METHOD FOR DETECTION OF CELLS BY REPETITIVE STAINING AND DESTAINING

Abstract

The invention is directed to a method for detecting a target moiety in a sample of biological specimens by providing a conjugate with the general formula (I)

##STR00001## characterized in contacting the sample of biological specimens with at least one conjugate (I), thereby labeling the target moiety recognized by an antigen recognizing moiety with the conjugate (I); exciting the labelled target moieties with light having a wavelength within the absorbance spectrum of the fluorescent moiety FL; detecting the labelled target moieties by detecting the fluorescence radiation emitted by the fluorescent moiety FL and degrading the fluorescent moiety FL of the labelled target moieties by irradiating the conjugate with light having a wavelength within the absorbance spectrum of fluorescent moiety FL for a time sufficient to deliver enough energy to reduce the fluorescence radiation emitted by the fluorescent moiety FL at least by 75% of the initial fluorescence radiation.

Claims

1. A method for detecting a target moiety in a sample of biological specimens by: providing a conjugate with the general formula (I): ##STR00006## wherein Ar, MU and L1 are repeating units of a polymer and wherein Ar is a aryl or heteroaryl group, MU is a polymer modifying unit or band gap modifying unit that is evenly or randomly distributed along the polymer main chain, L1 is an aryl or a heteroaryl group evenly or randomly distributed along the polymer, L2 is an aryl or a heteroaryl group located on the ends of the polymer, FL is a fluorescent moiety, G1 and G2 stand for hydrogen, halogen or an antigen recognizing moiety, with the provision than at least one of G1 or G2 is an antigen recognizing moiety, and a is 10 to 100 mol %, b is 0.1 to 50 mol % c is 0 to 90 mol % d is 1 to 10,000 with the provisio that a+b+c=100 mol %, contacting the sample of biological specimens with at least one conjugate (I), thereby labeling the target moiety recognized by an antigen recognizing moiety with the conjugate (I); exciting the labelled target moieties with light having a wavelength within the absorbance spectrum of the fluorescent moiety FL; detecting the labelled target moieties by detecting the fluorescence radiation emitted by the fluorescent moiety FL and degrading the fluorescent moiety FL of the labelled target moieties by irradiating the conjugate with light having a wavelength within the absorbance spectrum of fluorescent moiety FL for a time sufficient to deliver enough energy to reduce the fluorescence radiation emitted by the fluorescent moiety FL at least by 75% of the initial fluorescence radiation.

2. The method according to claim 1, characterized in that Ar is a aryl or heteroaryl group repeat unit substituted with a non-ionic side chain selected from the groups of an ethylene glycol oligomer side, dextran or glycerol.

3. The method according to claim 1, characterized in that MU is a polymer modifying unit or band gap modifying unit that is evenly or randomly distributed along the polymer main chain and is optionally substituted with one or more optionally substituted substituents selected from halogen, hydroxyl, C1-C12 alkyl, C2-C12 alkene, C2-C12 alkyne, C3-C12 cycloalkyl, C1-C12 haloalkyl, C1-C12 alkoxy, C2-C18 (hetero)aryloxy, C2-C18 (hetero) arylamino, a C2-C18 (hetero)aryl group and (CH2)x′, (OCH2CH2)y′ OCH3 where x′ is independently an integer from 0-20 and y′ is independently an integer from 0 to 50.

4. The method according to claim 1, characterized in that L1 is an aryl or a heteroaryl group evenly or randomly distributed along the polymer main chain and is substituted with one or more pendant chains terminated with: i) a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazine, azide, alkyne, aldehyde, thiol, and protected groups thereof for conjugation to a molecule or biomolecule; or ii) an attached conjugated organic dye as acceptor dye, or iii) a biomolecule.

5. The method according to claim 1, characterized in that L2 is an aryl or a heteroaryl group located on the ends of the polymer main chain and is substituted with one or more pendant chains terminated with: i) a functional group selected from amine, carbamate, carboxylic acid, carboxylate, maleimide, activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazine, azide, alkyne, aldehyde, thiol, and protected groups thereof for conjugation to a molecule or biomolecule; or ii) an attached organic dye as acceptor dye, or iii) a biomolecule.

6. The method according to claim 1, characterized in that FL is selected from the group consisting of Fluorescein, Fluorescein-Derivatives, Rhodamine, Tetramethylrhodamine, Silicon-Rhodamine (SiR), Coumarines, Resorufines, Pyrenes, Anthracenes, Phenylenes, Phthalocyanines, Cyanines, Xanthenes, Amidopyrylium-Fluorophores, Oxazine, Quadrain-Farbstoffe, Carbopyronine, 7-Nitrobenz-2-Oxa-1,3-Diazol (NBD) Fluorophore, BODIPY™ Fluorophores (Molecular Probes, Inc.), ALEXA™ Fluorophore (Molecular Probes, Inc.), DY™ Fluorophores (Dyomics GmbH), Benzopyrylium Fluorophores, Benzopyrylium-Polymethine Fluorophores, Lanthanid-Chelate, Metalloporhyrines, Rhodol dyes, Carborhodol dyes, Naphthalimides and Porphyrines.

7. The method according to claim 1, characterized in that G1 and G2 are both independently chosen from the group consisting of hydrogen, halogen or an antigen recognizing moiety at least one is biomolecule selected from the group onsisting of an antibody, an fragmented antibody, an fragmented antibody derivative, peptide/MHC-complexes, receptors for cell adhesion or costimulatory molecules, receptor ligands, antigens, hapten binders, avidin, streptavidin, travidin, aptamers, primers and ligase substrates, peptide/MHC complexe targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules or artificial engineered binding molecules.

8. The method according to claim 1, characterized in providing a conjugate according to general formula (II) ##STR00007## With R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, each same or independently ═H, SO.sub.2CF.sub.3, SO.sub.2R.sup.a, CF.sub.3, CCl.sub.3, CN, SO.sub.3H, NO.sub.2, NR.sup.aR.sup.bR.sup.c+, CHO, COR.sup.a, CO.sub.2R.sup.a, COCl, CONR.sup.aR.sup.b, F, Cl, Br, I, R.sup.a, OR.sup.a, SR.sup.a, OCOR.sup.a, NR.sup.aR.sup.b, NHCOR.sup.a, CCR.sup.a, aryl-, heteroaryl-, C.sub.6H.sub.4OR.sup.a or C.sub.6H.sub.4NR.sup.aR.sup.b, with R.sup.a-c independently hydrogen, alkyl-, alkenyl-, alkinyl-, heteroalkyl-, aryl-, heteroaryl-, cycloalkyl-, alkylcycloalkyl-, heteroalkylcycloalkyl-, heterocycloalkyl-, aralkyl- or a heteroaralkyl residue, or two residues both as part of a cycloalkyl- or heterocycloalkyl ring system and each residue is made of 1 to 100 atoms. x is an integer between 1 and 100, y is an integer between 0 and 100, a is 10 to 100 mol %, b is 0.1 to 50 mol % c is 0 to 90 mol % d is 1 to 10,000 with the provisio that a+b+c=100 mol %

9. The method according to claim 1, wherein the fluorescent moiety FL of the labelled target moieties is further degraded by adding oxidative agents.

10. Use of the method of any of the claims 1 to 8, in fluorescence microscopy, flow cytometer, fluorescence spectroscopy, cell separation, pathology or histology.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Various exemplary details are described with reference to the following figures, wherein:

[0031] FIG. 1. shows a schematic curve obtained for photodegradation of a xanthene dye;

[0032] FIG. 2. shows photodegrading decay curves for a series of dyes; and

[0033] FIG. 3 shows photodegradation curves obtained for a rhodamine dye.

[0034] It should be understood that the drawings are not necessarily to scale, and that like numbers may refer to like features.

DETAILED DESCRIPTION

[0035] In the following, the conjugate according to formula (1) is referred to CP-FL with polymer backbone CP as defined as followed to which one or more fluorescent moieties

##STR00003##

FL are attached.

[0036] In the method of the invention, the sample is irradiated with light having a wavelength within the absorbance spectrum of the fluorescent moiety FL in order to reduce the fluorescence radiation emitted by the fluorescent moiety so much that any residual fluorescence radiation from a first staining cycle does not interfere with subsequent staining and detection cycles. In general, reduction by at least 75% of the initial fluorescence radiation is deemed sufficient, but in order to achieve a higher quality of detection i.e. to reduce background radiation not originating from the staining step of interest, it is preferred to reduce fluorescence radiation by at least by 85%, more preferred at least by 95% and most preferred by at least 99%. While a reduction of 100% would be best, there is a trade-off with quenching quality and overall process duration.

[0037] In an alternative definition, degrading the fluorescent moiety FL attached to a conjugated polymer (CP) of the labelled target moieties is performed by irradiating the conjugate with light having a wavelength within the absorbance spectrum of fluorescent moiety FL or of CP or both (e.g. using white light) for a time sufficient to deliver enough energy to reduce the half-life of the fluorescence radiation emitted by the fluorescent moiety. The degradation rate given by the value of k from the mono-exponential decay fit analysis of the fluorescent moiety FL be at least 1.02 and up to 10.000.000 fold higher compared to the k obtained for the same fluorescent moiety non-conjugated to the conjugated polymer (CP).

[0038] Fluorescent moiety FL and antigen recognizing moiety can be bound covalently or quasi-covalently to CP. The terms “covalently or quasi-covalently” refers bonds between FL and CP and Y having a dissociation constant of greater or equal than 10.sup.−9 M.

[0039] The process of the invention may be performed in one or more sequences of the steps a) to d). After each sequence, the fluorescent moiety is degraded by irradiation with light. The terms “degrading”, “quenching” or “bleaching” are used interchangeably herein, and should be understood to mean the diminution of fluorescence intensity from the labeled biological sample, as result of an alteration of the fluorophore by radiation. For example, “quenching” or “bleaching” of the fluorescent moiety FL may be achieved by oxidation initiated by the radiation and/or by cleaving the fluorescent moiety FL from CP and removing the unbound fluorescent moiety from the labelled target by washing.

[0040] The bleaching system used in the present invention may be provided with more than one light sources emitting radiation of different wavelengths. For example the bleaching system may be provided with 1-5 light sources which have a combined emission spectrum in the range of 350-850 nm, preferable 400-650 nm. The emission of the light sources may optically combined to irradiate the sample simultaneously or subsequently. For example, the bleaching system may be provided with four light sources emitting in the ranges 380-410 (violet), 450-500 nm (blue), 520-560 nm (green) and 630-650 nm (red). In another embodiment only one light source is provided, emitting light in the range 200-1000 nm (white light), preferable 350-850 nm, and most preferable 400-650 nm. The advantage of separate light sources is that the sample is exposed to radiation only necessary to bleach (eliminate) the fluorescence dye thereby avoiding unnecessary exposure of the sample to radiation with other wavelengths. The radiation of the separate light sources may be combined by appropriate devices like mirrors or optical waveguide like optical fiber.

[0041] After and/or before each sequence, a washing step may be performed to remove unwanted material like unbound conjugates moieties and/or unbound fluorescent moieties FL from the sample.

[0042] The bleaching process as described may be further enhanced by adding oxidative agents. Oxidative agents may be for example O.sub.2, H.sub.2O.sub.2, peroxides or DMSO. The oxidative agents added may generate the active oxidative species, which, calculated as O, should be present in concentrations of 0.1 to 5 ppm, preferable 2 to 5 ppm.

Target Moiety

[0043] The target moiety to be detected with the method of the invention can be on any biological specimen, like tissues slices, cell aggregates, suspension cells, or adherent cells. The cells may be living or dead. Preferable, target moieties are antigens expressed intracellular or extracellular on biological specimen like whole animals, organs, tissues slices, cell aggregates, or single cells of invertebrates, (e.g., Caenorhabditis elegans, Drosophila melanogaster), vertebrates (e.g., Danio rerio, Xenopus laevis) and mammalians (e.g., Mus musculus, Homo Sapiens).

Fluorescent Moiety FL

[0044] Suitable fluorescent moieties FL are those known from the art of immunofluorescence technologies, e.g., flowcytometry or fluorescence microscopy. In the method of the invention, the target moiety labelled with the conjugate is detected by exciting the CP backbone or the the fluorescent moiety FL or both and detecting the resulting emission (photoluminescence) of FL or CP.

[0045] Useful fluorescent moieties FL might be protein based, such as phycobiliprotein, small organic molecule dyes, such as xanthenes, like fluorescein, or rhodamines, cyanines, oxazines, coumarins, acridines, oxadiazoles, pyrenes, pyrromethenes, pyridyloxazole or metallo-organic complexes, such as Ru, Eu, Pt complexes. Besides single molecule entities, clusters of fluorescent proteins or small organic molecule dyes, as well as nanoparticles, such as quantum dots, upconverting nanoparticles, gold nanoparticles, dyed polymer nanoparticles can also be used as fluorescent moieties.

[0046] In another embodiment of the invention the target labelled with the conjugate is not detected by radiation emission, but by absorption of UV, visible light, or NIR radiation. Suitable light-absorbing detection moieties are light absorbing dyes without fluorescence emission, such as small organic molecule quencher dyes like N-aryl rhodamines, azo dyes, and stilbenes. In another embodiment, the light-absorbing fluorescent moieties FL can be irradiated by pulsed laser light, generating an photoacoustic signal.

[0047] In a variant of the invention, the fluorophore FL is substituted with one more water solubility imparting substituents selected from the group consisting of sulfonates, phosphonates, phosphates, polyethers, sulfonamides and carbonates. It is particularly advantageous to use fluorescent moieties with sulfonate substituents, such as dyes of the Alexa Fluor family provided by Thermo Fisher Scientific Inc. The degree of sulfonate substitution per fluorophore may be 2 or more, i.e., for rhodamine dyes or cyanine dyes.

[0048] Suitable commercial available fluorescent moieties may be purchased from the product line “Vio” from Miltenyi Biotec BV & Co. KG, or FITC, or Promofluor, or Alexa Dyes and/or Bodipy dyes from Thermofisher, or Cyanines from Lumiprobe or DY™ Fluorophore from Dyomics GmbH or Star Dyes from Abberior GmbH.

[0049] Antigen Recognizing Moiety

[0050] The term “antigen recognizing moiety” refers to any kind of antibody, fragmented antibody or fragmented antibody derivatives, directed against the target moietiesexpressed on the biological specimens, like antigens expressed intracellular or extracellular on cells. The term relates to fully intact antibodies, fragmented antibody or fragmented antibody derivatives, e. g., Fab, Fab′, F(ab′)2, sdAb, scFv, di-scFv, nanobodies. Such fragmented antibody derivatives may be synthesized by recombinant procedures including covalent and non-covalent conjugates containing these kind of molecules. Further examples of antigen recognizing moieties are peptide/MHC-complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for costimulatory molecules, artificial engineered binding molecules, e.g., peptides or aptamers which target, e.g., cellsurface molecules.

[0051] The conjugate used in the method of the invention may comprise up to 100, preferable 1-20 antigen recognizing moieties Y. The interaction of the antigen recognizing moiety with the target antigen can be of high or low affinity. Binding interactions of a single low-affinity antigen recognizing moiety is too low to provide a stable bond with the antigen. Low-affinity antigen recognizing moieties can be multimerized by conjugation to the enzymatically degradable spacer to furnish high avidity. When the spacer is enzymatically cleaved, the low-affinity antigen recognizing moieties will be monomerized which results in a complete removal of the fluorescent marker.

[0052] Preferable, the term “Antigen recognizing moiety” refers to an antibody directed against antigen expressed by the biological specimens (target cells) intracellular, like IL2, FoxP3, CD154, or extracellular, like CD19, CD3, CD14, CD4, CD, CD25, CD34, CD56, and CD133. The antigen recognizing moieties G1, G2, especially antibodies, can be coupled to CP through side chain amino or sulfhydryl groups. In some cases the glycosidic side chain of the antibody can be oxidized by periodate resulting in aldehyde functional groups.

[0053] The antigen recognizing moiety can be covalently or non-covalently coupled. Methods for covalent or non-covalent conjugation are known by persons skilled in the art and the same as mentioned for conjugation of the fluorescent marker.

[0054] The method of the invention is especially useful for detection and/or isolation of specific cell types from complex mixtures and may comprise more than one sequentialsequences of the steps a)-d). The method may use a variety of combinations of conjugates. For example, a conjugate may comprise antibodies specific for two different epitopes, like two different anti-CD34 antibodies. Different antigens may be addressed with different conjugates comprising different antibodies, for example, anti-CD4 and anti-CD8 for differentiation between two distinct T-cell-populations or anti-CD4 and anti-CD25 for determination of different cell subpopulations like regulatory T-cells.

Cell Detection Methods

[0055] Targets labelled with the conjugate are detected by exciting either the fluorescent moiety FL or the backbone CP and analysing the resulting fluorescence signal. The wavelength of the excitation is usually selected according to the absorption maximum of the fluorescent moiety FL or CP and provided by LASER or LED sources as known in the art. If several different detection moieties FL are used for multiple colour/parameter detection, care should be taken to select fluorescent moieties having not overlapping absorption spectra, at least not overlapping absorption maxima. In case of fluorescent moieties the targets may be detected, e.g., under a fluorescence microscope, in a flow cytometer, a spectrofluorometer, or a fluorescence scanner. Light emitted by chemiluminescence can be detected by similar instrumentation omitting the excitation.

Use of the Method

[0056] The method of the invention can be used for various applications in research, diagnostics and cell therapy, like in fluorescence microscopy, flow cytometer, fluorescence spectroscopy, cell separation, pathology or histology.

[0057] In a first variant of the invention, biological specimens like cells are detected for counting purposes i.e. to establish the amount of cells from a sample having a certain set of antigens recognized by the antigen recognizing moieties of the conjugate. In another variant, the biological specimens detected by the conjugate in step c) are separated from the sample by optical means, electrostatic forces, piezoelectric forces, mechanical separation or acoustic means. For this purpose, the biological specimens detected by the conjugate in step d) are separated from the sample according to their detection signal to one or more populations simultaneously or subsequent before performing step d) by optical means, electrostatic forces, piezoelectric forces, mechanical separation or acoustic means.

[0058] In another variant of the invention, the location of the target moieties like antigens on the biological specimens recognized by the antigen recognizing moieties of the conjugate is determined. Such techniques are known as “Multi Epitope Ligand Cartography”, “Chip-based Cytometry” or “Multiomyx” and are described, for example, in EP0810428, EP1181525, EP 1136822 or EP1224472. In this technology, cells are immobilized and contacted with antibodies coupled to fluorescent moiety. The antibodies are recognized by the respective antigens on the biological specimen (for example on a cell surfacd) and after removing the unbound marker and exciting the furescentieties, the location of the antigen is detected by the fluorescence emission of the fluorescent moieties. In certain variants, instead of antibodies coupled to fluorescent moieties, antibodies coupled to moieties detectable for MALDI-Imaging or CyTOF can be used. The person skilled in the art is aware how to modify the technique based on fluorescent moiety to work with these detection moieties.

[0059] The location of the target moieties is achieved by a digital imaging device with a sufficient resolution and sensitivity in for the wavelength of the fluorescence radiation, The digital imaging device may be used with or without optical enlargement for example with a fluorescence microscope. The resulting images are stored on an appropriate storing device like a hard drive, for example in RAW, TIF, JPEG, or HDF5 format.

[0060] In order to detect different antigens, different antibody-conjugates having the same or different fluorescent moiety or antigen recognizing moiety can be provided. Since the parallel detection of fluorescence emission with different wavelengths is limited, the antibody-fluorochrome conjugates are utilized sequentially individually or in small groups (2-10) after the other.

[0061] In yet another variant of the method according to the invention, the biological specimens especially suspension cells of the sample are immobilized by trapping in microcavities or by adherence.

[0062] In general, the method of the invention can be performed in several variants. For example, the conjugate not recognized by a target moiety can be removed by washing for example with buffer before the target moiety labelled with the conjugate is detected.

[0063] In a variant of the invention, at least two conjugates are provided simultaneously or in subsequent staining sequences, wherein each antigen recognizing moiety recognizes different antigens. In an alternative variant, at least two conjugates can be provided to the sample simultaneously or in subsequent staining sequences. In both cases, the labelled target moieties can be detected simultaneously or sequentially.

Examples

[0064] The following compounds were investigated for their absorption behavior:

[0065] CP is

##STR00004##

[0066] with n=0.9,

[0067] m=0.1 and

[0068] x=11, and CP-FL is

##STR00005##

[0069] with n=0.9,

[0070] m=0.1 and

[0071] x=11.

[0072] With FL=fluorescein rhodamines, cyanines or carbopyronine

[0073] In order to illustrate the general kinetics involved in photodegradation, FIG. 1 shows a schematic curve obtained for photodegradation of a xanthene dye by light fitted with a mono-exponential decay curve such as f(x)=y0*exp(−k*x), where tau=1/k and t.sub.1/2=tau*ln(2) is the half-life. To measure the kinetics of photodegradation, organic fluorophores (i. e. coumarines, xanthenes, rhodamines, cyanines, among others) were either dissolved in DMSO and diluted in PBS or directly dissolved in PBS such that the concentration was adjusted to obtain an absorbance at their respective maxima of ca. 0.3 A.U. with a path-length of 1.00 cm. This way all solutions are normalized by their absorbance and comparable. Then the solution was placed inside a 3 way window fluorescence quartz cuvette with low head space and an air-tight top to avoid evaporation and sample concentration. The sample in the cuvette was then irradiated for fixed amount of times and both the absorbance and mission spectra were recorded. Intensity values and their maxima were used to plot vs. irradiation time and then a mathematical fit to mono-exponential decays was performed by appropriate computer software to obtain the curves as one shown in FIG. 1, with an absorption high absorption of the CP at roughly 400 nm and a redshifted absorption of the FL. Characteristic decay times (k) were used to calculate half-life among other parameters.

[0074] FIG. 2. shows photodegrading decay curves for a series of dyes belonging to a different chemical classification according to the nature of their chromophore (i.e. fluorescein rhodamines, cyanines, carbopyronine) where each dye is covalently attached to a CP moiety.

[0075] The data shown in FIG. 2 show that all dye classes are sensitive to photodegradation when they are attached to a CP as defined below and the rate of photodegradation is higher for constructs of this application than for FL itself.

[0076] FIG. 3 shows photodegradation curves obtained for a rhodamine dye under three different conditions: i) not bound to anything and free in solution, ii) attached to branched PEG as described in US2019/0162721A1 iii) covalently attached to a linear CP (according to this invention).

[0077] The results of table 1 show the different constructs of CP-FL compared to small molecule moieties (FL) show an increase in the bleaching constant K for the different constructs by a factor of 94 (Fluorescein), 22.5 (Rhodamine) and 5.2 (Cyanine), while the half-life of the fluorophores is reduced by a factor of 38 (Fluorescein), 156 (Rhodamine), 98 (Cyanine).

[0078] As shown in FIG. 3 the herein presented invention (e.g. CP-Rhodamine) shows an increase in the bleaching constant of factor 46 over the constructs from US2019/0162721A1 (Branched PEG Rhodamine) using a multimerization of FL on branched PEG as the state of the art. This higher bleaching constant leads to shorter bleaching times and less background e.g. in cycling imaging applications.

TABLE-US-00001 TABLE 1 Comparison of bleaching behavior of small molecule dyes and small molecule dyes as the FL part bound to CP Dye Fluorescein Rhodamine Cyanine Construct CP-FL FL CP-FL FL CP-FL FL k [min.sup.−1] 1.622 152.1 0.1789 4.02 1.417 5.16 τ [min] 0.43 16.2 3.87 606 0.49 48

[0079] While various details have been described in conjunction with the exemplary implementations outlined above, various alternatives, modifications, variations, improvements, and/or substantial equivalents, whether known or that are or may be presently unforeseen, may become apparent upon reviewing the foregoing disclosure. Accordingly, the exemplary implementations set forth above, are intended to be illustrative, not limiting.