Enhancing neutrophil cytotoxicity of a panel of clinical EGFR antibodies by Fc engineering to IgA3.0.

The epidermal growth factor receptor (EGFR) plays an essential role in cellular signaling pathways that regulate cell growth, proliferation and survival, and is often found dysregulated in cancer. Several monoclonal IgG antibodies have been clinically tested over the years which exert their function via blocking the ligand binding domain (thereby inhibiting downstream signaling) and induction of Fc-related effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). However, these IgG antibodies do not optimally recruit neutrophils, by far the most abundant white blood cell population in humans. Therefore, we reformatted six therapeutic EGFR antibodies (cetuximab, panitumumab, nimotuzumab, necitumumab, zalutumumab, and matuzumab) into the IgA3.0 format, which is an IgA2 isotype that has been adapted for clinical application. Reformatting these antibodies preserved Fab-mediated functions such as EGFR binding, growth inhibition and ligand blockade. Additionally, whole leukocyte ADCC was significantly increased when using this panel of IgA3.0 antibodies compared to their respective IgG counterparts, with no major differences between IgA3.0 antibodies. In vivo, IgA3.0 matuzumab outperformed the other antibodies, resulting in the strongest suppression of tumor outgrowth in a long intraperitoneal model. We show that neutrophils are important for the suppression of tumor outgrowth. IgA3.0 matuzumab exhibited reduced receptor internalization compared to the other antibodies, possibly accounting for its superior in vivo Fc-mediated tumor cell killing efficacy. In conclusion, reformatting EGFR antibodies into an IgA3.0 format increased Fc-mediated killing while retaining Fab-mediated functions and could therefore be a good alternative for the currently available antibody therapies.

Enhancing IgA-mediated neutrophil cytotoxicity against neuroblastoma by CD47 blockade.

BACKGROUND: Approximately half of the neuroblastoma patients develop high-risk neuroblastoma. Current treatment involves a multimodal strategy, including immunotherapy with dinutuximab (IgG ch14.18) targeting GD2. Despite achieving promising results, the recurrence rate remains high and poor survival persists. The therapeutic efficacy of dinutuximab is compromised by suboptimal activation of neutrophils and severe neuropathic pain, partially induced by complement activation. METHODS: To enhance neutrophil cytotoxicity, IgG ch14.18 was converted to the IgA isotype, resulting in potent neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), without complement activation. However, myeloid checkpoint molecules hamper neutrophil cytotoxicity, for example through CD47 that is overexpressed on neuroblastomas and orchestrates an immunosuppressive environment upon ligation to signal regulatory protein alpha (SIRPα) expressed on neutrophils. In this study, we combined IgA therapy with CD47 blockade. RESULTS: In vitro killing assays showed enhanced IgA-mediated ADCC by neutrophils targeting neuroblastoma cell lines and organoids in comparison to IgG. Notably, when combined with CD47 blockade, both IgG and IgA therapy were enhanced, though the combination with IgA resulted in the greatest improvement of ADCC. Furthermore, in a neuroblastoma xenograft model, we systemically blocked CD47 with a SIRPα fusion protein containing an ablated IgG1 Fc, and compared IgA therapy to IgG therapy. Only IgA therapy combined with CD47 blockade increased neutrophil influx to the tumor microenvironment. Moreover, the IgA combination strategy hampered tumor outgrowth most effectively and prolonged tumor-specific survival. CONCLUSION: These promising results highlight the potential to enhance immunotherapy efficacy against high-risk neuroblastoma through improved neutrophil cytotoxicity by combining IgA therapy with CD47 blockade.

MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C.

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.