Researchers at UCSD and Memorial Sloan Kettering Cancer Center used Excellos healthy donor starting material to carry out a fascinating study on ultrasonic activation of cancer immunotherapy.
CAR-T cell therapy is a paradigm shifting therapeutic with the potential to have an immense impact on cancer treatment options. The number of CAR-T therapies in clinical trial is rapidly increasing, as scientists search for ways to make the treatments safer, more efficient, and effective against a wider range of cancer types.
Cancer immunotherapy in solid tumors
While the treatment of leukemia and other blood borne cancers with CAR-T has been highly successful, the treatment of solid tumors is lagging behind. The research being developed in this study would provide a non-invasive way of activating live CAR-T cells on site. The ability to remotely control CAR expression would give doctors a method of precisely controlling where and when therapeutic cells are activated, which is particularly significant for the cancer immunotherapy in solid tumors.
Methods of controlling genetic activation do already exist. These often rely on chemical induction, radio waves, or magnetic or light activation. These methods are generally imprecise regarding location, require signal amplification, and, as far as light activation goes, only applicable at near-surface locations. There is an unmet need for alternative and more accurate targeting methods.
The concept driving this current research forward is intriguing. The scientists were focused on developing a method whereby therapeutic T cells are engineered to sense ultrasound stimulation that is applied remotely, activating the cells to begin CAR expression. To achieve this, they made use of the Piezo 1 ion channel protein, a natural mechanically activated sensor found in tissues throughout the human body. The system was designed so that low-frequency ultrasonic stimulation of Piezo 1 would in turn activate a transcription factor and drive expression of engineered target genes. These genes express protein receptors that recognize antigens that are highly specific to cancer cells.
Ultrasonic Stimulation
In this study, ultrasonic stimulation was mediated by the use of “microbubbles” mechanically coupled to Piezo 1 on the surface of the therapeutic target cell in order to amplify the signal being generated and elicit a more robust response. Microbubbles were coated with streptavidin and coupled to biotinylated RGD peptides, which were then engaged with membrane receptor proteins and Piezo 1. This provided a reporting system for the ultrasonic activation. Once this basic set up was in place, the research group proceeded to test their new ultrasonic gene activation system.
Ultrasound power was precisely kept to a range that would mechanically stimulate the cells without causing any damage. The researchers first used a human embryonic kidney cell line (HEK293T) to show that they could successfully activate the Piezo 1 ion channel in the presence of calcium. Cells without Piezo 1 did not respond, and neither did cells with Piezo 1 but without microbubble signal amplification. Only cells contained both the Piezo 1 ion channel and microbubble amplification responded by allowing calcium influx into the cell.
The next step was to examine whether the ultrasound-induced calcium influx could be used to control gene expression. For this part of the study, the research group created a modular genetic construct that would allow them to switch out transcription response and promotor elements. The research group could then compare the efficiency and specificity of various genetic elements. The team chose 6 potential construct candidates that could be linked to either luciferase or fluorescent reporter genes. After analyzing the results, the scientists chose their strongest candidate, a genetic construct that could clearly drive green fluorescent protein (GFP) expression when stimulated by the ultrasound signal. This construct would be used for the most important part of the study; determining whether they could drive CD19 targeted CAR expression remotely.
Obtaining Human Primary Lymphocytes
An immortalized human T cell line (Jurkat T cells) and primary human T cells (within the isolated PBMC preparation) were both used for the next series of experiments. To obtain human primary lymphocytes, healthy donor apheresis material was obtained from Excellos, after which PBMC were isolated and used as starting material for ultrasound-activated anti-CD19 CAR cells.
Preliminary experiments showed that both Jurkat cells and primary human T cells could successfully respond to ultrasound stimulation with robust expression of a GFP reporter gene. The next step was to test Jurkat cells modified to express the anti-CD19 CAR construct. Anti-CD19 CAR expression was also significantly upregulated upon ultrasonic activation. Next, the modified Jurkat cells were incubated with CD19 antigen expressing Toledo cells. Toledo cells are a cancer cell line used to model non-Hodgkin lymphomas. Results showed that ultrasound-induced signaling could be tightly controlled in regard to both timing and location, and that the Jurkat cells could successfully be induced to engage with the tumor cells.
The World’s First Ultrasonic Cancer Immunotherapy
The final proof-of-concept was dependent on the response of primary human T cells. Primary human T cells modified to express the ultrasound sensitive anti-CD19 CAR construct were incubated with Toledo cancer cells. Upon remote ultrasound activation, calcium influx into the PBMCs was clearly observed. The cells were evaluated via functional assays for cytotoxic efficiency. The researchers determined that the ultrasound activated CAR-T cells caused significantly more cytotoxic activity than either modified PBMCs not exposed to ultrasound or non-modified PBMCs exposed to ultrasound. Because the whole gene construct system is modular, the research group feel that the technology can continue to evolve for even greater efficiency and wider application.
This pioneering work was published just a few years ago. Today, the vision and hard work that went into this research is coming to fruition and the world’s first ultrasonic cancer immunotherapy treatment center is now a reality. [2]
At Excellos, we are proud to support this type of groundbreaking research with advanced, highly characterized cellular starting materials. Please contact us to learn more.
References
- Pan, Y., Yoon, S., Sun, J., Huang, Z., Lee, C., Allen, M., Wu, Y., Chang, Y. J., Sadelain, M., Shung, K. K., Chien, S., & Wang, Y. (2018). Mechanogenetics for the remote and noninvasive control of cancer immunotherapy. Proceedings of the National Academy of Sciences of the United States of America, 115(5), 992–997. https://doi.org/10.1073/pnas.1714900115
- University of Virginia. 2022. Barney J. World’s first focused ultrasound cancer immunotherapy center will be at UVA. May 2022. [online] Available at: https://news.virginia.edu [Accessed: 26 July 2022]
