Senescent cell marker and method for using same

Abstract

The present disclosure provides a senescent cell marker. The present disclosure also provides: a method for targeting or detecting senescent cells in vitro or in vivo; and a composition for use in said method. The present disclosure further provides a method for isolating or concentrating senescent cells. The present disclosure still further provides a method for identifying a surface antigen of a cell exhibiting autofluorescence or a subpopulation thereof.

Description

Senescent cell markers and their usage

[0001] This disclosure relates to senescent cell markers and methods for using them.

[0002] More specifically, this disclosure relates to senescent cell markers. This disclosure also relates to in vitro or in vivo methods for targeting or detecting senescent cells, and compositions for use in such methods. This disclosure further relates to methods for isolating or concentrating senescent cells. This disclosure further relates to methods for identifying surface antigens in autofluorescent cells or subpopulations thereof.

[0003] Methods for identifying senescent cells are being developed. To date, DPP4, uPAR, PD-L1, GPNMB, NKG2DL, NOTCH1, and other factors have been identified as senescent cell-related markers (see Non-Patent Documents 1-7).

[0004] Kim KM, et al., Genes Dev. 31, 15, 1529-1534 (2017)Amor C, et al., Nature 583, 7814, 127-132 (2020)Wang TW, et al., Nature 611, 7935, 358-364 (2022)Suda M, et al., Nature Aging 1, 12, 1117-1126 (2021)Sagiv A, et al., Aging (Albany NY) 8, 2, 328-344 (2016)Hoare M, et al., Nat. Cell Biol. 18, 9, 979-992 (2016)Althubiti M, et al., Cell Death Dis. 5, 11, e1528 (2014)

[0005] The aforementioned report did not search for senescent cell-related markers expressed on the cell surface, and therefore the complete picture of senescent cell-related markers expressed on the cell surface remains unclear.

[0006] This disclosure provides novel senescent cell markers and senescent cell markers expressed on the cell surface. This disclosure also provides in vitro or in vivo methods for targeting or detecting senescent cells, and compositions for use in such methods. This disclosure further provides methods for isolating or concentrating senescent cells. This disclosure further also provides methods for identifying surface antigens in autofluorescent cells or subpopulations thereof.

[0007] According to this disclosure, for example, the following inventions may be provided: (1) an in vitro or in vivo method for targeting or detecting senescent cells, comprising contacting senescent cells with means that recognize one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74; (2) the method according to (1), which is an in vitro method; (3) the method according to (1) or (2), wherein the means that recognizes senescent antigens is bound to one or more selected from the group consisting of CD85k, CD215, CD83, CXCL16, MSC&NPC antigen, CD184, CD46, CD79b, CD199, and CD74. (4) The method according to any one of (1) to (3) above, wherein the means for recognizing the aging antigen is bound to CXCL16. (5) The method according to any one of (1) to (4) above, wherein the means for recognizing the aging antigen is labeled. (6) The method according to any one of (1) to (5) above, wherein detection of binding between the means for recognizing the aging antigen and isolated senescent cells indicates that the cells are senescent cells. (7) The method according to any one of (1) to (6) above, wherein the means for recognizing the aging antigen is in the form of a conjugate with a cytotoxic agent, and binding between the means for recognizing the aging antigen and isolated senescent cells results in cytotoxicity to the senescent cells. (8) The method according to any one of (1) to (7) above, wherein the means for recognizing the aging antigen is labeled, the label is analyzed by a spectral analyzer, the spectrum of autofluorescence of senescent cells (autofluorescence spectrum) is used as a background, and detection of binding between the means for recognizing the aging antigen and isolated senescent cells is indicated by the presence of a difference from the background. (9) A composition comprising means for recognizing one or more aging antigens for use in any of the methods described in (1) to (8) above.(10) A method for isolating or concentrating isolated senescent cells, comprising contacting the isolated senescent cells with means for recognizing one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74. (11) A method for identifying a surface antigen selectively expressed in a subpopulation of cells that exhibit autofluorescence, comprising: contacting a cell population that exhibits autofluorescence with a labeled antibody that binds to a candidate surface antigen; measuring the label of the cell population to which the labeled antibody is bound using a spectral analyzer to obtain a measured spectrum of the cell population to which the labeled antibody is bound; measuring the cell population that has not been reacted with the labeled antibody using a spectral analyzer to obtain a background spectrum; subtracting the background spectrum from the measured spectrum to obtain a difference spectrum; and identifying the antigen as a surface antigen selectively expressed in a subpopulation of cells that exhibits autofluorescence, based on the selective detection of the difference spectrum in the subpopulation of cells that exhibit autofluorescence. (12) A method for identifying a surface antigen expressed on autofluorescent cells, comprising: contacting autofluorescent cells with a labeled antibody that binds to a candidate surface antigen; measuring the label of the cells to which the labeled antibody has bound using a spectral analyzer to obtain a measured spectrum of the cells to which the labeled antibody has bound; measuring the cells that have not been reacted with the labeled antibody using a spectral analyzer to obtain a background spectrum; subtracting the background spectrum from the measured spectrum to obtain a difference spectrum; and identifying the antigen as a surface antigen expressed on the cells based on the detection of a difference spectrum in the cells. (13) The method according to (11) or (12) above, wherein the autofluorescent cells are senescent cells.(14) A method for treating senescent cells, comprising inhibiting the expression or function of one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74 in the senescent cells. (15) A composition for use in the method of (14) above, comprising an expression inhibitor or a functional inhibitor for the senescent antigen.

[0008] (21) A composition for use in an in vitro or in vivo method for targeting or detecting senescent cells, comprising contacting senescent cells with means that recognize one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74. (22) The composition according to (21) above, which is an in vitro method. (23) The composition according to (21) or (22) above, wherein the means that recognizes senescent antigens is bound to one or more selected from the group consisting of CD85k, CD215, CD83, CXCL16, MSC&NPC antigen, CD184, CD46, CD79b, CD199, and CD74. (24) The composition according to any one of (21) to (23), wherein the means for recognizing the aging antigen is bound to CXCL16. (25) The composition according to any one of (21) to (24), wherein the means for recognizing the aging antigen is labeled. (26) The composition according to any one of (21) to (25), wherein detection of binding between the means for recognizing the aging antigen and isolated senescent cells indicates that the cells are senescent cells. (27) The composition according to any one of (21) to (26), wherein the means for recognizing the aging antigen is in the form of a conjugate with a cytotoxic agent, and binding between the means for recognizing the aging antigen and isolated senescent cells results in cytotoxicity to the senescent cells. (28) The composition according to any one of (21) to (27) above, wherein the means for recognizing aging antigens is labeled, the label is analyzed by a spectral analyzer, the spectrum of autofluorescence of senescent cells (autofluorescence spectrum) is used as the background, and the detection of binding between the means for recognizing aging antigens and isolated senescent cells is indicated by the presence of a difference from the background.(30) A composition for use in a method for isolating or concentrating isolated senescent cells, comprising contacting isolated senescent cells with means for recognizing one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74. (31) A composition for use in a method for identifying a surface antigen selectively expressed in a subpopulation of a cell population including autofluorescent cells, comprising: contacting an autofluorescent cell population with a labeled antibody that binds to a candidate surface antigen; measuring the label of the cell population to which the labeled antibody is bound using a spectral analyzer to obtain a measurement spectrum of the cell population to which the labeled antibody is bound; measuring the cell population that has not been reacted with the labeled antibody using a spectral analyzer to obtain a background spectrum; subtracting the background spectrum from the measurement spectrum to obtain a difference spectrum; and identifying the antigen as a surface antigen selectively expressed in a subpopulation of an autofluorescent cell population based on the selective detection of the difference spectrum in the subpopulation of the autofluorescent cell population. (32) A composition comprising a labeled antibody for use in a method for identifying a surface antigen expressed on autofluorescent cells, the method comprising: contacting autofluorescent cells with a labeled antibody that binds to a candidate surface antigen; measuring the label of the cells to which the labeled antibody has bound using a spectral analyzer to obtain a measured spectrum of the cells to which the labeled antibody has bound; measuring the cells that have not been reacted with the labeled antibody using a spectral analyzer to obtain a background spectrum; subtracting the background spectrum from the measured spectrum to obtain a difference spectrum; and identifying the antigen as a surface antigen expressed on the cells based on the detection of a difference spectrum in the cells. (33) The composition according to (31) or (32), wherein the autofluorescent cells are senescent cells.(34) A composition for use in a method for treating senescent cells, comprising inhibiting the expression or function of one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74 in the senescent cells, wherein the method includes an expression inhibitor or a function inhibitor for the senescent antigen (e.g., an antibody (e.g., an antagonist antibody), or an antigen-binding fragment thereof).

[0009] This document outlines a screening system for identifying senescent cell-specific surface antigens. It shows the Z'-factor in the established screening system. It displays the fluorescence intensity and type of 16 senescent cell markers that were hit in the established screening system. It shows the surface expression of each of the 16 hit senescent markers on senescent cells. The same applies to the previous section. It shows the surface expression of CXCL16 on senescent IMR-90 cells, senescent TIG-3 cells, senescent MDA-MB-231 cells, and senescent PANC-1 cells. It also shows the expression levels of senescence-associated secretory factors (SASP factors) in sorted CXCL16-positive and negative cells. Finally, it shows the effect of CXCL16 knockdown on SASP factor expression by senescent IMR-90 cells. This study shows the effects of CXCL16 knockdown on SASP factor expression in senescent MDA-MB-231 cells. It also shows the effects of CXCL16 knockdown on the survival of senescent IMR-90 cells, the effects of CXCL16 knockdown on the cell survival of senescent MDA-MB-231 cells, and the correlation between CXCL16 expression levels and survival time in cancer patients.

[0010] <Definition of Terms> In this specification, "senescent cells" are cells in which the cell cycle has been arrested under physiological conditions and cell proliferation has been inhibited, and include cells having any of the following characteristics: Abnormalities of cell cycle-related factors: p16, a cyclin-dependent kinase (CDK) inhibitor that inhibits the progression of the cell cycle. ＩＮＫ４ａ or p21 ＣＩＰ１／ＷＡＦ１The following characteristics are observed: Increased expression of the following: Activation of DNA damage response: The DNA damage response (DDR) pathway is activated, and accumulation of γ-H2AX and 53BP1 is observed. Telomere shortening: Telomere length is shortened, and abnormal expression of telomere-related proteins TRF2 or POT1 is observed. Morphological changes: Morphological changes characterized by increased cell volume and flattening are observed. Increased aging-related β-galactosidase (SA-β-Gal) activity: β-Gal activity present in lysosomes is detected. Aging-related secretory phenomena (SASP): Secretion of inflammatory cytokines (IL-6, IL-8, TNF-α), chemokines (CCL2, CXCL1), and matrix metalloproteinases (MMP-3, MMP-9) increases. Metabolic changes: Decreased mitochondrial function and accumulation of reactive oxygen species (ROS) are observed. In this specification, "senescent cells" include cells having any one or more of the above characteristics. Senescent cells are typically caused by endogenous aging-inducing factors (such as aging, stress response, and oncogene activation) or exogenous aging-inducing factors (such as DNA damage inducers, oxidative stress, radiation, and infection).

[0011] In this specification, "senescence-associated antigen" refers to an antigen that is specifically or selectively expressed on senescent cells. Senescence antigens include antigens expressed intracellularly (e.g., in the nucleus or cytoplasm) and antigens expressed on the cell surface. Antigens expressed on the cell surface can be detected from outside the cell by detection means and can be preferably utilized as targets for detection. Antigens expressed on the cell surface can also be accessed from outside the cell by therapeutic means and can be preferably utilized as therapeutic targets (particularly as targets for drug delivery or recognition targets for immune cells). Examples of antigens expressed on the cell surface of senescent cells include antigens related to immune evasion such as HLA-E, HLA-G, CD47, and PD-L1, as well as antigens related to phagocytosis promotion such as calreticulin and NKG2D ligand.

[0012] In this specification, "in vivo" means performed inside a living organism, and "in vitro" means performed outside a living organism.

[0013] In this specification, "antigen-binding means" refers to means capable of binding to an antigen, preferably capable of binding specifically, selectively, or preferentially to the antigen. Antigen-binding means are not particularly limited, but include, for example, antibodies (e.g., full-length antibodies, bispecific antibodies, human chimeric antibodies, humanized antibodies, human antibodies), antigen-binding fragments of antibodies (e.g., F(ab')2, Fab, scFv, monobodies, nanobodies, etc.), affimers, affitamers, affibodies, aptamers (e.g., peptide aptamers, nucleic acid aptamers, etc.), DARPin, adnectin, actimers, small molecule compounds, ligand mimics, and molecular glue and molecules containing these. These antigen-binding means can be appropriately prepared by conventional methods by those skilled in the art. Humanized antibodies are particularly useful when administered to humans.

[0014] In this specification, "means for recognizing aging antigens" are antigen-binding means capable of binding to aging antigens, and preferably capable of binding specifically, selectively, or preferentially to aging antigens. It is preferable that the means for recognizing aging antigens recognize senescent cells and not recognize non-senescent cells. A person skilled in the art can appropriately prepare means for recognizing aging antigens using conventional methods.

[0015] In this specification, “isolated” means that the substance, cell, or molecule of interest has been removed from its natural or original environment and has been removed from at least some or all of the other components present in that natural environment. Isolation is achieved by physical, chemical, or biological methods. For example, “isolated senescent cells” include cells that have been separated from living tissue, bodily fluids, or culture environments and from which at least some non-senescent or heterologous cells have been removed. The degree of isolation does not necessarily limit the degree of purification and may include a purity of 50% or higher.

[0016] In this specification, "concentrated" or "enriched" refers to a state in which the proportion of the target substance, cell, or molecule has increased compared to the original mixture or environment. "Concentration" does not absolutely guarantee the purity of the target component, but rather means a relative increase in a specific component. For example, a "concentrated senescent cell population" includes a cell population in which the proportion of senescent cells in the total cell population has increased. The degree of concentration includes, for example, an increase of 10% or more to those reaching a purity of 99% or more.

[0017] In this specification, a "cytotoxic agent" refers to a compound, protein, or physical means that acts on cells and has the effect of reducing cell viability or inducing cell death. Cytotoxic agents act through mechanisms such as suppression of cell proliferation, apoptosis (cell suicide), necrosis (death), and autophagy (self-eating). In the present invention, the "cytotoxic agent" includes, but is not limited to, those classified into any of the following categories. Examples of chemical cytotoxic agents include alkylating agents (cyclophosphamide, busulfan), antimetabolites (5-fluorouracil, methotrexate), topoisomerase inhibitors (doxorubicin, etoposide), microtubule inhibitors (paclitaxel, vincristine), oxidative stress-inducing agents (hydroxyurea, paraquat), and proteasome inhibitors (bortezomib). Examples of biological cytotoxic agents include cytotoxic antibodies (rituximab, trastuzumab), cytotoxic peptides or proteins (perforin, granzyme B), immune checkpoint inhibitors (anti-PD-1 antibody, anti-CTLA-4 antibody), cytotoxic viruses (oncolytic viruses), radiation, and physical cytotoxin factors. Radionuclides used as cytotoxic agents include those that emit α-rays, β-rays, or Auger electrons and induce cell death. For example, 2２3 Ra (radium-223), 212 Pb / 212 Bi, 213 Bi (bismuth-213), 225 Ac (actinium-225), 227 Th (thorium-227), 131 I (iodine-131),90 Y (Yttrium-90), 177 Lu (Lutetium-177), 67 Cu (copper-67), 188 Re (Rhenium-188), 153 Sm (Samarium-153), 123 I (iodine-123), 125 I (iodine-125), 111 It may be In (indium-111). These radionuclides can be used in radiopharmaceuticals, targeted radiotherapy (RIT), and radiopharmaceutical therapy.

[0018] In this specification, “label” means an identifiable component added to identify, detect, or track a particular molecule, cell, or structure. A “label” is one that, by binding to or being incorporated into the target object, makes its presence, location, quantity, or dynamics measurable, and includes, but is not limited to, any of the following types of labels: Examples of labels include fluorescent labels (fluorescent dyes such as FITC, AlexaFluor, Cy3, Cy5, Rhodamine, GFP, RFP, and quantum dots), radioactive labels (beta-emitting radionuclides, etc.). 3 H, 14 C, 32 P, 35 S) and gamma-ray emitting radionuclides ( 99 mTc, 123 I, 131 I, 111 In, 18 Examples of labeling methods include F), enzyme labeling (horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc.), chemiluminescent labeling (luminol, acridinium ester, etc.), metallic labeling (colloidal gold, lanthanide labeling, etc.), and magnetic labeling (superparamagnetic iron oxide nanoparticles, etc.).

[0019] In this specification, "cellular autofluorescence" refers to the phenomenon in which endogenous molecules present inside or on the cell surface spontaneously emit fluorescence in response to external excitation light. Because autofluorescence occurs without the introduction of specific fluorescent labels, it can affect fluorescence detection methods such as fluorescence microscopy and flow cytometry. In this invention, the main molecules that exhibit "autofluorescence" include, but are not limited to, the following. Senescent cells exhibit strong autofluorescence, which can be caused by, for example, lipofuscin, NADH (reduced nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), fluorescent oxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) due to lipid oxidation by reactive oxygen species (ROS), accumulation of porphyrins due to abnormalities in the heme synthesis pathway, and accumulation of elastin and collagen. Due to these factors, senescent cells are often excited by ultraviolet to blue light and emit yellow to red autofluorescence.

[0020] In this specification, a "spectral cell analyzer" is a device that analyzes the fluorescence spectral information of cells, and compared to conventional flow cytometers, it has the characteristic of being able to acquire more detailed fluorescence data (especially fluorescence spectra in a specific wavelength range). In particular, it is used as a device for performing spectral flow cytometry (SFC). A spectral cell analyzer can also measure the entire fluorescence spectrum, and background spectra such as autofluorescence of cells can be separated through principal component analysis or machine learning.

[0021] <Senosis Cell Markers> According to this disclosure, senescent cells express one or more senescence antigens (senescence cell markers) selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74. In a preferred embodiment, senescent cells or a population thereof express one or more senescence antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, MSC&NPC antigen, CD184, CD46, CD79b, CD199, and CD74. In a preferred embodiment, senescent cells or a population thereof express at least CXCL16.

[0022] These senescence cell markers are characterized by their expression on the cell surface of senescent cells. Therefore, these senescence cell markers are suitable for targeting and detecting from outside the cell. It is also possible to isolate and concentrate senescent cells using these senescence cell markers. Furthermore, they are advantageous because they allow for the targeting and detection of senescent cells simply by bringing a means of recognizing senescence antigens into contact with the senescent cells.

[0023] CD85k (Cluster of Difference 85k) is a type of immune receptor, also known as LILRB4 (Leukocyte Immunoglobulin-Like Receptor B4). CD85k is an immunosuppressive receptor belonging to the LILRB family, expressed particularly in monocytes and dendritic cells, and plays a role in suppressing the immune response. CD85k primarily interacts with MHC class I molecules such as HLA-G, and is involved in maintaining immune tolerance by suppressing the activation of immune cells. An example of CD85k is human CD85k, which may have the amino acid sequence registered as NCBI Reference Sequence: NP_001265355.2. CD85k can have native mutations. CD85k may also include CD85k having 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means for recognizing CD85k are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include ZM4.1, ZM3.8, REA141, 293623, F2W8T, F2P5N, 128-3, or 16742-MM05.

[0024] CD215 is an alternative name for interleukin-15 receptor α chain (IL-15Rα, Interleukin-15 Receptor Alpha). IL-15Rα is a specific receptor subunit of interleukin-15 (IL-15), and this subunit plays an important role in the function of IL-15. IL-15Rα alone does not have the ability to transmit signals. By associating with the β chain (CD122, IL-2 / IL-15Rβ) and the common γ chain (CD132, IL-2Rγc), it initiates the signal transduction of IL-15. An example of CD215 is human CD215, and human CD215 may have an amino acid sequence registered in NCBI Reference Sequence: NP_002180.1, NP_001230468.1, NP_001243694.1, NP_751950.2, or NP_001338024.2. CD215 may have natural mutations. CD215 may also include those having a sequence identity of 90% or more, or 95% or more with the above amino acid sequence. The means for recognizing CD215 is not particularly limited, but for example, it can be an antibody (for example, a polyclonal antibody, or particularly a monoclonal antibody, especially a humanized antibody, etc.) when administered in vivo). Such antibodies are not particularly limited, but for example, JM7A4 (or eBioJM7A4), DNT15Ra (clone for mouse), DM206, R3S27, or FAB5511A (clone 151303) can be mentioned.

[0025] CD83 is a Type I transmembrane glycoprotein and belongs to the Ig superfamily. CD83 is widely used as a maturation marker for dendritic cells (DC). DCs expressing CD83 are effector T cells (CD4 ＋ and CD8 ＋It promotes the proliferation of ) and contributes to the activation of adaptive immunity. Examples of CD83 include human CD83, which may have amino acid sequences registered in NCBI Reference Sequence: NP_001035370.1, NP_001238830.1, or NP_004224.1. CD83 may have native mutations. CD83 may also include CD83 having 90% or more, or 95% or more, sequence identity with the above amino acid sequences. Means of recognizing CD215 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or monoclonal antibodies, especially when administered in the body, particularly humanized antibodies, etc.). Examples of such antibodies are not particularly limited, but include HB15e, HB15, REA714, F5, Michel-19 (mouse clone), 38101, 3D11, or 10B2.

[0026] CXCL16 is a chemokine belonging to the CXC chemokine family and is a distinctive molecule that exists in two forms: a cell membrane-bound form and a soluble form. CXCL16 is mainly involved in immune responses, inflammation, cancer, and arteriosclerosis, and is thought to play a role in recruiting specific immune cells. An example of CXCL16 is human CXCL16, which may have the amino acid sequence registered in GenBank: AAH17588.1. CXCL16 may have native mutations. CXCL16 may also include CXCL16 with 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means of recognizing CXCL16 are not particularly limited, but may include, for example, antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies, though not limited to them, include 22104, 11648, 21115 (R&D Systems), REA887 (Miltenyi Biotec), CXCL16-1, 61308, 12.1, or F-8.

[0027] CD26, also known as dipeptidyl peptidase IV (DPP4), is a cell surface glycoprotein and a multifunctional protein possessing both enzymatic activity and cell signaling capabilities. CD26 is a 90-110 kDa transmembrane glycoprotein present on the cell membrane and secreted into the bloodstream as a soluble form (sCD26). CD26 is expressed in a variety of cells, including T cells, NK cells, B cells, fibroblasts, endothelial cells, intestinal epithelial cells, and adipocytes. Examples of CD26 include human CD26, which may have amino acid sequences registered in NCBI Reference Sequence: NP_001926.2, NP_001366533.1, NP_001366534.1, or NP_001366535.1. CD26 may have natural mutations. CD26 may also include CD26 having 90% or more, or 95% or more, sequence identity with the above amino acid sequences. Means for recognizing CD26 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies, though not limited to them, include BA5b, M-A261, H226-db, H194-112, 202.1, REA1165, D1-9, or EPR19658.

[0028] CD107b is a glycoprotein present in the lysosomal membrane and is thought to be mainly involved in membrane transport of intracellular organelles, autophagy, and immune responses. CD107b is particularly known as an indicator of the cell degradation process and the degranulation reaction of cytotoxic T cells (CTL) and natural killer (NK) cells. Examples of CD107b include human CD107b, which may have an amino acid sequence registered in NCBI Reference Sequence: NP_001116078.1, NP_002285.1, or NP_054701.1. CD107b may have natural mutations. CD107b may also include CD107b having 90% or more, or 95% or more sequence identity with the above amino acid sequence. The means for recognizing CD107b is not particularly limited, but for example, it can be an antibody (for example, a polyclonal antibody, or particularly a monoclonal antibody, especially a humanized antibody, etc.) when administered in vivo. Such antibodies include, but are not particularly limited to, H4B4, V9, REA407, H4B4-E10, ABL-93, GL2A7, L17, or EPR23136.

[0029] The MSC&NPC antigen (also referred to as the W4A5 antigen) is an antigen recognized by the monoclonal antibody W4A5. As an example of an anti-human MSC and NPC antibody, the monoclonal antibody W4A5 is commercially available (AB_940642 (BioLegend Cat. No. 330806)). The means for binding to the MSC&NPC antigen (also referred to as the W4A5 antigen) is not particularly limited, but for example, it can be an antibody (for example, a polyclonal antibody, or particularly a monoclonal antibody, especially a humanized antibody, etc.) when administered in vivo. Such antibodies include, but are not particularly limited to, W4A5, C40-1457, ME20.4, HB-8737, MLR2, 20Plus, or EPR23145.

[0030] CD184 is a seven-transmembrane G protein-coupled receptor (GPCR) that is thought to be primarily involved in cell migration, immune responses, hematopoietic stem cell maintenance, and cancer metastasis. CD184 is known to be involved in hematopoietic stem cell (HSC) homing, T cell and B cell immune responses, and cancer cell metastasis, particularly through its interaction with CXCL12 (SDF-1). An example of CD184 is human CD184, which may have amino acid sequences registered in NCBI Reference Sequence: NP_003458.1, NM_001008540.2, NM_001348056.2, NM_001348059.2, or NM_001348060.2. CD184 may have native mutations. CD184 may include CD184 having 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means for recognizing CD184 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include 12G5, 1D9, REA649, 2B11, 44708, UMG1, 6H8, or EPR18108.

[0031] CD95 is a type I transmembrane death receptor and is thought to be primarily involved in inducing apoptosis, regulating immune responses, and maintaining cellular homeostasis. CD95 is known to induce programmed cell death (apoptosis) particularly through its interaction with Fas ligand (FasL, CD95L). An example of CD95 is human CD95, which may have amino acid sequences registered in NCBI Reference Sequence: NP_000034.1, NP_001307548.1, NP_690610.1, or NP_690611.1. CD95 may have native mutations. CD95 may also include CD95 with 90% or more, or 95% or more, sequence identity with the above amino acid sequences. The means of recognizing CD95 are not particularly limited, but may include, for example, antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include DX2, EOS9.1, UB2, CH11, APO-1, Jo2, REA711, or EPR5700.

[0032] CD46 is a transmembrane complement regulatory protein (C46) primarily involved in regulating complement activity, modulating immune responses, and functioning as a pathogen infection receptor. CD46 is known to interact particularly with complement C3b and C4b, protecting autologous cells from complement attack by suppressing the complement cascade. Some viruses (such as measles virus) and bacteria also utilize CD46 as a receptor to induce infection. An example of CD46 is human CD46, which may have the amino acid sequence registered in NCBI Reference Sequence: NP_002380.3. CD46 can exhibit natural mutations. CD46 may also include molecules with 90% or more, or even 95% or more, sequence identity with the above amino acid sequence. The means of recognizing CD46 are not particularly limited, but may include, for example, antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include E4.3, TRA-2-10, MEM-258, M177, JSB-1, MCP9, REA759, or EPR4014.

[0033] CD9 is a transmembrane cell surface glycoprotein belonging to the tetraspanin family, and is thought to be mainly involved in cell adhesion, cell migration, signal transduction, and membrane protein organization. CD9 is particularly known to be involved in regulating intercellular interactions and controlling cell adhesion and migration in cooperation with the integrin family. CD9 is also involved in cell fusion and exosome formation, and influences germ cell fertilization and immune responses. An example of CD9 is human CD9, which may have an amino acid sequence registered in NCBI Reference Sequence: NP_001760.1. CD9 can have native mutations. CD9 may also include CD9 with 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means of recognizing CD9 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies, though not limited to them, include HI9a, ML-13, ALB6, M-L13, KMC8, MZ3, REA1071, or EPR2949.

[0034] CD79b is a transmembrane glycoprotein that constitutes part of the B cell antigen receptor (BCR) complex and is thought to be mainly involved in B cell signaling, activation, and differentiation. CD79b is particularly known to form a heterodimer with CD79a (Igα) and to be responsible for B cell receptor (BCR) signaling. Furthermore, CD79b plays an important role in the maturation process of B cells and its overexpression is sometimes observed in malignant diseases such as B cell lymphoma. An example of CD79b is human CD79b, which may have the amino acid sequence registered in NCBI Reference Sequence: NP_001267752.1 or NP_001267753.1. CD79b may have native mutations. CD79b may also include CD79b with 90% or more, or 95% or more, sequence identity with the above amino acid sequences. The means of recognizing CD79b are not particularly limited, but may include, for example, antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies, not particularly limited, include CB3-1, SN8, 3A2-2E7, 24C2.5, HM79-11, REA715, ZL7-4, or EPR6862.

[0035] CD199 is another name for C-C chemokine receptor 6 (CCR6, C-C Motif Chemokine Receptor 6) and is classified as a seven-transmembrane G protein-coupled receptor (GPCR). CD199 is thought to be mainly involved in the regulation of immune cell migration, inflammatory responses, and mucosal immunity. CD199 is known to regulate the recruitment of dendritic cells (DCs), T cells, B cells, and neutrophils, particularly through its interaction with CCL20 (MIP-3α). CD199 is also involved in intestinal immunity and inflammatory diseases such as autoimmune diseases (rheumatoid arthritis, inflammatory bowel disease), and also influences the regulation of the tumor microenvironment. An example of CD199 is human CD199, which may have the amino acid sequence registered in NCBI Reference Sequence: NP_004354.1. CD199 may have natural mutations. CD199 may also include CD199 having 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means of recognizing CD199 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include L053E8, 112509, 9B1, CW1.2, 7E7, 248621, REA505, or EPR24344.

[0036] CD230 is a cell surface marker for prion proteins (PrP, PRNP) and is thought to be involved in maintaining neuronal homeostasis, signal transduction, and the development of prion diseases. CD230, particularly as a normal prion (PrP^C), is known to regulate synaptic plasticity, antioxidant activity, and intercellular communication. Conversely, when converted to an abnormal prion (PrP^Sc), it causes prion diseases such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. An example of CD230 is human CD230, which may have the amino acid sequence registered in NCBI Reference Sequence: NP_001073590.1. CD230 can have natural mutations, which can alter susceptibility to prion diseases. CD230 may also include CD230 having 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means for recognizing CD230 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include 3F4, 6H4, SAF32, 8H4, 8G8, 6D11, BAR221, or EPR4041.

[0037] CD82 is a transmembrane glycoprotein belonging to the tetraspanin family and is thought to be involved in cell adhesion, signal transduction, and suppression of cancer metastasis. CD82 is known to regulate cell motility and proliferation, particularly through interactions with integrins and growth factor receptors, and to function as a tumor suppressor (metastasis suppressor). Furthermore, CD82 is involved in the activation of immune cells and exosome formation, influencing the regulation of infectious diseases and immune responses. An example of CD82 is human CD82, which may have the amino acid sequence registered in NCBI Reference Sequence: NP_002226.2. CD82 can have native mutations. CD82 may also include CD82 with 90% or more, or 95% or more, sequence identity with the above amino acid sequence. The means of recognizing CD82 are not particularly limited, but may include, for example, antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies, not particularly limited, include ASL-24, B-L2, TS82, IA4, 405020, C33, REA1020, or EPR4112.

[0038] CD74 is an MHC class II-related transmembrane glycoprotein thought to be involved in antigen presentation, immune response regulation, and intracellular signal transduction. CD74 is particularly known to function as a chaperone for MHC class II molecules, facilitating the transport and stabilization of newly synthesized peptides until they properly bind to MHC class II. CD74 also functions as a receptor for macrophage migration inhibitors (MIFs), participating in immune response regulation and inflammation promotion. CD74 expression is high in B cells, macrophages, and dendritic cells, and it has been reported to be associated with cancers (B-cell lymphoma, multiple myeloma) and autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus). An example of CD74 is human CD74, which may have the amino acid sequence registered in NCBI Reference Sequence: NP_004346.1. CD74 can have native mutations. CD74 may include CD74 having 90% or more, or 95% or more, sequence identity with the above amino acid sequence. Means for recognizing CD74 are not particularly limited, but may include antibodies (e.g., polyclonal antibodies, or especially monoclonal antibodies, particularly humanized antibodies, when administered into the body). Examples of such antibodies are not particularly limited, but include LN2, M-B741, By2, 5-359, In-1, PIN. 1, REA627, or EPR4064.

[0039] As described above, one or more senescence antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74 are expressed on the surface of senescent cells.

[0040] Some aging antigens are expressed only in certain cells within a cell population that includes senescent cells. Examples of such antigens include one or more selected from the group consisting of CD85k, CD215, CD83, CXCL16, MSC&NPC antigens, CD184, CD46, CD79b, CD199, and CD74. These aging antigens may be expressed in subpopulations that exhibit a stronger aging phenotype. These subpopulations, in particular, may show high expression of the SASP factor, for example, compared to cells that are negative for the above markers (e.g., senescent cells).

[0041] <Method for Targeting, Detecting, or Isolating Senescent Cells> The present disclosure provides a method for targeting, detecting, or isolating senescent cells. In this embodiment, the method for targeting or detecting may be an in vitro or in vivo method. The method for isolating may be an in vitro method. The isolating may include concentration.

[0042] Senescent cells may be cells that express senescence-associated secretory phenotype (SASP) factors. SASP factors may include one or more selected from the group consisting of interleukin (IL)-6, IL-8, IL-1α, IL-1β, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), CCL2, CCL5, CXCL1, MMP-1, MMP-3, MMP-9, CXCL10, and CXCL16.

[0043] The method in this embodiment may include contacting senescent cells with means for recognizing one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74.

[0044] In one preferred embodiment, the means for recognizing senescence antigens binds to one or more selected from the group consisting of CD85k, CD215, CD83, CXCL16, MSC&NPC antigen, CD184, CD46, CD79b, CD199, and CD74, and more preferably to CXCL16. In this embodiment, the means can bind only to a group of cells in a population designated as senescent cells that particularly exhibit a strong senescence phenotype (e.g., secretion of SASP factor).

[0045] When a means for recognizing senescence antigens is brought into contact with senescence cells, the means for recognizing senescence antigens binds to the senescence cells and is recruited to the vicinity of the surface of the senescence cells. By this method, those skilled in the art can target senescence cells and detect or isolate them using the means for recognizing senescence antigens.

[0046] Means for recognizing aging antigens can target senescent cells, and in some embodiments, by conjugating a pharmaceutical compound (e.g., a cytotoxic agent) to the means, the pharmaceutical compound can be delivered to the senescent cells and treated with the pharmaceutical compound specifically, selectively, or preferentially. The pharmaceutical compound is not particularly limited, but may include, for example, nucleic acids, proteins, small molecule compounds, and other physiologically active substances. For example, a cytotoxic agent may be used as the pharmaceutical compound. It is believed that by delivering a cytotoxic agent to senescent cells, the senescent cells can be killed specifically, selectively, or preferentially. In some embodiments, the pharmaceutical compound includes a reprogramming factor or a nucleic acid encoding a reprogramming factor. Reprogramming factors may also be used to rejuvenate senescent cells. For this purpose, the means for recognizing aging antigens may be a conjugate of a cytotoxic agent, a reprogramming factor, or a nucleic acid encoding a reprogramming factor. The conjugate links the means with or without a linker. The linker may be a cleavable or non-cleavable linker. A cleavable linker is a linker that is cleaved under certain conditions, and may be, for example, a linker that cleaves under a decrease in pH (e.g., under a lysosomal environment), a linker that cleaves under a reducing environment (e.g., in the presence of 1 M glutathione), or a linker that cleaves in the presence of a peptidase (e.g., Val-Cit).

[0047] Means of recognizing aging antigens can target senescent cells, and in some embodiments, the means of recognizing aging antigens may be chimeric antigen receptors (CARs). Chimeric antigen receptors are chimeric molecules having an antigen-binding fragment of an antibody (particularly scFv) and an activation domain of an immune cell. Generally, a CAR is a molecule consisting of scFv, an extracellular hinge domain, a transmembrane domain (e.g., CD8α or CD28), and an activation signaling domain (e.g., CD3ζ) linked together. CARs can be introduced into cells and expressed on the cell surface. Cells expressing CARs can be targeted against specific antigens. For example, a CAR can be introduced into immune cells such as T cells or NK cells, and these immune cells can target target cells such as cancer cells. In first-generation CARs, the scFv, extracellular hinge domain, transmembrane domain (e.g., CD8α or CD28), and activation signaling domain (e.g., CD3ζ) were linked. Second-generation CARs further include a co-stimulatory molecule signaling domain for the activation of immune cells into which the CAR has been introduced. Co-stimulatory factors such as CD28, 4-1BB, OX40, CD27, and ICOS are used as the co-stimulatory molecule signaling domain. In third-generation CARs, multiple co-stimulatory factors (e.g., combinations of the above co-stimulatory factors) are incorporated. Thus, improvements have been made to CARs to enable the sustained proliferation of immune cells into which the CAR has been introduced in vivo. Preferably, all domains other than the scFv portion are derived from human proteins. The scFv includes the heavy chain variable region and the light chain variable region of the antibody, where the heavy chain variable region and the light chain variable region are linked via a flexible linker (e.g., a GS linker and a Whitlow linker). CAR can be expressed in immune cells, etc., meaning that the means of recognizing aging antigens may be cells that express CAR (especially pluripotent stem cells, stem cells, hematopoietic stem progenitor cells, immune cells, etc.). Immune cells are not particularly limited, but examples include T cells (e.g., cytotoxic T cells and helper T cells), NK cells, macrophages, neutrophils, etc.

[0048] Means for recognizing aging antigens can target senescent cells, but in some embodiments, the means for recognizing aging antigens may be T-cell recruiting antibodies. T-cell recruiting antibodies have domains that bind to antigens on T cells (e.g., the CD3 complex, or its subunits (e.g., CD3ε)) and tumor antigens, respectively. The domains that bind to antigens on T cells (e.g., the CD3 complex, or its subunits (e.g., CD3ε)) are not particularly limited but may include, for example, the heavy chain variable region and the light chain variable region of OKT3. Examples of T-cell recruiting antibodies include whole IgG antibodies and non-whole IgG antibodies. Whole IgG antibodies have the structure of an IgG antibody (IgG1, IgG2, or IgG4), and specifically include a Fab containing a domain that binds to a tumor antigen and a Fab containing a domain that binds to an antigen on a T cell, with each heavy chain having a structure in which the Fc region of the IgG antibody is linked via a hinge. Non-Whole IgG antibodies do not contain the structure of IgG antibodies, but rather have a structure in which, for example, a domain that binds to a tumor antigen (e.g., scFv) and a domain that binds to an antigen on a T cell (e.g., scFv), and these are linked via a linker (e.g., a flexible linker).

[0049] For types and manufacturing methods of T cell recruiting antibodies, see Lejeune M. et al., Frontiers in Immunology, 11, Article 762, 2020, Blanco B. et al., Clinical Cancer Research, 27(20), 5457-5164, 2021, and Madsen A. et al., Frontiers in Bioengineering and Biotechnology, 12, Article 1352014, 2024. For example, T cell recruiting antibodies may have reduced or lost ability to bind to Fcγ receptors.Mutations employed in T cell recruitment antibodies are not particularly limited, but include, for example, amino acid mutations that reduce or eliminate binding to the Fcγ receptor: amino acid substitution mutations to L234 and L235 (e.g., double mutation of L234A / L235A (LALA mutation), double mutation of L234F / L235E, triple mutation of L234F / L235E / D265A; FEA mutation), amino acid mutations to D265 (e.g., D265A), amino acid mutations to G236 (e.g., Mutations that reduce or eliminate the ability to bind to complement C1q: amino acid mutations to P329 (e.g., P326G); Mutations that eliminate N-linked glucosylation: amino acid mutations to N297 (e.g., N297A); Mutations that alter the Fc structure and exhibit anti-inflammatory effects: amino acid mutations to F241 (e.g., F241A); Mutations that increase the ability to bind to the neonatal Fc receptor (FcRn): amino acid substitution mutations to M428 (e.g., M428L), amino acid substitutions to N434 Acid substitution mutations (e.g., N434S), amino acid substitution mutations to M428 and N434 (e.g., double mutation of M428L / N434S), amino acid substitution mutations to M252 (e.g., M252Y), amino acid substitution mutations to S254 (e.g., S254T), amino acid substitution mutations to T256 (e.g., T256E), and triple amino acid substitution mutations to M252, S254, and T256 (e.g., triple acid mutation of M252Y / S245T / T256E); IgG4 sub Examples of double-strand stabilizing mutations in the class include amino acid substitution mutations to S228 (e.g., S228P); amino acid mutations that selectively reduce complement activity include amino acid substitution mutations to K322 (e.g., K322A); knob-into-hole mutations include introducing T366W as the knob and T366S, L368A, and Y407V as the hole; and Duomab mutations include one, two, or more combinations of F405L, K409R, F412A, and Y436F. The heavy chain C-terminus K447 may be deleted to ensure antibody homogeneity, etc. In addition, additional mutations preferred for T cell recruitment antibodies may be added. Furthermore, the presence of various mutations is acceptable as long as they do not significantly inhibit the activity of the T cell recruitment antibody.

[0050] Examples of reprogramming factors include the Yamanaka trifactory (Oct4, Sox2, Klf4), the Yamanaka quadruplefactory (Oct4, Sox2, Klf4, c-myc), and the Thomson factor (Oct4, Sox2, Nanog, Lin28). Other examples of reprogramming factors include HDAC inhibitors such as Valproic acid (VPA), GSK3β inhibitors such as CHIR99021, MEK inhibitors such as PD0325901, cAMP activators such as Forskolin, TGF-β inhibitors such as RepSox, Esrrb, Tbx3, Glis1, Nr5a2, Utf1, Bcl2, and L-myc, as well as combinations thereof, which can be appropriately selected by those skilled in the art.

[0051] In one embodiment, the means can label senescent cells by linking the label. The label may be a fluorescent label, radioactive label, enzyme label, chemiluminescent label, metallic label, magnetic label, or tag label. A tag label refers to a molecule, chemical group, or physical label (e.g., a detection label, identification label, or separation label) that is applied for the purpose of detecting, identifying, or separating an object. The tag label enables the recognition or manipulation of the target substance by having specific interactions, physicochemical properties, or optical properties. Examples of tag labels, though not particularly limited, include fluorescent labels, biotin or avidin labels, tag peptide labels, and chemical labels. It is also possible to target senescent cells by targeting the label. Targeting of the label can be achieved by an antigen-binding means to the label.

[0052] Means for recognizing senescence antigens can also be used to detect senescent cells. In some embodiments, if the means are labeled, it is possible to determine whether a cell is a senescent cell or not based on the presence or absence of binding of the label to the cell, and thus it can be used for detecting senescent cells. As the label has been described above, we will refer to the description of the label above and omit redundant explanations.

[0053] In one embodiment, the means for recognizing senescence antigens is fluorescently labeled. The fluorescent labeling may be, for example, a fluorescent dye or a fluorescent protein. The fluorescently labeled means can bind to senescent cells, thereby indirectly binding the fluorescent label to the senescent cells. Senescent cells with fluorescent labeling can be determined to be fluorescently labeled, for example, using a spectral analyzer (e.g., a spectral cell analyzer), thereby enabling detection of senescent cells. A cell sorter equipped with a spectral analyzer can analyze the spectrum emitted by cells in real time and separate senescent cells with fluorescent labeling from the remaining cells. Since senescent cells emit strong autofluorescence, preferably, the autofluorescence spectrum (i.e., background spectrum) is subtracted from the measured spectrum to obtain a difference spectrum, and the difference spectrum can be used to determine whether or not the cells are fluorescently labeled, and cells determined to be fluorescently labeled can be separated. The difference spectrum may contain fluorescence wavelengths derived from the fluorescent labeling. If the difference spectrum contains fluorescence wavelengths derived from the fluorescent labeling, it can be evaluated that binding between the means for recognizing senescence antigens and isolated senescent cells has been detected. The main reason for using a spectral analyzer is to separate the autofluorescence spectrum from the spectrum derived from the detection or separation label; therefore, a wide range of devices or means for separating the autofluorescence spectrum from the spectrum derived from the detection or separation label can be used.

[0054] In other words, the present disclosure provides a method for separating or concentrating isolated senescent cells. This method may include contacting a cell composition containing senescent cells with means that bind to a senescent antigen. The senescent cells bind to the means and are affixed with a label provided by the means. The labeled senescent cells can be separated or concentrated based on the presence or absence of the label. In one embodiment, the label may be a fluorescent label, and senescent cells with the fluorescent label can be separated or concentrated. Separation and concentration can be carried out using a cell sorter with a spectral cell analyzer (e.g., a spectral cell sorter), preferably based on the presence of a characteristic fluorescence wavelength derived from the fluorescent label in a difference spectrum obtained by subtracting the autofluorescence spectrum from the measured spectrum. Examples of senescent antigens include one or more selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigens, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74.

[0055] Means for recognizing senescence antigens can also be used to isolate senescent cells from other cells. In some embodiments, if the means are labeled, it is possible to determine whether a cell is senescent based on the presence or absence of binding of the label to the cell, thereby enabling the isolation of senescent cells from other cells. The label may be fluorescent, radioactive, enzyme, chemiluminescent, metallic, magnetic, or tagged. Isolating senescent cells from other cells may lead to the enrichment of senescent cells.

[0056] <Methods for treating senescent cells according to this disclosure> According to this disclosure, methods for treating senescent cells (e.g., in vivo or in vitro methods) are provided. According to this disclosure, methods for treating senescent cells in subjects having senescent cells (e.g., human subjects) are also provided.

[0057] Inhibiting the expression or function of any of the above senescence markers (preferably CXCL16) in senescent cells may result in decreased expression of SASP factors, improvement of senescence, and / or removal of senescent cells. Therefore, in this embodiment, the method may include inhibiting the expression or function of any of the above senescence markers (preferably CXCL16) in senescent cells. This may preferably result in decreased expression of SASP factors, improvement of senescence in senescent cells, and / or removal of senescent cells (e.g., cell death of senescent cells). In some embodiments, senescent cells may be senescent cancer cells. In some embodiments, senescent cells may be senescent non-cancerous cells.

[0058] Inhibition of the expression of any of the above aging markers (preferably CXCL16) can be achieved by an expression inhibitor for the above aging marker (e.g., a knockdown nucleic acid). Knockdown nucleic acids may be siRNA, shRNA, antisense oligonucleotides (ASOs), microRNAs (miRNAs), gapmers, etc. siRNA consists of an antisense strand and a sense strand for target mRNA. siRNA usually has an overhang of about 2 nucleotides at its 3' end. siRNA is about 20 to 25 nucleotides long and can reduce the expression of target mRNA. siRNA can be sequenced to reduce mRNA expression. shRNA has a structure in which the antisense strand and sense strand of siRNA are linked by a loop about 4 to 10 nucleotides long. shRNA can be expressed from an shRNA expression vector. Antisense oligonucleotides (ASOs) are single-stranded short nucleic acids (oligonucleotides) that specifically bind to target mRNA and suppress translation or induce degradation. Antisense oligonucleotides (ASOs) are designed to regulate gene expression and are primarily used as RNA-targeted therapeutics and functional analysis tools. When an ASO binds to a specific complementary sequence of mRNA, it can activate RNaseH and degrade the mRNA. ASOs are not particularly limited, but may contain modified nucleic acids (modified nucleic acids) with modifications such as phosphorothioate modification, 2'-O-methyl modification, 2'-O-methoxyethyl modification, phosphoramide morpholino oligo modification, and bridging nucleic acid modification (LNA). MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by repressing or degrading the translation of target mRNA. miRNAs can be single-stranded RNAs of approximately 21-25 nucleotides. Gapmers are a type of antisense oligonucleotide (ASO) that have a structure that promotes mRNA degradation via RNaseH. They typically have a central DNA region (gap) and a structure modified with chemically modified RNA at both ends, efficiently inducing the degradation of target mRNA. The central gap region can be DNA approximately 8 to 10 nucleotides long.The gap region induces degradation of target mRNA by RNaseH. The gapmer has regions containing modified nucleic acids at both ends of the gap region. The regions containing modified nucleic acids are not particularly limited, but may include nucleic acids with modifications such as 2'-O-methyl modification, 2'-O-methoxyethyl modification, or cross-linked nucleic acid modification (LNA). These expression inhibitors may be linked to means for recognizing the aging antigen. This is thought to enable the expression inhibitor to be delivered specifically, selectively, or preferentially to senescent cells. These expression inhibitors may be encapsulated in lipid nanoparticles, liposomes, polyion complexes, etc.

[0059] Inhibition of the expression of any of the above aging markers (preferably CXCL16) can be achieved by a functional inhibitor of the above aging marker (for example, an antibody or its antigen-binding fragment (particularly an antagonist antibody or its antigen-binding fragment having antagonist activity)). Antibodies or their antigen-binding fragments (particularly antagonist antibodies or its antigen-binding fragment having antagonist activity) can be appropriately prepared by those skilled in the art.

[0060] Accordingly, the methods of the present disclosure may include contacting senescent cells with an expression inhibitor or functional inhibitor of the senescence marker (e.g., an antibody or its antigen-binding fragment (particularly an antagonist antibody or its antigen-binding fragment having antagonist activity)). The in vivo methods of the present disclosure may also include administering an effective amount of the expression inhibitor or functional inhibitor of the senescence marker (e.g., an antibody or its antigen-binding fragment (particularly an antagonist antibody or its antigen-binding fragment having antagonist activity)) to the subject (e.g., a human subject).

[0061] Furthermore, the method of this disclosure may include contacting senescent cells with a conjugate of means that recognize senescent antigens for the above-mentioned senescence markers and a pharmaceutical compound (e.g., a cytotoxic agent). This is expected to induce cytotoxicity in senescent cells and remove them. The in vivo method of this disclosure may include administering an effective amount of the conjugate of means that recognize senescent antigens for the above-mentioned senescence markers and a pharmaceutical compound (e.g., a cytotoxic agent) to a subject having senescent cells (particularly a human subject). The conjugate of means that recognize senescent antigens and a pharmaceutical compound (e.g., a cytotoxic agent) is as described above, so we will refer to that above and omit any redundant explanation here.

[0062] <Method for Identifying Surface Antigens Expressed on Autofluorescent Cells> The present disclosure provides a method for identifying surface antigens expressed on autofluorescent cells. Preferably, a fluorescently labeled antigen-binding means may be used in this method. Normally, when a fluorescently labeled means is bound to autofluorescent cells, the fluorescent label is obscured by the autofluorescence, making it difficult to detect the fluorescent label. In contrast, a spectral analyzer (e.g., a spectral cell analyzer) can be used to obtain a measurement spectrum, subtract the autofluorescence spectrum, and detect the difference spectrum. By detecting the fluorescence derived from the fluorescent label in the difference spectrum, the presence or absence of the fluorescent label can be detected with high sensitivity. Cells exhibiting autofluorescence may preferably be senescent cells. In some embodiments, other examples of autofluorescent cells include macrophages, dendritic cells, hepatocytes, nerve cells, lung epithelial cells, fibroblasts, erythrocytes and related cells containing porphyrins, cancer cells, and plant cells.

[0063] The present disclosure also provides a method for identifying surface antigens selectively expressed in subpopulations within a cell population including autofluorescent cells. Preferably, this method may utilize a fluorescently labeled antigen-binding means. Normally, when a fluorescently labeled means is bound to autofluorescent cells, the fluorescent label is obscured by the autofluorescence, making detection difficult. In contrast, by using a spectral analyzer (e.g., a spectral cell analyzer) to obtain a measurement spectrum, subtracting the autofluorescence spectrum to obtain a difference spectrum, and analyzing the fluorescence wavelength derived from the fluorescent label in the difference spectrum, the presence or absence of the fluorescent label can be detected with high sensitivity. Autofluorescent cells are preferably senescent cells (e.g., senescent cancer cells and senescent non-cancerous cells). In some embodiments, autofluorescent cells also include, for example, macrophages, dendritic cells, hepatocytes, nerve cells, lung epithelial cells, fibroblasts, porphyrin-containing erythrocytes and related cells, cancer cells, and plant cells.

[0064] <Compositions for Use in the Methods of the Disclosure> The Disclosure provides compositions for use in the methods described above. These compositions may include antigen-binding means (preferably means for recognizing aging antigens). Whether labeling is preferred in each method is specifically described. In methods in which labeled means are used, the means are labeled.

[0065] More specifically, the present disclosure provides a composition comprising means for recognizing aging antigens for use in methods for targeting, detecting, or isolating senescent cells.

[0066] In one embodiment, a composition is provided comprising means for recognizing aging antigens for use in a method for targeting senescent cells. In this embodiment, the means is labeled. The labeling may be any of the above-mentioned fluorescent, radioactive, enzymatic, chemiluminescent, metallic, magnetic, or tagged. The means may also be linked to a pharmaceutical compound. The pharmaceutical compound is not particularly limited, but may include, for example, nucleic acids, proteins, small molecules, or other physiologically active substances, as described above.

[0067] In one embodiment, a composition is provided comprising means for recognizing aging antigens for use in a method for isolating or concentrating senescent cells. In this embodiment, the means is, for example, tag-labeled. The tag labeling is not particularly limited, but may include, as described above, fluorescent labeling, biotin or avidin labeling, tag peptide labeling, chemical labeling, etc.

[0068] This disclosure provides the use of means for recognizing aging antigens for use in the above-described method. This disclosure also provides the use of means for recognizing aging antigens in the manufacture of compositions for use in the above-described method.

[0069] This disclosure provides compositions for use in methods for treating senescent cells (e.g., in vivo or in vitro methods). These compositions may comprise knockdown nucleic acids, or antibodies or antigen-binding fragments thereof (particularly antagonist antibodies or antigen-binding fragments thereof having antagonist activity). This disclosure provides the use of knockdown nucleic acids, or antibodies or antigen-binding fragments thereof (particularly antagonist antibodies or antigen-binding fragments thereof having antagonist activity) in the preparation of compositions for use in the above methods.

[0070] Materials and Methods Cell Culture and Induction of Cellular Senescence (Figure 1-13) Human normal fibroblasts (IMR-90; CCL-186) and human pancreatic cancer cell lines (PANC-1; CRL-1469) were obtained from the American Type Culture Collection (ATCC). Human breast cancer cell line (MDA-MB-231) was obtained from the human cancer cell panel JFCR39. Human normal fibroblasts (TIG-3; JCRB0506) were obtained from the JCRB Cell Bank.

[0071] IMR-90 cells were cultured in RPMI-1640 medium (Fujifilm Wako Pure Chemical Industries; 189-02025) containing 10% fetal bovine serum (FBS) (Biosera; 515-99055) and 100 U / μg / mL penicillin / streptomycin (Fujifilm Wako Pure Chemical Industries; 168-23191). PANC-1 cells, MDA-MB-231 cells, and TIG-3 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Nacalai Tesque; 08458-16) containing 10% fetal bovine serum (FBS) (Gibco, 10437-028) and 100 U / μg / mL penicillin / streptomycin (Fujifilm Wako Pure Chemical Industries; 168-23191). Cells were cultured at 37°C and 5% CO2. ２ Cells were cultured under environmental conditions. IMR-90 cells and TIG-3 cells with a Population Doubling Level (PDL) of less than 45 were used as normal cells. Cellular senescence was induced in IMR-90 cells by treating them with 200 ng / mL doxorubicin (Fujifilm Wako Pure Chemical Industries; 040-21521) and culturing them for 11-14 days. Cellular senescence was induced in PANC-1 cells by treating them with 50 nM gemcitabine (Selleck; S1149) and culturing them for 12 days. Cellular senescence was induced in MDA-MB-231 cells by treating them with 50 nM doxorubicin, then treating them again with the same concentration of doxorubicin 3-4 days later, and culturing them for a further 6-7 days. For the TIG-3 cells that underwent replication senescence, we used cells in which proliferation had stopped due to a PDL level exceeding 80.

[0072] Screening of senescent cell-specific surface antigens (Figure 1-4) Normal or senescent IMR-90 cells were detached by treatment with trypsin (using a 10-fold dilution of Fujifilm Wako Pure Chemical Industries; 208-17251), washed once with PBS, and then screened with LEGENDSScreen. ＴＭThe fluorescently labeled antibody library of cell surface antigens contained in the human PE antibody kit (BioLegend; 700011) was dispensed into a 96-well plate, with 1.5 x 10⁶ of antibodies per well. ４ The cells were dispensed individually and allowed to stand for 30 minutes at 4°C in the dark to stain the cell surface antigens. The cells were then washed once with Cell Staining Buffer (BioLegend; 420201), fixed with Fixation Buffer, and allowed to stand for 10 minutes at room temperature in the dark. After washing twice with Cell Staining Buffer, the cells were suspended in 160 μL / well of Cell Staining Buffer. The stained cell samples and the unstained cell samples for autofluorescence spectroscopy were each loaded into a Spectral Cell Analyzer SA3800 (SONY; LE-SA3800GA), and 4,000 events were measured spectrally. The fluorescence signal intensity of the PE was calculated by removing the autofluorescence spectrum using WLSM unmixing. To assess the accuracy of the screening system, the PE fluorescence signal intensity was measured for cell samples stained with PE anti-human CD90 (Thy1) antibody (BioLegend; 328109) as the positive symmetry group (N = 8) and cell samples stained with PE Mouse IgG1k Isotype control antibody (BioLegend; 400113) as the negative symmetry group (N = 8). The Z'-factor, calculated using the following formula, was then determined as an indicator of the screening system's accuracy.

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[0074] In senescent cells, the fluorescence signal intensity is log １０ Antigens with a (PE-Area) > 4 and exhibiting a fluorescence signal intensity at least four times higher in senescent cells compared to normal cells were selected as candidate surface antigens specific to senescent cells.

[0075] Evaluation of CXCL16 expression levels on the cell surface (Figure 5-8): Proliferating or senescent cells were detached by Accutase (NACALAI TESQUE; 12679-54), washed once with MACS buffer (0.5% BSA, 2 mM EDTA in PBS), resuspended in MACS buffer, and 1 / 100 (v / v) of PE anti-human CXCL16 antibody (BioLegend; 360803) or PE Mouse IgG1k Isotype control antibody (BioLegend; 400114) was added. Staining was performed at 4°C in the dark for 30 minutes, washed once with MACS buffer, and resuspended in MACS buffer. The stained cell samples and unstained cell samples for autofluorescence spectroscopy prepared in this manner were each loaded into a SA3800 spectral cell analyzer, and spectral measurements were performed for 10,000 events. The fluorescence signal intensity of PE was calculated by removing the autofluorescence spectrum using WLSM unmixing.

[0076] Sorting of senescent cells based on CXCL16 expression levels (Figure 9) Using doxorubicin-induced senescent IMR-90 cells, stained cell samples were prepared according to the method described in the previous section, "Evaluation of CXCL16 expression levels on the cell surface," and these were sorted together with unstained cell samples for autofluorescence spectroscopy using a spectral cell sorter FACSDiscover. ＴＭ The cells were incorporated into S8 (BD; 665158). More than 50,000 cells were separated from both the CXCL16-positive and CXCL16-negative populations.

[0077] Gene expression repression using siRNA (Figure 10-13) Gene expression repression using siRNA is achieved by lipofectamine ＴＭThe RNAiMAX transmission agent (Invitrogen; 13778500) was used, and the procedure was performed according to the manufacturer's protocol. As siRNAs, ON-TARGETplus Human CXCL16 siRNA (Horizon Discovery; J-007876-07, J-007876-08, J-007876-05), which targets the CXCL16 mRNA sequence, and ON-TARGETplus Non-targeting Control siRNA #1 (Horizon Discovery; D-001810-01) or ON-TARGETplus Non-targeting Control siRNA #2 (Horizon Discovery; D-001810-02) were used at a final concentration of 10 Treat with nM, at 37°C and 5% CO2. ２ After culturing for 3-4 days under the specified conditions, the culture medium was replaced with fresh medium, and a second siRNA treatment was performed under the same conditions as the first, at 37°C and 5% CO2. ２ The cells were cultured for an additional 3-4 days under the specified conditions. Gene expression repression efficiency was evaluated by RT-qPCR.

[0078] Measurement of cell viability (Figures 12, 13): Cells cultured in a 96-well plate were mixed with approximately 1 / 10 the volume of Cell Counting Kit-8 (DOJINDO; 343-07623), and the cells were incubated at 37°C and 5% CO2. ２ After culturing for approximately 2 hours under the specified conditions, the absorbance at 450 nm was measured using Varioskan LUX (Thermo; VL0L00D0). The absorbance in wells containing only culture medium and Cell Counting Kit-8 (without seeding cells) was subtracted as the background value.

[0079] Evaluation of mRNA expression levels by RT-qPCR (Figure 9-11) RNA extraction from cells is performed using mirVana. ＴＭ The procedure was performed using the miRNA Isolation Kit (Invitrogen; AM1561) according to the manufacturer's protocol. PrimeScript was applied to the extracted RNA. ＴＭcDNA was prepared by reverse transcription using RT Master Mix (TaKaRa; RR036A). RT-qPCR was performed using the StepOnePlus PCR system (Applied Biosystems) with SYBR Premix Ex Taq II (TaKaRa; RR820A). The quantity of all samples was determined by the Standard Curve method, and GAPDH or PPIA was used as the internal standard gene. The sequences of the primers used are as follows.

[0080]

[0081] Correlation analysis between gene expression levels and cancer prognosis (Figure 14) Using Survival Analysis on GEPIA 2 (http: / / gepia2.cancer-pku.cn / #index), a web server capable of analyzing RNA sequence expression data from tumor and normal samples obtained from the TCGA and GTEx projects, we analyzed the correlation between high and low CXCL16 gene expression levels and the patients' overall survival in each cancer type dataset (Brain Lower Grade Glioma, LGG; Lung squamous cell carcinoma, LUSC; Uveal Melanoma, UVM).

[0082] The analysis parameters were set as follows: Group cutoff, “Median”; cutoff-high (%), 50; cutoff-low (%), 50; hazards ratio (HR), “Yes”; 95% confidence intermediate, “Yes”; axis units, “Months”.

[0083] As a result, we first established a screening system for senescent cell-specific surface antigens. As shown in Figure 1, we created normal cells and doxorubicin-induced senescent cells, stained their surface antigens, and measured the fluorescence signal after removing the autofluorescence spectrum using a spectral analyzer to search for surface antigens specific to senescent cells. Antigens that produced a sufficiently strong fluorescence signal in senescent cells and had a higher fluorescence signal intensity compared to normal cells were identified as senescent cell-specific surface antigens.

[0084] In the screening system shown in Figure 1, cells were stained using a PE-labeled isotype control antibody as the negative control group and a PE-labeled anti-human CD90 antibody as the positive control group. The autofluorescence spectrum was removed using a spectral cell analyzer, and the fluorescence signal intensity was measured. The results are shown in Figure 2. In Figure 2, the Z'-Factor, an index indicating the accuracy of the assay system, was calculated. As shown in Figure 2, this screening system meets the standard of Z' > 0.5, which is generally considered to have sufficient accuracy, demonstrating that it is a system capable of detecting cell surface antigens with sufficient accuracy.

[0085] Screening results yielded senescent cell-specific surface antigens. Sufficient signal strength (Log) was observed in senescent cells. １０ Sixteen cell surface antigens were identified as those that yield (PE-Area) > 4) and have a higher fluorescence signal intensity compared to normal cells (mean fluorescence intensity ratio (senescent cells / normal cells) > 4) (see Figure 3).

[0086] Figure 4 shows histograms of fluorescence intensity for each of these antigens during screening. Among the senescent cell-specific surface antigens identified by screening, it was confirmed that some surface antigens are expressed throughout the entire senescent cell population, while others are expressed only in subpopulations.

[0087] Doxorubicin-induced senescent cells were stained with PE-labeled anti-CXCL16 antibody. Human normal fibroblasts IMR-90 were treated with 200 ng / mL doxorubicin to induce cellular senescence. Surface expression of CXCL16, which was identified as a senescent cell-specific surface antigen, was stained using PE-labeled anti-CXCL16 antibody, and the fluorescence signal was observed after removing the autofluorescence spectrum using a spectral cell analyzer. High expression of CXCL16 was confirmed only in a subset of senescent cells (see Figure 5).

[0088] Human normal fibroblasts TIG-3 were passaged to induce cellular senescence. Surface expression of CXCL16, identified as a senescent cell-specific surface antigen, was stained using a PE-labeled anti-CXCL16 antibody. Autofluorescence spectra were removed using a spectral cell analyzer, and the fluorescence signal was observed. High expression of CXCL16 was confirmed only in a subset of senescent cells (see Figure 6).

[0089] Human breast cancer cells MDA-MB-231 were treated with 50 nM doxorubicin to induce cellular senescence. Surface expression of CXCL16, identified as a senescent cell-specific surface antigen, was stained using a PE-labeled anti-CXCL16 antibody. Autofluorescence spectra were removed using a spectral cell analyzer, and the fluorescence signal was observed. High expression of CXCL16 was confirmed only in a subset of senescent cells (see Figure 7).

[0090] Human pancreatic cancer cells (PANC-1) were treated with 50 nM gemcitabine to induce cellular senescence. Surface expression of CXCL16, identified as a senescent cell-specific surface antigen, was stained using a PE-labeled anti-CXCL16 antibody. Autofluorescence spectra were removed using a spectral cell analyzer, and the fluorescence signal was observed. High expression of CXCL16 was observed only in a subset of senescent cells (see Figure 8).

[0091] As described above, CXCL16 consistently showed high expression only in certain cell populations of senescent cells.

[0092] Human normal fibroblasts IMR-90 were treated with 200 ng / mL doxorubicin to induce cellular senescence. CXCL16-positive and CXCL16-negative senescent cells were separated, and mRNA expression of the SASP gene, a known senescence-related gene, was confirmed by qRT-PCR. The results showed that SASP gene expression was significantly higher in CXCL16-positive cells than in CXCL16-negative cells (see Figure 9).

[0093] Human normal fibroblasts IMR-90 were treated with 200 ng / mL doxorubicin to induce cellular senescence. CXCL16 expression was suppressed using siRNA, and mRNA expression of the SASP gene, a known senescence-related gene, was confirmed by qRT-PCR. The results showed that suppression of CXCL16 expression significantly reduced the expression level of the SASP gene (see Figure 10).

[0094] Human breast cancer cells MDA-MB-231 were treated with 50 nM doxorubicin to induce cellular senescence. CXCL16 expression was suppressed using siRNA, and mRNA expression of the SASP gene, a known senescence-related gene, was confirmed by qRT-PCR. The results showed that suppression of CXCL16 expression significantly reduced the expression level of the SASP gene (see Figure 11).

[0095] Human normal fibroblasts IMR-90 were treated with 200 ng / mL doxorubicin to induce cellular senescence. Cell viability was then checked by suppressing CXCL16 expression using siRNA. As a result, it was confirmed that suppression of CXCL16 expression significantly reduced the viability of senescent cells (see Figure 12).

[0096] Human breast cancer cells MDA-MB-231 were treated with 50 nM doxorubicin to induce cellular senescence. CXCL16 expression was suppressed using siRNA, and cell viability was examined. The results showed that suppression of CXCL16 expression significantly reduced the viability of senescent cells (see Figure 13).

[0097] Using Survival Analysis on GEPIA 2 (http: / / gepia2.cancer-pku.cn / #index), a web server capable of analyzing RNA sequence expression data from tumor and normal samples obtained from the TCGA and GTEx projects, we analyzed the correlation between high and low CXCL16 gene expression levels and the overall survival of patients in each cancer type dataset (Brain Lower Grade Glioma; LGG, Lung squamous cell carcinoma; LUSC, Uveal Melanoma; UVM). As shown in Figure 14, a tendency for shorter survival times was observed in the high-expression group of CXCL16 compared to the low-expression group in each cancer type.

Claims

1. An in vitro or in vivo method for targeting or detecting senescent cells, comprising contacting senescent cells with means that recognize one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74.

2. The method according to claim 1, which is an in vitro method.

3. The method according to claim 1 or 2, wherein the means for recognizing aging antigens is bound to one or more selected from the group consisting of CD85k, CD215, CD83, CXCL16, MSC&NPC antigen, CD184, CD46, CD79b, CD199, and CD74.

4. The method according to any one of claims 1 to 3, wherein the means for recognizing the aging antigen is bound to CXCL16.

5. The method according to any one of claims 1 to 4, wherein the means for recognizing aging antigens is labeled.

6. The method according to any one of claims 1 to 5, wherein detection of binding between means for recognizing senescence antigens and isolated senescent cells indicates that the cells are senescent cells.

7. The method according to any one of claims 1 to 6, wherein the means for recognizing the aging antigen is in the form of a conjugate with a cytotoxic agent, and the binding of the means for recognizing the aging antigen to isolated senescent cells results in cytotoxicity to the senescent cells.

8. The method according to any one of claims 1 to 7, wherein the means for recognizing aging antigens is labeled, the label is analyzed by a spectral analyzer, the spectrum of autofluorescence of senescent cells (autofluorescence spectrum) is used as a background, and the detection of binding between the means for recognizing aging antigens and isolated senescent cells is indicated by the presence of a difference from the background.

9. A composition comprising means for recognizing one or more aging antigens for use in the method according to any one of claims 1 to 8.

10. A method for isolating or concentrating isolated senescent cells, comprising contacting the isolated senescent cells with means for recognizing one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74.

11. A method for identifying a surface antigen selectively expressed in a subpopulation within a cell population containing autofluorescent cells, comprising: contacting an autofluorescent cell population with a labeled antibody that binds to a candidate surface antigen; measuring the label of the cell population to which the labeled antibody is bound using a spectral analyzer to obtain a measured spectrum of the cell population to which the labeled antibody is bound; measuring the cell population that has not been reacted with the labeled antibody using a spectral analyzer to obtain a background spectrum; subtracting the background spectrum from the measured spectrum to obtain a difference spectrum; and identifying the antigen as a surface antigen selectively expressed in a subpopulation within an autofluorescent cell population based on the selective detection of the difference spectrum in the subpopulation within the autofluorescent cell population.

12. A method for identifying a surface antigen expressed on autofluorescent cells, comprising: contacting autofluorescent cells with a labeled antibody that binds to a candidate surface antigen; measuring the label of the cells to which the labeled antibody has bound using a spectral analyzer to obtain a measured spectrum of the cells to which the labeled antibody has bound; measuring the cells that have not been reacted with the labeled antibody using a spectral analyzer to obtain a background spectrum; subtracting the background spectrum from the measured spectrum to obtain a difference spectrum; and identifying the antigen as a surface antigen expressed on the cells based on the detection of a difference spectrum in the cells.

13. The method according to claim 11 or 12, wherein the cells exhibiting autofluorescence are senescent cells.

14. A method for treating senescent cells, comprising inhibiting the expression or function of one or more senescent antigens selected from the group consisting of CD85k, CD215, CD83, CXCL16, CD26, CD107b, MSC&NPC antigen, CD184, CD95, CD46, CD9, CD79b, CD199, CD230, CD82, and CD74 in the senescent cells.

15. A composition comprising an expression inhibitor or a functional inhibitor for the aging antigen, for use in the method according to claim 14.

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