Listen to this article on the DDW Podcast:
By Mark White, Associate Director of Biopharma Product Marketing at Bio-Rad and Marwan Alsarraj, Biopharma Segment Manager, Digital Biology Group.
Until about 25 years ago, the best treatment option for someone with blood cancer was either toxic radiation and chemotherapy or a bone marrow transplant that posed additional life-threatening risks to the patient1. But decades of research led to improvements in cancer therapy, centred around the idea that one’s immune system could aid in treatment. Next, the advent of antibody treatments in 1997 created the first targeted cancer therapy2. Finally, in 2017, after years of improving T cell engineering techniques, the first chimeric antigen receptor (CAR) T cell therapy received FDA approval. CAR T cells are T cells that have been trained to attack tumours. Since 2017, five CAR T cell therapies have been approved and have shown incredible success in the clinic. Their continued promise has led to an exponential increase in research on their utility, function, risks, and potential benefits. And as researchers study all of these factors, they are also discovering that CAR T cell development and manufacturing require a new approach to quality control to ensure their safety and efficacy.
Developing the first CAR T cell therapy
The discovery of CART cell therapy resulted from decades of research dedicated to understanding the immune system and its role in treating cancer. The immune system typically identifies foreign entities such as bacteria and viruses and activates lymphocytes such as T cells to destroy them with overwhelming force. However, tumour cells actively suppress the immune system, which allows the cells to proliferate virtually unhindered. Immunotherapies like CAR T therapy are designed to overcome the immunosuppressive behaviour of tumour cells and engage the immune system in the fight against cancer.
The first major success in treating cancer using the immune system was in the1960s, with the advent of the bone marrow transplant3. Fundamentally, a bone marrow transplant supplies the body with additional immune cells, including T cells. Later, scientists took advantage of antibodies’ natural tendency to seek out specific antigens and created antibody therapies that guide T cells towards the cells they are supposed to destroy. These treatments have shown success in treating a variety ofcancers4. In 1987, researchers developed the chimeric antigen receptor (CAR) as an alternative means to identify cancer cells and direct T cells to attack them. Exactly three decades later, the first CAR T cell therapy hit the market. And now, with more than 600 CAR T trials recruiting or ongoing, these cells represent a majority of the cellular immunotherapies in development today.5,6
Training T cells to target tumours
CAR T cells are designed to seek out tumour-associated antigens and destroy the cells that express them. Unmodified T cells fail to attack tumours because, to them, tumours are invisible. Normally, T cells only recognise foreign cells by identifying unfamiliar forms of a cell surface marker called a major histocompatibility complex (MHC). However, cancer cells inhibit the expression of MHCs and effectively hide from detection. To overcome this evasion tactic, the CAR protein was developed to recognise other markers on cancer cells.
CAR is a fusion protein composed of an extracellular antibody fragment that recognises antigens and an intracellular domain that directs the T cell to the tumour. Most of the CAR T cell therapies today target the CD19 receptor, a protein exclusively present on B cells. As a result, CD19-targeted CAR T cells show significant success in treating B cell lymphomas, leading to complete remission rates of up to 40%7. To create these cells, a physician draws a patient’s blood and sends it to a laboratory where the cells are isolated, engineered to express the CAR protein, and amplified in a bioreactor before being infused back into the patient. Physicians can trust that this therapy will destroy the right cells because the CAR protein was designed to seek out a particular antigen.
Identifying the right CAR T cell target
Selecting the right target is key to ensuring the safety and efficacy of CAR T therapy. The best target is present in high concentrations on the surface of tumour cells and does not appear in other tissues. For example, CD19 offers a sensitive and specific target for B cell lymphomas, but it does not appear on all tumour cells; therefore, researchers need to identify other targets to address other cancers.
Cells from solid tumours are genetically heterogeneous both between and within tumours, making it challenging to identify a single, selective antigen target for treatment. For example, many antigens found on solid tumours are also present at low levels on healthy tissue, opening the door to off-target effects. If CAR T cells attack non-tumour tissue, they can cause significant adverse events leading to organ dysfunction and death8.
To overcome the challenge of identifying the ideal target, scientists are using several creative methods. For instance, some therapies in development target combinations of antigens to improve the effectiveness and specificity of the therapy9. Other developers are installing kill switches that allow clinicians to deactivate a therapy that has begun to produce adverse reactions. For example, one clinical trial examined the safety and efficacy of a CAR T cell that includes a suicide gene, which produces a toxic protein that causes the CAR T cells to die upon exposure to a drug10. Once scientists identify the correct target and build their CAR T cells for testing, biomanufacturers then need to address the challenge of manufacturing these cells.
The challenge of manufacturing CAR T cells
CAR T cells are made by transfecting T cells with a transgene that encodes the CAR protein using either a virus, a transposon system, or mRNA. Unfortunately, transfection is not a predictable process. Efficiencies and success rates vary from lab to lab, protocol to protocol, and experiment to experiment. Transfection efficiency with lentiviruses can range from 30 to 80%11.
This variability stems from manufacturers’ inability to completely control the transfection process at the molecular level. For example, it is challenging to regulate transgene copy numbers in each T cell, posing safety risks. If the gene does not integrate into the genome, the cell will not become a CAR T cell. But if the cell accepts too many copies of the gene, the cell can cause cytokine release syndrome (CRS) or a ‘cytokine storm’. CRS can cause fevers, low blood pressure, reduced oxygen saturation, low levels of healthy blood cells, abnormal blood clotting, organ dysfunction, and a deadly form of immune over-activation12.
To prevent these potentially fatal side effects, the US FDA recommends that manufacturers who generate CAR T cells using retroviral or lentiviral vectors screen out batches that contain more than four transgene copies per cell13.
Furthermore, even if the transgene enters the cell in the correct numbers, the location where the gene integrates into the genome is somewhat random. Although transgenes show a preference for certain sites, the CAR transgene could integrate into the T cell genome at any location, including within a sequence that regulates oncogene expression, thereby increasing cancer risk. The gene could also bury itself in a silenced region of the genome, leaving the gene dormant. Therefore, it is important to identify the location of these integrations to predict the safety and efficacy of the treatment. The manufacturing process can also introduce replication-competent viruses (RCVs)to batches of cells. Although this type of contamination has not yet been reported in humans, RCVs can form rapidly growing T cell neoplasms, another type of cancer. To prevent this from happening, the FDA recommends that manufacturers test clinical vector lots, manufactured cell products, and patients who have received treatment with CAR T cell therapy14.
Fortunately, because all these elements can be tracked by detecting and quantifying nucleic acids, manufacturers can assess the safety using Droplet Digital PCR (ddPCR) technology. How ddPCR technology detects and quantifies DNA ddPCR technology is a highly sensitive and accurate tool used to detect and quantify known genetic sequences. Unlike qPCR, which requires users to generate a standard curve to estimate the concentration of DNA or RNA in a sample, ddPCR technology quantifies nucleic acids directly. The platform uses water-oil emulsion technology to fractionate a 10-microliter sample into roughly 20,000 nanoliter-sized droplets, each containing one or a few nucleic acid strands. Each droplet essentially functions as a separate reaction tube. Using primers and probes similar to those employed by other PCR techniques, users can measure amplification events across thousands of reactions taking place simultaneously across the droplets. A primer specifically targeted at the template strand will enable amplification within the droplets that contain the target strand. When amplification takes place, sequence-specific probes are cleaved and release a fluorescent signal. Meanwhile, droplets that do not contain the template strand only emit weak fluorescence. Users then pass the droplets through a detector that counts the number of fluorescent droplets and uses Poisson statistics to derive the nucleic acid concentration in the original sample.
This technique can be used to quantify copy number variation by measuring the number of copies of a nucleic acid sequence in a sample and calculating the average number of copies per cell. Its sensitivity also makes ddPCR technology suitable for detecting rare sequences, such as those found in RCVs and the small concentrations of CAR T cells in the human circulatory system following infusion. Overall, scientists use ddPCR technology when they need high accuracy and precision, such as in the development of cellular therapies that need to be safe and effective in humans.
ddPCR assays for CAR T cell copy number quantification
Quantifying transgene copy number is central to the development of a safe CAR T cell therapy. Still, the gold-standard nucleic acid measurement tool—qPCR—is not suitable for this purpose. Unlike qPCR, ddPCR technology can quantify nucleic acids down to one copy per genome, making it an effective tool for quantifying CAR transgene copy number15. National Institutes of Health researcher Ping Jin, Ph.D. demonstrated that ddPCR assays could quantify transgenes following lentivirus-or retrovirus-mediatedtransduction17. Her team first assessed how variability in manufacturing protocols affected copy number. ddPCR assays revealed that copy number remained consistent regardless of whether the cells were frozen before testing and whether any one of three technicians collected the cells.
Jin’s team also used ddPCR technology to study the relationship between multiplicity of infection (MOI) and copy number. MOI is the ratio between virus concentration and cell concentration, and with ddPCR assays, the researchers found that as MOI increased, so did copy number. Finally, ddPCR assays revealed that at low MOIs, centrifuging the cell/virus mixture increased copy number per cell. Since transfected genes show a preference for specific sites in the genome, scientists can design custom ddPCR assays that can detect whether integrations have occurred at those sites, saving them the time they would need to sequence the entire cell genome.
Detecting replication-competent viruses
Replication-competent retroviral vectors have caused lymphomas in primates. They’ve also induced lymphomas in humans when delivered directly in the form of gene therapy17. Given the risks, it is important to detecta nd screen out RCVs in CAR T cell therapy batches. The tools manufacturers currently use to detect RCVs pose several limitations that can be overcome by ddPCR technology. Cell-based assays often require considerable time to produce results. qPCR delivers results quickly, but manufacturers cannot necessarily use it to guarantee a CAR T cell batch’s safety due to its limited sensitivity. Researchers at the Mayo Clinic have designed a ddPCR assay that targets the stomatitis virus G glycoprotein envelope sequence, which is required for competent viral particles to assemble. Compared to qPCR, this ddPCR assay has a lower limit of detection (10 copies per microlitre) and reduces hands-on time and human error by removing the need for a standard curve18.
Measuring the pharmacokinetics of CAR T cells
Once CAR T cells are designed to target the correct tissue, have been successfully manufactured, and are free of harmful contaminants, they are ready for administration to patients. However, in the clinic, a whole new set of challenges arise. One factor that determines a CAR T cell therapy’s impact in the clinic is its persistence in the blood. If CAR T cells degrade too quickly, they will not be effective, but if they persist in the body for too long, they can induce a prolonged inflammatory response even after the cancer is gone12. Therefore, clinical researchers must investigate the nature of CAR T cell persistence in patients to ensure proper dosing, predict outcomes, optimise follow-up regimens, and understand the causes of treatment failure and adverse events.
Scientists have not yet adopted a standard method for monitoring CAR T cell persistence in the blood. However, given ddPCR technology’s capacity to detect minute quantities of nucleicacids in liquid samples, it is highly suitable for use in serial monitoring of CAR T cell kinetics and its impact on patient outcomes.
In a recent study, researchers in Germany tested the hypothesis that a ddPCR assay could be used to predict therapeutic efficacy in lymphoma by quantifying CAR T cell kinetics15. In a set of positive controls containing various known CART cell genomes concentrations, the scientists’ assay detected down to one transgene copy per genome. They then tested 54 samples from seven patients taken between four weeks and nine months following treatment and found that CAR T cell kinetics varied considerably from patient to patient, demonstrating the unpredictability of CAR T cell therapy and the need for serial monitoring.
The researchers also found that CAR T cell kinetics correlated with clinical responses. For example, one patient’s CAR transgene levels dropped to undetectable levels at day 75 and showed no clinical improvement, whereas patients whose transgene levels were still detectable after nine months went into remission. Furthermore, the patient who expressed the highest peak transgene levels experienced severe side effects. These data mirrored the molecular data seen in the laboratory. They also revealed another variable in the pursuit of developing safe and effective cell therapies: dosing is not so straightforward. Overall, the results demonstrated that serial monitoring of CAR T cells could be critical to ensuring their safety and efficacy in the clinic.
Can CAR T cells be made available off-the-shelf?
There are multiple ways to make CAR T cells safer and more effective, but one change that could make an especially big difference for all eligible patients is the development of allogeneic, or off-the-shelf, CAR T cells. All of today’s approved CAR T cells are autologous, meaning that each treatment is derived from an individual patient’s blood. While this approach minimises the risk of immune reactions to the treatment, it comes with a development turnaround time of weeks—time that many cancer patients cannot afford to lose.
According to the results of a recent simulation, even modest delays can hinder these cells’effectiveness19. To reduce wait times, scientists seek to develop CAR T cells that could be manufactured in advance. Allogeneic CAR T cells could potentially shorten the time between diagnosis and treatment.
However, allogeneic cells pose their own risks. The donor cells could potentially attack healthy tissues, thereby inducing graft-vs-host disease. On the other hand, the patient’s immune system might attack the foreign CAR T cells and render the treatment inert. One possible way to overcome this challenge is to bypass T cells entirely and instead transduce the CAR transgene into a different lymphocyte, the natural killer (NK) cell. NK cells naturally attack tumour cells and do not need to be genetically modified to target a specific antigen. Moreover, they not only attack tumour cells on their own, but they also release signals that activate the adaptive immune system. Data suggests that allogeneic NK cells are safer than CAR T cells, enabling the creation of an off-the-shelf option for patients20. However, the immunosuppressive behaviour of tumour cells often causes NK cells to become dysfunctional. This is where the CAR protein comes in. Scientists are developing allogeneic CAR NK cells that might more easily overcome immune suppression in the tumour micro environment. CAR NK cells are still in the early stages of development, but they can offer a cheaper, more flexible solution for patients21.
The next 25 years of CAR T cell therapy development
CAR T cells already offer many people with cancer a second chance at life. While these cells still face significant discovery and development challenges, hundreds of clinical trials and a significant number of preclinical studies are underway to uncover new benefits of CAR T cells and to find ways to overcome their limitations. In addition, a deeper understanding of the immune system and its relationship with cancer will lead to further advancements in CAR T cell therapy and bring relief to more people with different types of cancer. As new CAR T cell therapies reach the market, quality control will become even more important. In general, cell therapies represent a new kind of ‘drug’—one that is living, and therefore cannot be fully predicted or controlled. Tools like ddPCR technology will help cell biologists and manufacturers keep a close eye on these cells’ development and activity by measuring copy number, detecting the presence of RCVs, and monitoring CAR T cell persistence in patients’ blood.
The sheer size of the CAR T cell industry that has materialised over the past decade is emblematic of the rapidly growing optimism for this burgeoning field. Plus, regulators are promoting this growth. For example, the FDA supports the development of safe and effective T cell therapies through their guidances on cell therapy manufacturing22. As a result, the global CAR T cell market may reach $8 billion in size by 202823.With basic research leading to the identification of new target antigens, b) biomanufacturers developing more efficient development protocols, and regulators offering the support, there is virtually no limit to how far CAR T cells will progress over the next 25 years.
Volume 23 – Issue 3, Summer 2022
About the authors
Mark White is the Associate Director of Biopharma Product Marketing at Bio-Rad. He has played a key role in the development of multiple core technology capabilities and assays alongside a multidisciplinary team of biologists and engineers at Bio-Rad and previously at Berkeley Lights.
Marwan Alsarraj is the Biopharma Segment Manager at Bio-Rad. He has been at the forefront of developing, marketing, and commercialising technologies in the past 15 years in the life science research industry. Marwan obtained his M.S. in Biology at the University of Texas, El Paso.
REFERENCES:
1: Treatment Options for Lymphoma Patients: When to Consider Transplant or CAR-T. BMTinfonet.org. https://www.bmtinfonet.org/video/treatment-options-lymphoma-patients-when-consider-transplant-or-car-t
2: 2T Pierpont, C Limper, K Richards. Past, Present, and Future of Rituximab—The World’s First Oncology Monoclonal Antibody Therapy. Frontiers in Oncology. (2018) 4;8:163
3: First Successful Bone Marrow Transplant Led to Today’s Immmunotherapies. University of Wisconsin-Madison. https://www.med.wisc.edu/quarterly/volume-20-number-2/golden-anniversary-bone-marrow-transplant/
4: D Zahavi, L Weiner. Monoclonal Antibodies in Cancer Therapy.Antibodies (Basel). (2020) 10.3390/antib9030034
5: Studies found for: car-t | Recruiting, Active, not recruiting, Enrolling by invitation Studies,” U.S. National Library of Medicine. Clinicaltrials.gov.https://clinicaltrials.gov/ct2/results?term=cart&Search=Apply&recrs=a&rcrs=f&recrs=d&age_v=&gndr=&type=&rslt=
6: Cancer cell therapy landscape.Cancer Research Institute. https://www.cancerresearch.org/scientists/immuno-oncology-landscape/cancer-cell-therapy-landscape
7: J Brundo, N Lam, D Vanasse, et al.Safety and feasibility of anti-CD19CAR T cells with fully-human binding domains in patients with B-cell lymphoma. Nature Methods. (2020)26(2): 270–280
8: R Morgan, J Yang, M Kitano, et al. Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2. Molecular Therapy. (2010) 18(4):843–851.
9: S Yu, M Yi, S Qin et al. Next generationchimeric antigen receptor T cells:safety strategies to overcome toxicity.Molecular Cancer. (2019) 18: 125
10: M Casucci, L Falcone, B Camisa, etal. Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene. Frontiers in Immunolology. (2018) 9(507).
11: Zhang, S Qiu, X Zhang. Optimized DNA electroporation for primary human T cell engineering. BMC Biotechnology.(2018) 18(1).
12: Brundo, J Kochenderfer. Toxicities of chimeric antigen receptor T cells:recognition and management. Blood (2016) 127 (26): 3321f–3330
13: Y Zhao, H Stepto, C Schneider. Development of the First World Health Organization Lentiviral Vector Standard: Toward the Production Control and Standardization of Lentivirus-Based Gene Therapy Products. Human GeneTherapy Methods (2017) 28:4.
14: Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up: Guidance for Industry. Food and Drug Administration. https://www.fda.gov/media/113790/download
15: T Mika, A Maghnouj, S Klein-Scory, et al.Digital-Droplet PCR for Quantification of CD19-Directed CAR T-Cells. Frontiers Molecular Biosciences. (2020) 7:84.
16: A Lu, H Liu, R Shi, et al. Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCRT cell products. Journal of Translational Medicine. (2020) 18:191.
17: S Hacein-Bey-Abina, A Garrigue, GP Wang, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. Journal of Clinical Investigators. (2008) 118:3132–3142.
18: T Wiltshire, D Milosevic, E Jacob, etal. Sensitive detection of integrated and free transcripts in chimeric antigen receptor T-cell manufactured cell products using droplet digital polymerase chain reaction. Cytotherapy(2021) 23(5): 452-458.
19: S Tully, Z Feng, K Grindrod, et al. Impact of Increasing Wait Times on Overall Mortality of Chimeric Antigen Receptor T-Cell Therapy in Large B-Cell Lymphoma: A Discrete Event Simulation Model. JCO Clinical Cancer Informatics.(2019) 3, 1-9.
20: Liu S et al, V Galat, Y Galat, et al. NK cell-based cancer immunotherapy: from basic biology to clinical development.Journal Hematology and Oncololgy.(2021) 14:7.
21: N Albinger, J Hartmann, E Ullrich. Current status and perspective of CAR-T and CAR-NK cell therapy trials in Germany. Gene Therapy. (2021) 28:513–527
22: Cellular & Gene Therapy Guidances. Food and Drug Administration https://www.fda.gov/vaccines-blood-biologics/biologics-guidances/cellular-gene-therapy-guidances
23: Global CAR-T Cell Therapy Market to be Worth US$ 8 Billion by 2028.Coherent Market Insights. 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
