Designing Better Therapeutic mAbs: An Assay for Rapid, Parallel Screening of Fc/ FcɣR Interactions

The first monoclonal antibody (mAb) was produced in a lab 1975, and the first therapeutic mAb was introduced in the United States to prevent kidney transplant rejection in 1986. The first mAb used in cancer treatment the anti-CD20 mAb, rituximab, was used to treat non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. Today therapeutic mAbs have become a mainstay of cancer, autoimmune disease, and metabolic disease therapies and include HERCEPTIN® used to treat certain forms of breast cancer, Prolia used to treat bone loss in post-menopausal women, and Stelara used to treat autoimmune diseases like psoriatic arthritis and severe Crohn disease, among many others. Therapeutic mAbs bind targets with high specificity and affinity and they can recruit effector cells to drive target elimination through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), making them highly specific, effective therapies.

3D rendering of a Lumit Assay which can be used  for plate-based screening assay to measure the affinities of Fc interactions of therapeutic mAbs.
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Behind the Scenes at Promega with the Brazil Young Researchers Award Winners

“What I’ve learned in science is that we don’t do things alone. Everything is connected,” says Marcos da Silva Regueira Neto.

Marcos is a post-doctoral research at the Federal University of Pernambuco in Recife, Brazil. His project is part of a large interdisciplinary study, so he is no stranger to collaboration and welcomes opportunities to gain knowledge outside his specialty. Earlier this year, Marcos travelled nearly 5,000 miles to take advantage of one such opportunity.  

In May 2022, Marcos and eight other young Brazilian scientists spent a week in the United States experiencing a unique behind-the-scenes dive into Promega. Their trip included stops in New York City, Madison, and Chicago. For most of the students, this was their first look into new areas where science could lead them.

“I’ve spent most of my life in academia,” Marcos says. “I want to see the other side – the industry side. I want to learn new things and expand my knowledge.”

Over the course of a week, the students presented their research project to Promega leaders, got hands-on experience with emerging technologies alongside the scientists who invented them, and played with human-sized proteins in a virtual reality space.

They also took a picture with a six-foot duck.

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CCNE1-Amplified Cancers Targeted Through Synthetic Lethality Involving PKMYT1 Kinase

For cancers that have proven challenging to target with traditional therapies, one emerging option is an approach called synthetic lethality. Synthetic lethality arises when inactivation of two gene products together lead to cell death but where inactivation of one does not (1, 2). Targeting a gene that is synthetic lethal to a cancer-related mutation creates an opening to specifically kill cancer cells while leaving healthy cells untouched.

In a recent study in Nature, scientists found that cells with amplification of CCNE1 are sensitive to inhibition of PKYMT1 kinase and identified a small molecule that is a selective inhibitor of PKYMT1 (3). When mice with tumor xenografts derived from CCNE1­-high cell lines were dosed with the drug, researchers observed significantly slower tumor growth, and in some cases where the drug was co-dosed with another chemotherapeutic, tumor growth was completely halted.

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Use of ProAlanase Digestion Increases Number of Identified Methylglyoxal (MGO)-Modified Proteins in Whole-Cell Lysates

space filling structural model methylglyoxal (MGO)
Methylglyoxal is responsible for post translational protein modifications, that result in advanced glycation endproducts (AGEs), which are associated with aging and disease.

Post-translational modifications (PTM) of proteins are essential for the function of many proteins, but aberrant modification of protein residues also can interfere with protein function. PTMs occur in two ways. Proteins may be modified through the activity of enzymes such as kinases, phosphorylases, glycosylases and others that add or remove specific chemical moieties to amino acid residues. PTMs can also result from non-enzymatic reaction between electrophilic compounds and nucleophilic arginine and lysine residues within a protein. Metabolites and metabolic by products produced during glycolysis, especially glyoxal and methylglyoxal (MGO), are examples of such electrophilic compounds. These compounds can react with arginine and lysine to produce advanced glycation end products (AGEs), which are biomarkers associated with aging and degenerative diseases such as Alzheimer disease, diabetes and others. MGO is also elevated in tumors that have switched from oxidative phosphorylation to glycolysis as their main energy production pathway.

Only limited information is available about site-specific MGO PTMs in mammal cells, and most studies have focused on measuring the amount of MGO modifications in a treatment scenario compared to a control. Donnellan and colleagues recently published work to identify specific MGO protein modifications.  They used a “bottom-up” proteomic analysis of WIL2-NS B lymphoblastoid whole-cell lysates to identify specific MGO-modified proteins. In particular, the group was looking for modifications in proteins that might explain how MGO activity contributes to aneuploidy.

For the study, 100µg of cellular protein extract was reduced with dithiothreitol and then alkylated with chloroacetamide. The sample was diluted to reduce urea concentration. Trypsin Gold was added and samples were digested for 8 hours at 37°C. Digestion was terminated by adding formic acid. For ProAlanase digestion, 20µg of protein was reduced, alkylated and diluted to reduce urea concentration before adding digesting with ProAlanase for 4 hours at 37°C.

The authors identified 519 MGO-modified proteins.  Most of the modifications were identified in the trypsin digestion reactions; however, ProAlanase digestion increased the number of MGO modifications identified by approximately 25% (with less than 4% of the modification sites being detected in both the ProAlanase and trypsin digestion reactions. The authors suggest that ProAlanase increased sequence coverage to reveal sites not detected in the trypsin digestions. Therefore, they conclude that ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

ProAlanase can be used along with trypsin digestion to increase the identification of MGO modifications.

MGO-modified proteins from the WIL2-NS whole cell lysates included proteins involved in glycolysis, translation initiation, protein folding, mRNA splicing, cell-to-cell adhesion, heat response, nucleosome assembly, protein SUMOylation and the G2/M cell cycle transition. More work to further characterize the sites of these modifications and their potential effects on the function of the modified proteins is ongoing.


Read more about ProAlanase, a new site-specific endoprotease from Promega.


Literature Cited

Donnelian, L et al. (2022) Proteomic Analysis of Methylglyoxal Modifications Reveals Susceptibility of Glycolytic Enzymes to Dicarbonyl Stress Int. J. Mol. Sci. 23(7), 3689 doi.org/10.3390/ijms23073689

Monkeypox—The Latest Zoonotic Virus Making Headlines

Monkeypox has been making the news lately, and it has a lot of people wondering what it is, how it spreads and if they should be concerned. Understandably, we are all a little jumpy when we start hearing about a new viral outbreak, but monkeypox isn’t new. While the virus gained its unfortunate name from its discovery in monkeys in 1958 (1), it exists in a wide range of mammals including rodents, anteaters, hedgehogs, prairie dogs, squirrels and shrews (2) and can spread to humans through close contact with an infected animal.

Artists rendering of monkeypox on a torso.

A member of the Poxviridae family, monkeypox is closely related to the variola virus that causes smallpox; however, monkeypox causes milder symptoms and is rarely fatal (3). The genetic variant of the virus that is causing the recent outbreaks has a fatality rate of <4% (4). In contrast, smallpox fatality rate was close to 30% (4). Symptoms can include fever, headache, muscle and back pain, swollen lymph nodes, chills and exhaustion (2). The most distinguishing symptom is the blister-like rash.

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Making “Scents” of the Mysterious Science of Plant Odors

Corpse flower in bloom
Photo credit: “titan arum 01” by illumine23 is licensed under CC BY-NC-ND 2.0.

When it comes to plant aromas, we tend to forget that we, as humans, are not the target audience, and these odors were not designed with us in mind—we are really passive spectators to a show that luckily most of us happen to enjoy. This past spring Mother Nature demonstrated just what it means to have a target olfactory audience at Madison’s Olbrich Botanical Gardens. For the first time in about 12 years, one of the four massive titan arum (Amorphophallus titanum) plants that reside at Olbrich bloomed, an event that typically only happens for 24-48 hours at a time and 4-5 times total throughout this plant’s roughly 40 year lifespan. More informally (and aptly) known as the “corpse flower” due to its carcass-adjacent coloring and distinctly foul odor, hundreds of plant enthusiasts and hopeful spectators queued for hours to catch a glimpse and whiff of the pungent plant. Until rare events like this happen, it can be easy to forget just how interesting and complex plants really are. We romanticize and lend meaning to flowers and relish in the sweet fragrance they provide, while often completely overlooking the intricate biological and chemical processes that comprise the science of floral scent.

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The BTC Institute: Serving Youth Skills and Science for Summer

World Youth Skills Day provides a unique opportunity to emphasize the importance of equipping young people with experiences, skills, and opportunities in the workforce. This celebratory day falls on July 15th and was officially declared by the United Nations General Assembly in 2014.

At Promega, we are constantly adhering to invest in the future generations of science—and the BioPharmaceutical Technology Center Institute (BTC Institute) serves this mission best. The BTC Institute is a non-profit organization that provides educational, scientific, and cultural opportunities for people of all ages. Each summer, the organization hosts a wide range of experiences including camps, programs, and field trips to support individuals interested in science. In the spirit of World Youth Skills Day, let’s take a look at some experiences that are offered for young learners in summer 2022.

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Promega Highlights Innovative Work with Brazil Young Researcher Award


In late May 2022, Promega invited the nine finalists for the Promega Brazil Young Researcher Award to present their work at a Student Research Symposium on the Promega Madison campus.

Scientists from around Brazil recently traveled to Madison, WI, USA as part of the Brazil Young Researcher Award

The Brazil Young Researcher Award program was created to acknowledge exceptional work by Brazilian students utilizing Promega products in their research. These student researchers were recognized for their achievements and were given the opportunity to present their innovative research to Promega scientists as part of a week-long immersive experience on the Promega campus.

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PROTAC Virus Vaccines: A New Approach to Vaccine Development

Vaccine research and development is a major area of focus for life scientists across the globe. Clinical trials have shown that vaccines that target tumors show promise for cancer treatment. Additionally, the emergence of new zoonotic diseases has revealed a need to develop vaccines quickly as the world becomes more global and human populations interact more often with each other and wild habitats. Importantly, these vaccines need to be suitable for distribution in a variety of settings, including those that do not have easy access to refrigeration.

Influenza Virus. Si et al used influenza as a model to engineer and test PROTAC Virus vaccines

There are many ways to classify the different types of vaccines that are currently available. The National Institute of Allergy and Infectious Diseases in the United States, categorizes vaccines as: whole pathogen vaccines, subunit vaccines, and nucleic acid vaccines—based on how the antigen that stimulates the immune response is delivered to the host.

Whole-pathogen vaccines, which include many of vaccines used in clinical settings, use the entire pathogen (organism that causes the disease) that has been either weakened or killed to elicit a protective immune response. Killed vaccines are what their name implies: the pathogen has been killed so that it cannot cause disease, but enough of its structure remains to generate antibody response. Often, the immune response generated with killed vaccines is not as robust as that generated with other kinds of vaccines. 

Weakened or attenuated vaccines use whole pathogens that have been weakened in the laboratory through long-term culture or other means. Our modern MMR (measles, mumps and rubella) vaccine is an example of an attenuated vaccine. These vaccines tend to generate strong, long-lasting immune responses, but have increased risk for immunocompromised individuals.

Engineering an Influenza A PROTAC Virus Vaccine

A recent paper by Si et al published in Nature Biotechnology describes a new type of live-attenuated whole pathogen vaccine: the PROTAC virus. PROTAC viruses are prevented from replicating by targeting critical viral proteins for degradation using the host cell protein degradation pathway. The vaccine is live-attenuated by the host cells that degrade critical proteins.

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Getting To Know D.O.O.R.S. Scholar Anusha Ray Dey

“I want to make sure that what I think I want to do, truly is what I want to do.”

Anusha Ray Dey started working in a research lab as an undergraduate at the University of Wisconsin-Madison because she imagined it would improve her future application to medical school. However, there was one twist she didn’t see coming – Anusha realized she really enjoyed working in research. Now graduated, she’s earning a Masters degree to gain more experience before making her next decision.

“I could go into industry and do research, or even be a research coordinator. But maybe I’ll decide to still go on to medical school,” Anusha says. “My experiences in research definitely did shift my plans.”

Anusha Ray Dey
Photo courtesy of Anusha Ray Dey

Supporting Mental Health on Campus

Anusha Ray Dey completed an undergraduate thesis searching for chemical signals in the urine of male orangutans. She has a black belt in tae kwon do and she loves to draw. For an honors project, she drew all of the illustrations for an animated video on Alzheimer’s Disease.

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