Complement activation drives antibody-mediated transfusion-related acute lung injury via macrophage trafficking and formation of NETs.

ABSTRACT: Transfusion-related acute lung injury (TRALI) is one of the leading causes of transfusion-related fatalities and, to date, is without available therapies. Here, we investigated the role of the complement system in TRALI. Murine anti-major histocompatibility complex class I antibodies were used in TRALI mouse models, in combination with analyses of plasma samples from patients with TRALI. We found that in vitro complement activation was related to in vivo antibody-mediated TRALI induction, which was correlated with increased macrophage trafficking from the lungs to the blood in a fragment crystallizable region (Fc)-dependent manner and that this was dependent on C5. Human immunoglobulin G 1 variants of the murine TRALI-inducing antibody 34-1-2S, either unable to activate complement and/or bind to Fcγ receptors (FcγRs), revealed an essential role for the complement system, but not for FcγRs, in the onset of 34-1-2S-mediated TRALI in mice. In addition, we found high levels of complement activation in the plasma of patients with TRALI (n = 53), which correlated with elevated neutrophil extracellular trap (NET) markers. In vitro we found that NETs could be formed in a murine, 2-hit model, mimicking TRALI with lipopolysaccharide and C5a stimulation. Collectively, this reveals a critical role of Fc-mediated complement activation in TRALI, with a direct relation to macrophage trafficking from the lungs to the blood and an association with NET formation, suggesting that targeting the complement system may be an attractive therapeutic approach for combating TRALI.

IgG Fab Glycans Hinder FcRn-Mediated Placental Transport.

Abs can be glycosylated in both their Fc and Fab regions with marked effects on Ab function and binding. High levels of IgG Fab glycosylation are associated with malignant and autoimmune conditions, exemplified by rheumatoid arthritis and highly Fab-glycosylated (∼90%) anti-citrullinated protein Abs (ACPAs). Important properties of IgG, such as long half-life and placental transport, are facilitated by the human neonatal Fc receptor (hFcRn). Although it is known that glycosylation of Abs can affect binding to Fc receptors, little is known on the impact of IgG Fab glycosylation on hFcRn binding and transplacental transport. Therefore, we analyzed the interaction between hFcRn and IgG with and without Fab glycans in vitro with various methods as well as in vivo by studying placental transfer of Fab-glycosylated Abs from mothers to newborns. No effect of Fab glycosylation on IgG binding to hFcRn was found by surface plasmon resonance and hFcRn affinity chromatography. In contrast, studies in a cell membrane context revealed that Fab glycans negatively impacted IgG-hFcRn interaction. In line with this, we found that Fab-glycosylated IgGs were transported ∼20% less efficiently across the placenta. This appeared to be a general phenomenon, observed for ACPAs, non-ACPAs, as well as total IgG in rheumatoid arthritis patients and healthy controls. Our results suggest that, in a cellular context, Fab glycans inhibit IgG-hFcRn interaction and thus negatively affect the transplacental transfer of IgG. As Fab-glycosylated Abs are frequently associated with autoimmune and malignant disorders and may be potentially harmful, this might encompass a regulatory mechanism, limiting the half-life and transport of such Abs.

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.

Afucosylated IgG Targets FcγRIV for Enhanced Tumor Therapy in Mice.

Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (FcγR) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine FcγRIV, the mouse orthologue of human FcγRIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in FcγRIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies.

C-Reactive Protein Enhances IgG-Mediated Cellular Destruction Through IgG-Fc Receptors in vitro.

Antibody-mediated blood disorders ensue after auto- or alloimmunization against blood cell antigens, resulting in cytopenia. Although the mechanisms of cell destruction are the same as in immunotherapies targeting tumor cells, many factors are still unknown. Antibody titers, for example, often do not strictly correlate with clinical outcome. Previously, we found C-reactive protein (CRP) levels to be elevated in thrombocytopenic patients, correlating with thrombocyte counts, and bleeding severity. Functionally, CRP amplified antibody-mediated phagocytosis of thrombocytes by phagocytes. To investigate whether CRP is a general enhancer of IgG-mediated target cell destruction, we extensively studied the effect of CRP on in vitro IgG-Fc receptor (FcγR)-mediated cell destruction: through respiratory burst, phagocytosis, and cellular cytotoxicity by a variety of effector cells. We now demonstrate that CRP also enhances IgG-mediated effector functions toward opsonized erythrocytes, in particular by activated neutrophils. We performed a first-of-a-kind profiling of CRP binding to all human FcγRs and IgA-Fc receptor I (FcαRI) using a surface plasmon resonance array. CRP bound these receptors with relative affinities of FcγRIa = FcγRIIa/b = FcγRIIIa > FcγRIIIb = FcαRI. Furthermore, FcγR blocking (in particular FcγRIa) abrogated CRP's ability to amplify IgG-mediated neutrophil effector functions toward opsonized erythrocytes. Finally, we observed that CRP also amplified killing of breast-cancer tumor cell line SKBR3 by neutrophils through anti-Her2 (trastuzumab). Altogether, we provide for the first time evidence for the involvement of specific CRP-FcγR interactions in the exacerbation of in vitro IgG-mediated cellular destruction; a trait that should be further evaluated as potential therapeutic target e.g., for tumor eradication.

Afucosylated IgG characterizes enveloped viral responses and correlates with COVID-19 severity.

Immunoglobulin G (IgG) antibodies are crucial for protection against invading pathogens. A highly conserved N-linked glycan within the IgG-Fc tail, which is essential for IgG function, shows variable composition in humans. Afucosylated IgG variants are already used in anticancer therapeutic antibodies for their increased activity through Fc receptors (FcγRIIIa). Here, we report that afucosylated IgG (approximately 6% of total IgG in humans) are specifically formed against enveloped viruses but generally not against other antigens. This mediates stronger FcγRIIIa responses but also amplifies brewing cytokine storms and immune-mediated pathologies. Critically ill COVID-19 patients, but not those with mild symptoms, had high concentrations of afucosylated IgG antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), amplifying proinflammatory cytokine release and acute phase responses. Thus, antibody glycosylation plays a critical role in immune responses to enveloped viruses, including COVID-19.

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.

FcγR Binding and ADCC Activity of Human IgG Allotypes.

Antibody dependent cellular cytotoxicity (ADCC) is an Fc-dependent effector function of IgG important for anti-viral immunity and anti-tumor therapies. NK-cell mediated ADCC is mainly triggered by IgG-subclasses IgG1 and IgG3 through the IgG-Fc-receptor (FcγR) IIIa. Polymorphisms in the immunoglobulin gamma heavy chain gene likely form a layer of variation in the strength of the ADCC-response, but this has never been studied in detail. We produced all 27 known IgG allotypes and assessed FcγRIIIa binding and ADCC activity. While all IgG1, IgG2, and IgG4 allotypes behaved similarly within subclass, large allotype-specific variation was found for IgG3. ADCC capacity was affected by residues 291, 292, and 296 in the CH2 domain through altered affinity or avidity for FcγRIIIa. Furthermore, allotypic variation in hinge length affected ADCC, likely through altered proximity at the immunological synapse. Thus, these functional differences between IgG allotypes have important implications for therapeutic applications and susceptibility to infectious-, allo- or auto-immune diseases.

Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering.

Hybridoma technology is instrumental for the development of novel antibody therapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotype for therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibody function in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domains to obtain recombinant hybridomas, which secrete antibodies in the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab' fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibody products are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibody products via chemoenzymatic modification. We believe that this versatile platform facilitates antibody engineering for the entire scientific community, empowering preclinical antibody research.

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 VH-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn. We observed that FcγR binding was not required for transport and that FcRn transported less IgG2 than IgG1. Transport of IgG1 with a shortened lower hinge (ΔGly236, absent in germline IgG2), was reduced to levels equivalent to IgG2. Conversely, transport of IgG2 + Gly236 was increased to IgG1 levels. Gly236 is not a contact residue between IgG and FcRn, suggesting that its absence leads to an altered conformation of IgG, possibly due to a less flexible Fab, positioned closer to the Fc portion. This may sterically hinder FcRn binding and transport. We conclude that the lack of Gly236 is sufficient to explain the reduced FcRn-mediated IgG2 transcytosis and accounts for the low maternal/fetal IgG2 ratio at term.

FcγR interaction is not required for effective anti-PD-L1 immunotherapy but can add additional benefit depending on the tumor model.

Immunomodulatory antibodies blocking interactions of coinhibitory receptors to their ligands such as CTLA-4, PD1 and PD-L1 on immune cells have shown impressive therapeutic efficacy in clinical studies. The therapeutic effect of these antibodies is mainly mediated by reactivating antitumor T cell immune responses. Detailed analysis of anti-CTLA4 antibody therapy revealed that an optimal therapeutic efficacy also requires binding to Fc receptors for IgG, FcγR, mediating depletion of intratumoral regulatory T cells. Here, we investigated the role of Fc binding in anti-PD-L1 antibody therapy in the MC38 C57BL/6 and CT26 BALB/c colon adenocarcinoma tumor models. In the MC38 tumor model, all IgG subclasses anti-PD-L1 showed similar therapeutic efficacy when compared to each other in either wild-type mice or in mice deficient for all FcγR. In contrast, in the CT26 tumor model, anti-PD-L1 mIgG2a, the IgG subclass with the highest affinity for activating FcγR, showed stronger therapeutic efficacy than other IgG subclasses. This was associated with a reduction of a myeloid cell subset with high expression of PD-L1 in the tumor microenvironment. This subclass preference for mIgG2a was lost in C57BL/6 × BALB/c F1 mice, indicating that the genetic background of the host may determine the additional clinical benefit of the high affinity antibody subclasses. Based on these data, we conclude that FcγR are not crucial for anti-PD-L1 antibody therapy but might play a role in some tumor models.

Decoding the Human Immunoglobulin G-Glycan Repertoire Reveals a Spectrum of Fc-Receptor- and Complement-Mediated-Effector Activities.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

Identification of sequence variants influencing immunoglobulin levels.

Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10-55, ß = -0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10-8, ß = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.

Enhanced Effector Functions Due to Antibody Defucosylation Depend on the Effector Cell Fcγ Receptor Profile.

Abs of the IgG isotype are glycosylated in their Fc domain at a conserved asparagine at position 297. Removal of the core fucose of this glycan greatly increases the affinity for FcγRIII, resulting in enhanced FcγRIII-mediated effector functions. Normal plasma IgG contains ∼94% fucosylated Abs, but alloantibodies against, for example, Rhesus D (RhD) and platelet Ags frequently have reduced fucosylation that enhances their pathogenicity. The increased FcγRIII-mediated effector functions have been put to use in various afucosylated therapeutic Abs in anticancer treatment. To test the functional consequences of Ab fucosylation, we produced V-gene-matched recombinant anti-RhD IgG Abs of the four different subclasses (IgG1-4) with and without core fucose (i.e., 20% fucose remaining). Binding to all human FcγR types and their functional isoforms was assessed with surface plasmon resonance. All hypofucosylated anti-RhD IgGs of all IgG subclasses indeed showed enhanced binding affinity for isolated FcγRIII isoforms, without affecting binding affinity to other FcγRs. In contrast, when testing hypofucosylated anti-RhD Abs with FcγRIIIa-expressing NK cells, a 12- and 7-fold increased erythrocyte lysis was observed with the IgG1 and IgG3, respectively, but no increase with IgG2 and IgG4 anti-RhD Abs. Notably, none of the hypofucosylated IgGs enhanced effector function of macrophages, which, in contrast to NK cells, express a complex set of FcγRs, including FcγRIIIa. Our data suggest that the beneficial effects of afucosylated biologicals for clinical use can be particularly anticipated when there is a substantial involvement of FcγRIIIa-expressing cells, such as NK cells.