Bright and releasable labels for cell staining based on conjugates with several sites of fluorophore release

Abstract

To provide methods for cell detection by labeling a target cell with a conjugate having a detection moiety and an antigen recognition moiety linked via two different enzymatically degradable spacers.SOLUTION: The invention is directed to a conjugate characterized by high brightness to enable detection of rarely expressed epitopes and release of the label from the epitope to enable downstream applications such as sequential imaging or cell sorting, and has the general formula (I) Yn-P1(P2-Xm)o, (where X: detection moiety; P1: first enzymatically degradable spacer; P2: second enzymatically degradable spacer; Y: antigen recognizing moiety; and n, m, o are integers between 1 and 100; with the provision that first spacer P1 and second spacer P2 are not degradable by the same enzyme).SELECTED DRAWING: None

Description

[Technical Field]

[0001] background The present invention discloses a method for cell detection by labeling target cells with a conjugate having a detection portion and an antigen recognition portion linked via two different enzymatically degradable spacers.

[0002] Cell analysis and separation techniques such as fluorescence microscopy, flow cytometry, and cell sorting are crucial for detailed analysis and specific isolation of target cells from biological specimens in both research and clinical applications. These techniques rely on the fluorescent labeling of epitopes using fluorescent conjugates.

[0003] Regarding the use of conventional fluorescent conjugates for cell labeling, there are several technical limitations, particularly the following: - Achieving high fluorescence intensity of conjugates that enable the detection of rarely expressed epitopes, - Release of labels from epitopes that would enable downstream applications such as sequential imaging or cell sorting.

[0004] In particular, in techniques based on a continuous label-detection-removal cycle with high multiplexing potential for mapping protein networks, for example, the removal of fluorescent signals is essential.

[0005] Several approaches to releaseable labeling have been developed in recent years. For example, European Patent Application Publication No. 3037821 discloses a conjugate that allows for specific cell labeling but can be cleaved and washed away after the experiment, enabling subsequent staining and imaging.

[0006] International Publication No. 20080918100 discloses a conjugate in which an enzyme acts as an activator of a fluorescent dye, and which can be released from target cells by radioactive cleavage of the enzyme.

[0007] However, these reagents rely on a single enzymatically cleavable spacer. Since all enzymatic reactions have an equilibrium, the release of the dye is never complete, and some residual fluorescence will remain in the sample.

[0008] Summary Therefore, an object of the present invention is to label and detect a target in or on a sample of a biological specimen, and subsequently remove the detection moiety to provide a reversible label and / or be more highly released, i.e., reduce the residual fluorescence after release, and further enable different labeling and detection cycles by providing a staining reagent.

[0009] To solve the aforementioned problems regarding the release of fluorescent labels, a new approach for designing releasable conjugates has been proposed. This is based on a conjugate having the general structure (I) Y n -P1(P2-X m ) o (wherein X: detection moiety; P1 and P2 are enzymatically degradable spacers that cannot be cleaved by the same enzyme).

[0010] The subject of the present invention is a conjugate having the general formula (I) Y n -P1(P2-X m ) o (wherein X: detection moiety; P1: first enzymatically degradable spacer; P2: second enzymatically degradable spacer; Y: antigen recognition moiety, and n, m, o are integers from 1 to 100, provided that the first spacer P1 and the second spacer P2 cannot be cleaved by the same enzyme).

[0011] The general formula (I) requires that P1 binds to one or more (1 to "o" units) of P2. For clarity, the general formula (I) will be referred to hereinafter as Y n -P1-(P2-X m ) o and can be so called.

[0012] The design of such conjugates enables the following: - High degree of polymerization of phosphors to avoid their significant self-quenching (which is often difficult to achieve using a single polymer backbone); - Efficient release of pigment from the binder through double digestion of a spacer between the binder and the pigment (often difficult when using a single digestible spacer).

[0013] Another subject of the present invention is, a) General formula (I)Y n -P1(P2-X m ) o To supply at least one conjugate having (wherein X: detection portion; P1: first enzymatically degradable spacer; P2: second enzymatically degradable spacer; and Y: antigen recognition portion, and n, m, o are integers from 1 to 100) b) Contacting a sample of a biological specimen with at least one conjugate, thereby labeling the target region recognized by the antigen-recognizing portion Y. c) Detecting the conjugate-labeled target portion with detection portion X, and d) Supply a first enzyme capable of degrading the first spacer P1 and / or a second enzyme capable of degrading the second spacer enzyme P2, thereby cleaving the detection portion X from the conjugate, wherein the first enzyme is unable to degrade the second spacer P2 and the second enzyme is unable to degrade the first spacer P1. This is a method for detecting a target portion within a biological specimen.

[0014] The coupling of spacers P1 and P2 can be carried out using standard coupling chemistry. For example, coupling an oligonucleotide as P2 to dextran as P1 can be achieved by thiol-maleimide chemistry. Simultaneously, an antigen recognition site, such as an antibody or antibody fragment, can be attached to dextran as P1 using an orthogonal reaction. These conjugates can be used as templates for desired modifications. For example, the functionalization of each oligonucleotide can be carried out via covalent modification with a phosphor as detection site X, or by hybridization with a functionalized complementary oligonucleotide linked to detection site X. The resulting conjugate contains two sites for the release mechanism, e.g., dextran and oligonucleotides for dextranase and DNase, respectively, ensuring efficient removal of staining from the labeled antigen.

[0015] Using the conjugate and method of the present invention, more complete "enzymatic destaining" of the detection portion can be achieved, and therefore residual staining and, consequently, residual background signal in continuous imaging can be reduced.

[0016] Detailed explanation The detection portion X and the antigen recognition portion Y may be covalently or quasi-covalently bound to the enzymatically degradable spacers P1 and P2. The term "covalently or quasi-covalently" refers to a dissociation constant of 10 -9 This refers to combinations between X and P1, between Y and P2, and between P1 and P2, where M is less than or equal to M.

[0017] The term "cutting detection portion X" means that the bond between X and P2 and / or between Y and P1 is severed, and detection portion X can be removed from the target, for example, by washing.

[0018] Figure 1 schematically illustrates a conventional method of specific labeling of target cells as biological specimens using a conjugate having an antigen recognition portion Y, enzymatically degradable spacers P1 and P2, and a detection portion X.

[0019] The method of the present invention can be carried out in one or more sequences of labeling and decolorization. After each sequence, the detection moiety is released (removed) from the target moiety. In particular, when the biological sample is a living cell that has to be further processed, the method of the present invention has the advantage of providing non-labeled cells.

[0020] After each step of labeling and decolorization, a washing step can be carried out to remove unwanted materials such as unbound conjugates or released detection moieties from the sample.

[0021] target area The target moiety detected by the method of the present invention can be on any biological sample such as a tissue slice, cell aggregate, suspended cell, or adherent cell. The cells can be either alive or dead. Preferred target moieties are antigens expressed intracellularly or extracellularly on biological samples such as whole animals, organs, tissue sections, cell aggregates or single cells of invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., Danio rerio, Xenopus laevis) and mammals (e.g., Mus musculus, Homo sapiens).

[0022] Detection part The detection moiety X of the conjugate can be any moiety having a property or function that can be used for detection purposes, such as those selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, a light absorbing moiety, a radioactive moiety, and a transition metal isotope mass tag moiety.

[0023] Suitable fluorescent moieties are known from the field of fluorescence techniques, such as flow cytometry or fluorescence microscopy. In these embodiments of the present invention, the target moiety labeled with the conjugate is detected by exciting a detection moiety X and detecting the resulting emission (photoluminescence). In this embodiment, the detection moiety X is a preferred fluorescent moiety.

[0024] Useful fluorescent moieties may be protein-based, such as phycobiliproteins; polymers, such as polyfluorenes; small organic molecular dyes, such as xanthenes, such as fluorescein; or metal-organic complexes, such as rhodamine, cyanine, oxazine, coumarin, acridine, oxadiazole, pyrene, pyromethene, or Ru, Eu, Pt complexes. In addition to single molecular entities, clusters of fluorescent proteins or small organic molecular dyes, as well as nanoparticles such as quantum dots, upconvertible nanoparticles, gold nanoparticles, and stained polymer nanoparticles, can also be used as fluorescent moieties.

[0025] Another group of photoluminescence detection parts is the phosphorescent part, which exhibits time-delayed emission after excitation. The phosphorescent part includes nanoparticles incorporating metal-organic composites such as Pd, Pt, Tb, and Eu composites, or phosphorescent pigments such as lanthanide-doped SrAl2O4.

[0026] In another embodiment of the present invention, the conjugate-labeled target is detected without prior excitation by irradiation. In this embodiment, the detected portion may be radioactively labeled. These may be in the form of radioactive isotope labeling by replacing non-radioactive isotopes with their radioactive counterparts, such as tritium, 32P, 35S, or 14C, or covalent labeling such as 125I bound to tyrosine, 18F in fluorodeoxyglucose, or by introducing a metal-organic complex, i.e., 99Tc-DTPA.

[0027] In another embodiment, the detection portion can induce chemiluminescence, i.e., wasabi peroxidase labeling, in the presence of luminol.

[0028] In another embodiment of the present invention, the conjugate-labeled target is not detected by radiation emission but by absorption of UV, visible, or NIR radiation. Suitable light-absorbing detection portions are non-fluorescent light-absorbing dyes such as N-arylrhodamine, azo dyes, and small organic molecule quenching dyes like stilbene.

[0029] In another embodiment, the light absorption detection portion X can be illuminated by pulsed laser light to generate a photoacoustic signal.

[0030] In another embodiment of the present invention, the conjugate-labeled target is detected by mass spectrometry detection of transition metal isotopes. Transition metal isotope mass tagging can be introduced as a covalently bonded metal-organic composite or nanoparticle component. Lanthanide isotope tags and adjacent late transition elements are known in the art.

[0031] The detection portion X may be covalently or acovalently bonded to the spacer P2. Methods for covalent or acovalent bonding are known to those skilled in the art. When covalent bonding occurs between the detection portion X and the spacer P2, direct reactions are possible between any activated group on the detection portion or spacer P2 and any functional group on spacer P2 or detection portion X, or via a heterobifunctional linker molecule that reacts first with one and then with the other bonding partner.

[0032] For example, numerous heterobifunctional compounds can be used for linking to entities. Exemplary entities include azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl sverate, dimethyl adipimidate, disuccinimidyl tartrate, Ny-maleimidobutyryloxysuccinimidate, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, succinimidyl-[(N-maleimidopropionamide)polyethylene glycol]ester (NHS-PEG-MAL), and succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Preferred linking groups are 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester (SMCC), which have a reactive sulfhydryl group in the detection portion and a reactive amino group in the spacer portion.

[0033] The quasi-covalent bond of the detected portion X to the spacer has a dissociation constant of 10. -9 M or less can be achieved, for example, by using a binding system that provides a biotin-avidin binding interaction.

[0034] Enzyme-degradable spacers P1 and P2 Enzyme-degradable spacers can be any molecule that can be cleaved by a specific enzyme, particularly a hydrolase. Suitable enzyme-degradable spacers P1 and P2 include, for example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids, and their derivatives.

[0035] Suitable polysaccharides include, for example, dextran, pullulan, inulin, amylose, cellulose, hemicellulose, such as xylan or glucomannan, pectin, chitosan, or chitin, which can be derivatized to provide functional groups for the covalent or non-covalent bonding of the detection portion X and the antigen recognition portion Y. Various such modifications are known in the art, for example, an imidazolylcarbamate group can be introduced by reacting the polysaccharide with N,N'-carbonyldiimidazole. Subsequently, an amino group can be introduced by reacting the imidazolylcarbamate group with hexanediamine. The polysaccharide can also be oxidized with periodate to give an aldehyde group, or with N,N'-dicyclohexylcarbodiimide and dimethyl sulfoxide to give a ketone group. Aldehyde or ketone functional groups can subsequently react with diamines to yield an amino group, preferably under reductive amination conditions, or directly with amino substituents on the protein-binding moiety. Carboxymethyl groups can be introduced by treating polysaccharides with chloroacetic acid. Activating the carboxyl group in a manner known in the art to produce activated esters such as N-hydroxysuccinimide or tetrafluorophenyl esters allows it to react with any amino group of a diamine or directly with an amino group on the protein-binding moiety to yield an amino group. In general, alkyl-containing functional groups can be introduced by treating polysaccharides with halogen compounds under alkaline conditions. For example, allyl groups can be introduced using allyl bromide. Allyl groups can further be used in thiol-ene reactions with thiol-containing compounds such as cysteamines to introduce an amino group, or in direct reactions with protein-binding moieties having thiol groups that are liberated by reduction of disulfide bonds or introduced by thiolation with, for example, 2-iminothiolane.

[0036] Proteins, peptides, and depsipeptides used as enzymatically degradable spacers can be functionalized via amino acid side-chain functional groups to attach the detection site X to the antigen recognition site Y. Suitable side-chain functional groups for modification are, for example, amino groups supplied by lysine or thiol groups supplied by cysteine ​​after reduction of the disulfide bridge.

[0037] Polyesters and polyesteramides used as enzymatically degradable spacers can be synthesized using comonomers that provide side-chain functional groups, or they can be functionalized afterward. In the case of branched polyesters, functionalization can be carried out via carboxyl or hydroxyl terminal groups. Functionalization after polymerization of polymer chains can be carried out, for example, via addition to unsaturated bonds, i.e., via thiolene reactions or azido-alkyne reactions, or via introduction of functional groups by radical reactions.

[0038] Nucleic acids used as enzymatically degradable spacers are preferably synthesized using functional groups at the 3' and 5' ends that are suitable for binding the detection moiety X and the antigen recognition moiety Y. For example, suitable phosphoramidite structural blocks for nucleic acid synthesis that impart amino or thiol functionality are known in the art.

[0039] An enzymatically degradable spacer may consist of multiple different enzymatically degradable units that can be broken down by the same or different enzymes.

[0040] Antigen recognition part Y The term “antigen-recognition moiety Y” refers to any type of antibody, nanobody, fragmented antibody, or fragmented antibody derivative directed toward a target moiety expressed on a biological specimen, such as an antigen expressed intracellularly or extracellularly on a cell. This term refers to completely intact antibodies, fragmented antibodies, or fragmented antibody derivatives, e.g., Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, and nanobodies. Such fragmented antibody derivatives can be synthesized by recombinant procedures involving covalent and non-covalent conjugates containing these types of molecules. Further examples of antigen-recognition moieties include peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, and artificially engineered binding molecules, such as peptides or aptamers targeting cell surface molecules.

[0041] The conjugate used in the method of the present invention may contain up to 100, preferably 1 to 10, antigen-recognizing moieties Y. The interaction between the antigen-recognizing moieties and the target antigen may be high-affinity or low-affinity. The binding interaction of a single low-affinity antigen-recognizing moiety is too low to provide stable binding to the antigen. Low-affinity antigen-recognizing moieties can be polymerized by binding to an enzymatically cleavable spacer to provide high binding activity. When the spacer is enzymatically cleaved in step d), the low-affinity antigen-recognizing moieties are monomerized, resulting in the complete removal of the detection moiety X, the spacer, and the antigen-recognizing moiety Y. High-affinity antigen-recognizing moieties provide stable binding, and as a result, the detection moiety X and the spacer are removed during step d).

[0042] Preferably, the term “antigen-recognition region Y” refers to any antibody directed against an antigen expressed by a biological specimen (target cell), such as IL2, FoxP3, CD154, or on the cell surface, such as CD3, CD4, CD8, CD14, CD25, CD34, CD56, CD133, and EGFR.

[0043] The antigen-recognition portion Y, particularly the antibody, can be bound to spacer P via a side-chain amino group or sulfhydryl group. In some cases, the glycoside side chain of the antibody may be oxidized by periodate to produce an aldehyde functional group.

[0044] The antigen-recognizing portion Y can be covalently or noncovalently linked to the spacer P. Methods for covalent or noncovalent linkage are known to those skilled in the art and are the same as those described for the linkage of the detection portion X.

[0045] The method of the present invention is particularly useful for detecting and / or isolating specific cell types from complex mixtures and may comprise two or more sequential or parallel sequences of staining and destaining. This method can utilize various combinations of conjugates. For example, the conjugate may comprise antibodies specific to two different epitopes, such as two different anti-CD34 antibodies. Different antigens may be addressed with different conjugates comprising different antibodies, for example, anti-CD4 and anti-CD8 for differentiation between two different T cell populations, or anti-CD4 and anti-CD25 for determining different cell subpopulations, such as regulatory T cells.

[0046] enzyme The selection of the enzyme as the release agent is determined by the chemical properties of the enzymatically degradable spacers P1 and P2, and may be one enzyme or a mixture of different enzymes. The enzyme is preferably a hydrolase, but lyase or reductase is also possible. For example, if spacer P1 or P2 is a polysaccharide, glycosidase (EC3.2.1) is most suitable as the release agent. Glycosidases that recognize specific glycoside structures are preferred, such as dextranase (EC3.2.1.11) which cleaves dextran at the (1->6) bond, pullulanase which cleaves either the (1->6) bond (EC3.2.1.142) or both the (1->6) and (1->4) bond (EC3.2.1.41) of pullulan, neopullulanase (EC3.2.1.135) and isopullulanase (EC3.2.1.57) which cleave the (1->4) bond of pullulan. Amylase (EC3.2.1.1) and maltose-producing amylase (EC3.2.1.133) that cleave the (1->4) bond of amylose, inulinase (EC3.2.1.7) that cleaves the β(2->1) fructoside bond of inulin, cellulase (EC3.2.1.4) that cleaves the (1->4) bond of cellulose, xylanase (EC3.2.1.8) that cleaves the (1->4) bond of xylan, pectinase, for example Endopectin lyase (EC4.2.2.10) which cleaves the (1->4)D-galacturonan methyl ester bond detachably, or polygalacturonase (EC3.2.1.15) which cleaves the (1->4)D-galactosidurone bond of pectin, chitosanase (EC3.2.1.132) which cleaves the (1->4) bond of chitosan, and endochitinase (EC3.2.1.14) for cleaving chitin.

[0047] Proteins and peptides can be cleaved by proteinases that need to be sequence-specific to avoid degradation of target structures on cells. Sequence-specific proteases include, for example, TEV protease (EC3.4.22.44), a cysteine ​​protease that cleaves at the sequence ENLYFQ\S; enteropeptidase (EC3.4.21.9), a serine protease that cleaves after the sequence DDDDK; factor Xa (EC3.4.21.6), a serine endopeptidase that cleaves after the sequence IEGR or IDGR; or HRV3C protease (EC3.4.22.28), a cysteine ​​protease that cleaves at the sequence LEVLFQ\GP.

[0048] Depsipeptides, or polyesters, which are peptides containing ester bonds in their peptide backbone, can be cleaved by esterases such as porcine liver esterase (EC3.1.1.1) or porcine pancreatic lipase (EC3.1.1.3). Nucleic acids can be cleaved by sequence-specific endonucleases such as restriction enzymes (EC3.1.21.3, EC3.1.21.4, EC3.1.21.5) such as EcoRI, HindII, or BamHI, or by more general endonucleases such as DNase I (EC3.1.21.1) that cleave phosphodiester bonds adjacent to pyrimidines.

[0049] The amount of enzyme added must be sufficient to substantially degrade the spacer within the desired time. Typically, the detection signal is reduced by at least about 80%, more commonly at least about 95%, and preferably at least about 99%. The conditions for release can be empirically optimized with respect to temperature, pH, presence of metal cofactors, reducing agents, etc. Degradation is usually completed in at least about 15 minutes, more commonly at least about 10 minutes, and usually not longer than about 30 minutes.

[0050] Cell detection method The method and apparatus for detecting a conjugate-labeled target of the present invention are determined by the detection portion X.

[0051] In one variant of the present invention, the detection portion X is a fluorescent portion. A target labeled with a fluorescent dye conjugate is detected by exciting the fluorescent portion X and analyzing the resulting fluorescence signal. The excitation wavelength is typically selected according to the absorption maximum of the fluorescent portion X and is supplied by a laser or LED light source known in the art. When multiple different detection portions X are used for multicolor / parameter detection, care should be taken to select fluorescent portions whose absorption spectra do not overlap, or at least whose absorption maxima do not overlap. In the case of a fluorescent portion as the detection portion, the target can be detected, for example, in a flow cytometer, spectrofluorometer, or fluorescence scanner under a fluorescence microscope. Light emitted by chemiluminescence can be detected by similar devices without excitation.

[0052] In another variant of the present invention, the detected portion is a light-absorbing portion, which is detected by the difference between the irradiated light intensity and the transmitted or reflected light intensity. The light-absorbing portion may also be detected by photoacoustic imaging, which uses the absorption of a pulsed laser beam to generate acoustics such as ultrasonic signals.

[0053] The radioactive detection area is detected by radiation emitted by radioactive isotopes. Suitable equipment for detecting radioactive radiation includes, for example, a scintillation counter. In the case of beta-ray emission, an electron microscope may be used for detection.

[0054] The transition metal isotope mass tag portion is detected by mass spectrometry methods such as ICP-MS, and this is incorporated into a mass cytometry instrument.

[0055] Method of Use The method of the present invention can be used in a variety of applications in research, diagnostics, and cell therapy.

[0056] In the first variant of the present invention, a biological specimen, such as cells, is detected for counting purposes, i.e., to determine the quantity of cells from a specimen having a specific set of antigens recognized by the antigen-recognizing portion of the conjugate.

[0057] In the second variant, one or more populations of biological specimens are detected from the sample and isolated as target cells. This variant may be used, for example, for the purification of target cells in clinical research, diagnostics, and immunotherapy. In this variant, one or more sorting steps may be performed after either a staining and destaining step, and optionally after a washing step.

[0058] Suitable for such separation are flow sorters, such as FACS or MEMS-based cell sorter systems, such as those disclosed in European Patent No. 14187215.0 or European Patent No. 14187214.3.

[0059] In another variant of the present invention, the location of a target region on a biological specimen, such as an antigen, recognized by the antigen-recognizing portion of the conjugate, is determined. Such techniques are known as "multiepitope ligand mapping," "chip-based cytometry," or "Multioymx," and are described, for example, in European Patent No. 0810428, European Patent No. 1181525, European Patent No. 1136822, or European Patent No. 1224472. In this technique, cells are immobilized and brought into contact with an antibody conjugated to a fluorescent portion. The antibody is recognized by each antigen on the biological specimen (e.g., on the cell surface), unbound markers are removed, and the fluorescent portion is excited, after which the location of the antigen is detected by the fluorescence emission of the fluorescent portion. In certain variants, instead of an antibody conjugated to a fluorescent portion, an antibody conjugated to a portion detectable for MALDI imaging or CyTOF may be used. Those skilled in the art know how to modify techniques based on fluorescent portions to use these detection portions.

[0060] The position of the target area is determined by a digital imaging device with sufficient resolution and sensitivity to the wavelength of fluorescence emission. The digital imaging device can be used, for example, with or without optical magnification using a fluorescence microscope. The generated images are saved to a hard drive or other suitable storage device, such as in RAW, TIF, JPEG, or HDF5 format.

[0061] To detect different antigens, different antibody conjugates having the same or different fluorescent or antigen-recognition moieties Y may be supplied. Because there are limitations to parallel detection of fluorescence emission at different wavelengths, antibody-fluorescent dye conjugates are used sequentially, individually or in small groups (2-10).

[0062] In yet another variant of the method according to the present invention, the biological specimen of the sample—particularly suspension cells—is immobilized by trapping it in a microcavity or by attachment.

[0063] Furthermore, following the conjugate of the present invention, it is possible to provide an additional conjugate having general formula (II)Xn-P'-Ym (wherein X, Y, n, and m have the same meanings as in formula (I), where X and Y are covalently or noncovalently bound to P', but P' may be a spacer that is not enzymatically degraded, such as a PEG spacer). In another variant, at least one non-enzymatically degradable conjugate having general formula (III)Xn-Ym (wherein X, Y, n, and m have the same meanings as in formula (I)) may be supplied. The non-enzymatically degradable conjugates having general formulas (II) and (III) remain present after a cleavage step and can be used for further detection. The non-enzymatically degradable conjugates may be quenched by oxidative or radioactive destruction of the fluorescent moiety.

[0064] In general, the method of the present invention can be carried out in several variants. For example, conjugates that are not recognized by the target portion can be removed, for example, by washing with a buffer before the target portion labeled with the conjugate is detected.

[0065] In the variant of the present invention, at least two conjugates are supplied simultaneously or in a subsequent staining sequence, where each antigen-recognizing moiety Y recognizes a different antigen. Here, the labeled target moieties can be detected simultaneously or sequentially. Sequential detection includes simultaneous enzymatic degradation of spacer molecules P1 / P2, or subsequent enzymatic degradation of spacer molecules P1 / P2, with optional intermediate removal (washing) of unbound moieties.

[0066] Another variant of the present invention involves the removal of fluorescence emission by a combination of enzymatic degradation and oxidative bleaching. The chemicals required for bleaching are known from the above publications relating to "multiepitope ligand mapping," "tip-based cytometry," or "Multioymx" technologies. [Brief explanation of the drawing]

[0067] [Figure 1] This figure shows staining and destaining with a conjugate having one site of detection partial emission, as well as the limitations of this approach. [Figure 2] This figure shows an exemplary conjugate according to the claims, which can be broken down by two different enzymes in two ways. The first enzyme cleaves spacer P1, and the other enzyme cleaves spacer P2. [Figure 3] This figure shows flow cytometry dot plots of SUP-T1 cell staining using a dual-emission conjugate. They exhibit brighter staining (measured by MFI) compared to their counterparts used with conventional cell staining instruments. [Figure 4]This figure shows confocal laser scanning microscope images of SUP-T1 cells stained with a dual-emission conjugate. These images demonstrate brighter staining compared to conventional cell staining conjugates. [Figure 5] This figure shows a flow cytometry dot plot of SUP-T1 cell staining using a dual-release conjugate. It demonstrates that simultaneous cleavage of P1 and P2 is more efficient than cleavage of each of them individually. It also shows that cleavage can be performed orthogonally.

[0068] Examples Preparation of conjugates: Step A: Preparation of reactive oligonucleotides (oligo-sulfhydryls) C6-SS oligonucleotides were dissolved in water at a concentration of 2 μM and reduced at room temperature (RT) for 2 hours with a 100-fold molar excess of tris-(2-carboxyethyl)-phosphine (TCEP) solution in 100 mg / mL water. The solution was then purified under normal conditions by size exclusion chromatography (SEC) to remove residual TCEP and C6-S protecting groups.

[0069] Step B: Preparation of reactive aminodextran (Dex-maleimide) A solution of aminodextran in phosphate-buffered saline (PBS) at a concentration of 20 mg / mL and pH 7.4 was incubated with 50-fold molar excess succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) dissolved in dimethyl sulfoxide (DMSO) for 1 hour at RT. This solution was then purified from residual linker via SEC under normal conditions. The resulting compound contained 28.7 units of maleimide available for conjugation.

[0070] Step C: Preparation of oligonucleotide-dextran (Dex-oligo) Dex-maleimide (4.5 mg / mL) obtained in step B was incubated in 40-fold molar excess with oligo-sulfhydryl (2.4 mg / mL in PBS) obtained in step A at RT for 1 hour, and purified from the unconjugated compound via SEC under normal conditions.

[0071] Step C: Preparation of reactive antibody (Ab-DBCO) A solution of antibody in PBS (5.2 mg / mL) was treated with a solution of dibenzocyclooctin-N-hydroxysuccinimidyl ester (NHS-DBCO, 20 mg / mL) in DMSO in a 5-fold molar excess at RT for 1 hour. This solution was then purified from residual linker via SEC under normal conditions.

[0072] Step D: Preparation of reactive oligonucleotide-dextran (N3-Dex-oligo) The Dex-oligo (1 mg / mL) obtained in step C was incubated in PBS at RT for 1 hour in a 200-fold molar excess using a solution of NHS azidoacetic acid (2 mg / mL) in DMSO. Subsequently, it was purified from residual linker via SEC under normal conditions.

[0073] Step E: Preparation of antibody-dextran-oligoconjugate (Ab-Dex-oligo) The N3-Dex-oligo (0.3 mg / mL) obtained in step D was incubated in 50-fold molar excess with Ab-DBCO (10.3 mg / mL in PBS) obtained in step C at RT for 2 hours, and purified from the unconjugated compound via SEC under normal conditions. The resulting compound consisted of 1.6 units of antibody and 21.4 units of oligonucleotide per dextran.

[0074] Step F: Preparation of biotin-functionalized antibody-dextran-oligoconjugate (Ab-Dex-ds oligo-bio) Complementary biotinylated oligonucleotides (84 μM in water) were added to Ab-Dex-oligonucleotides (250 nM) in PBS in a 43-fold excess, incubated at 60°C for 10 minutes, and then cooled to RT within 1 hour under constant shaking. The crude product was used in the next step without further purification.

[0075] Step G: Preparation of staining reagent (Ab-Dex-ds oligo-bio-α biotin-dye) Crude Ab-Dex-ds oligo-bio was incubated in PBS with a biotin antibody dye conjugate in a 4x molar excess for 30 minutes, and then the cells were stained in the dark.

[0076] Step H: Cell staining PBMCs in PBS / EDTA / BSA buffer were stained at 4°C for 10 minutes using the staining reagent obtained in step G. The cells were washed with cold PBS / EDTA-BSA buffer and analyzed by flow cytometry. For reversibility of dextran-mediated fluorescent labeling, the cells were incubated at RT for 10 minutes with dextranase, washed with PBS / EDTA-BSA buffer, and analyzed by flow cytometry. For reversibility of DNA-mediated fluorescent labeling, the cells were incubated at RT for 5 minutes with DNase I in 10 mM Hepes buffer at pH 7.4 containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2, then washed with PBS / EDTA-BSA buffer and analyzed by flow cytometry. To ensure the reversibility of fluorescent labeling with DNA and dextran, cells were incubated with DNase I and dextranase in 10 mM Hepes buffer at pH 7.4 containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2 for 5 minutes at RT, followed by washing with PBS / EDTA-BSA buffer and analysis by flow cytometry.

Claims

1. General formula (I)Y ｎ -P1(P2-X ｍ ) ｏ wherein X: a detection moiety; P1: a first enzyme-degradable spacer; P2: a second enzyme-degradable spacer; Y: an antigen recognition moiety, and n, m, and o are integers from 1 to 100, with the proviso that the first spacer P1 and the second spacer P2 cannot be degraded by the same enzyme.

2. The conjugate of claim 1, wherein the first spacer P1 and the second spacer P2 are selected from the group consisting of polysaccharides, proteins, peptides, depsipeptides, polyesters, and nucleic acids, with the proviso that the first spacer P1 and the second spacer P2 are not selected from members of the same group.

3. 3. The conjugate according to claim 1 or 2, characterized in that the antigen-recognizing moiety Y is an antibody, an antibody fragment, an antibody fragment derivative, a peptide / MHC complex targeting a TCR molecule, a cell adhesion receptor molecule, a receptor for a costimulatory molecule or an artificially engineered binding molecule.

4. 3. The conjugate of claim 1 or 2, wherein the detection moiety is selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, a light-absorbing moiety, a radioactive moiety, a transition metal and an isotope mass tag moiety.

5. 1. A method for detecting a target moiety in a sample of a biological specimen, comprising: a) General formula (I)Y ｎ -P1(P2-X ｍ ) ｏ providing at least one conjugate having the formula: (wherein X: detection moiety; P1: first enzyme-degradable spacer; P2: second enzyme-degradable spacer; and Y: antigen recognition moiety, and n, m, and o are integers from 1 to 100); b) contacting a sample of the biological specimen with at least one conjugate, thereby labeling the target moiety recognized by the antigen recognition moiety Y; c) detecting the conjugate-labeled target moiety with detection moiety X; and d) enzymatically degrading the first spacer P1 and / or the second spacer enzyme P2 by providing a first enzyme capable of degrading the first spacer P1 and / or a second enzyme capable of degrading the second spacer enzyme P2, thereby cleaving the detection moiety X from the conjugate, wherein the first enzyme is incapable of degrading the second spacer P2 and the second enzyme is incapable of degrading the first spacer P1. A method characterized by:

6. 6. The method of claim 5, wherein the first and second spacers P1 and P2 are enzymatically degraded to cleave the antigen recognition moiety Y from the targeting moiety.

7. The method of claim 5 or 6, wherein the enzymes used to degrade the first spacer P1 and the second spacer P2 are selected from the group consisting of glycosidases, dextranases, pullulanases, amylases, inulinases, cellulases, hemicellulases, pectinases, chitosanases, chitinases, proteinases, esterases, lipases, reductases, and nucleases, provided that the enzymes used to degrade the first spacer P1 and the second spacer P2 are not identical or are not isozymes.

8. 7. The method according to claim 5 or 6, characterized in that in a subsequent sequence comprising steps a) to d), a sample of the biological specimen is contacted with at least two conjugates having different detection moieties X and / or different enzymatically degradable spacers P and / or different antigen recognition moieties Y.

9. 7. The method according to claim 5 or 6, characterized in that in step b) a sample of the biological specimen is simultaneously contacted with at least two conjugates having different detection moieties X and / or different enzymatically degradable spacers P and / or different antigen recognition moieties Y, and in subsequent steps c) and d) detection and cleavage of each conjugate are carried out.

10. 7. The method according to claim 5 or 6, characterized in that in step b) a sample of the biological specimen is simultaneously contacted with at least two conjugates having different detection moieties X and / or different enzymatically degradable spacers P and / or different antigen recognition moieties Y, and in steps c) and d) detection and cleavage of each conjugate are carried out simultaneously.

11. 7. The method according to claim 5 or 6, characterized in that a sample of a biological specimen is contacted with at least two conjugates having different detection moieties X and / or different enzymatically degradable spacers P and / or different antigen recognition moieties Y in a subsequent sequence comprising steps a) to c), and cleavage of the conjugates is carried out simultaneously in a single step d).

12. 7. The method according to claim 5, wherein a sample of a biological specimen is contacted with at least two conjugates having different detection moieties X and / or different enzymatically degradable spacers P and / or different antigen recognition moieties Y in a subsequent sequence comprising steps a) to c), and cleavage of each conjugate is carried out in a subsequent step d).

13. 7. The method according to claim 5 or 6, characterized in that the cell sample is contacted with at least two conjugates having different detection moieties X and / or different enzymatically degradable spacers P and / or different antigen recognition moieties Y in a subsequent sequence comprising steps a) to d), wherein step d) of the first conjugate and step b) of the second conjugate are carried out simultaneously.

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