Abstract Head and neck squamous cell carcinoma (HNSCC) is a solid tumor type that arises in the squamous epithelial cells lining the mucosal surfaces of the upper aerodigestive tract. Long-term survival of patients with advanced disease stage remains disappointing with current treatment options. We show that tissue factor is abundantly expressed on patient-derived HNSCC cell lines, xenograft tumor material, and tumor biopsies from patients with HNSCC. Tisotumab vedotin (TV) is an antibody–drug conjugate (ADC) directed to tissue factor, a protein expressed in many solid tumors. HNSCC cells and xenograft tumors were efficiently eliminated in vitro and in vivo with TV-monotherapy compared with treatment with a control antibody conjugated to monomethyl auristatin E (MMAE). Antitumor activity of TV was also tested in vivo in combination with chemoradiotherapy, standard of care for patients with advanced stage HNSCC tumors outside the oral cavity. Preclinical studies showed that by adding TV to chemoradiotherapy, survival was markedly improved, and TV, not radiotherapy or chemotherapy, was the main driver of antitumor activity. Interestingly, TV-induced cell death in xenograft tumors showed an influx of macrophages indicative of a potential immune-mediated mode-of-action. In conclusion, on the basis of these preclinical data, TV may be a novel treatment modality for patients suffering from head and neck cancer and is hypothesized to improve efficacy of chemoradiotherapy. Significance: This work shows preclinical in vitro and in vivo antitumor activity of the antibody–drug conjugate Tisotumab vedotin in head and neck cancer models, and enhanced activity in combination with chemoradiotherapy, supporting further clinical development for this cancer type.

IC50 values in vitro killing assays

Improved efficacy of chemotherapy when combined with tisotumab vedotin

Tumor growth curves FaDu xenografts

Median survival combined treatment TV and radiotherapy

Immunoglobulin A (IgA) is generally considered as a non-inflammatory regulator of mucosal immunity, and its importance in diversifying the gut microbiota is increasingly appreciated. IgA autoantibodies have been found in several autoimmune or chronic inflammatory diseases, but their role in pathophysiology is ill-understood. IgA can interact with the Fc receptor FcαRI on immune cells. We now established a novel IgA autoimmune blistering model, which closely resembles the human disease linear IgA bullous disease (LABD) by using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that presence of IgA anti-collagen XVII, - the auto-antigen in LABD-, resulted in neutrophil activation and extravasation from blood vessels into skin tissue. Continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented disease, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage.

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.

The immunosuppressive character of head and neck cancers may explain the relatively low response rates to antibody therapy targeting a tumor antigen, such as cetuximab, and anti-PD-1 checkpoint inhibition. Immunostimulatory agents that overcome tumor-derived inhibitory signals could augment therapeutic efficacy, thereby enhancing tumor elimination and improving patient survival. Here, we demonstrate that cetuximab treatment combined with immunostimulatory agonists for Toll-like receptor (TLR) 2 induces profound immune responses. Natural killer (NK) cells, isolated from healthy individuals or patients with head and neck cancer, harbored enhanced cytotoxic capacity and increased tumor-killing potential in vitro. Additionally, combination treatment increased the release of several pro-inflammatory cytokines and chemokines by NK cells. Tumor-bearing mice that received cetuximab and the TLR2 ligand Pam3CSK4 showed increased infiltration of immune cells into the tumors compared to mice that received cetuximab monotherapy, resulting in a significant delay in tumor growth or even complete tumor regression. Moreover, combination treatment resulted in improved overall survival in vivo. In conclusion, combining tumor-targeting antibody-based immunotherapy with TLR stimulation represents a promising treatment strategy to improve the clinical outcomes of cancer patients. This treatment could well be applied together with other therapeutic strategies such as anti-PD-(L)1 checkpoint inhibition to further overcome immunosuppression.

IgA binding to FcαRI (CD89) is rapidly enhanced by cytokine induced inside-out signaling. Dephosphorylation of serine 263 in the intracellular tail of FcαRI by PP2A and PI3K activation are instrumental in this process. To further investigate these signaling pathways, we targeted downstream kinases of PI3K. Our experiments revealed that PI3K activates PKCζ, which subsequently inhibits GSK-3, a constitutively active kinase in resting cells and found here to be associated with FcαRI. We propose that GSK-3 maintains FcαRI in an inactive state at homeostatic conditions. Upon cytokine stimulation, GSK-3 is inactivated through a PI3K-PKCζ pathway, preventing the maintenance of phosphorylated inactive FcαRI. The concomitantly activated PP2A is then able to dephosphorylate and activate FcαRI. Moreover, FRAP and FLIP studies showed that FcαRI activation coincides with an increased mobile fraction of the receptor. This can enhance FcαRI valency and contribute to stronger avidity for IgA immune complexes. This tightly regulated inside-out signaling pathway allows leukocytes to respond rapidly and efficiently to their environment and could be exploited to enhance the efficacy of future IgA therapeutics.

Immunoglobulin A (IgA) is generally considered a non-inflammatory regulator of mucosal immunity. Conversely, we previously demonstrated that IgA is a potent proinflammatory stimulus for neutrophils after crosslinking of the Fc receptor FcαRI in vitro. The role of FcαRI in IgA-mediated diseases is, however, poorly understood, mostly due to the lack of suitable mouse models. We now established a novel linear IgA bullous disease (LABD) mouse model using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that injection of anti-collagen XVII IgA, which are also present in patients, resulted in neutrophil activation and extravasation from the blood vessels into skin tissue. Moreover, continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented thickening of ear tissue as measure of inflammation, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage due to unrestrained neutrophil activation in patients.

The cross-linking of the IgA Fc receptor (Fc alpha RI) 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 FcaRI sequences. CLIPS technology was used to stabilize three-dimensional structures and to increase peptides' half-life. IgA and Fc alpha RI 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 Fc alpha RI. In conclusion, a cream containing IgA or Fc alpha RI 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.

The individual role of pathogen-binding Toll-like receptors (TLRs) and antibody-binding Fc receptors (FcRs) during pathogenic infections has been studied extensively. However, combined activation of these different receptor classes has received little attention, even though they are triggered simultaneously when immune cells bind antibody-opsonized pathogens. In the last few years, it has become evident that joined activation of TLRs and FcRs substantially tailors inflammatory immune responses, which is an efficient and controlled mechanism of the host to act upon invading pathogens. In this review, we discuss the mechanisms of cross-talk between different TLRs and FcRs and the resulting inflammatory immune responses. Furthermore, we propose how chronic activation via this cross-talk might be detrimental in inflammatory (auto) immune diseases. We conclude with the potential exploitation of the interplay between TLRs and FcRs for monoclonal antibody therapy to target tumors. Future interests in this field of research include establishing a more detailed and mechanistic understanding of the mode of action of TLR and FcR cross-talk and exploration of its physiological importance in health and disease. This may furthermore open up novel therapeutic options for intervention in inflammatory diseases or cancer.

During secondary immune responses, Ab-opsonized bacteria are efficiently taken up via FcRs by dendritic cells. We now demonstrate that this process induces cross-talk between FcRs and TLRs, which results in synergistic release of several inflammatory cytokines, as well as altered lipid metabolite profiles. This altered inflammatory profile redirects Th1 polarization toward Th17 cell responses. Interestingly, GM-CSF–producing Th cells were synergistically evoked as well, which suggests the onset of polyfunctional Th17 cells. Synergistic cytokine release was dependent on activation via MyD88 and ITAM signaling pathways through TLRs and FcRs, respectively. Cytokine regulation occurred via transcription-dependent mechanisms for TNF-α and IL-23 and posttranscriptional mechanisms for caspase-1–dependent release of IL-1β. Furthermore, cross-talk between TLRs and FcRs was not restricted to dendritic cells. In conclusion, our results support that bacteria alone initiate fundamentally different immune responses compared with Ab-opsonized bacteria through the combined action of two classes of receptors and, ultimately, may refine new therapies for inflammatory diseases.

Polymorphonuclear cells (neutrophils) are the first cells that arrive at sites of infections. According to the current dogma, they are involved in eliminating bacteria, after which they die through apoptosis. We now demonstrate that enhanced IgA-induced phagocytosis of bacteria or beads by neutrophils led to increased cell death. Nuclear changes and positivity for the general cell death marker 7-aminoactinomycin D were observed, but the absence of annexin V membrane staining supported that neutrophils did not die via apoptosis, in contrast to neutrophils that had not phagocytosed bacteria. Moreover, increased release of neutrophil extracellular traps (NETs) was observed, which was most likely due to augmented production of reactive oxygen species after uptake of IgA-opsonized particles. Blocking the IgA Fc receptor FcαRI abrogated phagocytosis and NET formation. Thus, FcαRI triggering on neutrophils resulted in a rapid form of cell death that is referred to as NETosis, as it is accompanied by the release of NETs. As such, IgA may play a prominent role in mucosal inflammatory responses, where it is the most prominent Ab, because it enhanced both phagocytosis of bacteria and formation of NETs, which are effective mechanisms that neutrophils employ to eliminate pathogens.

Targeted therapies like treatment with monoclonal antibodies (mAbs) have entered the arsenal of modern anticancer drugs. mAbs combine specificity with multiple effector functions that can lead to reduction of tumour burden. Direct mechanisms of action, including induction of apoptosis or growth inhibition, depend on the biology of the target antigen. Fc tails of mAbs have furthermore the potential to initiate complement-dependent lysis as well as immune effector cell-mediated tumour cell killing via binding to Fc receptors. Natural killer cells can induce apoptosis via antibody-dependent cellular cytotoxicity (ADCC), whereas macrophages are able to phagocytose mAb-opsonized tumour cells (antibody-dependent cellular phagocytosis; ADCP). Finally, neutrophils can induce non-apoptotic tumour cell death, especially in the presence of immunoglobulin A (IgA) antitumour mAbs. In spite of promising clinical successes in some malignancies, improvement of mAb immunotherapy is required to achieve overall complete remission in cancer patients. New strategies to enhance Fc receptor-mediated mechanisms of action or to overcome the immunosuppressive microenvironment of the tumour in mAb therapy of cancer are therefore currently being explored and will be addressed in this chapter.

ABStrACt Immunoglobulin A (IgA) represents the most prominent antibody class at mucosal surfaces and plays an important role in mucosal immunity. However, we recently demonstrated that cross-linking of the IgA Fc receptor (FcaRI; CD89) by IgA autoantibodies of patients with autoimmune skin blistering diseases induced massive recruitment of neutrophils, which resulted in severe tissue damage. Currently, there is no specific therapy for IgA-induced blistering diseases. Therefore, we developed specific peptides, which were based on either IgA or FcaRI sequences, and investigated whether these peptides could inhibit neutrophil migration. One linear IgA peptide and one FcaRI peptide effectively reduced neutrophil migration, and were further developed with CLIPS technology, which stabilizes the three-dimensional structure and increases peptides' half-life by introduction of covalently linked scaffolds. CLIPS peptides and linear peptides with the best blocking capacity were tested in ex vivo skin migration experiments. IgA-induced neutrophil migration was completely blocked in the presence of these peptides. Furthermore, Cetomacrogol cream, containing an IgA CLIPS peptide was developed, and penetration of the peptide in an ex vivo skin model was investigated after topical administration. Moreover, minimal penetration of peptides into the skin was observed in the absence of dodecyl-2-N, N-dimethylaminopropionate (DDAIP), which is a permeation enhancer. However, peptide penetration was increased in a dose dependent manner in the presence of DDAIP. Minimal levels of peptides were observed in the receptor fluid, which is a measure of systemic delivery. Thus, topical application of a cream containing peptides may safely block IgA-induced neutrophil migration in the skin, without systemic exposure. This may decrease severe morbidity and improve quality of life of patients with IgA-mediated blistering diseases.

Department of Molecular Cell Biology and Immunology, VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands Department of Dermatology, University of Freiburg, Hauptstrasse 7, 79104 Freiburg, Germany Department of Dermatology, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands Department of Plastic and Reconstructive Surgery, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands Department of Surgery, VU University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands

Immunoglobulin A (IgA) represents the most prominent antibody class at mucosal surfaces and has an important role in mucosal immunity. However, we recently demonstrated in an ex vivo human skin model that cross-linking of the neutrophil IgA Fc receptor (FcaRI) by IgA autoantibodies of linear IgA bullous disease (LABD) patients induced deranged neutrophil recruitment, resulting in tissue damage. LABD is a chronic autoimmune skin blistering disorder characterised by anti-collagen XVII IgA autoantibodies and large neutrophil infiltrates. In the present study, we developed a novel LABD-mouse model to investigate the role of FcaRI in IgA-induced tissue damage in vivo. Therefore, we generated a hybridoma producing human IgA (hIgA) monoclonal antibodies (mAb) directed against mouse collagen XVII (mCOL17). When cryosections of mouse ears were incubated with these mAbs, binding to the basement membrane of the skin was observed. To analyze the role of FcaRI in IgA-induced neutrophil migration in vivo, FcaRI transgenic mice were crossbred with mice containing neutrophils expressing LysEGFP. When anti-mCOL17 hIgA mAbs were injected in the ears of these mice, intravital imaging demonstrated rolling and extravasating neutrophils, which was not observed when human serum IgA was injected as control. Staining cryosections of mouse ears with the granulocyte marker GR-1 confirmed the presence of a large neutrophil infiltrate in response to anti-mCOL17 hIgA, which was not found in cryosections from ears of non-transgenic littermates. Importantly, no rolling or extravasation of neutrophils was observed when mice were injected with an FcaRI blocking antibody. This indicates that IgA-induced neutrophil activation and migration is dependent on FcaRI in vitro and in vivo. Thus, developing agents that specifically block IgA-FcaRI interactions may represent a promising novel approach for IgA-induced blistering diseases.