A method for detecting cells by repeated staining and destaining

Abstract

The present invention relates to a method for detecting a target moiety in a sample of a biological sample by providing a conjugate having the general formula (I), characterized in that a) a sample of a biological sample is brought into contact with at least one conjugate (I), whereby a target moiety recognized by the antigen recognizing moiety is labeled with the conjugate (I); b) the labeled target moiety is excited with light having a wavelength within the absorption spectrum of the fluorescent moiety FL; c) the labeled target moiety is detected by detecting the fluorescent radiation emitted by the fluorescent moiety FL, and d) the fluorescent moiety FL of the labeled target moiety is degraded by irradiating the conjugate with light having a wavelength within the absorption spectrum of the fluorescent moiety FL for a time sufficient to transfer sufficient energy to the fluorescent moiety FL to reduce the fluorescent radiation emitted by the fluorescent moiety FL by at least 75% of the initial fluorescent radiation.

Description

Technical Field

[0001] The present invention relates to a method for detecting or identifying a target part or target cell from a cell sample by labeling the target part or target cell with a conjugate having a detection part and an antigen recognition part, wherein after the target part is detected, the detection part is degraded by light irradiation, thereby enabling subsequent labeling and detection. Background Technology

[0002] Fluorescent moieties conjugated to one or more antibodies are commonly used in immunofluorescence analysis. Over the past two decades, numerous variants have been developed in antibodies, fluorescent moieties, flow cytometry, flow cytoscopy sorters, and fluorescence microscopy to enable the specific detection and separation of target cells.

[0003] It is known to use fluorescent fractions for the detection of isolated target cells, where the fluorescent fraction can be removed or destroyed after detection. For example, US7776562 discloses a reversible fluorescent labeling method based on indirect, non-covalent labeling of target cells using reversible peptide / MHC-multimers or Fab-streptamers.

[0004] To reduce fluorescence radiation after detection, GB 2372256 discloses a method for quenching fluorescence radiation by providing a conjugate comprising multiple fluorescent moieties attached to an antibody via a linker. The high density of the fluorescent moieties quenches the fluorescence signal. Furthermore, GB 2372256 describes enzymatic degradation of the linker to release the fluorescent moieties from the conjugate. The released fluorescent moieties do not undergo self-quenching, resulting in a stronger fluorescence signal, i.e., better resolution.

[0005] Elimination of the fluorescence signal is essential for immunofluorescence techniques based on sequentially stained samples. These techniques have shown greater multiplexing potential compared to standard procedures that use labeling and detection simultaneously. However, these techniques rely on the oxidative destruction of the antibody-conjugated fluorescent portion through chemical bleaching procedures (US7741045B2, EP0810428B1, or DE10143757), or, in the case of photobleaching-based methods, bleaching rates are slower than those presented here. US2019 / 0162721A1 illustrates photobleaching of a dye following polymerization on branched PEG.

[0006] Conjugated polymers (CPs) with small molecule dyes are also used for signal amplification, and are used as bright fluorescent components in examples such as US10126302B2 or US10481161B2. However, enhanced photobleaching is not mentioned.

[0007] The primary intent of existing technologies is to provide dyes, or conjugates containing such dyes, that emit the most intense fluorescence possible, i.e., with the highest quantum yield. To provide reliable and reproducible signals, the dyes are designed to be as stable as possible. While these properties are advantageous for cell detection and cell separation methods such as FACS, they prevent cells from being repeatedly stained and detected. Summary of the Invention

[0008] Accordingly, there is a need to develop methods for staining and destaining target portions labeled with bright fluorescent fractions, wherein the staining process provides a signal that is as bright as possible, and that the signal can be completely removed as quickly as possible during the destaining process. It has been found that a very efficient staining / destaining process can be achieved after appropriately selecting certain conjugates and developing a custom method.

[0009] The object of this invention is a method for detecting a target moiety in a biological sample by providing a conjugate having the general formula (I). Ar, MU, and L1 serve as repeating units in the polymer. Ar is an aryl or heteroaryl group. MU refers to polymer modification units or band gap modification units that are uniformly or randomly distributed along the polymer backbone. L1 represents aryl or heteroaryl groups that are uniformly or randomly distributed along the polymer. L2 is an aryl or heteroaryl group located at the end of the polymer. FL is the fluorescent component. G1 and G2 represent hydrogen, halogen, or antigen recognition moieties, provided that at least one of G1 or G2 is an antigen recognition moieties. and a is 10 to 100 mol%. b is 0.1 to 50 mol%. c is 0 to 90 mol%. d is from 1 to 10,000. The condition is that a + b + c = 100 mol%. Its features a) Contacting a biological sample with at least one conjugate (I) thereby labeling the target portion recognized by the antigen recognition portion with the conjugate (I); b) Excite the labeled target portion with light whose wavelength is within the absorption spectrum of the fluorescent portion FL; c) The labeled target portion is detected by detecting the fluorescent radiation emitted by the fluorescent portion FL, and d) By irradiating the conjugate with light of wavelength within the absorption spectrum of the fluorescent portion FL for a time sufficient to transfer enough energy, the fluorescent portion FL of the labeled target portion is degraded, and the energy causes the fluorescent radiation emitted by the fluorescent portion FL to decrease by at least 75% of the initial fluorescent radiation.

[0010] Another object of the present invention is the use of the method in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell separation, pathology or histology. Attached Figure Description

[0011] Figure 1 The single exponential decay curve is shown as follows: f (x) = y0*exp(- k *x) fitted, schematic curves obtained for the photodegradation of thallium dye by light, where tau = 1 / k and t 1 / 2 = tau*ln(2) is the half-life.

[0012] Figure 2 The photodegradation decay curves of a series of dyes belonging to different chemical classifications are shown based on the properties of their chromophores (i.e., fluorescein rhodamine, cyanine, carbopyronine), with each dye covalently attached to the CP moiety.

[0013] Figure 3 The photodegradation curves of the rhodamine dyes obtained under three different conditions are shown: i) unbound and free in solution, ii) attached to branched PEG as described in US2019 / 0162721A1, and iii) covalently attached to linear CP (according to the present invention). Detailed Implementation

[0014] In the following text, the conjugate according to formula (1) is referred to as CP-FL, which has one or more fluorescent moieties FL attached to a polymer backbone CP as defined below.

[0015] In the method of this invention, the sample is irradiated with light of a wavelength within the absorption spectrum of the fluorescent portion (FL) to reduce the fluorescence radiation emitted by the fluorescent portion so much that any residual fluorescence radiation from the first staining cycle does not interfere with subsequent staining and detection cycles. Generally, a reduction of at least 75% in initial fluorescence radiation is considered sufficient, but for higher detection quality, i.e., reduction of background radiation not originating from the target staining step, a reduction of at least 85%, more preferably at least 95%, and most preferably at least 99% is preferred. While a 100% reduction would be optimal, there is a trade-off between quenching quality and overall method duration.

[0016] In an alternative definition, degradation of the fluorescent portion FL attached to the labeled target portion of the conjugate polymer (CP) is performed by irradiating the conjugate with light (e.g., white light) with a wavelength within the absorption spectrum of the fluorescent portion FL or CP, or both, for a sufficient time to transfer sufficient energy, wherein the energy reduces the half-life of the fluorescent radiation emitted by the fluorescent portion. This is achieved with the same fluorescent portion not conjugated to the conjugate polymer (CP). k In contrast, analysis using single-exponential decay fitting from the fluorescent fraction FL... k The values ​​given are for degradation rates of at least 1.02 times and up to 10,000,000 times.

[0017] The fluorescent fraction (FL) and the antigen-recognizing fraction can covalently or quasi-covalently bind to the antigen-recognizing fraction (CP). The term "covalently or quasi-covalently" refers to a bond strength (greater than or equal to 10) between FL and CP and Y. -9 The dissociation constant of M.

[0018] The method of the present invention can be performed in one or more sequences of steps a) to d). After each sequence, the fluorescent portion is degraded by irradiation with light. The terms “degradation,” “quenching,” or “bleaching” are used interchangeably herein and should be understood to mean a reduction in fluorescence intensity from the labeled biological sample due to changes in the fluorophore caused by radiation. For example, “quenching” or “bleaching” of the fluorescent portion FL can be achieved by radiation-induced oxidation and / or cleaving the fluorescent portion FL from the CP and removing unbound fluorescent portions from the labeled target by washing.

[0019] The bleaching system used in this invention can provide more than one light source emitting radiation of different wavelengths. For example, the bleaching system can provide 1-5 light sources having a combined emission spectrum in the range of 350-850 nm, preferably 400-650 nm. The emission of the light sources can be optically combined to simultaneously or subsequently irradiate the sample. For example, the bleaching system can provide four light sources emitting in the ranges of 380-410 nm (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 of 200-1000 nm (white light), preferably 350-850 nm, and most preferably 400-650 nm. The advantage of separate light sources is that the sample is exposed only to the radiation required to bleach (eliminate) the fluorescent dye, thereby avoiding unnecessary exposure of the sample to radiation of other wavelengths. The radiation of the separate light sources can be combined by suitable means such as mirrors or optical waveguides such as optical fibers.

[0020] A washing step may be performed after and / or before each sequence to remove unwanted materials from the sample, such as unbound conjugate portions and / or unbound fluorescent portions (FL).

[0021] The bleaching process described above can be further enhanced by adding an oxidizing agent. The oxidizing agent can be, for example, O2, H2O2, peroxide, or DMSO. The added oxidizing agent should generate an active oxidizing agent, which, as oxygen, should be present at a concentration of 0.1 to 5 ppm, preferably 2 to 5 ppm.

[0022] Target section The target portion to be detected by the method of the present invention can be on any biological sample, such as tissue sections, cell aggregates, suspension cells, or adherent cells. The cells may be alive or dead. Preferably, the target portion is an antigen expressed intracellularly or extracellularly on a biological sample, such as a whole animal, organ, tissue section, cell aggregate, or a single cell of invertebrates (e.g., *Caenorhabditis elegans*, *Drosophila melanogaster*), vertebrates (e.g., zebrafish (*Danio rerio*, *Xenopus laevis*), and mammals (e.g., house mouse (*Mus musculus*, *Homo sapiens*)).

[0023] Fluorescent part FL Suitable fluorescent fractions (FLs) are those known in the field of immunofluorescence techniques, such as flow cytometry or fluorescence microscopy. In the method of the present invention, the target fraction labeled by the conjugate is detected by exciting the CP backbone or the fluorescent fraction FL, or both, and detecting the emission of the resulting FL or CP (photoluminescence).

[0024] Useful fluorescent moieties (FLs) can be protein-based, such as phycobiliproteins; small organic dyes, such as xanthines (e.g., fluorescein); or rhodamine, anthocyanins, oxazines, coumarins, acridines, oxadiazoles, pyrene, pyrromethene, pyridyloxazoles; or metal-organic complexes, such as Ru, Eu, and Pt complexes. Besides single-molecule entities, clusters of fluorescent proteins or small organic dyes, as well as nanoparticles such as quantum dots, upconversion nanoparticles, gold nanoparticles, and stained polymer nanoparticles, can also be used as fluorescent moieties.

[0025] In another embodiment of the invention, the target labeled with the conjugate is detected not by radiation emission, but by absorption of UV, visible, or NIR radiation. A suitable light-absorbing detection portion is a light-absorbing dye that does not emit fluorescence, such as small organic molecule quencher dyes, like N-arylrhodamine, azo dyes, and stilbene. In another embodiment, the light-absorbing fluorescent portion FL can be irradiated with a pulsed laser to generate a photoacoustic signal.

[0026] In a variation of the invention, the fluorophore FL is substituted with one or more substituents that impart water solubility, said substituents being selected from sulfonates, phosphonates, phosphates, polyethers, sulfonamides, and carbonates. Particularly advantageous are the use of fluorescent moieties having sulfonate substituents, such as dyes from the Alexa Fluor family supplied by Thermo Fisher Scientific Inc. The degree of sulfonate substitution for each fluorophore can be 2 or higher, i.e., for rhodamine dyes or cyanide dyes.

[0027] Suitable commercially available fluorescent components can be purchased from Miltenyi Biotec BV & Co. KG, FITC, or Promofluor's "Vio" product line; or Thermofisher's Alexa dyes and / or Bodipy dyes; or Lumiprobe's cyanine dyes; or Dyomics GmbH's DY dyes. TM Fluorophores, or Star dyes from Abberior GmbH.

[0028] Antigen recognition part The term "antigen recognition moiety" refers to any type of antibody, fragmented antibody, or fragmented antibody derivative that expresses a target moiety on a biological sample, such as an antigen expressed intracellularly or extracellularly. This term encompasses fully intact antibodies, fragmented antibodies, or fragmented antibody derivatives, such as Fab, Fab', F(ab')2, sdAb, scFv, di-scFv, and nanobodies. Such fragmented antibody derivatives can be synthesized via recombinant processes, including covalent and non-covalent conjugates containing these molecular types. Further examples of antigen recognition moieties are peptide / MHC complexes targeting TCR molecules, cell adhesion receptor molecules, receptors for co-stimulatory molecules, and artificially engineered binding molecules such as peptides or aptamers targeting, for example, cell surface molecules.

[0029] The conjugates used in the method of this invention can contain up to 100, preferably 1-20, antigen recognition moieties Y. The interaction between the antigen recognition moieties and the target antigen can be high-affinity or low-affinity. The binding interaction of a single low-affinity antigen recognition moieties is too weak to provide a stable bond with the antigen. Low-affinity antigen recognition moieties can be polymerized by conjugation with an enzymatically degradable spacer group to provide high affinity. When the spacer group is enzymatically cleaved, the low-affinity antigen recognition moieties are monomerized, resulting in the complete removal of the fluorescent label.

[0030] Preferably, the term "antigen recognition moiety" refers to an antibody against an antigen expressed intracellularly (e.g., IL2, FoxP3, CD154) or extracellularly (e.g., CD19, CD3, CD14, CD4, CD, CD25, CD34, CD56, and CD133) of a biological sample (target cell). The antigen recognition moiety G1, G2, and especially antibodies, can be coupled to CP via a side-chain amino or thiol group. In some cases, the glycosidic side chain of the antibody can be oxidized by periodate, resulting in an aldehyde functional group.

[0031] The antigen recognition portion can be covalently or non-covalently coupled. Methods for covalent or non-covalent conjugation are known to those skilled in the art and are the same as those mentioned for conjugation of fluorescent labels.

[0032] The method of the present invention can be particularly used to detect and / or isolate specific cell types from complex mixtures, and may include more than one sequential sequence of steps a)-d). The method can use various combinations of conjugates. For example, the conjugates may contain antibodies specific to two different epitopes, such as two different anti-CD34 antibodies. Different conjugates containing different antibodies can be used to respond to different antigens; for example, anti-CD4 and anti-CD8 can be used to distinguish two different T cell populations, or anti-CD4 and anti-CD25 can be used to identify different cell subsets such as regulatory T cells.

[0033] Cell detection methods A target labeled with a conjugate is detected by exciting the fluorescent fraction (FL) or the main chain (CP) and analyzing the resulting fluorescence signal. The excitation wavelength is typically selected based on the maximum absorption of the fluorescent fraction (FL) or CP and is provided by a laser or LED light source as known in the art. If several different detection fractions (FL) are used for multiple color / parameter detection, careful selection should be made of the fluorescent fraction having a non-overlapping absorption spectrum, at least a non-overlapping maximum absorption. In the case of the fluorescent fraction, the target can be detected, for example, under a fluorescence microscope, in a flow cytometer, spectrophotometer, or fluorescence scanner. Light emitted by chemiluminescence can be detected by similar instruments that omit the excitation.

[0034] Uses of the method The method of the present invention can be used in a variety of applications in research, diagnosis and cell therapy, such as in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell separation, pathology or histology.

[0035] In a first variant of the invention, biological samples, such as cells, are detected for counting purposes, i.e., to establish the cell count from a sample having a set of antigens recognized by the antigen recognition portion of the conjugate. In another variant, the biological samples detected by the conjugate in step c) are separated from the sample by optical, electrostatic, piezoelectric, mechanical separation, or acoustic means. For this purpose, before performing step d) by optical, electrostatic, piezoelectric, mechanical separation, or acoustic means, the biological samples detected by the conjugate in step d) are simultaneously or successively separated from the sample into one or more populations based on their detection signals.

[0036] In another variation of the invention, the location of a target portion, such as an antigen, on a biological sample is determined by the antigen-recognizing portion of the conjugate. Such techniques are referred to as “Multi-Epitope Ligand Cartography,” “Chip-Based Cell Counting,” or “Multiomyx,” and are described, for example, in EP0810428, EP1181525, EP 1136822, or EP1224472. In this technique, cells are immobilized and contacted with an antibody conjugated to a fluorescent portion. The antibody is recognized by the corresponding antigen on the biological sample (e.g., on the cell surface), and the location of the antigen is detected by the fluorescence emission of the fluorescent portion after the removal of unbound markers and excitation of the fluorescent portion. In some variations, instead of an antibody conjugated to a fluorescent portion, an antibody conjugated to a portion detectable by MALDI-Imaging or CyTOF can be used. Those skilled in the art know how to modify fluorescent portion-based techniques to work on these detection portions.

[0037] The target location is determined using a digital imaging device with sufficient resolution and sensitivity for the wavelength of fluorescence radiation. The digital imaging device may be used with or without optical magnification, such as with a fluorescence microscope. The resulting images are stored in RAW, TIF, JPEG, or HDF5 format on a suitable storage device, such as a hard disk drive.

[0038] To detect different antigens, different antibody-dye conjugates with the same or different fluorescent or antigen-recognizing moieties can be provided. Since parallel detection of fluorescence emission at different wavelengths is limited, antibody-fluorescent dye conjugates are used sequentially and individually or in groups (2-10).

[0039] In another variation of the method according to the invention, biological samples, particularly suspended cells of the sample, are immobilized by capturing or adhering in the microcavities.

[0040] Generally, the method of the present invention can be implemented in several variations. For example, before detecting the target portion labeled by the conjugate, conjugates not recognized by the target portion can be removed, for example, by washing with a buffer solution.

[0041] In a variant of the invention, at least two conjugates are provided simultaneously or in a subsequent staining sequence, wherein each antigen-recognizing portion recognizes a different antigen. In an alternative variant, at least two conjugates may be provided to the sample simultaneously or in a subsequent staining sequence. In both cases, the labeled target portions may be detected simultaneously or sequentially. Example

[0042] The following compounds were studied for their absorption behavior: CP is in n = 0.9, m = 0.1, and x = 11, And CP-FL is in n = 0.9, m = 0.1, and x = 11.

[0043] Where FL = fluorescein rhodamine, anthocyanin or carbopyronine.

[0044] To illustrate the general kinetics involved in photodegradation, Figure 1 The single exponential decay curve is shown as follows: f (x) = y0*exp(- k *x) fitted, schematic curves obtained for the photodegradation of thallium dye by light, where tau = 1 / k and t 1 / 2= tau*ln(2) is the half-life. To measure the photodegradation kinetics, organic fluorophores (i.e., especially coumarin, xanthan, rhodamine, and anthocyanins) were dissolved in DMSO and diluted in PBS, or directly dissolved in PBS, such that the concentration was adjusted to obtain an absorbance of approximately 0.3 AU at their respective maximum values, with a path length of 1.00 cm. In this way, all solutions were normalized and comparable by their absorbance. The solutions were then placed in 3-window fluorescent quartz cuvettes with a low headspace and an airtight top to avoid evaporation and sample concentration. The samples in the cuvettes were then irradiated for a fixed amount of time, and both absorbance and emission spectra were recorded. The intensity values ​​and their maximum values ​​were plotted relative to the irradiation time, and then a mathematical fit to a single exponential decay was performed using appropriate computer software to obtain, as shown in the figure. Figure 1 The curves shown exhibit high absorption at approximately 400 nm in the CP region and a redshift absorption in the FL region. The characteristic decay time (k) is used to calculate the half-life and other parameters.

[0045] Figure 2 The photodegradation decay curves of a series of dyes belonging to different chemical classifications are shown based on the properties of their chromophores (i.e., fluorescein rhodamine, cyanine, carbopyronine), with each dye covalently attached to the CP moiety.

[0046] Figure 2 The data shown indicates that when all dye classes are attached to CP as defined below, they are all sensitive to photodegradation, and the photodegradation rate is higher for the constructs of this application than for FL itself.

[0047] Figure 3 The photodegradation curves of the rhodamine dyes obtained under three different conditions are shown: i) unbound and free in solution, ii) attached to branched PEG as described in US2019 / 0162721A1, and iii) covalently attached to linear CP (according to the present invention).

[0048] The results in Table 1 show that, compared to the small molecule fraction (FL), different constructs of CP-FL exhibited bleaching constants that increased by 94-fold (fluorescein), 22.5-fold (rhodamine), and 5.2-fold (anthocyanin) for different constructs. K The half-life of the fluorophore is reduced to 1 / 38 (fluorescein), 1 / 156 (rhodamine), and 1 / 98 (anthocyanin).

[0049] like Figure 3As shown, the invention presented herein (e.g., CP-Rhodamine) exhibits a 46-fold increase in bleaching constant compared to the construct (branched PEG-Rhodamine) from US2019 / 0162721A1, which is a prior art invention, and the construct utilizes FL polymerization on branched PEG. This higher bleaching constant results in shorter bleaching times and less background, for example, in cyclic imaging applications.

[0050] Table 1: Comparison of bleaching behavior of small molecule dyes and small molecule dyes that are FL moieties that bind CP.

Claims

1. A method for detecting a target moiety in a biological sample by providing a conjugate thereof, ， FL is the fluorescent component. n = 0.9， m = 0.1, and x = 11， Its features a) Contact the biological sample with the conjugate to mark the target portion recognized by the antigen recognition portion with the conjugate; b) Excite the labeled target portion with light whose wavelength is within the absorption spectrum of the fluorescent portion FL; c) The labeled target portion is detected by detecting the fluorescent radiation emitted by the fluorescent portion FL, and d) By irradiating the conjugate with light of wavelength within the absorption spectrum of the fluorescent portion FL for a time sufficient to transfer enough energy, the fluorescent portion FL of the labeled target portion is degraded, and the energy causes the fluorescent radiation emitted by the fluorescent portion FL to decrease by at least 75% of the initial fluorescent radiation.

2. The method according to claim 1, characterized in that... FL is selected from fluorescein, fluorescein derivatives, rhodamine, coumarin, halogenated fluorescein, pyrene, anthracene, phenylene, phthalocyanine, anthocyanin, xanthan, amide-pyranium-fluorophore, oxazine, Quadrain-Farbstoffe, Carbopyronine, 7-nitrobenzene-2-oxa-1,3-diazole (NBD) fluorophore, and BODIPY. TM Molecular Probes, Inc., ALEXA TM Fluorophore (Molecular Probes, Inc.), DY TM Fluorophores (Dyomics GmbH), benzopyranium fluorophores, benzopyranium-polymethyne fluorophores, lanthanide chelates, Rhodol dyes, Carborhodol dyes, naphthalenedicarboximide, and porphyrins.

3. The method according to claim 1, characterized in that... FL is selected from tetramethylrhodamine, silylrhodamine (SiR), and metalloporphyrin.

4. The method according to any one of claims 1 to 3, wherein the fluorescent portion FL of the labeled target portion is further degraded by adding an oxidant.

5. Use of the method according to any one of claims 1 to 3 in fluorescence microscopy, flow cytometry, fluorescence spectroscopy, cell isolation, pathology, or histology.

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