Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.
T-cell engagers (TCEs) direct cytotoxic T-cell response towards tumor cells by binding simultaneously to a tumor-associated antigen (TAA) on target cells and to CD3 on T-cells, thereby forming an artificial immune synapse. They have been shown to be very potent anti-tumor drugs, as exemplified by blinatumomab, an α-CD19 x α-CD3 bispecific. However, the development of TCEs for hematological and solid tumors has been hampered by several factors, amongst them severe toxicity, elicited by on-target/off-tumor recruitment of T-cells and cytokine release syndrome (CRS). In order to overcome this challenge, an anti-CD3 Prodrug DARPin® (CD3-PDD) has been developed, consisting of a mouse cross-reactive EGFR-binder and a CD3-binder, linked via a protease-cleavable linker to an anti-idiotypic anti-CD3 binder (termed blocker hereafter). This α-EGFR x α-CD3 x blocker Prodrug is unable to bind and recruit T-cells in its non-cleaved state, but is designed to become activated in the tumor microenvironment upon cleavage of the linker by tumor-associated proteases. A control Prodrug DARPin® with a non-cleavable linker showed neither tumor cell killing, nor T-cell activation at concentrations >1'000-fold over the EC50 of the active, non-blocked TCE DARPin® in in vitro tumor cell killing and T-cell activation assays. In contrast, a CD3-PDD containing a cleavable linker was partially activated by proteases secreted from the tumor cells (HCT 116). Pre-treatment of the CD3-PDD with recombinant protease prior to the in vitro assay fully activated the molecule, with EC50 values comparable to the active, non-blocked TCE. Next, an in vivo proof-of-principle study was performed in a human colon carcinoma xenograft model (HCT 116) using immunodeficient mice humanized with hematopoietic stem cells (CD34+) and optimized for the presence of human myeloid cells. Due to the mouse cross-reactivity of the EGFR-binder, this animal model allowed to assess both anti-tumor efficacy and safety (therapeutic window). The cleavable CD3-PDD demonstrated a robust anti-tumor activity, similar to the one observed with active, non-blocked TCE. Most importantly, while the active, non-blocked TCE elicited strong toxicity, leading to loss of animals and requiring treatment stop, the cleavable CD3-PDD could be dosed without significant safety findings. In summary, a conditionally activated CD3-PDD shows similar efficacy but none of the toxicity of the active, non-blocked TCE. Our approach therefore holds great promise for the development of future CD3-PDD as therapeutics, enabling the utilization of less tumor-specific targets for highly potent TCEs. Ultimately, the ability of the versatile DARPin® technology to generate tailor-made anti-idiotypic DARPin® molecules can unlock novel therapeutic design spaces, which we are exploring beyond the conditionally activated CD3-PDD format. Citation Format: Andreas Bosshart, Julia Katharina Ahlskog, Aline Eggenschwiler, Dieter Schiegg, Yvonne Grübler, Sandra Wandel, Simon Fontaine, Maria Paladino, Susanne Mangold, Tanja Hospodarsch, Alexandra Neculcea, Chloé Iss, Christel Herzog, Bernd Schlereth. A solution to T-cell engager toxicity: An anti-CD3 Prodrug DARPin (CD3-PDD) shows no toxicity, but potent anti-tumor activity in a humanized mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1890.
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