Target removal of senescent cells by gamma delta T cells

Abstract

Target elimination of senescent cells by gamma delta T cells Senescent cells are, in certain aspects, associated with age-related decline in health and can contribute to certain diseases. Embodiments include compositions and methods for treating aging-related diseases and disorders by administering gamma delta (γδ) T cells. Gamma delta T cells target senescent cells preferentially over non-senescent cells. These methods can also be used to slow the aging process and / or reduce signs of aging.

Description

Technical Field

[0001] The present invention relates to treatment, and more specifically, to a treatment method using gamma delta T cells for treating aging-related diseases and disorders.

Background Art

[0002] Aging can be defined as the process of growing older. In humans, aging represents the accumulation of changes over time and can include physical, psychological, and social changes. Advanced age is the greatest risk factor for many chronic diseases. More than 90% of adults over 65 experience at least one chronic disease such as cancer, diabetes, cardiovascular disease. The phenotypes and pathologies of aging include a variety of aging-related diseases and disorders and are causally related to the accumulation of senescent cell burden with age.

[0003] Senescent cells are characterized by irreversible cell cycle arrest of proliferation-competent cells, morphological and metabolic changes, changes in gene expression, chromatin reorganization, and a unique inflammatory senescence-associated secretory phenotype (SASP). Replicative senescence is activated during successive passages of cells in culture (or as cells age within an organism). Senescence can also be induced by various insults, including activation of oncogenes, radiation exposure, and exposure to chemotherapeutic drugs. Furthermore, there are several drugs (such as CDK4 / CDK6 inhibitors like palbociclib) that induce senescence.

[0004] Aged cells in the elderly are associated with many diseases (such as cancer and fibrosis) and contribute to chronic inflammation and damage to surrounding tissues. Aged cells can become resistant to apoptosis and may have upregulation of anti-apoptosis pathways that protect them against their own inflammatory senescence-associated secretory phenotype (SASP). This enables aged cells to survive despite killing adjacent cells. It has been shown that genetically removing aged cells by genetic manipulation in transgenic mouse models can prevent or delay tissue dysfunction, improve age-related pathologies, and extend healthspan. This suggests that the removal of the aged cell burden in the elderly is worthy of further research as an interesting therapeutic target for the treatment and prevention of age-related diseases.

[0005] Efforts to develop therapies for age-related diseases have focused on ways to eliminate aged cells without affecting non-aged cells. Some senolytic compounds have shown promising results in mouse and human cell culture models. Known compounds include dasatinib, and quercetin, piperlongumine, and Bcl2 family inhibitors such as ABT263 and ABT737. These agents have had some success in selectively targeting aged cells, but they have limitations. Due to significant side effects, they cannot be administered at effective doses to achieve the desired effect. For example, ABT263 (also known as navitoclax) has dose-limiting thrombocytotoxicity that presents a risk of causing thrombocytopenia. Navitoclax, fisetin, and dasatinib and quercetin (D+Q) have been reported to destroy macrophages. Therefore, there is a need to identify new methods and compounds with aged cell-removing properties.

[0006] Recent research has begun to elucidate the ability of the immune system to recognize senescent cells and target them for elimination / destruction. Innate immunity, or non-specific immunity, refers to the defense system that protects an individual from any antigen. Innate immunity is associated with barriers that prevent harmful substances from entering the body. These barriers form the first line of defense in the immune response. In contrast, the adaptive immune system (i.e., the acquired immune system) is a subsystem of the immune system that includes specialized systemic cells and processes for eliminating pathogens or preventing their growth.

[0007] Natural killer (NK) cells appear to be the most important cell type involved in this process, although neutrophils and abT cells have also been reported to selectively kill senescent cells (SCs), and macrophages may also be involved to some extent. In addition to conventional αβT cells, other subclasses of T cells include mucosal-associated invariant T cells (MAIT), invariant natural killer T cells (iNKT), germline-encoded mycobacterial lipid-reactive T cells (GEMT), and γδT cells. αβ-T cells in peripheral blood account for approximately 50% of lymphocytes, while a smaller proportion (approximately 0.5 - 10%) of CD3+ cells are γδ-T cells. γδT cells recognize their target antigens regardless of MHC haplotype and mediate anti-tumor responses without causing graft-versus-host disease (GvHD). These exert cytotoxic activity via the granzyme-perforin axis or via antibody-dependent cell cytotoxicity (ADCC). γδT cells can also release cytokines such as TNF-α and IFN-γ. Furthermore, these cells can also phagocytose tumor antigens and apoptotic or live cancer cells (possibly via the scavenger receptor CD36) and induce the maturation of dendritic cells by increasing the production of TNF-α. Additionally, γδT cells can interact with B cells to promote class switching of immunoglobulins and cross-present antigens to CD8+ T cells.

[0008] In humans, gamma delta (γδ) T cells constitute a minor subset among T lymphocytes and make up 1% - 10% of mature circulating T cells. Unlike the majority of αβ T cells, most γδ T cells (>70%) are CD4-CD8-, some (about 30%) are CD8+CD4-, and a very small number (<1%) are CD4+CD8-. Gamma delta T cells are a subset of T cells that bring about the coordination of innate and adaptive immune responses. These cells undergo rearrangement of V-(D)-J segments to produce antigen-specific gamma delta T cell receptors (γδTCRs) and gamma delta T cells. Gamma delta T cells represent a small proportion of the total T cell population in mammals, which is about 1 - 5% of the T cells in peripheral blood and lymphoid organs, and are mainly expressed in compartments rich in epithelial cells such as the skin, liver, gastrointestinal tract, respiratory tract, and reproductive tract. Different from the αβ TCRs that recognize antigens bound to the major histocompatibility complex (MHC), γδ TCRs are bacterial antigens, viral antigens, stress antigens expressed by affected cells, and important components of complete proteins or non-peptide compounds.

[0009] There is a need for improved methods for identifying senescent cells and targeting them for apoptosis. The present invention includes the use of gamma delta T cells for selectively targeting senescent cells with high specificity and reliability. Also included are methods for treating age-related diseases and conditions by the selective removal of senescent cells.

Summary of the Invention

[0010] The invention described and claimed herein has many attributes and embodiments, including but not limited to those described, explained, or referenced in this brief summary. The invention described and claimed herein is not defined by or limited to the features or embodiments identified in this summary, which is included for illustrative purposes only and not intended to be limiting.

[0011] The present invention relates to gamma delta T cells and their therapeutic uses, as well as methods for generating (i.e., isolating / enriching) gamma delta T cells.

[0012] Described are methods of using gamma delta (γδ) T cells and therapeutic products to target senescent cells (SCs). These methods can be used to treat senescence-related diseases or disorders. These methods can also be used to slow the aging process and / or reduce signs of aging. In embodiments, the gamma delta T cells are modified, for example, by T cell receptor (TCR) gene transfer or chimeric antigen receptor (CAR) expression.

[0013] Accordingly, embodiments include methods of selectively inducing apoptosis of senescent cells to treat senescence-related diseases or disorders using gamma delta T cells. Embodiments also include methods for selectively inducing apoptosis of senescent cells to slow the aging process and / or reduce signs of aging.

[0014] Embodiments also include treatments for senescence-related diseases and disorders using gamma delta (γδ) T cells. In these aspects, the treatments can delay tissue dysfunction, improve age-related conditions, and / or extend healthspan.

[0015] In embodiments, the methods described herein include pharmaceutical formulations comprising a therapeutic agent that selectively kills senescent cells (i.e., selectively kills senescent cells more than or compared to non-senescent cells). The therapeutic agent can comprise gamma delta (γδ) T cells. In embodiments, the gamma delta (γδ) T cells are enriched and / or modified.

[0016] Embodiments also include methods of treating a disease (i.e., an aging-related disease or disorder), or methods of delaying the aging process / reducing signs of aging. The methods can include (a) a step of concentrating gamma-delta (γδ) T cells, and (b) a step of therapeutically administering the gamma-delta (γδ) T cells. The methods can also include a step of modifying the gamma-delta (γδ) T cells. In these embodiments, the γδ T cells are obtained from a subject. In these embodiments, the γδ T cells are obtained from one or more donors (e.g., from PBMCs).

[0017] Embodiments also include methods of removing senescent cells for diagnostic and / or therapeutic purposes.

[0018] Embodiments also include methods of combination therapy, since gamma-delta (γδ) T cells can be administered in combination with known senescent cell-removing or senescent cell-inhibiting agents. Thus, embodiments include methods of treating an aging-related disease or disorder that include administering gamma-delta (γδ) T cells together with one or more senescent cell-removing agents (e.g., small molecules) to selectively kill senescent cells over non-senescent cells. The senescent cell-removing agent can be, for example, dasatinib, quercetin, fisetin, or navitoclax.

[0019] Embodiments also include methods of identifying senescent cells for targeted therapy. Embodiments also include methods of removing senescent cells from diseased tissue of a subject.

[0020] Embodiments include methods of reducing senescent cell burden. Embodiments also include methods of treating, reducing the likelihood of occurrence of, or delaying the onset of an aging cell-related disease or disorder. A formulation containing gamma-delta T cells can be administered intravenously to a subject.

[0021] Embodiments also include methods of generating gamma-delta T cells and enhancing and / or potentiating their function(s). In an aspect, the method of isolating and / or enriching gamma-delta T cells includes supplementing the cells with interleukin-2 (IL-2) and zoledronic acid (ZOL).

[0022] One aspect is a method of isolating and / or enriching gamma-delta T (γδ) T cells. The method includes: (a) culturing a cell population containing γδ T cells with a phosphoantigen to expand the γδ T cells; (b) culturing the expanded γδ T cells with an artificial antigen-presenting cell expressing an Fc receptor and an anti-CD3 antibody; and (c) modifying the γδ T cells to express a chimeric antigen receptor (CAR), wherein the CAR includes an extracellular antigen-binding domain of NKG2D. In an aspect, the Fc receptor is CD64. In an aspect, the phosphoantigen is zoledronic acid. In an aspect, the cell population is peripheral blood mononuclear cells (PBMC).

[0023] In an aspect, the isolated / enriched gamma-delta T cells are administered to a subject for treating an aging-related disease or disorder. In these aspects, the isolated / enriched gamma-delta T cells are administered to a subject to reduce the number of senescent cells and / or reduce signs of aging.

[0024] In an aspect, the γδ T cells can be gene-edited to improve their therapeutic potential. Such gene editing may be performed by any means known in the art, such as by using artificial nuclease(s). Such gene editing can redirect the specificity of the γδ T cells via the expression of a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Such gene editing can improve the efficacy of the γδ T cells by improving homing, cytokine production, recycling death, and / or transplantation.

[0025] In one embodiment, a pharmaceutical composition is provided that includes the cell composition of this embodiment and a pharmaceutically acceptable carrier.

[0026] In one embodiment, provided is a method of treating a disease in a patient, comprising administering an effective amount of the cell composition or pharmaceutical composition described herein. In these embodiments, the disease is associated with aging.

[0027] In one embodiment, provided is a method of treating a disease in a patient, comprising producing a cell composition according to the method of an embodiment of the present invention and administering an effective amount of the cell composition to a patient in need thereof.

[0028] In some embodiments, provided is a method for treating an individual having a medical condition, comprising providing an effective amount of cells from the cell population described herein, administered at multiple times in some embodiments, for example, at intervals of at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or more.

[0029] In one embodiment, a composition comprising a cell population or pharmaceutical composition of an embodiment of the present invention is provided for use in the treatment of aging-related diseases in a patient. In one aspect, the cell composition can be allogeneic to the patient. In another aspect, the cell composition can be autologous to the patient. In another embodiment, provided is the use of a cell population of an embodiment of the present invention in the manufacture of a medicament for treating a disease.

[0030] The accompanying drawings illustrate embodiments of the present invention.

Brief Description of the Drawings

[0031]

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[0032] Definition References to "an embodiment / aspect" or "embodiments / aspects" in this specification mean that the particular features, structures, or characteristics described in connection with the embodiment / aspect are included in at least one embodiment / aspect of the present disclosure. The use of the phrases "in one embodiment / aspect" or "in another embodiment / aspect" in various places in this specification does not necessarily refer to all the same embodiments / aspects, nor are separate or alternative embodiments / aspects mutually exclusive of other embodiments / aspects. Further, various features are described that may be shown by some embodiments / aspects and not by others. Similarly, various requirements are described that may be requirements for some embodiments / aspects but not for others. Embodiments and aspects may be used interchangeably in some cases.

[0033] The terms used in this specification generally have their ordinary meanings in the art, within the context of the disclosure, and in the particular context in which each term is used. Specific terms used to describe the disclosure are discussed below or elsewhere in the specification to provide additional guidance to practitioners regarding the description of the disclosure. It will be understood that the same thing can be said in multiple ways.

[0034] Accordingly, alternative languages and synonyms may be used for any one or more of the terms discussed herein. Also, no particular importance is placed on whether a term is detailed or discussed in this specification. Synonyms for certain terms are given. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any of the terms discussed herein, is for illustration only and is not intended to further limit the scope or meaning of the present disclosure or any of the terms exemplified. Similarly, the present disclosure is not limited to the various embodiments described herein.

[0035] Without further limiting the scope of the present disclosure, examples of devices, apparatuses, methods, and the results associated therewith according to embodiments of the present disclosure are shown below. Note that topics and subtopics may be used in the examples for the convenience of the reader, but this is not intended to limit the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. In case of conflict, this specification, including definitions, will prevail.

[0036] The term "aging" refers to the gradual decline of functional characteristics in an organism. Cellular aging is often defined as the permanent cell cycle arrest of previously replication-competent cells, which is often caused by stress. The effects of senescent cells can be considered beneficial or harmful with respect to the host's physiological functions and diseases. In some situations, senescent cells can affect disease states in complex ways that can both promote and interfere with certain conditions.

[0037] As described herein, senescent cells can be, for example, senescent fibroblasts, senescent preadipocytes, senescent epithelial cells, senescent chondrocytes, senescent neurons, senescent smooth muscle cells, senescent mesenchymal cells, senescent macrophages, or senescent endothelial cells.

[0038] The term "aging-related disease or disorder" refers to diseases associated with aging, and may include, for example, atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataracts, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis. Other diseases related to aging or senescence (including aging-related conditions) include gray hair, sarcopenia, obesity, neurogenesis, fibrosis, and glaucoma.

[0039] Still other diseases related to aging or senescence include cardiovascular diseases (such as atherosclerosis, angina pectoris, arrhythmia, cardiomyopathy, congestive heart failure, coronary artery disease, carotid artery disease, endocarditis, coronary thrombosis, myocardial infarction, hypertension, aortic aneurysm, cardiac diastolic dysfunction, hypercholesterolemia, hyperlipidemia, mitral valve prolapse, peripheral vascular disease, cardiac stress tolerance, cardiac fibrosis, cerebral aneurysm, and stroke). Aging-related diseases or disorders can also be inflammatory or autoimmune diseases or disorders (such as osteoarthritis, osteoporosis, oral mucositis, inflammatory bowel disease, or kyphosis). Aging-related diseases or disorders can also be neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, mild cognitive impairment, or motor neuron dysfunction). Aging-related diseases or disorders can be metabolic diseases (such as diabetes, diabetic ulcers, metabolic syndrome, or obesity). Aging-related diseases or disorders can also be lung diseases (such as pulmonary fibrosis, chronic obstructive pulmonary disease, asthma, cystic fibrosis, emphysema, bronchiectasis, or age-related loss of lung function). Aging-related diseases or disorders can also be eye diseases or disorders (such as macular degeneration, glaucoma, cataracts, presbyopia, or vision loss). Aging-related diseases or disorders can also be kidney diseases, renal insufficiency, frailty, hearing loss, muscle fatigue, skin diseases, skin wound healing, liver fibrosis, pancreatic fibrosis, oral submucous fibrosis, or sarcopenia. Aging-related diseases or disorders can also be skin diseases or disorders (such as eczema, psoriasis, pigmentation, moles, rashes, atopic dermatitis, urticaria, photosensitivity, or diseases or disorders related to photoaging).

[0040] The terms "senescence-associated β-galactosidase", "SA-β-gal", or "SABG" refer to a putative hydrolase enzyme that catalyzes the hydrolysis of β-galactoside to monosaccharides only in senescent cells. Senescence-associated beta-galactosidase, together with p16Ink4A, can be used as a biomarker of cellular senescence.

[0041] The term "senolytic" or "senolytic agent" refers to a therapeutic agent such as a small molecule that can selectively or preferentially induce the death of senescent cells. A senolytic agent can kill senescent cells by inducing (activating, stimulating, or removing the inhibition of) the apoptotic pathway leading to cell death. Senolytic agents can be useful for the treatment of senescence-related diseases or disorders. For example, drugs such as dasatinib, quercetin, fisetin, and navitoclax have potential senolytic activity.

[0042] The term "biomarker" generally refers to a DNA-, RNA-, protein-, carbohydrate-, or glycolipid-based molecular marker whose expression or presence in a sample can be detected by standard methods (or the methods disclosed herein) and that predicts or foretells the effective responsiveness or susceptibility of a mammalian subject having a disease. A biomarker can be present in a test sample but not in a control sample, not present in a test sample but present in a control sample, or the amount of the biomarker can differ between a test sample and a control sample. For example, a protein biomarker can be present in such a sample but not in a control sample, or a particular biomarker can be seropositive in a sample but seronegative in a control sample. Also, the expression of such a biomarker can be determined to be higher than the expression observed from a control sample. The terms "marker" and "biomarker" are used interchangeably herein.

[0043] The amount of biomarker is measured in a test sample and can be compared to a "normal control level" using techniques such as reference limits, discrimination limits, risk definition thresholds, etc. to define a cut-off point and abnormal values for a certain disease. The normal control level means the level of an indicator of one or more biomarkers or a combination of biomarkers typically found in subjects not suffering from the disease. Such normal control levels and cut-off points can vary depending on whether the biomarker is used alone or in a formulation combining with other biomarkers into one indicator. Alternatively, the normal control level can be a database of biomarker patterns from previously tested subjects who did not experience the disease during a clinically relevant period.

[0044] Tests for measuring biomarkers and biomarker panels can be implemented in various diagnostic test systems. A diagnostic test system is a device typically equipped with means for obtaining test results from a biological sample. Examples of such means include modules for automating tests (e.g., chemical, immunological, nucleic acid detection assays). Some diagnostic test systems are designed to handle multiple biological samples and can be programmed to perform the same or different tests on each sample. A diagnostic test system typically comprises means for collecting, storing, and / or tracking test results for each sample, usually in a certain data structure or database. Examples include well-known physical and electronic data storage devices (e.g., hard drives, flash memories, magnetic tapes, paper printouts). It is also typical for a diagnostic test system to be equipped with means for reporting test results. Examples of reporting means include visual displays, links to data structures or databases, printers. The reporting means can be a data link for transmitting test results to external devices such as data structures, databases, visual displays, printers.

[0045] The terms "detect" or "determine" with respect to a biomarker value include the use of both an apparatus required to observe and record a signal corresponding to the biomarker value and the material(s) required to generate that signal. In various embodiments, the biomarker value is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared spectroscopy, Raman spectroscopy, atomic force microscopy, scanning tunneling microscopy, electrochemical detection, nuclear magnetic resonance, quantum dots.

[0046] The terms "treat" or "treatment" refer to one or more of (1) inhibiting a disease, condition or disorder in an individual experiencing or presenting the pathology or symptoms of the disease, condition or disorder (i.e., preventing further progression of the pathology and / or symptoms), and (2) improving a disease, condition or disorder in an individual experiencing or presenting the pathology or symptoms of the disease, condition or disorder (i.e., reversing the pathology and / or symptoms), for example reducing the severity of the disease.

[0047] The term "administer" refers to the introduction of an amount of a given substance into a patient by a particular suitable method. The compositions disclosed herein may be administered via any of the common routes, such as inhalation, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or rectal administration, as long as they can reach the desired tissue.

[0048] The term "subject" refers to a person who is susceptible to a disease (e.g., a disease associated with aging), or a person suspected of having the disease, or a person diagnosed with the disease. However, all subjects treated with the treatment methods described herein are included without limitation.

[0049] The term "T cell" refers to a type of lymphocyte. T cells are one of the important white blood cells of the immune system and play a central role in the adaptive immune response. T cells can be distinguished from other lymphocytes by the presence of T cell receptors (TCRs) on their cell surface. There are three main types of T cells: cytotoxic, helper, and regulatory. Each of them has a different role in the immune response.

[0050] T cells can also be classified as conventional adaptive T cells and natural T cells. Conventional adaptive T cells include helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, and regulatory CD4+ T cells. Natural T cells include natural killer T cells, mucosal-associated invariant T cells, and gamma-delta T cells.

[0051] Cytotoxic T cells (also known as TC, killer T cells, or cytotoxic T lymphocytes (CTLs)) are a subgroup of T cells that induce the death of cells infected with viruses (and other pathogens), or otherwise damaged or dysfunctional cells. Cytotoxic T cells have a co-receptor (i.e., CD8) on their cell surface. CD8 associates with the T cell receptor and MHC class I molecules, which enables cytotoxic T cells to recognize normal cells infected with pathogens. When a cytotoxic T cell recognizes an infected cell, it becomes activated and produces molecules that kill the infected cell, destroying the pathogen in the process.

[0052] CD4+ lymphocytes, also known as "helper" T cells, are mediators of the immune response and play an important role in establishing and maximizing the ability of the adaptive immune response. Helper T cells have different co-receptors (i.e., CD4) on their cell surface. CD4 also associates with the T cell receptor but interacts with MHC class II molecules rather than MHC class I molecules. This enables helper T cells to recognize pathogen peptides presented by antigen-presenting cells. When helper T cells recognize peptides on antigen-presenting cells, they become activated and initiate the production of molecules called cytokines that send signals to other immune cells. Thus, these cells do not have cytotoxic or phagocytic activity and cannot kill infected cells or remove pathogens, but they "manage" the immune response essentially by instructing other cells to perform these tasks.

[0053] The term "unconventional T cells" includes lymphocytes that express an αδ TCR and are commonly found in epithelial environments such as the skin, gastrointestinal tract, or urogenital tract. Their role is to recognize infections and cancer cells and regulate the inflammatory responses that occur in these tissues. Unconventional T cells include CD1-restricted T cells, MR1-restricted mucosal-associated invariant T cells (MAIT cells), MHC class Ib-reactive T cells, and γδ T cells. These T cells can recognize lipids, small molecule metabolites, and specially modified peptides, in contrast to major histocompatibility complex (MHC)-reactive T cells.

[0054] Gamma delta T cells (γδ T cells) are a distinct subset of T cells that are rare in secondary lymphoid organs but abundant in many peripheral tissues such as the skin, intestine, and lung. By rapidly producing large amounts of cytokines, γδ T cells make important contributions to immune responses in these tissues. Gamma delta T cells have an alternative T cell receptor (TCR) as opposed to CD4+ and CD8+ αβ T cells and share characteristics of helper T cells, cytotoxic T cells, and natural killer cells. Like other "unconventional" T cell subsets that possess invariant TCRs, such as CD1d-restricted natural killer T cells, γδ T cells exhibit features that place them at the interface between innate and adaptive immunity. On the one hand, γδ T cells can be considered components of adaptive immunity in that they rearrange their TCR genes by V(D)J recombination, which also generates junctional diversity and develops a memory phenotype. On the other hand, various subsets can be considered part of the innate immune system in which restricted TCRs or NK receptors can be used as pattern recognition receptors. Human gamma delta T cells can be activated by phosphoantigens and amino bisphosphonates such as zoledronate.

[0055] The major gamma delta T cell subtype in peripheral blood is the Vγ9Vδ2 T cell found in humans, which constitutes 0.5 - 10% of lymphocytes in human blood. Most Vγ9Vδ2 T cells are double negative (DN) for the coreceptors CD4 and CD8, approximately 20 - 30% are CD8 single positive, and 0.1 - 7% express CD4. Since Vγ9Vδ2 T cells recognize antigens in an HLA-independent manner, the functional roles of these coreceptors in the context of gamma delta T cells remain unclear.

[0056] Vγ9Vδ2 T cells respond to phosphoantigens produced by various microorganisms in a T cell receptor (TCR)-dependent manner. Vγ9Vδ2 T cells have been proposed as cells that can carry out tumor cell elimination and intracellular defense against parasitic bacteria and parasites without being restricted by major histocompatibility antigens. Furthermore, cancer treatment using Vγ9Vδ2 T cells obtained from human peripheral blood is currently being studied.

[0057] The term "phosphoantigen" or "PAg" refers to small molecules that are metabolites of the methylerythritol phosphate pathway in microbial pathogens and the eukaryotic mevalonate (MVA) pathway in tumor cells. Phosphoantigens such as isopentenyl pyrophosphate (IPP) play important roles in γδT cell-mediated cytotoxicity against pathogens and cancerous cells. The binding of IPP to the intracellular domain of BTN3A causes a structural change, which alters the extracellular domain and enables recognition by the γδTCR. PAg stimulates Vγ9Vδ2 cells in the presence of antigen-presenting cells, suggesting a strict requirement for dedicated antigen-presenting molecules.

[0058] The term "butyrophilin-3A" or "BTN3A" refers to a group of immunoglobulins present on the surface of various types of cells, including natural and cancer cells. Recent studies have identified butyrophilin (BTN) 3A1 as a molecule necessary to stimulate Vγ9Vδ2 cells. Butyrophilin 3A (BTN3A) has been widely studied as a ligand that is upregulated in many types of cancer cells and mediates the activation of γδT cells. The rate-limiting step in the mevalonate pathway is the conversion of HMGCoA to mevalonate via the HMGCR enzyme. HMGCR activity and downstream products in the mevalonate pathway can therefore be inhibited by statins.

[0059] The term "lysosome-associated membrane protein 1" or "LAMP-1" is also known as membrane glycoprotein 1 bound to lysosomes and CD107a (cluster of differentiation 107a), and is a protein encoded by the LAMP1 gene in humans.

[0060] The term "tripeptidyl peptidase 1" or "TPP-1" is also known as a lysosomal pepstatin-insensitive protease and is an enzyme encoded by the TPP1 gene in humans.

[0061] The term "antibody-dependent cell-mediated cytotoxicity", "ADCC" or "antibody-dependent cellular cytotoxicity" refers to a mechanism of cell-mediated immune defense by which effector cells of the immune system actively lyse target cells whose membrane surface antigens are bound by specific antibodies. This is one of the mechanisms by which antibodies can act, as part of the humoral immune response, to limit and contain infection.

[0062] The term "adoptive cell therapy", "ACT" or "adoptive transfer" refers to a treatment that uses a cancer patient's own T lymphocytes with anti-tumor activity that are expanded in vitro and reinfused into a patient with cancer. This approach involves harvesting naturally occurring T cells that have already infiltrated the patient's tumor and then activating and expanding them. Subsequently, large numbers of these activated T cells are reinfused into the patient, where they can then seek out and destroy the tumor.

[0063] The term "FC" or "flow cytometry" refers to a technique used to detect and measure the physical and chemical properties of a population of cells or particles. The term "FACS" or "fluorescence-activated cell sorting" refers to a technique used to sort cells in which a specific cell type is separated from other cell types present in a sample based on its physical or biological properties (e.g., size, morphological parameters, viability and protein expression). The homogeneous cell population obtained after sorting can be further studied.

[0064] All numerical designations, for example, pH, temperature, time, concentration, and molecular weight, are to be understood as approximate values following general practice in the art, including ranges. As used herein, the term "about" may include differences of (+) or (-) 1%, 5%, 10%, 15%, or 20% of the recited amount, as appropriate and taking into account the context. Although not necessarily explicitly indicated, it should be understood that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0065] Many known and useful compounds, among others, are found in Remington’s Pharmaceutical Sciences (13th Ed), Mack Publishing Company, Easton, PA, a standard reference for various types of administration. As used herein, the term "formulation(s)" refers to a combination of at least one active ingredient and one or more other components, often referred to as excipients, which may be independently active or inactive. The term "formulation" may or may not refer to a pharmaceutically acceptable composition for administration to humans or animals and may include compositions that are useful intermediates for storage or research purposes.

[0066] Other technical terms used herein have their ordinary meanings in the art in which they are used, as exemplified by various technical dictionaries. The specific values and configurations discussed in these non-limiting examples may be varied and are merely cited to illustrate at least one embodiment and are not intended to limit the scope.

DETAILED DESCRIPTION OF THE INVENTION

[0067] It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide a further description of the technology of the claimed subject matter. Additional features and advantages of the subject technology are described in the following description, and some of them are apparent from the description or can be learned by the practice of the subject technology. The advantages of the subject technology are realized and achieved by the structures particularly pointed out in the forms for carrying out the described invention and the claims.

[0068] Various types of immune cells, such as macrophages, NK cells, and T cells, are known to remove senescent cells (SCs). NK cells identify SCs via NKG2D ligands such as MICA and ULBP2 and secrete perforin and granzyme for the cytotoxic effector function in the elimination of SCs. On the other hand, αβ T cells recognize pathogens via the TCR receptor, which requires peptide antigen presentation to exert their cytotoxic activity. The main limitation of NK cells is that their effects can be hindered by inhibitory ligands such as NKG2A, while αβ T cells are restricted by the need for antigen presentation and the inhibitory ligand HLA-E. Gamma delta T cells (γδ T cells) can overcome the limitations of NK cells and αβ T cells because, unlike NK cells, they do not have receptors for inhibitory ligands and, unlike αβ T cells, they do not require antigen presentation. Furthermore, γδ T cells have both acquired and innate characteristics that make them particularly suitable for regulating the early response to the elimination of SCs.

[0069] Gamma delta T cells (γδ T cells) are T cells that express a unique T cell receptor (TCR) composed of one γ chain and one δ chain. These are found in the intestinal mucosa, skin, lung, and uterus and are involved in the initiation and propagation of the immune response. They are present in relatively low abundance in the body. In humans, γδ T cells are a small subset among T lymphocytes and constitute 1% - 10% of circulating mature T cells.

[0070] Unlike most αβ T cells, most gamma delta T cells (i.e., more than 70%) are CD4-CD8-. Approximately 30% are CD8+CD4-, and a small minority (i.e., less than 1%) are CD4+CD8-. Similar to αβ T cells and B cells, the structural diversity of gamma delta T cells depends on V(D)J somatic recombination, which generates a very diverse set of receptors for antigen recognition. However, the repertoire of gamma delta T cells is more restricted compared to αβ T cells and B cells. This diversity mainly occurs in the complementarity-determining region 3 (CDR3) of the TCR. Among the various subtypes, Vγ9Vδ2 T cells represent approximately 50-95% of the peripheral gamma delta T cells in circulation. The present applicants have proposed the use of Vγ9Vδ2 T cells for therapeutic use, particularly for senescent cell depletion therapy.

[0071] Recent studies have reported that Vγ9Vδ2 T cells have potent and broad tumor cell cytotoxicity through MHC-independent as well as recognition of targets based on phosphoantigens and MICA. These cells have a relatively low tendency to secrete IL-17 and are also less sensitive to immunosuppressive checkpoints such as PD-1. Vγ9Vδ2 T cells are stimulated by the end products of the mevalonate isoprenoid pathway of the prokaryotic isoprenoid pathway. The levels of these natural metabolites are very low to be detected as danger signals by Vγ9Vδ2 T cells in normal cells, while the dysfunctional metabolism of malignant tumor cells can lead to the accumulation of endogenous phosphoantigens.

[0072] Since gamma delta T cells can function as innate immune cells and there is some overlap in effector functions with NK cells, it was investigated whether gamma delta T cells are also involved in the immune surveillance of senescent cells. Gamma delta T cells were isolated and enriched from human peripheral blood mononuclear cells (PBMC), and then the effectiveness of those cells for immune-mediated removal of senescent cells was demonstrated.

[0073] Gamma-delta T cells were concentrated from less than 5% to a maximum of 85%. Furthermore, flow cytometry analysis further revealed that Vγ9Vδ2 T cells were the main cells in which enrichment occurred. Additionally, it was observed that during co-culture with senescent IMR-90 cells, gamma-delta T cells eliminated senescent cells in a dose-dependent manner, while having only a minimal effect on non-senescent cells.

[0074] The results shown below indicate that γδ T cells can eliminate multiple types of SC. One of the main limitations of senolytic drugs is non-specific toxicity to non-SC, which is due to their non-specific inhibition of cell survival pathways and those other metabolic pathways on which non-senescent cells also depend. In contrast, the unique combination of NKG2D and γδTCR on γδ T cells enables the implementation of specific cytotoxicity based on the presence of phosphoantigens and NKG2D ligands that are specific for cancer, infection, senescence, and other damaged cell types. These results also show that the enrichment and activation of γδ T cells by zoledronate treatment of target cells make them specifically cytotoxic to senescent cells (SC) rather than non-senescent cells. Furthermore, γδ T cells do not have a long-term cytolytic effect because they have a short half-life. In summary, the data herein show that specific activation of γδ T cells can take advantage of their lower cytotoxicity to normal cells and destruction of SC to provide a basis for a more specific approach to eliminating SC. It is shown that it can provide a basis for a more specific approach for eliminating SC, taking advantage of the lower cytotoxicity to normal cells and destruction of SC.

[0075] Examples The following non-limiting examples are provided for illustrative purposes only to more fully and readily understand the representative embodiments contemplated herein. These examples are intended to be merely a subset of all possible situations in which the components of the formulation can be combined. Accordingly, these examples should not be construed as limiting any of the embodiments described herein, including those related to the types and amounts of the components of the formulation and / or its methods and uses.

[0076] Methods Cell culture IMR-90 fibroblasts (ATCC, USA: catalog number CCL-186) were maintained at 37 °C in humidified air containing 5% CO2 and 3% O2. Fibroblasts were used at population doubling level (PDL) 30 - 47 and maintained in Dulbecco's Modified Eagle Medium (DMEM) (Corning; catalog number 10-013-CV) supplemented with 10% fetal bovine serum (FBS) (Millipore Sigma, USA; catalog number F4135) and 1X penicillin-streptomycin (Corning; catalog number 30-001-CI) in DMEM complete medium. Cumulative PDL was calculated using the following formula:

Equation

[0077] Induction of Senescence Human IMR-90 fibroblasts were treated with 300 nM doxorubicin hydrochloride (Millipore Sigma, USA; catalog number 504042) in DMEM complete medium for 24 hours and maintained in culture as described above.

[0078] Isolation and Enrichment of Gamma Delta (γδ) T Cells γδ T cells were isolated and enriched from human blood as described in the published protocol using zoledronate and interleukin-2 (see, for example, Kondo, M. et al. Expansion of human peripheral blood gamma delta T cells using zoledronate. J Vis Exp). Blood samples were obtained from healthy donors (n = 5, age range 20 - 42 years) in heparin-coated vacuum blood collection tubes. All subjects provided written informed consent. Inclusion criteria for healthy individuals included those who were not taking medications that could affect immunity (e.g., corticosteroids) and had no clinical signs of immunodeficiency. Blood samples were diluted with 1XPBS (Corning; catalog number 21 - 031 - CV), combined with lymphocyte separation medium (Corning; catalog number 25 - 072 - CI), and then density gradient centrifugation was performed according to the manufacturer's instructions. An aliquot of PBMCs was analyzed for the proportion of γδ T cells by flow cytometry. The remaining PBMCs were cultured in RPMI complete medium containing 20% fetal bovine serum (FBS) (Millipore Sigma, USA; catalog number F4135), 500 IU / ml human rIL - 2 (recombinant interleukin - 2) (catalog number TECIN teceleukin; Bulk Ro23 - 6019), and 5 μM zoledronic acid (Zol) (Tocris, USA catalog number 6111) for 10 days with rIL - 2, with fresh medium being replaced every other day, and then co - cultured with either senescent or non - senescent IMR - 90 fibroblasts.

[0079] Flow cytometry The cells were resuspended in 100 μl of PBS. The cells were then incubated with APC-conjugated anti-human CD3 antibody (Miltenyi Biotec; catalog number 130-113-135). FITC-conjugated anti-human gdTCR antibody (Miltenyi Biotec; catalog number 130-113-503), PE-conjugated TCRVδ1 antibody (Miltenyi Biotec; catalog number 130-120-440), FITC-conjugated TCRVδ2 antibody (Miltenyi Biotec; catalog number 130-111-009), FITC-conjugated CD56 antibody (Miltenyi Biotec; catalog number 130-113-312) and APC-conjugated CD56 antibody (Miltenyi Biotec; catalog number 130-113-312) were placed on ice for 30 minutes. The cells were washed with 1 ml of ice-cold PBS and resuspended in 100 μl of ice-cold PBS. Data were collected by a flow cytometer (DB Accuri C6). Cell viability was determined by propidium iodide (PI) staining, and live cells were gated for downstream analysis. Data were analyzed using Flowlogic software (Miltenyi Biotech, Germany).

[0080] Impedance measurement For the measurement of background values, 50 μl of complete medium was added to E-Plates 96 (Agilent). Senescent and non-senescent cells were seeded at a cell density of 10,000 cells / well in a total of 200 μl of medium per well. gdT cells were added on top of senescent and non-senescent cells at different effector-to-target (E:T) ratios. Cell viability was monitored using an RTCA MP (Agilent) instrument and RTCA software (Agilent). Cells treated with 0.2% Triton X-100 were used as 100% dead cell positive controls for the cytotoxicity assay. The cell index reflecting the number of viable cells and cytotoxicity was recorded every 15 minutes. All experiments were performed at least three times. To analyze the acquired data, the CI values and the percentage of cytotoxicity were exported.

[0081] Aging-related β-galactosidase SA-β Gal activity was detected using a commercially available kit (Biovision®).

[0082] Immunofluorescence Cells were plated at a density of 10,000 cells / well using a 96-well plate, and aging was induced as described above. On the 8th day after aging induction, non-aged controls were added to the plate at 10,000 cells / well. Ten days after aging induction, the cells were fixed with 4% PFA at room temperature for 15 minutes. The cells were then permeabilized with 0.2% Triton X-100 for 15 minutes and blocked with 5% goat serum in PBS for 1 hour. Primary antibodies were added and left overnight at 4°C on a shaker. The primary antibodies used were gamma γH2AX (PSer1393F2Novus) (1:1000 dilution) and anti-HMGB1 antibody (ab18256Abcam) (1:1000 dilution). CD277 immunofluorescence was performed on non-permeabilized cells using a BTN3A1 polyclonal antibody (Proteintech, 1:1000 dilution). After incubation with the primary antibody, the cells were incubated with fluorescent secondary antibodies and Hoechst (Invitrogen H3570) (1:2000 dilution) for 1 hour at room temperature in the dark. The secondary antibodies used were Invitrogen goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 546 (catalog number A-11010) and goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor® 488 (catalog number A-11029). Images were obtained using an EVOS cell imaging system at magnifications of 20x and 40x. ImageJ software was used for image analysis.

[0083] Western blot The cells were lysed in RIPA buffer (Cell Signaling technology, catalog number 5871) containing a protease phosphatase inhibitor cocktail (Cell Signaling technology, catalog number 9806S). The cell suspension was then incubated on ice for 15 minutes and sonicated for 30 seconds. The suspension was cleared of debris by centrifugation at 4 °C for 15 minutes. Protein concentration was determined by the BCA protein assay (Thermo Scientific, catalog number 23227). Electrophoresis was performed with 20 μg of protein per condition and transferred to a PVDF membrane (Bio-Rad, catalog number 1620177) using a wet transfer apparatus (Invitrogen™ XCell II™ Blot Module). The membrane was incubated with the primary antibody overnight at 4 °C and then with the secondary antibody for 1 hour at room temperature. The blot was developed using Azure Radiance Plus (SKU: AC2103) and visualized with an Azure500 chemiluminescence imaging system.

[0084] Isolation and enrichment of gamma delta (γδ) T cells: γδ T cells were isolated and enriched from human blood using a previously published protocol (Kondo et al, Cytotherapy 10, 842 - 856, 2008). Blood samples were obtained from healthy donors (n = 5, age range 20 - 42 years) in heparin-coated vacuum blood collection tubes. All subjects provided written informed consent. Inclusion criteria for healthy individuals included those not taking medications that could affect immunity (e.g., corticosteroids) and those without clinical signs of immunodeficiency. Blood samples were diluted with 1XPBS, combined with lymphocyte separation medium, and then density gradient centrifugation was performed according to the manufacturer's instructions. An aliquot of PBMCs was analyzed for the proportion of γδ T cells by flow cytometry. The remaining PBMCs were cultured in RPMI complete medium containing 20% fetal bovine serum (FBS), 500 IU / ml human rIL-2 (recombinant interleukin-2), and 5 μM zoledronic acid (Zol) for 24 hours. The cells were expanded with rIL-2 for 10 days while changing the fresh medium every other day, and then co-cultured with either senescent or non-senescent IMR-90 fibroblasts. After 10 days of expansion, γδ T cells were negatively selected using the Miltenyi Biotec™ γδ T cell isolation kit.

[0085] Flow cytometry The cells were resuspended in 100 μl of PBS. The cells were then incubated with an APC-conjugated anti-human CD3 antibody. FITC-conjugated anti-human γδTCR antibody, PE-conjugated TCRVδ1 antibody, FITC-conjugated TCRVδ2 antibody, FITC-conjugated CD56 antibody, and APC-conjugated CD56 antibody were added on ice for 30 minutes. The cells were washed with 1 ml of ice-cold PBS and resuspended in 100 μl of ice-cold PBS. Data were collected by a flow cytometer (MACSQuant10, Miltenyi Biotec™). Cell viability was determined by PI staining, and live cells were gated for downstream analysis. Data were analyzed using Flowlogic™ software.

[0086] Real-time cytotoxicity assay (xCELLigence (registered trademark)) 50 μL of medium was added to E-Plates 96 for background value measurement. SC and each non-senescent cell were seeded at a density of 10,000 cells / well in an additional 100 μL of medium. Cell adhesion was monitored using an RTCA MP instrument and RTCA software until the plateau phase, which was usually about 24 hours later, was reached. Floating cells were removed, and γδT cells were added on top of the senescent and non-senescent cells at different E:T ratios. Cells were treated with 0.2% Triton X-100 as a 100% dead cell positive control for the cytotoxicity assay. Impedance measurements were taken every 15 minutes for up to 36 hours upon addition of gdT cells. All experiments were performed at least three times in three independent experiments for each donor and cell type. Changes in impedance were represented as cell index (CI) values, which were obtained from the relative change in impedance corresponding to the cell coverage of the electrode sensor and normalized to the baseline impedance value of the medium only. The cytotoxicity rate was determined based on the relative CI values. To analyze the acquired data, the CI values were exported, and the lysis rate was calculated by comparing with control cells without any gdT cells present.

[0087] Quantification and statistical analysis Statistical analysis was performed using Graph Pad Prism 9. All data were represented as mean ± SEM. All cell culture data sets represent the mean of at least three experiments. Comparisons between groups were performed using appropriate corrections for multiple comparisons as needed, using two-sided Student's t-test, one-way ANOVA, or two-way ANOVA. Statistical parameters are found in the figure legends.

[0088] Example 1 Enrichment of γδT cells from human PBMC To determine the proportion of γδ T cells in human PBMCs, blood was collected from healthy donors (n = 5, age 20 - 42 years), and PBMCs were isolated. FACS analysis of human PBMCs showed that less than 5% of total PBMCs were positive for γδ TCR and CD3 (Figure 5A). There are several classes of γδ T cells, among which Vγ9Vδ2 T cells are the main class in PBMCs. The proportion of Vγ9Vδ2 T cells (hereinafter, "Vδ2") in human PBMCs was determined, and it was found that approximately 3% of total PBMCs were positive for Vγ9Vδ2 TCR (Figure 5B). Cells expressing Vγ9Vδ1 in PBMCs were not observed (Figure 5C and Figure 5D).

[0089] Vδ2 T cells have been shown to have potent and broad tumor cell cytotoxicity and to be less sensitive to immune inhibitory checkpoints such as PD-1 (see, for example, Kunkele et al, Cells 9, 2020). Since less than 5% of PBMCs are Vδ2 T cells, previously published protocols were used to enrich Vδ2 T cells (Kondo et al., Cytotherapy, 10 842 - 856, 2008) so that their effects on the immune surveillance of SC could be investigated.

[0090] Human Vδ2 T cells respond to phosphoantigens such as isopentenyl pyrophosphate (IPP) that are produced in eukaryotic cells through the mevalonate pathway. IPP can be induced to accumulate in monocytes when PBMCs are treated with amino bisphosphonates such as pamidronate and zoledronate. Amino bisphosphonates act on IPP to inhibit farnesyl pyrophosphate synthase (FPPS), an enzyme that produces downstream products in the mevalonate pathway. Monocytes efficiently take up zoledronate and accumulate IPP, becoming antigen-presenting cells that stimulate Vδ2 T cells in peripheral blood. A similar protocol was used. This protocol involved culturing freshly isolated PBMCs from healthy donors in RPMI medium with interleukin-2 (IL-2) and zoledronate (ZOL) for 24 hours and then maintaining the cells with IL-2 only for 10 days (Figure 1A). Separate cultures of PBMCs were also maintained using IL-2 only as a control. After 10 days of expansion, flow analysis was performed to examine the enrichment of γδ T cells. These results showed a substantial enrichment of γδTCR- and CD3-expressing T cells from less than 10% in IL-2-supplemented PBMCs to 86% in PBMCs treated with IL-2 and ZOL (Figures 2B and 2C). As expected, 86% of the CD3+ cells also expressed Vγ9Vδ2 TCR and none of them expressed Vγ9Vδ1 TCR, thus indicating that the enriched cells are Vδ2 T cells (Figures 1E and 1F). It was also shown that γδTCR-expressing cells could be enriched from samples isolated from multiple donors using this protocol (Figure 1E). Furthermore, flow cytometry analysis showed that approximately 85% of the cells were double-positive for γδTCR and CD56 (Figure 1F). Since CD56 expression is often associated with the degree of activation, these data suggest that the protocol for enrichment of γδ T cells results in activation of these cells along with their enrichment.

[0091] Example 2 γδ T cells are cytotoxic to senescent cells. To examine whether Vδ2T cells enriched from human PBMCs can selectively kill SCs, a doxorubicin model of senescence induction was used. Human fibroblasts (IMR90) were cultured and treated with doxorubicin to induce senescence. A strong senescent phenotype was confirmed using multiple markers. Untreated proliferating IMR90 cells were used as non-senescent controls. Separately, PBMCs from healthy human donors were divided into two cultures, one maintained with IL-2 and the other treated with ZOL to enrich for Vδ2T cells. Senescent or non-senescent IMR90 cells were seeded into wells, and PBMCs or enriched Vδ2T cells were added to these wells at various target-to-effector ratios. The xCELLigence® platform was used to measure cell impedance as a means of continuously monitoring the real-time motility behavior indicating cell number and attachment. Triton-X was used as a positive control for cytotoxicity. Since immune cells remain in suspension and do not attach to the wells, they have little effect on impedance.

[0092] xCELLigence® software was used to calculate the cell lysis rate and to compare the relative efficacy of enriched Vδ2T cells against senescent and non-senescent IMR90 target cells (Figure 2A). Co-culture of PBMCs with senescent and non-senescent IMR90 cells did not show selective cytotoxicity against SCs. Similarly, co-culture of enriched Vδ2T cells with non-senescent IMR90 cells showed minimal cell death at two target:effector ratios of 0.5:1 and 1:1. In contrast, co-culture of similarly enriched Vδ2T cells with senescent IMR90 cells induced a significant dose- and time-dependent increase in the lysis of senescent fibroblasts (Figure 2A). Furthermore, even at the lowest target:effector (E:T) ratio of 0.5:1, enriched Vδ2T cells were significantly more cytotoxic against SCs (Figure 2A).

[0093] This finding was confirmed by repeating the experiments using PBMCs or enriched Vδ2T cells from multiple donors. These results showed that enriched Vδ2T cells from multiple donors selectively killed SCs. To determine whether Vδ2T cells kill SCs from other cell type origins, senescence was induced in primary human endothelial cells by doxorubicin treatment, and the co-culture assay was repeated. The inventors found that Vδ2T cells from multiple donors selectively killed senescent endothelial cells. These results suggest that enriched Vδ2T cells can preferentially induce cytotoxicity in senescent cells rather than non-SCs.

[0094] Example 3 Isolation of γδT cells after enrichment improves specificity. Vδ2T cells from multiple donors showed high cytotoxicity against SCs compared to non-SCs, but it was observed that Vδ2T cells enriched from some donors also had relatively high cytotoxicity against non-SCs. The inventors were interested in knowing the cause of this secondary cytotoxicity and hypothesized that non-specific cytotoxicity might be due to the contamination of αβT cells. To test this hypothesis, flow analysis of Vδ2T cells enriched from multiple donors was performed to determine the level of αβT cell contamination. The inventors found that there was donor-to-donor variability in the Vδ2T enrichment rate after ZOL treatment. For example, in one donor, ZOL treatment resulted in 90% pure Vδ2T cells and less than 10% αβT cells, while in another donor, it was only 55% Vδ2T cells and 35% αβT cells. Even in a donor with 85% Vδ2T, there were 5% CD3-CD56+ (NK cells) and 8.5% CD3+γδTCR-CD56+ cytotoxic cells (αβT cells), which may contribute to non-specific cytotoxicity against non-SCs.

[0095] Next, it was examined whether the enrichment of γδ T cells was inversely correlated with non-specific cytotoxicity against non-SCs. Indeed, donors with highly enriched γδ T cells showed lower cytotoxicity against non-SCs compared to donors with lowly enriched γδ T cells. Conversely, donors with high levels of CD8+ T cell contamination tended to have higher cytotoxicity against non-SCs.

[0096] Finally, it was investigated whether depletion of αβ T cells after enrichment improved non-specific cytotoxicity against non-SCs. For this, all γδ TCR− cells were first depleted from a donor having only 55% Vδ2 T cells and 35% αβ T cells using the Miltenyi biotech® γδ T cell isolation kit, and isolated to further purify the Vδ2 T cells. By this method, the contamination of ab T cells in the cells from this donor could be drastically reduced from 35% to 1.5%, and the enrichment of γδ T cells could be further increased from 55% to 95%. This protocol was repeated with cells from other donors to similarly deplete αβ T cells and further enrich γδ T cells. Next, the cytotoxicity against senescent or non-SC cells of either the Vδ2 T cells enriched using the first method or the Vδ2 T cells with αβ T cells depleted as described above was compared. As expected, the enriched Vδ2 cells and the Vδ2 cells with αβ depleted showed cytotoxicity against SCs equivalent to that of the Vδ2 cells enriched using the previous protocol, but showed significantly lower cytotoxicity against non-SCs.

[0097] Example 4 The TCR and NKG2D receptors are required for the killing of senescent cells by Vδ2 T cells. Human γδ T cells have been reported to use several different mechanisms to kill pathogens and tumor cells. γδ T cells recognize pathogens based on cell contact-dependent mechanisms mediated by the NK receptor NKG2D or via the γδ TCR. The cytotoxicity of γδ T cells can then be mediated through the perforin-granzyme pathway, secretion of inflammatory cytokines such as TNF-α and IFN-γ, or via apoptosis-promoting molecules such as FasL and TRAIL. Here, we investigated whether NKG2D and γδ TCR are required for the cytotoxicity of γδ T cells against SCs.

[0098] By individually blocking the NKG2D or γδ TCR receptor and co-culturing them with senescent IMR90 cells treated with doxorubicin, we evaluated the mechanisms involved in the recognition of SCs by Vδ2 T cells. The cytotoxicity of Vδ2 T cells against SCs was significantly inhibited by either anti-γδ TCR or anti-NKG2D neutralizing antibodies, but Vδ2 T cells retained considerable cytotoxicity even after individually blocking each receptor. Therefore, we investigated whether simultaneously blocking γδ TCR and NKG2D would further reduce cytotoxicity. Indeed, simultaneously blocking NKG2D and γδ TCR significantly reduced cytotoxicity compared to blocking each receptor alone. This indicates that both NKG2D and TCR can be involved in the recognition and killing of SCs by Vδ2 T cells, but the remaining activity suggests that some additional mechanism of cytotoxicity is also involved.

[0099] Example 5 The mevalonate pathway of senescent cells is important for the cytotoxic activity of Vδ2 T cells. NKG2D recognizes target cells based on their surface expression of ligands such as MICA. Previous studies have shown that SCs express high levels of MICA on their surfaces (e.g., Sagiv et al, Againg, 8, 328 - 244, 2016). γδTCR further recognizes targets based on the sensing of phosphoantigens, which depends on the transmembrane proteins butyrophilin 3A1 (BTN3A1) and BTN2A1. Briefly, phosphoantigens such as IPP (an intermediate metabolite of the mevalonate pathway) bind to the intracellular domain of BTN3A, resulting in a structural change in the extracellular domain that is recognized by γδTCR. The inventors have shown that SCs have high levels of BNT3A on their surfaces. To determine the role of the mevalonate pathway, lovastatin was used to inhibit the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) in SCs, and the effect on the cytotoxicity by Vδ2T cells was tested. The inventors have found that inhibition of the mevalonate pathway in SCs significantly reduces the cytotoxicity mediated by Vδ2T cells against them. This indicates that the mevalonate pathway is required for the destruction of SCs by Vδ2T cells, consistent with the role of phosphoantigens such as IPP in SC immunosurveillance by this cell type.

[0100] Example 6 Therapeutic removal of senescent cells Studies have shown that the removal of senescent cells extends healthspan in a progeria mouse model and that the transplantation of a relatively small number of senescent cells into previously healthy animals induces multi-organ dysfunction similar to that seen in aged animals. Furthermore, it has been found that the correlation between the accumulation of senescent cells and disease extends to humans and that the senescent cell burden can be safely reduced in a clinical setting.

[0101] Adoptive cell transfer (ACT) is the transplantation of cells into a patient. The cells can be patient-derived or from another individual. These cells most commonly are derived from the immune system with the aim of improving immune function and characteristics. In autologous cancer immunotherapy, T cells are extracted from a patient, genetically modified, cultured in vitro, and returned to the same patient. In contrast, allogeneic therapy involves cells isolated and expanded from a donor other than the patient receiving the cells.

[0102] In this example, a patient (65-year-old male) presents to the clinic with signs and symptoms of inflammation, atherosclerosis, and hypertension. Healthcare providers consider that targeted removal of the patient's senescent cells may improve the patient's cardiovascular disease.

[0103] In this example, gamma delta T cells are obtained from the peripheral blood mononuclear cells (PBMCs) of 10 volunteer donors. Gamma delta T cells are isolated and enriched from human blood using zoledronic acid and interleukin-2 as described above (see also, e.g., Kondo, M. et al. J Vis Exp, 2008). Blood samples are diluted with 1XPBS, combined with lymphocyte separation medium, and then density gradient centrifugation is performed. For the proportion of gamma delta T cells, aliquots of PBMCs are analyzed by flow cytometry. The remaining PBMCs are cultured in RPMI complete medium containing 20% fetal bovine serum (FBS) with 500 IU / ml human rIL-2 (recombinant interleukin-2) and 5 μM zoledronic acid (Zol) for 24 hours. The cells are expanded for 10 days with rIL-2 while changing the fresh medium every other day, and then co-cultured with either senescent or non-senescent IMR-90 fibroblasts.

[0104] The patient is administered a single intravenous dose (i.e., half of the concentrated sample) of gamma delta T cells. The second dose (i.e., the remaining dose) is administered 30 days later. The γδT cells selectively target the destruction of senescent cells in the patient. Furthermore, the gamma delta T cells also slow down the aging process and reduce the signs of aging. Within two weeks of the second dose, the patient's blood pressure improved. The healthcare provider continuously monitors for inflammation, atherosclerosis, and hypertension.

[0105] Example 7 Gamma delta cells for reducing senescent cell burden Cellular senescence is a major aging process and tumor suppressor mechanism characterized by irreversible growth arrest, apoptosis resistance, production of the senescence-associated secretory phenotype (SASP), mitochondrial dysfunction, and DNA and chromatin alterations. In preclinical aging models, the accumulation of senescent cells is associated with multiple chronic diseases and disorders, geriatric syndromes, multimorbidity, and accelerated aging phenotypes. In animals, genetic and pharmacological reduction of the senescent cell burden results in the prevention, delay, and / or alleviation of various age-related diseases and sequelae. Initial clinical trials have thus far focused much more on the safety and target engagement of senolytic agents that remove senescent cells. In this example, γδT cells are administered to patients to remove senescent cells.

[0106] Senescent cell burden can be determined by markers that are present at increased levels in senescent cells. In addition to p16 and p21, a number of other markers can be used to identify senescent cells, but the sensitivity and specificity vary (see, for example, Gasek et al, Nature Aging 1, 870-892, 2021). The applicant has identified LAMP-1 and TPP-1 as surface biomarkers on senescent cells. Also, morphological features are suggestive of senescence in vitro and can be evaluated using approaches such as brightfield microscopy. Compared to their control cells, senescent cells have increased size and granularity, presumably reflecting their altered metabolism and organellar homeostasis.

[0107] In this example, a patient (a 65-year-old female) presents to internal medicine with signs and symptoms of senescent cell burden. Specifically, the patient presents with chronic inflammation, osteoarthritis, and signs / symptoms of frailty. Healthcare providers believe that targeted removal of the patient's senescent cells will improve the patient's overall health. In addition to those internal homeostatic fluctuations, senescent cells can induce inflammatory states that, via their SAS, induce local and systemic inflammation and tissue damage.

[0108] In this example, gamma delta T cells are obtained from the peripheral blood mononuclear cells (PBMCs) of 10 volunteer donors and concentrated as described above. The patient is administered a single intravenous dose (i.e., half of the concentrated sample) of gamma delta T cells. A second dose (i.e., the remaining dose) is administered 30 days later. The γδT cells selectively target the destruction of senescent cells in the patient. This reverses / slows the aging process. The healthcare provider continuously monitors the patient's inflammation and osteoarthritis. After treatment, the patient reports relief of pain due to inflammation. The patient also feels stronger and more agile.

[0109] Method of Use Embodiments include a method of treating a disease (i.e., an aging-related disease or disorder) and / or slowing the aging process or reducing signs of aging. The method can include concentrating a solution of gamma delta T cells and administering this to a subject. Embodiments also include a treatment for treating an aging-related disease or disorder and slowing the aging process. In one embodiment, the method includes administering a pharmaceutical formulation comprising a therapeutic agent that selectively kills senescent cells (i.e., selectively kills senescent cells over or compared to non-senescent cells). The treatment regimen can include administering the pharmaceutical formulation for a time and in an amount sufficient to selectively kill senescent cells. The pharmaceutical formulation can include gamma delta T cells and other senescent cell ablation agents.

[0110] The therapeutic methods of this specification may include the step of administering a drug product (e.g., gamma delta T cells) in a pharmaceutically effective amount. The total daily dosage is determined by appropriate medical judgment of a physician and needs to be administered once or multiple times. The specific therapeutically effective dosage level for any particular patient may vary depending on various factors well known in the medical field, including the type and degree of response to be achieved, the specific composition depending on whether other agents are used in combination, the patient's age, weight, health status, gender, as well as diet, administration time and route, the secretion rate of the composition, the treatment period, other drugs used in combination with or simultaneously with the compositions disclosed herein, and similar factors well known in the medical field.

[0111] In yet another aspect, this specification provides the use of a pharmaceutical composition comprising the above composition in the preparation of a drug for the prevention or treatment of an aging-related disease or disorder and / or for delaying the aging process / reducing the signs of aging.

[0112] In one embodiment, the dosage of the composition may be administered daily, every other week, weekly, biweekly, or monthly. The duration of treatment may be 1 week, 2 weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 1 year, or longer. The initial dosage may be more than the maintenance dosage. In one embodiment, the dosage is a weekly dosage in the range of at least 0.01 mg / kg, at least 0.25 mg / kg, at least 0.3 mg / kg, at least 0.5 mg / kg, at least 0.75 mg / kg, at least 1 mg / kg, at least 2 mg / kg, at least 3 mg / kg, at least 4 mg / kg, at least 5 mg / kg, at least 6 mg / kg, at least 7 mg / kg, at least 8 mg / kg, at least 9 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, or at least 30 mg / kg. In one embodiment, the weekly dosage can be up to 1.5 mg / kg, up to 2 mg / kg, up to 2.5 mg / kg, up to 3 mg / kg, up to 4 mg / kg, up to 5 mg / kg, up to 6 mg / kg, up to 7 mg / kg, up to 8 mg / kg, up to 9 mg / kg, up to 10 mg / kg, up to 15 mg / kg, up to 20 mg / kg, up to 25 mg / kg, or up to 30 mg / kg. In certain embodiments, the weekly dosage can be in the range of 5 mg / kg to 20 mg / kg. In another embodiment, the weekly dosage can be in the range of 10 mg / kg to 15 mg / kg.

[0113] This specification also provides a pharmaceutical composition for administration to a subject. The pharmaceutical composition disclosed herein may further comprise a pharmaceutically acceptable carrier, excipient, or diluent. As used herein, the term "pharmaceutically acceptable" means that the composition is sufficient to achieve a therapeutic effect without adverse side effects, and can be readily determined according to the type of disease, the age, weight, health status, gender, and drug sensitivity of the patient, the route of administration, the mode of administration, the frequency of administration, the duration of treatment, the drugs used in combination with the compositions disclosed herein, or drugs used simultaneously, and other factors known in medicine.

[0114] The pharmaceutical composition containing the agent(s) disclosed in this specification may further contain a pharmaceutically acceptable carrier. In the case of oral administration, the carrier may include, but is not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and flavoring agents, etc. In the case of injectable preparations, the carrier may include buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers. In the case of preparations for topical administration, the carrier may include bases, excipients, lubricants, and preservatives.

[0115] The disclosed composition may be formulated into various dosage forms in combination with the aforementioned pharmaceutically acceptable carrier. For example, in the case of oral administration, the pharmaceutical composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups, or wafers. In the case of injectable preparations, the pharmaceutical composition may be formulated into ampoules as single-dose or multi-dose containers. The pharmaceutical composition may also be formulated into solutions, suspensions, tablets, pills, capsules, and long-acting preparations.

[0116] On the other hand, examples of carriers, excipients, and diluents suitable for pharmaceutical preparations include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, the pharmaceutical preparation may further contain fillers, anticoagulants, lubricants, humectants, flavoring agents, and preservatives.

[0117] Furthermore, the pharmaceutical composition disclosed in this specification may have any preparation selected from tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized preparations, and suppositories.

[0118] The composition may be formulated into a single dosage form suitable for the patient's body, preferably formulated into a preparation useful as a peptide drug according to the usual methods in the pharmaceutical field, and thereby administered by oral or parenteral routes such as skin, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, vaginal, or rectal administration, but not limited thereto.

[0119] The composition can be used by blending with various pharmaceutically acceptable carriers such as physiological saline or organic solvents. Carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers may be used to enhance stability or absorbability.

[0120] The dosage and frequency of administration of the pharmaceutical composition disclosed herein are determined by various factors such as the type of active ingredient, the disease being treated, the route of administration, the age, sex, and weight of the patient, and the severity of the disease.

[0121] The total effective dose of the composition disclosed herein may be administered to the patient as a single dose, or may be administered in multiple doses over a long period according to a divided treatment protocol. In the pharmaceutical composition disclosed herein, the content of the active ingredient may vary depending on the severity of the disease. Preferably, the total daily dose of the peptide disclosed herein may be about 0.0001 μg to 500 mg per kg of the patient's body weight. However, the effective dose of the peptide is determined considering various factors including the route of administration and treatment frequency of the pharmaceutical composition, as well as the patient's age, weight, health status, sex, disease severity, diet, and secretion rate. Considering this, those skilled in the art can easily determine the effective dose suitable for a specific use of the pharmaceutical composition disclosed herein. The pharmaceutical composition disclosed herein is not particularly limited in its formulation, route of administration, and mode of administration as long as it exhibits a suitable effect.

[0122] Furthermore, the pharmaceutical composition may be administered alone or in combination with or simultaneously with other pharmaceutical formulations that exhibit a prophylactic or therapeutic effect.

[0123] In view of the teachings and guidance provided herein, one of ordinary skill in the art will understand that the formulations described herein are equally applicable to many types of biopharmaceuticals, including those exemplified and others known in the art. In view of the teachings and guidance provided herein, one of ordinary skill in the art will also understand that, for example, the selection of one or more excipients, surfactants and / or any type(s) and / or amount(s) of optional components can be made based on the chemical and functional compatibility with the biopharmaceutical being formulated and / or the mode of administration, as well as other chemical, functional, physiological and / or medical factors well known in the art. For example, non-reducing sugars exhibit favorable excipient properties when used with polypeptide biopharmaceuticals as compared to reducing sugars. Accordingly, exemplary formulations are further exemplified herein with reference to polypeptide biopharmaceuticals. However, the scope of application, chemical and physical properties, considerations, and methodologies applicable to polypeptide biopharmaceuticals can equally apply to biopharmaceuticals other than polypeptide biopharmaceuticals.

[0124] Compositions according to the embodiments described herein have desired properties such as desired solubility, viscosity, injectability, and stability. Lyophilates according to the embodiments described herein also have desired properties such as desired recovery, stability, and reconstitution.

[0125] In one embodiment, the pH of the pharmaceutical formulation is at least about 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, or 9.

[0126] In one embodiment, the pH of the pharmaceutical preparation is from about 3 to about 9, from about 4 to about 9, from about 5 to about 9, from about 6 to about 8, from about 6 to about 7, from about 6 to about 9, from about 5 to about 6, from about 5 to about 7, from about 5 to about 8, from about 4 to about 9, from about 4 to about 8, from about 4 to about 7, from about 4 to about 6, from about 4 to about 5, from about 3 to about 8, from about 3 to about 7, from about 3 to about 6, from about 3 to about 5, from about 3 to about 4, from about 7 to about 8, from about 7 to about 9, from about 7 to about 10.

[0127] The dosage can be a single dose or a cumulative dose (i.e., continuous dosing) and can be readily determined by one of ordinary skill in the art. For example, the treatment of an aging-related disease or disorder can include a single dose of the effective amount of the pharmaceutical composition disclosed herein. Alternatively, the treatment of an aging-related disease or disorder can include multiple doses of the effective amount of the pharmaceutical composition, administered over various periods, such as once a day, twice a day, three times a day, once every few days, or once a week. The timing of administration can vary from individual to individual depending on factors such as the severity of the individual's symptoms. For example, the effective amount of the pharmaceutical composition disclosed herein can be administered to an individual once a day for an indefinite period or until the individual no longer requires treatment. One of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and the effective amount of the pharmaceutical composition disclosed herein administered accordingly can be adjusted.

[0128] In one embodiment, the therapeutic agent (e.g., a solution of enriched γδ T cells) disclosed herein can reduce signs / symptoms of an aging-related disease or disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to patients not receiving the same treatment. In other embodiments, the therapeutic agent can reduce the number of signs / symptoms of an aging-related disease or disorder in, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% of individuals compared to patients not receiving the same treatment.

[0129] In one embodiment, the therapeutic agents disclosed herein can reduce the signs / symptoms of an individual suffering from an aging-related disease or disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% compared to a patient not receiving the same treatment. In another aspect of this embodiment, the therapeutic agent can reduce the signs / symptoms of an individual suffering from an aging-related disease or disorder by, for example, about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 10% to about 90%, about 20% to about 90%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 10% to about 80%, about 20% to about 80%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, or about 60% to about 80%, about 10% to about 70%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, or about 50% to about 70% compared to a patient not receiving the same treatment.

[0130] In a further embodiment, the therapeutic agent and its derivatives have a half-life of 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, or longer.

[0131] In one embodiment, the administration period of the therapeutic agent is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In a further embodiment, the period during which administration is stopped is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer.

[0132] In an aspect of this embodiment, a therapeutically effective amount of a therapeutic agent (e.g., a γδ T cell preparation) disclosed herein reduces signs / symptoms in an individual suffering from an aging-related disease or disorder by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. In another aspect of this embodiment, a therapeutically effective amount of a therapeutic agent disclosed herein reduces signs / symptoms by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In yet another aspect of this embodiment, a therapeutically effective amount of a therapeutic agent disclosed herein reduces signs / symptoms by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0133] In other aspects, a therapeutically effective amount of the therapeutic agent disclosed herein reduces the aging process in an individual and / or reduces signs of aging by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100%. In other aspects of this embodiment, a therapeutically effective amount of the therapeutic agent disclosed herein reduces the aging process and / or signs of aging by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95% or up to 100%. In yet other aspects of this embodiment, a therapeutically effective amount of the therapeutic agent disclosed herein reduces the aging process and / or signs of aging by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%.

[0134] In other aspects, a therapeutically effective amount of the therapeutic agents disclosed herein (e.g., a formulation enriched in γδ T cells) reduces the senescent cell burden in, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% of individuals. In other aspects of this embodiment, a therapeutically effective amount of the therapeutic agents disclosed herein reduces the senescent cell burden by, for example, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, or up to 100%. In still other aspects of this embodiment, a therapeutically effective amount of the therapeutic agents disclosed herein reduces the senescent cell burden by, for example, about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, or about 30% to about 50%. In an aspect, the senescent cell burden is measured by using biomarkers or comparing cell morphology. In an aspect, the decrease in the senescent cell burden delays / reverses the signs of aging.

[0135] Certain embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. It will of course be apparent to those skilled in the art upon reading the foregoing description that modifications of the described embodiments will occur to them. The inventors expect those skilled in the art to appropriately employ such modifications, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise indicated herein or clearly contradicted by context, any combination of the above-described embodiments in all possible variations is included within the scope of the invention.

[0136] Groupings of alternative embodiments, elements, or steps of the invention should not be construed as limitations. Each group member may be referred to and claimed individually, or in any combination with other group members disclosed herein. For reasons of convenience and / or patentability, it is anticipated that one or more members of a group may be included in, or deleted from, the group. If any such inclusion or exclusion occurs, the specification is amended and, accordingly, the group is considered to include that group as modified so as to satisfy all marker group descriptions used in the appended claims.

[0137] Unless otherwise indicated, all numerical values representing features, items, quantities, parameters, characteristics, terms, etc. used in this specification and the claims should be understood to be modified in all cases by the term "about." As used herein, the term "about" means that the feature, item, quantity, parameter, characteristic, or term so qualified encompasses a range of plus or minus 10 percent above and below the value of the feature, item, quantity, parameter, characteristic, or term being described. Accordingly, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximate values that can vary. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical indication should be construed in light of the reported significant digits and in accordance with ordinary rounding techniques. Although the numerical ranges and values setting forth the broad scope of the invention are approximations, the numerical ranges and values set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain errors necessarily resulting from the standard deviation found in the respective test measurements. The recitation of numerical ranges herein is merely intended to serve as a convenient method of referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each individual value within a numerical range is incorporated herein as if it were individually recited herein.

[0138] The specific embodiments disclosed herein may be further limited in the claims by the language "consisting of" or "consisting essentially of." As used in the claims, the transitional term "consisting of," whether used at the time of filing or added by amendment, excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the claim to the specified material or step and those that do not substantially affect the basic and novel characteristics (s). Embodiments of the invention so claimed are described and realized herein either essentially or explicitly.

[0139] Groupings of alternative embodiments, elements, or steps of the present invention should not be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. For reasons of convenience and / or patentability, it is contemplated that one or more members of a group may be included in or deleted from the group. If any such inclusion or deletion occurs, this specification is amended and, thus, considered to include the group as fulfilling the description of all Markush groups used in the appended claims.

[0140] All patents, patent publications, and other publications cited and identified herein are hereby incorporated by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for the disclosure prior to the filing date of this application. Nothing in this regard shall be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the date or representation of the content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the date or content of these documents.

[0141] Finally, while the aspects of this specification are emphasized by reference to particular embodiments, those skilled in the art should understand that these disclosed embodiments are merely illustrative examples of the principles of the subject matter disclosed herein. Accordingly, it should be understood that the disclosed subject matter is in no way limited to the particular methodologies, protocols, and / or reagents, etc. described herein. Thus, various modifications or alterations, or alternative configurations, to the disclosed subject matter can be made without departing from the spirit of this specification, in accordance with the teachings herein. Finally, the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is defined only by the claims. Accordingly, the invention is not strictly limited to what is shown and described.

Claims

1. A formulation comprising a therapeutic dose of gamma delta T cells for treating a disease or disorder in a subject.

2. A formulation comprising isolated gamma delta T cells or enriched gamma delta T cells for the treatment of age-related diseases or disorders in a subject, or for slowing the aging process, or for reducing signs of aging.

3. The formulation according to claim 1 or 2, wherein the gamma delta T cells selectively target senescent cells for destruction.

4. The formulation according to claim 1 or 2, wherein the gamma delta T cells target secondary senescent cells.

5. The formulation according to claim 1 or 2, wherein the gamma delta T cells target primary senescent cells and secondary senescent cells.

6. The formulation according to claim 1, wherein the disease or disorder is an aging-related disease or disorder.

7. The formulation according to claim 2 or 6, wherein the aging-related disease or disorder is one or more of atherosclerosis, osteoarthritis, osteoporosis, hypertension, arthritis, cataract, cancer, Alzheimer's disease, chronic obstructive pulmonary disease (COPD), as well as idiopathic pulmonary fibrosis, graying hair, sarcopenia, obesity, neuropathy, fibrosis, and glaucoma.

8. The formulation according to claim 1 or 2, wherein the gamma delta T cells are modified by T cell receptor (TCR) gene transfer or chimeric antigen receptor (CAR) expression.

9. The formulation according to claim 1 or 2, wherein the formulation further comprises a senescent cell remover.

10. The formulation according to claim 9, wherein the senescent cell scavenging agent is selected from dasatinib, quercetin, fisetin, and navitoclax.

11. The formulation according to claim 1 or 2, wherein the gamma delta T cells are activated by zoledronate or adoptive transplantation.

12. The formulation according to claim 1 or 2, wherein the gamma delta T cells are isolated and / or enriched from PBMCs of a plurality of human donors.

13. The formulation according to claim 1 or 2, wherein the formulation is administered intravenously to a subject.

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