragments (Fab) and the glycans present in various glycoforms. These components affect receptor binding interactions that mediate immune activation. To design new antibody drugs, a robust in silico method for linking stability to structural changes is necessary. In this work, all-atom simulations were used to compare the dynamic behavior of the four structural variants arising from presence or absence of the hinge and glycans. We expressed the simplest of these constructs, the 'minimal Fc' with no hinge and no glycans, in Escherichia coli and report its crystal structure. The 'maximal Fc' that includes full hinge and G0F/G1F glycans is based on a previously reported structure, Protein Data Bank (PDB) ID: 5VGP. These, along with two intermediate structures (with only the glycans or with only the hinge) were used to independently measure the stability effects of the two structural variables using umbrella sampling simulations. Principal component analysis (PCA) was used to determine free energy effects along the Fc's dominant mode of motion. This work provides a comprehensive picture of the effects of hinge and glycans on Fc dynamics and stability.Communicated by Ramaswamy H. Sarma.

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.)

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab') 2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunoglobulin G (IgG) antibodies contain a complex N-glycan embedded in the hydrophobic pocket between its heavy chain protomers. This glycan contributes to the structural organization of the Fc domain and determines its specificity for Fcγ receptors, thereby dictating distinct cellular responses. The variable construction of this glycan structure leads to highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported synthetic nanobodies that distinguish IgG glycoforms. Here, we present the structure of one such nanobody, X0, in complex with the Fc fragment of afucosylated IgG1. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a 'glycan sensor', forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-FcγRIIIa interactions and rescue mice in a model of dengue virus infection., (© 2023. The Author(s).)

Breast milk immunoglobulin G (IgG) plays an important role in the transfer of passive immunity in early life and in shaping the neonatal immune system through N-glycan-mediated effector functions. Currently, there are no protocols available to analyze breast milk IgG-Fc glycosylation in mouse models. Therefore, we developed and validated a glycoproteomic workflow for the medium-throughput subclass-specific nano-LC-MS analysis of IgG enriched from small milk volumes of lactating mice. With the established methods, the IgG glycopatterns in a mouse model of antibiotic use during pregnancy and increased asthma susceptibility in the offspring were analyzed. Pregnant BALB/c mice were treated with vancomycin during gestation days 8-17 and IgG1F, IgG2, and IgG3-Fc glycosylation was subsequently analyzed in maternal serum, maternal breast milk, and offspring serum on postnatal day 15. The IgG glycosylation profiles of mouse maternal milk and serum revealed no significant differences within the glycoforms quantified across subclasses. However, vancomycin use during pregnancy was associated with changes in IgG-Fc glycosylation in offspring serum, shown by the decreased relative abundance of the IgG1F-G1 and IgG3-G0 glycoforms, together with the increased relative abundance of the IgG3-G2 and S1 glycoforms. The workflow presented will aid in the emerging integrative multi-omics- and glycomics-oriented milk analyses both in rodent models and human cohorts for a better understanding of mother-infant immunological interactions., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

We have investigated the dynamics of two [Formula: see text]-immunoglobulin molecules, IgG1 and IgG4, using long all atom molecular dynamics simulations. We first show that the de novo structures of IgG1 and IgG4 predicted using AlphaFold, with no interactions between the fragment crystallizable (Fc) domain and the antigen fragment binding domain (Fab), eventually relaxes to a state with persistent Fc-Fab interactions that mirrors experimentally resolved structures. We quantified the conformational space sampled by antibody trajectories spawned from six different initial structures and show that the individual trajectories only sample states bound by a local minimum and display very little mixing in their conformational states. Furthermore, the dynamics of the individual Fab domains are strongly dependent on the initial crystal structure and isotype. In all conditions, we observe non-identical dynamics between the Fab arms in an antibody. For a six-bead coarse grained model, we show that non-covalent Fc-Fab interactions can modulate the stiffnesses associated with Fc-Fab distances, angles, and dihedral angles by up to three orders of magnitude. Our results clearly illustrate the inherent complexities in studying antibody dynamics and highlight the need to include non-identical Fab dynamics as an inherent feature in computational models of therapeutic antibodies., (© 2023. The Author(s).)

Engineering of Fc has been adapted as an efficient method for enhanced or reduced affinity towards Fc receptors in the development of therapeutic antibodies. S239D/I332E mutation of Fc induces approximately two logs greater affinity to the FcγRIIIa receptor and has been extensively employed in various Fc engineering studies. It is known that the mutation gives rise to the formation of salt bridges between the mutated residues of Fc and FcγRIIIa, but the overall effect of the mutation in the binding interface of the Fc-FcγRIIIa complex is still unclear. In this study, the molecular interactions in the binding interface of mutant Fc and FcγRIIIa were analyzed and compared with those of wild-type Fc binding through residue interaction network (RIN) analysis and molecular dynamics (MD) simulation. RIN analysis identified specific molecular interactions and Hub residues in the interfaces, and their numbers were increased by introducing the mutation, with maintaining most of the molecular interactions in the wild-type complex. MD simulation study revealed that the numbers of stable electrostatic interactions and stable Hub residues in the mutant complex were higher than those in the wild-type complex. The introduced mutations were shown to form further charge-charge attractive interactions in addition to the identified salt bridges without generating any repulsive interactions. These results are expected to provide further structural insight into Fc variants' design based on the S239D/I332E mutation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Antigen-binding Fc (Fcab™) fragments, where a novel antigen binding site is introduced by the mutagenesis of the C-terminal loops of the C H 3 domain, function as parts of bispecific IgG-like symmetrical antibodies when they replace their wild-type Fc. Their homodimeric structure typically leads to bivalent antigen binding. In particular, biological situations monovalent engagement, however, would be preferred, either for avoiding agonistic effects leading to safety issues, or the attractive option of combining a single chain (i.e., one half) of an Fcab fragment reactive with different antigens in one antibody. We present the strategies for construction and selection of yeast libraries displaying heterodimeric Fcab fragments and discuss the effects of altered thermostability of the basic Fc scaffold and novel library designs that lead to isolation of highly affine antigen binding clones., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (&lt;37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins., (© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Protein glycosylation is a crucial mediator of biological functions and is tightly regulated in health and disease. However, interrogating complex protein glycoforms is challenging, as current lectin tools are limited by cross-reactivity while mass spectrometry typically requires biochemical purification and isolation of the target protein. Here, we describe a method to identify and characterize a class of nanobodies that can distinguish glycoforms without reactivity to off-target glycoproteins or glycans. We apply this technology to immunoglobulin G (IgG) Fc glycoforms and define nanobodies that specifically recognize either IgG lacking its core-fucose or IgG bearing terminal sialic acid residues. By adapting these tools to standard biochemical methods, we can clinically stratify dengue virus and SARS-CoV-2 infected individuals based on their IgG glycan profile, selectively disrupt IgG-Fcγ receptor binding both in vitro and in vivo, and interrogate the B cell receptor (BCR) glycan structure on living cells. Ultimately, we provide a strategy for the development of reagents to identify and manipulate IgG Fc glycoforms.

Cancer therapy using immune checkpoint inhibitor antibodies has markedly shifted the paradigm of cancer treatment. However, methods completely eliminating the effector function of these signal-regulating antibodies is urgently required. The heterogeneity of glycan chains in antibodies limits their use as therapeutic agents due to their variability; thus, the development of uniform glycan chains is necessary. Here, we subjected the anti-programmed cell death protein (PD)-1 antibody nivolumab, a representative immune checkpoint inhibitor, to GlycoDelete (GD) engineering to remove the antibody-dependent cellular cytotoxicity (ADCC) of the antibody, leaving only one glycan in the Fc. Glyco-engineered CHO cells were prepared by overexpressing endo-β-N-acetyl-glucosaminidase (Endo T) in CHO cells, in which N-acetyl-glucosaminyl-transferase I was knocked out using Cas9. GD IgG1 nivolumab and GD IgG4 nivolumab were produced using GD CHO cells, and glycan removal was confirmed using mass spectrometry. Target binding and PD-1 inhibition was not altered; however, ADCC decreased. Furthermore, the IgG4 form, determined to be the most suitable form of GD nivolumab, was produced in a plant GD system. The plant GD nivolumab also reduced ADCC without affecting PD-1 inhibitory function. Thus, CHO and plant GD platforms can be used to improve signal-regulating antibodies by reducing their effector function., (© 2022. The Author(s).)

The presence of T regulatory (Treg) cells in the tumor microenvironment is associated with poor prognosis and resistance to therapies aimed at reactivating anti-tumor immune responses. Therefore, depletion of tumor-infiltrating Tregs is a potential approach to overcome resistance to immunotherapy. However, identifying Treg-specific targets to drive such selective depletion is challenging. CCR8 has recently emerged as one of these potential targets. Here, we describe GS-1811, a novel therapeutic monoclonal antibody that specifically binds to human CCR8 and is designed to selectively deplete tumor-infiltrating Tregs. We validate previous findings showing restricted expression of CCR8 on tumor Tregs, and precisely quantify CCR8 receptor densities on tumor and normal tissue T cell subsets, demonstrating a window for selective depletion of Tregs in the tumor. Importantly, we show that GS-1811 depleting activity is limited to cells expressing CCR8 at levels comparable to tumor-infiltrating Tregs. Targeting CCR8 in mouse tumor models results in robust anti-tumor efficacy, which is dependent on Treg depleting activity, and synergizes with PD-1 inhibition to promote anti-tumor responses in PD-1 resistant models. Our data support clinical development of GS-1811 to target CCR8 in cancer and drive tumor Treg depletion in order to promote anti-tumor immunity., Competing Interests: JAB, MW, TR, AM, VS, MP, SJ, AS, KK, YE, MAM, DW and MG are employees and own stock of Jounce Therapeutics. JDW, ECS, FD, BK, CF, RK, LM, KL, CX, YZ, CH, DU, CX, YZ and DRS were Jounce Therapeutics employees at the time of the study. DSA, MRK and BMW are employees and own stock of Gilead Sciences., (© 2022 Jounce Therapeutics, Inc. Published with license by Taylor & Francis Group, LLC.)

Introduction: Glioma is the most common and most invasive primary central nervous system tumour, and it is urgent to develop new specific therapeutic targets. Studies have confirmed that epithelial-derived tumour cells promote tumour cell proliferation and metastasis by secreting a large number of immunoglobulins (Igs), but the role of tumour-derived Igs in glioma has never been reported., Methods: The Gene Expression Profiling Interactive Analysis and Chinese Glioma Genome Atlas databases were used to analyse the Ig transcription and its correlation with the prognosis of patients with glioma. Immunohistochemistry and immunofluorescence were used to detect the protein expression of IgG and IgM in the glioma tissues of patients and glioma cell lines. When IgG was knocked down by small interfering RNA or knocked out by CRISPR-Cas9, the function of proliferation and migration of glioma cells were analysed by CCK-8, clone formation, wound healing, and transwell assays. Changes in proteins and their phosphorylation in signalling pathways were detected by western blotting. The nude mouse subcutaneous tumour-bearing model was established to analyse the effect of IgG in vivo., Results: The transcriptional level of IgG was pretty high in glioma tissues and was positively correlated with high WHO grade, recurrence, and poor prognosis. The expression of IgG and IgM was found in tumour tissues and human glioma cell lines U87 and U251, and the main expression form was secreted. Decreased IgG inhibited the proliferation and migration of glioma cells. Knockout or knockdown of IgG downregulated the phosphorylation of the key molecules in the MAPK and PI3K/Akt pathway through the HGF/SF-Met or FAK/Src pathway. In vivo tumourigenesis mouse model confirmed that reduced IgG expression inhibited glioma growth., Conclusion: Ig was expressed in glioma tissues and cell lines, and a high expression level predicted a poor prognosis of patients. Glioma-derived IgG promoted glioma cell proliferation and migration through the HGF/SF-Met or FAK/Src pathway., Competing Interests: Conflict of interest statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease for which prediction of long-term prognosis from disease's outset is not clinically feasible. The importance of immunoglobulin G (IgG) and its Fc N-glycosylation in inflammation is well-known and studies described its relevance for several autoimmune diseases, including RA. Herein we assessed the association between IgG N-glycoforms and disease prognosis at 2 years in an early inflammatory arthritis cohort., Methods: Sera from 118 patients with early inflammatory arthritis naïve to treatment sampled at baseline were used to obtain IgG Fc glycopeptides, which were then analyzed in a subclass-specific manner by liquid chromatography coupled to mass spectrometry (LC-MS). Patients were prospectively followed and a favorable prognosis at 2 years was assessed by a combined index as remission or low disease activity (DAS28 < 3.2) and normal functionality (HAQ ≤ 0.25) while on treatment with conventional synthetic DMARDs and never used biologic DMARDs., Results: We observed a significant association between high levels of IgG2/3 Fc galactosylation (effect 0.627 and adjusted p value 0.036 for the fully galactosylated glycoform H5N4F1; effect -0.551 and adjusted p value 0.04963 for the agalactosylated H3N4F1) and favorable outcome after 2 years of treatment. The inclusion of IgG glycoprofiling in a multivariate analysis to predict the outcome (with HAQ, DAS28, RF, and ACPA included in the model) did not improve the prognostic performance of the model., Conclusion: Pending confirmation of these findings in larger cohorts, IgG glycosylation levels could be used as a prognostic marker in early arthritis, to overcome the limitations of the current prognostic tools., (© 2022. The Author(s).)

Antibody-based therapeutics (ABTs) are used to treat a range of diseases. Most ABTs are either full-length IgG1 antibodies or fusions between for instance antigen (Ag)-binding receptor domains and the IgG1 Fc fragment. Interestingly, their plasma half-life varies considerably, which may relate to how they engage the neonatal Fc receptor (FcRn). As such, there is a need for an in-depth understanding of how different features of ABTs affect FcRn-binding and transport behavior. Here, we report on how FcRn-engagement of the IgG1 Fc fragment compare to clinically relevant IgGs and receptor domain Fc fusions, binding to VEGF or TNF-α. The results reveal FcRn-dependent intracellular accumulation of the Fc, which is in line with shorter plasma half-life than that of full-length IgG1 in human FcRn-expressing mice. Receptor domain fusion to the Fc increases its half-life, but not to the extent of IgG1. This is mirrored by a reduced cellular recycling capacity of the Fc-fusions. In addition, binding of cognate Ag to ABTs show that complexes of similar size undergo cellular transport at different rates, which could be explained by the biophysical properties of each ABT. Thus, the study provides knowledge that should guide tailoring of ABTs regarding optimal cellular sorting and plasma half-life., (© 2022. The Author(s).)

Regulatory T cells (T regs ) are major drivers behind immunosuppressive mechanisms and present a major hurdle for cancer therapy. T regs are characterized by a high expression of CD25, which is a potentially valuable target for T reg depletion to alleviate immune suppression. The preclinical anti-CD25 (αCD25) antibody, clone PC-61, has met with modest anti-tumor activity due to its capacity to clear T regs from the circulation and lymph nodes, but not those that reside in the tumor. The optimization of the Fc domain of this antibody clone has been shown to enhance the intratumoral T reg depletion capacity. Here, we generated a stable cell line that produced optimized recombinant T reg -depleting antibodies. A genome engineering strategy in which CRISPR-Cas9 was combined with homology-directed repair (CRISPR-HDR) was utilized to optimize the Fc domain of the hybridoma PC-61 for effector functions by switching it from its original rat IgG1 to a mouse IgG2a isotype. In a syngeneic tumor mouse model, the resulting αCD25-m2a (mouse IgG2a isotype) antibody mediated the effective depletion of tumor-resident T regs , leading to a high effector T cell (T eff ) to T reg ratio. Moreover, a combination of αCD25-m2a and an αPD-L1 treatment augmented tumor eradication in mice, demonstrating the potential for αCD25 as a cancer immunotherapy.

Immunosuppressants and biologicals are widely used therapeutics for various chronic inflammatory diseases (CID). To gain more detailed insight into their downstream effects, we examined their impact on serum immunoglobulin G (IgG) glycosylation. We analyzed IgG subclass-specific fragment crystallizable (Fc) N-glycosylation in patients suffering from various CID using the LC-MS approach. Firstly, we compared IgG Fc N-glycosylation between 128 CID patients and 204 healthy controls. Our results replicated previously observed CID-related decrease in IgG Fc galactosylation (adjusted p -value range 1.70 × 10 -2 -5.95 × 10 -22 ) and sialylation (adjusted p -value range 1.85 × 10 -2 -1.71 × 10 -18 ). Secondly, to assess changes in IgG Fc N-glycosylation associated with therapy and remission status, we compared 139 CID patients receiving either azathioprine, infliximab, or vedolizumab therapy. We observed an increase in IgG Fc galactosylation (adjusted p -value range 1.98 × 10 -2 -1.30 × 10 -15 ) and sialylation (adjusted p -value range 3.28 × 10 -6 -4.34 × 10 -18 ) during the treatment. Furthermore, patients who reached remission displayed increased Fc galactosylation levels ( p -value range 2.25 × 10 -2 -5.44 × 10 -3 ) in comparison to patients with active disease. In conclusion, the alterations in IgG Fc glycosylation and the fact these changes are even more pronounced in patients who achieved remission, suggest modulation of IgG inflammatory potential associated with CID therapy.

A well-defined and controlled glycosylation pattern is important to maintain quality and safety of therapeutic proteins. Glycosylation is strongly dependent on the host cell line used for recombinant protein expression. Cetuximab, which is produced in mouse myeloma cells has been shown to harbour Fab glycans, which contain non-human like features and hence, can potentially cause an immunogenic response in patients. In light of the advent of biosimilar and biobetter development, we produced cetuximab variants in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. A combination of orthogonal chromatographic modes such as hydrophilic interaction, size exclusion and strong cation exchange chromatography with various detection strategies was employed to characterise the three different cetuximab variants and to compare the in-house produced HEK and CHO variants with the reference drug product. While Fc galactosylation and sialic acid content of the drug product and the HEK variant were highly similar, the CHO product showed lower galactosylation on Fc glycans and a comparatively low sialic acid content in the Fab region. The elevated high-mannose content of CHO cetuximab also suggests potential rapid clearence from circulation. The combination of multiple chromatographic separation modes has proven powerful for the characterisation of expression system dependent protein quality attributes such as N-glycosylation., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

We recently demonstrated that capturing human monoclonal antibodies (hmAbs) using high affinity anti-human Fc (AHC) antibodies allows reliable characterization of antibody-antigen interactions. Here, we characterized six human Fc specific mouse monoclonal antibodies (mAbs) and compared their binding profiles with three previously characterized goat AHC polyclonal antibodies (pAbs), exhibiting properties of a good capture reagent. All six mouse AHC mAbs specifically bound with high affinity to the Fc region of hIgG1, hIgG2, hIgG4 and to 43 different hIgG variants, containing substitutions and/or mutations in the hinge and/or Fc region, that have been reported to exhibit modified antibody effector function and/or pharmacokinetics. Biacore sensor surfaces individually derivatized with mouse AHC mAbs exhibited >2.5-fold higher hIgG binding capacity compared to the three goat AHC pAb surfaces and reproducibly captured hIgG over 300 capture-regeneration cycles. The results of the capture kinetic analyses performed on 31 antibody-antigen interactions using surfaces derivatized with either of the two highest affinity AHC mAbs (REGN7942 or REGN7943) were in concordance with those performed using goat AHC pAb surfaces. Our data demonstrate that AHC mAbs such as REGN7942 and REGN7943 that have properties superior than the three goat AHC pAbs are highly valuable research reagents, especially to perform capture kinetic analyses of antibody-antigen interactions on optical biosensors., (Copyright © 2021 Regeneron Pharmaceuticals. Published by Elsevier Inc. All rights reserved.)

Immunoglobulin G (IgG) monoclonal antibodies are a prominent and expanding class of therapeutics used for the treatment of diverse human disorders. The chemical composition of the N-glycan on the fragment crystallizable (Fc) region determines the effector functions through interaction with the Fc gamma receptors and complement proteins. The chemoenzymatic synthesis using endo-β-N-acetylglucosaminidases (ENGases) emerged as a strategy to obtain antibodies with customized glycoforms that modulate their therapeutic activity. We discuss the molecular mechanism by which ENGases recognize different N-glycans and protein substrates, especially those that are specific for IgG antibodies, in order to rationalize the glycoengineering of immunotherapeutic antibodies, which increase the impact on the treatment of myriad diseases., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recently, we developed a platform strategy for hinge-deficient human immunoglobulin G1 (IgG1) Fc fusion as a non-immunostimulatory Fc fusion system. As a starting point to establish a promising approach for generating hinge-deficient Fc fusion proteins in Escherichia (E.) coli, we selected a CH2-CH3 scaffold as a model protein for evaluation. Recombinant CH2-CH3, expressed as inclusion bodies, was solubilized with various denaturants (urea, sarkosyl, sodium dodecyl sulfate (SDS), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or Triton X-100) in neutral (phosphate-buffered saline (PBS), pH 8) or alkaline (50 or 500 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), pH 11) buffer at 25 °C. Similar to the authentic CH2-CH3 produced in Chinese hamster ovary (CHO) cells, all denaturants, except urea in CAPS buffer but not in PBS, were found to elicit the dimer formation of solubilized CH2-CH3 on SDS-polyacrylamide gel electrophoresis (PAGE). After dialysis with PBS, sarkosyl-soluble CH2-CH3 inclusion bodies were successfully purified using protein G-Sepharose, indicating their successful refolding. Compared to the purified CH2-CH3 from its sarkosyl-soluble inclusion bodies in neutral buffer, that in 500 mM CAPS alkaline buffer revealed substantial structure-related similarities, such as secondary structures and thermal stabilities, as measured by circular dichroism spectroscopy, to authentic CH2-CH3. Native PAGE analysis also supported the above data. Therefore, solubilization at alkaline pH is an essential factor that promotes the refolding of CH2-CH3. Dimer formation of CH2-CH3 on SDS-PAGE may act as a surrogate marker for its protein refolding status. Our observations may provide important hints toward downstream processing of Fc-fusion production in E. coli.

Bispecific antibodies have emerged as therapeutic molecules with a multitude of modes of action and applications. Here, we present a novel approach to solve the light-chain problem for the generation of bispecific Ig-like antibodies using the second constant domain of IgE (EHD2) genetically modified to force heterodimerization. This was achieved by introducing a C14S mutation in one domain and a C102S mutation in the other domain, which removed of one of the crossover disulfide bonds. Substituting the C H 1 and C L domains of an antigen binding fragment (Fab) with these heterodimerizing EHD2 (hetEHD2) domains resulted in Fab-like building blocks (eFab). These eFabs were used to generate different bispecific antibodies of varying valency and molecular composition employing variable domains with different specificities and from different origins. Formats included bivalent bispecific IgG-like molecules (eIgs) and Fc-less Fab-eFab fusion proteins, as well as tri- and tetravalent Fab-eIg fusion proteins. All proteins, including bispecific antibodies for dual receptor targeting and for retargeting of T cells, efficiently assembled into functional molecules. Furthermore, none of the hetEHD2-comprising molecules showed binding to the two Fcε receptors and are thus most likely do not induce receptor cross-linking and activation. In summary, we established the eIg technology as a versatile and robust platform for the generation of bispecific antibodies of varying valency, geometry, and composition, suitable for numerous applications. Abbreviations: antibody drug conjugate (ADC), acute lymphocytic leukemia (ALL), constant domain of IgE (Cε), receptor of Cε (CεRI or CεRII), cluster of differentiation (CD), constant domain of heavy chain (C H ), constant domain of light chain (C L ), (single-chain) diabody ((sc)Db), diabody-immunoglobulin (Db-Ig), dynamic light scattering (DLS), Fragment antigen-binding (Fab), Fab with hetEHD2 (eFab), Fab-EHD2 with T121G in chain 1 and S10I in chain 2 (EFab), bispecific Ig domain containing hetEHD2 (eIg), extracellular domain (ECD), epidermal growth factor receptor 1, 2, 3 (EGFR, HER2, HER3), heavy chain domain 2 of IgE (EHD2), EHD2 domain with C102S (EHD2-1), EHD2 domain with C14S and N39Q (EHD2-2), (human or mouse) fragment crystalline ((hu or mo)Fc), heavy chain (HC), heterodimerized second domain of IgE (hetEHD2), high molecular weight (HMW), immunoglobulin (Ig), light chain (LC), liquid chromatography-mass spectrometry (LC-MS), mesenchymal epithelial transition factor (MET), heavy chain domain 2 of IgM (MHD2), peripheral blood mononuclear cell (PBMC), prolactin receptor (PRLP), Stokes radius (R S ), single-chain Fragment variable (scFv), tumor necrosis factor (TNF), TNF receptor 2 (TNFR2), single-chain TNF-related apoptosis-inducing ligand (scTRAIL), variable domain of heavy chain (V H ), variable domain of light chain (V L ).

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been described in the literature but none of them completely eliminates binding to all of the Fcγ receptors. Here we describe a set of novel variants having specific amino acid substitutions in the Fc region at L234 and L235 combined with the substitution G236R. They show no detectable binding to Fcγ receptors or to C1q, are inactive in functional cell-based assays and do not elicit inflammatory cytokine responses. Meanwhile, binding to FcRn, manufacturability, stability and potential for immunogenicity are unaffected. These variants have the potential to improve the safety and efficacy of therapeutic antibodies and Fc fusion proteins., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The subject matter of this publication is also disclosed in patent applications GB2007613.9 and PCT/GB2021/051233 (‘Modified immunoglobulin Fc regions’) which are assigned to mAbsolve Limited. mAbsolve has issued licences to biotechnology and pharma companies to develop antibodies and Fc fusion proteins described in the patent applications. IW and GH are founders and directors of mAbsolve Limited. Each of them owns more than 5% and less than 20% of the shares of mAbsolve. SA owns fewer than 5% of the shares of mAbsolve. This does not alter our adherence to PLOS ONE policies on sharing data and materials. No other authors declare a competing interest.

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients., Competing Interests: The authors have declared that no competing interests exist.

As a lymphoid organ, the spleen hosts a wide range of immune cell populations, which not only remove blood-borne antigens, but also generate and regulate antigen-specific immune responses. In particular, the splenic microenvironment has been demonstrated to play a prominent role in adaptive immune responses to enveloped viral infections and alloantigens. During both types of immunizations, antigen-specific immunoglobulins G (IgGs) have been characterized by the reduced amount of fucose present on N-linked glycans of the fragment crystallizable (Fc) region. These glycans are essential for mediating the induction of immune effector functions. Therefore, we hypothesized that a spleen may modulate humoral responses and serve as a preferential site for afucosylated IgG responses, which potentially play a role in immune thrombocytopenia (ITP) pathogenesis. To determine the role of the spleen in IgG-Fc glycosylation, we performed IgG subclass-specific liquid chromatography-mass spectrometry (LC-MS) analysis of Fc glycosylation in a large cohort of individuals splenectomized due to trauma, due to ITP, or spherocytosis. IgG-Fc fucosylation was consistently increased after splenectomy, while no effects for IgG-Fc galactosylation and sialylation were observed. An increase in IgG1- and IgG2/3-Fc fucosylation level upon splenectomy has been reported here for the first time, suggesting that immune responses occurring in the spleen may be particularly prone to generate afucosylated IgG responses. Surprisingly, the level of total IgG-Fc fucosylation was decreased in ITP patients compared to healthy controls. Overall, our results suggest a yet unrecognized role of the spleen in either the induction or maintenance of afucosylated IgG responses by B cells., (© 2021. The Author(s).)

Currently, monoclonal antibodies (mAbs) are the most used biopharmaceuticals for human therapy. One of the key aspects in their development is the control of effector functions mediated by the interaction between fragment crystallizable (Fc) and Fcγ receptors, which is a secondary mechanism of the action of biotherapeutics. N-glycosylation at the Fc portion can regulate these mechanisms, and much experimental evidence suggests that modifications of glycosidic chains can affect antibody binding to FcγRIIIa, consequently impacting the immune response. In this work, we try to elucidate via in silico procedures the structural role exhibited by glycans, particularly fucose, in mAb conformational freedom that can potentially affect the receptor recognition. By using adalimumab, a marketed IgG1, as a general template, after rebuilding its three-dimensional (3D) structure through homology modeling approaches, we carried out molecular dynamics simulations of three differently glycosylated species: aglycosylated, afucosylated, and fucosylated antibody. Trajectory analysis showed different dynamical behaviors and pointed out that sugars can influence the overall 3D structure of the antibody. As a result, we propose a putative structural mechanism by which the presence of fucose introduces conformational constraints in the whole antibody and not only in the Fc domain, preventing a conformation suitable for the interaction with the receptor. As secondary evidence, we observed a high flexibility of the antibodies that is translated into an asymmetric behavior of Fab portions shown by all the simulated biopolymers, making the dynamical asymmetry a new, to our knowledge, molecular aspect that may be further investigated. In conclusion, these findings can help understand the contribution of sugars on the structural architecture of mAbs, paving the way to novel strategies of pharmaceutical development., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Congenital tremor (CT) type A-II in piglets is caused by an emerging atypical porcine pestivirus (APPV), which is prevalent in swine herds and a serious threat to the pig production industry. This study aimed to construct APPV E2 subunit vaccines fused with Fc fragments and evaluate their immunogenicity in piglets. Here, APPV E2Fc and E2ΔFc fusion proteins expressed in Drosophila Schneider 2 (S2) cells were demonstrated to form stable dimers in SDS-PAGE and western blotting assays. Functional analysis revealed that aE2Fc and aE2ΔFc fusion proteins could bind to FcγRI on antigen-presenting cells (APCs), with the affinity of aE2Fc to FcγRI being higher than that of aE2ΔFc. Moreover, subunit vaccines based on aE2, aE2Fc, and aE2ΔFc fusion proteins were prepared, and their immunogenicity was evaluated in piglets. The results showed that the Fc fusion proteins emulsified with the ISA 201VG adjuvant elicited stronger humoral and cellular immune responses than the IMS 1313VG adjuvant. These findings suggest that APPV E2 subunit vaccines fused with Fc fragments may be a promising vaccine candidate against APPV.