Immunoglobulin G Molecules Research Articles

The importance of IgG glycosylation-What did we learn after analyzing over 100,000 individuals.

All four subclasses of immunoglobulin G (IgG) antibodies have glycan structures attached to the protein part of the IgG molecules. Glycans linked to the Fc portion of IgG are found in all IgG antibodies, while about one-fifth of IgG antibodies in plasma also have glycans attached to the Fab portion of IgG. The IgG3 subclass is characterized by more complex glycosylation compared to other IgG subclasses. In this review, we discuss the significant influence that glycans exert on the structural and functional properties of IgG. We provide a comprehensive overview of how the composition of these glycans can affect IgG's effector functions by modulating its interactions with Fcγ receptors and other molecules such as the C1q component of complement, which in turn influence various immune responses triggered by IgG, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In addition, the importance of glycans for the efficacy of therapeutics like monoclonal antibodies and intravenous immunoglobulin (IVIg) therapy is discussed. Moreover, we offer insights into IgG glycosylation characteristics and roles derived from general population, disease-specific, and interventional studies. These studies indicate that IgG glycans are important biomarkers and functional effectors in health and disease.

Recombinant streptococcal protein G-modified metal-organic framework ZIF-8 for the highly selective purification of immunoglobulin G from human serum

A novel solid phase extractant His-rSPG@ZIF-8 was prepared by covalently coupling recombinant streptococcal protein G (His-rSPG) with ZIF-8. The His-rSPG@ZIF-8 composite was characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Due to the specific binding between the immunoglobulin binding region of His-rSPG and the Fc region of immunoglobulin G (IgG), the His-rSPG@ZIF-8 composite demonstrated exceptional selectivity in adsorbing IgG. In Britton-Robinson buffer (BR buffer) with a salt concentration of 500 mmol L−1 (0.04 mol L−1, pH 8.0), the His-rSPG@ZIF-8 composite exhibited a remarkable adsorption efficiency of 99.8 % for 0.05 mg of the composite on 200 μL of IgG solution (100 μg mL−1). The adsorption behavior of the His-rSPG@ZIF-8 composite aligns with the Langmuir adsorption model, and the theoretical maximum adsorption capacity is 1428.6 mg g−1. The adsorbed IgG molecules were successfully eluted using a SDS solution (0.5 %, m/m), resulting in a recovery rate of 91.2 %. Indeed, the His-rSPG@ZIF-8 composite was successfully utilized for the isolation and purification of IgG from human serum samples. The obtained IgG exhibited high purity, as confirmed by SDS-PAGE analysis. Additionally, LC-MS/MS analysis was employed to identify the human serum proteins following the adsorption and elution process using the His-rSPG@ZIF-8 composite material. The results revealed that the recovered solution contained an impressive content of immunoglobulin, accounting for 62.4 % of the total protein content. Furthermore, this process also led to the significant enrichment of low abundance proteins such as Serpin B4 and Cofilin-1. Consequently, the His-rSPG@ZIF-8 composite holds great promise for applications such as IgG purification and immunoassays. At the same time, it expands the application of metal-organic frameworks in the field of proteomics.

Effects of flexibility in coarse-grained models for bovine serum albumin and immunoglobulin G.

We construct a coarse-grained, structure-based, low-resolution, 6-bead flexible model of bovine serum albumin (BSA, PDB: 4F5S), which is a popular example of a globular protein in biophysical research. The model is obtained via direct Boltzmann inversion using all-atom simulations of a single molecule, and its particular form is selected from a large pool of 6-bead coarse-grained models using two suitable metrics that quantify the agreement in the distribution of collective coordinates between all-atom and coarse-grained Brownian dynamics simulations of solutions in the dilute limit. For immunoglobulin G (IgG), a similar structure-based 12-bead model has been introduced in the literature [Chaudhri et al., J. Phys. Chem. B 116, 8045 (2012)] and is employed here to compare findings for the compact BSA molecule and the more anisotropic IgG molecule. We define several modified coarse-grained models of BSA and IgG, which differ in their internal constraints and thus account for a variation of flexibility. We study denser solutions of the coarse-grained models with purely repulsive molecules (achievable by suitable salt conditions) and address the effect of packing and flexibility on dynamic and static behavior. Translational and rotational self-diffusivity is enhanced for more elastic models. Finally, we discuss a number of effective sphere sizes for the BSA molecule, which can be defined from its static and dynamic properties. Here, it is found that the effective sphere diameters lie between 4.9 and 6.1nm, corresponding to a relative spread of about ±10% around a mean of 5.5nm.

An Fc variant with two mutations confers prolonged serum half-life and enhanced effector functions on IgG antibodies

The pH-selective interaction between the immunoglobulin G (IgG) fragment crystallizable region (Fc region) and the neonatal Fc receptor (FcRn) is critical for prolonging the circulating half-lives of IgG molecules through intracellular trafficking and recycling. By using directed evolution, we successfully identified Fc mutations that improve the pH-dependent binding of human FcRn and prolong the serum persistence of a model IgG antibody and an Fc-fusion protein. Strikingly, trastuzumab-PFc29 and aflibercept-PFc29, a model therapeutic IgG antibody and an Fc-fusion protein, respectively, when combined with our engineered Fc (Q311R/M428L), both exhibited significantly higher serum half-lives in human FcRn transgenic mice than their counterparts with wild-type Fc. Moreover, in a cynomolgus monkey model, trastuzumab-PFc29 displayed a superior pharmacokinetic profile to that of both trastuzumab-YTE and trastuzumab-LS, which contain the well-validated serum half-life extension Fcs YTE (M252Y/S254T/T256E) and LS (M428L/N434S), respectively. Furthermore, the introduction of two identified mutations of PFc29 (Q311R/M428L) into the model antibodies enhanced both complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity activity, which are triggered by the association between IgG Fc and Fc binding ligands and are critical for clearing cancer cells. In addition, the effector functions could be turned off by combining the two mutations of PFc29 with effector function-silencing mutations, but the antibodies maintained their excellent pH-dependent human FcRn binding profile. We expect our Fc variants to be an excellent tool for enhancing the pharmacokinetic profiles and potencies of various therapeutic antibodies and Fc-fusion proteins.

Nonenzymatic glycosylation of isolated human immunoglobulin-G by D-ribose.

Glycation is vital in terms of its damaging effect on macromolecules resulting in the formation of end products, which are highly reactive and cross-linked irreversible structures, known as advanced glycation end products (AGEs). The continuous accumulation of AGEs is associated with severe diabetes and its associated ailments. Saccharides with their reducing ends can glycate amino acid side chains of proteins, among them glucose is well-known for its potent glycating capability. However, other reducing sugars can be more reactive glycating agents than glucose. The D-ribose is a pentose sugar-containing an active aldehyde group in its open form and is responsible for affecting the biological processes of the cellular system. D-ribose, a key component of many biological molecules, is more reactive than most reducing sugars. Protein glycation by reducing monosaccharides such as D-ribose promotes the accelerated formation of AGEs that could lead to cellular impairments and dysfunctions. Also, under a physiological cellular state, the bioavailability rate of D-ribose is much higher than that of glucose in diabetes, which makes this species much more active in protein glycation as compared with D-glucose. Due to the abnormal level of D-ribose in the biological system, the glycation of proteins with D-ribose needs to be analyzed and addressed carefully. In the present study, human immunoglobulin G (IgG) was isolated and purified via affinity column chromatography. D-ribose at 10and 100 mM concentrations was used as glycating agent, for 1-12 days of incubation at 37°C. The postglycation changes in IgG molecule were characterized by UV-visible and fluorescence spectroscopy, nitroblue tetrazolium assay, and various other physicochemical analyses for the confirmation of D-ribose mediated IgG glycation.

Neuronal internalization of immunoglobulin G injected into the mouse brain by a novel absorption strategy to avoid unwanted interaction with immune complex using centrifugal filtration.

Monoclonal immunoglobulin-G (IgG) antibodies are now emerging as therapeutic tools to tackle various disorders, including those affecting the brain. However, little is known about how these IgG molecules behave in the brain. To better understand the potential behavior of IgG molecules in the brain, here we established a specific protocol to immunolocalize rat IgG injected into mouse striatum with an anti-rat IgG antibody. Using double immunolabeling, IgG-like immunoreactivity (IR) was mainly found in neurons but scarcely observed in glia 1h after intrastriatal injection of IgG, whereas some surrounding glia contained IgG-like IR 24h after injection. However, preabsorption with a large excess of rat IgG to confirm the authenticity of this labeling failed to eliminate this neuronal IgG-like IR but rather exhibited nuclear staining in glial cells. Because this unexpected nuclear staining escalated with increasing amount of absorbing IgG, we postulated that this nuclear staining is due to formation of immune complex IgG-anti-IgG, which can be removed by centrifugal filtration. As expected, this nuclear staining in glial cells was eliminated after centrifugal filtration of the IgG/anti-IgG mixture, and authentic IgG-like IR was chiefly detected in the cytoplasm of neurons around the injection channel. This study is the first demonstration of neuronal redistribution of injected IgG in the mouse brain. Neuronal internalization of exogenous IgG may be advantageous especially when the therapeutic targets of monoclonal IgG are intraneuronal such as neurofibrillary tangles or Lewy bodies.

Kinetic and molecular insight into immunoglobulin G binding to immobilized recombinant protein A of different orientations

Mechanistic understanding of immunoglobulin G (IgG) binding to protein A is crucial for the design and development of high-performance protein A chromatography. In this work, the IgG binding domain (Z) of protein A from Staphylococcus aureus was genetically modified by introducing a cysteine residue at the N-terminus (Cys-Z) or a cysteine-lysine dipeptide at the C-terminus (Z-Cys), and the two ligands were used to unravel the IgG binding mechanism by means of binding kinetics and different single molecule measurements. Surface plasma resonance (SPR) measurement of the binding kinetics of mouse myeloma IgG2a (mIgG2a) to the two ligands indicated that oriented ligand immobilization significantly increased the association rate constant of mIgG2a, and Z-Cys had the highest binding affinity to mIgG2a among the three ligands (Cys-Z, Z-Cys and Z). This was attributed to the synergistic contribution of the high association rate constant and low dissociation rate constant to mIgG2a. Furthermore, quartz crystal microbalance with energy dissipation monitoring (QCM-D) measurement provided the maximum adsorption densities of IgGs on the Z-Cys-immobilized chip as zeta potentials of IgGs were nearly zero. The QCM-D investigation revealed that the adsorbed layer was dependent on ligand type and density, and IgG. Moreover, Z-Cys and Cys-Z induced IgG binding in flipped orientations, as evidenced by the antigen-antibody reaction. Finally, rectangular DNA origami tiles were introduced to analyze the molecular orientation of adsorbed IgG. Single-molecule imaging showed that mIgG2a was associated with flexible Z-Cys on the tiles predominantly in side-on and end-on orientations. The research has provided molecular insight into the binding mechanism of IgG molecules at liquid–solid interfaces and would help design new protein A-based ligands and high-capacity adsorbents.

Platelet-activating anti-PF4 disorders: An overview

Platelet factor 4 (PF4) is a highly cationic tetrameric protein that can be targeted by platelet-activating anti-PF4 antibodies of immunoglobulin G (IgG) class. Certain features of PF4, including its multivalent nature (duplicate antigen sites per tetramer), the ability of many PF4 tetramers to undergo close approximation through charge neutralization, and the dimeric binding of IgG molecules, results in formation of IgG-containing immune complexes in situ on platelets, neutrophils, and monocytes, resulting in Fcγ receptor-mediated pancellular activation that also activates hemostasis (potential for disseminated intravascular coagulation). This review discusses 4 anti-PF4 disorders: classic heparin-induced thrombocytopenia ([HIT]; triggered by heparin and certain other polyanionic pharmaceuticals, featuring predominantly heparin-dependent antibodies), autoimmune HIT (aHIT; severe subtype of HIT that features both heparin-dependent and heparin-independent platelet-activating antibodies), and spontaneous HIT (non-heparin triggers such as knee replacement surgery and infection; predominantly heparin-independent platelet-activating antibodies). Most recently, a novel fourth anti-PF4 disorder, vaccine-induced immune thrombotic thrombocytopenia (VITT), was identified as an ultrarare complication of adenovirus vector vaccines. VITT is characterized by thrombocytopenia, disseminated intravascular coagulation, a high frequency of thrombosis—including in unusual sites (cerebral veins, splanchnic veins)—and highly pathogenic anti-PF4 antibodies with heparin-independent platelet-activating properties.

Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion.

Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

Rapid Production of Bispecific Antibodies from Off-the-Shelf IgGs with High Yield and Purity.

Bispecific antibodies (BsAb) refer to a class of biomacromolecules that are capable of binding two antigens or epitopes simultaneously. This can elicit unique biological effects that cannot be achieved with either individual antibody or two unlinked antibodies. Bispecific antibodies have been used for targeting effector cells to tumor cells, preferential targeting of cells expressing two target biomarkers over cells expressing either target biomarker individually, or to couple two molecular targets on the same cell surface to trigger unique intracellular signaling pathways. Here, we present two related methods that enable direct, rapid assembly of bispecific antibodies from any two "off-the-shelf" Immunoglobulin G (IgG) antibodies, in as little as 1 day. Both workflows can be summarized into two steps: (1) attach a small photoreactive antibody binding domain (pAbBD) fused to SpyCatcher or SpyTag (peptide-protein partners derived from the S. pyogenes fibronectin-binding protein FbaB) to each component IgG, respectively; (2) assemble the BsAb through the spontaneous isopeptide bond formation that occurs between SpyTag and SpyCatcher. These approaches enable production of BsAbs from any two IgG molecules without the need to elucidate their amino acid sequences or genetically alter their structure. Binding assays and T cell-mediated cytolysis assays were performed to validate the binding and functional properties of Trastuzumab × Cetuximab BsAb and Cetuximab × OKT3 BsAb, respectively. This approach enables rapid, low-cost production of highly homogeneous tetravalent BsAbs in a modular fashion, presenting an opportunity to quickly evaluate antibody pairs in a BsAb format for unique or synergistic functionalities.

Arginine and its Derivatives Suppress the Opalescence of an Antibody Solution

Opalescence is a problem concerned with the stability of an antibody solution. It occurs when a high concentration of a protein is present. Arginine (Arg) is a versatile aggregation suppressor of proteins, which is among the candidates that suppress opalescence in antibody solutions. Here, we investigated the effect of various types of small molecular additives on opalescence to reveal the mechanism of Arg in preventing opalescence in antibody solution. As expected, Arg suppressed the opalescence of the immunoglobulin G (IgG) solution. Arg also concentration dependently inhibited the formation of microstructures in IgG molecules. Interestingly, the intrinsic fluorescence spectra of highly concentrated IgG solutions differed from those having low concentrations, even though IgG retained a distinct tertiary structure. Arginine ethylester was more effective in suppressing the opalescence of IgG solutions than Arg, whereas lysine and γ-guanidinobutyric acid were less effective. These results indicated that positively charged groups of both α-amine and guanidinium actively influence Arg as an additive for suppressing opalescence. Diols, which are the suppressors of the liquid–liquid phase separation of proteins were also effective in suppressing the opalescence. These results therefore provide insight into the control of opalescence of antibody solutions at high concentrations using solution additives.

P–591 Mass spectrometry-based glycomic profiling of the follicular fluid total immunoglobulin G and proteome N-glycomes reveals deregulated inflammatory processes associated with specific controlled ovarian stimulation protocols

Abstract Study question Is there a significant inflammatory-related difference between analyzed follicular fluid (FF) glycome profiles regarding the ovarian stimulation protocol used in patients? Summary answer Observed differences between analyzed glycome profiles from patients that underwent different controlled ovarian stimulation (COS) protocols point to deregulated inflammatory processes associated with specific COS. What is known already Successful physiological folliculogenesis and ovulation require an adequate inflammatory response. On the other hand, COS application in ART relies on induced hormonal activation of systemic inflammatory processes. Several studies have confirmed a rise in inflammatory cytokines, CRP, and other markers of inflammation in patients subjected to different COS protocols, pointing to an enhanced inflammatory response during ovulation stimulation. Glycoproteins and glycans have an indisputable role in immune response modulation: proper glycosylation of glycoproteins plays a pivotal role in the regulation of normal physiological processes, and aberrant glycosylation of glycoproteins has been associated with various pathological states, including inflammation. Study design, size, duration Study design: Cross sectional – FFs from patients that underwent ART in modified natural cycle (MNC group) versus FFs from patients that underwent ART under GnRH antagonist COS (COS group). Size: 20 FFs from 20 patients undergoing ART. Duration: One year. Sampling procedure: Each FF was aspirated from the dominant follicle. In the COS group, only the fluid from the first aspirated follicle of each patient was collected. Participants/materials, setting, methods Study included 20 FF samples from 20 patients divided into two groups according to the applied ovarian stimulation protocol: MNC group (n = 10) and COS group (n = 10). The immunoglobulin G (IgG) was isolated from FF samples by immunoaffinity chromatography. The N-linked glycans derived from IgG molecule and the remaining FF total proteomes were enzymatically cleaved and subjected to derivatization procedure. N-glycomes of FF-isolated IgG and total proteomes were analyzed separately by MALDI-TOF-MS. Main results and the role of chance FF IgG N-glycome profiling The MALDI-TOF-MS based comparative analysis of the individual glycan relative abundances, revealed several significantly deregulated glycoforms between analyzed groups whose levels were significantly elevated (p˂0.05) in the COS vs. MNC group. Furthermore, additional low abundant N-glycan species were also found to be deregulated between the analyzed groups: two monogalactolysed and monosialylated N-glycan compositions were only identified in the COS group. The comparative analysis of FF IgG N-glycome features revealed statistically relevant differences in the levels of two derived traits: galactosylation and bigalactosylation levels of the FF IgG N-glycome, both significantly downregulated (p˂0.05) in the MNC vs. COS profile. Comparative analysis of FF total proteome N-glycome The majority of identified glycan compositions were complex type N-glycans representing more than 98% of the total N-glycome profiles in both analyzed groups. The comparative analysis of individual glycan relative abundances revealed relevant differences in regulation of ten N-glycan species between the two analyzed profiles. In the MNC group, six N-glycan species showed significantly increased abundances (p˂0.05) compared with the COS group. Moreover, two compositions were exclusively identified in the MNC group, while two compositions were identified only in the COS group. Limitations, reasons for caution Since this preliminary study was conducted on relatively small sample size, all results should be verified on a larger sample set. Moreover, focused glycosylation analysis of a panel of individual FF acute phase blood serum derived proteins and immunoglobulins, might additionally clarify the inflammatory mechanisms underlying different ART stimulation protocols. Wider implications of the findings: While glycome profiling of human FFs was conducted for the first time, previous evidence supports the shown association of aberrant inflammation in diverse ART stimulation protocols and in development of various pathological states (i.e. OHSS). Obtained results are in line with previous similar studies performed in the human plasma. Trial registration number uniri-biomed–18–161 1310

Biophysical characterization of dynamic structures of immunoglobulin G.

Immunoglobulin G (IgG) is a major antibody and functions as a hub linking specific antigen binding and recruitment of effector molecules typified by Fcγ receptors (FcγRs). These activities are associated primarily with interactions involving its Fab and Fc sites, respectively. An IgG molecule is characterized by a multiple domain modular structure with conserved N-glycosylation in Fc. The molecule displays significant freedom in internal motion on various spatiotemporal scales. The consequent conformational flexibility and plasticity of IgG glycoproteins are functionally significant and potentially important factors for design and engineering of antibodies with enhanced functionality. In this article, experimental and computational approaches are outlined for characterizing the conformational dynamics of IgG molecules in solution. In particular, the importance of integration of these approaches is highlighted, as illustrated by dynamic intramolecular interactions between the pair of N-glycans and their proximal amino acid residues in Fc. These interactions can critically affect effector functions mediated by human IgG1 and FcγRIII. Further improvements in individual biophysical techniques and their integration will advance understanding of dynamic behaviors of antibodies in physiological and pathological conditions. Such understanding will provide opportunities for engineering antibodies through controlling allosteric networks in IgG molecules.

Intravenous immunoglobulin significantly reduces exposure of concomitantly administered anti-C5 monoclonal antibody tesidolumab.

Awareness of drug-drug interactions is critical in organ transplant recipient management. However, biologic agents interfering with monoclonal antibodies is not widely considered. We report the effect of high-dose intravenous immunoglobulin (IVIg) on safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of the human anti-C5 monoclonal antibody tesidolumab (LFG316) in end-stage renal disease patients awaiting kidney transplant. In this single-center, phase 1, open-label, parallel-group study, 8 patients were assigned to receive either single-dose tesidolumab+IVIg or tesidolumab alone, with 56-day follow-up. Within-group PK parameters were consistent. Mean tesidolumab exposure decreased 34%, clearance increased 63%, and half-life decreased 41% comparing tesidolumab+IVIg to tesidolumab alone. IVIg influence on tesidolumab elimination was most evident in the first 3weeks. Complete suppression of both total and alternative complement activities was maintained for 4weeks in the tesidolumab alone group and for 2weeks in the tesidolumab+IVIg group. Tesidolumab was well tolerated. IVIg infused before tesidolumab affected tesidolumab PK and PD, resulting in a shortened period of full complement activity inhibition. These findings suggest a clinically relevant impact of IVIg on monoclonal antibody clearance and indirectly hint at an IVIg mechanism of action in treating autoimmune diseases and allosensitization by accelerating pathogenic IgG antibody degradation. Trial registration number: NCT02878616.

Antagonism of the Neonatal Fc Receptor as an Emerging Treatment for Myasthenia Gravis.

Myasthenia gravis is an autoimmune disease in which immunoglobulin G (IgG) autoantibodies are formed against the nicotinic acetylcholine receptor (AChR) or other components of the neuromuscular junction. Though effective treatments are currently available, many commonly used therapies have important limitations and alternative therapeutic options are needed for patients. A novel treatment approach currently in clinical trials for myasthenia gravis targets the neonatal Fc receptor (FcRn). This receptor plays a central role in prolonging the half–life of IgG molecules. The primary function of FcRn is salvage of IgG and albumin from lysosomal degradation through the recycling and transcytosis of IgG within cells. Antagonism of this receptor causes IgG catabolism, resulting in reduced overall IgG and pathogenic autoantibody levels. This treatment approach is particularly intriguing as it does not result in widespread immune suppression, in contrast to many therapies in routine clinical use. Experience with plasma exchange and emerging phase 2 clinical trial data of FcRn antagonists provide proof of concept for IgG lowering in myasthenia gravis. Here we review the IgG lifecycle and the relevance of IgG lowering to myasthenia gravis treatment and summarize the available data on FcRn targeted therapeutics in clinical trials for myasthenia gravis.

Minimal B Cell Extrinsic IgG Glycan Modifications of Pro- and Anti-Inflammatory IgG Preparations in vivo.

Select residues in the biantennary sugar moiety attached to the fragment crystallizable of immunoglobulin G (IgG) antibodies can modulate IgG effector functions. Thus, afucosylated IgG glycovariants have enhanced cytotoxic activity, whereas IgG glycovariants rich in terminal sialic acid residues can trigger anti-inflammatory effects. More recent evidence suggests that terminal α2,6 linked sialic acids can be attached to antibodies post IgG secretion. These findings raise concerns for the use of therapeutic antibodies as they may change their glycosylation status in the patient and hence affect their activity. To investigate to what extent B cell extrinsic sialylation processes modify therapeutic IgG preparations in vivo, we analyzed changes in human intravenous IgG (IVIg) sialylation upon injection in mice deficient in B cells or in mice lacking the sialyltransferase 1, which catalyzes the addition of α2,6 linked sialic acid residues. By performing a time course of IgG glycan analysis with HILIC-UPLC-FLR (plus MS) and xCGE-LIF our study suggests that therapeutic IgG glycosylation is stable upon injection in vivo. Only a very small fraction of IgG molecules acquired sialic acid structures predominantly in the Fab- but not the Fc-portion upon injection in vivo, suggesting that therapeutic antibody glycosylation will remain stable upon injection in vivo.

Molecular docking explores heightened immunogenicity and structural dynamics of acetaldehyde human immunoglobulin G adduct.

Acetaldehyde is a metabolite of ethanol, an important constituent of tobacco pyrolysis and the aldehydic product of lipid peroxidation. Acetaldehyde induced toxicity is mainly due to its binding to cellular macromolecules resulting in the formation of stable adducts accompanied by oxidative stress. The aim of this study was to characterize structural and immunological alterations in human immunoglobulin G (IgG) modified with acetaldehyde in the presence of sodium borohydride, a reducing agent. The IgG modifications were studied by various physicochemical techniques such as fluorescence and CD spectroscopy, free amino group estimation, 2,2-azobis 2-amidinopropane (AAPH) induced red blood cell hemolysis as well as transmission electron microscopy. Molecular docking was also employed to predict the preferential binding of acetaldehyde to IgG. The immunogenicity of native and acetaldehyde-modified IgG was investigated by immunizing female New Zealand white rabbits using native and modified IgG as antigens. Binding specificity and cross reactivity of rabbit antibodies was screened by competitive inhibition ELISA and band shift assays. The modification of human IgG with acetaldehyde results in quenching of the fluorescence of tyrosine residues, decrease in free amino group content, a change in the antioxidant property as well as formation of cross-linked structures in human IgG. Molecular docking reveals strong binding of IgG to acetaldehyde. Moreover, acetaldehyde modified IgG induced high titer antibodies (>1:12800) in the experimental animals. The antibodies exhibited high specificity in competitive binding assay toward acetaldehyde modified human IgG. The results indicate that acetaldehyde induces alterations in secondary and tertiary structure of IgG molecule that leads to formation of neo-epitopes on IgG that enhances its immunogenicity.

Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes.

Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7-PEG3400-MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 μM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7-PEG4-MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7-PEG3400-IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI-PEG3400-MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) for targeted antibody blocking.

Targeted antibody blocking enables characterization of binding sites on immunoglobulin G (IgG), and can efficiently eliminate harmful antibodies from organisms. In this report, we present a novel peptide-denoted as a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF)-for targeted blocking of antibodies. Synthesis of DCAF was achieved by native chemical ligation, and the molecule consists of three functional parts: a specific antigenic peptide, a linker and the Fc-III mimetic peptide, which has a high affinity toward the Fc region of IgG molecules. We demonstrate that DCAF binds the cognate antibody with high selectivity by simultaneously binding to the Fab and Fc regions of IgG. Animal experiments revealed that DCAF molecules diminish the antibody-dependent enhancement effect in a dengue virus infection model, and rescue the acetylcholine receptor by inhibiting the complement cascade in a myasthenia gravis model. These results suggest that DCAFs could have utility in the development of new therapeutics against harmful antibodies.

Fabrication of N-Type Silicon Nanowire Biosensor for Sub-10-Femtomolar Concentration of Immunoglobulin

A simple fabrication process of an n-type silicon nanowire (SiNW) biosensor for sub-10 femtomolar (fM) concentration immunoglobulin detection was presented in this work. The SiNWs with different widths of 80-190 nm were fabricated using electron beam lithography and reaction ion etching techniques. The electrical characteristics of SiNWs with various widths were measured. And it can be observed that thin SiNW has high resistance, which is in agreement with electrical resistance theory. Furthermore, the surface of the fabricated SiNW was functionalized by 3-aminopropyltriethoxysilane for making the biosensor device to detect the binding of immunoglobulin G (IgG) molecules. The responsivity of the biosensor was investigated by observing electrical performance in response due to IgG with various concentration from 6 fM to 600 nanomolar (nM). The resistance changing ratio based on the current voltage (I-V) characteristics was analyzed and it increased with increasing of the IgG concentration. As a result, it demonstrated that the n-type SiNW biosensor has the ability to detect the IgG molecules with low concentration of 6 fM.