Chimeric antigen receptor (CAR) T cell therapy represents a major advance toward providing personalized cancer treatment. Unlike chemotherapy that can affect both healthy and cancerous cells, CAR-T cell therapy specifically targets tumor cells for destruction by the patient’s own immune system. Producing effective CAR-T cell therapies requires that cell number and viability be closely monitored throughout the entire development and biomanufacturing process. The LUNA-FX7™ provides the high level of accuracy required to meet strict quality and regulatory guidelines, making it an ideal cell counter for CAR-T cell therapy research.
CAR-T cell therapy combines gene therapy and personalized immunotherapy to produce a “living drug”. It involves isolating a cancer patient’s T cells and modifying them to express chimeric antigen receptors at the cell surface. When the engineered CAR-T cells are reintroduced to the patient, they are able to recognize and bind specific proteins expressed on tumor cells, leading to their destruction by the immune system. Two CAR-T cell therapies are currently FDA-approved (Kymriah™ for B cell acute lymphoblastic leukemia and Yescarta™ for large B cell lymphoma, both of which target the CD19 B cell marker) and many more are being developed.
A main advantage of CAR-T cell therapy over chemotherapy is that patients suffer fewer harmful side effects. CAR-T cell therapy also requires fewer trips to the hospital for treatment. Typically, a single round of lymphodepletion chemotherapy is administered shortly before CAR-T infusion to improve acceptance of the modified cells; these are then delivered intravenously and left to circulate.
CAR-T cell therapy development can be broken down into several main steps. First, the patient provides a blood sample, from which the white blood cells (WBCs) are separated by leukapheresis. Next, the T cells are isolated from the WBC population and activated using antibody-coated beads, before being engineered to express the CAR of interest. After this, the modified cells are allowed to proliferate before undergoing a battery of tests to assess their quality and functionality. Only once strict regulatory criteria have been met can the CAR-T cells be infused into the patient.
Image: The CAR-T cell development and biomanufacturing process
Knowing the concentration and viability of WBCs in whole blood is essential to CAR-T cell production since it provides insights into the nature of the starting material. For example, previous chemotherapy treatment may have caused substantial WBC losses that could impact on CAR-T cell quality and yield. Equally important is the ability to evaluate WBC number and health after leukapheresis, not only to ascertain whether cell loss or damage has occurred during the separation process, but also to distinguish WBCs from non-cellular debris that could generate false positives further downstream. Accurate cell counting and viability measurements are also essential to CAR transduction/transfection, during scale-up, and to correctly determine dosing prior to patient infusion.
Image: Cell concentration and viability of WBC in whole blood samples. Prior to leukapheresis, a whole blood sample was diluted 1:100 with PBS, stained with AO/PI, and counted. Tiled images of Brightfield (BF), Green channel (GF), and Red channel (RF) (A). Overlay images of tagged (live/dead nucleated cells) images (B, C). Total WBC cell numbers and viability of the 1/100 diluted blood (D).
The LUNA-FX7™ provides the high degree of accuracy required for CAR-T cell therapy research and development. This is attributable to several noteworthy features, not least the quick and reliable autofocusing ability and enhanced optics. In combination, these produce clearer, more uniform images to increase detection sensitivity and the dynamic range, providing reliable counts across both high and low cell concentrations. At the same time, the upgraded counting algorithm more precisely distinguishes viable and non-viable cells within aggregates.
Another key element of the LUNA-FX7™ is its compatibility with different slide formats (including 1, 2, 3, and 8-channel slides) that make it suitable for almost any cell counting need, whether that be accuracy, convenience, or high-throughput. For example, the 1-channel slides enable counting volumes of up to almost 5 µL, translating to lower CV and greater precision. Additionally, the LUNA-FX7™ offers a customizable cell-detection protocol and reanalysis function, allowing users to adapt counting to particular cell characteristics and confirm any results.
Data generated using the LUNA-FX7™ can be stored and transferred via USB, Wi-Fi, or ethernet connection and exported as various file formats. These include both a CSV summary and a comprehensive PDF report with raw and tagged images that makes it easier to check and manage results.
Lastly, the LUNA-FX7™ is equipped with separate features meeting the rigorous QC and regulatory guidelines that cell therapy manufacturing facilities require. These include a Bioprocess mode for monitoring the progression of multiple batches, a Quality Control feature with unique validation slides, and innovative CountWire™ software that complies with 21 CFR Part 11 and GMP requirements.
To learn how the LUNA-FX7™ can benefit your CAR-T cell therapy research and development, visit logosbio.com