Pathogenic Activation and Therapeutic Blockage of FcαR-Expressing Polymorphonuclear Leukocytes in IgA Pemphigus

Pathomechanisms in IgA pemphigus are assumed to rely on Fc-dependent cellular activation by antigen-specific IgA autoantibodies; however, models for the disease and more detailed pathophysiologic data are lacking. In this study, we aimed to establish in vitro models of disease for IgA pemphigus, allowing us to study the effects of the interaction of anti-keratinocyte IgA with cell surface FcαRs. Employing multiple in vitro assays, such as a skin cryosection assay and a human skin organ culture model, in this study, we present mechanistic data for the pathogenesis of IgA pemphigus, mediated by anti–desmoglein 3 IgA autoantibodies. Our results reveal that this disease is dependent on FcαR-mediated activation of leukocytes in the epidermis. Importantly, this cell-dependent pathology can be dose-dependently abrogated by peptide-mediated inhibition of FcαR:IgA-Fc interaction, as confirmed in an additional model for IgA-dependent disease, that is, IgA vasculitis. These data suggest that IgA pemphigus can be modeled in vitro and that IgA pemphigus and IgA vasculitis are FcαR-dependent disease entities that can be specifically targeted in these experimental systems

Pathogenic Activation and Therapeutic Blockage of FcαR-Expressing Polymorphonuclear Leukocytes in IgA Pemphigus

Pathomechanisms in IgA pemphigus are assumed to rely on Fc-dependent cellular activation by antigen-specific IgA autoantibodies; however, models for the disease and more detailed pathophysiologic data are lacking. In this study, we aimed to establish in vitro models of disease for IgA pemphigus, allowing us to study the effects of the interaction of anti-keratinocyte IgA with cell surface FcαRs. Employing multiple in vitro assays, such as a skin cryosection assay and a human skin organ culture model, in this study, we present mechanistic data for the pathogenesis of IgA pemphigus, mediated by anti–desmoglein 3 IgA autoantibodies. Our results reveal that this disease is dependent on FcαR-mediated activation of leukocytes in the epidermis. Importantly, this cell-dependent pathology can be dose-dependently abrogated by peptide-mediated inhibition of FcαR:IgA-Fc interaction, as confirmed in an additional model for IgA-dependent disease, that is, IgA vasculitis. These data suggest that IgA pemphigus can be modeled in vitro and that IgA pemphigus and IgA vasculitis are FcαR-dependent disease entities that can be specifically targeted in these experimental systems.</p

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors

This research was in part funded by: "From CARs to TRUCKs: Induction of a concerted anti-tumor immune response by engineered T cells" (Deutsche Krebshilfe/German Cancer Aid-Priority Program in Translational Oncology #111975), "The Thomsen-Friedenreich antigen CD176: New target of chimeric antigen receptor (CAR)-modified immune cells in adoptive cancer immunotherapy" (Deutsche Kinderkrebsstiftung, Projekt DKS 2020.17), and Glycotope GmbH. AcknowledgmentsIntroduction: Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Methods: Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2 generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8 T cells upon co-cultivation with CD176 tumor cell lines as models for CD176 blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Results: Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176 cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176 cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176 control cells, their response proved to be target-specific. Discussion: Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity