Reduced FcRn-mediated transcytosis of IgG2 due to a missing Glycine in its lower hinge.

Neonatal Fc-receptor (FcRn), the major histocompatibility complex (MHC) class I-like Fc-receptor, transports immunoglobuline G (IgG) across cell layers, extending IgG half-life in circulation and providing newborns with humoral immunity. IgG1 and IgG2 have similar half-lives, yet IgG2 displays lower foetal than maternal concentration at term, despite all known FcRn binding residues being preserved between IgG1 and IgG2. We investigated FcRn mediated transcytosis of V-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn.

Human IgG lacking effector functions demonstrate lower FcRn-binding and reduced transplacental transport.

We have previously generated human IgG1 antibodies that were engineered for reduced binding to the classical Fcγ receptors (FcγRI-III) and C1q, thereby eliminating their destructive effector functions (constant region G1Δnab). In their potential use as blocking agents, favorable binding to the neonatal Fc receptor (FcRn) is important to preserve the long half-life typical of IgG. An ability to cross the placenta, which is also mediated, at least in part, by FcRn is desirable in some indications, such as feto-maternal alloimmune disorders.

Igg Subclasses Targeting the Flagella of Serovar Typhimurium Can Mediate Phagocytosis and Bacterial Killing.

Invasive non-typhoidal are a common cause of invasive disease in immuno-compromised individuals and in children. Multi-drug resistance poses challenges to disease control, with a critical need for effective vaccines. Flagellin is an attractive vaccine candidate due to surface exposure and high epitope copy number, but its potential as a target for opsonophacytic antibodies is unclear.

Human IgG1 antibodies suppress angiogenesis in a target-independent manner.

Aberrant angiogenesis is implicated in diseases affecting nearly 10% of the world's population. The most widely used anti-angiogenic drug is bevacizumab, a humanized IgG1 monoclonal antibody that targets human VEGFA. Although bevacizumab does not recognize mouse Vegfa, it inhibits angiogenesis in mice.

Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines.

An investigation into IgE-facilitated allergen recognition and presentation by human dendritic cells.

Background: Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcεRI and FcεRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs.

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets.

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture.

Fc gamma receptor 3A polymorphism and risk for HIV-associated cryptococcal disease.

Unlabelled: Cryptococcus neoformans is one of the most common causes of fungal disease in HIV-infected persons, but not all of those who are infected develop cryptococcal disease (CD). Although CD4(+) T cell deficiency is a risk factor for HIV-associated CD, polymorphisms of phagocytic Fc gamma receptors (FCGRs) have been linked to CD risk in HIV-uninfected persons. To investigate associations between FCGR2A 131 H/R and FCGR3A 158 F/V polymorphisms and CD risk in HIV-infected persons, we performed PCR-based genotyping on banked samples from 164 men enrolled in the Multicenter AIDS Cohort Study (MACS): 55 who were HIV infected and developed CD and a matched control group of 54 who were HIV infected and 55 who were HIV uninfected.

Recombinant HPA-1a antibody therapy for treatment of fetomaternal alloimmune thrombocytopenia: proof of principle in human volunteers.

Fetomaternal alloimmune thrombocytopenia, caused by the maternal generation of antibodies against fetal human platelet antigen-1a (HPA-1a), can result in intracranial hemorrhage and intrauterine death. We have developed a therapeutic human recombinant high-affinity HPA-1a antibody (B2G1Δnab) that competes for binding to the HPA-1a epitope but carries a modified constant region that does not bind to Fcγ receptors. In vitro studies with a range of clinical anti-HPA-1a sera have shown that B2G1Δnab blocks monocyte chemiluminescence by >75%.

Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells.

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes.

Expression of human FcgammaRIIIa as a GPI-linked molecule on CHO cells to enable measurement of human IgG binding.

The efficacy of a therapeutic IgG molecule may be as dependent on the optimisation of the constant region to suit its intended indication as on the selection of its variable regions. A crucial effector function to be maximised or minimised is antibody-dependent cell-mediated cytotoxicity by natural killer cells. Traditional assays of ADCC activity suffer from considerable inter-donor and intra-donor variability, which makes the measurement of antibody binding to human FcgammaRIIIa, the key receptor for ADCC, an attractive alternative method of assessment.

Developing recombinant HPA-1a-specific antibodies with abrogated Fcgamma receptor binding for the treatment of fetomaternal alloimmune thrombocytopenia.

Fetomaternal alloimmune thrombocytopenia (FMAIT) is caused by maternal generation of antibodies specific for paternal platelet antigens and can lead to fetal intracranial hemorrhage. A SNP in the gene encoding integrin beta3 causes a clinically important maternal-paternal antigenic difference; Leu33 generates the human platelet antigen 1a (HPA-1a), whereas Pro33 generates HPA-1b. As a potential treatment to prevent fetal intracranial hemorrhage in HPA-1a alloimmunized pregnancies, we generated an antibody that blocks the binding of maternal HPA-1a-specific antibodies to fetal HPA-1a1b platelets by combining a high-affinity human HPA-1a-specific scFv (B2) with an IgG1 constant region modified to minimize Fcgamma receptor-dependent platelet destruction (G1Deltanab).

Intravascular survival of red cells coated with a mutated human anti-D antibody engineered to lack destructive activity.

Alloimmune feto-maternal destruction of blood cells is thought to be mediated by binding of alloantibodies to Fc receptors on effector cells. Blocking the antigen using inert antibodies might prolong cell survival. We have performed a "proof of principle" study in volunteers to measure the intravascular survival of autologous red cells coated with human recombinant IgG antibody containing a novel constant region, G1Deltanab, devoid of in vitro cytotoxic activity.

A chimeric antibody to varicella-zoster virus glycoprotein e.

Varicella-Zoster virus (VZV) immune globulin (VZIG) derived from pooled human serum is currently used in immunotherapy of VZV-associated complications of chickenpox and shingles. We developed a mouse-human chimeric antibody against a VZV glycoprotein E (gE) epitope as a safer replacement for VZIG. Variable (V) heavy- and V kappa light-chain exons, derived from an anti-VZV gE antibody secreting mouse hybridoma cell line, were cloned into expression vectors containing an immunoglobulin promoter and enhancer, and human IgG1 or kappa constant (C) region genes.

The effect of recombinant IgG antibodies against the leucine-33 form of the platelet beta3 integrin (HPA-1a) on platelet function.

Recombinant HPA-1a antibodies with Fc, mutated to remove destructive effector functions, have been developed as a potential therapy for fetomaternal alloimmune thrombocytopenia (FMAIT), via blockade of binding of human HPA-1a polyclonal antibodies to fetal HPA-1a1b platelets. We have assessed the effect of the IgG1 HPA-1a antibody B2G1 and two mutated derivatives in various functional assays in resting and agonist-stimulated platelets of the three HPA-1 genotypes. With HPA-1a1b platelets (fetal genotype), the antibodies did not activate signalling or CD62P expression in resting platelets, did not change in vitro bleeding time (IVBT), and did not inhibit platelet adhesion to collagen in flowing blood.

Differential binding to human FcgammaRIIa and FcgammaRIIb receptors by human IgG wildtype and mutant antibodies.

We are investigating the interactions of recombinant human IgG antibodies with Fc receptors to enable selection of a constant region giving minimal depletion of antigen-bearing cells. Eight variant constant regions were made by substituting motifs between human IgG subclasses in the lower hinge region and/or a specially close loop of the CH2 domain. Mutations in the lower hinge region were shown to eliminate FcgammaRI binding and monocyte activation [Eur.