MSI Analysis and the Application of Therapies Based on 2018 Nobel Immuno-Oncology Work

The 2018 Nobel Prize in Physiology and Medicine was awarded to James P. Allison of the United States and Tasuku Honjo of Japan for their work to identify pathways in the immune system that can be used to attack cancer cells (1). Although immunotherapy for cancer has been a goal for many decades, Dr. Allison and Dr. Honjo succeeded through their manipulation of “checkpoint inhibitor” pathways to target cancer cells.

Immune checkpoint inhibitor drugs have been effective in cancers such as aggressive metastatic melanoma, some lung cancers, kidney, bladder and head and neck cancers. These therapies have succeeded in pushing many aggressive cancers below detectable limits, though these cases are notably not relapse-free or necessarily “cured” (2,3).

One challenge in implementing immunotherapy in a cancer treatment regime is the need to understand the genetic makeup of the tumor. Certain tumors, with specific genetic features, are far more likely to respond to immune checkpoint therapy than others. For this reason, Microsatellite Instability (MSI) analysis has become an increasingly relevant tool in genetic and immuno-oncology research.

What is MSI Analysis?

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What Could You Do with a Faster, More Consistent ADCC Reporter Bioassay?

Fc receptor-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MOA) by which antibodies target diseased cells for elimination. Traditional methods for measuring ADCC require primary donor peripheral blood mononuclear cells (PBMCs) or purified natural killer (NK) cells that express Fc receptors on the cell surface. Killing of target cells is an endpoint of this pathway activation and is used in classic ADCC bioassays.

PBMCs and NK cells are notoriously difficult to isolate and culture. Furthermore, cultured cells can be a source of variability.

There is a Better Way

Watch this video to learn why traditional ADCC assays can be problematic. You’ll also learn a solution. Find out how  to not only save time but also reduce assay variability.

For more details on the benefits of working with ADCC Reporter Bioassays go to the product page.

There you’ll see how standardized reagents in Promega ADCC Reporter Bioassays ensure better results and better consistency in an ADCC Reporter Bioassay that saves you time.

Overcoming Challenges When Scaling Antibody Production

Tradeoffs are a constant source of challenge in any research lab. To get faster results, you will probably need to use more resources (people, money, supplies). The powerful lasers used to do live cell imaging may well kill those cells in the process. Purifying DNA often leaves you to choose between purity and yield.

Robot performing autosamplingWorking with biologics also involves a delicate balancing act. Producing compounds in biological models rather than by chemical synthesis offers many advantages, but it is not without certain challenges. One of those tradeoffs results from scaling up; the more plasmid that is produced, the greater probability of endotoxin contamination.

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Bacteria and Viruses as Cancer Treatments

Over a hundred years ago William B Coley, the “Father of Immunotherapy”, discovered that injection of bacteria or bacterial toxins into tumors could cause those tumors to shrink. The introduction of bacteria had the side-effect of stimulating the immune system to attack the tumor. The field of cancer immunotherapy research—which today includes many different approaches for generating anti-tumor immune responses—originated with these early experiments.

Use of bacteria is one way to stimulate the immune system to attack cancer cells, others include use of cytokines, immune checkpoint blockades and vaccines. This Nature animation provides a simple overview of these methods.

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Factors Influencing Compound Potency in Biochemical and Cellular Assays

Late in 2017, a group here at Promega launched an exciting new assay, the NanoBRET™ Target Engagement (TE) Intracellular Kinase Assay.

It’s easy for me to call this assay exciting; I was an editor on the project team. But judging by the reviews on the SelectScience® web site, others are excited about NanoBRET™ Target Engagement Intracellular Kinase Assay too.

A review of the NanoBRET TE Kinase assay from SelectScience® .

A review of the NanoBRET TE Kinase assay from SelectScience® .

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A Cell Viability Assay for Today

Valued for ease of use and scalability, plate-based, bioluminescent cell viability assays are widely used to support research in biologics, oncology and drug discovery.

Cell viability assays are a bread-and-butter method for many researchers using cultured cells —everyday lab tools that are a part of many newsworthy papers, but rarely make news themselves.

Over time, cell viability assays have become easier to use and more “plug ‘n play”. Among modern assays, luminescent plate-reader based systems have been a favorite for several years because of their superior sensitivity, robustness, simple protocols and uncomplicated equipment requirements (all you need is a plate-reading luminometer). These qualities combine to allow easy scalability and adaptability from bench research to high throughput applications.

CellTiter-Glo® Luminescent Cell Viability Assay is an accepted go-to viability assay for many researchers. The assay measures ATP as an indicator of metabolically active cells. A quick search on Google Scholar returns 3,990 CellTiter-Glo results for 2017 and over 500 so far in January and February of 2018. A sampling of these recent publications gives a snapshot of some of the ways the CellTiter-Glo assay is used to support key areas of research today.

Does a treatment kill cells?

The obvious application of a cell viability assay is to understand whether cells are alive. In cancer research, the CellTiter-Glo assay is often used to confirm killing of tumor cells and to verify that normal cells survive. Therefore, these assays are a key part of the evaluation and screening of drug candidates and other therapies for cancer. Many papers reporting use of CellTiter-Glo are developing and evaluating the effectiveness of novel anti-cancer treatments. Continue reading

Determination of Antibody Mechanism of Action Using IdeS

Monoclonal antibodies (mAbs) have been widely used to eliminate undesired cells via various mechanisms, including antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and programmed cell death (PCD). Unlike the Fc-dependent mechanism of ADCC and CDC, certain antibody–antigen interactions can evoke direct PCD via apoptosis or oncosis. Previously, researchers have reported the specific killing of undifferentiated human embryonic stem cells (hESC) by mAb84 (IgM) via oncosis (1)

In a recent publication (2), a monoclonal antibody (mAb), TAG-A1 (A1), was generated to selectively kill residual undifferentiated human embryonic stem cells (hESC). One of the many experimental tools used to characterize the mechanism of oncosis was the fragmention of the A1 antibody with IdeS and papain.

Papain digestion of IgG produces Fab fragments in the presence of reducing agent. F(ab)2 fragments of A1 were produced using IdeS Protease.

The results indicate that both Fab_A1 and F(ab)2_A1 bind to hESC but only F(ab)2_A1 retained hESC killing. Hence bivalency, but not Fc-domain, is essential for A1 killing on hESC.

  1. Choo, A.B. et al. (2008) Selection against undifferentiated human embryonic stem cells by a cytotoxic antibody recognizing podocalyxin-like protein-1. Stem Cells  26, 1454.
  2. Zheng, J.Y. et al. (2017) Excess reactive oxygen species production mediates monoclonal antibody-induced human embryonic stem cell death via oncosis. Cell Death and Differentiation 24, 546–58.

Further reading about IdeS Protease is available here.

Analysis of a biosimilar mAb using Mass Spectrometry

Several pharmaceutical companies have biosimilar versions of therapeutic mAbs in development. Biosimilars can promise significant cost savings for patients, but the unavoidable differences
between the original and thencopycat biologic raise questions regarding product interchangeability. Both innovator mAbs and biosimilars are heterogeneous populations of variants characterized by differences in glycosylation,oxidation, deamidation, glycation, and aggregation state. Their heterogeneity could potentially affect target protein binding through the F´ab domain, receptor binding through the Fc domain, and protein aggregation.

As more biosimilar mAbs gain regulatory approval, having clear framework for a rapid characterization of innovator and biosimilar products to identify clinically relevant differences is important. A recent reference (1) applied a comprehensive mass spectrometry (MS)-based strategy using bottom-up, middle-down, and intact strategies. These data were then integrated with ion mobility mass spectrometry (IM-MS) and collision-induced unfolding (CIU) analyses, as well as data from select biophysical techniques and receptor binding assays to comprehensively evaluate biosimilarity between Remicade and Remsima.

The authors observed that the levels of oxidation, deamidation, and mutation of individual amino acids were remarkably similar. they found different levels of C-terminal truncation, soluble protein aggregates, and glycation that all likely have a limited clinical impact.  Importantly, they identified more than 25 glycoforms for each product and observed glycoform population differences.

Overall the use of mass spectrometry-based analysis provides rapid and robust analytical information vital for biosimilar development. They demonstrated the utility of our multiple-attribute monitoring workflow using the model mAbs Remicade and Remsima and have provided a template for analysis of future mAb biosimilars.

1. Pisupati, K. et. al. (2017) A Multidimensional Analytical Comparison of Remicade and the Biosimilar Remsima. Anal. Chem 89, 38–46.

Don’t Let These Three Common Issues Hurt Your Luminescent Assay Results

4621CAThere is a lot riding on your luminescent assay results. Each plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate?  Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for.  Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work.  Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments. Continue reading

Widening the Proteolysis Bottleneck: A New Protein Sample Preparation Tool

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

The poster featured in this blog provides background information and data on development of Rapid Digestion-Trypsin.

Improvements in Protein Bioprocessing

As more and more protein-based therapeutics enter research pipelines, more efficient protocols are needed for characterization of protein structure and function, as well as means of quantitation. One main step in this pipeline, proteolysis of these proteins into peptides, presents a bottleneck and can require optimization of multiple steps including reduction, alkylation and digestion time.

We have developed a new trypsin reagent, Rapid Digestion–Trypsin, that streamlines the protein sample preparation process, reducing the time to achieve proteolysis to about 1 hour, a remarkable improvement over existing overnight sample preparation times.

How Does it Work?

With this new trypsin product, proteolysis is performed at 70°C, incorporating both denaturation and rapid digestion. The protocol can be used with multiple protein types, including pure proteins and complex mixtures, and is compatible with digestion under native, reduced or nonreduced conditions.

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