WO2025242912A1 - Conjugates for mass spectrometry imaging - Google Patents

Abstract

The present invention pertains to the provision of conjugates for the detection of target molecules by using mass spectrometry imaging. In particular, disclosed herein a particularly innovative conjugate for the detection of target molecules that is usable in the four following techniques of MSI: MALDI-MSI, DESI-MSI, nano-DESI- MSI and SpiderMass (MSI).

Description

Description

Conjugates for Mass Spectrometry Imaging

Technical Field

[1] The present invention pertains to the provision of conjugates for the detection of target molecules by using mass spectrometry imaging (MSI).

Background Art

[2] Mass spectrometry imaging (MSI) is a powerful technique for visualizing the spatial distribution of molecules, including endogenous ones such as metabolites, lipids, peptides, or proteins and biomarkers. In MSI the molecules are visualized by their molecular weight through the measurement of their mass to charge (m/z) ratio across the entire tissue. MSI provides valuable information on the molecular composition of cells and tissues, thus revealing molecular alterations associated to pathophysiological changes occurring in diseases, such as cancer, neurodegenerative diseases, metabolic diseases, infections... MSI is also useful for studying the distribution and metabolism of drugs and their metabolites in biological samples or screen for the presence of xenobiotics in tissues in relation to exposome.

[3] MSI is based on the principle of mass spectrometry (MS), which is a method of identifying and quantifying molecules. MS instruments comprise at least a source to turn the analytes into gas phase ions, a mass analyzer which separates the ions according to their m/z and a detection system. Not all MS sources can be used to perform MSI. Moreover, each MSI compatible source shows its own characteristics in terms of molecules that can be studied, spatial resolution attainable and has its own specificity in terms of sample preparation. These differences are inherently linked to the process used to turn the molecules into gas phase ions in relation to their physicochemical properties. Among the different ion sources which can be used for MSI, the first that was introduced is Secondary Ion Mass Spectrometry (SIMS). However, among the most popular nowadays are Matrix-Assisted Laser Desorption/lonization (MALDI), Desorption Electrospray Ionization (DESI) and Nanospray Desorption Electrospray Ionization (nano-DESI). Alternatively, novel ambient ionization methods such as SpiderMass have shown to enable MSI.

[4] MALDI-MSI (matrix-assisted laser desorption/ionization mass spectrometry imaging) uses a MALDI (matrix-assisted laser desorption/ionization) ion source in which a laser beam promotes a desorption/ionization mechanism thanks to energy absorption by the MALDI matrix present in large excess in the system. The MALDI matrix is a small organic molecule that absorbs at the laser wavelength, ensuring an efficient energy transfer into the system while preventing the analytes from extensive fragmentation. MALDI-MSI can detect and identify both small and large molecules, such as proteins, peptides, lipids, glycans, nucleic acids (DNA, RNA), drugs, toxins and other metabolites, and has a spatial resolution which can reach down to 0.5 micrometers.

[5] DESI-MSI (desorption electrospray ionization mass spectrometry imaging) is based on DESI and uses spray of charged droplets of solvents produced by an electrospray mechanism to extract molecules from the sample surface and get them ionized thanks to a desolvation mechanisms similar to electrospray ionization (ESI). DESI-MSI is well suited for small molecules, such as metabolites and lipids but detection of peptides and proteins was also demonstrated, and can achieve a spatial resolution down to 20 pm and even less.

[6] Nano-DESI-MSI (nanospray desorption electrospray ionization mass spectrometry imaging) uses ambient ionization technique based on localized liquid extraction. It allows sensitive and quantitative analysis of molecules directly from a sample surface without requiring extensive sample preparation as for conventional DESL In Nano-DESI, analytes are desorbed from a sampling surface into a liquid bridge formed between two capillaries. This technique improves spatial resolution in mass spectrometry imaging applications, typically down to 10 pm or less.

[7] Recently, SpiderMass (MSI), an emerging ambient ionization laser-based mass spectrometry technique, which is mini-invasive and designed for in vivo real-time molecular analysis, has been described (WO2016046748, Fatou, B., Saudemont, P., Leblanc, E. et al. In vivo Real-Time Mass Spectrometry for Guided Surgery Application. Sci Rep 6, 25919 (2016). https://doi.org/10.1038/srep25919). SpiderMass (MSI) can detect and identify various types of molecules, such as proteins, peptides, metabolites, or lipids, and has so far been demonstrated for spatial resolution down to less than a hundred micrometers (50 pm). SpiderMass (MSI) can also provide the topography image of the sample surface and plot the molecular data directly to the 3D topographical image without the need for image fusion.

[8] These several complementary techniques of MSI exist and are very useful for detecting and mapping target molecules. Yet, for each technique a conjugate that is usable in the specific technique has to be synthesized, which is time-consuming.

Summary

[9] The inventors have developed a particularly innovative conjugate for the detection of target molecules that is usable in the four following techniques of MSI: MALDI-MSI, DESI-MSI, nano-DESI- MSI and SpiderMass.

[10] The first object of the invention is thus a conjugate of formula (A-Ue-(Li )f-v_g-B-Wh-(L2)i-Xj-)_nC, wherein

- A is a tag of known mass,

- u, v, w, x are linkers,

- Li and L2 are spacer arms,

- B is a cleavable o-nitrobenzyl moiety,

- n is an integer of 1 to 25,

- e, f, g, h, i and j are independently selected from 0 or 1 , and

- C is a binding molecule that binds to a target molecule.

[11] The invention also pertains to a method for detecting a target molecule in a tissue section, wherein said method comprises: a) providing a tissue section; b) contacting said tissue section with a conjugate according to the invention; c) cleaving the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A; d) detecting the tag A released by mass spectrometry imaging; wherein said mass spectrometric imaging is MALDI-MSI, DESI-MSI, nano-DESI-MSI or SpiderMass.

[12] This imaging method based on the conjugate according to the invention is suited for the detection of any target for which a binding molecule can be designed such as among other proteins, miRNA, ncRNA, to lipids, small molecules and drugs. This method presents i) remarkable specificity, ii) remarkable sensitivity and Hi) high multiplexing capabilities while remaining flexible and cost- effective since it does not require the acquisition of a specific mass spectrometry instrumentation.

Detailed Description

[13] Mass Spectrometry Imaging (MSI) is a technology with which the skilled person is completely familiar (see for review Buchberger et al., Analytical chemistry 90.1 (2018): 240). MSI techniques can vary depending on the ionization source used. Most common ionization technologies in the field of MSI are DESI (desorption electrospray ionization)-MSI, nano-DESI (nanospray desorption electrospray ionization)-MSI and MALDI (matrix-assisted laser desorption ionization)-MSI.

[14] Recently, another technique of MSI called “SpiderMass” has been developed (WO2016046748, Fatou, B., Saudemont, P., Leblanc, E. et al. In vivo Real-Time Mass Spectrometry for Guided Surgery Application. Sci Rep 6, 25919 (2016). https://doi.org/10.1038/srep25919).

[15] The first object of the invention is a conjugate of formula (A-Ue-(Li )f-v_g-B-Wh-(L2)i-Xj-)_nC, wherein

- A is a tag,

- u, v, w, x are linkers,

- Li and L2 are spacer arms,

- B is a cleavable o-nitrobenzyl moiety,

- n is an integer of 1 to 25, preferably of 2 to 25,

- e, f, g, h, i and j are independently selected from 0 or 1 , and

- C is a binding molecule that binds to a target molecule.

[16] According to the invention, a tag refers to a molecule of known molecular weight that is detectable by mass spectrometry imaging using MALDI-MSI, DESI-MSI, nano-DESI-MSI and SpiderMass.

[17] The skilled person is completely familiar with the synthesis of tags suitable for detection by MSI. The skilled person knows how to select the most appropriate tag depending on the mass spectrometry technology used. For instance, when MALDI mass spectrometry is used, tags preferably have a m/z <5000 u. Alternatively, when DESI is used, tags preferably have a m/z <2000 u. Alternatively, when SpiderMass is used, tags preferably have a m/z ratio <5000 u. [18] According to an embodiment, in the conjugate of the invention the tag A is selected from any molecules known to presents a good MS detection with the according ion source i.e. a molecule with heteroatoms in the composition and chemical groups such as amines, amides, carboxylic acids, alcohols, phosphates, sulfates, iminium. Thus, this includes peptides, amino-acids, nucleic acids, guanidinium derivatives, sugars, polymers, lipids, metabolites and fluorophores such as cyanines or rhodamines and their derivatives thereof. In particular, arginine and its derivatives thereof can be advantageously used as tags.

[19] Arginine derivatives that can be used as tags are for example L-Arginine alkyl ester di hydrochlorides, in particular L-Arginine methyl ester di hydrochlorides, L-Arginine ethyl ester di hydrochlorides, L-Arginine isopropyl ester dihydrochlorides, L-Arginine butanyl ester dihydrochlorides or L-Arginine isoamyl ester dihydrochlorides.

[20] Alternatively, instead of arginine derivatives, dipeptide or tripeptide derivatives can be used as tags (see Figure 1 ).

[21] According to a particular embodiment, isotopic forms of the tags can be used, in particular deuterated compounds, or ¹³C or ¹⁵N isotopic forms of tags. For example, isotopic forms of arginine and arginine derivatives can be used as tags. For example, isotopic forms of dipeptide and tripeptide derivatives can be used as tags.

[22] In one embodiment, the u, v, w, x linkers comprise ester, amide, phosphate, ether, amine, carbonyl, succinimide, thiourea, carbamate or carbonate moieties and derivatives thereof.

[23] In the context of the invention, the term “ester” refers to a compound that carries a functional group of formula -COOR. The term “amide” refers to a compound that carries a functional group of formula -CONRR’. The term “phosphate” refers to a compound that carries a functional group of formula -PC>3(OH). The term “ether” refers to a compound of formula R-O-R’. The term “amine” refers to a compound of formula R-NR’-R”. The term “carbonyl” refers to a compound that carries a functional group of formula -C=O. The term “succinimide” refers to a compound that carries a cyclic functional group of formula (CH2)2(CO)2NR. The term “thiourea” refers to a compound that carries a functional group of formula RHN-C(=S)-NHR’. The term “carbamate” refers to a compound that carries a functional group of formula -O-C(=O)-NHR. The term “carbonate” refers to a compound that carries a functional group of formula -O-C(=O)-O.

[24] In one embodiment, the Li , L2 spacer arms comprise a linear or branched C2 to C20 alkyl chain, or a polyethylene glycol (PEG) chain with a number m of repeating units from 1 to 20, preferably of 1 to 12, said PEG chains being optionally substituted with hydrophilic substituents such as alcohols or sulfonates. The L2 spacer arm can comprise a triazole moiety or a diazine moiety which can be advantageously synthesized by click chemistry or biorthogonal chemistry.

[25] In the context of the invention, the term “alkyl” refers to C1-20 linear (i.e., "straight-chain"), branched, or cyclic, saturated hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, groups. The term “polyethylene glycol (PEG) chain” refers to a polyether compound with a structure commonly expressed as R-(O-CH2-CH2)m-R’, m being the number of repeating units. The term “alcohol” refers to a compound that carries an hydroxyl group (-OH) bound to a saturated carbon atom. The term “sulfonate” refers to a compound that carries a functional group of formula -SOa-.

[26] According to an embodiment, the cleavable o-nitrobenzyl moiety B can be substituted on the benzylic position and/or at least one aromatic position. In particular, the benzylic position of the o- nitrobenzyl moiety can be substituted by an alkyl, such as a methyl. Aromatic positions of the o- nitrobenzyl moiety can be substituted by an O-Alkyl group such as a methoxy or an ethoxy group.

[27] According to an embodiment, in the conjugate of the invention the binding molecule C is chosen in the group consisting of lectins, aptamers, antibodies and derivatives thereof, such as antibodies fragments, or nanobodies, nucleic acids, in particular oligonucleotides, peptides, proteins, in particular receptors, ligands, enzymes, antibodies and derivatives thereof, such as antibodies fragments, or nanobodies, antigens and organic compounds.

[28] According to an embodiment, in the conjugate of the invention the binding molecule C may be a lectin, an aptamer, an antibody or an oligonucleotide.

[29] According to an embodiment, the binding molecule C is an antibody. The term antibody as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses whole antibody molecules such as four-chain antibodies comprising 2 heavy chains and 2 light chains, such as polyclonal antibodies, monoclonal antibodies or recombinant antibodies. It may also refer to derivatives such as antibodies fragments or nanobodies.

[30] According to an embodiment, the binding molecule C is an aptamer. The term aptamer as used herein refers to a short, single-stranded DNA or RNA sequence that can bind selectively to a specific target molecule. The use of aptamer makes it possible to target among others, drugs, peptide molecules, proteins, proteins complexes, proteins interactomes or DNA-protein interactomes, RNA- protein, lipids, free or associated glycans, volatile elements, metabolites, mRNAs, circulating tumor cells, non-coding RNAs and ghost proteins. In this embodiment, in order to obtain the conjugate of the invention, the aptamer may bear a reactive tail consisting of an assemblage of multiple reactive monomers. These monomers are reactive as they bear reactive chemical function for click-chemistry or bio-orthogonal reactions such as Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) or Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC) or IEDDA (Inverse Electron-Demand Diels- Alder). In particular, these reactive monomers may be modified amino acids, more particularly Lysine; natural or modified nucleosides/nucleotides, more particularly uridine nucleotides, thymine nucleotides or adenine nucleotides; or other synthetic monomers, more particularly modified peptidic nucleic acids.

[31] According to the invention, a metabolite refers to an intermediate or end product of metabolism.

[32] In a particular embodiment, when the binding molecule, such as an aptamer, has been modified by an enzyme to bear a reactive tail, then the enzymatic reaction can have produced a mixture of binding molecules bearing tails of different lengths. In that case, the number n in the formulas of the invention must be understood as a number average n.

[33] According to the invention, a target molecule means a molecule of interest that is capable to specifically bind to another molecule, which is referred to as a "binding molecule". Such tandem target/binding molecules are very well known by the skilled person - W02007000669A2.

[34] According to an embodiment, the target molecule is chosen in the group consisting of drugs, volatile elements, metabolites, lipids, free or associated glycans, peptides, proteins and alternative proteins, proteins complexes, proteins interactomes or DNA-protein interactomes, RNA-protein, mRNAs, miRNA, non-coding RNAs, circulating tumor cells and extracellular vehicles. [35] According to an embodiment, the conjugate can be selected from

wherein the binding molecule is the binding molecule C as previously defined.

[36] According to an embodiment, the conjugate can be selected from

[37] Another object of the invention is a reaction intermediate of formula A-u_e-(Li )f-v_g-B-Wh -L3-N3, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- e, f, g and h are independently selected from 0 or 1 ,

- B is a cleavable o-nitrobenzyl moiety, and

- N3 is an azide moiety,

A, u, v, w, e, f, g, h, Li and B being as previously defined.

[38] Another object of the invention is a reaction intermediate of formula A-u_e-(Li jf-Vg-B-Wh-Ls- Alkyne, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- e, f, g and h are independently selected from 0 or 1 , and

- B is a cleavable o-nitrobenzyl moiety,

A, u, v, w, e, f, g, h, Li , L3 and B being as previously defined.

[39] In the context of the invention, the term “alkyne” refers to an unsaturated hydrocarbon containing at least one carbon-carbon triple bond. [40] In one embodiment, the L3 spacer arm comprises a linear or branched C2 to C12 alkyl chain, or a polyethylene glycol (PEG) chain.

[41] The structure of the reaction intermediates of formula A-u_e-(Li )f-v_g-B-Wh-l_3-N3 or A-u_e-(Li)f-v_g-B- Wh-La-Alkyne makes them useful as building blocks for the synthesis of a conjugate of the invention of formula (A-Ue-(Li )f-v_g-B-Wh-L2-Xj-)_nC. Indeed, said conjugate can be synthesized via a Click Chemistry or Bioorthogonal Chemistry reaction, in particular via a Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) or a Strain-Promoted Azide-Alkyne Cycloaddition (SPAAC). These reactions have the major advantages of being highly efficient, selective and specific, while exhibiting fast kinetics in water in physiologically relevant conditions of temperature and pH. By reacting a reaction intermediate of formula A-u_e-(Li )f-v_g-B-Wh-L3-Ns with a C binding molecule derivatized with n alkyne moiety, a conjugate of formula (A-u_e-(Li)f-v_g-B-Wh-L2-Xj-)nC can be easily synthesized. In the same manner, by reacting a reaction intermediate of formula A-u_e-(Li)f-v_g-B-Wh-L3-Alkyne with a C binding molecule derivatized with n azide moiety, a conjugate of formula (A-u_e-(Li)f-v_g-B-Wh-L2-Xj-)_nC can be easily synthesized. Said (A-u_e-(Li)f-v_g-B-Wh-L2-Xj-)_nC conjugate would then bear a L2 spacer arm comprising a triazole moiety resulting from the cycloaddition reaction.

[42] Another object of the invention is a reaction intermediate of formula A-u_e-(Li )f-v_g-B-Wh-l_3- Tetrazine, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- e, f, g and h are independently selected from 0 or 1 , and

- B is a cleavable o-nitrobenzyl moiety,

A, u, v, w, e, f, g, h, Li , L3 and B being as previously defined.

[43] In the context of the invention, the term “tetrazine” refers to a compound comprising a sixmembered aromatic ring containing four nitrogen atoms of molecular formula C2H2N4.

[44] Another object of the invention is a reaction intermediate of formula A-u_e-(Li )f-v_g-B-Wh-l_3- Alkene, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- e, f, g and h are independently selected from 0 or 1 , and

- B is a cleavable o-nitrobenzyl moiety,

A, u, v, w, e, f, g, h, Li , L3 and B being as previously defined.

[45] In the context of the invention, the term “alkene” or “olefin” refers to an unsaturated hydrocarbon containing at least one carbon-carbon double bond.

[46] The structure of the reaction intermediates of formula A-u_e-(Li )f-v_g-B-Wh-l-3-Tetrazine or A-u_e- (L1 )f-v_g-B-Wh-L3-Alkene makes them useful as building blocks for the synthesis of a conjugate of the invention of formula (A-u_e-(Li )f-v_g-B-Wh-L2-Xj-)_nC. Indeed, said conjugate can be synthesized via a Bioorthogonal Chemistry reaction, in particular via an Inverse Electron-Demand Diels-Alder (IEDDA) reaction. These reactions have the major advantages of being highly efficient, selective and specific, while exhibiting fast kinetics in water in physiologically relevant conditions of temperature and pH. By reacting a reaction intermediate of formula A-u_e-(Li)f-v_g-B-Wh-L3-Tetrazine with a C binding molecule derivatized with n alkene moiety, a conjugate of formula (A-u_e-(Li )f-v_g-B-Wh-L2-x_r)_nC can be easily synthesized. In the same manner, by reacting a reaction intermediate of formula A-u_e-(Li)f-v_g- B-Wh-La-Alkenewith a C binding molecule derivatized with n tetrazine moiety, a conjugate of formula (A-u_e-(Li )f-v_g-B-Wh-L2-Xj-)_nC can be easily synthesized. Said (A-u_e-(Li)f-v_g-B-Wh-L2-Xj-)_nC conjugate would then bear a L2 spacer arm comprising a diazine moiety resulting from the IEDDA reaction.

[47] Another object of the invention is a method for detecting a target molecule in a tissue section, wherein said method comprises: a) providing a tissue section; b) contacting said tissue section with a conjugate according to the invention; c) cleaving the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A; d) detecting the tag A released by mass spectrometry imaging; wherein said mass spectrometric imaging is MALDI-MSI, DESI-MSI, nano-DESI-MSI or SpiderMass.

[48] According to the invention, a "tissue section" preferably has the following properties: it may be frozen or paraffin-embedded, its thickness is preferably in the order of a mammalian cell diameter, thus comprised between 3 and 20 pm. In the case of a frozen section that was obtained from a frozen tissue using a cryostat, OCT (optimal cutting temperature polymer) is preferably used only to fix the tissue, but the frozen tissue is not embedded in OCT, so that tissue sections were not brought into contact with OCT to avoid contamination by polymers.

[49] The inventors have developed a conjugate that comprises a o-nitrobenzyl cleavable moiety which makes the conjugate usable in MALDI-MSI, DESI-MSI, nano-DESI and SpiderMass for the detection of target molecules.

[50] The skilled person is completely familiar with MALDI-MSI technique (see for review: Tuck and al.; Front. Chem. 2022, 10, 904688 doi.org/10.3389/fchem.2022.904688).

[51] When using MALDI-MSI, at step c) of the method of the invention, the MALDI laser cleaves the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A. Typically, the MALDI Laser has a wavelength in the range from 200 nm to 500 nm, in particular in the range from 300 nm to 400 nm, more particularly 337 or 355 nm.

[52] According to an embodiment, MALDI-MSI is used in the method of the invention and the cleavable o-nitrobenzyl moiety B is cleaved by the MALDI Laser. [53] The skilled person is completely familiar with DESI-MSI technique (see for review: doi: 10.1021/acs. accounts.3c00382; doi: 10.1039/b925257f; doi.org/10.1016/j.ijms.2006.08.003 ; doi.org/10.1002/mas.21360).

[54] When using DESI-MSI, at step c) of the method of the invention, under acidic conditions the electrosprayed charged solvent droplets cleaves the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A. Typically, solvents for the electrospray may be Acetonitrile, Methanol, Isopropanol, Chloroform, Tetrahydrofuran, 1 ,4-dioxane or combinations of these solvents in various proportions. Those solvents or combinations thereof are mixed with varying amounts of water to achieve the desired acidity level.

[55] According to an embodiment, DESI-MSI is used in the method of the invention and the cleavable o-nitrobenzyl moiety B is cleaved by the electrospray under acidic conditions.

[56] The skilled person is completely familiar with nano-DESI-MSI technique (see for review: doi:10.1039/C0AN00312C).

[57] When using nano-DESI-MSI, at step c) of the method of the invention, under acidic conditions the nanospray capillary charged solvent droplets cleaves the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A. Typically, solvents for the nanospray capillary may be Acetonitrile, Methanol, Isopropanol, Chloroform, Tetrahydrofuran, 1 ,4-dioxane or combinations of these solvents in various proportions. Those solvents or combinations thereof are mixed with varying amounts of water to achieve the desired acidity level.

[58] According to an embodiment, nano-DESI-MSI is used in the method of the invention and the cleavable o-nitrobenzyl moiety B is cleaved by the nanospray under acidic conditions.

[59] Alternatively, according to an embodiment, the cleavable o-nitrobenzyl moiety B of the conjugate may be cleaved by a specific device prior to MALDI-MSI, DESI-MSI, or nano-DESI-MSI analysis. For example, the specific device may be a UV lamp having a wavelength in the range from 200 nm to 500 nm, in particular in the range from 300 nm to 400 nm, more particularly 365 nm. For example, the specific device may be a sprayer or micro-spotter using solvents such as Acetonitrile, Methanol, Isopropanol, Chloroform, Tetrahydrofuran, 1 ,4-dioxane or combinations of these solvents in various proportions. Those solvents or combinations thereof are mixed with varying amounts of water to achieve the desired acidity level.

[60] As mentioned above, recently, the SpiderMass technique, a mini-invasive IR laser-based MS technique designed for in vivo real-time molecular analysis, has been described (WO2016046748, Fatou, B., Saudemont, P., Leblanc, E. et al. In vivo Real-Time Mass Spectrometry for Guided Surgery Application. Sci Rep 6, 25919 (2016). https://doi.org/10.1038/srep25919). SpiderMass is a water- assisted laser desorption/ionization mass spectrometry (WALDI-MS) technique that enables in vivo and real-time analysis of biological tissues. It uses a fibered infrared laser that excites the water molecules in the tissue, resulting in the desorption and ionization of target molecules that are transferred to a mass spectrometer. [61] The detailed protocol for SpiderMass is described in the following publication: Ogrinc et al. Nat Protoc 14, 3162-3182 (2019). doi.org/10.1038/s41596-019-0217-8.

[62] When using SpiderMass, at step c) of the method of the invention, the SpiderMass Laser cleaves the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A. Typically, the SpiderMass Laser has a wavelength is the range from 2800 nm to 3100 nm, in particular 2900 nm to 3000 nm, more particularly 2940 nm. This range for the SpiderMass Laser is to excite the most intense vibrational band (O-H) of water. It is alternatively possible to perform cleavage by fragmentation in SpiderMass.

[63] According to an embodiment, SpiderMass is used in the method of the invention and the cleavable o-nitrobenzyl moiety B is cleaved by the SpiderMass Laser.

[64] The conjugate according to the invention can be used in SpiderMass, therefore it can be used in in vivo and real-time surface analyses of biological tissues.

[65] According to an embodiment, the method of the invention can be used in multiplexed mass spectrometric imaging of tissues.

[66] As described in W02007000669A2, in the context of a multiplexed mass spectrometric imaging of tissues, multiple target molecules can be detected in a single mass spectrometric imaging readout. In particular, more than 100 distinct target molecules may be mapped simultaneously in the same tissue section. In this context, the tissue section is placed into contact with multiple conjugates, each specific for one target molecule. The conjugates used in this context comprise distinct binding molecule (each specific for one of the target molecules) and distinct tags, thus displaying distinct molecular weights, to allow for the detection of several distinct target molecules. Using tag with widely dispersed molecular weights, it is thus possible to map simultaneously the expression of many distinct target molecules in the same tissue section. In particular embodiments, the detection encompasses at least 2, at least 3, at least 5, at least 8, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 75, or at least 100 target molecules.

[67] The method according to the invention may comprise the use of several conjugates wherein the tag A for MS detection has the same molecular weight but a different isotopic composition. The tags are differentiated at the time of fragmentation, as in the TMT/SILAC approach (DOI: 10.1002/mas.21709 ; dx.doi.org/10.1021/pr500880b).

[68] The method according to the present invention may be applied to biology- and clinics- related applications. It may be useful in target applications such as: pharmacokinetics, forensic, toxicology and animal/plant biology.

[69] The method according to the present invention may be adapted for MS Profiling, i.e. the detection of a target of interest in a region without necessarily making the image.

[70] The present invention will now be illustrated in view of the examples below. These examples are not to be construed as limiting the scope of the invention.

Description of figure [71] Figure 1 . : Structures of azides wherein the tag is an arginine derivative, a dipeptide derivative or a tripeptide derivative.

Examples

[72] Chemicals and Instruments

[73] PC Azido-NHS Ester is available from Clickchemistry Tools (reference CCT-1161 ). Water (H2O), ethanol (EtOH), acetic acid, dimethyl sulfoxide (DMSO), methanol (MeOH), ammonium bicarbonate, and 96-well ELISA plates (439454) were obtained from Thermo Fischer Scientific (Courtaboeuf, France). The 99% pure trifluoroacetic acid (TFA), HEPES, Tween 20, 5-DBCO-PEG4-dUTP linker and 2,5-dihydroxybenzoic acid (DHB) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Acetonitrile (ACN) with HPLC Plus grade was purchased Carlo ERBA Reagent. Isopropanol HPLC was purchased from VWR Chemicals. The tris used was supplied by Interchim, the PBS by Gibco and the milk powder by Regilait. The DBCO-PEG5-NHS ester linker was supplied by Click Chemistry Tools. The rtdt enzyme and 5X tdt buffer were purchased from Promega (Madison, Wl, USA).

[74] Thin layer chromatography was performed on MERCK silica gels, KIESELGEL 60 GF254 and revealed under UV light (254 nm and 365 nm). The different conjugates were purified using KIESELGEL 60 GF254 1.0 mm SIL G-100 I UV 254 pre-coated TLC plates. Proton NMR spectra were performed on a BRUKER AM 300 WB (at 300 MHz) using tetramethylsilane as internal reference. Tissues were cut using a CryoStat (Leica Microsystems, Nanterre, France). The ITO (Indium Tin Oxide) slides were purchased from LaserBio Labs (Valbonne, France), while the polylysine coated slides came from EprediaTM (Braunschweig, Germany). The matrix to perform MALDI MS imaging was deposited on tissue using an HTX M5-Sprayer™ (HTX Technologies, Carboro, NC). Conjugate cleavage was activated by exposure to UV light at 365 nm (LED Cube 100 IC from Honle UV Technology, Marlboro, MA). Mass spectrometry analyses were performed on a Rapiflex Tissuetyper MALDI TOF (Bruker Daltonics, Billerica, MA) equipped with a 3D Smart Beam laser.

[75] 1. Chemical synthesis

[76] Synthesis of Arginine Tags: Esterification of the carboxylic acid function.

[77] Arginine hydrochloride (1.0 eq) was dissolved in the alcohol of interest (0.01 M) before the solution was cooled to 0°C with an ice bath. A solution of thionyl chloride (2.0 eq) was added dropwise. The reaction mixture was stirred under reflux for 90 min until a yellow solution was obtained before being returned to room temperature for 16 hours. The solvent was removed in vacuo, and the crude reaction was transferred to 15mL of alcohol to repeat the procedure twice.

[78] The residue was taken up in 1 mL of methanol, and diethyl ether was added to the solution until it became cloudy. The mixture was kept at room temperature for 4-5 hours before being put under 4°C to complete the crystallisation. After removing the solvents, the arginine ester is obtained as a dihydrochloride salt (white solid).

[79] Table 1 : Arginine tags synthesised from the corresponding alcohol.

[80] Azide synthesis.

[81] In an inert atmosphere, 1.5 equivalents of L-Arginine alkyl ester dihydrochloride and one equivalent of PC Azido-NHS Ester were dissolved in anhydrous dichloromethane. The reaction mixture was stirred at room temperature and 3 equivalents of triethylamine were added dropwise. The reaction mixture was kept in the dark under stirring at room temperature for 18 hours.

[82] Excess L-Arginine alkyl ester dihydrochloride was filtered off and the solvent removed in vacuo. The triethylamine salts were removed by precipitation using cold acetone, then the reaction crude was purified by chromatography on silica with dichloromethane and methanol.

[83] Purification: DCM/MeOH (95/5). [84] Yellow oil.

[85] Table 2 : Azides synthesized from the corresponding arginine tags.

[86] Cleavage of the azides.

[87] The samples (liquids for ELISA experiments and tissue for immunolabelling) are exposed for at least 10 minutes under the UV lamp. [88] Data regarding sensitivity of the azides

[89] The inventors obtained data showing the sensitivity of the 10 MS reporters (T1-T10, see Table 2) which show similar signal indicating an improvement in terms of quantification (by comparison to Ambergen)

[90] The sensitivity of probes with closely related chemical formula (using stable isotope and various ester) is enhanced due to the intrinsic proximity of structural elements within the molecule. This arrangement facilitates efficient detection of the MS reporter after cleavage, thereby enabling the potential for quantification by mass spectrometry.

[91] Therefore, a correction factor could be applied, as is already done in fluorescence spectroscopy, particularly in FRET-based techniques or with internal reporter probes. [92] By analogy, the use of calibrated tags or internal isotopic standards could allow for more accurate quantification in MSI or targeted MS, even in complex biological contexts.

[93] The inventors also showed the detection of the MS reporter after chemical cleavage in acid condition of one of the Tag normally used for photocleavage.

[94] The photo-cleavable probes (azides) can also be cleaved under mild acidic conditions, notably in the presence of 10% TFA (trifluoroacetic acid), which is compatible with the solvents used for sample ionization via electrospray.

[95] This ionization technique is commonly applied in mass spectrometry systems coupled with DESI or nano-DESI sources operating directly on tissue.

[96] This demonstrates the chemical and analytical compatibility of these probes (azides) with ambient ionization mass spectrometry platforms, thus supporting their use in molecular imaging workflows under near-native conditions.

[97] 2. Preparation of molecular conjugates

[98] Antibodies

[99] Derivation of antibodies by DBCO-PEG5-NHS

[100] The antibody used for detection was derived in 40pL of PBS by adding 10pL of antibody at a concentration of 1 mg/mL and 2pL of a 50mM solution of DBCO-PEG5-NHS in DMSO. The reaction was incubated at 37°C overnight.

[101] For the indirect ELISA tests, the detection antibody used was the Polyclonal Rabbit AntiHuman IL-10 antibody from Peprotech (Catalog Number:500-P20).

[102] For multiplex MALDI immunohistochemistry imaging, the following detection antibodies were used:

Anti-Glial Fibrillary Acidic Protein Antibody, clone GA5 de chez merckmillipore (catalog num-ber : MAB360)

Anti-NeuN Antibody, clone A60 de chez merckmillipore (catalog number : MAB377)

Anti-Beta III Tubulin Antibody de chez merckmillipore (catalog number : MAB1637)

Human/Mouse/Rat MBP Antibody de chez bio-techne (catalog number : MAB42282-SP)

[103] Coupling of derived antibodies with azides by click chemistry

[104] From the derived antibody solution, 48pL of PBS and 2pL of 10mM azides in DMSO were added. Then, the reaction was incubated at room temperature overnight.

[105] Just before use, a neutralisation buffer is added to quench unreacted NHS-ester. To do this, TrisHCI buffer pH 8 is added to give a final concentration of 50 - 100 mM Tris. Incubate at room temperature for 5min.

[106] Verification of conjugate quality by SDS-Page Gel [107] The functionalization efficiency and click chemistry of the antibodies are checked on SDS- PAGE gels at 4% for the concentration gel and 12% for the separation gel according to the following protocol:

[108] Table 1 : production protocol for polyacrylamide concentration and separation gels

[109] Samples were prepared to the same concentration and placed in 1X Laemmli buffer before gel deposition. The ThermoScientific PageRuler Broad Protein Ladder was used as a molecular weight control. Migration was carried out in 1X TBS (Tris Buffered Saline) buffer at 70 V for 15 minutes, then at 120 V for 45 minutes. After reading the fluorescence, the gel was immediately stained with colloidal blue in a suitable container.

[110] Synthesis of final antibody-conjugate by click-chemistry

[111] The modification of the antibody by the NHS-PEG5-DBCO linker in the first instance and then the click chemistry of the azide on the DBCO in the second instance will induce a difference in mass between the different stages. These can be detected and monitored on a polyacrylamide gel with sufficient resolution to ensure that the reaction has taken place. A reading of the fluorescence emitted by the Alexa Fluor 488 tag will check its binding to the antibody.

[112] The antibody used in this study is a high molecular weight protein of approximately 150 kDa. This antibody was difficult to migrate on a polyacrylamide gel (denaturing, non-reducing) because its molecular weight and three-dimensional structure are too high. Bovine serum albumin (BSA) has a lower molecular weight of 67 kDa and 59 lysine residues, 30 to 35 of which are primary amines capable of reacting with the NHS ester. This protein can therefore replace antibodies and serve as a model for testing the effectiveness of tag modification. Indeed, according to the results, the inventors find the bands corresponding to the molecular weight of the unmodified BSA, the BSA activated by the linker and the BSA-linkerwith the fluorescent tag. There is a slight difference in mass between the unmodified BSA and the linker-tag BSA, indicating that the protein has been modified by several tags. It is necessary to confirm that the tag has been attached to the protein by fluorescence revelation of the gel where it can be seen that the BSA-linker-tag bands emit fluorescence, confirming that the tag has been attached to the protein.

[113] The streptavidin was also modified by attachment of a tag for use in the streptavidin-coupled antibody-biotin detection method. The results show a Coomassie Blue band at 50 kDa corresponding to the modified streptavidin. The gel shows fluorescence only at the deposit, corresponding to the click chemistry between the streptavidin-linker and the Alexa Fluor tag. [114] In conclusion, the interpretation of the polyacrylamide gel shows that the modifications made to the BSA by the NHS-PEG5-DBCO linker, and the tag worked well. As a result, this technique, under these operating conditions, can be applied to antibodies for use in an ELISA test.

[115] Aptamers

[116] Aptamers activation

[117] Functionalisation of the aptamer is an enzymatic reaction that must be carried out on the aptamer allowing 3'-OH elongation with a specific enzyme (rTdT) to chemically add activated nucleotides from dUTP-PEG4-DBCO.

[118] To do this, the aptamer is thawed, then heated at 95°C for 5 min and placed 15 min at room temperature (this step allows the aptamer to be conformed correctly).

[119] The following reaction mix was then prepared, in which 4 pL of 5X TdT, 5 pL of 1 mM dUTP- PEG4-DBCO in DMSO, 0.7 pL of rTdT (30 U/pL) and 40.3 pL of H20 were added to 2 pL of 10 pM aptamer. The mixture was incubated at 37°C for 30 minutes to obtain the poly U tail on our aptamer.

[120] Coupling of derived aptamers with azides by click chemistry

[121] 44pL of PBS and 4pL of 1 mM Tag in DMSO were added to the above reaction mix. The mixture was incubated overnight at room temperature.

[122] Checking conjugate quality using agarose gel

[123] The efficiency of aptamer elongation and click chemistry was checked on a 2% agarose gel. The samples were prepared in such a way as to deposit the same quantity and were placed in 1X Blue Silver buffer. 3 pL of 100 base pair mass control was deposited. The agarose gel was incubated for at least 10 minutes in a solution of Sybr Gold 1X before reading under UV in the Vilber Lourmat instrument using Gel Smart V7.5 software.

[124] Detection of the fluorescence emitted by the Alexa Fluor 488 tag is read directly on an AMERSHAM IMAGER 600 fluorescence reader.

[125] Gel control of aptamer modification

[126] In order to study the behavior of the aptamer with respect to elongation reactions, it was decided to first carry out tests with unmodified dNTPs. The bands visible on the agarose gel showed that elongation of the aptamer by the dNTPs was successful. In addition, the time taken for the elongation reaction influenced the number of nucleotides incorporated into the tail added to the aptamer, making it possible to control the size of the poly-nucleotide tail generated. At 5 minutes of reaction, no amplification was visible, at 30 minutes a band at 150 base pairs (bp) was observed, and a band at 200 bp at 60 minutes. However, when the aptamer was elongated by the dUTP-PEG4- DBCO nucleotide, it was found that the reaction time had no effect on the size of the poly-nucleotide tail synthesized. Indeed, the bands observed on the gel are all around 150 bp. This can be explained by reduced recognition of the dUTP-PEG4-DBCO modified nucleotide by the rTdT enzyme, or steric hindrance that blocks elongation. A second step was carried out by adding an Alexa Fluor 488 tag which binds to the DBCO group of the nucleotides added to the aptamer. The fluorescent tag was successfully attached to the modified aptamer, with the observation of fluorescent bands. This demonstrates the elongation and click chemistry of a tag on an aptamer. This conjugate aptamertag according to the invention can be used for various ELISA tests, MALDI imaging, DESI imaging, SpiderMass imaging, and nano-DESI imaging.

[127] Immuno-absorbent test

[128] Indirect ELISA

[129] The antigen was fixed to the bottom of the wells of an ELISA plate by the deposition of 50 pL of human IL-10 at 5 pg/mL, and the plate was incubated for 45 minutes at 37°C or overnight at 4°C. The wells were saturated by adding 100 pL of blocking buffer (PBS with 5% skim milk and 0.05% Tween-20) and incubation for 30 minutes at 37°C. The wells were emptied and 50 pL of anti-IL-10 primary antibody (produced in rabbit) at 20 pg/mL were added to each well and incubated for 30 minutes at 37°C. The secondary detection antibody reaction was performed by depositing 50 pL at 0.1 mg/mL of secondary antibody anti-rabbit (from goat) coupled to a guanidinium tag and incubating for 30 minutes at 37°C. The washing steps were repeated between each step and performed as follows the wells were emptied and washed three times with 150 pL of PBS buffer and 0.05% Tween 20. In the last step, the wells were rinsed three times with 150 pL of H2O and then 50 pL of H2O was added to each well before the UV irradiation step.

[130] Each sample was analysed by mass spectrometry to verify the presence or absence of the tag. To do this, 1 pL of each well was taken and deposited on a MALDI plate with a DHB matrix in MeOH:TFA 0.1% (70:30, v:v). Once the plate had been dried under vacuum, the spots were analysed by MALDI-TOF using the Rapiflex TissueTyper instrument in the positive ion mode, using a method parameterised for m/z range 160 to 1000, in positive mode with reflectron, 1000 shots I pixel with a spatial resolution of 60pm. The resulting MS spectra were recorded after each acquisition and analysed using FlexAnalysis (Bruker Daltonics, Billerica, MA) to access the mass measurement, intensity, and area of each peak.

[131] Demonstration that the addition of the Tags on the AB structure doesn’t modify its binding properties and allow quantification

[132] An indirect ELISA was performed to detect and quantification of interleukin-10 (IL-10). The primary antibody used was an antibody directed against IL-10, while the secondary antibody, an antirabbit goat which was functionalized with the Tags developed.

[133] This functionalization was carried out with the T2 probe (azide T2, see table 2), and with the T4 probe (azide T4, see table 2). The appearance of mass peaks corresponding to m/z 203.100 (T2) and 217.127 (T4) was observed in the experimental samples, while these signals were absent in the controls. This demonstrates the good sensitivity of the conjugates, as well as their effectiveness in detecting antibody-antigen interaction without impairing antibody recognition capabilities. Additionally, a dilution of the IL-10 antigen was performed in a 96 well-plate and the ELISA with the MS tags performed. By measuring the intensity of the MS reporter signal on the MS spectra it was possible to reconstruct a quantification curve demonstrating that the reporter signal is well proportional to the quantity of antigen in the well.

[134] Chemical synthesis of the aptamer-based conjugates : obtained results.

[135] Two aptamer-based conjugates were successfully synthesized. Azides T4 and T5 were coupled with aptamers via click-chemistry as described in the experimental section :

[136] Conjugate A: azide T4 + aptamer against IL-6

[137] Conjugate B: azide T5 + aptamer against IL-10

[138] 3. Multiplex immunohistochemistry imaging

[139] For this study, fresh-frozen rat brains were sectioned in a cryostat set at -20°C. Consecutive 12 pm sections were mounted onto ITO conductive slides for multiplex immunohistochemistry imaging by MALDI-TOF MS and on poly-lysine coated slide for DESI and SpiderMass MS Imaging. The slides were then stored at -80°C before use.

[140] The slides to be analysed underwent several tissue preparation steps, including thawing for 1 min in a desiccator, fixation with 2% PFA for 10 min at 4°C, and delipidation to make the protein sites more accessible. Delipidation was carried out by immersing the slides in a series of consecutive baths as follows: 30 sec EtOH 70%, 30 sec EtOH 100%, 2 min Carnoy (3 :6 :1 , v/v/v CHCI3:EtOH:Acetic Acid), 30 sec EtOH 100%, 30 sec H2O, and 30 sec EtOH 100%.

[141] The tissue was then dried under vacuum before a hydrophobic barrier was applied around the tissue. A blocking buffer (5% rat and rabbit serum, 2% BSA, 0.3% triton X-100 and PBS) was next applied to the tissue for a minimum of 2 hours at room temperature. The buffer was then aspirated and the primary antibody (antibody conjugated with the TAG, previously prepared) was applied at a concentration of 10 pg/mL in blocking buffer. During this step, the primary antibody can also be replaced by an aptamer conjugated with the TAG on the polyU tail at a concentration of 0.5 pM. After incubation overnight at 4°C in a humidity chamber, the antibody or aptamer solution was removed from the tissue before a series of washes. The slides were then immersed in two baths of PBS containing 0.1% Tween-20 for 1 minute, followed by three 5-minutes baths of miliQ water. The slides were then dried under vacuum for at least 1 hour.

[142] For the MALDI MS imaging, the slides were exposed to UV light at a wavelength of 365 nm for 20 min, to activate the photo-cleavage of the conjugates.

[143] A) MALDI IMAGING

[144] In order to carry out MALDI imaging, a 20 mg/mL DHB matrix, taken up in 0.1% MeOH:TFA (70:30, v/v), was sprayed onto the tissue using the HTX M5 automated system. The parameters used for matrix deposition included a spray temperature of 65°C, a plate temperature of 55°C, a pressure of 10 psi and a flow rate of 0.1 mL/min for 12 passes. The slides were then analysed using a MALDI TOF Rapiflex for MS Imaging. The MS spectra were obtained in positive reflectron mode, in the m/z 140-800 range, with 500 laser shots per pixel at a frequency of 5000 Hz, for a spatial resolution of 20 pm and a continuous scan of 20 pm. [145] The spectral data were processed using Fleximaging software (Bruker Daltonics, Billerica, MA), applying TIC normalisation. It was then possible to visualise the spatial distribution, individually or in multiplex, of each tag deposited on the tissue by selecting the corresponding masses.

[146] Results of MALDI-MS Imaging with one antibody directed against the GFAP and 5 different Tags from fresh frozen samples

[147] MALDI-Mass spectrometry imaging (MSI) was performed on a fresh frozen rat brain section, using an anti-GFAP antibody independently functionalized with 5 of the different azides (T1 , T2, T3, T4 and T5) to obtain 5 conjugates according to the invention. The antibody is directed against GFAP, a well-established astrocyte marker. It was observed that the antibody’s recognition capacity was preserved, as indicated by the consistent localization of the signal across each MSI image, regardless of the probe used.

[148] Images were obtained. All five images were imported into SCiLS Lab software to visualize and analyze the corresponding mass spectra. For each MSI image linked to a specific probe, the corresponding m/z signal was only detected in the image associated with that probe, and no detectable signal was present in the MALDI-MSI of the other probes.

[149] This confirms both the specificity of each conjugates according to the invention and the sufficient signal intensity, ensuring reliable detection without cross-reactivity

[150] Results of MALDI-MS Imaging with one antibody directed against the GFAP and 5 different Tags from FFPE tissues

[151] The same experiment was also conducted on FFPE (Formalin-Fixed, Paraffin-Embedded) tissues from rat brain which is the regular type of samples used in clinics.

[152] 5 conjugates according to the invention were synthesized with T6, T7, T8, T9 and T10 azides (table 2).

[153] The observations made on fresh frozen tissues were found to be transferable to paraffin- embedded sections, with specific MSI signals detected for each conjugate used. The signal is well found in the region where the astrocytes are known to be localized in the brain.

[154] This indicates that antibody functionalization remains effective on formalin-fixed tissues, despite a potential reduction in the accessibility of antigen recognition sites, which are typical limitations in human clinical samples. An antigen retrieval step was added in the sequence though as conventionally performed for IHC on FFPE tissues.

[155] Thus, the compatibility of this approach with archived pathological tissues is demonstrated, paving the way for translational and diagnostic applications using mass spectrometry imaging.

[156] Results of MALDI-MS MS Imaging in a 10-plex experiment from rat brain against 10 different targets (Tubulin, NeuroDI, Calnexin, Ncad, Ecad, Furin, GFAP, Synapsin, NeuN, Myelin) [157] 10 different synthesized tags were used to functionalize 10 different antibodies (Tubulin, NeuroDI , Calnexin, Ncad, Ecad, Furin, GFAP, Synapsin, NeuN, Myelin), each being linked to a distinct photocleavable tag to enable multiplexed detection. The inventors synthesized 10 different antibody-based conjugates according to the invention, from T1 , T2, T3, T4, T5, T6, T7, T8, T9 and T10 azides.

[158] This experiment was conducted on a single sagittal section of rat brain, demonstrating the compatibility of all probes within a single biological sample. Images were obtained. The absence of cross-signal interference between the specific m/z peaks corresponding to each probe confirms the feasibility of multiplex labeling in tissue contexts. The results obtained in a fresh frozen section demonstrate that the tags are shown individually on 10 different MALDI-MS images showing a specific localization for each of them.

[159] The superposition on one image of 3 markers at the same time (NCad, blue; GFAP, red; Calnexin, Green). Other images obtained show the same experiment performed from a FFPE rat brain section. Each Tag is presented on an individual MALDI MS image for the 10 different markers. Another image obtained shows the superposition on one image of 3 markers at the same time.

[160]

[161] 10-plex MALDI-IHC experiment with spatial resolution at the single cell (5 pm)

Another experiment (markers and tags except for calnexin) like the previous experiment was conducted. Yet the experiment was performed here on a TimsTof FleX instrument with a spatial resolution of images down to 5 pm which is single cell. This demonstrates that the high sensitivity of these probes enables data collection at single-cell resolution. This high-resolution capability highlights their potential for targeted molecular microscopy, particularly for detecting subcellular structures or rare cell populations.

[162]

[163] MALDI-IHC against Ki67 on FFPE tissue of a glioblastoma patient. Comparison of the clickable conjugates according to the invention to the Miralys from Ambergen.

[164] A comparative evaluation was performed between the conjugate according to the invention and the Ambergen Tags (commercialized under Myralis).

[165] The T6 azide (table 2) was conjugated to the Anti-Ki67 antibody [SP6] ab16667, targeting the same antigen as the Ambergen commercial probe, which was already pre-conjugated to an anti-Ki67 antibody by the supplier. The commercial probe is characterized by a signal detectable at m/z 1320.76. Images were obtained. An image corresponds to the detection of our Tag (m/z 245.32) and another image correspond to Myralis (m/z 1320.76).

[166] The comparison was carried out on two consecutive tissue sections from the same human glioblastoma, both subjected to identical preparation protocols, including parallel immunohistochemistry (IHC) under identical conditions and at an antibody concentration of 1 pg/mL

[167] Images were obtained. Those images show that the conjugate according to the invention synthesized from the T6 azide was clearly detected with strong sensitivity, while the commercial probe produced no detectable signal, suggesting lower sensitivity under the same experimental conditions.

[168] Demonstration of MALDI-IHC using lectin instead of antibody

[169] The conjugates of the invention developed also proved compatible with other types of recognition molecules, such as lectins, as illustrated in images obtained with MALDI-MSI. In this experiment, the lectin used was Concanavalin A (Con A), known for its ability to recognize mannose residues in a configuration, present in the "central oligosaccharide" motif of many serum and membrane glycoproteins. Specific detection of the conjugates via MSI confirms both the labelling capacity of Con A and the compatibility of the probes with recognition agents other than antibodies. This enables tissue glycome to be explored using a multiplexed, targeted mass spectrometry approach. The experiment was performed from sagittal fresh frozen rat brain tissue section. Images were obtained. One image corresponds to the MALDI image reconstructed based on the signal of the Tag 10 (T10, m/z 233.22). Another image is the control for MALDI-IHC against GFAP.

[170] Demonstration of the sensitivity of the MALDI-IHC Tags

[171] It was also verified that immunohistochemistry (IHC) could be performed using different incubation methods and durations, including shorter protocols at 37 °C, similar to those used in fluorescent IHC. [172] These reduced incubation times may help lower background signal, provided the target protein is sufficiently expressed to enable effective antibody recognition. However, such conditions require the use of reporter molecules with high sensitivity to compensate for reduced antigen-antibody interaction times. The conjugates of the invention have demonstrated compatibility with these modified conditions confirming their ability to yield detectable signals in accelerated IHC workflows.

[173] Images were obtained. These images present the result of the MALDI-IHC against GFAP from the cerebellum part of a rat brain sagittal section. The images were reconstructed selecting the signal of T6 (m/z 231 .26). One image corresponds to an incubation of the primary AB at 37°C for 1 H and another image corresponds to an incubation overnight at 4°C. Another image corresponds to the control (no antibody) and does not show any signal for the Tag in the MS spectra.

[174] Demonstration of the application of Tagged antibody in multiplex on clinical samples.

[175] The conjugates’ compatibility with both multiplexed detection and FFPE tissue sections, as demonstrated above, enables their application in pathological contexts to investigate complex biological phenomena. 10-plex MALDI-IHC MSI on Breast Cancer has been performed.

[176] In this breast cancer example, various biomarkers were targeted, revealing critical insights into tumor-immune system interactions. Tumor proliferation, identified by Ki67 expression, was shown to dominate the immune response, with deeper tissue infiltration and the presence of immunosuppressive signaling. This immune evasion was reflected by TIM3 expression (a marker of T-cell exhaustion), co-expression of CD11 b on CD4-/CD8- cells, and PDL1 expression on PD1- positive immune cells. As a result, immune activity appeared to be suppressed and excluded from the tumor core, as indicated by peripheral localization of CD47, a marker known for promoting immune escape. The experiment was performed from a single FFPE tissue section of a patient which is HR- and HER2-. The 10 tagged antibody (10 conjugates according to the invention) were used in the same IHC experiment.

[177] Images were obtained. The images show the individual localization of each of the 10 markers. They also show the morphological histology (HPS staining) of the tissue. Another image where 3 of the Tags are reported simultaneously has been obtained. [179] Demonstration of the possible use of multiplexing with several IHC cycles (eg 2 cycles of 9-plex for 18-plex total)

[180]

[181]

482] This approach was also implemented across different experimental cycles of multiplexing, using a combination of washing and photobleaching steps, a strategy inspired by methods commonly applied in fluorescence spectroscopy. The goal was to increase the number of biomarkers that could be visualized on a single tissue section by allowing for the sequential removal of probes after each imaging cycle. In this case, the method was applied due to the limited number of synthesized probes. However, if a broader panel of probes becomes available, this strategy could be adapted to enable simultaneous multiplexing, thereby minimizing the number of imaging cycles while maintaining high detection capacity. The experiments were performed on a FFPE breast cancer tissue section (HR- and HER2-).

[183] Images were obtained. They show a first 9-plex MALDI-IHC against a panel of 9 markers. Other images show the result of the second MALD-IHC (same tissue) after washing out the MALDI matrix of the tissue and a second run of 9-plex MALDI IHC against 9 additional markers. Each image corresponds to the distribution of the ion corresponding to one specific tag.

[184] Aptamer-based conjugates - Multiplex IHC with MALDI-MS Imaging : obtained results.

[185] The inventors have demonstrated that the aptamer-based conjugate of the invention provides great performance in multiplex immunohistochemistry (IHC) with MALDI -MS imaging. [186] The experiment was performed on a section of rat brain sagittal section from a rat that were submitted to trauma brain injury (TBI), inducing a cerebral inflammatory response.

[187] The inventors used the two conjugates A and B according to the invention, as described above. In details, two aptamers against IL-6 and 11-10 were functionalized respectively with the T4 probe (for IL-6) and the T5 probe (for IL-10), two key cytokines in immune inflammation processes which are expected to be found from TBI samples.

[188] The MALDI-MS images obtained illustrate the detection of these two markers despite their low relative concentration in tissues. Three images were obtained. The first image shows the distribution of IL-6 based on the distribution of the m/z 217 (T4) and the second image shows the distribution of the 11-10 based on the distribution of the m/z 231 signal in the MS spectra (T5). The third image corresponds to the overlay of the distribution of the T4 and T5 signal on the same image.

[189] Indeed, interleukins IL-6 and IL-10 are generally expressed at low levels in tissues in an acute inflammatory context, of the order of 500 to 1000 pg/mL, while constitutive proteins, such as GFAP, can reach concentrations of 1 ,000 to 100,000 pg/mL. Detection of these weakly expressed targets underlines the high sensitivity of the probes used, as well as their compatibility with other classes of biomolecules such as aptamers.

[190] Another image representing the data from this experiment after their importation in SCILS software for image processing has been obtained by the inventors. Very importantly the MS spectra obtained from the aptamer IHC MALDI MS imaging in the m/z range 200-245 show that the elevated intensities at m/z 217 and 231 in the tissue treated with the functionalized aptamer are indeed due to specific target recognition by the labeled aptamers (conjugates according to the invention) and that these signals are not found in the MS spectra of the controls.

[191] B) DESI immunohistochemistry imaging

[192] DESI solvents: The solutions were prepared fresh, particularly for the DESI spray, before the analysis. MeOH/H2O:50/50, MeOH/H2O:90/10, MeOH/H2O:95/5, ACN/H20:50/50, ACN/H2O with 1% acetic acid :50/50, MeOH/H2O with 0,1% TFA:50/50, ACN/H20 with 0,1% TFA:50/50 were tested as solvent spray.

[193] DESI imaging

[194] The mass spectrometer is a Quadrupole Time-of-Flight (Q-TOF), Waters Synapt G2-Si ion mobility enabled. The calibration of the mass spectrometer is performed with the ESI source using sodium formate 0.5 mM diluted in 90/10: isopropanol/HPLC grade water. The ESI source is replaced by the DESI source from Waters, with the High-Definition Imaging (HDImaging) software. The syringe pump used is Harvard Apparatus, Pump 11 Elite. The syringes used is SGE Luer-lock of 1 mL or 2.5 mL with respectively internal diameter of 4.606 mm and 7.284 mm.

[195] The DESI source is installed, with all the connections. The nitrogen gas pressure is set to around 0.5 MPa. The initial setup of the DESI source was performed using methanol as spray solvent with a flow rate of 2pL/min. The gas used is nitrogen at a pressure of 5 bar. The spray angle was set to 55°. The MS inlet capillary, also named bazooka, is placed as close as possible to the sample surface. The spray tip is placed at approximatively 1 mm above the surface. The distance between the spray tip and the bazooka is approximatively 3-4mm.

[196] The MassLynx software is used to control the DESI and mass spectrometer during the analysis. Imaging are setup with the software High-Definition Imaging (HDI). DESI images are always acquired in a rectangular shape. To acquire an image, different steps are necessary: select and scan the sample, load the image into HDI and select the area desired for imaging, select the parameters desired for this analysis. This information is saved and exported to Masslynx, the method of analysis is loaded into MassLynx and the experiment is started. For lipid analysis, the m/z 100-1000 range was chosen. The data was acquired in sensitivity mode, in both positive and negative mode. The MS inlet temperature was set up at T=150 °C. The voltage is constant at V=4.5 kV. The gas pressure was fixed at 5 bar but can be modified to enhance the analysis.

[197] Image processing

[198] The visualization of DESI images of a selected mass is possible with the HDI software package. DESI images are processed from the raw data. The processing involves peak picking to preserve the high mass accuracy data, to reduce the low background peaks and to reduce the file size. After normalization of the data by total ion count, it is possible to visualize individual m/z images or overlay of images. HDI Software allows to export images with different processing methods and export mass list. Region of interest analysis of multiple images can be performed. Hierarchical clustering analysis can be implemented with HDI. Multivariate statistical analysis, namely Principal Component Analysis (PCA), can be performed, if there are many data points, to search regions of interest in the tissue.

[199] Demonstration of the use of the photocleavable tags on antibodies using nanoDESI.

[200] Acid-induced cleavage was previously validated, and spectra were subsequently acquired using nanoDESI.

[201] Data acquisition was performed directly on tissue, following immunohistochemistry (IHC) in which an anti-Synapsin 2 antibody was functionalized with probe T10 (m/z 233.1296). The solvent used for electrospray ionization consisted of a 50:50 methanol/water mixture with 1% TFA. As shown in images and data obtained by the inventors, the spectrum acquired on tissue clearly reveals the appearance of a peak at 233.13 m/z corresponding to the probe, whereas no signal is detected outside the tissue, as illustrated in the bottom spectrum.

[202] These results confirm both the compatibility of the conjugates of the invention with acid conditions and its efficiency in tissue-based nanoDESI acquisition workflows.

[203] Given the proximity of DESI and nanoDESI, since the conjugates have shown good results in nanoDESI-MSI, the inventors are of the opinion that they are very likely to work very well in DESI- MSI.

[204] C) Spider MASS immunohistochemistry imaging

[205] SpiderMass analysis [206] The overall layout of the instrument setup has already been covered elsewhere (Ogrinc et al. Nat Protoc 14, 3162-3182 (2019). doi.org/10.1038/s41596-019-0217-8). In brief, the system is made up of three parts: the mass spectrometer itself, a laser system standing remotely from the MS instrument for micro-sampling of tissues and a transfer line allowing for the transfer of the microsampled material back to the MS instrument. The first component consists of a pulsed Nd:YAG laser (pulse duration: 5 ns, = 1064 nm, Quantel, Les Ulis, France) pumping a tunable wavelength OPO (Radiant version 1.0.1 , OPOTEK Inc., Carlsbad, CA, USA). A handpiece with a 4 cm focusing lens is attached to the end of the biocompatible laser fiber, which is connected to the laser system output and has an inner diameter of 450 microns and a length of 1 m. In these studies, the laser intensity was set to 4 mJ/pulse for a fixed irradiation time of 10 s, resulting in a laser fluence of approximately 3 J/cm2. The second component of the system is a 2 m transfer line made of Tygon ND 100-65 tubing (Akron, Ohio, USA, 2.4 mm inner diameter, 4 mm outer diameter). The transfer line is directly connected to the mass spectrometer (Xevo, Waters, Manchester, United Kingdom) from which the conventional electrospray source was removed and replaced by a REIMS interface on one side and is attached to the laser handpiece on the other. A 200 pL/min infusion of isopropanol was administered before each acquisition. 200 pg/mL of Leucine enkephalin was added to the infusion to play the role of a lockmass. The sampling position was determined based on the histopathological annotations. The acquisition was composed of a burst of 10 laser shots resulting in an individual spectrum. Spectral acquisition was performed both in positive and negative ion mode in sensitivity mode with a scan time of 1 s. The mass range was set to m/z 50-1000.

[207] SpiderMass MS Imaging

[208] The SpiderMass setup was described in the previous section. To perform imaging analysis, the Spider-Mass microprobe (conjugate) was coupled to a stiff robotic arm described in a previous work (Ogrinc, N., Kruszewski, A., Chaillou, P., Saudemont, P., Lagadec, C., Salzet, M., Duriez, C., and Fournier, I. (2021 ). Robot-Assisted SpiderMass for In Vivo Real-Time Topography Mass Spectrometry Imaging. Anal. Chem. 93, 14383-14391 . 10.1021 /acs.analchem.1 c01692). The spatial step size was set to 250 pm to achieve imaging by oversampling and reach 250 pm spatial resolution for the images. The final spatial resolution was divided by two thanks to interpolation algorithm. The mass-range was fixed between m/z 100-1000. The acquisition sequence was composed of 3 consecutive laser shots and 3 seconds between each step. The laser bursts and the spectrometer acquisition were automatically triggered through a MATLAB in-house user interface developed for the robotic WALDI-MSI (1. Ogrinc, N., Kruszewski, A., Chaillou, P., Saudemont, P., Lagadec, C., Salzet, M., Duriez, C., and Fournier, I. (2021 ). Robot-Assisted SpiderMass for In Vivo Real-Time Topography Mass Spectrometry Imaging. Anal. Chem. 93, 14383-14391.

10.1021/acs.analchem.1c01692). The data was acquired in positive and sensitivity ion mode.

[209] Demonstration of the detection of the MS reporter after SpiderMass analysis through irradiation with 2.94 pm mid-IR laser using one of the Tag normally used for photocleavage. [210] The probes (azides) were analyzed using the SpiderMass system, which is equipped with a mid-infrared laser (2.94 pm, few nanoseconds pulse duration, few mJ/shot), as illustrated with probe (azide) T2.

[211] A distinct mass peak at m/z 203.16 was detected, a signal absent in the control spectrum (shown in red), indicating that cleavage of the probe and release of the tag occurred upon laser irradiation. The tag is released when increasing the used laser energy/shot. This result highlights the particular sensitivity of ortho-nitrobenzyl groups to mid-IR radiation, making them effective for targeted photochemical release under infrared laser exposure. This is highly interesting considering that SpiderMass technology is designed for in vivo real-time analysis opening the way of targeted detection of known markers with Tag probes (conjugates) in vivo.

[212] 4. ISH with oligonucleotide-based conjugate : obtained results.

[213] The strategy previously described for performing immunohistochemistry-mass spectrometry (IHC-MS) using aptamers was found to be compatible with a broad range of oligonucleotide structures. This approach was extended to in situ hybridization (ISH) to target transcriptomic content by employing complementary single-stranded DNA (cDNA).

[214] In this experiment, a cDNA targeting actin mRNA was enzymatically elongated via terminal deoxynucleotidyl transferase (TdT), incorporating click-compatible dUTP-DBCO units. A subsequent click chemistry reaction was carried out to attach the T6 probe (T6-Azide, see Table 2) (m/z 245.217) to the oligonucleotide.

[215] The inventors have thus synthesized a conjugate as follows :

Conjugate C : azide T5 + cDNA having a “poly-U-DBCO” tail.

[216] A control sample lacking cDNA (but containing all other reaction components) was processed in parallel. Images were obtained. The resulting images show that: the sagittal rat brain section (centered on the cerebellum) revealed a structured molecular signal consistent with actin expression in the experimental condition, whereas only background noise was observed in the control.

[217] These results demonstrate the adaptability of the clickable probe strategy to other recognition molecules, and more specifically to single-stranded oligonucleotides for targeted transcript imaging by ISH-MS.

Claims

Claims

[Claim 1] Conjugate of formula (A-Ue-(Li)f-v_g-B-Wh-(L2)i-Xj-)_nC, wherein

- A is a tag,

- u, v, w, x are linkers, - Li and L2 are spacer arms,

- B is a cleavable o-nitrobenzyl moiety,

- n is an integer of 1 to 25,

- e, f, g, h, i and j are independently selected from 0 or 1 , and

- C is a binding molecule that binds to a target molecule.

[Claim 2] Conjugate according to claim 1 , wherein the tag A is chosen in the group consisting of peptides, amino-acids, nucleic acids, guanidinium derivatives, sugars, polymers, lipids, metabolites and fluorophores.

[Claim 3] Conjugate according to claim 1 or 2, wherein the tag A is chosen in the group consisting of arginine and derivatives thereof.

[Claim 4] Conjugate according to any one of claims 1 to 3, wherein the binding molecule C is an aptamer, an antibody, a lectin or an oligonucleotide.

[Claim 5] Conjugate according to any one of claims 1 to 4 selected from

[Claim 6] Conjugate according to any one of claims 1 to 5 selected from

[Claim 7] Reaction intermediate of formula A-u_e-(Li)f-v_g-B-Wh-L3-N3, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- B is a cleavable o-nitrobenzyl moiety,

- e, f, g and h are independently selected from 0 or 1 , and

- N3 is an azide moiety.

[Claim 8] Reaction intermediate of formula A-u_e-(Li )f-v_g-B-Wh-l_3-Alkyne, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- e, f, g and h are independently selected from 0 or 1 , and

- B is a cleavable o-nitrobenzyl moiety.

[Claim 9] Reaction intermediate of formula A-u_e-(Li)f-v_g-B-Wh-l-3-Tetrazine, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms,

- e, f, g and h are independently selected from 0 or 1 , and

- B is a cleavable o-nitrobenzyl moiety.

[Claim 10] Reaction intermediate of formula A-u_e-(Li jf-Vg-B-Wh-La-Alkene, wherein

- A is a tag,

- u, v, w are linkers,

- Li and L3 are spacer arms, - e, f, g and h are independently selected from 0 or 1 , and

- B is a cleavable o-nitrobenzyl moiety.

[Claim 11] A method for detecting a target molecule in a tissue section, wherein said method comprises: a) providing a tissue section; b) contacting said tissue section with a conjugate according to any one of claims 1 to 6; c) cleaving the cleavable o-nitrobenzyl moiety B of the conjugate so as to release the tag A; d) detecting the tag A released by mass spectrometry imaging; wherein said mass spectrometric imaging is MALDI-MSI, DESI-MSI, nano-DESI-MSI, or SpiderMass.