The design and manufacture of chimeric antigen receptor (CAR) T cells is fraught with complex hurdles. Some of these are encountered during production while others can only be inferred weeks or months after the cells are infused into a patient. These challenges have driven stepwise improvements to the functional design of CAR constructs, bringing the technology ever closer to adoption as a frontline treatment for specific cancer types.

Once T cells have been isolated from a donor, the cells must be transduced with new genetic material, whether in the form of mRNA or DNA. The new nucleic acid molecule, representing the CAR transgene can then be expressed by the cell’s transcription/translation machinery. The individual CAR components produced include an extracellular binding domain, a transmembrane domain, and intracellular signaling domains.[1]

Figure 1. Chimeric antigen receptor T-cell. Dimitri, Herbst & Fraietta, 2022.

The extracellular domain enables the engineered T cell to recognize a specific antigen and its design is crucial to success. It is a single chain variable fragment (scFv) made from an antibody against a specific antigen molecule. Selection of the antigen target is itself a scientifically challenging task that weighs whether a given antigen is consistently present on the target tumor type against how widespread its expression is in other tissues in the body. The risk of therapeutic toxicity climbs if other cell types also express the target antigen because they may be attacked by the CAR-T cells. In addition, the specific affinity level of the scFv to its target requires modulation. A very high-affinity scFv is more likely to target any cell that is even weakly expressing the antigen molecule on its surface, including non-cancer cells. A CAR construct whose scFv has a relatively weaker affinity for its target will tend to trigger attacks only on cells with high concentrations of the antigen on their surface, reducing toxicity towards healthy tissues.[2]

Immediately below the scFv lies the spacer region (Figure 1). The length of the spacer region can influence the efficacy of the scFv through steric hindrance effects, especially when it comes to larger epitopes on target antigen molecules.[2] The physical distance between the CAR-T cell and the tumor cell, determined by the length of the CAR’s spacer region during their interaction, will also impact the ability of granzymes and perforins to diffuse within that space to lyse and kill the tumor cell. [2]

Figure 2. Variations in the CAR transmembrane component design. Jayaraman, Mellody & Hou, et al. 2020.

The transmembrane domain is adjacent to the spacer region (Figure 1). This component is the CAR’s anchor and numerous design variants exist (Figure 2). It must effectively transmit signals from the extracellular portion of the CAR to the cytoplasmic components, while maintaining the stability of the whole CAR structure. The transmembrane design can greatly influence the CAR’s efficacy and expression level on the cell membrane.

Figure 3. The evolution of CAR constructs. Yeo, Giardina, Saxena & Rasko, 2022.

The intracellular domains of the CAR complex kick-start the T cell’s response when encountering the target and several generations of intracellular CAR designs have now been tested (Figure 3). The first generation CARs generally used the TCR-derived CD3ζ chain[1] and that is true of many downstream-generation designs as well. The number and variation of additional co-stimulatory motifs used since the first-generation CARs provides enough literature for a blog post covering that topic alone. Their addition has enabled researchers to use the initial antigen detection event to trigger multiple, simultaneous signaling cascades[3], to boost signal strength and speed[2], to make the T cell’s attack more lethal against tumor cells[5], and to increase cell survival and expansion within patients’ bodies[4].

CAR-T technology will continue to advance, especially as more CAR-T therapies win regulatory approval for cancer types that otherwise have little to no alternative treatment options. Already, research has moved into simultaneous targeting of two antigens with a single CAR design[5]. It is intriguing to speculate on how the design of CAR-T therapeutics will evolve: Will individual T cells bear dozens of CAR constructs? Will patients receive infusions consisting of cell cocktails including other modified immune cell types as well? Until cancer survival rates see drastic increases, there will be a need for breakthrough, cutting-edge therapies like these to come to the fore and provide a lifeline to patients in need.



  1. Dimitri, A., Herbst, F. & Fraietta, J.A. Engineering the next-generation of CAR T-cells with CRISPR-Cas9 gene editing. Mol Cancer 21, 78 (2022).
  2. Jayaraman, J., Mellody, M.P., Hou, A.J., et al. CAR-T design: Elements and their synergistic function, EBioMedicine, Volume 58, 2020, 102931, ISSN 2352-3964,
  3. Yeo, D., Giardina, C., Saxena, P., Rasko, J.E.J. The next wave of cellular immunotherapies in pancreatic cancer. Molecular Therapy: Oncolytics Vol. 24 March 2022.
  4. Feins, S, Kong, W, Williams, EF, Milone, MC, Fraietta, JA. An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer. Am J Hematol. 2019; 94: S3– S9.
  5. Mei, H., Li, C., Jiang, H. et al. A bispecific CAR-T cell therapy targeting BCMA and CD38 in relapsed or refractory multiple myeloma. J Hematol Oncol 14, 161 (2021).