As outlined in AIM3 of UNBIAS, we conducted experiments to determine whether neutralizing ASC specks—molecular remnants that persist after pyroptotic cell death—could prevent platelet bias in hematopoietic stem cells (HSCs) in inflammation-trained mice and mitigate the heightened inflammation associated with inflammageing. This work was carried out in collaboration with Prof. Fernando Cunha at the Medical Faculty of the University of São Paulo. We initially subjected wild-type mice to six weeks of a Western diet (WD) to induce a state of chronic inflammation. The mice were then treated with a single dose of the nanobody VHHASC or an unrelated nanobody (VHHNP) as a control, followed by a lipopolysaccharide (LPS) injection (10 mg/kg) to trigger an acute inflammatory response. We monitored survival rates, clinical symptoms, systemic cytokine levels, and markers of liver damage (ALT and urea).
While VHHASC demonstrated limited effects in mice with WD-induced inflammation when faced with LPS shock, a surprising discovery emerged in the control group. In mice fed a standard chow diet, VHHASC was significantly effective in reducing the impact of LPS-induced endotoxemia compared to the control nanobody. To further explore this effect, we repeated the experiments without the influence of the Western diet, directly testing the resistance of mice treated with VHHASC or the control nanobody following LPS challenge. In two subsequent trials, VHHASC consistently reduced inflammation and improved clinical outcomes, highlighting its potential as a therapeutic agent against sepsis, a severe and often fatal inflammatory condition. Unfortunately, we were unable to advance the studies involving WD due to the complex, systemic effects of the diet, which impact multiple organs and complicate the interpretation of results. Nonetheless, these findings open new possibilities for VHHASC in treating sepsis.
IP protection:
Our nanobodies targeting ASC have confirmed the pathogenic role of extracellular ASC specks in rheumatoid arthritis and gout, demonstrating their therapeutic potential in these diseases. Additionally, they have shown promising benefits in treating sepsis, a life-threatening condition. Building on these findings, we will design, develop, and test novel nanobody structures with optimized properties to ensure uniqueness and patentability. AI-assisted design will enhance this process, enabling the generation of improved nanobody variants, including VHHs with varying affinities for ASC and bi-specific VHHs that bind multiple ASC domains simultaneously to enhance therapeutic efficacy. To secure global market protection, we have consulted with IP specialists from Ascenion GmbH and will conduct comprehensive freedom-to-operate (FTO) analyses.
Development and Implementation
A key focus of our development strategy is ensuring regulatory alignment to facilitate the transition from preclinical to clinical applications. Since different disease indications require tailored preclinical data packages, we will engage proactively with regulatory authorities to optimize our approach. Early consultations with the Innovation Task Force of the European Medicines Agency (EMA) and the Innovation Office of BfArM will help define approval pathways and ensure that our preclinical safety, pharmacokinetic, and pharmacodynamic studies meet regulatory expectations.
