Conjugates Having an Enzymatically Releasable Detection Moiety and a Barcode Moiety

Abstract

The present invention relates to a compound represented by general formula (I): X n -PY m B o (I) wherein X is a detection moiety, P is a spacer unit, Y is an antigen recognition moiety, B is an oligonucleotide comprising 2 to 300 nucleotide residues, n, m, and o are independent integers between 1 and 100, P and B are covalently linked to Y, X is covalently linked to P, and X is erasable. The invention is further directed to libraries of such conjugates and methods of detecting target cells utilizing the conjugates or libraries of conjugates.

Description

[Technical field]

[0001] The present invention is directed to conjugates comprising a detection moiety and an antigen recognition moiety, optionally linked via an enzymatically degradable spacer, comprising an oligonucleotide as a barcode, and the use of such conjugates for the detection or identification of a target moiety or target cell from a cell sample.

[0002] Cell detection by conjugate that can be removed from cell after detection is a known process.For example, US Patent No. 7,776,562 discloses a reversible fluorescent labeling process that removes conjugate from cell by adding a competing molecule that breaks the non-covalent bond in the conjugate.

[0003] A variety of approaches to reversible fluorescent labeling are disclosed in EP 3037821 A1, in which the conjugate is provided with an enzymatically degradable spacer. By adding an appropriate enzyme, the conjugate is destroyed and both the detection and antigen recognition moieties of the conjugate are removed from the cell.

[0004] Known processes allow the detection of different cells in a cell sample, for example by repeated staining, detection and destaining with conjugates bearing different antigen recognition moieties, it is possible to detect or distinguish several phenotypes of cells and their location in tissues.

[0005] However, it is not possible to analyze the genetic information of a single isolated cell.

[0006] In different technical fields, it is known to identify genetic information obtained from a single cell by conjugating the cell with a polynucleotide as a barcode. These methods involve synthesizing a library of different polynucleotides that can be sequenced to identify a single cell.

[0007] The biology and necessary hardware for isolating cells is disclosed, for example, in U.S. Patent Nos. 9,388,456 and 9,695,468. However, this technique focuses on single isolated cells, not on cells in the context of tissues or cell cultures with cell-cell interactions.

[0008] It was therefore an object of the present invention to provide conjugates that allow the identification of single cells and their location / localization on or within tissues and their phenotype. Furthermore, the means for identification shall be erasable so as to allow multiple identification steps.

[0009] It has been found that conjugates composed of a) a barcode moiety and b) an antigen-binding moiety linked to a removable detection moiety are suitable for single cell identification.

[0010] The removable detection moiety allows the biological specimen to be subjected again to the same or different conjugates without interference from previous labels. All barcode moieties remain on the cells, thereby allowing sequencing to make a link between the phenotype of the cell detected by the binder and the genetic information of a single cell detected by sequencing.

[0011] The object of the present invention is therefore to provide a compound of general formula (I) X n -PY m B o (I) [In the formula, X is a detection moiety, P is a spacer unit, Y is an antigen recognition moiety, B is an oligonucleotide comprising 2 to 300 nucleotide residues; n, m, and o are independent integers from 1 to 100; P and B are covalently bonded to Y, X is covalently bonded to P, and X is erasable. is a conjugate having the formula:

[0012] In a preferred embodiment, the spacer unit P is enzymatically degradable.

[0013] Yet another object of the present invention is a method for detecting a target moiety on a cell, comprising the steps of: a) General formula (I) X n -PY m B o (I) [In the formula, X is a detection moiety, P is a spacer unit, Y is an antigen recognition moiety, B is an oligonucleotide comprising 2 to 300 nucleotide residues; n, m, and o are independent integers from 1 to 100; P and B are covalently bonded to Y, X is covalently bonded to P, and X is erasable. providing at least one conjugate having 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 target moiety labeled by the conjugate with a detection moiety X; d) isolating the cells labeled with the conjugate by detection moiety X; e) erasing the detected portion X; This is the method. [Brief description of the drawings]

[0014] [Figure 1] Comparison between CD3 FITC staining after 2 min cross-linking with 0.25% PFA (top) and 2% PFA (bottom), magnification x20. [Diagram 2]Flow cytometry analysis of cells labeled with antibody-fluorochrome-oligo against CD3 (PE), CD4 (APC) and CD8 (FITC) and cross-linked with 0.25% PFA (Fig. 2a) or 0.5% PFA (Fig. 2b) followed by mechanical force detachment. [Diagram 3] Flow cytometry analysis of cells labeled with antibody-fluorochrome-oligo against CD3 (PE), CD4 (APC) and CD8 (FITC) and cross-linked with 1% PFA (Figure 3a) or 2% PFA (Figure 3b) followed by mechanical force detachment. [Figure 4] Flow cytometric analysis of cells labeled with antibody-fluorochrome-oligos against CD3 (PE), CD4 (APC) and CD8 (FITC) and cross-linked with 0.25% PFA (Figure 4a) or 0.5% PFA (Figure 4b) followed by enzymatic detachment. [Diagram 5] Flow cytometric analysis of cells labeled with antibody-fluorochrome-oligos against CD3 (PE), CD4 (APC) and CD8 (FITC) and cross-linked with 1% PFA (Figure 5a) or 2% PFA (Figure 5b) followed by enzymatic detachment. [Figure 6] Flow cytometric analysis of cells labeled with antibody-fluorochrome-oligo against CD3 (PE), CD4 (APC) and CD8 (FITC) and cross-linked with 0.25% PFA (Fig. 6a) or 0.5% PFA (Fig. 6b) followed by enzymatic treatment and mechanical force detachment. [Figure 7] Flow cytometric analysis of cells labeled with antibody-fluorochrome-oligos against CD3 (PE), CD4 (APC) and CD8 (FITC) and cross-linked with 1% PFA (Figure 7a) or 2% PFA (Figure 7b) followed by enzymatic treatment and mechanical force detachment. [Figure 8]PBMCs labeled with DAPI and a CD4-specific antibody bound to a) a barcode moiety (oligonucleotide) and b) a detectable moiety that can be erased and c) a crosslinkable moiety. The oligonucleotide was hybridized with an antisense oligonucleotide labeled with Cy5. The image shows the fluorescent signal of DAPI. [Figure 9] PBMCs labeled with DAPI and a CD4-specific antibody bound to a) a barcode moiety (oligonucleotide) and b) a detectable moiety that can be erased and c) a crosslinkable moiety. The oligonucleotide was hybridized with an antisense oligonucleotide labeled with Cy5. The image shows the fluorescent signal of Cy5. [Figure 10] PBMCs labeled with DAPI and a CD4-specific antibody bound to a) a barcode moiety (oligonucleotide) and b) a detectable moiety that can be erased and c) a crosslinkable moiety. The oligonucleotide was hybridized with an antisense oligonucleotide labeled with Cy5. The image shows an overlay of the fluorescent signals of Cy5 (red) and DAPI (green).

[0015] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "covalent bond" refers to a bond having a dissociation constant of 10-9 M or less. The term "quenching detection moiety X" refers to the removal of fluorescent emission by X. This can be accomplished, for example, by either removing the ability of X to be detected, where X is one of a chromophore, fluorescent, phosphorescent, luminescent, or light absorbing moiety, or by destroying or decomposing the chemistry of X such that emission is undetectable.

[0016] Such destruction or degradation of the chemical nature of X can be achieved, for example, by enzymatic degradation, radiation or oxidative bleaching. The chemicals required for bleaching are known from the publications mentioned above for "Multi Epitope Ligand Cartography", "Chip-Based Cytometry" or "Multioymx" technology.

[0017] A further way to "eliminate the detection moiety X" is to remove X from the conjugate. This can be achieved by equipping the conjugate according to general formula (I) with an enzymatically degradable spacer unit P. When the enzymatically degradable spacer P is digested by adding a suitable enzyme (after detection of X), X is no longer bound to the conjugate and can be removed, for example, by washing.

[0018] Depending on the nature of the antigen recognition moiety Y, this embodiment can have the effect that the antigen recognition moiety Y is or can be removed from the antigen, leaving the target cell untouched, after enzymatic degradation of the spacer P. Antigen recognition moieties Y, e.g. FAB, that require several binding sites to provide stable binding to the antigen, tend to be removed from the antigen upon enzymatic degradation of the spacer P.

[0019] If it is not desired to remove the antigen recognition moiety Y from the antigen, this can be prevented by a further embodiment of the invention, in which the antigen recognition moiety Y and / or the oligonucleotide B comprises a cross-linker unit. In such a variant of the method of the invention, the antigen recognition moiety Y and / or the oligonucleotide B comprises a cross-linker unit capable of providing a covalent bond to the cell (preferably the antigen) recognised by the antigen recognition moiety Y, and in which the covalent binding of the cross-linker to the cell (preferably the antigen) is initiated by radiation, a chemical reaction or an enzymatic reaction.

[0020] Covalent attachment of the cross-linking agent to the cell or antigen can be initiated by radiation, chemical or enzymatic reactions.

[0021] The method of the invention, i.e. steps a) to e), can be subsequently repeated with at least two conjugates having different antigen recognition moieties Y. Alternatively, steps a) to e) can be subsequently repeated with at least two conjugates having different antigen recognition moieties Y and different oligonucleotides B.

[0022] In a further embodiment of the invention, steps a) to e) are carried out by the following steps: f) isolating the cells labeled with at least two conjugates bearing a detection moiety X. g) Lysing the cells h) Adding a second barcode to Oligo B i) adding the same second barcode to the genetic information of the cell (wherein g) and h) can be performed simultaneously, and h) and i) can be performed in a different order). Repeat at least once in addition to the above.

[0023] Optionally, step h) can be performed by hybridizing an antisense oligonucleotide to the sequence representing the genetic information and oligo B, whereby the antisense oligonucleotide is covalently linked to the second barcode. Oligonucleotide B' comprising 2 to 100 nucleotide residues can be used as the second barcode.

[0024] Preferentially, an oligo(dT)-containing oligonucleotide is used to hybridize with the oligo(dA) sequence stretches contained in the poly(A) and oligo(B) of the mRNA of said cell, whereby the oligo(dT) is covalently linked to the second barcode.

[0025] target area The target moiety detected by the method of the present invention can be on any biological specimen, such as tissue sections, cell aggregates, suspension cells or adherent cells. The cells can be live or dead. Preferably, the target moiety is an antigen that is expressed intracellularly or extracellularly on whole animals, organs, tissue sections, cell aggregates or single cells of biological specimens, such as invertebrates (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., Danio rerio, Xenopus laevis) and mammals (e.g., Mus musculus, Homo sapiens).

[0026] Barcode section In the present application, oligonucleotides B with different sequences are called "barcodes" because their unique sequences allow for the identification of a single target. The barcode portion B comprises an oligonucleotide containing 2 to 300 nucleotide residues, preferably 5 to 70 nucleotide residues. As nucleotide residues, natural cytosine (C), adenine (A), guanine (G) and thymine (T) are preferred. By randomly polymerizing these units, a library of oligonucleotides with different sequences can be obtained. For example, a library that randomly produces oligonucleotides containing 10 nucleotide residues can be obtained by polymerizing 4 10 = 1,048,576 members.

[0027] Techniques for generating oligonucleotides and libraries thereof are well known to those of skill in the art, as are techniques for amplifying isolated oligonucleotides to obtain larger quantities thereof. U.S. Patent No. 9,388,465 summarizes these techniques.

[0028] It is therefore a further object of the present invention to provide a library of the conjugates already contemplated, comprising at least 10, preferably at least 100, preferably at least 1000, preferably at least 10000 conjugates with oligonucleotides B having different sequences.

[0029] 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 one selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a light absorbing moiety, a radioactive moiety and a transition metal isotope mass tag moiety.

[0030] Suitable fluorescent moieties are those known in the art of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In these embodiments of the invention, the target moiety labeled with the conjugate is detected by exciting the detection moiety X and detecting the resulting light emission (photoluminescence). In this embodiment, the detection moiety X is preferably a fluorescent moiety.

[0031] Useful fluorescent moieties can be protein-based, e.g., phycobiliproteins, polymers, e.g., polyfluorenes, organic small molecule dyes, e.g., xanthenes, e.g., fluorescein or rhodamine, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethenes, or metal-organic complexes, e.g., Ru, Eu, Pt complexes. In addition to single molecular entities, fluorescent protein or organic small molecule dye clusters and nanoparticles, e.g., quantum dots, upconversion nanoparticles, gold nanoparticles, dyed polymer nanoparticles, can also be used as fluorescent moieties.

[0032] Another group of photoluminescent detection moieties are phosphorescent moieties that have a time-delayed emission of light after excitation. Phosphorescent moieties include metal-organic complexes, such as Pd, Pt, Tb, Eu complexes, or nanoparticles incorporating phosphorescent pigments, such as lanthanide-doped SrAl2O4.

[0033] In another embodiment of the present invention, the target labeled with the conjugate is detected without prior excitation by irradiation. In this embodiment, the detection moiety can be a radioactive label. They can be non-radioactive isotopes or their radioactive isotopes, e.g., tritium, 32 P, 35 S or 14 C or covalently attached labels, e.g., tyrosine 125 I, in fluorodeoxyglucose 18 F or organometallic complexes, i.e. 99 It can be in the form of a radioisotope label by incorporating Tc-DTPA.

[0034] In another embodiment, the detection moiety is capable of generating chemiluminescence, ie, a horseradish peroxidase label in the presence of luminol.

[0035] In another embodiment of the invention, targets labeled with the conjugates are detected by absorption of UV, visible or NIR radiation rather than by radioactive emission. Suitable light-absorbing detection moieties are light-absorbing dyes without fluorescent emission, such as organic small molecule quencher dyes, such as N-arylrhodamines, azo dyes and stilbenes.

[0036] In another embodiment, the optically absorbing detection moiety X can be illuminated with pulsed laser light to generate a photoacoustic signal.

[0037] In another embodiment of the present invention, the conjugate-labeled target is detected by mass spectrometry detection of the transition metal isotope. The transition metal isotope mass tag label can be introduced as a covalently bound metal-organic complex or nanoparticle component. Isotopic tags of lanthanides and adjacent late transition elements are known in the art.

[0038] The detection moiety X can be covalently attached to the spacer P by direct reaction of an activating group on either the detection moiety or on the spacer P with a functional group on either the spacer P or on the detection moiety X, or via a heterobifunctional linker molecule that reacts first with one and then the other binding partner.

[0039] For example, a large number of heterobifunctional compounds are available for linking to an entity. Exemplary entities include azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartrate, Ny-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl [4-azidophenyl]-1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl]aminobenzoate, glutaraldehyde, succinimidyl-[(N-maleimidopropionamido)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 on the detection moiety and a reactive amino group on the spacer P.

[0040] The quasi-covalent attachment of the detection moiety X to the spacer P is -9 This can be achieved by a binding system that provides a dissociation constant equal to or less than M, for example, a biotin-avidin binding interaction.

[0041] Spacer P Generally, any spacer P known in the art of antigen recognition conjugates can be used, for example, LCLC or PEG oligomers.

[0042] In a preferred embodiment, the conjugate according to the invention comprises an enzymatically degradable spacer unit P. The enzymatically degradable spacer P can be any molecule that can be cleaved by a specific enzyme, in particular a hydrolase. For example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids and derivatives thereof are suitable as enzymatically degradable spacers P.

[0043] Suitable polysaccharides are, for example, dextran, pullulan, inulin, amylose, cellulose, hemicellulose, such as xylan or glucomannan, pectin, chitosan or chitin. They can be derivatized to provide functional groups for covalent or non-covalent attachment of detection moiety X and antigen recognition moiety Y. A variety of such modifications are known in the art, for example, imidazolyl carbamate groups can be introduced by reacting the polysaccharide with N,N'-carbonyldiimidazole. Amino groups can then be introduced by reacting the imidazolyl carbamate groups with hexanediamine. Polysaccharides can also be oxidized using periodate to provide aldehyde groups or N,N'-dicyclohexylcarbodiimide and dimethylsulfoxide to provide ketone groups. The aldehyde or ketone functional groups can then be reacted with diamines, preferably under conditions of reductive amination, to provide either amino groups or directly with amino substituents on the proteinaceous binding moiety. Carboxymethyl groups can be introduced by treating the polysaccharide with chloroacetic acid. The carboxy groups can be activated by methods known in the art to produce activated esters, such as N-hydroxysuccinimide esters or tetrafluorophenyl esters, to either react with amino groups of diamines to provide amino groups or directly with amino groups of the proteinaceous binding moiety. Generally, alkyl-bearing functional groups can be introduced by treating the polysaccharide with halogen compounds under alkaline conditions. For example, allyl groups can be introduced by using allyl bromide. Additionally, allyl groups can be used in thiol-ene reactions with thiol-containing compounds, e.g., cysteamine, to introduce amino groups, or can be used directly in thiol-ene reactions with proteinaceous binding moieties with thiol groups liberated by reduction of disulfide bonds or introduced by thiolation, e.g., with 2-iminothiolane.

[0044] Proteins, peptides and depsipeptides used as enzymatically degradable spacers P can be functionalized via the side chain functional groups of amino acids for the attachment of detection moieties X and antigen recognition moieties Y. Side chain functional groups suitable for modification are, for example, the amino groups provided by lysine or the thiol groups provided by cysteine ​​after reduction of disulfide bridges.

[0045] The polyesters and polyesteramides used as enzymatically degradable spacers P can either be synthesized with comonomers providing side chain functionality or be subsequently functionalized. In the case of branched polyesters, functionalization can be via the carboxyl or hydroxyl end groups. Post-polymerization functionalization of the polymer chains can be, for example, by addition to unsaturated bonds, i.e., by thiolene or azide-alkyne reactions, or by the introduction of functional groups by radical reactions.

[0046] Nucleic acids used as enzymatically degradable spacers P are preferably synthesized with functional groups at the 3' and 5' ends suitable for attachment of detection moieties X and antigen recognition moieties Y. For example, phosphoramidite building blocks suitable for nucleic acid synthesis that provide amino or thiol functional groups are known in the art.

[0047] The enzymatically degradable spacer P may be composed of two or more different enzymatically degradable units which are degradable by the same or different enzymes.

[0048] Antigen recognition part Y The term "antigen recognition moiety Y" refers to any type of antibody, antibody fragment or antibody derivative fragment against a target moiety expressed on a biological specimen, for example an antigen expressed intracellularly or extracellularly on a cell. This term relates to fully intact antibodies, antibody fragments or antibody derivative fragments, for example Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, nanobodies. Such antibody fragment derivatives can be synthesized by recombinant techniques, including covalent and non-covalent conjugation containing these types of molecules. Further examples of antigen recognition moieties are peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, artificially engineered binding molecules, for example peptides or aptamers targeting cell surface molecules.

[0049] The conjugates used in the methods of the invention may comprise up to 100, preferably 1 to 20, preferably 2 to 10 antigen recognition moieties Y.

[0050] The interaction between the antigen recognition moiety and the target antigen can be of high affinity or low affinity. The binding interaction of a single low affinity antigen recognition moiety is too low to provide stable binding with the antigen. The low affinity antigen recognition moiety can be multimerized by conjugation to an enzymatically degradable spacer P to provide high binding activity. When the spacer P is enzymatically cleaved or degraded, the low affinity antigen recognition moiety will be monomerized, which will result in the complete removal of the detection moiety X, the spacer P and the antigen recognition moiety Y. The high affinity antigen recognition moiety provides stable binding, which will result in the removal of the detection moiety X and the spacer P.

[0051] Preferably, the term "antigen recognition moiety Y" refers to an antibody against an antigen expressed intracellularly of a biological specimen (target cell), such as IL2, FoxP3, CD154, or an antigen expressed extracellularly thereof, such as CD3, CD14, CD4, CD8, CD25, CD34, CD56 and CD133.

[0052] The antigen-recognizing moiety Y, in particular an antibody, can be attached to the spacer P via a side chain amino or sulfhydryl group. Optionally, the glycosidic side chains of the antibody can be oxidized with periodate to provide aldehyde functional groups.

[0053] The antigen recognition moiety Y can be covalently or non-covalently bound to the spacer P. Methods for covalent or non-covalent conjugation are known to those skilled in the art and are the same as those mentioned for the conjugation of the detection moiety X.

[0054] The method of the present invention is particularly useful for the detection and / or isolation of a specific cell type from a complex mixture and can include two or more sequential or parallel sequences of steps a) to e). Various combinations of conjugates can be used in the method. For example, the conjugate can include antibodies specific for two different epitopes, such as two different anti-CD34 antibodies. Different antigens can be addressed with different conjugates that include different antibodies, such as anti-CD4 and anti-CD8 for differentiation between two separate T cell populations, or anti-CD4 and anti-CD25 for determining different cell subpopulations, such as regulatory T cells.

[0055] enzyme The choice of enzyme as releasing agent is determined by the chemical nature of the enzymatically degradable spacer P and can be one or a mixture of different enzymes. The enzyme is preferably a hydrolase, but also a lyase or a reductase. For example, if the spacer P is a polysaccharide, glycosidases (EC 3.2.1) are most suitable as releasing agents. Glycosidases that recognize specific glycosidic structures, such as dextranase (EC 3.2.1.11), which cleaves at the α(1→6) bond in dextran; pullulanases that cleave either the α(1→6) bond (EC 3.2.1.142) or the α(1→6) and α(1→4) bonds (EC 3.2.1.41) in pullulan; neopullulanase (EC 3.2.1.135) and isopullulanase (EC 3.2.1.57), which cleave the α(1→4) bond in pullulan; α-amylase (EC 3.2.1.1) and maltogenic amylase (EC 3.2.1.133), which cleave the α(1→4) bond in amylose; and β(2→1) fructoside in inulin. Preferred are inulinases (EC 3.2.1.7), which cleave the β(1→4) bond in cellulose, cellulases (EC 3.2.1.4), which cleave the β(1→4) bond in xylan, xylanases (EC 3.2.1.8), which cleave the β(1→4) bond in xylan, pectinases, e.g., endo-pectin lyases (EC 4.2.2.10), which eliminate cleave the α(1→4) D-galacturonan methyl ester bond, or polygalacturonase (EC 3.2.1.15), which cleaves the α(1→4) D-galactosiduronic bond in pectin, chitosanases (EC 3.2.132), which cleave the β(1→4) bond in chitosan, and endo-chitinases (EC 3.2.1.14), which cleave chitin.

[0056] Proteins and peptides can be cleaved by proteinases that must be sequence-specific to avoid degradation of target structures on cells. Sequence-specific proteases are, for example, TEV protease (EC 3.4.22.44), a cysteine ​​protease that cleaves at the sequence ENLYFQ\S, enteropeptidase (EC 3.4.21.9), a serine protease that cleaves after the sequence DDDDK, factor Xa (EC 3.4.21.6), a serine endopeptidase that cleaves after the sequence IEGR or IDGR, or HRV3C protease (EC 3.4.22.28), a cysteine ​​protease that cleaves at the sequence LEVLFQ\GP.

[0057] Depsipeptides or polyesters, which are peptides containing ester bonds in the peptide backbone, can be cleaved by esterases, such as porcine liver esterase (EC 3.1.1.1) or porcine pancreatic lipase (EC 3.1.1.3). Nucleic acids can be cleaved by endonucleases that can be sequence specific, such as restriction enzymes (EC 3.1.21.3, EC 3.1.21.4, EC 3.1.21.5), such as EcoRI, HindII or BamHI, or more generally, such as DNAse I (EC 3.1.21.1), which cleaves phosphodiester bonds adjacent to pyrimidines.

[0058] The amount of enzyme added should be sufficient to substantially degrade the spacer within a desired time. Usually, the detection signal is reduced by at least about 80%, more usually, by at least about 95%, and preferably, by at least about 99%. The release conditions can be empirically optimized in terms of temperature, pH, the presence of metal cofactors, reducing agents, etc. Degradation will usually be complete in at least about 15 minutes, more usually, at least about 10 minutes, and usually within about 30 minutes.

[0059] Cell detection methods The methods and devices for detecting targets labeled with the conjugates of the invention are determined by the detection moiety X.

[0060] In one variant of the present invention, the detection moiety X is a fluorescent moiety. The target labeled with a fluorescent dye-conjugate is detected by exciting the fluorescent moiety X and analyzing the resulting fluorescent signal. The wavelength of excitation is usually selected according to the absorption maximum of the fluorescent moiety X and is provided by a laser or LED source known in the art. When multiple different detection moieties X are used for multiple color / parameter detection, care should be taken to select fluorescent moieties whose absorption spectra do not overlap, or at least whose absorption maxima do not overlap. In the case of a fluorescent moiety as a detection moiety, the target can be detected, for example, under a fluorescent microscope, in a flow cytometer, a spectrofluorometer or a fluorescent scanner. The light emitted by chemiluminescence can be detected by a similar device that omits excitation.

[0061] In another variation of the invention, the detection moiety is a light absorbing moiety, which is detected by the difference between the irradiated light intensity and the transmitted or reflected light intensity. Light absorbing moieties can also be detected by photoacoustic imaging, which uses the absorption of a pulsed laser beam to generate an acoustic, e.g., ultrasound, signal.

[0062] Radioactive detection moieties are detected by the radiation emitted by the radioisotope. Suitable instruments for detecting radioactive radiation include, for example, scintillation counters. In the case of beta emission electron microscopy, it can also be used for detection.

[0063] The transition metal isotope mass tag moieties are detected by mass spectrometry, for example ICP-MS, integrated into a mass cytometry instrument.

[0064] Use of the method The methods of the present invention can be used for a variety of applications in research, diagnostics and cell therapy.

[0065] In a first variant of the invention, a biological specimen, e.g., cells, are detected for enumeration purposes, i.e., to establish the amount of cells from the sample that have a particular set of antigens recognized by the antigen recognition portion of the conjugate.

[0066] In the second variant, one or more populations of biological specimens are detected from the sample and separated as target cells.This variant can be used for purification of target cells, for example, in clinical research, diagnosis and immunotherapy.In this variant, one or more selection steps can be performed after any of steps a), b), c), d) and optionally washing step e).

[0067] In another variant of the invention, the location of the antigen on the biological specimen recognized by the targeting moiety, for example the antigen recognition moiety of the conjugate, is determined. Such techniques are known as "multi-epitope ligand mapping", "chip-based cytometry" or "Multioymx" and are described, for example, in EP 0810428, EP 1181525, EP 1136822 or EP 1224472. In this technique, cells are fixed and contacted with an antibody bound to a fluorescent moiety. The antibody is recognized by each antigen on the biological specimen (for example on the cell surface) and, after removing unbound markers and exciting the fluorescent moiety, the location of the antigen is detected by the fluorescence emission of the fluorescent moiety. In a particular variant, instead of an antibody bound to a fluorescent moiety, an antibody bound to a moiety detectable for MALDI imaging or CyTOF can be used. The skilled artisan knows how to modify this technique based on the fluorescent moiety to work with these detection moieties.

[0068] The location of the target portion is achieved by a digital imaging device having sufficient resolution and sensitivity for the wavelength of the fluorescent radiation. The digital imaging device can be used with or without optical magnification, for example, using a fluorescent microscope. The obtained image is stored in a suitable storage device, for example, a hard drive, for example, in RAW, TIF, JPEG or HDF5 format.

[0069] To detect different antigens, different antibody conjugates can be provided with the same or different fluorescent or antigen recognition moieties Y. Because parallel detection of fluorescent emissions with different wavelengths is limited, antibody-fluorochrome-conjugates are utilized after one another, either sequentially, individually or in small groups (2-10 species).

[0070] In yet another variant of the method according to the invention, the biological specimens of the sample, in particular the cells in suspension, are immobilized by entrapment in microcavities or by adhesion.

[0071] Working Example The following examples are intended to illustrate the invention in more detail, without however limiting the invention to these examples.

[0072] Example 1: Isolation of cells after cycle immunofluorescence analysis for downstream sequencing analysis of nucleic acid information and oligonucleotide barcodes of antibodies used in immunofluorescence analysis Freshly isolated peripheral blood mononuclear cells (PBMCs) were spun at 100×g on a 24-well plate (1E06 PBMCs per well) and fixed with 4% PFA for 10 min. The fixed cells were washed three times and stained for 10 min with a conjugate consisting of a) an antigen-binding moiety (antibody-fluorochrome-oligo conjugate) bound to a barcode moiety and b) a detectable moiety that can be erased, and c) a crosslinking moiety (primary amino group). The antigen-binding moieties were antibodies specific for the proteins CD8, CD3 and CD4 (CD8-FITC-oligo, CD3-PE-oligo, CD4-APC-oligo). The antibody-fluorochrome-oligo conjugates were then crosslinked to the cells for 2 min with either 0.25% paraformaldehyde (PFA), 0.5% PFA, 1% PFA or 2% PFA. All wells were prepared in triplicate.

[0073] Labeling of cells was controlled by microscopy. Figure 1 shows an example of a comparison between CD3 FITC staining after 2 min cross-linking with 0.25% PFA (top) and 2% PFA (bottom).

[0074] Cells were detached from the microscopy using one of three methods: Variant 1: mechanical detachment by scraping the cells; Variant 2: enzymatic detachment by incubation with elastase; Variant 3: combined mechanical and enzymatic detachment using elastase and scraping. After detachment, cells were harvested and analyzed by flow cytometry.

[0075] For variant 1 (scraping), a lot of debris could be detected in the dot plots. Different percentages of PFA for cross-linking did not result in significant differences (Figures 2, 3).

[0076] For variant 2 (elastase) only very little debris could be detected. Again, there was no difference between the four cross-linking levels (Figure 4, Figure 5).

[0077] For variant 3 (elastase + scraping), there was a moderate amount of debris. Again, there was no difference between the four levels of post-fixation (Figure 6, Figure 7).

[0078] As an example, the dot plot in the left part (0.25%) of Figure 3 (variant 2, elastase) shows two distinct populations along each axis resembling APC (CD4) vs. FITC (CD8) labeled cells. As expected, no double labeled cells could be detected. CD8-FITC cells were gated at 11% of the population, and CD4-APC cells at 5.22%, which correlates with expectations when analyzing PBMCs. Both CD8-FITC and CD4-APC positive cells correlate well with CD3-PE, and separate populations of non-CD8-FITC and non-CD4-APC cells are similarly along the CD3-PE axis.

[0079] These results demonstrate that it is possible to detach and analyze cells from microscope slides without losing cell integrity and labeling. The degree of antibody-fluorochrome-oligo cross-linking did not affect the detachment of intact cells. Enzymatic detachment is clearly superior in preserving cell integrity.

[0080] Example 2: Testing the functionality of oligonucleotide-barcodes on antibody-fluorochrome-oligonucleotide conjugates Freshly isolated peripheral blood mononuclear cells (PBMCs) were spun at 100×g on a 24-well plate (1E06 PBMCs per well) and fixed with 4% PFA for 10 min. Fixed cells were washed three times and stained with DAPI 1:20 for 10 min at room temperature in the dark. Cells were washed five times and then stained for 10 min with a conjugate consisting of a) an antigen-binding moiety (antibody-fluorochrome-oligo conjugate) bound to a barcode moiety and b) a detectable moiety that can be erased, and c) a cross-linking moiety (primary amino group). The antigen-binding moieties were antibodies specific for the proteins CD8, CD3 and CD4.

[0081] The conjugate was then crosslinked to the cells with 0.25% PFA for 2 min. The antibody-fluorochrome-oligo conjugate was hybridized to an antisense oligonucleotide labeled with Cy5. The labeling of the cells was controlled by microscopy (Figure 8-X).

[0082] Figure 8 shows the nuclear staining of cells using DAPI (nuclear staining). Figure 9 shows the labeling of cells using CD4 specific antibody-oligo conjugates with respective CY5-labeled antisense oligos as an example (cell surface staining). It is clearly visible that a subpopulation of cells is labeled on the surface by the antibody-oligo conjugates. It can therefore be concluded that crosslinking of oligonucleotides to cells does not inhibit hybridization of antisense oligonucleotides, i.e., the function of the oligonucleotides as barcodes is preserved. Figure 10 shows an overlay of Figures 8 and 9, which confirms the specificity of labeling by antibody-oligonucleotide conjugates.

Claims

1. A library of conjugates comprising oligonucleotides having different sequences, the conjugates comprising: General formula (I) X ｎ -P-Y ｍ B ｏ (I) [In the formula, X is a detection moiety; P is a spacer unit, Y is an antigen recognition moiety, B is an oligonucleotide containing 2 to 300 nucleotide residues, n, m, and o are independent integers from 1 to 100; P and B are covalently bonded to Y, X is covalently bonded to P, and X is erasable. having A library, characterized in that the antigen recognition moiety Y and / or the oligonucleotide B comprises a crosslinker unit capable of providing a covalent bond to a cell recognized by the antigen recognition moiety Y, and the covalent bond of the crosslinker unit to the cell is initiated by radiation, a chemical reaction or an enzymatic reaction.

2. The library according to claim 1 , characterized in that the spacer unit P is enzymatically degradable.

3. 3. The library according to claim 1, wherein the detection portion X is removable by radiation or by enzymatic decomposition of the spacer unit P.

4. 4. The library of claim 1, wherein the detection moiety is selected from the group consisting of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a light absorbing moiety, a radioactive moiety, a transition metal and an isotopic mass tag moiety.

5. A library described in any one of claims 1 to 4, characterized in that the spacer unit P is selected from the group consisting of polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids and derivatives thereof.

6. The library according to any one of claims 1 to 5, characterized in that the antigen recognition portion 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 a peptide or aptamer targeting a cell surface molecule.

7. A library described in any one of claims 1 to 6, comprising at least 10 conjugates.

8. 1. A method for detecting a target moiety on a cell, comprising: a) the conjugate is General formula (I) X ｎ -P-Y ｍ B ｏ (I) [In the formula, X is a detection moiety; P is a spacer unit, Y is an antigen recognition moiety, B is an oligonucleotide containing 2 to 300 nucleotide residues, n, m, and o are independent integers from 1 to 100; P and B are covalently bonded to Y, X is covalently bonded to P, and X is erasable. wherein said antigen recognition moiety Y and / or said oligonucleotide B comprises a crosslinker unit capable of providing a covalent bond to a cell recognized by the antigen recognition moiety Y, and said covalent bond of said crosslinker unit to said cell is initiated by radiation, a chemical reaction or an enzymatic reaction, b) contacting a sample of a biological specimen with the conjugate, thereby labeling the target moiety recognized by the antigen recognition moiety Y; c) detecting the target moiety labeled with the conjugate by the detection moiety X; d) erasing the detected portion X; method.

9. 9. The method according to claim 8, characterized in that steps a) to d) are subsequently repeated.

10. 10. The method according to claim 8 or 9, characterized in that steps a) to d) are subsequently repeated, with the proviso that each combination of conjugates has a different antigen recognition moiety Y.

11. 11. The method according to any one of claims 8 to 10, characterized in that after step c), the cells labeled with the conjugate carrying the detection moiety X are isolated in step e).

12. 12. The method according to claim 8, wherein the detection moiety X is eliminated by radiation or by enzymatic cleavage of the spacer unit P.

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