Fc Peptide Research Articles

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Extensive substrate recognition by the streptococcal antibody-degrading enzymes IdeS and EndoS

Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a “flipped-out” conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes.

Intravenous immunoglobulin induces IgG internalization by tolerogenic myeloid dendritic cells that secrete IL-10 and expand Fc-specific regulatory T cells

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in many inflammatory conditions including Multisystem Inflammatory Syndrome-Children (MIS-C) and Kawasaki disease (KD). However, the exact mechanisms underlying its anti-inflammatory action are incompletely characterized. Here, we show that in KD, a pediatric acute vasculitis that affects the coronary arteries, IVIG induces a repertoire of natural Treg that recognize immunodominant peptides in the Fc heavy chain constant region. To address which antigen-presenting cell (APC) populations present Fc peptides to Treg, we studied the uptake of IgG by innate cells in subacute KD patients 2 weeks after IVIG and in children 1.6–14 years after KD. Healthy adults served as controls. IgG at high concentrations was internalized predominantly by two myeloid dendritic cell (DC) lineages, CD14+ cDC2 and ILT-4+ CD4+ tmDC mostly through Fcγ receptor (R) II and to a lesser extent FcγRIII. Following IgG internalization, these two DC lineages secreted IL-10 and presented processed Fc peptides to Treg. The validation of IVIG function in expanding Fc-specific Treg presented by CD14+ cDC2 and ILT-4+ CD4+ tmDC was addressed in a small cohort of patients with MIS-C. Taken together, these results suggest a novel immune regulatory function of IgG in activating tolerogenic innate cells and expanding Treg, which reveals an important anti-inflammatory mechanism of action of IVIG.

Immune Regulatory Functions of IgG in the Ontogeny of Human Natural Regulatory T Cells

The heavy constant region of IgG (Fc) is highly immunogenic for human natural regulatory T cells (nTreg). Mature IgG+ B cells prime Fc-specific Treg via recycling of surface immunoglobulin with an antigen-processing pathway that is very efficient in presenting immunodominant Fc peptides to Treg. Some of these peptides are pan-HLA binders, explaining the presence of Fc-specific Treg in circulation in healthy pediatric and adult subjects. Following IgG+ B cell priming, further Treg expansion occurs with the presentation of Fc peptides following IgG uptake and processing by CD14+ myeloid dendritic cells type 2 (cDC2) and CD4+ immunoglobulin-like transcript 4 (ILT-4+) tolerogenic DC that secretes IL-10 when stimulated by the Fc that enters cells prevalently via Fcg receptor II. Fc-specific Treg are important in regulating naive T cell differentiation and account for a key mechanism of success for intravenous immunoglobulin therapy (IVIG) in several inflammatory conditions, including Kawasaki disease (KD) a pediatric acute vasculitis of the coronary arteries.

Human Natural Regulatory T Cells Recognize Peptides of the Heavy Constant Region of IgG with High Sequence Homology with Peptides Derived from Pathogens

Human natural regulatory T cells (nTreg) that control the differentiation of pro-inflammatory T cells recognize the heavy constant region of immunoglobulins G (Fc). We defined 16 peptides, 15 amino acids long, out of the whole Fc sequence immunogenic in healthy donors and patients with Rheumatoid Arthritis, a systemic autoimmune disease. In Immunology it is still debated how the thymic selection of T cells that recognize “self” peptides occurs as these T cells could be potentially harmful. However, nTreg recognize “self” and are very important in controlling the immune homeostasis. Here we show by screening the Immune epitope Data Base (IEDB) that the immunodominant Fc peptides that stimulate nTreg have a very high sequence homology with “non-self” peptides derived from a variety of pathogens. These results suggest that T cells with T cell receptors that recognize “self” peptides could be rescued from the negative selection in the thymus by engaging antigenic peptides with high sequence homology and or that pathogens may inhibit the immune regulation by jeopardizing nTreg expansion by T cell receptor antagonism.

IgG epitopes processed and presented by IgG+ B cells induce suppression by human thymic-derived regulatory T cells

Abstract We described a human regulatory T cell (Treg) population activated by IgG+ B cells presenting peptides of the heavy constant region (Fc) via processing of the surface IgG underlying a model for B cell-Treg cooperation in the human immune regulation. Functionally, Treg inhibited the polarization of naive T cells toward a pro-inflammatory phenotype in both a cognate and a non-cognate fashion. Their fine specificities were similar in healthy donors and patients with rheumatoid arthritis (RA), a systemic autoimmune disease. Four immunodominant Fc peptides bound multiple HLA class II alleles and were recognized by most subjects in the two cohorts. The presentation of Fc peptides that stimulate Treg through the processing of IgG by dendritic cells occurred in myeloid dendritic cells cDC1 and cDC2. Different routes of antigen processing of the IgG impacted Treg expansion in RA patients.

IgG Epitopes Processed and Presented by IgG+ B Cells Induce Suppression by Human Thymic-Derived Regulatory T Cells.

We described a human regulatory T cell (Treg) population activated by IgG+ B cells presenting peptides of the heavy C region (Fc) via processing of the surface IgG underlying a model for B cell-Treg cooperation in the human immune regulation. Functionally, Treg inhibited the polarization of naive T cells toward a proinflammatory phenotype in both a cognate and a noncognate fashion. Their fine specificities were similar in healthy donors and patients with rheumatoid arthritis, a systemic autoimmune disease. Four immunodominant Fc peptides bound multiple HLA class II alleles and were recognized by most subjects in the two cohorts. The presentation of Fc peptides that stimulate Treg through the processing of IgG by dendritic cells (DC) occurred in myeloid DC classical DC 1 and classical DC 2. Different routes of Ag processing of the IgG impacted Treg expansion in rheumatoid arthritis patients.

Stress Temperature Studies in Small Scale Hastelloy® Drug Substance Containers Lead to Increased Extent of and Increased Variability in Antibody-Drug Conjugate and Monoclonal Antibody Aggregation: Evidence for Novel Oxidation-Induced Crosslinking in Monoclonal Antibodies.

Health authorities require that suitable stability of the drug substance be shown in relevant materials of construction. ICH Q1A(R2) explicitly states that "stability studies should be conducted on drug substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution". Stainless steel containers are commonly used for the long-term storage of frozen bulk drug substances (DSs). Hastelloy®-based metal containers are sometimes used due to their higher corrosion resistance and significantly lower iron content to mitigate the potential corrosion-related risks associated with high salt formulations. Despite their benefits, we have found that elevated temperature stability studies in small scale Hastelloy® containers can lead to degradation that is not representative of degradation under typical storage conditions relevant to the manufacturing process. We provide evidence for an oxidation-induced aggregation mechanism that is based on Fenton chemistry with peroxide being supplied by the autoxidation of polysorbate at stress temperatures. Further, variation in the rates of iron leaching between individual small scale containers is shown to be the cause of the variable rates of degradation through strong correlations between leached iron levels and the extents of oxidation and aggregation. The addition of a metal chelator or the removal of polysorbate from the formulation mitigates the oxidation and the non-representative behavior. Extended characterization by LC-MS and 18O labeled peptide mapping shows that a significant portion of the aggregate formed under these conditions is covalently crosslinked and that the predominant covalent species is either a dityrosine or tyrosine-tryptophan crosslink between an Fc peptide and a Fab peptide. This report is the first time either of these two crosslinks have been reported for antibodies with detailed analytical characterization. Because the behavior observed in these studies is not representative of degradation under typical storage conditions relevant to the manufacturing process, this study demonstrates that small scale stress studies in metal containers should be performed with caution and that extended incubation times can lead to non-representative degradation mechanisms.

Remarkable Morphology Transformation from Fiber to Nanotube of a Histidine Organogel in Presence of a Binuclear Iron(III)–Sulfur Complex

Ferrocene (Fc)–peptide conjugates are an interesting class of compounds that have been explored extensively due to the ability of Fc to act as a scaffold to support peptide secondary structural motifs. Fc–peptide conjugates have also displayed self-assembly and gelation behaviour forming an interesting class of new biomaterials. Here, a Fc–phenylalanine–histidine–OMe conjugate has been synthesized and tested for supramolecular gelation. Interestingly, this compound forms a supramolecular gel in the presence of the binuclear iron–sulfur (Fe–S) cluster and displays interesting properties including redox-responsiveness and morphological changes from fibers to spherical vesicles. The gelation in the presence of the Fe–S cluster has future potential to study prebiotic self-assembly, prebiotic cofactor relationships and biomimetic active sites.

Human natural regulatory T cells recognize peptides of the heavy constant region of immunoglobulins presented by IgG+ B cells

Abstract Human natural regulatory T cells (nTreg) recognize conserved peptides derived from the heavy constant region of IgG (Fc). The fine specificity of nTreg and the immunodominance of peptides was determined in healthy donors and rheumatoid arthritis (RA) patients. Eight Fc peptides were identified as highly immunogenic in all subjects with similar ranking and four of eight bound multiple class II HLA alleles. These Fc peptides are presented by IgG+ B cells by internalization and processing of surface IgG and are recognized by Fc-specific nTreg from both healthy donors and RA patients. However, exogenous IgG processed by antigen presenting cells (APC) led to the expansion of this nTreg population only in healthy donors. While nTreg from patients with RA responded to Fc peptides, the response to exogenous IgG presented by APC was compromised. These results demonstrate a hole in the central adaptive immune regulation in RA.

Restricted Proteolysis and LC-MS/MS To Evaluate the Orientation of Surface-Immobilized Antibodies.

The molecular orientation of antibodies immobilized on solid surfaces plays a significant role in the sensitivity of immunoassays and efficiency of protein isolation using antibody-decorated nanoparticles. Optimally, nearly all antibody binding sites should be available to bind. Here we report for the first time an LC-MS/MS approach to probe antibody orientation directly, utilizing sterically restricted proteolysis. Trypsin-decorated magnetic beads (MBs, 1.5 μm) were much larger than average antibody-free areas (55 × 55 nm) of oriented antibodies on MBs, restricting proteolysis to mainly Fab regions. Randomly attached antibodies on MB surfaces served as controls. The tryptic-hydrolyzed peptides were quantified using LC-MS/MS peptide analysis as markers for average positions of Fc and Fab of antibodies on the beads. Different patterns of digestion rates were found due to proteolysis of the oriented and nonoriented antibodies on MBs. For oriented antibodies, the peptides from outer Fab regions gave a much higher digestion rate than those from Fc regions, while for randomly immobilized antibodies digestion rates for Fab and Fc peptides were similar. This novel approach is a useful and convenient tool to characterize antibody orientation for immunoassays and other applications. The relative degree of orientation can be assessed using a metric Ro denoting amount of Fab marker peptides found divided by Fc + Fab marker peptides × 100%. Oriented antibodies on the MBs also provided more efficient antigen capture compared to randomly immobilized antibodies.

Synthetic Glycopeptides Allow for the Quantitation of Scarce Nonfucosylated IgG Fc N-Glycans of Therapeutic Antibody.

Glycans attached to the IgG Fc domain affect strongly biological activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of therapeutic antibodies. However, molecular mechanism in the glycoform-dependent functional modulation of the IgGs remains elusive. The present study communicates that selected reaction monitoring (SRM)-based assay of tryptic IgG Fc glycopeptides is a promising approach for the characterization of antibodies when combined with structure-defined synthetic Fc peptides having a focused N-glycoform as a calibration standard. We describe a novel synthetic approach to the human IgG1 Fc peptide having a bisected decasaccharide and its nonbisected counterpart compound, the signatures of antibodies involving Fc domain with rare N-glycans expected to show much higher ADCC/CDC than abundant IgG N-glycans, and their application to the SRM-based quantitative glycoproteomics. Use of a key intermediate, phenyl (2-O-benzyl-4,6-O-benzylidine-β-d-mannopyranosyl)-(1 → 4)-3,6-di-O-benzyl-2-azido-2-deoxy-1-thio-β-d-glucopyranoside, derived from locust bean gum galactomannan, facilitated greatly the synthesis of a bisected nonasaccharide as a stable precursor of oxazoline derivative needed for the enzymatic trans-glycosylation with Fc nonapeptide carrying a GlcNAc at Asn297 residue, while the coupling reaction catalyzed by mutant endo-M-N175Q proceeded very slowly. Strikingly, SRM assay using the synthetic Fc glycopeptides as calibration standards uncovered the occurrence of the targeted IgG1 Fc fragment carrying a nonfucosylated and bisected (315 fmol, 0.20%) and its nonbisected counterpart (1154 fmol, 0.73%) in the tryptic digests from 158 pmol of anticancer antibody Herceptin (trastuzumab). The results suggest that aberrantly glycosylated IgG Fc variants may contribute to the total biological activities of the therapeutic antibodies.

Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins.

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

Abstract O.19: Fine Specificity Of Natural Regulatory T Cells That Modulate Vascular Inflammation

Objective: We recently defined that a critical mechanism of IVIG therapy in KD patients is the activation and expansion of natural regulatory T cells (nTreg) that recognize the heavy constant region (Fc) of IgG1. Lack of circulating Fc-specific nTreg in the sub-acute phase of KD strongly correlates with arterial complications. Notably, Fc-specific nTreg are always detectable in circulation in healthy donors, suggesting their importance in maintaining vascular homeostasis. Here we aim at the characterization of the fine specificity of these nTreg by testing the immunogenicity of synthetic peptides (> 97% purity) 15 amino acid long, overlapping 10 amino acids, spanning the whole Fc protein. Methods: To define the immunodominat Fc sequences for nTreg expansion, T cell lines have been established with pools of two consecutive overlapping peptides been generated from PBMC ex vivo from: 1) healthy donors, 2) sub-acute KD subjects (2 weeks after IVIG), and 3) KD subjects that received IVIG 1-2 years earlier. IL-10 secretion and CD4+ CD25high T cell expansion have been the read-outs in these experiments. Results: 14 peptides have been defined immunogenic in all the 3 cohorts with several peptides recognized by the large majority of the subject studied, including patients with arterial complication that did not respond to the whole Fc protein. The algorithm prediction of the HLA binding affinity of these nTreg peptide epitopes suggests that these immunodominant regions of the Fc protein are promiscous for HLA binding and very good candidate for future therapeutic use. Clinical relevance of the findings: Immunodominat Fc peptides are an optimized, low cost alternative to IVIG, potentially capable to overcome the lack of nTreg responses in KD patients that develop arterial complications.

Irisin: A myokine with locomotor activity

The mechanisms underlying alterations in brain functions in response to physical exercise are not fully understood. The present study examined the central effect of irisin, a 112 amino acid polypeptide hormone secreted from the skeletal muscle after exercise, on the locomotion in rats. Central administration of irisin significantly increased the locomotion. Relative to control animals treated with IgG Fc peptide, rats receiving irisin demonstrated a marked increase in total travel distance, ambulatory counts and time, and vertical counts and time. These changes were associated with a significant decrease in resting time. Central treatment of irisin also induced significant increases in oxygen consumption, carbon dioxide production and heat production, indicating an increase in metabolic activity. Our study suggests that physical activity may signal to the central nervous system to coordinate locomotion with metabolic activity via irisin.

Fine specificities of natural regulatory T cells after IVIG therapy in patients with Kawasaki disease

The activation of natural regulatory T cells (nTreg) recognizing the heavy constant region (Fc) of IgG is an important mechanism of action of intravenous immunoglobulin (IVIG) therapy in Kawasaki disease (KD). Lack of circulating Fc-specific nTreg in the sub-acute phase of KD is correlated with the development of coronary artery abnormalities (CAA). Here, we characterize the fine specificity of nTreg in sub-acute (2- to 8-week post-IVIG) and convalescent (1- to 10-year post-IVIG) KD subjects by testing the immunogenicity of 64 peptides, 15 amino acids in length with a 10 amino acid-overlap spanning the entire Fc protein. About 12 Fc peptides (6 pools of 2 consecutive peptides) were recognized by nTreg in the cohorts studied, including two patients with CAA. To test whether IVIG expands the same nTreg populations that maintain vascular homeostasis in healthy subjects, we compared these results with results obtained in healthy adult controls. Similar nTreg fine specificities were observed in KD patients after IVIG and in healthy donors. These results suggest that T cell fitness rather than T cell clonal deletion or anergy is responsible for the lack of Fc-specific nTreg in KD patients who develop CAA. Furthermore, we found that adolescents and adults who had KD during childhood without developing CAA did not respond to the Fc protein in vitro, suggesting that the nTreg response induced by IVIG in KD patients is short-lived. Our results support the concept that peptide epitopes may be a viable therapeutic approach to expand Fc-specific nTreg and more effectively prevent CAA in KD patients.

Quantitative electrochemical detection of cathepsin B activity in complex tissue lysates using enhanced AC voltammetry at carbon nanofiber nanoelectrode arrays

The proteolytic activity of a cancer-related enzyme cathepsin B is measured with alternating current voltammetry (ACV) using ferrocene (Fc) labeled tetrapeptides attached to nanoelectrode arrays (NEAs) fabricated with vertically aligned carbon nanofibers (VACNFs). This combination enables the use of high AC frequencies (~1kHz) with enhanced electrochemical signals. The specific proteolysis of the Fc–peptide by cathepsin B produces decay in the ACV peak current versus the reaction time. The exponential component of the raw data can be extracted and defined as the “extracted proteolytic signal” which allows consistent quantitative analyses using a heterogeneous Michaelis–Menten model. A “specificity constant” kcat/KM=(3.68±0.50)×104M−1s−1 for purified cathepsin B was obtained. The detections of cathepsin B activity in different concentrations of whole lysate of human breast tissue, tissue lysate spiked with varied concentrations of cathepsin B, and the tissue lysate after immunoprecipitation showed that there is ~13.4nM higher cathepsin B concentration in 29.1µgmL−1 of whole tissue lysate than the immunoprecipitated sample. The well-defined regular VACNF NEAs by e-beam lithography show a much faster kinetics for cathepsin B proteolysis with kcat/KM=9.2×104M−1s−1. These results illustrate the potential of this technique as a portable multiplex electronic system for cancer diagnosis by rapid protease profiling of serum or blood samples.

Naturally occurring glycan forms of human immunoglobulins G1 and G2

High resolution glycan mapping was performed on human immunoglobulin G (IgG) obtained from individual healthy subjects and from a combined sample of healthy subjects. In addition to the commonly known complex glycans, a variety of minor glycans are described and quantified, including high mannose forms and several previously unreported hybrid forms. Fc specific glycan analysis was also performed through peptide mapping with LC/MS/MS. Differences in the glycan linked Fc peptide masses allowed glycan profiles to be analyzed and quantified from IgG1 and IgG2 simultaneously for each subject within the same sample. Glycan profiles differed between subtypes, with greater levels of more fully galactosylated species found on IgG1 (e.g. G2F, SG2F) than IgG2. These results also show that Gal attachment on G1F is biased to the Man (α1 → 6) arm for IgG1 and on the Man (α1 → 3) arm for IgG2 from individual healthy subjects.

Synthesis and characterization of new ferrocene peptide conjugates

Three classes of diamine linked ferrocene (Fc)-conjugates were prepared and their properties were investigates in solution and the solid state: (a) acyclic diamine-linked 1,n'-Fc conjugates, (b) acyclic diamine-linked 1,n'-Fc peptide conjugates, and (c) cyclic 1,n'-Fc-peptide diamine conjugates. In all cases, the synthetic procedure started from 1,1'-ferrocenecarboxylic acid methyl ester or 1,1'-ferrocene dicarboxylic acid or their amino acid conjugates followed by coupling with diaminoalkanes. The resulting conjugates exhibit H-bonding as is evident by temperature and, in some cases, concentration-dependent NMR shifts and in the solid-state structure of one of the conjugates. Our studies show that the structural properties of Fc-diamine-linked systems are different from those of the related cystamine conjugates, presumably due to the enhanced flexibility of the conjugates.

Gene therapy of diabetes using a novel GLP-1/IgG1-Fc fusion construct normalizes glucose levels in db/db mice

Glucagon-like peptide (GLP-1), a major physiological incretin, plays numerous important roles in modulating blood glucose homeostasis and has been proposed for the treatment of type 2 diabetes. The major obstacles for using native GLP-1 as a therapeutic agent are that it must be delivered by a parenteral route and has a short half-life. In an attempt to develop a strategy to prolong the physiological t(1/2) and enhance the potency of GLP-1, a fusion protein consisting of active human GLP-1 and mouse IgG(1) heavy chain constant regions (GLP-1/Fc) was generated. A plasmid encoding an IgK leader peptide-driven secretable fusion protein of the active GLP-1 and IgG(1)-Fc was constructed for mammalian expression. This plasmid allows for expression of bivalent GLP-1 peptide ligands as a result of IgG-Fc homodimerization. In vitro studies employing purified GLP-1/Fc indicate that the fusion protein is functional and elevates cAMP levels in insulin-secreting INS-1 cells. In addition, it stimulates insulin secretion in a glucose concentration-dependent manner. Intramuscular gene transfer of the plasmid in db/db mice demonstrated that expression of the GLP-1/Fc peptide normalizes glucose tolerance by enhancing insulin secretion and suppressing glucagon release. This strategy of using a bivalent GLP-1/Fc fusion protein as a therapeutic agent is a novel approach for the treatment of diabetes.