Drug Discovery 2019

Ageing is a complex process that has been observed in all biological systems at every level of organisation. Some anti-ageing interventions have demonstrated life-extending effects in model organisms. However, the translation of these interventions in human clinical practice remains limited due to the absence of comprehensive ageing biomarkers. Recent studies suggest that a set of biomarkers, rather than any individual biomarker, constitute the most effective means of assessing the health status. The presentation will cover the development of comprehensive and robust biomarkers of ageing using deep learning and blood biochemistry, transcriptomics, and even imaging data, to be able to track the effectiveness of the various interventions we are developing.

Learning Objectives: 

1. Different AI Approaches to aging biomarker development
2. How AI can revolutionise aging research and how aging research can accelerate AI research

The last two decades have seen an explosion in the volume of oncology data generated using next-generation sequencing (NGS) and multi-omics techniques. As a result, there is a growing need for computational tools that are powerful enough to generate meaningful hypotheses about the biological mechanisms underlying cancer. In this presentation, we demonstrate how two of QIAGEN’s bioinformatics solutions – Ingenuity Pathway Analysis (IPA) software and the OmicSoft OncoLand database – can be combined to generate testable hypotheses in an immuno-oncology case study (GSE67501). This study sought to understand the mechanism underlying the failure of anti-PD-1 therapy in advanced renal cell carcinoma patients. The study data, along with tens of thousands of other public-domain datasets (including TCGA, SRA, GEO, GTEx and others), have been reprocessed using OmicSoft’s bioinformatics pipeline, and are readily available in OmicSoft’s OncoLand platform. Analysis of these data in OncoLand revealed that UGT1 family members and immune genes were differentially regulated in responder vs non-responder populations, similar to as described in the original publication. These data were then interpreted in IPA, which has millions of findings from peer-reviewed publications. Immunologic, cell cycle, metabolite transport and solute related pathways and functions were found to be enriched. Using IPA’s analytic and predictive capabilities, along with the public data curated from TCGA and GEO and other sources present in OncoLand, we demonstrate how these QIAGEN solutions can be used to generate hypotheses for mechanism of action, and to discover potential therapeutic targets and biomarkers.

Learning Objectives: 

1. Introduction to databases backing Ingenuity Pathway Analysis and Oncoland
2. Studying potential biomarkers and targets through Oncoland’s data mining and comparison tools
3. Generation of peer-reviewed literature backed hypotheses through Ingenuity Pathway Analysis

The Chain of Custody in our labs is the unbroken link between the objects used in your research: your people, the animal models, the cell lines, reagents, compounds; the lab instruments and devices; the data records your store your data in; the link between the analytics and the data collected. In the Smart Lab – there is no place for uncertainty. SMART requires your research and data to be Specific, measurable, auditable, repeatable BUT, preclinical research is a long way from being SMART.

Learning Objectives: 

1. Manual recording of experimental data, manual input of results data – they leave the process open to human error, transcription issues and  do not provide audit trails and lack transparency. In the late 80,  early 90s there was an often quoted saying in the early days of data science: Garbage in; Garbage out. 
2. There is significant excitement, rightfully so, about Machine Learning and AI. But if you are training your algorithms on poor quality data – really how good are your algorithms? The key strategic advantage of a SmartLab based on digital ID and automated data collection is the data authenticity, accuracy and reliability.  And what you will see is the higher quality of data will drive insights faster. You can even start to move toward near real time discovery of  exceptions, of errors, of trends.

Second harmonic generation (SHG) is a biophysical method that sensitively measures real-time conformational change of biomolecules attached to membranes. SHG has recently been applied to detect protein conformational changes in the presence of biological ligands and small molecules. I will describe implementation of SHG as a primary screening platform to identify novel fragment ligands to oncogenic KRas. KRas is the most frequently mutated driver of pancreatic, colon, and lung cancers; however, there are few well-characterized small molecule ligands due to a lack of deep binding pockets. Using SHG, we identified a novel fragment binder to KRasG12D and then used 1H 15N HSQC NMR to characterize its binding site as a pocket adjacent to the Switch II region. The unique sensitivity of SHG furthered our study by revealing distinct conformations induced by our hit fragment compared to DCAI, a Ras ligand previously described to bind the same pocket, identified by fragment based NMR screening. This study highlights SHG as a high-throughput screening platform that reveals structural insights in addition to ligand binding.

Learning Objectives: 

1. Ras proteins, once considered “undruggable”, are now being subjected to a variety of new chemical and biophysical techniques to identify potential drugs that bins to shallow pockets in the protein
2. The biophysical phenomenon referred to as second harmonic generation (SHG) can be used as a tool for discovering molecules that bind to proteins of interest

Multiplexing IHC is a technique that is increasing in demand as more and more researchers and physicians are being tasked with doing more with less. With multiplexing, you have the advantage to view different targets and their levels of expression in relation to one another. In this presentation, we will review the different technologies and techniques that are currently available to assist with developing a multiplex assay. After a brief overview of the technologies, we will apply some of those technologies and walk through an example of optimizing a multiplex assay. Although digital analysis of multiplexed slides can be an important factor in the interpretation of a multiplexed assay, this presentation will focus on and emphasize the staining aspects of the assay optimization.

Learning Objectives: 

1. Understand the utility and methods for multiplexing
2. Understand the principles behind multiplex assay development

Recent clinical successes in cancer genome-inspired personalized medicine have been a major breakthrough in drug discovery. However, 98% of the patients have an incomplete therapeutic response. These patients with residual disease, progress upon therapy and develop acquired resistance and derive limited survival. Tumors are proficient at developing resistant to therapies, and the molecular mechanisms of therapeutic resistance are critical to pinpointing strategies to prevent these escape routes and to obtain long-lasting clinical responses. Using clinical specimen of disease progression, a majority of genetic alterations have been characterized and shown to co-exists upon acquired resistance, hence targeting single genetic driver would be futile to combat poly-clonal resistance. Therefore, there is an increasing need to find a targetable approach to prevent the evolution of resistance and yield a complete therapeutic response to abrogate the residual disease.

To address this, we have developed the in-vitro models of acquired resistance (AR models). We further used these AR models to identify therapeutic strategies that could overcome resistance. This drug screen revealed that aurora kinase inhibitors exhibited strong synergy with EGFR-TKI to abrogate cell proliferation and induced potent apoptosis. Furthermore, long-term pharmacological inhibition of Aurora pathway forestalled the emergence of resistance, inhibit the growth of residual disease and abrogate the acquired resistance. Since overactivation of aurora pathway was known to cause mitotic errors and polyploidy, we surveyed for mitotic defects induced by EGFR-TKI treatment and in acquired resistant cells. EGFR-TKI treatment resulted in a marked accumulation of errors in centrosome biogenesis, spindle assembly, and chromosome segregation resulting in polyploid cells with abnormal DNA content. These errors were present in acquired resistant lines indicating persistent mitotic stress is a feature of EGFR inhibition. Exogenous overexpression of AURKA or TPX2 in parental cells phenocopied these defects, implying causation. Therefore, we conclude that the abnormal activation of TPX2/AURKA leaves a signature of defects associated with mitotic stress, a prominent feature of genomic instability. Our results indicate that heightened TPX2/AURKA activation is required for tumor cells to transition out of sensitive cell state and persist upon drug pressure. This deregulation creates a highly genomically unstable environment and gives rise to genomic heterogeneity providing a fertile ground for the subsequent survival of fittest sub-clones.  

Learning Objectives: 

1. Define the limitation of therapeutic resistance in drug discovery
2. Design the strategy to overcome resistance
3. Identify basis of resistance evolution

Over the past 25 years surgically dependent animal models have expanded from a small, almost niche area in pharmaceutical research to an important component of most research portfolios.  In this current environment the importance for researchers to understand the effects of anatomic and physiologic differences between the species, the requirement for aseptic technique to avoid confounding complications and the requirement for trained and conscientious surgeons is ever more critical.  This presentation will discuss some of the subtle ways that the performance of a surgical procedure can significantly affect the experimental results, the importance of proper model, species and strain selection and some common mistakes that the author has observed over the past quarter century.

Learning Objectives: 

1. Demonstrate the need for a high level of surgical technique and attention to asepsis, anesthesia and analgesia to provide valid and reproducible data 
2. Describe a methodology to identify, design, and plan a high quality surgical protocol

An aptamer (also known as a synthetic antibody) is a stable DNA, RNA, or peptide ligand that binds with high affinity and specificity to targets such as small molecules, peptides, proteins, biomarkers, cells, and tissues. Typical aptamer affinities are in the nanomolar to picomolar range for several target types.  However, unlike the traditional antibody, aptamers have impressive specificities against target antigens, thereby eliminating cross-reactivity with closely related targets and avoiding false-positive results.  A classic example is an aptamer that exhibits greater than 10,000-fold binding affinity for theophylline over caffeine, which differ from one another in structure by only a single methyl group. Additional benefits of aptamers include: manufacturing costs and time are all lower compared to that of monoclonal antibody production. Once the aptamer (nucleic acid) sequence is known, the aptamer can be easily synthesized using a DNA (oligo) synthesizer.  These aptamer oligos are easy to label with reporters, enzymes, or fluorescent tags. In addition, aptamers can be engineered into molecular switches or apta-switches for signal generation in a drug discovery-based assay or to produce an output in a diagnostic test.

Learning Objectives: 

1. To learn how Aptamers are used in Diagnostics 
2. To learn how Aptamers are used in Drug Discovery

Advanced Cell Diagnostics’ RNAscope products and assay services are utilized widely for tissue-based gene expression analysis in early target validation, preclinical animal efficacy and safety studies and in biomarker development.  RNAscope is clinically validated and performs reliably on routine FFPE archival human tissues and clinical biopsies and any animal model tissues.  Detection limits are at single molecule level, enabling quantitative, cell-specific expression analysis within tissue microenvironments.  There are over 15,000 RNAscope probes in use and over 1,800 publications – more than 2-per-day.  Continuing advances in ACD RNA ISH technology are enable a wide range of applications, including:

-    Multiplexed RNA ISH
-    Dual ISH/IHC for target analysis combined with cell marker staining
-    Isoform-specific assays with exon junction probes
-    Tissue-based gene edit detection
-    Viral RNA and DNA genome detection
-    AAV gene therapy biodistribution analysis
-    CAR-TCR T cell detection and tumor infiltration assays with lentiviral probes

RNAscope technologies are enabling faster and more rigorous tissue-based target and biomarker research with cell-specific, single-molecule sensitive RNA detection in the context of complex disease microenvironments.  The technology, methods and its applications will be illustrated.

Learning Objectives:

1. How RNAscope® in situ hybridization is utilized for tissue-based, cell-specific expression analysis
2. How RNAscope® is enabling gene therapy and cell therapy tissue biodistribution analysis

Surgery is the first line of treatment for Stage III melanoma. Often adjuvant therapy is administered post-surgery, which can include weeks of radiation, chemotherapy, targeted drug therapy, or immunotherapy. Previous preclinical studies suggest that treatment with neoadjuvant immune checkpoint blockade may lead to increased survival compared to adjuvant treatment1. This webinar will cover recent data from parallel but independent clinical trials explored the efficacy of anti-PD-1 and/or anti-CLTA4 combination immunotherapy plus surgery in patients with early stage melanoma2,3. 

In each study, safety of the treatment regimen and evidence of immune activity in response to treatment was evaluated. In addition to standard H&E and immunohistochemistry (IHC) staining before and after treatment each study also employed NanoString’s GeoMx™ Digital Spatial Profiler to perform high-plex proteomic analysis with spatial resolution. GeoMx DSP enabled the discovery of markers of antigen presentation (β2M) and T cells (CD3) present on pretreatment tumor biopsies that correlated with clinical efficacy in each study. Additionally, the responses to therapy in each study were strong enough to merit larger trials and continued investigation of the most effective immunotherapy strategy.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Learning Objectives: 

1. Gain an appreciation for how multiplexed spatial profiling via GeoMx can aid in tumor characterization
2. Understand how GeoMx profiling was used in the 2 studies presented to identify biomarkers of response to immunotherapy and complement other molecular profiling strategies

NanoMIPs, often called “plastic antibodies”, are made using a proprietary self-assembly process where the template molecule is incubated with monomers, each of which is free in solution to adopt its optimal binding position, followed by a polymerisation step. By closely controlling the polymerisation, we have the ability to manufacture discrete nanoparticles typically with a single binding site, or imprint, of the template molecule. Each imprint is the correct “size and shape” for the target molecule and also have the corresponding complementary chemical functionality to enhance binding: H-bond acceptor for H bond donor etc. Once synthesised the nanoMIPs can be collected according to their affinity for the target (typically in the low nM range), or indeed to a selectivity target to meet the needs of the application. Furthermore, having a polyethylene backbone, nanoMIPs are extremely robust and highly resistant to pH, solvents and temperature. It is even possible to autoclave nanoMIPs without loss of function. 

Learning Objectives: 

1. Learn how synthetic polymer based nanoparticles can be developed as an alternative affinity reagent to antibodies.
2. Learn how these nanoMIPs have been applied to a range of different projects from epitope mapping, imaging and sensor development to drug delivery

Studies in drug development and on hematological and solid Tumors with a Thymidine Kinase 1 immunoassay

There are many tumor biomarkers but, they mostly reflect tumor mass or past growth, when what may be more important would be to obtain an indication of future growth and therapy response.

Proliferation biomarkers e.g. KI67, are mostly immunohistological biomarkers which are labor intensive, subjective and only show a small part of the tumor. The ability to measure a proliferation biomarker in an easily available body fluid could reduce this problem and provide new opportunities for studying tumor growth and therapy response. 

Thymidine Kinase 1 (TK1) has the potential to be such as biomarker. 

This presentation will demonstrate how the immunoassay of TK1, using a unique monoclonal antibody directed against the TK 210 epitope (AroCell TK 210 ELISA) can provide new opportunities for the study of tumor growth, therapy response and in-vitro modelling of drug effects.

Thymidine Kinase 1 (TK1) is a key enzyme in DNA synthesis and it is up-regulated in the S phase of cell growth. It is released into the extracellular medium and, eventually, the blood during cell turnover.

TK1 enzyme activity has been used for many years as a biomarker for hematological malignancies but the complexity of the assay, plus the fact that much of serum TK1 is inactive has restricted its use, particularly for solid tumors. However, all TK1 forms express the TK 210 epitope.

In studies on hematological malignancies, the AroCell TK 210 ELISA provides similar information to that of the established enzyme activity assays but in studies on solid tumors, where much of the TK1 is enzymatically inactive, the AroCell TK1 ELISA is more sensitive, without losing specificity.

The AroCell TK 210 ELISA kit can be profitably combined with biomarkers of tissue mass, e.g. CA15-3 increasing diagnostic accuracy.

In in-vitro studies on the effects of anti-cancer drugs, the induction and release of TK1 following drug exposure are similar to those seen in-vivo making the assay of TK1 with the AroCell TK 210 ELISA kit a potentially valuable translational biomarker.

Learning Objectives: 

1. Most current tumor biomarkers provide a linked to tumor mass and/or cell death and are backward looking (past growth).
2. When planning future therapy and making prognoses, biomarkers of current cell growth (proliferation biomarkers) can add valuable additional information.
3. The immunoassay of Thymidine Kinase 1 can be such a biomarker

Too frequently the investigator will provide “scientific” reasons to justify precluding analgesics from experimental procedures that may involve more than momentary pain, often with solid literature citations.  What is too frequently dismissed is the fact that unalleviated pain itself constitutes an uncontrollable experimental variable that may, in many cases, be more impactful than the effect that analgesics could have in the experimental results.  Yes, analgesics may alter immune function as well as certain molecular pathways.  However, pain also alters immune function and has a profound influence on the hypothalamic-pituitary-adrenal axis and therefore the animal’s overall physiology.

Through this presentation, Dr. Perret-Gentil will demonstrate the effect that unmitigated pain may have on research, and by doing so, he hopes to persuade the scientific community to give the issue of pain and analgesics greater consideration before dismissing their use within their study design.  Practical suggestions will be presented for effective control of both physical and mental pain (distress).

Learning Objectives: 

1. Demonstrate the effect that unmitigated pain may have on research outcomes. This effect can be more profound than pain-relieving drugs may have on research data 
2. Provide practical suggestion for the minimization of physical and mental pain

Isolated hepatocytes or their enzymes comprise the basis of most in vitro DMPK and toxicity assays used to predict human hepatic outcomes. Because hepato-specific functions are typically lost in the first hours or days of culture in many in vitro hepatic models, such models are of limited use in instances where meaningfully predictive simulation of a compound’s properties require that the in vitro model maintain such function stably over longer time periods, such as where compounds clear slowly, where metabolites are generated over extended periods of time, where membrane polarization is required for biliary canalicular formation, or where cellular conditions may cumulate after repeated dosing, such as in cholestatic toxicities. To address this limitation, more advanced liver models have been developed and extensively characterized.  These long-term models have been shown to more accurately predict intrinsic clearance, metabolite generation, and toxicity profiles.

Learning Objectives:

1.  Learn about advanced liver models for improved in vitro prediction.  
2.  Learn about the next wave of organ-on-chip with microfluidic advancements.  

Cellular research is optimal when using physiologically-relevant cell phenotypes and genotypes of human origin. This assertion has accelerated the adoption of primary cells, stem cells, and increasingly, patient-specific cells in drug discovery. Over the last five years, technological improvements in three-dimensional (3D) cell culture technology, to better mimic in vivo physiology, have also accelerated, not only in the areas of cancer and neurological research, but also in the assessment of clinical candidates for metabolic and toxicological liabilities. In this presentation, existing, and future, developments in 3D cell culture technology, (including the use of phenotypically-relevant human iPS cells) on fundamental research and drug discovery will be addressed. 

Learning Objectives: 

1. Understanding current status of 3D technologies  
2. Understanding applications to disease modeling and drug discovery

Small size, highly selective nature, flexible chemistry and ease of manufacturing make nucleic acid aptamers attractive for multiple applications in human diagnostics and drug discovery.  In this presentation, we will describe the aptamer selection process and describe key challenges that have been overcome to identify aptamers that selectively recognize small molecule targets.  Finally, we will share examples of aptamers used for biomarker discovery, drug delivery, therapy, treatment monitoring, pharmacokinetics/dosing studies, and diagnosis.

Learning Objectives: 

1. Understand the ways in which aptamers can provide advantages over traditional affinity agents in drug discovery and diagnostic product development processes.
2. Learn about healthcare applications of aptamers for small molecule targets and cell surface proteins. 

Whether you are performing killing assays, immunophenotyping of TILs, characterizing the tumor microenvironment, or investigating immunogenicity, primary cells are essential for the discovery of new therapeutics. Conclusive and relevant assay results depend on reliable sample processing, including cell separation and tumor dissociation. We have developed flexible automated solutions for primary cell isolation from various starting materials to ensure the best fit for every drug discovery program. Join us to learn about the best combination of cell isolation instruments and reagents for your research.

Learning Objectives:

1. Efficient and reproducible automated isolation of primary cells 
2. Use of primary cells in drug discovery assays

Dramatic responses observed with targeted agents in the ‘right’ genomic context have fueled large scale sequencing efforts to identify ‘effective’ treatments for a given patient. In order to expeditiously evaluate a targeted agent against a potential ‘driver’ genetic aberration, so-called Basket trials are conducted. Basket trials enroll patients with multiple tumor types that carry a common genetic aberration and evaluate a single targeted treatment regimen. I will discuss the rationale, design, and challenges in conducting Basket trials.

Learning Objectives:

1. Learn about when to consider conducting a Basket trial and how to design it
2. Learn about the promises and challenges of identifying patients based on genomic aberrations
 

In-vitro epithelial barrier models that are more representative of in-vivo tissues are urgently needed. Here we present extracellular matrix-supported intestinal tubules in a perfused microfluidic platform. These intestinal tubules exhibiting tissue (polarization) markers ErbB1, ErBb2, Ezrin, Glut-2, MRP2 and ZO-1, and crypt-like morphology. Drug-induced toxicity was assessed by apical exposure of the intestinal barriers to model compounds. This microfluidic platform is readily implemented in routine laboratories and allows for recording of response to pharmacological stimuli in real-time using automated imaging techniques. The co-culture capabilities of the platform can be explored to create complex tissue configurations, for example by incorporating mesenchymal and immune cells in the ECM adjacent to the epithelial tubes. In conclusion, intestinal tubules cultured in the OrganoPlate® are suitable for high-throughput toxicity screening, trans-epithelial transport studies, and complex co-culture models to recreate an in vivo-like microenvironment.

Learning Objectives: 

1. Identify challenges in standard in-vitro models 
2. Be able describe the method of seeding, growing and investigating intestinal tubular structures on the OrganoPlate® format 
3. Be able to translate this knowledge in generating high-throughput studies for drug development and discovery

In today's presentation I will give an overview to developing NextGen ADCs. We will discuss everything from the beginning stages of development to the varying ADC combination therapies available. (There are currently 23 ADCs in total being studied in 45 combination trials with 10 different  immune checkpoint modulators).

Learning Objectives: 

1. This presentation will discuss past, present challenges associated with developing antibody-drug conjugates (ADCs), a novel modality of targeted cancer chemotherapy
2. Presentation will also highlight key factors that influence in developing next generation ADCs.