Effect of posttranslational modifications and subclass on IgG activity: from immunity to immunotherapy.

Humoral immune responses are characterized by complex mixtures of polyclonal antibody species varying in their isotype, target epitope specificity and affinity. Posttranslational modifications occurring during antibody production in both the antibody variable and constant domain create further complexity and can modulate antigen specificity and antibody Fc-dependent effector functions, respectively. Finally, modifications of the antibody backbone after secretion may further impact antibody activity. An in-depth understanding of how these posttranslational modifications impact antibody function, especially in the context of individual antibody isotypes and subclasses, is only starting to emerge. Indeed, only a minute proportion of this natural variability in the humoral immune response is currently reflected in therapeutic antibody preparations. In this Review, we summarize recent insights into how IgG subclass and posttranslational modifications impact IgG activity and discuss how these insights may be used to optimize therapeutic antibody development.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

Immune Monitoring of Trans-endothelial Transport by Kidney-Resident Macrophages.

Small immune complexes cause type III hypersensitivity reactions that frequently result in tissue injury. The responsible mechanisms, however, remain unclear and differ depending on target organs. Here, we identify a kidney-specific anatomical and functional unit, formed by resident macrophages and peritubular capillary endothelial cells, which monitors the transport of proteins and particles ranging from 20 to 700 kDa or 10 to 200 nm into the kidney interstitium. Kidney-resident macrophages detect and scavenge circulating immune complexes "pumped" into the interstitium via trans-endothelial transport and trigger a FcγRIV-dependent inflammatory response and the recruitment of monocytes and neutrophils. In addition, FcγRIV and TLR pathways synergistically "super-activate" kidney macrophages when immune complexes contain a nucleic acid. These data identify a physiological function of tissue-resident kidney macrophages and a basic mechanism by which they initiate the inflammatory response to small immune complexes in the kidney.

IgG Fc domains that bind C1q but not effector Fcγ receptors delineate the importance of complement-mediated effector functions.

Engineered crystallizable fragment (Fc) regions of antibody domains, which assume a unique and unprecedented asymmetric structure within the homodimeric Fc polypeptide, enable completely selective binding to the complement component C1q and activation of complement via the classical pathway without any concomitant engagement of the Fcγ receptor (FcγR). We used the engineered Fc domains to demonstrate in vitro and in mouse models that for therapeutic antibodies, complement-dependent cell-mediated cytotoxicity (CDCC) and complement-dependent cell-mediated phagocytosis (CDCP) by immunological effector molecules mediated the clearance of target cells with kinetics and efficacy comparable to those of the FcγR-dependent effector functions that are much better studied, while they circumvented certain adverse reactions associated with FcγR engagement. Collectively, our data highlight the importance of CDCC and CDCP in monoclonal-antibody function and provide an experimental approach for delineating the effect of complement-dependent effector-cell engagement in various therapeutic settings.

Regulation of autoantibody activity by the IL-23-TH17 axis determines the onset of autoimmune disease.

The checkpoints and mechanisms that contribute to autoantibody-driven disease are as yet incompletely understood. Here we identified the axis of interleukin 23 (IL-23) and the TH17 subset of helper T cells as a decisive factor that controlled the intrinsic inflammatory activity of autoantibodies and triggered the clinical onset of autoimmune arthritis. By instructing B cells in an IL-22- and IL-21-dependent manner, TH17 cells regulated the expression of ß-galactoside α2,6-sialyltransferase 1 in newly differentiating antibody-producing cells and determined the glycosylation profile and activity of immunoglobulin G (IgG) produced by the plasma cells that subsequently emerged. Asymptomatic humans with rheumatoid arthritis (RA)-specific autoantibodies showed identical changes in the activity and glycosylation of autoreactive IgG antibodies before shifting to the inflammatory phase of RA; thus, our results identify an IL-23-TH17 cell-dependent pathway that controls autoantibody activity and unmasks a preexisting breach in immunotolerance.

Keeping T-he Killers at Bay via FcγRIIb.

Understanding how cytotoxic T cells are regulated is key for enhancing or suppressing their activity in tumor or transplantation settings. A study by Morris et al. (2020) in this issue of Immunity shows that crosslinking of inhibitory Fc receptor FcγRIIb by the suppressive cytokine Fgl2 limits cytotoxic CD8+ T cell responses by inducing T cell apoptosis.

Dissection of complement and Fc-receptor-mediated pathomechanisms of autoantibodies to myelin oligodendrocyte glycoprotein.

Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) have recently been established to define a new disease entity, MOG-antibody-associated disease (MOGAD), which is clinically overlapping with multiple sclerosis. MOG-specific antibodies (Abs) from patients are pathogenic, but the precise effector mechanisms are currently still unknown and no therapy is approved for MOGAD. Here, we determined the contributions of complement and Fc-receptor (FcR)-mediated effects in the pathogenicity of MOG-Abs. Starting from a recombinant anti-MOG (mAb) with human IgG1 Fc, we established MOG-specific mutant mAbs with differential FcR and C1q binding. We then applied selected mutants of this MOG-mAb in two animal models of experimental autoimmune encephalomyelitis. First, we found MOG-mAb-induced demyelination was mediated by both complement and FcRs about equally. Second, we found that MOG-Abs enhanced activation of cognate MOG-specific T cells in the central nervous system (CNS), which was dependent on FcR-, but not C1q-binding. The identification of complement-dependent and -independent pathomechanisms of MOG-Abs has implications for therapeutic strategies in MOGAD.

Functional Fc receptors are crucial in antibody-mediated protection against cytomegalovirus.

Human cytomegalovirus is a medically important pathogen. Previously, using murine CMV (MCMV), we provided evidence that both neutralizing and nonneutralizing antibodies can confer protection from viral infection in vivo. In this study, we report that serum derived from infected animals had a greater protective capacity in MCMV-infected RAG-/- mice than serum from animals immunized with purified virus. The protective activity of immune serum was strictly dependent on functional Fcγ receptors (FcγR). Deletion of individual FcγRs or combined deletion of FcγRI and FcγRIV had little impact on the protection afforded by serum. Adoptive transfer of CD115-positive cells from noninfected donors demonstrated that monocytes represent important cellular mediators of the protective activity provided by immune serum. Our studies suggest that Fc-FcγR interactions and monocytic cells are critical for antibody-mediated protection against MCMV infection in vivo. These findings may provide new avenues for the development of novel strategies for more effective CMV vaccines or antiviral immunotherapies.

Suppression of Fcγ-receptor-mediated antibody effector function during persistent viral infection.

Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control.

Toward Understanding How Staphylococcal Protein A Inhibits IgG-Mediated Phagocytosis.

IgG molecules are crucial for the human immune response against bacterial infections. IgGs can trigger phagocytosis by innate immune cells, like neutrophils. To do so, IgGs should bind to the bacterial surface via their variable Fab regions and interact with Fcγ receptors and complement C1 via the constant Fc domain. C1 binding to IgG-labeled bacteria activates the complement cascade, which results in bacterial decoration with C3-derived molecules that are recognized by complement receptors on neutrophils. Next to FcγRs and complement receptors on the membrane, neutrophils also express the intracellular neonatal Fc receptor (FcRn). We previously reported that staphylococcal protein A (SpA), a key immune-evasion protein of Staphylococcus aureus, potently blocks IgG-mediated complement activation and killing of S. aureus by interfering with IgG hexamer formation. SpA is also known to block IgG-mediated phagocytosis in absence of complement, but the mechanism behind it remains unclear. In this study, we demonstrate that SpA blocks IgG-mediated phagocytosis and killing of S. aureus and that it inhibits the interaction of IgGs with FcγRs (FcγRIIa and FcγRIIIb, but not FcγRI) and FcRn. Furthermore, our data show that multiple SpA domains are needed to effectively block IgG1-mediated phagocytosis. This provides a rationale for the fact that SpA from S. aureus contains four to five repeats. Taken together, our study elucidates the molecular mechanism by which SpA blocks IgG-mediated phagocytosis and supports the idea that in addition to FcγRs, the intracellular FcRn is also prevented from binding IgG by SpA.

Cell targeting and immunostimulatory properties of a novel Fcγ-receptor-independent agonistic anti-CD40 antibody in rhesus macaques.

Targeting CD40 by agonistic antibodies used as vaccine adjuvants or for cancer immunotherapy is a strategy to stimulate immune responses. The majority of studied agonistic anti-human CD40 antibodies require crosslinking of their Fc region to inhibitory FcγRIIb to induce immune stimulation although this has been associated with toxicity in previous studies. Here we introduce an agonistic anti-human CD40 monoclonal IgG1 antibody (MAB273) unique in its specificity to the CD40L binding site of CD40 but devoid of Fcγ-receptor binding. We demonstrate rapid binding of MAB273 to B cells and dendritic cells resulting in activation in vitro on human cells and in vivo in rhesus macaques. Dissemination of fluorescently labeled MAB273 after subcutaneous administration was found predominantly at the site of injection and specific draining lymph nodes. Phenotypic cell differentiation and upregulation of genes associated with immune activation were found in the targeted tissues. Antigen-specific T cell responses were enhanced by MAB273 when given in a prime-boost regimen and for boosting low preexisting responses. MAB273 may therefore be a promising immunostimulatory adjuvant that warrants future testing for therapeutic and prophylactic vaccination strategies.

Immunomodulation with romiplostim as a second-line strategy in primary immune thrombocytopenia: The iROM study.

Thrombopoietin receptor agonists (TPO-RAs) stimulate platelet production, which might restore immunological tolerance in primary immune thrombocytopenia (ITP). The iROM study investigated romiplostim's immunomodulatory effects. Thirteen patients (median age, 31 years) who previously received first-line treatment received romiplostim for 22 weeks, followed by monitoring until week 52. In addition to immunological data, secondary end-points included the sustained remission off-treatment (SROT) rate at 1 year, romiplostim dose, platelet count and bleedings. Scheduled discontinuation of romiplostim and SROT were achieved in six patients with newly diagnosed ITP, whereas the remaining seven patients relapsed. Romiplostim dose titration was lower and platelet count response was stronger in patients with SROT than in relapsed patients. In all patients, regulatory T lymphocyte (Treg) counts increased until study completion and the counts were higher in patients with SROT. Interleukin (IL)-4, IL-9 and IL-17F levels decreased significantly in all patients. FOXP3 (Treg), GATA3 (Th2) mRNA expression and transforming growth factor-ß levels increased in patients with SROT. Treatment with romiplostim modulates the immune system and possibly influences ITP prognosis. A rapid increase in platelet counts is likely important for inducing immune tolerance. Better outcomes might be achieved at an early stage of autoimmunity, but clinical studies are needed for confirmation.

B-cell modulation with anti-CD79b antibodies ameliorates experimental autoimmune encephalitis in mice.

B cells play a major role in the pathogenesis of many autoimmune diseases like MS, rheumatoid arthritis, or systemic lupus erythematosus. Depletion of B cells with anti-CD20 antibodies is an established therapy for MS. However, total B-cell depletion will also affect regulatory B cells that are known to suppress autoimmune responses. In our studies, we describe an alternative approach based on targeting CD79b that induces only partial B-cell depletion and achieves therapeutic effects by B-cell modulation. Prophylactic and therapeutic treatment with an antibody against CD79b and also a deglycosylated variant of this antibody, lacking effector function like antibody-dependent cellular cytotoxicity or complement activation, significantly reduced the development and progression of EAE in mice. Our data show that modulation of B cells via CD79b is equally effective as almost complete B-cell depletion with anti-CD20 antibodies and may constitute an alternative approach to treat MS.

Impaired B Cell Expression of the Inhibitory Fcγ Receptor IIB in Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease in which pathogenic immunoglobulin G antibodies bind to acetylcholine receptors (or to functionally related molecules at the neuromuscular junction). B cell expression of the inhibitory immunoglobulin G receptor, Fc-gamma receptor (FcγR) IIB, maintains peripheral immune tolerance, and its absence renders B cells hyperresponsive to autoantigen. Here, we report that FcγRIIB expression levels are substantially reduced in B lineage cells derived from immunotherapy-naïve patients with acetylcholine receptor antibody-positive early-onset MG. In contrast, genetic variants associated with impaired FcγRIIB expression are not enriched in MG, indicating post-transcriptional dysregulation. FcγR-targeted therapies could have therapeutic benefits in MG. ANN NEUROL 2022;92:1046-1051.

Next-generation antibody-based therapies in neurology.

Antibody-based therapeutics are now standard in the treatment of neuroinflammatory diseases, and the spectrum of neurological diseases targeted by those approaches continues to grow. The efficacy of antibody-based drug platforms is largely determined by the specificity-conferring antigen-binding fragment (Fab) and the crystallizable fragment (Fc) driving antibody function. The latter provides specific instructions to the immune system by interacting with cellular Fc receptors and complement components. Extensive engineering efforts have enabled tuning of Fc functions to modulate effector functions and to prolong or reduce antibody serum half-lives. Technologies that improve bioavailability of antibody-based treatment platforms within the CNS parenchyma are being developed and could invigorate drug discovery for a number of brain diseases for which current therapeutic options are limited. These powerful approaches are currently being tested in clinical trials or have been successfully translated into the clinic. Here, we review recent developments in the design and implementation of antibody-based treatment modalities in neurological diseases.

Response to Oswald et al.: ST6Gal1 in plasma is dispensable for IgG sialylation.

The presence or absence of terminal sialic acid residues in the sugar moiety attached to the Fc-domain of IgG molecules modulates IgG activity and is associated with autoimmune or infection related inflammation. In a recent paper, Oswald and colleagues suggest that IgG sialylation may occur post IgG secretion from plasma cells, which would be a major issue for therapeutic antibodies injected into patients. In contrast, we argue that previous work rather demonstrates that IgG sialylation occurs within B cells and that the experimental system used by the authors is not suitable to address this critical question.

Siglec-15 on Osteoclasts Is Crucial for Bone Erosion in Serum-Transfer Arthritis.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin, which is expressed on osteoclasts. Deficiency of Siglec-15 leads to an impaired osteoclast development, resulting in a mild osteopetrotic phenotype. The role of Siglec-15 in arthritis is still largely unclear. To address this, we generated Siglec-15 knockout mice and analyzed them in a mouse arthritis model. We could show that Siglec-15 is directly involved in pathologic bone erosion in the K/BxN serum-transfer arthritis model. Histological analyses of joint destruction provided evidence for a significant reduction in bone erosion area and osteoclast numbers in Siglec-15-/- mice, whereas the inflammation area and cartilage destruction was comparable to wild-type mice. Thus, Siglec-15 on osteoclasts has a crucial function for bone erosion during arthritis. In addition, we generated a new monoclonal anti-Siglec-15 Ab to clarify its expression pattern on immune cells. Whereas this Ab demonstrated an almost exclusive Siglec-15 expression on murine osteoclasts and hardly any other expression on various other immune cell types, human Siglec-15 was more broadly expressed on human myeloid cells, including human osteoclasts. Taken together, our findings show a role of Siglec-15 as a regulator of pathologic bone resorption in arthritis and highlight its potential as a target for future therapies, as Siglec-15 blocking Abs are available.