Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction

Senescent cells accumulate in organisms over time because of tissue damage and impaired immune surveillance and contribute to age-related tissue decline1,2. In agreement, genetic ablation studies reveal that elimination of senescent cells from aged tissues can ameliorate various age-related pathologies, including metabolic dysfunction and decreased physical fitness3–7. While small-molecule drugs capable of eliminating senescent cells (known as ‘senolytics’) partially replicate these phenotypes, many have undefined mechanisms of action and all require continuous administration to be effective. As an alternative approach, we have developed a cell-based senolytic therapy based on chimeric antigen receptor (CAR) T cells targeting uPAR, a cell-surface protein upregulated on senescent cells, and previously showed these can safely and efficiently eliminate senescent cells in young animals and reverse liver fibrosis8. We now show that uPAR-positive senescent cells accumulate during physiological aging and that they can be safely targeted with senolytic CAR T cells. Treatment with anti uPAR CAR T cells ameliorates metabolic dysfunction by improving glucose tolerance and exercise capacity in physiological aging as well as in a model of metabolic syndrome. Importantly, a single administration of a low dose of these senolytic CAR T cells is sufficient to achieve long-term therapeutic and preventive effects.


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Cellular senescence is a stress response program characterized by stable cell cycle arrest 9,10 and 49 the production of the senescence-associated secretory phenotype (SASP), which includes 50 proinflammatory cytokines and matrix remodeling enzymes 11 . In physiological conditions in young 51 individuals (e.g., wound healing, tumor suppression), the SASP contributes to the recruitment of 52 immune cells, whose role is to clear the senescent cells and facilitate restoration of tissue 53 homeostasis 11 . However, during aging, the combination of increased tissue damage and 54 decreased function of the immune system leads to the accumulation of senescent cells 1,2 , thereby 55 generating a chronic pro-inflammatory milieu that leads to a range age-related tissue 56 pathologies 5,12-14 . As such, senolytic strategies to eliminate senescent cells from aged tissues 57 have the potential to dramatically improve healthspan.

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Most efforts to develop senolytic approaches have focused on the development of small molecule 60 therapies that target as yet poorly defined molecular dependencies present in senescent cells 61 and that must be administered repeatedly over time 15 . In contrast, chimeric antigen receptor T 62 cells (CAR T cells) are a form of cellular therapy that redirects T cell specificity towards cells 7 fraction of the senescent-cell burden in these tissues (67-90% in liver, 92-66% in adipose tissue 124 and 76-63% in pancreas) (Fig.1i,k,m and Extended Data Fig. 3h,k,n). Note that while our 125 analysis could not evaluate pancreatic beta cells, analysis of published data revealed that 126 expression of Plaur was significantly upregulated in senescent beta cell populations isolated from 127 aged animals and subjected to bulk RNA-seq 7 .

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Finally, to ascertain whether uPAR was expressed in senescent cells that accumulate with age in 130 human tissues, we analyzed available datasets of human pancreas collected from young (0-6 131 year old) and aged (50-76 year old) individuals 29 . While we were limited to an analysis of Plaur 132 transcript abundance in these settings, we found that the fraction of Plaur-expressing cells was

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To determine the tolerability and therapeutic activity of uPAR-targeting CAR T cells on 144 physiologically aged mice, we intravenously infused aged C57BL/6 mice (18-20 months old) with 145 our previously developed murine second-generation CAR T cells targeting mouse uPAR 8 146 ). m.uPAR-m.28z CAR T cells contain an anti-mouse uPAR single-chain variable 147 fragment (scFV) linked to mouse CD28 costimulatory and mouse CD3z signaling domains and 148 are therefore fully murine CAR T cells that allow for syngeneic studies 8 . Importantly, the CAR T cells were generated from CD45.1 mice and infused into C57BL/6 mice which are CD45.2, thus 150 allowing for CAR T cells to be differentiated from endogenous T cells and therefore monitored 151 over time (Fig. 2a). As controls, parallel cohorts of sex and aged matched mice were infused with 152 the same dose of either untransduced T (UT) cells or T cells expressing a murine CAR targeting 153 human CD19 (h.19-m.28z) that does not recognize the murine CD19 protein but encompasses 154 the exact same signaling structure thus controlling for non-specific T cell cytotoxicity. We opted 155 to test a dose of 0.5x10 6 CAR-positive cells, which we previously found to balance safety and

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Despite detectable expression of uPAR in some normal tissues, our previous work indicates that 168 a dose of 0.5x10 6 m.uPAR-m.28z CAR T cells is well tolerated in young mice 8 . As was the case 169 in young animals, the dose of 0.5x10 6 m.uPAR-m.28z CAR T cells was well tolerated in aged 170 mice (18-20 months old), all of whom remained active without observable signs of morbidity, 171 weight loss, or relevant alterations in serum chemistry or complete blood counts (Extended Data 172 Fig. 6). In addition, microscopic evaluation of tissues did not reveal tissue damage secondary to 173 toxicity in aged tissues obtained from whole body necropsies of m.uPAR-m.28z CAR T treated 174 mice when compared to age-matched control treated animals (Extended Data Fig. 7).

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One prominent feature of aging in humans and mice is the emergence of age-related metabolic 177 dysfunction, which is a collection of phenotypes linked to impaired glucose tolerance 7,31 and 178 decreased exercise capacity 3,32 . Interestingly, we observed that aged m.uPAR-m.28z CAR T 179 treated mice had significantly decreased fasting glucose levels compared with UT or h.19-m.28z-180 treated controls (Fig. 2d). Upon challenge with an intraperitoneal bolus of glucose (2 g/kg), 181 m.uPAR-m.28z CAR T treated aged but not young mice presented significantly lower plasma 182 glucose levels than controls for over 2 hours after administration (Fig. 2e,f and Extended Data 183 Fig. 8a,b). Furthermore, m.uPAR-m.28z CAR T treated mice had lower basal insulin levels after 184 fasting that was followed by a significant increase in insulin levels 15 minutes after the glucose 185 load, indicative of improved pancreatic b cell function (Fig. 2g). Of note, m.uPAR-m.28z CAR T 186 treated aged mice also presented improved peripheral insulin sensitivity, suggesting a 187 coordinated multiorgan improvement in glucose homeostasis (Extended Data Fig. 8c,d). In 188 addition, most aged mice with m.uPAR-m.28z CAR T showed improvements in their exercise 189 capacity at 2.5 months after treatment compared to pretreatment levels (Fig. 2h,i).

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Importantly, the improvement in metabolic function noted in m.uPAR-m.28z CAR T cell-treated 192 old mice was accompanied by a significant expansion of m.uPAR-m.28z CAR T cells and their 193 trafficking to several organs such as liver and spleen as assessed by flow cytometry (Fig. 2j,k).

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Collectively, these results show that uPAR CAR T cells can safely and effectively remove 202 senescent uPAR-positive cells in the tissues of naturally aged mice and ameliorate age-203 dependent metabolic and physical dysfunction.

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Persistence and preventive activity of uPAR CAR T cells during physiological aging.

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Unlike small molecules, CAR T cells can persist in the organism and exert their effects over time 17 .

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Indeed, in human cancer patients cured of disease, the presence of CAR T cells has been noted 208 as much as 10 years after the initial infusion 17 . Such persistence raises the question of whether 209 the administration of uPAR CAR T cells in young animals would prevent or delay the development 210 of age-triggered phenotypes later in life. To explore this possibility, we infused young mice (3 211 months old) with one dose of 0.5x10 6 m.uPAR-m.28z CAR T, h.19-m.28z CAR T or UT cells and 212 monitored the mice over their natural lifespan (Fig. 3). Despite the initially lower numbers of 213 uPAR-positive cells compared to aged animals (see above), uPAR CAR T cells were detectable 214 in the spleens and livers of treated mice 12 months after the initial single infusion at significantly 215 higher levels than the low number of persisting UT or h.19 CAR T controls (Fig. 3a,b). Consistent 216 with their persistent activity, flow cytometry of the spleen and livers of uPAR CAR T cell treated 217 mice indicated that the persisting cells were mostly cytotoxic CD8 T cells harboring a memory 218 and effector phenotype in the spleens (Extended Data Fig. 9e-h). Therefore, uPAR CAR T cells 219 persist and expand over the lifespan of the animal, presumably owing to increased antigen 220 stimulation as the frequency of target uPAR positive cells increases over time.

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As was observed in aged animals upon therapeutic treatment, prophylactic uPAR CAR T cell 223 administration in young mice limited metabolic decline in old age. Specifically, uPAR CAR T 224 treated mice had significantly lower fasting glucose levels (Fig. 3c), improved glucose tolerance 225 (Fig. 3d,e) and enhanced pancreatic b cell function as assessed by glucose-stimulated insulin 226 secretion (Fig. 3f) than mice treated with either UT or h.19-m.28z. In terms of fitness, mice that in their youth had been treated with m.uPAR-m.28z CAR T cells, compared with control-treated 228 mice, showed higher exercise capacity at 9 months of age (Fig. 3g,h), although this waned over 229 time (Extended Data Fig. 9i,j). These phenotypes correlated with a significant decrease in both 230 SA-b-Gal-positive and uPAR-positive cells in pancreas, liver, and adipose tissue (Fig. 3i and   231 Extended Data Fig. 10). Taken together, these results show that uPAR CAR T cells can not only 232 treat, but also prevent, features of age-dependent metabolic decline.

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Therapeutic and preventive potential of uPAR CAR T cells in metabolic syndrome

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Many of the features associated with metabolic syndrome in aged mice can be recapitulated in 236 young animals given a high fat diet 33 and, indeed, obesity has been described to accelerate the 237 "aging clock" 34 . Indeed, as in aged animals, such treatment leads to the accumulation of 238 senescent cells 7 (Extended Data Fig. 11a-d). To test the therapeutic potential of uPAR CAR T 239 cells in this context, we modeled metabolic syndrome by feeding mice a high-fat diet (HFD), which 240 induces obesity and metabolic stress 35 . After two months on HFD, mice were treated with 0.5x10 6 241 m.uPAR-m.28z CAR T or UT cells and continued on the diet (Fig. 4a). At 20 days after infusion, 242 mice treated with uPAR CAR T cells displayed significantly lower body weight, better fasting blood 243 glucose levels and improvements in both glucose and insulin tolerance compared to controls ( Fig.   244 4b-g). This therapeutic effect persisted through the period of monitoring (2.5m after cell infusion)

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and was accompanied by decreased senescent cell burden in pancreas, liver and adipose tissue 246 as assessed by SA-b-gal (Fig. 4h,I and Extended Data Fig. 11e-h). Thus, uPAR CAR T therapy 247 produced a similar improvement to metabolic dysfunction in the context of metabolic syndrome in 248 young animals as was observed in naturally aged mice.

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To test whether prophylactic administration of uPAR CAR T cells could impede the development 251 of metabolic disorders in young mice given HFD, we administered 0.5x10 6 m.uPAR-m.28z CAR 252 T 1.5 months before placement on HFD (Fig. 4j). Remarkably, m.uPAR-m.28z CAR T cells (but 253 not treatment with UT cells) acted prophylactically to blunt the accumulation of senescent cells 254 over time, an effect that was also associated with decreased weight gain and glucose levels 3.5 255 months after infusion (Extended Data Fig. 9i-l and Fig. 4k-n). At this time, m.uPAR-m.28z CAR

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T were detectable and enriched in the spleens and livers of treated mice, they again were 257 composed mostly of CD8 T cells with an effector phenotype (Extended Data Fig. 12). This 258 preventive effect on metabolic dysfunction was sustained for at least 5.5 months after cell infusion 259 despite continuous exposure to high fat diet (Fig. 4o,p).

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Overall, these data highlight the contribution of uPAR-positive cells to metabolic dysfunction in 262 aged and obese mice and raise the possibility that targeting these cells through CAR T cells could 263 have therapeutic benefit in humans.

Discussion 266
Our study provides proof-of-principle evidence that senolytic cell therapies can ameliorate 267 symptoms associated with physiological aging. We previously showed that uPAR targeting CAR 268 T cells could safely and effectively eliminate senescent cells in the livers of young animals 8 . Here, 269 focusing on metabolic dysfunction as one prominent age-related pathology, we show that: (i) the 270 fraction of uPAR-positive cells increases with age, (ii) that these cells significantly contribute to 271 the senescence burden in aged tissues, (iii) uPAR-positive cells with senescence signatures 272 consist of both immune and non-immune populations, the latter consisting of a range of cell types 273 that are organ dependent, (iv) uPAR CAR T cells can be effective at eliminating uPAR-positive 274 senescent cells; (v) and their effect is not associated with pathology in tissues or alterations of 275 hepatic and renal functional parameters in aged mice. Finally, (vi), the action of uPAR CAR T 276 cells is associated with improved glucose homeostasis and metabolic fitness in both physiological 277 aging and high fat diet. Importantly, at doses used to produce these therapeutic benefits, we noted no overt toxicities of uPAR CAR T cells, which could persist and expand for over 15 months 279 as mice progressed from a youthful to an aged state.

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Perhaps the most striking observations of the current work was the ability of uPAR CAR T cells 282 to act prophylactically to blunt age-and diet-induced metabolic decline. Unlike senolytic 283 approaches based on small molecules, uPAR CAR T cells have long-lasting effects after the 284 administration of a single low dose, causing a marked impairment in age-or high fat diet-induced 285 metabolic syndrome when mice were treated during youth or administration of high fat diet, 286 respectively. Our findings are consistent with those of an earlier study that explored vaccination 287 against GPNMB on senescent cells to address age-related pathology 36 , although with our cellular 288 therapy, both effect sizes and duration were substantially larger. In fact, our results demonstrate 289 a protective effect for over a year in the context of physiological aging in the laboratory mouse, a 290 species with an average lifespan of 2 years.

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Studies using genetic or pharmacological approaches to senolysis have been equivocal as to 293 whether elimination of senescent cells will significantly extend longevity 3,4,32 . Our current studies 294 are not sufficiently powered to draw conclusions on longevity at this stage. As senescent cells 295 contribute to a range of age-related tissue pathologies, studying the impact of senolysis in aged 296 animals provides an opportunity to interrogate multiple co-morbidities under similar conditions.

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Future studies will evaluate the potential of uPAR CAR T cells (or other senolytic cell therapies) 298 in additional aging and related tissue-damage pathologies, the latter disease contexts providing   However, there are also reports suggesting that targeting senescent cells in adipose tissue 31 or 305 even immune-cell senescence 37 may also play a role. In this regard, recent studies also suggest 306 that the elimination of macrophage populations with senescent features can also improve tissue 307 decline in mice 38,39 . Whether or not these macrophages are truly 'senescent' or have an alternative 308 cell state is a topic of debate; regardless, given that we observe a fraction of uPAR-expressing 309 macrophages that also co-express SA-b-gal and senescence-associated transcriptional 310 signatures accumulating in aged tissues it seems likely that their elimination may contribute to the 311 phenotypes we observe.

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While the mechanism of action of most current small molecules is often inferred or poorly 314 understood, senolytic CAR T cells have a clear underlying rationale based on the expression of 315 a specific surface antigen. While toxicity issues are invariably a concern, cellular therapy harbors 316 the versatility to simultaneously target several surface antigens through AND gate approaches 16 , 317 modulate persistence through different CAR designs 40 and/or incorporate safety switches, 41 all of 318 which provide avenues to mitigate side effects that are not possible through vaccination strategies 319 or small molecule approaches 41 . Indeed, in another recent report, it has been shown that mice 320 and primates tolerate CAR T cells that target an NK cell ligand that is upregulated on senescent 321 cells and other cell types 42 . Taken together, these efforts could result in the identification of tissue-322 specific senolytic antigens that could be targeted with cellular therapy to treat different age-related 323 phenotypes. The persistence of the uPAR-targeted CAR T cells and the durability of the effects 324 after a single low-dose treatment highlight the clinical potential of the senolytic CAR T cell 325 approach for the treatment of chronic pathologies.

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In short, adipose tissue was isolated and placed in a digestion solution consisting of 4 mg/ml 415 collagenase, type II (Sigma) in DPBS (Life Technologies) supplemented with 0.5% BSA (Sigma) and 10 mM CaCl2 digested at 37° C for 20 min in a rotational shaker (200 rpm). Afterwards, 417 samples were mechanically dissociated with a 10-ml serological pipette, filtered through a 40-μm 418 strainer and washed with PBS and 2 mM EDTA, then red blood cells were lysed by ACK lysing 419 buffer (Lonza). Pancreata were placed into cold DMEM with 10% FBS and penicillin and 420 streptomycin. The pancreata were minced in this media on ice into 2-to 4-mm fragments so that 421 they would pass through the end of 1-ml pipette tip with ease. The minced tissue was collected in

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Sequencing data was demultiplexed, mapped, and processed into gene/cell expression matrices 457 using 10X Genomics' Cell Ranger software v7.1.0 (https://support.10xgenomics.com/single-cell-458 gene-expression/software/pipelines/latest/what-is-cell-ranger). Gene expression reads were 459 aligned to the mouse reference genome version gex-mm10-2020-A, available from the 10X 460 Genomics website. We kept cells using "min.cells > 10, nFeature_RNA > 500, nCount_RNA > 461 2500, percent.mt < 15". Gene expression count data were normalized using SCTransform to 462 regressed out percent mitochondrial RNA. The R package BBKNN was used to remove batch 463 effects between mouse samples, and 0.5 was used as expression cutoff to define uPAR High cell 464 populations. Clusters were identified using resolution = 0.8, and cell types were annotated using 465 R packages celldex, SingleR, Azimuth, and custom gene sets 24,25 . Known markers for each cell 466 type were plotted using DotPlot function in Seurat. Senescence gene sets from 21,28 were used to 467 calculate signature scores using AddModuleScore function in Seurat, and a signature score cutoff 468 of 0.05 was used to define Senescence High cell populations.  For the fluorescent SA-β-gal labelling, tissue slides were exposed to the C12RG substrate at 518 37°C according to manufacturer's instructions (ImaGene Red C12RG lacZ Gene Expression Kit, Molecular Probes, I2906) 48,49 . Subsequently, for IF analysis, slides were fixed with 4% PFA for 10 520 minutes at room temperature and proceed with regular IF as performed following standard 521 protocols and previously described 8 . The following antibodies were used: anti-mouse uPAR 522 (R&D, AF534, 1:100) and anti-mouse F4/80 (Bio Rad, CI:A3-1). For quantification 5 high power 523 fields per section were counted and averaged to quantify the percentage of SA-β-gal+, uPAR+

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Results are from 2 independent experiments (b-e;k-n) or 1 independent experiment (f-i; o-p).

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Data are mean ± s.e.m.; p values derived from two-tailed unpaired Student's t-test (b-i; k-p).

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Representative histology of normal lungs showed dense aggregates of lymphocytes and fewer 868 plasma cells and macrophages around bronchioles or vasculature (inset: pulmonary lobes). d.

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The liver from aged mice showed accumulation of lymphocytic and histiocytic aggregates in portal