Single domain antibody / polymer tandem fluorescent dye conjugates and methods of making and using same - Patents.com

Abstract

Single domain antibody / polymeric fluorescent tandem dye conjugates are provided. Aspects of the conjugates provided include a single domain antibody having one or more polymeric tandem fluorescent dyes conjugated thereto. In some examples, the polymeric tandem fluorescent dye comprises (i) a polymer backbone composed of non-conjugated repeat units, (ii) a plurality of pendant donor chromophore groups each independently linked to a non-conjugated repeat unit of the polymer backbone, and (iii) one or more pendant acceptor fluorophores linked to a non-conjugated repeat unit of the polymer backbone, the pendant donor fluorophore and the pendant acceptor fluorophore being in an energy transfer relationship. Also provided are methods of using the conjugates, for example in methods of evaluating a sample for the presence of a target analyte, and kits comprising the conjugates that find use in embodiments of the methods.

Description

[Technical field]

[0001] Fluorescent dyes are compounds that, when irradiated with light of a wavelength that they absorb, emit light of (usually) a different wavelength. Fluorescent dyes find use in various applications in biochemistry, biology and medicine, for example in diagnostic kits, in microscopy or in drug screening. Fluorescent dyes are characterized by a number of parameters, which allow the user to select the appropriate dye depending on the desired purpose. Parameters of interest include excitation wavelength maximum, emission wavelength maximum, Stokes shift, extinction coefficient, fluorescence quantum yield and fluorescence lifetime. Dyes can be selected according to the application of interest, for example to allow penetration of excitation radiation into biological samples, to minimize background fluorescence and / or to achieve a high signal-to-noise ratio. [Background technology]

[0002] Molecular recognition involves the specific binding of two molecules. Molecules with binding specificity for target biomolecules find use in a variety of research and diagnostic applications, such as analyte labeling and separation, 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 [Means for solving the problem]

[0003] Single domain antibody / polymeric fluorescent tandem dye conjugates are provided. Aspects of the conjugates provided include single domain antibodies having one or more polymeric tandem fluorescent dyes conjugated thereto. In some examples, the polymeric tandem fluorescent dyes include (i) a polymer backbone composed of non-conjugated repeat units, (ii) a plurality of pendant donor chromophore groups each independently linked to the non-conjugated repeat units of the polymer backbone, and (iii) one or more pendant acceptor fluorophores linked to the non-conjugated repeat units of the polymer backbone, wherein the pendant donor fluorophores and the pendant acceptor fluorophores are in an energy transfer relationship. Also provided are methods of using the conjugates, for example in methods of evaluating a sample for the presence of a target analyte, and kits comprising the conjugates that find use in embodiments of the methods. [Brief description of the drawings]

[0004] The invention may be best understood from the following detailed description when read in conjunction with the accompanying drawing figures, in which:

[0005] [Figure 1A] FIG. 1 provides a diagram of a single domain antibody / polymer fluorescent tandem dye conjugate according to one embodiment of the present invention. [Figure 1B] FIG. 1 provides a diagram of a single domain antibody / polymer fluorescent tandem dye conjugate according to one embodiment of the present invention. [Figure 2A] As further described in the Experimental Section below, a schematic diagram of an assay employing a single domain antibody / polymer fluorescent tandem dye conjugate is provided. [Figure 2B] As further described in the Experimental Section below, a schematic diagram of an assay employing a single domain antibody / polymer fluorescent tandem dye conjugate is provided. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] definition As used herein, the terms "chemoselective functional group" and "chemoselective tag" are used interchangeably and refer to a functional group that can selectively react with another compatible functional group to form a covalent bond, in some cases after optionally activating one of the functional groups.Chemoselective functional groups of interest include, but are not limited to, thiol and maleimide or iodoacetamide, amine and carboxylic acid or its active ester, and groups that can react with each other via click chemistry, such as azide and alkyne groups (e.g., cyclooctyne group), tetrazine, transcyclooctene, diene and dienophile, and azide, sulfur (VI) fluoride exchange chemistry (SuFEX), sulfonyl fluoride, and hydroxyl, hydrazide, hydrazino, aldehyde, ketone, azide, alkyne, phosphine, epoxide, etc.

[0007] As used herein, the term "sample" refers to a material or mixture of materials, in some cases in liquid form, that contains one or more analytes of interest. In some embodiments, the term used in its broadest sense refers to any plant, animal or bacterial material that contains cells or produces cellular metabolic products, such as tissues or fluids isolated from an individual (including but not limited to plasma, serum, cerebrospinal fluid, lymphatic fluid, tears, saliva and tissue sections) or from in vitro cell culture components, as well as samples from the environment. The term "sample" can also refer to "biological sample". As used herein, the term "biological sample" refers to a whole organism, or a subset of its tissues, cells or component parts (e.g., bodily fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "Biological sample" can also refer to homogenates, lysates or extracts, or fractions or portions thereof, prepared from a whole organism or a subset of its tissues, cells or components, including, but not limited to, plasma, serum, spinal fluid, lymphatic fluid, outer parts of the skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk, blood cells, tumors and organs. In certain embodiments, the sample has been removed 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 an organism, both eukaryotic and prokaryotic organisms, having at least a nucleus and a cell membrane. In certain embodiments, the cell includes a prokaryotic cell, such as from a bacterium. In other embodiments, the cell includes a eukaryotic cell, such as a cell obtained from a biological sample from an animal, a plant or a fungus.

[0008] The terms "bound to a carrier" and "linked to a carrier" are used interchangeably and refer to a moiety (e.g., a specific binding member) that is covalently or non-covalently bound to a carrier of interest. Covalent binding can include a chemical reaction of two compatible functional groups (e.g., two chemically selective functional groups, such as an electrophile and a nucleophile) to form a covalent bond between two moieties of interest (e.g., a carrier and a specific binding member). In some cases, non-covalent binding can include a specific bond between two moieties of interest (e.g., two affinity moieties such as a hapten and an antibody, or a biotin moiety and streptavidin). In certain cases, non-covalent binding can include absorption to a substrate.

[0009] The term "polypeptide" refers to a polymeric form of amino acids of any length, including peptides ranging from 2 to 50 amino acids in length, and polypeptides greater than 50 amino acids in length. The terms "polypeptide" and "protein" are used interchangeably herein. The term "polypeptide" includes coded and non-coded amino acids, polymers of amino acids that have been modified or derivatized chemically or biochemically, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with a non-naturally occurring or synthetic backbone. A polypeptide can be of any convenient length, such as, for example, 2 or more amino acids, such as 4 or more amino acids, such as 10 or more amino acids, such as 20 or more amino acids, such as 50 or more amino acids, such as 100 or more amino acids, such as 300 or more amino acids, such as up to 500 or 1000 or more amino acids. A "peptide" can be 2 or more amino acids, such as 4 or more amino acids, such as 10 or more amino acids, such as 20 or more amino acids, such as up to 50 amino acids. In some embodiments, a peptide is 5 to 30 amino acids in length.

[0010] As used herein, the term "isolated" refers to a moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which it is associated prior to purification.

[0011] A "plurality" includes at least two members. In certain 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.

[0012] Numeric ranges are inclusive of the numbers defining the range.

[0013] The term "specific binding" refers to a direct association between two molecules due to covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including, for example, interactions such as salt bridges and water bridges. A specific binding member describes a member of a pair of molecules that has binding specificity for one another. Members of a specific binding pair can be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface or a cavity that specifically binds to it and is thus complementary to a particular spatial and polar organization of the other member of the pair of molecules. Thus, the members of the pair have the property of specifically binding to one another. Examples of specific binding member pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The specific binding members of a binding pair exhibit high affinity and binding specificity for binding to one another. Typically, the affinity between the specific binding members of a pair is greater than 10 -6 M or less, e.g. 10 -7 M or less, and 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -13 M or less, 10 -14 M or less, and 10 -15 K below M dThe affinity is characterized by a dissociation constant (KD). "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule to another molecule is determined, for example, by measuring the binding kinetics of the interaction at 25°C. "Affinity" refers to the strength of binding, and an increase in binding affinity correlates with a lower KD. In one embodiment, affinity is determined by surface plasmon resonance (SPR), for example, as used by the Biacore system. The affinity of one molecule to another molecule is determined, for example, by measuring the binding kinetics of the interaction at 25°C.

[0014] The method described herein may include multiple steps. Each step may be performed after a predetermined time has elapsed between steps, if necessary. Thus, the time between each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and 5 hours or more. In certain embodiments, each subsequent step is performed immediately after the completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after the completion of the previous step, such as a waiting time of several minutes to overnight.

[0015] As used herein, the terms "evaluating," "determining," "measuring," and "assessing," and "assaying" are used interchangeably and include both quantitative and qualitative determinations.

[0016] The term "separating" as used herein refers to the physical separation of two elements (e.g., by size or affinity, etc.), as well as the degradation of one element while leaving the other intact.

[0017] The term "linker" or "link" refers to a linking moiety that connects two groups and has a backbone that is 100 atoms or less in length. A linker or linkage can be a covalent bond that connects two groups or chains of 1 to 100 atoms in length, for example, a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, and the linker can be linear, branched, cyclic, or a single atom. In some cases, a linker is a branched linker, which refers to a linking moiety that connects three or more groups. In certain cases, one, two, three, four, or five or more carbon atoms of the linker backbone can be optionally substituted with sulfur, nitrogen, or oxygen heteroatoms. In some cases, the linker backbone includes a linking functional group such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester, or imine. The bonds between backbone atoms can be saturated or unsaturated, and in some cases, there are one, two, or no more than three unsaturated bonds in the linker backbone. The linker can include one or more substituents, such as alkyl, aryl, or alkenyl groups. The linker can include, but is not limited to, polyethylene glycol; ether, thioether, tertiary amine, which can be linear or branched, alkyl, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), and the like. The linker backbone can include a cyclic group, such as an aryl, heterocyclic, or cycloalkyl group, and two or more atoms of the cyclic group, such as two, three, or four atoms, are included in the backbone. The linker can be cleavable or non-cleavable.

[0018] As used herein, the terms "water-solubilizing 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, to impart improved water solubility to the molecule to which it is attached. The WSG can increase the solubility of the multichromophore, primarily in aqueous solution, compared to a control multichromophore lacking the WSG. The water-solubilizing group can be any convenient hydrophilic group that is well solvated in an aqueous environment.

[0019] The terms "polyethylene oxide", "PEO", "polyethylene glycol" and "PEG" are used interchangeably and represent a compound of the formula --(CH2----O--) n - or derivatives thereof. In some embodiments, "n" is 5000 or less, e.g., 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, e.g., 3 to 15, or 10 to 15. It is understood that the PEG polymer group can be of any convenient length and can include a variety of end groups and / or further substituents, including, but not limited to, alkyl, aryl, hydroxyl, amino, acyl, acyloxy, amide end and / or substituents. PEG groups that may be adapted for use in the subject multichromophores include those PEGs described by S. Zalipsky “Functionalized poly(ethylene glycol) for preparation of biologically relevant conjugates”, Bioconjugate Chemistry 1995, 6(2), 150-165; and Zhu et al “Water-Soluble Conjugated Polymers for Imaging, Diagnosis, and Therapy”, Chem. Rev., 2012, 112(8), pp 4687-4735.

[0020] 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 propan-1-yl or propan-2-yl, and butyl, such as butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, or 2-methyl-propan-2-yl. In some embodiments, an alkyl group contains 1 to 20 carbon atoms. In some embodiments, an alkyl group contains 1 to 10 carbon atoms. In certain embodiments, a lower alkyl group contains 1 to 6 carbon atoms, such as 1 to 4 carbon atoms. This term includes, by way of example, straight-chain and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl (CH3)3CCH2-).

[0021] The term "substituted alkyl" refers to an alkyl group, as defined herein, in which one or more carbon atoms in the alkyl chain is replaced with an -O-, -N-, -S-, -S(O) n-(n is 0-2), -NR- (R is hydrogen or alkyl), and optionally substituted with heteroatoms such as alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NR a R b wherein 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.

[0022] "Alkoxy" refers to the group -O-alkyl, where alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. The term "alkoxy" also refers to alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0023] The term "substituted alkoxy" refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-, where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl, and substituted alkynyl are as defined herein.

[0024] "Alkenyl" refers to a monoradical, branched or straight chain, cyclic or acyclic hydrocarbonyl group with a carbon-carbon double bond. Examples of alkenyl groups include ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl, hexadecenyl, eicosenyl, and tetracosenyl. In some cases, the alkenyl group comprises 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 having 1 to 6 carbon atoms.

[0025] "Alkynyl" or "alkyne" refers to a straight or branched chain monovalent hydrocarbyl group having from 2 to 6 carbon atoms, preferably from 2 to 3 carbon atoms, and having at least 1, and preferably 1 to 2, sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CHC≡CH).

[0026] The term "substituted alkynyl" or "substituted alkyne" refers to an alkynyl group, as defined herein, having from one to five substituents, or from one to three substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0027] "Heterocyclyl" refers to a monoradical, cyclic group that contains a heteroatom (e.g., O, S, N) as a ring atom and is not aromatic (i.e., distinguishing heterocyclyl groups from heteroaryl groups). Examples of heterocyclyl groups include piperidinyl, tetrahydrofuranyl, dihydrofuranyl, and thiocanyl.

[0028] "Amino" refers to the group -NH2. The term "substituted amino" refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl, and where at least one R is not hydrogen.

[0029] "Aryl" by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of an aromatic ring system. Aryl groups of interest include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like. In certain embodiments, the aryl group contains 6 to 20 carbon atoms. In certain embodiments, the aryl group contains 6 to 12 carbon atoms. Examples of aryl groups are phenyl and naphthyl.

[0030] Unless otherwise constrained by the definition of the aryl substituent, "substituted aryl" refers to an aryl group substituted with one to five substituents, or one to three 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, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl.

[0031] "Heteroaryl" refers to a monovalent heteroaromatic radical derived by removing one hydrogen atom from a single atom of a heteroaromatic ring system, either by itself or as part of another substituent.Heteroaryl groups of interest include, but are not limited to, acridine, arsindol, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, triazole, benzotriazole, thiophene, triazole, xanthene, benzodioxole, etc. In certain embodiments, the heteroaryl group is a 5-20 membered heteroaryl. In certain embodiments, the heteroaryl group is 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.

[0032] "Heterocycle", "heterocyclic", "heterocycloalkyl" and "heterocyclyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, including fused bridged and spiro ring systems, having 3-20 ring atoms and containing 1-10 heteroatoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, and in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, and the point of attachment is through a non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocycle group are optionally oxidized to provide an N-oxide, -S(O)-, or -SO2- moiety.

[0033] Examples of heterocycles and heteroaryls include azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, These include, but are not limited to, phenoxazine, phenothiazine, 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 called thiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0034] "Substituted heteroaryl" refers to a heteroaryl group substituted with one to five substituents or one to three 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, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and trihalomethyl, unless otherwise constrained by the definition of the substituents.

[0035] The terms "alkaryl" or "aralkyl" refer to the groups -alkylene-aryl and substituted alkylene-aryl, where alkylene, substituted alkylene, and aryl are defined herein.

[0036] "Alkylene" refers to either a straight or branched chain, -O-, -NR 10 -, -NR 10 C(O)-, -C(O)NR 10 -, and the like. This term includes, by way of example and without limitation, methylene (-CH-), ethylene (-CHCH-), n-propylene (-CHCHCH-), iso-propylene (-CHCH(CH)-), (-C(CH)CHCH-), (-C(CH)CHC(O)-), (-C(CH)CHC(O)NH-), (-CH(CH)CH-), and the like. "Substituted alkylene" refers to an alkylene group having 1 to 3 hydrogens replaced with substituents as described for carbon in the definition of "substituted" below.

[0037] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced with the same or different substituent(s). Substituents of interest include alkylenedioxy (such as methylenedioxy), -M, -R 60 , -O - , =O, -OR 60 , -SR 60 , -S - , =S, -NR 60 R 61 , =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - , -S(O)2OH, -S(O)2R 60 , -OS(O)2O - , -OS(O)2R 60 , -P(O)(O - )2, -P(O)(OR 60 )(O - ), -OP(O)(OR60 )(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 M is a 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 attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring; R 64 and R 65 are 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 attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring. In certain embodiments, the substituents are -M, -R 60 , =O, -OR 60 , -SR 60 , -S - , =S, -NR60 R 61 , =NR 60 , -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2R 60 , -OS(O)2O - , -OS(O)2R 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 , -CF3, -CN, -NO2, -S(O)2R 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 , -CF3, -CN, -NO2, -S(O)2R 60 , -OP(O)(OR 60 )(OR 61 ), -C(O)R 60 , -C(O)OR 60 , -C(O)O - Including R 60 , R 61and R 62 is as defined above. For example, the substituent may bear a methylenedioxy substituent, or one, two or three substituents selected from a halogen atom, a (1-4C) alkyl group and a (1-4C) alkoxy group. When the substituted group is an aryl or heteroaryl group, the substituent(s), such as those described herein, may be referred to as "aryl substituent(s)".

[0038] In all of the above defined substituents, it is understood that polymers arrived at by defining the substituents themselves to have further substituents (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted with a substituted aryl group, etc.) are not intended to be included herein. In such cases, the maximum number of such substitutions is 3. For example, tandem substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0039] "Acyl" refers to a group of formula -C(O)R, where R is alkyl, alkenyl, or alkynyl. For example, an acetyl group has the formula -C(O)CH3.

[0040] "Halo" and "halogen" refer to chloro, bromo, fluoro, and iodo groups.

[0041] "Carboxyl", "carboxy", and "carboxylate" refer to the -CO2H group and salts thereof.

[0042] "Sulfonyl" refers to the group -SO2R, where R is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and substituted versions thereof. Exemplary sulfonyl groups include -SO2CH3 and -SO2(C6H5).

[0043] Unless otherwise specified, a reference to an atom is meant to include all isotopes of that atom. For example, a reference to H is1 H, 2 H (i.e. D) and 3 H (i.e. T) is meant to include C. 12 C and all isotopes of carbon ( 13 C, etc. Additionally, any group described includes all stereoisomers of that group.

[0044] Unless otherwise indicated, the nomenclature of substituents not explicitly defined herein is accomplished by naming the terminal portion of the functional group followed by the adjacent functional group toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-OC(O)-.

[0045] With respect to any of the groups disclosed herein that contain one or more substituents, it is of course understood that such groups do not contain any substitutions or substitution patterns that are sterically impractical and / or synthetically impractical. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0046] Detailed Description Single domain antibody / polymeric fluorescent tandem dye conjugates are provided. Aspects of the conjugates provided include single domain antibodies having one or more polymeric tandem fluorescent dyes conjugated thereto. In some examples, the polymeric tandem fluorescent dyes include (i) a polymer backbone composed of non-conjugated repeat units, (ii) a plurality of pendant donor chromophore groups each independently linked to the non-conjugated repeat units of the polymer backbone, and (iii) one or more pendant acceptor fluorophores linked to the non-conjugated repeat units of the polymer backbone, wherein the pendant donor fluorophores and the pendant acceptor fluorophores are in an energy transfer relationship. Also provided are methods of using the conjugates, for example in methods of evaluating a sample for the presence of a target analyte, and kits comprising the conjugates that find use in embodiments of the methods.

[0047] Before describing the exemplary embodiments in more detail, the following definitions are set forth to illustrate and define the meaning and scope of terms used in the description.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, for clarity and ease of reference, certain terms are defined below. In addition, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, but representative exemplary methods and materials are described herein.

[0049] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any otherwise stated or intervening value in that stated range, is included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0050] A range is presented herein, and numerical values ​​are preceded by the term "about". The term "about" is used herein to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number it precedes. When determining whether a number is close to or approximately equal to a specifically stated number, the unstated number that is close or approximate may be a number that, in the context in which it is presented, results in a substantial equivalence to the specifically stated number.

[0051] 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 describe and explain the methods and / or materials in connection with which the publications are cited. The 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.

[0052] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prior basis for using exclusive terminology such as "solely," "only," or "negative" limitations in connection with the recitation of claim elements.

[0053] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0054] Although the apparatus and methods have been described or will be described for grammatical fluidity with functional descriptions, unless expressly formulated under 35 U.S.C. § 112, the claims should not be construed as necessarily limited in any way by means- or step-limitation constructions, but should be given the full scope of meaning and equivalents of the definitions provided by the claims under the doctrine of equivalents, and where a claim is expressly formulated under 35 U.S.C. § 112, it should be expressly understood that the full statutory equivalents under 35 U.S.C. § 112 are accorded.

[0055] To further describe various embodiments of the present invention, the single domain antibody / polymer fluorescent tandem dye conjugates will first be discussed in more detail, followed by a discussion of uses and methods of making the conjugates, and a discussion of kits containing the conjugates.

[0056] Single domain antibody / polymer fluorescent tandem dye conjugates As summarized above, the present disclosure provides single domain antibody / polymer fluorescent tandem dye conjugates. The dye conjugates of the present invention comprise a single domain antibody and one or more polymer tandem fluorescent dyes conjugated thereto. In other words, the dye conjugates of the present invention comprise at least one polymer tandem fluorescent dye conjugated to a single domain antibody, and 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 can vary, in some examples ranging from 2 to 5, such as 2 to 4, including 2 to 3 polymer tandem fluorescent dyes. In some examples, the single domain antibody / polymer fluorescent tandem dye conjugates of the present invention have a low molecular weight. In some embodiments, the molecular weight of the single domain antibody / polymeric fluorescent tandem dye conjugate ranges from 20-200 kDa, such as 25-150 kDa, such as 30-75 kDa, such as 30-70 kDa, such as 30-65 kDa. Figures 1A and 1B provide an illustration of a single domain antibody / polymeric fluorescent tandem dye conjugate according to one embodiment of the invention. In the embodiment shown in Figure 1A, the single domain antibody / polymeric fluorescent tandem dye conjugate comprises a VhH nanobody conjugated to two polymeric fluorescent tandem dyes (each labeled with a "pendant chromophore"). Figure 1B shows the same dyes as shown in Figure 1A, with the structure of each of the polymeric fluorescent tandem dyes provided. As shown, each polymeric fluorescent tandem dye comprises a peptide scaffold having multiple pendant donor fluorophores and pendant acceptor fluorophores, where the fluorophores of the tandem dyes are in an energy transfer relationship. The different components of the single domain antibody / polymer fluorescent tandem dye conjugates of the present embodiments will now be considered in more detail.

[0057] Single Domain Antibodies As used herein, a single domain antibody, which may be referred to herein as a nanobody and designated as an sdAb, is an antibody fragment that contains a single monomeric variable antibody domain that can specifically bind to an antigen. In some examples, the "single domain antibody" or "sdAb" present in the conjugate of the present invention comprises a single antigen-binding polypeptide with three complementary determining regions (CDRs). The sdAb alone can specifically bind to a target antigen without pairing with a corresponding CDR-containing polypeptide.

[0058] Single domain antibodies, i.e. nanobodies, may comprise a heavy chain variable domain or a light chain variable domain. In some examples, the single domain antibodies of the embodiments of the present application comprise a heavy chain variable domain. Single domain antibodies are also known to those of the Camelidae (V H H fragment) or cartilaginous fish (V NAR Alternatively, single domain antibodies can be derived from the splitting of dimeric variable domains from IgG into monomers.

[0059] Single domain antibodies comprise variable regions, which are primarily responsible for antigen recognition and binding, and framework regions. The "variable regions", also called "complementarity determining regions" (CDRs), comprise loops that vary widely in size and sequence based on antigen recognition. CDRs are generally responsible for the binding specificity of the nanobody. Separate from the CDRs are the framework regions. The framework regions are relatively conserved and aid in the overall protein structure. The framework regions may comprise large solvent-exposed surfaces consisting of beta-sheets and loop structures. They may comprise a signal sequence, as known in the art, which is then cleaved from the mature nanobody.

[0060] A single domain antibody that "binds" to an antigen of interest is a single domain antibody that binds to the antigen with sufficient affinity and does not significantly cross-react with other proteins such that the single domain antibody or binding molecule is useful for targeting the desired antigen. In such embodiments, the extent of binding of single domain antibodies, i.e. nanobodies, or other binding molecules to non-target antigens is typically 10% or less, as determined, for example, by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0061] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence and are used in the binding and specificity of each particular variable domain for its particular antigen. However, variability is not uniformly distributed throughout the variable domains. It is concentrated in the hypervariable regions. The more highly conserved portions of the variable domains are called framework regions (FR). As used herein, the term "hypervariable region" refers to amino acid residues involved in antigen binding. A hypervariable region may comprise amino acid residues from "complementarity determining regions" or "CDRs" and / or those residues from "hypervariable loops". "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein.

[0062] Immunoglobulin sequences, such as antibodies and antigen-binding fragments derived therefrom (e.g., immunoglobulin single variable domains or ISVs), are used to specifically target each of the antigens disclosed herein. HHThe generation of immunoglobulin single variable domains such as s or ISVs can include selection from phage display or yeast display, for example ISVs can be selected by utilizing a surface display platform where the cell or phage surface displays a synthetic library of ISVs in the presence of a tagged antigen. A fluorescent secondary antibody directed against the tagged antigen is added to the solution, thereby labeling the cells that bind to the antigen. The cells are then sorted using any cell sorting platform of interest, for example magnetic activated cell sorting (MACS) or fluorescence activated cell sorting (FACS). The sorted clones are amplified, resulting in an enriched library of clones expressing ISVs that bind to the antigen. The enriched library is then screened again with the antigen to further enrich for the antigen-binding ISVs displayed on the surface. These clones are then sequenced to identify the sequences of the ISVs of interest, which can then be transferred to other heterologous systems for large-scale protein production.

[0063] Unless otherwise indicated, the term "immunoglobulin single variable domain" or "ISV" refers to an immunoglobulin single variable domain (IVV), HH Domain or V H Or V L It is used as a general term to include, but not be limited to, antigen-binding domains or fragments such as domains. HH Domains are of interest in embodiments of the present invention. The terms antigen-binding molecule or antigen-binding protein are used interchangeably and include the term NANOBODIES®. Immunoglobulin single variable domains are comprised of a light chain variable domain sequence (e.g., V L sequence) or a heavy chain variable domain sequence (e.g., V Hsequences), more particularly they can be heavy chain variable domain sequences derived from traditional four-chain antibodies or heavy chain variable domain sequences derived from heavy chain antibodies. Thus, immunoglobulin single variable domains can be single domain antibodies, or immunoglobulin sequences suitable for use as single domain antibodies, "dAbs", or immunoglobulin sequences suitable for use as dAbs, or NANOBODIES™ (V HH The sequence may be, but is not limited to,

[0064] Immunoglobulin single variable domains include immunoglobulin sequences of different origins, including but not limited to immunoglobulin sequences from mouse, rat, rabbit, donkey, human, camelid, cartilaginous fish such as shark, lamprey, etc. Immunoglobulin single variable domains include fully human, humanized, otherwise sequence-optimized or chimeric immunoglobulin sequences. Immunoglobulin single variable domains, and the structure of immunoglobulin single variable domains, can be considered to consist of four framework regions or "FRs", 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", respectively, which are interrupted by three complementarity determining regions or "CDRs", referred to in the art as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. It should be noted that the term nanobody or nanobodies is a registered trademark of Ablynx NV and therefore may be referred to as NANOBODY® or NANOBODIES®, respectively.

[0065] An amino acid sequence, e.g., an immunoglobulin single variable domain or polypeptide according to the invention is said to be a "VHH1 type immunoglobulin single variable domain" or a "VHH type 1 sequence" if the VHH1 type immunoglobulin single variable domain or VHH type 1 sequence has 85% identity to the VHH1 consensus sequence and has a forced cysteine ​​at position 50, i.e., C50 (using Kabat numbering) (using the blast algorithm using the VHH1 consensus sequence as the query sequence and standard settings, i.e., the blosom62 scoring matrix). HH See domains from Camelids in the article of Riechmann and Muyldermans, J. Immunol. Methods 2000 Jun 23;240(1-2):185-195.

[0066] Such immunoglobulin single variable domains may be derived in any suitable manner and from any suitable source, for example naturally occurring V HH sequences (i.e. from a suitable species of Camelidae, e.g. llama), or synthetic or semi-synthetic VHs or VLs (e.g. from humans). Such immunoglobulin single variable domains may include "humanized" or otherwise "sequence-optimized" VHHs, "camelized" immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences, i.e. camelized VHs), as well as human VHs, human VLs, camelized VHHs, which have been modified by affinity maturation (e.g. starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, fragment combinations derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to those skilled in the art, or any suitable combination of any of the foregoing as further described herein.

[0067] Immunoglobulin single variable domains are naturally occurring V HHThe amino acid sequence of the naturally occurring V domain is HH One or more amino acid residues in the amino acid sequence of the sequence (particularly in the framework sequence) may be replaced with a V from a conventional four-chain antibody from a human (e.g., as shown above). H The humanized immunoglobulin single variable domain of the present invention may comprise a humanized amino acid sequence by substituting one or more of the amino acid residues occurring at the corresponding position(s) in the domain. This can be carried out in a manner known per se, which will be clear to the skilled person, for example on the basis of the further explanations herein and the prior art on humanization referred to herein. Again, such a humanized immunoglobulin single variable domain of the present invention can be obtained in any suitable manner known per se, and thus can be obtained by using as starting material a naturally occurring V HH It should be noted that the use of a polypeptide comprising a domain is not strictly limited to the resulting polypeptide.

[0068] Another class of immunoglobulin single variable domains of the invention is the naturally occurring V H The amino acid sequence of the V domain of a heavy chain antibody is "camelized" HH Naturally occurring V from a conventional four-chain antibody by one or more of the amino acid residues occurring at the corresponding position(s) in the domain H The immunoglobulin single variable domains have an amino acid sequence that has been "camelized" by replacing one or more amino acid residues in the amino acid sequence of the domain. This can be done in a manner known per se and will be clear to the skilled person, for example on the basis of the description herein. Such "camelization" replacements can be H -V L Forming an interface, and / or V H -V LThe V domains may be inserted at amino acid positions present at the interface and / or at so-called camelid hallmark residues (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). H The sequence is preferably a V H Sequence, more preferably V H 3 sequences of human V H However, such camelized immunoglobulin single variable domains of the invention can be obtained in any suitable manner known per se and thus can be obtained from naturally occurring V H It should be noted that the invention is not strictly limited to polypeptides obtained using a polypeptide comprising the domain as a starting material.

[0069] The single domain antibodies employed in the embodiments of the present invention can be engineered to provide ease of manufacture. To facilitate the assembly of dye conjugates, single domain antibodies can be engineered to have specific reactive sites for the covalent attachment of one or more polymeric tandem fluorescent dyes. By utilizing engineered reactive sites on the periphery of the single domain antibody structure (rather than sites in the interior of the single domain antibody protein), the unique binding affinity of the single domain antibody is retained after dye conjugation. These reactive sites can be selected and placed from a list of commonly used reactive groups for bioconjugation, including but not limited to thiol, transcyclooctene, and dibenzylcyclooctyne. The thiol group allows for site-specific maleimide addition of the polymeric fluorescent tandem dye to the single domain antibody. The transcyclooctene or dibenzylcyclooctyne allows for site-specific polymeric fluorescent tandem dye addition via strain-promoted cycloaddition. The single domain antibodies present in the conjugates of the invention may be of various sizes and in some instances have a molecular weight in the range of 10-25 kDa, such as 15-20 kDa, for example 15-17 kDa.

[0070] The single domain antibodies of the conjugates of the invention may specifically bind to a variety of different targets. In some instances, the single domain antibodies specifically bind to cell surface proteins or cell markers, i.e., cell receptors and cell surface antigens. In some cases, single domain antibodies may specifically bind to cell surface antigens, including but 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 alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71. In other embodiments, single domain antibodies may bind to intracellular targets. In such embodiments, the intracellular targets of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules, fusion proteins, modified proteins, such as proteins that are phosphorylated, glycosylated, ubiquitinated, sumoylated, or acetylated. Intracellular proteins of interest to which single domain antibodies may specifically bind include, but are not limited to, hormones, growth factors, transcription factors, receptors, enzymes, cytokines, osteoinductive factors, colony stimulating factors, and the like. In some embodiments, the single domain antibodies specifically bind to the primary antibody employed in a given assay, such as an immunoglobulin, e.g., a primary mouse or rat (rate) antibody, e.g., a single antibody domain / polymer tandem fluorescent dye is employed as the secondary label.

[0071] Polymeric fluorescent tandem dyes As summarized above, the single domain antibody / polymeric fluorescent tandem dye conjugates of the present embodiments comprise a single domain antibody conjugated (e.g., covalently linked) to one or more polymeric fluorescent tandem dyes, e.g., as described above. As discussed above, in some examples the conjugate comprises a single domain antibody conjugated to multiple polymeric tandem fluorescent dyes, e.g., two or more, e.g., three or more, and four or more, e.g., five or more polymeric tandem fluorescent dyes. In some examples the conjugate comprises 2-4 polymeric tandem fluorescent dyes, e.g., 2-3 polymeric tandem fluorescent dyes, e.g., 2 polymeric tandem fluorescent dyes.

[0072] The polymeric fluorescent tandem dyes found in the conjugates of the present invention may vary. In some examples, the polymeric fluorescent tandem dyes found in the conjugates of the present invention include a polymer backbone, a plurality of pendant donor fluorophores, each independently linked to a repeat unit of the polymer backbone, one or more pendant acceptor fluorophores linked to a repeat unit of the polymer backbone, the pendant donor fluorophores and the pendant acceptor fluorophores being in an energy transfer relationship. Thus, the polymeric tandem dyes of the present invention include one or more pendant donor fluorophores and one or more pendant acceptor fluorophores configured in energy transfer proximity to the one or more pendant donor fluorophores, e.g., both are linked to a common polymer backbone. In some embodiments, there are multiple pendant donor fluorophores configured in energy transfer proximity to the pendant acceptor fluorophore(s), in some examples the multiple pendant donor fluorophores range from 2 to 20, e.g., 2 to 15, e.g., 2 to 10. The term "pendant group" refers to a side group that is connected to the backbone but is not part of the backbone itself. In embodiments of the polymer tandem dye, the donor fluorophore can transfer energy to the linked acceptor fluorophore. Thus, the subject tandem dyes include a linked acceptor signaling fluorophore in energy-receiving proximity to the donor fluorophore system, i.e., in energy-receiving proximity to at least one linked donor fluorophore. The particular configuration of the pendant group can be determined and controlled by the arrangement of the repeating units of the underlying polymer backbone (also referred to herein as the "modular scaffold" to which the pendant groups are attached). The dye conjugate can include multiple water-solubilizing groups attached to the scaffold and / or the pendant groups at any convenient location to provide a water-soluble polymer dye. The polymer backbone, i.e., the modular scaffold, can be composed of repeating units that form a polymer backbone with side groups to which the pendant groups can be attached.The repeat units can be arranged in a variety of configurations to provide tandem dyes with desired spectral properties. The distance and location between the sites for covalent attachment of the pendant donor fluorophore and acceptor fluorophore(s), if present, can be controlled to provide the desired energy transfer process.

[0073] In some examples, the polymer backbone is composed of non-conjugated repeat units having any convenient configuration, such as linear, branched, or dendrimer configuration. The polymer backbone can be a linear polymer. The polymer backbone can be branched. In some examples, the dye conjugate comprises a plurality of pendant donor chromophore groups, each independently linked to a non-conjugated repeat unit of the polymer backbone. The configuration of the pendant groups can be installed during or after the synthesis of the polymer backbone. Incorporation of the pendant groups can be achieved in a random configuration, a block configuration, or in a sequence-specific manner via stepwise synthesis, depending on the particular method of synthesis utilized.

[0074] The term "unit" refers to a structural subunit of a polymer. The term unit is meant to include monomers, comonomers, coblocks, repeat units, and the like. A "repeating unit" or "repeat unit" is a subunit of a polymer defined by the minimum number of distinct structural features required for the unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes a polymer or a block thereof. In some cases, a polymer may contain two or more different repeat units, for example, when the polymer is a multiblock polymer, a random arrangement or a defined sequence of units, each block may define a separate repeat unit. It is understood that various arrangements of repeat units or blocks are possible, and in the depicted formulas of the polymer backbones described herein, any convenient linear arrangement of various lengths can be included within the structure of the overall polymer. It is understood that a polymer may also be represented by a formula in terms of the mole percent values ​​of each unit in the polymer, and that such formulas may represent various arrangements of repeat units, such as random or multiblock polymers or defined sequences of residues. In some cases, the repeat unit of the polymer includes a single monomer group. In certain instances, the repeat unit of the polymer includes two or more monomer groups, i.e., comonomer groups (e.g., two, three, four or more comonomer groups). The term "comonomer" or "comonomer group" refers to a structural unit of a polymer that may itself be part of the repeat unit of the polymer.

[0075] The polymer backbone may have a random configuration of non-conjugated repeat units. The polymer backbone may comprise a block or conblock configuration of non-conjugated repeat units. Alternatively, the polymer backbone may comprise specific defined sequences of non-conjugated repeat units, e.g., 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.

[0076] "Non-conjugated" means that at least some of the repeat units contain saturated backbone groups (e.g., groups with two or more consecutive single covalent bonds), which excludes π-conjugation or extended delocalized electronic structures along the polymer backbone from one repeat unit to the next. Even if one repeat unit cannot be conjugated to an adjacent repeat unit, it is understood that such a repeat unit can contain one or more isolated unsaturated groups, including unsaturated bonds (e.g., of an alkenylene or alkynylene group) and / or aryl or heteroaryl groups, which can be part of the backbone. In some cases, each repeat unit of the polymer backbone contains a side chain that includes a linking pendant group or a chemoselective tag for linking to a pendant group.

[0077] In certain embodiments of the tandem dye, the polymer backbone is a linear polymer. In certain cases, the linear polymer is selected from a peptide, a peptoid, a hydrocarbon polymer, and a PEG polymer. In certain cases, the linear polymer is a peptide. In certain cases, the linear polymer is a peptoid. In certain other cases, the polymer is a hydrocarbon polymer. In certain other cases, the polymer is a PEG polymer. Further details regarding polymer backbones that may be employed in embodiments of the present invention can be found in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, and International Application No. PCT / US2020 / 019510, published as WO2020 / 222894, the disclosures of which are incorporated herein by reference.

[0078] The tandem dyes of the present invention may include a linear polymer backbone of any number of units. As used herein, the term "unit" refers to a structural subunit of a polymer. The term unit is meant to include monomers, comonomers, coblocks, segments, repeat units, and the like. A "repeat unit" is a subunit of a polymer defined by the minimum number of distinct structural features required for the unit to be considered a monomer, such that when the unit is repeated n times, the resulting structure describes a polymer or a block thereof. In some cases, the polymer may include two or more different repeat units, such as when the polymer is a multiblock polymer, or a random arrangement of units, each block may define a distinct repeat unit, e.g., n blocks and m blocks. It is understood that various arrangements of n and / or m repeat units or blocks are possible, and any convenient linear arrangement of coblocks of various lengths is included within the overall structure of the polymer. A polymer may also be represented by a formula in terms of the mole percent values ​​of each unit in the polymer, with it being understood that such formulas may represent various arrangements of repeat units, such as random or multiblock polymers. In some cases, the repeat unit of a polymer includes a single monomer group. In certain examples, the repeat unit of a polymer includes two or more monomer groups, i.e., comonomer groups (e.g., two, three, four or more comonomer groups). As used herein, the term "comonomer" or "comonomer group" refers to a structural unit of a polymer that may itself be part of the repeat unit of the polymer.

[0079] In certain instances, the tandem dye comprises a linear peptide backbone of 2-100 amino acids, e.g., 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, or 2-30 amino acids. In some cases, the linear peptide backbone comprises two 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 100 amino acids. In certain cases, the tandem dye comprises a linear peptide backbone of 5-30 amino acids, e.g., 5-25, 5-20, 5-15, or 5-10 amino acids.

[0080] 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 be in the range of 2 to 500,000, e.g., 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, e.g., 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 instances, the specific number of monomer repeat units or segments of the backbone can be in the range of 2 to 1,000, e.g., 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 certain cases, the specific number of monomer repeat units or segments of the backbone can be in the range of 2 to 500, e.g., 2 to 400, 2 to 300, 2 to 200, or 2 to 100 units or segments. In certain cases, the specific number of monomer repeat units or segments of the backbone can be in the range of 2 to 100 repeat monomer units, e.g., 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.

[0081] As summarized above, the tandem dye of the present invention comprises one or more pendant donor fluorophores linked to a polymer backbone. Any convenient pendant donor fluorophore can be linked to the polymer backbone, and the donor fluorophores of interest include, but are not limited to, BODIPY fluorophores, aryl fluorophores, heteroaryl fluorophores, and the like. In some examples, the donor fluorophore is a BODIPY fluorophore. The term "BODIPY fluorophore" refers to the pendant donor fluorophore of the tandem dye that comprises a chromophore having the boron-dipyrromethene (BODIPY) core structure shown below.

[0082] [ka]

[0083] Wherein Q is C or N, and each R is any convenient boron substituent. In some cases, Q is C. In some examples, 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 can be optionally further substituted, for example, with water-solubilizing groups and / or aryl or heteroaryl substituents that impart desirable light absorption properties to the chromophore. Further description of BODIPY fluorophores that can be employed as donor fluorophores in embodiments of the invention is described in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, the disclosure of which is incorporated herein by reference. In some embodiments, the donor fluorophore is a fluorophore that includes an aryl or heteroaryl chromophore group. Aryl or heteroaryl chromophore groups of interest that find use 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, silole, biphenyl, and bridged biphenyl. The aryl or heteroaryl chromophore groups can be optionally further substituted, for example with water-solubilizing groups and / or aryl or heteroaryl substituents that impart desirable light absorption properties to the aryl or heteroaryl groups. In some embodiments, the donor fluorophore comprises a fused tricyclic aryl or heteroaryl. In some embodiments, the donor fluorophore comprises one or more groups selected from fluorene, carbazole, silole, biphenyl, and bridged biphenyl. The fused tricyclic chromophore is a group that comprises a tricyclic aromatic group having three fused rings in a configuration in which two aryl or heteroaryl six-membered rings are fused to a central five- or six-membered carbocyclic or heterocyclic ring. In some cases, the fused tricyclic group contains two benzo or pyrido rings fused to a central five- or six-membered carbocyclic or heterocyclic ring. The fused tricyclic group can be linked to the side chains of comonomers in the polymer backbone through any convenient ring atom of the fused ring.The central five- or six-membered ring may be carbocyclic or heterocyclic, aromatic or partially saturated, and may further include side chain substituents such as WSG and / or chemoselectivity tags or linkers to the comonomer side chains. The bridged biphenyl comonomer is a fused tricyclic group with a biphenyl group, and the two phenyl rings are further linked to each other via a central six-membered carbocyclic or heterocyclic ring. Further description of aryl / heteroaryl fluorophores that may be employed as donor fluorophores in embodiments of the present invention is described in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, the disclosure of which is incorporated herein by reference. The number of donor water-solvated light-harvesting chromophore repeat units having linked acceptor signaling fluorophore groups can vary, and in some examples, 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 repeat units.

[0084] In addition to the pendant donor fluorophore(s), the tandem dyes of the present invention include one or more acceptor fluorophores. In the polymer tandem dyes, any convenient fluorescent dye may be utilized as the acceptor fluorophore. 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, naphthalimide, phycobiliproteins, peridinum chlorophyll proteins, conjugates thereof, and combinations 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 examples, the acceptor fluorophore (A) is selected from DY431, DY485XL, DY500XL, DY610, DY640, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY778, DY782, DY800, DY831, Biotium CF555, Cy3.5, and diethylaminocoumarin. Fluorescent dyes of interest include fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a protein), and 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 568, 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, BODIPY530 / 550, BODIPY558 / 568, BODIPY564 / 570, BODIPY576 / 589, BODIPY581 / 591, BODIPY630 / 650, BODIPY650 / 665, BODIPY R6G, BODIPY TMR, BODIPY TR, conjugates thereof, and combinations thereof. Lanthanide chelates of interest include, but are not limited to, europium chelates, terbium chelates, and samarium chelates. In some embodiments, the polymeric tandem dye comprises multiple chromophores linked to an acceptor fluorophore selected from Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Alexa488, Alexa647, and Alexa700. In certain embodiments, the polymer tandem dye is a Dyomics dye (such as DY431, DY485XL, DY500XL, DY530, DY610, DY633, DY640, DY651, DY654, DY682, DY700, DY701, DY704, DY730, DY731, DY732, DY734, DY752, DY754, DY778, DY782, DY800, or DY831), Biotium CF555, Cy3.5, and diethylaminocoumarin. In certain cases, the acceptor fluorophore (A) is fluorescein, 6-FAM, rhodamine, Texas Red, California Red, iFluor594, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G, carboxyrhodol, carboxyrhodamine 110, cascade blue, cascade yellow, coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight350, DyLight405, DyLight488, DyLight549, DyLight594, DyLight633, DyLight649, DyLight680, DyLight750, DyLight800, phycoerythrin, PerCP (peridinin 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® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa The fluorine-containing amine is selected from Fluor® 680, 7-amino-4-methylcoumarin-3-acetic acid, BODIPY® FL, BODIPY® FL-Br2, BODIPY® 530 / 550, BODIPY® 558 / 568, BODIPY® 564 / 570, BODIPY® 576 / 589, BODIPY® 581 / 591, BODIPY® 630 / 650, BODIPY® 650 / 665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugates thereof, and combinations thereof.

[0085] As discussed above, if desired, the polymer backbone and / or the pendant fluorophores (i.e., donor and acceptor fluorophores) may include one or more water-solubilizing groups (WSGs). In some cases, the WSGs are pendant groups attached directly to the polymer backbone, e.g., as side chains of the polymer backbone. In certain cases, the WSGs are substituents attached to the pendant donor fluorophore or the pendant acceptor fluorophore. In some examples, each of the pendant donor fluorophore groups is substituted with one or more WSGs. As used herein, the terms "water-solubilizing group," "water-soluble group," and WSGs are used interchangeably and refer to a group or substituent that is sufficiently solvated in an aqueous environment, e.g., under physiological conditions, and confers improved water solubility to the molecule to which it is attached. The WSGs can increase the solubility of a given polymer tandem dye, primarily in aqueous solutions, compared to a control dye lacking the WSG. The water-solubilizing group can be any convenient hydrophilic group that is sufficiently solvated in an aqueous environment. The water-soluble tandem dyes of the present disclosure have solubility under aqueous conditions that make them particularly suitable for application in a variety of biological assays. A variety of water-soluble polymeric groups can be adapted for use with the subject dyes in WSGs. Any convenient water-solubilizing group (WSG) can be included in the dyes described herein to enhance water solubility. The increase in solubility can vary, but in some instances the increase is 2-fold or more (compared to a compound without a WSG(s)), e.g., 5-fold, 10-fold, 25-fold, 50-fold, 100-fold or more. In some cases, the hydrophilic water-solubilizing group is, for example, positively charged or negatively charged. In certain cases, the hydrophilic water-solubilizing group is a neutral hydrophilic group. In some embodiments, the WSG is branched (e.g., as described herein). In certain instances, the WSG is linear. In some embodiments, the WSG is a hydrophilic polymer, such as polyethylene glycol, modified PEG, peptide sequences, peptoids, carbohydrates, oxazolines, polyols, dendrons, dendritic polyglycerols, cellulose, chitosan, or derivatives thereof.Water solubilizing groups of interest include, but are not limited to, carboxylates, phosphonates, phosphates, sulfonates, sulfates, sulfinates, sulfonium, esters, polyethylene glycols (PEGs) and modified PEGs, hydroxyls, amines, amino acids, ammonium, guanidinium, pyridinium, polyamines and sulfonium, polyalcohols, linear or cyclic sugars, primary, secondary, tertiary, or quaternary amines and polyamines, phosphonate groups, phosphinate groups, ascorbate groups, glycols, and polyethers, -COOM', -SO3M', -PO3M', -NR3. + , Y', (CH2CH2O) p R and mixtures thereof, Y' can be any halogen, sulfate, sulfonate, or oxygen-containing anion, p can be 1 to 500, each R can independently be H or alkyl (e.g., methyl), M' is a cationic counterion or hydrogen, --(CH2CHO) yy CH2CH2XR yy , --(CH2CH2O) yy CH2CH2X--, --X(CH2CH2O) yy CH2CH2--, glycol, and polyethylene glycol, where yy is selected from 1 to 1000, and X is O, S, or NR ZZ Selected from R ZZ and R YY are independently H and C 1-3 In some cases, WSG is selected from (CH2) x (OCH2CH2) y OCH3, where 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-solubilizing group comprises a non-ionic polymer (e.g., a PEG polymer) terminally substituted with an ionic group (e.g., a sulfonate). Further details regarding suitable WSG groups that may be present in the dyes of embodiments of the present invention can be found in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, the disclosure of which is incorporated herein by reference.

[0086] As summarized above, the polymeric fluorescent tandem dyes of the present invention include a plurality of pendant donor fluorophores and one or more pendant acceptor fluorophores. In some examples, the number of donor fluorophores exceeds the number of acceptor fluorophores. In certain embodiments of the subject tandem dyes, 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 certain cases, the ratio of donor fluorophores to acceptor fluorophores is 5:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 6:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 7:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 8:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 9:1. In certain cases, the ratio of donor fluorophores to acceptor fluorophores is 10:1.

[0087] As mentioned above, in the polymeric fluorescent tandem dyes of the present invention, the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship. Thus, in embodiments of the present invention, excitation of the donor can lead to energy transfer to and emission from the covalently linked acceptor signaling fluorophore. Mechanisms for energy transfer between the donor chromophore and the linked acceptor signaling fluorophore include, for example, resonance energy transfer (e.g., Forster (or fluorescence) resonance energy transfer, FRET), quantum charge exchange (Dexter energy transfer), and the like. These energy transfer mechanisms can be relatively short-range, i.e., the proximity of the chromophores of the light-harvesting multichromophore system to each other and / or to the acceptor fluorophore provides efficient energy transfer. Under conditions for efficient energy transfer, amplification of emission from the acceptor fluorophore may occur, where emission from the emitting acceptor fluorophore is stronger when the incident light ("pump light") is at a wavelength that is absorbed by and transferred from the chromophore of the light-harvesting chromophore than when the emitting acceptor fluorophore is directly excited by the pump light. "Efficient" energy transfer means 10% or more, e.g. 20% or more, 30% or more, 40% or more, 50% or more, of the energy recovered by the donor chromophore. "Amplification" means that when excited by energy transfer from the donor light-harvesting chromophore system, the signal from the acceptor fluorophore is 1.5 times or more compared to direct excitation of the acceptor fluorophore with incident light of equivalent intensity. The signal may 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 certain cases, a signal of 1.5 times or more refers to an increased signal-to-noise ratio.In certain embodiments of the tandem dyes, the acceptor fluorophore emission, 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 than compared to direct excitation of the acceptor fluorophore with incident light.

[0088] The tandem dyes of the present embodiments can be of any convenient molecular weight (MW). In some cases, the MW of the tandem dye can be expressed as an average molecular weight. In some examples, the tandem dye has an average molecular weight in the range of 500 to 500,000, such as 1,000 to 100,000, 2,000 to 100,000, 10,000 to 100,000, or even 50,000 to 100,000 Daltons. In some examples, the polymeric fluorescent dye has a molecular weight in the range of 5 to 75 kDa, such as 10 to 50 kDa, such as 15 to 45 kDa. In certain embodiments, the molecular weight of the polymeric fluorescent dye is in the range of 1 to 30 kDa, such as 2 to 25 kDa, and 2 to 20 kDa.

[0089] In some examples, 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, when the light-harvesting chromophore is directly excited with incident light. In some cases, the effective Stokes shift of the tandem dye is up to about 300 nm, e.g., 100-300 nm, 100-250 nm, or 100-200 nm.

[0090] The emission of the polymeric 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 more. In some examples, the polymeric tandem dye can have a quantum yield of 5×10 5 cm-1 M -1 Or more, for example, 6×10 5 cm -1 M -1 That's it, 7 x 10 5 cm -1 M -1 That's it, 8 x 10 5 cm -1 M -1 That's it, 9 x 10 5 cm -1 M -1 Or more, for example, 1×10 6 cm -1 M -1 That's it, 1.5 x 10 6 cm -1 M -1 That's it, 2 x 10 6 cm -1 M -1 That's it, 2.5 x 10 6 cm -1 M -1 That's it, 3 x 10 6 cm -1 M -1 That's it, 4 x 10 6 cm -1 M -1 That's it, 5 x 10 6 cm -1 M -1 That's it, 6 x 10 6 cm -1 M -1 That's it, 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 In some embodiments, the polymer tandem dye has an extinction coefficient of 5×10 5 M -1 cm -1 In certain embodiments, the tandem dye has a molar extinction coefficient of 1×10 or greater. 6 M -1 cm -1 It has a molar extinction coefficient of or greater.

[0091] In embodiments, the subject tandem dyes provide fluorescent emission from the acceptor fluorophore that is brighter than the emission possible from such fluorescent dye alone. The emission of the polymeric tandem dyes is at 50 mM -1 cm -1 More than 60mM, for example -1 cm -1 Above 70mM -1 cm -1 Above 80mM -1 cm -1 Above 90mM -1 cm -1 More than 100mM -1 cm -1 More than 150mM -1 cm -1 More than 200mM -1 cm -1 more than 250mM -1 cm -1 More than 300mM -1 cm -1 In certain examples, the emission of the tandem dye is at least 5 times greater than the brightness of the directly excited acceptor fluorophore, at least 10 times greater, at least 20 times greater, at least 30 times greater, at least 50 times greater, at least 100 times greater, at least 300 times greater, or even greater than the brightness of the directly excited acceptor fluorophore.

[0092] In addition to the attributes discussed above, the tandem dyes of embodiments of the present invention may have one or more additional desirable spectral properties, such as a particular emission maximum wavelength, extinction coefficient, quantum yield, etc. As discussed above, the subject tandem dyes in some examples provide a donor chromophore having an absorption maximum between 555 nm and 585 nm, e.g., between 555 nm and 575 nm, e.g., between 555 and 575 nm, e.g., between 555 and 574 nm, 555 and 573 nm, 555 and 572 nm, 555 and 571 nm, 555 and 570 nm, 555 and 569 nm, 555 and 568 nm, 555 and 567 nm, 555 and 566 nm, 555 and 565 nm, 555 and 564 nm, 555 and 563 nm, or 555 and 562 nm.

[0093] The various emission profiles depend on various factors such as the comonomers, linking groups, substituents, and linked acceptor fluorophores selected from which the tandem dyes are constructed. In some embodiments, the chromophores have emission maximum wavelengths in the range of 300-900 nm, e.g., 350-850 nm, 350-600 nm, 360-500 nm, 370-500 nm, 380-500 nm, 390-500 nm, or 400-500 nm, with specific examples of emission maxima of interest including, but 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, and 805 nm ± 5 nm. In certain examples, the chromophore has an emission maximum wavelength selected from the group consisting of 395 nm, 460 nm, 490 nm, 550 nm, 560 nm, 605 nm, 650 nm, 680 nm, 700 nm, and 805 nm. In certain examples, the tandem dye has an emission maximum wavelength of 395 nm ± 5 nm. In some examples, the tandem dye itself has an emission maximum wavelength in the range of 375 to 900 nm (e.g., in the range of 380 nm to 900 nm, 390 nm to 900 nm, or 400 nm to 900 nm).

[0094] In some instances, the tandem dyes are 5×10 5 cm -1 M -1 Or more, for example, 6×10 5 cm -1 M -1 That's it, 7 x 10 5 cm -1 M -1 That's it, 8 x 10 5 cm -1 M -1 That's it, 9 x 10 5 cm -1 M -1 Or more, for example, 1×10 6 cm -1 M -1 , 1.5×10 6 cm -1 M -1 That's it, 2 x 10 6 cm -1 M-1 That's it, 2.5 x 10 6 cm -1 M -1 That's it, 3 x 10 6 cm -1 M -1 That's it, 4 x 10 6 cm -1 M -1 That's it, 5 x 10 6 cm -1 M -1 That's it, 6 x 10 6 cm -1 M -1 That's it, 7 x 10 6 cm -1 M -1 or more, or 8 x 10 6 cm -1 M -1 In such cases, the tandem may have 5 or more repeat units, such as 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more repeat units. In some embodiments, the tandem dye has an extinction coefficient of 5×10 or more. 5 M -1 cm -1 In certain embodiments, the chromophore has a molar extinction coefficient of 1×10 or greater. 6 M -1 cm -1 It has a molar extinction coefficient of or greater.

[0095] In some instances, the tandem dyes have 40,000 cm per repeat unit. -1 M -1 , 45,000 cm per repeat unit -1 M -1 , 50,000 cm per repeat unit -1 M -1 More than 55,000 cm per repeat unit -1 M -1 More than 60,000 cm per repeat unit -1 M -1 More than 70,000 cm per repeat unit -1 M -1 More than 80,000 cm per repeat unit -1 M -1 More than 90,000 cm per repeat unit-1 M -1 More than 100,000 cm per repeat unit -1 M -1 In some instances, the repeating unit has an extinction coefficient of 40,000 cm or more, or even more. -1 M -1 is the average extinction coefficient. In certain examples, the repeat unit of the chromophore may contain a single monomer, two comonomers, or three or more comonomers. In some examples, the chromophore has an average extinction coefficient of 40,000 cm per comonomer. -1 M -1 For example, 45,000 cm per unit -1 M -1 Above 50,000 cm per unit -1 M -1 Above 55,000 cm per unit -1 M -1 Above 60,000 cm per unit -1 M -1 Above 70,000 cm per unit -1 M -1 Above 80,000 cm per unit -1 M -1 Above 90,000 cm per unit -1 M -1 Above 100,000 cm per unit -1 M -1 In some cases, the extinction coefficient is 40,000 cm per comonomer or more. -1 M -1 The above are average extinction coefficients.

[0096] In some embodiments, the polymeric tandem dye can include a segment of formula (Ia).

[0097] [ka]

[0098] Here, each M 1 and M. 2are independently unsaturated comonomers (e.g., amino acid residues); Each S 1 and S 2 are independently non-conjugated spacer units; Each D 1 Independently, M 1 a pendant donor fluorophore (e.g., as described herein) linked to Each A 1 Independently, M 2 is an acceptor fluorophore linked to x is 75 mole % or more; y is 25 mol % or less.

[0099] The first (M 1 -S 1 ) and the second repeat unit (M 2 -S 2 ) can be arranged randomly or in a coblock configuration. In the particular case of formula (Ia), D of the first repeat unit 1 The pendant group includes two or more (e.g., two or three) distinct types of pendant light-absorbing chromophores that together provide a light-harvesting multichromophore system. In a particular example of formula (Ia), D of the first repeat unit 1 All of the pendant groups are the same.

[0100] In some examples 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 examples 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.

[0101] In some examples, the polymeric tandem dye comprises a segment of formula (IIa).

[0102] [ka]

[0103] Here, the polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, SM 1 , S.M. 2 and S.M. 3 Equipped with accessories; Each D 1 is independent, SM 1 a pendant donor chromophore linked to Each A 1 is independent, SM 2 is an acceptor fluorophore linked to each Z 2 is S.M. 3 is an optional side group linked to x is 50 mole % or more; y+z is 50 mol % or less.

[0104] Z 2 can be absent or any convenient side chain group, such as a light absorbing chromophore, a chemoselectivity tag, a linker, a linking biomolecule, an acceptor fluorophore, a WSG (e.g., as described in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, the disclosure of which is incorporated herein by reference). In the particular case of formula (IIa), SM 3 is Z 2 is an absent spacer comonomer. In a particular example of formula (IIa), SM 3 is the second pendant light-absorbing chromophore, Z 2 Each D 1 and each Z 2 Together, they provide a light-harvesting multichromophore system. In some cases, SM 3 is the second chemical selection tag (Z 2 ), e.g., Z 1 is a comonomer that contains a protected functional group or tag that is orthogonal to

[0105] In certain cases of formula (IIa), x is 60 mol% or more, for example 65 mol% or more, 70 mol% or more, 75 mol% or more, 80 mol% or more, 85 mol% or more, 90 mol% or more, 95 mol% or more, or even more. In certain examples of formula (IIa), y+z is 40 mol% or less, for example 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, 10 mol% or less, 5 mol% or less, or even less. In certain examples of formula (IIa), y is at least 1 mol% and 25 mol% or less, for example 20 mol% or less, 15 mol% or less, 10 mol% or less, 5 mol% or less, or even less. In certain examples of formula (IIa), z is at least 1 mol% and 10 mol% or less, for example 5 mol% or less, or even less.

[0106] In some examples, the polymeric tandem dye comprises a segment of formula (IIIa).

[0107] [ka]

[0108] Here, the polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, SM 1 and S.M. 2 Equipped with accessories; Each D 1 is independent, SM 1 a pendant donor chromophore linked to Each A 1 is independent, SM 2 is an acceptor fluorophore linked to x is 75 mole % or more; y is 25 mol % or less.

[0109] In certain embodiments of Formula (IIIa), SM 1 and S.M. 2 are each independently a saturated non-conjugated comonomer, e.g., a comonomer that provides only a single covalent C-C bond. In some embodiments of formula (IIIa), SM 1 and S.M.2 are each independently a partially saturated non-conjugated comonomer, e.g., a comonomer providing an isolated double C=C covalent bond in a backbone of saturated covalent bonds. The first and second repeat units (SM 1 and S.M. 2 ) can be arranged in a random configuration, in a block or coblock configuration, or in a specific sequence. In the specific case 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 a particular example of formula (IIIa), D of the first repeat unit 1 All of the pendant groups are the same.

[0110] In some examples of Formula (IIIa), 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 examples of Formula (IIIa), 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, or 1 mol% or less.

[0111] In a particular example, the polymeric tandem dye is of formula (IVa):

[0112] [ka]

[0113] Here, each D 1 are independently pendant donor chromophore groups; Each A 1 are independently acceptor fluorophores; Each L 1 and L 2 is a separate linker; p1 and q1 are independently 0 or 1, and p1+q1≦1; p2 and q2 are independently 0 or 1, and p1 + q1 ≦ 1; x is 75 mole % or more; y is 25 mol % or less; G 1 and G 2 are each independently selected from an end 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.

[0114] In some embodiments of formula (IVa), p1 and p2 are each 0 and q1 and q2 are each 1 (e.g., β3-amino acid residues). In some embodiments of formula (IVa), p1 and p2 are each 1 and q1 and q2 are each 0 (e.g., β2-amino acid residues). In some cases, p1, p2, q1 and q2 are each 0 and the polymeric tandem dye is of formula (Va).

[0115] [ka]

[0116] Here, each D 1 are independently pendant donor chromophore groups; Each A 1 are independently acceptor fluorophores; L 1 and L 2 are each independently a linker; x is 75 mole % or more; y is 25 mol % or less; G 1 and G 2 are each independently selected from an end group, a polymer segment, a light absorbing (e.g., donor) chromophore group, a linker, and a linker to a single domain antibody, e.g., as described above. It is understood that the tandem dyes described by formula (Va) include any convenient arrangement of the comonomers in a defined linear sequence, which collectively has a defined mole percent ratio of x and y. In some cases, the A containing comonomers 1are spaced throughout the sequence of the polymer backbone and are therefore always flanked on both sides by one or more D1 containing comonomers.

[0117] In a particular example of formula (Va), the polymeric tandem dye comprises a segment of formula (VIa).

[0118] [ka]

[0119] Here, each D 1 are independently pendant donor chromophore groups; Each A 1 are independently acceptor chromophores; Each L 1 and L 3 is a separate linker; n and p each independently represent an integer of 1 to 20, and n+p≧2; m is 1 or 2.

[0120] In some cases of Formula (VIa), n and p are each independently 1 to 10, such as 2 to 20, 3 to 10, or 3 to 6. In some examples of Formula (VIa), n+p is an integer from 2 to 20, such as 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.

[0121] The subject polymeric tandem dyes can include multiple segments of formula (VIa), each segment including a D 1 One isolated A containing comonomers flanked by blocks of 1 In some cases, the multichromophore comprises two or more segments of formula (VIa) arranged adjacent to each other and having 2 to 20 D containing comonomers. 1 D blocks of 3-20, 4-20, 5-20, 5-15 or 5-12, e.g. 1 Two isolated A-containing comonomers separated by a block of1 Thus, in certain embodiments, the polymeric tandem dye comprises q segments of a block copolymer and is of formula (VIIa):

[0122] [ka]

[0123] Here, for each (n) q and each (p) q is independently an integer from 1 to 20, and for each of the q segments, (n) q +(p) q ≧3, and q is an integer from 1 to 100.

[0124] In certain embodiments, the polymeric tandem dye has the formula (VIIIa):

[0125] [ka]

[0126] Here, each D 1 are independently pendant donor chromophore groups; Each A 1 are independently acceptor chromophores; Each L 1 and L 2 is a separate linker; x is 75 mole % or more; y is 25 mol % or less; G 1 and G 2 are each independently selected from an end group, a polymer segment, a donor chromophore group, a linker, and a linker to a single domain antibody, e.g., as described above.

[0127] In certain embodiments, the polymeric tandem dye has the formula (IXa):

[0128] [ka]

[0129] Here, each D 1 are independently pendant BODIPY donor chromophores; Each A 1 are independently acceptor fluorophores; Each L 1 and L 2 is a separate linker; x is 75 mole % or more; y is 25 mol % or less; G 1 and G 2 are each independently selected from the group consisting of an end 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.

[0130] In some examples of formulas (IVa), (Va), (XIIla) 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 examples of formulas (IVa), (Va), (XIIIa) and (IXa), 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.

[0131] In certain embodiments, the polymeric tandem dye has the formula (Xa):

[0132] [ka]

[0133] Here, each D 1 are independently pendant donor chromophores; Each A 1 are independently acceptor fluorophores; Each L 1 , L 2 and L 3is a separate linker; a, b and c are the mole percent values ​​for each comonomer; d represents the total polymerization or average length of the polymer (e.g., d is 2 to 1000, e.g., 2 to 500, 2 to 200, 2 to 100, or 2 to 50); WSG is a water solubilizing group (e.g., as described in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, the disclosure of which is incorporated herein by reference); G 1 and G 2 are each independently selected from the group consisting of an end 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.

[0134] In some examples of formula (Xa), c=0. In some examples of formula (Xa), a>0 and b>0. In some examples of formula (Xa), a 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 examples of formula (Xa), b 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, or 1 mol% or less. In some examples of formula (Xa), a is 65-95 mol%, b is 5-35 mol%, c is 0-30 mol%, and a+b+c=100%.

[0135] Any convenient end group (e.g., G 1 and G 2 ) may be utilized at the terminus of the tandem fluorescent dye. As used herein, the terms "end group" and "terminal group" are used interchangeably to refer to groups located at the termini of the polymeric structure of the light-harvesting chromophore, for example as described herein. 1 and G 2The groups include, but are not limited to, end-capping groups, π-conjugated segments, linkers, and linkers to single domain antibodies, for example as described above. In some embodiments, the end-capping group is a monovalent group conjugated to the backbone of the light-harvesting chromophore after polymerization. In certain examples, the end-capping group is an aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkyl, or substituted alkyl. In some cases, the end comonomer is directed to and linked to a chemoselective tag or linker. In certain cases, the end-capping group is derived from a terminal group of a monomer used in the polymerization method, such as a halogen (e.g., Br), a boronic acid, or a boronic ester, which can undergo further conjugation. In some examples, G 1 and / or G 2 is a π-conjugated segment. As used herein, π-conjugated segment refers to any convenient segment of a conjugated polymer to which a light-harvesting chromophore can be conjugated, i.e., allowing delocalization of π-electrons across adjacent units. In certain embodiments, G 1 and / or G 2 is a linker, such as a linker containing a functional group suitable for conjugation to a specific binding moiety. 1 and / or G 2 It is understood that the linker located at position G may be selected to be orthogonal to any other linker, including a chemoselectivity tag (e.g., as described herein), that may be present in the side chain of the light-harvesting chromophore. In certain embodiments, the amino functionality or a derivative thereof is 1 and / or G 2 In certain embodiments, the carboxylic acid functional group or a derivative thereof is included in 1 and / or G 2 Included in.

[0136] In some embodiments of the formulas described herein, G 1 and G 2 At least one of the -L 6 -Z 4 And L 6 is a linker (e.g., as described herein), and Z4 is a specific binding member (e.g., as described herein). In some embodiments of the formulas described herein, G 1 and G 2 At least one of them is -L 6 -Z 3 And L 6 is a linker (e.g., as described herein), and Z 3 is a chemoselectivity tag (e.g., as described herein). Any convenient chemoselectivity tag and conjugation chemistry can be adapted for use in the subject light-harvesting chromophores. Chemoselectivity 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, transcyclooctene, aldehydes, alkoxylamines, alkynes, cyclooctynes, azides, and the like. In some examples, Z 3 is selected from the group consisting of carboxylic acid, active ester (e.g., N-hydroxysuccinimidyl ester (NHS) or sulfo-NHS), amino, maleimide, iodoacetyl, and thiol. In certain embodiments of the formulas described herein, G 1 and G 2 At least one of the following is described by the following structure:

[0137] [ka]

[0138] where Ar is a π-conjugated aryl group, L is a linker, and Z is a chemoselectivity tag or specific binding member. In some cases, the LZ group can be attached towards the terminal comonomer. In certain embodiments of the formulas described herein, G 1 and G 2 At least one of the following is described by the following structure:

[0139] [ka]

[0140] where q is 0 or an integer from 1 to 12; L is an optional linker; Z is a chemoselectivity tag or specific binding member. Further details regarding WSG groups that may be found in the tandem dyes of the present embodiments can be found in International Application No. PCT / US2019 / 024662, published as WO2019 / 191482, the disclosure of which is incorporated herein by reference.

[0141] Representative single domain antibody / polymer fluorescent tandem dye conjugates 1A and 1B provide diagrams of a single domain antibody / polymer fluorescent tandem dye conjugate according to an embodiment of the present invention. In FIG. 1A, the illustrated single domain antibody / polymer fluorescent tandem dye conjugate comprises a V H H single domain antibodies (labeled "tandem 'cobalt' dyes"). The tandem dyes may be conjugated to any convenient residue of the single domain antibody, with residues of interest for conjugation or linkage being those that do not adversely affect the binding activity of the single domain antibody when linked to the dye. Residues that may be employed for conjugation to the dye may be naturally occurring or residues engineered into the single domain antibody, such as terminal cysteines or unnatural amino acids, as desired. Figure 1B provides a more detailed view of the conjugate shown in Figure 1A, showing the structure of the polymeric fluorescent tandem dyes, as well as the structure of the dyes when linked to VH single domain antibodies (labeled "tandem 'cobalt' dyes"). H H shows a PEG water-solubilizing linker for linking to a single domain antibody. The length of the linker can vary, but in some examples the linker is in the range of 10-50 nm, e.g., 15-40 nm, e.g., 20-30 nm. A given linker can be flexible, e.g., PEG, or can be rigid, e.g., a poly-glycine chain, e.g., in place of PEG, if desired.

[0142] method An embodiment of the invention includes a method of evaluating a sample for the presence of a target analyte. An embodiment of the method includes contacting the sample with a single domain antibody / polymer fluorescent dye conjugate that specifically binds the target analyte to generate an assay composition comprising the 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 bound to the target molecule, e.g., a primary antibody, depending on whether the single domain antibody / polymer fluorescent dye conjugate is employed as a primary or secondary label. Any convenient method may be used to contact the sample with the single domain antibody / polymer fluorescent tandem dye conjugate that specifically binds the target analyte to generate the assay composition. In some examples, the sample is contacted with the single domain antibody / polymer fluorescent tandem dye conjugate under conditions that specifically bind the single domain antibody component of the conjugate to the target analyte, if present. A suitable solution that maintains the biological activity of the sample and the components of the single domain antibody may be used to allow the single domain antibody of the conjugate to specifically bind to the target analyte. The solution may be a balanced salt solution, such as normal saline, PBS, Hank's balanced salt solution, etc., conveniently supplemented with fetal bovine serum, human platelet lysate or other factors, along with an acceptable buffer at a low concentration, such as 5-25 mM. Convenient buffers include HEPES, phosphate buffer, lactate buffer, etc. In some cases, a variety of media are commercially available, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., supplemented with fetal bovine serum or human platelet lysate, and may be used according to the nature of the target analyte. The final components of the solution may be selected depending on the components of the sample involved. The temperature at which specific binding of the single domain antibody of the conjugate to the target analyte occurs may vary, and in some instances may be in 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 instances, 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 particular examples, the temperature is 25° C., 30° C., 35° C. or 37° C. In particular 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 for the formation of a desired amount of binding complex, and in some examples can be 1 minute or more, such as 2 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, or even 6 hours or more.

[0143] Any convenient single domain antibody may be utilized in the conjugate employed in the method of the present invention. Single domain antibodies of interest include, but are not limited to, those single domain antibodies that specifically bind to cell surface proteins of various cell types, including, but 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 (e.g., HIV cells), NK cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and erythroid cells. Target cells of interest include cells that have a convenient cell surface marker or antigen that can be captured by a convenient specific binding member conjugate. In some embodiments, the target cells are selected from HIV-containing cells, Treg cells, antigen-specific T cell populations, 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 for specific binding to the conjugates employed in the subject methods, hi 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 cell may comprise 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 alpha / beta, T cell receptor gamma / delta, CD253, CD95, CD20, CD105, CD117, CD120b, Notch4, Lgr5 (N-terminus), SSEA-3, TRA-1-60 antigen, disialoganglioside GD2, and CD71.

[0144] Any convenient target may be selected for evaluation utilizing the subject method. Targets of interest include, but are not limited to, nucleic acids such as RNA, DNA, PNA, CNA, HNA, LNA or ANA molecules, fusion proteins, modified proteins, such as proteins that are phosphorylated, glycosylated, ubiquitinated, sumoylated or acetylated, or antibodies, peptides, aggregated biomolecules, cells, small molecules, vitamins and drug molecules. As used herein, the term "target protein" refers to all members of a target family, and fragments thereof. Target proteins can be any protein of interest, such as therapeutic or diagnostic 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 that can be prepared using any convenient recombinant expression method or using any convenient synthetic method, or can be purchased commercially. In some embodiments, the polymer-dye conjugate comprises an antibody or an antibody fragment. Any convenient target analyte that specifically binds to the antibody or antibody fragment of interest can be targeted in the subject methods.

[0145] In some embodiments, the target analyte is associated with a cell. In certain instances, the target analyte is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. In some instances, the target analyte is an intracellular target, and the method further comprises permeabilizing or lysing the cell. Thus, the single domain antibody of a given conjugate employed in the method of the present invention may target a cell surface or intracellular antigen. Alternatively, the single domain antibody of a given conjugate employed in the method of the present invention may target a primary antibody that in turn specifically binds to a target cell surface or intracellular antigen.

[0146] In some embodiments, the sample may comprise a heterogeneous cell population from which target cells are isolated. In some examples, the sample comprises peripheral whole blood, peripheral whole blood from which red blood cells have been lysed prior to cell isolation, umbilical cord blood, bone marrow, density gradient purified peripheral blood mononuclear cells, or homogenized tissue. In some cases, the sample comprises hematopoietic progenitor cells (e.g., CD34+ cells) in whole blood, bone marrow, or umbilical cord blood. In certain embodiments, the sample comprises tumor cells in peripheral blood. In certain examples, the sample is a sample that contains (or is suspected of containing) viral cells (e.g., HIV).

[0147] The single domain antibody / polymeric fluorescent tandem dye conjugates find use in the subject methods, for example, to label target cells, particles, targets, or analytes with the polymeric tandem fluorescent dye. For example, the single domain antibody / polymeric fluorescent tandem dye conjugates find use in labeling cells that are processed (e.g., detected, analyzed, and / or sorted) in a flow cytometer. The single domain antibody / polymeric fluorescent tandem dye conjugates may include, for example, single domain antibodies that specifically bind to cell surface proteins of various cell types (e.g., as described herein). The labeled single domain antibody / polymeric fluorescent tandem dye conjugates may be used to investigate various biological (e.g., cellular) 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. The labeled specific binding members may be used in any application that includes (or may include) antibody-mediated labeling of cells, particles, or analytes.

[0148] Aspects of the method include assaying the assay composition, i.e., the sample contacted with the single domain antibody / polymeric fluorescent tandem dye conjugate, for the presence of a single domain antibody / polymeric fluorescent tandem dye conjugate-target analyte binding complex to assess whether the target analyte is present in the sample. Once the sample is contacted with the single domain antibody / polymeric fluorescent tandem dye conjugate, any convenient method may be utilized in assaying the generated assay composition for the presence of a single domain antibody / polymeric fluorescent tandem dye conjugate-target analyte binding complex. The single domain antibody / polymeric fluorescent tandem dye conjugate-target analyte binding complex is a binding complex generated upon specific binding of the specific binding member of the conjugate to the target analyte, if present (or a primary binding member, e.g., a primary antibody to a target antigen, depending on the embodiment). Assaying the assay composition may include detecting a fluorescent signal, if present, from the binding complex. In some cases, assaying includes a separation step in which the target analyte, if present, is separated from the sample. A variety of methods may be utilized to separate the target analyte from the sample, for example via immobilization on a carrier. Assay methods of interest include, but are not limited to, any convenient method and assay format that finds interest in using pairs of specific binding members, such as avidin-biotin or hapten-antihapten antibodies.Interesting methods and assay formats that can be adapted for use with the subject compositions include, but are not limited to, flow cytometry, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography.

[0149] In certain embodiments, the method further comprises contacting the sample with a second specific binding member that specifically binds to the target analyte. In certain examples, the second specific binding member is carrier-bound. Any convenient carrier can be utilized to immobilize the components of the subject method (e.g., the second specific binding member). In certain examples, the carrier is a particle, such as a magnetic particle. In some examples, the second specific binding member and the polymer-dye conjugate generate a sandwich complex that can be isolated and detected, if present, using any convenient method. In some embodiments, the method further comprises analyzing the polymer-dye conjugate-target analyte binding complex, i.e., the fluorescently labeled target analyte, by flow cytometry. Assaying for the presence of the polymer-dye conjugate-target analyte binding complex can provide an assay result (e.g., qualitative or quantitative assay data) that can be used to evaluate whether the target analyte is present in the sample.

[0150] Any convenient carrier can be utilized in the subject method to immobilize any convenient component of the method, such as a labeled specific binding member, a target, a secondary specific binding member, etc. Carriers of interest include, but are not limited to, solid substrates, which can have a variety of configurations, such as, for example, sheets, beads, or other structures, such as wells, beads, polymers, particles, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, chromatography supports, etc. In some examples, the carrier is selected from the group consisting of particles, planar solid substrates, fibrous meshes, hydrogels, porous matrices, pins, microarray surfaces, and chromatography supports. The carrier can be incorporated into a system that provides cell isolation assisted by any convenient method, such as a manually operated syringe, a centrifuge, or an automated liquid handling system. In some cases, the carrier finds use in automated liquid handling systems for high throughput isolation of cells, such as a flow cytometer.

[0151] In some embodiments of the method, the separation step comprises applying an external magnetic field to immobilize the magnetic particles. Any convenient magnet can be used as the source of the external magnetic field (e.g., magnetic field gradient). In some cases, the external magnetic field is generated by a magnetic source, for example, by a permanent magnet or an electromagnet. In some cases, immobilizing the magnetic particles means that the magnetic particles accumulate near the surface closest to the magnetic field gradient source, i.e., the magnet.

[0152] The separating may further include one or more optional washing steps to remove unbound material of the sample from the carrier. Any convenient washing method may be used, such as washing the immobilized carrier with a biocompatible buffer that maintains the specific binding interaction between the polymer dye and the specific binding member. The separation and optional washing of the unbound material of the sample from the carrier, from which undesirable cells and materials may be removed, provides an enriched population of target cells.

[0153] In certain embodiments, the method includes detecting the labeled target analyte. Detecting the labeled target analyte may include exciting the polymeric fluorescent tandem dye with one or more lasers, followed by detecting the fluorescent emission from the polymeric fluorescent tandem dye using one or more optical detectors. The detection of the labeled target can be performed using any convenient instrument and method, including but not limited to flow cytometry, FACS system, fluorescence microscopy; fluorescence, luminescence, ultraviolet, and / or visible light detection using a plate reader; 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 the subject method can be performed using different types of fluorescent detection systems. In some cases, high throughput screening can be performed, for example, a system using 96 well or more microtiter plates. Various methods of performing assays on fluorescent materials are available, such as those described in Lakowicz, JR, Principles of Fluorescence 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.

[0154] 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 excitation optics. The excitation optics causes the excitation radiation to excite the sample. In response, fluorescently labeled targets in the sample emit radiation having a wavelength different from the excitation wavelength. The collection optics then collects the emission from the sample. The device can include a temperature controller to maintain the sample at a particular temperature while it is being scanned. In certain examples, a multi-axis translation stage moves a microtiter plate holding multiple samples to expose and position different wells. The multi-axis translation stage, temperature controller, autofocus function, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer. The computer can also convert data collected during the assay into another format for presentation.

[0155] In some embodiments, the method for evaluating the sample for the presence of the target analyte further comprises detecting fluorescence in a flow cytometer. In some embodiments, the method for evaluating the sample for the presence of the target analyte further comprises imaging the sample contacted with the labeling composition using fluorescence microscopy. Fluorescence microscopy imaging can be used to identify the polymer dye conjugate-target analyte binding complex in the contacted sample to evaluate whether the target analyte is present. Microscopy of interest that finds use in the subject method includes laser scanning confocal microscopy.

[0156] Figure 2A provides a schematic of one embodiment of a method in which a primary antibody is employed along with a single domain antibody / polymer fluorescent tandem dye conjugate that specifically binds to the primary antibody to detect intracellular cytokines, and Figure 2B illustrates a similar method in which an Alexa-fluor labeled nanobody is employed.

[0157] Also provided are methods for producing single domain antibody / polymeric fluorescent tandem dye conjugates, e.g., as described herein. In some embodiments, the methods include contacting a single domain antibody with a polymeric fluorescent tandem dye (e.g., as described herein) to generate a single domain antibody / polymeric fluorescent tandem dye conjugate, where the polymeric fluorescent tandem dye comprises a conjugation tag that covalently binds the dye to the single domain antibody. The term "conjugation tag" refers to a group that comprises a chemoselective functional group (e.g., as described herein) that can be covalently bonded to a compatible functional group of a single domain antibody after optional activation and / or deprotection. Any convenient conjugation tag may be utilized on the subject polymeric dye to conjugate the polymeric fluorescent tandem dye to the single domain antibody of interest. In some embodiments, the conjugation tag comprises a terminal functional group selected from amino, carboxylic acid or derivatives thereof, thiol, hydroxyl, hydrazine, hydrazide, azide, alkyne, and protein reactive groups (e.g., amino-reactive, thiol-reactive, hydroxyl-reactive, imidazolyl-reactive, or guanidinyl-reactive). Any convenient method and reagent can be adapted for use in the subject method to covalently attach the conjugation tag to the single domain antibody. Methods of interest for labeling the target include, but are not limited to, those described in Hermanson, Bioconjugate Techniques, Third edition, Academic Press, 2013. The contacting step can be carried out in an aqueous solution. In some examples, the conjugation tag comprises an amino functional group and the target molecule comprises an active ester functional group, such as an NHS ester or a sulfo-NHS ester, or vice versa. In a particular example, the conjugation tag comprises a maleimide functional group and the target molecule comprises a thiol functional group, or vice versa. In a particular example, the conjugation tag comprises an alkyne (e.g., a cyclooctyne group) functional group and the target molecule comprises an azide functional group, or vice versa, and can be conjugated via click chemistry.In some cases, the method includes a separation step in which the product single domain antibody / polymer fluorescent tandem dye conjugate is separated from the reaction mixture, e.g., excess reagents or unlabeled single domain antibody. A variety of methods may be utilized to separate the target from the sample, e.g., via immobilization on a support, precipitation, chromatography, etc.

[0158] In some examples, the method further comprises detecting and / or analyzing the product single domain antibody / polymer fluorescent tandem dye conjugate. In some examples, the method further comprises fluorescently detecting the single domain antibody / polymer fluorescent tandem dye conjugate. Any convenient method may be utilized to detect and / or analyze the labeled target molecule in conjunction with the subject methods and compositions. Methods of analyzing targets of interest that find use in the subject methods include, but are not limited to, flow cytometry, fluorescent microscopy, in-situ hybridization, enzyme-linked immunosorbent assay (ELISA), Western blot analysis, magnetic cell separation assay, and fluorescent dye purification chromatography. Detection methods of interest include, but are not limited to, fluorescent spectroscopy, fluorescent microscopy, nucleic acid sequencing, fluorescent in-situ hybridization (FISH), protein mass spectrometry, flow cytometry, and the like.

[0159] Detection can be achieved directly via the polymer tandem dye or indirectly by a secondary detection system. The latter can be based on any one principle 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 known in the fields of immunocytochemistry, molecular biology, light, fluorescence, and electron microscopy, cell immunophenotyping, cell sorting, flow cytometry, cell visualization, detection, counting, and / or signal output quantification. More than one antibody of specific and / or non-specific nature can be labeled and used simultaneously or sequentially to enhance target detection, identification, and / or analysis.

[0160] system Aspects of the invention further include systems for use in carrying out the subject methods and compositions. The sample analysis system can include a sample field 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 including a flow cytometer including a flow path and a composition in the flow path, the composition comprising 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 fluorescent microscope system, including a fluorescent microscope with a sample field and a composition disposed in the sample field, the composition comprising a sample and a labeled specific binding member (e.g., as described herein).

[0161] In certain embodiments of the system, the composition further comprises a second specific binding member that is bound to the carrier and specifically binds to the target analyte. In some cases, the carrier comprises magnetic particles. Thus, in certain examples, the system may also comprise a controllable external paramagnetic field configured to be applied to the assay region of the flow channel.

[0162] The sample may include cells. In some examples, the sample is a cell-containing biological sample. In some examples, the sample includes a labeled specific binding member that is specifically bound to a target cell. In certain examples, the target analyte that is specifically bound by the specific binding member is a cell surface marker of the cell. In certain cases, the cell surface marker is selected from a cell receptor and a cell surface antigen.

[0163] In certain embodiments, the system may also include a light source configured to direct light to the assay region or sample field of the flow channel. The system may include a detector configured to receive a signal from the assay region or sample field of the flow channel, the signal being provided by the fluorescent composition. Optionally, the sample analysis system may further include one or more additional detectors and / or light sources for detection of one or more additional signals.

[0164] In certain embodiments, the system may further comprise a computer-based system configured to detect the presence of fluorescent signals. "Computer-based system" refers to the hardware means, software means, and data storage means 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), input means, output means, and data storage means. Those skilled in the art can easily understand that any one of the currently available computer-based systems is suitable for use in the subject system. The data storage means may include any product that contains a record of the information as described above, or a memory access means that can access such a product.

[0165] "Recording" data, programming, or other information on a computer-readable medium refers to a process for storing information, using any such methods known in the art. Any convenient data storage structure may be selected based on the means used to access the stored information. A variety of data processor programs and formats may be used for storage, such as, for example, word processing text files, database formats, and the like.

[0166] A "processor" refers to any combination of hardware and / or software that performs the functions required of it. For example, any processor herein may be a programmable digital microprocessor, such as those available in the form of an electronic controller, mainframe, server, or personal computer (desktop or portable). If the processor is programmable, the appropriate programming may be transmitted to the processor from a remote location or may be stored in advance in a computer program product (such as a portable or fixed computer-readable storage medium, whether magnetic, optical, or solid-state device-based). For example, a magnetic medium or optical disk may carry the programming and can be read by an appropriate reader that communicates with each processor at its corresponding station.

[0167] In addition to sensor devices and signal processing modules, e.g., as described above, the systems of the present invention may include a number of additional components, such as data output devices, e.g., monitors and / or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, etc.

[0168] In some embodiments, the system comprises a flow cytometer. Suitable flow cytometry systems include, but are not limited to, those described in 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 ed., Wiley-Liss (1995); Virgo et al. (2012) Ann Clin Biochem. Jan; 49(pt1): 17-28; Linden et al., Semin Thromb 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 disclosures of which are incorporated herein by reference.In particular examples, flow cytometry systems of interest include BD Biosciences FACSCanto™ flow cytometers, BD Biosciences FACSCanto™ II flow cytometers, BD Accuri™ flow cytometers, BD Accuri™ C6 Plus flow cytometers, BD Biosciences FACSCelesta™ flow cytometers, BD Biosciences FACSLyric™ flow cytometers, BD Biosciences FACSVerse™ flow cytometers, BD Biosciences FACSymphony™ flow cytometers, BD Biosciences LSRFortessa™ flow cytometers, BD Biosciences LSRFortessa™ X-20 flow cytometers, BD Biosciences FACSPresto™ flow cytometers, BD Biosciences FACSVia™ flow cytometers, and BD Biosciences FACSCalibur™ cell sorters, BD Biosciences FACSCount™ cell sorters, BD Biosciences FACSLyric™ cell sorters, BD Cell sorters and cell sorters used in cell sorting include the BD Biosciences Via™ cell sorter, BD Biosciences Influx™ cell sorter, BD Biosciences Jazz™ cell sorter, BD Biosciences Aria™ cell sorter, BD Biosciences FACSAria™ II cell sorter, BD Biosciences FACSAria™ III cell sorter, BD Biosciences FACSAria™ Fusion cell sorter and BD Biosciences FACSMelody™ cell sorter, BD Biosciences FACSymphony™ S6 cell sorter, and the like.

[0169] In some embodiments, the subject systems may be implemented using the same or similar technology as disclosed in U.S. Patent 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; 10,302,5 No. 45; No. 10,145,793; No. 10,113,967; No. 10,006,852; No. 9,952,076; No. 9,933,341; No. 9 ,726,527;No.9,453,789;No.9,200,334;No.9,097,640;No.9,095,494;No.9,092,034 and flow cytometry systems such as those described in Nos. 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 by reference in their entireties herein.

[0170] In certain examples, the flow cytometry system of the present invention may be implemented using the methods disclosed in Diebold et al., Nature Photonics Vol. 7(10); 806-810 (2013) and U.S. Patent 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; 10,408,758; 10,451,538; 10,620,110; No. 2017 / 0328826; 2017 / 0350803; 2018 / 0275042; 2019 / 0376895; and 2019 / 0376894.

[0171] Other systems may find use in carrying out the subject methods. In certain aspects, the system may be a fluorometer or microscope loaded with a sample having the fluorescent composition of any of the embodiments described herein. The fluorometer or microscope may include a light source configured to direct light to an assay region or sample field of view of the flow channel. The fluorometer or microscope may also include a detector configured to receive a signal from the assay region or field of view of the flow channel, the signal being provided by the fluorescent composition.

[0172] kit Aspects of the invention further include kits for use in carrying out the subject methods and compositions. The compositions of the invention can be included as reagents, either as starting materials or in kits provided for use in, for example, the methodologies described above. The kits 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 the single domain antibody / polymer fluorescent tandem dye conjugate. In such instances, the polydispersity between the multiple copies of the single domain antibody / polymer fluorescent tandem dye conjugate can be low. Any convenient container can be utilized, such as a tube, a bottle, or a well in a multi-well strip or plate, a box, a bag, an insulated container, etc. The subject kits can further include one or more components selected from a primer specific binding member for a given target analyte, a carrier-bound specific binding member, a cell, a carrier, a biocompatible aqueous elution buffer, and instructions for use. A given kit may contain reagents suitable for the detection of a single target analyte, or multiple reagents suitable for the detection of two or more different target analytes, e.g., if a given kit is configured for multiplex detection applications.

[0173] In certain embodiments, the kit finds use in evaluating a sample for the presence of a target analyte, such as an intracellular target.Thus, in some examples, the kit includes one or more components suitable for permeabilizing or lysing cells.One or more additional components of the kit may be provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0174] In certain embodiments, the kit further comprises a reagent for performing flow cytometry assay. Reagents of interest include, but are not limited to, buffers for reconstitution and dilution, buffers for contacting chromophores with cell samples, washing buffers, control cells, control beads, fluorescent beads for flow cytometer calibration, and combinations thereof. The kit may also comprise one or more cell fixation reagents, such as paraformaldehyde, glutaraldehyde, methanol, acetone, formalin, or any combination or buffer thereof. In addition, the kit may comprise a cell permeabilization reagent, such as methanol, acetone, or detergent (e.g., triton, NP-40, saponin, tween20, digitonin, leucoperm, or any combination or buffer thereof). Other protein transport inhibitors, cell fixation reagents, and cell permeabilization reagents well known to those skilled in the art are within the scope of the subject kit.

[0175] The compositions of the kit may be provided in a liquid composition, such as any suitable buffer. Alternatively, the compositions of the kit may be provided in a dry composition (e.g., lyophilized), and the kit may optionally include one or more buffers for reconstituting the dry composition. In certain embodiments, the kit may include aliquots of the compositions provided in separate containers (e.g., separate tubes, bottles, or wells in a multi-well strip or plate).

[0176] In addition, one or more components may be combined in a single container, such as a glass or plastic vial, tube or bottle. In certain examples, the kit may further include a container (e.g., a box, bag, insulated container, bottle, tube, etc.) in which all components (and their separate containers) are present. The kit may further include packaging that is separate from or attached to the kit container and is printed with information about the kit, the components and / or instructions for use of the kit.

[0177] In addition to the above components, the subject kits may further include instructions for carrying out the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as information printed on a suitable medium or substrate, such as a sheet or sheets of paper on which the information is printed, kit packaging, package inserts, and the like. Yet another means may be a computer readable medium, such as a diskette, CD, DVD, portable flash drive, and the like on which the information is stored. Yet another means may be present is a website address that may be used via the Internet to access the information at the destination site. Any convenient means may be present in the kit.

[0178] Utilities The single domain antibody / polymer fluorescent tandem dye conjugates, compositions, methods, and systems described herein may find use in a variety of applications, including diagnostic and research applications, where labeling, detection, and / or analysis of a target of interest is desired. Such applications include methodologies such as cytometry, microscopy, immunoassays (e.g., competitive or non-competitive), free analyte assessment, receptor-bound ligand assessment, and the like. The compositions, systems, and methods described herein may be useful for the analysis of any of a number of samples, including, but not limited to, biological fluids, cell culture samples, and tissue samples. In certain embodiments, the compositions, systems, and methods described herein may find use in methods in which an analyte is detected in a sample when present, for example, using fluorescent labels in fluorescence-activated cell sorting or analysis, immunoassays, immunostaining, and the like. In certain examples, the compositions and methods find use in applications in which evaluation of a sample for the presence of a target analyte is of interest.

[0179] In some cases, the methods and compositions find use in any assay format in which detection and / or analysis of a target from a sample is of interest, including, but 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. In certain examples, the methods and compositions find use in any application in which fluorescent labeling of a target molecule is of interest. The subject compositions can be adapted for use in any convenient application in which pairs of specific binding members, such as biotin-streptavidin and hapten-antihapten antibodies, find use.

[0180] The following examples are offered by way of illustration and not by way of limitation.

[0181] [Example] The following examples are written to provide those 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. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. 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. Standard abbreviations may be used, such as, for example, bp, base pair(s); kb, kilobase(s); pl, picoliters(s); s or sec, seconds(s); min, minutes(s); h or hr, hours(s); aa, amino acid(s); nt, nucleotide(s), etc.

[0182] I. Reagent Preparation A. VHH anti-mouse lgG1-RB780 preparation: Tandem dye conjugation to VHH anti-mouse lgG1 (Chromotek Nanobodies CTK103, CTK104) RB780™ tandem dye was modified to contain terminal maleimide groups for maleimide-thiol attachment to the N- and C-terminal cysteines of the target engineered nanobody CTK103 (Chromotek). The resulting purified RB780-maleimide dye was then added to a buffer solution (10 mM HEPES pH 7.0 500 mM NaCl) at a concentration of 20 mg / mL. Nanobodies were received frozen in aqueous buffer (10 mM HEPES pH 7.0, 500 mM NaCl, 1 mM TCEP). Prior to conjugation, the frozen nanobody solution was thawed to 4° C., centrifuged, and the supernatant transferred to a reaction vessel. An amount of RB780-maleimide dye solution corresponding to 2.7 molar equivalents of nanobody was then added to the solution containing the thawed nanobody. The mixture was vortexed, placed in an ice bath, and agitated with a magnetic stirrer for 2 hours. The resulting crude reaction mixture was then purified using a Superdex SEC column with standard elution buffer. The fractions of the desired product were pooled and characterized. Equimolar amounts of CTK103-RB780 conjugate and CTK104-RB780 were mixed in solution before staining with primary antibodies.

[0183] RB780™ was modified to contain terminal maleimide groups for maleimide-thiol attachment to the N- and C-terminal cysteines of the target engineered nanobody CTK104 (Chromotek). The resulting purified RB780-maleimide dye was then added to a buffer solution (10 mM HEPES pH 7.0 500 mM NaCl) at a concentration of 20 mg / mL. The nanobodies were received frozen in aqueous buffer (10 mM HEPES pH 7.0, 500 mM NaCl, 1 mM TCEP). Prior to conjugation, the frozen nanobody solution was thawed to 4° C., centrifuged, and the supernatant was transferred to a reaction vessel. An amount of RB780-maleimide dye solution corresponding to 2.7 molar equivalents of nanobody was then added to the solution containing the thawed nanobody. The mixture was vortexed, placed in an ice bath, and agitated with magnetic stirring for 2 hours. The resulting crude reaction mixture was then purified using a Superdex SEC column with standard elution buffer. The fractions of the desired product were pooled and characterized. Equimolar amounts of CTK103-RB780 conjugate and CTK104-RB780 were mixed in solution prior to staining with the primary antibody.

[0184] B. VHH anti-mouse lgG2b-RB780 preparation: Tandem dye conjugation to VHH anti-mouse lgG2b (Chromotek Nanobodoies CTK105, CTK106) RB780™ was modified to contain terminal maleimide groups for maleimide-thiol attachment to the N- and C-terminal cysteines of the target engineered nanobody CTK105 (Chromotek). The resulting purified RB780-maleimide dye was then added to a buffer solution (10 mM HEPES pH 7.0 500 mM NaCl) at a concentration of 20 mg / mL. The nanobodies were frozen and received in aqueous buffer (10 mM HEPES pH 7.0, 500 mM NaCl, 1 mM TCEP). Prior to conjugation, the frozen nanobody solution was thawed to 4° C., centrifuged, and the supernatant was transferred to a reaction vessel. An amount of RB780-maleimide dye solution corresponding to 2.7 molar equivalents of nanobody was then added to the solution containing the thawed nanobody. The mixture was vortexed, placed in an ice bath, and agitated with a magnetic stirrer for 2 hours. The resulting crude reaction mixture was then purified using a Superdex SEC column with standard elution buffer. The fractions of the desired product were pooled and characterized. Equimolar amounts of CTK105-RB780 conjugate and CTK106-RB780 were mixed in solution before staining with primary antibodies.

[0185] RB780™ was modified to contain terminal maleimide groups for maleimide-thiol conjugation to the N- and C-terminal cysteines of the target engineered nanobody CTK106 (Chromotek). The resulting purified RB780-maleimide dye was then added to a buffer solution (10 mM HEPES pH 7.0 500 mM NaCl) at a concentration of 20 mg / mL. The nanobodies were frozen and received in aqueous buffer (10 mM HEPES pH 7.0, 500 mM NaCl, 1 mM TCEP). Prior to conjugation, the frozen nanobody solution was thawed to 4° C., centrifuged, and the supernatant was transferred to a reaction vessel. An amount of RB780-maleimide dye solution corresponding to 2.7 molar equivalents of nanobody was then added to the solution containing the thawed nanobody. The mixture was vortexed, placed in an ice bath, and agitated with magnetic stirring for 2 hours. The resulting crude reaction mixture was then purified using a Superdex SEC column with standard elution buffer. Fractions of the desired product were pooled and characterized. Equimolar amounts of CTK105-RB780 conjugate and CTK106-RB780 were mixed in solution prior to staining with the primary antibody.

[0186] II. Further Research A. Preparation of RB780-labeled nanobodies 1. Modification of RB780 22 milligrams of RB780 derivative bearing amine reactive sites were dissolved in degassed 1x phosphate buffered saline (VWR catalog number 97063-660) at a concentration of 10 mg / mL. 5.55 milligrams of SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) were 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. To remove unreacted SMCC, the crude reaction mixture was purified by size exclusion membrane (MilliporeSigma™ UFC900308) and lyophilized overnight on a Schlenk line. Yield and purity were assessed by ion exchange chromatography (Thermo Scientific Ultimate3000 with Propoac SAX-10 4x250mm).

[0187] 2. Nanobody Disulfide Reduction Nanobody samples previously stored in buffer at -80°C were thawed and buffer exchanged into 10 mM Hepes pH 7.0, 500 mM NaCl, 1 mM TCEP using a size exclusion membrane (MilliporeSigma™ UFC900308). The buffer exchanged solution containing the freshly reduced nanobodies was then centrifuged to remove precipitates and aggregates.

[0188] 3. Dye Conjugation to Nanobodies Nanobody CTK0103 (ProteinTech) was concentrated to 2mg / mL in 10mM Hepes pH7.0, 500mM NaCl, 1mM TCEP and kept below 4°C via ice bath. RB780-maleimide was dissolved in amine-free DMF (Fisher Scientific Catalog No. AA43465K7) at 20mg / mL and cooled to about 4°C via ice bath. RB780-maleimide solution was added to the nanobody solution at a ratio of 2.7 moles RB780-maleimide per mole of nanobody and stirred for 2 hours at about 4°C. The crude conjugation mixture was purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE & UV-Vis.

[0189] Nanobody CTK0104 (ProteinTech) was concentrated to 2mg / mL in 10mM Hepes pH7.0, 500mM NaCl, 1mM TCEP and kept below 4°C via ice bath. RB780-maleimide was dissolved in amine-free DMF (Fisher Scientific Catalog No. AA43465K7) at 20mg / mL and cooled to about 4°C via ice bath. RB780-maleimide solution was added to the nanobody solution at a ratio of 2.7 moles RB780-maleimide per mole of nanobody and stirred for 2 hours at about 4°C. The crude conjugation mixture was purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE & UV-Vis.

[0190] Nanobody CTK0105 (ProteinTech) was concentrated to 2mg / mL in 10mM Hepes pH7.0, 500mM NaCl, 1mM TCEP and kept below 4°C via ice bath. RB780-maleimide was dissolved at 20mg / mL in amine-free DMF (Fisher Scientific Catalogue No. AA43465K7) and cooled to about 4°C via ice bath. RB780-maleimide solution was added to the nanobody solution in a ratio of 2.7 moles RB780-maleimide per mole of nanobody and stirred for 2 hours at about 4°C. The crude conjugation mixture was purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE & UV-Vis.

[0191] Nanobody CTK0106 (ProteinTech) was concentrated to 2mg / mL in 10mM Hepes pH7.0, 500mM NaCl, 1mM TCEP and kept below 4°C via ice bath. RB780-maleimide was dissolved at 20mg / mL in amine-free DMF (Fisher Scientific Catalogue No. AA43465K7) and cooled to about 4°C via ice bath. RB780-maleimide solution was added to the nanobody solution in a ratio of 2.7 moles RB780-maleimide per mole of nanobody and stirred for 2 hours at about 4°C. The crude conjugation mixture was purified by size exclusion chromatography (GE AKTA FPLC with Superdex) and characterized by SDS-PAGE & UV-Vis.

[0192] B. Cell Preparation / Staining & Flow Cytometry 1. Surface Staining 100 μl of well-mixed anticoagulated whole blood was incubated with unlabeled primary antibody, human CD4 (clone SK3, mouse lgG1) for 20-30 min in the dark at room temperature. Then 2 ml of lysis solution was added and incubated at room temperature in the dark. After 10 min, cells were washed and secondary reagent, nanobody anti-mouse lgG1-RB780, was added (nanobodies CTK0103 and CTK0104 from ProteinTech) and incubated at room temperature in the dark for 20-30 min. A final wash was performed and 200-500 μl of wash buffer was added for flow cytometry analysis or refrigerated storage at 2-8 °C.

[0193] 100 μl of well-mixed anticoagulated whole blood was incubated with unlabeled primary antibody, human CD56 (clone NCAM16.2, mouse lgG2b) for 20-30 min in the dark at room temperature. Then 2 ml of lysis solution was added and incubated at room temperature in the dark. After 10 min, cells were washed and the secondary reagent, nanobody VHH anti-mouse lgG2b-RB780 (nanobodies CTK0105 and CTK0106 from ProteinTech), was added and incubated at room temperature in the dark for 20-30 min. A final wash was performed and 200-500 μl of wash buffer was added before flow cytometry analysis or refrigerated storage at 2-8 °C.

[0194] 2. Intracellular Staining Peripheral blood mononuclear cells (PBMCs) were activated, fixed and permeabilized according to standard protocols. 100 μl of PBMCs were incubated with unlabeled primary antibody, human IFN-γ (clone B27, mouse lgG1), for 30 min at 4 °C in the dark. Cells were then washed twice and the secondary reagent, nanobody VHH anti-mouse lgG1-RB780 (nanobodies CTK0103 and CTK0104 from ProteinTech), was added, followed by incubation for 30 min at 4 °C in the dark. A final wash was performed and 200-500 μl of wash buffer was added prior to flow cytometry analysis or refrigerated storage at 2-8 °C.

[0195] C. Result: 1. Surface Staining Positive staining in lymphocytes corresponding to the CD4 positive population was observed. The signal when using nanobodies CTK0103 and CTK0104 (either used individually or pooled together) was fainter compared to the signal observed with the same existing nanobodies conjugated with AlexaFluor647 (CTK0103 and CTK0104 pooled from ProteinTech). Comparisons were performed at both mass and molar equivalents.

[0196] We also observed positive staining in lymphocytes corresponding to the CD56 positive population. The signal when using nanobodies CTK0105 and CTK0106 (either used individually or pooled together) was fainter compared to the signal observed with the same existing nanobodies conjugated with AlexaFluor647 (CTK0105 and CTK0106 pooled from ProteinTech). Comparisons were performed at both mass and molar equivalents.

[0197] 2. Intracellular staining: Positive staining in lymphocytes corresponding to the IFN-γ positive population was observed. The signal when using nanobodies CTK0103 and CTK0104 (either used individually or pooled together) was fainter compared to the signal observed with the same existing nanobodies conjugated with AlexaFluor647 (CTK0103 and CTK0104 pooled from ProteinTech). Comparisons were performed at both mass and molar equivalents.

[0198] III. Further Observations The single domain antibody / polymer fluorescent tandem dye conjugates of the present embodiments offer many advantages. Prior to the work reported herein, there were two general classes of commercialized sdAb+fluorescent dye reagents. The first of these are sdAb+fluorescent proteins, such as GFP, GYP, GRP, etc. Typically these reagents are recombinantly produced as fusion proteins. While these reagents hold advantages in manufacturability and performance due to their small size and in vivo expression, their usefulness is limited because they are relatively dim and they are not tandem dyes and do not allow for the multiplexing advantages of tandem dyes. The second class of sdAb+fluorescent dye reagents consists of sdAbs conjugated to small molecule dyes or a small number of small molecule dyes, such as AlexaFluor647. These reagents also have size-based advantages, but are limited in several ways. As reagents, they have brightness limitations associated with self-quenching behavior. If too much AlexFluor647 dye is attached to the sdAb, the fluorescence of the reagent can actually decrease. Furthermore, like the fusion proteins described above, these small molecule dyes are not tandem dyes and do not allow for the multiplexing advantages of tandem dyes.

[0199] The single domain antibody / polymeric fluorescent dye conjugate reagents of the present embodiment combine the advantages of compactness and improved manufacturing with additional advantages that may include one or more increased reagent brightness and compatibility with high parameter panel design. These advantages are achieved by using polymeric fluorescent tandem dyes instead of single molecule dyes. Polymeric fluorescent tandem dyes are brighter per molecule than small molecule dyes and do not exhibit appreciable inter-dye quenching of small molecule dyes when conjugated to antibodies. Polymeric fluorescent tandem dyes also represent a tandem dye platform that is specifically intended for optimizing multicolor flow cytometry. Thus, the conjugates of the present invention can be employed in multiplexed applications using a panel of dyes.

[0200] Notwithstanding the claims appended hereto, the present disclosure is also defined by the following clauses. 1. A single domain antibody / polymer fluorescent tandem dye conjugate comprising: (a) a single domain antibody, and (b) one or more polymeric tandem fluorescent dyes conjugated to said single domain antibody. wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeat units; (ii) a plurality of pendant donor fluorophores, each independently linked to a non-conjugated repeat unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores linked to non-conjugated repeat units of the polymer backbone; wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship. Conjugate. 2. The conjugate described in clause 1, wherein said single domain antibody comprises a heavy chain or a light chain. 3. The single domain antibody is H The conjugate according to clause 2, comprising an H domain. 4. The single domain antibody is NAR The conjugate according to clause 2, comprising a domain. 5. A conjugate according to any of the preceding claims, wherein said single domain antibody specifically binds to an intracellular antigen. 6. A conjugate according to any of the preceding claims, wherein said single domain antibody specifically binds to a cell surface antigen. 7. A conjugate according to any preceding claim, wherein the single domain antibody specifically binds to a primary antibody. 8. A conjugate according to any of the preceding claims, wherein the conjugate comprises two or more polymeric tandem fluorescent dyes conjugated to the single domain antibody. 9. The polymer tandem fluorescent dye has the formula [ka] Described by, wherein the polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, S 1 and S.M. 2 Equipped with accessories, Each D 1 is independent, SM 1 is a pendant donor fluorophore linked to Each A 1 is independent, SM 2 is a pendant acceptor fluorophore linked to x is 75 mol % or more; y is 25 mol% or less; A conjugate according to any of the preceding claims. 10. The conjugate of clause 9, wherein the repeat units of the polymer backbone have a defined linear sequence. 11. S&M 1 and S.M. 2 The conjugate according to clause 10, wherein is a comonomer derived from an amino acid and a peptoid monomer. 12. The conjugate of clause 11, wherein the polymer backbone is a polypeptide having a defined sequence of α- and / or β-amino acid residues. 13. The conjugate of any preceding claim, wherein the polymer backbone is a linear polymer. 14. A conjugate according to any preceding claim, wherein the pendant donor fluorophores are configured in energy transfer proximity to one another. 15. The conjugate of any of the preceding paragraphs, wherein the polymer tandem dye has a Stokes shift of 100 nm or more. 16. The conjugate of any of the preceding claims, wherein the pendant donor fluorophore is selected from a fused tricyclic aryl, a fused tricyclic heteroaryl, and a BODIPY fluorophore. 17. The conjugate according to claim 16, wherein the pendant donor fluorophore is a BODIPY fluorophore. 18. A conjugate according to any preceding claim, wherein the pendant donor fluorophore is substituted with a water-solubilizing group. 19. The conjugate of clause 18, wherein the water-solubilizing group comprises polyethylene glycol. 20. A conjugate according to any preceding claim, wherein the acceptor fluorophore is a small molecule fluorophore. 21. The conjugate according to clause 20, wherein the acceptor fluorophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes and acridine dyes. 22. A method for evaluating a sample for the presence of a target analyte, comprising: (a) combining the sample with a single domain antibody / polymer fluorescent tandem dye conjugate that specifically binds the target analyte to generate an assay composition; (b) assaying the assay composition for the presence of any conjugate-target analyte binding complexes to assess whether the target analyte is present in the sample; wherein the conjugate comprises (1) single domain antibodies, and (2) one or more polymeric tandem fluorescent dyes conjugated to said single domain antibody; wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeat units; (ii) a plurality of pendant donor fluorophores, each independently linked to a non-conjugated repeat unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores linked to non-conjugated repeat units of the polymer backbone; wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship; method. 23. The method of claim 22, wherein the target analyte is associated with a cell. 24. The method of clause 23, wherein the target analyte is a cell surface marker. 25. The method of claim 24, wherein the cell surface marker is selected from the group consisting of a cell receptor and a cell surface antigen. 26. The method of claim 23, wherein the target analyte is an intracellular target. 27. The method of claim 26, wherein the method further comprises permeabilizing the cells. 28. The method of any of clauses 22-27, wherein the assaying comprises analyzing the assay composition by flow cytometry. 29. The method of any one of clauses 22 to 28, wherein the single domain antibody comprises a heavy chain domain or a light chain domain. 30. The single domain antibody is H 30. The method of clause 29, comprising an H domain. 31. The single domain antibody is NAR 30. The method of claim 29, comprising the domain. 32. The method of any of clauses 22-31, wherein the conjugate comprises two or more polymeric tandem fluorescent dyes conjugated to the single domain antibody. 33. The polymer tandem fluorescent dye has the formula [ka] Described by, wherein the polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, S 1 and S.M. 2 Equipped with accessories, Each D 1 is independent, SM 1 is a pendant donor fluorophore linked to Each A 1 is independent, SM 2 is a pendant acceptor fluorophore linked to x is 75 mol % or more; y is 25 mol% or less; 33. The method according to any one of clauses 22 to 32. 34. The method of claim 33, wherein the repeat units of the polymer backbone have a defined linear sequence. 35. S&M 1 and S.M. 2 The method according to clause 34, wherein is a comonomer derived from an amino acid and a peptoid monomer. 36. The method of clause 35, wherein the polymer backbone is a polypeptide having a defined sequence of α-amino acid residues and / or β-amino acid residues. 37. The method of any one of clauses 22 to 36, wherein the polymer backbone is a linear polymer. 38. The method of any of clauses 22-37, wherein the pendant donor fluorophores are configured in energy transmitting proximity to each other. 39. The method of any one of clauses 22 to 38, wherein the polymer tandem fluorescent dye has a Stokes shift of 100 nm or more. 40. The method of any one of clauses 22 to 39, wherein the pendant donor fluorophore is selected from a fused tricyclic aryl, a fused tricyclic heteroaryl, and a BODIPY fluorophore. 41. The method of claim 40, wherein the pendant donor fluorophore is a BODIPY group. 42. The method of any one of clauses 22 to 41, wherein the pendant donor fluorophore is substituted with a water-solubilizing group. 43. The method of claim 42, wherein the water-solubilizing group comprises polyethylene glycol. 44. The method of any one of clauses 22 to 43, wherein the acceptor fluorophore is a small molecule fluorophore. 45. The method of claim 44, wherein the acceptor fluorophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes and acridine dyes. 46. ​​A single domain antibody / polymer fluorescent tandem dye conjugate, comprising: (a) a single domain antibody, and (b) one or more polymeric tandem fluorescent dyes conjugated to said single domain antibody. wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeat units; (ii) a plurality of pendant donor fluorophores, each independently linked to a non-conjugated repeat unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores linked to non-conjugated repeat units of the polymer backbone; wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship; kit. 47. The kit of clause 46, wherein the single domain antibody comprises a heavy chain or a light chain. 48. The single domain antibody is H The kit of clause 47, comprising an H domain. 49. The single domain antibody is NAR 48. The kit of clause 47, comprising a domain. 50. The kit of any of clauses 46 to 49, wherein the single domain antibody specifically binds to an intracellular antigen. 51. The kit of any of clauses 46 to 50, wherein the single domain antibody specifically binds to a cell surface antigen. 52. The kit of any of clauses 46 to 51, wherein the single domain antibody specifically binds to a primary antibody. 53. The kit of any of clauses 46-52, wherein the conjugate comprises two or more polymeric tandem fluorescent dyes conjugated to the single domain antibody. 54. The polymer tandem fluorescent dye has the formula [ka] Described by, wherein the polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, S 1 and S.M. 2 Equipped with accessories, Each D 1 is independent, SM 1 is a pendant donor fluorophore linked to Each A 1 is independent, SM 2 is a pendant acceptor fluorophore linked to x is 75 mol % or more; y is 25 mol% or less; 54. The kit according to any one of clauses 46 to 53. 55. The kit of clause 54, wherein the repeating units of the polymer backbone have a defined linear sequence. 56. S&M 1 and S.M. 2 56. The kit according to clause 55, wherein is a comonomer derived from an amino acid and a peptoid monomer. 57. The kit of clause 56, wherein the polymer backbone is a polypeptide having a defined sequence of α- and / or β-amino acid residues. 58. The kit of any one of clauses 46 to 57, wherein the polymer backbone is a linear polymer. 59. The kit of any of clauses 46-58, wherein the pendant donor fluorophores are configured in energy transmitting proximity to one another. 60. A kit according to any one of clauses 46 to 59, wherein the polymer tandem fluorescent dye has a Stokes shift of 100 nm or more. 61. The kit of any of clauses 46-60, wherein the pendant donor fluorophore is selected from a fused tricyclic aryl, a fused tricyclic heteroaryl, and a BODIPY fluorophore. 62. The kit of clause 61, wherein the pendant donor fluorophore is a BODIPY group. 63. The kit of any of clauses 46-62, wherein the pendant donor fluorophore is substituted with a water-solubilizing group. 64. The kit of clause 63, wherein the water-solubilizing group comprises polyethylene glycol. 65. The kit of any one of clauses 46 to 64, wherein the acceptor fluorophore is a small molecule fluorophore. 66. The kit of claim 65, wherein the acceptor fluorophore is selected from cyanine dyes, rhodamine dyes, xanthene dyes, coumarin dyes, polymethines, pyrenes, dipyrromethene boron difluorides, naphthalimides, thiazine dyes and acridine dyes.

[0201] In at least some of the above-described embodiments, one or more elements used in an embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter defined by the appended claims.

[0202] In general, those skilled in the art will understand that the terms used herein, and particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "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 "including" should be interpreted as "including but not limited to," etc.). Those skilled in the art will further understand that if a specific number of recitations of an introduced claim are intended, such intent will be explicitly set forth in the claim, and that in the absence of such recitation, no such intent exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the recitations of the claim. However, the use of such phrases should not be construed as meaning that the introduction of a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to embodiments that include only one such recitation, even if that claim includes the introductory phrase "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 to mean "at least one" or "one or more"), and the same applies to the use of a definite article used to introduce a claim recitation. Moreover, even if a specific number of introduced claim recitations is explicitly recited, one of ordinary skill in the art will recognize that such recitations should be interpreted to mean at least the recited number (e.g., the bare recitation "two recitations" without any other modifier means at least two recitations, or more than two recitations).Furthermore, in those instances where a conventional expression similar to "at least one of A, B, and C, etc." is used, such a structure is generally intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a conventional expression similar to "at least one of A, B, or C, etc." is used, such a structure is generally intended in the sense that one of ordinary skill in the art would understand the conventional expression (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually any alternative word and / or phrase, whether in the specification, claims, or drawings, presenting two or more alternative terms, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0203] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is thereby described in terms of any individual members or subgroups of members of the Markush group.

[0204] As will be understood by those skilled in the art, all ranges disclosed herein encompass all possible subranges and combinations of subranges for any and all purposes, including in terms of providing a written description. Any recited range can be readily recognized as fully describing and allowing the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, a middle third, and an upper third, etc. As will also be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," "less than," etc., refers to a range that includes the recited numbers and can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 to 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0205] Although the foregoing invention has been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0206] Thus, the above merely describes the principles of the invention. It will be appreciated that those skilled in the art can devise various configurations that embody the principles of the invention and fall within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts the inventors contribute to furthering the art, and should not be construed as being limited to such specifically recited examples and conditions. Furthermore, all statements herein describing the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Moreover, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any developed elements that perform the same function, regardless of structure. Furthermore, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is expressly set forth in the claims.

[0207] Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied in the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined to apply to a limitation of a claim only if the exact phrase "means for" or the exact phrase "step for" is prefaced in such limitation, and if such exact phrase is not used in a limitation in a claim, then 35 U.S.C. §112(f) or 35 U.S.C. §112(6) does not apply. CROSS-REFERENCE TO RELATED APPLICATIONS

[0208] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of U.S. Provisional Patent Application No. 63 / 326,104, filed March 31, 2022, the disclosure of which is incorporated herein by reference in its entirety.

Claims

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

2. The conjugate of claim 1 , wherein the single domain antibody specifically binds to an intracellular or cell surface antigen.

3. The conjugate of claim 1 or 2, wherein the single domain antibody specifically binds to a primary antibody.

4. The conjugate according to any one of claims 1 to 3, comprising two or more polymeric tandem fluorescent dyes conjugated to the single domain antibody.

5. The polymeric tandem fluorescent dye has the formula 【Chemistry 1】 Described by wherein the polymer backbone of each non-conjugated repeat unit is independently a non-conjugated comonomer, SM １ and S.M. ２ Equipped with accessories, Each D １ became independent and became S.M. １ is a pendant donor fluorophore linked to Each A １ became independent and became S.M. ２ is a pendant acceptor fluorophore linked to x is 75 mol % or more; y is 25 mol% or less; The conjugate according to any one of claims 1 to 4.

6. A conjugate according to any preceding claim, wherein the pendant donor fluorophores are configured in energy transferring proximity to one another.

7. The conjugate of any one of claims 1 to 6, wherein the pendant donor fluorophore is substituted with a water-solubilizing group.

8. The conjugate of any one of claims 1 to 7, wherein the acceptor fluorophore is a small molecule fluorophore.

9. 1. A method for evaluating a sample for the presence of a target analyte, comprising: (a) combining the sample with a single domain antibody / polymer fluorescent tandem dye conjugate that specifically binds the target analyte to generate an assay composition; (b) assaying the assay composition for the presence of any conjugate-target analyte binding complexes to assess whether the target analyte is present in the sample; wherein the conjugate comprises (1) single domain antibodies, and (2) one or more polymeric tandem fluorescent dyes conjugated to said single domain antibody; wherein the one or more polymer tandem fluorescent dyes comprise: (i) a polymer backbone comprising non-conjugated repeat units; (ii) a plurality of pendant donor fluorophores, each independently linked to a non-conjugated repeat unit of the polymer backbone; and (iii) one or more pendant acceptor fluorophores linked to non-conjugated repeat units of the polymer backbone; wherein the pendant donor fluorophore and the pendant acceptor fluorophore are in an energy transfer relationship.

10. The method of claim 9 , wherein the target analyte is associated with a cell.

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

12. The method of any of claims 9 to 11, wherein said assaying comprises flow cytometric analysis of said assay composition.

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

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