Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical fluorescence techniques.

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG, and contributes to their long plasma half-life. We recently identified an FcRn-dependent recycling pathway from macropinosomes in macrophages; however, little is known about the dynamics of intracellular FcRn-ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin-FcRn interactions in living bone marrow-derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn-mCherry fusion protein with endocytosed Alexa Fluor 488-labeled human serum albumin (HSA-AF488) in BMDMs, and raster image correlation spectroscopy (RICS) analysis of single fluorescent-labeled albumin molecules to monitor the diffusion kinetics of internalized albumin. Our data identified a major fraction of immobile HSA-AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM-FRET following endocytosis, including detection of FRET in tubular-like structures. A nonbinding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn-ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand-receptor interactions.

Therapeutic exploitation of neutrophils to fight cancer.

Antibody-based immunotherapy is a promising strategy in cancer treatment. Antibodies can directly inhibit tumor growth, induce complement-dependent cytotoxicity and induce Fc receptor-mediated elimination of tumor cells by macrophages and natural killer cells. Until now, however, neutrophils have been largely overlooked as potential effector cells, even though they are the most abundant type of immune cells in the circulation. Neutrophils display heterogeneity, especially in the context of cancer. Therefore, their role in cancer is debated. Nevertheless, neutrophils possess natural anti-tumor properties and appropriate stimulation, i.e. specific targeting via antibody therapy, induces potent tumor cell killing, especially via targeting of the immunoglobulin A Fc receptor (FcαRI, CD89). In this review we address the mechanisms of tumor cell killing by neutrophils and the role of neutrophils in induction of anti-tumor immunity. Moreover, possibilities for therapeutic targeting are discussed.

Fc Receptor Variants and Disease: A Crucial Factor to Consider in the Antibody Therapeutics in Clinic.

The fragment crystallizable (Fc) domain of antibodies is responsible for their protective function and long-lasting serum half-life via Fc-mediated effector function, transcytosis, and recycling through its interaction with Fc receptors (FcRs) expressed on various immune leukocytes, epithelial, and endothelial cells. Therefore, the Fc-FcRs interaction is a control point of both endogenous and therapeutic antibody function. There are a number of reported genetic variants of FcRs, which include polymorphisms in (i) extracellular domain of FcRs, which change their affinities to Fc domain of antibodies; (ii) both cytoplasmic and intracellular domain, which alters the extent of signal transduction; and (iii) the promoter region of the FcRs gene, which affects the expression level of FcRs, thus being associated with the pathogenesis of disease indications. In this review, we firstly describe the correlation between the genetic variants of FcRs and immunological disorders by individual differences in the extent of FcRs-mediated regulations. Secondly, we discuss the influence of the genetic variants of FcRs on the susceptibility to infectious diseases or cancer in the perspective of FcRs-induced effector functions. Overall, we concluded that the genetic variants of FcRs are one of the key elements in the design of antibody therapeutics due to their variety of clinical outcomes among individuals.

Pathogenic Activation and Therapeutic Blockage of FcαR-Expressing Polymorphonuclear Leukocytes in IgA Pemphigus.

Pathomechanisms in IgA pemphigus are assumed to rely on Fc-dependent cellular activation by antigen-specific IgA autoantibodies; however, models for the disease and more detailed pathophysiologic data are lacking. In this study, we aimed to establish in vitro models of disease for IgA pemphigus, allowing us to study the effects of the interaction of anti-keratinocyte IgA with cell surface FcαRs. Employing multiple in vitro assays, such as a skin cryosection assay and a human skin organ culture model, in this study, we present mechanistic data for the pathogenesis of IgA pemphigus, mediated by anti-desmoglein 3 IgA autoantibodies. Our results reveal that this disease is dependent on FcαR-mediated activation of leukocytes in the epidermis. Importantly, this cell-dependent pathology can be dose-dependently abrogated by peptide-mediated inhibition of FcαR:IgA-Fc interaction, as confirmed in an additional model for IgA-dependent disease, that is, IgA vasculitis. These data suggest that IgA pemphigus can be modeled in vitro and that IgA pemphigus and IgA vasculitis are FcαR-dependent disease entities that can be specifically targeted in these experimental systems.

Targeting Fc effector function in vaccine design.

INTRODUCTION: Antibodies mediate pathogen neutralization in addition to several cytotoxic Fc functions through engaging cellular receptors and recruiting effector cells. Fc effector functions have been well described in disease control and protection against infectious diseases including HIV, Ebola, malaria, influenza and tuberculosis, making them attractive targets for vaccine design. AREAS COVERED: We briefly summarize the role of Fc effector functions in disease control and protection in viral, bacterial and parasitic infectious diseases. We review Fc effector function in passive immunization and vaccination, and primarily focus on strategies to elicit and modulate these functions as part of a robust vaccine strategy. EXPERT OPINION: Despite their known correlation with vaccine efficacy for several diseases, only recently have seminal studies addressed how these Fc effector functions can be elicited and modulated in vaccination. However, gaps remain in assay standardization and the precise mechanisms of diverse functional assays. Furthermore, there are inherent difficulties in the translation of findings from animal models to humans, given the difference in sequence, expression and function of Fc receptors and Fc portions of antibodies. However, overall it is clear that vaccine development to elicit Fc effector function is an important goal for optimal prevention against infectious disease.

Fetal mast cells mediate postnatal allergic responses dependent on maternal IgE.

Mast cells (MCs) are central effector cells in allergic reactions that are often mediated by immunoglobulin E (IgE). Allergies commonly start at an early age, and both MCs and IgE are detectable in fetuses. However, the origin of fetal IgE and whether fetal MCs can degranulate in response to IgE-dependent activation are presently unknown. Here, we show that human and mouse fetal MCs phenotypically mature through pregnancy and can be sensitized by maternal IgE. IgE crossed the placenta, dependent on the fetal neonatal Fc receptor (FcRN), and sensitized fetal MCs for allergen-specific degranulation. Both passive and active prenatal sensitization conferred allergen sensitivity, resulting in postnatal skin and airway inflammation after the first allergen encounter. We report a role for MCs within the developing fetus and demonstrate that fetal MCs may contribute to antigen-specific vertical transmission of allergic disease.

A cross-reactive antibody protects against Ross River virus musculoskeletal disease despite rapid neutralization escape in mice.

Arthritogenic alphaviruses cause debilitating musculoskeletal disease and historically have circulated in distinct regions. With the global spread of chikungunya virus (CHIKV), there now is more geographic overlap, which could result in heterologous immunity affecting natural infection or vaccination. Here, we evaluated the capacity of a cross-reactive anti-CHIKV monoclonal antibody (CHK-265) to protect against disease caused by the distantly related alphavirus, Ross River virus (RRV). Although CHK-265 only moderately neutralizes RRV infection in cell culture, it limited clinical disease in mice independently of Fc effector function activity. Despite this protective phenotype, RRV escaped from CHK-265 neutralization in vivo, with resistant variants retaining pathogenic potential. Near the inoculation site, CHK-265 reduced viral burden in a type I interferon signaling-dependent manner and limited immune cell infiltration into musculoskeletal tissue. In a parallel set of experiments, purified human CHIKV immune IgG also weakly neutralized RRV, yet when transferred to mice, resulted in improved clinical outcome during RRV infection despite the emergence of resistant viruses. Overall, this study suggests that weakly cross-neutralizing antibodies can protect against heterologous alphavirus disease, even if neutralization escape occurs, through an early viral control program that tempers inflammation.

FcRn is a CD32a coreceptor that determines susceptibility to IgG immune complex-driven autoimmunity.

IgG immune complexes (ICs) promote autoimmunity through binding fragment crystallizable (Fc) γ-receptors (FcγRs). Of these, the highly prevalent FcγRIIa (CD32a) histidine (H)-131 variant (CD32aH) is strongly linked to human autoimmune diseases through unclear mechanisms. We show that, relative to the CD32a arginine (R)-131 (CD32aR) variant, CD32aH more avidly bound human (h) IgG1 IC and formed a ternary complex with the neonatal Fc receptor (FcRn) under acidic conditions. In primary human and mouse cells, both CD32a variants required FcRn to induce innate and adaptive immune responses to hIgG1 ICs, which were augmented in the setting of CD32aH. Conversely, FcRn induced responses to IgG IC independently of classical FcγR, but optimal responses required FcRn and FcγR. Finally, FcRn blockade decreased inflammation in a rheumatoid arthritis model without reducing circulating autoantibody levels, providing support for FcRn's direct role in IgG IC-associated inflammation. Thus, CD32a and FcRn coregulate IgG IC-mediated immunity in a manner favoring the CD32aH variant, providing a novel mechanism for its disease association.

The neonatal Fc receptor in cancer FcRn in cancer.

Since the neonatal IgG Fc receptor (FcRn) was discovered, it was found to be involved in immunoglobulin recycling and biodistribution, immune complexes routing, antigen presentation, humoral immune response, and cancer immunosurveillance. The latest data show that FcRn plays a part in cancer pathophysiology. In various types of cancers, such as lung and colorectal cancer, FcRn has been described as an early marker for prognosis. Dysregulation of FcRn expression by cancer cells allows them to increase their metabolism, and this process could be exploited for passive targeting of cytotoxic drugs. However, the roles of this receptor depend on whether the studied cell population is the tumor tissue or the infiltrating cells, bringing forward the need for further studies.

The biodistribution of therapeutic proteins: Mechanism, implications for pharmacokinetics, and methods of evaluation.

Therapeutic proteins (TPs) are a diverse drug class that include monoclonal antibodies (mAbs), recombinantly expressed enzymes, hormones and growth factors, cytokines (e.g. chemokines, interleukins, interferons), as well as a wide range of engineered fusion scaffolds containing IgG1 Fc domain for half-life extension. As the pharmaceutical industry advances more potent and selective protein-based medicines through discovery and into the clinical stages of development, it has become widely appreciated that a comprehensive understanding of the mechanisms of TP biodistribution can aid this endeavor. This review aims to highlight the literature that has advanced our understanding of the determinants of TP biodistribution. A particular emphasis is placed on the multi-faceted role of the neonatal Fc receptor (FcRn) in mAb and Fc-fusion protein disposition. In addition, characterization of the TP-target interaction at the cell-level is discussed as an essential strategy to establish pharmacokinetic-pharmacodynamic (PK/PD) relationships that may lead to more informed human dose projections during clinical development. Methods for incorporation of tissue and cell-level parameters defining these characteristics into higher-order mechanistic and semi-mechanistic PK models will also be presented.

The protective potential of Fc-mediated antibody functions against influenza virus and other viral pathogens.

In recent years, there has been a renewed interest in utilizing antibody fragment crystallizable (Fc) functions to prevent and control viral infections. The protective and therapeutic potential of Fc-mediated antibody functions have been assessed for some clinically important human viruses, including HIV, hemorrhagic fever viruses and influenza virus. There is mounting evidence that influenza-specific antibodies with Fc-mediated functions, such as antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis, can aid in the clearance of influenza virus infection. Recent influenza challenge studies and intravenous immunoglobulin G therapy studies in humans suggest a protective role for Fc effector functions in vivo. Broadly reactive influenza antibodies with Fc-mediated functions are prevalent in the human population and could inform the development of a universally protective influenza vaccine or therapy. In this review, we explore the utility of antibodies with Fc-mediated effector functions against viral infections with a focus on influenza virus.

Serum IgA Fc effector functions in infectious disease and cancer.

Immunoglobulin (Ig) A is the most abundant antibody isotype present at mucosal surfaces and the second most abundant in human serum. In addition to preventing pathogen entry at mucosal surfaces, IgA can control and eradicate bacterial and viral infections through a variety of antibody-mediated innate effector cell mechanisms. The role of mucosal IgA in infection (e.g. neutralization) and in inflammatory homeostasis (e.g. allergy and autoimmunity) has been extensively investigated; by contrast, serum IgA is comparatively understudied. IgA binding to fragment crystallizable alpha receptor plays a dual role in the activation and inhibition of innate effector cell functions. Mounting evidence suggests that serum IgA induces potent effector functions against various bacterial and some viral infections including Neisseria meningitidis and rotavirus. Furthermore, in the era of immunotherapy, serum IgA provides an interesting alternative to classical IgG monoclonal antibodies to treat cancer and infectious pathogens. Here we discuss the role of serum IgA in infectious diseases with reference to bacterial and viral infections and the potential for IgA as a monoclonal antibody therapy.

Integration of Ontogeny Into a Physiologically Based Pharmacokinetic Model for Monoclonal Antibodies in Premature Infants.

An understanding of pediatric pharmacokinetics (PK) is essential for first-in-pediatric dose selection and clinical trial design. At present, there is no reliable way to scale the PK of monoclonal antibodies and immunoglobulin G drug products from adults to young children or to premature infants-a vulnerable population with a rapidly growing drug development pipeline. In this work, pediatric physiologically based PK models are constructed in PK-Sim and Mobi to explore the PK of pagibaximab, palivizumab, MEDI8897, and intravenous immunoglobulin in preterm infants. In addition to considering ontogeny in pediatric organ volumes, organ composition, blood flow rates, and hematocrit, advanced ontogeny is applied for 3 key parameters: capillary surface area, hematopoietic cell concentration, and lymph flow rate. The role and importance of each parameter for determining pediatric clearance (CL) and volume of distribution at steady state (VSS ) are quantitatively assessed with a local sensitivity analysis. In addition, the uncertainty around parameters with limited information in pediatrics is addressed (eg, free neonatal Fc receptor concentration). The full ontogeny parameterization yields pediatric PK predictions that are within 1.5-fold prediction error >90% of the time for preterm infants, with an absolute average fold error of 1.05. This result suggests that many of the key factors related to ontogeny are appropriately addressed. Overall, this study makes a first step toward developing a platform pediatric physiologically based PK model for monoclonal antibodies and immunoglobulin G drug products by solidifying existing parameterizations, integrating new concepts, and drawing attention to unmet needs for physiologic knowledge in children.

FcRn mediates fast recycling of endocytosed albumin and IgG from early macropinosomes in primary macrophages.

The neonatal Fc receptor (FcRn) rescues albumin and IgG from degradation following endocytosis and thereby extends the half-life of these plasma proteins. However, the pathways for the uptake of these soluble FcRn ligands, and the recycling itinerary of the FcRn-ligand complexes, have not been identified in primary cells. Here, we have defined the recycling of human albumin and IgG in primary mouse macrophages selectively expressing the human FcRn. Albumin is internalised by macropinocytosis; in the absence of FcRn, internalised albumin is rapidly degraded, while in the presence of FcRn albumin colocalises to SNX5-positive membrane domains and is partitioned into tubules emanating from early macropinosomes for delivery in transport carriers to the plasma membrane. Soluble monomeric IgG was also internalised by macropinocytosis and rapidly recycled by the same pathway. In contrast, the fate of IgG bound to surface Fcγ receptors differed from monomeric IgG endocytosed by macropinocytosis. Overall, our findings identify a rapid recycling pathway for FcRn ligands from early macropinosomes to the cell surface of primary cells.

Mesangial Deposition Can Strongly Involve Innate-Like IgA Molecules Lacking Affinity Maturation.

BACKGROUND: IgA nephropathy (IgAN) often follows infections and features IgA mesangial deposition. Polymeric IgA deposits in the mesangium seem to have varied pathogenic potential, but understanding their pathogenicity remains a challenge. Most mesangial IgA1 in human IgAN has a hypogalactosylated hinge region, but it is unclear whether this is required for IgA deposition. Another important question is the role of adaptive IgA responses and high-affinity mature IgA antibodies and whether low-affinity IgA produced by innate-like B cells might also yield mesangial deposits. METHODS: To explore the effects of specific qualitative variations in IgA and whether altered affinity maturation can influence IgA mesangial deposition and activate complement, we used several transgenic human IgA1-producing models with IgA deposition, including one lacking the DNA-editing enzyme activation-induced cytidine deaminase (AID), which is required in affinity maturation. Also, to explore the potential role of the IgA receptor CD89 in glomerular inflammation, we used a model that expresses CD89 in a pattern observed in humans. RESULTS: We found that human IgA induced glomerular damage independent of CD89. When comparing mice able to produce high-affinity IgA antibodies with mice lacking AID-enabled Ig affinity maturation, we found that IgA deposition and complement activation significantly increased and led to IgAN pathogenesis, although without significant proteinuria or hematuria. We also observed that hinge hypoglycosylation was not mandatory for IgA deposition. CONCLUSIONS: In a mouse model of IgAN, compared with high-affinity IgA, low-affinity innate-like IgA, formed in the absence of normal antigen-driven maturation, was more readily involved in IgA glomerular deposition with pathogenic effects.

Human Neonatal Fc Receptor Is the Cellular Uncoating Receptor for Enterovirus B.

Enterovirus B (EV-B), a major proportion of the genus Enterovirus in the family Picornaviridae, is the causative agent of severe human infectious diseases. Although cellular receptors for coxsackievirus B in EV-B have been identified, receptors mediating virus entry, especially the uncoating process of echovirus and other EV-B remain obscure. Here, we found that human neonatal Fc receptor (FcRn) is the uncoating receptor for major EV-B. FcRn binds to the virus particles in the "canyon" through its FCGRT subunit. By obtaining multiple cryo-electron microscopy structures at different stages of virus entry at atomic or near-atomic resolution, we deciphered the underlying mechanisms of enterovirus attachment and uncoating. These structures revealed that different from the attachment receptor CD55, binding of FcRn to the virions induces efficient release of "pocket factor" under acidic conditions and initiates the conformational changes in viral particle, providing a structural basis for understanding the mechanisms of enterovirus entry.

Immunoglobulin A: magic bullet or Trojan horse?

BACKGROUND: Neutrophils participate in the first line of defense by executing several killing mechanisms, including phagocytosis, degranulation and the release of neutrophil extracellular traps. Additionally, they can orchestrate the adaptive immune system by secreting cytokines and chemokines. Opsonization with antibodies aids in the recognition of pathogens, via binding to Fc receptors on the neutrophil surface. Immunoglobulin A (IgA) is the most abundant antibody at mucosal sites and has multiple functions in homeostasis and immunity. Neutrophils and IgA can interact via the IgA Fc receptor Fc?RI (CD89), leading to pro- or anti-inflammatory responses. AIMS: The aim of this review is to give a concise overview of the interplay between IgA, Fc?RI and neutrophils and to explore potential therapies for autoimmune diseases and cancer. RESULTS: Crosslinking of FcαRI by IgA-immune complexes yields potent neutrophil activation and pro-inflammatory effector functions, including the recruitment of neutrophils. This can lead to neutrophil accumulation and tissue destruction during IgA-autoantibody mediated diseases. Conversely, for cancer treatment, the myriad of powerful neutrophil effector functions after targeting FcαRI may contribute to effective immunotherapy. CONCLUSION: By interfering with or actively promoting the interaction between IgA and FcαRI, therapies for multiple maladies could be developed.