Although T cells expressing chimeric antigen receptors with CD28/CD3ζ or 4-1BB/CD3ζ signaling domains have similar signaling cascade mechanisms with “nearly identical protein phosphorylation events” according to a study published in Science Signaling, the CD28-based domains were found to be less robust when it came to their ability to eliminate lymphoma across mouse models of disease.1

Liquid chromatography/mass spectroscopy data was used to compare signaling across these two distinct, commonly used signaling domains. According to corresponding study author Stanley Riddell, MD, professor at the Fred Hutchinson Cancer Research Center in Seattle, Washington, the CD19 CAR developed by Kite Pharma/Gilead (axicabtagene ciloleucel) harnesses CD28, whereas the Novartis and Juno Therapeutics/Celgene CD19 CARs, such as those used to make tisagenlecleucel, rely on the 4-1BB signaling domain.

Investigators determined that stimulation of CD28/CD3ζ spurred increased phosphorylation after activation, and that the signal intensity linked to this stimulatory domain was stronger compared with the intensity and kinetics of the signals generated from a CAR 4-1BB/CD3ζ domain.

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Enhanced signal intensity was also associated with increased short-term effector responses. This translated into a reduction in T-cell memory formation via the CD28/CD3ζ pathway, which was subsequently linked to a reduction in the CAR’s in vivo antitumor activity through this signaling complex. In bone marrow, CD28/CD3ζ CAR-T cells expressed higher levels of PD-1 and other biomarkers that are correlated with an exhausted T-cell phenotype.

Plus, rapid phosphorylation signaling in CD28/CD3ζ produced more robust T cell cytokine production; the investigators wrote that encoding a fully functional CD28 signaling domain on a CAR polypeptide chain may actually cause excessive stimulation, which in turn could potentially increase the likelihood of cytokine release syndrome and T-cell exhaustion, and reduce CAR-T cell persistence.

Researchers hypothesized that CD28 signaling domain intensity was associated with a greater Lck, and that the CD28 domains could feasibly be altered to weaken the signal intensity, subsequently improving the CAR-T’s tumor-killing ability. “Crafting safer and more effective CAR-T cells might be accomplished by modifying CAR design, but optimization is hindered by the rudimentary understanding of how signaling by synthetic CARs directs T cell functional outputs and cell fate decisions,” they wrote.1

However, the investigators warned that the signaling cascades of synthetic CARs cannot be predicted solely based on the signaling domain selected for each CAR-T construct, as synthetic CARs “initiate a plethora of signals” that surpass the signals observed in naturally occurring signaling domains.

The researchers noted that although investigators have been selecting CAR constructs for drug development based on the associated domain’s ability to quickly prompt T-cell effector functions and T-cell proliferation, this approach may not actually be the best way to predict an investigational CAR-T’s in vivo efficacy over time. “Future optimizations to CAR design should consider both the signaling domains contained within the receptor, interactions with potential binding partners, and how modifications of costimulatory domains might tailor overall signal strength for downstream T cell functional outputs,” the authors concluded.1

“We do not yet claim that one signaling domain should be preferred over the other in any disease setting. Rather, the focus of our study was to understand at a deeper level how these fully synthetic (and unnatural) receptor designs actually signal T cells, and perhaps identify what underlies the differences in behavior of T cells engineered with different receptors,” Dr Riddell wrote to Cancer Therapy Advisor. “We caution interpreting preclinical data and preclinical models to infer clinical outcomes, but believe that our data provide insights into how receptors might be designed to provide quantitatively different signals that could be important in efficacy and toxicity.”

Disclosures: This research was supported by the National Institutes of Health and the Fred Hutchinson Cancer Research Center. For a full list of competing interests, please see the original study.


  1. Salter AI, Ivey RG, Kennedy JJ, et al. Phosphoproteomic analysis of chimeric antigen receptor signaling reveals kinetic and quantitative differences that affect cell function. Sci Signal. 2018;11(544):eaat6753. doi: 10.1126/scisignal.aat6753