Augmented antibody-based anticancer therapeutics boost neutrophil cytotoxicity.

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.

Glycan-Modified Apoptotic Melanoma-Derived Extracellular Vesicles as Antigen Source for Anti-Tumor Vaccination

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

Fc gamma receptor IIa suppresses type I and III interferon production by human myeloid immune cells.

Type I and type III interferons (IFNs) are fundamental for antiviral immunity, but prolonged expression is also detrimental to the host. Therefore, upon viral infection high levels of type I and III IFNs are followed by a strong and rapid decline. However, the mechanisms responsible for this suppression are still largely unknown. Here, we show that IgG opsonization of model viruses influenza and respiratory syncytial virus (RSV) strongly and selectively suppressed type I and III IFN production by various human antigen-presenting cells. This suppression was induced by selective inhibition of TLR, RIG-I-like receptor, and STING-dependent type I and III IFN gene transcription. Surprisingly, type I and III IFN suppression was mediated by Syk and PI3K independent inhibitory signaling via FcγRIIa, thereby identifying a novel non-canonical FcγRIIa pathway in myeloid cells. Together, these results indicate that IgG opsonization of viruses functions as a novel negative feedback mechanism in humans, which may play a role in the selective suppression of type I and III IFN responses during the late-phase of viral infections. In addition, activation of this pathway may be used as a tool to limit type I IFN-associated pathology.

FcγRIIIb Restricts Antibody-Dependent Destruction of Cancer Cells by Human Neutrophils.

The function of the low-affinity IgG-receptor FcγRIIIb (CD16b), which is uniquely and abundantly expressed on human granulocytes, is not clear. Unlike the other Fcγ receptors (FcγR), it is a glycophosphatidyl inositol (GPI) -anchored molecule and does not have intracellular signaling motifs. Nevertheless, FcγRIIIb can cooperate with other FcγR to promote phagocytosis of antibody-opsonized microbes by human neutrophils. Here we have investigated the role of FcγRIIIb during antibody-dependent cellular cytotoxicity (ADCC) by neutrophils toward solid cancer cells coated with either trastuzumab (anti-HER2) or cetuximab (anti-EGFR). Inhibiting FcγRIIIb using CD16-F(ab')2 blocking antibodies resulted in substantially enhanced ADCC. ADCC was completely dependent on FcγRIIa (CD32a) and the enhanced ADCC seen after FcγRIIIb blockade therefore suggested that FcγRIIIb was competing with FcγRIIa for IgG on the opsonized target cells. Interestingly, the function of neutrophil FcγRIIIb as a decoy receptor was further supported by using neutrophils from individuals with different gene copy numbers of FCGR3B causing different levels of surface FcγRIIIb expression. Individuals with one copy of FCGR3B showed higher levels of ADCC compared to those with two or more copies. Finally, we show that therapeutic antibodies intended to improve FcγRIIIa (CD16a)-dependent natural killer (NK) cell ADCC due to the lack of fucosylation on the N-linked glycan at position N297 of the IgG1 heavy chain Fc-region, show decreased ADCC as compared to regularly fucosylated antibodies. Together, these data confirm FcγRIIIb as a negative regulator of neutrophil ADCC toward tumor cells and a potential target for enhancing tumor cell destruction by neutrophils.

DCs facilitate B cell responses against microbial DNA via DC-SIGN.

Microbial DNA is highly immunostimulatory and is sensed by endosomal pattern recognition receptors after release from internalized microbes. It is unclear how extracellular DNA released from dead microbes is delivered to endosomal PRRs to induce immune responses. Here we have investigated the ability of DCs to bind and internalize extracellular E.coli DNA as well as synthetic DNA. DCs internalized E.coli and synthetic DNA, which was dependent on the C-type lectin receptor DC-SIGN. Notably, endosomal uptake of DNA by DCs enhanced TLR9-dependent responses of B cells against DNA. Hence, we have identified DC-SIGN as a cell surface receptor for DNA that facilitates immune responses directed against DNA.

New insights in the pathogenesis of immunoglobulin A vasculitis (Henoch-Schönlein purpura).

Immunoglobulin A vasculitis (IgAV), also referred to as Henoch-Schönlein purpura, is the most common form of childhood vasculitis. The pathogenesis of IgAV is still largely unknown. The disease is characterized by IgA1-immune deposits, complement factors and neutrophil infiltration, which is accompanied with vascular inflammation. Incidence of IgAV is twice as high during fall and winter, suggesting an environmental trigger associated to climate. Symptoms can resolve without intervention, but some patients develop glomerulonephritis with features similar to IgA nephropathy that include hematuria, proteinuria and IgA deposition in the glomerulus. Ultimately, this can lead to end-stage renal disease. In IgA nephropathy immune complexes containing galactose-deficient (Gd-)IgA1 are found and thought to play a role in pathogenesis. Although Gd-IgA1 complexes are also present in patients with IgAV with nephritis, their role in IgAV is disputed. Alternatively, it has been proposed that in IgAV IgA1 antibodies are generated against endothelial cells. We anticipate that such IgA complexes can activate neutrophils via the IgA Fc receptor FcαRI (CD89), thereby inducing neutrophil migration and activation, which ultimately causes tissue damage in IgAV. In this Review, we discuss the putative role of IgA, IgA receptors, neutrophils and other factors such as infections, genetics and the complement system in the pathogenesis of IgA vasculitis.

Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA-induced human neutrophil activation and migration.

The cross-linking of the IgA Fc receptor (FcαRI) by IgA induces release of the chemoattractant LTB4, thereby recruiting neutrophils in a positive feedback loop. IgA autoantibodies of patients with autoimmune blistering skin diseases therefore induce massive recruitment of neutrophils, resulting in severe tissue damage. To interfere with neutrophil mobilization and reduce disease morbidity, we developed a panel of specific peptides mimicking either IgA or FcαRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and FcαRI peptides reduced phagocytosis of IgA-coated beads, as well as IgA-induced ROS production and neutrophil migration in in vitro and ex vivo (human skin) experiments. Since topical application would be the preferential route of administration, Cetomacrogol cream containing an IgA CLIPS peptide was developed. In the presence of a skin permeation enhancer, peptides in this cream were shown to penetrate the skin, while not diffusing systemically. Finally, epitope mapping was used to discover sequences important for binding between IgA and FcαRI. In conclusion, a cream containing IgA or FcαRI peptide mimetics, which block IgA-induced neutrophil activation and migration in the skin may have therapeutic potential for patients with IgA-mediated blistering skin diseases.

Immunoglobulin A: magic bullet or Trojan horse?

BACKGROUND: Neutrophils participate in the first line of defense by executing several killing mechanisms, including phagocytosis, degranulation and the release of neutrophil extracellular traps. Additionally, they can orchestrate the adaptive immune system by secreting cytokines and chemokines. Opsonization with antibodies aids in the recognition of pathogens, via binding to Fc receptors on the neutrophil surface. Immunoglobulin A (IgA) is the most abundant antibody at mucosal sites and has multiple functions in homeostasis and immunity. Neutrophils and IgA can interact via the IgA Fc receptor Fc?RI (CD89), leading to pro- or anti-inflammatory responses. AIMS: The aim of this review is to give a concise overview of the interplay between IgA, Fc?RI and neutrophils and to explore potential therapies for autoimmune diseases and cancer. RESULTS: Crosslinking of FcαRI by IgA-immune complexes yields potent neutrophil activation and pro-inflammatory effector functions, including the recruitment of neutrophils. This can lead to neutrophil accumulation and tissue destruction during IgA-autoantibody mediated diseases. Conversely, for cancer treatment, the myriad of powerful neutrophil effector functions after targeting FcαRI may contribute to effective immunotherapy. CONCLUSION: By interfering with or actively promoting the interaction between IgA and FcαRI, therapies for multiple maladies could be developed.

The era of the immunoglobulin A Fc receptor FcαRI; its function and potential as target in disease.

Immunoglobulin A (IgA) is the most prevalent antibody at mucosal sites, and has an important role in defense by preventing invasion of pathogens. Traditionally, IgA has been thought of as a non-inflammatory antibody that helps to maintain homeostasis in the mucosa. However, in the last decade it has become clear that IgA is a very potent stimulus to initiate pro-inflammatory cellular processes, especially after triggering the IgA Fc receptor (FcαRI) on neutrophils. It was furthermore described that FcαRI acts as a regulator between anti- and pro-inflammatory responses of IgA. Although neutrophil activation is beneficial in (mucosal) infections, abnormal or excessive IgA immune complexes can induce disproportionate neutrophil migration and in this way initiate a perpetuating neutrophil recruitment and activation loop, which will result in severe tissue damage. Increasing evidence on this process plays a detrimental role in several diseases, including autoimmune IgA blistering diseases, a subtype of rheumatoid arthritis and ulcerative colitis. Inhibiting FcαRI-mediated activation may dampen inflammation in these patients. This process also opens up the possibility of targeting FcαRI in antibody immunotherapy of cancer. Thus, interfering with IgA-mediated FcαRI activation may represent an attractive novel therapeutic strategy for multiple maladies.

The cytochrome bd-type quinol oxidase is important for survival of Mycobacterium smegmatis under peroxide and antibiotic-induced stress.

Targeting respiration and ATP synthesis has received strong interest as a new strategy for combatting drug-resistant Mycobacterium tuberculosis. Mycobacteria employ a respiratory chain terminating with two branches. One of the branches includes a cytochrome bc1 complex and an aa3-type cytochrome c oxidase while the other branch terminates with a cytochrome bd-type quinol oxidase. In this communication we show that genetic inactivation of cytochrome bd, but not of cytochrome bc1, enhances the susceptibility of Mycobacterium smegmatis to hydrogen peroxide and antibiotic-induced stress. The type-II NADH dehydrogenase effector clofazimine and the ATP synthase inhibitor bedaquiline were bacteriostatic against wild-type M. smegmatis, but strongly bactericidal against a cytochrome bd mutant. We also demonstrated that the quinone-analog aurachin D inhibited mycobacterial cytochrome bd at sub-micromolar concentrations. Our results identify cytochrome bd as a key survival factor in M. smegmatis during antibiotic stress. Targeting the cytochrome bd respiratory branch therefore appears to be a promising strategy that may enhance the bactericidal activity of existing tuberculosis drugs.