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www.biopharminternational.com Quality and Regulatory Sourcebook eBook March 2022 BioPharm International 7 Quality and Regulatory Sourcebook Quality: Analytics of the virus (9). The limit of detection for their assay was 10 copies/μL. The technique, overall, provided sensi- tive, specif ic, and reproducible RCL detection, expediting quality-control assessments compared to traditional cell-based or qPCR-based approaches. T. Wiltshire et al.'s results suggest that ddPCR could be used to screen out CAR-T cells that contain RCL so that these cells never reach patients. DETERMINING PERSISTENCE IN THE BODY Phy sicia ns in f use pat ients w it h a specif ic dose of CAR-T cells that is intended to deliver a therapeutic ben- efit without causing harm to patients. However, there is no way to f u l ly predict CAR-T cell kinetics once the cells enter the body. Consequently, physicians must monitor CAR-T cell concentration in the blood on a serial basis to ensure the cells are present in the appropriate concentration for the right amount of time. CAR-T cells are supposed to survive in the body for a few months, but, if they persist for too long, they can elicit a prolonged inf lammatory response even after they've effectively treated the patient's cancer (4). Conversely, if the cells degrade too quickly, they will not do their job. Therefore, physicians must monitor CAR-T cell kinetics to optimize clinical follow-up regimens, understand treatment failure, and understand the cause of adverse events. There are currently no standard meth- ods for measuring CAR-T cell kinetics, but ddPCR assays present an attractive option. Because ddPCR technology can detect small concentrations of nucleic acid sequences in liquid samples, it can be used to precisely quantify CAR-T cell concentration in the blood. Furthermore, because liquid biopsies based on ddPCR technology are minimally invasive, these tests can be run on a serial basis without causing further harm to patients. T. Mika et al. in Germany demon- strated the feasibility of using ddPCR technology for this purpose in patients with various types of lymphoma (10). Two CAR-T therapies, tisagenlecleu- cel and axicabtagene ciloleucel (axi- cel), are currently approved to treat this disease. In patients with lym- phoma treated with CAR-T cell ther- apy, plasma CAR-T cell concentration correlates w ith treatment response and patient outcomes, indicating that CAR-T cell k inetics can ser ve as a predictive biomarker of therapeutic efficacy (11–13). To test this hypoth- esis, researchers developed a ddPCR assay that examines axi-cell kinetics. First, they tested it on positive con- trol samples containing various known concentrations of CAR-T genomes. Their assay reliably detected one CAR gene per genome. The resea rchers then tested 54 samples from seven patients between four weeks and nine months following treatment. They found that CAR-T cell kinetics varied greatly bet ween patients, demonstrating the importance of serial monitoring. Looking closer at each patient, the researchers discov- ered that CAR-T cell persistence and peak expansion correlated with clini- cal responses. For example, the patient whose CAR levels dropped to unde- tectable levels at day 75 after treatment showed no clinical improvement, while the patients whose CAR levels were still detectable after nine months went into remission. At the other end of the spec- trum, the patient with the highest peak CAR copy levels experienced severe side effects following therapy. Altogether, these data revealed the variability in CAR-T cell kinetics and the impor- tance of serial monitoring. ddPCR AND CAR-T CELL THERAPY GO HAND-IN-HAND ddPCR technology has already proven useful in multiple research areas over the past decade. CAR-T cell therapy is a key current example that demon- strates the technology's full potential to help biopharmaceutical manufacturers and physicians keep patients safe and healthy. CAR-T cell therapy develop- ment is not simple, but by quantifying CAR transgenes and other genetic ele- ments using ddPCR assays throughout the process, CAR-T developers will be more successful in developing therapies that will achieve regulatory approval, and physicians can be more confident that they can make a meaningful dif- ference in their patients' lives. REFERENCES 1. University of Pittsburgh Medical Center Hillman Cancer Center, "FDA-approved CAR T-Cell Therapies," hillman.upmc.com, accessed June 10, 2021. 2. Cancer Research Institute, "Cancer Cell Therapy Landscape," cancerresearch.org, accessed Feb. 16, 2022. 3. Research and Markets, CAR T-cell Therapy Market - Global Industry Analysis, Size, Share, Growth, Trends, and Forecast, 2021-2031, researchandmarkets.com, September 2021. 4. J. Brudno, et al., Blood. 127 (26) 3321–3330 (2016). 5. Y. Zhao, et al., Human Gene Therapy Methods. 28 (4) 205–214 (2017). 6. Y. Lou, et al., J Mol Diag. 22 (5) 699–707 (2020). 7. A. Lu, et al., J Trans Med. 18 (191) (2020). 8. US Department of Health and Human Services, FDA, CBER, Hum Gen Ther. 12 (3) 315–320 (2001). 9. T. Wiltshire, et al., Cytotherapy. 23 (5) 452–458 (2021). 10. T. Mika, et al., Front Mol Biosci. 7, 84 (2020). 11. S. Neelapu, et al. N Engl J Med. 377, 2531–2544 (2017). 12. J Park, et al., N Engl J Med. 378, 449–459 (2018). 13. S. Schuster, et al., N Engl J Med. 380, 45–56 (2019). BP Physicians infuse patients with a specific dose of CAR-T cells ... to deliver a therapeutic benefit without causing harm to patients.