Chimeric antigen receptor (CAR) T cells culminate from multiple significant advances in science, from tumor sequencing to genetic engineering and immunotherapy. Unsurprisingly, CAR T-cell therapy was one of the most anticipated oncological approaches since immunotherapy first entered the clinic. The approach exploits T cells’ natural ability to seek out foreign invaders in the body and destroy them, with an added boost of selectivity through targeted genetic engineering. The FDA has approved three CAR T therapies already and has its eye on many more, as more than 650 CAR T-cell trials are currently active or recruiting, according to ClinicalTrials.gov.1 One forecast predicts that the global CAR T-cell therapy market could reach $8 billion by 2028.2
With just a few years of commercial sales under their belts, CAR T-cell therapy manufacturers are still ironing out the kinks. CAR T cells are not typical drugs and producing them presents unique challenges. They are living cells, which means manufacturers, in part, depend on the cells' physiological workings to integrate and express the CAR gene in the appropriate quantity. Since this process is not entirely under a manufacturer’s control, the process of generating safe and effective CAR T cells requires meticulous quality control.
Reducing the risk of harm to patients
The CAR T-cell manufacturing process begins with extracting T cells from a patient’s blood. A scientist then uses either viral vectors, transposon systems, or direct mRNA transduction to insert the CAR gene into the cells' DNA. This allows them to express the CAR protein on their surface. Finally, a scientist amplifies the engineered cells in a bioreactor, and a physician infuses a patient with their own modified T cells. Manufacturers need to monitor this process to ensure the cells are functioning appropriately and won’t cause harm to patients.
One challenge presents itself during the transfection or transduction step: once the CAR gene is delivered to the cell, the manufacturer cannot control where it will integrate into the cell’s genome. It could embed itself in a DNA stretch that regulates oncogene expression, thereby promoting tumor growth. Or, it could integrate into a silenced region of the genome and never get expressed.
In addition to controlling the site of integration, it can be challenging to control how many copies of the CAR gene integrate into the T cell’s genome. This impacts the safety and function of the cell. If the gene doesn’t integrate, the cell won’t fulfill its intended purpose. Conversely, if too many copies of the gene integrate, the cell can become toxic. The protein it produces could induce a systemic inflammatory response, called cytokine release syndrome or “cytokine storm,” that can damage organs and cause death. To regulate this, the US Food and Drug Administration (FDA) recommends that when manufacturers are using retroviral/lentiviral vectors, the CAR gene copy number per cell does not exceed four.3 Consequently, manufacturers must monitor CAR gene copy numbers, as well.
Using ddPCR for CAR T-cell QC
Today, scientists mostly rely on quantitative polymerase chain reaction (qPCR) to quantify CAR gene copy numbers, but this technique can only deliver relative results. qPCR cannot directly quantify gene copies; instead, scientists need to compare their result to a standard curve. This, in turn, needs to be generated using serial dilutions, which is a time-consuming and unreliable process. The variability inherent to qPCR makes it nearly impossible to detect genes present in only one copy per cell.
In contrast, droplet digital PCR (ddPCR) can detect gene copies down to one per cell. While ddPCR and qPCR both employ the same chemistry, ddPCR quantifies gene sequences directly, which delivers far greater sensitivity. In practice, ddPCR divides a sample into tens of thousands of nanoliter-sized droplets and performs independent PCR reactions on the nucleic acid strands contained within each one. The CAR gene will amplify and emit a fluorescent signal, lighting up the droplets where it is present, while the remaining droplets remain dark. This provides the scientist with a binary result for each droplet to precisely quantify those containing the target sequence. From there, one can calculate the concentration of the gene in the sample and derive the average gene copy number among the T cells in that batch.
By quantifying nucleic acid sequences directly, ddPCR removes the need for standard curves. Also, since it is sensitive enough to detect one CAR gene copy per cell, manufacturers can tell with certainty whether their transfection or transduction method was successful.
Using ddPCR and flow cytometry to study CAR transduction
At the National Institutes of Health (NIH) Clinical Center, Ping Jin, PhD, demonstrated that ddPCR could reliably quantify CAR gene sequences in T cells following transduction using either lentiviral or retroviral vectors. Jin and her team transduced a batch of T cells with the CAR gene and then isolated and purified several CAR T cells' genomes and assessed the transduction protocol's success.4 They measured the transduction efficiency using flow cytometry and quantified the CAR gene copy number per genome using ddPCR.
In this study, ddPCR was consistent in quantifying CAR copy number. They found the same result whether the sample was tested immediately or after three or six weeks of freezing. The results were also consistent across three different technicians, demonstrating the technique's high reliability and robustness.
The researchers examined the impact of two variables, multiplicity of infection (the ratio of viral vectors to T cells) and centrifugation, on transduction efficiency and copy number. Using flow cytometry and a ddPCR assay, respectively, they found that transduction efficiency and copy number increased as multiplicity of infection (MOI) – the virus:cell ratio – increased, and that transduction efficiency and copy number were directly correlated. Finally, using ddPCR, they found that the low copy numbers seen at the lower range of MOIs were increased with high-speed centrifugation.
Finally, they mapped the locations where the CAR transgene integrated along the genome using next-generation sequencing (NGS). In the future, Jin’s group hopes to use ddPCR for this step as well. Although transfected or transduced genes integrate randomly, they show a preference for particular sites along the genome. Once these sites are determined, Jin and her team plan to design dedicated PCR probes, enabling them to detect integrations at those sites. Overall, ddPCR offers a shorter turnaround time than NGS.
Taken together, Jin’s data shows ddPCR reliably quantifies transgene copy number in clinical CAR T products and can complement flow cytometry in supporting the refinement of CAR T manufacturing protocols. While the researchers used flow cytometry to measure the relationship between MOI and transduction efficiency, ddPCR enabled them to study the relationship between MOI and copy number, and subsequently, relate copy number and transduction efficiency. With a set of tools that includes ddPCR, CAR T-cell manufacturers can be more confident that their products are safe and that they will be effective in treating cancer.
Jin’s work only highlights one area where ddPCR can assist in CAR T-cell manufacturing, but given its versatility, it might one day help in all aspects of the process, both upstream and downstream.
1. Search of: "car+t": Active, not recruiting Studies - List Results. ClinicalTrials.gov. https://clinicaltrials.gov/ct2/results?term=%22car%2Bt%22&Search=Apply&recrs=d&age_v=&gndr=&type=&rslt=. Accessed March 19, 2021.
2. Coherent Market Insights. Global CAR-T cell therapy market to be worth US$ 8 billion by 2028. GlobeNewswire News Room. https://www.globenewswire.com/news-release/2018/05/31/1514897/0/en/Global-CAR-T-Cell-Therapy-Market-to-be-Worth-US-8-Billion-by-2028-Coherent-Market-Insights.html. Published May 31, 2018. Accessed March 19, 2021.
3. Zhao Y, Stepto H, Schneider CK. Development of the first World Health Organization lentiviral vector standard: toward the production control and standardization of lentivirus-based gene therapy products. Hum Gene Ther Methods. 2017;28(4):205-214. doi:10.1089/hgtb.2017.078
4. Lu A, Liu H, Shi R, et al. Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products. J Transl Med. 2020;18(1). doi:10.1186/s12967-020-02358-0
About the author:
Mark White is the Director of Biopharma Product Marketing at Bio-Rad Laboratories, Digital Biology Group.