Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody.

Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C-C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740).

Cellenium-a scalable and interactive visual analytics app for exploring multimodal single-cell data.

SUMMARY: Multimodal single-cell sequencing data provide detailed views into the molecular biology of cells. To allow for interactive analyses of such rich data and to readily derive insights from it, new analysis solutions are required. In this work, we present Cellenium, our new scalable visual analytics web application that enables users to semantically integrate and organize all their single-cell RNA-, ATAC-, and CITE-sequencing studies. Users can then find relevant studies and analyze single-cell data within and across studies. An interactive cell annotation feature allows for adding user-defined cell types. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are freely available under an MIT license and are available on GitHub (https://github.com/Bayer-Group/cellenium). The server backend is implemented in PostgreSQL, Python 3, and GraphQL, the frontend is written in ReactJS, TypeScript, and Mantine css, and plots are generated using plotlyjs, seaborn, vega-lite, and nivo.rocks. The application is dockerized and can be deployed and orchestrated on a standard workstation via docker-compose.

Meta-Analysis of Human Cancer Single-Cell RNA-Seq Datasets Using the IMMUcan Database.

The development of single-cell RNA sequencing (scRNA-seq) technologies has greatly contributed to deciphering the tumor microenvironment (TME). An enormous amount of independent scRNA-seq studies have been published representing a valuable resource that provides opportunities for meta-analysis studies. However, the massive amount of biological information, the marked heterogeneity and variability between studies, and the technical challenges in processing heterogeneous datasets create major bottlenecks for the full exploitation of scRNA-seq data. We have developed IMMUcan scDB (https://immucanscdb.vital-it.ch), a fully integrated scRNA-seq database exclusively dedicated to human cancer and accessible to nonspecialists. IMMUcan scDB encompasses 144 datasets on 56 different cancer types, annotated in 50 fields containing precise clinical, technological, and biological information. A data processing pipeline was developed and organized in four steps: (i) data collection; (ii) data processing (quality control and sample integration); (iii) supervised cell annotation with a cell ontology classifier of the TME; and (iv) interface to analyze TME in a cancer type-specific or global manner. This framework was used to explore datasets across tumor locations in a gene-centric (CXCL13) and cell-centric (B cells) manner as well as to conduct meta-analysis studies such as ranking immune cell types and genes correlated to malignant transformation. This integrated, freely accessible, and user-friendly resource represents an unprecedented level of detailed annotation, offering vast possibilities for downstream exploitation of human cancer scRNA-seq data for discovery and validation studies. SIGNIFICANCE: The IMMUcan scDB database is an accessible supportive tool to analyze and decipher tumor-associated single-cell RNA sequencing data, allowing researchers to maximally use this data to provide new insights into cancer biology.

Parallel single-cell and bulk transcriptome analyses reveal key features of the gastric tumor microenvironment.

BACKGROUND: The tumor microenvironment (TME) has been shown to strongly influence treatment outcome for cancer patients in various indications and to influence the overall survival. However, the cells forming the TME in gastric cancer have not been extensively characterized. RESULTS: We combine bulk and single-cell RNA sequencing from tumors and matched normal tissue of 24 treatment-naïve GC patients to better understand which cell types and transcriptional programs are associated with malignant transformation of the stomach. Clustering 96,623 cells of non-epithelial origin reveals 81 well-defined TME cell types. We find that activated fibroblasts and endothelial cells are most prominently overrepresented in tumors. Intercellular network reconstruction and survival analysis of an independent cohort imply the importance of these cell types together with immunosuppressive myeloid cell subsets and regulatory T cells in establishing an immunosuppressive microenvironment that correlates with worsened prognosis and lack of response in anti-PD1-treated patients. In contrast, we find a subset of IFNγ activated T cells and HLA-II expressing macrophages that are linked to treatment response and increased overall survival. CONCLUSIONS: Our gastric cancer single-cell TME compendium together with the matched bulk transcriptome data provides a unique resource for the identification of new potential biomarkers for patient stratification. This study helps further to elucidate the mechanism of gastric cancer and provides insights for therapy.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

Characterization of BAY 1905254, an Immune Checkpoint Inhibitor Targeting the Immunoglobulin-Like Domain Containing Receptor 2 (ILDR2).

The immunoglobulin-like domain containing receptor 2 (ILDR2), a type I transmembrane protein belonging to the B7 family of immunomodulatory receptors, has been described to induce an immunosuppressive effect on T-cell responses. Besides its expression in several nonlymphoid tissue types, we found that ILDR2 was also expressed in fibroblastic reticular cells (FRC) in the stromal part of the lymph node. These immunoregulatory cells were located in the T-cell zone and were essential for the recruitment of naïve T cells and activated dendritic cells to the lymph nodes. Previously, it has been shown that an ILDR2-Fc fusion protein exhibits immunomodulatory effects in several models of autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and type I diabetes. Herein, we report the generation and characterization of a human/mouse/monkey cross-reactive anti-ILDR2 hIgG2 antibody, BAY 1905254, developed to block the immunosuppressive activity of ILDR2 for cancer immunotherapy. BAY 1905254 was shown to promote T-cell activation in vitro and enhance antigen-specific T-cell proliferation and cytotoxicity in vivo in mice. BAY 1905254 also showed potent efficacy in various syngeneic mouse cancer models, and the efficacy was found to correlate with increasing mutational load in the cancer models used. Additive or even synergistic antitumor effects were observed when BAY 1905254 was administered in combination with anti-PD-L1, an immunogenic cell death-inducing chemotherapeutic, or with tumor antigen immunization. Taken together, our data showed that BAY 1905254 is a potential drug candidate for cancer immunotherapy, supporting its further evaluation.

Hepatocyte Growth Factor Receptor overexpression predicts reduced survival but its targeting is not effective in unselected HNSCC patients.

BACKGROUND: MET has emerged as target in head and neck squamous cell carcinoma (HNSCC). However, clinical data on MET inhibition in HNSCC are limited. METHODS: HNSCC biopsies and cell lines were tested for MET activity. The response of cell lines to BAY-853474 was tested in proliferation assays. The prognostic value of MET expression was also analyzed. RESULTS: HNSCC cell lines do not respond to MET inhibition. MET-dependent gastric cancer cell lines have much higher levels of MET expression and phosphorylation than HNSCC cell lines. Clinical samples of HNSCC contain much less MET than responsive models. CONCLUSIONS: No clinical response to MET inhibitors in monotherapy may be expected in unselected cases of HNSCC. Only selected patients with MET amplifications should be treated with MET inhibitors. Patients with increased MET immunoreactivity have shorter overall survival. MET might be useful as marker for the detection of patients with more aggressive types of HNSCC.

Drug2Gene: an exhaustive resource to explore effectively the drug-target relation network.

BACKGROUND: Information about drug-target relations is at the heart of drug discovery. There are now dozens of databases providing drug-target interaction data with varying scope, and focus. Therefore, and due to the large chemical space, the overlap of the different data sets is surprisingly small. As searching through these sources manually is cumbersome, time-consuming and error-prone, integrating all the data is highly desirable. Despite a few attempts, integration has been hampered by the diversity of descriptions of compounds, and by the fact that the reported activity values, coming from different data sets, are not always directly comparable due to usage of different metrics or data formats. DESCRIPTION: We have built Drug2Gene, a knowledge base, which combines the compound/drug-gene/protein information from 19 publicly available databases. A key feature is our rigorous unification and standardization process which makes the data truly comparable on a large scale, allowing for the first time effective data mining in such a large knowledge corpus. As of version 3.2, Drug2Gene contains 4,372,290 unified relations between compounds and their targets most of which include reported bioactivity data. We extend this set with putative (i.e. homology-inferred) relations where sufficient sequence homology between proteins suggests they may bind to similar compounds. Drug2Gene provides powerful search functionalities, very flexible export procedures, and a user-friendly web interface. CONCLUSIONS: Drug2Gene v3.2 has become a mature and comprehensive knowledge base providing unified, standardized drug-target related information gathered from publicly available data sources. It can be used to integrate proprietary data sets with publicly available data sets. Its main goal is to be a 'one-stop shop' to identify tool compounds targeting a given gene product or for finding all known targets of a drug. Drug2Gene with its integrated data set of public compound-target relations is freely accessible without restrictions at http://www.drug2gene.com.

Inferring epigenetic and transcriptional regulation during blood cell development with a mixture of sparse linear models.

MOTIVATION: Blood cell development is thought to be controlled by a circuit of transcription factors (TFs) and chromatin modifications that determine the cell fate through activating cell type-specific expression programs. To shed light on the interplay between histone marks and TFs during blood cell development, we model gene expression from regulatory signals by means of combinations of sparse linear regression models. RESULTS: The mixture of sparse linear regression models was able to improve the gene expression prediction in relation to the use of a single linear model. Moreover, it performed an efficient selection of regulatory signals even when analyzing all TFs with known motifs (>600). The method identified interesting roles for histone modifications and a selection of TFs related to blood development and chromatin remodelling. AVAILABILITY: The method and datasets are available from http://www.cin.ufpe.br/~igcf/SparseMix. CONTACT: igcf@cin.ufpe.br SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Transcription factor binding predictions using TRAP for the analysis of ChIP-seq data and regulatory SNPs.

The transcription factor affinity prediction (TRAP) method calculates the affinity of transcription factors for DNA sequences on the basis of a biophysical model. This method has proven to be useful for several applications, including for determining the putative target genes of a given factor. This protocol covers two other applications: (i) determining which transcription factors have the highest affinity in a set of sequences (illustrated with chromatin immunoprecipitation-sequencing (ChIP-seq) peaks), and (ii) finding which factor is the most affected by a regulatory single-nucleotide polymorphism. The protocol describes how to use the TRAP web tools to address these questions, and it also presents a way to run TRAP on random control sequences to better estimate the significance of the results. All of the tools are fully available online and do not need any additional installation. The complete protocol takes about 45 min, but each individual tool runs in a few minutes.

Predicting gene expression in T cell differentiation from histone modifications and transcription factor binding affinities by linear mixture models.

BACKGROUND: The differentiation process from stem cells to fully differentiated cell types is controlled by the interplay of chromatin modifications and transcription factor activity. Histone modifications or transcription factors frequently act in a multi-functional manner, with a given DNA motif or histone modification conveying both transcriptional repression and activation depending on its location in the promoter and other regulatory signals surrounding it. RESULTS: To account for the possible multi functionality of regulatory signals, we model the observed gene expression patterns by a mixture of linear regression models. We apply the approach to identify the underlying histone modifications and transcription factors guiding gene expression of differentiated CD4+ T cells. The method improves the gene expression prediction in relation to the use of a single linear model, as often used by previous approaches. Moreover, it recovered the known role of the modifications H3K4me3 and H3K27me3 in activating cell specific genes and of some transcription factors related to CD4+ T differentiation.

A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk.

Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 × 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.

TransFind--predicting transcriptional regulators for gene sets.

The analysis of putative transcription factor binding sites in promoter regions of coregulated genes allows to infer the transcription factors that underlie observed changes in gene expression. While such analyses constitute a central component of the in-silico characterization of transcriptional regulatory networks, there is still a lack of simple-to-use web servers able to combine state-of-the-art prediction methods with phylogenetic analysis and appropriate multiple testing corrected statistics, which returns the results within a short time. Having these aims in mind we developed TransFind, which is freely available at http://transfind.sys-bio.net/.

CpG-depleted promoters harbor tissue-specific transcription factor binding signals--implications for motif overrepresentation analyses.

Motif overrepresentation analysis of proximal promoters is a common approach to characterize the regulatory properties of co-expressed sets of genes. Here we show that these approaches perform well on mammalian CpG-depleted promoter sets that regulate expression in terminally differentiated tissues such as liver and heart. In contrast, CpG-rich promoters show very little overrepresentation signal, even when associated with genes that display highly constrained spatiotemporal expression. For instance, while approximately 50% of heart specific genes possess CpG-rich promoters we find that the frequently observed enrichment of MEF2-binding sites upstream of heart-specific genes is solely due to contributions from CpG-depleted promoters. Similar results are obtained for all sets of tissue-specific genes indicating that CpG-rich and CpG-depleted promoters differ fundamentally in their distribution of regulatory inputs around the transcription start site. In order not to dilute the respective transcription factor binding signals, the two promoter types should thus be treated as separate sets in any motif overrepresentation analysis.

PASTAA: identifying transcription factors associated with sets of co-regulated genes.

MOTIVATION: A major challenge in regulatory genomics is the identification of associations between functional categories of genes (e.g. tissues, metabolic pathways) and their regulating transcription factors (TFs). While, for a limited number of categories, the regulating TFs are already known, still for many functional categories the responsible factors remain to be elucidated. RESULTS: We put forward a novel method (PASTAA) for detecting transcriptions factors associated with functional categories, which utilizes the prediction of binding affinities of a TF to promoters. This binding strength information is compared to the likelihood of membership of the corresponding genes in the functional category under study. Coherence between the two ranked datasets is seen as an indicator of association between a TF and the category. PASTAA is applied primarily to the determination of TFs driving tissue-specific expression. We show that PASTAA is capable of recovering many TFs acting tissue specifically and, in addition, provides novel associations so far not detected by alternative methods. The application of PASTAA to detect TFs involved in the regulation of tissue-specific gene expression revealed a remarkable number of experimentally supported associations. The validated success for various datasets implies that PASTAA can directly be applied for the detection of TFs associated with newly derived gene sets. AVAILABILITY: The PASTAA source code as well as a corresponding web interface is freely available at http://trap.molgen.mpg.de.

Statistical modeling of transcription factor binding affinities predicts regulatory interactions.

Recent experimental and theoretical efforts have highlighted the fact that binding of transcription factors to DNA can be more accurately described by continuous measures of their binding affinities, rather than a discrete description in terms of binding sites. While the binding affinities can be predicted from a physical model, it is often desirable to know the distribution of binding affinities for specific sequence backgrounds. In this paper, we present a statistical approach to derive the exact distribution for sequence models with fixed GC content. We demonstrate that the affinity distribution of almost all known transcription factors can be effectively parametrized by a class of generalized extreme value distributions. Moreover, this parameterization also describes the affinity distribution for sequence backgrounds with variable GC content, such as human promoter sequences. Our approach is applicable to arbitrary sequences and all transcription factors with known binding preferences that can be described in terms of a motif matrix. The statistical treatment also provides a proper framework to directly compare transcription factors with very different affinity distributions. This is illustrated by our analysis of human promoters with known binding sites, for many of which we could identify the known regulators as those with the highest affinity. The combination of physical model and statistical normalization provides a quantitative measure which ranks transcription factors for a given sequence, and which can be compared directly with large-scale binding data. Its successful application to human promoter sequences serves as an encouraging example of how the method can be applied to other sequences.

Predicting transcription factor affinities to DNA from a biophysical model.

MOTIVATION: Theoretical efforts to understand the regulation of gene expression are traditionally centered around the identification of transcription factor binding sites at specific DNA positions. More recently these efforts have been supplemented by experimental data for relative binding affinities of proteins to longer intergenic sequences. The question arises to what extent these two approaches converge. In this paper, we adopt a physical binding model to predict the relative binding affinity of a transcription factor for a given sequence. RESULTS: We find that a significant fraction of genome-wide binding data in yeast can be accounted for by simple count matrices and a physical model with only two parameters. We demonstrate that our approach is both conceptually and practically more powerful than traditional methods, which require selection of a cutoff. Our analysis yields biologically meaningful parameters, suitable for predicting relative binding affinities in the absence of experimental binding data. AVAILABILITY: The C source code for our TRAP program is freely available for non-commercial use at http://www.molgen.mpg.de/~manke/papers/TFaffinities/