Conclusion

T cells are white blood cells that make up the army of the immune system. They use their receptors to bind to proteins presented on the surface of disease-causing pathogens and toxic cells to stimulate an immune response. Chimeric Antigen Receptor T cells (CAR-T) are T cells with receptors that have been engineered by applied molecular engineers to target these specific proteins. Throughout this article I will discuss how CAR T-cells are manufactured.

There are three main steps: Extraction and Collection, CAR-T Preparation, and finally Release Testing and Patient delivery.

The patient’s blood sample is taken at a hospital and shipped to a manufacturing facility. T- cells from patient is extracted via leukapheresis. This is a procedure where white blood cells are separated from red blood cells in a blood sample by destroying all the red blood cells, platelets, and other non-white blood cell contents. Specific T-cells with receptors are then selected.

First the extracted T cells must be activated, after which the genes encoding the CAR proteins specific to the disease-causing pathogen are inserted. Next T-cells that have successfully taken up the genes are grown en-masse.

1. T cell activation

Some of the T-cells that are extracted from the patient are inactive, they are known as naïve T cells. Naïve T cells often lack a receptor required for CAR gene delivery, as a retroviral vector needs to interact with specific receptors in order to enter and deliver CAR genes into the T-cells. Therefore, these T-cells need to be activated.

Once these naïve T cells are extracted, they are activated either via a cell-based method or a beads-based method. A cell-based method involves culturing the T-cells with Artificial Antigen Presenting Cells (AAPCs). The beads-based method uses magnetic beads which can be coated with antibodies, or with anti-CD3+ antibodies or with Expamer technology a (recently developed reagent). These methods are designed to stimulate the growth of a specific receptor(s) which are indicative of active T-cells, the receptor chosen depends on the disease-causing protein you are targeting.

2. Vector selection and CAR gene delivery

At this stage, the gene encoding the engineered receptor (CAR) is subcloned into a plasmid, then inserted into a vector, which will deliver the gene to the activated T cells. These transfected cells will then express the CAR receptor on their surface.

Vectors range from retroviral vectors to transposons. In some cases, vectors are not used, rather the mRNA coding for the CAR genes are inserted directly into the cytoplasm of the T-cells. However, this method is not as efficient as other vectors due to the susceptibility of mRNA to degrade very quickly within the cell and before administration, which is not ideal if you are looking for a long-term expression of the CAR receptor. Retroviral vectors, on the other hand, are chosen and used more often. Due to their proven delivery efficiency resulting in the high and longer-term expression of delivered genes.

3. Growing the successfully transformed T-cells.The cultured T cells are then grown in a bioreactor, in order to provide a large quantity of CAR-T cells. This will allow for an effective targeting and destruction of the disease-causing pathogen or toxic cell in the patient. Examples of Bioreactors used are the G-rex bioreactors, or Prodigy Bioreactors.

Throughout and after the drugs have been manufactured, there are many tests conducted to ensure safety of the manufacturing process, the purity of the product formed, to confirm the identity of the product, and to assess the potency of the drug.

Assessing the safety of manufacturing process involves testing of the raw materials used in manufacturing the drug. This also includes testing by the vendor of the raw materials, to meet the CAR-T manufacturer’s specifications, after which further testing then occurs at the CAR-T cell manufacturing site before the use of the vectors to deliver these genes.

The identity of the product is assessed by detecting if the T-cells are expressing the CAR receptor on the surface of the modified T-cells.

The purity of the product is assessed by measuring the concentration of CAR-receptor-expressing T cells there are present in the culture, relative to impurities, using criteria established by the FDA.

How potent the CAR-T cells formed are is assessed via interferon-𝛾-secretion and in-vitro cytotoxic T-lymphocyte assays.

After these tests, the CAR T- cells are packaged and delivered to a hospital to be administered to a patient.

Overall CAR — T cell manufacturing is a multi-step process, with intricate testing stages throughout the process to assess safety of raw materials and to ensure the best possible product is formed. This process begins with the extraction and separation of T-cells from a patient’s blood sample. These extracted T-cells are then activated to prepare them for CAR receptor gene delivery and transfected by vectors carrying these genes. Finally, the successfully transfected T-cells are selected and grown in large volume, tested to ensure purity, potency and identity and shipped back to the hospital to be administered to a patient.

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