Immunotherapy technologies are rapidly advancing, as are the methods of ensuring their safety and reliability. ICON explores the tools used in cell and gene therapy (CGT) trials.
CGT involves the use of genes or cells to treat a disease. With cell-based therapies the cells can either be derived from the patient (autologous) or from a donor (allogeneic). Cell-based therapies such as CAR-T cells are engineered using viral vectors to express receptors that recognize tumor antigens resulting in attack and destruction of the tumor cells. Patients receiving CAR-T therapies first undergo chemotherapeutic pre-treatment before transduced cells are infused into their bodies. This pre-treatment results in lymphodepletion which helps prolong the persistence of infused therapeutic cells. These patients then face a period of immune system reconstitution; throughout this time, there is a series of follow-up visits to monitor the patient’s health and determine the expansion and persistence of the CAR-T cells.
PCR and qPCR assays for PK and safety monitoring
ICON’s CGT team have developed polymerase chain reaction (PCR) assays that have applications in a myriad of cell therapies, from patient selection (to identify the cell therapy target in solid tumors) to baseline assessments, and pharmacokinetic (PK) monitoring of clinical drug products.
PCR tools enable the rapid amplification of a segment of DNA or cDNA. The PCR exponentially amplify the number of copies of a specific DNA fragment or transcript, which allows for detection and quantification of the nucleic acid sequences that are of interest.
Quantitative PCR (qPCR) and digital droplet PCR (ddPCR) differ in performance characteristics and are therefore used for different applications. Unlike qPCR, ddPCR offers absolute quantitation without the need for calibrators. ddPCR is more sensitive than qPCR, which improves precision and reproducibility at low inputs. However, qPCR is an established and robust technology that offers a wide dynamic range and higher throughput for sample testing.
ddPCR is an emerging technology used for the accurate and sensitive quantification of viral vector sequences that are found in CAR-T and other cell-based therapies. The detection and quantification of vector copy numbers (VCN) in DNA extracted from a whole blood or PBMC sample can be used to monitor the persistence and potential expansion of the engineered cell therapy in the patient’s bloodstream following infusion.
One challenge that the lab faces when performing VCN testing is the low DNA yields from blood samples that are collected at early timepoints post-infusion. The low DNA yields are a result of lymphodepletion. In addition to low yields, the isolated DNA can be fragmented, due to apoptosis caused by the chemotherapeutics. This fragmentation greatly reduces the quality of the DNA in the sample. Therefore, ICON’s researchers have developed methods and strategies to overcome this challenge: one strategy involves the performance of a reduced mass input (RMI) study during assay validation. ICON’s RMI studies involve the testing of several mass inputs that are below the standard input to assess the lowest possible input allowed that will not compromise the safety or the reliability of the data.
Monitoring the safety of gene therapies
Chimeric antigen receptor T (CAR T) cell therapy requires a retrovirus or lentivirus to introduce the CAR gene into activated T cells. A safety concern with using retroviral/lentiviral vectors for this treatment is the potential for replication competent retrovirus or lentivirus (RCR/RCL) to arise. The emergence of RCR or RCL could result in development of vector-associated malignancies. The FDA has posed safety concerns with the possibility of RCR/RCL being created and recommend monitoring of patients who have received CAR-T therapy. qPCR assays are used for RCR and RCL monitoring.
The FDA offers guidance on the testing of replication-competent retrovirus (RCR) during the manufacturing of these therapy products and the follow-up with patients who have received these treatments. The testing schedule, as suggested by the FDA, includes pre-treatment testing and post-treatment testing three times throughout the first year and yearly for up to 15 years following treatment. At the manufacturing stage, the FDA guidance includes the identification of sample type (PBMC) and required sensitivity of the test (50 copies/µg DNA) as well as general testing methods (qPCR).
Looking forward with ICON
ICON’s cell and gene therapy team leverages their experience in the industry to deliver all phases of clinical trials, from the early phase to commercialisation. ICON has had a role in over 130 CGT trials in 23 countries, and as an early pioneer of CGT, ICON was also part of four of the seven approved CAR-T therapy programs available today.
The rapid and continued growth of CGTs over the last decade shows great promise for those suffering from serious diseases. But as new developments arise, so will safety considerations and concerns; however, ICON’s innovative CGT team will be there to help create a safer and more efficient treatment landscape for our sponsors and their patients.
Learn more about ICON’s work in cell and gene therapies here
Listen to our on-demand webinar: Best practices for laboratory solutions relating to cell and gene therapy clinical trials.