Biology subjects

Bald, T.

Publications and source records attributed to Bald, T..

11 recordsLinked to original sources

bioRxiv2026

Engineered endothelial cell grafts form functional anastomoses and enable recruitment of intravenously delivered human T cells in CAM tumor models

The development of cancer immunotherapies requires preclinical models that capture intravenous delivery, immune cell recruitment and intratumoral T cell activation. Conventional 3D in vitro systems lack perfused vascular networks, whereas mouse models are limited by throughput. The chick chorioallantoic membrane (CAM) assay enables rapid growth of vascularized tumors derived from human cancer cells in ovo, but its application to human T cell-based immunotherapy testing is constrained by CAM-derived vascularization and species-specific barriers between human immune cells and avian endothelium. Here, we establish an endothelial graft-enhanced CAM tumor model that incorporates an immortalized murine endothelial cell line capable of anastomosing with the chick vasculature. This generates a perfused and branched mammalian vascular interface within human tumor xenografts growing on the CAM. Human ICAM-1 expression on the grafted endothelial cells further enhances recruitment of intravenously delivered human T cells into CAM tumors. Using this platform, we demonstrate target-dependent intratumoral T cell activation by bispecific T cell engagers (TCEs) across tumor models, including evaluation of the clinically approved DLL3-targeting TCE tarlatamab in small cell lung cancer models.

bioengineering↗

bioRxiv2026

Evolutionary algorithms accelerate de novo design of potent Nectin-4-specific cancer biologics

Recent advances in AI-based structural biology have made de novo protein binder design increasingly effective, yet performance remains highly target dependent. For instance, the cancer surface antigen Nectin-4, an immunoglobulin-like cell adhesion protein, proved particularly challenging for RFdiffusion-based minibinder generation, yielding substantially fewer high-quality candidates than related targets. To address this bottleneck, we integrated an evolutionary genetic algorithm (GA) with AI-driven design. GA selection with tunable stringency was coupled with diversification via partial diffusion or direct sequence editing, enabling efficient exploration of sequence-structure space and rapid enrichment of promising candidates. This AI-GA pipeline quickly produced large and diverse minibinder panels with very good in silico quality metrics and is compatible with inputs from multiple design algorithms. Pooled, large-scale experimental screening identified highly stable Nectin-4 minibinders with single-digit nanomolar down to subnanomolar affinities. Lead binders were further engineered into Nectin-4-specific flow cytometry detection reagents and potent bispecific T cell engagers, demonstrating functional activity beyond binding. Together, these results show that evolutionary refinement can unlock challenging targets and accelerate de novo protein design for next-generation cancer biologics.

bioengineering↗

bioRxiv2026

Longitudinal spatial profiling of neutrophils during adoptive T cell therapy in murine melanoma reveals distinct lymph node infiltration patterns across anatomical sites

Reactive neutrophil infiltration can restrain CD8+ T cell expansion in lymph nodes during adoptive T cell therapy (ACT), yet its spatiotemporal regulation remains incompletely understood. Levaraging flow cytometry and multiplex immunofluorescence data, we performed a time-resolved quantitative assessment of immune cell dynamics in tumor-draining lymph node (tdLN) and non-tumor-draining lymph node (non-tdLN) in a melanoma mouse model receiving ACT. Transferred tumor-reactive CD8+ T cells accumulated and expanded early after treatment initiation, showing the highest frequency of a favorable central memory CD8+ T cell phenotype in the tdLN. Enhancing innate immune signaling in melanomas increased neutrophil influx into lymph nodes, particularly the non-tdLN; however, within the tdLN, neutrophils were enriched in the T cell zone, which also contained the largest absolute reservoir of transferred CD8+ T cells. Together, these findings indicate that tdLN and non-tdLN differ in early neutrophil dynamics and compartmentalization during ACT, influenced by the strength of innate immune signaling in the tumor.

immunology↗

bioRxiv2025

Development of AI-designed protein binders for detection and targeting of cancer cell surface proteins

Artificial intelligence (AI)-based protein design opens new avenues for the rapid generation of new research tools and therapeutics, but experimental validation lags behind the computational design throughput. Here, we present a scalable workflow for the discovery and validation of AI-designed protein binders. Leveraging the RFdiffusion protein design pipeline with a custom filter for stable alpha-helical bundle folds, we construct libraries of thousands of AI-binders against cancer-associated surface proteins. Mammalian cell-surface and phage display screening yield multiple high-affinity PD-L1 binders but fewer hits for CD276 (B7-H3) and VTCN1 (B7-H4), reflecting the target-dependent efficiency of RFdiffusion in generating high-quality designs. Using our experimentally validated AI-designed binder libraries, we benchmark freely available structure prediction models. We find that interface predicted template modelling (ipTM) scores by Chai-1 with ESM embedding correlate well with experimental success and even predict deleterious effects of binding interface mutations. To demonstrate the versatility of AI-binders as research tools, we deploy them in CAR-T cells and also assemble them with fluorophore-labeled streptavidin into tetravalent quattrobinders, which achieve antibody-comparable staining of endogenous PD-L1 by flow cytometry. With high production yields and accessible structural models, AI-designed quattrobinders are versatile and cost-effective research tools amenable to community-driven validation and optimization.

bioengineering↗

bioRxiv2025

NKG7 is a stable marker of cytotoxicity across immune contexts and within the tumor microenvironment

Cytotoxicity is a cornerstone of immune defense, critical for combating tumors and infections. This process relies on the coordinated action of granzymes and pore-forming proteins, with Granzyme B (GZMB) and Perforin (PRF1) being key markers and the most widely studied molecules pertaining to cytotoxicity. However, other human granzymes and cytotoxic components remain underexplored, despite growing evidence of their distinct, context-dependent roles. Natural Killer Cell Granule Protein 7 (NKG7) has recently emerged as a crucial cytotoxicity regulator, yet its expression patterns and function are poorly understood. Using large publicly available single-cell RNA sequencing atlases, we performed a comprehensive profiling of cytotoxicity across immune subsets and tissues. Our analysis highlights NKG7 expression as a strong marker of cytotoxicity, exhibiting a strong correlation with overall cytotoxic activity (r = 0.97) and surpassing traditional markers such as Granzyme B and Perforin in reliability. Furthermore, NKG7 expression is notably consistent across diverse immune subsets and tissues, reinforcing its versatility and robustness as a cytotoxicity marker. These findings position NKG7 as an invaluable tool for evaluating immune responses and a reliable indicator of cytotoxic functionality across biological and clinical contexts.

immunology↗

bioRxiv2024

Type I interferon drives a cellular state inert to TCR-stimulation and could impede effective T-cell differentiation in cancer

Head and neck squamous cell carcinoma (HNSCC) arises from the mucosal epithelium of the oral cavity, pharynx, or larynx and is linked to exposure to classical carcinogens and human papillomavirus (HPV) infection. Due to molecular, immunological, and clinical disparities between HPV+ and HPV-HNSCC, they are recognized as distinct cancer types. While immune checkpoint inhibition (ICI) has demonstrated efficacy in recurrent/metastatic HNSCC, response variability persists irrespective of HPV status. To gain insights into the CD8+ T-cell landscape of HPV-HNSCC, we performed multimodal sequencing (RNA and TCR) of CD8+ tumor-infiltrating lymphocytes (TILs) from treatment-naive HPV-HNSCC patients. Additionally, we subjected cells to ex vivo TCR-stimulation, facilitating the tracing of clonal transcriptomic responses. Our analysis revealed a subset of CD8+ TILs highly enriched for interferon-stimulated genes (ISG), which were found to be clonally related to a subset of granzyme K (GZMK)-expressing cells. Trajectory inference suggests ISG transition via GZMK cells towards terminal effector states. However, unlike GZMK cells, which rapidly an effector-like phenotype in response to TCR stimulation, ISG cells remain transcriptionally inert. Consequently, ISG cells may impede effective T-cell differentiation within the TME. Although, the functional consequences of ISG cells are poorly understood, we revealed that they possess receptors and ligands enabling cell-cell communication networks with key TME immunomodulators such as dendritic cells. Additionally, ISG cells were found to be a core feature across various tumor entities and were specifically enriched within tumor tissue. Thus, our findings illuminate the complexity of T-cell heterogeneity in HPV-HNSCC and reveal an overlooked population of IFN-stimulated CD8+ TILs. Further exploration of their functional significance may offer insights into therapeutic strategies for HPV-HNSCC and other cancer types.

immunology↗

bioRxiv2023

Oncogenic drivers dictate immune control of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a genetically heterogeneous, aggressive hematological malignancy induced by distinct oncogenic driver mutations. The effect of specific AML oncogenes on immune activation or suppression has not been investigated. Here, we examine immune responses in genetically distinct models of AML and demonstrate that specific AML oncogenes dictate immunogenicity, the quality of immune response and immune escape through immunoediting. Specifically, expression of NrasG12D alone is sufficient to drive a potent anti-leukemia response through increased MHC Class II expression that can be overcome with increased expression of Myc. These data have important implications for the design and implementation of personalized immunotherapies for patients with AML. Statement of SignificanceThe endogenous immune response against acute myeloid leukemia (AML) is determined by leukemia-specific oncogenic driver mutations. Mutant Nras drives immunological selection of AML.

cancer biology↗

bioRxiv2022

Melanoma cells appropriate pericyte:endothelial cell crosstalk during perivascular invasion in the brain

Post brain colonization, cancer cells may adhere to and spread along the abluminal surface of the vasculature providing advantageous access to oxygen, nutrients, and vessel-derived paracrine factors. For example, brain-tropic melanoma cells are well-known to invade along or within blood vessels at the invasive front, but the molecular mechanisms that guide this process are not well-characterized. We have used melanoma cells of different phenotypic states (melanocytic versus mesenchymal) to characterize different modes of perivascular invasion in the brain. We find that Sox9hi mesenchymal state melanoma cells undergo pericyte-like spreading along brain blood vessels via a Snail1-dependent process; in contrast, Sox10hi melanocytic state melanoma form proliferative, perivascular clusters. Snai1 deletion in mesenchymal-state melanoma cells diminishes Tgf{beta}-induced expression of Pdgfr{beta} which impairs Tgf{beta}/Pdgf ligand-driven motility along the brain microvasculature and dramatically reduces perivascular dispersal of melanoma cells throughout the brain post-colonization. These data suggest that, depending on their transcriptional/phenotypic state, some melanoma cells may appropriate signals that typically mediate endothelial cell:pericyte cross talk as an adaptive mechanism for maintaining vessel proximity and motility within the brain microenvironment.

cancer biology↗

bioRxiv2020

Discovery of drugs to treat cytokine storm-induced cardiac dysfunction using human cardiac organoids

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined, but could be direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory cytokine-storm, a cocktail of interferon gamma, interleukin 1{beta} and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids and hearts of SARS-CoV-2 infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCO and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the FDA breakthrough designated drug apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

cell biology↗

bioRxiv2020

Microbial activation converts neutrophils into anti-tumor effectors

Neutrophils infiltrate most solid tumors and their presence is usually correlated with suppression of anti-tumor responses, metastasis and poor prognosis. Here we used microbial bioparticles administered into the tumor microenvironment to transform neutrophils into anti-tumor effectors. Microbially activated neutrophils acquired an effector phenotype associated with pathogen killing and lost vascular endothelial growth factor expression associated with tumor growth and metastasis. They became the dominant immune cell infiltrating the tumor and inhibited tumor growth. Using intravital two-photon microscopy microbially activated neutrophils could be seen forming close contacts with tumor cells resulting in tumor tissue remodeling and tumor cell death. Thus, microbial bioparticle treatment can endow neutrophils with anti-tumor properties, suggesting that neutrophil plasticity in cancer could be exploited for tumor killing. These data highlight a pathway for the rational development of neutrophil-based cancer therapy.

immunology↗

bioRxiv2019

Experimental and stochastic models of melanoma T-cell therapy define impact of subclone fitness on selection of antigen loss variants

Antigen loss is a key mechanism how tumor cells escape from T-cell immunotherapy. Using a mouse model of melanoma we directly compared antigen downregulation by phenotypic adaptation with genetically hardwired antigen loss. Unexpectedly, genetic ablation of Pmel, the melanocyte differentiation antigen targeted by adoptively transferred CD8+ T-cells, impaired melanoma cell growth in untreated tumors due to competitive pressure exerted by the bulk wild-type population. This established an evolutionary scenario, where T-cell immunotherapy imposed a dynamic fitness switch on wild-type melanoma cells and antigen loss variants, which resulted in highly variable enrichment of the latter in recurrent tumors. Stochastic simulations by an individual-based continuous-time Markov process suggested variable fitness of subclones within the antigen loss variant population as the most likely cause, which was validated experimentally. In summary, we provide a framework to better understand how subclone heterogeneity in tumors influences immune selection of genetic antigen loss variants through stochastic events.

systems biology↗