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HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain,HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies,independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro,HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation,cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies,HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens,in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation,cytokine secretion,and cytotoxic activity in vitro compared to either compound alone. In conclusion,HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner,without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade. View Publication

Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor family transcription factor (TF) critical for adipogenesis,lipid metabolism,insulin sensitivity,and inflammation. It has also been known to play essential roles in trophoblast development and placentation. Dysregulation of PPARG in trophoblast differentiation has been implicated in pregnancy complications,such as pre-eclampsia and gestational diabetes. However,the molecular mechanisms of PPARG-dependent target gene regulation and its interactions with other regulatory factors during human trophoblast differentiation remain unclear. Using human trophoblast stem cells (TSCs),mimicking placental cytotrophoblasts (CTs),and their differentiation into extravillous trophoblasts (EVTs) as our models,we reveal that PPARG has cell-type-specific targets in TSCs and EVTs. We also find that while PPARG is essential for both TSC self-renewal and EVT differentiation,only its role in EVT differentiation is ligand sensitive and requires ligand-binding domain (LBD)-mediated transcriptional activity,whereas its function in TSC self-renewal appears to be ligand insensitive. Combined analysis with chromosomal targets of previously defined key TFs in TSCs and EVTs shows that PPARG forms trophoblast cell-type-specific regulatory circuitries,leading to differential target gene regulation via transcriptional re-wiring during EVT differentiation. Additionally,the enhanced invasiveness of EVTs treated with a PPARG agonist suggests a potential connection between PPARG pathways and human placenta accreta. View Publication

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71,encoded by the UL82 gene,plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance,standardized monoclonal antibodies (mAbs) for pp71 remain limited,prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study,we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae,and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones,one designated ID2 exhibited high sensitivity and specificity for pp71,as demonstrated by flow cytometry,immunofluorescence,an enzyme-linked immunosorbent assay (ELISA),and biolayer interferometry (BLI). Conclusions: Collectively,these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics. View Publication

uveal melanoma (UM) is the most common primary intraocular tumor in adults,with metastasis being the leading cause of death. However,effective treatments for metastatic UM remain limited. Emerging evidence suggests that cholesterol metabolism plays a role in cancer progression,but its impact on UM metastasis is not well understood. we investigated the effects of miR-181a on UM metastasis using multiple UM cell lines and a suprachoroidal injection mouse model. Functional assays,including migration,invasion,and cancer stem-like cell (CSC) formation,were performed. The target of miR-181a was identified through bioinformatics,luciferase assays,and western blotting. Cholesterol levels were measured,and in vitro and in vivo studies assessed the therapeutic potential of combining miR-181a with crizotinib. miR-181a significantly decreases UM cell migration,invasion,and metastasis. Mechanistically,miR-181a was found to target sterol regulatory element-binding protein 2 (SREBP2),thereby inhibiting cholesterol biosynthesis. This decrease in cholesterol levels hindered reduced epithelial-to-mesenchymal transition (EMT) and led to a decline in cancer stem-like cell (CSC) populations in UM. Furthermore,elevated cholesterol or overexpression of SREBP2 abrogated the anti-metastatic effects of miR-181a. Additionally,a combination of miR-181a and crizotinib significantly inhibited metastasis,both in vitro and in vivo. miR-181a inhibits UM metastasis by targeting SREBP2 and reducing cholesterol biosynthesis. Its combination with crizotinib may provide a promising therapeutic strategy for metastatic UM. The online version contains supplementary material available at 10.1186/s13046-025-03459-8. View Publication

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide,with a clinical presentation that varies between sexes. In late-stage AMD,choroidal neovascularization (CNV) triggers retinal inflammation and degeneration,processes that are exacerbated by an overactive response of retinal microglial cells. Short-chain fatty acids (SCFAs) have emerged as potential treatments for AMD due to their anti-inflammatory properties. In this study,we investigate the effects of SCFA treatment in a laser-induced CNV mouse model,focusing on sex-dependent differences in disease progression and microglial response. Our findings demonstrate distinct sex-specific patterns in the development of CNV and associated pathological hallmarks. SCFA treatment resulted in a slight increase in density of Iba1 + microglial cells in females at 3 days post-laser (3dpl),while it prevented an increase in males at 7 dpl,with both sexes showing enhanced microglial ramification. The dynamics of microglial density were likely linked to protective effects on CNV lesion,leakage size,and inflammation,which occurred earlier in females and later in males. At transcriptional level,SCFA showed mixed effects,mainly targeting inflammation resolution,mitochondrial support,and neuronal repair in a sex-dependent manner. In vitro,SCFAs reduced microglial phagocytosis of retinal debris,suggesting a potential anti-inflammatory action. This study underscores the importance of considering sex-specific responses in the development of AMD treatments,such as SCFAs,and highlights the need for personalized therapeutic strategies. The online version contains supplementary material available at 10.1186/s12974-025-03508-1. View Publication

Organoids are pivotal for bridging cellular-level and organism-level biological studies; however,significant challenges persist in their three-dimensional (3D) visualization. This study presents a nylon mesh chip designed to overcome these obstacles specifically for intestinal organoids (IOs). The chip,meticulously fabricated and assembled,comprises an upper glass layer,a nylon mesh,and a lower glass layer. We cultured IOs from mouse intestinal crypts and performed fluorescent labeling on the chip. For enhanced visualization,fluorescent labeling combined with 3D reconstruction techniques was employed. Results demonstrate that the chip’s structure stabilizes IOs in liquid environments. While conventional fluorescence imaging is limited by mesh interference,laser confocal 3D reconstruction achieves high-quality visualization by effectively filtering out redundant signals. The nylon mesh chip is a robust tool for 3D visualization of IOs and holds potential for other budding organoid types. This innovation is poised to advance organoid 3D visualization research. The online version contains supplementary material available at 10.1038/s41598-025-12015-5. View Publication

During lung cancer metastasis,tumor cells undergo epithelial-to-mesenchymal transition (EMT),enabling them to intravasate through the vascular barrier and enter the circulation before colonizing secondary sites. Here,a human in vitro microphysiological model of EMT-driven lung cancer intravasation-on-a-chip was developed and coupled with machine learning (ML)-assisted automatic identification and quantification of intravasation events. A robust EMT-inducing cocktail (EMT-IC) was formulated by augmenting macrophage-conditioned medium with transforming growth factor-β1. When introduced into microvascular networks (MVNs) in microfluidic devices,EMT-IC did not affect MVN stability and physiologically relevant barrier functions. To model lung cancer intravasation on-a-chip,EMT-IC was supplemented into co-cultures of lung tumor micromasses and MVNs. Wihin 24 h of exposure,EMT-IC facilitated the insertion of membrane protrusions of migratory A549 cells into microvascular structures,followed by successful intravasation. EMT-IC reduced key basement membrane and vascular junction proteins - laminin and VE-Cadherin - rendering vessel walls more permissive to intravasating cells. ML-assisted vessel segmentation combined with co-localization analysis to detect intravasation events confirmed that EMT induction significantly increased the number of intravasation events. Introducing metastatic (NCI-H1975) and non-metastatic (BEAS-2B) cell lines demonstrated that both,baseline intravasation potential and responsiveness to EMT-IC,are reflected in the metastatic predisposition of lung cancer cell lines,highlighting the model's universal applicability and cell-specific sensitivity. The reproducible detection of intravasation events in the established model provides a physiologically relevant platform to study processes of cancer metastasis with high spatio-temporal resolution and short timeframe. This approach holds promise for improved drug development and informed personalized patient treatment plans. View Publication

Chronic myeloid leukemia (CML) remains a therapeutic challenge,particularly in patients who develop resistance to standard tyrosine kinase inhibitors (TKIs) such as imatinib. Here,we present the first demonstration of the potent anti-leukemic activity of the histone deacetylase (HDAC) inhibitor martinostat in both TKI-sensitive and TKI-resistant CML. Structural and biochemical analyses confirmed the efficient and selective binding of martinostat to HDAC isoenzyme ligand-binding pockets,resulting in histone and tubulin hyperacetylation in both imatinib-sensitive and resistant CML cells,outperforming vorinostat,a clinically used HDAC inhibitor (HDACi). It selectively impaired CML cell proliferation and viability and induced apoptosis across various CML models,including resistant cell models and patient blasts,with minimal toxicity to healthy cells and low developmental toxicity in zebrafish. In addition to its single-agent efficacy,martinostat demonstrated enhanced anticancer effects when combined with imatinib,both in vitro and in vivo,significantly reducing tumor growth in resistant CML xenograft models. Mechanistically,mRNA-seq data showed that martinostat disrupted key survival signaling pathways and amplified apoptotic responses,contributing to its anticancer activity. These findings highlight the potential of martinostat as a selective,low-toxicity HDACi that,combined with TKIs,could provide an effective strategy to overcome drug resistance in CML and improve therapeutic outcomes. The online version contains supplementary material available at 10.1186/s13148-025-01921-0. View Publication

Secondary and tertiary lymphoid structures are a critical target of suppression in many autoimmune disorders,protein replacement therapies,and in transplantation. Although antigen-specific regulatory T cells (Tregs),such as chimeric antigen receptor (CAR) Tregs,generally persist longer and localize to target tissues more effectively than polyclonal Tregs in animal models,their numbers still progressively decline over time. A potential approach to maximize Treg activity in vivo is the expression of chemokine receptors such as CXCR5,which would enable localization of a greater number of engineered cells at sites of antigen presentation. Indeed,CXCR5 expression on follicular T helper cells and follicular Tregs enables migration toward lymph nodes,B cell zones,and tertiary lymphoid structures that appear in chronically inflamed non-lymphoid tissues. In this study,we generated human and murine CXCR5 co-expressing engineered receptor Tregs and tested them in preclinical mouse models of allo-immunity and hemophilia A,respectively. Additionally,we engineered a murine CXCR5 co-expressing clotting factor VIII (FVIII) specific T cell receptor fusion construct epsilon (FVIII TRuCe CXCR5) Treg to suppress anti-drug antibody development in a model of FVIII protein replacement therapy for hemophilia A. In vitro,anti-HLA-A2 CXCR5+ CAR-Tregs showed enhanced migratory and antigen-specific suppressive capacities compared to untransduced Tregs. When injected into an NSG mouse model of HLA-A2+ pancreatic islet transplantation,anti-HLA-A2 CXCR5+ CAR-Tregs maintained a good safety profile allowing for long-term graft survival in contrast to anti-HLA-A2 CXCR5+ conventional CAR-T (Tconv) cells that eliminated the graft. Similarly,FVIII TRuCe CXCR5 Treg demonstrated increased in vivo persistence and suppressive capacity in a murine model of hemophilia A. Collectively,our findings indicate that CXCR5 co-expression is safe and enhances in vivo localization and persistence in target tissues. This strategy can potentially promote targeted tolerance without the risk of off-target effects in multiple disease models. View Publication

CRISPR/Cas9 genome editing has emerged as a promising treatment for genetic diseases like β-thalassemia. Editing γ-globin promoters to disrupt ZBTB7A/LRF or BCL11A binding sites has shown potential for reactivating fetal hemoglobin and treating sickle cell disease. However,its application to β 0 -thalassemia/HbE disease remains unclear. This study utilized CRISPR/Cas9 to disrupt these sites in mobilized CD34 + hematopoietic stem /progenitor cells from healthy donors and β 0 -thalassemia/HbE patients. The editing efficiency for the BCL11A site (75–92%) was higher than for the ZBTB7A/LRF site (57–60%). Both disruptions similarly increased fetal hemoglobin production in healthy donors ( BCL11A 26.2 ± 1.4%,ZBTB7A/LRF 27.9 ± 1.5%) and β 0 -thalassemia/HbE cells ( BCL11A 62.7 ± 0.9%,ZBTB7A/LRF 64.0 ± 1.6%). Off-target effects were absent in BCL11A -edited cells but observed at low frequencies in ZBTB7A/LRF -edited cells. Neither disruption significantly affected erythroid differentiation. These findings highlight the comparable contributions of ZBTB7A/LRF and BCL11A binding sites to γ-globin reactivation. CRISPR/Cas9 editing of either site may offer a potential therapeutic strategy for β 0 -thalassemia/HbE disease. View Publication

Reticular dysgenesis (RD) is a rare genetic disorder caused by mutations in the adenylate kinase 2 ( AK2 ) gene. It is characterized by a T − B − severe combined immunodeficiency,agranulocytosis,and sensorineural deafness. We established and characterized a haematopoiesis‐specific conditional Ak2 ‐knockout mouse model to provide a model system to study the molecular pathophysiology of RD. As expected from the human phenotype of RD,haematopoiesis‐specific AK2‐deficient embryos had a small,atrophic thymus consisting mainly of epithelial cells. No recognizable T‐cell component was observed,but B‐cell lineage precursor cells were present in the foetal liver. The effects of AK2 deficiency on myelopoiesis were less severe in mice than in humans. The absolute numbers of monocytes,macrophages,granulocytes and megakaryocytes in foetal liver as well as colony‐forming precursors were not reduced. In contrast to humans,haematopoiesis‐specific Ak2 ‐knockout mice exhibit embryonic lethality between E13 and E15 due to severe anaemia caused by an early block in definitive erythropoiesis. Murine erythroid progenitors mainly express AK2 and only low levels of functionally related kinases,which are unable to compensate for AK2 deficiency,in contrast to human erythroid progenitors. View Publication

This work focused on generating a three-dimensional (3D) in vitro dynamic model to study chronic lymphocytic leukemia (CLL) cell dissemination,homing,and mechanisms of therapy resistance. We used a gelatin-based,hard porous biomaterial as a support matrix to develop 3D tissue-like models of the human lymph node and bone marrow,which were matured inside bioreactors under dynamic perfusion of medium. Comparing static and dynamic cultures of these 3D constructs revealed that perfusion promoted a tissue-like internal organization of cells,characterized by the expression of specific functional markers and deposition of an intricate extracellular matrix protein network. Recirculation of CLL cells within the dynamic system led to changes in leukemic cell behavior and in the expression of key markers involved in tumor progression. These findings suggest that the model is well suited for investigating the pathophysiological mechanisms of CLL and potentially other hematological malignancies. View Publication