The structure of a CAR construct
Chimeric Antigen Receptors (CARs) are human-made molecules expressed on the surface of T cells, key cells of the immune system.
These CARs are targeted at markers on cancer cells. T cells with CARs on their surface are then directed straight towards cancer cells, enabling for targeted, specific killing.
CARs are genetically engineered fusion proteins, proteins consisting of at least two domains encoded by separate genes that have been joined so they are transcribed and translated as a single unit, producing a single polypeptide.
It is useful to think of the CAR molecule as trying to get our T cells to react and activate in ways that mimic what would happen under natural conditions during an immune response, but armed with ‘unnatural’ targeting capacities that allow them to ‘home in’ directly on cancer cells.
T cells are distinguished as such due to the presence of T cell receptors (TCRs) on their surface, which bind to fragments of antigens on foreign cells and initiate their destruction.
CARs can be viewed as manufactured and altered T cell receptors.
They consist of an ectodomain, transmembrane domain and an endodomain.
The ectodomain sits outside of the T cell in the extracellular space and consists of a targeting element (signal peptide) of a single chain variable region domain (scFv) formed by variable regions of immunogloblin (antibody). The signal peptide is targeted to the cancer-specific marker on tumour cells (CARs are targeted to different markers).
The transmembrane domain is attached to the ectodomain by the spacer domain. The transmembrane spans the T cell membrane and connects to the endodomain, the functional end of the CAR molecule embedded inside the T cell.
CAR constructs are classified as being in one of 4 GENERATIONS based on the structure of their endodomain.
The first-generation CAR molecule, generated in 1993 by Dr Eshhar and colleagues, had an endodomain consisting of two CD3 zeta-chain molecules (or CD3 ζ), which is one of the four peptides that forms the T cell receptor CD3 complex initiating intracellular signaling that leads to T cell activation. It is still the most common component of CAR endodomains today.
After limited success with the first-generation CARs, scientists realised that the CAR constructs worked better when they were designed with a co-stimulatory molecule (as well as the CD3 zeta molecules on the first gen CARs) to help with intracellular signaling and encourages the CAR-T cells to remain active and proliferate once in the body.
Dr Michel Sadelain and colleagues went on to develop second-generation CAR-T cells with a co-stimulatory signaling domain CD28, one of the proteins expressed on T cells that provides co-stimulatory signals required for T cell activation and survival.
Third-generation CARs were produced by incorporating two or more co-stimulatory domains (usually CD28 and 41BB or OX40) into the CAR construct.
Fourth-generation CARs, known as ‘T cell redirected for universal cytokine-mediated killing’ (TRUCKs) were generated by adding IL-2 (Interleukin 2, a type of cytokine signaling molecule in the immune system) to the base of the second-generation constructs. TRUCKs have shown improvements in anti-tumour efficacy.
Now, researchers have many options to choose from when building their CAR constructs – adding some elements in, taking some out, modifying others.