In recent years, chimeric antigen receptor (CAR) T-cell therapy has emerged as one of the most promising immunotherapy for the treatment of cancer. This therapy relies on CARs, which are engineered receptors designed to recognize cancer antigens and activate the T cell receptor.

The Structure of CAR-T

The general structure of the CAR-T consists of three main parts, (i) an extracellular domain containing the scFv antigen-binding and spacer domain, (ii) a transmembrane domain anchoring to the cell membrane, and (iii) an intracellular domain responsible for initiating the signalling cascade. 

The antigen-binding domain is typically derived from the variable regions of a monoclonal antibody. This domain is a chimeric protein made up of light (VL) and heavy (VH) chains connected with a short linker peptide to form a single-chain variable fragment (scFv). The antigen-binding domain targets the specific antigen on the cancer cells and activates the T cells. The specificity and affinity of this domain are important factors for optimal therapeutic efficiency.

The spacer domain links the antigen-binding domain to the transmembrane domain, providing flexibility to reduce the spatial constraints between the CAR and its target antigen.

The transmembrane domain anchors the CAR to the T cell membrane, bridging the extracellular domain with the intracellular domain. It can also influence membrane stability and CAR-T efficiency.

The intracellular domain contains the costimulatory signalling domains that transmit the signal inside the T cell once it is activated. CD3 ζ, a T cell co-receptor is commonly used as the main CAR signaling domain. In addition to CD3 ζ, T cell also requires co-stimulatory molecules to sustain the response. Several co-stimulatory molecules have been successfully tested, including CD28, CD134 (OX40), and CD137 (4-1BB).

The design of the receptors has evolved over the years, and it is mainly the changes in the intracellular signalling domains that define the generation of the CAR-T cell. To date, five generations of CAR have been developed according to the organization of their intracellular signalling domains.

From First to Fifth Generations of CAR Cells

First-generation CARs contained a single CD3 ζ domain to induce T-cell activation. These first-generation CARs had limited signalling and expansion capability. Therefore, given their weak response, first-generation CARs had to be supplemented with exogenous IL-2 to ensure an efficient response.

Second-generation CARs were designed with additional costimulatory signalling domains (e.g., CD28, 4-1BB/CD137). This leads to improved activation, enhanced survival, and effective expansion of the modified T cells.

To improve the outcomes, third-generation CARs were made by adding multiple costimulatory signalling domains. Most commonly, both CD28 and 4-1BB are used. Although these third-generation CARs displayed increased persistence and proliferation, the efficacy is not significantly enhanced in comparison to second-generation CAR-T cells.

The fourth-generation CARs, also called TRUCKs (T-cells redirected for universal cytokine-mediated killing) are armoured second-generation CARs with additional transgenes to enhance anti-tumour activity via expression and secretion of cytokines (e.g., IL-12).

Fifth-generation CARs are the next-gen CARs. It is based on the second-generation CARs, which consist of a novel costimulatory domain to activate other signalling pathways such as the IL‑2‑Rβ intracellular binding domain of signal transducer and activator of transcription 3/5 (STAT3/5). This signal together with the costimulatory signals on the second-generation CARs are required to drive full T cell activation and proliferation. Additional variants of CARs, including dual CARs, split CARs, and inducible-split CARs, have also been designed to further enhance the specificity and control of the transfused T cells.

The structure of CARs has progressed rapidly in the past years, to improve the safety and efficacy of these therapies. So far, FDA-approved CART-T therapies are based on second-generation CARs. It is important to note that recent the generation of novel CAR designs and improvements is mainly based on in vitro or pre-clinical animal data. Therefore, further studies are warranted to determine which design would provide the best clinical outcome.

References

Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11, 69 (2021). https://www.nature.com/articles/s41408-021-00459-7#citeas

Tomasik J, Jasiński M and Basak GW (2022) Next generations of CAR-T cells – new therapeutic opportunities in hematology? Front. Immunol. 13:1034707. doi: 10.3389/fimmu.2022.1034707