Use of the glycans as senescent cell surface markers

Abstract

The present invention provides a method of detecting the presence or absence of senescent cells and / or vesicles thereof in a sample, the method comprising: providing a sample; determining, in the sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of: (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine (GalNAc) residues, (v) terminal fucose residues, (vi) terminal N-acetylglucosamine (GlcNAc) residues, and (vii) internal N-acetyllactosamine residues; comparing the level of display of the glycan in the sample with a reference value; and detecting the presence of apoptotic cells and / or vesicles thereof, or detecting the absence of apoptotic cells and / or vesicles in the sample when based on the comparison.

Description

[0001] SENESCENT CELL SURFACE MARKERS

[0002] Field of the Invention

[0003] The present invention relates to methods and kits for detecting senescent cells and / or vesicles thereof, as well as methods and kits for enriching non-senescent cells and / or vesicles thereof. The invention also extends to probe-conjugates, and medical and nonmedical uses thereof. The invention also encompasses the use of an extracellular or extravesicular surface displayed glycan as a senescent cell biomarker.

[0004] Background

[0005] Senescent cells are live cells that have stopped dividing or have a reduced rate of division, and their distinct state is associated with various biomarkers such as expression of P-galactosidase, DNA damage markers such as yH2AX and the activation of pl6INK4A. Senescent cells may occur in vitro, ex vivo, and in vivo, and they tend to accumulate in organs and tissues over time. Cellular senescence in vivo is believed to play a role in several age-related diseases, including, for example, cancer, atherosclerosis, and osteoarthritis as well as in aging. A better understanding of the role that senescent cells play in age-related diseases could lead to improved therapeutic treatments.

[0006] For example, mesenchymal stem cells (MSCs) have the potential to treat age-related diseases because of their stem cell nature (namely their ability to self-renew and differentiate into different lineages), immune-modulatory abilities, and their paracrine effects. Many mesenchymal stem cell (MSC)-based products are currently under development. The expansion of MSCs in culture is often necessary to produce adequate numbers of cells for therapy. In case of autologous therapies, the donors are usually older and so have fewer stem cells. However, MSCs undergo replicative senescence when they are expanded too heavily. Senescent MSCs have diminished proliferation, differentiation, immunomodulatory and migration potentials. This has a significant negative impact on their therapeutic efficacy.

[0007] Moreover, it has been found that senescence not only abrogates the therapeutic potential of MSCs when administered in vivo, but it can also lead to the development of age- related diseases when such senescent cells are transplanted into an individual. Most importantly, in addition to withdrawing from the cell cycle (i.e., no longer dividing), senescent cells are believed to possess distinct markers and exhibit a discrete secretome called senescence associated secretory phenotype (SASP). However, despite some biomarkers of cell senescence being described in the literature, there are currently only a few cell surface biomarkers available.

[0008] Thus, there is a need for a specific and sensitive biomarker for cellular senescence, as well as a cell surface marker for cellular senescence.

[0009] Statements of the Invention

[0010] According to a first aspect of the invention, there is provided a method of detecting senescent cells and / or vesicles thereof in a test sample, the method comprising: providing a test sample; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residue selected from the group consisting of:

[0011] (i) terminal sialic acid residues,

[0012] (ii) terminal galactose residues,

[0013] (iii) terminal mannose residues,

[0014] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0015] (v) terminal fucose residues,

[0016] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0017] (vii) internal N-acetyllactosamine residues; comparing the level of display of the glycan in the test sample with the level of display in a reference sample; wherein a difference in the level of display of the glycan in the test sample compared to a reference level of display of the glycan in a reference sample is indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof.

[0018] The test sample may comprise any cell type. Exemplary cell types include cells of the connective tissue (e.g. a fibroblast), a stem cell (e.g. a mesenchymal stem cell such as a bone marrow-derived mesenchymal stem cells (BMMSC) and / or dental pulp-derived mesenchymal stem cells (DP-MSC) and / or an endothelial cell (e.g. a vascular endothelial cell, such as a human umbilical vein endothelial cell, HUVEC). A terminal fucose may be, for example, an a(1^2) linked fucose linked (e.g. directly linked) to a galactose, e.g. a galactose that is a more internal relative to said terminal fucose. Some examples include Fuc (al, 2) Gal (pi,3) GlcNAc and / or Fuc (al, 2) Gal (Pl,4), with Fuc (al, 2) Gal (P 1,3) GlcNAc being a preferred example. Said terminal fucose may preferably be referred to as a terminal glycan residue capable of being bound by an agglutinin, more preferably Ulex Europaeus Agglutinin I (UEA-I). Thus, a probe for a terminal fucose, such as an a( 1— >2) linked fucose linked (e.g. directly linked) to galactose (e.g. Fuc (al, 2) Gal (Pl,3) GlcNAc and / or Fuc (al, 2) Gal (pi,4)) may be UEA-1. UEA-1 may be said to be specific for terminal a( 1— >2) fucose linkages linked (e.g. directly linked) to galactose. The term Fuc (al, 2) Gal (P 1,3) GlcNAc may also be presented as Fuc al-2 Gal bl-3 GlcNAc; the term Fuc (al, 2) Gal (Pl,4) may also be presented as Fuc al-2 Gal bl -4 GlcNAc; the term a(1^2) linked fucose may also be presented as Fuc al-2.

[0019] As reflected by the preceding sentence, the skilled person would appreciate that the manner in which certain glycans are presented in writing can vary; and would understand that language such as “Fuc al-2” can be used to “a(l— >2) linked fucose” for convenience, and so on. Such alternative presentations may be used herein.

[0020] A terminal fucose may be an a( 1^6), an a( 1— >2), an a( 1^3), and / or an a( 1— >4) linked fucose (each of said alternatives being linked to an N-acetylglucosamine (GlcNAc), e.g. a GlcNAc that is more internal relative to said terminal fucose). More preferably, a terminal fucose may be an a(1^6) fucose, linked to N-acetylglucosamine (GlcNAc)). Some examples include Fuc (al, 6) GlcNAc and Fuc al-2 Gal [6S] bl-4 Glc; with Fuc (al, 6) GlcNAc being a particularly preferred example. Said terminal fucose may preferably be referred to as a terminal glycan residue capable of being bound by Aleuria Aurantia Lectin (AAL). Thus, a probe for a terminal fucose, such as an a(1^6), an a(1^2), an a(1^3), and / or an a(1^4) fucose (linked to N-acetylglucosamine (GlcNAc), e.g. a GlcNAc that is more internal relative to said terminal fucose), may be AAL. AAL can bind terminal fucose, preferably a( 1— >6) linkages. AAL may be said to recognise fucose (e.g. terminal fucose) stemming from the base of glycans where GlcNAc is more internal relative to said terminal fucose. The terms a(1^6), an a(1^2), an a(1^3), and / or an a( 1— >4) linked fucose may also be presented as Fuc al- 6, Fuc al-2 and Fuc al-3 (respectively); the term Fuc (al, 6) GlcNAc may also be presented as Fuc al-6 GlcNAc.

[0021] A terminal sialic acid may be an alpha-2, 3-linked sialic acid, preferably a terminal NeuAc a2-3 Gal b 1-3 GalNAc residue. Said terminal sialic acid may preferably be referred to as a terminal glycan residue capable of being bound by Maackia Amurensis Lectin II (MAL-II). Thus, a probe for a terminal sialic acid (such as an alpha-2, 3-linked sialic acid, for example a terminal NeuAc a2-3 Gal bl -3 GalNAc residue) may be MAL- II.

[0022] A terminal sialic acid may be an alpha-2, 6-linked sialic acid, preferably a terminal NeuAc a2-6 Gal bl -4 GlcNAc residue. Said terminal sialic acid may preferably be referred to as a terminal glycan residue capable of being bound by Sambucus Nigra Lectin (SNA). Thus, a probe for a terminal sialic acid (such as an alpha-2, 6-linked sialic acid, for example a terminal NeuAc a2-6 Gal bl -4 GlcNAc residue) may be SNA.

[0023] A terminal N-acetylglucosamine (e.g. P(l,4)-linked GlcNAc) may be a monomer of N- acetylglucosamine and / or an oligomer of N-acetylglucosamine, such as Chitin and / or (G1CNAC)3. Said terminal N-acetylglucosamine may preferably be referred to as a terminal glycan residue capable of being bound by Solanum Tuberosum Lectin (STL). Thus, a probe for a terminal N-acetylglucosamine (such as a P(l,4)-linked GlcNAc) may be STL.

[0024] An internal N-acetyllactosamine (e.g. LacNAc) described herein may be referred to as a disaccharide having galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P- 1,4 bonds, and examples include poly-LacNAc. Said internal N-acetyllactosamine may preferably be referred to as a terminal glycan residue capable of being bound by Solanum Tuberosum Lectin (STL). Thus, a probe for internal N-acetyllactosamine (such as LacNAc) may be STL. Throughout this specification, where STL is referred to, it is preferred that such references are construed as being in the context of being a glycan that binds (e.g. is capable of binding) internal N-acetyllactosamine, unless the context requires otherwise.

[0025] A terminal mannose may be a terminal alpha- 1, 6-linked mannose, preferably alpha-1, 6- linked Man3GlcNAc2 and / or alpha- 1, 6-linked Man5GlcNAc2. Said terminal alpha-1,6- linked mannose may preferably be referred to as a terminal glycan residue capable of being bound by Narcissus Pseudonarcissus Lectin (NPL). Thus, a probe for a terminal mannose (such as a terminal alpha- 1,6-linked mannose, preferably alpha- 1,6-linked Man3GlcNAc2 and / or alpha- 1,6-linked Man5GlcNAc2) may be NPL.

[0026] A terminal mannose residue may be a terminal alpha-1, 3-linked mannose, preferably alpha-1, 3-linked Man3GlcNAc2. Said terminal alpha-1, 3-linked mannose may preferably be referred to as a terminal glycan residue capable of being bound by Galanthus Nivalis Lectin (GNA). Thus, a probe for a terminal mannose residue (such as a terminal alpha-1, 3-linked mannose, preferably alpha-1, 3-linked Man3GlcNAc2) may be GNA.

[0027] A terminal galactose may be a galactosyl (P-1,3) N-acetylgalactosamine, for example, Fuc al-2( Gal b 1-3 GalNAc al-3) Gal bl-4 GlcNAc. Said terminal galactose may preferably be referred to as a terminal glycan residue capable of being bound by Peanut Agglutinin (PNA). Thus, a probe for a terminal galactose (such as a galactosyl (P-1,3) N-acetylgalactosamine, for example, Fuc al-2( Gal bl -3 GalNAc al-3) Gal bl- 4 GlcNAc) may be PNA.

[0028] A terminal galactose may be Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc, that may preferably be referred to as a terminal glycan residue capable of being bound by Styphnolobium japonicum (Japanese pagoda) lectin (SJA). Thus, a probe for a terminal galactose (such as Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc) may be SJA.

[0029] A terminal N-acetylgalactosamine may be GalNAc bl-4 GlcNAc bl -3 GalNAc bl-4 GlcNAc. Said terminal N-acetylgalactosamine may preferably be referred to as a terminal glycan residue capable of being bound by Styphnolobium japonicum (Japanese pagoda) lectin (SJA). Thus, a probe for a terminal N-acetylgalactosamine (such as GalNAc bl-4 GlcNAc bl -3 GalNAc bl-4 GlcNAc) may be SJA.

[0030] Said lectin “SJA” is referred to at various points (e.g. in the context of various embodiments) throughout this disclosure. For each reference to “SJA” throughout this specification, it is preferred that the SJA is “B-SJA-I”, noting that an alternative subunit of SJA (i.e. B-SJA-II) has also been previously described. The experiments in the examples section made use of B-SJA-I. Advantageously, it may be “bispecific” as indicated by the two paragraphs directly preceding this paragraph, thus can be used for probing two sugars within the “terminal galactose” family.

[0031] The skilled person will understand that the following glycans (mentioned above) can be said to fall within a ‘genus’ of glycans that can be called “terminal galactose”, with the following sugars representing ‘species’ of said genus:

[0032] GalNAc b 1 -4 GlcNAc b 1 -3 GalNAc b 1 -4 GlcNAc; this is referred to herein as a “terminal GlcNac”, e.g. with a GlcNac being understood to represent an amino acid sugar derivative of galactose (hence falling within the genus of “terminal galactose”), this can be bound by SJA;

[0033] Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc; this is referred to herein as a “terminal galactose”, this can (also) be bound by SJA; and

[0034] Fuc al-2( Gal bl -3 GalNAc al-3) Gal bl-4 GlcNAc; this is referred to herein as a “terminal galactose”, this can be bound by PNA.

[0035] Thus, items (ii) “terminal galactose residues” and (iv) “terminal N-acetylgalactosamine (GalNAc) residues” in the above-mentioned ‘first aspect’ (and any corresponding list of any other aspect / embodiment described herein) may optionally be grouped together and referred to as a “a genus of terminal galactose residues”, comprising the ‘species’ listed above. That being said, there are embodiments in which discussing the individual ‘species’ may be advantageous, which will be discussed in more detail below.

[0036] As mentioned above, it is preferred that STL is used for binding internal N- acetyllactosamine (e.g. LacNAc). As such, an embodiment of list (i)-(vi) may optionally be absent “terminal N-acetylglucosamine (GlcNAc) residues”. Thus, the glycan list of the above-mentioned ‘first aspect’ (and any corresponding list of any other aspect / embodiment described herein) may optionally be absent item (vi) “terminal N- acetylglucosamine (GlcNAc) residues”. Indeed any reference to “terminal N- acetylglucosamine (GlcNAc) residues” throughout this disclosure is totally optionally and can optionally be omitted from the associated aspect / embodiment. Thus methods of the invention may comprise determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residue selected from the group consisting of:

[0037] (i) terminal sialic acid residues,

[0038] (ii) terminal galactose residues, (iii) terminal mannose residues,

[0039] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0040] (v) terminal fucose residues, and

[0041] (vi) internal N-acetyllactosamine residues.

[0042] Although the term “terminal N-acetylglucosamine (GlcNAc) residues” may be absent as per the list directly above, the list of course embraces “terminal galactose residues” and “internal N-acetyllactosamine residues” that may happen to comprise a GlcNAc component.

[0043] The level of display of more than one glycan may be determined in a method of the invention. For example, as will be discussed in more detail below, the level of one or more glycan may be determined by challenging the test sample with a probe or binding partner having affinity for said one or more glycan (e.g. an appropriate lectin, or appropriate lectins) and detecting a level of biomarker-binding partner complex.

[0044] The invention is based on the observation that senescent cells and / or vesicles thereof, and non-senescent cells and / or vesicles thereof, differentially display glycans on their cell surface (specifically on the extracellular surface of the membrane). Consequently, known bio-physical separation techniques can be used as part of the invention to detect, enrich, isolate, remove and target (e.g., kill) senescent cells. Therefore, unlike the prior art, the present invention does not require cells to be lysed in order to detect the relevant biomarkers. Thus, in a preferable embodiment, the sample (e.g. test sample) has not been subjected to a cell lysis step (such as mechanical disruption, liquid homogenization, high frequency sound waves (sonication), freeze / thaw cycles, or manual grinding). In a more preferable embodiment, the test sample has not been subjected to a cell lysis step, or to a permeabilization step (e.g. detergent treatment), or to an acid treatment step (most preferably the test sample has not been subjected to any of such cell lysis / permeabilization / acid treatment steps). In a suitable embodiment, a method of the invention is practiced using a sample comprising, or consisting essentially of, intact cells. Therefore, a difference in the level of display of a glycan detected in a test sample compared to a reference level of display of the glycan in a reference sample is indicative that the test sample comprises a senescent cell and / or a vesicle thereof.

[0045] As discussed in the examples section, such difference has been detected across multiple, and diverse, cell types that have become senescent (where artificially induced, else following extended culture ‘passaging’). Thus, particularly where the “reference level” is indicative of value for said glycan in a reference sample of non-senescent cells (e.g. viable cells), a difference whether ‘higher’ or Tower’ is reliably detectable across multiple cell types. Thus, even at such broad level, simply detecting a “difference” provides a readout that indicates the presence of senescent cells. Naturally, the invention may also utilise alternative references, for example the “reference level” may be indicative of value for said glycan in a reference sample of senescent cells (preferably stage-matched cells). When employing such reference value, detecting ‘no’ difference may indicate the presence of senescent cells in the test sample. As an alternative to the ‘wherein’ clause of the above-described first aspect may be: wherein ‘no’ difference in the level of display of the glycan in the test sample compared to a reference level of display of the glycan in a reference sample is indicative that the test sample comprises one or more senescent cells and / or vesicles thereof.

[0046] Taken together, an aspect of the invention may read as: a method of detecting the presence or absence of senescent cells and / or vesicles thereof in a test sample (e.g. test sample of cells), the method comprising: providing a test sample; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0047] (i) terminal sialic acid residues,

[0048] (ii) terminal galactose residues,

[0049] (iii) terminal mannose residues,

[0050] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0051] (v) terminal fucose residues,

[0052] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0053] (vii) internal N-acetyllactosamine residues; comparing the level of display of the glycan in the test sample with the level of display in a reference sample; and determining that the sample comprises senescent cells and / or vesicles thereof, or determining that the sample does not comprise senescent cells and / or vesicles thereof when based on the comparison.

[0054] Further details on how individual glycans may be compared with a particular reference value will be provided below.

[0055] An aspect of the invention provides a method of assaying a test sample having or suspected of having senescent cells and / or vesicles thereof, the method comprising: providing a test sample; and assaying the test sample for a level of extracellular and / or extravesicular surface display of a glycan comprising one or more types of residues selected from the group consisting of:

[0056] (i) terminal sialic acid residues,

[0057] (ii) terminal galactose residues,

[0058] (iii) terminal mannose residues,

[0059] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0060] (v) terminal fucose residues,

[0061] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0062] (vii) internal N-acetyllactosamine residues; optionally wherein a test sample having senescent cells (and / or vesicles thereof) demonstrates a different level of extracellular and / or extravesicular surface display of a glycan than a sample that lacks senescent cells (and / or vesicles thereof), or wherein a test sample that does ‘not’ have senescent cells (and / or vesicles thereof) does ‘not’ demonstrate a different level of extracellular and / or extravesicular surface display of a glycan than a sample that lacks senescent cells (and / or vesicles thereof).

[0063] In one embodiment, the method comprises detecting, in a test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:

[0064] (i) terminal sialic acid residues,

[0065] (ii) terminal galactose residues,

[0066] (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0067] (v) terminal fucose residues,

[0068] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0069] (vii) internal N-acetyllactosamine residues; wherein detection of a glycan comprising residues according to one or more of

[0070] (i) to (vii) is indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof. Therefore, detection of a glycan comprising residues according to one or more of (i) to (vii) may be indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof.

[0071] In another embodiment, the method may comprise: determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:

[0072] (i) terminal sialic acid residues,

[0073] (ii) terminal galactose residues,

[0074] (iii) terminal mannose residues,

[0075] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0076] (v) terminal fucose residues,

[0077] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0078] (vii) internal N-acetyllactosamine residues; comparing this level of display determined in the test sample with the level of display in a reference sample that does ‘not’ comprise a senescent cell or vesicle thereof (e.g. does not comprise senescent cells or vesicles thereof), wherein a difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof, or wherein ‘no’ difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample does 'not’ comprise one or more senescent cells and / or a vesicles thereof.

[0079] In another embodiment, the method may comprise; determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of: (i) terminal sialic acid residues,

[0080] (ii) terminal galactose residues,

[0081] (iii) terminal mannose residues,

[0082] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0083] (v) terminal fucose residues,

[0084] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0085] (vii) internal N-acetyllactosamine residues; comparing this level of display determined in the test sample with the level of display in a reference sample that ‘does’ comprise senescent cells (and / or vesicles thereof), wherein ‘no’ difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof, or wherein a difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample is indicative that the test sample does ‘not’ comprise one or more senescent cells and / or a vesicles thereof.

[0086] Therefore, the method according to the invention may comprise comparing the level of display of the glycan detected in the test sample with a reference level for the glycan in a reference sample that does ‘not’ comprise senescent cells and / or vesicles thereof (or alternatively a reference sample that ‘does’ comprise senescent cells and / or vesicles thereof), wherein a difference in the level of display of the glycan in the test sample relative to the reference level of display of the glycan in the reference sample is indicative that the test sample comprises (or alternatively does ‘not’ comprise) senescent cells and / or vesicles thereof.

[0087] Throughout this disclosure, a reference sample that ‘does’ comprise senescent cells (and / or vesicles thereof) preferably means that the reference sample is indicative of the level of display of the glycan on senescent cells that are at the same stage (of senescence) as those cell for which the presence (or absence) is to be detected. For example, a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof) has preferably been subjected to the same number (optionally + / - about 5) of ‘passages’, or ‘cell culture passages’ as the test sample. The skilled person would understand that a “reference sample” is used as a control, and that fundamental characteristics such as the cell type (and species from which it derives) may be matched across the test sample and reference sample e.g. where the test sample is a sample of ‘fibroblasts’ the reference sample is also a sample of ‘fibroblasts’; and where the test sample is of “human” origin, so too is the reference sample.

[0088] The difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be a higher level of display or a lower level of display. A higher level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof. The difference may be a higher level of display of a glycan comprising residues selected from the group consisting of (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine, (v) terminal fucose residues, (vi) terminal N-acetylglucosamine, and (vii) internal N- acetyllactosamine residues. The lower level of display may be a lower level of display of a glycan comprising terminal fucose residues, such as Fuc al -2 Gal bl -4 GlcNAc or Fuc al-2 Gal [6S] bl-4 Glc. The lower level of display may be a lower level of display of a glycan comprising terminal fucose residues, such as an a(1^2) linked fucose linked to a galactose; or to a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a(1^6) GlcNAc).

[0089] In one embodiment, the ‘presence’ of senescent cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha- 2,3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0090] However, where an increase (‘higher’ level) of said glycan when compared to a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof) has been established to correlate with the ‘presence’ of a senescent cell (and / or vesicle thereof) the skilled person will understand that no change (or indeed even a Tower level’), when compared to a reference sample that does 'not’ comprise a senescent cell (or vesicles thereof) may be indicative of the ‘absence’ of a senescent cell (and / or vesicle thereof). Likewise, the skilled person would understand that ‘no’ change (or indeed even a ‘higher’ level) of said glycan when compared to a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof) may be indicative of the ‘presence’ of a senescent cell (and / or vesicle thereof). The skilled person would also understand that a decrease (Tower level’) of said glycan when compared to a reference sample that ‘does’ comprise a senescence cell (and / or vesicle thereof) may be indicative of the ‘absence’ of a senescent cell (and / or vesicle thereof).

[0091] Thus, in one embodiment, the ‘absence’ of senescent cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N-acetylglucosamine is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0092] In one embodiment, the ‘presence’ of senescent cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha- 2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N-acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0093] In one embodiment, the ‘absence’ of senescent cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha- 2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N-acetylglucosamine is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0094] In embodiments (e.g. the embodiments above) that refer to detecting the presence or absence of “senescent cells”, exemplary cell types include cells of the connective tissue (e.g. an extracellular matrix cell, more preferably a fibroblast), a stem cell (e.g. a mesenchymal stem cell such as a bone marrow-derived mesenchymal stem cell (BMMSC) and / or dental pulp-derived mesenchymal stem cell (DP-MSC)) and / or an endothelial cell (e.g. a vascular endothelial cell, such as a human umbilical vein endothelial cell, HUVEC).

[0095] In any embodiment described herein directed to detecting the presence or absence of a senescent cell of any particular cell type, it may be said that the sample (e.g. test sample) is one having or suspected of having cells of such cell type.

[0096] In one embodiment, the ‘presence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0097] In one embodiment, the ‘absence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0098] In one embodiment, the ‘presence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0099] In one embodiment, the ‘absence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0100] In one embodiment, the ‘presence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a( 1— >2) linked fucose linked to a galactose is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0101] In one embodiment, the ‘absence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a( 1— >2) linked fucose linked to a galactose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0102] In one embodiment, the ‘presence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a( I— >2) linked fucose linked to a galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0103] In one embodiment, the ‘absence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a( I— >2) linked fucose linked to a galactose is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0104] In one embodiment, the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0105] In one embodiment, the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof). In one embodiment, the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0106] In one embodiment, the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0107] In one embodiment, the ‘presence’ of senescent endothelial cells, senescent connective tissue cells (such as fibroblasts) and / or senescent BMMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0108] Throughout this specification (in the context of any aspect or embodiment), reference to (e.g. senescent) connective tissue cells may preferably mean (e.g. senescent) extracellular matrix cells, more preferably (e.g. senescent) fibroblasts.

[0109] In one embodiment, the ‘absence’ of senescent endothelial cells, senescent connective tissue cells (such as fibroblasts) and / or senescent BMMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof). In one embodiment, the ‘presence’ of senescent endothelial cells, senescent connective tissue cells (such as fibroblasts) and / or senescent BMMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0110] In one embodiment, the ‘absence’ of senescent endothelial cells, senescent connective tissue cells (such as fibroblasts) and / or senescent BMMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0111] In one embodiment, the ‘presence’ of senescent DPMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0112] In one embodiment, the ‘absence’ of senescent DPMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0113] In one embodiment, the ‘presence’ of senescent DPMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0114] In one embodiment, the ‘absence’ of senescent DPMSC cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0115] In one embodiment, the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0116] In one embodiment, the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0117] In one embodiment, the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0118] In one embodiment, the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0119] In one embodiment, the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0120] In one embodiment, the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof).

[0121] In one embodiment, the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0122] In one embodiment, the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

[0123] The more glycan types that are detected in the test sample, the greater the accuracy and reliability with which senescent cells and / or vesicles thereof can be identified. Therefore, the method may comprise detecting two or more, three or more, four or more, five or more, six or more, or seven or more different glycans described herein in the sample. In embodiments in which the method comprises detecting two different glycans, the glycans may comprise terminal sialic acid residues and terminal mannose residues. In embodiments in which the method comprises detecting three different glycans, the glycans may comprise terminal sialic acid residues, terminal mannose residues, and terminal N-acetylglucosamine residues. Glycans comprising terminal sialic acid residues may be alpha-linked sialic acids, such as alpha-2, 3-linked sialic acid or alpha- 2,6-linked sialic acid. Glycans comprising terminal mannose residues may be alphalinked mannose, such as alpha- 1,6-linked mannose. The method may comprise detecting the presence of a glycan comprising terminal alpha-linked sialic acid residues and terminal alpha-linked mannose residues. The method may comprise detecting one or more types of the glycans referred to herein in a sample together with other known biomarkers (e.g., proteins).

[0124] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal sialic acid residues (e.g., an alpha-linked sialic acid, such as an alpha-2, 3-linked sialic acid or an alpha-2, 6-linked sialic acid), and optionally one or more monosaccharide residues selected from the group consisting of:

[0125] (i) terminal galactose residues,

[0126] (ii) terminal mannose residues,

[0127] (iii) terminal N-acetylgalactosamine (GalNAc) residues,

[0128] (iv) terminal fucose residues,

[0129] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0130] (vi) internal N-acetyllactosamine residues.

[0131] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more galactose residues, and optionally one or more monosaccharide residues selected from the group consisting of:

[0132] (i) terminal sialic acid residues,

[0133] (ii) terminal mannose residues,

[0134] (iii) terminal N-acetylgalactosamine (GalNAc) residues,

[0135] (iv) terminal fucose residues,

[0136] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0137] (vi) internal N-acetyllactosamine residues. The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal mannose residues (e.g., an alpha-linked mannose, such as alpha- 1,6-linked mannose) and optionally one or more terminal monosaccharide residues selected from the group consisting of:

[0138] (i) terminal sialic acid residues,

[0139] (ii) terminal galactose residues,

[0140] (iii) terminal N-acetylgalactosamine (GalNAc) residues,

[0141] (iv) terminal fucose residues,

[0142] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0143] (vi) internal N-acetyllactosamine residues.

[0144] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal N- acetylgalactosamine (GalNAc) residues and optionally one or more terminal monosaccharide residues selected from the group consisting of:

[0145] (i) terminal sialic acid residues,

[0146] (ii) terminal galactose residues,

[0147] (iii) terminal mannose residues,

[0148] (iv) terminal fucose residues and

[0149] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0150] (vi) internal N-acetyllactosamine residues.

[0151] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal fucose residues (e.g., an alpha linked fucose residue) and optionally one or more terminal monosaccharide residues selected from the group consisting of:

[0152] (i) terminal sialic acid residues,

[0153] (ii) terminal galactose residues,

[0154] (iii) terminal mannose residues,

[0155] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0156] (v) terminal N-acetylglucosamine (GlcNAc) residues, and

[0157] (vi) internal N-acetyllactosamine residues. The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more terminal N- acetylglucosamine residues (e.g., Chitin, or (GlcNAc)3) and optionally one or more terminal monosaccharide residues selected from the group consisting of:

[0158] (i) terminal sialic acid residues,

[0159] (ii) terminal galactose residues,

[0160] (iii) terminal mannose residues,

[0161] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0162] (v) terminal fucose residues and

[0163] (vi) internal N-acetyllactosamine residues.

[0164] The method may comprise detecting, in the test sample, extracellular and / or extravesicular surface display of a glycan comprising one or more internal N- acetyllactosamine residues and optionally one or more terminal monosaccharide residues selected from the group consisting of:

[0165] (i) terminal sialic acid residues,

[0166] (ii) terminal galactose residues,

[0167] (iii) terminal mannose residues,

[0168] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0169] (v) terminal fucose residues, and

[0170] (vi) terminal N-acetylglucosamine (GlcNAc) residues.

[0171] The method may comprise detecting in the sample the level of extracellular and / or extravesicular surface display of a glycan comprising one or more terminal monosaccharide residues selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1,6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, alpha linked fucose residues, such as Fuc al- 2 Gal bl -4 GlcNAc or Fuc al -2 Gal [6S] bl -4 Glc, alpha linked fucose residues (such as an a(1^2) linked fucose linked to a galactose; or a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc)), and terminal N-acetylglucosamine residues. Alternatively, or additionally, the method may comprise detecting in the sample the level of extracellular and / or extravesicular surface display of a glycan comprising internal N- acetyllactosamine residues. Surprisingly it has been found, as demonstrated herein, that the level of display of the one or more glycans referred to herein acts as biomarkers for senescent cells and / or vesicles thereof. The inventors investigated the level of display of a large number of glycan molecules with reference to their terminal monosaccharide residues, and internal N-acetyllactosamine residues, and were surprised to observe that a number of glycans exhibit a higher level of display in senescent cells and / or vesicles thereof compared to non-senescent cells (e.g., glycans comprising terminal alpha-linked sialic acid residues, terminal alpha-linked mannose residues, terminal N-acetylgalactosamine residues and terminal N-acetylglucosamine residues). Glycans comprising terminal fucose residues or galactose residues may also exhibit a higher level of display in senescent cells and / or vesicles thereof compared to non-senescent cells and / or vesicles thereof. The inventors also observed that a number of glycans exhibit a lower level of display in senescent cells and / or vesicles thereof compared to non-senescent cells and / or vesicles thereof (e.g., glycans comprising terminal alpha-linked fucose residues).

[0172] Also disclosed herein is a kit that may be used to perform the method according to the invention.

[0173] Thus, according to second aspect, there is provided a kit for identifying senescent cells and / or vesicles thereof, or for diagnosing a senescence-related disease, the kit comprising: a probe for a glycan, wherein the glycan comprises one or more residues selected from the group consisting of:

[0174] (i) terminal sialic acid residues,

[0175] (ii) terminal galactose residues,

[0176] (iii) terminal mannose residues,

[0177] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0178] (v) terminal fucose residues,

[0179] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0180] (vii) internal N-acetyllactosamine residues; preferably wherein the probe of the kit is used to determine, in a test sample, the level of display of one or more of the glycans, wherein a difference in the level of display of one or more of the glycans in the test sample relative to a level of display of one or more of the glycans in a reference sample, indicates the presence of senescent cells and / or vesicles thereof in the test sample, optionally this can be used in the diagnosis of a senescence-related disease. The reference sample may not contain senescent cells and / or vesicles thereof.

[0181] A kit of the invention may comprise (e.g. as a physical feature / component of the kit) technical instructions to use the kit to detect the presence or absence of senescent cells (and / or vesicles thereof) by a method described herein.

[0182] In one embodiment, the kit comprises: a probe for a glycan, wherein the glycan comprises monosaccharide residues selected from the group consisting of:

[0183] (i) terminal sialic acid residues, such as alpha-2, 3-linked sialic acid residues and / or alpha-2, 6-linked sialic acid residues,

[0184] (ii) terminal galactose residues,

[0185] (iii) terminal mannose residues, such as alpha- 1, 6-linked mannose residues and / or alpha-1, 3-linked mannose residues,

[0186] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0187] (v) terminal fucose residues,

[0188] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0189] (vii) internal N-acetyllactosamine residues; preferably wherein the probe of the kit is used to detect, in a test sample, the level of display of one or more of the glycans.

[0190] When levels of the glycans in the test sample are compared to the levels in a reference sample, wherein the reference sample does not contain senescent cells and / or vesicles thereof, if a difference is observed it may then be concluded that the test sample contains senescent cells and / or vesicles thereof, this may help diagnosis of a senescence-related disease. The group from which terminal monosaccharide residues may be selected from optionally further consists of: NeuAc a2-3 Gal bl -3 GalNAc, NeuAc a2-6 Gal bl- 4 GlcNAc, Fuc al-2( Gal b 1-3 GalNAc al-3) Gal bl-4 GlcNAc, fuc al-3( Fuc al- 2( Gal al -3) Gal bl -4) GlcNAc, GalNAc bl-4 GlcNAc bl-3 GalNAc bl-4 GlcNAc, Man3GlcNAc2, Man5GlcNAc2, an a(1^2) linked fucose linked to a galactose (e.g. Fuc al -2 Gal bl-4 GlcNAc), and Fuc al -2 Gal [6S] bl-4 Glc. In addition, or alternatively, the probe may be for internal N-acetyllactosamine residues (e.g. LacNAc) such as Chitin or (GlcNAc)3 (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds). In addition, or alternatively, the probe may be for internal N-acetyllactosamine residues (e.g. LacNAc) such poly- LacNAc (e.g. as part of a disaccharide having of galactose (Gal) and N- acetylglucosamine (GlcNAc) linked by P-1,4 bonds).

[0191] The difference in the level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be a higher level of display in the test sample or a lower level of display in the test sample.

[0192] A higher level of display of the glycan in the test sample relative to the level of display of the glycan in the reference sample may be indicative that the test sample comprises one or more senescent cells and / or vesicles thereof.

[0193] The probe may be for binding a glycan displayed on the extracellular surface of cells and / or vesicles thereof. The probe may be a lectin referred to herein. The probe may be for a glycan referred to herein. The probe may be for use in enriching for senescent cells and / or vesicles thereof.

[0194] A kit described herein may comprise a probe for terminal fucose, preferably a probe for a terminal a( 1— >2) linked fucose linked to a galactose (e.g. an alpha-glycosidic linked fucose). Said probe for terminal fucose may preferably be Ulex Europaeus Agglutinin I (UEA-I), optionally conjugated to an imaging agent.

[0195] Additionally or alternatively, a kit described herein may comprise a probe for a terminal Fuc a(1^6) GlcNAc, Fuc a(1^2) GlcNAc, Fuc a(l— >3) GlcNAc, and / or Fuc a(1^4) GlcNAc (preferably Fuc a(1^6) GlcNAc) (e.g. a terminal fucose). Said probe for a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1 — >6) GlcNAc) may preferably be Aleuria Aurantia Lectin (AAL), optionally conjugated to an imaging agent.

[0196] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha-2, 3-linked sialic acid, preferably a probe for terminal NeuAc a2-3 Gal bl- 3 GalNAc residue. Said probe for terminal sialic acid may preferably be Maackia Amurensis Lectin II (MAL-II), optionally conjugated to an imaging agent. Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha-2, 6-linked sialic acid, preferably a probe for a terminal NeuAc a2-6 Gal bl- 4 GlcNAc residue. Said probe for a terminal alpha-2, 6-linked sialic acid may preferably be Sambucus Nigra Lectin (SNA), optionally conjugated to an imaging agent.

[0197] Additionally or alternatively, a kit described herein may comprise a probe for a terminal N-acetylglucosamine (e.g. as monomer and / or oligomer), preferably a probe for ( 1 ,4)- linked GlcNAc. Said probe for a terminal N-acetylglucosamine may preferably be Solanum Tuberosum Lectin (STL), optionally conjugated to an imaging agent.

[0198] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha- 1, 6-linked mannose, preferably a probe for alpha- 1, 6-linked Man3GlcNAc2 and / or alpha- 1, 6-linked Man5GlcNAc2. Said probe for a terminal alpha- 1, 6-linked mannose may preferably be Narcissus Pseudonarcissus Lectin (NPL), optionally conjugated to an imaging agent.

[0199] Additionally or alternatively, a kit described herein may comprise a probe for a terminal alpha-1, 3-linked mannose, preferably a probe for alpha-1, 3-linked mannose Man3GlcNAc2. Said probe for a terminal alpha-1, 3-linked mannose may preferably be Galanthus Nivalis Lectin (GNA), optionally conjugated to an imaging agent.

[0200] Additionally or alternatively, a kit described herein may comprise a probe for a terminal galactose, preferably a probe for a galactosyl (P-1,3) N-acetylgalactosamine, such as Fuc al -2 ( Gal bl -3 GalNAc a 1-3) Gal bl -4 GlcNAc.

[0201] The kit may further comprise a control sample and / or reference sample. The kit may further comprise a positive control and / or a negative control sample. The positive control may be a sample that comprises senescent cells and / or vesicles thereof. The negative control may be a sample that does not comprise or contain senescent cells and / or vesicles thereof (e.g., non-senescent cells).

[0202] The reference sample may be a sample that comprises cells that do not display any of the glycans referred to herein, or low, undetectable levels of the glycan referred to herein. The reference sample may comprise dividing cells or cells that are capable of dividing. The reference sample may comprise cells that do not have biomarkers such as P-galactosidase, DNA damage markers such as yH2AX or activate pl6INK4A. The kit may comprise a reference sample.

[0203] The kit may comprise an imaging agent. Thus, an imaging agent may be conjugated or attached to the probe. The imaging agent may be a fluorophore, a radioisotope, or a dye (preferably a fluorophore). The kit may comprise a conjugating agent. The conjugating agent may be used to conjugate the probe with the imaging agent. The kit may comprise a probe, or probes, conjugated to an imaging agent (or imaging agents).

[0204] According to a third aspect of the invention, there is provided a method of enriching or removing non-senescent or senescent cells and / or vesicles thereof from the sample, the method comprising: contacting the sample, which comprises a mixture of non-senescent cells and senescent cells and / or vesicles thereof, with a probe for a glycan; and enriching for the non-senescent or the senescent cells and / or vesicles thereof using the probe, wherein the probe recognises one or more types of residues of the glycan selected from the group consisting of:

[0205] (i) terminal sialic acid residues,

[0206] (ii) terminal galactose residues,

[0207] (iii) terminal mannose residues,

[0208] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0209] (v) terminal fucose residues,

[0210] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0211] (vii) internal N-acetyllactosamine residues.

[0212] Exemplary cell types include cells of the connective tissue (e.g. a fibroblast), a stem cell, such as a mesenchymal stem cell (e.g. a BBMSC and / or DPMSC) and / or an endothelial cell (e.g. a vascular endothelial cell, such as a human umbilical vein endothelial cell, HUVEC).

[0213] The method may comprise contacting the sample with a probe for a glycan comprising terminal monosaccharide residues selected from the group consisting of terminal alpha- 2,3-linked sialic acid residues, terminal alpha-2, 6-linked sialic acid residues, alpha-1,6- linked mannose residues, alpha-1, 3-linked mannose residues, NeuAc a2-3 Gal bl-

[0214] 3 GalNAc, NeuAc a2-6 Gal bl -4 GlcNAc, Fuc al-2( Gal bl -3 GalNAc al -3) Gal bl-

[0215] 4 GlcNAc, fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc, GalNAc bl-4 GlcNAc bl-

[0216] 3 GalNAc bl-4 GlcNAc, Man3GlcNAc2, Man5GlcNAc2, a( 1— >2) linked fucose linked to a galactose, a Fuc a( 1— >6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), Fuc al -2 Gal bl-

[0217] 4 GlcNAc, and Fuc al -2 Gal [6S] bl-4 Glc. In addition, or alternatively, the probe may be for internal N-acetyllactosamine residues such as Chitin, (GlcNAc)3 (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds). In addition, or alternatively, the probe may be for internal N- acetyllactosamine residues such as poly-LacNAc (e.g. as part of a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds).

[0218] The method may comprise contacting the sample with a probe for a glycan comprising one or more terminal monosaccharide residues selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, alpha linked fucose residues, such as Fuc al -2 Gal bl-4 GlcNAc or Fuc al -2 Gal [6S] bl-4 Glc, and terminal N-acetylglucosamine residues. Alternatively, or additionally, the method may comprise contacting the sample with a probe for a glycan comprising internal N- acetyllactosamine residues.

[0219] It will be appreciated that the method according to the invention may be used to remove senescent or non-senescent cells and / or vesicles thereof from the sample. The sample may be an in vivo, in vitro or ex vivo sample (preferably in vitro or ex vivo). In embodiments in which the invention is in vivo, the method may be used to remove senescent cells or non-senescent cells and / or vesicles thereof from a subject. In embodiments in which the invention is ex vivo, the method may be used to treat a disease or a condition in a subject.

[0220] The method according to the invention may be repeated multiple times on a single sample in order to improve the purity of the sample. Thus, the method according to the invention may be performed twice, three times or four or more times on a single test sample. It will be appreciated that performing the contacting step leads to the probe being bound to the glycan(s) displayed on the senescent cells and / or vesicles thereof, or the probe being bound to the glycan(s) displayed on the non-senescent cells. The probe may be for a glycan referred to herein. The probe may be specific or selective for the glycan.

[0221] The method according to the invention may further comprise the step of providing a sample (prior to the contacting step). The step of providing the sample may comprise providing the cells in culture (prior to the contacting step). Thus, the method according to the invention may or may not comprise extracting the cells from a subject. Thus, the method according to the invention may comprise providing cells that have (already) been obtained from a subject.

[0222] The enriching step of the invention comprises using the probe to separate or isolate either non-senescent cells and / or vesicles thereof in the sample or senescent cells and / or vesicles thereof in the sample. Thus, the enriching step may comprise using the probe to separate / isolate non-senescent cells from senescent cells, or separate / isolate senescent cells from non-senescent cells.

[0223] Enrichment of non-senescent cells and / or vesicles thereof may be performed by a probe for a glycan binding to and removing senescent cells and / or vesicles thereof (thus leaving behind unbound non-senescent cells). The glycan may comprise one or more residues selected from the group consisting of sialic acid residues, galactose residues, mannose residues, N-acetylgalactosamine residues, fucose residues, N-acetylglucosamine residues and / or N-acetyllactosamine residues. The probe may be a lectin. In one embodiment, the lectin may be a selection of one or more of MAL-II, SNA, NPL, GNA, PNA, SJA and STL. Alternatively, enrichment of non-senescent cells and / or vesicles thereof may be performed by a probe binding to and isolating the non-senescent cells and / or vesicles thereof. The probe may be a lectin, such as UEA-I or AAL. UEA-I binds to terminal fucose residues, for example on endothelial cells such as HUVECs.

[0224] Similarly, enrichment of senescent cells may be performed by a probe for a glycan binding to and removing non-senescent cells (thus leaving behind unbound senescent cells). The glycan may be a terminal alpha-linked fucose, for example on endothelial cells such as HUVECs. The probe may be a lectin. The lectin may be UEA-I or AAL. Alternatively, enrichment of senescent cells may be performed by a probe for a glycan binding to and isolating the senescent cells. The glycan may comprise one or more residues selected from the group consisting of sialic acid residues, galactose residues, mannose residues, N-acetylgalactosamine residues, fucose residues, N-acetylglucosamine residues, and N-acetyllactosamine residues. The probe may be a lectin. The lectin may be a selection of one or more of MAL-II, SNA, NPL, GNA, PNA, SJA, AAL, UEA-I and STL.

[0225] The terminal sialic acid residues may be terminal alpha-linked sialic acid residues, such as alpha-2, 3-linked sialic acid residues or terminal alpha-2, 6-linked sialic acid residues. The terminal mannose residues may be terminal alpha-linked mannose acid residues, such as alpha- 1, 6-linked mannose residues or alpha-1, 3-linked mannose residues. The terminal fucose residues may be alpha-linked fucose residues.

[0226] Enrichment may be performed using one or more probes for a glycan comprising terminal sialic acid residues, terminal galactose residues, terminal mannose residues, terminal N-acetylgalactosamine residues, terminal fucose residues, terminal N- acetylglucosamine residues, and / or internal N-acetyllactosamine residues.

[0227] The skilled person will appreciate that although the level of display of certain glycans generally increases with time, the probes (e.g., lectins) specific for these glycans are still capable of specifically binding to the glycan as long as they are used selectively (i.e., at the correct concentration). The concentration to be used will vary depending on a number of factors, including the type of cell to be bound / enriched. The skilled person would appreciate that, when using a lectin probe, the lectin concentration used during staining would be one that provides staining without causing significant agglutination / cell death / saturation. Lectin titration to appropriate concentrations for ‘staining’ is well within standard abilities of the skilled person.

[0228] Enrichment may be performed using any technique known in the art, including for example, flow cytometry sorting, magnetic bead separation, separation columns, a bioreactor, or other flow-based technique. Therefore, enrichment may be performed using magnetic bead separation. Magnetic bead separation may allow cells bound to a probe conjugated to a magnetic particle to either i) be pulled out of a sample by a magnet and removed from the unbound cells, or ii) allow the bound cells to be held in the sample by a magnet whilst unbound cells are washed away. Enrichment may be performed on live / intact cells.

[0229] According to fourth aspect, there is provided a kit for enriching or removing nonsenescent or senescent cells and / or vesicles thereof from a sample, the kit comprising: a separation means for separating non-senescent cells and / or vesicles thereof from senescent cells and / or vesicles thereof, or a separation means for separating senescent cells and / or vesicles thereof from non-senescent cells and / or vesicles thereof; and a probe for a glycan, wherein the glycan comprises one or more residues selected from the group consisting of:

[0230] (i) terminal sialic acid residues,

[0231] (ii) terminal galactose residues,

[0232] (iii) terminal mannose residues,

[0233] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0234] (v) terminal fucose residues,

[0235] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0236] (vii) internal N-acetyllactosamine residues; wherein the probe binds to senescent cells and / or vesicles thereof that extracellularly and / or extravesicularly display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (non-senescent) cells from bound (senescent) cells and / or vesicles thereof, thereby enriching the non-senescent cells and / or vesicles thereof within the test sample, or wherein the probe binds to non-senescent cells and / or vesicles thereof that extracellularly display on their surface a glycan comprising terminal residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (senescent) cells and / or vesicles thereof from bound (non-senescent) cells and / or vesicles thereof, thereby enriching the senescent cells and / or vesicles thereof within the test sample.

[0237] In one embodiment, the kit comprises: a probe for a glycan; wherein the glycan comprises residues selected from the group consisting of:

[0238] (i) terminal sialic acid residues, (ii) terminal galactose residues,

[0239] (iii) terminal mannose residues,

[0240] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0241] (v) terminal fucose residues,

[0242] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0243] (vii) internal N-acetyllactosamine residues; wherein the probe binds to senescent cells and / or vesicles thereof that display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (non-senescent) cells from bound (senescent) cells and / or vesicles thereof, thereby enriching the nonsenescent cells and / or vesicles thereof within the test sample, or wherein the probe binds to non-senescent cells and / or vesicles thereof that display on their surface a glycan comprising terminal residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (senescent) cells and / or vesicles thereof from bound (non-senescent) cells and / or vesicles thereof, thereby enriching the senescent cells and / or vesicles thereof within the test sample.

[0244] In another embodiment, the glycan comprises terminal monosaccharide residues selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6- linked sialic acid residues, alpha linked mannose residues, alpha- 1,6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, terminal N-acetylglucosamine residues, and alpha linked fucose residues. Alternatively, or additionally, the glycan comprises internal N-acetyllactosamine residues.

[0245] The separation means may be used with a probe to separate cells bound or capable of being bound by the probe (e.g., senescent cells) from cells unbound or incapable of being bound by the probe (e.g., non-senescent cells). For example, the separation means may be magnetic separation beads.

[0246] The separation means may be conjugated to the probe to create a conjugated probe. The kit may therefore comprise a means for conjugating the separation means to the probe, so as to create a conjugated probe. The kit may comprise a conjugating agent. The conjugating agent may be used to conjugate the probe with the magnetic beads. The kit may comprise a conjugated probe, or probes, conjugated to one or more separation means.

[0247] The probe may be for binding a glycan displayed on an extracellular surface or an extravesicular surface of senescent cells, particularly the terminal residues of the glycan. The probe may be for enriching senescent cells and / or vesicles thereof. The probe may be for binding a glycan displayed on an extracellular surface of nonsenescent cells and / or vesicles thereof. The probe may be for a glycan referred to herein. Thus, the probe may be for enriching non-senescent cells and / or vesicles thereof.

[0248] The choice of an appropriate probe depends on whether the method comprises (i) binding (e.g. immobilising or isolating) senescent cells and / or vesicles thereof via binding of the probe to the same, or (ii) binding (e.g. immobilising or isolating) nonsenescent cells and / or vesicles thereof via binding of the probe to the same. Suitable probes for either scenario are discussed below.

[0249] For example, any probe (e.g. lectin) described herein may be for binding to (e.g. preferentially binding to, or specifically binding to) senescent cells, as at least senescent cell types are associated with ‘higher’ levels (vs non-senescent reference) of any given glycan. That being said, it may be advantageous to use particular probes depending on cell type, as outlined below.

[0250] Reference to a probe for “binding to non-senescent cells (and / or vesicles thereof)” preferably means that the probe preferentially binds to non-senescent cells, with “preferentially binds” meaning that the probe binds a higher number of (i) nonsenescent cells (and / or vesicles thereof) than (ii) senescent cells (and / or vesicles thereof) in a mixed population comprising both (i) and (ii). Similarly, reference to a probe for “binding to senescent cells (and / or vesicles thereof)” preferably means that the probe preferentially binds to senescent cells, with “preferentially binds” meaning that the probe binds a higher number of (i) senescent cells (and / or vesicles thereof) than (ii) non-senescent cells (and / or vesicles thereof) in a mixed population comprising both (i) and (ii). Reference to said term “higher number” may mean that the probe binds at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100% more of (i) than (ii). A preferred probe for binding to senescent cells (and / or vesicles thereof) may be a probe for binding extracellular (and / or extravesicular) displayed terminal alpha-2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and / or terminal N- acetylglucosamine. As such, a probe for binding to senescent cells may be MAL-II, SNA, NPL, GNA, and / or STL. Advantageously, extracellular and / or extravesicular surface display of these glycans have been demonstrated to be consistently ‘higher’ (vs non-senescent reference) in senescence across multiple cell types.

[0251] A preferred probe for binding to non-senescent endothelial cells (and / or vesicles thereof) may be a probe for binding extracellular (and / or extravesicular) displayed terminal a(1^2) linked fucose linked to a galactose. As such, a probe for binding nonsenescent endothelial cells (and / or vesicles thereof) may be UEA-1.

[0252] A preferred probe for binding to senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof may be a probe for binding extracellular (and / or extravesicular) displayed terminal a(1^2) linked fucose linked to a galactose. As such, a probe for binding connective tissue cells (such as fibroblasts) and / or vesicles thereof may be UEA-1.

[0253] A preferred probe for binding to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof may be a probe for binding extracellular (and / or extravesicular) displayed terminal a(1^2) linked fucose linked to a galactose. As such, a probe for binding senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof may be UEA-1.

[0254] A preferred probe for binding to senescent endothelial cells, senescent fibroblasts and / or senescent BMMSC cells (and / or vesicles thereof) may be a probe for binding extracellular (and / or extravesicular) displayed terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc). As such, a probe for binding to senescent endothelial cells, senescent fibroblasts and / or senescent BMMSC cells (and / or vesicles thereof) may be A AL. A preferred probe for binding to non-senescent DPMSC cells (and / or vesicles thereof) may be a probe for binding extracellular (and / or extravesicular) displayed terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc). As such, a probe for binding to non-senescent DPMSC cells (and / or vesicles thereof) may be AAL.

[0255] A preferred probe for binding to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be a probe for binding extracellular (and / or extravesicular) displayed terminal N-acetylgalactosamine. As such, a probe for binding to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be SJA.

[0256] Another preferred probe for binding to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be a probe for binding extracellular (and / or extravesicular) displayed terminal galactose. As such, a probe for binding to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be PNA.

[0257] The kit may further comprise a control sample. The control sample may be a sample of cells. The kit may further comprise a positive control and / or a negative control sample. The positive control may be a sample that comprises senescent cells and / or vesicles thereof. The negative control may be a sample that does not comprise or contain senescent cells (e.g., thus comprises non-senescent cells) and / or vesicles thereof. The reference sample may be a sample that does not comprise cells and / or vesicles that do not display any of the glycans referred to herein, or low, undetectable levels of the glycans referred to herein. The reference sample may or may not comprise dividing cells or cells that are capable of dividing. The reference sample may or may not comprise cells that do not have biomarkers such as P-galactosidase, DNA damage markers such as yH2AX or activate pl6INK4A. The reference sample may be a sample for a healthy subject.

[0258] The kit may comprise a reference sample.

[0259] The invention may be used to enrich for non-senescent cells and / or vesicles thereof. According to another aspect of the invention, there is provided a method of treating or preventing a disease or disorder in a subject, the method comprising: enriching non-senescent cells and / or vesicles thereof in a sample comprising a mixture of senescent and non-senescent cells and / or vesicles thereof using a method or a kit according to the invention; and administering the enriched sample of non-senescent cells and / or vesicles thereof to the subject to treat the disease or disorder.

[0260] The sample may be an in vitro, an in vivo or an ex vivo sample. The disease or disorder may be any disease or disorder referred to herein. The sample may be a sample of heterologous cells or a sample of autologous cells.

[0261] According to fifth aspect of the invention, there is provided a method of diagnosing a senescence-related disease or disorder in a subject, the method comprising: providing a sample obtained from a subject; detecting, in the sample, extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:

[0262] (i) terminal sialic acid residues,

[0263] (ii) terminal galactose residues,

[0264] (iii) terminal mannose residues,

[0265] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0266] (v) terminal fucose residues,

[0267] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0268] (vii) internal N-acetyllactosamine residues; wherein detection, in the sample, of a glycan comprising residues selected from the group consisting of (i) to (vii) is indicative that the subject has a senescence-related disease or disorder.

[0269] In an embodiment, the glycan comprises terminal monosaccharide residues selected from the group consisting of: alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, terminal N-acetylglucosamine residues, and alpha linked fucose residues. Alternatively, or additionally, the glycan comprises internal N-acetyllactosamine residues. Senescence-related diseases or disorders may include but are not limited to osteoporosis, frailty, cardiovascular diseases, osteoarthritis, pulmonary fibrosis, renal diseases, neurodegenerative diseases (e.g., Alzheimer’s disease), hepatic steatosis, metabolic dysfunction, COPD, and macular degeneration. Senescence-related diseases or disorders may also be other age-related diseases.

[0270] The invention may further provide a method of treating a senescence related disease or disorder by administering an enriched sample of non-senescent cells and / or vesicles thereof to the subject to treat the disease or disorder and / or the step of administering a probe-agent conjugate according to the invention to the subject to treat the disease or disorder. The enriched samples may be prepared using a method according to the invention.

[0271] The terms N-acetylgalactosamine and GalNAc are used interchangeably herein.

[0272] The terms N-acetylglucosamine and GlcNAc are used interchangeably herein.

[0273] The term “detecting” referred to herein can mean determining the presence of one or more of the glycans referred to herein in a sample or subject. The detecting may comprise contacting the test sample with one or more probes that bind to the glycans referred to herein. Thus, detecting / detection may comprise contacting a mixture of nonsenescent cells and senescent cells and / or vesicles thereof within a sample with one or more probes that bind to the glycans referred to herein. It is preferred that the probe is a “binding partner” (e.g. complementary binding partner) having affinity for said one or more glycan (e.g. an appropriate lectin, or appropriate lectins) and that the “detecting” step comprises detecting a level of glycan-binding partner complex (e.g. as a readout of the glycan residue level) formed following contact of the test sample with the one or more probe / binding partner. For example, the probe / binding partner may comprise a detectable agent (optionally as a conjugate) such as a fluorophore, a radioisotope or a dye (preferably a fluorophore). The level of such detectable agent can provide be used to measure the level of the complex.

[0274] The probe may be a probe referred to herein. Detection may be performed using any conventional assay or technique known in the art, including for example, but not limited to: flow cytometry; immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), an enzyme immunoassay (EIAs), radioimmunoassay (RIAs), Western Blots, immuno-precipitation or immunohistochemistry; chromogenic (enzyme activity) assays; fluorometric imaging plate reader (FLIPR) assay; or high performance liquid chromatography (HPLC) tandem mass spectrometry (MS / MS), or in vivo detection techniques, such as magnetic resonance imaging (MRI), positron emission tomography (PET) or radioactive labelling of lectins. Detection may be performed using flow cytometry or immunofluorescence. Detection may be performed using flow cytometry. Flow cytometry may be preferred because detection can be performed on the extracellular surface of live / intact cells or vesicles thereof.

[0275] When flow cytometry is used, a level of extracellular and / or extravesicular surface display of a glycan in the sample may be represented as percentage of cells (within a cell population) that are ‘positive’ for said extracellular and / or extravesicular glycan. In other words, a cell (and / or vesicle) that displays the glycan extracellularly (and / or extravesicularly) may be said to be “glycan positive”. For example, a “higher” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a higher percentage of “glycan positive” cells in the sample / test sample when compared with the reference sample. Similarly, an “lower” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a lower percentage of “glycan positive” cells in the sample / test sample when compared with the reference sample.

[0276] Additionally or alternatively, and preferably where the probe is conjugated to a detectable agent (e.g. fluorophore), a level of extracellular and / or extravesicular surface display of a glycan in the sample may be represented as a “mean fluorescence intensity” (MFI) value. This term is well known to the skilled person in the art, and such MFI value is understood to be indicative of antigen abundance (e.g. for the presence circumstances, glycan abundance). For example, a “higher” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a higher “MFI” value in the sample / test sample when compared with the reference sample. Similarly, a “lower” level of extracellular and / or extravesicular surface display of a glycan in a sample (e.g. test sample) vs. a reference sample may be detected as a lower “MFI” value in the sample / test sample when compared with the reference sample. When reviewing both “percentage positive” measurements (as per the paragraph directly preceding this paragraph) and “MFI” values (e.g. from the same or similar cell population), it is preferred that more weight is placed on the MFI value (e.g. to determine whether a glycan level is ‘higher’ or ‘lower’ than a reference.

[0277] The term “mean” here (in the context of MFI) can be used interchangeably with the terms “median”, “arithmetic mean”, “geometric mean” and “mode”.

[0278] The “sample ” or “test sample ” may be a biological sample or a bodily sample of a subject (a sample taken from a subject, such as human or any other organism). The sample may be a bodily sample. Thus, the invention may be carried out in vitro. The sample may comprise blood (e.g., umbilical cord blood), plasma, serum, bone marrow, amniotic fluid, adipose or other tissues, or combinations thereof. Thus, the sample may be recovered from blood, bone marrow, amniotic fluid, dental pulp of molar cells, an umbilical cord blood, adipose tissue, or any other tissue or bodily fluid or combinations thereof. Alternatively, the sample may be an ex vivo sample or an in vitro sample. Therefore, the cells may be in a tissue or organ sample (for ex vivo based tests) or the cells may be grown in culture (an in vitro sample) or in the blood, tissue or body (an in vivo sample). The sample may or may not have undergone in vitro expansion. The sample may be “a primary cell culture ”. The methods disclosed herein may performed in vivo (e.g., via blood plasma apheresis system or PET imaging). The sample or test sample may comprise a mixture of non-senescent cells and senescent cells and / or vesicles thereof. All the cells may be live cells. In embodiments wherein the invention is performed in vivo, the test sample may be the subject per se. The sample may comprise a mixture of cells. The sample may comprise a mixture of senescent cells and / or vesicles thereof, which display a glycan on their external surface, together with non-senescent cells and / or vesicles thereof.

[0279] The reference sample or negative control may be a sample that does not comprise or contain senescent cells and / or vesicles thereof (e.g., non-senescent cells). The reference sample (or negative control) may be a sample that comprises cells and / or vesicles that do not display any of the glycans referred to herein, or low, undetectable levels of the glycans referred to herein. The reference sample may comprise dividing cells or cells that are capable of dividing. The reference sample may be from the same or a different subject as the test sample. The test sample may comprise the same type of cells as the reference sample. The test sample may be matched with a reference sample of the same age. There may be about at 1 to 5 -year difference between the reference sample and the test sample.

[0280] According to sixth aspect, there is provided a senescent cell probe-agent conjugate comprising: a probe for targeting and / or binding to a glycan comprising one or more residues selected from the group consisting of:

[0281] (i) terminal sialic acid residues,

[0282] (ii) terminal galactose residues,

[0283] (iii) terminal mannose residues,

[0284] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0285] (v) terminal fucose residues,

[0286] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0287] (vii) internal N-acetyllactosamine residues, conjugated to an agent.

[0288] The probe may be any probe referred to herein (e.g., a lectin, an antibody, an Fc fusion, an aptamer, a radionucleotide, a bead, a drug, a nanoparticle composition or a cell). The glycan may comprise a terminal monosaccharide residue selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylglucosamine residues, N- acetylgalactosamine residues, and alpha linked fucose residues. Alternatively, or additionally, the glycan may comprise internal N-acetyllactosamine residues.

[0289] The senescent cell probe-agent conjugate may be used to kill a senescent cell or alter its function. The conjugate referred to herein may be bispecific. In embodiments in which the conjugate is used to kill a senescent cell, the agent may be a cytotoxic agent, which kills senescent cells. The cytotoxic agent may be or may comprise a drug, a prodrug, a toxin, a toxic peptide, an antibody or a cell. The cytotoxic agent may be or may comprise a senolytic drug. The senolytic may be navitoclax. In embodiments in which the cytotoxic agent is a cell, the cell targeting agent may be a CAR-NK or CAR- T cell, such that the senescent cell probe conjugate can be used to perform perforin- based or other forms of cell killing. In embodiments in which the agent is an antibody, the antibody may initiate antibody-dependent cellular cytotoxicity or cell killing.

[0290] In embodiments in which the conjugate is used to alter the function of a senescent cell, the agent may be a drug. Thus, the conjugate may be used to deliver a drug to senescent cells. In embodiments in which the conjugate is used to deliver a drug to a senescent cell, the agent may be a drug that modulates (e.g., increases or decreases) an activity of the bound senescent cell. The drug may not be cytotoxic. The drug may be a senomorphic drug.

[0291] The conjugate may be used to visualise a senescent cell, or tag it for detection, elimination or enrichment. In embodiments in which the conjugate is used to visualise a senescent cell or tag it for detection, the agent may be an imaging agent. The imaging agent may be a fluorophore, a radioisotope, a dye or another agent used to image cells.

[0292] According to a seventh aspect of the invention, there is provided a senescent cell probeagent conjugate according to the invention, for use as a medicament.

[0293] According to an eighth aspect of the invention, there is provided a senescent cell probeagent conjugate according to the invention for use in the prevention or treatment of a disease or disorder of a subject.

[0294] According to a ninth aspect, there is provided a method of preventing or treating a disease or disorder of a subject, the method comprising administering a senescent cell probe-agent conjugate according to the invention to the subject.

[0295] The disease or disorder referred to herein may be one or more selected from the group consisting of: aging, sarcopenia, cancer, cachexia, COPD, osteoporosis, frailty, a cardiovascular disease, osteoarthritis, pulmonary fibrosis, renal diseases, neurodegenerative diseases (e.g., Alzheimer’s disease), hepatic steatosis, metabolic dysfunction, and macular degeneration, or others.

[0296] The agent may be a therapeutic agent. The therapeutic agent may be used to prevent or treat the disease or disorder. According to a tenth aspect, there is provided a method of killing senescent cells, the method comprising: determining the presence of a senescent cell in a subject using a method or kit according to the invention; and administering, to the subject, a therapeutically effective amount of the probeagent conjugate according to the invention in order to kill the senescent cells.

[0297] The method according to the invention may be used to kill senescent cells in vivo, in vitro or ex vivo.

[0298] According to an eleventh aspect, there is provided use of an extracellular or extravesicular surface displayed glycan as a senescent cell biomarker, wherein the glycan comprises one or more residues selected from the group consisting of:

[0299] (i) terminal sialic acid residues,

[0300] (ii) terminal galactose residues,

[0301] (iii) terminal mannose residues,

[0302] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0303] (v) terminal fucose residues,

[0304] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0305] (vii) internal N-acetyllactosamine residues.

[0306] In an embodiment, the glycan comprises terminal monosaccharide residues selected from the group consisting of: alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, terminal N-acetylglucosamine residues, and alpha linked fucose residues. Alternatively, or additionally, the glycan comprises internal N-acetyllactosamine residues.

[0307] According to a twelfth aspect, there is provided a method of killing a cell of a sample, the method comprising: contacting the sample comprising the cell with a probe for a glycan in order to create a cell-bound probe; and contacting the cell-bound probe with an immune cell, thereby causing the immune cell to bind to the probe and kill the cell, wherein the residues of the glycan comprise one or more residue(s) selected from the group consisting of:

[0308] (i) terminal sialic acid residues,

[0309] (ii) terminal galactose residues,

[0310] (iii) terminal mannose residues,

[0311] (iv) terminal N-acetylgalactosamine (GalNAc) residues,

[0312] (v) terminal fucose residues,

[0313] (vi) terminal N-acetylglucosamine (GlcNAc) residues, and

[0314] (vii) internal N-acetyllactosamine residues.

[0315] In an embodiment, the glycan comprises terminal monosaccharide residues selected from the group consisting of alpha-2, 3-linked sialic acid residues, alpha-2, 6-linked sialic acid residues, alpha linked mannose residues, alpha- 1, 6-linked mannose residues, alpha-1, 3-linked mannose residues, galactose residues, N-acetylgalactosamine residues, terminal N-acetylglucosamine residues, and alpha linked fucose residues. Alternatively, or additionally, the glycan comprises internal N-acetyllactosamine residues.

[0316] According to a thirteenth aspect, there is provided use of a probe to kill a cell in a sample.

[0317] The probe may be any probe referred to herein. Thus, the probe may be an antibody, lectin, an Fc fusion, an aptamer, a radionucleotide, a bead, a drug, a nanoparticle composition, a cell, or something similar. An Fc fusion protein is a recombinant protein that may comprise an extracellular domain of a receptor (e.g., a lectin) that is combined with an Fc region of an antibody. The antibody may be used as antibody-dependent Cell-mediated Cytotoxicity (ADCC) therapy to kill a live cell. ADCC may comprise using an immune cell to bind to the live cell via the probe. The immune cell referred to herein may be a cell that is capable of degranulation. The immune cell may be capable of causing apoptosis or necrosis of a live cell (or via an alternative way of killing a cell). Thus, the method according to the invention may comprise the immune cell killing the live cell by degranulating onto the live cell. The invention may comprise the immune cell causing the death of the live cell (e.g., by apoptosis or necrosis). The immune cell may be a cell that is capable of triggering apoptosis of a live cell. The immune cell may be an NK cell, a dendritic cell, a T-cell, and / or a macrophage. The live cell may be a senescent cell. According to a fourteenth aspect, there is provided use of a lectin to detect senescent cells and / or vesicles thereof, enrich senescent cells and / or vesicles thereof, isolate senescent cells and / or vesicles thereof, remove senescent cells and / or vesicles thereof or target senescent cells and / or vesicles thereof.

[0318] In one embodiment, the invention comprises use of a selection of one or more of MAL- II, SNA, GNA, STL, AAL, UEA-I, PNA, SJA and NPL to detect, enrich, isolate, remove or target senescent cells and / or vesicles thereof. In another embodiment, the invention comprises use of a selection of one or more of MAL-II, SNA, GNA, STL, PNA, SJA and NPL to enrich, isolate, remove or target senescent cells and / or vesicles thereof. In another embodiment, the invention comprises use UEA-I and / or AAL to detect, enrich, isolate, remove or target non-senescent cells and / or vesicles thereof.

[0319] The term “terminal” may refer to the last five, last four, last three or last two residues of the glycan. The term “terminal” may refer to the last or final residue of the glycan. The term “terminal” may refer to the residues located at the terminus situated furthest from the relevant cell or vesicle. The term "terminal" may refer to the last or final residue of the glycan, which is located at the terminus situated furthest from the relevant cell or vesicle.

[0320] The term “internal” for example “internal N-acetyllactosamine residues” refers to residues that lie underneath the terminal residue.

[0321] The probe is capable of binding to the glycan(s) displayed on the surface of senescent cells and / or vesicles thereof, the probe may bind to the terminal residues thereof. The probe may be specific or selective for one or more of the glycans referred to herein, or the terminal residues thereof. The probe may be contacted with a sample of cells to produce cells bound by the probe and cells not bound by the probe.

[0322] The probe may be specific or selective for the glycan. The probe may be a lectin, an antibody, an Fc fusion, an aptamer, a radionucleotide, a bead, a drug, a nanoparticle composition or a cell. The probe may most preferably be a lectin (either itself or attached to other structures) e.g., a lectin selected from the group consisting of: AAL, GNA, MAL-II, NPL, UEA-I, SNA, STL, SJA and PNA. The probe may be referred to as a “binding partner”; following challenge of the test sample with the probe / binding partner, methods of the invention may comprise detecting a level of probe / binding partner-glycan complex.

[0323] A glycan detected by methods of the invention may be referred to by reference to a complementary lectin having affinity for the glycan. For example, the one or more residues (of the glycan) described herein may be capable of being bound by a lectin selected from the group consisting of: AAL, GNA, MAL-II, NPL, UEA-I, SNA, STL, SJA and PNA.

[0324] The skilled person understands which glycans said lectins bind to, and would thus understand the what the “one or more residues (of the glycan)” is when described with reference to the lectin it is bound by. However, for the avoidance of doubt, and to summarise what was outlined above: terminal fucose “a(l— >2) linked fucose linked to a galactose” may be referred to as a terminal glycan residue capable of being bound by Ulex Europaeus Agglutinin I (UEA- i); terminal fucose “Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc)” may be referred to as a terminal glycan residue capable of being bound by AAL; terminal sialic acid that is an “alpha-2, 3-linked sialic acid” (preferably a terminal NeuAc a2-3 Gal bl -3 GalNAc residue) may be referred to as a terminal glycan residue capable of being bound by MAL-II; terminal sialic acid that is an alpha-2, 6-linked sialic acid (preferably a terminal NeuAc a2-6 Gal bl -4 GlcNAc residue) may be referred to as a terminal glycan residue capable of being bound by SNA; terminal N-acetylglucosamine (preferably a P(l,4)-linked GlcNAc) and / or an oligomer thereof (e.g. Chitin and / or (GlcNAc)3) may preferably be referred to as a terminal glycan residue capable of being bound by STL; terminal mannose that is an “alpha- 1, 6-linked mannose” (preferably alpha- 1, 6-linked Man3GlcNAc2 and / or alpha- 1, 6-linked Man5GlcNAc2) may be referred to as a terminal glycan residue capable of being bound by NPL; terminal mannose that is an “alpha-1, 3-linked mannose” (preferably alpha- 1,3 -linked Man3GlcNAc2) may be referred to as a terminal glycan residue capable of being bound by GNA; terminal galactose “Fuc al-2( Gal bl -3 GalNAc al -3) Gal bl -4 GlcNAc” may be referred to as a terminal glycan residue capable of being bound by PNA; terminal galactose “Fuc al-3( Fuc al-2( Gal al-3) Gal bl-4) GlcNAc” may be referred to as a terminal glycan residue capable of being bound by SJA; terminal N-acetylgalactosamine (preferably GalNAc bl-4 GlcNAc b 1 -3 GalNAc bl-4 GlcNAc) may be referred to as a terminal glycan residue capable of being bound by SJA.

[0325] The lectin may be an agglutinin. The agglutinin may be Ulex Europaeus Agglutinin I (UEA-I). UEA-I binds to glycans comprising a terminal fucose, such as an alpha- glycosidic linked fucose, for example a( 1— >2) linked fucose linked to a galactose (e.g. Fuc al-2 Gal bl-4 GlcNAc).

[0326] The lectin may be Aleuria Aurantia Lectin (AAL). AAL can bind a Fuc a( 1 — >6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc). AAL may bind to terminal alpha-linked fucose glycans such as Fuc al-2 Gal [6S] bl-4 Glc.

[0327] The lectin may be Maackia Amurensis Lectin II (MAL-II). MAL-II binds to glycans comprising terminal sialic acids, such as an alpha glycosidic-linked sialic acid, for example an alpha-2, 3-linked sialic acid. MAL-II may bind to a terminal NeuAc a2-

[0328] 3 Gal bl -3 GalNAc residue.

[0329] The lectin may be Sambucus Nigra Lectin (SNA). SNA binds to glycans comprising terminal sialic acids, such as an alpha glycosidic-linked sialic acid, for example an alpha-2, 6-linked sialic acid. SNA may bind to a terminal NeuAc a2-6 Gal bl-

[0330] 4 GlcNAc residue.

[0331] The lectin may be Solanum Tuberosum Lectin (STL). STL binds to glycans comprising terminal N-acetylglucosamine, such as N-acetylglucosamine and / or oligomers of N- acetylglucosamine, and / or to glycan comprising internal N-acetyllactosamine. STL may bind to Chitin and / or (GlcNAc)3. The lectin may be Narcissus Pseudonarcissus Lectin (NPL). NPL binds to glycans comprising a terminal alpha glycosidic-linked mannose, such as an alpha- 1,6-linked mannose. NPL may bind to Man3GlcNAc2 and / or Man5GlcNAc2.

[0332] The lectin may be Galanthus Nivalis Lectin (GNA). GNA binds to glycans comprising a terminal alpha glycosidic-linked mannose, such as an alpha-1, 3-linked mannose. GNA may bind to Man3GlcNAc2.

[0333] The lectin may be Peanut Agglutinin (PNA). PNA binds to glycans comprising a terminal galactose, for example Fuc al-2(Gal bl -3 GalNAc al -3) Gal bl -4 GlcNAc.

[0334] The lectin may be Styphnolobium japonicum (Japanese pagoda) lectin (SJA). SJA binds to glycans comprising a terminal galactose or terminal N-acetylgalactosamine, for example Fuc al-3(Fuc al-2(Gal al-3) Gal bl-4) GlcNAc or GalNAc bl-4 GlcNAc bl- 3 GalNAc bl-4 GlcNAc.

[0335] The probe or lectin may be one or more selected from the group comprising MAL-II; SNA; UEA-I; STL; NPL; AAL; GNA; PNA; SJA; and combinations thereof.

[0336] Thus, one or more lectins selected from the group consisting of MAL-II, SNA, NPL, GNA, AAL, UEA-I, PNA, SJA and STL may be used to detect, identify, enrich senescent cells or non-senescent cells and / or vesicles thereof or isolate senescent cells or non-senescent cells and / or vesicles thereof. One or more lectins selected from the group consisting of MAL-II, SNA, NPL, GNA, PNA, SJA and STL may be used to detect, enrich senescent cells and / or vesicles thereof or isolate senescent cells and / or vesicles thereof. AAL and / or UEA-I may be used to detect, enrich senescent cells or negatively isolate non-senescent cells.

[0337] The inventors have determined that senescent cells and vesicles thereof display higher levels of certain glycans compared to non-senescent cells. The glycans include (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine residues, (v) terminal fucose residues, (vi) terminal GlcNAc residues, and (vii) internal N-acetyllactosamine residues. The glycans may comprise terminal alpha-2, 3-linked sialic acids residues, terminal alpha- 2,6-linked sialic acids, terminal alpha-linked fucose residues, terminal N- acetylgalactosamine residues, terminal N-acetylglucosamine residues, terminal alpha- 1,3 linked mannose residues, and terminal alpha- 1,6 linked mannose residues, terminal galactose residues and internal N-acetyllactosamine residues. Thus, the methods and kits referred to herein may be performed by detecting such glycans or using probes for such glycans.

[0338] The “subject” referred to herein may be an animal or a plant. The animal may be a vertebrate, a mammal, or a domestic animal. The subject may be any animal of veterinary interest, for instance, a cat, dog, horse etc. The subject may be a human.

[0339] Furthermore, “senescent cells” (and / or vesicles thereof), for example as described in methods of the invention, may be animal or a plant cells. The “senescent cells” (and / or vesicles thereof) may be vertebrate, mammal, or domestic animal cells. The “senescent cells” (and / or vesicles thereof) may be from any animal of veterinary interest, for instance, a cat, dog, horse etc. Preferably, the “senescent cells” (and / or vesicles thereof) are human cells. All of these standard taxonomic definitions may likewise apply to a “non-senescent” cell described herein.

[0340] Vesicles referred to herein may be a structure that is located inside or outside a cell or fused with a cell and comprises a lipid bilayer that encloses a liquid, such as a cytoplasm, and / or bioactive molecules, and / or RNA. Vesicles referred to herein may be one or more structures selected from the group consisting of lysosomes, extra-cellular vesicles, microvesicles, nano-vesicles, exosomes, oncosomes, enucleated cells, artificial vesicles or similar structures.

[0341] Preferably, a “vesicle” comprises an outer membrane derived from the outer membrane of senescent cells from which the vesicle is released. This allows advantage to be taken of the observation that outer cell-membrane glycans provide a readout of senescence. The term “senescent cells ” can refer to live cells that display one or more senescence biomarkers. The term “non-senescent cells ” can refer to live cells that do not display any senescence biomarkers (e.g., P-galactosidase, DNA damage markers, such as yH2AX, or pl6INK4A). While it is possible to assay senescent cells and / or vesicles thereof, it is preferred that it is the cells that are assayed in the present invention (e.g. the term “senescent cells and / or vesicles thereof’ may mean “senescent cells”).

[0342] It will be appreciated that the term “treatment” and “treating” as used herein means the management and care of a subject for the purpose of combating a condition, such as a disease or a disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, including alleviating symptoms or complications, delaying the progression of the disease, disorder or condition, alleviating or relieving the symptoms and complications, and / or to cure or eliminating the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a subject for the purpose of combating the disease, condition, or disorder and includes the administration of the ligand to prevent the onset of the symptoms or complications.

[0343] The methods, kits, and conjugates according to the invention rely on extracellular or extravesicular surface display of a glycan comprising terminal monosaccharide residues selected from the group consisting of: (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine residues, (v) terminal fucose residues, (vi) terminal N-acetylglucosamine residues, and (vii) internal N-acetyllactosamine residues. In a specific embodiment, the glycan may comprise terminal monosaccharide residues selected from the group consisting of alpha- 2,3-linked sialic acid, alpha-2, 6-linked sialic acid residues, alpha- 1,6-linked mannose residues, alpha-1, 3-linked mannose residues, N-acetylglucosamine residues, alphalinked fucose residues, galactose residues and / or N-acetylgalactosamine residues. Alternatively, or additionally, internal N-acetyllactosamine residues.

[0344] The glycan referred to herein may be a homopolymer or a heteropolymer of monosaccharide residues. While the invention involves assaying the level of “extracellular and / or extravesicular surface display” of a glycan, the skilled person would understand that there are scenarios in which the glycan may first be 'liberated’ from the exterior surface of a cell (and / or vesicle thereof) before its level is determined. Such scenarios still allow for assaying a glycan that has been (e.g. pre-liberation solely for measurement / assay purposes) bound to the exterior surface of a cell or vesicle surface, thus continue to allow for assaying the level of “extracellular and / or extravesicular surface display” of a glycan. That being said, the inventors have succeeded in assaying the glycans as they are bound to the cell (or vesicle thereof) surface, e.g. via an advantageous cell sorting / flow cytometry approach. Thus, it is preferred that the glycan referred to herein is extracellular or extravesicular and thus preferably bound to the exterior surface of a cell or vesicle surface.

[0345] It may be said that the glycan is present as an integral part of the outer membrane of the cell (or outer membrane of a vesicle thereof) in the test sample. The glycan may be bound directly or indirectly. The glycan may, for example, be bound indirectly via a cell surface protein or lipid. Thus, the glycan may be part of a glycoprotein or a glycolipid. The glycan may be branched or linear. In embodiments in which the glycan is branched, either / any branch of the glycan may consist of one or more residues of: (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine residues, (v) terminal fucose residues, (vi) terminal N-acetylglucosamine residues, and / or (vii) internal N-acetyllactosamine residues.

[0346] In some embodiments, the method disclosed herein may comprise detecting the level of extracellular and / or extravesicular surface display of a plurality of the glycans disclosed herein. In such embodiments, the pattern of display, where glycans are displayed at either higher or lower (for example significantly higher or significantly lower) levels in a test sample compared to a reference sample may be referred to as the “glycan signature ” . The glycan signature represents a useful and robust marker for the presence or absence of senescent cells and / or vesicles thereof. Thus, a glycan signature may be used to indicate whether a sample comprises senescent cells and / or vesicles thereof.

[0347] The invention may be used in vivo, in vitro or ex vivo (preferably in vitro or ex vivo). Thus, the method disclosed herein may be performed in in vivo, in vitro or ex vivo. The kit may be used in vivo, in vitro or ex vivo.

[0348] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Figure legends

[0349] For a better understanding of the invention, and to show how embodiments of the invention may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which: -

[0350] Figure 1 - Flow cytometry analysis of senescent markers on HUVEC cells after DOXO treatment.

[0351] HUVEC from three different donors, at passage 4 or 5, were pooled and subjected to DOXO treatment for senescence induction. Young and DOXO treated HUVEC were analyzed for beta-galactosidase activity with DDAO (A, B); pl6 (C, D) and cell size with forward scatter (FSC) (E). Percentage positive DDAO or P16 cells are shown in histograms (A-D). Young (red) and DOXO treated (blue) HUVEC are shown in forward (FSC) vs side (SSC) scatter dot-plot (E). Geometric mean of forward scatter, FSC-A, is shown (E). An alternative view of (E) is shown in (F).

[0352] Figure 2 - Flow cytometry analysis of senescent markers on fibroblasts after DOXO treatment. Fibroblasts from three different donors, at passage 5, were pooled before DOXO induction of senescence. Young and DOXO treated fibroblasts were analyzed for beta-galactosidase activity with DDAO (A, B) and cell size with FSC (C). Expression levels of DDAO in both young (A) and DOXO treated fibroblasts (B) are shown in histograms. Percentage positive DDAO cells are shown in histograms (A, B). Young (red) and DOXO treated (blue) fibroblasts are shown in forward (FSC) vs side (SSC) scatter dot-plot and their geometric mean of FSC-A, is shown (C). An alternative view of (C) is shown in (D).

[0353] Figure 3 - Flow cytometric analysis of senescence marker panel on MSC. Flow cytometry was used to confirm the establishment of senescence in MSC upon replicative- and induced-senescence. Levels of beta-galactosidase activity (A), pl6 (B), yH2AX (C) and ki-67 (D) were analysed at early (P5), intermediate (‘Interim’ Pl 1-13) and late (P16-25) passages, as well as after induction of senescence with DOXO. Values are expressed as mean ± SD of n=5 donors. One-Way Anova test was used for replicative senescence (early vs interim vs late passages) and unpaired, parametric student t-test for induced senescence (early vs DOXO-treated). Figure 4 - Luminex multiplex analysis of MSC SASP upon replicative- and induced-senescence. MSC secretion levels of IL-6 (A), IL-8 (B) and IGFBP-2 (C), all part of SASP, at early (P4-5), intermediate (‘Interim’ Pl 1-13) and late (P16-25) passages, as well as after induction of senescence with DOXO. Values are expressed as mean ± SD of n=5 donors. One-Way Anova test was used for replicative senescence (early vs interim vs late passages) and unpaired, parametric student t-test for induced senescence (early vs DOXO-treated).

[0354] Figure 5 - Percentage positive flow cytometry analysis of lectin binding to youngproliferating and DOXO treated primary cells. Flow cytometry analysis of lectin binding to three different cell types following DOXO treatment. (HUVEC: top, fibroblasts: middle, BMMSC: bottom), expressed as percentage positivity. Lectins used: MAL-II, SNA, NPL, GNA, AAL, UEA-I, STL, SJA and PNA. Three donors were pooled per cell type. Percentages as indicated.

[0355] Figure 6 - MFI flow cytometry analysis of lectin binding to young-proliferating and Doxo-treated primary cells. Flow cytometry analysis of lectin binding to three different cell types following DOXO treatment. (HUVEC: top, fibroblasts: middle, BMMSC: bottom), expressed as MFI. Three donors were pooled per cell type. MFI as indicated.

[0356] Figure 7 - Percentage positive flow cytometry analysis of lectin binding to DPMSC upon replicative senescence. Flow cytometry analysis of lectin binding to human DPMSC at early (P5) and late (P22-24) passage, expressed as percentage positive binding, n = 5 donors. Data expressed as means ± SD and unpaired, parametric student t-test was used.

[0357] Figure 8 - MFI flow cytometry analysis of lectin binding to DPMSC upon replicative senescence. Flow cytometry analysis of lectin binding to human DPMSC at early (P5) and late (P22-24) passage, expressed as MFI. n = 5 donors. Data expressed as means ± SD and unpaired, parametric student t-test was used.

[0358] Figure 9 - Percentage positive flow cytometry analysis of lectin binding to DOXO induced senescent DPMSC. Flow cytometry analysis of lectin binding to youngproliferating (P5) and DOXO-treated (P6) human DPMSC, presented as percentage positive. MAL-II, STL, SJA, UEA-I, AAL, GNA (n = 3 donors for each of the previous lectins); SNA (n = 6 donors); NPL (n = 1 donor). Data is expressed as means ± SD and unpaired, parametric student t-test was used.

[0359] Figure 10 - MFI of lectin panel flow analysis of DPMSC upon induction of senescence. Lectin data of young-proliferating (P5) and Doxo-treated (P6) DPMSC is presented as MFI. MAL-II, STL, SJA, UEA-I, AAL, GNA (n = 3 donors for each of the previous lectins); SNA (n = 6 donors); NPL (n = 1 donor). Data is expressed as means ± SD and unpaired, parametric student t-test was used.

[0360] Figure 11 - Young and senescent cell purification by lectins. Young cells were stained with cell tracer violet and senescent cells (Doxo-treated) were stained with cell tracer red. These stained cells were mixed in 1 : 1 ratio. Cells were incubated with either individual lectins at different concentrations as indicated, or a mixture of lectins and subsequently bound to magnetic affinity beads prior to magnetic separation. Preseparation cells (black bars) as well as flow-through cells (green bars) and bound cells (red bars) were analyzed by flow cytometry. Only data from one round of purification was captured. Analysis was performed on HUVEC (A), fibroblasts (B) and BMMSC (C). The ratio of young to old cells in either the flow-through population or the bound population are expressed as a percentage relative to that in the pre-sort population. Cells from three donors were pooled and used in a single experiment.

[0361] Materials and Methods

[0362] Cell culture

[0363] Mesenchymal stem cells derived from human dental pulp (DP-MSCs, Leipzig University) or from bone marrow (Biorooster), were grown in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% of Fetal Bovine Serum (FBS; 12,483,040; Thermo Fisher Scientific). Human endothelial cells (HUVEC; PromoCell) were cultured using a ready to use endothelial cell growth medium (C- 22010; Promocell). Human fibroblasts (StemNovate) were cultured using a ready to use fibroblast growth medium (C-23025; Promocell). All different cell types were incubated in standard condition (37°C with 5% CO2). Surface marker expression - cell identity

[0364] Dental pulp derived MSC were tested for the expression of specific surface markers by flow cytometry, using the MSC phenotyping kit (130-095-198; MiltenyiBiotec). This kit consists of two antibody cocktails: phenotyping (CD73-APC, CD90-FITC, CD 105- PE, and CD34 / CD45 / CD14 / CD20-PerCP) as an isotype control cocktail. Briefly, MSCs (5 x 105cells) were suspended in staining buffer consisting of 2mM EDTA and 3% FBS in PBS and incubated for 10 min at 4°C with either phenotype or isotype antibody cocktails. Data was acquired with the BD FACSCanto™ II (BD Biosciences) flow cytometer and analysed using Flowjo software version 10 (Flowjo). A compensation matrix was generated using single antibodies from the kit. Doublets were excluded using FSA vs FSH gating for all samples, before gating on total live cells, negative for V450 viability dye (562247; BD Biosciences). Bone marrow MSC were tested in the same manner but just for the following markers: CD45, CD73, CD90, CD105 and found to be positive for CD73, CD90 and CD105 and negative for CD45 (Table 1), as is common for MSC. For the HUVEC characterization we used the following markers: CD31, CD45 and CD 146 and found to be positive for CD31 and CD 146 and negative for CD45 (Table 1), as is common for endothelial cells. Finally, the human fibroblasts were tested positive for the CD90 and negative for CD45 surface markers (Table 1), as is common for fibroblasts.

[0365] Table 1. Immunophenotype of HUVEC, fibroblasts, BMMSC and DP-MSC by flow cytometry. The HUVEC and the fibroblast populations from 3 different donors were pooled. BMMSC (n=3 individual donors) and DPMSC (n=5 individual donors). Senescence induction

[0366] Senescence was induced by exposure of proliferating cells to 200 nM Doxorubicin (Doxo, 5927S; Cell Signalling Technology) dissolved in DMSO for 48 hours. Vehicle control samples were treated with equal concentration of DMSO. The cells were washed with PBS following incubation with either the drug or DMSO for 2 days and then cultured with fresh drug-free culture media for 10 additional days before determining senescence level. All experiments were replicated and performed with appropriate controls.

[0367] Flow cytometric senescence assessment

[0368] Cells were subjected to flow cytometry analysis for senescence assessment with the simultaneous study of any the following markers; beta-galactosidase activity with DDAO (11540346; Fisher scientific), expression levels of pl6 protein (ab209579; Abeam), yH2AX protein (pS 139; BD Biosciences), ki-67 protein (561283; BD biosciences) and cell size with forward scatter (FSC). The detailed procedure as well as the concentrations used for the senescence assessment with the above markers has been described in detail at Adeolu et al., 2020.

[0369] SASP secretion

[0370] The secretion of IL8, IL6, and MCP1 proteins in the culture medium was measured using Luminex technology according to the manufacturer’s instructions (R&D Systems). The cells at different stages of senescence were cultured for 2 days in serum - free culture medium (A1033201; ThermoFisher). Then, the culture medium for each condition was collected, and centrifuged at 300x g for 5 min, and that supernatant was stored at -80C until analysis. Data acquisition was performed on a Luminex MAGPIX™ Multiplex Reader (BioRad). Data was normalised to cell number for each condition.

[0371] Lectin staining and flow cytometry analysis

[0372] To assay cells, after washing adhering cells, adherent cells were mildly detached using known treatment protocols (for detachment) and briefly incubated at culture temperature prior to lectin staining. Lectins were used to stain glycans on the surface of different cell populations followed by incubation of cells with PE conjugated secondary antibody, prior to flow cytometry analysis. This was conducted following standard parameters with normal configurations suited to flow cytometry. The lectins were titrated to prevent agglutination (as per standard practice). Flow cytometric analysis was normalised by subtracting secondary only control staining.

[0373] Briefly, MSC (Bone marrow and dental pulp), HUVECs or fibroblasts were detached from flasks and cells washed twice in PBS (300g, 5min) prior determination of cell numbers. Cells were then incubated in blocking buffer for lOmin and washed twice (300g, 5min). Subsequently, cells were incubated in blocking buffer containing viability dye (554061; BD Biosciences) for 15min and washed (300g, 5min). In the meantime, lectins were diluted in blocking buffer at the appropriate concentrations for the best signal between conditions. Following staining with the viability dye, about 5x 105cells per condition were washed twice in blocking buffer (300g, 5min) and incubated in lectin containing buffers for 30min at 4°C. Cells were then washed once in blocking buffer and incubated with PE-conjugated secondary antibody in blocking buffer for further incubation of 30min at 4°C. Finally, cells were washed (300g, 5min) and resuspend in blocking buffer prior analysis with the flow cytometer.

[0374] MSC magnetic isolation and quantification

[0375] Cell number of young or Doxo-treated cells was determined using an automated cell counter (NucleoCounter®NC-3000™, Chemometec) following the manufacturers’ recommendations. After centrifugation (300g, 5 min) the supernatant was removed, and the young cells were stained with CellTrace Violet (CTV; C34571; Thermofisher) in PBS, while the Doxo-treated cells were stained with CellTraceFarRed (C34572; Thermofisher) in PBS for 15min at 37°C. Cells were then washed in culture medium (300g, 5 min), resuspended in blocking buffer and incubated for lOmin at room temperature. After a wash with PBS, both young and Doxo-treated cells were mixed in 1 : 1 ratio, incubated with different lectins previously diluted in blocking buffer for 30min at 4°C. Subsequently, the mixed population was washed in blocking buffer, resuspended in lOOpl of cold autoMACS buffer (130-091-221; MiltenyiBiotec) supplemented with 0,5% BSA (A9418; Sigma-Aldrich) and incubated with lOpl affinity microbeads for 15min at 4°C. Cells were washed and resuspended in 600pl BSA in autoMACs buffer and were passed twice through an MS column (130-042-201; MiltenyiBiotec) attached to an OctoMACS separator (130-042-109; MiltenyiBiotec), following company’s recommendations. The mixed population before the magnetic separation, as well as the cells from the flow-through (passed through the magnet) and the bound cells (preserved in the column), were all subject to analysis by flow cytometry.

[0376] Statistical analysis

[0377] Statistical analyses were performed using Prism 8 (Graph Pad) using Student’s t-test or one-way Anova when appropriate. Significance was reached at p<0.05. Additional significances are shown as * p < 0.05 ** p < 0.01 ** p < 0.001 **** p < 0.0001. Examples

[0378] Example 1- Successful drug induction of senescence in endothelial cells (HUVEC) and fibroblasts

[0379] Human primary endothelial cells, HUVEC, and human primary fibroblasts were subjected to doxorubicin (DOXO) treatment, to induce senescence. Flow cytometry analysis of three markers on senescent HUVEC show elevation post DOXO treatment: (1) beta-galactosidase activity as measured by DDAO (Figure 1A, B); (2) pl6 protein levels (Figure 1C, D) and (3) cell size as indicated by forward scatter (Figure IE). Same analysis in DOXO treated fibroblasts demonstrated elevated (1) beta-galactosidase activity (Figure 2A, B) and (2) cell size (Figure 2C), in comparison to the young, untreated cells. These data show DOXO treatment of HUVEC and fibroblasts induces senescence conforming to accepted markers.

[0380] Example 2 - Confirmation of MSC senescence upon prolonged culture expansion (replicative senescence) and after DOXO treatment

[0381] Human primary MSC isolated from either bone-marrow (BMMSC) or dental pulp (DP- MSC), were either culture expanded for various passages or treated with DOXO for senescence induction and were subjected to senescence assessment (Figure 3). Flow cytometric analysis of four markers of senescence demonstrated an increase in beta-galactosidase activity (Figure 3 A) as well as in pl6 (Figure 3B) and yH2AX (Figure 3C) protein levels but a decrease in ki-67 (Figure 3D) protein levels in both replicative- and induced-senescence. To further confirm the establishment of senescence upon replicative- and induced-senescence of MSC, we studied the secretion levels of IL-6 (Figure 4A), IL-8 (Figure 4B) and IGFBP-2 (Figure 4C), which are all part of the senescence-associated secretory phenotype (SASP), with Luminex technology. These secreted proteins all show significant elevation upon prolonged expansion and DOXO treatment (Figure 4). All the above changes are in accordance with the literature and clearly demonstrate senescence establishment (Adeolu et al., 2020).

[0382] Example 3 - Cell surface binding by lectins differentiates senescent cells from young cells.

[0383] Having established confidence in the drug-induced and replicative senescence models, the inventors applied a panel of lectins targeting specific terminal carbohydrate residues by flow cytometry to detect cell surface binding differences between senescent and young cells. The inventors demonstrate that for three different cell types, HUVEC, fibroblasts and MSC. One lectin, UEA-I, while showing weak binding for BMMSC and fibroblasts, show strong binding on young HUVEC cells (Figure 5 and 6). By contrast to other lectins, UEA-I exhibit reduced binding upon DOXO-treated HUVECs (Figure 5 and 6). Both AAL and UEA-I bind fucose residues, but differ in the linkage between fucose and the underlying carbohydrate residue. It is interesting, how drastically different UEA-I binding is between the cell types while AAL is binding consistently. It is very significant that UEA-I declines rather than increases upon senescence in HUVEC cells.

[0384] Replicative senescence for DPMSC induces a similar profile of lectin binding changes, where MAL-II, SNA, GNA, UEA-I, STL and SJA all exhibit increased binding with increased passages (Figure 7 and 8). UEA-I binds weakly but also increases upon DOXO treatment on DPMSCs (Figure 7 and 8). DOXO treatment of DPMSC in comparison produces the same lectin changes, apart from AAL binding which consistently goes down (Figure 9 and 10). Finally, DOXO treatment of DPMSC increases NPL binding (Figure 9 and 10).

[0385] These data present a consistent trend of increased lectin binding in MAL-II, SNA, NPL, GNA, AAL, STL, SJA and PNA upon replicative senescence. In induced senescence, the binding of all the above lectins is consistent, except AAL which significantly drops after DOXO treatment in DPMSC. UEA-I is showing weak binding to fibroblasts and BMMSCs but changes upon senescence for DPMSC and HUVECs.

[0386] Example 4 - Purification of young or senescent cells based on their lectin content

[0387] Following the study of lectin binding in different cell types upon senescence, the inventors selected the following five lectins to attempt to enrich young cells from a 1 : 1 ratio of young and DOXO treated cells: AAL, NPL, MAL-II, GNA and SNA (Figure 11). These lectins were used independently or as a mixture to isolate cells using magnetic affinity beads (Figure 11). The inventors achieved enrichment of young cells to 150% compared to the old cells in the flow-through population for HUVEC (Figure 11A). Over 300% enrichment was seen for fibroblasts (Figure 11B) and 200% of BMMSC (Figure 11C). These data show that lectins independently or as a mixture can enrich young cells from a mixed population. References

[0388] Adeolu et al., (2020). Multiparameter flow cytometric detection and quantification of senescent cells in vitro. Biogerontology. 21(6):773-786 (PMID: 32776262).

Claims

Claims1. A method of detecting senescent cells and / or vesicles thereof in a test sample, the method comprising: providing a test sample (e.g. obtained from a subject); determining, in the test sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues, wherein a difference in the level of display of the glycan in the test sample relative to a level of display of the glycan detected in a reference sample is indicative that the test sample comprises one or more senescent cells and / or a vesicles thereof.

2. A method of detecting the presence or absence of senescent cells and / or vesicles thereof in a sample, the method comprising: providing a sample (e.g. obtained from a subject); determining, in the sample, the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues; comparing the level of display of the glycan in the sample with a reference value; anddetecting the presence of apoptotic cells and / or vesicles thereof, or detecting the absence of apoptotic cells and / or vesicles in the sample when based on the comparison.

3. A kit for identifying senescent cells and / or vesicles thereof, or for diagnosing a senescence-related disease, the kit comprising: a probe for a glycan; wherein the glycan comprises one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues, wherein the probe of the kit is used to detect, in a test sample, a difference in the level of display of one or more glycans relative to a level of display of the glycans detected in a reference sample, and thus identify senescent cells and / or vesicles thereof, or help diagnose a senescence-related disease.

4. The method of claim 1 or claim 2, or the kit of claim 3, wherein the difference in the level of display of the glycan in the test sample relative to the level of display of the glycan detected in the reference sample may be a higher level of display in the test sample or a lower level of display in the test sample.

5. The method of claim 2 or claim 4, wherein:(i) the ‘presence’ of senescent cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, a terminal mannose and a terminal N-acetylglucosamine is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linkedsialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N- acetylglucosamine is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N- acetylglucosamine is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of a terminal alpha-2, 3-linked sialic acid, a terminal alpha-2, 6-linked sialic acid, terminal mannose and terminal N- acetylglucosamine is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

6. The method any one of claims 2 or claim 4-5, wherein:(i) the ‘presence’ of senescent endothelial cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or Tower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent endothelial cells (and / or vesicles thereof) may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘higher’ whencompared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

7. The method any one of claims 2 or claim 4-6, wherein:(i) the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

8. The method any one of claims 2 or claim 4-7, wherein:(i) the ‘presence’ of senescent endothelial cells, senescent connective tissue cells and / or senescent BMMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc) residue is ‘higher’ when comparedwith the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent endothelial cells, senescent connective tissue cells and / or senescent BMMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent endothelial cells, senescent connective tissue cells and / or senescent BMMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent endothelial cells, senescent connective tissue cells and / or senescent BMMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc) residue is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

9. The method any one of claims 2 or claim 4-8, wherein:(i) the ‘presence’ of senescent DPMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent DPMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is‘the same’ or ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent DPMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘the same’ or ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent DPMSC cells (and / or vesicles thereof) is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc) residue is ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

10. The method any one of claims 2 or claim 4-9, wherein:(i) the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof may be detected when the levelof extracellular and / or extravesicular surface display of a glycan comprising a terminal N-acetylgalactosamine residue is ‘lower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

11. The method any one of claims 2 or claim 4-10, wherein:(i) the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof may be detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal galactose residue is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

12. The method of any one of claims 1-2 or 4-11, further comprising the step of recording on a suitable data carrier, data obtained in the step of determining the level of extracellular and / or extravesicular surface display of a glycan in the sample.

13. A method of enriching or removing non-senescent cells or senescent cells from a test sample, the method comprising: contacting the test sample, which comprises a mixture of non-senescent cells and senescent cells and / or vesicles thereof, with one or more probes for one or more glycans; and enriching for the non-senescent or the senescent cells using the probe, wherein the probe is specific for residues on the glycan selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues.

14. The method according to claim 13, wherein the method is repeated multiple times on a single sample.

15. The method according to claim 13 or claim 14, wherein the method is repeated twice, three times or four times on a single sample.

16. A kit for enriching or removing senescent cells or non-senescent cells from a sample, the kit comprising: a separation means for separating non-senescent cells from senescent cells and / or vesicles thereof; and a probe for a glycan; wherein the probe is directed to glycans comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues; wherein the probe is used to detect the presence of senescent cells and / or vesicles thereof that extracellularly and / or extravesicularly display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (non-senescent) cells from bound (senescent) cells and / or vesicles thereof, thereby enriching the non-senescent cells and / or vesicles thereof within the sample, or wherein the probe is used to detect the presence of non-senescent cells and / or vesicles thereof that extracellularly and extravesicularly display on their surface a glycan comprising residues selected from the group consisting of (i) to (vii), and the separation means uses the probe to separate unbound (senescent) cells and / or vesicles thereof from bound (non-senescent) cells and / or vesicles thereof, thereby enriching the senescent and / or vesicles thereof cells within the sample.

17. The method or kit according to any one of claims 13-16, wherein the probe binds to senescent cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed terminal alpha-2, 3-linked sialic acid, terminal alpha-2, 6-linked sialic acid, terminal mannose and / or terminal N- acetylglucosamine, optionally wherein the probe is selected from the list consisting of MAL-II, SNA, NPL, GNA, and STL; the probe binds to non-senescent endothelial cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed terminal a(1^2) linked fucose linked to a galactose, optionally where the probe is UEA-1; the probe binds to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably DPMSC cells) and / or vesicles thereof by binding extracellular (and / or extravesicular) displayed terminal a(1^2) linked fucose linked to a galactose, optionally wherein the probe is UEA-1; the probe binds to senescent endothelial cells, senescent connective tissue cells and / or senescent BMMSC cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed terminal a Fuc a( 1— >6) GlcNAc, a Fuc a( 1— >2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a( 1— >4) GlcNAc (preferably a Fuc a( 1— >6) GlcNAc), optionally wherein the probe is AAL; the probe binds to senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof by binding extracellular (and / or extravesicular) displayedterminal a(1^2) linked fucose linked to a galactose, optionally wherein the probe is UEA-1; the probe binds to non-senescent DPMSC cells (and / or vesicles thereof) by binding extracellular (and / or extravesicular) displayed terminal a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a(1^6) GlcNAc), optionally wherein the probe is AAL; the probe binds to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells) and / or vesicles thereof by binding extracellular (and / or extravesicular) displayed terminal N-acetylgalactosamine, optionally wherein the probe is SJA; and / or the probe binds to senescent stem cells, such as mesenchymal stem cells (e.g. DPMSC and / or BMMSC cells, preferably BMMSC cells) and / or vesicles thereof by binding extracellular (and / or extravesicular) displayed terminal galactose, optionally wherein the probe is PNA.

18. A method of diagnosing a senescence-related disease or disorder in a subject, the method comprising: detecting, in the sample, extracellular and / or extravesicular surface display of a glycan comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues, wherein detection, in the sample, of a glycan comprising terminal residues selected from the group consisting of (i) to (vii) is indicative that the subject has a senescence-related disease or disorder.

19. The method of claim 18, wherein the senescence-related disease or disorder is one or more selected from aging, sarcopenia, cancer, cachexia, COPD, osteoporosis, frailty, a cardiovascular disease, osteoarthritis, pulmonary fibrosis, renal diseases,neurodegenerative diseases (e.g., Alzheimer’s disease), hepatic steatosis, metabolic dysfunction, and macular degeneration, or others.

20. A method of treating or preventing a disease or disorder in a subject, the method comprising: enriching non-senescent cells and / or vesicles thereof in a sample comprising a mixture of senescent and non-senescent cells and / or vesicles thereof using the method of any one of claims 13 to 15 or 17, or the kit according to claim 16 or 17; and administering the enriched sample of non-senescent cells and / or vesicles thereof to the subject to treat the disease or disorder.

21. A senescent cell probe-agent conjugate, the conjugate comprising: a probe for targeting and / or binding to a glycan comprising one or more residues selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues, conjugated to an agent.

22. A senescent cell probe-agent conjugate according to claim 21, for use as a medicament.

23. A senescent cell probe-agent conjugate according to claim 21 for use in the prevention or treatment of a disease or disorder of a subject.

24. A method of preventing or treating a disease or disorder of a subject, the method comprising administering a senescent cell probe-agent conjugate according to claim 21 to the subject.

25. The method according to claim 24, wherein the disease or disorder is one or more selected from the group consisting of: aging, sarcopenia, cancer, cachexia, COPD,osteoporosis, frailty, a cardiovascular disease, osteoarthritis, pulmonary fibrosis, renal diseases, neurodegenerative diseases (e.g., Alzheimer’s disease), hepatic steatosis, metabolic dysfunction, and macular degeneration, or others.

26. The conjugate for use according to claim 22 or 23, or the method according to claim 24, wherein the agent is a therapeutic agent.

27. A method of killing senescent cells, the method comprising: determining the presence of a senescent cell in a subject using a method according to claim 1 or claim 2 or a kit according to claim 3; and administering, to the subject, a therapeutically effective amount of the probeagent conjugate according to claim 21.

28. The method according to claim 24, wherein the agent is a cytotoxic agent, such as a senolytic drug.

29. The method according to claim 26, wherein the method is used to kill senescent cells in vivo, in vitro or ex vivo.

30. Use of an extracellular or extravesicular surface displayed glycan as a senescent cell biomarker.

31. The use according to claim 30, wherein the glycan comprises one or more monosaccharide residues selected from the group consisting of (i) terminal sialic acid residues, (ii) terminal galactose residues, (iii) terminal mannose residues, (iv) terminal N-acetylgalactosamine residues, (v) terminal fucose residues, (vi) terminal N- acetylglucosamine residues, and (vii) internal N-acetyllactosamine.

32. A method of killing a live cell of a sample, the method comprising: contacting the sample comprising the live cell with a probe for a glycan in order to create a cell-bound probe; and contacting the cell-bound probe with an immune cell, thereby causing the immune cell to bind to the probe and kill the live cell, wherein the monosaccharide residues of the glycan comprise one or more residue(s) selected from the group consisting of:(i) terminal sialic acid residues,(ii) terminal galactose residues,(iii) terminal mannose residues,(iv) terminal N-acetylgalactosamine (GalNAc) residues,(v) terminal fucose residues,(vi) terminal N-acetylglucosamine (GlcNAc) residues, and(vii) internal N-acetyllactosamine residues.

33. Use of a probe according to claim 21 to kill a live cell in a sample.

34. Use of a lectin to detect senescent cells and / or vesicles thereof, enrich senescent cells and / or vesicles thereof, isolate senescent cells, and / or vesicles thereof remove senescent cells and / or vesicles thereof or target senescent cells and / or vesicles thereof.

35. The kit according to claim 3, 4, 16 or 17, the conjugate according to claim 21, the conjugate for use according to claim 22, 23, or 26, the method according to any one of claims 24 to 28 or 32, the use of claims 33 or 34, wherein the probe is a lectin.

36. The kit, conjugate, conjugate for use, method or use of claim 35, wherein the lectin is one or more selected from the group comprising MAL-II; SNA; UEA-I; STU; NPU; AAU; GNA; PNA; SJA; any other lectin that recognises the same glycan as one or more of MAU-II, SNA, UEA-I, STL, NPL, AAL, GNA, PNA, and SJA; and combinations thereof.

37. The method, kit, conjugate, probe, use or method of any preceding claim wherein the sialic acid residues are alpha linked sialic acid residues.

38. The method, kit, conjugate, probe, use or method of claim 37, wherein the alpha linked sialic acid residues are alpha-2, 3-linked sialic acids,39. The method, kit, conjugate, probe, use or method of claim 38, wherein the alpha- 2, 3-linked sialic acids are NeuAc a2-3 Gal b 1-3 GalNAc residues.

40. The method, kit, conjugate, probe, use or method of claim 37, wherein the alpha linked sialic acid residues are alpha-2, 6-linked sialic acids.

41. The method, kit, conjugate, probe, use or method of claim 40, wherein the alpha- 2,6-linked sialic acid residues are NeuAc a2-6 Gal bl -4 GlcNAc.

42. The method, kit, conjugate, probe, use or method of any preceding claim wherein the galactose residues are Fuc al-2(Gal bl -3 GalNAc al -3) Gal bl -4 GlcNAc or Fuc al-3( Fuc al-2( Gal al-3) Gal b 1-4) GlcNAc.

43. The method, kit, conjugate, probe, use or method of any preceding claim wherein the mannose residues are alpha linked mannose residues, such as alpha- 1,6-linked mannose residue or an alpha-1, 3-linked mannose residue which can optionally be Man3GlcNAc2 or Man5GlcNAc2.

44. The method, kit, conjugate, probe, use or method of any preceding claim wherein the N-acetylgalactosamine residues are GalNAc bl -4 GlcNAc bl -3 GalNAc bl -4 GlcNAc.

45. The method, kit, conjugate, probe, use or method of any preceding claim wherein the fucose residues are alpha-linked fucose residues, such as a(1^2) linked fucose linked to a galactose or a Fuc a(1^6) GlcNAc, a Fuc a(1^2) GlcNAc, a Fuc a(l— >3) GlcNAc, and / or a Fuc a(1^4) GlcNAc (preferably a Fuc a( 1^6) GlcNAc).

46. The method, kit, conjugate, probe, use or method of any preceding claim wherein the N-acetylglucosamine residues are Chitin or (GlcNAcjs.

47. The method, kit, conjugate, probe, use or method of any preceding claim wherein the internal N-acetyllactosamine residues are referred to as a disaccharide having of galactose (Gal) and N-acetylglucosamine (GlcNAc) linked by P-1,4 bonds.

48. The method of claim 18 or claim 19, further comprising administering an enriched sample of non-senescent cells to the subject to treat the disease or disorder and / or administering a probe-agent conjugate according to claim 21 to the subject to treat the disease or disorder.

49. The method of claim 1, 2, 4-12 or any one of claims 18-19, wherein detecting comprises contacting the test sample with one or more probes that bind the glycan.

50. The method of claim 1, 2-12 or any one of claims 18-19, wherein detecting is performed using by flow cytometry.

51. The method of any one of claims 2, 4-12, 37-47 or 49-50, wherein:(i) the ‘presence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose residue is ‘higher’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(ii) the ‘absence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose residue is ‘the same’ or Tower’ when compared with the level of display in a reference sample that does ‘not’ comprise a senescent cell (or vesicle thereof);(iii) the ‘presence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose residue is ‘the same’ or ‘higher’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof); or(iv) the ‘absence’ of senescent connective tissue cells (such as fibroblasts) and / or vesicles thereof is detected when the level of extracellular and / or extravesicular surface display of a glycan comprising a terminal a(1^2) linked fucose linked to a galactose residue is Tower’ when compared with the level of display in a reference sample that ‘does’ comprise a senescent cell (or vesicle thereof).

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