Blog Post – The Role of Flow Cytometry in CAR-T Therapy and Clinical Trials

Welcome back! In our last post, we covered some key background on what flow cytometry is and general perspective on how to best utilize it within a clinical trial setting. In this post, we’re going to focus on a specific clinical application where flow cytometry is having a big impact: immunotherapy, in particular adoptive cell therapies like CAR-T.

Introduction to CAR-T Therapy:

CAR-T (Chimeric Antigen Receptor T-cell) therapy has become a major player in the fight against cancer, especially for blood cancers like B-cell lymphomas and acute lymphoblastic leukemia (ALL). This cutting-edge therapy involves modifying a patient’s T cells, allowing them to better identify and attack cancer cells (Ref.1,2). There are two types of CAR-T therapies:

• Autologous CAR-T therapy: Uses the patient’s own T cells. This approach reduces the risk of immune rejection but takes longer time to prepare the CAR-T cells and could be expensive for the patients. 
• Allogeneic CAR-T therapy: Uses any healthy donor T cells, which can be used as an “off-the-shelf” option, so the CAR-T cells are available to treat patients immediately. These cells have a higher risk of rejection since the cells come from a donor and are shown to have less persistence 

Figure 1: Steps for Production of Autologous vs. Allogeneic CAR-T Cell Therapies: Orange arrows indicate steps unique to Autologous CAR-T (patient-derived T cells).  Green arrows indicate steps unique to Allogeneic CAR-T (healthy donor-derived T cells). Black arrows indicate shared steps between the two therapies.

How Flow Cytometry Plays a Key Role in CAR-T Therapy:

While the CAR-T cells themselves occupy most of the limelight, flow cytometry frequently makes appearances throughout the CAR-T process—from manufacturing the modified T-cells to ensuring safety and tracking the therapy’s success. Flow cytometry assays are an optimal approach to assess individual cell behavior and ensure the CAR-T treatment is effective and safe for each patient (Ref. 3). Much like the back-stage crew of a show, flow cytometry makes sure everything runs smoothly.

Here are some of the many roles flow cytometry can play during the different stages of a CAR-T study:

1. CAR-T Cell Manufacturing Process
   • Transduction Efficiency: After T cells are modified to express the CAR, flow cytometry can be used to check how well the modification worked. This check is usually done by tagging T cells with a fluorescent antibody that specifically binds to the CAR and using flow cytometry to measure how many cells were successfully engineered.
   • Phenotypic Characterization: It’s not just about numbers. Flow cytometry also can be used to ensure the CAR-T cells have the right characteristics, such as surface markers that tell us whether these cells are likely to stick around and do their job in the patient’s body.
2. Quality Control and Release Testing
   • Purity and Viability: Flow cytometry can help answer several key questions prior to infusion: Are the CAR-T cells pure and viable? Are the cells ready to fight (i.e expressing biomarkers of activation)? 
   • Functional Assays: Flow cytometry is used to make sure the CAR-T cells can do what they’re supposed to—kill cancer cells. By running cytotoxicity tests, flow cytometry can quantify how effective the CAR-T cells are in killing cancer cells.
   • Safety Testing: Flow cytometry helps detect any unwanted cells that could cause harmful effects, ensuring the therapy is safe for the patient.
3. Monitoring CAR-T Therapy in Clinical Trials
   • Post-Infusion Monitoring: After the CAR-T cells are infused, flow cytometry continues to be a powerful tool for monitoring their behavior in the bloodstream. It tracks their expansion, persistence, and phenotype, helping clinicians understand how long the therapeutic effect might last and whether any adverse events, like cytokine release syndrome (CRS), are developing.
   • Biomarker Discovery and Immune Profiling: Flow cytometry is also used to profile the patient’s immune response to the CAR-T therapy, helping researchers discover biomarkers that could predict how well a patient will respond or whether they might develop resistance.
   • Assessing Minimal Residual Disease (MRD): In blood cancers, flow cytometry can assess MRD, providing a clear picture of how well the CAR-T therapy has eliminated cancer cells. This information is crucial for deciding if more treatments are needed.

Figure 2: Flow Cytometry Assessment of CAR-T Cell Therapy Before and After Infusion. Flow cytometry enables evaluation of CAR-T cell therapy at various stages, both before and after infusion. This figure highlights the key assessment points throughout the CAR-T process using flow cytometry.

At Cerba Research, we make full use of the power of advanced flow cytometry assays to monitor CAR-T Therapy in clinical trials. Our real-time monitoring tracks how CAR-T cells expand, persist, and interact with the immune system, helping to spot any issues early and fine-tune treatments for safer, more effective results.

Success Stories in CAR-T Therapy

Several CAR-T therapies have been approved by the FDA, including well-known names like Kymriah, Yescarta, Tecartus, Abecma, Breyanzi, and Carvykti (Ref.4,5).

Let’s dive deeper into Carvykti (ciltacabtagene autoleucel), one of the newer FDA-approved CAR-T therapies, and how flow cytometry played a key role in its development. Approved in 2022 for relapsed or refractory multiple myeloma, Carvykti targets BCMA (B-cell maturation antigen) on cancerous plasma cells.

Flow cytometry was crucial during the clinical trials of Carvykti for several reasons:

• Assessing Transduction Efficiency: In the early stages of manufacturing, flow cytometry was used to confirm that the T cells were successfully engineered to express the CAR that targets BCMA. This step ensured that the therapy would be effective in recognizing and attacking the cancer cells.
• Monitoring CAR-T Cells in Real Time: During the clinical trial, flow cytometry allowed researchers to track how well the CAR-T cells expanded in the patient’s body after infusion. It gave real-time insights into how the cells were behaving, helping clinicians understand how long the CAR-T cells were sticking around and how effectively they were eliminating the cancer.
• Evaluating MRD and Patient Outcomes: Flow cytometry was also used to assess whether any cancer cells remained in the patients after treatment, known as minimal residual disease (MRD). This helped researchers and doctors determine how effective the therapy was and make decisions about additional treatments.

Overall, flow cytometry was instrumental in showing that Carvykti could induce deep and lasting responses in patients with advanced multiple myeloma, making it a powerful addition to the CAR-T therapy landscape.

Conclusion

Flow cytometry is the unsung hero behind the success of CAR-T therapies, providing critical data at every step—from ensuring the CAR-T cells are engineered correctly to monitoring their behavior in clinical trials. With its ability to provide real-time insights, flow cytometry continues to play a key role in making these therapies safer and more effective for patients.

If you’re working in CAR-T research or clinical trials, flow cytometry should be in your toolkit; it certainly is in ours!

References

1. Wei, J., Guo, Y., Wang, Y., Wu, Y., Qin, Y., and Ye, Y. (2021). Advances in CAR-T Cell Therapy for Cancer: Factors Affecting Therapeutic Efficacy and Resistance Mechanisms. Frontiers in Immunology, 12:658314. https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2021.658314/full.
2. Nature Portfolio. (2023). The Next Generation of Cancer Treatments: CAR-T Therapy. https://www.nature.com/articles/d42473-023-00226-1
3. CERBA Research. E-book: Flow Cytometry and CAR-T Cell Therapy. https://cerbaresearch.com/white-papers/e-book-flow-cytometry-car-t/.
4. Hillman Cancer Centre. FDA-Approved CAR-T Cell Therapies. https://hillman.upmc.com/mario-lemieux-center/treatment/car-t-cell-therapy/fda-approved-therapies.
5. U.S. Food and Drug Administration (FDA). https://www.fda.gov/.

Illustrations created by Sanaz Koosha using Servier Medical Art by Servier, licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).