阅读说明：本技术 使用具有用于增加荧光亮度的接头和酶促可释放荧光部分的缀合物的可逆细胞检测 (Reversible cell detection using conjugates with linkers for increasing fluorescence brightness and enzymatically releasable fluorescent moieties ) 是由 C·杜斯 J·潘克拉茨 于 2019-04-23 设计创作，主要内容包括：本发明针对用于在细胞上标记目标部分的缀合物,其特征为具有通式(I)(X-(o)-L)-(n)–P–Y-(m),其中Y：识别目标部分的抗原识别部分,P：酶促可降解间隔基,X：荧光部分,L：包含一个或多个聚乙二醇残基的接头单元,n、m：1到100之间的整数,o：1到100之间的整数,其中L共价结合荧光部分X和酶促可降解间隔基P,且Y共价地结合至酶促可降解间隔基P,且其中酶促可降解间隔基P选自多糖、聚酯、核酸及其衍生物。在生物样本样品中用缀合物检测目标部分的方法。(The present invention is directed to conjugates for labeling a target moiety on a cell characterized by having the general formula (I) (X) o ‑L) n –P–Y m Wherein Y: an antigen recognition moiety that recognizes a target moiety, P: enzymatically degradable spacer, X: fluorescent moiety, L: a linker unit comprising one or more polyethylene glycol residues, n, m: integer between 1 and 100, o: 1 to 100, wherein L is covalently bound to the fluorescent moiety X and the enzymatically degradable spacer P, and Y is covalently bound to the enzymatically degradable spacer P, and wherein the enzymatically degradable spacer P is selected from the group consisting of polysaccharides, polyesters, nucleic acids and derivatives thereof. A method for detecting a target moiety with a conjugate in a biological sample.)

1. A conjugate for labeling a target moiety on a cell, characterized by having the general formula

(I) (X_o-L)_n – P – Y_m，

Wherein Y: an antigen recognition moiety that recognizes the target moiety,

p: the spacer can be enzymatically degraded by an enzyme,

x: a fluorescent moiety that is capable of emitting light,

l: a linker unit comprising one or more polyethylene glycol residues,

n, m: an integer between 1 and 100, and,

o: an integer between 1 and 100, and,

wherein L is covalently bound to the fluorescent moiety X and the enzymatically degradable spacer P, and Y is covalently bound to the enzymatically degradable spacer P, and wherein the enzymatically degradable spacer P is selected from the group consisting of polysaccharides, polyesters, nucleic acids and derivatives thereof.

2. The conjugate according to claim 1, characterized in that the linker unit L comprises one or more polyethylene glycol residues bound to at least one core unit selected from the group consisting of polyhydroxy compounds, polyamino compounds, polysulfides.

3. A conjugate according to claim 1 or 2, characterized in that the linker unit L comprises one or more polyethylene glycol residues having from 2 to 500 ethylene glycol repeating units.

4. A conjugate according to any one of claims 1 to 3, characterized in that the antigen recognition moiety Y is an antibody, a fragmented antibody, a derivative of a fragmented antibody, 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, a peptide, a lectin or aptamer, RNA, DNA, an oligonucleotide and the like.

5. A conjugate according to any one of claims 1 to 4, characterised in that the fluorescent moiety is selected from xanthene dyes, rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, perylene dyes,Oxazine dyes, pyridinesAzole dyes, pyromethylene dyes, acridine dyes,Oxadiazole dyes, pyronine dyes, benzopyrylium, fluorene dyes, fluorescent oligomers or fluorescent polymers.

6. A conjugate according to any one of claims 1 to 5, characterized in that it is according to general formula (X)_o-L)_n – P(L)_l(X)_x – Y_mThe enzymatically degradable spacer P is further provided with at least one covalently bound linker unit L which does not bind to the fluorescent moiety X and/or at least one covalently bound fluorescent moiety X which does not bind to the linker unit L, wherein L and X are integers between 0 and 100.

7. A method of detecting a target moiety in a biological sample by:

a) providing at least one conjugate having the general formula I

(I) (X_o-L)_n – P – Y_m

Wherein Y: an antigen recognition moiety that recognizes the target moiety,

p: the spacer can be enzymatically degraded by an enzyme,

x: a fluorescent moiety that is capable of emitting light,

l: a linker unit comprising one or more polyethylene glycol residues,

n, m: an integer between 1 and 100, and,

o: an integer between 1 and 100, and,

wherein L is covalently bound to the fluorescent moiety X and the enzymatically degradable spacer P, and Y is covalently bound to the enzymatically degradable spacer P,

b) contacting the biological sample with a conjugate according to formula (I) to label the target moiety recognized by the antigen-recognizing moiety Y

c) Detecting the target moiety labelled with a conjugate having a fluorescent moiety X.

8. The method according to claim 7, characterized in that in step d) the enzymatically degradable spacer P is enzymatically degraded, thereby cleaving the fluorescent moiety X from the labeled target moiety.

9. The method according to claim 7 or 8, characterized in that in step d) the enzymatically degradable spacer P is enzymatically degraded, thereby cleaving the fluorescent moiety from X and the antigen recognition moiety Y from the labeled target moiety.

10. The method according to any one of claims 7 to 9, characterized in that the enzyme for degrading the enzymatically degradable spacer P is selected from the group consisting of glycosidases, glucanases, pullulanases, amylases, inulinases, cellulases, hemicellulases, pectinases, chitosanases, chitinases, proteases, esterases, lipases and nucleases.

11. The method according to any one of claims 7 to 10, characterized in that at least one conjugate of the general formula II is further provided

(II) (X)_n – P – Y_m

Wherein Y: an antigen recognition moiety that recognizes the target moiety,

p: the spacer can be enzymatically degraded by an enzyme,

x: a fluorescent moiety that is capable of emitting light,

n, m: an integer between 1 and 100, and,

wherein X and Y are covalently bound to an enzymatically degradable spacer P and the biological sample is contacted with a conjugate according to formula (II) thereby labeling the target moiety recognized by the antigen recognition moiety Y.

12. The process according to any one of claims 7 to 11, characterized in that it is carried out according to the general formula (X)_o-L)_n – P(L)_l(X)_x – Y_mFurther providing the enzymatically degradable spacer P with at least one covalently bound linker unit L and/or at least one non-covalently bound linker unit L which is not bound to the fluorescent moiety XA covalently bound fluorescent moiety X bound to a linker unit L, wherein L and X are integers between 0 and 100.

Background

The present invention is directed to a process for detecting a target moiety in a biological sample by labeling the target moiety with a conjugate having an antigen recognition moiety and a fluorescent moiety linked by an enzymatically degradable spacer and a hydrophilic linker group comprising polyethylene glycol, wherein upon detection or isolation of the target moiety, the degradable spacer is enzymatically degraded, thereby releasing target cells from at least the fluorescent moiety.

Immunofluorescence and immunomagnetic labeling are important for detailed analysis and specific isolation of target cells from biological samples, both in research and clinical applications. This technique combines specific labeling of the target moiety with a conjugate having a detectable unit, such as a magnetic particle, to be retained in a magnetic field and thereby separate the cells, or such as a fluorescent dye or transition metal isotope mass label, to detect and characterize the cells by microscopy or cytometry. For immunofluorescence analysis, a number of variants have been developed over the last two decades in view of antibodies, fluorescent dyes, flow cytometers, flow sorters, and fluorescence microscopy to achieve target cell-specific detection and separation. One problem in immunofluorescence techniques is the detection threshold and the brightness of the fluorescence emission, which can be enhanced by, for example, better detectors, filter systems, lasers, or modified fluorescent dyes (i.e., with better quantum yields). Immunofluorescent conjugates typically contain multiple dyes to increase fluorescence intensity, but brightness is limited by a self-quenching mechanism resulting from the formation of dimers, trimers, or multimers.

Recently, with the development of reversible labeling technologies, flexibility has developed with respect to downstream applications as well as successive detection or separation cycles for various applications (such as magnetic cell enrichment, flow sorting, or fluorescence microscopy). Those techniques allow for the removal of fluorescent or magnetic labels after cell sorting or cell analysis. In particular for technologies based on successive cycles of label-detection-elimination with high multiplexing potential for mapping, e.g. protein networks, elimination of the fluorescent signal is essential. However, these techniques are based on oxidative destruction of the conjugated fluorescent moiety by photobleaching or chemical bleaching procedures (US 7741045B 2, EP 0810428B 1 or DE10143757) and are hindered by the antibodies left on the sample.

In this regard, several methods have been developed over the last years for bright immunofluorescent conjugates and for reversible labeling with immunoconjugates.

For example, it is known to use PEG as a linker to reduce fluorescence quenching as disclosed by y. Guo et al, j. Am. chem. soc. 2012, 134, 19338-. Herein, the use of PEG as a linker to inhibit the annoying interactions of fluorescent dyes with biomolecules and to improve quantum yield is described. However, the use of PEG in multimerization is not indicated. Each fluorescent dye is attached to the RGD peptide via the PEG linker.

EP3098269 a1 teaches the multimerization of fluorescent dyes on a branched polyether backbone. The 20 to 200 atom core moiety serves as a tether for multiple PEG linkers carrying a fluorescent dye at the other end of the linker chain. The multimerized polyether backbone may be conjugated to an antibody. The polyether backbone prevents quenching and non-specific binding of the fluorescent dye. However, this publication does not teach any reversible label or method of label release. The core moiety is too small to allow enzymatic degradation of the polyether backbone and monomerization of the fluorescent dye. Thus, EP3098269 a1 is directed to providing bright fluorescent labels by multimerization of unquenched fluorescent dyes, but does not disclose a method for releasing the labels.

WO 96/31776 describes a method for releasing magnetic particles from target cells after separation by enzymatic cleavage of the coating layer portion of the particles or of a portion present in the linker between the coating layer and the antigen recognition moiety. An example is the use of magnetic particles coated with dextran and/or linked to an antigen recognition moiety through dextran. The separated target cells are then lysed from the magnetic particles by adding the glucan degrading enzyme glucanase. Thus, WO 96/31776 is directed to the release of magnetic labels from target moieties by enzymatic digestion, but does not disclose a method of fluorescent labeling.

A similar approach is disclosed in EP3037821, wherein a conjugate with an enzymatically degradable spacer for reversible fluorescent labeling is used to detect and isolate a target moiety based on, for example, a fluorescent signal.

Embodiments of EP3037821 are directed to covalent multimerization strategies for low affinity antigen recognition moieties. This strategy provides a low affinity antigen recognition moiety and a detection moiety (such as a fluorescent dye) that are covalently linked and thus covalently multimerized by an enzymatically degradable spacer. Covalent attachment enables stable and unambiguous multimerization and multiparameter labeling options. During enzymatic degradation of the spacer, the detection moiety is released and the low affinity antigen recognition moiety is monomerized. Thus, EP3037821 is directed to the release of fluorescent labels from target moieties by enzymatic digestion and discloses a method for reversible covalent multimerization of low affinity antigen recognition moieties, but does not provide a method to prevent fluorescence quenching or to enhance fluorescence brightness but maintain releasability.

US 5,719,031 describes dextran-fluorescent dye-conjugates in which the degree of labelling is high enough to provide fluorescence quenching. Thus, degradation is accompanied by an increase in the fluorescence emission signal, which is used for quantification of the enzymatic digestion process. Thus, US 5,719,031 discloses a method in which fluorescence quenching in dextran-fluorescent dye conjugates is desirable, rather than prevented.

Fluorescence quenching is also described in GB 2372256. Cells were stained with a conjugate comprising a plurality of fluorescent dyes linked to an antibody by a linker. Since fluorescent signals are quenched by high density fluorescent dyes, GB2372256 describes enzymatic degradation of the linker for releasing the fluorescent dye from the conjugate. The released fluorescent dye does not undergo self-quenching, resulting in a more intense fluorescent signal (i.e., better resolution). However, since the fluorescent signal is detected after release from the target, it is not possible to identify the target moiety on the cell surface using the method according to GB 2372256. Furthermore, it is not possible to detect more than one target simultaneously, since the resulting mixed fluorescent signal cannot be assigned to a particular conjugate and/or target.

US9023604 discloses a method based on reversible labeling of receptor molecules indirectly, non-covalently, on target cells with reversible multimers. A receptor binding reagent characterized by an off-rate constant for binding partner C of about 0.5x 10-4/sec or greater is multimerized by a multimerizing reagent having at least two binding sites Z that reversibly, non-covalently interact with binding partner C to provide a complex with high affinity for an antigen of interest. The detectable label is bound to the multivalent binding complex. Reversibility of multimerization is initiated after disruption of the binding between binding partner C and binding site Z of the multimerization reagent. An example of this strategy is the Fab-StreptagII/Streptactin multimer, where multimerization can be reversed by the competitor biotin. Thus, US9023604 discloses a method for reversible non-covalent multimerization of low affinity antigen recognition moieties, but does not mention strategies for reversible covalent multimerization and multiparameter labeling or strategies to enhance fluorescence brightness or maintain releasability.

As mentioned, EP3037821 describes conjugates of the general formula Xn-P-Ym consisting of a detection moiety X, an enzymatically degradable spacer P and an antigen recognition moiety Y, which enable multiparameter fluorescent labeling and cleavage of the detection moiety by enzymatic degradation of the spacer P.

WO2007109364 takes a different approach in which a releasable conjugate of a fluorescent dye is disclosed that has quenching when bound to a target. The conjugate comprises a "protease cleavage site", i.e. a spacer unit that is only degradable by proteases. After digestion of the "protease cleavage site", the fluorescent dye is free to emit radiation for detection purposes. This method is intended for indirect detection of cells and is not intended for localization of targets on the cell surface.

The challenge in the development of these immunofluorescent conjugates for reversible labeling is to ensure maximum fluorescence brightness and high reversibility. Theoretically, an increase in the extent of labeling with the detection moiety on the enzymatically degradable spacer P could enhance the fluorescence emission intensity. However, this development shows two limiting factors, since the increased degree of labeling and the proximity of fluorescent dyes leads to fluorescence quenching and thus a decrease in fluorescence intensity, as well as a decrease in enzymatic cleavage efficiency. That is, increasing the amount of fluorescent label does not result in a proportional increase in fluorescent signal intensity, and also reduces enzymatic release by sterically hindering access of the enzyme to the substrate.

SUMMARY

It is therefore an object of the present invention to provide conjugates and methods for specific labeling, detection and de-labeling of target moieties in biological sample samples to achieve further labeling, which avoids fluorescence quenching.

Surprisingly, it has been found that the use of a PEG linker between the enzymatically degradable unit P and the fluorescent moiety X protects the fluorescence of the fluorescent moiety (which would otherwise be lost due to quenching), allowing the use of a lower degree of labelling, which in turn increases the release by enzymatic cleavage.

It should be noted that the conjugates according to the invention emit fluorescent radiation when bound or even not bound to the target cells, i.e. do not show quenched fluorescence as the dyes disclosed in WO 2007109364. Without being bound to this theory, the quenched fluorescence may originate from the dendrimers used in WO2007109364, which sterically hinders the excitation/emission process. After separation from the dendrimer by enzymatic degradation of the spacer, the fluorescent ability of the dye was restored. Since "quenched fluorescence" is not present in the present conjugate, the conjugate according to WO2007109364 is chemically different from the conjugate of the present invention.

Accordingly, the present invention is directed to conjugates for labeling a target moiety on a cell characterized by the general formula

(I) (X_o-L)_n – P – Y_m，

Wherein Y: an antigen recognition moiety that recognizes the target moiety,

p: the spacer can be enzymatically degraded by an enzyme,

x: a fluorescent moiety that is capable of emitting light,

l: a linker unit comprising one or more polyethylene glycol residues,

n, m: an integer between 1 and 100, and,

o: an integer between 1 and 100, and,

wherein L is covalently bound to the fluorescent moiety X and the enzymatically degradable spacer P, and Y is covalently bound to the enzymatically degradable spacer P, and wherein the enzymatically degradable spacer P is selected from the group consisting of polysaccharides, polyesters, nucleic acids and derivatives thereof.

The conjugates used in the present invention may, for example, have the general sequence "fluorochrome (X) -peg (l) -dextran (P) -antibody (Y)" or "fluorochrome (X) -peg (l) -dextran (P) -fab (Y)". Specific conjugates thereof are described in the examples.

The conjugates of the invention show an increase in fluorescence intensity achieved by linker L compared to prior art conjugates and are suitable for multi-parameter labeling targeting more than one target moiety in a biological sample. Since the fluorescent moiety of the conjugate can be removed from the target cells by adding an enzyme, it is possible to re-label the cells with a different antigen recognition moiety carrying the same fluorescent moiety, which provides additional possibilities for cell analysis or isolation. The present method enables a fast and less invasive solution compared to the prior art and avoids the use of reactive oxygen species, high energy or heat, which may be harmful to the target subject.

Furthermore, the object of the present invention is a method for detecting a target moiety in a biological sample by:

a) providing at least one conjugate having the general formula I

(I) (X_o-L)_n – P – Y_m

Wherein Y: an antigen recognition moiety that recognizes the target moiety,

p: the spacer can be enzymatically degraded by an enzyme,

x: a fluorescent moiety that is capable of emitting light,

l: linker unit comprising one or more polyethylene glycol residues

n, m: an integer between 1 and 100, and,

o: an integer between 1 and 100

Wherein L is covalently bound to the fluorescent moiety X and the enzymatically degradable spacer P, and Y is covalently bound to the enzymatically degradable spacer P.

b) Contacting the biological sample with a conjugate according to formula (I) thereby labelling the target moiety recognised by the antigen recognition moiety Y.

c) Detecting the target moiety labelled with the conjugate having the fluorescent moiety X.

Brief Description of Drawings

Fig. 1 shows schematically the process of the invention as follows: a target moiety on a cell as a biological sample is specifically labeled and released using a conjugate of a high affinity (a) or low affinity (b) antigen recognition moiety Y, an enzymatically degradable spacer P and a fluorescent moiety X conjugated to the enzymatically degradable spacer P via a linker unit L.

Figure 2 shows exemplary results of absorption and fluorescence emission of dextran-PEG-coumarin-dye and dextran-coumarin-dye of different degrees of labeling at constant concentration of dextran.

Fig. 3 shows an exemplary histogram of the results of a single parameter labeled flow cytometry analysis with different anti-CD 4-Fab-dextran-PEG-coumarin-dye conjugates (a-c) according to the present invention compared to the anti-CD 4-Fab-dextran-coumarin-dye conjugate (d).

Detailed description of the invention

The methods and conjugates of the invention are preferably used for the in vitro detection of target cells.

For the purposes of the present invention, a covalent bond is defined as a bond between atoms sharing an electron pair or a quasi-covalent bond having an equilibrium dissociation constant between non-covalent interaction partners of less than 10E-9M. Non-covalent bonds are defined as bonds having an equilibrium dissociation constant greater than 10E-9M.

The method of the invention may involve removing the antigen recognition moiety Y from the target moiety. The method may thus involve a step d) in which the enzymatically degradable spacer P is enzymatically degraded, thereby cleaving the fluorescent moiety X from the labeled target moiety.

In this respect, the invention encompasses two embodiments by using conjugates with high affinity (a) or low affinity (b) antigen recognition moiety Y.

Fig. 1 schematically shows these embodiments of the invention as follows: a target moiety is specifically labelled on target cells as a biological sample using a conjugate of a high affinity (a) or low affinity (b) antigen recognition moiety Y, an enzymatically degradable spacer P, a linker unit L and a fluorescent moiety X.

The high affinity antigen recognition moiety Y is represented by 1: a ratio of 1 stably binds to the target moiety, i.e. n =1 in formula (I). When the spacer is enzymatically degraded, the high affinity antigen recognition moiety provides a stable bond which results in the removal of the fluorescent moiety X, linker moiety L and spacer P.

In a variant of the method according to the invention, in step d) the enzymatically degradable spacer P is enzymatically degraded, thereby cleaving the fluorescent moiety from X and the antigen recognition moiety Y from the labeled target moiety.

This can be achieved by providing conjugates of the antigen recognition moiety Y with low affinity. Such low affinity antigen recognition moieties do not provide a binding affinity for the target moiety of 1: 1, but some low affinity antigen recognition moieties may multimerize in one conjugate and thus bind to the target moiety, i.e. n >1 in formula (I). During degradation, the low affinity antigen recognition moiety will be monomerized. Thus, after the dissociation of the monomeric low-affinity antigen-recognizing moiety, the target moiety is taken out from the fluorescent moiety X, the linker moiety L, the spacer P and the antigen-recognizing moiety Y. The stability of non-covalent bonds can be described by balancing the dissociation constant (KD), dissociation rate constant (k (off)), and association rate constant (k (on)), where KD = k (off)/k (on). The low affinity antigen recognition portion can be characterized by an equilibrium dissociation constant (KD) in the range of 0.5E-08M or greater and a dissociation rate constant (k (off)) in the range of 1E-03/sec or greater, preferably an equilibrium dissociation constant (KD) in the range of between 0.5E-08M and 1E-04M and a dissociation rate constant (k (off)) in the range of between 1E-03/sec and 1E-00/sec.

In a further embodiment of the invention, according to the general formula (X)_o-L)_n – P(L)_l(X)_x – Y_mThe enzymatically degradable spacer P is further provided with at least one covalently bound linker unit L not bound to the fluorescent moiety X and/or at least one covalently bound fluorescent moiety X not bound to the linker unit L, wherein L and X are integers between 0 and 100 and n, o, m have the meaning as already disclosed.

In other words, it is possible that one or more fluorescent moieties X are coupled to the enzymatically degradable spacer P without a linker L and/or one or more linkers L are coupled to the enzymatically degradable spacer P without a fluorescent moiety X, both variants having the proviso: at least one (X)_o-L) the unit is covalently bound to an enzymatically degradable spacer P.

For example, the conjugate can have the general formula (X)_o-L)_n – P(L)_l – Y_mWherein l is an integer ranging from 1 to 100; or (X)_o-L)_n – P(X)_x – Y_mWherein x is an integer ranging from 1 to 100; or (X)_o-L)_n – P(L)_l(X)_x – Y_mWherein l and m are integers in the range of 1-100.

Target portion

The target moiety to be detected using the methods of the invention may be on any biological sample, such as a tissue section, cell aggregates, suspended cells, or adherent cells. The cells may be living or dead. The target moiety is preferably an antigen expressed intracellularly or extracellularly on a biological sample such as whole animals, organs, tissue sections, cell aggregates or individual cells of invertebrates such as Caenorhabditis elegans (Caenorhabditis elegans), Drosophila melanogaster (Drosophila melanogaster), vertebrates such as zebrafish (Danio rerio), Xenopus laevis (Xenopus laevis) and mammals such as mice (Mus musculus), Homo sapiens.

Fluorescent moieties

Suitable fluorescent moieties X are those known from the field of immunofluorescence techniques such as flow cytometry or fluorescence microscopy. In these embodiments of the invention, the detection of the conjugate-labeled target moiety is carried out by exciting the fluorescent moiety X and detecting the resulting emission (photoluminescence).

Useful fluorescent moieties may be small organic molecular dyes, such as xanthene dyes (e.g., fluorescein), or rhodamine dyes, coumarin dyes, cyanine dyes, pyrene dyes, perylene dyes, and the like,Oxazine dyes, pyridinesAzole dyes, pyromethylene (pyrom)ethyl ene) dye, acridine dye,Oxadiazole dyes, pyronine (carbopyronine) dyes, benzopyrylium (benzopyrylium) dyes, fluorene dyes, or metal organic complexes, such as ruthenium, europium, platinum complexes. In addition to single molecule entities, small organic molecule dye clusters, fluorescent oligomers or fluorescent polymers (e.g., polyfluorenes) can also be used as the fluorescent moiety. Furthermore, the fluorescent moiety may be protein-based, such as phycobiliprotein; nanoparticles such as quantum dots, upconversion nanoparticles, gold nanoparticles, dyed polymeric nanoparticles.

The fluorescent moiety X may be covalently coupled to the linker unit. Methods for covalent conjugation are known to those skilled in the art. It is possible that the activating group on the fluorescent moiety X or on the linker unit L reacts directly with the functional group on the linker unit L or on the fluorescent moiety X, or through a hetero-bifunctional linker molecule which can react first with one binding partner and second with the other binding partner.

For example, fluorescent dyes are available having groups reactive with amino or thiol groups, e.g., active esters that react with amino groups on the linker unit, such as N-hydroxysuccinimide ester (NHS), sulfodichlorophenyl ester (SDP), tetrafluorophenyl ester (TFP), and pentafluorophenyl ester (PFP), or Michael acceptors or haloacetyl groups that react with thiol groups on the linker unit, e.g., maleimide groups, iodoacetamide groups, and bromomaleimide groups. A wide variety of heterobifunctional compounds can be used to attach to an entity. Illustrative entities include: azidobenzoyl hydrazine, N- [4- (p-azidosalicylamido) butyl ] -3'- [2' -pyridyldithio ] propionamide), disulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyl tartrate, N-y-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 N-hydroxysuccinimide 3- (2-pyridyldithio) propionate (SPDP) or N-hydroxysuccinimide 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC) with a reactive thiol group on the fluorescent moiety and a reactive amino group on the linker unit.

The conjugates used in the method of the invention may comprise 1 to 100, preferably 2-30 fluorescent moieties X.

Antigen recognition moiety Y

The term "antigen recognition moiety Y" refers to any kind of molecule that binds to a target moiety expressed on a biological sample (e.g., an antigen expressed intracellularly or extracellularly on a cell). The term "antigen recognition moiety Y" especially relates to antibodies, fragmented antibody derivatives, peptides/MHC-complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for co-stimulatory molecules or artificially engineered binding molecules, peptides, lectins or aptamers, RNA, DNA, oligonucleotides and analogues thereof.

Fragmented antibody derivatives are, for example, 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 species of molecules.

The conjugate used in the method of the invention may comprise 1 to 100, preferably 1 to 20 antigen recognition moieties Y. The interaction of the antigen recognition moiety with the target moiety may be of high or low affinity. The binding interaction of a single low affinity antigen recognition moiety is too low to provide a stable bond to the antigen. The low affinity antigen recognition moiety may be multimerized by conjugation with an enzymatically degradable spacer P to provide high avidity.

The term "antigen recognition moiety Y" preferably refers to an antibody or Fab directed against an antigen expressed intracellularly in a biological sample (target cells), such as IL2, FoxP3, CD154, or an extracellularly expressed antigen, such as CD3, CD14, CD4, CD8, CD25, CD34, CD56, and CD 133.

The antigen recognition moiety Y, particularly an antibody, may be coupled to the spacer P via a side chain amino group or a thiol group. In some cases, the glycoside (glyosidic) side chain of the antibody can be oxidized by periodate to form an aldehyde functional group.

The antigen recognition moiety Y may be covalently or non-covalently coupled to the spacer P. Methods of 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 fluorescent moiety X.

Enzymatically degradable spacers P

The enzymatically degradable spacer P can be any molecule which can be cleaved by a particular enzyme, such as a hydrolase. Suitable as enzymatically degradable spacers P are, for example, polysaccharides, proteins, peptides, depsipeptides, polyesters, nucleic acids and derivatives thereof.

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 linker L and antigen recognition moiety Y. Various such modifications are known in the art, for example imidazolyl carbamate groups can be introduced by reacting the polysaccharide with N, N' -carbonyldiimidazole. Subsequently, an amino group can be introduced by reacting the imidazolylcarbamate group with hexamethylenediamine. The polysaccharide may also be oxidized with periodate to provide aldehyde groups, or with N, N' -dicyclohexylcarbodiimide and dimethyl sulfoxide to provide ketone groups. The aldehyde or ketone functional group can then be reacted with diamines to provide an amino group, or directly with an amino substituent on the binding moiety of the protein, preferably under reductive amination conditions. The carboxymethyl group can be introduced by treating the polysaccharide with chloroacetic acid. The carboxyl group is activated using methods known in the art to produce activated esters such as N-hydroxysuccinimide ester or tetrafluorophenyl ester, allowing reaction with the amino group of a diamine to provide an amino group, or directly with an amino group on a binding moiety of a protein. The functional group having an alkyl group can be introduced by treating the polysaccharide with a halogen compound under an alkaline condition in general. For example, allyl groups can be introduced by using allyl bromide. The allyl groups can be further used in a mercapto-ene reaction with a mercapto-containing compound, such as cysteamine, to introduce amino groups, or directly reacted with a binding moiety of a protein that has a mercapto group that is released by reduction of a disulfide bond or introduced by thiolation (e.g., using 2-imine thiacyclopentane).

Proteins, peptides and depsipeptides used as enzymatically degradable spacers P can be functionalized by side chain functionalities of amino acids to be linked to the linker L and the antigen recognition moiety Y. Side chain functional groups suitable for modification are, for example, amino groups provided by lysine or thiol groups provided by cysteine after reduction of the disulfide bridge.

The polyesters and polyester amides used as enzymatically degradable spacers P can be synthesized from comonomers that provide pendant functionality, or subsequently functionalized. In the case of branched polyesters, the functionalization may be via terminal carboxyl groups or terminal hydroxyl groups. After multimerization, functionalization of the polymer chains can be carried out, for example, by addition to unsaturated bonds (i.e., a mercaptoene reaction or an azide-alkyne reaction) or by introducing functional groups in a free radical reaction.

The nucleic acid used as the enzymatically degradable spacer P is preferably synthesized using functional groups suitable for the linkage of the binding moiety B and the antigen recognition moiety a at the 3 'and 5' ends. Suitable phosphoramidite building blocks for nucleic acid synthesis which provide, for example, amino or thiol functionality are known in the art.

The enzymatically degradable spacer P may consist of more than one different enzymatically degradable unit, which units may be degraded by the same or different enzymes.

Joint L

Linker L is a polar hydrophilic oligomer comprising from 2 to 500, preferably from 4 to 30 ethylene glycol repeating units.

The linker group L may be linear to allow attachment of a single fluorescent moiety X. The linker moiety may comprise functional or activating groups at each end of the oligomer to react with the activating or functional groups on the fluorescent moiety and with the activating or functional groups on the enzymatically degradable spacer P, either directly or through prior reaction with a heterobifunctional crosslinker. The methods and groups employed are the same as described for the covalent attachment of the fluorescent moiety X. Alternatively, the fluorescent moiety X may already comprise a polyethylene glycol chain with an activating or functional group that can be conjugated to the enzymatically degradable spacer P. In this case, a polyethylene glycol chain is used as the linker L.

In a particular practical embodiment of the invention, a commercially available hetero-bifunctional polyethylene glycol may be reacted at one end with an activated fluorescent moiety and at the other end activated for reaction with an enzymatically degradable spacer P.

In another embodiment, the linker group L may be branched to allow attachment of multiple fluorescent moieties. In this embodiment, the linker unit L comprises one or more polyethylene glycol residues that bind to at least one (e.g., one to six) polyhydroxy branching unit selected from a core unit selected from a polyhydroxy compound, a polyamino compound, a polythio compound. Preferred as core unit are, for example, glycerol having three hydroxyl groups as the point of attachment of the 3 polyether residues via ether bonds; pentaerythritol having four hydroxyl groups as attachment points for 3 to 4 polyether residues via ether linkages; dipentaerythritol having six hydroxyl groups as attachment points for 3 to 6 polyether branches via ether linkages; tripentaerythritol or hexaglycerol having eight hydroxyl groups which serve as attachment points for 3 to 8 polyether branches via ether linkages. In this embodiment, linker L comprises a total of 3 to 500 ethylene glycol repeat units.

In a particular practical embodiment of the invention, a commercially available multi-arm polyethylene glycol (branched PEG) is used as the linker, which includes a branching moiety and a polyether branch. The arm ends of the branched PEG are functionalized or activated to allow covalent attachment to a fluorescent moiety or enzymatically degradable spacer P as described previously. Multi-arm polyethylene glycols are commercialized by, for example, Nanocs inc.

The linker L may be covalently or quasi-covalently coupled to the enzymatically degradable spacer P. Methods of covalent or quasi-covalent conjugation are known to those skilled in the art and are the same as those mentioned for the conjugation of the fluorescent moiety X. Quasi-covalent binding of the fluorescent moiety X to the linker unit L can be achieved using a binding system that provides an equilibrium dissociation constant of ≦ 10-9M, such as a biotin-avidin binding interaction.

Method of the invention

A preferred embodiment of the method of the invention comprises a step d) wherein the enzymatically degradable spacer P is enzymatically degraded, thereby cleaving the fluorescent moiety X from the labeled target moiety.

In another embodiment of step d), the enzymatically degradable spacer P is degraded by an enzyme, thereby cleaving the fluorescent moiety from X and the antigen recognition moiety Y from the labeled target moiety.

The term "enzymatically degrades the spacer P, thereby cleaving the fluorescent moiety X from the conjugate" means cleaving by degrading the spacer P in the following manner (X)_o-L)_n – P – Y_mCovalent bond of fragment: at least the fluorescent moiety X and the linker unit L are removed from the target moiety.

In a variant of the invention, the enzymatically degradable spacer P is degraded by an enzyme, thereby cleaving the fluorescent moiety from X and the antigen recognition moiety Y from the labeled target moiety. The variants are initiated by the use of low affinity antigen recognition moieties (e.g., FAB) and/or at m >1 (e.g., 2-5).

The process of the invention may be carried out in one or more sequences of steps a) to d). After each sequence, the fluorescent moiety and linker L, and optionally the antigen recognition moiety, are released (removed) from the target moiety. The method of the invention has the advantage of providing unlabelled cells, especially when the biological sample is living cells which should be further processed.

After and/or before each of steps a) -d), one or more washing steps may be carried out to remove unwanted substances, such as unbound conjugate (I), or released parts of the conjugate, such as fluorescent moiety X or antigen recognition moiety Y or reagents for cleavage. The term "washing" means that the biological sample is separated from the environmental buffer by a suitable procedure, such as sedimentation, centrifugation, drainage or filtration. Prior to this separation, a wash buffer may be added and optionally incubated for a period of time. After this separation, the sample can be refilled or resuspended with buffer.

The method of the invention provides high flexibility for the use of specific labels of the conjugates and the release of the conjugates, providing a variety of different detection strategies.

Any step may be monitored qualitatively or quantitatively depending on the fluorescent moiety X used or by other suitable quantitative or qualitative methods known to those skilled in the art, such as by visual counting. This may be useful for determining the efficiency of the various steps provided by the method of the present invention.

Such methods for labeling are known to the person skilled in the art, such as the use of non-degradable conjugates according to the general formulae (III) to (VI) as illustrated below.

Step a)

In step a) of the method, at least one conjugate of general formula (I) is provided. For the detection of different target moieties or the same target moiety by different detection moieties, different conjugates having the general formula (I) may be provided, wherein the conjugates and their components Y, P, L, X, o, n, m have the same meaning, but may be the same or different kinds and/or numbers of antigen recognition moieties Y and/or linker units L and/or enzymatically degradable spacers P and/or fluorescent moieties X. In a further embodiment of the method, the biological sample may be labeled with an enzymatically degradable conjugate that does not contain a linker L.

In one of these embodiments, at least one conjugate having the general formula II is provided.

(II) (X)_n – P – Y_m

Wherein Y: an antigen recognition moiety that recognizes the target moiety,

p: the spacer can be enzymatically degraded by an enzyme,

x: a fluorescent moiety that is capable of emitting light,

n, m: an integer between 1 and 100, and,

wherein X and Y are covalently bound to an enzymatically degradable spacer P and the biological sample is contacted with a conjugate according to formula (II) thereby labeling the target moiety recognized by the antigen recognition moiety Y.

Furthermore, conjugates other than those having the general formula (I) or (II) may be provided which do not comprise an enzymatically degradable spacer P and which can survive the optional cleavage step d). Such conjugates can be used to label a biological sample during or after any of steps a) -d) for qualitative or quantitative monitoring.

Such further conjugates may have general formulae (III) and (IV):

(X_o-L)_n – P’ – Y_m(III) and/or X_n – P’ – Y_m(IV); wherein Y, L, X, n, m have the same chemical meaning as in formula (I), but wherein P' is a non-enzymatically degradable spacer. X, Xo-L, P' and Y may be covalently or non-covalently bound.

Further, at least one conjugate having the general formulae (V) and (VI) may be provided:

(X_o-L)_n – Y_m(V) and/or X_n – Y_m(VI); wherein Y, X, n, m have the same meanings as in formula (I). X, X_oL and Y may be bound to each other covalently or non-covalently.

The method can use a variety of conjugate combinations. For example, the conjugate may comprise antibodies specific for two different epitopes, such as two different anti-CD 34 antibodies. Different antigens may be addressed with different conjugates comprising different antibodies, e.g. anti-CD 4 and anti-CD 8 for distinguishing two distinct T cell populations or anti-CD 4 and anti-CD 25 for determining different cell subsets, such as regulatory T cells.

Step b)

In step b), the target portion of the biological sample is labeled with a conjugate according to formulae (I) to (VI).

In a variant of the invention, the contacting with more than one conjugate of general formula (I) can be carried out simultaneously or subsequently in more than one step b).

Furthermore, the conjugate not recognized by the target moiety may be removed by washing with, for example, a buffer, before the target moiety labeled with the conjugate is detected or separated in step c), or before the next contacting step b).

In a variant of the invention, a plurality of steps b) may be carried out. In addition to the conjugate according to formula (I), step b) may comprise at least one conjugate of general formulae (II) - (VI), which may be incubated simultaneously or subsequently.

The conditions during incubation are known to those skilled in the art and can be optimized empirically with respect to time, temperature, pH, and the like. The incubation time is typically up to 1 hour, more typically up to 30 minutes and preferably up to 15 minutes. The temperature is typically 4-37 deg.C, more typically less than 37 deg.C.

Step c)

Methods and apparatus for detecting a target moiety labeled with a conjugate by a fluorescent moiety X are provided.

The targets labeled with the conjugates are detected by exciting the fluorescent moiety X and analyzing the resulting fluorescent signal. The excitation wavelength is typically selected based on the absorption maximum of the fluorescent moiety X, as is known in the art, and is provided by a laser or LED source. If several different fluorescent moieties X are used for the detection of multiple colors/parameters, care should be taken to select fluorescent moieties that do not have overlapping absorption and emission spectra, at least do not have overlapping absorption and emission maxima. The target can be detected in a flow cytometer, a fluorescence spectrophotometer, or a fluorescence scanner, for example, under a fluorescence microscope. Chemiluminescence-emitted light can be detected by similar instruments, omitting excitation.

The methods of the invention can be used not only to detect target moieties (i.e., target cells expressing such target moieties), but also to isolate target cells from a biological sample based on the fluorescent moiety X. In the methods of the invention, the term "detecting" comprises "isolating".

For example, detection of target moieties by fluorescence can be used to initiate an appropriate separation process by optical means, electrostatic forces, piezoelectric forces, mechanical separation, or acoustic means.

In one variant of the invention, a flow sorter (such as FACS or TYTO) or a MEMS-based cell sorting system, in particular, suitable for such separation on the basis of fluorescent signals, is disclosed, for example, in EP14187215.0 or EP 14187214.3.

In a further variant of the invention, at least one detection and/or separation step c) can be combined simultaneously or in a subsequent step.

In addition, the contaminating non-labeled portion of the biological sample may be removed by washing with, for example, a buffer, during or after separation of the target portion.

Step d)

After detection and/or isolation of the target moiety in step c), in step d) the spacer P is enzymatically degraded, thereby cleaving at least the fluorescent moiety X, the linker unit L from the conjugate.

Depending on the antigen recognition moiety Y, when the spacer P is enzymatically cleaved, the low affinity antigen recognition moiety will be monomerized and possibly dissociated, which results in complete removal of the fluorescent moiety X, linker unit L, spacer P and antigen recognition moiety Y. The high affinity antigen recognition moiety provides a stable bond, which results in the removal of the fluorescent moiety X, linker unit L and spacer P.

In a variant of the invention, step d) may be carried out outside the detection system (e.g. in a tube in solution with the target moiety).

In another variant, the enzymatic degradation may be accomplished in a detection device. For example, the disruption may occur during signal detection, such as during fluorescence microscopy, cytometry, or photometry. The reduction in the detection signal can thus be monitored in real time.

Optionally, after the splitting in d), there may be a further step c) with detection or separation of target moieties.

The fluorescent moiety X and linker unit L and/or enzymatically degraded spacer P and/or antigen recognition moiety Y and/or remaining target moiety still labeled by the conjugate (I) or the non-cleaved part of the conjugate (I) and/or the reagent used for enzymatic degradation in c) can be isolated from the sample e.g. by washing or using the method as described in step c).

Those optional one or more detection and/or separation steps provide the possibility to separate the released target portion or to determine the efficiency of the splitting step d).

Another variant of the invention comprises the elimination of the fluorescence emission by a combination of enzymatic degradation and oxidative bleaching. The necessary chemicals for bleaching are known from the publications referred to above in connection with "multiepitopic ligand mapping", "chip-based cytometry" or "Multioymx" technology.

Enzymes for degrading the spacer P

The enzymatically degradable spacer P is degraded by addition of a suitable enzyme. The choice of enzyme as the release agent is determined by the chemical nature of the enzymatically degradable spacer P and may be one enzyme or a mixture of different enzymes.

The enzyme is preferably a hydrolase, but may also be a lyase or a reductase. Preferred enzymes may be selected from glycosidases, glucanases, pullulanases, amylases, inulinases, cellulases, hemicellulases, pectinases, chitosanases, chitinases, proteases, esterases, lipases and nucleases.

For example, if the spacer P is a polysaccharide, glycosidases (EC 3.2.1) are most suitable as release agents. Preferred are glycosidases that recognize specific glycosidic structures, such as glucanase (EC3.2.1.11), which cleaves at the α (1- >6) junction of glucan; pullulanase which cleaves alpha (1- >6) linkage (EC 3.2.1.142) or alpha (1- >6) and alpha (1- >4) linkage (EC 3.2.1.41) of pullulan; a neopullulanase (EC 3.2.1.135) and an isopullulanase (EC 3.2.1.57) which cleave α (1 — >4) linkages in pullulan; alpha-amylase (EC 3.2.1.1) and maltogenic amylase (EC 3.2.1.133), which cleave the alpha (1- >4) linkage in amylose; inulinase (EC 3.2.1.7), which cleaves β (2 >1) fructoside linkages in inulin; cellulase (EC 3.2.1.4), which cleaves at the β (1 — >4) junction of cellulose; xylanases (EC 3.2.1.8), which cleave at the β (1 — >4) junction of xylan; pectinases such as endo-pectolyase (EC 4.2.2.10) which eliminate cleavage at the α (1- >4) D-polygalacturonic acid (D-galacturonan) methyl ester junction; or polygalacturonase (EC 3.2.1.15) which cleaves at the alpha (1 ≧ 4) D-galacturonal (D-galactosiduronic) junction of pectin; chitosanase (EC 3.2.1.132), which cleaves at the β (1 — >4) junction of chitosan; and endochitinase (EC 3.2.1.14) for chitin cleavage.

Proteins and peptides can be cleaved by proteases, which need to 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 ENLYFQ \ S sequence; enteropeptidase (EC 3.4.21.9), a serine protease that cleaves after DDDDK sequence; factor Xa (EC 3.4.21.6), a serine endopeptidase that cleaves after the IEGR or IDGR sequences; or HRV3C protease (EC3.4.22.28), a cysteine protease that cleaves at the LEVLFQ \ GP sequence.

Depsipeptides, which are peptides comprising ester bonds in the peptide backbone, or polyesters can be cleaved by esterases, such as pig liver esterase (EC 3.1.1.1) or pig pancreatic lipase (EC 3.1.1.3). The nucleic acid may be cleaved by an endonuclease, which may be sequence specific, such as restriction enzymes (EC 3.1.21.3, EC 3.1.21.4, EC 3.1.21.5), e.g.EcoRI、HindII orBamHI or more generally, such as dnase i (EC 3.1.21.1), which cleaves phosphodiester linkages adjacent to pyrimidines.

The amount of enzyme added needs to be sufficient to substantially degrade the spacer over the desired period of time. Typically the efficiency is at least about 80%, more typically at least about 95%, preferably at least about 99%. The conditions for release can be optimized empirically based on temperature, pH, presence of metal cofactors, reducing agents, and the like. Degradation will typically be complete in at least about 15 minutes, more typically in at least about 10 minutes, and typically no longer than about 2 hours.

Complete degradation of the spacer P is not necessary. For the method of the present invention, it is necessary to degrade the spacer P as much as possible so that the fluorescent moiety X or the fluorescent moiety X and the antigen recognition moiety Y can be removed from the labeled target moiety by washing or dissociation.

Sequence of steps a) to d)

The method of the invention is particularly useful for detecting and/or isolating specific target moieties from complex mixtures and may be carried out in one or more of the sequences of steps a) to d). After each sequence, the fluorescent moiety, and optionally the antigen recognition moiety Y, is released (removed) from the target moiety. Furthermore, the sequence may have a combination of any of steps a) to d). The sequence may stop at any of steps a) to d). Additional cleaning steps may be performed.

In a variant of the invention, at least two conjugates are provided, wherein each antigen recognition moiety Y recognizes a different antigen, either simultaneously or in a subsequent staining sequence. In a further variant of the invention, at least two conjugates are provided, simultaneously or in a subsequent staining sequence, wherein each conjugate comprises a different fluorescent moiety X. In an alternative variant, the sample may be provided with at least two conjugates, wherein each conjugate comprises a different enzymatically degradable spacer P cleaved by a different enzyme, simultaneously or in a subsequent staining sequence. In all cases, the target portions of the markers may be detected simultaneously or sequentially. Sequential detection may involve simultaneous enzymatic degradation of the spacer molecule P, or subsequent enzymatic degradation of the spacer molecule P with optional intermediate removal (washing) of the non-bonded portion.

Embodiments of the sequence of steps a) to d)

The process of the invention may be carried out in the following embodiments:

in all variants and embodiments, the conjugates of general formula (I) can be used in a mixture and/or, if used in different sequences, in combination with one or more conjugates according to general formulae (II), (III), (IV), (V) and (VI).

Embodiment A of the invention is characterized in that steps a) to d) are carried out in at least one sequence, wherein in each sequence a conjugate of the general formula (I) or (II) is used. In this embodiment, in at least one sequence the biological sample is contacted with one conjugate in step b), the detection is performed in step c), and the conjugate is cleaved in step d). Thus, embodiment a comprises a single or multiple cycles of using one conjugate. In each cycle X, L, P, Y and o, n, m of the conjugate used may be the same or different kinds or numbers of antigen recognition moiety Y and/or linker unit L and/or fluorescent moiety X and/or enzymatically degradable spacer P.

An example of the use of this variant for a single cycle with a single conjugate is the separation of a cell population defined by the conjugate from a biological sample by fluorescence-based flow sorting, where the fluorescent label is eliminated after sorting, providing different downstream applications.

An example of the use of this variant for multiple cycles with a single conjugate is the sequential detection of different target moieties by using different antigen recognition moieties and the same fluorescent moiety in cycles of label-detection-elimination, which achieves a high multiplexing potential for mapping proteins on cells as by microscopy. Another example is that in a sequential sorting cycle using the same fluorescent moieties, cell subsets are separated from biological sample samples by fluorescence-based flow sorting. In a further example, in a first cycle, the same target moiety may be addressed with a conjugate having a fluorescent moiety suitable for flow sorting purposes, and after release of this fluorescent moiety, the target moiety may be re-addressed with a conjugate having another fluorescent moiety particularly suitable for analysis by fluorescence microscopy.

Embodiment B of the invention is characterized in that steps a) to d) are carried out in at least one sequence, wherein at least a first and a second conjugate of the general formula (I) or (II) are used in each sequence. In this embodiment, in at least one sequence, the biological sample is contacted with at least a first and a second conjugate in a simultaneous or subsequent step b), the detection is performed in a simultaneous or subsequent step c), and the conjugates are cleaved in a subsequent or simultaneous step d). Thus, embodiment B comprises a single or multiple cycles using multiple conjugates. In each cycle X, L, P, Y and o, n, m of the conjugate used may be the same or different kinds or numbers of antigen recognition moiety Y and/or linker unit L and/or fluorescent moiety X and/or enzymatically degradable spacer P.

An example of the use of this variant for a single cycle with multiple conjugates is simultaneous labeling with different conjugates, which enables differentiation of different cell subsets by flow cytometry analysis and separation of defined subsets by fluorescence-based flow sorting, where the fluorescent label is eliminated after sorting, providing different downstream applications.

An example of the use of this variant for multiple cycles with multiple conjugates is the sequential detection of different target moieties by using different antigen recognition moieties and different fluorescent moieties in the cycle of label-detection-elimination, which achieves even higher multiplexing potential.

Embodiment C of the invention is characterized in that steps a) to C) are carried out in at least two sequences, wherein one conjugate of the general formula (I) or (II) is used in each sequence, and step d) is carried out thereafter. In this embodiment, in at least two sequences, the biological sample is contacted with one conjugate in step b) and the detection is performed in step c). After at least two of those sequences, the conjugate is cleaved in a subsequent or simultaneous step d). Thus, embodiment C comprises single or multiple cycles a) -C) and step d) using one conjugate. In each cycle X, L, P, Y and o, n, m of the conjugate used may be the same or different kinds or numbers of antigen recognition moiety Y and/or linker unit L and/or fluorescent moiety X and/or enzymatically degradable spacer P.

Embodiment D of the invention is characterized in that steps a) to c) are carried out in at least two sequences, wherein at least a first and a second conjugate of the general formula (I) or (II) are used in each sequence, and step D) is carried out thereafter. In this embodiment, in at least two sequences, the biological sample is contacted with at least the first and second conjugates in a simultaneous or subsequent step b) and the detection is performed in a simultaneous or subsequent step c). After at least two of those sequences, the conjugate is cleaved in a subsequent or simultaneous step d). Thus, embodiment D comprises a single or multiple cycles a) -c) using multiple conjugates, as well as step D). In each cycle, the conjugates used may be the same or different, X, L, P, Y and o, n, m may be the same or different amounts of antigen recognition moiety Y and/or linker unit L and/or enzymatically degradable spacer P and/or fluorescent moiety X.

Embodiment E of the invention is characterized in that steps a) to b) are carried out in at least two sequences, wherein one conjugate of the general formula (I) or (II) is used in each sequence, and steps c) and d) are carried out thereafter. In this embodiment, the biological sample is contacted with one conjugate in step b) in at least two sequences. After at least two of those sequences, the detection is carried out in a subsequent or simultaneous step c) and the conjugate is cleaved in a subsequent or simultaneous step d). Thus, embodiment E comprises single or multiple cycles a) -b) and steps c) and d) using one conjugate. In each cycle X, L, P, Y and o, n, m of the conjugate used may be the same or different kinds or numbers of antigen recognition moiety Y and/or linker unit L and/or fluorescent moiety X and/or enzymatically degradable spacer P.

Embodiment F of the invention is characterized in that steps a) to b) are carried out in at least two sequences, wherein at least a first and a second conjugate of the general formula (I) or (II) are used in each sequence, and steps c) and d) are carried out thereafter. In this embodiment, in at least two sequences, the biological sample is contacted with at least the first and second conjugates in a simultaneous or subsequent step b). After at least two of those sequences, the detection is carried out in a simultaneous or subsequent step c) and the conjugate is cleaved in a subsequent or simultaneous step d). Thus, embodiment D comprises a single or multiple cycles a) -b) and steps c) and D) using multiple conjugates. In each cycle X, L, P, Y and o, n, m of the conjugate used may be the same or different kinds or numbers of antigen recognition moiety Y and/or linker unit L and/or fluorescent moiety X and/or enzymatically degradable spacer P.

An example of embodiments C to F is the stepwise analysis of individual target moieties in a biological sample with sequential overlaying of signals, wherein after a certain number of cycles the signals can be completely or only partially eliminated such that further cycles can be achieved. Those embodiments provide greater flexibility than embodiments a and B.

Embodiment G of the invention is characterized in that steps a) to d) are carried out in at least two staggered sequences, wherein in each sequence a conjugate of the general formula (I) or (II) is used. In this embodiment, the biological sample is contacted with one conjugate in step b), the detection is performed in step c), and the conjugate is lysed in step d) in at least two sequences, wherein step d) of the first cycle and step b) of the second cycle are combined in one simultaneous step. Thus, embodiment G comprises interleaved multiple cycles using one conjugate per cycle. X, L, Y and o, n, m of the conjugates used in each cycle may be the same or different kinds or numbers of antigen recognition moieties Y and/or linker units L and/or fluorescent moieties X. At least every second cycle, the enzymatically degradable spacer P is of a different kind.

Embodiment H of the invention is characterized in that steps a) to d) are carried out in at least two staggered sequences, wherein at least a first and a second conjugate of the general formula (I) or (II) are used in each sequence. In this embodiment, the biological sample is contacted with at least a first and a second conjugate in a simultaneous or subsequent step b) in at least two sequences, the detection is performed in a simultaneous or subsequent step c), and the conjugates are lysed in a subsequent or simultaneous step d), wherein step d) of the first cycle and step b) of the second cycle are combined in one simultaneous step. Thus, embodiment G comprises multiple cycles of interleaving using multiple conjugates. In each cycle X, L, Y and o, n, m of the conjugate used may be the same or different kinds or numbers of antigen recognition moieties Y and/or linker units L and/or fluorescent moieties X. At least every second cycle, the enzymatically degradable spacer P is of a different kind.

The process according to embodiment G or H provides a reduction in the time of the multiple cycles of labeling, detection and enzymatic degradation of the spacer P compared to embodiments a and B. The requirement of these embodiments is the use of at least two different enzymatically degradable spacers P and correspondingly different enzymes as release agents which can be used orthogonally to each other.

Use of the method

The methods of the invention can be used in a variety of applications in research, diagnostics, and cell therapy.

In a first use of the invention, a biological sample (e.g., cells) is detected or isolated for enumeration purposes, i.e., the number of cells is established from a sample having a set of antigens recognized by the antigen recognition portion of the conjugate.

In a second use, one or more biological sample populations are isolated for purification of target cells. Those isolated, purified cells can be used for a variety of downstream applications, such as molecular diagnostics, cell culture, or immunotherapy.

In other uses of the invention, the location of a moiety of interest (e.g., an antigen) on a biological sample that is recognized by an antigen recognition moiety of a conjugate is determined. Advanced imaging methods are known as "multiepitope ligand mapping", "chip-based cytometry" or "multiymx" and are described, for example, in EP 0810428, EP1181525, EP 1136822 or EP 1224472. In this technique, a sample of the biological sample is contacted in sequential cycles with an antigen recognition moiety coupled to a fluorescent moiety, the location of the antigen is detected by the fluorescent moiety, and the fluorescent moiety is subsequently eliminated. Thus, subsequent cycles of label-detection-elimination using at least one fluorescent moiety provide the possibility to map protein networks, locate different cell types, or analyze disease-related changes in proteomes.

Examples

Example 1-conjugation of dextran-PEG-coumarin-dye and dextran-coumarin-dye and burst fluorescence Determination of extinct

To prepare conjugates according to the invention, small organic molecule dyes such as coumarin-dyes like Pacific Blue NHS ester (available from siemer fly's science)) are dissolved in DMSO and carboxy-PEG-amines like ca (PEG)24 (available from siemer fly's science)) dissolved in DMSO are added. The reaction mixture was stirred at room temperature for 2 hours. The carboxy-PEG-coumarin-dye is then activated at room temperature overnight by the addition of EDC and NHS (available from e.g. merck).

In the next step, according to the invention (X) was prepared by incubating aminodextran (70 kilodaltons) at a concentration of 10mg/mL (available from Fina Biosolutions) with activated NHS-PEG-coumarin-dye and NHS-coumarin-dye (such as Pacific Blue) each in different molar ratios (dextran: NHS-coumarin-dye = 1: 10 to 1: 24)_o-L)_n-P toAnd according to the prior art (X)_n-dextran-fluorochrome-conjugate of P. After an incubation time of 60 minutes at room temperature, the dextran-fluorescent dye-conjugate was purified by size exclusion chromatography using PBS/EDTA buffer. The amount of conjugated coumarin-dye as well as the degree of labelling (DOL) was determined by the absorbance at a specific wavelength of the fluorescent dye (416 nm for coumarin-dye). DOL is 4.1, 6.5 and 8.6 for dextran-PEG-coumarin-dye and 3.7, 5.0, 7.8 for dextran-coumarin-dye.

dextran-PEG-coumarin-dye-and dextran-coumarin-dye-conjugates were diluted to the same dextran concentration to determine the dependence of fluorescence quenching on the degree of labeling. The absorbance at a specific wavelength of the fluorescent dye (416 nm for the coumarin dye) and the emission intensity after excitation at 416 nm were determined.

Figure 2 shows the absorption and emission intensities of exemplary dextran-PEG-coumarin-dye-and dextran-coumarin-dye-conjugates. For dextran-coumarin-dyes, the fluorescence emission intensity only increases minimally with increasing absorbance and DOL, respectively, indicating a strong quenching of the fluorescence of the coumarin molecule on the dextran molecule. In contrast, for dextran-PEG-coumarin-dye, the fluorescence emission intensity is higher at comparable DOL. The intensity increased with increasing absorbance and DOL, respectively, indicating that the PEG-linker prevents quenching of the coumarin molecule.

Example 2-conjugation with Fab-dextran-coumarin-dye-and Fab-dextran-PEG-coumarin-dye Cell surface reversible staining and flow cytometry analysis of Compounds

For the preparation of (X) according to formula (I)_o-L)_n – P – Y_mOr formula (II) (X)_n – P – Y_mThe antibody-or Fab-dextran-fluorescent dye-conjugate of (a), dextran-PEG-coumarin-dye-and dextran-coumarin-dye-conjugates are activated by incubation with SMCC for 60 minutes at room temperature and purified by size exclusion chromatography using PBS/EDTA buffer. Antibodies or Fab (e.g., anti-CD 4) were reduced with 10 mM DTT in MES buffer. At 90 minutesAfter a room temperature incubation time, the antibodies were purified by size exclusion chromatography using PBS/EDTA buffer. For conjugation of antibody-or Fab-dextran-fluorochrome-conjugates, activated Fab or antibody is added to the activated dextran. After an incubation time of 60 minutes at room temperature, a molar excess of β -mercaptoethanol followed by N-ethylmaleimide is added sequentially to block unreacted maleimide-or thiol-functional groups. Antibody-or Fab-dextran-fluorochrome-conjugates were purified by size exclusion chromatography using PBS/EDTA buffer. The concentration of antibody or Fab and fluorescent moiety is determined by the absorbance at 280 nm and the absorbance at the specific wavelength of the fluorescent dye.

Cell surface staining

PBMCs in PBS/EDTA/BSA buffer were stained with anti-CD 4-Fab-dextran-coumarin-dye-conjugate DOL 5.0 or with anti-CD 4-Fab-dextran-PEG-coumarin-dye-conjugate DOL 4.1, 6.5 and 8.6 for 10 min at 4 ℃. Cells were washed with cold PBS/EDTA-BSA buffer and analyzed by flow cytometry. For reversibility of the fluorescent label, cells were incubated with dextranase for 10 min at 21 ℃, washed with PBS/EDTA-BSA buffer and analyzed by flow cytometry.

FIG. 3 shows an exemplary histogram of the results of single parameter labeled flow cytometry analysis using different anti-CD 4-Fab-dextran-PEG-coumarin-dyes (a-c) and anti-CD 4-Fab-dextran-coumarin-dye-conjugates (d) (pre-gating of lymphocytes by propidium iodide and exclusion of dead cells, upper right: mean fluorescence intensity of CD4+ T cell population). Depending on DOL, the brightness of cells stained with anti-CD 4-Fab-glucan-PEG-coumarin-dye was 2.3-3.8 times higher than cells stained with anti-CD 4-glucan-coumarin-dye. The fluorescence intensity remaining for the labeled CD4+ T cell population after addition of the glucan degrading enzyme dextranase was within the detection limit.