Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Aspergillus fumigatus is an important airborne fungal pathogen and a major cause of invasive fungal infections. Susceptible individuals become infected via the inhalation of dormant conidia. If the immune system fails to clear these conidia, they will swell, germinate and grow into large hyphal structures. Neutrophils are essential effector cells for controlling A. fumigatus infection. In general, opsonization of microbial particles is crucial for efficient phagocytosis and killing by neutrophils. Although the antibodies present in human serum do bind to all fungal morphotypes, we observed no direct antibody-mediated phagocytosis of A. fumigatus. We show that opsonization, phagocytosis and killing by neutrophils of A. fumigatus is complement-dependent. Using human sera depleted of key complement components, we investigated the contribution of the different complement initiation pathways in complement activation on the fungal surface. We describe the classical complement pathway as the main initiator of complement activation on A. fumigatus swollen conidia and germ tubes. Antibodies play an important role in complement activation and efficient innate recognition, phagocytosis and killing of A. fumigatus by neutrophils.

Broad application of cell therapies like CAR-T have been hampered by a lack of tumor-specific targets. Gadeta leverages the natural HLA-independent tumor recognition capabilities of γδTCRs combined with the proliferative capacity and robust tumor killing of αβT cells to develop tumor-specific cell therapies. γδT cells expressing Vγ9Vδ2 TCRs are the most common subtype found in peripheral blood. They sense the presence of phosphoantigens (pAgs) upregulated in malignant cells due to a dysregulated mevalonate pathway. The Vγ9Vδ2 TCR expressed by GDT002 was isolated from a PBMC-derived γδT cell of a healthy donor and was selected for its broad and strong tumor reactivity. GDT002 is currently being evaluated in a multicenter first-in-human phase 1/2 study for the treatment of Multiple Myeloma (NCT04688853). Here we describe studies carried out to identify potential solid tumor indications which could be effectively targeted by GDT002. Tumor reactivity was tested against cell lines from different cancer types in the presence of pamidronate, a clinically approved N-bisphosphonate that boosts pAgs levels. Significant GDT002 reactivity, in terms of cytotoxicity and cytokine release, was observed against most tumor targets. In a more complex ex vivo 3D co-culture system, GDT002 reactivity was also observed towards a broad panel of patient-derived tumor organoids. In contrast, there was no or very limited GDT002 reactivity against primary healthy cells. From our broad screening of solid tumor types, we identified ovarian cancer as a promising indication for GDT002. GDT002 displayed significant reactivity towards 4/5 ovarian cell lines and 9/10 patient-derived ovarian cancer organoids in the presence of pamidronate. To study GDT002 in vivo, we investigated the biodistribution of i.v. administered pamidronate. In tumor-bearing mice, intra-tumor pamidronate concentrations were detected during the 7-day observation period, allowing targeting by GDT002. Taken together, our results demonstrate that GDT002 has a potent anti-tumor activity across a broad spectrum of solid and liquid tumor types. We have identified ovarian cancer as a potential indication to explore the clinical effect of GDT002 against solid tumors. Citation Format: Esther Drent, Andrea Bisso, Sjoerd Baardman, Dagmar Verweij, Estefania Salcedo, Chris Coomans, Steven Braem, Sander van de Weg, Sara Melief, Haakan Norell, Marleen van Loenen, Stefania Gobessi, Mark Throsby. Targeting solid tumors with GDT002, a first-in-class γδTCR-based T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2818.

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