Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria.

Monoclonal IgG antibodies constitute the fastest growing class of therapeutics. Thus, there is an intense interest to design more potent antibody formats, where long plasma half-life is a commercially competitive differentiator affecting dosing, frequency of administration and thereby potentially patient compliance. Here, we report on an Fc-engineered variant with three amino acid substitutions Q311R/M428E/N434W (REW), that enhances plasma half-life and mucosal distribution, as well as allows for needle-free delivery across respiratory epithelial barriers in human FcRn transgenic mice.

Potent TRIM21 and complement-dependent intracellular antiviral immunity requires the IgG3 hinge.

Humans have four IgG antibody subclasses that selectively or differentially engage immune effector molecules to protect against infections. Although IgG1 has been studied in detail and is the subclass of most approved antibody therapeutics, increasing evidence indicates that IgG3 is associated with enhanced protection against pathogens. Here, we report that IgG3 has superior capacity to mediate intracellular antiviral immunity compared with the other subclasses due to its uniquely extended and flexible hinge region, which facilitates improved recruitment of the cytosolic Fc receptor TRIM21, independently of Fc binding affinity.

Prevention of Fetal/Neonatal Alloimmune Thrombocytopenia in Mice: Biochemical and Cell Biological Characterization of Isoforms of a Human Monoclonal Antibody.

Maternal alloantibodies toward paternally inherited Ags on fetal platelets can cause thrombocytopenia and bleeding complications in the fetus or neonate, referred to as fetal and neonatal alloimmune thrombocytopenia (FNAIT). This is most commonly caused by Abs against the human platelet Ag (HPA)-1a in Caucasians, and a prophylactic regimen to reduce the risk for alloimmunization to women at risk would be beneficial. We therefore aimed to examine the prophylactic potential of a fully human anti-HPA-1a IgG1 (mAb 26.

Antibody-mediated delivery of T-cell epitopes to antigen-presenting cells induce strong CD4 and CD8 T-cell responses.

Targeted delivery of antigen to antigen-presenting cells (APCs) enhances antigen presentation and thus, is a potent strategy for making more efficacious vaccines. This can be achieved by use of antibodies with specificity for endocytic surface molecules expressed on the APC. We aimed to compare two different antibody-antigen fusion modes in their ability to induce T-cell responses; first, exchange of immunoglobulin (Ig) constant domain loops with a T-cell epitope (Troybody), and second, fusion of T-cell epitope or whole antigen to the antibody C-terminus.

An engineered human albumin enhances half-life and transmucosal delivery when fused to protein-based biologics.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn).

Publisher Correction: Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

In the Supplementary Information file originally published with this Article, the Supplementary references 48-62 were missing; the amended file has now been uploaded.

Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies.

Although repetitive patterns of antigens are crucial for certain immune responses, an understanding of how antibodies bind and dynamically interact with various spatial arrangements of molecules is lacking. Hence, we introduced a new method in which molecularly precise nanoscale patterns of antigens are displayed using DNA origami and immobilized in a surface plasmon resonance set-up. Using antibodies with identical antigen-binding domains, we found that all the subclasses and isotypes studied bind bivalently to two antigens separated at distances that range from 3 to 17 nm.

A human endothelial cell-based recycling assay for screening of FcRn targeted molecules.

Albumin and IgG have remarkably long serum half-lives due to pH-dependent FcRn-mediated cellular recycling that rescues both ligands from intracellular degradation. Furthermore, increase in half-lives of IgG and albumin-based therapeutics has the potential to improve their efficacies, but there is a great need for robust methods for screening of relative FcRn-dependent recycling ability. Here, we report on a novel human endothelial cell-based recycling assay (HERA) that can be used for such pre-clinical screening.

Multivalent pIX phage display selects for distinct and improved antibody properties.

Phage display screening readily allows for the identification of a multitude of antibody specificities, but to identify optimal lead candidates remains a challenge. Here, we direct the antibody-capsid fusion away from the signal sequence-dependent secretory SEC pathway in E. coli by utilizing the intrinsic signal sequence-independent property of pIX to obtain virion integration.

TRIM21 Immune Signaling Is More Sensitive to Antibody Affinity Than Its Neutralization Activity.

Ab-coated viruses can be detected in the cytosol by the FcR tripartite motif-containing 21 (TRIM21), which rapidly recruits the proteasomal machinery and triggers induction of immune signaling. As such, TRIM21 plays a key role in intracellular protection by targeting invading viruses for destruction and alerting the immune system. A hallmark of immunity is elicitation of a balanced response that is proportionate to the threat, to avoid unnecessary inflammation.

Enhanced FcRn-dependent transepithelial delivery of IgG by Fc-engineering and polymerization.

Monoclonal IgG antibodies (Abs) are used extensively in the clinic to treat cancer and autoimmune diseases. In addition, therapeutic proteins are genetically fused to the constant Fc part of IgG. In both cases, the Fc secures a long serum half-life and favourable pharmacokinetics due to its pH-dependent interaction with the neonatal Fc receptor (FcRn).

Characterization of a human platelet antigen-1a-specific monoclonal antibody derived from a B cell from a woman alloimmunized in pregnancy.

Human platelet Ag (HPA)-1a, located on integrin β3, is the main target for alloantibodies responsible for fetal and neonatal alloimmune thrombocytopenia (FNAIT) in the white population. There are ongoing efforts to develop an Ab prophylaxis and therapy to prevent or treat FNAIT. In this study, an mAb specific for HPA-1a, named 26.

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

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 FcγRs and complement factor C1q, has not been studied in detail.

A pilot study showing differences in glycosylation patterns of IgG subclasses induced by pneumococcal, meningococcal, and two types of influenza vaccines.

The presence of a carbohydrate moiety on asparagine 297 in the Fc part of an IgG molecule is essential for its effector functions and thus influences its vaccine protective effect. Detailed structural carbohydrate analysis of vaccine induced IgGs is therefore of interest as this knowledge can prove valuable in vaccine research and design and when optimizing vaccine schedules. In order to better understand and exploit the protective potential of IgG antibodies, we carried out a pilot study; collecting serum or plasma from volunteers receiving different vaccines and determining the IgG subclass glycosylation patterns against specific vaccine antigens at different time points using LC-ESI-MS analysis.

Structural features and complement fixing activity of polysaccharides from Codonopsis pilosula Nannf. var. modesta L.T.Shen roots.

Two pectic polysaccharides, 50 WCP-II-I and 100 WCP-II-I, were obtained from 50 and 100 °C water extracts of Codonopsis pilosula roots by ion exchange chromatography and gel filtration. The study of the sub-fractions obtained after pectinase degradation showed that the complement fixation activities of these pectins are expressed mainly by their ramified regions. The structure studies of native and sub-fractions showed the 50 WCP-II-I is a pectic polysaccharide, with long homogalacturonan regions (some of the galacturonic acid units were methyl esterified), interrupted by one short rhamnogalacturonan I (RG-I) region.

Enzyme inhibition, antioxidant and immunomodulatory activities, and brine shrimp toxicity of extracts from the root bark, stem bark and leaves of Terminalia macroptera.

Ethnopharmacological Relevance: The root bark, stem bark and leaves of Terminalia macroptera have been traditionally used against a variety of ailments such as wounds, hepatitis, malaria, fever, cough, and diarrhea as well as tuberculosis and skin diseases in African folk medicine. Boiling water extracts of Terminalia macroptera, administered orally, are the most common preparations of this plant used by the traditional healers in Mali. This study aimed to investigate the inhibition of the activities of α-glucosidase, 15-lipoxygenase and xanthine oxidase, DPPH scavenging activity, complement fixation activity and brine shrimp toxicity of different extracts obtained by boiling water extraction (BWE) and by ASE (accelerated solvent extraction) with ethanol, ethanol-water and water as extractants from different plant parts of Terminalia macroptera.

Complement activity of polysaccharides from three different plant parts of Terminalia macroptera extracted as healers do.

Ethnopharmacological Relevance: Water decoctions of the root bark, stem bark and leaves of Terminalia macroptera are used by traditional healers in Mali to cure a wide range of illnesses, such as wounds, hepatitis, malaria, fever, cough and diarrhea as well as tuberculosis. Plant polysaccharides isolated from crude water extracts have previously shown effects related to the immune system. The aims of this study are comparing the properties of the polysaccharides among different plant parts, as well as relationship between chemical characteristics and complement fixation activities when the plant material has been extracted as the traditional healers do, with boiling water directly.

Complement fixing polysaccharides from Terminalia macroptera root bark, stem bark and leaves.

The root bark, stem bark and leaves of Terminalia macroptera were sequentially extracted with ethanol, 50% ethanol-water, and 50 °C and 100 °C water using an accelerated solvent extractor. Ten bioactive purified polysaccharide fractions were obtained from those crude extracts after anion exchange chromatography and gel filtration. The polysaccharides and their native extracts were characterized with respect to molecular weight, chemical compositions and effects in the complement assay.

Characterisation and immunomodulating activities of exo-polysaccharides from submerged cultivation of Hypsizigus marmoreus.

Exo-polysaccharides were purified and characterized from the fermentation broth of Hypsizigus marmoreus, a popular edible mushroom consumed in Asia. Among them, B-I-I and B-II-I exhibited potent complement fixating activity, meanwhile, B-N-I, B-I-I, B-II-I and B-II-II exhibited significant macrophage stimulating activity. Molecular weights of the four exo-polysaccharides were determined to be 6.

Immunomodulating pectins from root bark, stem bark, and leaves of the Malian medicinal tree Terminalia macroptera, structure activity relations.

The root bark, stem bark, and leaves of Terminalia macroptera were sequentially extracted with ethanol, 50% ethanol-water, and 50°C water using an accelerated solvent extractor (ASE). Six bioactive purified pectic polysaccharide fractions were obtained from the 50°C crude water extracts after anion exchange chromatography and gel filtration. The root bark, stem bark, and leaves of T.

Immunomodulating polysaccharides from Lessertia frutescens leaves: isolation, characterization and structure activity relationship.

Ethnopharmacological Relevance: Sutherlandia frutescens (syn. Lessertia frutescens) is an indigenous plant in Southern Africa and has been extensively studied from the ethnobotanical point of view. Amongst the various traditional uses, several illnesses involving the immune system have been reported.

Hot-water extracts from the inner bark of Norway spruce with immunomodulating activities.

The inner bark of Norway spruce (Picea abies) was sequentially extracted with hot water at 100°C, 140°C and 160°C. The hot-water extracts (IB 100°C, IB 140°C and IB 160°C) contained pectic polysaccharides and showed immunostimulating activities. Structural analyses of their carbohydrate content, including glycosidic linkage analyses, revealed the presence of pectins with a large rhamnogalacturonan RG-I domain ramified with highly-branched arabinans.

Polysaccharides with immunomodulating properties from the bark of Parkia biglobosa.

The bark of Parkia biglobosa is used in traditional medicine to cure a wide range of illnesses. Polysaccharides were extracted from the bark with 50% ethanol-water, 50°C and 100°C water, and seven active fractions obtained by anion exchange chromatography and gel filtration. The complement fixation and macrophage stimulating activities of the different fractions were determined.

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy.

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1.

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn.