Conjugates with enzymatically releasable detection moieties and barcode moieties

Abstract

The present invention relates to a conjugate X having the general formula (I) n -P-Y m B o (I), wherein X is a detection moiety, P is a spacer unit, Y is an antigen recognizing moiety, B is an oligonucleotide comprising 2 to 300 nucleotide residues, and n, m, o are independent integers between 1 and 100, wherein P and B are covalently bound to Y, and X is covalently bound to P, and wherein X is erasable. Further, the present invention relates to a library of such conjugates, and to a method for detecting a target cell using the conjugate or the library of conjugates.

Description

TECHNICAL FIELD

[0001] The present invention relates to a conjugate comprising a detection moiety and an antigen recognizing moiety, and the use of such conjugate for detecting or identifying a target moiety or a target cell or for detecting or identifying a target moiety or a target cell from a cell sample, the detection moiety and the antigen recognizing moiety being linked, optionally via an enzymatically degradable spacer, wherein the antigen recognizing moiety has an oligonucleotide as a barcode. BACKGROUND

[0002] Cellular detection with conjugates that can be removed from the cell after detection is a known procedure. For example, US7776562 discloses a reversible fluorescent labeling procedure, wherein the non-covalent binding in the conjugate is broken by the addition of a competitor molecule, removing the conjugate from the cell.

[0003] A different reversible fluorescent labeling method is disclosed in EP3037821A1, wherein the conjugate has an enzymatically degradable spacer. By adding the appropriate enzyme, the conjugate is destroyed and the detection moiety and the antigen recognizing moiety of the conjugate are removed from the cell.

[0004] Known procedures enable the detection of different cells in a cell sample. For example, by repeated staining, detection and destaining with conjugates having different antigen recognizing moieties, several phenotypes of cells and their location in a tissue can be detected or distinguished.

[0005] However, the genetic information of single, isolated cells cannot be analyzed.

[0006] In a different technical field, it is known to identify genetic information obtained from single cells by conjugating the cells with polynucleotides as barcodes. These methods involve the synthesis of libraries of different polynucleotides, which can be sequenced in order to identify the single cells.

[0007] For example, the biology and the necessary hardware to isolate cells are disclosed in US9388456 or US9695468. However, this technology focuses on single isolated cells and does not involve cells in the context of a tissue or cell culture with intercellular interactions. SUMMARY

[0008] It is therefore an object of the present invention to provide a conjugate that enables the identification of single cells and their localization / position on or in a tissue and their phenotype. Further, the means for identification should be erasable in order to allow for multiple identification steps.

[0009] It was found that a conjugate consisting of an antigen binding moiety coupled to a) a barcode moiety and b) a detection moiety that can be erased is suitable for single cell identification.

[0010] Due to the erasable detection moiety, the biological specimen can be subjected to the same or a different conjugation again without interference of the previous respective labels. All barcode moieties remain on the cell, enabling to link via sequencing the phenotype of the cell detected by the binder to the genetic information of the individual cell detected by sequencing.

[0011] It is therefore an object of the present application a conjugate having the general formula (I)

[0012] X n -P-Y m B o (I),

[0013] wherein X is a detection moiety,

[0014] P is a spacer unit,

[0015] Y is an antigen recognizing moiety,

[0016] B is an oligonucleotide comprising 2 to 300 nucleotide residues,

[0017] and n, m, o are independent integers between 1 and 100,

[0018] wherein P and B are covalently bound to Y and X is covalently bound to P, and wherein X is erasable.

[0019] In a preferred embodiment, the spacer unit P is enzymatically degradable.

[0020] It is a further object of the present application a method for detecting a target moiety on a cell by

[0021] a) providing at least one conjugate having the general formula (I)

[0022] X n -P-Y m B o (I),

[0023] wherein X is a detection moiety,

[0024] P is a spacer unit,

[0025] Y is an antigen recognizing moiety,

[0026] B is an oligonucleotide comprising 2 to 300 nucleotide residues,

[0027] and n, m, o are independent integers between 1 and 100,

[0028] wherein P and B are covalently bound to Y and X is covalently bound to P, and wherein X is erasable; b) contacting a sample of a biological specimen with the at least one conjugate, thereby labeling the target moiety recognized by the antigen recognizing moiety Y;

[0029] c) detecting the target moiety labeled with the conjugate having the detection moiety X;

[0030] d) isolating the cell labeled with the conjugate having the detection moiety X;

[0031] e) erasing the detection moiety X. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A comparison between CD3 FITC staining at 20x magnification after cross-linking with 0.25% PFA (top) and 2% PFA (bottom) for two minutes is shown.

[0033] Figure 2 shows flow cytometric analysis of mechanically detached cells after labeling of CD3 (PE), CD4 (APC) and CD8 (FITC) with antibody-fluorochrome-oligonucleotide and cross-linking with 0.25% PFA ( Figure 2a ) or 0.5% PFA ( Figure 2b ).

[0034] Figure 3 shows flow cytometric analysis of mechanically detached cells after labeling of CD3 (PE), CD4 (APC) and CD8 (FITC) with antibody-fluorochrome-oligonucleotide and cross-linking with 1% PFA ( Figure 3a ) or 2% PFA ( Figure 3b ).

[0035] Figure 4 shows flow cytometric analysis of enzymatically detached cells after labeling of CD3 (PE), CD4 (APC) and CD8 (FITC) with antibody-fluorochrome-oligonucleotide and cross-linking with 0.25% PFA ( Figure 4a ) or 0.5% PFA ( Figure 4b ).

[0036] Figure 5 shows flow cytometric analysis of enzymatically detached cells after labeling of CD3 (PE), CD4 (APC) and CD8 (FITC) with antibody-fluorochrome-oligonucleotide and cross-linking with 1% PFA ( Figure 5a ) or 2% PFA ( Figure 5b ).

[0037] Figure 6 shows flow cytometry analysis of cells detached by enzymatic treatment and mechanical force after labelling CD3 (PE), CD4 (APC) and CD8 (FITC) with antibody-fluorochrome-oligonucleotide and cross-linking with 0.25% PFA Figure 6a ) or 0.5% PFA Figure 6b ).

[0038] Figure 7 shows flow cytometry analysis of cells detached by enzymatic treatment and mechanical force after labelling CD3 (PE), CD4 (APC) and CD8 (FITC) with antibody-fluorochrome-oligonucleotide and cross-linking with 1% PFA Figure 7a ) or 2% PFA Figure 7b ).

[0039] Figure 8 PBMCs labelled with DAPI and CD4 specific antibodies coupled to a) a barcode moiety (oligonucleotide) and b) a detectable moiety able to be erased and c) a cross-linkable moiety. The oligonucleotide is hybridized with an antisense oligonucleotide labelled with Cy5. The image shows the fluorescent signal of DAPI.

[0040] Figure 9 PBMCs labelled with DAPI and CD4 specific antibodies coupled to a) a barcode moiety (oligonucleotide) and b) a detectable moiety able to be erased and c) a cross-linkable moiety. The oligonucleotide is hybridized with an antisense oligonucleotide labelled with Cy5. The image shows the fluorescent signal of Cy5.

[0041] Figure 10 PBMCs labelled with DAPI and CD4 specific antibodies coupled to a) a barcode moiety (oligonucleotide) and b) a detectable moiety able to be erased and c) a cross-linkable moiety. The oligonucleotide is hybridized with an antisense oligonucleotide labelled with Cy5. The image shows the overlay of the fluorescent signal of Cy5 (red) and DAPI (green). DETAILED DESCRIPTION

[0042] The term "covalent binding" refers to a bond with a dissociation constant < 10 -9 The term "eraseable detectable moiety X" refers to the elimination of the fluorescent emission by X. This can be achieved by eliminating the detectable ability of X (for example in case X is one of a chromophore moiety, a fluorescent moiety, a phosphorescent moiety, a luminescent moiety, a light absorbing moiety) or by destroying or degrading the chemical properties of X such that no emission is detectable upon excitation of X.

[0043] Such a chemical property that destroys or degrades X can be achieved by e.g. enzymatic degradation, radiation or oxidative bleaching. The chemicals required for bleaching can be learned from the above mentioned publications on "Multiepitope Ligand Mapping", "Chip-based cytometry" or "Multioymx" technology.

[0044] Another approach to "erasing the detection moiety X" is to remove X from the conjugate. This can be achieved by providing the conjugate according to general formula (I) with an enzymatically degradable spacer unit P. When (after detection of X) the enzymatically degradable spacer P is digested by adding the appropriate enzyme, X is no longer bound to the conjugate and can be removed by e.g. washing.

[0045] Depending on the nature of the antigen recognition moiety Y, this embodiment can have the effect that after enzymatic degradation of the spacer P, the antigen recognition moiety Y is or can be removed from the antigen without contacting the target cell. Antigen recognition moieties Y that require several binding sites to provide stable binding to an antigen (such as a FAB) are easily removed from the antigen upon enzymatic degradation of the spacer P.

[0046] 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 present invention, wherein the antigen recognition Y and / or the oligonucleotide B has a crosslinker unit. In such a variant of the method of the present invention, the antigen recognition Y and / or the oligonucleotide B has a crosslinker unit that is able to provide covalent binding to the cell (preferably the antigen) that is recognized by the antigen recognition moiety Y and the covalent binding of the crosslinker to the cell (preferably the antigen) is initiated by radiation, a chemical reaction or an enzymatic reaction.

[0047] The covalent binding of the crosslinker to the cell or the antigen can be initiated by radiation, a chemical reaction or an enzymatic reaction.

[0048] The method of the present 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.

[0049] In a further embodiment of the present invention, steps a) to e) are repeated at least once in addition to the following steps:

[0050] f) isolating the cell labeled with the at least 2 conjugates having a detection moiety X;

[0051] g) lysing the cell;

[0052] h) adding a second barcode to the oligonucleotide B;

[0053] 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 an alternating order).

[0054] Alternatively, step h) can be performed by hybridizing an antisense oligonucleotide to the sequence represented in the genetic information to the oligonucleotide B, whereby the antisense oligonucleotide is covalently linked to the second barcode. An oligonucleotide B' comprising 2 to 100 nucleotide residues can be used as the second barcode.

[0055] Preferably, Oligo(dT) containing oligonucleotides are used to hybridize to the poly(A) of the mRNA of the cell and to the oligo(dA) sequence comprised in Oligo B, wherein Oligo(dT) is covalently linked to the second barcode.

[0056] Target moiety

[0057] The target moiety to be detected with the method of the application can be on any biological specimen, such as a tissue section, a cell aggregate, a suspension cell or an adherent cell. The cell can be alive or dead. Preferably, the target moiety is an intracellular or extracellular antigen expressed on a biological specimen such as a whole animal, an organ, a tissue section, a cell aggregate, or a single cell of an invertebrate (e.g., Caenorhabditis elegans, Drosophila melanogaster), a vertebrate (e.g., Danio rerio, Xenopus laevis) and a mammal (e.g., Mus musculus, Homo sapiens).

[0058] Barcoding moiety

[0059] In the present application, oligonucleotides B having different sequences are referred to as "barcodes" because they allow the identification of individual targets by their unique sequence. The barcode moiety B comprises an oligonucleotide comprising 2 to 300 nucleotide residues, preferably 5 to 70 nucleotide residues. As nucleotide residues, naturally occurring cytosine (C), adenine (A), guanine (G) and thymine (T) are preferred. By randomly polymerizing these units, a library of oligonucleotides having different sequences can be obtained. For example, randomly generating a library of oligonucleotides comprising 10 nucleotide residues will have 4 10 = 1048576 members.

[0060] Techniques for generating oligonucleotides and libraries thereof, as well as techniques for amplifying isolated oligonucleotides to obtain larger amounts of oligonucleotides, are well known to the person skilled in the art. US 9388465 summarizes these techniques.

[0061] It is therefore a further object of the present application to provide a library of conjugates as already discussed, which library comprises at least 10, preferably at least 100, preferably at least 1000, preferably at least 10 000 conjugates with oligonucleotides B having different sequences.

[0062] Detection moiety

[0063] The detection moiety X of the conjugate can be any moiety having a property or function useful for detection purposes, such as those selected from the group consisting of chromophoric moieties, fluorescent moieties, phosphorescent moieties, luminescent moieties, light absorbing moieties, radioactive moieties and transition metal isotope mass tag moieties.

[0064] Suitable fluorescent moieties are those known in the art of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In these embodiments of the application, the target moiety labeled with the conjugate is detected by exciting the detection moiety X and detecting the resulting emission (photoluminescence). In this embodiment, the detection moiety X is preferably a fluorescent moiety.

[0065] Useful fluorescent moieties can be proteinaceous (such as phycobiliproteins), polymeric (such as polyfluorenes), small organic molecule dyes (such as xanthene, like fluorescein, or rhodamine, cyanine, oxazine, coumarin, acridine, oxadiazole, pyrene, methine pyrrole, or metal-organic complexes (such as Ru, Eu, Pt complexes). In addition to single molecular entities, clusters of fluorescent proteins or small organic molecule dyes, as well as nanoparticles (such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymeric nanoparticles) can also be used as fluorescent moieties.

[0066] Another group of photoluminescent detection moieties are phosphorescent moieties, which emit light with a long delay after excitation. Phosphorescent moieties include metal-organic complexes (such as Pd, Pt, Tb, Eu complexes), or nanoparticles doped with phosphorescent pigments (such as lanthanide-doped SrAl2O4).

[0067] In another embodiment of the application, the target labeled with the conjugate is detected without prior excitation by irradiation. In this embodiment, the detection moiety can be a radioactive label. The detection moiety can take the form of a radioactive isotope label, by exchanging a non-radioactive isotope for its radioactive isotope (such as tritium, 32 P, 35 S or 14 C), or by introducing a covalently bound label (such as 125 I) bound to tyrosine, fluorodeoxyglucose, 18 F or metal-organic complexes (i.e. 99 Tc-DTPA).

[0068] In another embodiment, the detection moiety is capable of causing chemiluminescence in the presence of luminol, i.e. horseradish peroxidase labelling.

[0069] In another embodiment of the application, the target labelled with the conjugate is not detected by emission of radiation, but by absorption of UV, visible or NIR radiation. Suitable light-absorbing detection moieties are light-absorbing dyes without fluorescence emission, such as small organic molecule quenching dyes like N-aryl rhodamines, azo dyes and stilbenes.

[0070] In another embodiment, the light-absorbing detection moiety X is capable of being irradiated with pulsed laser light, generating a photoacoustic signal.

[0071] In another embodiment of the application, the target labelled with the conjugate is detected by transition metal isotopes by mass spectrometry. Transition metal isotope mass tag labelling can be introduced as covalently bound metal-organic complex or nanoparticle component. Isotope labelling of lanthanides and adjacent late transition elements is known in the art.

[0072] The detection moiety X can be covalently coupled to the spacer P either directly by reaction of an activated group on the detection moiety or spacer P with a functional group on the spacer P or detection moiety X, or via a heterobifunctional linker molecule which reacts with one binding partner first and then with the other binding partner.

[0073] For example, a number of heterobifunctional compounds are available for attachment to entities. Exemplary entities include: azidobenzhydrazide, N-[4-(p-azidosalicylamido)butyl]-3'-[2'- pyridyldithio]propionamide), bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyl tartarate, N-y-maleimidobutyryloxy succinimide 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. A preferred linking group is 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) or 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N- hydroxysuccinimide ester (SMCC), wherein the detection moiety has an active thiol group and the spacer P has an active amino group.

[0074] Quasi-covalent binding of the detection moiety X to the spacer P can be achieved by providing <10 -9The dissociation constant of M is combined with a system, for example a biotin-avidin binding interaction.

[0075] Spacer P

[0076] In general, any spacer P known in the art of antigen recognizing conjugates, such as LCLC or PEG oligomers, can be used.

[0077] In a preferred embodiment, the conjugate according to the application 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. Suitable as enzymatically degradable spacer P are, for example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids and derivatives thereof.

[0078] Suitable polysaccharides are, 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 covalent or non-covalent binding of the detection moiety X and the antigen recognizing moiety Y. A variety of such modifications are known in the art, for example imidazolylcarbamate groups can be introduced by reacting the polysaccharide with N,N'-carbonyldiimidazole. Subsequently, amino groups can be introduced by reacting the imidazolylcarbamate groups with hexanediamine. The polysaccharide can also be oxidized using periodate to provide aldehyde groups, or with N,N'-dicyclohexylcarbodiimide and dimethyl sulfoxide to provide ketone groups. The aldehyde or ketone functional groups can subsequently be reacted with diamines, preferably under reductive amination conditions, to provide amino groups, or directly with amino substituents on the protein-containing binding moiety. Carboxymethyl groups can be introduced by treating the polysaccharide with chloroacetic acid. Activation of the carboxyl groups with methods known in the art to yield activated esters, such as N-hydroxysuccinimidyl esters or tetrafluorophenyl esters, allows reaction with amino groups of diamines to provide amino groups, or directly with amino groups of the protein-containing binding moiety. In general, functional group-containing alkyl groups can be introduced by treating the polysaccharide with halogen compounds under basic conditions. For example, allyl groups can be introduced by using allyl bromide. Allyl groups can further be used in thiol-ene reactions with thiol-containing compounds, such as cystamine, to introduce amino groups, or directly in thiol-ene reactions with the protein-containing binding moiety, wherein the thiol groups are liberated by reduction of disulfide bridges or introduced, for example, by alkylthiolation with 2-iminothiolane.

[0079] Proteins, peptides and depsipeptides used as enzymatically degradable spacer P can be functionalized via side chain functionalities of the amino acids for attachment of the detection moiety X and the antigen recognizing moiety Y. For example, suitable side chain functionalities for modification are the amino groups provided by lysine or the thiol groups provided by cysteine after reduction of disulfide bridges.

[0080] Polyesters and polyesteramides used as the enzymatically degradable spacer P can be synthesized with co-monomers that provide side chain functionality or can be functionalized subsequently. In the case of branched polyesters, functionalization can be performed via carboxyl or hydroxyl end groups. Post-polymerization functionalization of the polymer chain can be performed via addition of, for example, unsaturated bonds (i.e. thiol reaction or azide base reaction) or via the introduction of functional groups by free radical reactions.

[0081] Nucleic acids used as the enzymatically degradable spacer P are preferably synthesized using functional groups at the 3'- and 5'-ends that are suitable for attachment of the detection moiety X and the antigen recognizing moiety Y. For example, suitable phosphoramidite building blocks for the synthesis of nucleic acids provide amino or thiol functional groups, which are known in the art.

[0082] The enzymatically degradable spacer P can be composed of more than one different enzymatically degradable unit, which can be degraded by the same or different enzymes.

[0083] Antigen recognition moiety Y

[0084] The term "antigen recognizing moiety Y" refers to any kind of antibody, fragmented antibody or fragmented antibody derivative directed against a target moiety expressed on a biological specimen, such as an antigen expressed on a cell, intracellularly or extracellularly. The term relates to fully intact antibodies, fragmented antibodies or fragmented antibody derivatives, such as Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, nanobodies. Such fragmented antibody derivatives can be synthesized by recombinant procedures, including covalent and non-covalent conjugates containing these kinds of molecules. Further examples for antigen recognizing moieties are peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, artificially engineered binding molecules (e.g. peptides or aptamers targeting e.g. cell surface molecules).

[0085] The conjugate used in the method of the application can comprise up to 100, preferably 1-20, preferably 2-10 antigen recognizing moieties Y.

[0086] The interaction of the antigen recognizing moiety with the target antigen can be of high affinity or of low affinity. The binding interaction with a single low affinity antigen recognizing moiety is too low to provide a stable bond to the antigen. The low affinity antigen recognizing moiety can be multimerized by conjugation to the enzymatically degradable spacer P to provide a high affinity. When the spacer P is cleaved or enzymatically degraded, the low affinity antigen recognizing moiety will be monomerized, which results in the complete removal of the detection moiety X, the spacer P and the antigen recognizing moiety Y. The high affinity antigen recognizing moiety provides a stable bond, which results in the removal of the detection moiety X and the spacer P.

[0087] Preferably, the term "antigen recognizing moiety Y" refers to an antibody directed against an antigen expressed intracellularly by the biological specimen (target cell) such as IL2, FoxP3, CD154 or an antigen expressed extracellularly such as CD3, CD14, CD4, CD8, CD25, CD34, CD56 and CD133.

[0088] The antigen recognizing moiety Y, in particular an antibody, can be coupled to the spacer P via a side chain amino or thiol group. In some cases, the glycoside side chain of the antibody can be oxidized by periodate, thereby generating an aldehyde function.

[0089] The antigen recognizing moiety Y can be coupled covalently or non-covalently to the spacer P. Methods for covalent or non-covalent conjugation are known to the person skilled in the art and are identical to the methods mentioned for the conjugation of the detection moiety X.

[0090] The method of the application is particularly suitable for the detection and / or isolation of specific cell types from complex mixtures and can comprise more than one consecutive or parallel sequence of steps a) to e). The method can use various combinations of conjugates. For example, the conjugate can comprise antibodies specific for two different epitopes, such as two different anti-CD34 antibodies. Different antigens can be treated with different conjugates comprising different antibodies, for example, anti-CD4 and anti-CD8 for distinguishing two different T cell populations or anti-CD4 and anti-CD25 for determining different cell subpopulations such as regulatory T cells.

[0091] Enzyme

[0092] The choice of enzyme as a demasking agent is determined by the chemical nature of the spacers P that are enzymatically degradable and can be one enzyme or a mixture of different enzymes. The enzyme is preferably a hydrolase, but can also be a lyase or a reductase. If, for example, the spacers P are polysaccharides, glycosidases (EC 3.2.1) are most suitable as demasking agents. Preferred are glycosidases that recognize specific glycosidic structures, such as dextranase (EC 3.2.1.11), which cleaves at the alpha(1—>6) bonds of dextran; pullulanase (EC 3.2.1.142), which cleaves alpha(1—>6) bonds of pullulan, or pullulanase (EC 3.2.1.41), which cleaves alpha(1—>6) and alpha(1—>4) bonds of pullulan; neopullulanase (EC 3.2.1.135) and iso-pullulanase (EC 3.2.1.57), which cleave alpha(1—>4) bonds of pullulan. Alpha-amylase (EC 3.2.1.1) and maltogenic amylase (EC 3.2.1.133), which cleave alpha(1—>4) bonds in amylose; inulinase (EC 3.2.1.7), which cleaves beta(2—>1) fructosidic bonds in inulin; cellulase (EC 3.2.1.4), which cleaves at the beta(1—>4) bonds of cellulose; xylanase (EC 3.2.1.8), which cleaves at the beta(1—>4) bonds of xylan; pectinases such as endo-pectate lyases (EC 4.2.2.10), which cleave eliminatively at the alpha(1—>4) D-galacturonan methyl ester bonds; or polygalacturonase (EC 3.2.1.15), which cleaves at the alpha(1—>4) D-galactosiduronic acid bonds of pectin; chitosanase (EC 3.2.1.132), which cleaves at the beta(1—>4) bonds of chitosan; and endochitinase (EC 3.2.1.14), which is used to cleave chitin.

[0093] Proteins and peptides can be cleaved by proteases, which need to have sequence specificity to avoid degradation of target structures on the cell. As an example, sequence-specific proteases are: TEV protease (EC 3.4.22.44), which is a cysteine protease cleaving at the sequence ENLYFQ\; enterokinase (EC 3.4.21.9), which is a serine protease cleaving after the sequence DDDDK; Factor Xa (EC 3.4.21.6), which is a serine endopeptidase cleaving after the sequence IEGR or IDGR; or HRV3C protease (EC 3.4.22.28), which is a cysteine protease cleaving at the sequence LEVLFQ\GP.

[0094] A depsipeptide (i.e. a peptide containing ester bonds in the peptide backbone) or a polyester can be cleaved by an esterase such as pig liver esterase (EC 3.1.1.1) or pig pancreatic lipase (EC 3.1.1.3). A nucleic acid can be cleaved by an endonuclease which can be sequence specific such as a restriction enzyme (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 the phosphodiester bond adjacent to a pyrimidine.

[0095] The amount of enzyme added needs to be sufficient to degrade the spacer substantially within the desired time period. Typically, the detection signal is reduced by at least about 80%, more typically by at least about 95%, and preferably by at least about 99%. The conditions of the release can be empirically optimized with respect to temperature, pH, presence of metal cofactors, reducing agents, etc. The degradation will typically be completed within at least about 15 minutes, more typically within at least about 10 minutes, and will typically not exceed about 30 minutes.

[0096] Cell detection method

[0097] The method and the device for the detection of a target labeled with a conjugate of the application are determined by the detection moiety X.

[0098] In a variant of the application, the detection moiety X is a fluorescent moiety. The detection of a target labeled with a fluorescent dye conjugate is performed by exciting the fluorescent moiety X and analyzing the resulting fluorescent signal. The wavelength of excitation is typically chosen according to the absorption maximum of the fluorescent moiety X and is provided by a laser source or an LED source known in the art. If several different detection moieties X are used for multi-color / multi-parameter detection, care should be taken to select fluorescent moieties whose absorption spectra do not overlap, at least whose absorption maxima do not overlap. In the case where the fluorescent moiety is the detection moiety, the target can be detected under a fluorescence microscope, in a flow cytometer, a spectrofluorimeter or a fluorescence scanner, for example. The light emitted by a chemiluminescent moiety can be detected by similar instruments omitting the excitation.

[0099] In another variant of the application, the detection moiety is a light-absorbing moiety, which is detected by the difference between the intensity of the light of the illumination and the intensity of the light transmitted or reflected. The light-absorbing moiety can also be detected by photoacoustic imaging, which uses the absorption of a pulsed laser beam to generate an acoustic effect like an ultrasound signal.

[0100] A radioactive detection moiety is detected by the radiation emitted by the radioisotope. Suitable instruments for detecting radioactive radiation include, for example, a scintillation counter. In the case of beta emission, detection can also be performed using an electron microscope.

[0101] The transition metal isotope mass tag moiety is detected by mass spectrometry methods, such as ICP-MS integrated in a mass cytometry instrument.

[0102] Use of method

[0103] The method of the application can be used for various applications in research, diagnostics and cell therapy.

[0104] In a first variant of the application, the biological specimen (such as cells) are detected for the purpose of enumeration, i.e. to determine the amount of cells from a sample having a specific set of antigens recognized by the antigen recognizing moiety of the conjugate.

[0105] In a second variant, one or more populations of biological specimens are detected from a sample and separated as target cells. This variant can be used for the purification of target cells, for example in clinical research, diagnostics and immunotherapy. In this variant, one or more sorting steps can be performed after any of the steps a), b), c), d) and optionally the washing step e).

[0106] In another variant of the application, the location of a target moiety (such as an antigen) recognized by the antigen recognizing moiety of the conjugate on a biological specimen is determined. Such techniques are referred to as "multiplex ligand mapping", "chip-based cytometry" or "Multioymx" and are described in e.g. EP 0810428, EP 1181525, EP 1136822 or EP 1224472. In this technique, cells are fixed and brought into contact with antibodies coupled to fluorescent moieties. The antibodies are recognized by the corresponding antigens on the biological specimen (e.g. the cell surface) and, after removal of unbound label and excitation of the fluorescent moieties, the location of the antigens is detected by the fluorescent emission of the fluorescent moieties. In certain variants, antibodies coupled to detectable moieties other than fluorescent moieties can be used, such as MALDI imaging or CyTOF detectable moieties. The skilled person will understand how to modify the fluorescent moiety-based techniques to use these detection moieties.

[0107] The location of the target moiety is achieved by means of a digital imaging device having sufficient resolution and sensitivity for the wavelength of the fluorescent radiation. The digital imaging device can be used with e.g. a fluorescence microscope, with or without optical magnification. The generated images are stored on a suitable storage device (such as a hard drive) in e.g. RAW, TIF, JPEG or HDF5 format.

[0108] To detect different antigens, different antibody conjugates can be provided having the same or different fluorescent moieties or antigen recognizing moieties Y. Since parallel detection of fluorescent emissions having different wavelengths is limited, the antibody-fluorescent dye-conjugates are used individually in succession, or in small groups (2-10) in succession.

[0109] In yet another variant of the method according to the application, the biological specimen of the sample, in particular the suspended cells, are immobilized by capture in the microcavities or by adhesion.

[0110] Examples

[0111] The following examples are intended to explain the application in more detail, but the application is not limited to these examples.

[0112] Example 1: Nucleic acid information and downstream sequencing analysis of oligonucleotide barcodes of antibodies used in immunofluorescence analysis after separation of cells following cyclic immunofluorescence analysis Figure 1

[0113] Freshly isolated peripheral blood mononuclear cells (PBMC) were spun onto a 24 well plate with 100xg (1E06 PBMC per well) and fixed with 4% PFA for 10 minutes. Fixed cells were washed 3 times and stained for 10 minutes with conjugates consisting of an antigen binding moiety coupled to a) a barcode moiety, b) a detectable moiety capable of being erased (antibody-fluorochrome-oligonucleotide conjugate) and c) a cross-linkable moiety (primary amino group). The antigen binding moiety was an antibody specific for the proteins CD8, CD3 and CD4 (CD8-FITC-oligonucleotide, CD3-PE-oligonucleotide, CD4-APC-oligonucleotide). Then, the antibody-fluorochrome-oligonucleotide conjugate was cross-linked to the cells with 0.25% paraformaldehyde (PFA), 0.5% PFA, 1% PFA or 2% PFA for 2 minutes. All wells were prepared in triplicates.

[0114] The labeling of the cells was controlled by microscopy. Example 2: Functional testing of oligonucleotide barcodes on antibody-fluorochrome- oligonucleotide conjugates A comparative example between CD3 FITC staining after cross-linking with 0.25% PFA (top) and 2% PFA (bottom) for two minutes is shown.

[0115] The cells were detached from the microscope using one of the following three methods: Variant 1 : mechanical detachment by scraping off the cells; Variant 2: enzymatic detachment by incubation with elastase; Variant 3: a combination of enzymatic detachment using elastase and mechanical detachment using scraping.

[0116] After detachment, the cells were collected and analyzed by flow cytometry.

[0117] For Variant 1 (scraping), many fragments could be detected in the dot plot. Different percentages of PFA for cross-linking did not lead to significant differences (Fig. 2, Fig. 3).

[0118] For Variant 2 (elastase), only few fragments could be detected. Again, there was no difference between the four cross-linking levels (Fig. 4, Fig. 5).

[0119] For variant 3 (elastase plus scraping), there was a moderate amount of debris. Again, there was no difference between the four grades after fixation (Fig. 6, Fig. 7).

[0120] As an example, the dot plot of the left portion (0.25%) of Fig. 3 (variant 2, elastase) shows two distinct populations along each axis, similar to APC (CD4) versus FITC (CD8) labeled cells. As expected, no double-labeled cells could be detected. CD8-FITC cells accounted for 11% of the population, and CD4-APC cells accounted for 5.22%, which correlates with expectations when analyzing PBMCs. Both CD8-FITC and CD4-APC positive cells were closely associated with CD3-PE, and there were separate populations of non-CD8-FITC and non-CD4-APC cells along the CD3-PE axis.

[0121] These results show that cells can be detached and analyzed from microscope slides without loss of cell integrity and labeling. The degree of cross-linking of the antibody-fluorochrome-oligonucleotide had no effect on the detachment of intact cells. Enzymatic detachment was clearly superior in preserving cell integrity.

[0122] Figure 8-X

[0123] Freshly isolated peripheral blood mononuclear cells (PBMCs) were spun at 100xg onto a 24-well plate (1E06 PBMCs per well) and fixed with 4% PFA for 10 minutes. Fixed cells were washed 3 times and stained with DAPI 1 :20 for 10 minutes in the dark at room temperature. Cells were washed 5 times and then stained with conjugates for 10 minutes, the conjugates being coupled from an antigen-binding moiety with a) a barcode moiety, b) a detectable moiety capable of being erased (antibody-fluorochrome-oligonucleotide conjugate) and c) a cross-linkable moiety (primary amino group). The antigen-binding moiety was an antibody specific to the proteins CD8, CD3 and CD4.

[0124] The conjugates were then cross-linked to the cells with 0.25% PFA for 2 minutes. The antibody-fluorochrome-oligonucleotide conjugate was hybridized to an antisense oligonucleotide labeled with Cy5. Labeling of the cells was controlled by microscopy. Figure 8 ).

[0125] Figure 9 Nuclei staining of the cells is shown using DAPI (nuclear stain). Figure 10An example of the labeling of cells with CD4 specific antibody-oligonucleotide conjugates with the corresponding CY5 labeled antisense oligonucleotide (cell surface staining) is shown. It is clearly visible that a subpopulation of cells is labeled on the surface by the antibody-oligonucleotide conjugate. Thus, it can be concluded that the crosslinking of the oligonucleotide to the cell does not inhibit the hybridization of the antisense oligonucleotide, i.e. the function of the oligonucleotide as a barcode is preserved. Figure 8 An overlay of the images shown in Figure 9 and ​ demonstrates the specificity of the labeling by the antibody-oligonucleotide conjugate.

Claims

1. A conjugate having the general formula (I) X n -P-Y m B o (I), Where X is the detection part. P is the spacer subunit. Y is the antigen recognition part. B is an oligonucleotide containing 2 to 300 nucleotide residues. And n, m, and o are independent integers between 1 and 100. P and B are covalently bonded to Y, and X is covalently bonded to P, wherein X is erasable; The antigen recognition portion Y and / or the oligonucleotide B have cross-linking units capable of providing covalent binding to cells recognized by the antigen recognition portion Y; and The covalent binding of the crosslinking agent to the cell is initiated by radiation, a chemical reaction, or an enzymatic reaction.

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

3. The conjugate according to claim 1 or 2, characterized in that, The detection portion X can be erased by radiation or by enzymatic degradation of the spacer unit P.

4. The conjugate according to claim 1 or 2, characterized in that, The detection component is selected from the group consisting of: chromophore component, fluorescent component, phosphorescent component, luminescent component, light-absorbing component, radioactive component, and transition metal and isotope mass label component.

5. The conjugate according to claim 2, characterized in that, The enzymatically degradable spacer unit P is selected from the group consisting of: polysaccharides, proteins, peptides, phenolic peptides, polyesters, nucleic acids and their derivatives.

6. The conjugate according to claim 1 or 2, characterized in that, The antigen recognition portion Y is an antibody, a fragmented antibody, a fragmented antibody derivative, a peptide / MHC complex targeting a TCR molecule, a cell adhesion receptor molecule, a receptor for a co-stimulatory molecule, or an artificially engineered binding molecule.

7. A library of conjugates according to any one of claims 1 to 6, said library comprising at least 10 conjugates having oligonucleotide B with different sequences.

8. A method for detecting a target motif on a cell by: a) Provide at least one conjugate having the general formula (I). X n -P-Y m B o (I), Where X is the detection part. P is the spacer subunit. Y is the antigen recognition part. B is an oligonucleotide containing 2 to 300 nucleotide residues. And n, m, and o are independent integers between 1 and 100. P and B are covalently bonded to Y, and X is covalently bonded to P, wherein X is erasable; b) Contacting a sample of a biological specimen with at least one of the conjugates to label the target portion recognized by the antigen recognition portion Y; c) Detecting the target portion marked with the conjugate having the detection portion X; d) Erase the detection portion X; The antigen recognition portion Y and / or the oligonucleotide B have cross-linking units capable of providing covalent binding to cells recognized by the antigen recognition portion Y; and The covalent binding of the crosslinking agent to the cell is initiated by radiation, a chemical reaction, or an enzymatic reaction.

9. The method according to claim 8, characterized in that, Under the condition that at least two of the said conjugates have oligonucleotide B with different nucleotide residue sequences, steps a) to d) are then repeated.

10. The method according to claim 8 or 9, characterized in that, Under the condition that at least two of the said conjugates have different antigen recognition portions Y, steps a) to d) are then repeated.

11. The method according to claim 8 or 9, characterized in that, Following step c), in step e), cells labeled with the conjugate having the detection portion X are separated.

12. The method according to claim 8 or 9, characterized in that, The detection portion X is erased by radiation or by enzymatic degradation of the spacer unit P.

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