Staffan Eriksson is Professor in Medical and Physiological Chemistry at the Swedish University of Agricultural Sciences. He is also a founder of AroCell AB and one of the inventors of the TK 210 ELISA test for measuring cell growth and turnover. This candid informal interview with Staffan Eriksson captures his insight into key historical milestones of the product and company, as well as his ideas and hopes for the coming year
You have published over 120 scientific publications so far in the course of your career and research, and the first study on Thymidine Kinase was published in 1996. How did your interest in TK get started?
My basic interest is in medical biochemistry and how DNA building blocks are made. There are four different components needed to make DNA. One system that has been known for many years involves Thymidine Kinase as a key enzyme in reusing one type of these building blocks. When you study cell proliferation you need to measure how cells are growing and duplicating their DNA. The big breakthrough was in the early 50s, when you could label the newly made DNA with Thymidine. That was the way to define the process of cells going through different cycles phases – with an isotopic label on the Thymidine that was being incorporated into newly made DNA.
Researchers also understood at this very early stage that there must be an enzyme that puts a phosphate on Thymidine to produce a form that can be incorporated into DNA, and this enzyme was Thymidine Kinase. So TK was known for many years as a key enzyme in labeling DNA, and in following cell proliferation. A lot of pioneering work on Thymidine Kinase was done by the Stanford Nobel prizewinner Arthur Kornberg’s group using classical biochemistry with the E-coli bacterium. It took a long time for people to realize what the enzyme looked like in animals. This was finally accomplished at the Karolinska Institute, where I worked with a Danish guest scientist, and we finally purified it to determine the exact size of the protein.
This was the starting point, in the early 80s, for being able to make very specific antibodies against this protein. Some of these antibodies could be used to study the TK enzyme, for instance to determine how much is present in the cell during the cell cycle, where it is located in the cell and which processes it is involved in. When we had made these antibodies we realized that they could have some commercial applications. At that time, research profiles such as Simon Gronowitz in Uppsala showed that increased TK activity in the blood was an important indicator of cell proliferation in cancer patients. All of this early research focused on how DNA is synthesized and the role of the Thymidine Kinase enzyme in healthy and diseased cells.
You are one of the original founders of AroCell, which was started under the name “Xi Bao Research AB” in 2003. Can you share more about how it all got started?
In the 1990’s we realized that we were good at detecting TK at a cellular level, but if you looked in the more complex mixture of blood you could not see TK with these antibodies. In the case of animals such as rabbits the TK protein was not sufficiently immunogenic, and we never got antibodies that could bind to serum TK with high specificity.
With Xi Bao Research, we were struggling to get this working, and by using chickens, where the antibodies are isolated from the egg yolks, reasonably good antibodies against serum TK were obtained. A test based on a manual dot blot method was established at the Karolinksa Institute and then scaled up in China by a new start up company (SSTK Biotech Inc). The research group there also used a different method to make antibodies, with slightly longer TK1 peptides as antigens. We thought it might be better initially to do the development work together. Using these antibodies, made in rather large scale from chickens, a manual dot blot tests was established and launched in some parts of China. The main use was for health screening purposes. In China, your employer can pay health screening costs, but if you become ill you are in principle responsible for all medical costs. The SSTK dot blot tests have been used in China for more than 10 years and by many thousands of people, but it is difficult and costly to produce similar antibody batches from the different immunizations. This is the main reason why this product is not used in other markets with higher wage- and production-related costs and other regulations.
However, so far SSTK have not been able to produce monoclonal antibodies against serum TK1 with sufficient specificity. Thus, they still lack a source of specific antibodies with defined protein sequence and binding properties ,which can be continuously produced. This is a unique property of the AroCell’s ELISA test, and has been the core of the patenting processes during recent years.
A few years later, the company was renamed AroCell and product development started. What were some of the most challenging first milestones in your opinion?
It was the development of our two monoclonal antibodies. This was accomplished in two stages, the first one was developed starting in China, and it was reasonably good but only bound to one exposed region of the protein, in serum TK1 That worked well but we never got another one that could complement it. The real milestone came during the last 5 years when we developed a pre-treatment method, that is, a method to treat the serum so that we can have two antibodies with different specificities binding to the protein. This provides sufficient sensitivity and specificity to be able to bring the test into the clinic. With only one or two of these three aspects missing, the test would be inefficient.
In your opinion, what is some of the most exciting research that has been published on TK 210 ELISA in recent years?
About 2 years ago we published proof that these antibodies can be used to fish out the protein from blood (using affinity-chromatography) and define it and examine it on the molecular level, by so-called western blotting, which is a research analysis method. One monoclonal antibody was used to fish out the Thymidine Kinase 1 protein from serum and a second antibody is used to detect it in the western blot. We used a polyclonal antibody earlier, but now we have two monoclonal antibodies in an ELISA test. This enabled us to do studies to confirm that it is the correct protein, and that there are different levels of this protein in healthy subjects compared to diseased patients with breast cancer, prostate cancer and several other types of cancers. One of the most interesting things about Thymidine Kinase is that it is not tumor-specific but specific to cell proliferation and turnover, which means that it can be used to detect proliferation in solid tumors as well as in blood-based malignancies. High cell proliferation is a key feature in the development of the highly aggressive form of cancer, which is no longer treatable.
The AroCell TK 210 ELISA test can thus detect proliferation in blood from patients with hematological malignancies as well as solid tumors. There is much more variability in the TK that leaks from solid tumors – you can find protein with or without enzyme activity. This allows for the activity and protein levels to be measured and compared, and we now have a method to measure TK protein as well as activity with the required specificity. We have seen that the specific activity (activity per TK protein unit) in many solid tumors is different from that in hematological tumors.
What do you see as the next scientific milestones for TK 210 ELISA in research?
The first is establishing the cut-off levels that distinguish between healthy persons and those with disease. The other aspect is investigating the contribution that the TK 210 ELISA could make to management of the most prevalent cancer forms for women and men, i.e. breast cancer and prostate cancer. There has been a revolution during the last 10-15 years with 80 – 90 percent of patients living over 5 years with these diseases. But there are subgroups in breast cancer and prostate cancer, where the disease still progresses relatively rapidly, even though the spreading of malignant disease can now often be successfully treated. However, if you divide the breast and prostate patients into subgroups, you will see that patients in some groups have a very good prognosis, and should just be closely monitored, while patients in other subgroups have a rapidly progressive disease that needs efficient treatment and frequent follow-up.
Last question: if you had a crystal ball and could see 12 months into AroCell’s future, what would you expect to see, and what would you most like to see?
We are already analyzing a big study on breast cancer initiated by the Karolinska Institute called the Promix study, which will provide a lot of information. It involved over 150 women with large tumors about 5 cm in size. I am looking forward to the finalization of the study.
Other studies in collaboration with the clinical chemists in Ljubljana, Slovenia, are in progress and some results with breast cancer are already in press . We will continue to determine the blood levels of TK, both by the activity and the TK 201 ELISA protein tests, before treatment and after treatment and relate the results to the stages of the breast cancer disease and the clinical effects of different treatments.. Our initial findings show that the activity-based Thymidine kinase test has a lower sensitivity compared to the TK protein test and we look forward to see how the two tests can predict the overall results of different treatments.
AroCell will be very active this year at many different scientific conferences, and I welcome independent researchers to start using the TK 210 ELISA test. I am convinced that these studies will show that we have a sensitive and reliable test that can aid in the management of several important cancer diseases.