The idea of engineering T cells with a synthetic receptor to more effectively kill cancer cells began more than three decades ago. Since the 1990s, chimeric antigen receptor T (CART) cell therapy has been refined and resulted in several FDA-approved therapies. In 2017, two CD19-targeting CART products, Kymriah (tisagenlecleucel) and Yescarta (axicabtagene ciloleucel), were FDA-approved after showing unprecedented success in subsets of patients with leukemia and lymphoma. The key difference between the two products is the costimulatory domain (CSD), a part of the CAR which enhances the anti-tumor activity of CART cells. Tisagenlecleucel is equipped with 4-1BB costimulation (BBζ), while axicabtagene ciloleucel is equipped with CD28 costimulation (28ζ).

The choice of CSD contributes to distinct CART cell behavior; generally, studies have found that BBζ generates a milder but longer-lasting response, while 28ζ has a shorter but stronger response against cancer cells. Further analysis suggests that 28ζ is more prone to exhaustion, a well-known dysfunctional T cell state, than BBζ. However, regardless of the CSD used, more than half of patients treated with CART cells relapsed within the first year of treatment.

Exhaustion is a widely accepted reason for CART cell failure. However, exhaustion is not the only dysfunctional T cell phenotype. T cell senescence is another suboptimal T cell fate, but its role in CART cell failure had not been studied. T cell senescence develops as a response to several stressors commonly seen in patients with cancer, including chemotherapy, radiation, strong stimulation, and extended interaction with cancer cells. Although both T cell exhaustion and senescence result in impaired T cell activity, the underlying mechanisms and clinical treatment can be quite different. Therefore, it is imperative to assess the role of senescence in CART cell failures.

Due to the aggressive, relapsing nature of cancer, CART cells repeatedly interact with malignant cells. To simulate this chronic CART cell activation in the lab, CART cells are commonly activated with cancer cells in multiple coculture cycles in vitro. During these activation cycles, we adapted this model with a key difference to distinguish between exhaustion and senescence. Exhaustion is often considered reversible, while senescence is a permanent cell fate. In our model, we introduced a rest time between activation cycles to generate activated/rested CART cells. The rest time proved critical to allowing exhaustion marker expression to fall back to baseline levels. Despite expressing similar levels of exhaustion markers, repeatedly activated/rested CART cells showed impaired T cell activity compared to basal CART cells in vitro and in vivo, suggesting that CART cells can be impacted through mechanisms beyond exhaustion.

Further analysis with activated/rested CART cells revealed that CART cells undergo several changes which are similar to those seen in senescent T cells. These changes are more prominent in BBζ than 28ζ and coincided with MYC activation in only BBζ. MYC is an oncogene, and oncogene activation is a well-known senescence inducer. We found that ectopic MYC activation seemed to impair only BBζ. Alternatively, we induced senescence by mildly irradiating CART cells. To our surprise, the efficacy of 28ζ, but not BBζ, improved upon irradiation-induced senescence. These results were consistent with published patient data which showed that senescent 28ζ but not senescent BBζ correlated with better clinical outcomes.

In conclusion, we studied the role of senescence in CART cell dysfunction through multiple models, including a fast and scalable model using repeated activation/resting cycles. This model allows senescent T cells to be studied and provides a platform to form a more complete understanding of CART cell failures. Our study identifies senescence as a failure mechanism mainly in BBζ. This study not only provides a more comprehensive understanding of CART cell failures but also provides opportunities to generate more durable CART cell treatment protocols through removal of senescent cells. Our study sought to better understand CART cell failure, with the ultimate goal of developing more durable CART cell therapy and improved patient outcomes.

 Study Title: Differential susceptibility and role for senescence in CART cells based on costimulatory domains

 Published: June 2025, Molecular Cancer
Read it here: https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-025-02371-1