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Deep-sea creatures offer insights into efficacy of immunotherapies
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Researchers used bioluminescence from marine animal luciferases as the basis for a novel assay designed to determine the effectiveness of cancer therapies.
“One of the most promising areas in cancer research is immunotherapy, including chimeric antigen receptor T cells (CAR T cells). It also is one of the most difficult because the methods for testing immunotherapies are not ideal,” Preet M. Chaudhary, MD, PhD, professor of medicine at Keck School of Medicine at USC, chief of the division of hematology and Center for the Study of Blood Diseases, and director for bone marrow transplant at USC Norris Comprehensive Cancer Center, said in a press release.
“Radioactive chromium release assay is the gold standard for testing whether an immunotherapy kills cancer cells,” Chaudhary added. “This method is expensive, complicated and requires special disposal practices. Other available methods also suffer from limitations and don’t allow scientists to rapidly screen immunotherapeutic agents to find the best candidates.”
Chaudhary and colleagues aimed to develop a test based on luciferases — enzymes responsible for bioluminescence — from certain small crustaceans and deep-sea shrimp.
These luciferases — which leak out of cells when they die, creating a visible glow that can be measured with a luminometer — became the basis for the Matador assay.
Chaudhary and colleagues evaluated the assay’s ability to measure cell death using several types of cancer cells, including acute myelogenous leukemia, chronic myeloid leukemia, Burkitt’s lymphoma and solid tumors. These cells had been treated with forms of immunotherapy, including monoclonal antibodies, CAR T cells and bispecific T-cell engagers.
The researchers determined the assay could detect the death of a single cell, demonstrating a sensitivity that greatly exceeds existing assays. The Matador also is fast — detecting cell death in as little as a half-hour — and inexpensive, and it can be performed in a 384-well plate format, according to researchers.
HemOnc Today spoke with Chaudhary about how the assay was developed, the effectiveness observed so far, and what still must be confirmed in subsequent research.
Question: How did this research come about?
Answer: The chimeric antigen receptor and cellular immunotherapy is a hot area of research, so we got into it around 3 or 4 years ago. When we entered the field, we saw that some of the assays people were running to find out if CAR T cells were functioning had a number of limitations. The chromium release assay, for example, was developed nearly 50 years ago. It required radioactivity, was hazardous and expensive, and was not very sensitive. One of the first things we decided to do was develop an assay that was sensitive, specific, economical and fast, and could be done in any lab.
Q: How did you develop the Matador assay?
A: Once we decided we want to develop an assay, we tested a number of marine luciferases because they happened to be very sensitive, small in size, nontoxic and extremely stable under culture conditions. We tested several combinations. The final configuration that we developed worked beautifully when we tested it in cell lines. It exceeded our expectations. We then went on to develop 70 to 80 different target cancer cell lines. Because we had this easy-to-use, high-throughput assay in our hand — even high school students can routinely run it — we could use it to start testing CAR T cell therapies we were developing in our lab. We have tested several thousand CAR T cells using this assay. We have used it both in hematologic malignancies and solid tumor-derived cell lines.
Q: How does this assay work?
A: The radioactive chromium release assay, which is the gold standard, involves labeling the cells with radioactive chromium. Imagine a cell being a bag loaded with radioactive chromium. If the cell dies, radioactivity leaks out in the culture medium, where it can be measured to calculate how many cells have died. In our assay, we have stably engineered the cells to express luciferases inside the cell. Then — when you add an antibody or a CAR T cell to those target cells — if the cell dies, the luciferase, because it is small in size, leaks out. Additionally, the substrate can enter the cell more easily and react with any luciferase still trapped inside. The resulting reaction between the luciferase and its substrate releases luminescence, which you can measure using a standard lab luminometer. The setup of this assay is extremely easy and fast. You just put the target cells with the CAR T cells, culture them together for an appropriate time, add the substrate and read the result. There is no loading of target cells with radioactive chromium or centrifugation step involved. This not only makes the assay fast but also lessens its variability. The sensitivity is extremely high and is down to a single cell level, which also translates into how quickly you can run the assay. In fact, the assay can be run after as little as 30 minutes of co-culture time. This is important for CAR T cell manufacturing, as it can shorten the time needed to test the final product for potency before it can be released for administration to the patient.
Q: What are the early indications of efficacy?
A: As I mentioned, we have run it on more than 70 cell lines and nearly thousands of CAR constructs. The sensitivity is extremely high — much higher than any of the assays currently in use. We believe it has a lot of utility, not only in research and development but also in manufacturing of cellular therapy products. If you are developing an antibody candidate or CAR T cell, you can use an assay like this to screen hundreds of candidates quickly to find the best candidate for further development. The assay also has applications in CAR T-cell manufacturing. CAR T cells are custom-made for each patient and can cost approximately half million dollars. You need a robust assay to make sure this product works. In fact, the FDA requires a potency test to be included in the manufacturing of all cellular therapy products. We believe our assay — with its speed, sensitivity, consistency, ease of use and economy — will have utility in CAR T-cell manufacturing.
Q: Are there any validation studies planned?
A: We have already validated it in our own labs on the research side. Hopefully other groups will take this on and validate it further for manufacturing and clinical applications. – by Rob Volansky
Reference:
Matta H, et al. Sci Rep. 2018;doi:10.1038/s41598-017-18606-1.
For more information:
Preet M. Chaudhary, MD, PhD, can be reached at the 1441 Eastlake Ave., Health Sciences Campus, Los Angeles, CA 90033; email: pchaudha@usc.edu.
Disclosure: Chaudhary is the inventor on several patent applications related to the Matador assay and chimeric antigen receptors.