ELRIG-Forum 2021: Abstracts
Katrin Beeman, Merck KGaA, Darmstadt
The quantification of gene expression is an essential step to understand the function of a gene in a physiological context. In recent years transcriptomic solutions have been developed to investigate the gene expression levels of different tissues and cell types associated with diseases e.g. oncology. Bioinformatic tools have been heavily used for the identification of biomarker and the identification of novel drug targets. Based on these investigations a specific gene expression analysis is frequently part of an early assay cascades within drug discovery projects. Therefore, we at Merck decided to implement a high throughput RT-qPCR (=Reverse transcription-quantitative real-time) workflow to address higher demands on early compound profiling and screening application. The presentation describes different assay approaches, automation solutions and application examples. Additionally, another application for high throughput RT-qPCR emerged due to the circumstances early 2020, when the fight against the COVID-19 pandemic started. We at Merck identified the synergies of the existing automated RT-qPCR workflow and adapted it to the gold standard for SARS-CoV-2 virus testing.
Martin Lange, Nuvision Innovation Campus Berlin
This study describes the identification and target deconvolution of novel small molecule inhibitors of oncogenic YAP1/TAZ activity with potent anti-tumor activity in vivo. A high-throughput screen of 3.8 million compounds was conducted using a cellular YAP1/TAZ reporter assay. Target deconvolution studies, including cellular thermal shift assays and CRISPR/Cas9-KO screens identified PGGT1B, a subunit of the geranylgeranyltransferase‑I (GGTase‑I) complex, as the direct target of YAP1/TAZ pathway inhibitors. The novel small molecule inhibitors blocked the activation of Rho-GTPases at the cell membrane, leading to subsequent inactivation of YAP1/TAZ. YAP1/TAZ target genes AXL/CAV1 identified as selection biomarker candidates based on cell line response profiles. Multi-parameter optimization resulted in BAY 2814593, an in vivo probe with favorable in vivo PK properties, which demonstrated potent anti-tumor activity and blockade of YAP1/TAZ signaling in vivo.
Janina Caspar, Biontech, Mainz
T-cell receptor (TCR) engineered T cells offer an attractive option for adoptive cell therapy of various advanced cancer types. However, due to a low frequency of reactive T-cell clones, TCR-discovery from patient and healthy donor material still represents a massive bottleneck, especially for personalized TCR therapy approaches. One potential solution might be the high throughput validation of isolated and cloned TCR sequences. In order to develop therapeutic TCR products, a rapid validation of a plethora of TCRs is urgently needed. Validation of TCRs using primary PBMC derived T cells is associated with many challenges and is very time consuming. Therefore, we use an NFAT-reporter encoding effector T-cell line that helps us to overcome these difficulties and allows further assay optimization. Moreover, we customized a robotics-based automation assay platform, comprising all necessary experimental steps and devices. This high throughput TCR-validation platform, which is more cost effective and requires less hands-on time, currently enables the validation of up to 80 TCRs against 20 different tumor targets per week.
Marion Nikolas Gaeve, Fraunhofer IPA, Stuttgart
For the past decades, cell based assays and animal models have represented the gold standard test systems for fundamental scientific research and pre-clinical validation of novel therapeutic agents. Despite their crucial role in these fields, both systems harbor individual disadvantages in the establishment, maintenance, application and relevance and are often only partially adequate for implementation.
Organoids represent a novel, uprising test system by combining the advantages of both standards. The microspheres typically consist of human pluripotent stem cells or patient-derived tumor cells developed into specific organ-like tissues within days to weeks. The cells naturally aggregate and grow in 3D formation, and the resulting organoids can be subsequently used, e.g. for high-quality research or drug screening. Organoids resemble a complex environment similar to human in-vivo tissues with superior species and physiological relevance compared to conservative 2D cell based assays or animal models.
Lukas Königer, Universitätsklinikum Würzburg, Lehrstuhl Tissue Engineering und Regenerative Medizin
The discovery and design of specific drugs and therapeutic agents are major drivers for increasing investment in healthcare worldwide. The costs for their development are mainly determined by the expenditures of the clinical phase. On average, only one in ten drug candidates passes clinical trials despite having shown good results in the preclinical phase. One strategy to reduce this discrepancy in the drug evaluation might be to use in vitro testing systems with improved predictive power in research before a drug candidate is used in clinical trials. Already two-dimensional (2D) cell culture enables the evaluation of drugs and their impact on human cells of the tissue of interest. However, a monolayer of human cells does not resemble the complexity and functionality of a whole organ or tissue. Thus, essential tissue functions are not represented in a 2D cell culture.
