Single domain antibody / polymer tandem fluorescent dye conjugates and methods of making and using same

Abstract

Provided herein are single domain antibody / polymer fluorescent tandem dye conjugates. Aspects of the conjugates provided herein include single domain antibodies having one or more polymer tandem fluorescent dyes conjugated thereto. In some cases, a polymer tandem fluorescent dye includes: (i) a polymer backbone consisting of non-conjugated repeating units; (ii) a plurality of pendant donor chromophore groups, each group independently attached to a non-conjugated repeating unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores attached to non-conjugated repeating units of the polymer backbone wherein the pendant donor and acceptor fluorophores are in an energy transfer relationship. Also provided herein are methods of using the conjugates, for example, methods for assessing the presence of a target analyte in a sample, and kits comprising conjugates useful in embodiments of the methods.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. §119(e), this application claims priority to the filing date of U.S. Provisional Patent Application Serial No. 63 / 326,104, filed on March 31, 2022, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0003] Fluorescent dye is such a compound, when the light of the wavelength absorbed by it is irradiated, it emits (usually) light of different wavelengths. Fluorescent dye has multiple applications in biochemistry, biology and medicine, for example, for diagnostic kits, microscopy or drug screening. Fluorescent dye is characterized by allowing the user to select multiple parameters of suitable dye according to the desired purpose. The parameter of interest includes excitation wavelength maximum, emission wavelength maximum, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. Dye can be selected according to the application of interest, so as to allow the excitation radiation to penetrate into the biological sample, thereby minimizing background fluorescence and / or realizing high signal to noise ratio.

[0004] Molecular recognition involves the specific binding of two molecules. Molecules with binding specificity for a target biomolecule can be used in a variety of research and diagnostic applications, such as labeling and separation of analytes, flow cytometry, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation, and chromatography. Target biomolecules can be detected by labeling with fluorescent dyes. Summary of the invention

[0005] Single domain antibody / polymer fluorescent tandem dye conjugates are provided herein. Aspects of the conjugates provided herein include: single domain antibodies having one or more polymer tandem fluorescent dyes conjugated thereto. In some cases, the polymer tandem fluorescent dye includes: (i) a polymer backbone consisting of non-conjugated repeating units; (ii) a plurality of pendant donor chromophore groups, each independently attached to the non-conjugated repeating units of the polymer backbone; and (iii) one or more pendant acceptor fluorophores, which are attached to the non-conjugated repeating units of the polymer backbone, wherein the pendant donor and acceptor fluorophores are in an energy transfer relationship. Also provided herein are methods of using the conjugates, e.g., methods for assessing the presence of a target analyte in a sample, and kits comprising conjugates of embodiments that can be used for the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The present invention can be better understood by reading the following detailed description in conjunction with the accompanying drawings, which include the following figures:

[0007] Figure 1A and 1BA schematic representation of a single domain antibody / polymer fluorescent tandem dye conjugate according to an embodiment of the invention is provided.

[0008] Figure 2A and 2B Schematic diagrams of assays employing single domain antibody / polymer fluorescent tandem dye conjugates and prior art single domain antibody reagents are provided, as further described in the experimental section below. DETAILED DESCRIPTION

[0009] definition

[0010] As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that, in certain cases, can selectively react with another compatible functional group to form a covalent bond after optionally activating one of the functional groups. Chemoselective functional groups of interest include, but are not limited to, thiols and maleimides or iodoacetamides, amines and carboxylic acids or their active esters, and groups that can react with each other via click chemistry, such as azides and alkynyl groups (e.g., cyclooctynyl), tetrazines, trans-cyclooctenes, dienes and dienophiles, as well as azides, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, and hydroxyls, hydrazides, hydrazines, aldehydes, ketones, azidos, alkynes, phosphines, epoxides, and the like.

[0011] As used herein, the term "sample" refers to a material or mixture of materials containing one or more analytes of interest, in some cases in liquid form. In some embodiments, the term, when used in its broadest sense, refers to any plant, animal, or bacterial material containing cells or producing cell metabolites, such as tissues or fluids separated from individuals (including but not limited to plasma, serum, cerebrospinal fluid, lymph, tears, saliva, and tissue sections) or tissues or fluids separated from in vitro cell culture components, as well as samples from the environment. The term "sample" may also refer to a "biological sample". As used herein, the term "biological sample" refers to a whole organism or a subset of its tissues, cells, or components (e.g., body fluids, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to a homogenate, lysate or extract prepared from the following substances, i.e. a subset or fraction or part thereof of a whole organism or its tissues, cells or components, including but not limited to plasma, serum, spinal fluid, lymph fluid, external parts of the skin, respiratory tract, intestinal tract and urogenital tract, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been taken out from an animal or plant. The biological sample may include cells. The term "cell" is used in its conventional sense to refer to the basic structural unit of a living organism (both eukaryotic and prokaryotic organisms), having at least a nucleus and a cell membrane. In certain embodiments, cells include prokaryotic cells, such as prokaryotic cells from bacteria. In other embodiments, cells include eukaryotic cells, such as cells obtained from biological samples from animals, plants or fungi.

[0012] The terms "support binding" and "connected to a support" are used interchangeably and refer to a portion (e.g., a specific binding member) that is covalently or non-covalently connected to a support of interest. Covalent bonding can involve a chemical reaction of two compatible functional groups (e.g., two chemoselective functional groups, an electrophilic group, and a nucleophilic group, etc.) to form a covalent bond between two portions of interest (e.g., a support and a specific binding member). In some cases, non-covalent bonding can involve specific bonding between two portions of interest (e.g., two affinity moieties, such as a hapten and an antibody or a biotin moiety and streptavidin, etc.). In some cases, non-covalent bonding can involve absorption of a substrate.

[0013] The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides with a length range of 2-50 amino acids and polypeptides with a length greater than 50 amino acids. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes polymers of coded amino acids and non-coded amino acids, polymers of chemically or biochemically modified or derived amino acids, and polypeptides with modified peptide backbones, wherein conventional backbones have been replaced by non-naturally occurring or synthetic backbones. Polypeptides can have any convenient length, for example, 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids." Peptide" can be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, the length of the peptide is 5 to 30 amino acids.

[0014] As used herein, the term "isolated" refers to a fraction of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, or even at least 99% free from other components with which the fraction of interest was associated prior to purification.

[0015] A "plurality" contains at least 2 members. In some cases, a plurality can have 5 or more, e.g., 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 300 or more, 1000 or more, 3000 or more, 10,000 or more, 100,000 or more members.

[0016] Numeric ranges are inclusive of the numbers defining the range.

[0017] The term "specific binding" refers to a direct association between two molecules, for example due to covalent, electrostatic, hydrophobic and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Specific binding members describe members of a pair of molecules that have specific binding to each other. The members of a specific binding pair can be naturally derived or produced in whole or in part synthetically. One member of the molecular pair has an area on its surface or cavity that specifically binds to and is therefore complementary to the specific spatial and polar organization of the other member of the molecular pair. Therefore, the members of the pair have the property of specifically binding to each other. Examples of specific binding member pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding members of the binding pair exhibit high affinity and binding specificity for binding to each other. Typically, the affinity between a pair of specific binding members is characterized by K d (dissociation constant) is 10 -6 M or lower, e.g. 10 -7 M or lower, including 10 -8 M or lower, e.g. 10 -9 M or lower, 10 -10 M or lower, 10 -11 M or lower, 10 - 12 M or lower, 10 -13 M or lower, 10 -14 M or lower, including 10 -15 M or lower. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In embodiments, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of a molecule to another molecule is determined by, for example, measuring the binding kinetics of the interaction at 25°C. "Affinity" refers to the strength of binding, and increased binding affinity is associated with a lower KD. In embodiments, affinity is determined by surface plasmon resonance (SPR), for example, as used by a Biacore system. The affinity of a molecule to another molecule is determined by, for example, measuring the binding kinetics of the interaction at 25°C.

[0018] Method described herein may include a plurality of steps. As required, each step may be performed after a predetermined amount of time between steps. Therefore, the time between performing each step may be 1 second or longer, 10 seconds or longer, 30 seconds or longer, 60 seconds or longer, 5 minutes or longer, 10 minutes or longer, 60 minutes or longer and include 5 hours or longer. In certain embodiments, each subsequent step is performed immediately after the previous step is completed. In other embodiments, step may be performed after the incubation or waiting time after completing the previous step, and this incubation or waiting time are for example the waiting time of several minutes to overnight.

[0019] As used herein, the terms "assess," "determine," "measure," "evaluate," and "measure" are used interchangeably and include both quantitative and qualitative determinations.

[0020] As used herein, the term "separation" refers to the physical separation of two elements (eg, by size or affinity, etc.) as well as the degradation of one element while leaving the other element intact.

[0021] The term "joint" or "connector" refers to a connecting portion that connects two groups and has a length of 100 atoms or less of a skeleton. A joint or a connector can be a covalent bond connecting two groups or a chain of 1 to 100 atoms in length, such as a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, wherein the joint can be a straight chain, a branched chain, a cyclic or a single atom. In some cases, the joint is a branched joint, and a branched joint refers to a connecting portion that connects three or more groups. In some cases, one, two, three, four or five or more carbon atoms of the joint skeleton can be optionally substituted by sulfur, nitrogen or oxygen heteroatoms. In some cases, the joint skeleton includes a connecting functional group, such as ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imines. The key between the skeleton atoms can be saturated or unsaturated, and in some cases, there is no more than one, two or three unsaturated bonds in the joint skeleton. The joint may include one or more substituents, such as alkyl, aryl or alkenyl groups. The joint may include, but is not limited to, polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, which may be linear or branched, such as methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), etc. The joint skeleton may include a cyclic group, such as an aryl, a heterocycle or a cycloalkyl group, wherein 2 or more atoms of the cyclic group, such as 2, 3 or 4 atoms, are included in the skeleton. The joint may be cleavable or non-cleavable.

[0022] As used herein, the terms "water-soluble group," "water-soluble group," and WSG are used interchangeably and refer to a group or substituent that is well solvated in an aqueous environment, e.g., under physiological conditions, and confers improved water solubility to the molecule to which it is attached. WSG can increase the solubility of the multichromophore in a solution that is predominantly aqueous compared to a control multichromophore lacking WSG. The water-soluble group can be any convenient hydrophilic group that is well solvated in an aqueous environment.

[0023] The terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably and refer to a polyethylene glycol having the formula --(CH 2 ----O--) n -described chain polymer group or its derivative. In some embodiments, "n" is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10 to 15. It should be understood that the PEG polymer group can have any convenient length and can include a variety of terminal groups and / or additional substituents, including but not limited to alkyl, aryl, hydroxyl, amino, acyl, acyloxy and amido terminal and / or substituents. PEG groups that may be suitable for use in the subject multichromophores include those described by S. Zalipsky in “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165 and by Zhu et al. in “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp 4687-4735.

[0024] The term "alkyl" by itself or as part of another substituent refers to a saturated branched or straight-chain monovalent hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent alkane. Alkyl groups of interest include, but are not limited to, methyl; ethyl, propyl, such as propane-1-yl or propane-2-yl; and butyl, such as butane-1-yl, butane-2-yl, 2-methyl-propane-1-yl or 2-methyl-propane-2-yl. In some embodiments, the alkyl group comprises 1 to 20 carbon atoms. In some embodiments, the alkyl group comprises 1 to 10 carbon atoms. In certain embodiments, the low alkyl group comprises 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. For example, the term includes straight and branched hydrocarbon groups, such as methyl (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl ((CH 3 )(CH 3 CH 2 )CH-), tert-butyl ((CH 3 ) 3 C-), n-pentyl (CH 3 CH 2 CH 2 CH 2 CH 2 -) and neopentyl ((CH 3 ) 3 CCH 2 -).

[0025] The term "substituted alkyl" refers to an alkyl group as defined herein in which one or more carbon atoms in the alkyl chain have been optionally replaced by a heteroatom (such as -O-, -N-, -S-), -S(O) n-(wherein n is 0 to 2), -NR-(wherein R is hydrogen or alkyl) and has 1 to 5 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thio, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -Aryl, -SO 2 -heteroaryl and -NR a R b , where R ’ and R ” and R and R may be the same or different and are selected from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocycle.

[0026] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. For example, alkoxy includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0027] The term "substituted alkoxy" refers to substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- groups, wherein substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0028] "Alkenyl" refers to a monovalent, branched or straight-chain, cyclic or acyclic hydrocarbon group containing a carbon-carbon double bond. Exemplary alkenyl groups include vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosene and tetracosene. In some cases, the alkenyl group includes 1 to 24 carbon atoms, such as 1 to 18 carbon atoms or 1 to 12 carbon atoms. The term "lower alkenyl" refers to an alkyl group with 1 to 6 carbon atoms.

[0029] "Alkynyl" or "alkyne" refers to a straight or branched monovalent hydrocarbon radical having 2 to 6 carbon atoms, preferably 2 to 3 carbon atoms, and having at least 1 and preferably 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl radicals include ethynyl (-C≡CH) and propargyl (-CH 2 C≡CH).

[0030] The term "substituted alkynyl" or "substituted alkyne" refers to an alkynyl group as defined herein having from 1 to 5 substituents or from 1 to 3 substituents selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxy, oxo, thioketo, carboxy, carboxyalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclyloxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -Aryl and -SO 2 -heteroaryl.

[0031] "Heterocyclyl" refers to a monoradical cyclic group that contains heteroatoms (e.g., O, S, N) as ring atoms and is not aromatic (ie, distinguishing heterocyclyl from heteroaryl). Exemplary heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocyanato.

[0032] "Amino" refers to the group -NH 2 The term "substituted amino" refers to the group -NRR, wherein each R is independently selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, with the proviso that at least one R is not hydrogen.

[0033] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by the removal of one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl groups of interest include, but are not limited to, those derived from aceanthrene, acenaphthene, acephenanthrene, anthracene, azulene, benzene, In some embodiments, the aryl group comprises 6 to 20 carbon atoms. In some embodiments, the aryl group comprises 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0034] "Substituted aryl", unless the definition of aryl substituents is otherwise limited, refers to aryl substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -Aryl, -SO 2 -heteroaryl and trihalomethyl.

[0035] "Heteroaryl" by itself or as part of another substituent refers to a monovalent heteroaryl radical derived by removing one hydrogen atom from a single atom of a heteroaromatic ring system. Heteroaryl groups of interest include, but are not limited to, groups derived from acridine, arsindene, carbazole, β-carboline, chroman, chromene, cinnamoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, oxazidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, and the like. In certain embodiments, the heteroaryl group is derived from a 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is derived from a 5-10 membered heteroaryl. In certain embodiments, the heteroaryl group is derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0036] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings (including fused bridged rings and spiro ring systems) and having 3 to 20 ring atoms (including 1 to 10 heteroatoms). The ring atoms are selected from nitrogen, sulfur or oxygen, wherein, in a fused ring system, one or more rings can be cycloalkyl, aryl or heteroaryl, provided that the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atoms of the heterocyclic group are optionally oxidized to provide N-oxides, -S(O)- or -SO 2 -part.

[0037] Examples of heterocyclic and heteroaryl groups include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indoline, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnamoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenanthroline, Thiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also known as thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0038] "Substituted heteroaryl", unless the definition of substituents is otherwise limited, refers to heteroaryl substituted with 1 to 5 substituents, or 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclyloxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -Aryl, -SO 2 -heteroaryl and trihalomethyl.

[0039] The term "alkaryl" or "aralkyl" refers to the groups -alkylene-aryl and substituted alkylene-aryl wherein alkylene, substituted alkylene and aryl are as defined herein.

[0040] "Alkylene" refers to a divalent aliphatic hydrocarbon group preferably having 1 to 6 and more preferably 1 to 3 carbon atoms, which is linear or branched and optionally substituted by one or more selected from -O-, -NR- 10 -、-NR 10 C(O)-、-C(O)NR 10 - etc. For example, the term includes methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), n-propylene (-CH 2 CH 2 CH 2 -), isopropylidene (-CH 2 CH(CH 3 )-)、(-C(CH 3 ) 2 CH 2 CH 2 -)、(-C(CH 3 ) 2 CH 2 C(O)-), (-C(CH 3 ) 2 CH 2 C(O)NH-), (-CH(CH 3 )CH 2 -), etc. "Substituted alkylene" refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described below in the definition of "substituted" for the carbon.

[0041] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituents. Substituents of interest include, but are not limited to, alkylenedioxy (such as methylenedioxy), -M, -R 60 , -O - ,=O,-OR 60 、-SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF 3 , -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 、-N 3 、-S(O) 2 O - 、-S(O) 2 OH, -S(O) 2 R 60 、-OS(O) 2 O -、-OS(O) 2 R 60 、-P(O)(O - ) 2 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(S)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 、-C(O)O - 、-C(S)OR 60 、-NR 62 C(O)NR 60 R 61 、-NR 62 C(S)NR 60 R 61 、-NR 62 C(NR 63 )NR 60 R 61 and -C(NR 62 )NR 60 R 61 , wherein M is halogen; R 60 , R 61 , R 62 and R 63 are independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally, R 60 and R 61 Together with the nitrogen atom to which they are bound, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R 64 and R 65 R is independently hydrogen, alkyl, substituted alkyl, aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl, or optionally R 64 and R 65 Together with the nitrogen atom to which they are bound, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF3 , -CN, -OCN, -SCN, -NO, -NO 2 , =N 2 、-N 3 、-S(O) 2 R 60 、-OS(O) 2 O - 、-OS(O) 2 R 60 、-P(O)(O - ) 2 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(S)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 、-C(O)O - 、-NR 62 C(O)NR 60 R 61 In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF 3 、-CN、-NO 2 、-S(O) 2 R 60 、-P(O)(OR 60 )(O - )、-OP(O)(OR 60 )(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)NR 60 R 61 ,-C(O)O - In certain embodiments, the substituents include -M, -R 60 ,=O,-OR 60 、-SR 60 、-NR 60 R 61 , -CF 3 、-CN、-NO 2 、-S(O) 2 R 60 、-OP(O)(OR 60)(OR 61 )、-C(O)R 60 、-C(O)OR 60 、-C(O)O - , where R 60 , R 61 and R 62 As defined above. For example, a substituted group may carry a methylenedioxy substituent or one, two or three substituents selected from halogen atoms, (1-4C)alkyl groups and (1-4C)alkoxy groups. When the substituted group is an aryl or heteroaryl group, the substituent (e.g., as described herein) may be referred to as an "aryl substituent".

[0042] It is understood that, in all of the substituents defined above, polymers obtained by defining a substituent as itself having further substituents (e.g., a substituted aryl having a substituted aryl as a substituent, which itself is substituted with a substituted aryl, which is further substituted with a substituted aryl, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is three. For example, sequential substitution of substituted aryl groups specifically contemplated herein is limited to substituted aryl-(substituted aryl)-substituted aryl.

[0043] "Acyl" refers to a group of the formula -C(O)R, where R is alkyl, alkenyl, or alkynyl. For example, an acetyl group has the formula -C(O)CH 3 .

[0044] "Halo" and "halogen" refer to chloro, bromo, fluoro and iodo groups.

[0045] "Carboxyl", "carboxyl" and "carboxylate" refer to -CO 2 H groups and their salts.

[0046] "Sulfonyl" refers to the group -SO 2 R, wherein R is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and substituted forms thereof. Exemplary sulfonyl groups include -SO 2 CH 3 and-SO 2 (C 6 H 5 ).

[0047] Unless otherwise specified, a reference to an atom is intended to include all isotopes of that atom. For example, a reference to H is intended to include 1 H. 2 H (i.e. D) and 3 H (i.e. T), and reference to C means including 12 C and all isotopes of carbon (e.g. 13C) In addition, any group described includes all stereoisomers of that group.

[0048] Unless otherwise indicated, nomenclature for substituents not specifically defined herein is derived by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkoxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0049] For any group containing one or more substituents disclosed herein, it is of course understood that such groups do not contain any sterically impractical and / or synthetically unfeasible substitutions or substitution patterns. In addition, the subject compounds include all stereochemical isomers resulting from substitutions of these compounds.

[0050] Single domain antibody / polymer fluorescent tandem dye conjugates are provided herein. Aspects of the conjugates provided herein include: single domain antibodies having one or more polymer tandem fluorescent dyes conjugated thereto. In some cases, the polymer tandem fluorescent dye includes: (i) a polymer backbone consisting of non-conjugated repeating units; (ii) a plurality of pendant donor chromophore groups, each independently attached to the non-conjugated repeating units of the polymer backbone; and (iii) one or more pendant acceptor fluorophores, which are attached to the non-conjugated repeating units of the polymer backbone, wherein the pendant donor and acceptor fluorophores are in an energy transfer relationship. Also provided herein are methods of using the conjugates, e.g., methods for assessing the presence of a target analyte in a sample, and kits comprising conjugates of embodiments that can be used for the method.

[0051] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the specification.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Nevertheless, for clarity and ease of reference, certain terms are defined below. In addition, representative illustrative methods and materials are described herein, although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0053] Where a numerical range is provided, it is understood that each intermediate value between the upper and lower limits of the range (unless the context clearly indicates otherwise, up to one tenth of the unit of the lower limit) and any other stated or intermediate value in the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also encompassed within the present invention, but are subject to the impact of any specifically excluded limitations in the stated range. When the stated range includes one or two limits, ranges excluding one or both of the included limits are also encompassed within the present invention.

[0054] Certain ranges of values ​​given herein are preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that follows it, as well as numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the close or approximate unrecited number may be a number that, in the context presented, provides a substantial equivalent to the specifically recited number.

[0055] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with the publication cited. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0056] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0057] As will be apparent to those skilled in the art after reading this disclosure, each of the various embodiments described and illustrated herein has discrete components and features that can be easily separated or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any described method can be executed in the order of events described or in any other order that is logically possible.

[0058] The apparatus and method have been or will be described for grammatical fluency and functional explanation, but it should be clearly understood that unless expressly stated in accordance with 35 U.S.C. §112, the claims should not be interpreted as being necessarily limited in any way to the interpretation of "means" or "step" limitations, but should be given the full scope of the meaning and equivalents provided by the claims under the doctrine of equivalents, and the case where the claims are expressly stated in accordance with 35 U.S.C. §112 should be given all legal equivalents under 35 U.S.C. §112.

[0059] In further describing various embodiments of the present invention, single domain antibody / polymer fluorescent tandem dye conjugates are first reviewed in more detail, and then methods of using and preparing the conjugates and kits including the conjugates are reviewed.

[0060] As described above, the present disclosure provides a single domain antibody / polymer fluorescent tandem dye conjugate. The dye conjugate of an embodiment of the present invention includes: a single domain antibody and a polymer tandem fluorescent dye conjugated thereto with one or more. In other words, the dye conjugate of an embodiment of the present invention includes at least one polymer tandem fluorescent dye conjugated to a single domain antibody, wherein the number of polymer tandem fluorescent dyes conjugated to a single domain antibody in a given single domain antibody / polymer fluorescent dye conjugate of the present invention may vary, ranging from 2 to 5 in some cases, such as 2 to 4, including 2 to 3 polymer tandem fluorescent dyes. In some cases, the single domain antibody / polymer fluorescent tandem dye conjugate of the present invention has a low molecular weight. In some embodiments, the molecular weight range of the single domain antibody / polymer fluorescent tandem dye conjugate is 20 to 200 kDa, such as 25 to 150 kDa, including 30 to 75 kDa, such as 30 to 70 kDa, such as 30 to 65 kDa. Figure 1A and 1B A schematic diagram of a single domain antibody / polymer fluorescent tandem dye conjugate according to an embodiment of the present invention is provided. Figure 1A In the embodiment shown, the single domain antibody / polymer fluorescent tandem dye conjugate comprises a VhH Nanobody conjugated to two polymer fluorescent tandem dyes (each labeled "pendant chromophore"). Figure 1B Shown with Figure 1A The same dye as shown in , wherein the structure of each polymer fluorescent tandem dye is provided. As shown, each polymer fluorescent tandem dye includes a peptide backbone having a plurality of pendant donor fluorophores and pendant acceptor fluorophores, wherein the fluorophores of the tandem dye are in an energy transfer relationship. The different components of the single domain antibody / polymer fluorescent tandem dye conjugates of embodiments of the present invention are now further reviewed in more detail.

[0061] Single domain antibodies

[0062] As used herein, single domain antibodies (also referred to herein as nanobodies, may be designated as sdAbs) are antibody fragments comprising a single monomeric variable antibody domain capable of specific binding to an antigen. In some cases, the "single domain antibody" or "sdAb" present in the conjugates of the present invention comprises a single antigen-binding polypeptide having three complementary determining regions (CDRs). An individual sdAb is capable of specific binding to a target antigen without pairing with a corresponding polypeptide containing CDRs.

[0063] Single domain antibodies, i.e. nanobodies, may include a heavy chain variable domain or a light chain variable domain. In some cases, the single domain antibodies of the embodiments of the present application include a heavy chain variable domain. The single domain antibodies may be derived from camelids (V H H fragment) or cartilaginous fish (V NAR Alternatively, single domain antibodies can be derived from splitting the dimeric variable domains of IgG into monomers.

[0064] Single domain antibodies include variable regions and framework regions that are primarily responsible for antigen recognition and binding. "Variable regions" are also called "complementarity determining regions" (CDRs), and include loops that vary greatly in size and sequence based on antigen recognition. CDRs are generally responsible for the binding specificity of nanobodies. Different from CDRs are framework regions. Framework regions are relatively conservative and contribute to the overall protein structure. Framework regions can include large solvent exposed surfaces consisting of beta sheets and loop structures. Signal sequences known in the art can be included, which are then cleaved from mature nanobodies.

[0065] A single domain antibody that "binds" an antigen of interest is an antibody with sufficient affinity to the antigen that allows the single domain antibody or binding molecule to be used to target the desired antigen and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of a single domain antibody, i.e., a nanobody, or other binding molecule to a non-targeted antigen is typically no more than 10%, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0066] The term "variable" refers to certain parts of the variable domain that are very different in sequence and are used for the binding and specificity of each specific variable domain to its specific antigen. However, the variability is not evenly distributed throughout the variable domain. It is concentrated in the hypervariable region. The more highly conserved parts of the variable domain are called framework regions (FRs). The term "hypervariable region" when used herein refers to the amino acid residues responsible for antigen binding. The hypervariable region can include amino acid residues from "complementarity determining regions" or "CDRs", and / or residues from "hypervariable loops". "Framework region" or "FR" residues are variable region residues other than the hypervariable region residues defined herein.

[0067] Immunoglobulin sequences, such as antibodies and antigen-binding fragments derived therefrom (e.g., immunoglobulin single variable domains or ISVs) are used to specifically target the corresponding antigens disclosed herein. HH The generation of ISVs (or ISVs) may involve selection from phage display or yeast display, for example, ISVs can be selected by utilizing a surface display platform, wherein cells or phages display a synthetic ISV library on the surface in the presence of a labeled antigen. A fluorescent secondary antibody directed against the labeled antigen is added to the solution, thereby marking cells that bind to the antigen. The cells are then sorted using any cell sorting platform of interest, such as magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS). The sorted clones are amplified to produce an enriched library of clones expressing ISVs that bind to the antigen. The enriched library is then rescreened with antigen to further enrich for surface displayed antigen-binding ISVs. These clones can then be sequenced to identify the sequence of the ISV of interest and further transferred to other heterologous systems for large-scale protein production.

[0068] Unless otherwise indicated, the term "immunoglobulin single variable domain" or "ISV" is used as a general term and includes, but is not limited to, antigen binding domains or fragments, such as V HH Domain or V H or V L Domain. V HH Domains are of interest in embodiments of the present invention. The terms antigen binding molecule or antigen binding protein are used interchangeably and also include the terms The immunoglobulin single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a heavy chain variable domain sequence (e.g., a V H sequences); more specifically, they may be heavy chain variable domain sequences derived from conventional four-chain antibodies or heavy chain variable domain sequences derived from heavy chain antibodies. Thus, the immunoglobulin single variable domain may be a single domain antibody, or an immunoglobulin sequence suitable for use as a single domain antibody, a "dAb", or an immunoglobulin sequence suitable for use as a dAb, or a nanobody, including but not limited to V HH sequence.

[0069] Immunoglobulin single variable domains include immunoglobulin sequences from different sources, including but not limited to immunoglobulin sequences of mice, rats, rabbits, donkeys, humans, camelids, cartilaginous fish such as sharks, lampreys, etc. Immunoglobulin single variable domains include fully humanized, humanized, sequence-optimized or chimeric immunoglobulin sequences. The structure of immunoglobulin single variable domains and immunoglobulin single variable domains can be considered (but not limited to) to consist of four framework regions or "FRs", respectively referred to in the art and herein as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4"; these framework regions are interrupted by three complementary determining regions or "CDRs", respectively represented in the art as "complementary determining region 1" or "CDR1"; "complementary determining region 2" or "CDR2"; and "complementary determining region 3" or "CDR3". It should be noted that the terms Nanobody or Nanobodies are registered trademarks of Ablynx N.V. and may also be referred to as or

[0070] If a VHH1 type immunoglobulin single variable domain or VHH type 1 sequence has 85% identity with a VHH1 consensus sequence (using the VHH1 consensus sequence as query sequence and using a blast algorithm with standard settings, i.e., the blosom62 scoring matrix) and must have a cysteine ​​at position 50, i.e., C50 (using Kabat numbering), then the amino acid sequence (e.g., an immunoglobulin single variable domain or polypeptide according to the invention) is referred to as a "VHH1 type immunoglobulin single variable domain" or "VHH type 1 sequence". See, for example, the article Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23; 240 (1-2): 185-195 for VHH1 from Camelids. HH Structural domain.

[0071] Such immunoglobulin single variable domains may be obtained in any suitable manner and from any suitable source, and may, for example, be naturally occurring V HHSequence (i.e. from a suitable camelid species, such as a camel) or a synthetic or semi-synthetic VH or VL (e.g. from a human). Such immunoglobulin single variable domains may include "humanized" or otherwise "sequence optimized" VHH, "camelized" immunoglobulin sequences (particularly camelized heavy chain variable domain sequences, i.e. camelized VH), and human VH, human VL, camelid VHH that have been altered by techniques such as affinity maturation (e.g. starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneer, combining fragments from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for modifying immunoglobulin sequences well known to those skilled in the art; or any suitable combination of the foregoing as further described herein.

[0072] An immunoglobulin single variable domain may comprise a V corresponding to a naturally occurring HH The amino acid sequence of the amino acid sequence of the structural domain, but the amino acid sequence has been "humanized", that is, by replacing the V of a conventional 4-chain antibody from a human H One or more amino acid residues at the corresponding positions in the structural domain replace the naturally occurring V HH The humanized immunoglobulin single variable domains of the present invention can be obtained in any suitable manner known per se and are therefore not strictly limited to the use of single variable domains comprising naturally occurring V HH A polypeptide having a structural domain is a polypeptide obtained as a starting material.

[0073] Another class of immunoglobulin single variable domains of the present invention includes single variable domains having a V H The amino acid sequence of the domain corresponds to the amino acid sequence of an immunoglobulin single variable domain, but the amino acid sequence has been "camelized", i.e., by replacing the naturally occurring V domains of a conventional four-chain antibody with H One or more of the amino acid sequences of the V domain of the heavy chain antibody are replaced by HH This can be done in a manner known per se, which will be clear to the skilled person, for example based on the description herein. Such "camelizing" substitutions can be inserted in the region forming and / or present in V H -V LThe amino acid positions at the camelid interface, and / or at the so-called camelid hallmark residues (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). V sequences used as starting material or starting point for generating or designing camelized immunoglobulin single variable domains H The sequence is preferably from a mammalian V H Sequence, more preferably human V H Sequence, such as V H 3 sequences. However, it should be noted that such camelized immunoglobulin single variable domains of the present invention can be obtained in any suitable manner known per se and are therefore not strictly limited to the use of single variable domains comprising naturally occurring V H A polypeptide obtained by using a polypeptide having a domain as a starting material.

[0074] The single domain antibodies used in the embodiments of the present invention can be engineered for ease of manufacture. In order to facilitate the assembly of dye conjugates, single domain antibodies can be engineered to have specific reaction sites for covalently connecting one or more polymer tandem fluorescent dyes. By utilizing the engineered reaction sites located at the periphery of the single domain antibody structure (rather than the sites located inside the single domain antibody protein), the unique binding affinity of the single domain antibody is retained after dye conjugation. These reaction sites can be selected and installed from a list of reactive groups commonly used for biological binding, wherein such groups are not limited to thiols, trans-cyclooctene and dibenzylcyclooctyne. The thiol group allows polymer fluorescent tandem dyes to be added to the single domain antibody by site-specific maleimide. Trans-cyclooctene or dibenzylcyclooctyne allow site-specific polymer fluorescent tandem dye attachment by strain-promoted cycloaddition reactions. The single domain antibodies present in the conjugates of the present invention may be of different sizes, and in some cases, their molecular weight ranges from 10 to 25 kDa, such as 15 to 20 kDa, such as 15 to 17 kDa.

[0075] The single domain antibodies of the conjugates of the invention can bind to a variety of different targets. In some cases, the single domain antibodies specifically bind to cell surface proteins or cell markers, such as cell receptors and cell surface antigens. In some cases, the single domain antibody can specifically bind to a cell surface antigen, wherein the cell surface antigen that the single domain antibody can bind to includes but is not limited to CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminal), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2 and CD71. In other embodiments, the single domain antibody can bind to an intracellular target. In such embodiments, intracellular targets of interest include, but are not limited to, nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules, proteins, such as fusion proteins, modified proteins, such as phosphorylated, glycosylated, ubiquitinated, SUMOylated or acetylated proteins, etc. Intracellular proteins of interest to which single domain antibodies can specifically bind include, but are not limited to, hormones, growth factors, transcription factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors, etc. In some embodiments, single domain antibodies specifically bind to immunoglobulins, such as primary antibodies used in a given assay, such as primary mouse or rat antibodies, for example, where single antibody domain / polymer tandem fluorescent dyes are used as secondary labels.

[0076] Polymer fluorescent tandem dyes

[0077] As described above, the single domain antibody / polymer fluorescent tandem dye conjugate of the embodiment of the present invention comprises a single domain antibody, for example, as described above, conjugated with (e.g., covalently bound to) one or more polymer fluorescent tandem dyes. As described above, in some cases, the conjugate includes a single domain antibody conjugated to multiple polymer tandem fluorescent dyes, for example, 2 or more, for example, 3 or more, including 4 or more, for example, 5 or more polymer tandem fluorescent dyes. In some cases, the conjugate includes 2 to 4 polymer tandem fluorescent dyes, for example, 2 to 3 polymer tandem fluorescent dyes, for example, 2 polymer tandem fluorescent dyes.

[0078] The polymer fluorescent tandem dyes found in the conjugates of the present invention may be different. In some cases, the polymer fluorescent tandem dyes found in the conjugates of the present invention include: a polymer backbone; a plurality of pendant donor fluorophores, each fluorophore independently connected to a repeating unit of the polymer backbone; and one or more pendant acceptor fluorophores, which are connected to a repeating unit of the polymer backbone, wherein the pendant donor fluorophore and the acceptor fluorophore are in an energy transfer relationship. Therefore, the polymeric tandem dyes of the present invention embodiments include one or more pendant donor fluorophores and one or more pendant acceptor fluorophores, which are configured to be close to one or more pendant donor fluorophores to receive energy, for example, both are connected to a common polymer backbone. In some embodiments, a plurality of pendant donor fluorophores are present and are configured to be close to the pendant acceptor fluorophore for energy transfer, wherein in some cases, the range of the plurality of pendant donor fluorophores is 2 to 20, for example 2 to 15, for example 2 to 10. The term "pendant group" refers to a pendant group that is connected to the backbone but is not part of the backbone itself. In the embodiment of the polymer tandem dye, the donor fluorophore is capable of transferring energy to the connected acceptor fluorophore. Therefore, the subject tandem dye includes a connected acceptor signaling fluorophore near the donor fluorophore system to receive energy, that is, near at least one connected donor fluorophore to receive energy. The specific configuration of the pendant group can be determined and controlled by the arrangement of the repeating units of the lower polymer backbone (also referred to as "modular scaffold" herein), and the pendant group is attached to the backbone. The dye conjugate can include multiple water-soluble groups, which are attached to any convenient position of the support and / or pendant group to provide a water-soluble polymer dye. The polymer backbone, i.e., modular scaffold, can be composed of repeating units, which form a polymer backbone with a side group, and the side group can be attached to the pendant group. Repeating units can be arranged into various configurations to provide tandem dyes with desired spectral characteristics. The distance and arrangement between the covalent attachment sites of the pendant donor fluorophore and the acceptor fluorophore (when present) can be controlled to provide the desired energy transfer process.

[0079] In some cases, the polymer backbone is composed of non-conjugated repeating units with any convenient configuration (e.g., linear, branched, or dendritic polymer configuration). The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some cases, the dye conjugate includes a plurality of pendant donor chromophore groups, each of which is independently connected to the non-conjugated repeating units of the polymer backbone. The configuration of the pendant group can be realized during or after the polymer backbone synthesis. The incorporation of the pendant group can be realized in a sequence-specific manner by random configuration, block configuration, or by stepwise synthesis, depending on the specific synthesis method used.

[0080] The term "unit" refers to a structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, coblocks, repeating units, etc. "Repeat unit" is a subunit of a polymer, which is defined by the unique structural features of the minimum number of units required for the unit to be considered as a monomer, so that when the unit is repeated n times, the resulting structure describes a polymer or its block. In some cases, a polymer may include two or more different repeating units, for example, when the polymer is a multi-block polymer, a random arrangement of units, or a defined sequence, each block may define a different repeating unit. It should be understood that the arrangement of repeating units or blocks is varied, and in the illustrated formula of the polymer backbone described herein, any convenient linear arrangement of various lengths may be included in the structure of the overall polymer. It should be understood that a polymer may also be represented by a formula represented by the mol% value of each unit in the polymer, and such a formula may represent a variety of arrangements of repeating units, such as random or multi-block polymers or defined residue sequences. In some cases, the repeating unit of a polymer includes a single monomer group. In some cases, the repeating unit of a polymer includes two or more monomer groups, i.e., comonomer groups, such as two, three, four or more comonomer groups. The term "comonomer" or "comonomer group" refers to a structural unit of a polymer, which itself can be part of a repeating unit of the polymer.

[0081] The polymer backbone may have a random configuration of non-conjugated repeat units. The polymer backbone may include a block or co-block configuration of non-conjugated repeat units. Alternatively, the polymer backbone may include a specific defined sequence of non-conjugated repeat units, such as the amino acid residues of a polypeptide sequence. These configurations may be characterized by polymer segments of repeat units (e.g., as described herein), which themselves may be repeated throughout the modular scaffold.

[0082] "Non-conjugated" means that at least a portion of the repeat units include saturated backbone groups (e.g., groups having two or more consecutive single covalent bonds) that exclude π conjugation or a delocalized electronic structure extending from one repeat unit to the next along the polymer backbone. It should be understood that even though a repeat unit may not be conjugated to an adjacent repeat unit, such a repeat unit may also include one or more separate unsaturated groups, including unsaturated bonds (e.g., alkenyl or alkynyl unsaturated bonds) and / or aryl or heteroaryl groups, which may be part of the backbone. In some cases, each repeat unit of the polymer backbone includes a side chain that includes a pendant group attached or a chemoselective tag for attachment to a pendant group.

[0083] In certain embodiments of the tandem dye, the polymer backbone is a linear polymer. In some cases, the linear polymer is selected from a peptide, a peptoid, a hydrocarbon polymer, and a PEG polymer. In some cases, the linear polymer is a peptide. In some cases, the linear polymer is a peptoid. In some cases, the polymer is a hydrocarbon polymer. In some other cases, the polymer is a PEG polymer. More details about polymer backbones that can be used in embodiments of the present invention can be found in PCT Application Serial No. PCT / US2019 / 024662 (published as WO2019 / 191482) and PCT Application Serial No. PCT / US2020 / 019510 (published as WO2020 / 222894); the disclosures of these applications are incorporated herein by reference.

[0084] The tandem dye of the embodiment of the present invention can include a linear polymer backbone with any number of units. As used herein, the term "unit" refers to the structural subunit of a polymer. The term "unit" is intended to include monomers, comonomers, coblocks, segments, repeating units, etc. "Repeat unit" is a subunit of a polymer, which is defined by the unique structural features of the minimum number of units required for the unit to be considered as a monomer, so that when the unit is repeated n times, the resulting structure describes a polymer or its block. In some cases, the polymer can include two or more different repeating units, for example, when the polymer is a random arrangement of a multi-block polymer or a unit, each block can define different repeating units, for example, n blocks and m blocks. It is understood that a variety of arrangements of n and / or m repeating units or blocks are possible, and any convenient linear arrangement of coblocks of various lengths is included in the structure of the overall polymer. It should be understood that the polymer can also be represented by the formula represented by the mol% value of each unit in the polymer, and this formula can represent a variety of arrangements of repeating units, such as random or multi-block polymers. In some cases, the repeating unit of a polymer includes a single monomer group. In some cases, the repeating unit of a polymer includes two or more monomeric groups, i.e., comonomer groups, such as two, three, four or more comonomer groups. As used herein, the term "comonomer" or "comonomer group" refers to a structural unit of a polymer, which itself can be part of a repeating unit of a polymer.

[0085] In some cases, the tandem dye includes a linear peptide backbone of 2 to 100 amino acids (e.g., 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 amino acids). In some cases, the linear peptide backbone includes 2 or more amino acids, e.g., 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, up to a maximum of 100 amino acids. In some cases, the tandem dye includes a linear peptide backbone of 5 to 30 amino acids (e.g., 5 to 25, 5 to 20, 5 to 15, or 5 to 10 amino acids).

[0086] The backbone of the tandem dye can be of any convenient length. In some cases, the specific number of monomer repeat units or segments of the chromophore can fall within the range of 2 to 500,000, such as 2 to 100,000, 2 to 30,000, 2 to 10,000, 2 to 3,000, or 2 to 1,000 units or segments, or such as 5 to 100,000, 10 to 100,000, 100 to 100,000, 200 to 100,000, or 500 to 50,000 units or segments. In some cases, the specific number of monomer repeating units or segments of the backbone may fall within the range of 2 to 1,000, such as 2 to 500, 2 to 100, 3 to 100, 4 to 100, 5 to 100, 6 to 100, 7 to 100, 8 to 100, 9 to 100, or 10 to 100 units or segments. In some cases, the specific number of monomer repeating units or segments of the backbone may fall within the range of 2 to 500, such as 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or segments. In some cases, the specific number of monomer repeating units or segments of the backbone may fall within the range of 2 to 100 repeating monomer units, such as 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, or 2 to 30 units or segments.

[0087] As described above, the tandem dyes of embodiments of the present invention include one or more pendant donor fluorophores connected to a polymer backbone. Any convenient pendant donor fluorophore can be connected to the polymer backbone, wherein the donor fluorophore of interest includes, but is not limited to: BODIPY fluorophore; aryl fluorophores, heteroaryl fluorophores, etc. In some cases, the donor fluorophore is a BODIPY fluorophore. The term "BODIPY fluorophore" refers to a pendant donor fluorophore of a tandem dye, which includes a chromophore having the following boron-dipyrromethene (BODIPY) core structure:

[0088]

[0089] Wherein Q is C or N, and each R is any convenient boron substituent. In some cases, Q is C. In some cases, each R is independently selected from F, OH, H, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, substituted alkoxy, alkynyl and substituted alkynyl. The BODIPY chromophore group may be optionally further substituted, for example, by a water-soluble group and / or an aryl or heteroaryl substituent that imparts the desired light absorption properties of the chromophore. Further description of the BODIPY fluorophore that can be used as a donor fluorophore in embodiments of the present invention is described in PCT Application Serial No. PCT / US2019 / 024662 (published as WO2019 / 191482); its disclosure is incorporated herein by reference. In some embodiments, the donor fluorophore is a fluorophore including an aryl or heteroaryl chromophore. The aryl or heteroaryl chromophores of interest that can be used in embodiments of the present invention include, but are not limited to, phenyl, biphenyl, benzoxazole, benzothiazole, polyphenylene and fused tricyclic groups, such as fluorene, carbazole, thiole, biphenyl and bridged biphenyl. The aryl or heteroaryl chromophore can optionally be further substituted, for example, by a water-soluble group and / or an aryl or heteroaryl substituent, thereby giving the desired light absorption properties of the aryl or heteroaryl. In some embodiments, the donor fluorophore includes a fused tricyclic aryl or heteroaryl. In some embodiments, the donor fluorophore includes one or more groups selected from fluorene, carbazole, thiole, biphenyl and bridged biphenyl. The fused tricyclic chromophore is a group including a tricyclic aromatic group with three fused rings, wherein two aryl or heteroaryl 6-rings are fused to a central 5-yuan or 6-yuan carbocyclic or heterocyclic ring. In some cases, the fused tricyclic group includes two benzo rings or pyridine rings fused to a central 5-yuan or 6-yuan carbocyclic or heterocyclic ring. The fused tricyclic group can be connected to the side chain of the comonomer in the polymer backbone through any convenient ring atom of the fused ring. The central 5-membered or 6-membered ring can be carbocyclic or heterocyclic, aromatic or partially saturated, and can also include side chain substituents, such as WSG and / or a linker to a chemoselective tag or comonomer side chain. The bridged biphenyl comonomer is a fused tricyclic group with a biphenyl group, in which the two benzene rings are further interconnected by a central 6-membered carbocyclic or heterocyclic ring. Further descriptions of aryl / heteroaryl fluorophores that can be used as donor fluorophores in embodiments of the present invention are described in PCT application serial number PCT / US2019 / 024662 (published as WO2019 / 191482); its disclosure is incorporated herein by reference.The number of repeating units of the donor water-solvated light-harvesting chromophore with an attached acceptor signal fluorophore group can vary, wherein in some cases the number ranges from 1 mol% to 50 mol%, e.g., 1 mol% to 25 mol%, 2 mol% to 25 mol%, 3 mol% to 25 mol%, 4 mol% to 25 mol%, 5 mol% to 25 mol%, or 10 mol% to 25 mol% of the repeating units.

[0090] In addition to the pendant donor fluorophore, the tandem dye of the present invention also includes one or more acceptor fluorophores. Any convenient fluorescent dye can be used as an acceptor fluorophore in the polymer tandem dye. The acceptor fluorophore (e.g., each A) can be a small molecule fluorophore. The acceptor fluorophore (e.g., each A) can be a dye molecule selected from rhodamine, perylene, diimide, coumarin, xanthene, cyanine, polymethine, pyrene, thiazine, acridine, dipyrromethene boron difluoride, naphthylimide, phycobiliprotein, dinoflagellate chlorophyll protein, its conjugate and combination thereof. In certain embodiments, the acceptor fluorophore (A) is a cyanine dye, a xanthene dye, a coumarin dye, a thiazine dye or an acridine dye. In some cases, the acceptor fluorophore (A) is selected from DY 431, DY 485XL, DY 500XL, DY 610, DY 640, DY 654, DY 682, DY 700, DY 701, DY 704, DY 730, DY731, DY 732, DY 734, DY 752, DY 778, DY 782, DY 800, DY 831, Biotium CF 555, Cy 3.5, and diethylaminocoumarin.Fluorescent dyes of interest include, but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, Coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinyl chlorophyll-a protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 550, Alexa Fluor 561, Alexa Fluor 576, Alexa Fluor 577, Alexa Fluor 578, Alexa Fluor 579, Alexa Fluor 580, Alexa Fluor 581, Alexa Fluor 582, Alexa Fluor 583, Alexa Fluor 584, Alexa Fluor 585 Fluor568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY FL, BODIPY FL-Br.sub.2, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY581 / 591, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, and conjugates and derivatives thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, the polymer tandem dye comprises a multichromophore linked to an acceptor fluorophore selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa 647, and Alexa700. In certain embodiments, the polymer tandem dye includes a multichromophore linked to an acceptor fluorophore selected from the group consisting of a Dyomics dye (e.g., DY 431, DY485XL, DY 500XL, DY 530, DY 610, DY 633, DY 640, DY 651, DY 654, DY 682, DY 700, DY701, DY 704, DY 730, DY 731, DY 732, DY 734, DY 752, DY 754, DY 778, DY 782, DY 800, or DY831), Biotium CF 555, Cy 3.5, and diethylaminocoumarin.In some cases, the acceptor fluorophore (A) is selected from fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodamine alcohol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow, coumarin, etc. Cy-Chrome, DyLight 350, DyLight 405, DyLight 488, DyLight549, DyLight 594, DyLight 633, DyLight 649, DyLight 680, DyLight 750, DyLight 800, Phycoerythrin, PerCP (Peridinyl Chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4′,5′-dichloro-2′,7′-dimethyloxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa 350. Alexa 430. Alexa 488. Alexa 532. Alexa 546. Alexa 568. Alexa 594. Alexa 633. Alexa 647、Alexa 660、Alexa 680, 7-amino-4-methylcoumarin-3-acetic acid, FL, FL-Br2, 530 / 550, 558 / 568, 564 / 570, 576 / 589, 581 / 591, 630 / 650, 650 / 665, R6G, TMR, TR, its conjugates and combinations thereof.

[0091] As mentioned above, if necessary, the polymer backbone and / or the pendant fluorophore (i.e., donor and acceptor fluorophores) may include one or more water-soluble groups (WSG). In some cases, WSG is a pendant group directly connected to the polymer backbone, such as a side chain of the polymer backbone. In some cases, WSG is a substituent attached to a pendant donor fluorophore or a pendant acceptor fluorophore. In some cases, each pendant donor fluorophore group is substituted by one or more WSG. As used herein, the terms "water-soluble group", "water-soluble group" and WSG are used interchangeably, and refer to a group or substituent that is fully solvated in an aqueous environment, such as under physiological conditions, and imparts improved water solubility to the molecule to which it is attached. Compared with a control dye lacking WSG, WSG can increase the solubility of a given polymer tandem dye in a solution based on an aqueous solution. The water-soluble group can be any convenient hydrophilic group that is well solvated in an aqueous environment. The solubility of the water-soluble tandem dye of the present disclosure under aqueous conditions makes it particularly suitable for application in a variety of biological assays. Various water-soluble polymer groups can be applicable to the WSG of the subject dye. Any convenient water-soluble group (WSG) may be included in the dye described herein to provide higher water solubility. Although the increase in solubility may be different, in some cases, the increase (compared to the compound without WSG) is 2 times or more, such as 5 times, 10 times, 25 times, 50 times, 100 times or more. In some cases, the hydrophilic water-soluble group is charged, such as positively charged or negatively charged. In some cases, the hydrophilic water-soluble group is a neutral hydrophilic group. In some embodiments, WSG is a branched (for example, as described herein). In some cases, WSG is linear. In some embodiments, WSG is a hydrophilic polymer, for example, polyethylene glycol, modified PEG, peptide sequence, peptoid, carbohydrate, oxazoline, polyol, dendritic polymer, dendritic polyglycerol, cellulose, chitosan or its derivatives. Water-soluble groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfoniums, esters, polyethylene glycols (PEGs) and modified PEGs, hydroxyls, amines, amino acids, ammonium, guanidinium, pyridinium, polyamines and sulfoniums, polyols, linear or cyclic sugars, primary, secondary, tertiary or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols including polyethers, -COOM', -SO 3 M′, -PO 3 M′, -NR 3 + , Y′, (CH 2 CH 2 O) pR and mixtures thereof, wherein Y′ can be any halogen, sulfate, sulfonate or oxyanion, p can be from 1 to 500, each R can independently be H or an alkyl group (e.g., methyl) and M′ can be a cationic counterion or hydrogen, --(CH 2 CH 2 O) yy CH 2 CH 2 XR yy 、--(CH 2 CH 2 O) yy CH 2 CH 2 X--、--X(CH 2 CH 2 O) yy CH 2 CH 2 --, ethylene glycol and polyethylene glycol, wherein yy is selected from 1 to 1000, and X is selected from O, S and NR ZZ , and R ZZ and R YY Independently selected from H and C 1-3 In some cases, WSG is (CH 2 ) x (OCH 2 CH 2 ) y OCH 3 , wherein each x is independently an integer from 0 to 20, and each y is independently an integer from 0 to 50. In some cases, the water-soluble group includes a nonionic polymer (e.g., a PEG polymer) substituted with an ionic group (e.g., a sulfonate) at the end. More detailed information about soluble WSG that may be present in the dye of embodiments of the present invention can be found in PCT Application Serial No. PCT / US2019 / 024662 (published as WO2019 / 191482); the disclosure of which is incorporated herein by reference.

[0092] As described above, the polymer fluorescent tandem dye of the embodiment of the present invention includes a plurality of pendant donor fluorophores and one or more pendant acceptor fluorophores. In some cases, the number of donor fluorophores exceeds the number of acceptor fluorophores. In certain embodiments of the tandem dye of the present invention, the ratio of donor fluorophores to acceptor fluorophores is selected from 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1 and 20: 2. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 5: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 6: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 7: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 8: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 9: 1. In some cases, the ratio of donor fluorophores to acceptor fluorophores is 10: 1.

[0093] As described above, in the polymeric fluorescent tandem dyes of the present invention, the donor and acceptor pendant fluorophores are in an energy transfer relationship. Thus, in embodiments of the present invention, excitation of the donor can result in energy transfer to and emission from the covalently attached acceptor signaling fluorophore. Energy transfer mechanisms between the donor chromophore and the attached acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), etc. These energy transfer mechanisms can be relatively short distances; that is, the proximity of the chromophores of the light-collecting multi-chromophore system to each other and / or to the acceptor fluorophore provides efficient energy transfer. Under conditions of efficient energy transfer, when the wavelength of the incident light ("pump light") is absorbed by the chromophore of the light-collecting chromophore and transferred from it, the emission of the luminescent acceptor fluorophore is more intense than when the luminescent acceptor fluorophore is directly excited by the pump light, and the emission of the acceptor fluorophore can be amplified. "Effective" energy transfer means that 10% or more (e.g., 20% or more or 30% or more, 40% or more, 50% or more) of the energy collected by the donor chromophore is transferred to the acceptor. "Amplification" means that when excited by energy transfer from the donor light-collecting chromophore system, the signal from the acceptor fluorophore is 1.5 times or more than that of directly exciting the acceptor fluorophore with incident light of equal intensity. The signal can be measured using any convenient method. In some cases, a signal of 1.5 times or more refers to the intensity of the emitted light. In some cases, a signal of 1.5 times or greater refers to an increased signal-to-noise ratio. In certain embodiments of tandem dyes, the acceptor fluorophore emission emitted when excited by the chromophore is 1.5 times or more, e.g., 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 8 times or more, 10 times or more, 20 times or more, 50 times or more, 100 times or more, or even greater, compared to direct excitation of the acceptor fluorophore with incident light.

[0094] The tandem dye of the present invention embodiment can have any convenient molecular weight (MW).In some cases, the MW of tandem dye can be expressed as mean molecular weight.In some cases, the mean molecular weight of tandem dye is in the range of 500 to 500,000, for example 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000 or even mean molecular weight is in the range of 50,000 to 100,000 dalton.In some cases, the molecular weight of polymer fluorescent dye is in the range of 5 to 75kDa, for example 10 to 50kDa, for example 15 to 45kDa.In some embodiments, the molecular weight range of polymer fluorescent dye is 1 to 30kDa, for example 2 to 25kDa, including 2 to 20kDa.

[0095] In some cases, when the light-harvesting chromophore is directly excited by incident light, the tandem dye exhibits an effective Stokes shift of 100 nm or more (e.g., 110 nm or more, 120 nm or more, 130 nm or more, 140 nm or more, 150 nm or more, 160 nm or more, 170 nm or more, 180 nm or more, 190 nm or more, 200 nm or more, 250 nm or more). In some cases, the tandem dye has an effective Stokes shift of up to about 300 nm, e.g., 100-300 nm, 100-250 nm, or 100-200 nm.

[0096] The emission of the polymer tandem dye can have a quantum yield of 0.03 or more, e.g., 0.04 or more, 0.05 or more, 0.06 or more, 0.07 or more, 0.08 or more, 0.09 or more, 0.1 or more, 0.15 or more, 0.2 or more, 0.3 or more, or even higher. In some cases, the extinction coefficient of the polymer tandem dye is 5x10 5 cm -1 M -1 or more, e.g. 6x 10 5 cm -1 M -1 or more, 7x 10 5 cm -1 M -1 or more, 8x10 5 cm -1 M -1 or more, 9x10 5 cm -1 M -1 or more, e.g. 1x 10 6 cm -1 M -1 or more, 1.5x 10 6 cm -1 M -1 or more, 2x 10 6 cm -1 M -1 or more, 2.5x 10 6 cm -1 M -1 or more, 3x 10 6 cm -1 M -1 or more, 4x 10 6 cm -1 M -1 or more, 5x 10 6 cm -1 M -1 or more, 6x106 cm -1 M -1 or more, 7x 10 6 cm -1 M -1 or more or 8x 10 6 cm -1 M -1 More. In some embodiments, the molar extinction coefficient of the polymer tandem dye is 5 x 10 5 M -1 cm -1 In some embodiments, the molar extinction coefficient of the tandem dye is 1 x 10 6 M -1 cm -1 or higher.

[0097] In embodiments, the subject tandem dyes provide brighter fluorescence emission from the acceptor fluorophore than such fluorescent dyes are capable of alone. The brightness of the emission of the polymer tandem dye may be 50 mM -1 cm -1 or more, e.g. 60 mM -1 cm -1 or more, 70mM -1 cm -1 or more, 80mM -1 cm -1 or more, 90mM -1 cm -1 or more, 100mM -1 cm -1 or more, 150mM -1 cm -1 or more, 200mM -1 cm -1 or more, 250mM -1 cm -1 or more, 300mM -1 cm -1 In some cases, the brightness of the emission of the tandem dye is at least 5 times higher than the brightness of the directly excited acceptor fluorophore, such as at least 10 times higher, at least 20 times higher, at least 30 times higher, at least 50 times higher, at least 100 times higher, at least 300 times higher, or even more times higher than the brightness of the directly excited acceptor fluorophore.

[0098] In addition to the above-mentioned properties, the tandem dyes of embodiments of the present invention may also have one or more additional desired spectral characteristics, such as a particular maximum emission wavelength, extinction coefficient, quantum yield, etc. As described above, in some cases, the subject tandem dye provides a donor chromophore having an absorption maximum of 555nm to 585nm, such as 555nm to 575nm, such as 555 to 575nm, such as 555 to 574nm, 555 to 573nm, 555 to 572nm, 555 to 571nm, 555 to 570nm, 555 to 569nm, 555 to 568nm, 555 to 567nm, 555 to 566nm, 555 to 565nm, 555 to 564nm, 555 to 563nm or 555 to 562nm.

[0099] Various emission spectra depend on various factors, such as the selected comonomers, linking groups, substituents and attached acceptor fluorophores contained in the tandem dye. In some embodiments, the maximum emission wavelength of the chromophore is in the range of 300 to 900 nm, such as 350 to 850 nm, 350 to 600 nm, 360 to 500 nm, 370 to 500 nm, 380 to 500 nm, 390 to 500 nm or 400 to 500 nm, wherein specific examples of the maximum emission wavelength of interest include, but are not limited to, 395 nm ± 5 nm, 460 nm ± 5 nm, 490 nm ± 5 nm, 550 nm ± 5 nm, 560 nm ± 5 nm, 605 nm ± 5 nm, 650 nm ± 5 nm, 680 nm ± 5 nm, 700 nm ± 5 nm, 805 nm ± 5 nm. In some cases, the maximum emission wavelength of the chromophore is selected from 395nm, 460nm, 490nm, 550nm, 560nm, 605nm, 650nm, 680nm, 700nm and 805nm. In some cases, the maximum emission wavelength of the tandem dye is 395nm ± 5nm. In some cases, the maximum emission wavelength of the tandem dye itself is in the range of 375 to 900nm (e.g., in the range of 380nm to 900nm, 390nm to 900nm, or 400nm to 900nm).

[0100] In some cases, the extinction coefficient of the tandem dye is 5 x 10 5 cm -1 M -1 or higher, e.g. 6x 10 5 cm -1 M -1 or higher, 7x 10 5 cm -1 M -1 or higher, 8x 10 5 cm -1 M -1or higher, 9x 10 5 cm -1 M -1 or higher, e.g. 1x 10 6 cm -1 M -1 or higher, 1.5x 10 6 cm -1 M -1 or higher, 2x 10 6 cm -1 M -1 or higher, 2.5x 10 6 cm -1 M -1 or higher, 3x 10 6 cm -1 M -1 or higher, 4x 10 6 cm -1 M -1 or higher, 5x 10 6 cm -1 M -1 or higher, 6x 10 6 cm -1 M -1 or higher, 7x 10 6 cm -1 M -1 or higher or 8x 10 6 cm -1 M -1 In this case, the tandem dye may have 5 or more repeating units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more repeating units. In some embodiments, the molar extinction coefficient of the tandem dye is 5 x 10 5 M -1 cm -1 In certain embodiments, the molar extinction coefficient of the chromophore is 1 x 10 6 M -1 cm -1 or higher.

[0101] In some cases, the extinction coefficient of the tandem dye is 40,000 cm per repeat unit. -1 M -1 or higher, e.g. 45,000 cm per repeating unit -1 M -1 Higher, 50,000cm per repeating unit -1 M -1 Higher, 55,000cm per repeating unit -1 M -1Higher, 60,000cm per repeating unit -1 M -1 Higher, 70,000cm per repeating unit -1 M -1 Higher, 80,000cm per repeating unit -1 M -1 Higher, 90,000cm per repeating unit -1 M -1 Higher, 100,000cm per repeating unit -1 M -1 In some cases, the 40,000 cm per repeat unit described herein is -1 M -1 or greater is the average extinction coefficient. In some cases, the repeating unit of the chromophore can include a single monomer, two comonomers, or three or more comonomers. In some cases, the extinction coefficient of the chromophore is 40,000 cm per comonomer. -1 M -1 or higher, for example 45,000 cm per comonomer -1 M -1 Higher, 50,000cm per comonomer -1 M -1 Higher, 55,000cm per comonomer -1 M -1 Higher, 60,000cm per comonomer -1 M -1 Higher, 70,000cm per comonomer -1 M -1 Higher, 80,000cm per comonomer -1 M -1 Higher, 90,000cm per comonomer -1 M -1 Higher, 100,000cm per comonomer -1 M -1 In some cases, 40,000 cm per comonomer -1 M -1 or higher is the average extinction coefficient.

[0102] In some embodiments, a polymeric tandem dye may include a segment of formula (Ia):

[0103]

[0104] in:

[0105] Each M 1 and M 2 are independently unsaturated comonomers (e.g., amino acid residues);

[0106] Each S 1 and S 2 are independently non-conjugated spacer units;

[0107] Each D 1 Independently for M 1 a linked pendant donor fluorophore (e.g., as described herein);

[0108] Each A 1 Independently for M 2 the attached acceptor fluorophore;

[0109] x is 75 mol% or more; and

[0110] y is 25 mol% or less.

[0111] The first repeating unit (M 1 -S 1 ) and the second repeating unit (M 2 -S 2 ) can be arranged in a random or co-block configuration. In certain cases of Formula (Ia), D of the first repeating unit 1 The pendant group includes two or more (e.g., two or three) different types of pendant light absorbing chromophores that together provide a light harvesting multichromophore system. In certain instances of Formula (Ia), D of the first repeating unit 1 The pendant groups are all identical.

[0112] In some cases of Formula (Ia), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some cases of Formula (Ia), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0113] In some cases, the polymer tandem dye comprises a segment of formula (IIa):

[0114]

[0115] in:

[0116] The polymer backbone of the non-conjugated repeating unit comprises SM 1 , SM 2 and SM 3 comonomers, each of which is independently a non-conjugated comonomer;

[0117] Each D 1 Independently for SM 1 a linked pendant donor chromophore;

[0118] Each A 1 Independently for SM 2 the attached acceptor fluorophore;

[0119] Each Z 2 Independently for SM 3 attached optional pendant groups;

[0120] x is 50 mol% or more; and

[0121] y+z is 50 mol% or less.

[0122] Z 2 may be absent or may be any convenient pendant group, such as a light absorbing chromophore, a chemoselective tag, a linker, a linked biomolecule, an acceptor fluorophore, WSG (e.g., as described in PCT Application Serial No. PCT / US2019 / 024662 (published as WO 2019 / 191482), the disclosure of which is incorporated herein by reference), etc. In certain cases of Formula (IIa), when Z 2 When it does not exist, SM 3 is a spacer comonomer. In certain cases of formula (IIa), SM 3 It contains Z 2 The Z 2 The group is a second pendant light absorbing chromophore, where each D 1 and each Z 2 Together they provide a light harvesting multichromophore system. In some cases, the SM 3 is a second chemoselective tag (Z 2 ) comonomers, for example, to provide a protected functional group for selective installation of a moiety of interest or to 1 Orthogonal labels.

[0123] In some cases of formula (IIa), x is 60mol% or more, for example 65mol% or more, 70mol% or more, 75mol% or more, 80mol% or more, 85mol% or more, 90mol% or more, 95mol% or more or even more. In some cases of formula (IIa), y+z is 40mol% or less, for example 30mol% or less, 25mol% or less, 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, or even less. In some cases of formula (IIa), y is at least 1mol% and 25mol% or less, for example 20mol% or less, 15mol% or less, 10mol% or less, 5mol% or less, or even less. In some cases of formula (IIa), z is at least 1mol% and 10mol% or less, for example 5mol% or less, or even less.

[0124] In some cases, the polymer tandem dye comprises a segment of formula (IIIa):

[0125]

[0126] in:

[0127] The polymer backbone of the non-conjugated repeating unit comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0128] Each D 1 Independently for SM 1 a linked pendant donor chromophore;

[0129] Each A 1 Independently for SM 2 the attached acceptor fluorophore;

[0130] x is 75 mol% or more; and

[0131] y is 25 mol% or less.

[0132] In certain embodiments of Formula (IIIa), SM 1 and SM 2 Each is independently a saturated non-conjugated comonomer, for example, a comonomer that provides only a single covalent CC bond. In some embodiments of Formula (IIIa), SM 1 and SM 2 Each is independently a saturated non-conjugated comonomer, for example, a comonomer that provides a single double C=C covalent bond in the saturated covalent bond backbone. The first and second repeating units (SM 1and SM 2 ) can be arranged in a random configuration, a block or co-block configuration or in a specific order. In certain cases of formula (IIIa), SM 1 D 1 The pendant group includes two or more (e.g., two or three) different types of pendant light absorbing chromophores that together provide a light harvesting multichromophore system. In certain instances of formula (IIIa), D of the first repeating unit 1 The pendant groups are all identical.

[0133] In some cases of formula (IIIa), x is 80 mol% or more, for example 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some cases of formula (IIIa), y is 20 mol% or less, for example 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0134] In some cases, the polymeric tandem dye has formula (IVa):

[0135]

[0136] in:

[0137] Each D 1 independently a pendant donor chromophore group;

[0138] Each A 1 independently for the acceptor fluorophore;

[0139] Each L 1 and L 2 independently a joint;

[0140] p 1 and q 1 are independently 0 or 1, where p 1 +q 1 ≤1;

[0141] p 2 and q 2 are independently 0 or 1, where p 1 +q 1 ≤1;

[0142] x is 75 mol% or more;

[0143] y is 25 mol% or less; and

[0144] G 1and G 2 Each is independently selected from a terminal group, a polymer segment, a light absorbing (eg, donor) chromophore group, an acceptor fluorophore, a linker, and a linker to a single domain antibody, eg, as described above.

[0145] In some embodiments of Formula (IVa), p 1 and p 2 Each is 0, and q 1 and q 2 Each is 1 (eg, β3-amino acid residue). In some embodiments of Formula (IVa), p 1 and p 2 Each is 1, and q 1 and q 2 Each is 0 (e.g., β2-amino acid residue). In some cases, p 1 、p 2 ,q 1 and q 2 Each is 0, and the polymer tandem dye has formula (Va):

[0146]

[0147] in:

[0148] Each D 1 independently a pendant donor chromophore group;

[0149] Each A 1 independently for the acceptor fluorophore;

[0150] L 1 and L 2 Each independently is a connector;

[0151] x is 75 mol% or more;

[0152] y is 25 mol% or less; and

[0153] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a light absorbing (e.g., donor) chromophore group, a linker, and a linker to a single domain antibody, such as described above. It will be appreciated that the tandem dye described by formula (Va) includes any convenient arrangement of comonomers in a defined linear sequence, these monomers having a total of defined x and y mol%. In some cases, the A-containing 1 The comonomers of D1 are spaced throughout the polymer backbone sequence, so that they are always flanked by one or more comonomers containing D1.

[0154] In certain instances of Formula (Va), the polymeric tandem dye comprises a segment of Formula (VIa):

[0155]

[0156] in:

[0157] Each D 1 independently a pendant donor chromophore group;

[0158] Each A 1 independently an acceptor chromophore;

[0159] Each L 1 and L 2 independently a joint;

[0160] n and p are each independently an integer from 1 to 20, wherein n+p≥2; and

[0161] m is 1 or 2.

[0162] In some instances of Formula (VIa), n and p are each independently 1 to 10, e.g., 2 to 20, 3 to 10, or 3 to 6. In some instances of Formula (VIa), n+p is an integer from 2 to 20, e.g., 3 to 20, 4 to 20, 5 to 20, 5 to 15, or 5 to 12. In certain embodiments of Formula (VIa), m is 1.

[0163] The subject polymer tandem dye may include multiple segments of formula (VIa), wherein each segment includes two D-containing 1 A single comonomer block containing A 1 In some cases, the multichromophore includes two or more segments of formula (VIa) adjacent to each other to provide a copolymer having 2-20 D-containing 1 The blocks of comonomers (e.g., 3 to 20, 4 to 20, 5 to 20, 5 to 15 or 5 to 12 containing D 1 Two separate A-containing blocks separated by 1 Comonomer. Thus, in certain embodiments, the polymer tandem dye comprises q segments of a block copolymer and has formula (VIIa):

[0164]

[0165] Where: Each (n) q and each (p) q are independently integers from 1 to 20, wherein for each of the q segments, (n) q +(p) q ≥3; and q is an integer from 1 to 100.

[0166] In certain embodiments, the polymer tandem dye has formula (VIIIa):

[0167]

[0168] in

[0169] Each D 1 independently a pendant donor chromophore group;

[0170] Each A 1 independently an acceptor chromophore;

[0171] Each L 1 and L 2 independently a joint;

[0172] x is 75 mol% or more;

[0173] y is 25 mol% or less; and

[0174] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a donor chromophore group, a linker and a linker to a single domain antibody, e.g. as described above.

[0175] In certain embodiments, the polymeric tandem dye has Formula (IXa):

[0176]

[0177] in:

[0178] Each D 1 Independently, the pendant BODIPY donor chromophore;

[0179] Each A 1 independently for the acceptor fluorophore;

[0180] Each L 1 and L 2 independently a joint;

[0181] x is 75 mol% or more;

[0182] y is 25 mol% or less; and

[0183] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a donor chromophore group, an acceptor fluorophore, a linker and a linker to a single domain antibody, e.g. as described above.

[0184] In some cases of Formula (IVa), (Va), (XIIIa) and (IXa), x is 80 mol% or more, e.g., 85 mol% or more, 90 mol% or more, 95 mol% or more, 96 mol% or more, 97 mol% or more, 98 mol% or more, or 99 mol% or more. In some cases of Formula (IV), (V), (XIII) and (IX), y is 20 mol% or less, e.g., 15 mol% or less, 10 mol% or less, 5 mol% or less, 4 mol% or less, 3 mol% or less, 2 mol% or less, 1 mol% or less.

[0185] In certain embodiments, the polymeric tandem dye has formula (Xa):

[0186]

[0187] in:

[0188] Each D 1 independently as pendant donor chromophores;

[0189] Each A 1 independently for the acceptor fluorophore;

[0190] Each L 1 , L 2 and L 3 independently a joint;

[0191] a, b and c are the mol% values ​​of each comonomer;

[0192] d represents the overall degree of polymerization or average length of the polymer (e.g., d is 2-1000, such as 2-500, 2-200, 2-100 or 2-50);

[0193] WSG is a water-soluble group (e.g., as described in PCT Application Serial No. PCT / US2019 / 024662 (published as WO2019 / 191482), the disclosure of which is incorporated herein by reference); and

[0194] G 1 and G 2 Each is independently selected from a terminal group, a polymer segment, a donor chromophore group, an acceptor fluorophore, a linker and a linker to a single domain antibody, e.g. as described above.

[0195] In some cases of formula (Xa), c=0. In some cases of formula (Xa), a>0 and b>0. In some cases of formula (Xa), a is 80mol% or more, such as 85mol% or more, 90mol% or more, 95mol% or more, 96mol% or more, 97mol% or more, 98mol% or more, or 99mol% or more. In some cases of formula (Xa), b is 20mol% or less, such as 15mol% or less, 10mol% or less, 5mol% or less, 4mol% or less, 3mol% or less, 2mol% or less, 1mol% or less. In some cases of formula (Xa), a is 65-95mol%, b is 5-35mol% and c is 0-30mol%, wherein a+b+c=100%.

[0196] Any convenient end group (e.g., G 1 and G 2 ) can be used to terminate the tandem fluorescent dye. As used herein, the terms "terminal group" and "terminal group" are used interchangeably to refer to a group located at the end of a polymer structure of a light-harvesting chromophore, such as described herein. G of interest 1 and G 2 Groups include, but are not limited to, terminal capping groups, π-conjugated segments, linkers, and linkers to single domain antibodies, such as described above. In some embodiments, the terminal capping group is a monovalent group that is conjugated to the backbone of the light-collecting chromophore after polymerization. In some cases, the terminal capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the terminal comonomer is directly connected to a chemoselective tag or linker. In some cases, the terminal capping group is derived from a monomer used in the polymerization method, for example, an end group that is capable of further conjugation, such as a halogen (e.g., Br), boronic acid, or borate. In some cases, G 1 and G 2 is a π-conjugated segment. As used herein, a π-conjugated segment refers to any convenient segment of a conjugated polymer to which a light-harvesting chromophore can be conjugated, i.e., allowing π electrons to be delocalized between adjacent units. In certain embodiments, G 1 and G 2 is a linker, for example, as a linker, comprising a functional group suitable for conjugation with a specific binding moiety. It is understood that G located at the light-harvesting chromophore 1 and / or G 2 The linker at the position of G can be selected so as to be orthogonal to any other linker that may be present on the side chain of the light-harvesting chromophore, including a chemoselective tag (e.g., as described herein). 1 and / or G 2The position includes an amino functional group or a derivative thereof. In certain embodiments, G 1 and / or G 2 The position includes a carboxylic acid functional group or a derivative thereof.

[0197] In some embodiments of the formulae described herein, G 1 and G 2 At least one of them is -L 6 -Z 4 , where L 6 is a linker (eg, as described herein) and Z 4 is a specific binding member (e.g., as described herein). In some embodiments of the formulae described herein, G 1 and G 2 At least one of them is -L 6 -Z 3 , where L 6 is a linker (eg, as described herein) and Z 3 is a chemoselective tag (e.g., as described herein). Any convenient chemoselective tag and conjugation chemistry may be suitable for use with the subject light-harvesting chromophore. Chemoselective tags of interest include, but are not limited to, amines, active esters, maleimides, thiols, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluorides, Diers Alder cycloaddition click reagents and click chemistry, tetrazines, trans-cyclooctenes, aldehydes, alkoxyamines, alkynes, cyclooctynes, azides, and the like. In some cases, Z 3 is selected from carboxylic acids, active esters (e.g., N-hydroxysuccinimide esters (NHS) or sulfo-NHS), amino groups, maleimides, iodoacetyl groups, and thiols. In certain embodiments of the formulae described herein, G 1 and G 2 At least one of is described by the following structure:

[0198] *-Ar-LZ

[0199] wherein Ar is a π-conjugated aromatic group, L is a linker, and Z is a chemoselective tag or specific binding member. In some cases, the LZ group can be directly attached to a terminal comonomer. In certain embodiments of the formula described herein, G 1 and G 2 At least one of is described by the following structure:

[0200]

[0201] in:

[0202] q is 0 or an integer from 1 to 12;

[0203] L is an optional linker; and

[0204] Z is a chemoselective tag or a specific binding member. More detailed information about the WSG group that can be found in the tandem dyes of embodiments of the present invention can be found in PCT application serial number PCT / US2019 / 024662 (published as WO2019 / 191482), the disclosure of which is incorporated herein by reference.

[0205] Representative single domain antibody / polymer fluorescent tandem dye conjugates

[0206] Figure 1A and 1B Provided are views of single domain antibody / polymer fluorescent tandem dye conjugates according to embodiments of the present invention. Figure 1A In the figure, the single domain antibody / polymer fluorescent tandem dye conjugate shown includes V conjugated to two polymer fluorescent dyes. H H single domain antibody (labeled as "tandem 'cobalt' dye"). Tandem dyes can be conjugated to any convenient residue of the single domain antibody, where the residue of interest for conjugation or attachment is a residue that does not adversely affect the binding activity of the single domain antibody when attached to the dye. Residues that can be used for conjugation with dyes can be naturally occurring residues or residues that are engineered into single domain antibodies as desired, such as terminal cysteine ​​or non-natural amino acids. Figure 1B Provided Figure 1A A more detailed view of the conjugate shown in Figure 2, showing the structure of the polymer fluorescent tandem dye and the attachment of the dye to V H H single domain antibody PEG water-soluble linker. Although the length of the linker can vary, in some cases, the linker ranges from 10 to 50 nm, such as 15 to 40 nm, such as 20 to 30 nm. Depending on the needs, a given linker can be flexible, such as PEG, or rigid, such as a polyglycine chain instead of PEG.

[0207] method

[0208] Aspects of the present invention include methods for assessing the presence of a target analyte in a sample. Aspects of the method include contacting a sample with a single domain antibody / polymer fluorescent dye conjugate that specifically binds to a target analyte to produce a determination composition comprising a sample contacted with the single domain antibody / polymer fluorescent dye conjugate. In the following sections, the target analyte may be a target molecule of interest or a reagent (e.g., an antibody) that binds to a target molecule, depending on whether the single domain antibody / polymer fluorescent dye conjugate is used as a first marker or a second marker. Any convenient method may be used to contact a sample with a single domain antibody / polymer fluorescent dye conjugate that specifically binds to a target analyte to produce a determination composition. In some cases, the sample is contacted with a single domain antibody / polymer fluorescent tandem dye conjugate under conditions where the single domain antibody component of the conjugate specifically binds to the target analyte (if present). In order to specifically bind the single domain antibody of the conjugate to the target analyte, an appropriate solution may be used to maintain the biological activity of the sample component and the single domain antibody. The solution can be a balanced salt solution, such as physiological saline, PBS, Hank's balanced salt solution, etc., which is conveniently supplemented with fetal bovine serum, human platelet lysate or other factors, combined with an acceptable buffer at a low concentration such as 5-25mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. Various media are commercially available and can be used according to the properties of the target analyte, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., supplemented with fetal bovine serum or human platelet lysate in some cases. The final component of the solution can be selected according to the components of the sample included. The temperature at which the single domain antibody of the conjugate specifically binds to the target analyte can vary, and in some cases can be within the range of 5°C to 50°C, such as 10°C to 40°C, 15°C to 40°C, 20°C to 40°C, such as 20°C, 25°C, 30°C, 35°C or 37°C (e.g., as described above). In some cases, the temperature at which specific binding occurs is selected to be compatible with the biological activity of the single domain antibody and / or the target analyte. In some cases, the temperature is 25°C, 30°C, 35°C or 37°C. In some cases, the temperature at which specific binding occurs is room temperature (e.g., 25°C), 30°C, 35°C or 37°C. Any convenient incubation time for specific binding can be selected to allow the formation of a desired amount of binding complex, and in some cases, can be 1 minute (min) or longer, such as 2min or longer, 10min or longer, 30min or longer, 1 hour or longer, 2 hours or longer or even 6 hours or longer.

[0209] Any convenient single domain antibody can be used in the conjugate of the label used in the method of the present invention. Interested single domain antibodies include but are not limited to those single domain antibodies that specifically bind to cell surface proteins of various cell types, and the cell types include but are not limited to stem cells, such as pluripotent stem cells, hematopoietic stem cells, T cells, T regulatory cells, dendritic cells, B cells such as memory B cells, antigen-specific B cells, granulocytes, leukemia cells, lymphoma cells, viral cells (such as HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells and erythrocytes. Interested target cells include cells with convenient cell surface markers or antigens that can be captured by convenient specific binding member conjugates. In some embodiments, target cells are selected from cells containing HIV, Treg cells, antigen-specific T cell colonies, tumor cells, or hematopoietic progenitor cells (CD34+) from whole blood, bone marrow or umbilical cord blood. Any convenient cell surface protein or cell marker can be targeted to specifically bind to the conjugate used in the subject method. In some embodiments, the target cell comprises a cell surface marker selected from a cell receptor and a cell surface antigen. In some cases, the target cells can include cell surface antigens such as CD11b, CD123, CD14, CD15, CD16, CD19, CD193, CD2, CD25, CD27, CD3, CD335, CD36, CD4, CD43, CD45RO, CD56, CD61, CD7, CD8, CD34, CD1c, CD23, CD304, CD235a, T cell receptor α / β, T cell receptor γ / δ, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N terminus), SSEA-3, TRA-1-60 antigen, disialylganglioside GD2, and CD71.

[0210] Any convenient target can be selected to utilize the subject method for assessment. Interested targets include but are not limited to nucleic acids, such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules, proteins such as fusion proteins, modified proteins, such as phosphorylation, glycosylation, ubiquitination, SUMOylation or acetylated proteins, or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins and drug molecules. As used herein, the term "target protein" refers to all members of the target family and fragments thereof. Target protein can be any protein of interest, such as treatment or diagnosis targets, including but not limited to: hormones, growth factors, transcription factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors and immunoglobulins. The term "target protein" is intended to include recombinant and synthetic molecules, which can be prepared using any convenient recombinant expression method or using any convenient synthesis method, or commercial purchase. In some embodiments, the polymer dye conjugate includes an antibody or an antibody fragment. In the subject method, any convenient target analyte that can be targeted specifically combined with an antibody of interest or an antibody fragment.

[0211] In some embodiments, the target analyte is associated with a cell. In some cases, the target analyte is a cell surface marker of a cell. In some cases, the cell surface marker is selected from a cell receptor and a cell surface antigen. In some cases, the target analyte is an intracellular target, and the method also includes permeabilizing or lysing the cell. Therefore, the single domain antibody of a given conjugate used in the method of the present invention can target a cell surface or an intracellular antigen. Alternatively, the single domain antibody of a given conjugate used in the method of the present invention can target a primary antibody, which in turn specifically binds to a target cell surface or an intracellular antigen.

[0212] In some embodiments, the sample may include a heterogeneous cell population from which a target cell is isolated. In some cases, the sample includes peripheral whole blood, peripheral whole blood in which red blood cells have been lysed before cell separation, cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells or homogenized tissue. In some cases, the sample includes hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow or cord blood. In certain embodiments, the sample includes tumor cells in peripheral blood. In some cases, the sample includes (or is suspected of including) viral cells (e.g., HIV).

[0213] Single domain antibody / polymer fluorescent tandem dye conjugates can be used in the subject method, for example, target cells, particles, targets or analytes are labeled with polymer tandem fluorescent dyes. For example, single domain antibody / polymer fluorescent tandem dye conjugates can be used to mark cells to be processed (e.g., detected, analyzed and / or sorted) in a flow cytometer. Single domain antibody / polymer fluorescent tandem dye conjugates can include single domain antibodies (e.g., as described herein) that specifically bind to cell surface proteins of, for example, a variety of cell types. Labeled single domain antibody / polymer fluorescent tandem dye conjugates can be used to study a variety of biological (e.g., cell) properties or processes, such as cell cycle, cell proliferation, cell differentiation, DNA repair, T cell signaling, apoptosis, cell surface protein expression and / or presentation, etc. Labeled specific binding members can be used for any application of antibody-mediated labeling including (or can include) cells, particles or analytes.

[0214] Aspects of the method include determining whether there is a single domain antibody / polymer fluorescent tandem dye conjugate-target analyte binding complex in the assay composition (i.e., a sample contacted with a labeled single domain antibody / polymer fluorescent tandem dye conjugate) to assess whether the target analyte is present in the sample. Once the sample is in contact with the single domain antibody / polymer fluorescent tandem dye conjugate, any convenient method can be used to determine the produced assay composition to determine the presence of the single domain antibody / polymer fluorescent tandem dye conjugate-target analyte binding complex. The single domain antibody / polymer fluorescent tandem dye conjugate-target analyte binding complex is a binding complex generated when the specific binding member of the conjugate is specifically bound to the target analyte (or the primary binding member for the target antigen, e.g., a primary antibody, depending on the embodiment). If present, the assay composition may include detecting a fluorescent signal from the binding complex. In some cases, the assay includes a separation step in which the target analyte (if present) is separated from the sample. A variety of methods can be used to separate the target analyte from the sample, such as by being fixed on a support. Assay methods of interest include, but are not limited to, any convenient methods and assay formats in which specific binding member pairs such as avidin-biotin or hapten-anti-hapten antibodies are used. Methods of interest and assay formats that may be suitable for use with the subject compositions include, but are not limited to, flow cytometry methods, in situ hybridization methods, enzyme-linked immunosorbent assays (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography.

[0215] In certain embodiments, the method also includes contacting the sample with the second specific binding members that specifically binds the target analyte. In some cases, the second specific binding members is supported. The component (for example, the second specific binding members) of the subject method can be fixed using any convenient support. In some cases, the support is a particle, such as a magnetic particle. In some cases, the second specific binding members and the polymer dye conjugate produce a sandwich complex, and any convenient method can be used to separate and detect the sandwich complex (if present). In some embodiments, the method also includes the target analyte binding complex of the conjugated dye by flow cytometry, i.e., fluorescently labeled target analyte. Determining the presence of polymer dye conjugate-target analyte binding complex can provide a determination result (for example, qualitative or quantitative determination data), which can be used to assess whether there is a target analyte in a sample.

[0216] In the subject method, any convenient support can be used to fix any convenient component of the method, such as the specific binding members, targets, second specific binding members of the labeling, etc. The support of interest includes but is not limited to: solid matrix, wherein the matrix can have a variety of structures, such as sheets, beads or other structures, such as plates with holes; beads, polymers, particles, fiber nets, hydrogels, porous substrates, pins, microarray surfaces, chromatographic supports, etc. In some cases, the support is selected from particles, planar solid substrates, fiber nets, hydrogels, porous substrates, pins, microarray surfaces and chromatographic supports. The support can be incorporated into the system, which provides the cell separation assisted by any convenient method (such as manually operated syringes, centrifuges or automatic liquid handling systems). In some cases, the support can be used for automated liquid handling systems, to achieve high-throughput separation of cells, such as flow cytometers.

[0217] In some embodiments of the method, the separation step includes applying an external magnetic field to fix the magnetic particles. Any convenient magnet can be used as the source of an external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is produced by a magnetic source, such as produced by a permanent magnet or an electromagnet. In some cases, fixing the magnetic particles means that the magnetic particles are gathered near the surface closest to the magnetic field gradient source (i.e., magnet).

[0218] Separation can also include one or more optional washing steps to remove the unbound material of the sample from the support. Any convenient washing method can be used, for example, washing the immobilized support with a biocompatible buffer that retains the specific binding interactions of the polymeric dye and the specific binding members. Separating and optionally washing the unbound material of the sample from the support provides an enriched target cell population in which unwanted cells and materials can be removed.

[0219] In certain embodiments, the method also includes detecting the target analyte of the marker.Detecting the target analyte of the marker can include exciting the polymer fluorescent tandem dye with one or more lasers, and then using one or more optical detectors to detect the fluorescence emission from the polymer fluorescent tandem dye.Detection of the marker target can be carried out using any convenient instrument and method, including but not limited to flow cytometry, FACS system, fluorescence microscopy; using a microplate reader for fluorescence, cold light, ultraviolet light and / or visible light detection; high performance liquid chromatography (HPLC); and mass spectrometry.When using fluorescently labeled components in the methods and compositions of the present disclosure, it is recognized that different types of fluorescence detection systems can be used to practice the subject method.In some cases, high throughput screening can be performed, such as a system using 96 wells or larger microtiter plates. A variety of methods for measuring fluorescent materials can be used, such as those described in Lakowicz, JR, Principles of Fluorescent Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, DL & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turro, NJ, Modern Molecular Photochemistry, Menlo Park: Benjamin / Cummings Publishing Col, Inc. (1978), pp. 296-361.

[0220] Fluorescence in a sample can be measured using a fluorometer. In some cases, excitation radiation from an excitation source having a first wavelength passes through an excitation optical device. The excitation optical device causes the excitation radiation to excite the sample. In response, the fluorescently labeled target in the sample emits radiation having a wavelength different from the excitation wavelength. The collection optical device then collects the emitted light from the sample. The device may include a temperature controller to keep the sample at a specific temperature while scanning the sample. In some cases, a multi-axis translation stage moves a microtiter plate holding multiple samples in order to locate different wells to be exposed. The multi-axis translation stage, temperature controller, autofocus function, and electronic devices associated with imaging and data collection can be managed by a properly programmed digital computer. The computer can also convert the data collected during the assay into another format for presentation.

[0221] In some embodiments, the method for assessing the presence of a target analyte in a sample further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method for assessing the presence of a target analyte in a sample further comprises imaging the sample contacted with the labeling composition using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify polymer dye conjugate-target analyte binding complexes in the contacted sample to assess whether the target analyte exists. Microscopic methods of interest that can be used for the subject method include laser scanning confocal microscopy.

[0222] Figure 2A A schematic diagram of an embodiment of the method is provided in which a primary antibody is used to detect intracellular cytokines in conjunction with a single domain antibody / polymer fluorescent tandem dye conjugate that specifically binds to the primary antibody. Figure 2B A similar approach was demonstrated using Alexa-fluor labeled nanobodies.

[0223] Also provided is a method for producing a single domain antibody / polymer fluorescent tandem dye conjugate, for example as described herein. In some embodiments, the method includes: contacting a single domain antibody with a polymer fluorescent tandem dye (for example as described herein) to produce a single domain antibody / polymer fluorescent tandem dye conjugate, wherein the polymer fluorescent tandem dye includes a conjugated tag that covalently connects the dye to the single domain antibody. The term "conjugated tag" refers to a group including a chemoselective functional group (for example, as described herein), which can be covalently connected to a compatible functional group of a single domain antibody after optional activation and / or deprotection. Any convenient conjugated tag can be used in the subject polymer dye to conjugate the polymer fluorescent tandem dye to the single domain antibody of interest. In some embodiments, the conjugated tag includes a terminal functional group selected from amino, carboxylic acid or its derivatives, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne and protein reactive groups (e.g., amino reactive, thiol reactive, hydroxy reactive, imidazole reactive or guanidinium reactive). Any convenient method and reagent can be applied to the subject method to covalently connect the conjugated tag to the single domain antibody. Interested methods for labeling targets include, but are not limited to, those methods and reagents described by Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contacting step can be carried out in an aqueous solution. In some cases, the conjugated label includes an amino functional group and the target molecule includes an activated ester functional group, such as an NHS ester or a sulfo-NHS ester, or vice versa. In some cases, the conjugated label includes a maleimide functional group and the target molecule includes a thiol functional group, or vice versa. In some cases, the conjugated label includes an alkyne (e.g., a cyclooctyne group) functional group and the target molecule includes an azide functional group, or vice versa, which can be conjugated by click chemistry. In some cases, the method includes a separation step, wherein the product single domain antibody / polymer fluorescent tandem dye conjugate is separated from the reaction mixture (e.g., excess reagent or unlabeled single domain antibody). A variety of methods can be used to separate the target from the sample, such as by being fixed on a support, precipitation, chromatography, etc.

[0224] In some cases, the method also includes detecting and / or analyzing product single domain antibody / polymer fluorescent tandem dye conjugates. In some cases, the method also includes fluorescent detection of single domain antibody / polymer fluorescent tandem dye conjugates. Any convenient method can be used in conjunction with the subject method and composition to detect and / or analyze the target molecule of the label. The method for analyzing the target of interest that can be used for the subject method includes but is not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay and fluorescent dye purification chromatography. Interested detection methods include but are not limited to fluorescence spectroscopy, fluorescence microscopy, nucleic acid sequencing, fluorescence in situ hybridization (FISH), protein mass spectrometry, flow cytometry, etc.

[0225] Detection can be achieved directly by polymerizing tandem dyes, or indirectly by auxiliary detection systems. The latter can be based on any one or a combination of several different principles, including but not limited to antibody-labeled anti-species antibodies and other forms of immunological or non-immunological bridging and signal amplification systems (e.g., biotin-streptavidin technology, protein A and protein G-mediated technology, or nucleic acid probes / anti-nucleic acid probes, etc.). Suitable reporter molecules can be those molecules known in the quantitative fields of immunocytochemistry, molecular biology, light, fluorescence and electron microscopy, cell immunophenotype, cell sorting, flow cytometry, cell visualization, detection, counting and / or signal output. More than one specific and / or nonspecific antibodies can be labeled and used simultaneously or sequentially to enhance target detection, identification and / or analysis.

[0226] system

[0227] Aspects of the present invention further include systems for practicing subject methods and compositions. The sample analysis system may include a sample field of view or a flow channel loaded with a sample and a single domain antibody / polymer fluorescent tandem dye conjugate. In some embodiments, the system is a flow cytometry system, comprising: a flow cytometer comprising a flow path; a composition in the flow path, wherein the composition comprises: a sample; and a single domain antibody / polymer fluorescent tandem dye conjugate (e.g., as described herein). In some embodiments, the system for analyzing a sample is a fluorescence microscope system, comprising: a fluorescence microscope comprising a sample field of view; and a composition disposed in the sample field of view, wherein the composition comprises a sample; and a labeled specific binding member (e.g., as described herein).

[0228] In certain embodiments of the system, the composition further comprises a second specific binding member that is support-bound and specifically binds to the target analyte. In some cases, the support comprises magnetic particles. Thus, in some cases, the system may further comprise a controllable external paramagnetic field configured to be applied to the assay region of the flow channel.

[0229] The sample may comprise cells. In some cases, the sample is a biological sample containing cells. In some cases, the sample comprises a labeled specific binding member that specifically binds to a target cell. In some cases, the target analyte specifically bound by the specific binding member is a cell surface marker of a cell. In some cases, the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0230] In some cases, the system may further include a light source configured to direct light to the assay region or sample field of view of the flow channel. The system may include a detector configured to receive a signal from the assay region or sample field of view of the flow channel, wherein the signal is provided by the fluorescent composition. Optionally, the sample analysis system may include one or more additional detectors and / or light sources for detecting one or more additional signals.

[0231] In some aspects, the system may further include a computer-based system configured to detect the presence of a fluorescent signal. "Computer-based system" refers to hardware devices, software devices, and data storage devices that are used to analyze the information of the present invention. The minimum hardware of the computer-based system of the present invention includes a central processing unit (CPU), an input device, an output device, and a data storage device. The technician can easily recognize that any currently available computer-based system is suitable for use in the subject system. The data storage device can include any product that includes any record of the present information as described above, or a storage accessor that can access this product.

[0232] "Recording" data, programming, or other information on a computer readable medium refers to the process of storing the information using any such method known in the art. Any convenient data storage structure may be selected based on the means used to access the stored information. Storage may be performed using a variety of data processor programs and formats, such as word processing text files, database formats, etc.

[0233] "Processor" refers to any hardware and / or software combination that performs its required functions. For example, any processor herein may be a programmable digital microprocessor, such as available in the form of an electronic controller, a mainframe, a server, or a personal computer (desktop or portable). In the case where the processor is programmable, suitable programming may be transmitted to the processor from a remote location, or may be pre-stored in a computer program product (such as a portable or fixed computer-readable storage medium, whether based on magnetic, optical, or solid-state devices). For example, a magnetic medium or an optical disc may carry a program and may be read by a suitable reader that communicates with each processor at its corresponding site.

[0234] In addition to the sensor device and the signal processing module, for example as described above, the system of the present invention may include many additional components, such as data output devices, such as displays and / or speakers, data input devices (such as interface terminals, keyboards, etc.), fluid processing components, power supplies, etc.

[0235] In certain aspects, the system comprises a flow cytometer. Suitable flow cytometry systems may include, but are not limited to, Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); Practical Flow Cytometry, 3rd edition, Wiley-Liss (1995); Virgo, et al. (2012) Ann Clin Biochem. Jan; 49 (pt 1): 17-28; Linden, et al., Semin Throm Hemost. 2004 Oct; 30 (5): 502-11; Alison, et al. J Pathol, 2010 Dec; 222 (4): 335-344; and Herbig, et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the disclosure of which is incorporated herein by reference. In some cases, flow cytometry systems of interest include BD Biosciences FACSCanto TM Flow cytometer, BD Biosciences FACSCanto TM II flow cytometer, BD Accuri TM Flow cytometer, BD Accuri TM C6 Plus flow cytometer, BD Biosciences FACSCelesta TM Flow cytometer, BD Biosciences FACSLyric TM Flow cytometer, BD Biosciences FACSVerse TM Flow cytometer, BD Biosciences FACSymphony TM Flow cytometer, BD Biosciences LSRFortessa TMFlow cytometer, BD Biosciences LSRFortessa TM X-20 flow cytometer, BD Biosciences FACSPresto TM Flow cytometer, BD Biosciences FACSVia TM Flow cytometer and BD Biosciences FACSCalibur TM Cell sorter, BD Biosciences FACSCount TM Cell sorter, BD Biosciences FACSLyric TM Cell sorter, BD Biosciences Via TM Cell sorter, BD Biosciences Influx TM Cell sorter, BD Biosciences Jazz TM Cell sorter, BD Biosciences Aria TM Cell sorter, BD Biosciences FACSAria TM II cell sorter, BD Biosciences FACSAria TM III cell sorter, BD Biosciences FACSAria TM Fusion Cell Sorter and BD Biosciences FACSMelody TM Cell sorter, BD Biosciences FACSymphony TM S6 cell sorter, etc.

[0236] In some embodiments, the subject system is a flow cytometry system, such as U.S. Pat. Nos. 10,663,476, 10,620,111, 10,613,017, 10,605,713, 10,585,031, 10,578,542, 10,578,469, 10,481,074, 10,302,545, 10,145,793, 10,113,967, 10,006,852, 9,952,076, 9,933,341, 9,726,527, 9,453,789, 9,200,334, 9,097,640, 9,947,834, and 9,113,967. those described in, for example, 4,084,975,595, 4,095,494, 9,092,034, 8,975,595, 8,753,573, 8,233,146, 8,140,300, 7,544,326, 7,201,875, 7,129,505, 6,821,740, 6,813,017, 6,809,804, 6,372,506, 5,700,692, 5,643,796, 5,627,040, 5,620,842, 5,602,039, 4,987,086, and 4,498,766, the disclosures of which are incorporated herein by reference in their entireties.

[0237] In some cases, the flow cytometry systems of the present invention are configured to image particles in a flow stream by fluorescence imaging using radio frequency labeled emission (FIRE), such as described in Diebold, et al. Nature Photonics Vol. 7(10); 806-810 (2013) and in U.S. Pat. Nos. 9,423,353, 9,784,661, 9,983,132, 10,006,852, 10,078,045, 10,036,699, 10,222,316, 10,288,546, 10,324,019, 10,40 8,758, 10,451,538, 10,620,111 and those described in U.S. Patent Publication Nos. 2017 / 0133857, 2017 / 0328826, 2017 / 0350803, 2018 / 0275042, 2019 / 0376895 and 2019 / 0376894, the disclosures of which are incorporated herein by reference.

[0238] Other systems can also be used to practice the subject method. In some aspects, the system can be a fluorometer or a microscope loaded with a sample of the fluorescent composition of any embodiment discussed herein. The fluorometer or microscope can include a light source configured to direct light to the assay area or sample visual field of the flow channel. The fluorometer or microscope can also include a detector configured to receive a signal from the assay area or visual field of the flow channel, wherein the signal is provided by the fluorescent composition.

[0239] Reagent test kit

[0240] Aspects of the present invention further include kits for practicing the subject methods and compositions. The composition of the present invention can be included in the kit as a reagent or as a starting material, or provided for use in, for example, the above-mentioned method. The kit can include a single domain antibody / polymer fluorescent tandem dye conjugate (e.g., as described herein) and a container. The single domain antibody / polymer fluorescent tandem dye conjugate can be provided as a composition of multiple copies of a single domain antibody / polymer fluorescent tandem dye conjugate. In this case, the polydispersity between multiple copies of the single domain antibody / polymer fluorescent tandem dye conjugate can be low. Any convenient container can be used, such as a tube, a bottle, or a hole in a porous strip or plate, a box, a bag, an insulated container, etc. The subject kit can further include one or more components selected from primer-specific binding members for a given target analyte, a specific binding member bound by a support, a cell, a support, a biocompatible aqueous elution buffer, and instructions for use. A given kit can include a reagent suitable for detecting a single target analyte, or a plurality of reagents suitable for detecting two or more different target analytes, for example, wherein a given kit is configured for multiplex detection applications.

[0241] In certain embodiments, the kit is used to assess the presence of a target analyte (such as an intracellular target) in a sample. Thus, in some cases, the kit comprises one or more components suitable for permeabilization or lysis of cells. One or more additional components of the kit can be provided in a separate container (e.g., a separate tube, bottle, or porous strip or plate well).

[0242] In some cases, the kit further comprises a reagent for performing flow cytometry. Reagents of interest include but are not limited to buffers for reconstruction and dilution, buffers for contacting cell samples with chromophores, washing buffers, control cells, control beads, fluorescent beads for flow cytometry calibration, and combinations thereof. The kit can also include one or more cell fixatives, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination thereof or buffer. In addition, the kit can include cell permeabilization reagents, such as methanol, acetone or detergents (for example, triton, NP-40, saponin, tween 20, digitonin, leucoperm, or any combination thereof or buffer. Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents familiar to those skilled in the art are within the scope of the subject kit.

[0243] The composition of the kit can be provided as a liquid composition, such as any suitable buffer. Alternatively, the composition of the kit can be provided as a dry composition (e.g., it can be lyophilized), and the kit can optionally include one or more buffers for reconstructing the dry composition. In some aspects, the kit can include aliquots of the composition, which are provided in separate containers (e.g., separate tubes, bottles, or porous strips or wells in plates).

[0244] In addition, one or more components can be combined into a single container such as a glass or plastic vial, tube or bottle. In some cases, the test kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all components (and their individual containers) are present. The test kit may further include a package that is separate from or attached to the test kit container and has information about the test kit, components and / or instructions for use of the test kit printed on the package.

[0245] In addition to the above-mentioned components, the subject test kit can further include instructions for practicing the subject method. These instructions can be present in the subject test kit in various forms, one or more of which can be present in the test kit. One of the forms in which these instructions can exist is as packaging, package inserts, etc. of printed information, test kit on suitable medium or substrate (for example, one or more sheets of paper printed with information). Another means can be a computer-readable medium, for example, a floppy disk, CD, DVD, portable flash drive, etc., with information recorded. Another means that can exist is a website address, which can be used to access the information at the removed site by the Internet. Any convenient means can be present in the test kit.

[0246] use

[0247] Single domain antibody / polymer fluorescent tandem dye conjugates, compositions, methods and systems as described herein can be used for a variety of applications, including diagnostic and research applications in which the labeling, detection and / or analysis of a target of interest is required. Such applications include methods such as cell counting, microscopy, immunoassays (e.g., competitive or non-competitive), assessment of free analytes, assessment of receptor binding ligands, etc. The compositions, systems and methods described herein can be used for any of a variety of samples, including but not limited to biological fluids, cell culture samples and tissue samples. In some aspects, the compositions, systems and methods described herein can be used for methods in which analytes (if present) in samples are detected using fluorescent markers, such as fluorescence activated cell sorting or analysis, immunoassays, immunostaining, etc. In some cases, compositions and methods are used for applications in which the presence of a target analyte in a sample is assessed with interest.

[0248] In some cases, the methods and compositions are used in any assay format where there is interest in detecting and / or analyzing a target from a sample, including but not limited to flow cytometry, fluorescence microscopy, in situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assays, and fluorescent dye purification chromatography. In some cases, the methods and compositions are used in any application where there is interest in fluorescently labeling a target molecule. The subject compositions can be adapted for any convenient application using specific binding member pairs such as biotin-streptavidin and hapten-anti-hapten antibodies.

[0249] The following examples are offered by way of illustration and not limitation.

[0250] Example

[0251] The following examples are presented to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; nt, nucleotide; etc.

[0252] I. Reagent Preparation

[0253] A. VHH anti-mouse IgG1-RB780 preparation:

[0254] Tandem dye conjugated with VHH anti-mouse IgG1 (Chromotek Nanobodies CTK103, CTK 104) RB780 TMThe tandem dye is modified to contain a terminal maleimide group for attaching maleimide-thiol to the N-terminal and C-terminal cysteine ​​of the target engineered nanobody CTK103 (Chromotek). The purified RB780-maleimide dye obtained is then added to a buffer solution (10mM HEPES pH 7.0500mM NaCl) at a concentration of 20mg / mL. The nanobody is frozen in an aqueous buffer (10mM HEPES pH 7.0, 500mM NaCl, 1mM TCEP). Before conjugation, the frozen nanobody solution is thawed to 4 ° C, centrifuged and the supernatant is transferred to a reaction vessel. Then the RB780-maleimide dye solution equivalent to the volume of 2.7 molar equivalents of nanobody is added to the solution containing the thawed nanobody. The mixture is vortexed, put into an ice bath and stirred for 2 hours with a magnetic stirrer. The resulting crude reaction mixture is then purified using a Superdex SEC column with a standard elution buffer. Fractions of the desired product were pooled and characterized. Prior to staining with the primary antibody, equimolar amounts of CTK103-RB780 conjugate and CTK104-RB780 were combined in solution.

[0255] RB780 TM Modified to contain terminal maleimide groups, for attaching maleimide-thiol to the N-terminal and C-terminal cysteine ​​of target engineered nanobody CTK104 (Chromotek). The purified RB780-maleimide dye obtained is then added to a buffer solution (10mM HEPES pH 7.0 500mM NaCl) at a concentration of 20mg / mL. Nanobodies are frozen in aqueous buffer (10mM HEPES pH 7.0, 500mM NaCl, 1mM TCEP). Before conjugation, the frozen nanobody solution is thawed to 4 ° C, centrifuged and the supernatant is transferred to a reaction vessel. Then the RB780-maleimide dye solution equivalent to the volume of 2.7 molar equivalents of nanobodies is added to the solution containing the thawed nanobody. The mixture is vortexed, put into an ice bath and stirred for 2 hours with a magnetic stirrer. The resulting crude reaction mixture is then purified using a Superdex SEC column with a standard elution buffer. Fractions of the desired product were pooled and characterized. Prior to staining with the primary antibody, equimolar amounts of CTK103-RB780 conjugate and CTK104-RB780 were combined in solution.

[0256] B. VHH anti-mouse IgG2b-RB780 preparation:

[0257] Tandem dye conjugated with VHH anti-mouse IgG2b (Chromotek Nanobodies CTK105, CTK 106) RB780TM Modified to contain terminal maleimide groups, for attaching maleimide-thiol to the N-terminal and C-terminal cysteine ​​of target engineered nanobody CTK105 (Chromotek). The purified RB780-maleimide dye obtained is then added to a buffer solution (10mM HEPES pH 7.0 500mM NaCl) at a concentration of 20mg / mL. Nanobodies are frozen in aqueous buffer (10mM HEPES pH 7.0, 500mM NaCl, 1mM TCEP). Before conjugation, the frozen nanobody solution is thawed to 4 ° C, centrifuged and the supernatant is transferred to a reaction vessel. Then the RB780-maleimide dye solution equivalent to the volume of 2.7 molar equivalents of nanobodies is added to the solution containing the thawed nanobody. The mixture is vortexed, put into an ice bath and stirred for 2 hours with a magnetic stirrer. The resulting crude reaction mixture is then purified using a Superdex SEC column with a standard elution buffer. Fractions of the desired product were pooled and characterized. Prior to staining with the primary antibody, equimolar amounts of CTK105-RB780 conjugate and CTK106-RB780 were combined in solution.

[0258] RB780 TM Modified to contain terminal maleimide groups, for attaching maleimide-thiol to the N-terminal and C-terminal cysteine ​​of target engineered nanobody CTK106 (Chromotek). The purified RB780-maleimide dye obtained is then added to a buffer solution (10mM HEPES pH 7.0 500mM NaCl) at a concentration of 20mg / mL. Nanobodies are frozen in aqueous buffer (10mM HEPES pH 7.0, 500mM NaCl, 1mM TCEP). Before conjugation, the frozen nanobody solution is thawed to 4 ° C, centrifuged and the supernatant is transferred to a reaction vessel. Then the RB780-maleimide dye solution equivalent to the volume of 2.7 molar equivalents of nanobodies is added to the solution containing the thawed nanobody. The mixture is vortexed, put into an ice bath and stirred for 2 hours with a magnetic stirrer. The resulting crude reaction mixture is then purified using a Superdex SEC column with a standard elution buffer. Fractions of the desired product were pooled and characterized. Prior to staining with the primary antibody, equimolar amounts of CTK105-RB780 conjugate and CTK106-RB780 were combined in solution.

[0259] II. Further Research

[0260] A. Preparation of RB780-labeled Nanobodies

[0261] 1. Modify RB780

[0262] 22 mg of the RB780 derivative with amine reactive sites was dissolved in degassed 1x phosphate buffered saline (VWR catalog number 97063-660) at a concentration of 10 mg / mL. 5.55 mg of SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) was dissolved in degassed amine-free DMF (Fisher Scientific catalog number AA43465K7). The SMCC solution was added to the solution containing RB780-amine and stirred at room temperature for 2 hours. The 50% SMCC solution was filtered through a size exclusion membrane (MilliporeSigma). TM The crude reaction mixture was purified by HPLC (UFC900308) to remove unreacted SMCC and freeze-dried overnight on a Schlenk line. Yield and purity were assessed by ion exchange chromatography (Thermo Scientific Ultimate 3000 and Propoac SAX-10 4x250 mm).

[0263] 2. Reduction of disulfide groups of nanobodies

[0264] Nanobody samples previously stored in -80°C buffer were thawed and separated using a size exclusion membrane (MilliporeSigma TM UFC900308) The buffer was exchanged to 10 mM Hepes pH 7.0, 500 mM NaCl, 1 mM TCEP. The buffer exchange solution containing the newly reduced Nanobodies was then centrifuged to remove precipitates and aggregates.

[0265] 3. Dye conjugation with nanobodies

[0266] Nanobody CTK0103 (ProteinTech) is concentrated to 2mg / mL in 10mM Hepes pH 7.0, 500mM NaCl, 1mM TCEP and kept below 4°C by ice bath. RB780-maleimide is dissolved in amine-free DMF (Fisher Scientific catalog number AA43465K7) with 20mg / mL and cooled to about 4°C by ice bath. RB780-maleimide solution is added to nanobody solution at a ratio of 2.7 moles of RB780-maleimide per mole of nanobody and stirred for two hours at about 4°C. The crude conjugated mixture is purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE and UV-Vis.

[0267] Nanobody CTK0104 (ProteinTech) is concentrated to 2mg / mL in 10mM Hepes pH 7.0, 500mM NaCl, 1mM TCEP and kept below 4°C by ice bath. RB780-maleimide is dissolved in amine-free DMF (Fisher Scientific catalog number AA43465K7) with 20mg / mL and cooled to about 4°C by ice bath. RB780-maleimide solution is added to nanobody solution at a ratio of 2.7 moles of RB780-maleimide per mole of nanobody and stirred for two hours at about 4°C. The crude conjugated mixture is purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE and UV-Vis.

[0268] Nanobody CTK0105 (ProteinTech) is concentrated to 2mg / mL in 10mM Hepes pH 7.0, 500mM NaCl, 1mM TCEP and kept below 4°C by ice bath. RB780-maleimide is dissolved in amine-free DMF (Fisher Scientific catalog number AA43465K7) with 20mg / mL and cooled to about 4°C by ice bath. RB780-maleimide solution is added to nanobody solution at a ratio of 2.7 moles of RB780-maleimide per mole of nanobody and stirred for two hours at about 4°C. The crude conjugated mixture is purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE and UV-Vis.

[0269] Nanobody CTK0106 (ProteinTech) was concentrated to 2 mg / mL in 10 mM Hepes pH 7.0, 500 mM NaCl, 1 mM TCEP and kept below 4°C by an ice bath. RB780-maleimide was dissolved in amine-free DMF (Fisher Scientific catalog number AA43465K7) at 20 mg / mL and cooled to about 4°C by an ice bath. The RB780-maleimide solution was added to the nanobody solution at a ratio of 2.7 moles of RB780-maleimide per mole of nanobody and stirred for two hours at about 4°C. The crude conjugated mixture was purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE and UV-Vis. B. Cell preparation / staining and flow cytometry

[0270] 1. Surface dyeing

[0271] 100 μl of well-mixed anticoagulated whole blood was incubated with unlabeled primary antibody human CD4 (clone SK3, mouse IgG1) at room temperature in the dark for 20-30 minutes. Then 2 ml of lysate was added and then incubated at room temperature in the dark. After 10 minutes, the cells were washed and the secondary anti-nano antibody anti-mouse IgG1-RB780 (nano antibodies CTK0103 and CTK0104 from ProteinTech) was added and then incubated at room temperature in the dark for 20-30 minutes. Before flow cytometry analysis or before storage in a 2-8 ° C refrigerator, a final wash was performed and 200-500 ul of washing buffer was added.

[0272] 100 μl of well-mixed anticoagulated whole blood was incubated with unlabeled primary antibody human CD56 (clone NCAM16.2, mouse IgG2b) at room temperature in the dark for 20-30 minutes. Then 2 ml of lysate was added, followed by incubation at room temperature in the dark. After 10 minutes, the cells were washed, and the secondary antibody reagent, nanobody VHH anti-mouse IgG2b-RB780 (nanobody CTK0105 and CTK0106 from ProteinTech) was added, followed by incubation at room temperature in the dark for 20-30 minutes. Before flow cytometry analysis or before storage in a 2-8°C refrigerator, a final wash was performed and 200-500 ul of wash buffer was added.

[0273] 2. Intracellular Staining

[0274] Peripheral blood mononuclear cells (PBMC) were activated and then fixed and permeabilized according to standard protocols. 100 μl PBMC was incubated with unlabeled primary antibody human IFN-γ (clone B27, mouse IgG1) at 4 ° C for 30 minutes in the dark. The cells were then washed twice, followed by the addition of secondary anti-nano antibody VHH anti-mouse IgG1-RB780 (nano antibodies CTK0103 and CTK0104 from Protein Tech), followed by incubation at 4 ° C for 30 minutes in the dark. The last wash was performed and 200-500 ul wash buffer was added before flow cytometry analysis or before storage in a 2-8 ° C refrigerator.

[0275] C. Results:

[0276] 1. Surface dyeing

[0277] We observed positive staining in lymphocytes corresponding to the CD4 positive population. Compared to the signal observed with the same existing nanobodies (pooled CTK0103 and CTK0104 from ProteinTech) conjugated to AlexaFluor647, the signal was fainter when using nanobodies CTK0103 and CTK0104 (used alone or pooled together). The comparison was performed in both mass equivalent and molar equivalent ways.

[0278] We also observed positive staining in lymphocytes corresponding to the CD56 positive population. Compared to the signal observed with the existing identical nanobodies (pooled CTK0105 and CTK0106 from ProteinTech) conjugated to AlexaFluor647, the signal was fainter when using nanobodies CTK0105 and CTK0106 (used alone or pooled together). The comparison was performed in both mass equivalent and molar equivalent ways.

[0279] 2. Intracellular staining:

[0280] We observed positive staining in lymphocytes corresponding to the IFN-γ positive population. Compared to the signal observed with the same existing nanobodies (pooled CTK0103 and CTK0104 from ProteinTech) conjugated to AlexaFluor647, the signal was fainter when using nanobodies CTK0103 and CTK0104 (used alone or pooled together). The comparison was performed in both mass equivalent and molar equivalent ways.

[0281] III. Additional Observations

[0282] The single domain antibody / polymer fluorescent tandem dye conjugate of the embodiment of the present invention has many advantages. Prior to the work reported in this article, there were two major categories of commercial sdAb+ fluorescent dye reagents. The first category is composed of sdAb+ fluorescent proteins (such as GFP, GYP, GRP, etc.). Typically, these reagents are recombinantly produced in the form of fusion proteins. Although these reagents retain advantages in manufacturability and performance due to their small size and in vivo expression, their practicality is limited because they are relatively dark, and because they are not tandem dyes, the multiplexing advantages of tandem dyes cannot be achieved. The second type of sdAb+ fluorescent dye reagents consists of sdAb conjugated with small molecule dyes or some small molecule dyes (such as AlexaFluor647). These reagents also have size-based advantages, but are limited in several aspects. As reagents, they have brightness limitations associated with self-quenching behavior. If too many AlexFluor647 dyes are attached to sdAb, the fluorescence of the reagent will actually decrease. In addition, like the above-mentioned fusion proteins, these small molecule dyes are not tandem dyes and the multiplexing advantages of tandem dyes cannot be achieved.

[0283] The single domain antibody / polymer fluorescent dye conjugate reagents of embodiments of the present invention combine the advantages of small size and improved manufacturing with other advantages, which may include one or more of increased reagent brightness and compatibility with high parameter panel designs. These advantages are achieved by using polymer fluorescent tandem dyes instead of single molecule dyes. Polymer fluorescent tandem dyes have higher per molecule brightness than small molecule dyes and do not exhibit significant inter-dye quenching of small molecule dyes when conjugated to antibodies. In addition, polymer fluorescent tandem dyes represent a tandem dye platform specifically designed for optimized multicolor flow cytometry. Therefore, the conjugates of the present invention can be used in multiplexed applications using dye panels.

[0284] Notwithstanding the appended claims, the present disclosure is also defined by the following clauses:

[0285] 1. A single domain antibody / polymer fluorescent tandem dye conjugate, the conjugate comprising:

[0286] (a) single domain antibodies; and

[0287] (b) one or more polymer tandem fluorescent dyes conjugated to the single domain antibody, wherein the one or more polymer tandem fluorescent dyes comprise:

[0288] (i) a polymer backbone comprising non-conjugated repeating units;

[0289] (ii) a plurality of pendant donor fluorophores, each pendant donor fluorophore independently attached to a non-conjugated repeat unit of the polymer backbone; and

[0290] (iii) one or more pendant acceptor fluorophores attached to a non-conjugated repeat unit of the polymer backbone, wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship.

[0291] 2. The conjugate according to item 1, wherein the single domain antibody comprises a heavy chain or a light chain.

[0292] 3. The conjugate according to item 2, wherein the single domain antibody comprises V H H domain.

[0293] 4. The conjugate according to item 2, wherein the single domain antibody comprises V NAR domain.

[0294] 5. The conjugate according to any one of the preceding items, wherein the single domain antibody specifically binds to an intracellular antigen.

[0295] 6. The conjugate according to any one of the preceding items, wherein the single domain antibody specifically binds to a cell surface antigen.

[0296] 7. The conjugate according to any one of the preceding items, wherein the single domain antibody specifically binds to a primary antibody.

[0297] 8. The conjugate according to any one of the preceding clauses, wherein the conjugate comprises two or more polymer tandem fluorescent dyes conjugated to the single domain antibody.

[0298] 9. The conjugate according to any one of the preceding clauses, wherein the polymer tandem fluorescent dye is described by the following formula:

[0299]

[0300] in:

[0301] The polymer backbone of the non-conjugated repeating units comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0302] Each D 1 Independently for SM 1 attached pendant donor fluorophore;

[0303] Each A 1 Independently for SM 2 a linked pendant acceptor fluorophore;

[0304] x is 75 mol% or more; and

[0305] y is 25 mol% or less.

[0306] 10. The conjugate according to item 9, wherein the repeating units of the polymer backbone have a defined linear sequence.

[0307] 11. The conjugate according to item 10, wherein SM 1 and SM 2 It is a comonomer derived from amino acids and peptoid monomers.

[0308] 12. The conjugate according to item 11, wherein the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues.

[0309] 13. The conjugate according to any one of the preceding items, wherein the polymer backbone is a linear polymer.

[0310] 14. The conjugate according to any of the preceding clauses, wherein the pendant donor fluorophores are configured to energy-transferring proximity to each other.

[0311] 15. The conjugate according to any one of the preceding items, wherein the polymer tandem dye has a Stokes shift of 100 nm or more.

[0312] 16. The conjugate according to any one of the preceding items, wherein the pendant donor fluorophore is selected from a fused tricyclic aryl, a fused tricyclic heteroaryl and a BODIPY fluorophore.

[0313] 17. The conjugate according to item 16, wherein the pendant donor fluorophore is a BODIPY fluorophore.

[0314] 18. The conjugate according to any one of the preceding items, wherein the pendant donor fluorophore is substituted with a water soluble group.

[0315] 19. The conjugate according to item 18, wherein the water soluble group comprises polyethylene glycol.

[0316] 20. The conjugate according to any of the preceding items, wherein the acceptor fluorophore is a small molecule fluorophore.

[0317] 21. The conjugate according to item 20, wherein the acceptor fluorophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluoride, naphthylimide, thiazine dyes and acridine dyes.

[0318] 22. A method of assessing the presence of a target analyte in a sample, the method comprising:

[0319] (a) combining the sample with a single domain antibody / polymer fluorescent tandem dye conjugate to produce an assay composition, the conjugate specifically binding to the target analyte, wherein the conjugate comprises:

[0320] (1) single domain antibodies; and

[0321] (2) one or more polymer tandem fluorescent dyes conjugated to the single domain antibody, wherein the one or more polymer tandem fluorescent dyes comprise:

[0322] (i) a polymer backbone comprising non-conjugated repeating units;

[0323] (ii) a plurality of pendant donor fluorophores, each pendant donor fluorophore independently attached to a non-conjugated repeat unit of the polymer backbone; and

[0324] (iii) one or more pendant acceptor fluorophores attached to a non-conjugated repeat unit of the polymer backbone, wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship; and

[0325] (b) assaying the assay composition for the presence of any conjugate-target analyte binding complex to assess the presence or absence of the target analyte in the sample.

[0326] 23. The method according to item 22, wherein the target analyte is associated with a cell.

[0327] 24. The method according to item 23, wherein the target analyte is a cell surface marker.

[0328] 25. The method according to item 24, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen.

[0329] 26. The method according to item 23, wherein the target analyte is an intracellular target.

[0330] 27. The method according to item 26, wherein the method further comprises permeabilizing the cells.

[0331] 28. The method according to any one of items 22 to 27, wherein said assaying comprises analyzing said assay composition by flow cytometry.

[0332] 29. The method according to any one of items 22 to 28, wherein said single domain antibody comprises a heavy chain domain or a light chain domain.

[0333] 30. The method according to item 29, wherein the single domain antibody comprises V H H domain.

[0334] 31. The method according to item 29, wherein the single domain antibody comprises V NAR domain.

[0335] 32. The method according to any one of items 22 to 31, wherein the conjugate comprises two or more polymer tandem fluorescent dyes conjugated to the single domain antibody.

[0336] 33. The method according to any one of items 22 to 32, wherein the polymer tandem fluorescent dye is described by the following formula:

[0337]

[0338] in:

[0339] The polymer backbone of the non-conjugated repeating unit comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0340] Each D 1 Independently for SM 1 attached pendant donor fluorophore;

[0341] Each A 1 Independently for SM 2 a linked pendant acceptor fluorophore;

[0342] x is 75 mol% or more; and

[0343] y is 25 mol% or less.

[0344] 34. The method according to item 33, wherein the repeating units of the polymer backbone have a defined linear sequence.

[0345] 35. The method according to item 34, wherein SM 1 and SM 2 It is a comonomer derived from amino acids and peptoid monomers.

[0346] 36. The method according to item 35, wherein the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues.

[0347] 37. The method according to any one of items 22 to 36, wherein the polymer backbone is a linear polymer.

[0348] 38. The method according to any one of items 22 to 37, wherein the pendant donor fluorophores are configured for energy transfer in proximity to each other.

[0349] 39. The method according to any one of items 22 to 38, wherein the polymer tandem fluorescent dye has a Stokes shift of 100 nm or more.

[0350] 40. The method according to any one of items 22 to 39, wherein the pendant donor fluorophore is selected from a fused tricyclic aryl, a fused tricyclic heteroaryl and a BODIPY fluorophore.

[0351] 41. The method according to item 40, wherein the pendant donor fluorophore is a BODIPY group.

[0352] 42. The method according to any one of items 22 to 41, wherein the pendant donor fluorophore is substituted with a water soluble group.

[0353] 43. The method according to item 42, wherein the water soluble group comprises polyethylene glycol.

[0354] 44. The method according to any one of items 22 to 43, wherein the acceptor fluorophore is a small molecule fluorophore.

[0355] 45. The method according to item 44, wherein the acceptor fluorophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluoride, naphthylimide, thiazine dyes and acridine dyes.

[0356] 46. ​​A kit comprising:

[0357] A single domain antibody / polymer fluorescent tandem dye conjugate, the conjugate comprising:

[0358] (a) single domain antibodies; and

[0359] (b) one or more polymer tandem fluorescent dyes conjugated to the single domain antibody, wherein the one or more polymer tandem fluorescent dyes comprise:

[0360] (i) a polymer backbone comprising non-conjugated repeating units;

[0361] (ii) a plurality of pendant donor fluorophores, each pendant donor fluorophore independently attached to a non-conjugated repeat unit of the polymer backbone; and

[0362] (iii) one or more pendant acceptor fluorophores attached to a non-conjugated repeat unit of the polymer backbone, wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship.

[0363] 47. The kit according to item 46, wherein the single domain antibody comprises a heavy chain or a light chain.

[0364] 48. The kit according to item 47, wherein the single domain antibody comprises V H H domain.

[0365] 49. The kit according to item 47, wherein the single domain antibody comprises V NAR domain.

[0366] 50. The kit according to any of items 46 to 49, wherein said single domain antibody specifically binds to an intracellular antigen.

[0367] 51. The kit according to any of items 46 to 50, wherein said single domain antibody specifically binds to a cell surface antigen.

[0368] 52. The kit according to any of items 46 to 51, wherein said single domain antibody specifically binds to a primary antibody.

[0369] 53. The kit according to any one of items 46 to 52, wherein the conjugate comprises two or more polymer tandem fluorescent dyes conjugated to the single domain antibody.

[0370] 54. The kit according to any one of items 46 to 53, wherein the polymer tandem fluorescent dye is described by the following formula:

[0371]

[0372] in:

[0373] The polymer backbone of the non-conjugated repeating unit comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer;

[0374] Each D 1 Independently for SM 1 attached pendant donor fluorophore;

[0375] Each A 1 Independently for SM 2 a linked pendant acceptor fluorophore;

[0376] x is 75 mol% or more; and

[0377] y is 25 mol% or less.

[0378] 55. The kit according to item 54, wherein said repeating units of said polymer backbone have a defined linear sequence.

[0379] 56. The kit according to item 55, wherein SM 1 and SM 2It is a comonomer derived from amino acids and peptoid monomers.

[0380] 57. Kit according to item 56, wherein the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues.

[0381] 58. Kit according to any of items 46 to 57, wherein said polymer backbone is a linear polymer.

[0382] 59. The kit according to any one of items 46 to 58, wherein the pendant donor fluorophores are configured for energy transfer in proximity to each other.

[0383] 60. The kit according to any one of items 46 to 59, wherein the polymer tandem fluorescent dye has a Stokes shift of 100 nm or more.

[0384] 61. The kit according to any one of items 46 to 60, wherein the pendant donor fluorophore is selected from a fused tricyclic aryl, a fused tricyclic heteroaryl and a BODIPY fluorophore.

[0385] 62. The kit according to item 61, wherein the pendant donor fluorophore is a BODIPY group.

[0386] 63. The kit according to any one of items 46 to 62, wherein the pendant donor fluorophore is substituted with a water soluble group.

[0387] 64. The kit according to item 63, wherein the water soluble group comprises polyethylene glycol.

[0388] 65. The kit according to any one of items 46 to 64, wherein the acceptor fluorophore is a small molecule fluorophore.

[0389] 66. The kit according to item 65, wherein the acceptor fluorophore is selected from the group consisting of cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluoride, naphthylimide, thiazine dyes and acridine dyes.

[0390] In at least some previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such replacement is technically infeasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0391] Those skilled in the art will appreciate that, in general, the terms used herein, and particularly in the appended claims (e.g., the bodies of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "comprising" should be interpreted as "including but not limited to", etc.). Those skilled in the art will also appreciate that if a specific number of introduced claim recitations is intended, such an intent will be explicitly recited in the claim, and in the absence of such a recitation, such an intent is not present. For example, to aid understanding, the following appended claims may contain the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be interpreted as implying that the introduction of a claim recitation by the indefinite article "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted as meaning "at least one" or "one or more"); the same applies to the use of definite articles to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, one skilled in the art will recognize that such recitation should be interpreted as indicating at least the number recited (e.g., the mere recitation "two recitations" without other modifiers means at least two recitations, or two or more recitations). In addition, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally, such construction is intended that one skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include, but is not limited to, systems including A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In addition, in those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally, such construction is intended that one skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, or C" will include, but is not limited to, systems including A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will also appreciate that virtually any transitional word and / or phrase presenting two or more alternative terms, whether in the specification, claims or drawings, should be understood to contemplate the possibility of including one, either or both terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0392] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0393] As will be understood by those skilled in the art, for any and all purposes, for example, in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily considered to be fully described and enables the same range to be decomposed into at least equal halves, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be easily decomposed into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all such as "until", "at least", "greater than", "less than" and similar languages ​​include the numbers listed and refer to the ranges that can be subsequently decomposed into sub-ranges as described above. Finally, as will be understood by those skilled in the art, the range includes each individual member. Therefore, for example, a group with 1-3 articles refers to a group with 1, 2 or 3 articles. Similarly, a group with 1-5 articles refers to a group with 1, 2, 3, 4 or 5 articles, and so on.

[0394] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art in light of the teachings of this invention that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0395] Therefore, the foregoing only illustrates the principles of the present invention.It should be understood that those skilled in the art will be able to think of various arrangements, although not explicitly described or shown herein, these arrangements embody the principles of the present invention and are included in its spirit and scope.In addition, all examples and conditional language recorded herein are mainly intended to help readers understand the principles of the present invention and the concepts contributed by the inventors to promote this area, and should be interpreted as not being limited to such specific examples and conditions of recording.In addition, all statements of the principles, aspects and embodiments of the present invention and their specific examples recorded herein are intended to cover their structural and functional equivalents.In addition, such equivalents are intended to include currently known equivalents and equivalents developed in the future, that is, no matter how the structure, any element of the developed performance of the same function.In addition, any content disclosed herein is not intended to be dedicated to the public, regardless of whether such disclosures are clearly recorded in the claims.

[0396] Therefore, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Instead, the scope and spirit of the present invention are embodied by the appended claims. In the claims, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is expressly defined as being cited for such a limitation in a claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of a certain limitation in a claim; if such exact phrase is not used in the limitation in a claim, 35 U.S.C. § 112 (f) or 35 U.S.C. § 112 (6) is not cited.

Claims

1. A single domain antibody / polymer fluorescent tandem dye conjugate, the conjugate comprising: (a) single domain antibodies; and (b) one or more polymer tandem fluorescent dyes conjugated to the single domain antibody, wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeating units; (ii) a plurality of pendant donor fluorophores, each pendant donor fluorophore independently attached to a non-conjugated repeat unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores attached to a non-conjugated repeat unit of the polymer backbone, wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship.

2. The conjugate according to any one of the preceding claims, wherein the single domain antibody specifically binds to an intracellular antigen or a cell surface antigen.

3. The conjugate according to any one of the preceding claims, wherein the single domain antibody specifically binds to a primary antibody.

4. The conjugate according to any one of the preceding claims, wherein the conjugate comprises two or more polymer tandem fluorescent dyes conjugated to the single domain antibody.

5. The conjugate according to any one of the preceding claims, wherein the polymer tandem fluorescent dye is described by the following formula: in: The polymer backbone of the non-conjugated repeating units comprises SM 1 and SM 2 comonomers, each of which is independently a non-conjugated comonomer; Each D 1 Independently for SM 1 attached pendant donor fluorophore; Each A 1 Independently for SM 2 a linked pendant acceptor fluorophore; x is 75 mol% or more; and y is 25 mol% or less.

6. The conjugate of any one of the preceding claims, wherein the pendant donor fluorophores are configured for energy transfer in proximity to each other.

7. The conjugate according to any one of the preceding claims, wherein the pendant donor fluorophore is substituted with a water soluble group.

8. The conjugate according to any one of the preceding claims, wherein the acceptor fluorophore is a small molecule fluorophore.

9. A method for assessing the presence of a target analyte in a sample, the method comprising: (a) combining the sample with a single domain antibody / polymer fluorescent tandem dye conjugate to produce an assay composition, the conjugate specifically binding to the target analyte, wherein the conjugate comprises: (1) single domain antibodies; and (2) one or more polymer tandem fluorescent dyes conjugated to the single domain antibody, wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeating units; (ii) a plurality of pendant donor fluorophores, each pendant donor fluorophore independently attached to a non-conjugated repeating unit of the polymer backbone; as well as (iii) one or more pendant acceptor fluorophores attached to the non-conjugated repeat units of the polymer backbone, wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship; as well as (b) assaying the assay composition for the presence of any conjugate-target analyte binding complex to assess the presence or absence of the target analyte in the sample.

10. The method of claim 9, wherein the target analyte is associated with a cell. The method of claim 10 , wherein the target analyte is a cell surface marker or an intracellular target.

12. The method of any one of claims 9 to 11, wherein the assay comprises analyzing the assay composition by flow cytometry.

13. A kit comprising: A single domain antibody / polymer fluorescent tandem dye conjugate, the conjugate comprising: (a) single domain antibodies; and (b) one or more polymer tandem fluorescent dyes conjugated to the single domain antibody, wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeating units; (ii) a plurality of pendant donor fluorophores, each pendant donor fluorophore independently attached to a non-conjugated repeat unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores attached to the non-conjugated repeat units of the polymer backbone, The pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship.

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