CAR T-cell therapy represents an advanced form of immunotherapy that utilizes a patient’s own immune cells to target and eliminate cancer. This treatment involves collecting T-cells, a type of white blood cell, from the patient and then genetically modifying them in a laboratory. The modified cells are designed to recognize specific markers on cancer cells, enhancing the body’s defenses against the disease. Once prepared, these re-engineered cells are returned to the patient, where they can seek out and destroy malignant cells.
Collecting Patient Cells
The initial step in CAR T-cell therapy involves collecting the patient’s T-cells, the raw material for manufacturing. This collection is performed through leukapheresis, a process similar to donating plasma. During leukapheresis, blood is drawn from a vein, typically in the arm or through a central line, and circulated through an apheresis machine.
The apheresis machine separates the white blood cells, including T-cells, from other blood components. The remaining blood, such as red blood cells and plasma, is then returned to the patient. This procedure usually takes several hours, typically 3 to 6 hours, and may be repeated over one or two days to ensure a sufficient number of T-cells are collected. Cell quality is a factor in manufacturing success.
Genetic Engineering and Cell Expansion
Once the T-cells are collected, they are transported to a specialized laboratory where genetic engineering and expansion occur. The first step involves isolating the T-cells from the collected material by washing the leukapheresis product to remove anticoagulants. These isolated T-cells are then activated, preparing them for genetic modification and growth. T-cell activation is achieved by stimulating specific pathways (e.g., CD3, CD28) with cytokine support.
Following activation, the T-cells undergo genetic modification, known as transduction, to express the Chimeric Antigen Receptor (CAR). This involves introducing a new gene into the T-cells that codes for the CAR, allowing the cells to recognize specific cancer antigens. Viral vectors, particularly lentiviruses or gamma-retroviruses, are commonly used for this gene delivery due to their efficiency in integrating the CAR gene into the T-cell’s DNA. Lentiviral vectors are often preferred as they can infect both dividing and non-dividing cells and offer stable, long-term gene expression.
After successful genetic modification, the modified CAR T-cells are expanded to achieve a therapeutic dose. This expansion occurs in specialized bioreactors or culture bags, which provide a controlled environment for cell growth, including temperature regulation, pH balance, and nutrient supply. Common bioreactor types include stirred-tank bioreactors, wave bioreactors, and G-Rex devices, each offering different advantages for scalability and control. The expansion process can take several weeks, during which the cells multiply to reach the millions or even billions needed for treatment.
Quality Control and Final Formulation
After the CAR T-cells have been genetically engineered and expanded, they undergo quality control testing to ensure they are safe, pure, and potent for administration. These tests are performed at various stages throughout the manufacturing process and are important before final product release. Sterility testing checks for microbial contamination, such as bacteria, fungi, and mycoplasma, which could compromise patient safety.
Viability testing confirms that a high percentage of the CAR T-cells are alive and functional, while identity testing verifies that the cells are the intended CAR T-cells. Potency assays are also performed to confirm the cells’ ability to effectively kill cancer cells, often by measuring their cytotoxicity and cytokine production in response to target cells. These checks ensure the product meets specifications for quality and effectiveness.
Once all quality control tests are passed, the CAR T-cell product is prepared for final formulation, which involves concentrating the cells to a volume suitable for infusion. The cells are often cryopreserved to maintain their viability during storage and transport. Cryopreservation typically involves mixing the cells with cryoprotectants like dimethyl sulfoxide (DMSO) and human serum albumin (HSA) before cooling them to very low temperatures, halting biological activity until use. This step also includes a precise cell count to ensure the patient receives the correct therapeutic dose.
Infusion Preparation and Administration
The final phase of CAR T-cell therapy involves preparing the manufactured cells for infusion and administering them to the patient. The cryopreserved CAR T-cells are shipped from the manufacturing facility to the treatment center in specialized containers, maintaining their frozen state. Upon arrival at the hospital or clinic, the cryopreserved product is carefully thawed.
Thawing usually occurs rapidly in a water bath heated to 37°C, with the CAR T-cell infusion bag placed in a secondary bag to prevent direct contact with the water. Infusion must occur soon after thawing, typically within 30 minutes, to maintain cell viability, as DMSO can be toxic at warmer temperatures. Before infusion, patients may receive pre-medications like acetaminophen or diphenhydramine to minimize allergic reactions to preservatives in the product.
The thawed CAR T-cells are then administered to the patient, typically through an intravenous (IV) line. This is often done via a central venous catheter, such as a tunneled chest catheter or a PICC line, or sometimes through a peripheral IV in the arm. The infusion process is similar to a blood transfusion and usually takes a short time, typically 15 to 30 minutes, depending on the volume. A nurse monitors the patient’s vital signs throughout the infusion for safe administration.