Restoration of p53 activity in immune cells

Abstract

The present disclosure provides a new approach to cancer immunotherapy, focusing on restoring p53 function in immune cells with TP53 mutations. This strategy seeks to counteract exhaustion and bolster the anti-tumor activity of immune cells, thereby enhancing the efficacy of current immune cell therapies for cancer. The present disclosure provides methods for restoring anti-tumor activity of an immune cell, including contacting the immune cell with a p53 reactivator, wherein the immune cell includes a TP53 gene mutation.

Description

RESTORATION OF P53 ACTIVITY IN IMMUNE CELLSBACKGROUND OF THE INVENTIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 654,579, filed May 31, 2024. The content of the prior application is considered part of an is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0002] The present disclosure generally relates to methods for restoring p53 activity in immune cells and more specifically to restoring p53 activity in cells that have TP53 mutations, thereby reactivating their anti-tumor functions.BACKGROUND INFORMATION

[0003] The transcription factor p53, encoded by the TP53 gene, is pivotal as a tumor suppressor, activated in response to cellular stressors such as DNA damage. This activation orchestrates the regulation of key genes involved in DNA repair, cell differentiation, cell cycle arrest, senescence and programmed cell death (apoptosis). Mutations in the TP53 gene are frequent in tumor cells, leading to diminished DNA binding capacity and, as a result, a reduced ability of p53 to act as tumor suppressor. In addition, p53 mutations can exert dominant-negative effect, further impairing any remaining functional p53.

[0004] These mutations endow the mutant p53 protein with oncogenic properties. p53 functions to either halt the cell cycle or induce apoptosis in response to stressors such as DNA damage, hypoxia, and oncogene activation. Both p53 -induced cell cycle arrest and apoptosis play roles in its tumor suppression capabilities. A significant number of human tumors produce a "mutant" p53 protein due to TP53 mutations, highlighting the critical need to restore wild-type p53 activity to suppress tumor growth. Tumor cells are especially sensitive to p53 reactivation, primarily because they are predisposed to apoptosis through oncogene activation, and mutant p53 proteins accumulate in high levels within them. Thus, restoring wild-type p53 in these cells, which are abundant and presumably in an "activated" state, could trigger substantial apoptotic responses in these already sensitive tumor cells, while sparing normal cells that maintain low or undetectable levels of p53, unless they are involved in processes that require p53.

[0005] Additionally, p53 modulates the innate immune system by secreting factors that influence macrophage functions to inhibit tumor growth. In the context of cancer, dysfunctional p53 impairs the activity and recruitment of T and myeloid cells, facilitating immune evasion. Utilizing immune cells to combat cancer is a potent strategy, largely dependent on the efficacy of tumor-reactive T cells. However, their cytotoxicity is often hindered in the tumor microenvironment, where interactions among myeloid-derived suppressor cells, macrophages, dendritic cells, and regulatory T cells intensify anti-tumor immunity. While TP53 mutations are prevalent in various tumor cells, they are rarely observed in immune cells, such as CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, macrophages, leaving the effects of mutant p53 protein in immune cells somewhat ambiguous.

[0006] In Acute Myeloid Leukemia (AML) and myelodysplastic syndromes (MDS), patients with mutated TP53 (TP53m) carry an extremely poor prognosis, with intensive or targeted therapies, with a median overall survival rate of merely 6.5 months, in stark contrast to 33.6 months for individuals with wild-type TP53 AML, depending on established risk factors. These challenges underscore the critical need for novel and effective treatment strategies for TP53m AML, highlighting the imperative for innovative approaches in addressing this particularly resistant subset of AML.

[0007] While immunotherapy has significantly improved the outcomes for patients with lymphomas and myelomas and has even replaced chemotherapy in certain B-cell lymphoid malignancies, the translation of these successes into the treatment of AML has been challenging. The obstacles to successful immunotherapies in AML are linked to several factors, including genetic and clonal heterogeneity, a scarcity of viable leukemic targets, and an immunosuppressive tumor microenvironment. In addition, little is known about mutations in immune cells. Previous studies have identified mutations in T-cells, such as TET2, IDH1 / 2, and DNMT3a, primarily considering these as indicators of clonal hematopoiesis rather than an key contributor to leukemia pathobiology or potential therapeutic targets.

[0008] The efficacy of immunotherapies and to a degree of chemo-and targeted therapies largely depends on the functional response of immune cells, including the ability of T cells and NK cells to eliminate cancer cells. There is a need to investigate the mutations in these immune cells, which could unveil novel insights into the mechanisms by which they impair the immune system's capacity to combat cancer, potentially unveiling new therapeutic strategies to enhance the efficacy of immunotherapy in AML patients.SUMMARY OF THE INVENTION

[0009] The present disclosure builds on the pivotal discovery that in patients with acute myeloid leukemia (AML) who have TP53 mutations, their autologous bone marrow-resident CD4+ and CD8+ T cells also exhibit corresponding p53 mutations, a mechanism by which p53 mutations may impair immune cell function against AML cells, potentially explaining the failure of immunotherapies in these patients. Leveraging this discovery, the present disclosure provides methods for reactivating the anti-tumor activity of TP53 mutant immune cells by restoring their wild-type TP53 functionality, offering a new approach to cancer treatment.

[0010] In one embodiment, the present disclosure provides a method for restoring anti-tumor activity of an immune cell, including: contacting the immune cell with a p53 reactivator, wherein the immune cell includes a TP53 gene mutation, thereby restoring the anti-tumor activity of the immune cell.

[0011] In some aspects, the immune cell is in a subject.

[0012] In some aspects, the subject has a cancer including a TP53 mutation.

[0013] In some aspects, the immune cell of the subject includes the TP53 mutation.

[0014] In some aspects, the TP53 mutation includes Y220C, P151A, R241G, P33R, K132N, or any combination thereof.

[0015] In some aspects, the immune cell is selected from a CD4+ T cell, a CD8+ T cell, an NK cell, a dendritic cell, a macrophage, or any combination thereof.

[0016] In some aspects, the immune cell is a bone marrow-resident CD4+ T cell or a bone marrow-resident CD8+ T cell.

[0017] In some aspects, the immune cell is a blood-circulating CD4+ T cell or a bloodcirculating CD8+ T cell.

[0018] In some aspects, the p53 reactivator is selected from a small molecule, a nucleic acid, a peptide, a protein, a saccharide, a lipid, or any combination thereof.

[0019] In some aspects, the p53 reactivator decreases exhaustion markers in TP53 mutant T cell.

[0020] In some aspects, the exhaustion markers of the TP53 mutant T cell are selected from PD- 1, TIM3, LAG3, TIGIT, or any combination thereof.

[0021] In some aspects, the TP53 mutant T cell expresses CD39.

[0022] In some aspects, the CD39 is an activation and an exhaustion marker of the TP53 mutant T cell.

[0023] In some aspects, the subject has an acute myeloid leukemia (AML).

[0024] In some aspects, an AML cancer cell from the subject has the same TP53 mutation as the immune cell.

[0025] In another embodiment, the present disclosure provides a method for enhancing the CAR-T cell therapy efficacy of cancer treatment in a subject with a cancer including a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the CAR-T cell, thereby enhancing the CAR-T cell therapy efficacy of cancer treatment in the subject.

[0026] In some aspects, the CAR-T cell includes the TP53 mutation.

[0027] In some aspects, the TP53 mutation includes Y220C, P151A, R241G, P33R, K132N, or any combination thereof.

[0028] In some aspects, the p53 reactivator is selected from a small molecule, a nucleic acid, a peptide, a protein, a saccharide, a lipid, or any combination thereof.

[0029] In some aspects, the p53 reactivator is the p53 reactivator is APR-246 (eprenetapopt), COTI-2, rezatapopt (PC14586), PRIMA-1, PK7088, andNSC59984, or a combination thereof. In one aspect, the p53 reactivator is rezatapopt (PC 14586).

[0030] In some aspects, the p53 reactivator decreases exhaustion markers in TP53 mutant CAR- T cell.

[0031] In some aspects, the exhaustion markers of theTP53 mutant CAR-T cell are selected from PD-1, TIM3, LAG3, TIGIT, or any combination thereof.

[0032] In some aspects, the TP53 mutant CAR-T cell expresses CD39.

[0033] In some aspects, the CD39 is an activation and an exhaustion marker of the TP53 mutant CAR-T cell.

[0034] In some aspects, the subject has an acute myeloid leukemia (AML).

[0035] In some aspects, an AML cancer cell from the subject has the same TP53 mutation as the CAR-T cell.

[0036] In another embodiment, the present disclosure provides a method for treating cancer in a subject, wherein the cancer includes a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in an immune cell of the subject, thereby treating the cancer in the subject.

[0037] In some aspects, the immune cell is selected from a CD4+ T cell, a CD8+ T cell, an NK cell, a dendritic cell, a macrophage, or any combination thereof.

[0038] In another embodiment, the present disclosure provides a method for preventing cancer in a subject, wherein the subject has a TP53 mutation, including: administering a p53 reactivatorto the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the subject, thereby preventing the cancer in the subject.

[0039] In another embodiment, the present disclosure provides a method for enhancing the CAR-NK cell therapy efficacy of cancer treatment in a subject with a cancer including a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the CAR-NK cell, thereby enhancing the CAR-NK cell therapy efficacy of cancer treatment in the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The new features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0041] FIG. 1A-1E are graphs illustrating single cell transcriptomics, proteomics, and secretomics in TP53 WT and TP53 mutant. FIG. 1A is a graph illustrating dimensional reduction using Uniform Manifold Approximation and Projection (UMAP). FIG. IB is a graph illustrating exhaustion score in naive T cells, cytotoxic T cells, and NK cells. FIG. 1C is a graph illustrating expression of exhaustion markers, TIGIT, PD-1, LAG-3, and TIM-3 in TP53 mutant samples compared to TP53 WT. FIG. ID is a graph illustrating expression of PD-1, TIGIT, and TIM-3 in NK cells from TP53m AML patients relative to both TP53wt AML patients and healthy donors. FIG. IE is a graph illustrating polyfunctional strength index (PSI) in both CD4+ and CD8+ T cells from TP53m AML patients.

[0042] FIG. 2A-2F are graphs illustrating that TP53 mutations occur in T and NK cells in AML patients and alter their phenotype. FIG. 2A is a graph illustrating that TP53 mutations were widely distributed across different immune cell lineages using integration of single-cell DNA sequencing data with UMAP clustering. FIG. 2B is a graph illustrating a genotype analysis of BM-resident T cells from AML patients with TP53 mutations using Tapestri technology. FIG. 2C is a graph illustrating frequency of TP53 mutant T and NK cells quantified across four AML patients at the single-cell resolution level. FIG. 2D is a graph illustrating that TP53mut T andNK cells displayed phenotypic alterations compared to their TP53 wild-type counterparts. FIG. 2E is a graphillustrating that CD4, CD8, and NK cells were distinctly separated in the UMAP, and TP53mt T and NK cells formed unique clusters. FIG. 2F is a graph illustrating expression of markers such as CD44, CD69, and CD71 in TP53 mutant NK cells compared to TP53 WT NK cells.

[0043] FIG. 3A-3B are graphs illustrating the detection of TP53 mutations in subsets of CD4+ and CD8+ BM-resident T cells of AML patients using digital-droplet PCR (ddPCR) on FACS- sorted T cells. FIG. 3 A is a graph illustrating blast purity and T cell purity. FIG. 3B is a graph illustrating TP53 mutations were detected in 52% of the analyzed T cells

[0044] FIG. 4A-4I are graphs illustrating the impact of mutant p53 in CAR-T cells phenotype. FIG. 4A is schematic illustrating control CAR-T cells generated without overexpression of mutant p53, serving as a baseline comparison. FIG. 4B is a graph illustrating CAR expression on over 85% of the transduced T cells. FIG. 4C is a graph illustrating that p53mt CAR-T cells proliferated more rapidly than control CAR-T cells under normoxic. FIG. 4D is a graph illustrating UMAP analysis of the phenotypic profile of the CAR-T cells characterized using CyTOF analysis. FIG. 4E is a graph illustrating expression levels of PD-1, LAG-3, TIM-3, TIGIT, and CD39 in both CD4 and CD8 p53mt CAR-T cells compared to control CAR-T cells. FIG. 4F is a schematic illustrating fresh Molml3 cells that were replenished every 48 hours which lacked mutant p53 expression. FIG. 4G is a graph illustrating UMAP clustering of rechallenged p53mt CAR-T cells. FIG. 4H is a graph illustrating Phenograph clustering analysis across 28 CAR-T cell samples collected at seven different time points. FIG. 41 is a graph illustrating the temporal evolution of exhaustion marker expression.

[0045] FIG. 5 is a graph illustrating an upregulation of CD27.

[0046] FIG. 6A-6G illustrate that mutant p53 impairs CAR-T cells anti-tumor function in vitro. FIG. 6A is a graph illustrating an incucyte Live-Cell analysis system to evaluate the cytotoxic capacity of p53mt CAR-T cells against their target cells. FIG. 6B is a graph illustrating a singlecell cytokine secretion assay was conducted to assess the polyfunctionality of these cells under stimulation. FIG. 6C is a graph illustrating production of TNF-a, IFN-y, and IL-2 in p53mt CAR- T cells using a flow cytometry-based approach. FIG. 6D is a schematic illustrating Timelapse Imaging Microscopy in Nanowell Grids (TIMING) to assess the functionality of p53 mutant (p53mt) CAR-T cells compared to control CAR-T cells. FIG. 6E are graphs illustrating time required for CAR-T cells to establish contact with tumor cells (tSeek). FIG. 6F are images illustrating the duration of contact between effector and target cells (tContact) for p53mt CAFUT cells compared to control CAR-T cells. FIG. 6G is a graph illustrating survival proportions of Molml3 tumor cells in 1:1 effector-to-target (E) ratio over time.

[0047] FIG. 7 is graph illustrating that CAR-T cells with the p53-R175H mutation exhibited the exhaustion-associated features and reduced cytotoxicity as the other p53 mutants.

[0048] FIG. 8A-8D illustrate that mutant P53 impairs CAR-T cell and eliminate AML cells in vivo. FIG. 8 A is a schematic illustrating mice injection with luciferase-transduced PDX cells, treatment with p53 mutant (p53mt) CAR-T cells, control CAR-T cells, or empty vector T cells via tail vein injection and tissue collection for bioluminescence imaging (BLI). FIG. 8B is a graph illustrating tumor burden using bioluminescence imaging (BLI). FIG. 8C is a graph illustrating quantification of AML cells by measuring human CD45 (hCD45) levels using flow cytometry in mouse blood at various time points. FIG. 8D is a graph illustrating survival analysis in mice treated with p53mt CAR-T cells compared to mice treated with control CAR-T cells.

[0049] FIG. 9 is a graph illustrating expression of PD-1, TIM-3, TIGIT, LAG-3, CD39, and CTLA-4 in CAR-T cells harvested from the bone marrow of p53mut-CAR-T cells treated mice compared to CAR-T cells from control animals.

[0050] FIG. 10A-10I are graphs illustrating the efficacy of the p53 reactivator in T cells. FIG. 10A is a graph illustrating that CD4 and CD8 T-cells exhibited reduced levels of mutant p53 in p53mt CAR-T cells following treatment with the p53 reactivator. FIG. 10B is a graph illustrating a time-dependent reduction in mutant p53 protein levels. FIG. 10C is a graph illustrating a proteinlevel differential expression analysis comparing mutant p53 -expressing CAR T-cells before and after treatment with a p53 reactivator. FIG. 10D is a graph illustrating a real-time cell killing assay performed using the InCuCyte system. FIG. 10E is a graph illustrating changes in the cellularproteomic landscape of mutant p53 CAR-T cells before and after p53 reactivation. FIG. 10F is a graph illustrating heatmap analysis of cluster profile. FIG. 10G is a graph illustrating phenotypic analysis of these clusters for mutant p53 CAR-T cells before and after p53 reactivaton. FIG. 10H is a graph illustrating consolidation of five meta-clusters based on their shared phenotypic characteristics in mutant p53 CAR-T cells before and after p53 reactivation. FIG. 101 is a graph illustrating exhaustion levels in p53mt CAR-T cells following p53 reactivator treatment.

[0051] FIG. 11 is an image illustrating western blot analysis of expression of p21 and MDM2 following p53 reactivator treatment.

[0052] FIG. 12 illustrates a comparison of the anti-tumor efficacy between untreated p53-mut CAR-T cells and pretreatment with the p53 reactivator using a Venetoclax -resistant AML mouse model. The survival rate of the mice receiving the indicated CAR-T cell regimen has been determined. p53-mut CAR-T cells were treated with a p53 reactivator for 3 days prior to injection into the mice.

[0053] FIG. 13A-13E are graphs illustrating that reactivation of mutant p53 in CAR-T cells prolongs mouse survival. FIG. 13A is a schematic illustrating mice treatment with T cells expressing anti-CD123 CAR and mutant p53 pre-treated with the p53 reactivator. FIG. 13B is a graph illustrating human cells in the blood identified using anti-human CD45 antibodies, followed by CD3 and CD33 staining to distinguish T cells from AML cells. FIG. 13C is a graph illustrating circulating AML cells in the reactivator pre-treated p53mt CAR-T cell group compared to untreated p53mt CAR-T cell-treated mice. FIG. 13D is a graph illustrating analysis of circulating CD3+ T cells for T-cell circulation in both the reactivator pre-treated and untreated p53mt CAR- T cell groups. FIG. 13E is a graph illustrating mouse survival when pre-treated with p53mt CAR- T cells compared to untreated p53mt CAR-T cells.DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention stems from the critical discovery that patients with acute myeloid leukemia (AML) who have TP53 mutations also show identical p53 mutations in their bone marrow-resident CD4+ and CD8+ T lymphocytes, a mechanism by which p53 mutations impair immune cell function against AML cells, potentially explaining the failure of immunotherapies in these patients. This finding highlights a direct link between TP53 mutations and diminished antitumor immunity in immune cells. By reactivating p53 in these mutant immune cells, the invention aims to restore their wild- type TP53 function, offering a promising approach for effective cancer treatment.

[0055] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular aspects only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0056] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0057] 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 invention, it will be understood that modifications andvariations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0058] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” include one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0059] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0060] The term “effective amount” or “therapeutically effective amount” as used herein refers to the amount of a therapeutic compound, a combination of therapeutic compounds or pharmaceutical compositions thereof provided herein, which is sufficient to result in the desired outcome.

[0061] The terms “subject” and “patient” may be used interchangeably. As used herein, in some aspects, a subject is a mammal. In some aspects, the subject is a human. In some aspects, the subject is a mammal, e.g., a human, diagnosed with a disease or disorder. In some aspects, the subject is a mammal, e.g., a human, at risk of developing a disease or disorder.

[0062] “Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration.

[0063] As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or disorder resulting from the administration of one or more therapies. Treating may be determined by assessing whether there has been a decrease, alleviation and / or mitigation of one or more symptoms associated withthe underlying disorder such that an improvement is observed with the patient, despite that the patient may still be afflicted with the underlying disorder. The term “treating” includes both managing and ameliorating the disease.

[0064] The terms “prevent,” “preventing,” and “prevention” refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptom(s).

[0065] In one embodiment, the present disclosure provides a method for restoring anti-tumor activity of an immune cell, including: contacting the immune cell with a p53 reactivator, wherein the immune cell includes a TP53 gene mutation, thereby restoring the anti-tumor activity of the immune cell.

[0066] As used herein the term “contacting a cell” refers to the physical interaction between an agent and a cell. The contact can be via diffusion, blood circulation, direct contact, or intercellular junctions.

[0067] The term “p53 reactivator” refers to a compound designed to restore the normal function of the p53 protein. Examples of p53 reactivators include but are not limited to APR-246 or eprenetapopt, targeting p53 mutations such as R175H and R273H; COTI-2, targeting p53 R175H mutation; rezatapopt (PC 14586), specifically targeting the Y220C mutant p53; PRIMA-1, converting to APR-246 in the body and reactivates mutant p53; PK7088, binding to the p53 Y220C mutant and stabilizing its structure; and NSC59984, reactivating mutant p53.

[0068] The TP53 gene can have numerous mutations. Examples of p53 mutations include but are not limited to Y220C, P151A, R241G, P33R, K132N, R175H, R248Q, R273H, G245S, R282W, and R249S.

[0069] In some aspects, the p53 reactivator provided herein is selected from a small molecule, a nucleic acid (e.g., oligonucleotide, polynucleotide, etc.), a peptide (e.g., a polypeptide, a protein, etc.), a saccharide (e.g., monosaccharide, oligosaccharide, polysaccharide, etc.), a lipid, and any combination thereof. In some aspects, the p53 reactivator provided herein is selected from a nucleotide, an oligonucleotide, a polynucleotide, an amino acid, a peptide, a protein, a small molecule, a synthetic molecule, an organic molecule, an inorganic molecule, a polymer, a synthetic polymer, or any combination thereof.

[0070] In some aspects, the p53 reactivator is the p53 reactivator is APR-246 (eprenetapopt), COTI-2, rezatapopt (PC14586), PRIMA-1, PK7088, andNSC59984, or a combination thereof. In one aspect, the p53 reactivator is rezatapopt (PC 14586).

[0071] In some aspects, the p53 reactivator provided herein increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or at least 90% of p53 activity, e.g., in a cell such as an immune cell. In some aspects, the p53 reactivator provided herein increases p53 activity by about 50%, e.g., in a cell such as an immune cell. The methods for measuring p53 activity and the increase thereof are well known in the art. For example, in some aspects, the increased activity corresponds to an increased level of transcription of p53 target genes, which may be studied by quantifying the mRNA increase. An increased level of mRNA may be determined by methods known in the art. In some aspects, mRNA is quantified by TaqMan analysis.

[0072] In some aspects, the p53 reactivator provided herein increases wild-type p53 activity. In some aspects, the p53 reactivator provided herein is capable of reactivating a mutant p53. In some aspects, the p53 reactivator provided herein is capable of increasing wild-type p53 activity and reactivating a mutant p53.

[0073] The p53 gene TP53 is a very common target for mutation in tumors. Around half of all human tumors carry mutations in TP53. p53 halts the cell cycle and / or triggers apoptosis in response to various stress stimuli, including DNA damage, hypoxia, and oncogene activation. Upon activation, p53 initiates the p53 -dependent biological responses through transcriptional transactivation of specific target genes carrying p53 DNA-b inding motifs.

[0074] Analyses of a large number of mutant p53 genes in human tumors have revealed a strong selection for mutations that inactivate the DNA binding function of p53; most mutations in tumors are point mutations clustered in the part encoding the core domain of p53 (residues 94-292) that harbors the DNA binding activity.

[0075] Both p53 -induced cell cycle arrest and apoptosis could be involved in p53 -mediated tumor suppression. While there are various ways to reverse p53 -induced cell cycle arrest, p53- induced cell death is irreversible. Indeed, evidence from animal in vivo models and human tumors indicates that p53 -dependent apoptosis plays a significant role in the elimination of emerging tumors, particularly in response to oncogenic signaling. Moreover, the ability of p53 to induce apoptosis often determines the efficacy of cancer therapy.

[0076] In addition to hyperproliferative diseases, such as cancer, it is also known in the art that deficient p53 function is involved in a number of other disease states, e.g., autoimmune diseases and cardiac diseases.

[0077] For example, human autoimmune diseases share the common feature of an imbalance between the production and destruction of various cell types, including lymphocytes (SLE), synovial cells (RA), and fibroblasts (scleroderma). Abnormal expression also occurs in genes that regulate apoptosis, such as TP53.

[0078] In some aspects, the p53 reactivator directly or indirectly targets a mutant p53 protein.

[0079] In some aspects, the mutant TP53 includes a missense mutation, which is a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid. Missense mutant p53 proteins can be broadly classified as DNA-contact mutants and structural mutants. The p53 DNA contact mutant contains mutations in amino acids that directly bind to DNA, such as single amino acid changes (R248Q, R248W, R273H, or R273C). R248Q indicates that the mutant has substituted a glutamine for the wild-type residue arginine in position 248. In p53 structural mutants, an amino acid replacement alters the overall structure and / or stability, preventing DNA binding. This has been demonstrated in mutants with R175H, Y220C, G245S, R249S, or R282W replacements.

[0080] In some aspects, the mutant TP53 includes a nonsense mutation. A nonsense mutation is a genetic mutation that changes a codon for an amino acid into a stop codon, resulting in a shorter, unfinished protein product. Nonsense mutations in TP53 are less frequent than missense mutations, but they still account for about 10% of all TP53 mutations in cancer. The most common TP53 nonsense mutation yields a truncated p53: R213X, aka R213*.

[0081] In some aspects, the p53 reactivator provided herein reactivates or restores at least part of the wild- type p53 activity of a mutant p53, for example, by promoting proper folding of the mutant p53 and restoring the normal p53 function of the mutant p53.

[0082] In some aspects, the p53 reactivator covalently binds to the mutant p53 protein to inhibit improper protein misfolding and / or promote proper protein folding of the mutant p53.

[0083] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein reacts as an electrophile with one or more thiols in the mutant p53.

[0084] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein covalently binds to one or more thiols in the mutant p53.

[0085] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with or binds to one or more thiols in the mutant p53 as an electrophile.

[0086] In some aspects, a metabolite or degradation product of the p53 reactivator) reacts with or binds to one or more thiols in the mutant p53.

[0087] In some aspects, reversible or irreversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53.

[0088] In some aspects, reversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53.

[0089] In some aspects, irreversible covalent bonds are formed between the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) and the mutant p53.

[0090] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) provided herein reacts with one or more thiols in the DNA binding domain of the mutant p53 to stabilize the mutant p53 conformation, thus restoring their transcriptional activities.

[0091] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with one or more thiols of cysteine residues in the core domain of wildtype p53 protein and stabilizes wild-type p53 conformation.

[0092] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) reacts with one or more thiols of cysteine residues in the core domain of mutant p53 and restores the mutant p53 conformation to a wild- type p53-like conformation.

[0093] In some aspects, the p53 reactivator (or a metabolite or degradation product of the p53 reactivator) shifts the equilibrium from unfolded mutant p53 conformation towards a wild-type p53-like conformation.

[0094] In some aspects, the p53 reactivator provided herein inhibits improper protein misfolding and / or promotes proper protein folding by non-covalent binding to the mutant p53 protein. These p53 reactivators include chaperones that can non- covalently stabilize the mutant p53 protein.

[0095] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and / or promotes proper protein folding by covalent binding to the mutant p53 protein, for example, by electrophiles binding to one or more thiols in the mutant p53 DNA binding domain to stabilize a folded conformation and restore their transcriptional activities.

[0096] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof binds to the thiol of cysteine residues in the core domain and stabilizes wildtype p53 conformation.

[0097] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof shifts the equilibrium from unfolded towards a wild-type-like p53 conformation.

[0098] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof binds to thiol groups in the core domain and restores wild-type conformation.

[0099] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof inhibits improper protein misfolding and / or promotes proper protein folding through non-covalent binding to the mutant p53 protein. Such p53 reactivators include chaperones that can non-covalently stabilize mutant p53 structures.

[0100] In some aspects, the p53 reactivator provided herein reactivates mutant p53 by interfering with the aggregation of misfolded p53 or by reducing the aggregation of mutant p53. Sometimes, p53 misfolds or unfolds into an aggregation-prone stage that loses its DNA-b inding capacity. Similarly, misfolded p53 may convert wild-type p53 to a misfolded form and accelerate p53 aggregation. Thus, in some aspects, the p53 reactivator provided herein may reactivate p53 by interfering with the aggregation of misfolded p53.

[0101] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, thereby reducing aggregation.

[0102] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof reactivates mutant p53 by interfering with the aggregation of misfolded p53 or by reducing the aggregation of mutant p53. Sometimes, p53 misfolds or unfolds into an aggregation-prone stage that loses its DNA-binding capacity. Similarly, misfolded mutant p53 may cause the accumulation of wild-type p53 in a misfolded form and accelerate p53 aggregation. Thus, in some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof may reactivate p53 by interfering with the aggregation of misfolded p53.

[0103] In some aspects, the p53 reactivator provided herein or a degradation product or metabolite thereof reduces non-folded or incorrectly folded mutant p53 that may otherwise aggregate, thereby reducing aggregation. An increased melting temperature correlates with shifting the equilibrium towards the folded structure and may be studied, for example, by Circular Dichroism (CD) or Differential Scanning Fluorimetry (DSF). Aggregation of the unfolded protein chain competes with folding and may be studied by intrinsic fluorescence measurements or light scattering, while the actual aggregates may be investigated by atomic force microscopy.

[0104] In some aspects, the mutant p53 includes at least one amino acid replacement in the core domain of the mutant p53 (between residues 94 and 292) caused by a TP53 mutation. In some aspects, the mutant p53 includes replacements selected from the group consisting of VI 73 A, S241F, R249S, R273H, R175H, R248Q, and Y220C. In some aspects, the mutant p53 includes one of the amino acid replacements of R175H and R273H.

[0105] In some aspects, the p53 reactivator provided herein can induce the reactivation of a mutant p53 protein. In some aspects, the p53 reactivator can result in the reactivation of a mutant p53 protein. In some aspects, the p53 reactivator is transformed into a metabolite or degradation product that reacts with a mutant p53 protein. In some aspects, the p53 reactivator is transformed into a metabolite or degradation product in vivo. In some aspects, the p53 reactivator is transformed into a metabolite or degradation product in tumor tissue.

[0106] Without being bound by any theory, in some aspects, the p53 reactivator or a degradation product or metabolite stabilizes wild-type p53 protein in a situation where its production has been induced as part of a normal physiological process, enhancing the effect of said wild- type p53 induction. Without being bound by any theory, in some aspects, the p53 reactivator provided herein or a degradation product or metabolite stabilizes wild- type p53 protein in a situation where its production has been induced as part of a normal physiological process, thereby enhancing the effect of said wild-type p53 induction.

[0107] In some aspects, the immune cell is in a subject. In some aspects, the immune cell of the subject includes the TP53 mutation. In some aspects, the immune cell is selected from a CD4+ T cell, a CD8+ T cell, an NK cell, a dendritic cell, a macrophage, or any combination thereof. In some aspects, the immune cell is a bone marrow-resident CD4+ T cell or a bone marrow-resident CD8+ T cell. In some aspects, the immune cell is a blood-circulating CD4+ T cell or a bloodcirculating CD8+ T cell.

[0108] In some aspects, the subject has a cancer including a TP53 mutation. Examples of such cancers include, but are not limited to, cancer, mesothelioma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, bone cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and / or duodenal) cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, testicular cancer, hepatocellular (hepatic and / or biliary duct) cancer, primary or secondary central nervous system tumor, primary or secondary brain tumor, Hodgkin's disease, chronic or acute leukemia, chronic myeloid leukemia, lymphocytic lymphoma, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, multiple myeloma, oral cancer, non-small-cell lung cancer, prostate cancer, small-celllung cancer, cancer of the kidney and / or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, nonHodgkin's lymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocortical cancer, gall bladder cancer, cancer of the spleen, cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma or a combination thereof.

[0109] In some aspects, the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, bone marrow cancer, cervical cancer, chronic lymphocytic leukemia, acute lymphocytic leukemia, colorectal cancer, esophageal cancer, hepatocellular cancer, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cell origin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovarian cancer, non-small-cell lung cancer, prostate cancer, small-cell lung cancer, and spleen cancer.

[0110] In some aspects, the cancer is a solid tumor cancer. In some aspects, the solid tumor cancer is selected from the group consisting of a carcinoma, an adenocarcinoma, an adrenocortical carcinoma, a colon adenocarcinoma, a colorectal adenocarcinoma, a colorectal carcinoma, a ductal cell carcinoma, a lung carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, a non-melanoma skin carcinoma, and a lung cancer.

[0111] In some aspects, the solid tumor malignancy is an advanced non-CNS-primary solid tumor. In some aspects, the solid tumor malignancy is selected from the group consisting of gastric / gastroesophageal junction (GE J) cancer, bladder / urothelial cancer, and non-small-cell lung cancer (NSCLC).

[0112] In some aspects, the subject has an acute myeloid leukemia (AML). In some aspects, an AML cancer cell from the subject has the same TP53 mutation as the immune cell.

[0113] In some aspects, the TP53 mutation includes Y220C, P151A, R241G, P33R, K132N, or any combination thereof.

[0114] In some aspects, the p53 reactivator decreases exhaustion markers in TP53 mutant T cell. In some aspects, the exhaustion markers of the TP53 mutant T cell are selected from PD-1, TIM3, LAG3, TIGIT, or any combination thereof. In some aspects, the TP53 mutant T cell expresses CD39. In some aspects, the CD39 is an activation and an exhaustion marker of the TP53 mutant T cell.

[0115] In another embodiment, the present disclosure provides a method for enhancing the CAR-T cell therapy efficacy of cancer treatment in a subject with a cancer including a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivatorreduces mutant p53 protein levels in the CAR-T cell, thereby enhancing the CAR-T cell therapy efficacy of cancer treatment in the subject.

[0116] In some aspects, the cytotoxic T lymphocytes used to prepare the CAR-T cells used in the methods described herein can be autologous cells, although heterologous cells can also be used, such as when the patient being treated has received high-dose chemotherapy or radiation treatment to destroy the patient’s immune system. In some aspects, allogenic cells can be used.

[0117] In some aspects, the cytotoxic lymphocytes T can be obtained from a patient by means well-known in the art. For example, cytotoxic T cells can be obtained by collecting peripheral blood from the patient, subjecting the blood to Ficoll density gradient centrifugation, and then using a negative T cell isolation kit (such as EasySep™ T Cell Isolation Kit) to isolate a population of cytotoxic T cells from the peripheral blood. In some aspects, the population of cytotoxic T lymphocytes need not be pure and may contain other cells such as other T cells, monocytes, macrophages, natural killer cells, and B cells. In some aspects, the population being collected can include at least about 90% of the selected cell type, at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the selected cell type.

[0118] In some aspects, after the cytotoxic T lymphocytes are obtained, the cells are cultured under conditions that promote their activation. In some aspects, the culture conditions may be such that the cells can be administered to a patient without concern for reactivity against components of the culture medium. For example, the culture conditions may not include bovine serum products, such as bovine serum albumin. In some aspects, the activation can be achieved by introducing known activators into the culture medium, such as anti-CD3 antibodies in the case of cytotoxic T cells. Other suitable activators include anti-CD28 antibodies. In some aspects, the population of lymphocytes can be cultured under conditions promoting activation for about 1 to about 4 days. In some aspects, the appropriate level of activation can be determined by cell size, proliferation rate, or activation markers determined by flow cytometry.

[0119] In some aspects, after the population of cytotoxic T lymphocytes has been cultured under conditions promoting activation, the cells can be transfected with an expression vector encoding a CAR. Suitable vectors and transfection methods are described above. In some aspects, after transfection, the cells can be immediately administered to the patient, or the cells can be cultured for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more days, or between about 5 and about 12 days, between about 6 and about 13 days, between about 7 and about 14 days, or between about 8 and about 15 days, for example, to allow time for the cells to recover from the transfection. Suitable culture conditions can be similar to the conditions under which thecells were cultured for activation, either with or without the agent that was used to promote activation.

[0120] In some aspects, the TP53 mutant CAR-T cell expresses CD39. In some aspects, the CD39 is an activation and an exhaustion marker of the TP53 mutant CAR-T cell.

[0121] In another embodiment, the present disclosure provides a method for treating cancer in a subject, wherein the cancer includes a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in an immune cell of the subject, thereby treating the cancer in the subject.

[0122] The amount of the p53 reactivator that will be effective in the prevention and / or treatment of a disease or condition can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of a disease or condition, and in some aspects, should be decided according to the judgment of the practitioner and each patient’s circumstances.

[0123] The dose administered to a subject in the context of the present disclosure should be sufficient to effect a therapeutic response. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the potency of the specific p53 reactivator, the age, condition, and body weight of the patient, as well as the stage or severity of the disease. The route (administration form), timing, and frequency of administration will also determine the dose. A dosing period, as used herein, is a period of time during which a p53 reactivator has been administered at least once. A dosing cycle can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In some aspects, the dosing cycle is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. In some aspects, a dosing period is a dosing cycle.

[0124] The p53 reactivator can be delivered as a single dose (e.g., a single bolus injection), or over time (e.g., continuous infusion over time or divided bolus doses over time). The p53 reactivator can be administered repeatedly, if necessary, for example, until the patient experiences stable disease or regression, or until the patient experiences disease progression or unacceptable toxicity. Stable disease or lack is determined by methods known in the art, such as evaluation of patient symptoms, physical examination, and visualization of the tumor that has been imaged using X-ray, CAT, PET, MRI scan, or other commonly accepted evaluation modalities.

[0125] The p53 reactivator can be administered once daily (QD) or divided into multiple daily doses, such as twice daily (BID), three times daily (TID), and four times daily (QID). In addition, the administration can be continuous (i.e., daily for consecutive days or every day) or intermittent, e.g., in cycles (i.e., including days, weeks, or months of rest without drug). As used herein, theterm “daily” is intended to mean that a therapeutic compound is administered once or more than once each day, for example, for a period of time. The term “continuous” is intended to mean that the p53 reactivator is administered daily for an uninterrupted period of, e.g., at least 10 days. The term “intermittent” or “intermittently” as used herein is intended to mean stopping and starting at either regular or irregular intervals. For example, intermittent administration of the p53 reactivator is administration for one to six days per week, administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week), or administration on alternate days.

[0126] In some aspects, the frequency of administration is in the range of about a daily dose to about a monthly dose. In some aspects, administration occurs once a day, twice a day, three times a day, four times a day, once every other day, twice a week, once every week, once every two weeks, once every three weeks, or once every four weeks.

[0127] In some aspects, the p53 reactivator is administered once per day from one day to six months, from one week to three months, from one week to four weeks, from one week to three weeks, or from one week to two weeks.

[0128] In some aspects, the cancer to be treated is a carcinoma, a sarcoma, a lymphoma, a melanoma, a mesothelioma, a nasopharyngeal carcinoma, a leukemia, an adenocarcinoma, or a myeloma. In some aspects, the cancer may be lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head, cancer of the neck, cutaneous melanoma, intraocular melanoma uterine cancer, ovarian cancer, endometrial cancer, rectal cancer, stomach cancer, colon cancer, breast cancer, triple negative breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, non-small cell lung cancer, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, prostate cancer, chronic leukemia, acute leukemia, a lymphocytic lymphoma, pleural mesothelioma, cancer of the bladder, Burkitt's lymphoma, cancer of the ureter, cancer of the kidney, renal cell carcinoma, carcinoma of the renal pelvis, a neoplasm of the central nervous system (CNS), primary CNS lymphoma, a spinal axis tumor, a brain stem glioma, a pituitary adenoma, or an adenocarcinoma of the gastroesophageal junction.

[0129] In some aspects, the cancer is a folate receptor expressing cancer. In some aspects, the cancer is an endometrial cancer, a non-small cell lung cancer, an ovarian cancer, or a triple-negative breast cancer. In some aspects, the cancer is benign. In some aspects, the cancer is malignant.

[0130] In another embodiment, the present disclosure provides a method for preventing cancer in a subject, wherein the subject has a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the subject, thereby preventing the cancer in the subject.

[0131] In some aspects, the TP53 mutation in the subject may be detected in various tissues and type of samples, depending on the clinical context and the purpose of the testing.

[0132] In some aspects, the TP53 mutation may be detected in the immune cells of the subject, wherein the immune cells include, but not limited to, CD4+ T cells, CD8+ T cells, NK cells, dendritic cells, macrophages, or any combination thereof.

[0133] In some aspects, the TP53 mutation may be detected in the blood of the subject. Blood samples from the subject may be used to detect germline TP53 mutations.

[0134] In some aspects, the TP53 mutation may be detected in tissue samples of the subject obtained from a tissue biopsy or a tissue removal during surgical procedures.

[0135] In some aspects, the TP53 mutation may be detected in bone marrow samples of the subject.

[0136] In some aspects, the TP53 mutation may be detected in saliva or buccal swaps.

[0137] In some aspects, the TP53 mutation may be detected in urine samples or cerebrospinal fluid (CSF) samples.

[0138] In some aspects, the TP53 mutation may be detected using sanger sequencing, nextgeneration sequencing (NGS), polymerase chain reaction (PCR), digital droplet PCR (ddPCR), and / or immunohistochemistry (IHC).

[0139] In another embodiment, the present disclosure provides a method for enhancing the CAR-NK cell therapy efficacy of cancer treatment in a subject with a cancer including a TP53 mutation, including: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the CAR-NK cell, thereby enhancing the CAR-NK cell therapy efficacy of cancer treatment in the subject.

[0140] In some aspects, the provided methods may be used for the treatment of residual cancers after cancer treatment.

[0141] In some aspects, the provided methods may be used to reactivate exhausted immune cells to regain anti-tumor functionality.

[0142] The following examples are provided to further illustrate the aspects of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLES

[0143] The invention is further illustrated by the following non-limiting examples.EXAMPLE 1T-Cell Cytokine Production and Genotypic Analysis in TP53-Mutant AML

[0144] This example describes the cytokine production variations in T cells derived from the bone marrows of patients with TP53 -mutant acute myeloid leukemia (AML) versus those with TP53 wild- type AML. A notable discovery was made that autologous bone marrow-resident CD4+ and CD8+ T cells in patients with TP53 mutations also possess corresponding p53 mutations. To further investigate, a single-cell cytokine secretion analysis (IsoPlexis) was conducted to assess the cytokine production in T cells from the bone marrows of patients with TP53-mutant AML compared to those from TP53 wild-type AML. This analysis indicated a significant reduction in the secretion of effector cytokines, including granzyme B, IFN-g, MIP- la, perforin, and TNF-a, as well as stimulatory cytokines such as IL-5 and IL-8, in T cells from TP53-mutant AML patients (FIG. IE). Concurrently, Tapestri technology was employed to perform genotype analysis of bone marrow-resident T cells from AML patients with TP53 mutations.EXAMPLE 2T and NK Cells Exhaustion is Associated with TP53 Mutations in AML Patients

[0145] AML is a heterogeneous bone marrow disorder resulting from the accumulation of complex mutations in hematopoietic stem cells (HSCs). While many therapies target various mutations in AML, TP53 mutations remain without any effective targeted treatments, and their impact on immune cells is still largely unknown. To address this, single-cell technologies, including transcriptomics, proteomics, and secretomics were employed, to dissect the bone marrow and its microenvironment from AML patients and comprehensively analyze the impact of p53 mutations on immune cells.

[0146] In a previous study, it was demonstrated that T-cells from AML patients harboring TP53 mutations exhibit impaired polyfunctionality, as evidenced by altered cytokine secretion profiles. To obtain a deeper understanding of the immune-cell landscape in TP53 -mutant AML, single-cell RNA sequencing was performed and an unsupervised analytical approach employed to investigatetranscriptional changes at the single-cell level of immune populations, specifically T and NK cells. For an unbiased comparison across patients, scRNA-seq data from the bone marrow of 24 newly diagnosed AML patients were pooled for downstream analysis. Dimensional reduction using Uniform Manifold Approximation and Projection (UMAP) (FIG. 1A) enabled the visualization of distinct immune cell clusters, while unsupervised clustering identified cell types based on lineagespecific gene expression. To assess T and NK cell functionality, an exhaustion score was calculated, defined as the mean expression of 21 exhaustion-associated markers (Table 1). Notably, this analysis revealed significantly elevated exhaustion scores in T andNK cells from TP53 -mutant AML patients compared to their TP53 wild-type counterparts (FIG. IB). Increased expression of canonical exhaustion markers, including TIGIT, PD-1, LAG-3, and TIM-3, was particularly evident in TP53 -mutant samples (FIG. 1C), suggesting a pronounced exhaustion phenotype. Further the protein-level expression of exhaustion markers PD-1, TIGIT, and TIM-3 was assessed using CyTOF analysis, which enables high-parametric single-cell protein quantification. To minimize the batch effect, live-cell barcoding approach was employed, allowing sample pooling after surface staining while preserving individual sample identity. A total of 38 AML bone marrow (BM) samples were analyzed, including 26 TP53-mutant (TP53m) and 12 TP53 wild-type (TP53wt) AML cases, along with 13 TP53wt chronic myelomonocytic leukemia (CMML) BM samples and four BM samples from healthy donors. CyTOF analysis confirmed the significant upregulation of PD-1 and TIGIT in CD3+ T cells from TP53m compared to TP53wt AML patients. Furthermore, NK cells from TP53m AML patients also exhibited increased expression of PD-1, TIGIT, and TIM-3 relative to both TP53wt AML patients and healthy donors (FIG. ID), further corroborating the transcriptional evidence of immune exhaustion. To assess the functional implications of these findings, cytokine secretion capacity was evaluated using a single-cell fluorescence-based ELISA assay (Isoplexis). This assay quantified the secretion of 32 cytokines at the single-cell level following stimulation with anti-CD3 / CD28 microbeads. Analysis revealed a significant reduction in the polyfunctional strength index (PSI) in both CD4+ and CD8+ T cells from TP53m AML patients, indicating impaired effector function. Notably, the decrease in PSI was driven primarily by reductions in granzyme B, IFN-y, MIP-la, perforin, and TNF-a, key mediators of cytotoxic T-cell responses (FIG. IE). Collectively, these findings demonstrate that T and NK cells from TP53 -mutant AML patients exhibit greatly increased exhaustion at both the transcriptional and functional levels, providing further insights into the immune dysfunction associated with TP53 mutations in AML.

[0147] Table 1: Exhaustion-associated MarkersEXAMPLE 3TP53 Mutations Occur in T and NK Cells in AML Patients and Alter Their Phenotype

[0148] While TP53 mutations in AML patients have been associated with altered immune response, only few studies have identified specifically addressed the presence of TP53 mutations in immune cells. Using digital droplet PCR (ddPCR), it was demonstrated that TP53 mutations in AML patients arise somatically in T cells, independent of Li-Fraumeni Syndrome (LFS). In this study, whole-exome sequencing (WES) was utilized to detect TP53 mutations in AML patients and confirmed these mutations in highly purified (>99%) CD3+CD34- FACS-sorted cells using digital droplet PCR (ddPCR). The present disclosure revealed that TP53 mutations were presentin over 20% of droplets, and the mutations identified in T cells were identical to those detected in leukemic blasts. Simultaneously, single-cell DNA sequencing integrated with surface antigen expression analysis was performed, enabling direct phenotype-genotype correlations from bone marrow (BM) samples of four AML patients with TP53 mutations detected by WES. Cells were clustered based on the expression of 45 oligo-conjugated antibodies and are colored by cell types. Using UMAP analysis, various cell types identified by surface antigen expression were visualized, identifying T cells as CD3-positive andNK cells as CD56-positive. Notably, by integrating singlecell DNA sequencing data with UMAP clustering, it was found that TP53 mutations were widely distributed across different immune cell lineages. These mutations were observed not only in myeloid lineages (e.g., monocytes and leukemic cells) but also in lymphoid cell subsets, including T, B, and NK cells. This platform enables the simultaneous analysis of single-cell DNA and surface antigens, thus facilitating direct correlations between phenotype and genotype (FIG. 2A and 2B). Importantly, TP53 mutations were detected in subsets of T and NK cells across all four AML patient samples, with more than 95% of mutations being monoallelic. (FIG. 2A). At the single-cell resolution level, the frequency of TP53 mutant T and NK cells was quantified across four AML patients. TP53 mutations were detected in CD4, CD8 T, and NK cells, with mean mutation frequencies of 32% in CD8 T cells (range: 7%— 91%), 36% in CD4 T cells (range: 4%- 89%), and 32% in NK cells (range: 9%-86%) (FIG. 2C). To further characterize the phenotypic impact of TP53 mutations on T and NK cells, a comprehensive analysis of surface antigen expression was conducted using a heatmap. Distinct phenotypic profiles were observed across different immune cell subsets, with CD4 and CD8 T cells displaying high CD5 and CD7 expression, while NK cells exhibited high CD 16 expression, as expected. Notably, TP53mut T and NK cells displayed phenotypic alterations compared to their TP53 wild- type counterparts (FIG. 2D). To further explore these differences, a comparative analysis of TP53mut and TP53wt was performed on T and NK cells. Unsupervised clustering was applied to analyze phenotypic differences, incorporating TP53 mutation status. Interestingly, CD4, CD8, and NK cells were distinctly separated in the UMAP, and TP53mt T and NK cells formed unique clusters, further underscoring their distinct phenotypic profiles (FIG. 2E). Indeed, differential expression of multiple immune -related markers was observed between TP53mt and TP53wt T and NK cells from the same patient sample. Specifically, CD69, CD71, and CD38 were significantly increased in TP53mt CD4 and CD8 T cells compared to their TP53wt counterparts, suggesting enhanced proliferative capacity in the presence of TP53 mutations. More importantly, CD2 expression, a key molecule for immunological synapse formation and T-cell anti-tumor functionl, wasmarkedly reduced in TP53AmtACD4 and CD8 T cells, suggesting a potential impairment in T-cell functionality. Similarly, TP53 mutant NK cells exhibited elevated expression of activation markers such as CD44, CD69, and CD71 (FIG. 2F), indicating a TP53 mutation-driven activation phenotype. These findings confirm that TP53 mutations occur in immune cells, including T and NK cells, and profoundly reshape the immune landscape in AML patients. While TP53 mutations enhance the proliferative and activation profiles of T and NK cells, they simultaneously impair T- cell functionality, potentially contributing to T-cell exhaustion. Based on these data, described herein is that monoallelic TP53 mutations may impair normal p53 function in T cells, which is essential for cell cycle regulation and proliferation control. Through a dominant-negative effect, these mutations may drive T-cell exhaustion and dysfunction, ultimately compromising antileukemic immune responses.

[0149] The results verified the presence of TP53 mutations in subsets of CD4+ and CD8+ bone marrow-resident T cells, mirroring those in the leukemic blasts from the same patients. To further confirm the occurrence of TP53 mutations within T cells, digital-droplet PCR (ddPCR) was executed on fluorescence-activated cell sorted (FACS) T cells of exceptional purity (> 99%), targeting specific TP53 mutations. Notably, TP53 mutations were detected in 52% of the analyzed T cells (FIG. 3A-3B), underlining the impact of TP53 mutations not only in the pathology of AML but also in the immune response.EXAMPLE 4Functional Impact of TP53 Mutations on CAR-T Cell Efficacy

[0150] CD 123 -targeted CAR-T cells overexpressing the mutant p53-Y220C were engineered using a lentiviral vector to specifically target AML cells. This specific p53 mutation was chosen because of its reversibility by PC 14586, a small molecule that could restore mutant p53 to its wildtype conformation and reactivate the functions of WT p53 protein. The overexpressed mutant p53 is expected to exert a dominant-negative effect on wild-type p53. Control CAR-T cells were generated without overexpression of mutant p53, serving as a baseline comparison (Fig. 4A). Notably, when wild-type p53 was overexpressed, these T cells underwent apoptosis. Prior to assessing the functionality of the p53mt CAR-T cells, the successful transduction of the engineered CAR construct into T cells was confirmed. Utilizing a specific CAR antibody, CAR expression on over 85% of the transduced T cells was observed. Remarkably, p53 protein was detected in 80% of the p53mt CAR-T cells, while p53 protein was detected at low level (<5%) in the control CAR-T cells, as expected (FIG. 4B). Subsequently, the autonomous growth of p53mt CAR-T cells in comparison to control CAR-T cells was tested. It was observed that p53mt CAR-T cells proliferated more rapidly than control CAR-T cells under normoxic (5% CO2 at 37°C) as well as hypoxic conditions (1% oxygen at 37°C) for 7 days (FIG. 4C). These results suggest that mutant p53 enhances the proliferative capacity of CAR-T cells and enable them to overcome hypoxic conditions.

[0151] Next, the phenotypic profile of the CAR-T cells was characterized using CyTOF analysis using a 50-parameter T-cell-focused panel. UMAP analysis revealed that p53 mutant (p53mt) CAR-T cells exhibited a distinct profile compared to both control CAR-T cells and empty vector T cells (V-T cells), particularly within the CD4 T-cells subset (FIG. 4D). To further investigate our hypothesis that mutant p53 induces T-cell exhaustion, we assessed the expression of exhaustion markers on p53mt CAR-T cells relative to control CAR-T cells. Notably, the expression levels of PD-1, LAG-3, TIM-3, TIGIT, and CD39 were significantly increased in both CD4 and CD8 p53mt CAR-T cells compared to control CAR-T cells (FIG. 4E). These results demonstrate that mutant p53 not only enhances the proliferative capacity of CAR-T cells but also induces a pronounced state of T-cell exhaustion.

[0152] To investigate the impact of CAR-T cells upon repeated antigenic exposure, an in vitro rechallenge model was developed. In this system, p53mt CAR-T cells were co-cultured with CD 123 -positive Molml3 cells at a 1:1 ratio in the absence of exogenous cytokine support. To mimic sustained antigen exposure, fresh Molml3 cells were replenished every 48 hours which lacked mutant p53 expression and served as a baseline comparator, underwent identical conditions (FIG. 4F).

[0153] To characterize the dynamic functional and phenotypic changes in p53mt CAR-T cells during AML interactions, cells were collected from the co-culture every two days for 14 days and analyzed by CyTOF using a previously described live-cell barcoding approach 12(Table 2). By day 14, cytotoxicity assays revealed that control CAR-T cells efficiently eradicated over 99% of Molml3 cells, while p53mt CAR-T cells exhibited markedly reduced cytotoxic activity, with 11% of Molml3 cells persisting in the culture. This significant impairment in cytolytic function under repeated antigenic stimulation suggests that mutant p53 compromises CAR-T cell effector capabilities, potentially through exhaustion-mediated dysfunction. Further analysis using UMAP clustering revealed that rechallenged p53mt CAR-T cells displayed a distinct proteomic signature compared to their unstimulated counterparts, indicative of chronic antigenic engagement triggering extensive proteomic reprogramming via CAR signaling (FIG. 4G). This shift suggests that prolonged AML exposure alters key functional states within p53mt CAR-T cells, likely impacting their persistence and therapeutic efficacy.

[0154] Table 2: T Cell Panel for CyTOF

[0155] To further delineate these proteomic changes, Phenograph clustering analysis was performed, a computational approach for resolving subpopulations within high-dimensional single-cell data. Across 28 CAR-T cell samples collected at seven different time points, 24 distinct clusters were identified (FIG. 4H). The proportions of these clusters remained stable in unstimulated p53mt CAR-T cells but exhibited notable shifts following antigenic rechallenge. Specifically, cluster 2 expanded from 2% on day 1 to 32% on day 13, characterized by elevated expression of exhaustion markers PD-1, TIGIT, TIM-3, and LAG-3. Conversely, cluster 15, enriched for high expression of Eomes and T-bet, key regulators of T-cell memory and effector functionl5, declined from 28% on day 1 to 0.2% on day 13, suggesting a substantial loss of memory-like and cytotoxic potential in p53mt CAR-T cells. This decline in memory-associated transcription factors likely contributes to impaired persistence and reduced long-term efficacy, exacerbating exhaustion-driven dysfunction. Additionally, CD27, a crucial marker for T-cell survival and activation, was markedly downregulated (FIG. 4H, FIG. 5). To assess the temporal evolution of exhaustion marker expression, p53 levels was examined over time, as its dysregulation has been implicated in T-cell exhaustion and impaired persistence. Tracking p53 expression dynamics allows us to determine whether its accumulation contributes directly to the upregulation of inhibitory receptors, thereby influencing CAR-T cell efficacy. In rechallenged p53mt CAR-T cells, p53 expression progressively increased, whereas no such trend was observed in unstimulated p53mt CAR-T cells. As expected, p53 expression remained undetectable in control CAR-'T cells, irrespective of antigen exposure. Exhaustion markers, including CD39, LAG-3, TIGIT, and TIM-3, were upregulated in both p53mt and control CAR-T cells upon repeated antigen stimulation. However, p53mt CAR-T cells exhibited an accelerated and heightened expression of these exhaustion markers compared to controls, potentially driven by aberrant activation of NF-KB and STAT3 signaling pathways. Notably, TIGIT expression demonstrated a pronounced increase, reaching twice the level observed in control CAR-T cells by the final time point. The mechanistic link between mutant p53 expression and TIGIT upregulation remains under investigation (FIG. 41). Collectively, these findings suggest that mutant p53 accelerates the acquisition of exhaustion markers, rendering p53mt CAR-T cells more prone to functional exhaustion upon sustained antigenic stimulation, thereby compromising their antitumor efficacy.EXAMPLE 5Mutant P53 Impairs CAR-T Cell Anti-Tumor Function In Vitro

[0156] Based on our phenotypic observations, it was hypothesized that mutant p53 impairs the anti-tumor function of CAR-T cells. To test this hypothesis, the Incucyte Live-Cell Analysis System was employed to evaluate the cytotoxic capacity of p53mt CAR-T cells against their target cells by continuously tracking the number of apoptotic and surviving target cells in real time. This real-time monitoring approach provides a more dynamic and precise assessment of cytotoxicity compared to traditional endpoint assays, allowing for a detailed understanding of temporal killing kinetics and functional differences between p53mt and control CAR-T cells. p53mt CAR-T cells exhibited a significant reduction in cytotoxicity compared to control CAR-T cells (FIG. 6A), suggesting that the anti-tumor function of p53mt CAR-T cells is compromised due to mutant p53 expression. Moreover, a single-cell cytokine secretion assay was conducted to assess the polyfunctionality of these cells under stimulation. The data revealed a marked reduction in the polyfunctional strength index (PSI) in both CD4 and CD8 p53mt CAR-T cells compared to control CAR-T cells, particularly for effector cytokines such as Granzyme B, IFN-y, MIP-la, perforin, and TNF-a, as well as IL-5 and IL-8, which are associated with T-cell activation (FIG. 6B). Notably, this cytokine downregulation aligns with well-established exhaustion signatures observed in dysfunctional CAR-T cellsl7, suggesting that mutant p53 may drive an early onset of exhaustion through altered transcriptional or metabolic pathways. Additionally, the production of TNF-a, IFN-y, and IL-2 in p53mt CAR-T cells was determined using a flow cytometry-based approach. The results confirmed that p53mt CAR-T cells produced lower levels of these cytokines compared to control CAR-T cells (FIG. 6C). These findings collectively indicate that the presence of mutant p53 not only impairs the cytotoxic function of CAR-T cells but also diminishes their cytokine secretion capacity, ultimately leading to reduced anti-tumor efficacy. To determine whether this phenomenon is specific to the p53-Y220C mutation or a broader effect of p53 mutations, CAR-T cells with the p53-R175H mutation was also engineered. These cells exhibited the same exhaustion-associated features and reduced cytotoxicity (FIG. 7). Mechanistically, this could be attributed to transcriptional repression via NF-KB, which is known to regulate cytokine expressionl8, or metabolic constraints imposed by mutant p53, limiting energy availability for cytokine production. Further studies are required to dissect these pathways and their contribution to CAR-T cell dysfunction.

[0157] Additionally, Timelapse Imaging Microscopy in Nanowell Grids (TIMING) was employed to assess the functionality of p53 mutant (p53mt) CAR-T cells compared to controlCAR-T cells, thereby dissecting functional heterogeneity at the single-cell level. TIMING offers a distinct advantage over traditional imaging or cytotoxicity assessment methods by enabling high-resolution, real-time tracking of individual cell interactions. This approach provides detailed insights into CAR-T cell engagement, synapse formation, and killing efficiency, allowing for a more precise evaluation of functional impairments in p53mt CAR-T cells. The cytotoxic process was delineated into three distinct phases: the time taken for CAR-T cells to seek out tumor cells (tSeek), the duration of contact between CAR-T cells and tumor cells (tContact), and the time required to induce tumor cell apoptosis (tDeath), at an effector-to-target (E) ratio of 1:1 (FIG. 6D). Interestingly, the time required for CAR-T cells to establish contact with tumor cells (tSeek) did not differ significantly between p53mt and control CAR-T cells (FIG. 6E). However, the duration of contact between effector and target cells (tContact) was significantly shorter for p53mt CAR-'T cells compared to control CAR-T cells, suggesting that p53mt CAR-T cells exhibit impaired synapse formation ability (FIG. 6F). This impairment in synapse formation is consistent with the observed decreased propensity of p53mt CAR-T cells to eliminate AML cells. This defect may be attributed to the downregulation of adhesion molecules, such as CD2, which were observed in AML patients samples (FIG. 2A-2F). Furthermore, the survival proportions of Mohn 13 tumor cells in 1:1 effector-to-target (E) ratio over time, as shown in the survival curves, indicated that Molml3 cells had a higher survival rate when co-cultured with p53mt CAR-T cells compared to control CAR-T cells. This increased survival could result from reduced cytolytic function, impaired tumor recognition, or a combination of both factors. Defects in immune synapse formation, diminished cytokine secretion, or altered metabolic fitness may contribute to the overall functional impairment of p53mt CAR-T cells. This finding further underscores the impaired cytotoxic function of p53mt CAR-T cells (FIG. 6G). Taken together, all in vitro data demonstrate that mutant p53 impairs the anti-tumor function of CAR-T cells by reducing cytokine secretion and downregulating synapse formation.EXAMPLE 6Mutant P53 Impairs CAR-T Cell Eliminate AML Cells In Vivo

[0158] To investigate the in vivo impact of mutant p53 on CAR-T cell functionality, a patient- derived xenograft (PDX) mouse model was generated using venetoclax-resistant AML patient cells, as described in the Methods section. Briefly, mice were intravenously injected with luciferase-transduced PDX cells. After confirming the engraftment of PDX cells by detecting 1% human CD45 in mouse blood, treatment was initiated with p53 mutant (p53mt) CAR-T cells, control CAR-T cells, or empty vector T cells via tail vein injection. Tumor burden was monitoredusing bioluminescence imaging (BLI) and overall survival was assessed. The BLI data revealed that mice treated with p53mt CAR-T cells exhibited an increased AML burden by day 47 posttreatment. In contrast, mice treated with control CAR-T cells maintained the suppression of AML cells until day 120 post-treatment (FIG. 8B). The BLI signals indicated a significantly higher tumor burden in mice treated with p53mt CAR-T cells compared to those treated with control CAR-T cells and empty vector T cells, as well as untreated PDX mice. This increased tumor burden may correlate with reduced CAR-T cell persistence or expansion in vivo, potentially due to the exhaustion phenotype observed in vitro. Further analysis of CAR-T cell engraftment, proliferation, and exhaustion marker expression in treated mice is necessary to gain deeper insight into the mechanisms underlying this impaired anti-tumor response.

[0159] To validate the BLI imaging findings and evaluate systemic tumor growth, circulating AML cells were quantified by measuring human CD45 (hCD45) levels using flow cytometry in mouse blood at various time points. Consistent with the BLI data, no human cells were detectable in the blood of mice treated with control CAR-T cells. However, mice treated with p53mt CAR- T cells showed a marked increase in circulating AML cells, with over 70% hCD45+ cells detected by day 123 post-treatment (FIG. 8C). This was in stark contrast to the control CAR-T cell-treated group, which had undetectable levels of hCD45+ cells. Crucially, survival analysis revealed that mice treated with p53mt CAR-T cells exhibited significantly worse survival outcomes compared to those treated with control CAR-T cells (FIG. 8D).

[0160] Consistent with the exhaustion phenotype observed in vitro, CAR-T cells harvested from the bone marrow of p53mut-CAR-T cells treated mice expressed markedly higher levels of PD-1, TIM-3, TIGIT, LAG-3, CD39, and CTLA-4 than CAR-T cells from control animals (FIG. 9). These findings collectively suggest that p53mt CAR-T cells are significantly less effective in controlling tumor growth compared to control CAR-T cells, highlighting the detrimental effects of mutant p53 on CAR-T cell efficacy in vivo.EXAMPLE 7Reactivating Mutant p53 in T Cells Enhances Anti-Tumor Activity

[0161] Based on the phenotypic and functional observations that mutant p53 drives T-cell exhaustion and impairs their anti-tumor function, it was hypothesized that correcting the structure of mutant p53 might restore T-cell functionality. Mechanistically, mutant p53 may contribute to exhaustion by dysregulating key transcriptional programs involved in T-cell proliferation. To test this hypothesis, a small molecule was utilized, PC 14586, which has been reported to fill the structural gap in mutant p53-Y220C protein, thereby restoring its conformation and transcriptionalactivity to that of the wild-type and reactivating its function. This molecule is currently under investigation in clinical trials, as well as in preclinical and most recently in a clinical trial in AML.

[0162] To evaluate the efficacy of the p53 reactivator in T cells, first the p53mt CAR-T cells were treated in vitro with the recommended dose (8 pM) added to the culture media. Then the expression levels of the mutant p53 protein was assessed in samples with and without the reactivator using a specific antibody that recognizes the mutant p53 protein. As expected, both CD4 and CD8 T-cells exhibited reduced levels of mutant p53 in p53mt CAR-T cells following treatment with the p53 reactivator (FIG. 10A). The reduction in mutant p53 protein levels is timedependent, with a 50% reduction in CD4 T-cells and a 38% reduction in CD8 T-cells observed at 72 hours (FIG. 10B). This finding was confirmed by Western blot analysis (FIG. 11), which showed increased expression of p21 and MDM2 following p53 reactivator treatment, suggesting that the reactivator either facilitates the degradation of misfolded mutant p53 or promotes its refolding into a wild-type-like conformation.

[0163] To further investigate the impact of mutant p53 on T-cell phenotype, a differential expression analysis comparing mutant p53 -expressing T-cell subsets to those not expressing the mutant protein was conducted. Interestingly, T-cells expressing mutant p53 exhibited significantly increased levels of exhaustion markers: PD-1, TIM-3, and CD39 in CD4 T-cells, and LAG-3, TIGIT, and PD-1 in CD8 T-cells (FIG. 10C). Additionally, there was a significant reduction in the expression of naive memory markers, CD45RA and CD62L, in CD8 T-cells. These findings corroborate the previous observations that mutant p53 induces T-cell exhaustion and terminal differentiation. To further investigate the functional impact of p53 reactivation on CAR-T cells, a real-time cell killing assay was performed using the InCuCyte system. The data demonstrated that p53 reactivator-treated p53mt CAR-T cells exhibited improved cytotoxic capacity (FIG. 10D), confirming that p53 reactivation enhances the anti-AML efficacy of CAR-T cells.

[0164] To explore the mechanisms underlying the functional restoration of p53mt CAR-T cells following p53 reactivator treatment, CyTOF analysis was conducted. Using high-dimensional reduction analysis via UMAP, cellular similarities were visualized on a two-dimensional map. The results demonstrated that p53mt CAR-T cells treated with the reactivator clustered predominantly in the top right of the map, while untreated cells were located in the bottom left, with some overlap between the two groups (FIG. 10E). This spatial distribution suggests a significant alteration in the proteomic landscape of p53mt CAR-T cells post-reactivator treatment. To further characterize the subpopulations within the CD4 and CD 8 T-cell compartments, we employed the unsupervised clustering algorithm Phenograph, which identified 13 distinct clusterswithin the entire T-cell population (FIG. 10E). Heatmap analysis revealed that each cluster exhibited a unique profile, and the proportions of these clusters were markedly altered following p53 reactivator treatment (FIG. 10F, 10G). Notably, the proportions of clusters 1, 3, and 5 were significantly reduced upon treatment. Phenotypic analysis of these clusters indicated that they were enriched for mutant p53 -positive subsets.

[0165] To gain deeper insights into the landscape changes, certain clusters were consolidated into five meta-clusters based on their shared phenotypic characteristics. This strategy facilitated a more comprehensive visualization of the modifications driven by p53 reactivator treatment. Metacluster 1, defined by elevated PD-1 and mutant p53 expression, declined from 43% to 25%. Metacluster 3, enriched in exhaustion markers including TIGIT, TIM-3, LAG-3, and CD39, decreased from 22% to 11%. Conversely, meta-cluster 2, lacking mutant p53 expression, expanded from 5% to 16%. Furthermore, meta-cluster 7, representing a less differentiated T-cell subset, grew from 4% to 11% (FIG. 10H). These findings were substantiated by the exhaustion score, as detailed in the methods section, which revealed a significant reduction in exhaustion levels in p53mt CAR-T cells following p53 reactivator treatment (FIG. 101). Taken together, these data indicate that the p53 reactivator rescues the anti-tumor efficacy of p53mt CAR-T cells by correcting mutant p53, alleviating exhaustion, and optimizing their phenotypic and functional properties, ultimately enhancing their therapeutic potential against AML.EXAMPLE 8Reactivation of Mutant p53 in CAR-T Cells Prolongs Mouse Survival

[0166] In vivo experiments further validated the therapeutic potential of modifying mutant p53 in CAR-T cells. Using a Venetoclax-resistant AML mouse model, the anti-tumor efficacy of untreated p53-mut CAR-T cells was compared to those pre- treated with the p53 reactivator. Mice treated with pre-treated p53-mut CAR-T cells demonstrated significantly improved survival rates, emphasizing the transformative impact of the p53 reactivator on the efficacy of CAR-T cell therapy against AML (FIG. 12). This approach not only alleviates T cell exhaustion but also significantly enhances their tumor-killing performance, representing a significant advancement in CAR-T cell treatment strategies.

[0167] To further evaluate the in vivo efficacy of the p53 reactivator in restoring the anti-tumor function of p53 mutant (p53mt) CAR-T cells, the venetoclax-resistant patient-derived xenograft (PDX) mouse model was employed. Mice received a single intravenous dose of 3 x 10A6 T cells expressing anti-CD123 CAR and mutant p53 pre-treated with the p53 reactivator. Control groups included T cells expressing anti-CD123 CAR with or without mutant p53, or empty vector-transduced T cells, administered on day 1 following confirmation of engraftment in mouse blood (FIG. 13A). Mouse survival and circulating AML cells were monitored via flow cytometry. Human cells in the blood were identified using anti-human CD45 antibodies, followed by CD3 and CD33 staining to distinguish T cells from AML cells (FIG. 13B).

[0168] Prior to T-cell administration, AML cell distribution was consistent across all groups. By day 20 post-treatment, AML cells were effectively eradicated in mice receiving reactivator pretreated p53mt CAR“,T cells, as well as those treated with control CAR-T cells. In contrast, mice receiving untreated p53mt CAR-T cells exhibited persistent AML burden at this time point. By day 80 post-treatment, circulating AML cells reappeared in the reactivator pre-treated p53mt CAR-T cell group but remained significantly lower compared to untreated p53mt CAR-T cell- treated mice (FIG. 13C). These results indicate that while the p53 reactivator enhances the antiAML function of p53mt CAR-T cells, their effects may wane over time. This decline could be attributed to progressive T-cell exhaustion, partial reversion of p53 activity, or downregulation of essential survival and metabolic pathways. Further studies are required to determine whether continuous or intermittent administration of the reactivator can sustain CAR-T cell function and improve long-term therapeutic outcomes.

[0169] Additionally, analysis of circulating CD3+ T cells revealed persistent T-cell circulation in both the reactivator pre-treated and untreated p53mt CAR-T cell groups. However, the reactivator pre-treated group exhibited a higher frequency of circulating T cells at both day 20 and day 80 (FIG. 13D), which correlated with improved mouse survival. This observation aligns with prior studies21-23 demonstrating that prolonged CAR-T cell persistence is associated with better therapeutic outcomes. Consistently, it was observed that pre-treated p53mt CAR-T cells significantly extended mouse survival compared to untreated p53mt CAR-T cells, while no significant survival difference was observed between the reactivator pre-treated group and the control CAR-T cell-treated group (FIG. 13E). Collectively, these findings provide compelling evidence that the p53 reactivator restores the anti-tumor function of p53mt CAR-T cells, leading to survival benefits comparable to those of control CAR-T cells.EXAMPLE 9Discussion

[0170] TP53 mutations, frequently observed in cancers, compromise the DNA binding capacity of p53 and diminish its tumor-suppressing abilities. In de novo acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS), TP53 mutations are found in approximately 5-10% of cases, with increases to 30-40% in therapy-related or relapsed cases. These mutations aresignificant prognostic factors in AML and MDS, invariably correlating with poor outcomes and resistance to standard chemotherapy. As a result, immunotherapeutic approaches are under investigation, including CD47-blocking therapies, immune checkpoint inhibitors, and T-cell and NK-cell-based cellular therapies. Despite variable success of these therapies in other malignancies, the clinical response of TP53-mutant AML, particularly cases with bi-allelic p53 or locus loss, has been dismal.

[0171] Currently, it is widely believed that the bone marrow microenvironment in leukemia is the primary mechanism behind the failure of immunotherapy. This belief has driven the development of numerous therapeutic strategies aimed at modifying the microenvironment, such as stromal-targeting agents, hypoxia-modifying drugs, and immune checkpoint inhibitors. While these approaches have shown promise in preclinical models and select patient cohorts, they have largely failed to achieve durable responses in TP53 -mutant AML. The present disclosure suggests that an alternative mechanism, specifically the intrinsic dysfunction of TP53-mutant immune cells, may also contribute significantly to immune escape and therapy resistance. However, based on described herein observations of immune cell alterations in bone marrow samples from AML patients carrying TP53 mutations, the present disclosure proposes a new mechanism. Specifically, it is suggested that TP53 mutations in immune cells induce their dysfunction, allowing AML cells to evade immune surveillance. Described herein is the first report linking TP53 mutations in T- cells to T-cell dysfunction.

[0172] Although some studies have detected TP53 mutations in T-cells from AML patients, they primarily identified these mutations as indicators of early pre-leukemic events. For instance, John Dick and his team reported that TP53 mutations in T-cells serve as markers for pre-leukemic mutations, without elucidating their functional impact. Similarly, Takahashi noted the presence of TP53 mutations in T-cells in the context of evolving pre-leukemic mutations and the development of AML. Another study published in Blood highlighted the persistence of TP53 mutations in T- cells but did not consider the potential impact on immune function. Collectively, these studies did not investigate whether TP53 mutations affect T-cell activity or contribute to therapeutic resistance. In contrast, the present disclosure addresses this critical gap by demonstrating that TP53 -mutant T cells exhibit impaired cytotoxicity and increased exhaustion, thereby promoting immune evasion and AML progression. Notably, this contrasts with a recent report by Garcia et al., which showed that certain mutations derived from T cell malignancies such as the CARD11- PIK3R3 fusion can enhance CAR-T cell efficacy. Together, these findings underscore the context-dependent effects of somatic mutations in T cells, which may either hinder or augment anti-tumor immunity depending on the specific mutation and its downstream signaling pathways.

[0173] Utilizing advanced single-cell technologies, including scDNA sequencing integrated with surface antigen expression profiling, a comprehensive analysis of TP53 mutations was conducted across diverse immune cell populations at single-cell resolution. Subsequent validation via ddPCR confirmed the presence of TP53 mutations in multiple immune subsets, including T cells, NK cells, B cells, and monocytes. Since most immunotherapies focus on activating and restoring T-cell function, the present disclosure focused on T-cells carrying TP53 mutations to understand their impact on immune cells. The described herein scDNA sequencing data revealed that TP53 -mutant T-cells predominantly harbored monoallelic mutations and exhibited a distinct proteomic profile compared to their TP53 wild-type (WT) counterparts. Notably, these mutant T- cells demonstrated heightened proliferation, a characteristic frequently associated with p53 loss- of- function. Given that the p53 protein functions as a tetramer, mutant p53 can bind to wild-type p53, potentially causing a dominant-negative effect, which impairs the function of the remaining wild-type allele.

[0174] Due to technological constraints, directly assessing the function of p53 mutant T-cells derived from patients was not feasible. To address this limitation, p53 mutant CAR-T cells were generated from healthy donor T-cells, enabling a controlled investigation into the effects of mutant p53 expression in T-cells. This approach allowed the evaluation of the anti-tumor functionality of T-cells harboring mutant p53 against AML cell targets. The described herein experimental data demonstrated that p53 mutant CAR-T cells exhibited significantly enhanced proliferation, as evidenced by elevated expression of Ki67, CD69, and CD1 lb, compared to control CAR-T cells lacking mutant p53 expression. Furthermore, these p53 mutant CAR-T cells displayed an increased expression of exhaustion markers, including PD-1, TIM3, TIGIT, LAG3, and CD39, aligning with the described herein scRNAseq analysis of T-cells from 24 AML patients. Specifically, T-cells from TP53 -mutant patients consistently exhibited higher exhaustion marker expression. Cytotoxicity assays, conducted both in vitro and in vivo, further reinforced the hypothesis that mutant p53 compromises T-cell anti-tumor functionality.

[0175] To substantiate the hypothesis that mutant p53 drives T-cell dysfunction, a targeted molecule, the p53-Y220C reactivator was employed, which selectively restores the wild- type conformation of the p53-Y220C mutant. Although this compound remains under clinical investigation, its ability to reestablish wild- type p53 functionality has been demonstrated in preclinical studies. Treatment of p53 mutant CAR-T cells with this reactivator resulted in a markedreduction in mutant p53 levels and a concomitant decrease in exhaustion marker expression in T- cells. Consequently, the anti-AML activity of p53 mutant CAR-T cells was significantly restored in both in vitro and in vivo models. These findings further support the hypothesis that mutant p53 actively contributes to T-cell dysfunction and that restoring its wild-type function can reverse this impairment.

[0176] The present disclosure reveals a new mechanism that may contribute to CAR-T therapy failure in AML. While previous research has focused on challenges such as the absence of ideal surface targets on AML cells and the suppressive tumor microenvironment, the role of mutations within the T-cells themselves has been largely overlooked. The present disclosure highlights TP53 mutations in T-cells from AML patients as a potential factor impairing T-cell anti-tumor function and leading to CAR-T cell therapy failure. Although TP53 mutations are present in only a subset of patient-derived T-cells, it was observed that these mutant T-cells exhibited significantly enhanced proliferative capacity compared to their wild-type counterparts under in vitro culture conditions. This unchecked proliferation raises concerns that TP53-mutant T-cells could expand disproportionately within CAR-T products prior to infusion, potentially leading to an exhausted and dysfunctional phenotype. Given that T-cell exhaustion is a well-established contributor to reduced CAR-T efficacy, the present disclosure suggest that the presence of mutant p53- expressing T-cells could directly compromise therapeutic outcomes. These results suggest that TP53 mutations not only serve as prognostic biomarkers but also actively drive immune dysfunction, a critical determinant of immunotherapy success. By demonstrating that mutant p53 can be selectively targeted to restore T-cell function, our research opens new avenues for therapeutic intervention. Collectively, the present disclosure provides compelling evidence that p53-mutant T-cells contribute to immune suppression in AML, and proof-of-concept that restoring wild-type p53 activity in these cells can reverse their dysfunction and reinvigorate their anti-tumor capabilities was established. This concept is being tested in a clinical trial of Rezatapopt (PC 14586) in patients with MDS and AML carrying TP53-y220c mutations.References1 Srivastava, S., Wang, S., Tong, Y. A., Hao, Z. M. & Chang, E. H. Dominant negative effect of a germ-line mutant p53: a step fostering tumorigenesis. Cancer Res 53, 4452-4455 (1993).2 Bejar, R. et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med 364, 2496-2506 (2011).313 Prochazka, K. T. et al. Clinical implications of subclonal TP53 mutaHons in acute myeloid leukemia. Haematologica 104, 516-523 (2019).4 Facon, T. et al. Daratumumab plus Lenalidomide and Dexamethasone for Untreated Myeloma. N Engl J Med 380, 2104-2115 (2019).5 Tian, C. & Chen, Z. Immune therapy: a new therapy for acute myeloid leukemia. Blood Sei 5, 15-24 (2023).6 Vadakekolathu, J. et al. TP53 abnormalities correlate with immune infiltration and associate with response to flotetuzumab immunotherapy in AML. Blood Adv 4, 5011-5024 (2020).7 Abolhalaj, M. et al. Transcriptional profiling demonstrates altered characteristics of CD8(+) cytotoxic T-cells and regulatory T-cells in TP53-mutated acute myeloid leukemia. Cancer Med 11, 3023-3032 (2022).8 Shlush, L. I. et al. Identification of pre-leukemic hematopoietic stem cells in acute leukemia. Nature 506, 328-333 (2014).9 Morita, K. et al. Clonal evolution of acute myeloid leukemia revealed by high-throughput single-cell genomics. Nat Commun 11, 5327 (2020).10 Daver, N. et al. Abstract LB-222: Polyfunctionality determined by single-cell proteomics of bone marrow-derived CD4 T cells from patients with acute myeloid leukemia identifies patients responding to anH-PD-1 -based therapy and discovers profound T cell defect in mutant TP53 disease. Cancer Research 79, LB-222-LB-222 (2019).11 Xu, C. et al. Automatic cell-type harmonization and integration across Human Cell Atlas datasets. Cell 186, 5876-5891 e5820 (2023).12 Muluoglu, M. et al. Extended live-cell barcoding approach for multiplexed mass cytometry. Sci Rep 11, 12388 (2021).13 Issa, G. C. et al. Somatic MutaHons in TP53 Identified in T-Cells of Patients with TP53- mutated AML. Blood 140, 6267-6269 (2022).14 Li, B. et al. Cis interactions between CD2 and its ligands on T cells are required for T cell Chavon. Sci Immunol 7, eabn6373 (2022).15 Intlekofer, A. M. et al. Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol 6, 1236-1244 (2005).16 Hendriks, J. et al. CD27 is required for generation and long-term maintenance of T cell immunity. Nat Immunol 1, 433-440 (2000).17 Andrews, L. P. et al. LAG-3 and PD-1 synergize on CD8(+) T cells to drive T cell exhaustion and hinder autocrine IFN-gamma-dependent anti-tumor immunity. Cell 187, 4355- 4372 e4322 (2024).18 Ishimaru, N., Kishimoto, H., Hayashi, Y. & Sprent, J. Regulation of naive T cell function by the NF-kappaB2 pathway. Nat Immunol 7, 763-772 (2006).19 Dumbrava, E. E. et al. First- in-human study of PC14586, a small molecule structural corrector of Y220C mutant p53, in patients with advanced solid tumors harboring a TP53 Y220C mutation. Journal of Clinical Oncology 40, 3003-3003 (2022).20 Carter, B. Z. et al. Selective Targeting of TP53-Y220C Mutant AML By PC14586 Results in TP53 Wild-Type Conformation and Synergetic Apoptosis Induction By Concomitant Inhibition of Xpo-1, MDM2, or Bcl-2. Blood 142, 2261-2261 (2023).21 Mu-Mosley, H. et al. Transgenic Expression of IL15 Retains CD 123 -Redirected T Cells in a Less Differentiated State Resulting in Improved Anti-AML Activity in Autologous AML PDX Models. Front Immunol 13, 880108 (2022).22 Maude, S. L. et al. Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 378, 439-448 (2018).23 Laetsch, T. W. et al. Three- Year Update of Tisagenlecleucel in Pediatric and Young Adult Patients With Relapsed / Refractory Acute Lymphoblastic Leukemia in the ELIANA Trial. J Clin Oncol 41, 1664-1669 (2023).24 Bernard, E. et al. Implications of TP53 allelic state for genome stability, clinical presentation and outcomes in myelodysplastic syndromes. Nat Med 26, 1549-1556 (2020).25 Cancer Genome Atlas Research, N. et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 368, 2059-2074 (2013).26 Kadia, T. M. et al. TP53 mutaHons in newly diagnosed acute myeloid leukemia: Clinicomolecular characteristics, response to therapy, and outcomes. Cancer 122, 3484-3491 (2016).27 Kim, K. et al. Outcomes of TP53-mutant acute myeloid leukemia with decitabine and venetoclax. Cancer 127, 3772-3781 (2021).28 Baryawno, N. et al. A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia. Cell 177, 1915-1932 el916 (2019).29 Portwood, S. et al. Activity of the hypoxia-activated prodrug, TH-302, in preclinical human acute myeloid leukemia models. Clin Cancer Res 19, 6506-6519 (2013).30 Daver, N. et al. Efficacy, Safety, and Biomarkers of Response to Azacitidine and Nivolumab in Relapsed / Refractory Acute Myeloid Leukemia: A Nonrandomized, Open-Label, Phase II Study. Cancer Dis cov 9, 370-383 (2019).31 Chitadze, G. et al. Somatic TP53 mutaHons are preleukemic events in acute lymphoblastic leukemia. Blood 141, 1640-1644 (2023).32 Garcia, J. et al. Naturally occurring T cell mutaHons enhance engineered T cell therapies. Nature 626, 626-634 (2024).33 Kato, S. et al. Understanding the function-structure and function-mutation relationship of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proc Natl Acad Sci U S A 100, 8424-8429 (2003).34 Hegi, M. E. et al. p53 transdominance but no gain of function in mouse brain tumor model. Cancer Res 60, 3019-3024 (2000).35 de Vries, A. et al. Targeted point mutaHons of p53 lead to dominant-negative inhibition of wild-type p53 function. Proc Natl Acad Sci U S A 99, 2948-2953 (2002).

[0177] While preferred aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the aspects of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered accordingly.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for restoring anti-tumor activity of an immune cell, comprising: contacting the immune cell with a p53 reactivator, wherein the immune cell comprises a TP53 gene mutation, thereby restoring the anti-tumor activity of the immune cell.

2. The method of claim 1, wherein the immune cell is in a subject.

3. The method of claim 2, wherein the subject has a cancer comprising a TP53 mutation.

4. The method of claim 2, wherein the immune cell of the subject comprises the TP53 mutation.

5. The method of claim 1, wherein the TP53 mutation comprises Y220C, P151A, R241G, P33R, K132N, or any combination thereof.

6. The method of claim 1, wherein the immune cell is selected from a CD4+ T cell, a CD8+ T cell, an NK cell, a dendritic cell, a macrophage, or any combination thereof.

7. The method of claim 6, wherein the immune cell is a bone marrow-resident CD4+ T cell or a bone marrow-resident CD8+ T cell.

8. The method of claim 6, wherein the immune cell is a blood-circulating CD4+ T cell or a bloodcirculating CD8+ T cell.

9. The method of claim 1 , wherein the p53 reactivator is selected from a small molecule, a nucleic acid, a peptide, a protein, a saccharide, a lipid, or any combination thereof.

10. The method of claim 9, wherein the p53 reactivator is APR-246 (eprenetapopt), COTI-2, rezatapopt (PC14586), PRIMA-1, PK7088, and NSC59984, or a combination thereof.

11. The method of claim 9, wherein the p53 reactivator is rezatapopt (PC14586).

12. The method of claim 1, wherein the p53 reactivator decreases exhaustion markers in TP53 mutant T cell.

13. The method of claim 12, wherein the exhaustion markers of the TP53 mutant T cell are selected from PD-1, TIM3, LAG3, TIGIT, or any combination thereof.

14. The method of claim 12, wherein the TP53 mutant T cell expresses CD39.

15. The method of claim 14, wherein the CD39 is an activation and an exhaustion marker of the TP53 mutant T cell.

16. The method of claim 2, wherein the subject has an acute myeloid leukemia (AML).

17. The method of claim 16, wherein an AML cancer cell from the subject has the same TP53 mutation as the immune cell.

18. A method for enhancing the CAR-T cell therapy efficacy of cancer treatment in a subject with a cancer comprising a TP53 mutation, comprising: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the CAR-T cell, thereby enhancing the CAR-T cell therapy efficacy of cancer treatment in the subject.

19. The method of claim 18, wherein the CAR-T cell comprises the TP53 mutation.

20. The method of claim 19, wherein the TP53 mutation comprises Y220C, P151 A, R241G, P33R, K132N, or any combination thereof.

21. The method of claim 18, wherein the p53 reactivator is selected from a small molecule, a nucleic acid, a peptide, a protein, a saccharide, a lipid, or any combination thereof.

22. The method of claim 18, wherein the p53 reactivator decreases exhaustion markers in TP53 mutant CAR-T cell.

23. The method of claim 22, wherein the exhaustion markers of the TP53 mutant CAR-T cell are selected from PD-1, TIM3, LAG3, TIGIT, or any combination thereof.

24. The method of claim 19, wherein the TP53 mutant CAR-T cell expresses CD39.

25. The method of claim 24, wherein the CD39 is an activation and an exhaustion marker of the TP53 mutant CAR-T cell.

26. The method of claim 18, wherein the subject has an acute myeloid leukemia (AML).

27. The method of claim 26, wherein an AML cancer cell from the subject has the same TP53 mutation as the CAR-T cell.

28. A method for treating cancer in a subject, wherein the cancer comprises a TP53 mutation, comprising: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in an immune cell of the subject, thereby treating the cancer in the subject.

29. The method of claim 28, wherein the immune cell is selected from a CD4+ T cell, a CD8+ T cell, an NK cell, a dendritic cell, a macrophage, or any combination thereof.

30. A method for preventing cancer in a subject, wherein the subject has a TP53 mutation, comprising: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the subject, thereby preventing the cancer in the subject.

1. A method for enhancing the CAR-NK cell therapy efficacy of cancer treatment in a subject with a cancer comprising a TP53 mutation, comprising: administering a p53 reactivator to the subject, wherein the p53 reactivator reduces mutant p53 protein levels in the CAR-NK cell, thereby enhancing the CAR-NK cell therapy efficacy of cancer treatment in the subject.

Images