A method of treating signs or symptoms of the aging process

Abstract

Provided herein are methods and pharmaceutical compositions for treating signs or symptoms of the aging process.

Description

A METHOD OF TREATING SIGNS OR SYMPTOMS OF THE AGING PROCESSPRIORITY CLAIM

[0001] This application ciaims priority to U.S. Provisional Application No. 63 / 625,531 filed 26 January 2024, which is hereby incorporated by reference herein in its entirety.FIELD OF THE DISCLOSURE

[0002] The instant disclosure relates generally to a method delaying or treating at least one sign or symptom of the aging process.BACKGROUND

[0003] Aging is the leading risk factor for chronic diseases that account for the bulk of morbidity , mortality, and health costs. Aging can be defined as a state of progressive functional decline at cellular, tissue, and organ level accompanied by an increase in mortality. During the aging process, specific molecular structures and pathways break down, there is a loss of homeostasis, and a failure in hemodynamics. Aging is the number one risk factor for many human disease or conditions, including diabetes, arthritis, cancer, cardiovascular diseases or conditions (e.g., hypertension, high cholesterol, ischemic / coronary heart disease heart failure), depression, chronic kidney disease, chronic obstructive pulmonary disease (COPD) and various neurodegenerative diseases or conditions (Alzheimer’s Disease, dementia, Parkinson’s Disease), to name only a few. In fact, owing to increased lifespan and subdued fertility, the world population aged 60 and over is anticipated to increase to 21.8% of the total population by 2050. Many of these people will be afflicted with diseases or conditions of the aging process. Chronological age is the major predictor for most of the diseases that account for the bulk of morbidity, mortality, and health costs across low-, middle- and high-income countries.

[0004] A fundamental aging mechanism that likely contributes to chronic age-related diseases or conditions is cellular senescence. Cellular senescence was first described by Hayflick and Moorfield in 1961 who observed that cultures of normal human fibroblasts had a limited replicative potential and eventually became irreversibly arrested. Most senescent cells assume a characteristic flattened and enlarged morphology, and over the years, many molecular phenotypes have been described, such as changes in gene expression, protein processing and chromatin organization. Conceptually, there are two broad categories of replicative cellular senescence. The first is initiated by dysfunctional myocatelomeres or other forms of genotoxic stress eliciting a DNA damage response mediated primarily by the p53 tumor suppressor pathway. The second, much less understood response, does not involve telomeres or DNA damage and is characterized by the upregulation of the CDKN2A gene (cyclin dependent kinase inhibitor pl6INK4a). These basic distinctions are however complicated by the fact that pl 6 can be upregulated by a wide variety of stresses, including some forms of genotoxic damage.

[0005] Senescent cells are viable. They survive even though they have active DNA damage responses, heightened metabolic flux, and increased local levels of Senescence- Associated Secretory Phenotype (SASP) inflammatory cytokines and other factors that can induce apoptosis. Indeed, senescent cells are better able to withstand stresses such as serum deprivation than non-senescent cells. In vivo, senescent cells appear to be removed by the immune system, rather than apoptosis or necrosis. Per one publication, it is hypothesized that (i) anti-apoptotic, pro-survival mechanisms could be upregulated in senescent cells and (ii) interfering with these protective mechanisms might achieve selective elimination of senescent cells. Based on these hypotheses, several clinically used drugs were identified that induce apoptosis preferentially of senescent cells in vitro and in vivo, leading to unproved cardiovascular function and exercise endurance, reduced osteoporosis and frailty, and extended health span in several murine systems. Senescent cells have much in common with cancer cells, such as active DNA damage responses, except senescent cells do not divide. Thus, pro-survival pathways, which when inhibited drive cancer cell apoptosis, might be good targets if the pathway is not linked to cell proliferation. Obesity is associated with increased senescent cell burden and neuropsychiatric disorders, including anxiety and depression.

[0006] Senescent cells can secrete pro-inflammatory cytokines, chemokines, and extracellular matrix proteases, which together constitute the SASP. SASP promotes chrome inflammation and can induce senescence in normal cells. At the same time, chronic inflammation accelerates the senescence of immune cells, resulting in weakened immune function and an inability to clear senescent cells and inflammatory factors, which creates a vicious cycle of inflammation and senescence. Persistently elevated inflammation levels in organs such as the bone marrow, liver, and lungs cannot be eliminated in time, leading to organ damage and aging-related diseases.

[0007] The SASP likely contributes to the correlation between senescent cell accumulation and local and systemic dysfunction and disease. Various roles and actions of SASP factors have been reported. In an autocrine maimer, SASP factors re-enforce cellular senescence of senescent cells themselves. SASP factors can also act in a paracrine manner, inducing senescence of surrounding cells, and this is termed as paracrine senescence. Tire released chemokines from senescent cells as SASP factors reportedly act on immune cells, such as NK cells, and macrophages that can scavenge senescent cells.

[0008] Though one of the important physiological roles of SASP is repairing damaged tissues, deleterious effects of SASP factors such as aging-associated inflammation and cancer have been suggested. SASP factors can globally be divided into the following major categories: soluble signaling factors (interleukins, chemokines, and growth factors), secreted proteases, and secreted insoluble components. SASP proteases can have three major effects: shedding of membrane- associated proteins resulting in soluble versions of membrane-bound receptors, cleavage, or degradation of signaling molecules, and degradation or processing of the extracellular matrix. The most prominent cytokine of the SASP is interleukin (IL)-6, a pleiotropic pro-inflammatory cytokine. Another interleukin signaling pathway that is upregulated by senescent cells is IL-1. Most senescentcells overexpress IL-8 (CXCL-8), along with GROa and GRO[i (CXCL-1 , -2; the murine CXCL-1 is named KC). Among CCL family members that are generally upregulated in senescent cells are: MCP- 2, -4, -1 (CCL-8, -13, -2), HCC-4 (CCL-16), eotaxin-3 (CCL-26), MIP-3a, and -la (CCL-20, -3). MCP-3 (CCL-7) is overexpressed by senescent liver stellate cells and by prostate and skin fibroblasts. Senescent endothelial, epithelial, and fibroblast cells express high levels of almost all the IGF-binding proteins (IGFBPs) including IGFBP-2, -3, -4, -5, and -6 and their regulators, IGFBP-rPl and IGFBP- rP2 (connective tissue growth factor — CTGF). Senescent cells also secrete increased levels of some matrix metalloproteinases (MMPs). The MMP family members that are consistently upregulated in human and mouse fibroblasts undergoing replicative or stress-induced senescence are stromelysin- 1 and -2 (respectively, MMP-3 and -10) and collagenase- 1 (MMP-1).

[0009] Moreover, there are a plethora of signs or symptoms related to aging. Examples include but certainly are not limited to chronic inflammation accompanied by cellular senescence, organ dysfunction, and age-related diseases. Immunosenescence, which is another example of a sign or symptom related to aging, is mainly manifested by a decrease of the body’s immune response to endogenous and exogenous antigens, leads to a decrease of the individual’s anti-tumor capacity and the ability to clear senescent cells. Immunosenescence is a multifactorial cascade of events with different types of immune cells exhibiting different sensitivities.

[0010] Senescence of Hematopoietic stem cells (HSCs) are the basis of immunosenescence. Senescent HSC differentiate into various types of dysfunctional immune cells, driving immunosenescence. Inflammation drives impaired self-renewal activity and accelerates aging of HSCs. Immunosenescence of neutrophils occurs in a low-grade inflammatory environment, with specific abnormalities in their metabolism and function, including decreased phagocytic capacity’, abnormalities in adhesion and chemotaxis, and increased apoptosis. The deterioration of macrophage function is a critical contributor to immunosenescence, where the capability of macrophages to effectively clear senescent cells from tissues reduces with aging. Age-related changes in B ceil composition are the main reason for decreased antibody response to vaccination and infection in older adults. With aging, the number of T helper cells (Th) and T regulatory cells (Treg) increases. The levels of cytokines secreted by Thl and Th2 cells diminish with age, making the body less able to defend itself against external pathogens. Elderly individuals exhibit increased expression of TGF-P receptor 3 (TGF[1R3) on naive CD4 cells.

[0011] Recent findings indicate that NK cells play a central role in the immune surveillance of aging cells, and that dysfunctional NK cell activity is associated with infections, malignant tumors, inflammatory diseases, and an increased burden of aging cells with advancing age. While age does not seem to affect the number of NK progenitors in the peripheral blood or bone marrow, most studies suggest that the aging process causes an elevation of the overall number of NK cells in older adults. However, this increase in NK cell number is accompanied by a decline in their ability to proliferate and kill targets. Due to age-related functional decline, NK cells from younger donorsexhibit a greater potential for expansion than those from older donors when subjected to in vitro stimulation with IL-2, underscoring the susceptibility of NK cells to age-related dysfunction. Also, the signs of reduced NK cell effector functions, such as decreased cytotoxicity, as well as lower expression of perforin and granzyme and reduced secretion of IFN-a and IFN-y but more IL-1, IL-4, IL-6, IL-8, IL- 10, and TNF-a are identified.

[0012] Other examples of molecular, phenotypical and genomic biomarkers related to aging include, but certainly are not limited to telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication, to name only a few.

[0013] Accordingly, what is needed is a method to modulate the signs or symptoms of aging in a way that modulates the biomarkers related thereto that prevents further aging or may reverse the aging process.

[0014] The citation of any reference herein should not be deemed as an admission that such reference is available as prior art to the instant disclosure.SUMMARY OF THE DISCLOSURE

[0015] Broadly, the present disclosure extends to a method for selectively killing senescent cells, comprising contacting the senescent cells with placenta-derived hematopoietic stem cell derived NK cells.

[0016] Also provided is a method of decreasing expression level of one or more stress ligands or one or more SASP cytokines by senescent cells, comprising contacting the senescent cells with placenta- derived hematopoietic stem cell derived NK cells such that the expression level of the one or more stress ligands or the one or more SASP cytokines by the NK cell contacted senescent cells is less than the expression level of the one or more stress ligands or the one or more SASP cytokines by the senescent cells prior to contact with the NK cells.

[0017] hi a method of the instant disclosure, killing of senescent ceils results in a decrease in the expression level of one or more stress ligands or one or more SASP cytokines in the senescent cells compared to the expression level of the one or more stress ligands or one or more SASP cytokines in the senescent cells prior to contact with the placenta-derived hematopoietic stem cell derived NK cells. Examples of one or more stress ligands include, but certainly are not limited to CD58, CD155, ULBP2 / 5 / 6, or MIC-A / b. Likewise, particular SASP cytokines whose expression level decreases in a method of the instant disclosure comprise IL- 1 alpha, IL -6, IL-8, CCL5, or any combination thereof.

[0018] Numerous placenta-derived hematopoietic stem cell derived NK cells have applications in a method of the instant disclosure. Particular placenta-derived hematopoietic stem cell derived NK cells having applications in a method of the instant disclosure are CYNK-001 NK cells, CYNK-201 NK cells, or a combination of CYNK-001 and CYNK-201 NK cells. In a particular embodiment of a method of the instant disclosure, the placenta-derived hematopoietic stem cell derived NK cells arehuman placenta-derived hematopoietic stem cell derived NK cells. Moreover, in a method of the instant disclosure, the senescent cells can be selectively killed in vivo, in vitro, or ex vivo, and in a particular embodiment, the senescent cells are cancer cells that have reached senescence.

[0019] The instant disclosure further extends to a method for treating at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering a therapeutically effective amount of placenta-derived hematopoietic stem cell derived natural killer (NK) cells to the subject in order to modulate a biomarker of aging in the subject.

[0020] Also provided is a method for treating at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering a therapeutically effective amount of a pharmaceutical composition of the instant disclosure (described infra)'.

[0021] Examples of signs and symptoms of aging include, but certainly not limited to an increased number of senescent cells in a subject of particular chronological age as compared to the number of senescent cells in the subject at an earlier chronological age than the particular chronological age, or as compared to the number of senescent cells in a second subject at an earlier chronological age than the particular chronological age, age-related inflammation, telomere attrition in cells of the subject, impaired homeostasis, epigenetic alterations of cells in the subject, genetic instability of cells in the subject, deregulated nutrient sensing; or mitochondrial dysfunction.

[0022] In a method of the instant disclosure, the at least one sign or symptom of the aging process includes treating or delaying tire onset of a disease or condition related to the aging process. Examples of at least one sign or symptom of the aging process in a subject of a particular chronological age that can be treated with a method of the instant disclosure include, but certainly are not limited to diabetes, arthritis, cancer, cardiovascular (hypertension, high cholesterol, ischemic / coronary heart disease heart failure), depression, chronic kidney disease, chronic obstructive pulmonary disease and various neurodegenerative conditions (Alzheimer’s Disease, dementia, Parkinson’s Disease), to name only a few.

[0023] Moreover, examples of biomarkers of aging that can be modulated in a method of the instant disclosure include, but certain are not limited to the length of telomeres in the subject's leukocytes, immunohistochemistry of gamma-H2Al , levels of senescence-associated beta-galactosidase levels of an inflammatory biomarker, levels of 8-isoprostaglandin f2alpha (8-isoprostane), a phenotype biomarker, levels of an SASP cytokine, as well as any combination of these biomarkers.Examples of phenotype biomarkers that can modulated in a method of tire instant disclosure include physical function, e.g., walking speed, chair stand, standing balance, grip strength, body mass index, waist circumference, muscle mass, and sarcopenia, to name only a few.Anthropometry biomarkers having applications in the instant disclosure comprise quantifying one or more facial features based on three-dimensional facial images, wherein such facial features comprise mouth width, nose width, eye comer droop, or any combination thereof.

[0024] hi a method of the instant disclosure, a biomarker of aging is modulated. This modulation comprises: (a) preventing telomere attrition in cells of the subject, (b) increasing the length of telomeres in cells of the subject, (c) decreasing levels of gamma H2A-X in cells of the subject, (d) decreasing the level of an inflammatory marker in cells of the subject, (e) decreasing the levels of 8- isoprostaglandin F 2-alpha ( 8-isoprostane ) in cells of the subject, (f) decreasing the level of the serum marker in cells of the subject, (f) decreasing levels of an SASP cytokine, or any combination (a)-(f).

[0025] Numerous types of cells can be senescent cells in a method of the instant disclosure. In a particular embodiment, the senescent cells are cancer cells that have reached senescence.

[0026] Furthermore, the instant disclosure extends to a pharmaceutical composition comprising human placenta-derived hematopoietic stem cells as disclosed herein, and a pharmaceutically acceptable carrier. In a particular an embodiment of a pharmaceutical composition of the instant disclosure, the human placenta-derived hematopoietic stem cells of a pharmaceutical composition of die instant disclosure can be CYNK-001 NK cells, CYNK-201 NK cells, or a combination of CYNK- 001 and CYNK-201 NK ceils. Such a pharmaceutical composition as disclosed herein can be used in a medicament for method for treating at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering a therapeutically effective amount of the medicament to the subject in order to modulate a biomarker of aging in the subject.

[0027] Furthermore, die instant disclosure extends to a method of decreasing expression level of one or more stress ligands or one or more SASP cytokines by senescent cells, comprising contacting the senescent cells with a pharmaceutical composition as disclosed herein, such diat die expression level of the one or more stress ligands or the one or more SASP cytokines by senescent cells contacted with a pharmaceutical composition of the distant disclosure is less tiian the expression level of the one or more stress ligands or the one or more SASP cytokines by die senescent cells prior to contact the pharmaceutical composition of die instant disclosure.

[0028] hi another embodiment, the instant disclosure extends to a mediod for treating at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering to die subject a therapeutically effective amount of a pharmaceutical composition of the instant disclosure in order to modulate a biomarker of aging in the subject. As explained above, examples of signs and symptoms of aging include, but certainly not limited to an increased number of senescent cells in a subject of particular chronological age as compared to the number of senescent cells in the subject at an earlier chronological age than the particular chronological age, or as compared to die number of senescent cells in a second subject at an earlier chronological age than the particular chronological age, age-related inflammation, telomere attrition in cells of the subject, impaired homeostasis, epigenetic alterations of cells in the subject, genetic instability of cells in the subject, deregulated nutrient sensing; or mitochondrial dysfunction. Administering a therapeutically effective amount of a pharmaceutical composition of the instant disclosure can be topical administration, intradermal

[0029] Numerous routes of administration of a pharmaceutical composition have applications in a method of the instant disclosure. Examples include topical administration as well as parenteral administration. Particular examples of parenteral routes of administration having applications in a method of the instant disclosure include subcutaneous administration, intramuscular administration, intravenous administration, and intrathecal administration. A particular objective of a route of administration having applications in a method of the instant disclosure is to enable a pharmaceutical composition of the instant disclosure to be delivered to the subject systemically.

[0030] Furthermore, a method for treating at least one sign or symptom of the aging process in a subject of a particular chronological age as disclosed herein can further comprise tire step of administering to the subject a therapeutically effective amount of a therapeutic agent. In a particular embodiment, tire therapeutic agent has applications in treating at least one sign or symptom of the aging process. The administration of the therapeutic agent can occur prior to, simultaneously, concomitantly or serially with administration of a pharmaceutical composition as described herein. Moreover, a pharmaceutical composition of the instant disclosure can further comprise the therapeutic agent such that it is a mixture of a pharmaceutical composition as disclosed herein and the therapeutic agent.

[0031] Numerous therapeutic agents have applications in a method or a pharmaceutical composition as described herein. Particular examples include, but certainly are not limited to, a non-steroidal antiinflammatory drug, e.g., ibuprofen, naproxen, aspirin, acetaminophen, a cyclooxygenase inhibitor (COX-1 or COX-2), hyaluronic acid, a steroid, a biologic or a biguanide antihyperglycemic agent, e.g. metformin.

[0032] Furthermore, NK cells having applications in a method and a pharmaceutical composition of the instant disclosure can be cryopreserved. Cryopreservation of cells and tissues is useful for the long-term storage of cell lines to provide an rmchanging population of cells for storage of populations of cells for research or medical purposes. Thus, cry opreservation enables the stability of the cells or a pharmaceutical composition of the instant disclosure to be maintained for a period of time sufficient to enable shipping of the ceils, and if necessary, storage of the cells until use. Prior to use, the ceils or a pharmaceutical composition of the instant disclosure can be thawed and prepared for administration to the subject. Numerous methods of cryopreserving cells have applications herein. Numerous methods of cryopreserving cells readily have applications in the instant disclosure and are well known to those of ordinary skill in art. Particular methods having applications herein are set forth in U.S. published patent application No. 20150225697, and issued US Patents 10292382 and 10472606, all of which are hereby incorporated by reference herein in their entireties.

[0033] The instant disclosure further extends to the use of a pharmaceutical composition disclosed herein in a medicament for treating or delaying onset of at least one sign or symptom of the aging process in a subject of a particular chronological age.

[0034] These and other aspects of the present disclosure will be better appreciated by reference to the following drawings and Detailed Description.DESCRIPTION OF THE FIGURESFIG. 1 are images showing the senescence associated [i -Galactosidase (SA-[3-GaI) enzymatic activity of non-treated A549 cells (NT), A549 cells treated with etoposide (Etop) as disclosed herein, and A549 cells treated with doxorubicin (Doxo) as described herein.FIG. 2 are histograms of flow cytometry data showing SA-P-Gal activity in non-treated A549 cells (NT), A549 cells treated with Etop, and A549 cells treated with Doxo as disclosed herein. The data were presented as a percentage of SA-|3-Gal positive cells and mean fluorescence intensity’ (MFI). Additionally, the mean ± SEM of the enzymatic activity for these 3 groups from 3 independent experiments is included.FIG. 3 are bar graphs showing the mean ± SEM levels of SASP cytokines IL-Ia, IL-6, IL-8, CCL5, and CCL2 measured by multiplex Luminex cytokine assays in conditioned medium derived from A549 cell culture with or without Etop- or Doxo-induced senescence from 2 independent experiments.FIG. 4 are bar graphs of the percent of cells expressing the indicated stress ligands in non-treated A549 cells (NT), or upon Etop or Doxo treatment as disclosed herein determined by flow cytometry analysis. The data were presented as the mean ± SEM percentage of stress ligand positive cells from 3 independent experiments.FIG. 5 are graphical representation of cytotoxicity activities of CYNK-001 (N=4) and CYNK-201 (N=3) cells against A549 cells with or without drug-induced senescence measured by flow cytometry after 24 hours of co-culture with various effector to target cell ratios (x-axis). Statistical significance (**, P<0.01, ****, P<0.0001) was analyzed by two-way ANOVA.FIG. 6 are bar graphs of the levels of the inflammatory cytokines G-CSF, GM-CSF, TNF-a and IFN-y as measured by multiplex Luminex cytokine assays in conditioned medium collected from non-treated A549 cells (NT-A549), Etop treated A549 cells (Etop-A549 cells), Doxo treated A549 cells (Doxo- A549 cells), A549 cells alone, or A549 cells co-cultured with CYNK-001 cells (N=6) for 24 hours. Statistical significance (*, P<0.05, ***, P<0.005 for CYNK-001 with Etop-A549 or with Doxo-A549 compared to CYNK-001 with NT-A549) was analyzed by two-way ANOVA.DETAILED DESCRIPTION

[0035] Broadly, the present disclosure extends to a method for treating at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering atherapeutically effective amount of placenta-derived hematopoietic stem cell derived natural killer (NK) cells to the subject in order to modulate a biomarker of aging in the subject.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0037] All numerical designations, e.g. volume, mass, number of resin particles, etc., are approximations which are varied by (+) or (-) by increments of 1.0 or 0.1, as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about".

[0038] Numerous terms and phrases are used throughout the instant specification and claims and are defined below.

[0039] “About” and “approximately” are interchangeable and mean plus or minus a percent (e.g., ±5%) of the number, parameter, or characteristic so qualified, which would be understood as appropriate by a skilled artisan to the scientific context in which the term is utilized.

[0040] As used herein, the singular form “a,” “an” and “the” include plural reference unless the context clearly dictates otherwise.

[0041] As used herein, the terms “comprising,” “comprises” and “comprise” are intended to mean that the compositions, preparations and methods disclosed herein include recited elements, but do not exclude others.

[0042] Therefore, if appearing herein, the following terms and phrases shall have the definitions set out below.

[0043] As used herein, the phrase “the aging process” and the term “aging” can be used interchangeably and refer to the time-related deterioration of the physiological functions necessary for survival and reproduction. The decline in the regenerative potential of tissues is one of the most obvious characteristics of the aging process. For example, hematopoiesis declines with age, resulting in immunosenescence, and in an increased incidence of anemia and myeloid malignancies. Studies on aged mice have revealed an overall decrease in cell-cycle activity of hematopoietic stem cells (HSCs), with old HSCs undergoing fewer cell divisions than young HSCs. Although deficient proliferation of stem and progenitor cells is obviously detrimental for the long-term maintenance of the organism, an excessive proliferation of stem and progenitor cells can also be deleterious by accelerating the exhaustion of stem cell niches. The importance of stem cell quiescence for the long-term functionality of stem cells has been compellingly demonstrated in the case of Drosophila intestinal stem cells, in which excessive proliferation leads to exhaustion and premature aging. Recent studies have shown that an increase in Fibroblast Growth Factor (FGF2) signaling in the aged muscle stem cell niche results in the loss of quiescence and eventually in stem cell depletion and diminished regenerative capacity, whereas suppression of this signaling pathway rescues these defects. Transplantation ofmuscle-derived stem cells from young mice to progeroid mice extends lifespan and improves degenerative changes of these animals. Furthermore, parabiosis experiments have demonstrated that the decline in neural and muscle stem cell function in old mice can be reversed by systemic factors from young mice.

[0044] As used herein the term “placenta-derived” with respect to a ceil means that the source of the cell is the placenta, which is typically obtained from animals (such as pigs or sheep) or, from human placental tissue.

[0045] As used herein, “stress ligands” refers to ligands expressed on a cell that are recognized by NKG2D, a single receptor that is expressed on natural killer (NK) cells, cytotoxic T cells and other T cell subsets. Examples of stress ligands includes, but is not limited to : (i) CD58; (ii) CD155; (iii) ULBP2 / 5 / 6; or (iii) MIC-A / B,

[0046] As used herein, the term “SASP cytokine" refers to cytokines that are expressed by a cell with Senescence- Associated Secretory Phenotype (SASP), a phenotype associated with senescent cells, which secrete high levels of inflammatory cytokines, immune modulators, growth factors, and proteases. Examples are (i) IL-lalpha; (ii) IL -6, (iii) IL-8; or (iv) CCL5, to name only a few.

[0047] As used herein, the phrase “chronological age” refers to the actual amount of time a person has existed, as opposed to “biological age,” which refers to refers to epigenetic alteration and DNA methylation which express on how able and functioning a person is and whether she / he has diseases or conditions related to old age

[0048] As used herein, a “sign” with respect to aging refers to objective evidence of disease, and in the case of the instant disclosure, evidence of aging.

[0049] As used herein, the term “symptom” refers to a physical or mental feature which is regarded as indicating a condition of disease, particularly such a feature that is apparent to the patient.

[0050] As used herein, the phrase “telomere attrition” refers to the decreasing length of telomeres during cell replication over time. Telomeres are specialized chromatin structures at the ends of eukaryotic chromosomes that prevent the chromosome ends from being recognized as a DNA break. They measure betw een 5 and 15 kb. The repeat unit DNA sequence is 5'TTAGGG3’ and its complementary DNA sequence is 5’CCCTAA3’. Telomerase, which comprises protein component TERT and RNA component TERC, elongates telomerases after replication, thereby maintaining telomere length. However, mammalian somatic cells do not express telomerase, and this leads to the progressive and cumulative loss of telomere-protective sequences from chromosome ends. At each somatic cell division cycle, telomeres shorten by 50-200 bp through incomplete synthesis of the lagging strand during the DNA replication. Telomere length is an important biomarker in accessing the biological age.

[0051] As used herein, the phrase “epigenetic changes” involves alterations in DNA methylation patterns, post-translational modification of histones, and chromatin remodeling. Increased histone H4K16 acetylation, H4K20 trimethylation or H3K4 trimethylation, as well as decreased H3K9methylation or H3K27 trimethylation, constitute age-associated epigenetic marks. Global loss of canonical histones is regarded as a common feature of aging in a range of organisms from yeast to humans. In general, histone levels are decreased, and aberrant nucleosome occupancies occur during aging. The accumulation of histone variants is another common feature in the aging process. In contrast, histone modifications such as acetylation, methylation, phosphorylation, and ubiquitylation are more complex.

[0052] The term “proteostasis” refers to aging and some aging-related diseases or conditions linked to impaired protein homeostasis. Proteostasis involves mechanisms for the stabilization of correctly folded proteins, most prominently the heat-shock family of proteins, and mechanisms for the degradation of proteins by the proteasome or the lysosome. Moreover, there are regulators of age- related proteotoxicity, such as MO AG-4, which act through an alternative pathway distinct from molecular chaperones and proteases. All these systems function in a coordinated fashion to restore the structure of misfolded polypeptides or to remove and degrade them completely, thus preventing the accumulation of damaged components and assuring the continuous renewal of intracellular proteins. Accordingly, many studies have demonstrated that proteostasis is altered with aging. Chronic expression of unfolded, misfolded or aggregated proteins contributes to the development of some age-related pathologies, such as Alzheimer’s disease, Parkinson’s disease and cataracts.[0(153 J As used herein, the phrase “deregulated nutrient sensing” refers to the somatotrophic axis in mammals that comprises the growth hormone (GH), which is produced by the anterior pituitary, and its secondary mediator, insulin-like growth factor 1 (IGF-1 ), produced in response to GH by many cell types, most notably hepatocytes. The intracellular signaling pathway of IGF-1 is the same as that elicited by insulin, which informs cells of the presence of glucose. For this reason, IGF-1 and insulin signaling are known as the “insulin and IGF-1 signaling” (IIS) pathway. Remarkably, the IIS pathway is the most conserved aging-controlling pathway in evolution, and among its multiple targets are the FOXO family of transcription factors and the mTOR complexes, which are also involved in aging and conserved through evolution. The other two nutrient sensors, AMPK and sirtuins, act in the opposite direction to IIS and mTOR, meaning that they signal nutrient scarcity and catabolism instead of nutrient abundance and anabolism. Dietary restriction (DR) increases lifespan or health span in all investigated eukaryote species, including nonhuman primates. Anabolic signaling accelerates aging and decreased nutrient signaling extends longevity.

[0054] As used herein, the phrase “mitochondrial dysfunction” refers to efficacy of the respiratory chain. As cells and organisms age, the efficacy of the respiratory chain tends to diminish, thus increasing electron leakage and reducing ATP generation. The mitochondrial free radical theory of aging proposes that the progressive mitochondrial dysfunction that occurs with aging results in increased production of Reactive Oxygen Species (ROS), which in turn causes further mitochondrial deterioration and global cellular damage. The primary' effect of ROS will be the activation of compensatory homeostatic responses. As chronological age advances, cellular stress and damageincrease and the levels of ROS increase in parallel in an atempt to maintain survival. Beyond a certain threshold. ROS levels betray their original homeostatic purpose and eventually aggravate, rather than alleviate, the age-associated damage. Numerous studies have shown that elderly people are under constant and increasing assault by ROS, as indicated by enhanced lipid peroxidation, protein oxidation, and alteration of antioxidant enzyme activities.

[0055] As used herein, the phrase “cellular senescence” refers to a stable arrest of the cell cycle coupled to stereotyped phenotypic changes. Besides telomere shortening, nontelemetric DNA damage and depression of the INK4 / ARF locus, which progressively occur with chronological aging, they are also capable of inducing senescence. Some studies have directly used senescence-associated P-galactosidase (SABG) to identify senescence in tissues. Senescent cells manifest dramatic alterations in their secretome, which is particularly enriched in proinflammatory cytokines and matrix metalloproteinases and is referred to as the “senescence-associated secretory phenotype.” This proinflammatory secretome may contribute to aging. “Acute” senescence occurs when senescent cells are promptly removed from organs and contribute to tissue repair. “Chronic” senescence occurs due to the persistence of senescent cells in tissues. It promotes tissue aging and inflammation, thus enhancing tumorigenesis. Overall, cellular senescence is a beneficial compensatory response to damage that becomes deleterious and accelerates aging when tissues exhaust their regenerative capacity. A moderate enhancement of the senescence-inducing tumor suppressor pathways may extend longevity, and at the same time, elimination of senescent cells in an experimental progeria model delays age-related pathologies.[0056 J As used herein, the phrase “stem cell exhaustion” refers to the decline in the regenerative potential of tissues, and a decline in stem cell numbers and renewal capacity. It is one of the well- known signs or symptoms of the aging process. For example, hematopoiesis declines with age, resulting in an increased incidence of anemia and myeloid malignancies. Studies on aged mice have revealed an overall decrease in cell-cycle activity of hematopoietic stem cells (HSCs), with old HSCs undergoing fewer cell divisions than young HSCs. Although deficient proliferation of stem and progenitor ceils is obviously detrimental for the long-term maintenance of the organism, an excessive proliferation of stem and progenitor cells can also be deleterious by accelerating the exhaustion of stem cell niches. The importance of stem cell quiescence for the long-term functionality of stem cells has been compellingly demonstrated in the case of Drosophila intestinal stem cells, in which excessive proliferation leads to exhaustion and premature aging. Recent studies have shown that an increase in Fibroblast Growth Factor (FGF2) signaling in the aged muscle stem cell niche results in the loss of quiescence and eventually in stem cell depletion and diminished regenerative capacity, whereas suppression of this signaling pathway rescues these defects. Transplantation of muscle- derived stem cells from young mice to progeroid mice extends lifespan and improves degenerative changes of these animals. Furthermore, parabiosis experiments have demonstrated that the decline inneural and muscle stem cell function in old mice can be reversed by systemic factors from young mice.

[0057] As used herein, the phrase “altered intercellular communication” refers to changes in signals between cells (intracellular communication) that can lead to some of the diseases, conditions, signs, and symptoms of aging.

[0058] As used herein, the term “sarcopenia” refers to an age related, involuntary loss of skeletal muscle mass and strength. Prevalence of sarcopenia correspond to about 5-10% of persons over 65. It increases with age and can be over 50% for people above 80 years old. The European Working Group for Sarcopenia (EWGSOP) defines sarcopenia as muscle atrophy (normalized for height) combined with muscle weakness (measured in hand-grip muscles) and / or reduced physical performance. Evidence from animal studies has reported that an accumulation of oxidative stress damage in mitochondria, proteins and DNA during aging is a potential cause of sarcopenia and skeletal muscle damage. Sarcopenia reduces mobility, diminishes quality of life, and can lead to fall- related injuries, which require costly hospitalization and extended rehabilitation. MRI and CT scans are considered the gold standards to measure muscle mass. Muscle strength is routinely measured in the hand-grip muscles with a dynamometer and shows a correlation with several age-related diseases or conditions. A short physical performance battery (SPPB) has emerged as a promising tool to evaluate functional capacity and incorporates standing balance, 6MWT, and chair stand test in addition to gait speed. SPPB has shown high sensitivity for sarcopenia and may be a favorable tool in clinical settings. SARC-F questionnaire has emerged as a useful tool for the rapid assessment of sarcopenia. It considers various measures of functional independence such as strength, assistance in walking, rising from a chair, climbing stairs and falls. Each component is awarded a score from 0 to 10, with a maximal score of 10 in the overall assessment. A score > 4 is considered as a predictor of sarcopenia.

[0059] As used herein, the term “frailty” refers to a geriatric syndrome characterized by weakness, weight loss, and low activity that is associated with adverse health outcomes. Frailty' manifests as an age-related, biological vulnerability to stressors and decreased physiological reserves yielding a limited capacity to maintain homeostasis. The validated and widely utilized five-item frailty criteria for screening involves self-reported exhaustion, slowed performance (by walking speed ), weakness (by grip strength), unintentional weight loss (10 lbs. in past year), and low physical activity are composite outcomes of multiple organ systems. One commonly used frailty assessment tool is the Clinical Frailty Scale (CFS). It is heavily weighted to evaluate function, including the ability to mobilize and perform activities of daily living. Biomarkers for sarcopenia include musculoskeletal changes such as muscle mass, grip strength, gait velocity, body weight; serum markers have been investigated, hemoglobin, glomerular filtration rate, and albumin levels; hormones (e.g..dehydroepiandrosterone [DHEA] sulfate, testosterone, insulin-like growth factor-1 [IGF1] and vitamin D [VitD]); inflammatory’ markers such as CRP, IL-6, and tumor necrosis factor alpha.

[0060] As used herein, the phrase “natural killer ceils” refers to white blood cells that destroy infected and diseased cells, like cancer cells. They are also a type of lymphocyte, like B-cells and T- ceils. NK cells can destroy harmful cells in the early stages, preventing viruses and cancer cells from spreading, hi a particular embodiment of a method of the instant disclosure, NK ceils are derived from hematopoietic stem cells that are themselves derived from placenta. Examples of such cells having applications herein are CYNK-001 and CYNK-201 NK cells. CYNK-001 is an allogeneic, culture-expanded natural killer (NK) cell population derived from human placental hematopoietic stem cells. Previously, CYNK-001 was manufactured as PNK-007 fresh NK cells. CYNK-001 cells are the cryopreserved NK cells. CYNK-001 consists of culture-expanded NK cells that are harvested and washed in Plasma-Lyte A, then packaged at 30.0 + / - 9.0 x 106cells / mL in a total volume of 20- mL of cryopreservation solution containing 10% (w / v) HSA, 5.5% (w / v) Dextran 40, 0.21% NaCl (w / v), 32% (v / v) Plasma-Lyte A, and 5% (v / v) dimethyl sulfoxide (DMSO). It is filled into the container closure, frozen using a controlled rate freezer, and cryopreserved. When required for administration by a site, CYNK-001 is shipped in vapor phase liquid nitrogen to the designated clinical site where it is processed for dose preparation in a standardized manner just prior to IV administration.

[0061] CYNK-001 is well-characterized with respect to key cellular attributes: identity, morphology, immunophenotype, and functionality7. The identity that defines the majority (> 85%) of CYNK-001 cells is CD56-r and CD3- , as measured by flow cytometry. CYNK-001 cells morphologically appear as large granular lymphocytes, and they are roughly spherical in shape with an average cell diameter of 9.5 ± 0.1pm. CYNK-001 contains very low to non-detectable levels of CD3-r T cells (< 1.0%) or CD19+ B cells (< 1.0%), as measured by flow cytometry.

[0062] CYNK-001 demonstrates a range of biological activities expected of NK cells, including expression of perforin and granzyme B cytotoxic granules, cytolytic activity against hematological tumor cells lines and GBM solid tumor cell lines, and secretion of immunomodulatory cytokines such as IFN-y, TNF-a and GM-CSF in the presence of tumors cell lines. These cells express the nominal NK surface phenotype CD3- , CD56+, CD19- and additionally express activating receptors including NKG2D+, NKp46+, NKp30+ and DNAM-1+, intracellular cytotoxic enzymes (perforin and granzyme B). NK cells exert cytotoxic effects by releasing granules containing cytotoxic proteins such as perforin and granzymes (i.e., granzyme B) onto their targets. Tire release of granules from NK cells is dependent upon the engagement of activating receptors present on their surface with the corresponding ligands present on tumor cells.

[0063] Regarding the homing to infected tissues, CYNK-001 cells have immediate localization to the lungs following intravenous (IV) injection in the non-obese diabetic (NOD)-scid IL2Rgammanull(NSG) immune deficient mice. It has been shown that CXCR3 expression on NK cells is involved in NK cell trafficking to the lung in Influenza virus infection. CXCR3 is also involved in CXCL10- directed NK cell homing to coronavirus infected tissues. Single-cell RNA sequencing (scRNAseq) demonstrated that CYNK-001 cells highly express the CXCR3 transcript. The biodistribution and persistence of CYNK-001 through IV route of administration was evaluated along with safety assessment in single and repeat dose toxicity studies in the NSG mouse. Single or repeat IV administration of CYNK-001 at 10 x 106cells / animal (approximately 4 x 108cells / kg) was well tolerated by NSG mice, and no clinical symptoms were observed. Histological analysis of the bone marrow, liver, spleen, kidney, lung, brain, eye, small intestine, ovaries, and heart did not reveal any pathological changes related to the administration of CYNK-001. Human gDNA was detected by quantitative polymerase chain reaction (qPCR) analysis in the lung, bone marrow, liver, spleen, kidney and heart on Day 7, Day 14, and Day 21 , but not on Day 28.

[0064] Functionally, CYNK-001 cells showed in vitro cytotoxicity against multiple human tumor cell lines of gastric cancer, B cell lymphoma and acute myeloid leukemia. In vivo, intracranial injection of CYNK-001 cells significantly reduced tumor burden in a murine model of glioblastoma (CELU-2018-001). CYNK-001 cells effectively recognize and eliminate influenza A virus-infected cells in vitro, as well as mediate protection against viral infection and alleviation of symptoms in a mouse model. More importantly, according to on-going nonclinical in vitro studies, CYNK-001 cells have demonstrated up to -30% of cytotoxicity specifically against senescent cells, in a cell-culture model by which senescence is induced by pharmacological stress.

[0065] In addition to the preclinical data, favorable safety for around 71 subjects through both PNK- 007 and CYNK-001 clinical studies have been demonstrated. PNK-007 was studied among a total of 25 patients at a dose range of 1 x 106cells / kg to 3 x 107cells / kg with well-tolerated safety among both Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) patients. CYNK-001 studies were conducted in three oncology indications: - 27 subjects in AML, 9 subjects in MM, and 3 subjects in Glioblastoma (GBM). Furthermore, CYNK-001 was also evaluated among 7 subjects with SARS-COVID. Three of these studies (MM, GBM, CO VID-19) were completed with clinical study reports being submitted to the FDA. The AML study is closed for enrollment and data analysis is in process. While the CYNK-001 studies in oncology indications were conducted with or without IL-2, the COVID-19 study was conducted without lymphodepletion and IL-2 administration. Overall, CYNK-001 cells demonstrated a well-tolerated safety profile in multiple dosing regimens up to 5.4 billion cells, in approximately 46 subjects with preliminary evidence of clinically meaningful efficacy in cancer and viral disease.

[0066] CYNK-201 cells are a human placental CD34+-derived, cryopreserved, off-the-shelf, allogeneic NK cell product genetically modified to express high IgG binding affinity, protease cleavage resistant CD 16 and secreted IL-15. With the expression of high IgG binding affinity’, protease cleavage resistant CD 16, CYNK-201 cells can display significantly improved antibodydependent cellular cytotoxicity (ADCC) activity against tumor cells when combined with the treatment of therapeutic antibodies. Since IL- 15 has been shown to be important for NK cell survival, proliferation and function, the ectopic expression of this cytokine will further support the in vivo persistence and functionality of CYNK-201 cells.

[0067] CYNK-201 is well-characterized with respect to key cellular attributes: identity, morphology, immunophenotype, and functionality. The identity that defines the majority of CYNK-201 cells is CD56+ and CD3- , as measured by flow cytometry. Similar to CYNK-001 cells, CYNK-201 cells morphologically appear as large granular lymphocytes, containing very low to non-detectable levels of CD3+ T cells or CD19-t- B cells, as measured by flow cytometry. CYNK-201 cells express the nominal NK surface phenotype CD3- , CD56+, CD19-, as well as activating markers or receptors including CD1 la, NKG2D, CD226 and NKp30. CYNK-201 cells express secreted IL-15 and high level of CD16, which is resistant of shedding upon phorbol 12-myristate 13-acetate plus ionomycin (PMAi) activation.

[0068] Functionally, CYNK-201 displayed enhanced in vitro ADCC against solid tumor cell line NCI-N87 and liquid tumor cell line Daudi as compared to that of unmodified CYNK cells with combination of therapeutic antibodies trastuzumab and rituximab, respectively. 14 days post infusion, CYNK-201 cells were detectable inNSG mice in significantly higher numbers compared to unmodified CYNK cells with supplementation of recombinant IL- 15. This enhanced persistence of CYNK-201 led to significant tumor reduction in NCI-N87 tumor model in combination with trastuzumab in vivo.

[0069] As used herein, the phrase “therapeutically effective amount” refers to an amount of a therapeutic composition sufficient to produce a measurable biological response. Actual dosage levels of NK cells of the instant disclosure can be varied so as to administer an amount that is effective to achieve the desired therapeutic response for a particular subject and / or application. Of course, the therapeutically effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other therapeutic agents or treatments, severity of the condition being heated, and the physical condition and prior medical history of the subject being treated. A minimal dose is administered, and the dose is escalated in the absence of dose-limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.

[0070] As used herein, the terms “carrier” and “excipient” can be used interchangeably, and refer to a diluent, adjuvant, or carrier with which a pharmaceutical composition of the instant disclosure is administered to subject. In general, sterile water, oil, saline, aqueous dextrose (glucose), polysorbate and related sugar solutions and glycols such as propylene glycol or polyethylene glycols, proteins (e.g., serum albumin), are suitable carriers for parenteral or transdermal solutions. Solutions oremulsions for parenteral or transdermal administration may further contain about 5-15% polysorbate 80 or lecithin, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as, but not limited to, sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also useful are citric acid and its salts, and sodium EDTA. In addition, parenteral and transdermal solutions may contain preservatives including, but not limited to, benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.

[0071] As used herein, “cryopreservation”, “cryopreserved” and “cryopreserving” refer to the storage of biological material, e.g. cells, tissues or organs, at temperatures below 4° C. Generally, the intention of the cryopreservation is to maintain the biological material in a preserved or dormant state, after which time the biological material is returned to a temperature above 4° C for subsequent use. Preferably, the cryop reserving temperature is below 0° C. For example, the cryopreserving temperature may be below -5° C„ -10° C., -20° C., -60° C, or in liquid nitrogen or liquid helium, carbon dioxide (‘dry-ice’), or slurries of carbon dioxide with other solvents. In some preferred embodiments, the cryopreserving temperature is about -20° C., about -80° C. or about -180° C.

[0072] The present disclosure may be better understood by reference to the following non-limiting examples, which are provided as exemplary of the disclosure. The following examples are presented in order to more fully illustrate particular embodiments of the disclosure. They should in no way be construed, however, as limiting the broad scope of the disclosure.EXAMPLECYNK-001 Generation

[0073] Human placental CD34+ cells were prepared from healthy full-term donors with informed consent, donor eligibility documentation, and qualification using a series of tests including serology and sterility. Blood was isolated from donor tissues and processed by red blood cell depletion. The resulting cells were magnetically labeled using Direct CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Cat# 170-076-711) and positively selected by CliniMACS Cell Separator (Miltenyi Biotec, Cat# 170-076-651) following manufacturer’s protocol. Placental CD34+ cells were cultivated in the presence of various human cytokines for 35 days to generate CYNK-001 cells under current good manufacturing practices standards, followed by release testing. A cytokine cocktail containing IL -2, IL-15, SCF and IL-7 were used for placental CD34+ cells expansion and differentiation. Cells were harvested following the 35-day expansion and differentiation process and then frozen as a drug substance.CYNK-201 Generation

[0074] Human placental CD34+ cells were prepared from healthy full-term donors with informed consent, donor eligibility documentation, and qualification using a series of tests including serology and sterility. Blood was isolated from donor tissues and processed by red blood cell depletion. Theresulting cells were magnetically labeled using Direct CD34 Progenitor Cell Isolation Kit (Miitenyi Biotec, Cat# 170-076-711) and positively selected by CliniMACS Cell Separator (Miitenyi Biotec, Cat# 170-076-651) following manufacturer’s protocol. Placental CD34+ cells were then cryopreserved and stored in liquid nitrogen before use. Placental CD34+ cells were transduced with a customized lentivirus vector expressing a high binding affinity and protease resistant CD 16 variant and secreted IL-15, then cultured for up to 35 days in the presence of cytokines, including IL-2, SCF and IL-7, but not IL- 15, to generate CYNK-201 cells.Senescence Induction

[0075] Senescence was induced in the non-small-cell lung carcinoma tumor cell line A549 by 24h treatment with chemotherapy drug etoposide or doxorubicin followed by a 6-day recovery period. Senescence in A549 cells was confirmed by staining fixed cells for senescence associated |i- galactosidase (SA-β-Gal) activity or by flow cytometry’ using a fluorescent reagent (Spider p-Ga 1 (Dojindo Laboratories)) for detection of the SA-[i-Gaf activity. Upon senescence induction in A549 cells, the expression of stress ligands was examined by flow cytometry, and the secretion of senescence associated secretory’ phenotype (SASP) cytokines was monitored by multiplex Luminex analysis in conditioned medium.Cytotoxicity Assays

[0076] A549 cells either treated with or without etoposide or doxorubicin were seeded as target cells for 24-hour coculture with CYNK-001 or CYKK-201 cells as the effector cells with indicated E:T ratios. Cytotoxicity activity’ of CYNK-001 and CYNK-201 against A549 cells with or without senescence induction was assessed by flow cytometry, while inflammatory cytokine secretion in the conditioned medium was also analyzed by multiplex Luminex analysis as additional evidence of cytotoxicity.Flow cytometry'

[0077] CYNK-001, CYNK-201 and A549 cells were analyzed by multicolor flow cytometry using fluorochrome-conjugated antibodies or the corresponding isotype controls according to the manufacturer’s protocol. For cell phenotyping, the viable cells were defined for the negative staining of the Liy'e / Dead Fixable Aqua Stain (Invitrogen, Cat# L34957) or 7- Aminoactinomycin D (7-AAD) (BD Biosciences, Cat# 559925). Data were acquired on BD Fortessa X20 flow cytometer (BD Biosciences) and were analyzed using FlowJo software. The data were presented as % positive cells and mean fluorescence intensity (MFI). Setting of the % positive gate was done using the corresponding isotype-stained samples as controls.Establish Senescence Model in A549 Cells

[0078] As explained above, chemotherapeutic dings doxorubicin (Doxo) and etoposide (Etop), which are known to act via DNA damage, were used to induce senescence in non-small-ceil lung carcinoma A549 cells. Based on our pharmacokinetic titration data, A549 cells were exposed to Doxo at a concentration of lOOnM or to Etop at 6.25pM for 24 hours before 6 days of recovery. Figure 1 illustrates induction of senescence by Etop or Doxo based on increased S A-[1-Gai activity, which was stained in green color, as well as morphological alterations such as enlargement, flattening, and granulation. The increased presence of S A-[3-Gal activity? in A549 cells upon Etop- or Doxo-treatment (22.0 ± 9.0% of cells positive after etoposide treatment and 25.8 ± 1.6% after doxorubicin treatment vs. 7.4 ± 2.8% of non-treated cells) was also detected by flow cytometry? (Figure 2), which further confirms the induced senescence in this tumor cell model.

[0079] Consistent with the induced senescence, chemotherapeutic drug-treated A549 cells also displayed increased secretion of SASP cytokines IL- la, IL -6, IL-8, CCL-5 and CCL-2 compared to non-treated A549 (Figure 3). Importantly, increased expression of stress ligands such as MIC-A / B and ULBP -2 / 5 / 6 was also detected in A549 cells (Figure 4). These proteins are known to be recognized by NKG2D expressed on the NK cells.CYNK-001 and CYNK-201 Cells Preferentially Eliminate Senescent A549 Cells

[0080] Both CYNK-001 and CYNK-201 cells demonstrated significantly enhanced cytotoxicity at 24h against A549 cells with chemotherapeutic drug induced-senescence (Figure 5). With CYNK-001, at the 5:1 effector to target ratio, there was 43.7 ± 11.6% cytolysis of etoposide treated cells and 37.2 ± 14% cytolysis of doxorubicin treated cells vs. 23.2 ± 13.8% cytolysis of control non-treated A549 cells. For CYNK-201, at the 2.5:1 effector to target ratio, there was 41.4 ± 4.8% cytolysis of etoposide treated cells and 37.4 ± 4.8% for doxorubicin treated cells vs. 26.4 ± 4.6% for control non treated A549 cells. Consistently, significantly increased secretion of inflammatory cytokines GM-CSF, G- CSF, IFN-y, and TNF-a was also observed following 24h CYNK co-culture with chemotherapeutic drug-induced senescent A549 cells compared to non-treated cells (Figure 6), further confirming the increased killing of senescent over non-senescent A549 cells.

[0081] The data discussed above and set forth in the figures readily support the instant invention, i.e., treating at least one sign or symptom of the aging process in a subject of a particular chronological age, particularly because it has been demonstrated that CYNK-001 and CYNK-201 are potent and selective senolytic agents. As explained above, treatment of subjects of a particular chronological age suffering from an age-related disease or condition with CYNK-001 and CYNK-201 will benefit from such treatment. Consequently, they will enjoy an improvement in their overall therapeutic outcomes regarding such age-related diseases or conditions or the onset of signs or symptoms of such diseases or the delay in the onset of such signs or symptoms.

[0082] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the disclosure in addition to those described herein willbecome apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of decreasing expression level of one or more stress ligands or one or more SASP cytokines by senescent cells, comprising contacting the senescent cells with placenta-derived hematopoietic stem cell derived NK cells such that the expression level of tire one or more stress ligands or one or more SASP cytokines by the contacted senescent cells is less than the expression level of the one or more stress ligands or the one or more SASP cytokines by the senescent cells prior to contact with the placenta-derived NK cells.

2. The method of Claim 1, wherein the at least one stress ligand comprise: (i) CD58; (ii) CD155; (iii) ULBP-2 / 5 / 6; or (iii) MIC-A / B.

3. The method of either of Claims 1 or 2, wherein the at least one SASP cytokine comprises (i) IL- 1 alpha; (ii) IL -6, (iii) IL-8, (iv) CCL5; or (v) CCL2.

4. The method of any one of Claims I -3, wherein the placenta-derived hematopoietic stem cell derived NK cells comprise: (a) CYNK-001 cells: (b) CYNK-201 cells; or (c) a combination of (a) and (b).

5. The method of any one of Claims 1-4, wherein the senescent cells are cancer cells that have reached senescence.

6. A method for treating or delaying onset of at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering a therapeutically effective amount of placenta-derived hematopoietic stem cell derived natural killer (NK) cells to the subject in order to modulate a biomarker of aging in the subject.

7. The method of Claim 6, wherein the signs and symptoms of aging comprise:(a) increased numbers of senescent cells in the subject compared to the number of such cells in:(i) the subject at an earlier chronological age than the particular chronological age;(ii) a second subject that is at an earlier chronological age than the particular chronological age of the subject;(b) age-related inflammation;(c) telomere attrition in cells of the subject; id) impaired homeostasis;(e) epigenetic alterations of cells in the subject;(f) genetic instability of cells in the subject;(g) deregulated nutrient sensing;(h) cancer;(i) mitochondrial dysfunction; or(j) any combination of (a) - (i).

8. The method of either of Claims 6 or 7, wherein the biomarker of aging comprises:(a) length of telomeres in the subject's leukocytes;(b) immunohistochemistry of gamma-H2Al;(c) levels of senescence-associated beta-galactosidase;( d) levels of an inflammatory biomarker;(e) levels of 8-isoprostaglandin F 2alpha (8-isoprostane);( fj a phenotype biomarker;(g) levels of a SASP cytokine; or(h) any combination of (a)-(g).

9. The method of Claim 8, wherein the phenotypic biomarker comprises;(a) physical function;(b) anthropometry; or(c) a combination of (a) and (b ).

10. The method of either of Claims 8 or 9, wherein the phenotype biomarker comprises:(a) walking speed;(b) chair stand;(c) standing balance;(d) grip strength;(e) body mass index;(f) waist circumference;(g) muscle mass;(h) sarcopenia; or(i) any combination of (a)-(h).

11. The method of any one of Claims 6-10, wherein modulating the biomarker of aging comprises;(a) preventing telomere attrition in cells of the subject;(b) increasing the length of telomeres in cells of the subject;(c) decreasing levels of gamma H2A-X in cells of the subject;( d) decreasing the level of an inflammatory' marker in cells of the subject;(e) decreasing the levels of 8-isoprostaglandin F 2-alpha (8-isoprostane) in cells of the subject;(f) decreasing the level of the serum markers in cells of the subject;(g) decreasing levels of a SASP cytokine; or(h) any combination of (a)-(g).

12. The method of any one of Claims 6-11 , wherein the senescent cells are cancer cells that have reached senescence.

13. The method of any one of Claims 6-12, wherein the placenta-derived hematopoietic cell derived NK cells comprise: (a) CYNK-001 cells; (b) CYNK-201 cells: or (c) a combination of (a) and (b).

14. The method of any one of Claims 6-13, wherein at least one sign or symptom of the aging process is a disease or condition comprising: (a) diabetes; (b) arthritis; (c) cancer; (d) a cardiovascular disease or condition; (e) depression; (f) chronic kidney disease; (g) chronic obstructive pulmonary’ disease; (h) a neurodegenerative disease or condition; (i) any combination of (a)-(h).

15. The method of Claim 14, wherein the neurodegenerative disease or condition is selected from the group consisting of: (a) Alzheimer’s Disease; (b) dementia; and (c) Parkinson’s Disease.

16. The method of Claim 14, wherein the cardiovascular disease or condition is selected from the group consisting of: (a) hypertension; (b) high cholesterol; (c) ischemic heart disease; (d) coronary heart disease; and (e) heart failure.

17. A pharmaceutical composition comprising placenta-derived hematopoietic cell derived NK cells and a pharmaceutically acceptable carrier.

18. A method for treating or delaying onset of at least one sign or symptom of the aging process in a subject of a particular chronological age, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of Claim 17.

19. The use of the pharmaceutical composition of Claim 17 in a medicament for treating or delaying onset of at least one sign or symptom of the aging process in a sub ject of a particular chronological age.

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