CAR-T Cell Therapy and the Importance of Vector Copy Number Determination
Jan 22, 2025 | Informative Articles
Chimeric Antigen Receptor T-cell (CAR-T) therapy represents a revolutionary advancement in personalized medicine, offering hope for patients with various serious diseases, particularly hematological malignancies. By engineering a patient’s own T-cells to target and destroy cancer cells, CAR-T therapy has achieved remarkable clinical success. However, the complexity of this therapy brings significant challenges in manufacturing and quality control. One of these is the precise determination of vector copy number.
The CAR-T Therapy Process
① Collecting T-cells: At first white blood cells are removed from the patient to isolate T-cells.
② Reprogramming T-cells: The T-cells are reprogrammed by introducing a genetic sequence enabling the T-cells to produce new surface receptors, called Chimeric Antigen Receptor (CAR). This is often done by using lentiviral vectors.
③ Multiplying CAR-T cells: CAR-T cells are expanded in the lab to create millions of copies.
④ Infusing CAR-T cells: Patients are often treated with a preparatory chemotherapy. The multiplied CAR-T cells are then infused back into the patient.
CAR-T cells can now recognise and attach to specific marker proteins on the cancer cells – signalling their destruction. They are able to multiply in the body and persist as a ‘living cancer drug’.
Lentiviral vectors are most commonly used in a CAR-T cell therapy process due to their efficiency and stability. They are a type of retrovirus that derive from HIV-1 and can deliver genes into both dividing and non-dividing cells. Lentiviral vectors have the capacity to integrate permanently into the host cell DNA, leading to a long-term and stable CAR expression in the T-cells.
What is Vector Copy Number and why is it important?
Several QC tests must be carried out during a CAR-T production process - regarding potency, purity and safety. This includes residual host cell DNA testing, mycoplasma and sterility testing as well as vector copy number determination.
Vector Copy Number (VCN) refers to the average number of copies of integrated viral vector per cell genome after ex vivo transduction. Accurate VCN determination is crucial for ensuring the safety and efficacy of the cell therapy product.
A sufficient number of integrated viral vectors is required for the T-cells to express enough CAR molecules to effectively target cancer cells. But vector integration can potentially alter expression of cellular genes and contribute to tumorgenicity, resulting in an increased risk of insertional mutagenesis if the VCN per cell is high. Therefore, vector integration is an important safety attribute for CAR-T cell release. Regulatory agencies mandate strict control over VCN to ensure balance between safety and efficacy. The FDA recommends that the VCN shall be < 5 copies per genome to minimize the risk of insertional mutagenesis. [1]
Discover Next-Generation VCN Testing
Digital PCR (dPCR) is a powerful and easy tool to determine VCN in a CAR-T cell product. Compared to real-time PCR, this technology does not rely on standard curves for absolute quantification, shows higher sensitivity and precision and is less affected by inhibitors, making it highly reliable for quality control.
MiQuant® CAR-T Cell Lenti – dPCR kits enable fast and accurate VCN determination and are compatible with various dPCR devices (QIAcuity®, ddPCR™, Quantstudio™ AbsoluteQ™). The copies of the lentiviral gene (psi region) are determined in relation to the human housekeeping gene PCBP2. Genomic DNA must first be extracted from transduced cells (e.g. with MiQuant® SP Beads).
After dPCR analysis the VCN can be calculated considering the ploidy and transduction frequency – manually or by using MiQuant® CAR-T Cell Data Analysis Application.
Both products can be tested. Simply request a free sample.
The MiQuant® CAR-T Cell – dPCR kits can be used for VCN determination during the production process as an in-process control, and prior to infusion into the patient as final QC release testing. In addition to these applications the kits can also be used to determine VCN in cell culture samples in research and development and are suitable for human or other mammalian cells such as mice or rats.
[1] https://www.sciencedirect.com/science/article/abs/pii/S1465324920303479