Periodic Reporting for period 1 - UNBIAS (Nanobodies to prevent hematopoietic stem cell bias and hyper-inflammation.)
Reporting period: 2023-08-01 to 2025-01-31
Inflammation is a fundamental immune response, but when persistent, it drives chronic inflammatory diseases (CIDs), which account for over half of global mortality. Most CIDs are caused by persistent protein aggregates that fuel long-term tissue damage and inflammation. Current treatments fail to address these aggregates, rely on broad immunosuppression, and often require lifelong administration, leading to significant side effects and limited efficacy. Prolonged inflammation also damages hematopoietic stem cells (HSCs), skewing their differentiation toward myeloid lineages, and contributing to a heightened inflammatory state known as inflammageing. Current anti-inflammatory therapies fail to eliminate the molecular remnants of inflammation, leaving an unmet medical need for targeted strategies. The UNBIAS project introduces a novel therapeutic approach using nanobodies to neutralize extracellular ASC specks, key inflammatory remnants that accumulate, and cause HSCs bias. This project supported our discovery of the inflammatory role of platelets in systemic monocyte inflammation, reinforcing the link between chronic inflammation and disrupted hematopoiesis. Moving forward, the project aims to establish nanobodies as a novel therapy, create a spin-off based on this technology, strengthen collaborations with industry leaders. By targeting the underlying mechanisms of chronic inflammation, UNBIAS has a transformative potential for immune health, age-related diseases, and inflammation-driven hematopoietic disorders.
The realization of UNBIAS gave us technical advances and ecouraging results.
Firstly, we designed a flow cytometry panel for the identification and sorting of LT-HSCs from human blood and mouse BM. Furhtermore, we have established protocols for isolating human HSCs from peripheral blood and from frozen PBMCs, and expanding LT-HSCs ex vivo for up to 7 days in culture. These advances expanded the sample repertoire we can work with, and allowed the identification of other HSPC populations such as ST-HSCs, MPPs and oligopotent progenitors (GMPs, CMPs and MEPs). Applying these methods, we managed to recover enough LT-HSCs for MFI measurements from total 20x106 BM cells, or 1x106 sorted Lin− cells from blood and use them for the identification of platelet-biased HSCs. Appearance of platelet-biased HSCs correlated with increased platelet counts in aged mice. We additionally showed that platelets amplify monocyte-driven cytokine production, establishing a self-perpetuating cycle of inflammation. This resulted in the publication by Hawwari et al. in EMBO Molecular Medicine (https://doi.org/10.1038/s44321-024-00093-3(opens in new window)).
As proposed in AIMs 1 and 2 of UNBIAS, we also conducted preliminary experiments of Wersten Diet in mice and evaluated the occurrence of platelet-biased hematopoiesis in the bone marrow HSCs. We found that platelet-biased HSCs accumulate with chronic inflammation, leading to increased platelet production at the cost of multilineage differentiation. Western diet (WD) and chronic immune stimulation promoted EM activation, increasing platelet markers (CD41, CD61) in bone marrow HSCs. We also observed a age-associated platelet bias which correlated with heightened IL-1β levels in bone marrow fluids, reinforcing the role of persistent inflammation in shaping hematopoiesis. Therefore, the prolonged exposure to IL-1 inflammation may dysregulate EM causing platelet bias and CIDs. Supporting this hypothesis, we found that the exposure of mouse BM cells to recombinant murine IL-1β increased the expression of CD41 and c-Kit in LT-HSCs.
Additionally, we performed experiments in vitro and in vivo to optimize and test ASC-nanobodies (VHHASC) for theic capacity to inhibit ASC inflammation in cells and in animal models of chronic WD-inflammation and hypersusceptibility to LPS shock. Through these experiments, we learned that VHHASC could be additionally used against LPS-induced sepsis, creating a novel hypothesis for the inflammatory roles of ASC and open novel applications for our nanobodies.
Firstly, we designed a flow cytometry panel for the identification and sorting of LT-HSCs from human blood and mouse BM. Furhtermore, we have established protocols for isolating human HSCs from peripheral blood and from frozen PBMCs, and expanding LT-HSCs ex vivo for up to 7 days in culture. These advances expanded the sample repertoire we can work with, and allowed the identification of other HSPC populations such as ST-HSCs, MPPs and oligopotent progenitors (GMPs, CMPs and MEPs). Applying these methods, we managed to recover enough LT-HSCs for MFI measurements from total 20x106 BM cells, or 1x106 sorted Lin− cells from blood and use them for the identification of platelet-biased HSCs. Appearance of platelet-biased HSCs correlated with increased platelet counts in aged mice. We additionally showed that platelets amplify monocyte-driven cytokine production, establishing a self-perpetuating cycle of inflammation. This resulted in the publication by Hawwari et al. in EMBO Molecular Medicine (https://doi.org/10.1038/s44321-024-00093-3(opens in new window)).
As proposed in AIMs 1 and 2 of UNBIAS, we also conducted preliminary experiments of Wersten Diet in mice and evaluated the occurrence of platelet-biased hematopoiesis in the bone marrow HSCs. We found that platelet-biased HSCs accumulate with chronic inflammation, leading to increased platelet production at the cost of multilineage differentiation. Western diet (WD) and chronic immune stimulation promoted EM activation, increasing platelet markers (CD41, CD61) in bone marrow HSCs. We also observed a age-associated platelet bias which correlated with heightened IL-1β levels in bone marrow fluids, reinforcing the role of persistent inflammation in shaping hematopoiesis. Therefore, the prolonged exposure to IL-1 inflammation may dysregulate EM causing platelet bias and CIDs. Supporting this hypothesis, we found that the exposure of mouse BM cells to recombinant murine IL-1β increased the expression of CD41 and c-Kit in LT-HSCs.
Additionally, we performed experiments in vitro and in vivo to optimize and test ASC-nanobodies (VHHASC) for theic capacity to inhibit ASC inflammation in cells and in animal models of chronic WD-inflammation and hypersusceptibility to LPS shock. Through these experiments, we learned that VHHASC could be additionally used against LPS-induced sepsis, creating a novel hypothesis for the inflammatory roles of ASC and open novel applications for our nanobodies.
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.
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.