Question

1. Chimeric antigen receptor (CAR) T-cells are a type of immunotherapy that utilizes a fusion protein to hyperactivate the immune system and target cancer cells. Your PI wants you to generate a CAR T-cell against CD19, a marker for B-cell lymphoma. Before doing the protocol, your PI asks you the following questions to ensure you mechanistically understand what a CAR T-cell is:
   1. Briefly run through the process of genetically engineering CAR T-cells. (Schematics would be preferred.)
   2. Why would encoding a fusion protein into a T-cell be beneficial to create a CAR T-cell?
   3. What specifically do you need to encode in your viral vector to create this specific CAR T-cell?

Answer 1

a) Overview of genetically engineering CAR T-cells:

1. Collection: T cells are collected from patients by apheresis, a process to withdraw blood from the body and remove its components (here, T cells) and the remaining blood is returned to the body.
2. Engineering: The isolated T cells are genetically engineered to express chimeric antigen receptors (CARs) on their surface. Chimeric antigen receptors are proteins which allow the T cells to recognize targeted tumor cell antigen .
3. Multiplication: The genetically modified CAR T cells are "expanded" by allowing them to grow.
4. Conditioning Therapy: Before the infusion of the CAR T cells, patients receive chemotherapy that helps their immune system to adapt for the infused CAR T cells to expand and proliferate.
5. Infusion: Then, patients are infused with the CAR T cells and monitored for several weeks for the response to treatment  and side effects.

b)  Encoding a fusion protein into a T-cell be beneficial to create a CAR T-cell:

CAR T-cell therapy involves genetic modification of patient’s autologous T-cells to express a CARs which are the fusion proteins of a T-cell receptor intracellular signaling domains specific for a tumor antigen and selected single-chain fragment variable from a specific monoclonal antibody. This T-cell genetic modification will recognize the specific tumor cells and kill them.

C) Encode in your viral vector to create this specific CAR T-cell:

The T-cell genetic modification has to be done by encoding the viral vector with the expression of CARs which are the fusion proteins of a T-cell receptor intracellular signaling domains specific for a tumor antigen and selected single-chain fragment variable from a specific monoclonal antibody. It is important to recognize the tumors cells specifically to kill it.