Biology subjects

Hölzel, M.

Publications and source records attributed to Hölzel, M..

6 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↗

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↗