Evotec (Germany)

The evolutionarily conserved RNA exosome complex modulates gene expression during development. Mutations in RNA exosome complex subunits have been implicated in various human brain disorders, suggesting that defects in RNA decay are linked to impaired neural development. In our study, we identified de novo variants of EXOSC10 in microcephalic individuals. The patient's phenotype can be replicated by heterozygous conditional knockout of Exosc10 in the developing mouse forebrain. The heterozygous loss of Exosc10 in the developing mouse cortex leads to increased cell cycle exit, with a premature differentiation of radial glial cells to intermediate progenitors and neurons in the embryonic cortex. This premature neurogenesis at the expense of neural stem cell proliferation coincides with a smaller cortical size. RNA sequencing and RNA immunoprecipitation sequencing revealed upregulation of many Sonic hedgehog (Shh) signalling genes. We further show the direct degradation of Shh pathway transcripts such as Scube1 and Scube3 by Exosc10. In Exosc10 mouse mutants, the reduced cortical size could be largely rescued by reducing Shh activity. We propose that increased Shh activity due to Exosc10 deficiency leads to premature neurogenesis and ultimately to microcephaly. These observations offer new insights into the neurodevelopmental role of Exosc10 and highlight the dosage-dependent regulation of Shh signalling by Exosc10 in cortical development.

CD4+ regulatory T (Treg) cells in tissues play crucial immunoregulatory and regenerative roles. Despite their importance, the epigenetics and differentiation of human tissue Treg cells are incompletely understood. Here, we performed genome-wide DNA methylation analysis of human Treg cells from skin and blood and integrated these data into a multiomic framework, including chromatin accessibility and gene expression. This analysis identified programs that governed the tissue adaptation of skin Treg cells. We found that subfamilies of transposable elements represented a major constituent of the hypomethylated landscape in tissue Treg cells. Based on T cell antigen receptor sequence and DNA hypomethylation homologies, our data indicate that blood CCR8+ Treg cells contain recirculating human skin Treg cells. Conversely, differences in chromatin accessibility and gene expression suggest a certain reversal of the tissue adaptation program during recirculation. Our findings provide insights into the biology of human tissue Treg cells, which may help harness these cells for therapeutic purposes.

Abstract Therapeutic antibodies modulating the immune response are frequently specific for human proteins but fail to cross-react with the corresponding rodent homologs. Hence, their preclinical development requires suitable “humanized” mouse models, i.e. mice bearing a human immune system, so that immunotherapies can be evaluated in a more relevant immune context in vivo. Such “humanized” mice are developed by reconstituting the immune system of immunodeficient animals with human immune cells from 2 possible origins: CD34+ hematopoietic stem cells (huCD34+ mice) isolated from cord blood, or mature peripheral blood mononuclear cells prepared from buffy coats (huPBMC mice). According to the compound to be tested and the goal of the research, one model may be preferred over the other. HuCD34+ mice exhibit multilineage immune populations allowing the evaluation of immune checkpoint inhibitors. We developed the human A375 melanoma model in huCD34+ NCG mice to evaluate the combination strategy of relatlimab (anti-LAG-3 antibody) and nivolumab (anti-PD-1 antibody) approved at the beginning of 2024 as a first-line treatment for specific melanoma indications. Anti-PD-1 single treatment tended to, albeit without statistical significance, decrease tumor growth compared to isotype control group while anti-LAG-3 alone had no impact on tumor growth. The combination strategy induced a significant decrease in tumor growth compared to single agents in line with the data observed in the RELATIVITY-047 clinical trial (Tawbi H., NEJM, 2022). We also developed huPBMC mice that exhibit exclusively T-cell lineage and are suitable option to evaluate T-cell engagers like the CD3xEpCAM bispecific antibody, solitomab. We observed a dose-dependency between the dose of PBMC injected and the obtained level of human immune cell reconstitution. We observed that the level of reconstitution did not impact engraftment or tumor progression of the A375 melanoma or the BT474-clone5 breast cancer model, and conversely, that the tumor growth did not impact T-cell reconstitution. Finally, in the BT474-Clone5 model, we confirmed a dose-dependent efficacy of solitomab in controlling tumor growth. Moreover, solitomab efficacy was further potentiated by combination treatment with an Evotec proprietary co-stimulatory bispecific antibody. Mice humanized for the immune system exhibiting either multiple lineages (CD34+ huNCG) or just T-cell lineage (huPBMC) proved themselves suitable tools to assess non-mouse cross-reactive anti-tumor immunotherapy assets. Expertise in handling such models and deep knowledge in their benefits and limitations are required to selecting the most valuable model to perform preclinical studies predictive of human therapeutic response. Citation Format: Lise Pasquet, Alice Marchand, Marion Mars, Sophie Chabot, Frédérique Dol-Gleizes, Sébastien Tabruyn, Dan Georgess, Mélanie Pichery, Thomas Eden, Welbeck Danquah, Gerhard Niederfellner, Pascale Lejeune. Mice humanized for the immune system as validated tool for therapeutic antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1290.

Huntington’s disease (HD) arises from a CAG expansion in the huntingtin (HTT) gene beyond a critical threshold. A major thrust of current HD therapeutic development is lowering levels of mutant HTT mRNA (mHTT) and protein (mHTT) with the aim of reducing the toxicity of these product(s). Human genetic data also support a key role for somatic instability (SI) in HTT’s CAG repeat – whereby it lengthens with age in specific somatic cell types – as a key driver of age of motor dysfunction onset. Thus, an attractive HD therapy would address both mHTT toxicity and SI, but to date the relationship between SI and HTT lowering remains unexplored. Here, we investigated multiple therapeutically-relevant HTT-lowering modalities to establish the relationship between HTT lowering and SI in HD knock-in mice. We find that repressing transcription of mutant Htt (mHtt) provides robust protection from SI, using diverse genetic and pharmacological approaches (antisense oligonucleotides, CRISPR-Cas9 genome editing, the Lac repressor, and virally delivered zinc finger transcriptional repressor proteins, ZFPs). However, we find that small interfering RNA (siRNA), a potent HTT-lowering treatment, lowers HTT levels without influencing SI and that SI is also normal in mice lacking 50% of total HTT levels, suggesting HTT levels, per se, do not modulate SI in trans. Remarkably, modified ZFPs that bind the mHtt locus, but lack a repressive domain, robustly protect from SI, despite not reducing HTT mRNA or protein levels. These results have important therapeutic implications in HD, as they suggest that DNA-targeted HTT-lowering treatments may have significant advantages compared to other HTT-lowering approaches, and that interaction of a DNA-binding protein and HTT’s CAG repeats may provide protection from SI while sparing HTT expression.

BackgroundThe PET (positron emission tomography) radioligand [11C]CHDI-180R was designed to target mutant huntingtin (mHTT) aggregates in Huntington's disease (HD) brains. It has been validated in preclinical HD models and has demonstrated promising kinetic properties in a first-in-human study.AimIn this investigation, focus was to identify mHTT proteoforms that CHDI-180 binds to, which is crucial for understanding its pharmacology and developing second generation tracers.MethodsProbes with photoreactive chloroalkane tag were designed to selectively isolate mHTT target(s). UV-induced covalent photo-cross-linkage is applied to secure the binding between probe and mHTT. Targeted mHTT proteoform(s) cross-linked to ligand is enriched through covalent capture onto beads. After capture, target-ligand complex is selectively released from beads for characterization by using mass spectrometry, mass photometry (MP), gel electrophoresis (AGERA) and negative stain electron microscopy (nsEM). MP and nsEM have been optimized for analysis of various mHTT proteoforms.ResultsWe show optimization of key steps, including coupling of ligands to beads and linker cleavage. Our results indicate that all 6 PAL ligands can be successfully coupled and cleaved. The extent of coupling and cleavage varied based on applied conditions and ligand investigated. Results from a time course of recombinantly derived aggregated mHTT analyzed by nsEM and MP will be presented.ConclusionsOur studies revealed that nsEM and MP are valuable tools to characterize mHTT aggregates. Optimized methods will be applied to characterize mHTT proteoforms to which CHDI-180 binds and to compare CHDI-180 and other HTT PET tracer target(s) in animals and human post-mortem brain samples.

Intersite communication in dimeric enzymes, triggered by ligand binding, represents both a challenge and an opportunity in enzyme inhibition strategy. Though often understestimated, it can impact on the in vivo biological mechansim of an inhibitor and on its pharmacokinetics. Thymidylate synthase (TS) is a homodimeric enzyme present in almost all living organisms that plays a crucial role in DNA synthesis and cell replication. While its inhibition is a valid strategy in the therapy of several human cancers, designing specific inhibitors of bacterial TSs poses a challenge to the development of new anti-infective agents. N,O-didansyl-l-tyrosine (DDT) inhibits both Escherichia coli TS (EcTS) and Lactobacillus casei TS (LcTS). The available X-ray structure of the DDT:dUMP:EcTS ternary complex indicated an unexpected binding mode for DDT to EcTS, involving a rearrangement of the protein and addressing the matter of communication between the two active sites of an enzyme dimer. Combining molecular-level information on DDT binding to EcTS and LcTS extracted from structural and FRET-based fluorometric evidence with a thermodynamic characterization of these events obtained by fluorometric and calorimetric titrations, this study unveiled a negative cooperativity between the DDT bindings to the two monomers of each enzyme dimer. This result, complemented by the species-specific thermodynamic signatures of the binding events, implied that communication across the protein dimer was triggered by the first DDT binding. These findings could challenge the conventional understanding of TS inhibition and open the way for the development of novel TS inhibitors with a different mechanism of action and enhanced efficacy and specificity.

Abstract Background: Volume depletion, sepsis, major surgery, and nephrotoxins are the most common causes of acute kidney injury (AKI). The classical markers serum creatinine (Scr) and urine output (UO) for the evaluation of kidney function are of limited value in critical ill patients because they reflect an already existing organ dysfunction. Our hypothesis is that the measurement of the functional biomarker Proenkephalin A 119-159 (penKid), which is freely filtered in the glomerulus and is used as a marker for estimating the glomerular filtration rate, contributes to the early identification of patients with subclinical kidney damage. Methods: This was a secondary analysis of the prospective multicenter LifePOC study. We evaluated critically ill patients admitted to the emergency department (ED) with suspected organ dysfunction based on the risk-stratification tool qSOFA , who developed AKI, defined as Scr ≥0.3 mg/dl from baseline, within 72 hours of enrolment. The primary endpoint was evolving AKI after 48 h. AKI after 24 h, AKI after 72 h and 28-day mortality were defined as secondary endpoints. Measurement and main results: Within 48 h, 88 out of 453 patients (19.4%) developed AKI. Patients with AKI showed increased penKid levels at admission in comparison to patients without AKI (111.5 [73.0-247.5] pmol/l vs. 74.8 [47.2-120.4] pmol/l, p<0.001). PenKid was a superior predictor for AKI within 24, 48 and 72 h in comparison to Scr (all p<0.05), and the advantage increased the later the renal events occurred. Regarding 28-day mortality prediction, penKid also outperformed Scr (p<0.05). The observed superiority of penKid persisted if the recently proposed PENK-Crea formula to estimate the GFR was applied and compared to the latest CKD-EPI formula. Conclusions: Early measurement and the trajectory of penKid predicts early AKI and 28-day mortality in patients with suspected organ dysfunction in the ED superior compared to the classical marker Scr. The results indicate that the superiority is attributed to an earlier rise in penKid compared to Scr. Trial registration: The trial was registered in the German Registry for Clinical Trials (DRKS00011188) on 20 October 2016.

BackgroundT-cell immunotherapy with chimeric antigen receptors (CARs) has evolved as part of the standard of care for several hematological malignancies and has transformed the oncology landscape. CAR-T-cell products are traditionally generated from autologous, patient-derived αβT-cells engineered with a CAR for tumor cell targeting. Generating autologous T-cell products for each patient is associated with manufacturing and logistical complexity and high product costs. Furthermore, manufacturing fails in a significant number of cases due to the poor quality and quantity of blood-derived T-cells and restrictions apply in terms of throughput to produce autologous cell products. Consequently, many patients are left without this treatment option, so it is crucial to develop strategies for off-the-shelf T-cell products. Immune cells derived from induced pluripotent stem cells (iPSCs) offer the opportunity to manufacture allogeneic T-cell products with consistently high quality and scalable quantities. Use of iPSCs as a starting material makes it easier to introduce several genetic modifications (e.g. to enhance both cell persistence and tumor infiltration) addressing tumor resistance mechanisms for both liquid and solid tumors.MethodsUsing a validated GMP iPSC line modified with an NY-ESO-1-specific T-cell receptor (TCR) knock-in, we have established a feeder-free differentiation protocol that enables robust production of iPSC-derived αβT-cells (iαβT). Flow cytometry and single cell transcriptome analysis ensured a stringent monitoring of all process stages. To demonstrate functional activity of our iαβT, we performed cytotoxicity and cytokine release assays against tumor cell lines presenting the NY-ESO-1 antigen.ResultsSuccessful knock-in of the NY-ESO-1-TCR-transgene cassette into the iPSC line was confirmed by marker-gene expression. Hematopoietic progenitor cells were induced from knock-in-enriched iPSCs and differentiated into iαβT. During the differentiation process, the T-cell markers CD45, CD5 and CD7 were displayed and cells started to express the TCR. After activation of iαβT, the fraction of NY-ESO-1-TCR-positive cells increased to over 95%. Importantly, iαβT expressed CD8α and CD8β which is crucial for the function of cytotoxic T-cells. Transcriptome analysis validated the efficient differentiation from pluripotent cells towards cells with a T-cell-specific gene expression profile. Co-culture experiments with NY-ESO-1 antigen presenting tumor cell lines confirmed cytotoxic activity of iαβT and their potential to release cytokines.ConclusionsOur scalable iαβT differentiation process enables us to generate CD8+ T-cells that secrete cytokines and show cytotoxic activity, indicating their potential as a promising cell source for TCR-T or CAR-T cancer immunotherapies.

The human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R), a class B G-protein-coupled receptor (GPCR) identified almost 30 years ago, represents an important pharmacological target in the areas of neuroscience, oncology, and immunology. Despite interest in this target, only a very limited number of small molecule modulators have been reported for this receptor. We herein describe the results of a drug discovery program aiming for the identification of a potent and selective hPAC1-R antagonist. An initial high-throughput screening (HTS) screen of 3.05 million compounds originating from the Bayer screening library failed to identify any tractable hits. A second, completely revised screen using native human embryonic kidney (HEK)293 cells yielded a small number of hits exhibiting antagonistic properties (4.2 million compounds screened). BAY 2686013 (1) emerged as a promising compound showing selective antagonistic activity in the submicromolar potency range. In-depth characterization supported the hypothesis that BAY 2686013 blocks receptor activity in a noncompetitive manner. Preclinical, pharmacokinetic profiling indicates that BAY 2686013 is a valuable tool compound for better understanding the signaling and function of hPAC1-R. SIGNIFICANCE STATEMENT: Although the human pituitary adenylate cyclase-activating polypeptide receptor (hPAC1-R) is of major significance as a therapeutic target with a well documented role in pain signaling, only a very limited number of small-molecule (SMOL) compounds are known to modulate its activity. We identified and thoroughly characterized a novel, potent, and selective SMOL antagonist of hPAC1-R (acting in an allosteric manner). These characteristics make BAY 2686013 an ideal tool for further studies.