Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences

The Fab region of IgG impairs the internalization pathway of FcRn upon Fc engagement

Binding to the neonatal Fc receptor (FcRn) extends serum half-life of IgG, and antagonizing this interaction is a promising therapeutic approach in IgG-mediated autoimmune diseases. Fc-MST-HN, designed for enhanced FcRn binding capacity, has not been evaluated in the context of a full-length antibody, and the structural properties of the attached Fab regions might affect the FcRn-mediated intracellular trafficking pathway. Here we present a comprehensive comparative analysis of the IgG salvage pathway between two full-size IgG1 variants, containing wild type and MST-HN Fc fragments, and their Fc-only counterparts. We find no evidence of Fab-regions affecting FcRn binding in cell-free assays, however, cellular assays show impaired binding of full-size IgG to FcRn, which translates into improved intracellular FcRn occupancy and intracellular accumulation of Fc-MST-HN compared to full size IgG1-MST-HN. The crystal structure of Fc-MST-HN in complex with FcRn provides a plausible explanation why the Fab disrupts the interaction only in the context of membrane-associated FcRn. Importantly, we find that Fc-MST-HN outperforms full-size IgG1-MST-HN in reducing IgG levels in cynomolgus monkeys. Collectively, our findings identify the cellular membrane context as a critical factor in FcRn biology and therapeutic targeting

Anti-hinge antibodies recognize IgG subclass- and protease-restricted neoepitopes

Anti-hinge Abs (AHAs) target neoepitopes exposed after proteolytic cleavage of IgG. In this study, we explored the diversity of protease- and IgG subclass-restricted AHAs and their potential as immunological markers in healthy donors (HDs) and patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). AHA reactivity against IgG-degrading enzyme of Streptococcus pyogenes (IdeS)- or pepsin-generated F(ab′)2 fragments of all four human IgG subclasses was determined. AHA reactivity against one or more out of eight F(ab′)2 targets was found in 68% (68 of 100) of HDs, 69% (68 of 99) of SLE patients, and 81% (79 of 97) of RA patients. Specific recognition of hinge epitopes was dependent on IgG subclass and protease used to create the F(ab′)2 targets, as confirmed by inhibition experiments with F(ab′)2 fragments and hinge peptides. Reactivity against IdeS-generated F(ab′)2 targets was found most frequently, whereas reactivity against pepsin-generated F(ab′)2 targets better discriminated between RA and HDs or SLE, with significantly higher AHA levels against IgG1/3/4. In contrast, AHA levels against pepsin-cleaved IgG2 were comparable. No reactivity against IdeS-generated IgG2-F(ab′)2s was detected. The most discriminatory AHA reactivity in RA was against pepsin-cleaved IgG4, with a 35% prevalence, ≥5.8-fold higher than in HDs/SLE, and significantly higher levels (p < 0.0001). Cross-reactivity for F(ab′)2s generated from different IgG subclasses was only observed for subclasses having homologous F(ab′)2 C termini (IgG1/3/4). For IgG2, two pepsin cleavage sites were identified; anti-hinge reactivity was restricted to only one of these. In conclusion, AHAs specifically recognize IgG subclass- and protease-restricted hinge neoepitopes. Their protease-restricted specificity suggests that different AHA responses developed under distinct inflammatory or infectious conditions and may be markers of, and participants in, such processes

Phagocytosis of platelets opsonized with differently glycosylated anti-HLA hIgG1 by monocyte-derived macrophages

Immune-mediated platelet refractoriness (PR) remains a significant problem in the setting of platelet transfusion and is predominantly caused by the presence of alloantibodies directed against class I human leukocyte antigens (HLA). Opsonization of donor platelets with these alloantibodies can result in rapid clearance after transfusion via multiple mechanisms, including antibody dependent cellular phagocytosis (ADCP). Interestingly, not all alloimmunized patients develop PR to unmatched platelet transfusions, suggesting variation in HLA-specific IgG responses between patients. Previously, we observed that the glycosylation profile of anti-HLA antibodies was highly variable between PR patients, especially with respect to Fc galactosylation, sialylation and fucosylation. In the current study, we investigated the effect of different Fc glycosylation patterns, with known effects on complement deposition and FcγR binding, on phagocytosis of opsonized platelets by monocyte-derived human macrophages. We found that the phagocytosis of antibody- and complement-opsonized platelets, by monocyte derived M1 macrophages, was unaffected by these qualitative IgG-glycan differences

Biological and structural characterization of murine TRALI antibody reveals increased Fc-mediated complement activation

Transfusion-related acute lung injury (TRALI) remains a leading cause of transfusionrelated deaths. In most cases, anti-leukocyte antibodies in the transfusion product trigger TRALI, but not all anti-leukocyte antibodies cause TRALI. It has been shown that the anti-major histocompatibility complex (MHC) class I antibody 34-1-2S (anti-H-2Kd) causes TRALI in BALB/c mice (MHC class I haplotype H-2Kd), whereas SF1.1.10 (anti-H-2Kd) does not. In C57BL/6 mice (MHC class I haplotype H-2Kb), TRALI only occurs when anti-MHC class I antibody AF6-88.5.5.3 (anti-H-2Kb) is administered together with a high dose of 34-1-2S. It remains unknown which specific antibody characteristics are responsible for eliciting TRALI. We therefore investigated several biological and structural features of 34-1-2S compared with other anti-MHC class I antibodies, which on their own do not cause TRALI: SF1.1.10 and AF6-88.5.5.3. No substantial differences were observed between the TRALIcausing 34-1-2S and the TRALI-resistant SF1.1.10 regarding binding affinity to H-2Kd. Regarding binding affinity to H-2Kb, only AF6-88.5.5.3 potently bound to H-2Kb, whereas 34-1-2S exhibited weak but significant cross-reactivity. Furthermore, the binding affinity to FcgRs as well as the Fc glycan composition seemed to be similar for all antibodies. Similar Fc glycosylation profiles were also observed for human TRALI-causing donor anti-HLA antibodies compared with human anti-HLA antibodies from control donors. 34-1-2S, however, displayed superior complement activation capacity, which was fully Fc dependent and not significantly dependent on Fc glycosylation. We conclude that TRALI induction is not correlated with Fab- A nd Fc-binding affinities for antigen and FcgRs, respectively, nor with the composition of Fc glycans; but increased Fc-mediated complement activation is correlated with TRALI induction

Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a large impact on global health, travel, and economy. Therefore, preventative and therapeutic measures are urgently needed. Here, we isolated monoclonal antibodies from three convalescent coronavirus disease 2019 (COVID-19) patients using a SARS-CoV-2 stabilized prefusion spike protein. These antibodies had low levels of somatic hypermutation and showed a strong enrichment in VH1-69, VH3-30-3, and VH1-24 gene usage. A subset of the antibodies was able to potently inhibit authentic SARS-CoV-2 infection at a concentration as low as 0.007 micrograms per milliliter. Competition and electron microscopy studies illustrate that the SARS-CoV-2 spike protein contains multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as non-RBD epitopes. In addition to providing guidance for vaccine design, the antibodies described here are promising candidates for COVID-19 treatment and prevention

Cross-reactivity of mouse IgG subclasses to human Fc gamma receptors: Antibody deglycosylation only eliminates IgG2b binding

Immunoglobulin G (IgG) antibodies are important for protection against pathogens and exert effector functions through binding to IgG-Fc receptors (FcγRs) on myeloid and natural killer cells, resulting in destruction of opsonized target cells. Despite interspecies differences, IgG subclasses and FcγRs show substantial similarities and functional conservation between mammals. Accordingly, binding of human IgG (hIgG) to mouse FcγRs (mFcγRs) has been utilized to study effector functions of hIgG in mice. In other applications, such as immunostaining with mouse IgG monoclonal antibodies (mAbs), these cross-reactivities are undesired and prone to misinterpretation. Despite this drawback, the binding of mouse IgG (mIgG) subclasses to human FcγR (hFcγR) classes has never been fully documented. Here, we report detailed and quantifiable characterization of binding affinities for all mIgG subclasses to hFcγRs, including functional polymorphic variants. mIgG subclasses show the strongest binding to hFcγRIa, with relative affinities mIgG2a = mIgG2c > mIgG3 >> mIgG2b, and no binding by mIgG1. hFcγRIIa/b showed general low reactivities to all mIgG (mIgG1> mIgG2a/c > mIgG2b), with no reactivity to mIgG3. A particularly high affinity was observed for mIgG1 to the hFcγRIIa-R131 polymorphic variant. hFcγRIIIa showed lower binding (mIgG2a/c > mIgG3), slightly favouring binding to the hFcγRIIIa-V158 over the F158 polymorphic variant. No binding was observed of mIgG to hFcγRIIIb. Deglycosylation of mIgG1 did not abrogate binding to hFcγRIIa-R131, nor did deglycosylation of mIgG2a/c and mIgG3 prevent hFcγRIa binding. Importantly, deglycosylation of the least cross-reactive mIgG subclass, mIgG2b, abrogated reactivity to all hFcγRs. Together, these data document for the first time the full spectrum of cross-reactivities of mouse IgG to human FcγRs