Stringent quality control during manufacturing yields safer, more effective therapies, advise Mark White, Associate Director of Biopharma Product Marketing, and Marwan Alsarraj, BioPharma Segment Manager, Bio-Rad Laboratories
In 1984, a patient received infusions of IL-2, triggering her own T cells to expand and target a metastatic melanoma tumour. The treatment halted her disease completely, enabling the patient to remain in remission for 29 years.
She was just the first of many to benefit from this type of treatment.1 Building on this and other immunotherapy successes, the latest CAR-T cell therapies further hone and strengthen the natural immune response by genetically modifying a patient’s T cells to make them into targeted cancer killers.2
According to ClinicalTrials.gov, this remarkable approach has inspired more than 350 currently active CAR-T trials.3 Recognising the promise of these designs, regulatory agencies intend to approve 10–20 cell and gene therapies per year by 2025.4
As the field of immunotherapy matures, developers must adapt. The field is still young and manufacturing a “living drug” is not fully standardised. Scientists need highly sensitive, accurate quality control and quality assurance tools to monitor dynamic, living cells as they are manipulated during the manufacturing process to ensure that the product will be safe and effective when infused into each patient.
To manufacture a CAR-T cell therapy, T cells are extracted from human blood and modified — typically using an adeno-associated virus or lentivirus — to insert a chimeric antigen receptor (CAR) transgene into the cell’s genome.
The cells are then grown in a bioreactor and allowed to multiply to an appropriate volume. Then, a physician transfuses them back to the patient where they express the CAR protein, circulate throughout the body and kill the target cancer cells.
When a therapy is proceeding through clinical trials, the manufactured cells can vary significantly from batch to batch. Developers use a variety of approaches to produce different CAR-T cell therapies, and every batch of every therapy is currently made with different starting material: the T cells of an individual patient who has most likely already received other treatments that may have affected their cells’ growth and behaviour.
To account for the increased possibility for variation, developers must monitor cells in production using stringent quality control measures to ensure safe, effective products.
Molecular methods are used to test for key elements such as CAR-T cell potency, persistence and the presence of contaminants. These quality control practices maximise the benefit, minimise the risk of harm to the patient and promote optimal clinical trial results to improve the chances of the therapy reaching the market.
Lab conditions and the fortitude of the patient’s cells affect how efficiently the CAR gene integrates in any given batch. Therefore, developers must monitor the cells’ CAR transgene copy number to avoid dosing a patient with cells that have too many or too few copies.
If CAR-T cells have too many copies of the CAR transgene, the patient is at increased risk of experiencing severe, potentially life-threatening toxicities.5
By contrast, if a batch of cells did not take up any copies, it would be ineffective. The US FDA has indicated that CAR-T cells should carry between one and four transgene copies, striking a balance.6
Transgene copy number is monitored using various methods, but developers have historically favoured qPCR for this purpose. Unfortunately, because this technique is not accurate or precise enough to track small concentrations of transgene DNA, this preference puts the patient’s health at increased risk.
qPCR can only estimate transgene copy number. The technique relies on a lab worker to perform serial dilutions of a sample to generate a standard curve — a process that is less sensitive because it is susceptible to human error and variability.
Because of this limitation, qPCR is incapable of measuring down to one gene copy per cell and cannot reliably measure whether CAR-T cells contain a safe copy number of the CAR transgene.
Conversely, the sensitivity required is attainable by Droplet Digital PCR (ddPCR), a method designed for absolute nucleic acid quantification. ddPCR’s precision and sensitivity make it amenable to quantify transgene copy number down to one copy and well-suited to address other challenges throughout CAR-T cell development and manufacturing, such as CAR-T persistence and batch contamination.7
To run a ddPCR assay, a 20 µL nucleic acid sample is partitioned into 20,000 nanolitre-sized droplets, with each droplet containing one or a few nucleic acid strands.
Effectively, these can be thought of as thousands of individual samples being run simultaneously, some of which contain a copy of the CAR transgene.
Independent reactions occur in every droplet, but amplification and the production of a fluorescent signal only occurs in droplets containing the transgene. The droplets are quantified in a droplet reader, which can be used to extrapolate the exact CAR transgene concentration in that batch of cells.
ddPCR’s superior sensitivity has been demonstrated at the bench.8 Researchers at the Huazhong University of Science and Technology in Wuhan, China, compared key performance parameters of ddPCR and qPCR.
They evaluated each method’s ability to quantify DNA standards and the blood samples of patients undergoing CAR-T cell therapy. They found that ddPCR was more sensitive when used to measure diluted CAR DNA standards, detecting down to 3.2 copies/mL, whereas qPCR could not detect such a low concentration of the transgene.
Similarly, when measuring CAR persistence in patient’s blood, qPCR’s limit of detection was 20 copies per reaction, whereas ddPCR’s was five copies per reaction and showed better repeatability and reproducibility.
In addition to potency, CAR-T cell persistence — the length of time the cells are alive in the patient’s body — must be fine-tuned to optimise therapeutic benefit.
After CAR-T cells are infused back into a patient, they circulate throughout the bloodstream, destroying any cancer cells they encounter. The cells must be active for a few months after treatment to have sufficient time to do their job.
However, it is important that they do not live too long. CAR-T cells that persist even after cancer has entered remission can lead to unwanted side-effects, such as neurologic adverse events.8
Physicians can track CAR-T cell persistence with time by using ddPCR to measure CAR-T cells in blood, applying the resulting data to optimise the safety and efficacy of treatment.9
Manufacturers must also ensure that the viral vectors used to produce their CAR-T cells cannot replicate and persist for an indefinite amount of time within the patient.
Such a scenario would lead to severe health consequences. This event has not yet been recorded in humans; but, as a safeguard, the FDA recommends that manufacturers test for replication-competent lentiviruses at several stages throughout the manufacturing process and in the patient’s blood after the therapy has been administered.10
Owing to its high sensitivity, ddPCR technology is an ideal tool to detect even extremely low levels of replication-competent virus, so any instances can be screened out before the cells are introduced to patients.11 ddPCR assays can also be used at each stage to screen batches of CAR-T cells for other microbial contaminants.
As the immunotherapy field evolves, CAR-T cell therapy development stands to become more nuanced, and it will require quality control testing methods to match.
Currently, five CAR-T therapies have been approved by the FDA for the treatment of multiple myeloma and lymphoma, but the field is gaining momentum, pitting CAR-T cells against solid tumours and developing new generations of CAR-T designs with an increased ability to address a wider variety of cancers.12–14
These new therapy strategies will need to be evaluated to predict their functionality within the body. At its core, ddPCR is a highly versatile and sensitive method to quantify nucleic acids, which can increase precision at every step of the manufacturing process, making it well-matched to ensure the safety and efficacy of current future CAR-T cell therapies alike.
Through rigorous manufacturing practices, quality control and beyond, CAR-T developers can work with regulators to continue raising the bar when it comes to CAR-T cell manufacturing rigour. Through their work, they will continue to bring even more innovative, life-saving therapies to market.