Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Binding affinities of human IgG1 and chimerized pig and rabbit derivatives to human, pig and rabbit Fc gamma receptor IIIA.

ABSTRACT: While pigs and rabbits are used as models for human immune diseases, Fc?R binding is poorly characterized in both test species. To evaluate antibody binding to Fc?RIIIA, a receptor involved in antibody-dependent cellular cytotoxicity, chimerized antibodies were generated by grafting the variable regions of a human IgG1 onto scaffolds from both species. The affinities of the parent and chimeric antibodies to the Fc?RIIIA proteins from all three species were determined. While the human IgG1 and rabbit IgG had similar affinities for each Fc?RIIIA with notable differences across species, pig IgG1 only bound pig Fc?RIIIA with appreciable affinity. Also, the functional pig and rabbit proteins described here can be used in future experiments, such as pharmacology and mechanism of action studies.

SUBMITTER: Bhatti MM

PROVIDER: S-EPMC6641210 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Binding affinities of human IgG1 and chimerized pig and rabbit derivatives to human, pig and rabbit Fc gamma receptor IIIA.

Bhatti Maryam M MM Cai Allen G AG Theunissen Jan-Willem JW

PloS one 20190719 7

While pigs and rabbits are used as models for human immune diseases, FcγR binding is poorly characterized in both test species. To evaluate antibody binding to FcγRIIIA, a receptor involved in antibody-dependent cellular cytotoxicity, chimerized antibodies were generated by grafting the variable regions of a human IgG1 onto scaffolds from both species. The affinities of the parent and chimeric antibodies to the FcγRIIIA proteins from all three species were determined. While the human IgG1 and ra ...[more]

PMID: 31323052

Similar Datasets

Fc Engineering of Human IgG1 for Altered Binding to the Neonatal Fc Receptor Affects Fc Effector Functions.

Project description:Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical Fc?Rs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical Fc?Rs and complement factor C1q, which ultimately results in alterations of cellular mechanisms such as Ab-dependent cell-mediated cytotoxicity, Ab-dependent cellular phagocytosis, and Ab-dependent complement-mediated cell lysis. Thus, engineering of the FcRn-IgG1 interaction may greatly influence effector functions, which has implications for the therapeutic efficacy and use of Fc-engineered hIgG1 variants.

| S-EPMC4432726 | biostudies-literature

Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors.

Project description:Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.

| S-EPMC1138517 | biostudies-other

Post-HDX Deglycosylation of Fc Gamma Receptor IIIa Glycoprotein Enables HDX Characterization of Its Binding Interface with IgG.

Project description:Protein glycosylation is a common and highly heterogeneous post-translational modification that challenges biophysical characterization technologies. The heterogeneity of glycoproteins makes their structural analysis difficult; in particular, hydrogen-deuterium exchange mass spectrometry (HDX-MS) often suffers from poor sequence coverage near the glycosylation site. A pertinent example is the Fc gamma receptor RIIIa (FcγRIIIa, CD16a), a glycoprotein expressed on the surface of natural killer cells (NK) that binds the Fc domain of IgG antibodies as a trigger for antibody-dependent cell-mediated cytotoxicity (ADCC). Here, we describe an adaptation of a previously reported method using PNGase A for post-HDX deglycosylation to characterize the binding between the highly glycosylated CD16a and IgG1. Upon optimization of the method to improve sequence coverage while minimizing back-exchange, we achieved coverage of four of the five glycosylation sites of CD16a. Despite some back-exchange, trends in HDX are consistent with previously reported CD16a/IgG-Fc complex structures; furthermore, binding of peptides covering the glycosylated asparagine-164 can be interrogated when using this protocol, previously not seen using standard HDX-MS.

| S-EPMC8906513 | biostudies-literature

Structural basis for binding of human IgG1 to its high-affinity human receptor Fc?RI.

Project description:Cell-surface Fc? receptors mediate innate and adaptive immune responses. Human Fc? receptor I (hFc?RI) binds IgGs with high affinity and is the only Fc? receptor that can effectively capture monomeric IgGs. However, the molecular basis of hFc?RI's interaction with Fc has not been determined, limiting our understanding of this major immune receptor. Here we report the crystal structure of a complex between hFc?RI and human Fc, at 1.80?Å resolution, revealing an unique hydrophobic pocket at the surface of hFc?RI perfectly suited for residue Leu235 of Fc, which explains the high affinity of this complex. Structural, kinetic and thermodynamic data demonstrate that the binding mechanism is governed by a combination of non-covalent interactions, bridging water molecules and the dynamic features of Fc. In addition, the hinge region of hFc?RI-bound Fc adopts a straight conformation, potentially orienting the Fab moiety. These findings will stimulate the development of novel therapeutic strategies involving hFc?RI.

| S-EPMC4423232 | biostudies-literature

Therapeutic Administration of Broadly Neutralizing FI6 Antibody Reveals Lack of Interaction Between Human IgG1 and Pig Fc Receptors.

Project description:Influenza virus infection is a significant global health threat. Because of the lack of cross-protective universal vaccines, short time window during which antivirals are effective and drug resistance, new therapeutic anti-influenza strategies are required. Broadly, cross-protective antibodies that target conserved sites in the hemagglutinin (HA) stem region have been proposed as therapeutic agents. FI6 is the first proven such monoclonal antibody to bind to H1-H16 and is protective in mice and ferrets. Multiple studies have shown that Fc-dependent mechanisms are essential for FI6 in vivo efficacy. Here, we show that therapeutic administration of FI6 either intravenously or by aerosol to pigs did not reduce viral load in nasal swabs or broncho-alveolar lavage, but aerosol delivery of FI6 reduced gross pathology significantly. We demonstrate that pig Fc receptors do not bind human IgG1 and that FI6 did not mediate antibody-dependent cytotoxicity (ADCC) with pig PBMC, confirming that ADCC is an important mechanism of protection by anti-stem antibodies in vivo. Enhanced respiratory disease, which has been associated with pigs with cross-reactive non-neutralizing anti-HA antibodies, did not occur after FI6 administration. Our results also show that in vitro neutralizing antibody responses are not a robust correlate of protection for the control of influenza infection and pathology in a natural host model.

| S-EPMC5928291 | biostudies-literature

Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn.

Project description:Susceptibility of methionine residues to oxidation is a significant issue of protein therapeutics. Methionine oxidation may limit the product's clinical efficacy or stability. We have studied kinetics of methionine oxidation in the Fc portion of the human IgG2 and its impact on the interaction with FcRn and Protein A. Our results confirm previously published observations for IgG1 that two analogous solvent-exposed methionine residues in IgG2, Met 252 and Met 428, oxidize more readily than the other methionine residue, Met 358, which is buried inside the Fc. Met 397, which is not present in IgG1 but in IgG2, oxidizes at similar rate as Met 358. Oxidation of two labile methionines, Met 252 and Met 428, weakens the binding of the intact antibody with Protein A and FcRn, two natural protein binding partners. Both of these binding partners share the same binding site on the Fc. Additionally, our results shows that Protein A may serve as a convenient and inexpensive surrogate for FcRn binding measurements.

| S-EPMC2708056 | biostudies-literature

Antigen-Induced Allosteric Changes in a Human IgG1 Fc Increase Low-Affinity Fcγ Receptor Binding.

Project description:Antibody structure couples adaptive and innate immunity via Fab (antigen binding) and Fc (effector) domains that are connected by unique hinge regions. Because antibodies harbor two or more Fab domains, they are capable of crosslinking multi-determinant antigens, which is required for Fc-dependent functions through associative interactions with effector ligands, including C1q and cell surface Fc receptors. The modular nature of antibodies, with distal ligand binding sites for antigen and Fc-ligands, is reminiscent of allosteric proteins, suggesting that allosteric interactions might contribute to Fc-mediated effector functions. This hypothesis has been pursued for over 40 years and remains unresolved. Here, we provide evidence that allosteric interactions between Fab and Fc triggered by antigen binding modulate binding of Fc to low-affinity Fc receptors (FcγR) for a human IgG1. This work opens the path to further dissection of the relative roles of allosteric and associative interactions in Fc-mediated effector functions.

| S-EPMC7288244 | biostudies-literature

Soluble monomeric IgG1 Fc.

Project description:Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.

| S-EPMC3365978 | biostudies-literature

Fc-gamma IIIa-V158F receptor polymorphism contributes to the severity of Guillain-Barre syndrome.

Project description:ObjectiveGuillain-Barré syndrome (GBS) is a rare, life-threatening disorder of the peripheral nervous system. Immunoglobulin G Fc-gamma receptors (FcγRs) mediate and regulate diverse effector functions and are involved in the pathogenesis of GBS. We investigated whether the FcγR polymorphisms FcγRIIa H/R131 (rs1801274), FcγRIIIa V/F158 (rs396991), and FcγRIIIb NA1/NA2, and their haplotype patterns affect the affinity of IgG-FcγR interactivity and influence GBS susceptibility and severity.MethodsWe determined FcγR polymorphisms in 303 patients with GBS and 302 ethnically matched healthy individuals from Bangladesh by allele-specific polymerase chain reaction. Pairwise linkage disequilibrium and haplotype patterns were analyzed based on D ́statistics and the genotype package of R statistics, respectively. Logistic regression analysis and Fisher's exact test with corrected P (Pc) values were employed for statistical comparisons.ResultsFcγRIIIa-V158F was associated with the severe form of GBS compared to the mild form (P = 0.005, OR = 2.24, 95% CI = 1.28-3.91; Pc = 0.015); however, FcγR genotypes and haplotype patterns did not show any association with GBS susceptibility compared to healthy controls. FcγRIIIa-V/V158 and FcγRIIIb-NA2/2 were associated with recent Campylobacter jejuni infection (P ≤ 0.001, OR = 0.36, 95% CI = 0.23-0.56; Pc ≤ 0.003 and P = 0.004, OR = 1.70, 95% CI = 1.18-2.44; Pc ≤ 0.012, respectively). Haplotype 1 (FcγRIIa-H131R- FcγRIIIa-V158F- FcγRIIIb-NA1/2) and the FcγRIIIb-NA2/2 genotype were more prevalent among anti-GM1 antibody-positive patients (P = 0.031, OR = 9.61, 95% CI = 1.24-74.77, Pc = 0.279; P = 0.027, OR = 1.62, 95% CI = 1.06-2.5, Pc = 0.081, respectively).InterpretationFcγR polymorphisms and haplotypes are not associated with susceptibility to GBS, though the FcγRIIIa-V158F genotype is associated with the severity of GBS.

| S-EPMC7317642 | biostudies-literature

Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells.

Project description:Many therapeutic monoclonal antibodies require binding to Fc γ receptors (FcγRs) for full effect and increasing the binding affinity increases efficacy. Preeminent among the five activating human FcγRs is FcγRIIIa/CD16a expressed by natural killer (NK) cells. CD16a is heavily processed, and recent reports indicate that the composition of the five CD16a asparagine(N)-linked carbohydrates (glycans) impacts affinity. These observations indicate that specific manipulation of CD16a N-glycan composition in CD16a-expressing effector cells including NK cells may improve treatment efficacy. However, it is unclear if modifying the expression of select genes that encode processing enzymes in CD16a-expressing effector cells is sufficient to affect N-glycan composition. We identified substantial processing differences using a glycoproteomics approach by comparing CD16a isolated from two NK cell lines, NK92 and YTS, with CD16a expressed by HEK293F cells and previous reports of CD16a from primary NK cells. Gene expression profiling by RNA-Seq and qRT-PCR revealed expression levels for glycan-modifying genes that correlated with CD16a glycan composition. These results identified a high degree of variability between the processing of the same human protein by different human cell types. N-glycan processing correlated with the expression of glycan-modifying genes and thus explained the substantial differences in CD16a processing by NK cells of different origins.

| S-EPMC7948478 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data