Biology subjects

Unger, T.

Publications and source records attributed to Unger, T..

8 recordsLinked to original sources

bioRxiv2025

Structure-based design of antibody repertoires with drug-like properties

Animal immunization is the prevalent strategy for discovering antibody therapeutics1, but it is a lengthy and poorly controlled process. As an alternative, synthetic antibody repertoires deliver antibodies without animal welfare concerns, but the resulting antibodies often fail to exhibit "drug-like" biophysical properties1,2. Modern repertoires have improved developability by using a handful of frameworks with desirable biophysical properties, but at the cost of reduced structural diversity3-6. We developed a principled structure- and energy-based strategy, called CADAbRe, to navigate the complex tradeoffs between developability and structural diversity in repertoire design. The designed repertoire comprises billions of antibodies that are predicted to be stable and foldable, built from hundreds of different frameworks and hundreds of thousands of designed CDR H3s. We also developed an economical and scalable strategy for synthesizing large antibody repertoires, and as a proof of concept, designed and synthesized a 500-million variant phage display repertoire. Selections against four unrelated targets produced structurally diverse binders that exhibited drug-like properties, two of which were readily formatted as bispecifics for functional studies. Furthermore, one of the binders targets a challenging, highly charged surface. The proof-of-concept repertoire is available for academic research. We envision that the CADAbRe approach and repertoire will accelerate and rationalize antibody discovery while addressing animal-welfare concerns7.

synthetic biology↗

bioRxiv2025

A pharmacological modality to sequester homomeric proteins

Molecules that can perturb protein-protein interactions have an immense impact on chemical biology and therapeutics. However, such compounds typically rely on accessory proteins to function, such as E3 ligases in the case of targeted degradation, which may restrict their target and tissue scope or lead to resistance. Here we alleviate the need for accessory proteins with a novel pharmacological modality to knock-down protein function. Our strategy exploits protein symmetry as a selective vulnerability, and is widely applicable owing to the ubiquitous nature of homomeric proteins in cellular systems. We target homomeric proteins with PINCHs (Polymerization Inducing Chimeras) - bifunctional molecules composed of two linked ligands that act as bridges between homomers and trigger their supramolecular assembly into insoluble polymers. We design PINCHs that achieve efficient polymerization of three homomeric targets. In cells, we observed that a PINCH targeting Keap1 exhibited a longer duration of action compared to its monomeric inhibitor, and a PINCH targeting BCL6 displayed selective and improved B cell toxicity compared to its monomeric parent. Our results highlight PINCHs as a novel and general strategy to modulate and knock out protein function.

biochemistry↗

bioRxiv2025

Differential effects of immobilized CCL21 and ICAM1 on TILs with distinct expansion properties

Adoptive T cell therapy (ACT), particularly tumor-infiltrating lymphocyte (TIL), holds great promise for cancer treatment, yet it still faces major challenges such as variability in expansion rates, cytotoxic potency and immune suppression. Recent studies suggest that a "synthetic immune niche" (SIN), composed of immobilized CCL21 and ICAM-1, enhances both the expansion and cytotoxicity of murine and patient-derived T cells. Here, we investigate the mechanism underlying the expansion variability by identifying morphological and molecular markers that distinguish low- and high-expanding TILs and predict their ex vivo expansion potential. We further developed novel SIN-based strategies that differentially reinforce the efficacy of both low- and high-expanding TILs. We demonstrate that a 14-day REP with feeder cells and SIN facilitates the proliferation of the low-expanding cells, while the high-expanding counterparts benefit from a sequential expansion protocol of 7 days with feeder cells only, followed by 7 days with SIN treatment. At the end of the REP both TIL populations display high levels of granzyme B and perforin and reduced levels of exhaustion markers. In conclusion, our findings demonstrate that the refined CCL21+ICAM1 SIN treatment improves expansion rates and activation profiles of both TIL populations, thereby enabling personalized SIN-enhanced protocols for TIL-based immunotherapy.

bioengineering↗

bioRxiv2023

The impact of molecular variants, crystallization conditions and space group on structure-ligand complexes: A case study on Bacterial Phosphotriesterase Variants and complexes

While attempting to study the 3D structure of proteins with bound ligands, one often encounters considerable difficulties. We illustrate, as an example, the bacterial enzyme phosphotriesterase and specifically examine the effects of multiple factors such as the molecular constructs, ligands used during protein expression and purification, crystallization precipitance, and space group on the visualization of molecular complexes of organophosphate ligands bound to the enzyme. We analyzed twelve crystal structures of the different phosphotriesterase constructs derived by directed evolution in both apo and holo forms (in complex with organophosphate analogs), with resolutions up to 1.38 [A]. Crystals obtained from three different crystallization conditions, crystallized in four space groups, with and without N-terminal tags, were utilized to investigate the impact of these factors on visualizing molecular complexes of ligands bound to the enzyme. The study revealed that residual tags used for protein expression can lodge in the active site and hinder ligand binding. Additionally, the space groups in which the proteins are crystallized can significantly impact the visualization of the organophosphate ligands bound to the phosphotriesterase. The study also reveals that the crystallization precipitants can compete with and even preclude ligand binding, leading to false positives or the incorrect identification of lead drug candidates, which is particularly crucial for ligands with pharmacological and toxicological contexts. Overall, this study provides valuable insights into the challenges and considerations involved in studying the 3D structure of proteins with bound ligands, highlighting the importance of careful experimental design and rigorous data analysis to ensure the accuracy and reliability of the resulting protein-ligand structures.

biochemistry↗

bioRxiv2022

How talin allosterically activates vinculin

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single molecule magnetic tweezer experiments, Molecular Dynamics simulations, actin bundling assays, and adhesion assembly experiments in live cells, we here discover a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding which reinforces the binding to talin at a rate of 0.03 s-1. This allosteric transition can compete with force-induced dissociation of vinculin from talin only at 7-10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The allosteric vinculin mutant is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere.

biophysics↗

bioRxiv2021

TMEM106B modifies TDP-43 pathology in human ALS brain and cell-based models of TDP-43 proteinopathy

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43) inclusions (FTLD-TDP) share the neuropathological hallmark of aggregates of TDP-43. However, factors governing the severity and regional distribution of TDP-43 pathology, which may account for the divergent clinical presentations of ALS and FTLD-TDP, are not well-understood. Here, we investigated the influence of genotypes at TMEM106B, a locus associated with risk for FTLD-TDP, and hexanucleotide repeat expansions in C9orf72, a known genetic cause for both ALS and FTLD-TDP, on global TDP-43 pathology and regional distribution of TDP-43 pathology in 899 postmortem cases from a spectrum of neurodegenerative diseases. We found that, among the 110 ALS cases, minor (C)- allele homozygotes at the TMEM106B locus sentinel SNP rs1990622 had more TDP-43 pathology globally, as well as in select brain regions. C9orf72 expansions similarly associated with greater TDP-43 pathology in ALS. However, adjusting for C9orf72 expansion status did not affect the relationship between TMEM106B genotype and TDP-43 pathology. In order to elucidate the direction of causality for this association, we directly manipulated TMEM106B levels in an inducible cell system that expresses mislocalized TDP-43 protein. We found that partial knockdown of TMEM106B, to levels similar to what would be expected in rs1990622 C allele carriers, led to development of more TDP-43 cytoplasmic aggregates, which were more insoluble, in this system. Taken together, our results support a causal role for TMEM106B in modifying the development of TDP-43 proteinopathy.

neuroscience↗

bioRxiv2020

Pharmacological depletion of microglia prevents vascular cognitive impairment in Ang II-induced hypertension

Rationale: Hypertension is a major risk factor for cerebral small vessel disease, the most prevalent cause of vascular cognitive impairment. As we have shown, hypertension induced by a prolonged Ang II infusion is associated with increased permeability of the blood-brain barrier (BBB) and chronic activation of microglia. In this study we therefore aim to determine the contribution of microglia to hypertension-induced cognitive impairment in an experimental hypertension model by a pharmacological depletion approach. Methods: For this study, adult Cx3Cr1gfp/wtxThy1yfp/0 reporter mice were infused for 12 weeks with Angiotensin II or saline and subgroups were treated with PLX5622, a highly selective CSF-1R inhibitor. Systolic blood pressure (SBP) was measured via tail-cuff. Short- and long-term spatial memory were assessed during an Object Location task and a Morris Water Maze task (MWM). At the end of the study, microglia depletion efficacy and BBB leakages were assessed using flow cytometry and immunohistochemistry. Results: SBP, heart weight and carotid pulsatility were increased by Ang II and were not affected by PLX5622. Short-term memory was significantly impaired in Ang II hypertensive mice, but not in Ang II mice treated with PLX5622. Histological and flow cytometry analyses revealed almost complete ablation of microglia upon CSF1R inhibition, while brain resident perivascular macrophages, were reduced by 60%. Number and size of BBB leakages were increased in Ang II hypertensive mice, but not altered by PLX5622 treatment. Conclusion: Our results show that depletion of microglia, and less so PVMs, by CSF1R inhibition prevents short-term memory impairment in Ang II induced hypertensive mice. This novel finding supports the critical role of brain immune cells, and most in particular microglia, in the pathogenesis of hypertension-related cognitive impairment.

neuroscience↗

bioRxiv2017

A dementia-associated risk variant near TMEM106B alters chromatin architecture and gene expression

Neurodegenerative diseases pose an extraordinary threat to the worlds aging population, yet no disease-modifying therapies are available. While genome-wide association studies (GWAS) have identified hundreds of novel risk loci for neurodegeneration, the mechanisms by which these loci influence disease risk are largely unknown. Indeed, of the many thousands of SNP-trait associations identified by GWAS over the past ~10 years, very few are understood mechanistically. Here, we investigate the association of common genetic variants at the 7p21 locus with risk for the neurodegenerative disease frontotemporal lobar degeneration. We show that variants associated with disease risk correlate with increased brain expression of the 7p21 gene TMEM106B, and no other genes. Furthermore, incremental increases in TMEM106B levels result in incremental increases in lysosomal phenotypes and cell toxicity. We then combine fine-mapping, bioinformatics, and bench-based approaches to functionally characterize all candidate causal variants at this locus. This approach identified a noncoding variant, rs1990620, which differentially recruits CTCF, influencing CTCF-mediated long-range chromatin looping interactions between multiple cis-regulatory elements, including the TMEM106B promoter. Our findings thus provide an in-depth analysis of the 7p21 locus linked by GWAS to frontotemporal lobar degeneration, nominating a causal variant and a causal mechanism for allele-specific expression and disease association at this locus. Finally, we show that genetic variants associated with risk for neurodegenerative diseases beyond frontotemporal lobar degeneration are enriched in brain CTCF-binding sites genome-wide, implicating CTCF-mediated gene regulation in risk for neurodegeneration more generally.

genetics↗