Immunogens with native conformation and a high-throughput IgG cloning platform are two of the most important key components for high quality lead antibody molecules discovery. DIMABIO’s DimAb® discovery technology platform seamlessly integrates a native transmembrane purification platform (DiMProTM) and a single B IgG cloning system for lead antibody molecule discovery. In this talk, we will elucidate the technical details on DiMProTM and DimAb® single B development procedure. At the end of talk, we will show how we utilized DIMABIO's platforms to successfully develop an anti-GPRC5D CAR construct with superior performance in pre-clinical evaluation for potential MM treatment.

1. Discuss how to produce active GPCR proteins from HEK293 cells.

2. Explain how to use single B IgG cloning system for the monoclonal antibody production.

3. State how to get high quality CAR construct for pre-clinical studies.

If you use a pipette on a daily basis, then this webinar is for you. Pipettes are used to prepare samples, blanks, and standards for a range of analytical experiments. This is why the wrong type of pipette, improper technique, or a poorly maintained pipette can have a negative impact in your data. 

In this webinar we will talk about how different pipetting techniques can influence your pipetting results and review tips and tricks on choosing the right pipettes and adjusting your technique for different liquids and conditions. Finally, we will go through the importance of regular pipette maintenance and calibration to ensure reliability of results and conformance to international standard ISO8655.  

Learning Objectives:

1. Explain why pipetting technique matters.

2. Discuss how to pipette challenging liquids.

3. Analyze the importance of regular maintenance and calibration.

In general, once a receptor drug target has been validated for the treatment of a particular condition, either a high throughput screen is carried out to identify potential hits, or existing drugs or drug candidates are investigated for potential repurposing. Either way, it is beneficial to thoroughly profile the molecular pharmacology of potential drug candidates prior to proceeding towards preclinical development. Do all the candidates have similar affinities/potencies across different binding/signalling assays or is there evidence of bias? Do some candidates result in distinct internalization/trafficking profiling? What does this mean, and do certain profiles correlate with distinct phenotypes observed later in pre-clinical testing? Using such technologies as bioluminescence resonance energy transfer (BRET), it is now possible to carry out profiling in real time and in live cells at a range of ligand concentrations to characterise the ligand binding properties of selected candidates, as well as assessing G protein coupling, arrestin recruitment and the nuances of receptor internalization/trafficking, all particularly relevant for studying the effects on G protein-coupled receptor pharmacology. Receptor tyrosine kinase receptors can also be investigated, utilising proximity to molecules such as Grb2. Heteromerization has considerable potential for modifying receptor pharmacology and so understanding how drug candidates are influenced by heteromerization of the receptor target with other receptors may well also be important.

Learning Objectives:

1. Explain how molecular pharmacology approaches can be used to profile potential drug candidates prior to preclinical development.

2. Explain how receptor internalization/trafficking can be investigated using bioluminescence resonance energy transfer.

3. Explain how heteromerization can influence receptor pharmacology.

Antibody-based therapeutics are helping patients across a wide range of disease areas, from cancer, such as Rituximab treatment of CD20-positive non-Hodgkin's lymphomas, to infectious diseases like SARS-CoV-2 and HIV. Conventional techniques used in antibody discovery workflows are often limited due to low-throughput sample acquisition, large sample volume requirements, and challenges around analyzing complex datasets. The development of novel antibody therapeutics relies on high-throughput technologies with powerful analysis capabilities that can reliably screen and profile drug candidates.

Learning Objectives:

1. Discuss combining the iQue® Advanced Flow Cytometry and Octet® BLI Label-Free Detection platforms to characterize antibodies during drug discovery.

2. Discuss using the iQue® and associated reagent kits for high-throughput analysis of live-cell antibody binding, competitive binding and Fc function

3. Discuss the determination of ligand and Fc-gamma receptor binding kinetics through robust measurements with the Octet® system

Learning Objectives:

1. Describe and discuss some of the unique aspect of in vivo studies.

2. Identify specific study practices to ensure high quality study data and animal welfare.

3. Discuss a specific case study that illustrates several of the avoidable pitfalls with these types of studies.

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic disorders affecting 25% of our global population. 20% of NAFLD patients go on to develop Non-alcoholic steatohepatitis (NASH), the most severe form of NAFLD, which can result in cirrhosis and liver cancer if left untreated. NASH is characterized by lipid accumulation, infiltration of immune cells, and liver fibrosis. Despite continued research and development efforts, there are currently no approved drug treatments for NASH. Traditional preclinical tests, mainly based on in vivo animal models, have continuously failed to predict human drug efficacy as they inadequately recreate the complexity and multifaceted nature of this human disease. There is a relevancy gap between preclinical models and humans, which human organ-on-a-chip models can address.

Building more predictive, human-relevant models is crucial to successfully bringing efficient anti-NASH therapies to the market. Microphysiological systems (MPS) recapitulate key aspects of human organs’ phenotype, architecture. MPSs extend culture longevity up to a month, enabling studies that were not previously possible, such as the ability to profile phase I and II drug metabolism, mimic key aspects of human diseases, identify potential metabolite-driven toxicity, or detect drug efficacy. In this presentation, we will discuss the use of a liver MPS to model different aspects of clinical NASH and its use to investigate the efficacy and mechanisms of actions of novel NASH therapeutics. 

Learning Objectives:

1. Identify preclinical drug development challenges for NASH studies.

2. Examine a liver MPS model for investigating NASH.

3. Demonstrate how this NASH liver MPS provide translatable insights into drug efficacy for NASH therapeutics.

Ninety percent of therapeutic agents reaching clinical trial fail to reach entry to market, with safety concerns being a  major cause for failures in phase I and II clinical trials. Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure in both Europe and US and is also one of the leading causes for cessation of drug development. This is predominantly due to traditional preclinical models failing to adequately predict human responses to a drug . Moreover, Drugs with human-specific modes of action, such as ASOs or siRNA, are less well served by in vivo animal models due to their human-specific modes of action.

Incorporating more predictive, human relevant models into drug development workflows is key to reducing drug attrition. Microphysiological systems (MPS), also known as organ-on-a-chip (OOC), bridge the gap between preclinical testing and the clinic. MPS enable us to better recapitulate human physiology via the use of human cells and circulating flow to simulate the blood stream. Yet, without the supply of good quality cells, validated for use in 3D assay formats, the advantages of MPS over current preclinical models can't be fully realized. This presentation will demonstrate the clinical predictability of a Liver MPS using OOC-validated liver cells and a validation set of mildly to severely hepatotoxic compounds, plus two new drug modalities (ASOs).

Learning Objectives

1. Discuss a Liver MPS for drug safety testing.

2. Demonstrate the critical need for 3D cell validation to reliably predict DILI.   

3. Explain the ability of MPS to improve safety assessment predictions earlier in drug development workflows.

G Protein Coupled Receptors (GPCRs) are therapeutic targets for many medications used to treat a variety of disease states. A large number of genetic variants have been identified in the genes coding for GPCRs and functional significance of these genetic variants continues to be explored. Changes to receptor function have already been associated with many of the identified genetic variations of these receptors. The effects of these GPCR genetic variants on the interaction of these receptors with the drugs that target them is an area of interest. Some changes in GPCR response to drugs in the presence of genetic variations of the receptors have been identified, but the application of GPCR pharmacogenomics in clinical practice is not yet fully realized. This is due to the complex nature of the associations between receptor genetic variability and drug response. More data is needed to fully understand the significance of GPCR genetic variants in patient responses to medications. With more information, GPCR pharmacogenomics may eventually prove to be useful in personalizing  medication use. Some current areas of GPCR exploration include medications used in cardiovascular disease and in some psychiatric condition. The current state of pharmacogenomics application in these areas of clinical practice will be discussed.

Learning Objectives:

1. Describe how GPCR variants may affect receptor function.

2. Describe how these genetic variants may affect drug response.

3. List GPCR genetic variants that are being evaluated for their potential clinical impact.

Novel immunotherapy has revolutionized the landscape of cancer therapy in multiple tumor types since Ipilimumab, the first ICB agent, was approved for the treatment of metastatic melanoma is 2011. Growing evidence has revealed the importance of TME and how it may impact the response to cancer immunotherapy. The dynamic and spatially heterogeneous TME is made up of malignant and nonmalignant cells such as endothelial cells, cancer-associated fibroblasts, immune cells in addition to vascular and lymphatic networks and the extracellular matrix. It is well established that a comprehensive characterization of the TME is needed to identify prognostic and predictive immune biomarkers. To this end, it has been demonstrated that the deep profiling and spatial characterization information provide by multiplex immunofluorescence (mIF) platform is a powerful tool. 

MultiOmyx is a proprietary mIF platform for the visualization and characterization of up to 60 protein biomarkers in a single FFPE section. Herein we reported to use different MultiOmyximmunoncology (IO) panels to characterize the TME in variety of tumor indications. Using these IO panels in combination with proprietary deep-learning based image analysis, we successfully studied the expression and spatial distribution of different subtypes of immune tumor infiltrates and immune modulators within the TME. In addition, the MultiOmyx panel also enables the detection of spatial morphological structures such as Tertiary lymphoid structures (TLS). Using MultiOmyx assay, we quantified TLS’s in 40 CRC samples and classified them by maturation stage based on biomarker expression and spatial organization of the immune markers.

The MultiOmyx assay provides a powerful tool to characterize the cellular composition and spatial organization of the TME. The rich datasets generated by the MultiOmyx assay can provide greater understanding of the immune contexture within the TME and deeper insights into the correlations between biomarkers. These findings may have broad application and help identify biomarker signatures with improved predictive performance to the efficacy of immunotherapeutics.

Learning Objectives:

1. Explain what a multiplex immunofluorescence (mIF) assay is and its advantages.

2. Describe spatial characterization of tumor microenvironment (TME) using mIF in combination with AI-based imaging analysis.

3. Explain using mIF and IA to support Immuno-oncology drug discovery.

Advances in tissue-based technologies over the past few years have created opportunities to identify patterns that can be used as biomarkers and prognostic indicators for different disease states. With the use of multiplex immunofluorescence (mIF) assays, the field has gone beyond visualizing a single protein or  cell type on a tissue section and there is now room for greater complexity of image analysis. mIF can not only allow complex phenotyping of multiple cell types on a single tissue section, but the spatial relationships within the tissue can be both visualized and quantified. The incorporation of same slide H&Es, can even further increase the amount of information present on a single tissue section. This technology offers a unique advantage of preserving the architectural features of the tissue and revealing the spatial relationships that allow a detailed characterization of specific cell phenotypes defined by co- or lack of expression of multiple markers that may help in predicting clinical responses and mechanisms of resistance to treatment.

Learning Objectives:

1. Examine the concept that both spatial relationships and cell phenotypes can influence factors such as patient outcome and response to treatment. 

2. Demonstrate how advanced image analysis software can be applied to discover cell types and populations within a morphological and spatial context.

3. Discuss real world examples of how whole slide image analysis can give insight into the immune environment and spatial relationships.

In this project, we optimized a high-throughput fluorescent-based assay to assess transporter activity by measuring uptake of the substrate, ASP+  4-(4-(dimethylamino)styryl)-N-methylpyridinium. ASP+ is the fluorescent derivative of the neurotoxin MPP+, both of which have been used to study various transporter properties. The ASP+ microplate method offers an easily teachable platform for pharmacy, graduate, or undergraduate students to perform meaningful, short-term projects. Confocal microscopy and radioligand binding techniques are informative, but are relatively low throughput, require extensive training, expensive equipment, and dedicated laboratory space, or can generate considerable hazardous risks. Our current work focuses on organic cation transporters (OCTs), proteins expressed in the brain and throughout the body that have vital roles in normal physiology and drug responses. OCTs have a low-affinity and high-capacity to transport substrates across the cell membrane, and can quickly take up a broad range of compounds that include pharmacological drugs, endogenous neurotransmitters like dopamine, norepinephrine, and serotonin, and experimental substrates like ASP+. OCT research is limited by the lack of selective ligands for each of the three OCT subtypes. Our initial data, generated by collaborators at RTI International, used an optimized ASP+ uptake for high-throughput screens (measuring 96 samples simultaneously). The goal of this collaborative project is to identify experimental selective inhibitors to propel the study of broadly expressed OCTs. Using human embryonic kidney cells that overexpressed human OCT3, nearly 1500 compounds were screened with the ASP+ uptake model and 105 compounds (used at 10 µM concentrations) were found to inhibit ASP+ uptake similarly to other known OCT3 inhibitors. Ongoing ASP+ experiments are screening the new OCT3 compounds to determine their IC50 values and activity at subtypes OCT1 and OCT2. The ASP+ microplate reader assay offers a rapid, adaptable, economically and environmentally favorable means for studies to identify novel selective ligands, and evaluate the impact of physiological environment, or genetic variations on the function of OCTs and other monoamine transporters.

Learning Objectives:

1. Define the fluorescent molecule, types of transport proteins, and expression system used in the study.

2. Identify 3 differences between radioligand binding and fluorescent substrate microplate detection. 

3. Outline how ASP+ can be used as a preclinical tool to screen transporter inhibitors using high throughput, microplate detection.

Many current technologies used in drug discovery and preclinical development programmes typically involve multi-step operations, extensive hands-on time, and lengthy liquid handling processes as well as low-throughput bioassays for functional validation and verification. We will discuss how microfluidic devices, with well-defined microchannel geometries and accurate flow control, can be used to generate uniform picolitre volume droplets (picodroplets) for use as miniaturized “test tubes” for high-throughput bioassays. 

Picodroplets can be used in a diverse range of applications. An important application of picodroplet microfluidics is single-cell analysis, in which single cells (and their corresponding secreted molecules) are encapsulated in a volume that is 100,000s of times lower than a typical-bulk volume method. This not only enables the rapid detection of secreted molecules and high-throughput sample processing but also time- and cost-effective screening compared to conventional assays.  More importantly, we have demonstrated that cells remain viable and functional in the picodroplets for several days, making the technology the ideal tool for a wide range of bioassays and research projects. 

Picodroplet-based technology can isolate and screen various types of cells, including bacteria, yeast, Chinese hamster ovary cells, HEK cells, primary B and T cells, and even stem cells. We will demonstrate how novel picodroplet bioassays can be used in a wide range of applications including biotherapeutic discovery, cell line development, cell therapy research and quality control, genome engineering (comprising CRISPR techniques) and synthetic biology.

Learning Objectives:

1. List the main categories of dispersed droplets, types of droplet generation, and manipulation of droplets.

2. Clarify the main advantages of droplet microfluidics.

3. Explain the workflow of an antigen-specific FRET assay in picodroplets for human plasma cells.

Novel annotated Patient-Derived tumor Xenografts (PDX) propagated in immunodeficient mice and Organoids (PDO) in Matrigel have become essential models to study inter- and intra-tumor heterogeneity, drug sensitivity and mechanisms of drug resistance. The establishment and utility of these models are shown from work with researchers at the Princess Margaret Cancer Centre (PM) and the PM living biobank, Toronto, Canada. Collectively, we have established over 1000 PDX models and 200 PDO models derived from tumors including lung, mesothelium, pancreas, bile duct, duodenum, esophagus, colon, breast, ovarian, and head and neck. Non-small cell lung cancer (NSCLC) is our largest clinically annotated and proteogenomics profiled set, which includes adenocarcinomas characterized by drug targetable alterations EGFR, ALK and KRAS. These models were derived from patient tumors with varying successes dependent on tumor cellularity of different tumor sources including fluids, biopsies and resections, as well as tumor stage and treatment exposure. These PDX models with activating mutations mirror patient tumors in targeted therapy responses including initial sensitivity, followed by an intermediate drug tolerant persister cell phase, and eventually developing resistance. Preliminary organoid drug sensitivity results show reliability of targeted drug effects on models with activating mutations. We continue to use PDX/PDO systems in parallel to evaluate their utility in recapitulating their parent tumor genotype and phenotype relationships. These models have allowed us to track drug action during treatment phases which are otherwise clinically challenging to monitor. 

Learning Objectives:

1. Apply current practices for PDX/PDO studies.

2. Describe experimental design and utility of PDX/PDO to study drug sensitivity and resistance mechanisms.

3. Identify resources to access models and support for PDX/PDO methodology.

Marine organisms produce a wide variety of primary and secondary metabolites with unique scaffolds that are biologically active. Manzamines are marine-derived polycyclic alkaloids. Approximately 100 manzamine-type alkaloids have been isolated from 16 marine sponge species, and some of these compounds are reported to have anti-inflammatory and neuritogenic properties. The goal of this study was to screen a subset of the manzamine compounds to identify novel neuroactive marine natural products for potential neuroscience drug development.  Calcium activity plays an important role in neurite outgrowth and connectivity, so we screened using FLIPR high-throughput assays. Minimal effects were observed on frequency and amplitude of calcium oscillations at 10 micromolar concentration, suggesting the manzamines do not directly modulate ion channels. Initial neurite outgrowth screens identified seven of the 21 compounds evaluated as potentially neuritogenic, based on total neurite outgrowth and complexity. Neurite outgrowth assays identified the following compounds for further analyses: manzamine A, 8-methoxymanzamine A, 12-propoxymanzamine A, 12-isobutoxymanzamine A, ATL 2-97, ircinin-1, and palinurin. Currently, we are assessing dose-response curves in neurite outgrowth assays for these compounds. Future directions include live-dead assays with propidium iodide and fluorescein diacetate using a dose-response curve to assess for neurotoxicity, and further analysis on potential pathways that may be contributing to neuritogenesis. Together, these studies will help identify novel neuroactive manzamine compounds for the marine natural products preclinical pipeline.

Learning Objectives:

1. Describe the drug development process and timeline.

2. Identify challenges associated with neuropharmacological drug development. 

3. Discuss how the marine environment contributes to unique metabolite structure.

4. Discuss upcoming marine natural products in preclinical and clinical use. 

The Human Neutrophil Lipocalin (HNL) also called NGAL, Lipocalin 2 etc. is produced by and secreted from epithelial cells and neutrophil granulocytes. In blood HNL exists in several molecular forms i.e. monomeric, dimeric and heteromeric forms. The latter form often in complex with MMP9. We purified dimeric HNL from human neutrophils and raised polyclonal and monoclonal against this native dimeric antigen. This procedure resulted in several unique antibodies which in combination uniquely detect the dimeric protein originating from neutrophils or the monomeric protein originating from epithelial cells. Several clinical studies were conducted and showed the immunoassays to be very useful diagnostic tools in the distinction between bacterial and viral infections, sepsis, sepsis with organ failure, sepsis monitoring, COVID-infections, acute kidney injury (AKI) and IBD/IBS. Depending on the disease in question and on the biological material obtained from the patient different formats of the HNL assays were used in order to optimize the clinical performance. In my presentation I will focus on the use of HNL immunoassays in infections and AKI.

Learning Objectives:

1. Discuss the fact that HNL exists in several different molecular forms with differences in cellular origins.

2. Explain how differences in antibody configurations of the HNL immunoassays identify different molecular forms of HNL and the impact on the clinical utilities of the assays.

3. Explain why depending on clinical question HNL is best measured after whole blood activation or in EDTA-plasma or in serum, which has been prepared in a standardized fashion, but also in urine and feces.

4. Conclude that the main clinical applications of HNL currently known are related to infections (bacterial-viral distinction and sepsis) and kidney injury, but more are to be discovered.