Tutorials | Podium Talks | Solutions Spotlights

Tutorials

Lab of the Future: Automating Best Practices in Protein-Protein Interaction Assay (PPI) Development
Wednesday, Feb 9, 8:30 – 10:00 am
Room #104B
Jen Carlstrom, PerkinElmer

Assay development often involves many manual steps with multiple iterations likely utilizing numerous software programs for protocol development, metadata management, subsequent analysis and visualization. Such manual assay handling, variability in conditions, and disintegrated software tools during assay optimization potentially lead to errors and unreliable data.

Here we report the development of an integrated, automated, and optimally configured end-to-end assay development solution for PPI. Upfront assay planning and protocol development was done through TIBCO^® Spotfire^® which generated a step-by-step transcript for the JANUS^® workstation. The workstation automates the manual steps of the workflow, including pipetting and plate reading, on the VICTOR^® Nivo^™ multimode plate reader. We created a recommendations engine within Signals™ VitroVivo which empowers the user with optimal combination and concentrations of reagents for the assay, as well as data analysis at the back end of the workflow.

Join us to learn more about this integrated and automated systems approach to assay development where we codified best practices for HTRF^® PPI assay development to enhance productivity and accuracy.

How to Build Confidence Earlier to Unleash your Drug Discovery Project
Wednesday, Feb 9, 11:30 - 12:30 pm
Room #104C
Helena Almqvist, Pelago Bioscience AB

Confirmation of target engagement in a relevant physiological environment ensures successful drug discovery and the right project prioritization. By applying CETSA^®, you can make better and more informed decisions at earlier stages in your drug discovery projects. Welcome to learn from the Pelago Bioscience Team on recent developments including:

• CETSA Navigate MS – A targeted proteomics approach for assessment of target engagement of compounds against target proteins without the requirements of detection antibodies.
• CETSA Navigate HT – A paradigm shift in lead generation, an efficient approach for identifying small molecule binders to challenging targets in true physiological settings.
• CETSA Explore – Unbiased proteome-wide target identification for target deconvolution, selectivity assessments, elucidation of the molecular MoA and biomarker discovery.

Nexcelom Bioscience: Large-batch Evaluation of Cell Counter Instrument-to-instrument Consistency for Cell Therapy Applications
Wednesday, Feb 9, 9:00 -10:00 am
Room #104C
Jordan Bell, Nexcelom Bioscience

Cell therapy has been hailed as a medical revolution, and its rise has only increased the importance of accuracy and precision in cell counting. Ensuring the identity, purity, and viability of cell therapy products is critical for increasing efficacy and avoiding potentially harmful side effects. The burden of sample analysis often rests on fleets of automated cell counters, leading to the need for confidence that multiple cell counters will give similar results.

In this work, we compare groups of two distinct cell counting instruments. The first type is a well-established single-sample counter (Cellometer K2), similar to those often found in R&D laboratories. The second type of instrument (Cellaca MX) is a high-throughput cell counter that is often more suitable for a manufacturing environment than its single-sample counterparts.

The consistency of these cell counters is tested using both live cells and fluorescent beads. Jurkat cell samples were used to simulate immunotherapy products and the beads prepared in fixed reference samples to allow the comparison of multiple instruments over extended periods. The beads were used to investigate agreement among sixty Cellometer K2 instruments and thirty Cellaca MX instruments, which we believe is the largest group of cell-counting instruments to ever be involved in a single comparison study.

Our work illustrates experimental approaches suitable for the comparison of multiple cell-counting methods. Consistency was measured among the cell counters, both within groups of similar instruments and between instrument types. This consistency encourages optimism for “future-proof” assays that can survive the transition from R&D to production with minimal adjustment.

Podium Talks

Next-Generation Pooled and Arrayed CRISPR-Cas9 Screens to Identify and Prioritise Drug Targets
Monday, Feb 7, 1:30 - 2:00 pm
Room #205ABC
Divya Malik, Horizon Discovery

CRISPR screening is a transformative technology that utilises the power and precision of CRISPR-Cas9 gene editing to reveal and validate novel drug targets or to study underlying causes of disease and to explore the effect of genetic mutations on drug resistance and patient responsiveness.

Horizon Discovery broadly employs two CRISPR screening formats in target ID and validation studies: pooled and arrayed, which can be utilized to answer a plethora of research questions.

Pooled screens involve introducing a ‘pool’ or mixture of sgRNA into a single population of cells which enables large genome-wide screens. However, these are most commonly performed using CRISPR-knockout, which limits the exploration of biology inaccessible to knockout screening. To address this limitation, we have evolved our pooled CRISPRko screening platform to include CRISPRi (interference) and CRISPRa (activation), which can be used to down- or up-regulate endogenous gene expression, respectively. Moreover, CRISPRi and CRISPRa can be combined to deliver dual loss-of-function or dual direction screening. This strategy enables researchers to explore drug mechanisms of action, enables the identification of novel biomarkers, and can provide compelling targets for the development of combination therapies. Additionally, Horizon has recently demonstrated the amenability of our platform for conducting next-generation in vivo pooled CRISPR screens in patient-derived tumor xenograft PDX mouse models, screens in spheroids/organoids and screens that utilise single-cell analysis.

In arrayed CRISPRko screening, only one gene is targeted per well within multi-well plates using CRISPR libraries containing guide RNAs using the latest sgRNA design algorithms. Arrayed screens are key for functional genomic screening with multiplexed phenotypic endpoints, complex co-culture assay and for the confirmation of hits identified in pooled screens. To address the challenges of scaling arrayed screens, we have developed a range of automated workflows to perform screens in both primary and secondary cells.

Finally, CRISPR-based functional studies in primary immune cells have recently received a lot of attention as it opens the doors to significant scientific discovery in crucial areas of research, specifically in immuno-oncology. However, the widespread application of these screens has been limited due to their extremely technically challenging nature. To address this, Horizon has developed an innovative approach to support the robust screening of primary human immune cells including gene-modified B and T cells. Any potential new target or biological behavior identified in these screens could translate more effectively and predictively from bench to bedside.

Here we present a range of these next-generation pooled and arrayed CRISPR screening platforms and illustrate how each type of screen can be used throughout the drug development pipeline to speed the identification of new therapeutics and reduce the chance of late-stage failures in the clinic.

Large-scale Validation of AlphaLISA SureFire kits to enable Target Engagement Quantification via High-throughput CETSA Screening
Wednesday, Feb 9, 11:00 - 11:30 am
Room #206AB
Benedikt von der Heyde, Pelago Bioscience AB

Validating target engagement of compounds utilizing a high-throughput CETSA® approach can generate valuable leads, discover novel chemistry and drive SAR with the benefit that hits are on the endogenous target within a physiological environment. The development of high-throughput CETSA® assays can be expedited by employing ‘off-the-shelf’ AlphaLISA® SureFire® Ultra kits for detection in a 384-well assay format. Previously, setting up optimal assay conditions for these kits has been limited to single or few targets. This study was conceived to tackle a large-scale kit validation approach, allowing for rapid assessment of many targets investigating whether the proteins are detectable, meltable and shiftable using commercially available tool molecules. The resultant findings allow for a wide overview of which target classes, proteins and tools are amenable ‘off the shelf’ for target engagement assessment. We first set out to determine the optimal cell type(s) in which most targets are expressed. Therefore, all targets were cross-referenced with their expression levels in all available cell lines of the Human Protein Atlas. This resulted in the selection of two cell types, THP1 and U2OS cells, and 46 intracellular proteins to be examined in both cell types in six-point temperature melting curves.

The selected proteins belong to diverse classes and are localized in different cellular compartments. 41 proteins demonstrated good detectability and qualified for full twelve-point temperature melt curves in a single cell line. Having pinpointed the optimal melting range, the proteins were examined for their ability to shift upon target engagement with up to three commercially available tool compounds. A thermal shift was observed for most targets using standard conditions, and a few selected examples were further followed-up with lysate experiments and thermal proteome profiling.

In summary, we demonstrate an approach that allows for rapid validation of large batches of AlphaLISA^® SureFire^® kits to test individual proteins for their ability to be detected, melted and shifted in high-throughput CETSA^®. Further, the newly validated kits were employed to focus on a few selected proteins illustrating the effects of compounds on pathways, and follow-up of selected compounds via thermal proteome profiling.

Solutions Spotlights

Next-Generation High-Performance HCS Image Analysis and Management
Monday, Feb 7, 2:30 - 2:50 pm
Exhibition Theater #2644
Tiao Xie, PerkinElmer

High-content screening and cellular imaging experiments generate huge amounts of image data. With phenotypic screening, cell painting, and 3D imaging which use more complex, physiologically relevant disease models, the volume of data continues to grow. To maximize this valuable data, scientists need a powerful solution for image processing, analysis, management, storage, and sharing of data.

Learn how the new Signals Image Artist^™ image analysis and management platform (launched October 2021) brings together cell imaging data from a wide range of sources and allows scientists to quickly process and analyze cellular images to get answers sooner - reducing time to results significantly.

High-Throughput Drug Discovery of the First Inhibitors of a Novel Oncogene in Cancer
Tuesday, Feb 8 from 2:30 - 2:50 pm
Exhibition Theater #2644
Dan LaBarbera, The University of Colorado Anschutz Medical Campus

Chromodomain Helicase DNA Binding Protein 1 Like (CHD1L), also known as Amplified in Liver Cancer 1 (ALC1), is a chromatin-remodeling enzyme and new oncogene implicated in tumor progression, multidrug resistance, and metastasis in many types of cancer. This presentation will discuss the high-throughput (HTS and HCS) drug discovery and lead validation of the first-in-class inhibitors of CHD1L.

Improving Therapeutic Discovery by Leveraging HT-SPR to Complement AlphaLISA
Wednesday, Feb 9, 12:30 - 12:50 pm
Exhibition Theater #2644
Noah Ditto, Carterra & Jen Carlstrom, PerkinElmer

Therapeutic discovery and development rely on numerous analytical techniques in order to best position candidate drugs for commercial success. Different analytical technologies are utilized for their respective strengths in maximizing the understanding of candidate properties. Reliable FcRn binding assays are used in both the discovery and development of therapeutic antibodies to predict the half-life in vivo. Here we show two orthogonal assays for measuring the binding of antibodies to FcRn. The AlphaLISA FcRn binding assay is a robust, high throughput, no-wash immunoassay that was used to measure relative affinities of therapeutic antibodies to FcRn. We then describe the power of HT-SPR as an orthogonal approach to reinforce AlphaLISA potency findings. The real-time, multiplexed, and label-free nature of HT-SPR enables a diverse range of measures to be obtained from a relatively simple set of experiments. Collectively, these studies highlight how AlphaLISA and HT-SPR can be used in conjunction to facilitate a more thoughtful understanding of therapeutic candidate attributes.