WO2024197007A1

WO2024197007A1 - Senotherapy for treating and preventing neurodegenerative disease

Info

Publication number: WO2024197007A1
Application number: PCT/US2024/020678
Authority: WO
Inventors: Aaron Raymond HAEUSLER; Davide Trotti; Adam Eugene Snook; Piera PASINELLI
Original assignee: Thomas Jefferson University
Current assignee: Thomas Jefferson University
Priority date: 2023-03-20
Filing date: 2024-03-20
Publication date: 2024-09-26
Anticipated expiration: 2025-09-20

Abstract

The present invention relates to the compositions comprising CAR cells, such as CAR-T cells, which target cell surface proteins. In certain compositions, the CAR cells target cell surface proteins associated with senescence. Accordingly, the invention also provides methods of administering said compositions for reducing the number senescent cells. These compositions and methods are of use in treatment of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS).

Description

SENOTHERAPY FOR TREATING AND PREVENTING NEURODEGENERATIVE DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/491,130, filed March 20, 2023, and U.S. Provisional Application No. 63/564,122, filed March 12, 2024, the contents of each of which are incorporated by reference herein in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED IN XML FORMAT The present application hereby incorporates by reference the entire contents of the XML file named “20596 l-0062-00WO_SequenceListing.xml” which was created on March 17, 2024, and is 18,214 bytes in size.

BACKGROUND

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), frequently show clinicopathological focal origins of pathology in the CNS that propagate to surrounding tissues and throughout the neuroaxis. While propagation mechanisms remain highly investigated, there is emerging evidence that neural senescence and subsequent secretion of proinflammatory molecules, referred to as SASP (senescence-associated secretory phenotype), may contribute to this process (Baker, D. J., et al., 2018, Journal of Clinical Investigation, 128: 1208-1216; Martinez-Cue, C., et al., 2020, Frontiers in Cellular Neuroscience, 14: Article 16; Saez-Atienzar, S., et al., 2020, Nature Reviews Neuroscience, 21 :433-444; Amor, C., et al., 2020, Nature, 583: 127-132). Classically defined as a state of arrested proliferation by cells in response to stressors, senescence is an aging-related process with pathophysiological hallmarks characterized by increased activation and levels of p 16/p21 signalling pathways, DNA damage, senescence- associated B-galactosidase activity, and increased SASP. These hallmarks of brain senescence are present in ALS models and patient post-mortem tissues (Porterfield, V., et al., 2020, Neurobiology of Aging, 90: 125-134; Trias, E., et al., 2019, Frontiers in Aging Neuroscience, 11 : Article 42; Tam, O. H., et al., 2019, Cell Reports, 29: 1164-1177). In C9orf72-linked ALS/FTD, the extent and contribution to the disease of senescence are unknown. As such, senescence and senescent cells (SCs) could play a role in the propagation of pathology throughout the CNS and the associated neurodegeneration is the most common genetic cause of ALS/FTD, the GGGGCC nucleotide repeat expansion (NRE) mutation in the C9orf72 (C9) gene.

Several therapeutic strategies, known as senotherapies, have been developed to eliminate tissue-specific cells undergoing senescence or tissue-specific senescent cells. However, almost none have been tested in models of ALS. Senescent cells provide ideal targets for cell-based immunotherapy (e.g., CAR-T cell immunotherapy). Several CAR-T cell immunotherapies are now FDA-approved for different types of cancer, and several are being tested in clinical trials. A handful of senotherapies have been developed to eliminate tissuespecific SCs selectively. However, CAR-T therapies for neurodegeneration have not been realized.

As such, current treatment options are inadequate due, in part, to the lack of clearly defined therapeutic targets. Thus, there is an unmet need for therapeutics that target senescent cells in the brain to treat neurodegeneration. The present invention meets this long felt, but unmet, need.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides methods of reducing the number of senescent cells, comprising contacting one or more senescent cells with a composition comprising one or more CAR-T cells, wherein the one or more CAR-T cells target one or more cell surface proteins associated with senescence. In some embodiments, the senescent cells are central nervous system cells. In some embodiments, the central nervous system cells are glial cells.

In various embodiments, the CAR-T cells have been modified to not express one or more interferons. In some embodiments, the one or more interferons are selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA11P, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1. In some embodiments, the interferon is interferon gamma (IFNG).

In various embodiments, the one or more cell surface proteins associated with senescence are selected from the group consisting of uPAR, DEP1, NT AL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, N0TCH1, N0TCH3, CD36, oxidized vimentin, and ICAM-1. In some embodiments, the cell surface protein associated with senescence is urokinase-type plasminogen activator receptor (uPAR).

In various embodiments, the method further comprises contacting the cells with one or more inhibitors of IL-6. In some embodiments, the one or more inhibitors of IL-6 are selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab. In some embodiments, the inhibitor of IL-6 is tocilizumab.

In one aspect, the disclosure provides methods of reducing the number of senescent cells in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising one or more CAR-T cells, wherein the one or more CAR-T cells target one or more cell surface proteins associated with senescence. In some embodiments, the senescent cells are central nervous system cells. In some embodiments, the central nervous system cells are glial cells.

In various embodiments, the CAR-T cells have been modified to not express one or more interferons. In some embodiments, the one or more interferons are selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA11P, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1. In some embodiments, the interferon is IFNG.

In various embodiments, the one or more cell surface proteins associated with senescence are selected from the group consisting of uPAR, DEP1, NT AL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, N0TCH1, N0TCH3, CD36, oxidized vimentin, and ICAM-1. In some embodiments, the cell surface protein associated with senescence is uPAR.

In another aspect, the disclosure provides methods of treating a neurodegenerative disease in a subject in need thereof comprising reducing the number of senescent central nervous system cells; wherein reducing the number of senescent cells comprises the step of administering to the subject a composition comprising one or more CAR-T cells, wherein the one or more CAR-T cells target one or more cell surface proteins associated with senescence. In some embodiments, the CAR-T cell has been modified not to express one or more interferons. In some embodiments, the one or more interferons are selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA1 IP, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNWl.In some embodiments, the interferon is IFNG.

In various embodiments, the method further comprises contacting the cells with one or more inhibitors of IL-6. In some embodiments, the one or more inhibitors of IL-6 are selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab. In some embodiments, the inhibitor of IL-6 is tocilizumab. In some embodiments, the tocilizumab is administered before, concurrently with, or after administration of the one or more CAR-T cells.

In various embodiments, the neurodegenerative disease being treated is amyotrophic lateral sclerosis (ALS).

In another aspect, the disclosure provides compositions for reducing the number of senescent cells comprising: a) a CAR-T cell; and b) tocilizumab; wherein the CAR-T cell targets a cell surface protein associated with senescence; and wherein the CAR-T cell has been modified not to express IFNG. In some embodiments, the cell surface protein associated with senescence is uPAR.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. Figure 1 depicts a graphical representation of senescence-associated genes upregulated in neurons expressing arginine-rich dipeptide repeats (DPRs). In the RNAseq analyses of data from Maor-nof, M et al. Cell, 2021, rat primary cortical neurons transiently transfected with polyproline-arginine (PR) show a time dependent increase in expression for a panel of senescent-associated genes compared to GFP or RNA-binding protein TDP-43 (TDP) (senescent qPCR gene panel obtained from Bio-Rad SAB target list, #10034121 with additional genes of interest included). The heatmap shows the list of senescent-associated genes with row hierarchal clustering on the left and a few top genes from the first two dimensions of a PCA are highlighted on the right as well as select senescence surfaceosome makers. The bar graph shows the cumulative RNA levels for the senescent panel for each day. Samples were performed in duplicate and all RNA levels were normalized to the internal average of ACTB+GAPDH for each sample to demonstrate feasibility of the gene panel when using a commercial or 96-well plate Senescence panel analyses format. All data was analyzed using R (heatmap or fviz) or Prism software.

Figure 2, comprising Figure 2A through Figure 2B, depicts representative increases of the levels of 13-galactosidase activity and IL-6 that indicate hallmarks of senescence in patient-derived C9-NRE cortical neurons. Figure 2A depicts a representative increase in 13- galactosidase (13-gal.) activity in aged C9-NRE i³neuronal cultures compared to controls. The 13-gal colorimetric staining shows robust visual/ quantitative detection of increased levels in C9- NRE neurons. n=l patient line per group, each point is indicating a different cortical i3neuronal differentiation. Figure 3B depicts a representative increase in IL-6 levels in an age-dependent manner in C9-NRE neurons. Secreted IL-6 levels were measured from the culture media over time in patient-derived C9-NRE and control cortical i³neurons using a single molecular assay (SIMOA) manufactured by Quanterix.

Figure 3, comprising Figure 3A and Figure 3C, depicts representative elevated transcript levels for senescence associated genes in cortical i³neurons derived from C9-NRE patients. Figure 3A depicts a principal component analysis (PCA) of differential gene expression (DGE), showing a robust separation between C9-NRE and control (CTRL) groups when filtered with a panel of senescence associated gene markers. The top ten contributing genes in the PCA are labeled and denoted by the arrow vectors and colored according to their respective contributions (Contrib). The results for K-means clustering for the two groups are plotted as 95% confidence interval ellipses. RNAseq data was analyzed for DGE using DESeq2 in R. Figure 3B depicts a representative heatmap comparison between WT and C9-NRE DGE, showing an overall increase in senescence-associated gene expression. Bar color values correspond to the rlog data transformation of the DGEs as provided in the colormap key, which includes a histogram of the total counts in each rlog bin. Figure 3C depicts representative quantification of the cellular senescence associated genes, p21 and pl 6, showing increased transcript levels in quantitative PCR analyses of RNA isolated from C9-NRE i³neurons. RNA levels were normalized to GAPDH RNA. For all results, n = 1 patient line per group with each point indicating a different cortical i³neuronal differentiation.

Figure 4, comprising Figure 4A through Figure 4G, depicts representative characterization of cerebral organoids. Figure 4A depicts an schematic overview of the protocol used to obtain cerebral organoids, including representative brightfield micrograph images of organoids overtime. Figure 4B depicts a representative graph bar showing the mean dimension over time of the COs. Figure 4C depicts exemplary micrographs showing different COs developing neuroepithelium. Figure 4D depicts representative bar graphs showing quantification over time of developmental-specific genes: Oct4, Nanog, Sox2 (stem cell markers); Pax6 (neural precursors marker); Tuj l, Tbrl (neural stage); Gfap (early neurons, glial cells). Figure 4E depicts exemplary immunofluorescence performed on a 60-day old CO. Tbrl is shown in green, NeuN is shown in red, Ctip2 is shown in magenta and DAPI is shown in blue. Figure 4F depicts exemplary light-sheet imaging performed on cleared DIV 60 cerebral organoids. GFAP is shown in blue, NeuN is shown in red and Sox2 is shown in green. Figure 4G depicts a schematic representation of microglia invasion. Figure 4H depicts exemplary light-sheet imaging performed on cleared DIV 60 cerebral organoids invaded with induced microglia. DAPI is shown in blue, Ctip is shown in red and Ibal is shown in yellow.

Figure 5 depicts a schematic diagram for the prevention of senolytic CAR-T cell-induced neurotoxicity. CAR-T cells have established cytokine-release syndrome (CRS) and neurotoxicity risk in cancer patients due to IFNy-induced IL-6 production by myeloid cells. CRISPR-Cas9 deletion of IFNG in CAR-T cells and IL-6 blockade with tocilizumab are emerging and well-established strategies to mitigate toxicity in cancer patients. Their application to senolytic CAR-T cells similarly prevents toxicity.

Figure 6, comprising Figure 6A through Figure 6C, depicts representative experimental results demonstrating that cortical i³Neurons derived from C9-NRE patients show hallmarks of age-related senescent phenotypes. Figure 6A depicts a heatmap comparison between control (CTRL) and C9-NRE DGE shows increased SA gene expression at 60 days post neuronal differentiation. The bar color values correspond to the Z-score for each gene. DGEs were calculated using DESeq2 in R. Figure 6B depicts a representative graph plotting a PC A of DGEs from Figure 6 A showing robust separation between C9-NRE and CTRL. The results for K-means clustering for the two groups are plotted as 95% confidence interval ellipses. Top contributing variables to the dimensions are shown. Figure 6C depicts representative results demonstrating that SA genes, p21 and pl 6, show increased RNA levels in C9-NRE i³neurons. QPCR analyses of RNA isolated i³neurons used GAPDH as a normalizer. DNA damage is elevated in C9-NRE cortical neurons versus CTRL. Comet assays were performed to quantify all DNA breaks. n=l line/group, with at least 3 separate differentiations. For all data, n = 1 line per group with each point indicating a separate differentiation.

Figure 7, comprising Figure 7A through Figure 7C, depicts representative experimental results demonstrating that cerebral organoids (COs) allow for cell-type specific modeling of senescence and senescence cell transitions. Figure 7A depicts a representative image of IF imaging from mature COs. Figure 7B depicts representative UMAP plots of single-cell data generated in SEURAT for mature COs derived from C9 and control patients. Senescence cells (SCs) can be annotated and mapped to clusters (black). The addition of microglia during organoid development in culture alters the prevalence of SCs. Figure 7C depicts a representative dot plot of annotated cell clusters from Figure 7B for upregulated SA genes.

Figure 8, comprising Figure 8A through Figure 8C, depicts representative experimental results demonstrating that SOD1^G93A mouse models exhibit increased temporal expression of SA gene markers in the spinal column. Figure 8A depicts a representative graph plotting a PCA that was performed on a panel of SA genes, revealing a growing temporal divergence of SOD1^G93A mice from aged, matched controls (WT). Each point in the 2D PCA plot represents an individual mouse (n = 3), with point size indicating Cos2 and ellipses representing the 95% confidence interval. The top 10 contributing variables to the PCA are shown as vectors. The expression profile Affymetrix array data was obtained from GSE18597. Figure 8B depicts representative box plots of RNA levels over time in SOD1^G93A and SOD1^WT mice for four SA markers shown. Figure 8C depicts representative images illustrating spatial transcriptomic data that demonstrates robust disease-linked temporal expression of candidate senescence genes in SOD1^G93A mice compared to age-matched controls. The data was obtained from als-st.nygenome.org. Rstudio was utilized for all analyses and plots.

Figure 9 depicts representative experimental results demonstrating that increased expression of senescence associated genes are found in the CNS of ALS/FTD patients. RNA sequencing data obtained from the cerebellum (CB) or frontal cortex (FCX) (GSE67196) and from the anterior horn (AH) or laser captured enrichment of spinal motor neurons (sMN) (GSE18920) from C9ALS, sALS and CTRLs were normalized and filtered for candidate senescence-associated genes. Representative box plots are shown for these top candidate genes identified in our preliminary in vitro and in vivo data. All p-adjusted values shown were calculated using a pairwise comparison with Bonferroni correction, with * < 0.05, and ** < 0.01.

Figure 10, comprising Figure 10A through Figure 10D, depicts representative experimental results demonstrating that there is increased expression of SA proteins in the cortices of ALS patients. Figure 10A depicts representative experimental results of western blot analyses of the gene PLAUR (alias, uPAR) demonstrate elevated protein levels in the mid motor cortex (Mid motor) of ALS patients compared to controls, while the “disease- spared” occipital cortex (Occ.) shows lower protein levels than the disease-burdened motor cortex. Each point in the bar graph represents an individual patient, with the size of the point corresponding to the patient's age at postmortem tissue collection. A representative western blot is shown below the bar graph. Figure 10B depicts representative images of western blots demonstrating that the protein levels for the senescence associated pl6 marker are increased in the frontal cortex (FC) and/or motor cortex (MC) tissues of C9 patients versus an aged control. HEK293T (HEK) cell lysate was included as a positive control for pl6 expression and total protein served as a loading control. Figure 10C depicts representative images from IF microscopy imaging illustrating increased levels of uPAR along with increased levels of activated microglia (Ibal+) in ALS/FTD patients. Figure 10D depicts representative images from immunofluorescence (IF) microscopy imaging showing that microglia (Ibal+) and astrocytes (GFAP+) can display elevated levels of uPAR and are found in regions with high uPAR levels.

Figure 11, comprising Figure 11 A through Figure 11C, depicts representative experimental results demonstrating that SOD1^G93A mouse models exhibit increased temporal gene expression of microglial cell surface markers and Plaur in the spinal column. Figure 11 A depicts a representative graph plotting a PC A that was performed on a panel of microglial genes (PMCID: PMC7424058) and Plaur, revealing a growing temporal divergence of SOD1^G93A mice from aged, matched controls (WT). Each point in the 2D PC A plot represents an individual mouse (n = 3), with point size indicating Cos2 and ellipses representing the 95% confidence interval. The top 10 contributing variables to the PC A are shown as vectors. The expression profile Affymetrix array data was obtained from GSE18597. Figure 1 IB depicts representative box plots of RNA levels over time in SOD1^G93A and SOD1^WT mice for several microglial cell surface markers and Plaur are shown. Figure 11C depicts representative images of spatial transcriptomic data demonstrating robust disease-linked temporal expression of candidate microglial cell surface genes in SOD1^G93A mice compared to age-matched controls. Plaur and other genes plotted in Figure 1 IB were not reported. The data was obtained from als-st.nygenome.org. Rstudio was utilized for all analyses and plots.

Figure 12, comprising Figure 12A through Figure 12B, depicts representative experimental results from RNA sequencing showing that PLAUR gene expression is undetectable in most cells from mouse brain and patient-derived cerebral organoids. Figure 12A depicts a representative graph of an analysis of snRNAseq datasets from Tabula Muris. Figure 12B depicts representative experimental results from single cell RNAseq on human organoids (CO) generated from healthy and C9-NRE patient-derived iPSCs. SEURAT was used to cluster and annotate cells

Figure 13, comprising Figure 13A through Figure 13B, depicts representative experimental results demonstrating that increased RNA levels for PLAUR and microglial cell surface markers are found in the CNS of ALS/FTD patients. Figure 13 A depicts representative box plots of RNA sequencing data obtained from the cerebellum (CB) or frontal cortex (FCX) (GSE67196) and from the anterior horn (AH) or laser captured enrichment of spinal motor neurons (sMN) (GSE18920) from C9ALS, sALS and CTRLs which were normalized and filtered for candidate microglial cell surface marker genes and PLAUR. Representative box plots are shown for these top candidate genes identified in these preliminary data. All p-adjusted values shown were calculated using a pairwise comparison with Bonferroni correction, with * < 0.05, and ** < 0.01. Figure 13B depicts a representative graph of single nuclei RNA sequencing results obtained from analyses of (PMCID: PMC10504300, brainome.ucsd.edu/C9_ALS_FTD) patient postmortem motor (MCX) and FCX showing PLAUR is predominantly expressed in high levels in cells within annotated microglia clusters compared to other annotated cell clusters in the UMAP plot. Nuclei with PLAUR relative expression range >1.10 are denoted.

Figure 14, comprising Figure 14A though Figure 14C, depicts representative experimental results demonstrating that there is increased microglial expression of uPAR in the cortices of ALS patients. Figure 14A depicts representative experimental results of western blot analyses of the gene PLAUR (alias, uPAR) showing elevated protein levels in the mid motor cortex (Mid motor) of ALS patients compared to controls, while the “disease- spared” occipital cortex (Occ.) shows lower protein levels than the disease-burdened motor cortex. Each point in the bar graph represents an individual patient, with the size of the point corresponding to the patient's age at postmortem tissue collection. A representative western blot is shown below the bar graph. Figure 14B depicts representative immunofluorescent images showing that appreciable uPAR expression is predominantly associated with microglia in disease. Figure 14C depicts a representative quantification showing that contrary to control, patient tissues have a significantly higher uPAR+activated microglia.

Figure 15 depicts representative images from multiplex fluorescence imaging microscopy evaluating uPAR cell- and context-specific expression in patient postmortem tissues: Mulitplex immunostaining of lumbar spinal section from an SOD1 patient using an Akoya Phenocycler fusion. Multiple zoom images of a cross-section of the dorsal, lateral and ventral horns. Specific markers are denoted in between upper and lower panels.

Figure 16, comprising Figure 16A through Figure 16B, depicts representative experiments for human uPAR binder screening. Figure 16A depicts a schematic diagram for a screening system to identify human uPAR binder candidates for CAR generation. CAR-T cells recognizing FITC were incubated with target cells and FITC-labeled uPAR binders including its ligand uPA and anti -uPAR antibodies. Target cell killing indicated cultures with suitable binders. Figure 16B depicts representative screening data using human A549 lung cancer target cells known to express uPAR plated (10,000 cells/well) in 96-well xCELLigence Real-Time Cytotoxicity Assay (RTCA) plates. Conventional CAR-T cells targeting irrelevant or known A549 antigens were used as negative and positive controls, respectively. Similarly, an antibody known to bind and induce killing in this system targeting EpCAM was used as a positive control. The data shows that both uPA and some anti -uPAR antibodies induced specific, dose and time (not shown) dependent killing.

Figure 17, comprising Figure 17A through Figure 17B, depicts representative experiments for human uPAR binder validation. Figure 17A depicts representative data employing human DLD1 cells. Figure 17B depicts representative data employing NCI-H1792 cells. Cancer target cells known to express uPAR were plated (10,000 cells/well) in 96-well xCELLigence Real-Time Cytotoxicity Assay (RTCA) plates. Conventional CAR-T cells targeting irrelevant or known antigens were used as negative and positive controls, respectively. Similarly, an antibody known to bind and induce killing in this system targeting the epithelial marker EpCAM (a-Epithelium) was used as a positive control. Isotype antibody served as a negative control. The data confirms that anti -uPAR antibody induced specific, dose and time (not shown) dependent killing. The data indicated killing -12-14 hrs after CAR-T addition. Figure 18 depicts representative results demonstrating human microglia killing. The human microglial cell line HMC3 was plated (10,000 cells/well) in 96-well xCELLigence Real-Time Cytotoxicity Assay (RTCA) plates. Conventional CAR-T cells targeting irrelevant or known antigens were used as negative and positive controls, respectively. Isotype control and antibody and antibody targeting the epithelial marker EpCAM (a-Epithelium) were used as negative controls. Data confirms that anti -uP AR antibody induced specific, dose and time (not shown) dependent killing. Data indicate killing ~16 hrs after CAR-T addition.

Figure 19 depicts a schematic of human uPAR CAR constructs. 28BBz-based designs of uPAR-directed CAR molecules using H2L or L2H scFv configurations used the sequenced antibody identified in Figure 16 through Figure 18.

Figure 20, comprising Figure 20A through Figure 20F, depicts representative experimental results establishing the efficacy of CAR-T cells targeting microglia expressing uPAR through a surrogate approach. Figure 20A depicts representative flowcharts illustrating the microglial (C-20) populations under various conditions, with P2RY12 plotted on the Y- axis and UPAR (CD87) fluorescence on the X-axis. Figure 20B depicts representative histograms displaying the cell count corresponding to UPAR intensity levels. Figure 20C depicts a representative bar graph indicating the percentage of UPAR+ cells within the P2RY12+ cell population. Figure 20D depicts a representative tSNE graph depicting the dimensionality reduction analysis performed on C-20 cells across all conditions tested. Figure 20E depicts representative overlay multi-color graphs presenting the intensity of each marker within different populations. Figure 20F depicts a representative tSNE graph showing the dimensionality reduction analysis on C-20 cells for each condition, with distinct colors representing different conditions.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that senescent central nervous system cells associated with neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), express one or more cell surface proteins associated with senescence (such as urokinase plasminogen activator receptor (uPAR)), and that CAR-T cells targeting the cell surface protein associated with senescence are able to reduce the number, or eliminate, senescent cells expressing one or more cell surface proteins associated with senescence. Accordingly, in some embodiments, the present invention provides compositions and methods for reducing the number of or eliminating senescent cells expressing one or more cell surface proteins associated with senescence by contacting the senescent cells with a CAR-T cell targeting the one or more cell surface proteins associated with senescence.

In some embodiments, the present invention provides methods of reducing the number of senescent cells in a subject comprising administering to the subject a composition comprising a CAR-T cell targeting one or more cell surface proteins associated with senescence (such as uPAR) to the subject. In some embodiments, the present invention provides methods of reducing or eliminating senescent cells in a subject comprising administering to the subject a composition comprising a CAR-T cell targeting a cell surface protein associated with senescence to the subject.

In one embodiment, the invention provides a method of treating a neurodegenerative disease, such as ALS or FTD, by reducing the number of senescent cells in the subject that are associated with the neurodegenerative disease. In another embodiment, the invention is a method of treating a neurodegenerative disease, such as ALS or FTD, by eliminating senescent cells in the subject that are associated with the neurodegenerative disease. Therefore, in some embodiments, the present invention provides methods of treating a neurodegenerative disease, such as ALS or FTD, by reducing the number of senescent cells associated with the neurodegenerative disease by administering to a subject a composition comprising a CAR-T cell targeting one or more cell surface proteins associated with senescence.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

As used herein, the term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule as defined herein. In one embodiment, the stimulatory molecule is the C, chain associated with the T cell receptor complex. In one embodiment, the intracellular signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is 4-1BB (i.e., CD137). In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and a cytoplasmic signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one embodiment the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the scFv domain during cellular processing and localization of the CAR to the cellular membrane. As used herein, the terms intracellular and cytoplasmic are used interchangeably.

The term “antibody,” as used herein, refers to a polypeptide of the immunoglobulin family that is capable of binding a corresponding antigen non-covalently, reversibly, and in a specific manner. For example, a naturally occurring IgG antibody is a tetramer comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term “antibody” includes, but is not limited to, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, and chimeric antibodies. The antibodies can be of any isotype/class (e.g., IgG, IgE, IgM, IgD, IgA and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).

The term “antibody fragment” refers to at least one portion of an intact antibody, or recombinant variants thereof, and refers to the antigen binding domain, e.g., an antigenic determining variable regions of an intact antibody that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, single chain or “scFv” antibody fragments, linear antibodies, single domain antbodies such as sdAb (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL- linker-Vu or may comprise Vn-linker-Vi.. The portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879- 5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment that comprises a scFv.

By the term “recombinant antibody” as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant or synthetic DNA or amino acid sequence technology which is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated, synthesized or can be derived from a biological sample, or it can be a macromolecule that is not necessarily a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-P, and/or reorganization of cytoskeletal structures, and the like.

A “stimulatory molecule,” as the term is used herein, means a molecule expressed by a T cell that provide the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some embodiment of the T cell signaling pathway. In one embodiment, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the invention include those derived from TCR FcR gamma, FcR beta, CD3 gamma, CD3 delta , CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d. In a specific CAR of the invention, the cytoplasmic signaling molecule in any one or more CARs of the invention comprises a cytoplasmic signaling sequence derived from CD3(^. In a specific CAR of the invention, the cytoplasmic signaling sequence derived from CD3(^ is the human sequence, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

“Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

A “lentiviral vector” is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other Examples or lentivirus vectors that may be used in the clinic as an alternative to the pELPS vector, include but not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

An “antigen presenting cell” or “APC” as used herein, means an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays foreign antigens complexed with major histocompatibility complexes (MHC's) on their surfaces. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

As used herein, the term “autologous” is meant to refer to any material derived from the same individual to whom it is later to be re-introduced.

“Allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

“Knockout” means having a specific gene or allele(s) of a gene disrupted from a genome by genetic manipulation. Accordingly, a “knockout” cell refers to a cell in which a single allele of a gene has been disrupted such that its gene product is not expressed.

“Measuring” or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of the substance or the sample. A “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals including human).

By the term "synthetic" as it refers to a nucleic acid or polypeptide, including an antibody, is meant a nucleic acid, polypeptide, including an antibody, which has been generated by a mechanism not found naturally within a cell. In some instances, the term "synthetic" may include and therefore overlap with the term "recombinant" and in other instances, the term "synthetic" means that the nucleic acid, polypeptide, including an antibody, has been generated by purely chemical or other means.

The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

By the term “specifically binds,” as used herein, is meant an antibody or antigen binding fragment thereof, or a ligand, which recognizes and binds with a cognate binding partner (e.g., a stimulatory and/or costimulatory molecule present on a T cell) protein present in a sample, but which antibody, antigen binding fragment thereof or ligand does not substantially recognize or bind other molecules in the sample.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description The invention is based, in part, on the discovery that senescent central nervous system cells associated with neurodegenerative disease, such as ALS or FTD, express cell surface proteins associated with senescence, such as uPAR, and that CAR-T cells targeting cell surface proteins associated with senescence, such as uPAR, are able to reduce the number of senescent cells expressing the cell surface protein. Accordingly, in some embodiments, the present invention provides compositions and methods reducing the number of senescent cells expressing a cell surface protein associated with senescence, such as uPAR, by contacting the senescent cells with a composition comprising a CAR-T cell targeting the cell surface protein associated with senescence.

In some embodiments, the invention comprises compositions and methods of treating a neurodegenerative disease, such as ALS or FTD, in a subject comprising reducing the number of (or eliminating) senescent cells associated with the neurodegenerative disease by administering to the subject a composition comprising a CAR-T cell targeting a cell surface protein associated with senescence.

Compositions

In some embodiments, the invention comprises compositions for reducing the number of, or eliminating, senescent cells.

In various embodiments, the composition comprises one or more CAR-T cell targeting one or more cell surface proteins. In some embodiments, the one or more cell surface proteins are one or more cell surface proteins associated with senescence. Examples of cell surface proteins associated with senescence include, but are not limited to, urokinase plasminogen activator receptor (uPAR), DEP domain-containing protein 1 A (DEP1), linker for activation of T-cells family member 2 (NTAL), TBC1 domain family member 10A (EBP50), syntaxin-4 (STX4), vesicle-associated membrane protein 3 (VAMP3), armadillo repeat-containing X-linked protein 3 (ARMCX3), beta-2-microglobulin (B2MG), glutathione S-transferase LANCL1 (LANCL1), 5’-3’ exonuclease PLD3(PLD3), vacuolar protein sorting-associated protein 26 (VPS26A), dipeptidyl peptidase 4 (DPP4), secretory carrier- associated membrane protein 4 (SCAMP4), tumor necrosis factor receptor super family member 10D (TNFRSF10D/CD264), neurogenic locus notch homolog protein 1 (N0TCH1), neurogenic locus notch homolog protein 3 (N0TCH3), cluster of differentiation 36 (CD36), oxidized vimentin, and intercellular adhesion molecule 1 (ICAM-1). In some embodiments, the cell surface protein associated with senescence is uPAR. In some embodiments, the CAR-T cell has been modified to not express IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA1 IP, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1. In some embodiments, the CAR-T cell modified to not express a gene may be referred to as a knock-out CAR-T cell. In one embodiment, the interferon is IFNG. In some embodiments, the CAR-T cell has been modified to not express interferon gamma (IFNG).

In some embodiments, the composition further comprises one or more inhibitors of IL-6. In some embodiments, the one or more inhibitors of IL-6 are one or more selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab In some embodiments, the inhibitor of IL-6 Is tocilizumab.

In one embodiment, the CAR-T cell of the invention comprises a chimeric antigen receptor (CAR). In one embodiment, the CAR comprises an antigen binding domain. In one embodiment, the antigen binding domain is a targeting domain, wherein the targeting domain directs the T cell expressing the CAR to a specific cell or tissue of interest. For example, in one embodiment, the targeting domain comprises an antibody, antibody fragment, or peptide that specifically binds to an antigen (e.g., uPAR, DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, N0TCH1, N0TCH3, CD36, oxidized vimentin, or ICAM-1) thereby directing the T cell expressing the CAR to a cell or tissue expressing the antigen. In one embodiment, the invention provides a nucleoside modified nucleic acid molecule encoding a uPAR-CAR.

In one embodiment, the invention relates to a delivery vehicle comprising an agent, wherein the agent comprises a recombinant nucleic acid sequence encoding a chimeric antigen receptor (CAR). In one embodiment, the agent comprises a modified nucleoside mRNA molecule encoding a chimeric antigen receptor (CAR). In one embodiment, agent comprises an mRNA molecule encoding a CAR. In one embodiment, agent comprises a nucleoside modified mRNA molecule encoding a CAR.

In one embodiment, the CAR comprises an antigen binding domain specific for binding to an antigen on a senescent cell. In one embodiment, the CAR comprises an antigen binding domain specific for binding to uPAR, DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, N0TCH1, NOTCH3, CD36, oxidized vimentin, or ICAM-1. In one embodiment, the CAR comprises an antigen binding domain specific for binding to uPAR.

In some embodiments, the antigen binding domain specific for binding to uPAR comprises one or more sequences as listed in Table 1. In some embodiments, the antigen binding domain specific for binding to uPAR comprises complementarity determining regions (CDRs) comprising the amino acid sequences listed in Table 1. In some embodiments, the antigen binding domain specific for binding to uPAR comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, an HCDR3 comprising the amino acid sequence of SEQ ID NO:3, a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:4, an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

Table 1 : uPAR Complementarity Determining Region Sequences

In some embodiments, the antigen binding domain specific for binding to uPAR comprises a variable heavy chain region (VH) and a variable light chain region (VL) comprising the amino acid sequences listed in Table 2. In some embodiments, the antigen binding domain specific for binding to uPAR comprise a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8.

Table 2: uPAR Variable Region Sequences

In some embodiments, the antigen binding domain specific for binding to uPAR comprises an scFv. In some embodiments, the scFv is in a heavy-to-light (H2L) orientation. In some embodiments, the scFv in H2L orientation comprises an amino acid sequence listed in Table 3. In some embodiments, the scFv in H2L orientation comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the scFv is in a light-to-heavy (L2H) orientation. In some embodiments, the scFv in L2H orientation comprises an amino acid sequence listed in Table 3. In some embodiments, the scFv in L2H orientation comprises the amino acid sequence of SEQ ID NO: 10.

Table 3: scFv Sequences

In some embodiments, the CAR comprises an amino acid sequence listed in Table 4. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CAR is encoded by a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CAR is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 12. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 13. In some embodiments, the CAR is encoded by a nucleic acid molecule comprising a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 13. In some embodiments, the CAR is encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 14.

Table 4: CAR Sequences

In various embodiments, the CAR can be a “first generation,” “second generation,” “third generation,” “fourth generation” or “fifth generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257: 127-133 (2014); Sharpe et al., Dis. Model Meeh. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32: 169-180 (2009)).

“First generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to a transmembrane domain, which is fused to a cytoplasmic/intracellular domain of the T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3^-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

“Second-generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to augment T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388- 398 (2013)). CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically borne by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory molecules, for example, CD28, 4- IBB, ICOS, 0X40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

“Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3(^ signaling domain. Preclinical studies have indicated that “Second Generation” CARs can improve the antitumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9): 1577-1583 (2012)).

“Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4- IBB domains, and activation, for example, by comprising a CD3(^ activation domain.

“Fourth generation” CARs provide co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3(^ signaling domain in addition to a constitutive or inducible chemokine component.

“Fifth generation” CARs provide co-stimulation, for example, by CD28 or 4- 1BB domains, and activation, for example, by a CD3(^ signaling domain, a constitutive or inducible chemokine component, and an intracellular domain of a cytokine receptor, for example, IL-2Rp.

In various embodiments, the CAR can be included in a multivalent CAR system, for example, a DualCAR or “TandemCAR” system. Multivalent CAR systems include systems or cells comprising multiple CARs and systems or cells comprising bivalent/bispecific CARs targeting more than one antigen.

In some embodiments, the CAR cells are regulated CAR cells. Examples of regulated CAR cells include, but are not limited to SynNotch-CAR cells.

In the embodiments disclosed herein, the CARs generally comprise an antigen binding domain, a transmembrane domain and an intracellular domain, as described above. In a particular non-limiting embodiment, the antigen-binding domain is an scFv.

Sources of cells for making CAR cells

In one embodiment, the CAR cells are derived from leukocytes. In one embodiment, the CAR cells are derived from one or more selected from the group consisting of macrophages, T cells, and natural killer (NK) cells. In one embodiment, the CAR cells can be autologous with respect to the recipient. In such an embodiment, a CAR cell may be derived from a cell isolated from a subject who is also to be the recipient and modified according to the methods of the invention prior to administration to the subject.

In an alternative embodiment, the modified CAR cells can be allogeneic or syngeneic with respect to the recipient. In such an embodiment, a CAR cell may be derived from cells isolated from a subject or obtained from a source and modified according to the methods of the invention prior to administration to the recipient, wherein the subject or source and the recipient are not the same.

Prior to expansion and modification, the cells may be obtained from a subject. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of cell lines available in the art, may be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi -automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. In another embodiment, Car cells are generated from cells isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of cells can be further isolated by positive or negative selection techniques. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a CAR cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immune-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface proteins present on the cells negatively selected.

For isolation of a desired population of cells by positive or negative selection, the concentration of cells, and available surface area for cell binding (e.g., particles such as beads), can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8⁺ T cells that normally have weaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4⁺ T cells express higher levels of CD28 and are more efficiently captured than CD8⁺ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5 * 10⁶/ml. In other embodiments, the concentration used can be from about 1 X 10⁵/ml to 1 x 10⁶/ml, and any integer value in between.

In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.

Cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1 °C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.

Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in CAR cell therapy for any number of diseases or conditions that would benefit from CAR cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities.

In some embodiments, the CAR cells are generated in vivo. In some embodiments, the CAR cells are generated in vivo by administering to the subject a composition comprising one or more CAR cell -generating agents. Examples of CAR cellgenerating agents include, but are not limited to, mRNA, plasmids, Cas proteins, guide RNAs, siRNAs, shRNAs, viral delivery vectors, and nonviral delivery vectors.

In some embodiments, the composition comprises a delivery vehicle. In some embodiments, the delivery vehicle is selected from the group consisting of microspheres, microparticles, nanoparticles, nanospheres, liposomes, and viral vectors. In some embodiments, the delivery vehicle comprises a targeting ligand. In some embodiments, the targeting ligand is for delivery to one or more selected from the group consisting of T cells, NK cells, and macrophages.

Methods of Reducing the Number Cells

In some embodiments, the invention comprises methods of reducing the number of cells. In some embodiments, the cells are senescent cells. In some embodiments, the invention comprises methods of eliminating senescent cells. In one embodiment, the method comprises introducing a composition comprising a CAR cell targeting one or more cell surface proteins associated with senescence.

In some embodiments, the CAR cells target one or more cell surface proteins. In some embodiments, the cell surface proteins are associated with senescence. In some embodiments, the cell surface protein associated with senescence is selected from the group consisting of urokinase plasminogen activator receptor (uPAR), DEP domain-containing protein 1A (DEP1), linker for activation of T-cells family member 2 (NT AL), TBC1 domain family member 10A (EBP50), syntaxin-4 (STX4), vesicle-associated membrane protein 3 (VAMP3), armadillo repeat-containing X-linked protein 3 (ARMCX3), beta-2-microglobulin (B2MG), glutathione S-transferase LANCL1 (LANCL1), 5’-3’ exonuclease PLD3 (PLD3), vacuolar protein sorting-associated protein 26 (VPS26A), dipeptidyl peptidase 4 (DPP4), secretory carrier-associated membrane protein 4 (SCAMP4), tumor necrosis factor receptor super family member 10D (TNFRSF10D/CD264), neurogenic locus notch homolog protein 1 (N0TCH1), neurogenic locus notch homolog protein 3 (N0TCH3), (CD36), oxidized vimentin, and intercellular adhesion molecule 1 (ICAM-1). In some embodiments, the senescence-specific cell surface protein is uPAR. In some embodiments, the one or more CAR-T cells have been modified to not express (e.g., knock-out), or to express reduced levels (e.g., knock-down), of one or more interferons. In one embodiment, the one or more interferons are one or more selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA11P, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1. In one embodiment, the interferon is IFNG.

In one embodiment, the invention provides methods of reducing the number of, or eliminating, cells. In one embodiment, the cells are senescent cells. In one embodiment, the senescent cells are one or more selected from the group consisting of stem cells, blood cells, central nervous system cells, muscle cells, cartilage cells, bone cells, skin cells, endothelial cells, epithelial cells, fat cells, sex cells, and immune system cells. In one embodiment, the senescent cells are central nervous system cells. In some embodiments, the central nervous system cells are neurons or glial cells. In some embodiments, the glial cells are oligodendrocytes, microglial cells, or astrocytes.

In some embodiments, the method further comprises contacting the senescent cells with one or more inhibitors of IL-6, before, concurrently or after contacting the senescent cell with a CAR-T cell of the invention. In some embodiments, the one or more inhibitors of IL-6 are one or more selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab. In some embodiments, the inhibitor of IL-6 Is tocilizumab.

In some embodiments, the invention provides methods of reducing the number of, or eliminating, cells in vitro, in vivo, or ex vivo. In some embodiments, the cells are senescent cells.

Methods of Targeted Delivery

In some embodiments, the invention provides methods of delivering a payload to target cells. In one embodiment, the payload to is delivered by CAR cells targeting the desired cells. In some embodiments, the CAR cells target one or more cells selected from the group consisting of stem cells, blood cells, central nervous system cells, muscle cells, cartilage cells, bone cells, skin cells, endothelial cells, epithelial cells, fat cells, and sex cells. In one embodiment, the senescent cells are central nervous system cells. In some embodiments, the cells are neurons or glial cells. In some embodiments, the glial cells are oligodendrocytes, microglial cells, or astrocytes. In some embodiments, the cells are senescent.

In some embodiments, the payload to be delivered is one or more selected from the group consisting of nucleic acids, proteins, peptides, carbohydrates, lipids, viruses, bacteria, and small molecules. In some embodiments, the nucleic acids are one or more selected from the group consisting of dsDNA, ssDNA, dsRNA, ssRNA, mRNA, rRNA, tRNA, miRNA, piRNA, piwiRNA, shRNA, siRNA, IncRNA, sncRNA, snoRNA, snRNA, and mtRNA.

Methods of Administering, Preventing and Treating

In some embodiments, the invention provides methods of treating or preventing diseases and/or disorders in a subject in need thereof. In some embodiments, the disease or disorder is a neurological disease or disorder. In some embodiments, the neurological disease or disorder is selected from the group consisting of neuroinflammatory diseases or disorders, neurodegenerative diseases or disorders, viral infections of the central nervous system, bacterial infections of the central nervous system, fungal infections of the central nervous system, and parasitic infections of the central nervous system. In some embodiments, the neurological disease or disorder is a neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Huntington’s disease, Lewy body disease, Parkinson’s disease, synucleinopathies, spinal muscular atrophy, frontotemporal dementia (FTD), multiple sclerosis (MS), spinal cerebellar ataxia (SCA), and spinal bulbar muscular atrophy (SBMA), and tauopathies. In certain embodiments, the neurodegenerative disease is ALS. In some embodiments, the method comprises administering to the subject a composition of the invention

In some embodiments, the invention comprises administering a composition of the invention to a subject that has, or is at risk of developing, a neurodegenerative disease.

In some embodiments, the method comprises administering to the subject a composition comprising a CAR-T cell, and, optionally, a composition comprising one or more inhibitors of IL-6. In some embodiments, the one or more inhibitors of IL-6 are one or more selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab. In some embodiments, the inhibitor if IL-6 Is tocilizumab. In one embodiment, the method comprises administering to the subject composition comprising a CAR-T cell before, during, or after administering a composition comprising one or more inhibitors of IL-6.

In one embodiment, the method comprises administering the composition comprising a CAR-T cell to the subject before administering a composition comprising one or more inhibitors of IL-6. Examples of administering a composition comprising a CAR-T cell to the subject before administering a composition comprising one or more inhibitors of IL-6 include, but are not limited to, administering a composition comprising a CAR-T cell 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, or 30 minutes or less, before administering a composition comprising one or more inhibitor of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject more than one time before administering a composition comprising one or more inhibitors of IL-6.

In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject concurrently with administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject one or more times while administering a composition comprising one or more inhibitors of IL-6. In one embodiment the method comprises administering composition to the subject continuously while administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering to the subject a composition comprising a CAR-T cell and one or more inhibitors of IL-6.

In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject after administering a composition comprising one or more inhibitors of IL-6. Examples of administering a composition comprising a CAR-T cell to the subject after administering a composition comprising one or more inhibitors of IL-6 include, but are not limited to, administering a composition comprising a CAR-T cell 24 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour, or 30 minutes or less, after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject more than one time after administering a composition comprising one or more inhibitors of IL-6.

In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject before and while administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell one or more times before administering a composition comprising one or more inhibitors of IL-6 and one or more times while administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell once before administering a composition comprising one or more inhibitors of IL-6 and a second time while administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject continuously starting before administering a composition comprising one or more inhibitors of IL-6 and continuing while administering a composition comprising one or more inhibitors of IL-6.

In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject before and after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject one or more times before administering a composition comprising one or more inhibitors of IL-6 and administering a composition comprising a CAR-T cell to the subject one or more times after administering a composition comprising one or more inhibitors of IL-6.

In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject while and after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject one or more times before administering a composition comprising one or more inhibitors of IL-6 and administering a composition comprising a CAR-T cell to the subject one or more times after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject continuously beginning while administering a composition comprising one or more inhibitors of IL-6 and ending after administering a composition comprising one or more inhibitors of IL-6.

In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject before, while, and after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject one or more times before administering a composition comprising one or more inhibitors of IL-6, administering a composition comprising a CAR-T cell to the subject one or more times while administering a composition comprising one or more inhibitors of IL-6, and administering the composition to the subject one or more times after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject continuously beginning before administering a composition comprising one or more inhibitors of IL-6 and ending after administering a composition comprising one or more inhibitors of IL-6. In one embodiment, the method comprises administering a composition comprising a CAR-T cell to the subject continuously beginning before administering a composition comprising one or more inhibitors of IL-6 and ending while administering a composition comprising one or more inhibitors of IL-6 and administering a composition comprising a CAR-T cell to the subject continuously beginning while administering a composition comprising one or more inhibitors of IL-6 and ending after administering a composition comprising one or more inhibitors of IL-6.

Pharmaceutical Compositions

In some embodiments, the present invention relates to pharmaceutical compositions for reducing the number of, or eliminating, senescent cells. Briefly, pharmaceutical compositions of the present invention may comprise a target CAR-T cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are in one embodiment formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

When “an effective amount,” “a therapeutically effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the CAR-T cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. CAR-T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

In certain embodiments, it may be desired to administer activated CAR-T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from lOcc to 400cc. In certain embodiments, T cells are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or lOOcc. Wishing not to be bound by theory, using this multiple blood draw/multiple reinfusion protocol, may select out certain populations of T cells.

The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the CAR-T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are administered by i.v. injection. The compositions of T cells may be injected directly into a desired region of the subject’s brain.

The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

Eliminating SCs is possible by genetically silencing critical genes in the senescence pathway (Baker, D. J., et al., 2018, Journal of Clinical Investigation, 128: 1208- 1216). This perspective has triggered profound interest in developing therapeutic strategies to remove SCs without genetic modifications (Martinex-Cue, C., et al., 2020, Frontiers in Cellular Neuroscience, 14: Article 16). These “senolytic” approaches eradicate SCs and the accompanying senescence-associated secretory phenotype (SASP) to benefit the local environment where SCs reside and could potentially inhibit SASP propagation and neural decline. Several small molecules have already been identified to exhibit senolytic activity, but often lack potency and/or produce substantial side effects (Kim, E.-C., et al., 2019, BMB Reports, 52:47-55). Described here is an approach to blunt and remove SCs, which involves engineering CAR-T cells directed against senescence-specific cell surface antigens. CARs are synthetic receptors that redirect T cells targeting specificity in different contexts. It is demonstrated that CAR-T cells can serve as senolytic agents in the context of C9orf72-linked ALS/FTD. Emerging studies demonstrated that targeting SCs benefits aging and age-related disease pathologies.

Example 1 : Nucleotide Repeat Expansion (NRE) in the C9orf72 Gene is Associated with Senescence and Senescence- Associated Secretory Phenotype (SASP)

Neurodegenerative diseases, including ALS and FTD, frequently show clinicopathological focal origins of pathology in the CNS that propagate to surrounding tissues and throughout the neuroaxis. While propagation mechanisms remain highly investigated, there is emerging evidence that neural senescence and subsequent secretion of proinflammatory molecules, referred to as SASP, may contribute to this process (Baker, D. J., et al., 2018, Journal of Clinical Investigation, 128: 1208-1216; Martinez-Cue, C., et al., 2020, Frontiers in Cellular Neuroscience, 14: Article 16; Saez-Atienzar, S., et al., 2020, Nature Reviews Neuroscience, 21 :433-444; Amor, C., et al., 2020, Nature, 583: 127-132). Classically defined as a state of arrested proliferation by cells in response to stressors, senescence is an aging-related process with pathophysiological hallmarks characterized by increased activation and levels of p 16/p21 signaling pathways, DNA damage, senescence-associated P- galactosidase activity, and increased SASP. It has been proposed that post-mitotic cells like neurons and proliferative cells such as astrocytes and microglia can enter a chronic senescence state and, through SASP, propagate the functional decline of the CNS tissue. There is cumulative evidence in patient iPSC-derived cells, rodent models, and patient postmortem tissues that hallmarks of senescence are present in ALS (Porterfield, V., et al., 2020, Neurobiology of Aging, 90: 125-134; Trias, E., et al., 2019, Frontiers in Aging Neuroscience, 11 : Article 42; Tam, O. H., et al., 2019, Cell Reports, 29: 1164-1177).

Preliminary data suggest that many senescence-associated mechanisms are discernible in the most common genetic form of ALS and FTD caused by C9-NRE mutation. In an in-vitro neuronal model of C9-ALS/FTD, which recapitulates the expression of the proteinaceous arginine (R)-rich dipeptide repeats (DPRs) produced from the unconventional translations of the C9-NRE, elevated levels of pl 6, p21, and a senescence-associated transcriptomic profile were identified. It has previously been shown that both the R-rich DPRs, poly PR and GR form nuclear inclusions in cells and display robust neurotoxicity (Wen, X., et al., 2014, Neuron, 84: 1213-1225). There is also evidence that R-rich DPR- mediated neurotoxicity occurs through p53 activation and the activation of the cyclin- dependent kinase inhibitor p21.

Interestingly, both R-rich DPR species localize to the nucleolus, a subnuclear membrane-less organelle, where ribosomal biogenesis occurs, before inducing neuronal death (Yank, K., et al., 2018, Cell Stress, 2: 125-140). Nucleolar or ribosomal stress, characterized by diverse cellular insult-induced abnormalities in nucleolar structure and function, ultimately leads to activation of p53 and alteration in cell behavioral phenotype. This stress response is multifaceted, but induction of p53 is potentially the most significant component (Slomnicki, L. P., et al., 2017, Scientific Reports, 7:el6652). Maor-Nof et al. recently showed that polyPR-mediated toxicity occurs through a p53-mediated and a cyclin-dependent kinase inhibitor p21 activation (Maor-Nof, M., et al., 2021, Cell, 184(3):689-708). PR expression leads to upregulation of numerous senescence-associated genes in primary neurons (Figure 1) suggesting that PR may likely initiate a senescence program, which could potentially drive the production of several cytokines, chemokines, growth factors, and proteases collectively known as SASP (Maor-Nof, M., et al., 2021, Cell, 184(3):689-708). The same study provided evidence that PR-induced p53 activates a downstream transcriptional program centered on the mitochondrial protein Puma to induce cytochrome c release, leading to a cascade of cleaved caspases and endonuclease activation in DNA fragmentation and neurodegeneration by apoptosis (Maor-Nof, M., et al., 2021, Cell, 184(3):689-708).

Apoptosis and senescence are two faces of the same coin; specific processes or stress responses may simultaneously engage these two pathways. The programming of each cell type decides which outcome - senescence or apoptosis - will occur first. In some circumstances, apoptosis results from overwhelming stress, whereas senescence results from more chronic damage (Kastenhuber, E. R., et al., 2017, Cell, 170: 1062-1078). Senescence has been recently revisited for a potential primary role in dementia (Baker, D. J., et al., 2018, Journal of Clinical Investigation, 128: 1208-1216). As aging is the leading risk factor for dementia-related neurodegeneration, changes in the timing or nature of the cellular hallmark of normal aging may be vital to understanding events that cause and accelerate neuronal death in neurodegenerative diseases. Therefore, cellular senescence is a candidate mechanism that may influence this process in the context of C9-ALS/FTD.

The pervasiveness of senescence in C9-ALS/FTD and its potential mechanistic contribution to disease pathogenesis is unknown. Although several significant pathways associated with increased cellular senescence have been described in C9-NRE patients and models, there is no data linking many seemingly disparate phenotypes to possible increases in cellular senescence or SASP. To this end, hallmarks of senescence were investigated in patient-derived cortical i³neuronal cultures (Femandopulle, M. S., et al., 2018, Current Protocols in Cell Biology, 79:e51). One of the most widely used senescence markers is the increased levels of senescence-associated B-galactosidase (SA-B-Gal, a lysosomal enzyme) activity. Using X-Gal as a chromogenic substrate, this enzymatic assay monitors the increased expression and activity of this lysosomal protein in SCs and indicates increased lysosomal mass (Kurz, D. J., et al., 2000, Journal of Cell Science, 113(20):3613-3622). This assay is colorimetric and observed with a light microscope. C9-NRE cortical i³neurons have increased B-galactosidase activity as well as increased levels of the SASP, interleukin 6 (IL- 6) with increasing cell age/maturation compared to controls (Figure 2). These results, with two obligatory senescence phenotypic hallmarks, support potentially accelerated agedependent increases in senescence phenotypes in neurons derived from patients carrying the C9-NRE mutation. Using these same patient-derived neurons, senescent signatures were further examined by filtering RNAseq data with previously identified senescence and SASP transcriptomic marker panels (Coppe, J. P., et al., 2010, Annual Review of Pathology, 5:99- 118; Fridman, A. L., et al., 2008, oncogene, 27:5975-5987). In the differential gene expression results the robust separation of C9-NRE from control groups can be seen in a principal component analysis with C9-NRE neurons showing an overall increase in many senescence-associated transcripts (Figures 3 A and 3B), such as pl6 and p21, which were confirmed by quantitative PCR (Figure 3C). Additionally, as seen in Figure 3B, several known senescence-associated cell surface biomarkers, such as ICAM1, ULBP1, and uPAR have low or limited transcript expression in controls but are highly expressed in C9-NRE (Rossi, M., et al., 2021, Cells, 10(7): 1740). Thus, these data confirm that senescence- associated phenotypic hallmarks and gene expression profiles are more prevalent in patient- derived C9-NRE neurons and that there is upregulation of specific candidate senescence- associated cell-surface biomarkers identified that could be further examined for targeted therapeutic approaches. One specific senescent cell surface biomarker, urokinase plasminogen activator receptor (uPAR, from the gene PLAUR), was recently targeted by CAR-T cells for effective elimination of SCs in an in-vivo model of liver fibrosis (Amor, C., et al., 2020, Nature, 583: 127-132). uPAR plays a multifaceted role in almost any process where migration of cells and tissue-remodeling is involved such as inflammation (Baart, V. M., et al., 2020, EJNMMI Research, 10:Article 87). Typically, uPAR is absent in healthy tissues, as reflected in the RNAseq data from cortical i³neuron tissue cultures (Figure 3). By its carefully orchestrated interaction with the protease urokinase plasminogen activator and its inhibitor (plasminogen activator inhibitor- 1), uPAR localizes a cascade of proteolytic activities, enabling (patho)physiologic cell migration. Moreover, via the interaction with a broad range of cell membrane proteins, like vitronectin and various integrins, uPAR plays a significant, but not yet completely understood, role in the differentiation and proliferation of cells, also affecting disease progression. The implications of these processes, either for diagnostics or therapeutics, have received much attention in oncology but only limited beyond that. Nonetheless, the role of uPAR in different diseases, including neurological and neurodegenerative conditions, provides ample opportunity to exploit new applications for treatment options (Merino, P., et al., 2017, Receptors & Clinical Investigation, 4(2):el552).

Although there remains a gap in knowledge between the observations of phenotypic hallmarks of neural senescence and their direct causative role in age-related neurodegeneration, harnessing the therapeutic potential of targeting senescence-related pathways to prevent age-related neurodegeneration could address an urgent clinical need for treatments. Several publications have highlighted the clearance of SCs as a potential therapeutic approach, with the recent demonstration that this approach in the CNS can attenuate tau pathology and cognitive decline in a proteinaceous tauopathy mouse model (Martinex-Cue, C., et al., 2020, Frontiers in Cellular Neuroscience, 14: Article 16; Amor, C., et al., 2020, Nature, 583: 127-132; Bussian, T. J., et al., 2018, Nature, 562:578-582). In C9- ALS/FTD, this has not been tested yet. Based on prior findings and this data, it is hypothesized that neural senescent-associated phenotypes originate early in ALS/FTD disease cascades. Therefore, targeting senescent neural cells constitutes a novel therapeutic approach to lessen disease progression.

Example 2: Characterization of Senescence Phenotype in a Cerebral Organoid Model of C9- ALS/FTD

Using immunofluorescence (IF), light-sheet and confocal microscopy, neurons, astrocytes, and microglia are imaged in cerebral organoids (COs) produced from the C9orf72 and control lines for evidence of senescence, neuroinflammation, and neurodegeneration beginning at day 52 DIV and every two to four weeks, as cellular senescence signatures emerge. Organoids can be maintained for more than one year in culture and thus model aspects of age-related events such as senescence (Ormel, P. R., et al., 2018, Nature Communications, 9(1):4167).

Because cellular senescence is multifaceted, the concurrent validation of multiple hallmarks is needed to confirm the senescent phenotype, such as altered cell function and morphology, which are driven by specific changes in gene expression and chromatin structure. Senescence in the COs in response to the disease-causative C9-NRE mutation is examined by IF in the different cell types constituting the organoids. The primary characteristics of senescence are assessed, starting with cell cycle arrest (pl6INK4a, p21, p53) and verified by the absence of proliferation (PCNA or Edu/BrdU incorporation). The expression of additional hallmarks of senescence is verified, focusing on structural changes associated with senescence, such as increased lysosomal mass (SA-B-galactosidase), changes in organelle structures (Lamin Bl downregulation), or markers of epigenetic changes such as senescence-associated heterochromatic foci (SAHFs). The presence of DNA damage response-related markers, increased ROS levels, or SASP expression is also assessed. The protocols to identify these markers of senescence are commonly published in literature (Gonzalez-Gualda, E., et al., 2021, FEBS Journal, 288:56-80; Crowe, E. P., et al., 2014, Methods in Molecular Biology, 1170:425-445). The SASP comprises several factors, such as cytokines, chemokines, and growth factors that are secreted from SCs. The SASP is thought to be a major reason SCs contribute to either tissue homeostasis or dysfunction, as these factors play many roles in immune signaling, cell-to-cell communication and neuroinflammation (Coppe, J. P., et al., 2010, Annual Review of Pathology, 5:99-118). Components of the SASP are identified by RT-qPCR (IL-la; IL-6; IL-8; CCL2; CXCL2; CXCL10, and other candidates shown in Figure 3), ELISA assay, and cytokine and chemokines arrays. Some of the factors most upregulated in SCs include the interleukins IL- la, IL-6 and IL- 15, the chemokines, IL-8, GRO-a and MIP-la, and others such as IFN-c, VEGF, ICAM-1 and GM-CSF17. Apoptosis exclusion from the putative SCs was evaluated by IF using an antibody against cleaved caspase 3 (CC3+). CC3+ staining also served as a marker of neuronal degeneration in the COs. Activation of microglia is monitored over time by IF (to measure the presence and length of processes by Ibal staining) and by FACS analysis; a recent publication has indeed shown that a specific population of microglia, which is increased in neuroinflammation, is detectable by FACS by gating the CD317⁺ MHC-II⁺ CD39^hlgh CD86⁺ cell population and their transcriptome profiled by RNAseq (Ajami, B., et al., 2018, Nature Neuroscience, 21 :541-551). Change in astrocyte reactive status is evaluated over time by IF assessing the percentage of GFAP+ and S100P+ cells. To confirm their activation status, microglia and astrocytes are FACS sorted from the organoid preparation (gating CD271- CD44⁺ cells for CD184⁺ to obtain cells with the astrocyte marker signature) and their transcriptome profiled by RNAseq (Janssens, S., et al., 2019, Current Protocols in Stem Cell Biology, 48:e65).

The urokinase-type plasminogen activator receptor (uPAR) was previously identified as a cell surface protein that is specifically up-regulated in senescent cells (Amor, C., et al., 2020, Nature, 583: 127-132). In the present RNAseq dataset (Figure 3), the PLAUR (the gene encoding uPAR) transcript was also upregulated in aging C9-ALS/FTD iPSC- derived neurons which are beginning to undergo senescence, as per the increase in IL-6 release and SA-P-galactosidase specific markers (Figure 2). The expression of uPAR is assessed in COs undergoing senescence over time and across genotypes by using IF and single-cell transcriptomic analysis and FACS sorting.

Senotherapeutics are classified as senolytics, which selectively kill SCs, and senomorphics which modulate SCs by blocking SASP4. By utilizing senomorphic and senolytic compounds, which respectively drive and inhibit senescence, and by employing biochemical, multi-omics, and imaging analyses, the effects of these compounds on cell viability, pathological hallmarks of senescence, and SASP protein markers are measured, as previously illustrated, in COs of C9-ALS/FTD and wild type control genotypes. Example 3: Senolytic Compounds Target Senescent Phenotype in a Cerebral Organoid Model of C9-ALS/FTD

Seven classes of compounds with evidence of senolytic activity have been reported, including the combination of dasatinib and quercetin, as well as BCL2 family inhibitors. In addition, a forkhead box protein 04 (F0X04)-interacting peptide, which blocks the association of F0X04 with p53, induces apoptosis of human SCs in vitro and reduces the expression of senescence markers while extending health span in vivo. The natural compounds fisetin, a quercetin related flavonoid, and piperlongumine also exhibit evidence of senolytic or senomorphic activity in certain cell types in vitro (Martinez-Cue, C., et al., 2020, Frontiers in Cellular Neuroscience, 14: Article 16). Clinically used compounds targeting the co-chaperone heat shock protein 90 (HSP90) were also identified as a novel class of potential senolytics, able to induce apoptosis of senescent murine and human cells in vitro and improve health span in vivo. Finally, the FDA-approved histone deacetylase inhibitor Panobinostat induces apoptosis of senescent tumor cells in vitro.

Senolytic candidates being tested are: Dasatinib/Quercetin; ABT-263; ABT- 737; A1331852 and A1155463; 17-AAG; Geldanamycin; Fisetin; Piperlongumine; Quercetin-3-D-galactose; UBX0101; Panobinostat; FOXO4-DRI peptide; Klotho (Kim, E - C., et al., 2019, BMB Reports, 52:47-55; Gonzalez-Gualda, E., et al., 2021, FEBS Journal, 288:56-80; Shaker, M. R., et al., 2021, NPJ Aging and Mechanisms of Diseases, 7:Article 18). These compounds are screened against the SASP in COs of C9-LS/FTD and wild type control genotypes. Optimum concentrations of these compounds are initially based on the literature and determined experimentally with dose-response and time-course experiments.

Example 4: Senomorphic Compounds Induce Senescent Phenotype in a Cerebral Organoid Model of C9-ALS/FTD

Several classes of senomorphics - drugs that suppress markers of senescence or their secretory phenotype without inducing apoptosis - have also been identified (Kim, E - C., et al., 2019, BMB Reports, 52:47-55; Gonzalez-Gualda, E., et al., 2021, FEBS Journal, 288:56-80). Senomorphics include inhibitors of IkB kinase (IKK) and nuclear factor (NF)- kB, free radical scavengers, and Janus kinase (JAK) pathway inhibitors. Even rapamycin acts as a senomorphic by reducing the SASP. Some compounds, for example, fisetin, have senomorphic effects on some cell types while having senolytic activity on others, at least in- vitro. Senomorphic candidates being tested are: NBD peptide; JAK inhibitor (ruxolitinib); KU-60019; JH4; Juglanin; Quercetin-3-O-P-D-glucuronide; (-)-Loliolide; Quercetagetin 3,4'-dimethyl ether; ESC-CM; Mmu-miR-291a-3p. Optimum concentrations of these compounds are initially based on the literature and determined experimentally with dose-response and time-course experiments.

Example 5: Immunotherapeutic Interventions Eliminate Senescent Cells in a Cerebral Organoid Model of C9-ALS/FTD

While multiple senotherapies are being explored, immune-mediated approaches have particular advantages, including life-long activity due to the formation of immune memory, the ability of T cells to eliminate senescent cells by inducing their apoptosis, and the high specificity of T cells. In that context, CAR-T cells targeting the senescent cell marker uPAR39 lysed senescent cells in vitro, eliminated them in vivo and successfully treated liver fibrosis in mice (Amor, C., et al., 2020, Nature, 583: 127-132). Similarly, CAR-T cells targeting fibroblast activation protein (FAP), a marker of activated fibroblasts, reduced cardiac fibrosis and improved cardiac function in a mouse model of cardiac injury (Rurik, J. G., et al., 2022, Science, 375:91-96). In the context of emerging data supporting senescent cells in ALS/FTD, COs are used herein to explore the ability of senolytic CAR-T cells to target senescent cells and mitigate the C9-ALS/FTD mediated neurodegenerative phenotype.

Senolytic CAR-T Cells Eliminate uPAR-Expressing Senescent Glia in vitro. “Senolytic” uPAR-directed CAR-T cells lysed SCs in vitro, eliminated them in vivo, and successfully treated liver fibrosis in mice (Amor, C., et al., 2020, Nature, 583: 127-132). uPAR is a biomarker of SCs underlying pathogenesis in C9-ALS/FTD (Figure 3). Here, the ability of uPAR-directed senolytic CAR-T cells to recognize and lyse uPAR-expressing senescent glia in-vitro is examined.

A well-established source of de-identified normal healthy donor PBMCs (peripheral blood mononuclear cells) for research purposes is available. PMBCs are isolated by Ficoll-Paque are cryopreserved for subsequent CAR-T cell generation using established methods (Magee, M. S., et al., 2018, Cancer Immunology Research, 6:509-516; Magee, M. S., et al., 2016, Oncoimmunology, 5:el227897; Neron, S., et al., 2006, Transfusion, 46:537- 544). T cells are isolated from thawed PBMCs, activated with anti-CD3/CD28 beads, and transduced with lentiviral vectors expressing 28BBz CARs targeting murine CD 19 (negative control) or uPAR (indicated as “control” or “uPAR” CAR-T cells throughout) (Magee, M. S., et al., 2018, Cancer Immunology Research, 6:509-516; Magee, M. S., et al., 2016, Oncoimmunology, 5:el227897). uPAR-directed CAR-T cells employ previously established designs with senolytic activity (Amor, C., et al., 2020, Nature, 583: 127-132). T cells are expanded for 10-14 days prior to functional studies (Magee, M. S., et al., 2018, Cancer Immunology Research, 6:509-516; Magee, M. S., et al., 2016, Oncoimmunology, 5:el227897; Lock, D., et al., 2017, Human Gene Therapy, 28:914-925).

CAR-T cell activity is validated by well-established assays including T-cell phenotype, activation marker upregulation, cytokine production, and cytolytic activity after stimulation with control and CT26 cells engineered to express uPAR and mouse KP lung cancer cells that were treated with combined MEK and CDK4/6 inhibition, an established model of senescence-induced uPAR expression (Magee, M. S., et al., 2018, Cancer Immunology Research, 6:509-516; Amor, C., et al., 2020, Nature, 583: 127-132; Magee, M. S., et al., 2016, Oncoimmunology, 5:el227897). Human iPSC-derived microglia-like cells treated with the senescence-inducing (senomorophic) compounds (inhibitors of IKK, NFKB, and JAK) as above, are employed to examine uPAR CAR-T cell recognition and killing of senescent glia (Abud, E. M., et al., 2017, Neuron, 94:278-293). T cell phenotype and composition are analyzed using fluorescent antibodies against CD62L, CD45RO, CD95, CD3, CD4, and CD8 to quantify naive (TN), stem-cell memory (TSCM), central-memory (TCM), effector-memory (TEM), and effector (TEFF) subsets (Lock, D., et al., 2017, Human Gene Therapy, 28:914-925). Activation marker and cytokine production are measured in control or CAR-T cells stimulated for 6 hrs with the above cell lines (media and PMA+Ionomycin will serve as negative and positive controls, respectively). Cells are stained with a LIVE/DEAD viability stain, antibodies to CD8 and CD4, the activation markers CD69, CD25, CD44, the cytokines IFNy, TNFa, IL-2, and MIPla, and the degranulation marker CD107a (Magee, M. S., et al., 2018, Cancer Immunology Research, 6:509-516). Antibody-stained cells are analyzed on a BD LSRFortessa flow cytometer using FlowJo software (Magee, M. S., et al., 2016, Oncoimmunology, 5:el227897). Cytolysis is determined using the xCELLigence real-time cytotoxicity system (RTCA; Acea Biosciences Inc.) (Magee, M. S., et al., 2018, Cancer Immunology Research, 6:509-516; Magee, M. S., et al., 2016, Oncoimmunology, 5:el227897; Lisby, A. N., et al., 2022, Methods in Cell Biology, 167:81-98). Senolytic CAR-T Cells Mitigate C9-ALS/FTD Pathogenicity in Organoids. Senolytic uPAR-directed CAR-T cells lysed senescent cells in vitro, eliminated them in vivo, and successfully treated liver fibrosis in mice (Amor, C., et al., 2020, Nature, 583: 127-132). The ability of uPAR-directed senolytic CAR-T cells to eliminate senescent cells and reduce C9-ALS/FTD pathogenicity in C9-ALS/FTD cerebral organoid cultures is examined. Generation of control and uPAR CAR-T cells is carried out as above. Fully matured COs incorporating microglia are generated and validated as above from human C9-ALS/FTD iPSC lines prior to the addition of control and uPAR CAR-T cells (Szebenyi, K., et al., 2021, Nature Neuroscience, 24(11): 1542-1554; Shaker, M. R., et al., 2021, NPJ Aging and Mechanisms of Disease, 7: Article 18). Biochemical, cellular, and imaging analyses are employed to measure the effects of uPAR CAR-T cells on neuron and glia viability, pathological hallmarks, and SASP protein makers as above.

IFNY Blockade Prevents IL-6 Production, CRS, and ALS/FTD Exacerbation. CAR-T cell therapy for cancer poses a known risk for neurotoxicity reflecting cytokine release syndrome (CRS) induced by myeloid cell production of IL-650 induced by IFNY produced by CAR-T cells (Bedoya, F., et al., 2017, Molecular Therapy, 25:314-320). Importantly, blockade of IL-6 is a well-established mitigation strategy and emerging data support IFNy blockade as a possibility without impacting antitumor activity (Maude, S. L., et al., 2014, Cancer Journal, 20: 119-122; Bailey, S. R., et al., 2021, Blood, 128(13): 1723-1734). Interestingly, emerging data strongly implicate IL-6 as an important factor in ALS/FTD, suggesting that senolytic CAR-T cell efficacy in ALS/FTD could be negatively impacted by unintended exacerbation by the CAR-T —> IFNy —> myeloid cell —> IL-6 axis (Figure 5) (Garbuzova-Davis, S., et al., 2018, International Journal of Molecular Science, 19(2):423; Ehrhart, J., et al., 2015, Journal of Neuroinflammation, 12: 127). Thus, the cytokine profiles (with a focus on IL-6) of CAR-T and organoid mixed cultures are examined for evidence of a CRS-like response and its amelioration by IL-6 blocking antibody and CRISPR-Cas9 deletion of IFNY iⁿ CAR-T cells.

CAR-T cell generation and organoid models are carried out as above. CRISPR/Cas9 deletion of IFNy (IFNG gene) in control uPAR CAR-T cells use well- established protocols based on electroporation of gRNA+Cas9 complexes based on a recent demonstration of IFNy deletion in human CAR-T cells (Bailey, S. R., et al., 2021, Blood, 128(13): 1723-1734; Salas-Mckee, J., et al., 2019, Human Vaccines & Immunotherapeutics, 15: 1126-1132). Deletion is verified by targeted genomic DNA sequencing and intracellular cytokine staining for IFNy as above. Scrambled gRNA is used as a negative control. Senescent cell elimination and C9-ALS/FTD pathogenicity is measured in organoid cultures treated with control and uPAR CAR-T cells gene-edited with control or IFNG gRNA complexes. Moreover, control and uPAR CAR-T cell treatments are carried out in the absence or presence of the IL-6 blocking antibody tocilizumab (van Blitterswijk, M., et al., 2013, Neurology, 81 : 1332-1341). The 71-Plex Human Cytokine/Chemokine Assay Array (Eve Technologies) is employed to quantify IFNy, IL-6, and 69 other cytokines and chemokines over time in mixed cultures of CAR-T cells and organoids ± IFNG deletion ± tocilizumab.

The materials and methods used are described here.

Cells

Three control (CS5NWTiCTR-n3-l, CS6GNGiCTR-n2, CS2AE8iCTR-n6;) and three C9orf72 (CS2DDGiALS-nl-l; CS5MNZiALS-n2; CS5ZGKiALS-n5-l) iPSC lines are cultured according to the protocol supplied by Answer ALS consortium (Cedars Sinai). These lines are characterized by staminality markers (Oct3/4, SOX2, NANOG, TRA-1-60, TRA-1-84, SSEA4), karyotyping, and other tests. Cells were acquired from the Answer ALS consortium.

Generation of Cerebral Organoids

Cerebral organoids (COs) are three-dimensional in-vitro cultures that can be generated from induced pluripotent stem cells (iPSCs). They present a unique opportunity to study the interplay between neural cells in-vitro. The current methodology allows generating COs based on the “self-assembly” of the cerebral structure from human iPSCs without external control. COs have the unique ability to resemble the human cerebral cortex, making them a relevant model for studying C9-ALS/FTD. COs are grown from iPSC lines derived from C9-ALS/FTD patients that recapitulate mature architecture and display C9orf72 ALS/FTD pathology (Lancaster, M. A., et al., 2014, Nature Protocols, 9:2329-2340). COs from iPSC lines derived from healthy, control individuals are used as reference. However, the protocol is not known to reliably incorporate microglia cells as they differentiate from mesodermal progenitors. COs are infiltrated with differentiated microglia (iMG) harvested from 2D cultures following an established protocol (Abud, E. M., et al., 2017, Neuron, 94:278-293; Brownjohn, P. W., et al., 2018, Stem Cell Reports, 10: 1294-1307). Fig.4 G, H shows the infiltration protocol. COs with infiltrated microglia are used as the experimental platform of choice starting at DIV 52 after the organoids are considered fully matured (Lancaster, M. A., et al., 2014, Nature Protocols, 9:2329-2340).

Data Analysis

Imaging data are analyzed with Imaged, R-studio, and IMARIS software. Fluorescence signals are quantified in an automated, unbiased manner. Automated thresholding is used to correct for background fluorescence. Western blots are normalized to total protein sample content and quantified by Imaged. Statistical analysis is done by one-way ANOVA or unpaired t-test, as appropriate and data presented as mean ± SEM. Statistical analyses are performed in GraphPad. Uncorrected Fisher LSD two-way ANOVA test is used to compare control vs. treatment conditions. When variability of the results within the same CO groups is high, a power analysis is performed to determine the appropriate sample size and ensure robustness of the results.

Example 6: Senescence-associated genes and markers are increased in ALS/FTD

Although there is a constellation for evidence for molecular pathways associated with senescence across neurodegenerative diseases, including ALS/FTD, a conclusive involvement of senescence in the pathogenesis of age-related diseases remains unsubstantiated. To unequivocally address these gaps in knowledge, hallmarks of senescence were examined in patient-derived cortical i³Neuronal cultures (Fernandopulle, M. S. et al., 2018, Curr Protoc Cell Biolz, 79, e51). One of the most common genetic causes of ALS and FTD, the nucleotide repeat expansion (NRE) mutation on the chromosome 9 open reading frame 72 gene (C9orf72, C9) (Renton, A. E. et al., 2011, Neuron, 72, 257-268; DeJesus- Hemandez, M. et al., 2011, Neuron, 72, 245-256), was chosen as a model. To understand the extent of senescence molecular signature in these models, new transcriptomic data was generated and filtered for senescence-associated (SA) genes from transcriptomic markers panels previously shown to be highly associated with senescence and/or SASP (Coppe, J. P., et al., 2010, Annu Rev Pathol, 5, 99-118; Fridman, A. L., et al., 2008, Oncogene, 27, 5975- 5987; Saul, D. et al., 2022, Nat Commun, 13, 4827). In the differentially expressed gene (DEG) results, C9-NRE i³Neurons show robust (Figure 6A and Figure 6B). These cortical- like neurons display well -documented phenotypic features of SCs, such as an overall increase in many senescence-associated (SA) transcripts like pl6 and p21, which were also confirmed by quantitative PCR (Figure 6C), and increased DNA damage levels (Figure 6C). These results suggest that cortical i³Neuronal models from patients with C9-NRE exhibit accelerated age-related senescence traits compared to controls. The findings reinforce the idea that senescence is a common feature in patient-derived models, at least for the most prevalent genetic causes of ALS and FTD. Additionally, several known SA cell surface biomarkers (Rossi, M., et al., 2021, Cells, 10), such as ICAML and PLAUR, have low or limited transcript expression in controls but are highly expressed in C9-NRE (Figure 6A), which makes these genes potentially more accessible for selective FACS or cell enrichment strategies. These data indicate that senescence signatures appear in cortical neuron-like cells and could drive neuronal dysfunction in ALS-FTD. To determine if senescence signatures would emerge in more complex multicellular patient-derived models, cerebral organoids (COs) were utilized. Immunofluorescence (IF) microscopy imaging was performed on COs (DIV 90-180) to visualize the cellular composition and organization within the organoids, setting the stage for further investigations into senescence-related processes at the cell-type level (Figure 7A). SEURAT analysis was also used to generate UMAP plots from single-cell data obtained from aged COs, derived from both C9-NRE mutation carriers and control patients. In the UMAP plots, the clusters, which were then unbiasedly annotated using the human Allen Brain Cell Atlas, show cell annotations consistent with what has been demonstrated with CO cultures (Figure 7B). When the curated SA gene panel was incorporated into the cellular annotations, SC clusters appear (shown in black). This suggests that COs can effectively model senescence and potentially SC transitions, consistent with recent evidence for cellular senescence in aged CO models (Aguado, J. et al., 2023, Nature Aging, 3: 1561-1575).

Therefore, this in-vitro model can recapitulate aspects of cellular aging processes. Interestingly, when microglia were added during the development of the COs in culture, larger changes in the prevalence of SCs were observed (Figure 7B). The alteration of SC prevalence upon microglia addition indicates a potential role of microglia in influencing senescence-related processes within the COs and could potentially mirror emerging mechanisms that were recently reported for microglia-facilitated senescence induction of motor neurons in nonhuman primate spinal cords (Sun, S. et al., 2023, Nature, 624:611-620). These findings in COs, highlight the importance of studying the interactions between different cell types in modeling senescence. Additionally, there was focus on the cell annotated clusters in the UMAP plots to examine the expression patterns of specific SA genes across various cell clusters (Figure 7C). The dot plot revealed the expression levels of upregulated SA genes in the different cell clusters, with oligodendrocytes and endothelial cells often being preferentially annotated as SCs. These preliminary data provide valuable insights into the molecular changes associated with senescence in specific cell types within the COs. Overall, these data demonstrate that COs offer a unique platform for investigating senescence and SC transitions with cell-type specificity. The presence of SCs in the organoids and their responsiveness to microglia addition validate the relevance and versatility of this in-vitro model for studying cellular aging processes, its potential to unravel novel insights into neurodegenerative diseases and serve as an important tool to investigate senescence and cell-type specific SC transitions.

The temporal gene expression of SA gene markers in the “gold standard” preclinical model for ALS, the SOD1^G93A mouse model (mutSODl), was then investigated. A PCA was once again utilized for an unbiased comparison of control versus mutSODl . The PCA shows distinct temporal patterns of gene expression changes in the mutSODl mouse, which leads to increased significant separation of mutSODl from control over time (Figure 8A). A close examination of several top candidate genes observed in both the in vitro and in vivo data sets, reveals significant differences in the temporal expression patterns of specific SA markers between the control and the mutSODl, with mutSODl levels showing increased temporal expression over disease progression (Figure 8B). Analyses of the SA gene, Plaur (Amor, C. et al., 2020, Nature, 583: 127-132), which encodes the urokinase plasminogen activator receptor (uPAR) and is also found to be expressed in ALS patients and in the mutSODl mouse model of ALS (Gias, M. et al., 2007, Exp Neurol, 207, 350-356), was also included. Interestingly, early inhibition with a synthetic inhibitor prolonged the life of the mutSODl mouse (Gias, M. et al., 2007, Exp Neurol, 207, 350-356). In this dataset, significant increases of Plaur in transcript levels following disease onset was also observed. Spatial transcriptomic data from the ALS-st.nygenome.org database was then utilized to investigate disease-linked temporal expression patterns of candidate senescence genes in mutSODl mice compared to age-matched controls (Figure 8C). The spatial transcriptomic dataset provides spatially resolved gene expression information, allowing identification of specific anatomical regions in the spinal column where candidate senescence genes are differentially expressed in mutSODl mice compared to age-matched controls. The robust disease-linked temporal expression of these genes indicates that senescence may play a significant role in the ALS pathology observed in these mice. Overall, these results suggest that the mutSODl mouse model exhibits increased temporal expression of SA gene markers in the spinal column, indicating a potential role for cellular senescence in ALS disease progression. RNA expression levels in different ALS-FTD patient tissues including cerebellum (CB), frontal cortex (FCX), anterior horn (AH), and laser-captured spinal motor neurons enriched data were then examined. These RNA expression datasets were obtained from publicly available sources (GSE18920) and (GSE67196) (Rabin, S. J. et al., 2010, Human molecular genetics, 19, 313-328; Prudencio, M. et al., 2015, Nat. Neurosci., 18, 1175- 1182). In Figure 9, the expression levels of candidate SA genes in ALS-FTD patients and controls, show several significant differences in gene expression levels between the two groups, which indicate their potential involvement in the pathogenesis of ALS-FTD. Notably, increased expression of these SA genes, such as p21 and PLAUR, is consistently observed in both sporadic ALS (sALS) and C9orf72 repeat expansion (C9-NRE)-linked ALS/FTD compared to controls. These findings further support the role of senescence pathways in disease progression in patients.

Immunofluorescence (IF) microscopy imaging and western blotting were performed to assess the protein levels of uPAR in tissue samples collected from ALS patients and controls. The western blot results showed significantly elevated levels of uPAR in the mid motor cortex of ALS patients compared to controls, while the disease-spared occipital cortex showed lower protein levels. (Figure 10A). This differential expression suggests a potential role of uPAR in the disease pathology specific to disease affected area(s) in ALS and ALS/FTD. As seen at the transcript level (CDKN2A), patients carrying the C9-NRE mutation showed elevated levels of pl6 protein in the motor and frontal cortices compared to control (Figure 10B). IF microscopy images also showed increased levels of uPAR along with increased levels of activated microglia in ALS/FTD patients, indicating a potential relationship between uPAR expression and microglial activation in the context of the diseases (Figure 10C). IF images further revealed elevated levels of uPAR in microglia and astrocytes, and these cells were frequently found in regions that associated with high uPAR levels (Figure 10D). This observation suggests that microglia and astrocytes may contribute to the increased uPAR expression observed in ALS/FTD patients.

Overall, these results indicate increased expression of SA genes and markers in both in vitro and in vivo ALS and ALS/FTD models as well as in the CNS of ALS/FTD patients. These findings shed light on the potential involvement of cellular senescence in the pathogenesis of ALS and ALS/FTD and provided valuable insights into the molecular and cellular mechanisms that can contribute to the progression of neurodegenerative diseases. Additional data are shown in the following examples. Example 7: uPAR, a valuable cell surface marker to develop CAR-T-, is unregulated in ALS- SOD1 mice as disease progresses uPAR is the surface receptor of the urokinase-type plasminogen activator (uPA), the extracellular matrix protein that, upon binding to uPAR, promotes remodeling of extracellular matrixes and the regulation of fibrinolysis in the bloodstream. In physiological conditions, uPAR is primarily expressed in a subset of myeloid cells and, at low levels, in bronchial epithelium, while its expression in all other cells in negligible (Amor, C. et al., 2020, Nature 583, 127-132). However, increased expression of uPAR has been described in senescence cells across different cell types including neurons and neuronal cells as well as during inflammation (Amor, C. et al., 2020, Nature 583, 127-132; Mehra, A., et al., 2016, Biochim Biophys Acta 1862, 395-402; Baart, V. M. et al., 2020, EJNMMI Res 10, 87). In fact, in recent years, research showed that the function of uPA/uPAR goes beyond the extracellular matrix and fibrinolysis. It has been reported that uPA/uPAR influence several cellular and molecular factors of inflammation, making it a key player in the regulation of neuroinflammation, including microglial activation (Baart, V. M. et al., 2020, EJNMMI Res 10, 87). uPAR expression in senescence and physiological aging, has been well studied in recent years, and uPAR is a well-defined marker of senescence and the ideal cell-surface marker for CAR-T cell-based therapy approaches (that require a cell-surface protein target) against aging. Indeed, a recent study showed that uPAR-positive senescent cells accumulate during aging in mice and that a senolytic therapy based on CAR-T cells targeting uPAR- positive senescent cells, prevents age-related metabolic dysfunction(s) and improves exercise capacity (Amor, C. et al., 2023, Research Square, 3385749).

Since Plaur (Ximerakis, M. et al., 2019, Nat Neurosci 22), the gene that encodes uPAR, was found to be expressed in ALS patients and in the mutSODl mouse model of ALS (Gias, M. et al., 2007, Exp Neurol 207, 350-356), the temporal gene expression of microglial markers and how they relate to uPAR? Plaur in the SOD1^G93A (mutSODl) was investigated. An initial principal component analysis (PCA) was conducted to impartially compare gene expression over time between control and mutSODl (Figure 11 A) revealing distinct temporal patterns of microglial gene expression alterations in the mutSODl mice in contrast to controls. Further analysis of these temporal changes for various microglial surface markers, including uPAR, showed a significant increase in uPAR/Plaur transcript levels and many of these markers following disease onset (Figure 1 IB). Spatial transcriptomic data from the ALS-st.nygenome.org database was then leveraged to investigate disease-linked temporal expression patterns of candidate microglia genes in mutSODl mice compared to age-matched controls (Figure 11C). The spatial transcriptomic dataset provides spatially resolved gene expression information, allowing identification of specific anatomical regions in the spinal column where candidate microglia-specific genes are differentially expressed in mutSODl mice compared to age-matched controls. Although, uPAR levels were not reported in these data, robust disease-linked temporal expression of other markers genes indicates that activated and harmful microglia play a significant role in disease progression. uPAR has minimal RNA expression in healthy human organoids and in adult mouse brain but its RNA levels increase in tissues of ALS and ALS-FTD patients

It was then determined if uPAR had minimal expression in the CNS of healthy in vitro and in vivo models. This is important at two levels: (a) when designing CAR-T immunotherapy, it is important to have minimal expression of the target of interest in cells other than the cells of interest because high or unwanted expression of the target protein could lead to unintended gross elimination of cells; (b) it is also important to confirm the cell specificity of the expression of the target protein. Therefore, single nuclei RNAseq datasets from the Tabula Muris database were first analyzed and it was determined that in mice, expression of uPAR/P/cw is low (Figure 12A), with some expression observed in endothelial cells at baseline. These data were confirmed in a recent publication showing that uPAR/P/cw expression is almost undetectable in young mice with a baseline expression in endothelial cells, but then it increases with age specifically in microglia cells (Ximerakis, M. et al., 2019, Nat Neurosci 22, 1696-1708). Single cell RNAseq was then performed on the human organoids generated from healthy and C9-NRE patient-derived iPSCs, and cells were clustered and unbiasedly annotated using SEURAT. As seen in Figure 12B, uPAR/P/cw expression is minimally expressed or undetectable across all the cell types present in human organoids, with the caveat that organoids do not possess detectable levels of microglia. RNA expression levels of uPAR in different ALS-FTD patient tissues including cerebellum (CB), frontal cortex (FCX), anterior horn (AH), and laser-captured enrichment of spinal motor neurons (sMN) from the AH were then examined. The RNA expression datasets were obtained from publicly available sources (GSE 18920) and (GSE67196) (Rabin, S. J. et al., 2010, Human molecular genetics 19, 313-328; Prudencio, M. et al., 2015, Nat. Neurosci. 18, 1175-1182). In Figure 13, it is evident that there is increased expression of uPAR RNA in both sporadic ALS (sALS) and C9orf72 repeat expansion (C9-NRE)-linked ALS/FTD tissues compared to controls. Interesting, expression levels correlated with the areas affected by disease in each genotype. Importantly, in sALS patients, the increased uPAR levels in the anterior horn of the spinal cord are not due to elevated expression in motor neurons, since RNA sequencing analysis of laser-captured motor neurons showed no difference in uPAR RNA levels between controls and sALS patients.

Taken all together, these data confirm that uPAR is potentially the ideal candidate target to develop gene-edited chimeric antigen receptor (CAR)-T cells, provided that we see a correlation between mRNA levels and the corresponding protein levels in tissues and cells of patients. uPAR protein levels are elevated in ALS and ALS-FTD patients specifically in activated microglia

Therefore, to confirm that the increase in uPAR mRNA levels observed in patients also correlates with higher protein levels, western blotting was first performed in tissue samples collected from the ALS patients and controls. The western blot results showed significantly elevated levels of uPAR in the mid motor cortex of ALS patients compared to controls, while the disease-spared occipital cortex showed lower protein levels (Figure 14A). This differential expression suggests a potential role of uPAR in the disease pathology specific to disease affected area(s) in ALS and ALS/FTD. Immunofluorescence (IF) in tissues from patients with ALS, ALS-FTD or control was then performed with the goal to determine the cellular localization of uPAR in disease starting first with the mid motor cortex, as in the western blot analysis, and a variety of patients with different genotypes was used, including SOD 1 -ALS, C90rf72- ALS/FTD, and sALS, all aged-matched (between 55 and 60 years of age). However, since it is known that uPAR expression is associated with senescence and increases in aging (Figure 12A) (Amor, C. et al., 2020, Nature 583, 127-132; Amor, C. et al., 2023, Research Square, 3385749; Ximerakis, M. et al., 2019, Nat Neurosci 22, 1696-1708), a 95-year-old non-neurological disease patient was used as a control. The goal was to examine whether there was a disease-specific “uPAR-response” and, specifically, a disease specific cellular response triggering increased uPAR expression irrespective of aging. The tissues were stained for neurons (not shown), microglia, and astrocytes (reactive-GFAP -positive astrocytes). Microscopy images showed increased levels of uPAR along with increased levels of activated microglia (as judged by ameboid-like morphology) in ALS/FTD patients, indicating a potential relationship between uPAR expression and microglial activation in the context of the disease (Figure 14B). IF images further revealed that in patients uPAR levels are specifically elevated in microglia with no staining in astrocytes (Figure 14B). Interestingly, in the 95-year-old control, the number of ameboid activated microglia was low and, although a slight increase in uPAR expression in resting microglia (0.03%) was identified, uPAR was practically undetectable in activated microglia (Figure 14C), confirming the increase of uPAR expression in microglia is disease specific. Consistent with this notion, in ALS patients, uPAR expression and the number of activated microglia increased (Figure 14C) with the majority of uPAR expressed in activated microglia with percentage of uPAR+activated microglia as follows: 77.02% in sALS; 74.6% in SOD1-ALS, and 41.73% in C9-ALS/FTD. Preliminary observation with multiplex fluorescence imaging microscopy, which allows cell and context-specific evaluation of protein expression, suggests uPAR enrichment in microglia in the spinal cord of SOD 1 -ALS patient (Figure 15). Together, these data clearly indicate that (a) uPAR is a surface maker of activated microglia in disease; (b) that its protein expression is low in normal tissue, and it is differentially expressed in the target microglia cells.

Therefore, uPAR is the ideal target to begin the design of a CAR-T cell therapy to specifically eliminate activated microglia in disease with minimal or no unintended off-target effects.

First generation human uPAR-directed CAR construct to specifically target human uPAR is successful in killing uPAR-expressing lung cancer cells

Human uPAR binders were screened, including anti -uPAR antibodies and the uPAR ligand, uPA, for successful binding and induction of CAR-T cell killing using the anti- FITC CAR-T system designed for binder screening (Figure 16). As seen in Figure 16, anti- FITC CAR-T cells kill human lung cancer cells, providing proof-of-concept efficacy data and identifying candidate binders for CAR design. Candidate antibodies were sequenced for CAR design and construction of gene-edited ZFNy-knock-out (KO) CAR-T cells, which cannot initiate inflammatory cascades (Bailey, S. R. et al., 2022, Blood Cancer Discov 3, 136-153), therefore making them more specific and reducing the unwanted off-target effects and achieving enhanced therapeutic effectiveness.

Example 8: uPAR binder screening and validation

Human DLD1 and NCI-H1792 cells, which are known to express uPAR, were used to validate the human uPAR binder (Figure 17). The cytotoxicity assay confirmed that anti-uP AR antibody induced specific, dose, and time-dependent killing. The results indicated killing about 12-14 hours after CAR-T cell addition. The human microglial cell line HMC3 was used to confirm human microglial killing (Figure 18). The cytotoxicity assay confirmed that anti -uP AR antibody induced specific, dose, and time-dependent killing. The results indicated killing about 16 hours after CAR-T cell addition. Human uPAR CAR constructs were designed (Figure 19). Features of the constructs are shown in Table 1.

The efficacy of CAR-T cells targeting microglia expressing uPAR is established through a surrogate approach (Figure 20).

Table 1. Anti-Human uPAR CAR constructs features

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

WHAT IS CLAIMED IS:

1. A method of reducing the number of senescent cells, comprising contacting one or more senescent cells with a composition comprising one or more chimeric antigen receptor (CAR)-T cells, wherein the one or more CAR-T cells target one or more cell surface proteins associated with senescence.

2. The method of claim 1, wherein the senescent cells are central nervous system cells.

3. The method of claim 2, wherein the central nervous system cells are glial cells.

4. The method of claim 2, wherein the CAR-T cells have been modified to not express one or more interferons.

5. The method of claim 4, wherein the one or more interferons are selected from the group consisting of interferon alpha 1 (IFNA1), interferon alpha 2 (IFNA2), interferon alpha 4 (IFNA4), interferon alpha 5 (IFNA5), interferon alpha 6 (IFNA6), interferon alpha 7 (IFNA7), interferon alpha 8 (IFNA8), interferon alpha 10 (IFNA10), interferon alpha 11 (IFNA11P), interferon alpha 12 (IFNA12P), interferon alpha 13 (IFNA13), interferon alpha 14 (IFNA14), interferon alpha 16 (IFNA16), interferon alpha 17 (IFNA17), interferon alpha 20 (IFNA20P), interferon alpha 21 (IFNA21), interferon alpha 22 (IFNA22P), interferon beta 1 (IFNB1), interferon gamma (IFNG), interferon kappa (IFNK), interferon lambda 1 (IFNL1), interferon lambda 2 (IFNL2), interferon lambda 3 (IFNL3), interferon nu 1 (IFNNP1), interferon omega 2 (IFNWP2), interferon omega 4 (IFNWP4), interferon omega 5 (IFNWP5), interferon omega 9 (IFNWP9), interferon omega 15 (IFNWP15), interferon omega 18 (IFNWP18), interferon omega 19 (IFNWP19), and interferon omega 1 (IFNW1).

6. The method of claim 5, wherein the interferon is interferon gamma (IFNG).

7. The method of claim 2, wherein the CAR of the CAR-T cells targets one or more cell surface proteins associated with senescence selected from the group consisting ofCD36, oxidized vimentin, and ICAM-1 urokinase plasminogen activator receptor (uPAR), DEP domain-containing protein 1A (DEP1), linker for activation of T-cells family member 2 (NT AL), TBC1 domain family member 10A (EBP50), syntaxin-4 (STX4), vesicle-associated membrane protein 3 (VAMP3), armadillo repeat-containing X-linked protein 3 (ARMCX3), beta-2-microglobulin (B2MG), glutathione S-transferase LANCL1 (LANCL1), 5’-3’ exonuclease PLD3(PLD3), vacuolar protein sorting-associated protein 26 (VPS26A), dipeptidyl peptidase 4 (DPP4), secretory carrier-associated membrane protein 4 (SCAMP4), tumor necrosis factor receptor super family member 10D (TNFRSF10D/CD264), neurogenic locus notch homolog protein 1 (N0TCH1), neurogenic locus notch homolog protein 3 (N0TCH3), cluster of differentiation 36 (CD36), oxidized vimentin, and intercellular adhesion molecule 1 (ICAM-1).

8. The method of claim 7, wherein the cell surface protein associated with senescence is uPAR.

9. The method of claim 8, wherein the antigen binding domain of the CAR for uPAR comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, an HCDR3 comprising the amino acid sequence of SEQ ID NO:3, an LCDR1 comprising the amino acid sequence of SEQ ID NO:4, an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

10. The method of claim 9, wherein the antigen binding domain of the CAR for uPAR comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8.

11. The method of claim 9, wherein the antigen binding domain of the CAR for uPAR comprises an scFv; wherein the scFv is in a heavy-to-light (H2L) orientation or light-to-heavy (L2H) orientation; wherein the scFv in the H2L orientation comprises the amino acid sequence of

SEQ ID NO: 9; wherein the scFv in the L2H orientation comprises the amino acid sequence of SEQ ID NO: 10.

12. The method of claim 9, wherein the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11 and 13.

13. The method of claim 9, wherein the CAR comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 11; a nucleotide sequence having at least 90% identity to SEQ ID NO: 12; the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 13; a nucleotide sequence having at least 90% identity to SEQ ID NO: 14; and the nucleotide sequence of SEQ ID NO: 14.

14. The method of claim 1, wherein the method further comprises contacting the cells with one or more inhibitors of IL-6.

15. The method of claim 14, wherein the one or more inhibitors of IL-6 are selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab.

16. The method of claim 15, wherein the inhibitor of IL-6 is tocilizumab.

17. A method of reducing the number of senescent cells in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising one or more CAR-T cells, wherein the one or more CAR-T cells target one or more cell surface proteins associated with senescence.

18. The method of claim 17, wherein the senescent cells are central nervous system cells.

19. The method of claim 18, wherein the central nervous system cells are glial cells.

20. The method of claim 17, wherein the CAR-T cells have been modified not to express one or more interferons.

21. The method of claim 20, wherein the one or more interferons are selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA11P, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1.

22. The method of claim 21, wherein the interferon is IFNG.

23. The method of claim 17, wherein the method further comprises contacting the cells with one or more inhibitors of IL-6.

24. The method of claim 23, wherein the one or more inhibitors of IL-6 are selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab.

25. The method of claim 24, wherein the inhibitor of IL-6 is tocilizumab.

26. The method of claim 18, wherein the one or more cell surface proteins associated with senescence are selected from the group consisting of uPAR, DEP1, NT AL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, and ICAM-1.

27. The method of claim 26, wherein the cell surface protein associated with senescence is uPAR.

28. The method of claim 27, wherein the antigen binding domain of the CAR for uPAR comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, an HCDR3 comprising the amino acid sequence of SEQ ID NO:3, an LCDR1 comprising the amino acid sequence of SEQ ID NO:4, an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

29. The method of claim 28, wherein the antigen binding domain of the CAR for uPAR comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8.

30. The method of claim 28, wherein the antigen binding domain of the CAR for uPAR comprises an scFv; wherein the scFv is in a heavy -to-light (H2L) orientation or light-to-heavy (L2H) orientation; wherein the scFv in the H2L orientation comprises the amino acid sequence of SEQ ID NO: 9; wherein the scFv in the L2H orientation comprises the amino acid sequence of SEQ ID NO: 10.

31. The method of claim 28, wherein the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11 and 13.

32. The method of claim 28, wherein the CAR comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 11; a nucleotide sequence having at least 90% identity to SEQ ID NO: 12; the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 13; a nucleotide sequence having at least 90% identity to SEQ ID NO: 14; and the nucleotide sequence of SEQ ID NO: 14.

33. A method of treating a neurodeg enerative disease in a subject in need thereof comprising reducing the number of senescent central nervous system cells; wherein reducing the number of senescent cells comprises the step of administering to the subject a composition comprising one or more CAR-T cells, wherein the one or more CAR-T cells target one or more cell surface proteins associated with senescence.

34. The method of claim 33, wherein the CAR-T cell has been modified not to express one or more interferons.

35. The method of claim 34, wherein the one or more interferons are selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA11P, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1.

36. The method of claim 35, wherein the interferon is IFNG.

37. The method of claim 33, wherein the method further comprises contacting the cells with one or more inhibitors of IL-6.

38. The method of claim 37, wherein the one or more inhibitors of IL-6 are selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab.

39. The method of claim 38, wherein the inhibitor of IL-6 is tocilizumab.

40. The method of claim 39, wherein the tocilizumab is administered before, concurrently with, or after administration of the one or more CAR-T cells.

41. The method of claim 33, wherein the one or more cell surface proteins associated with senescence are selected from the group consisting of uPAR, DEP1, NT AL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, NOTCH1, NOTCH3, CD36, oxidized vimentin, and ICAM-1.

42. The method of claim 41, wherein the cell surface protein associated with senescence is uPAR.

43. The method of claim 42, wherein the antigen binding domain of the CAR for uPAR comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO:2, an HCDR3 comprising the amino acid sequence of SEQ ID NO:3, an LCDR1 comprising the amino acid sequence of SEQ ID NO:4, an LCDR2 comprising the amino acid sequence of SEQ ID NO:5, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:6.

44. The method of claim 43, wherein the antigen binding domain for uPAR comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO: 8.

45. The method of claim 43, wherein the antigen binding domain for uPAR comprises an scFv; wherein the scFv is in a heavy -to-light (H2L) orientation or light-to- heavy (L2H) orientation; wherein the scFv in the H2L orientation comprises the amino acid sequence of SEQ ID NO: 9; wherein the scFv in the L2H orientation comprises the amino acid sequence of SEQ ID NO: 10.

46. The method of claim 43, wherein the CAR comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs: 11 and 13.

47. The method of claim 43, wherein the CAR comprises an amino acid sequence encoded by a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: a nucleotide sequence that encodes the amino acid sequence of SEQ ID

NO: 11; a nucleotide sequence having at least 90% identity to SEQ ID NO: 12; the nucleotide sequence of SEQ ID NO: 12; a nucleotide sequence that encodes the amino acid sequence of SEQ ID

N0: 13; a nucleotide sequence having at least 90% identity to SEQ ID NO: 14; and the nucleotide sequence of SEQ ID NO: 14.

48. The method of claim 47, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

49. A composition for reducing the number of senescent cells comprising a CAR-T cell; wherein the CAR-T cell targets a cell surface protein associated with senescence; and wherein the CAR-T cell has been modified not to express one or more interferons.

5

50. The composition of claim 49, wherein the composition further comprises a one or more inhibitors of IL-6.

51. The composition of claim 50, wherein the one or more inhibitors of IL-6 are selected from the group consisting of tocilizumab, siltuximab, sarilumab, olokixumab, elsilimomab, clazakizumab, sirukumab, and levilimab.

52. The method of claim 51, wherein the inhibitor of IL-6 is tocilizumab.

53. The composition of claim 49, wherein the one or more interferons are selected from the group consisting of IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA1 IP, IFNA12P, IFNA13, IFNA14, IFNA16, IFNA17, IFNA20P, IFNA21, IFNA22P, IFNB1, IFNG, IFNK, IFNL1, IFNL2, IFNL3, IFNNP1, IFNWP2, IFNWP4, IFNWP5, IFNWP9, IFNWP15, IFNWP18, IFNWP19, and IFNW1.

54. The composition of claim 53, wherein the interferon is interferon gamma (IFNG).

55. The composition of claim 49, wherein the one or more cell surface proteins associated with senescence are selected from the group consisting of uPAR, DEP1, NTAL, EBP50, STX4, VAMP3, ARMCX3, B2MG, LANCL1, PLD3, VPS26A, DPP4, SCAMP4, TNFRSF10D/CD264, N0TCH1, N0TCH3, CD36, oxidized vimentin, and ICAM-1.

56. The composition of claim 55, wherein the cell surface protein associated with senescence is uPAR.

57. The composition of claim 49, wherein the composition further comprises one or more selected from the group consisting of pharmaceutically acceptable carriers, antioxidants, chelating agents, adjuvants, and preservatives.

58. The composition of claim 49, wherein the composition further comprises one or more senolytic compounds.

59. The composition of claim 58, wherein the one or more senolytic compounds are selected from the group consisting of dasatinib and quercetin, ABT-263, ABT-737, A1331852 and Al 155463, 17-AAG, geldanamycin, fisetin, piperlongumine, quercetin-3-D-galactose, UBX0101, panobinostat, F0X04-DRI peptide, and klotho.

60. The composition of claim 59, wherein the one or more senolytic compounds are loaded in the CAR-T cell.