Unleashing CAR T cells to delay metabolic aging

Abstract

Amor and colleagues previously developed chimeric antigen receptor (CAR) T cells that can target and eliminate senescent cells. The utility of these senolytic CAR T cells is now expanded to show that they can combat age-related metabolic dysfunction, and that they can be used prophylactically and have effects that persist for months, thus opening the door to the development of long-term senolytic approaches.

Senescent cells accumulate with age and have been shown to have a causal role in multiple age-related pathological conditions, including physical dysfunction¹, metabolic dysfunction² and many others³. Therapeutic approaches to eliminate senescent cells have tremendous promise to improve overall healthspan. In this issue of Nature Aging, Amor et al. report on a senolytic treatment that harnesses the power of CAR T cells to target and eliminate senescent cells in vivo⁴. Importantly, one dose of the senolytic CAR T cells provided long-lasting health benefits and detectable levels of the engineered T cells in mice months after administration These findings expand the authors’ previous work on senolytic CAR T cells in the context of liver fibrosis⁵ to the aging domain and add important translational value for potential new treatments to combat metabolic dysfunction, as well as other diseases of aging.

Age is a leading risk factor for impaired glucose homeostasis, insulin resistance and the development of type 2 diabetes. Diabetes and these metabolic dysfunctions then lead to increased risk for other age-related conditions, such as myocardial infarction, stroke, cognitive dysfunction, visual impairment and functional impairment. Thus, targeting the metabolic dysfunctions and insulin resistance that develop with aging could provide overall improvements in healthspan in older adults. Age- and obesity-related metabolic dysfunction have recently been linked to increased cellular senescence burden^6–8. Cellular senescence refers to the irreversible proliferative arrest that occurs when cells experience a range of cellular stressors. These cells are metabolically active and secrete a variety of pro-inflammatory cytokines, chemokines and proteases that are termed the senescence-associated secretory phenotype (SASP)³. As we age, dysregulated immune responses and increased tissue damage leads to an accumulation of senescent cells in various tissues. Senescent cells are highly heterogeneous⁹ and their presence in diverse tissues has been causally associated with metabolic dysfunction, including β-cells in the pancreas⁶, preadipocytes, macrophages and endothelial cells in adipose tissue^2,7, and T cells⁸, using various transgenic mouse models and senolytics, which are small molecules that can specifically eliminate senescent cells.

Although transgenic models enable specific mechanistic studies, they lack translational therapeutic value. Senolytic drugs, on the other hand, have been successfully developed and are in the process of being tested in humans for various conditions^3,10. These drugs offer tremendous promise for eliminating senescent cells and improving overall health in humans. However, they also have limitations. Most senolytics to date target pathways associated with cellular senescence and the SASP, but it is important to note that some of these pathways are also present and active in other cells that are not senescent, which creates the potential for undesired off-target effects for these drugs. Further, most senolytic treatments to date have used a ‘hit and run’ approach that requires repeated administration over time, as their effects are only transient. Moreover, given the heterogeneous nature of senescent cells, it remains unclear what exact senescent cell populations are targeted and eliminated in vivo by different senolytic drugs. This leads to the possibility that multiple senolytic drugs might need to be combined to achieve better efficacy. Therefore, it is of great value and clinically significant to develop new senolytics to further improve their translation.

Amor and colleagues previously developed a senolytic approach using CAR T cells to target and kill senescent cells⁵. CAR T cell therapies were a breakthrough for cancer care; the first therapy was approved by the FDA in 2017. Now at least six different CAR T cell therapies are FDA-approved for cancer treatment¹¹ and other uses for CAR T cell therapies beyond cancer are under development¹². Chimeric T cell receptors were first developed to modify T cells to recognize antigens independently of major histocompatibility complex (MHC), the component that enables T cells to recognize foreign peptides and initiate specific effector responses. T cell activation requires both the T cell receptor to bind the MHC–peptide complex and co-stimulation. CAR T cells are modified so that they do not require the T cell receptor to bind the MHC–peptide complexes from antigen-presenting cells for activation. Rather, they use a single-chain variable fragment (scFv) derived from monoclonal antibodies to recognize the target antigen (for example, CD19 in B cell acute lymphoblastic leukemia). This enables scientists to direct specific T cell responses to the antigen of interest. CAR T cells have the major advantage of being ‘living drugs’¹² — for example, the engineered T cells can persist and mediate their effects in the long term.

Amor and colleagues previously showed that senescent cells have elevated expression of urokinase-type plasminogen activator receptor (uPAR) and leveraged CAR T cell technology to engineer T cells with an anti-mouse uPAR scFv to kill uPAR-expressing cells in mouse liver fibrosis models⁵. These findings provided crucial proof of concept, but only examined senescence cell clearance in young animals and did not study long-term effects. In their current study⁴, the authors first performed an extensive characterization of uPAR expression in 3-month-old and 20-month-old C57BL/6 mice. They determined that uPAR expression increased with age in multiple tissues, and also — more specifically — showed (using single-cell RNA sequencing) that uPAR-positive cells in the liver, adipose tissue and pancreas were mainly preadipocytes, endothelial cells, fibroblasts, dendritic cells and myeloid cells, and were enriched with other transcriptomic signatures of senescence. As senescent cells are heterogeneous, it was integral to show that uPAR was an appropriate marker for age-related senescence accumulation in multiple tissues.

Next, the authors generated uPAR-targeting CAR T cells from six-week-old CD45.1 mice, as they previously developed⁵. Aged C57BL/6 mice were then intravenously infused with uPAR-targeting CAR T cells or negative controls. uPAR-targeting CAR T cells reduced uPAR-positive and SA-β-Gal-positive cells in multiple tissues, as well as resulted in reduced systemic inflammation and improved glucose tolerance and overall metabolic function (Fig. 1). More specifically, uPAR-targeting CAR T cells led to an increase in time to exhaustion during treadmill running compared to the negative controls. The authors next sought to determine whether uPAR-targeting CAR T cells could be given prophylactically to young mice to prevent age-associated declines related to senescent cell accumulation. Notably, uPAR-targeting CAR T cells were still present and had expanded in the spleen and liver 12 months after infusion. These now-middle-aged mice (9–15 months old) infused with uPAR-targeting CAR T cells at three months old had improved metabolic and physical function compared to mice infused with negative control. These results suggest that uPAR-targeting CAR T cells can act prophylactically in young mice and persist to continually eliminate uPAR-expressing senescent cells that occur during physiological aging. The authors also examined the effect of uPAR-targeting CAR T cells in the context of obesity. Similar to their aging studies, uPAR-targeting CAR T cells could act both therapeutically to alleviate metabolic dysfunction following a high-fat diet, as well as prophylactically when administered before the start of the high-fat diet.

Fig. 1 |. uPAR-targeting CAR T cells eliminate uPAR-expressing senescent cells and reduce metabolic dysfunction.

Senescent burden and metabolic dysfunction increase markedly with age. Amor et al. developed CAR T cells that target and eliminate uPAR-expressing senescent cells and recover age-related declines in metabolic function. When given at an early age, the CAR T cells persist and prevent age-related metabolic dysfunction up to 12 months later in mice. When given later in life (when metabolic dysfunction is already present), the CAR T cells eliminate uPAR-expressing senescent cells and recover metabolic function in aged mice. Created with Biorender.com.

These experiments provide critical proof-of-principle confirmation that uPAR-targeting CAR T cells can persist in the long term in vivo to continually target and eliminate uPAR-expressing senescent cells and reduce age-associated and obesity-associated dysfunction, mainly related to metabolic and physical function. These findings are exciting for the potential of senolytic CAR T cells and add to the existing literature on senolytic therapeutics. CAR T cells that target natural killer group 2 member D ligands (NKG2DLs), another highly expressed factor on senescent cells, have also recently been shown to effectively eliminate senescent cells in aged mice and improve physical function¹³ Interestingly, these NKG2DL-targeting CAR T cells were also effective in eliminating senescent cells in aged nonhuman primates¹³. Thus, the utility of senescence-targeting CAR T cells has now been demonstrated with more than one approach.

A major question that remains is about the long-term safety of these senescence-targeting CAR T cells. Owing to the fact that many physiologically relevant nonsenescent cells could also express high levels of uPAR or NKG2DLs (and thus can be eliminated by these CAR T cells), it is likely that this strategy could lead to unexpected side effects. Although Amor and colleagues report no major acute toxicity, side effects or pathology, it will be important to follow the long-term effects of these CAR T cells with comprehensive assessments, including a lifespan analysis. Moreover, unlike mouse studies (which are performed in a very clean environments), humans are exposed to various pathogens and injuries on a regular basis. Given the known role of senescence in wound repair and cancer defense, it could be speculated that continuous and long-lasting senolytic action of CAR T cells may impair these positive roles of senescent cells and cause some harmful effects. By contrast, the hit-and-run approach used with small-molecule senolytics is titratable and can be designed to reduce the side effects of these drugs¹⁰. These important safety concerns warrant additional scrutiny as part of future translational efforts for senolytic CAR T cells. Excitingly, some of these concerns might be alleviated by the fact that recent CAR T cell developments refined the engineering of these cell therapies to have more ‘safety switches’ in place if needed to reduce side effects¹⁴.

Overall, the development of uPAR-targeting CAR T cells represents an exciting step towards long-lasting senolytic treatment that can prevent senescent cell accumulation with age and improve overall healthspan. With the aging population ever-growing, novel treatments such as these will be necessary to keep older adults healthy and help them to maintain their independence — but more investigations are essential in the future to demonstrate the long-term safety and clinical translation potential of CAR T cells.