Application of click chemistry for signal amplification in IHC and ISH assays
11860167 ยท 2024-01-02
Assignee
Inventors
Cpc classification
C07F9/6524
CHEMISTRY; METALLURGY
C07F9/5535
CHEMISTRY; METALLURGY
International classification
C07F9/553
CHEMISTRY; METALLURGY
Abstract
Applicants have developed an amplification system and methodology for IHC and ISH staining that utilizes click chemistry to covalently bind reporter molecules to tissue.
Claims
1. A conjugate having Formula (IIa): ##STR00087## wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; R.sup.1 is a group selected from phosphate, amide, urea, sulfate, ester, beta-lactam, or a sugar; R.sup.2 is a halide; R.sup.3, R.sup.5, and R.sup.6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms; R.sup.4 is a hydrogen, an aliphatic group having between 1 and 4 carbon atoms, or the group CH(R.sup.2)R.sup.7-[Linker]-A; and R.sup.7 is selected from the group consisting of (CH.sub.2).sub.wNH, O(CH.sub.2).sub.wNH, N(H)C(O)(CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2).sub.wNH, (CH.sub.2).sub.wO, O(CH.sub.2).sub.wO, O(CH.sub.2CH.sub.2O).sub.w, N(H)C(O)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2CH.sub.2O).sub.w, (CH.sub.2).sub.wS, O(CH.sub.2).sub.wS, N(H)C(O)(CH.sub.2).sub.wS, C(O)N(H)(CH.sub.2).sub.wS, (CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)N(H)(CH.sub.2)NHC(O)CH(CH.sub.3)(CH.sub.2).sub.wNH, or N(H)(CH2).sub.wNH, where w is an integer ranging from 1 to 12.
2. The conjugate of claim 1, wherein R.sup.6, R.sup.5, R.sup.4, and R.sup.3 are each hydrogen.
3. The conjugate of claim 1, wherein R.sup.1 is a phosphate.
4. The conjugate of claim 1, wherein R.sup.2 is fluorine.
5. The conjugate of claim 1, wherein R.sup.1 is a phosphate; R.sup.2 is fluorine; and R.sup.6, R.sup.5, R.sup.4, and R.sup.3 are each hydrogen.
6. The conjugate of claim 1, wherein Linker has the Formula (Ia): ##STR00088## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.
7. The conjugate of claim 6, wherein R.sup.a and R.sup.b are each hydrogen.
8. The conjugate of claim 7, wherein Q is oxygen.
9. The conjugate of claim 1, wherein R.sup.7 is C(O)N(H)(CH2).sub.wNH.
10. The conjugate of claim 1, wherein R.sup.1 is a phosphate and R.sup.7 is C(O)N(H)(CH2).sub.wNH, and w ranges from 2 to 10.
11. The conjugate of claim 10, wherein R.sup.2 is fluorine; and R.sup.6, R.sup.5, R.sup.4, and R.sup.3 are each hydrogen.
12. The conjugate of claim 1, wherein the sugar is selected from the group consisting of alpha-glucose, beta-glucose, alpha-galactose, beta-galactose, alpha-lactose, beta-lactose, alpha-glucuronic acid, and beta-glucuronic acid; and the ester is phosphodiester.
13. A conjugate having Formula (IId): ##STR00089## wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and w ranges from 1 to 12.
14. The conjugate of claim 13, wherein Linker has the Formula (Ia): ##STR00090## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.1); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.
15. The conjugate of claim 13, wherein w ranges from 1 to 8; and wherein R.sup.a and R.sup.b are each hydrogen.
16. The conjugate of claim 13, wherein A is dibenzocyclooctyne.
17. The conjugate of claim 13, wherein Linker comprises a PEG group.
18. A conjugate having Formula (Id): ##STR00091## wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and Tissue Reactive Moiety is selected from the group consisting of: ##STR00092## ##STR00093##
19. The conjugate of claim 18, wherein Linker has the Formula (Ia): ##STR00094## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided to the Office upon request and the payment of the necessary fee.
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DETAILED DESCRIPTION
(42) In general, the present disclosure is directed to click conjugates, as well as methods of employing click conjugates for detecting one or more targets present in a biological sample. In some embodiments, the click conjugates (or kits comprising one or more click conjugates) are used in a multiplex assay to detect multiple targets within a tissue sample, either simultaneously or sequentially. These and other aspects of the present disclosure are detailed more fully herein.
(43) Definitions
(44) As used herein, the singular terms a, an, and the include plural referents unless context clearly indicates otherwise. Similarly, the word of is intended to include and unless the context clearly indicates otherwise. The term includes is defined inclusively, such that includes A or B means including A, B, or A and B.
(45) The terms comprising, including, having, and the like are used interchangeably and have the same meaning. Similarly, comprises, includes, has, and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of comprising and is therefore interpreted to be an open term meaning at least the following, and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, a device having components a, b, and c means that the device includes at least components a, b and c. Similarly, the phrase: a method involving steps a, b, and c means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.
(46) As used herein, alkaline phosphatase (AP) is an enzyme that removes (by hydrolysis) and transfers phosphate group organic esters by breaking the phosphate-oxygen bond, and temporarily forming an intermediate enzyme-substrate bond. For example, AP hydrolyzes naphthol phosphate esters (a substrate) to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes.
(47) As used herein, the term antibody, occasionally abbreviated Ab, refers to immunoglobulins or immunoglobulin-like molecules, including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, (e.g., in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules. Antibody further refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies may be composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. The term antibody also includes intact immunoglobulins and the variants and portions of them well known in the art.
(48) As used herein, the phrase antibody conjugates, refers to those antibodies conjugated (either directly or indirectly) to one or more labels, where the antibody conjugate is specific to a particular target and where the label is capable of being detected (directly or indirectly), such as with a secondary antibody (an anti-label antibody). For example, an antibody conjugate may be coupled to a hapten such as through a polymeric linker and/or spacer, and the antibody conjugate, by means of the hapten, may be indirectly detected. As an alternative example, an antibody conjugate may be coupled to a fluorophore, such as through a polymeric linker and/or spacer, and the antibody conjugate may be detected directly. Antibody conjugates are described further in US Publication No. 2014/0147906 and U.S. Pat. Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191. By way of a further example, the term antibody conjugates includes those antibodies conjugated to an enzyme, e.g. HRP or AP.
(49) As used herein, the term antigen refers to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins.
(50) As used herein, the term a biological sample can be any solid or fluid sample obtained from, excreted by or secreted by any living organism, including without limitation, single celled organisms, such as bacteria, yeast, protozoans, and amoebas among others, multicellular organisms (such as plants or animals, including samples from a healthy or apparently healthy human subject or a human patient affected by a condition or disease to be diagnosed or investigated, such as cancer). For example, a biological sample can be a biological fluid obtained from, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for example, fluid obtained from an abscess or any other site of infection or inflammation), or fluid obtained from a joint (for example, a normal joint or a joint affected by disease). A biological sample can also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or can include a cell (whether a primary cell or cultured cell) or medium conditioned by any cell, tissue or organ. In some examples, a biological sample is a nuclear extract. In certain examples, a sample is a quality control sample, such as one of the disclosed cell pellet section samples. In other examples, a sample is a test sample. Samples can be prepared using any method known in the art by of one of ordinary skill. The samples can be obtained from a subject for routine screening or from a subject that is suspected of having a disorder, such as a genetic abnormality, infection, or a neoplasia. The described embodiments of the disclosed method can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as normal samples. Samples can include multiple targets that can be specifically bound by one or more detection probes.
(51) As used herein, the term chromophore refers to a molecule or a part of a molecule responsible for its color. Color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others. A molecule having an energy difference between two different molecular orbitals falling within the range of the visible spectrum may absorb visible light and thus be aptly characterized as a chromophore. Visible light incident on a chromophore may be absorbed thus exciting an electron from a ground state molecular orbital into an excited state molecular orbital.
(52) As used herein, the term conjugate refers to two or more molecules or moieties (including macromolecules or supra-molecular molecules) that are covalently linked into a larger construct. In some embodiments, a conjugate includes one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or more other molecules moieties.
(53) As used herein, the terms couple or coupling refers to the joining, bonding (e.g. covalent bonding), or linking of one molecule or atom to another molecule or atom.
(54) As used herein, haptens are small molecules that can combine specifically with an antibody, but typically are substantially incapable of being immunogenic except in combination with a carrier molecule. In some embodiments, haptens include, but are not limited to, pyrazoles (e.g. nitropyrazoles); nitrophenyl compounds; benzofurazans; triterpenes; ureas (e.g. phenyl ureas); thioureas (e.g. phenyl thioureas); rotenone and rotenone derivatives; oxazole (e.g. oxazole sulfonamides); thiazoles (e.g. thiazole sulfonamides); coumarin and coumarin derivatives; and cyclolignans. Additional non-limiting examples of haptens include thiazoles; nitroaryls; benzofurans; triperpenes; and cyclolignans. Specific examples of haptens include di-nitrophenyl, biotin, digoxigenin, and fluorescein, and any derivatives or analogs thereof. Other haptens are described in U.S. Pat. Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of which are incorporated herein by reference in their entirety. The haptens themselves may be suitable for direct detection, i.e. they may give off a suitable signal for detection.
(55) As used herein, horseradish peroxidase (HRP) is an enzyme that can be conjugated to a labeled molecule. It produces a colored, fluorimetric, or luminescent derivative of the labeled molecule when incubated with a proper substrate, allowing it to be detected and quantified. HRP acts in the presence of an electron donor to first form an enzyme substrate complex and then subsequently acts to oxidize an electronic donor. For example, HRP may act on 3,3-diaminobenzidinetrahydrochloride (DAB) to produce a detectable color. HRP may also act upon a labeled tyramide conjugate, or tyramide like reactive conjugates (i.e. ferulate, coumaric, caffeic, cinnamate, dopamine, etc.), to deposit a colored or fluorescent or colorless reporter moiety for tyramide signal amplification (TSA).
(56) As used herein, the terms multiplex, multiplexed, or multiplexing refer to detecting multiple targets in a sample concurrently, substantially simultaneously, or sequentially. Multiplexing can include identifying and/or quantifying multiple distinct nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) both individually and in any and all combinations.
(57) As used herein, the term primary antibody refers to an antibody which binds specifically to the target protein antigen in a tissue sample. A primary antibody is generally the first antibody used in an immunohistochemical procedure.
(58) As used herein, a quinone methide is a quinone analog where one of the carbonyl oxygens on the corresponding quinone is replaced by a methylene group (CH2) to form an alkene.
(59) As used herein, the term secondary antibody herein refers to an antibody which binds specifically to a primary antibody, thereby forming a bridge between the primary antibody and a subsequent reagent (e.g. a label, an enzyme, etc.), if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure.
(60) As used herein, the term specific binding entity refers to a member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10-3 M greater, 10-4 M greater or 10-5 M greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Particular examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.
(61) As used herein, the term target refers to any molecule for which the presence, location and/or concentration is or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens, such as haptens covalently bonded to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.
(62) Click Conjugates
(63) The present disclosure provides two general subsets of click conjugates. A first subset of click conjugates comprises a tissue reactive moiety coupled to a reactive functional group through an optional linker. In some embodiments, this first subset of click conjugates is used as first members of pairs of click conjugates. A second subset of click conjugates comprises one or more reporter moieties coupled to a reactive functional group through an optional linker. In some embodiments, this second subset of click conjugates are used as second members of pairs of click conjugates. It will be appreciated that the different subsets of click conjugates disclosed herein may serve as modular building blocks such that when any two conjugates having appropriate reactive function groups are combined (a pair of click conjugates), they may undergo a reaction and form a covalent bond, thereby coupling the two conjugates to form a click adduct having the desired structure or component parts.
(64) As will be described further herein, the click adducts formed may serve as species suitable for detecting targets in a biological assay. Without wishing to be bound by any particular theory, it is believed that the click conjugates disclosed herein are stable in aqueous media, and thus suitable for use in certain biological assays, including in IHC and ISH. Moreover, it is believed that the click conjugates have a large thermodynamic driving force that favors a fast reaction providing a single product. In addition, the solubility of any of the click conjugates described herein may be tuned to meet the requirements of any particular assay, and such tuning may be accomplished by, for example, introducing a water soluble linker or water soluble linker components into the conjugates. Moreover, the reactions comprising the click conjugates described herein may be carried out in a wide variety of buffers and thus at a wide variety of pHs, allowing the skilled artisan to choose the ideal conditions for reporter stability.
(65) In one aspect of the present disclosure are click conjugates of Formula (I):
ALinker
B(I), wherein A is a reactive functional group, Linker is an optional linking group, B is selected from a tissue reactive moiety or a reporter moiety.
(66) As used herein, the term tissue reactive refers to a moiety that is capable of reacting with an enzyme. As such, when a click conjugate comprising a tissue reactive moiety is reacted with an appropriate enzyme, the tissue reactive moiety portion of the click conjugate undergoes a structural, conformational, and/or electronic change, thereby providing a tissue reactive species (an intermediate, including radical intermediates) suitable for bonding directly or indirectly onto (or, to the extent possible, within) a biological sample. For example, where the tissue reactive moiety is a tyramide or derivative thereof, when the tyramide reacts with an appropriate enzyme (e.g. an HRP), a tyramide radical species (an intermediate) is formed. This highly reactive tyramide radical species is capable of bonding to tyrosine residues in biological samples. In a similar manner, a quinone methide precursory moiety, upon reaction with an appropriate enzyme (e.g. AP), is converted to a quinone methide, which is believed to be highly reactive with nucleophiles in a biological sample. The role of the tissue reactive moiety portion of any click conjugate, its interaction with a suitable enzyme, and the formation of an immobilized tissue-click conjugate complex is described further herein.
(67) In some embodiments, A is selected from the group consisting of dibenzocyclooctyne (DBCO), trans-cyclooctene (TCO), azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. In some embodiments, A is selected from a group capable of undergoing a photo-initiated reaction.
(68) The click conjugates optionally comprise a Linker. In some embodiments, a Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S. In some embodiments, the Linker comprises one or more groups selected from amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine groups. The linker chain may also comprise an aromatic group, including heteroaromatic groups, wherein the heteroaromatic groups comprise 1 to 4 heteroatoms selected from O, N, or S.
(69) In some embodiments, the Linker has the structure depicted in Formula (Ia):
(70) ##STR00024## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. In some embodiments, X and Y include carbonyl groups, amide groups, ester groups, ester groups, substituted or unsubstituted aryl groups, or any combination thereof. In other embodiments, d and e are integers ranging from 2 to 10. In yet other embodiments, d and e are integers ranging from 2 to 6.
(71) In some embodiments, the Linker has the structure depicted in Formula (Ib):
(72) ##STR00025## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently either 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.
(73) In some embodiments, X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 2 and 8 carbon atoms and optionally having one or more O, N, or S heteroatoms.
(74) In some embodiments, the Linker has the structure depicted in Formula (Ic):
(75) ##STR00026## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently either 0 or 1; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms.
(76) In other embodiments, d and e are integers ranging from 2 to 10. In yet other embodiments, d and e are integers ranging from 2 to 6.
(77) The alkylene oxide based Linker of Formulas (Ia), (Ib), and (Ic), are represented herein by reference to glycols, such as ethylene glycols. In some embodiments, the incorporation of such alkylene oxide linkers is believed to increase the hydrophilicity of the click conjugate. A person of ordinary skill in the art will appreciate that, as the number alkylene oxide repeat units in the linker increases, the hydrophilicity of the conjugate also may increase. Additional heterobifunctional polyalkyleneglycol spacers useful for practicing certain disclosed embodiments of the present disclosure are described in assignee's co-pending applications, including Nanoparticle Conjugates, U.S. patent application Ser. No. 11/413,778, filed Apr. 28, 2006; Antibody Conjugates, U.S. application Ser. No. 11/413,415, filed Apr. 27, 2006; and Molecular Conjugate, U.S. Provisional Patent Application No. 60/739,794, filed Nov. 23, 2005; all of which applications are incorporated herein by reference.
(78) Tissue Reactive Precursor Moiety Click Conjugates
(79) In some embodiments, the click conjugates of the present disclosure have the structure of Formula (Id):
(80) ##STR00027## wherein the Tissue Reactive Precursor Moiety is (i) a tyramide or a derivative or analog thereof, or (ii) a quinone methide precursor; and where A and Linker are as defined above. Exemplary quinone methide precursor derivatives suitable for incorporation into the disclosed click conjugates of Formula (I) include those recited in PCT/EP2015/053556, entitled Quinone Methide Analog Signal Amplification, having an international filing date of Feb. 20, 2015, the disclosure of which is hereby incorporated by reference herein in its entirety.
(81) Quinone Methide Click Conjugates
(82) In some embodiments, the compounds have Formula (II):
ALinker
U(II) wherein A is as defined above; Linker is an optional linking group as defined above; and U is a quinone methide precursor, derivative, or analog thereof.
(83) In some embodiments, the compounds of Formula (II) are a first member of a pair of click conjugates.
(84) As used herein, quinone methide precursors are a class of conjugated compounds that, when reacted with an appropriate enzyme (e.g. AP), are converted to a highly reactive quinone methide. As noted above, quinone methide precursors and their conversion to quinone methides are described in PCT/EP2015/053556, the disclosure of which is hereby incorporated by reference herein in its entirety.
(85) In some embodiments, the quinone methide precursor portion of the conjugate of Formula (II) is derived from one of the following quinone methide precursor derivatives:
(86) ##STR00028##
(87) In some embodiments, the conjugates of Formula (II) have the structure of Formula (IIa):
(88) ##STR00029## wherein Linker and A are as defined herein, R.sup.1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar; R.sup.2 is a halide; R.sup.3, R.sup.5, and R.sup.6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms; R.sup.4 is a hydrogen, an aliphatic group having between 1 and 4 carbon atoms, or the group CH(R.sup.2)R.sup.7-[Linker]-A; R.sup.7 is (CH.sub.2).sub.wNH, O(CH.sub.2).sub.wNH, N(H)C(O)(CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2).sub.wNH, (CH.sub.2).sub.wO, O(CH.sub.2).sub.wO, O(CH.sub.2CH.sub.2O).sub.w, N(H)C(O)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2CH.sub.2O).sub.w, (CH.sub.2).sub.wS, O(CH.sub.2).sub.wS, N(H)C(O)(CH.sub.2).sub.wS, C(O)N(H)(CH.sub.2).sub.wS, (CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)N(H)(CH.sub.2)NHC(O)CH(CH.sub.3)(CH.sub.2).sub.wNH, or N(H)(CH2).sub.wNH, where w is an integer ranging from 1 to 12. When R1 is a sugar, the sugar may be selected from glucose, -glucose, a-galactoside, -galactoside, a-glucuronose, neuraminide, or -glucuronose.
(89) In other embodiments, the conjugates of Formula (II) have the structure of Formula (IIb):
(90) ##STR00030## where R.sup.1 is selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar; and where R.sup.7 is (CH.sub.2).sub.wNH, O(CH.sub.2).sub.wNH, N(H)C(O)(CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2).sub.wNH, (CH.sub.2).sub.wO, O(CH.sub.2).sub.wO, O(CH.sub.2CH.sub.2O).sub.w, N(H)C(O)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2CH.sub.2O).sub.w, (CH.sub.2).sub.wS, O(CH.sub.2).sub.wS, N(H)C(O)(CH.sub.2).sub.wS, C(O)N(H)(CH.sub.2).sub.wS, (CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)(CH.sub.2CH.sub.2O), CH.sub.2CH.sub.2NH, C(O)N(H)(CH.sub.2)NHC(O)CH(CH.sub.3)(CH.sub.2).sub.wNH, or N(H)(CH2).sub.wNH, where w is an integer ranging from 1 to 12.
(91) In some embodiments of the conjugates of Formula (IIb), R.sup.1 is a phosphate and R.sup.7 is C(O)N(H)(CH2).sub.wNH, and w ranges from 2 to 10.
(92) In yet other embodiments, the conjugates of Formula (II) have the structure of Formula (IIc):
(93) ##STR00031## where R.sup.7 is (CH.sub.2).sub.wNH, O(CH.sub.2).sub.wNH, N(H)C(O)(CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2).sub.wNH, (CH.sub.2).sub.wO, O(CH.sub.2).sub.wO, O(CH.sub.2CH.sub.2O).sub.w, N(H)C(O)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2CH.sub.2O).sub.w, (CH.sub.2).sub.wS, O(CH.sub.2).sub.wS, N(H)C(O)(CH.sub.2).sub.wS, C(O)N(H)(CH.sub.2).sub.wS, (CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)N(H)(CH.sub.2)NHC(O)CH(CH.sub.3)(CH.sub.2).sub.wNH, or N(H)(CH2).sub.wNH, where w is an integer ranging from 1 to 12.
(94) In some embodiments, R.sup.7 is C(O)N(H)(CH.sub.2).sub.wNH and w is as defined above. In other embodiments, R.sup.7 is C(O)N(H)(CH.sub.2).sub.wNH and w ranges from 2 to 6.
(95) In yet further embodiments, the conjugates of Formula (II) have the structure of Formula (IId):
(96) ##STR00032## where w ranges from 1 to 12, and Linker and A are as defined above.
(97) In some embodiments w ranges from 1 to 8. In other embodiments, w ranges from 2 to 8. In yet other embodiments, w ranges from 2 to 6. In further embodiments, w is 6.
(98) Specific examples of the compounds of Formulas (II) include the following:
(99) ##STR00033##
(100) The quinone methide precursor click conjugates of Formula (II) may be synthesized according to any method as known to those of ordinary skill in the art. In some embodiments, a reagent comprising the desired reactive functional group and linker are merely coupled with a quinone methide precursor or derivative or analog thereof as illustrated in the reaction schemes which follow. For example, a quinone methide precursor having a terminal amine group may be coupled to a compound comprising an amine reactive group (e.g. active esters such as N-Hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like).
(101) In some of the specific examples below, a Click partner having an NHS-ester group is coupled with a quinone methide precursor having a terminal amine. In some embodiments, the reaction takes place in DMSO and is allowed to react for 60 minutes. The reaction is then diluted with methanol and directly purified by preparative HPLC.
(102) ##STR00034##
(103) Tyramide Click Conjugates
(104) In other embodiments, the compounds have Formula (III):
ALinker
M(III) wherein A is as defined above; Linker is an optional linking group as defined above; and M is tyramide or a derivative or analog thereof. In some embodiments, the compounds of Formula (III) are a first member of a pair of click partners.
(105) In some embodiments, the conjugates of Formula (III) comprise a tyramide derived from a compound having the structure of Formula (IIIa)
(106) ##STR00035## wherein each R group is independently selected from hydrogen or lower alkyl group (which may be straight chain or branched) having between 1 and 4 carbon atoms, and where Linker and A are as defined herein.
(107) In some embodiments, the compounds of Formula (III) comprise a tyramide derived from a compound having the structure of Formula (IIIb)
(108) ##STR00036## where A is selected from an azide, a thiol, a 1,3-nitrone, a hydrazine, or a hydroxylamine, and where Linker is as defined herein.
(109) In some embodiments, the compounds of Formula (III) comprise a moiety from a compound having the structure of Formulas (IIIc) or (IIId):
(110) ##STR00037##
(111) Non-limiting examples of particular tyramide click conjugates include the following:
(112) ##STR00038##
(113) The tyramide click conjugates of Formula (III) may be synthesized according to any method as known to those of ordinary skill in the art. In some embodiments, a reagent comprising the desired reactive functional group and linker are merely coupled with a tyramide or derivative or analog thereof as illustrated in the reaction schemes below. For example, a tyramide (having a terminal amine group) may be coupled to a compound comprising an amine reactive group (e.g. active esters such as N-Hydroxysuccinimide (NHS) or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like).
(114) In some of the specific examples below, a Click partner having an NHS-ester group is coupled with a tyramide. In some embodiments, the reaction takes place in DMSO and is allowed to react for 60 minutes. The reaction is then diluted with methanol and directly purified by preparative HPLC.
(115) ##STR00039##
(116) Reporter Moiety Click Conjugates
(117) In other embodiments, the click conjugates of the present disclosure have the structure of Formula (IV):
ALinker
Z(IV), wherein A is as defined above; Linker is an optional linking group as defined above; and Z comprises at least one reporter moiety (the terms reporter moiety and reporter are used interchangeably herein). In some embodiments, the compounds of Formula (IV) are a second member of a pair of click partners.
(118) In some embodiments, the conjugates of Formula (IV) comprise one reporter moiety, and thus the group Z is a reporter moiety coupled directly, or indirectly through a Linker, to the reactive functional group A. In other embodiments, the conjugates of Formula (IV) comprise a plurality of reporter moieties, and thus Z represents a group having two or more reporter moieties. In embodiments where Z represents a group having two or more reporter moieties, the group Z is coupled directly, or indirectly through a Linker, to the reactive functional group A.
(119) In some embodiments, Z comprises two reporters. In other embodiments, Z comprises four reporters. In other embodiments, Z comprises six reporters. In yet other embodiments, Z comprises greater than 6 reporters. In embodiments where Z comprises more than one reporter, the reporters may be the same or different. For example, Z may comprise two of the same chromogens (e.g. two TAMRA chromogens). Alternatively, Z may comprise two different chromogens (e.g. TAMRA and cy5).
(120) In some embodiments, Z comprises at least two reporter moieties, and the at least two reporter moieties are linked to each other via a straight chain or branched aliphatic group, optionally comprising one or more heteroatoms. In other embodiments, Z comprises at least two reporter moieties, and the at least two reporter moieties are linked to each other via a dendrimer or branched polymer.
(121) In some embodiments, the compounds of Formula (IV) have the structure of Formula (V):
ALinker
Scaffold
Z].sub.v(V), wherein Scaffold is a group capable of coupling multiple reporter moieties, and v is an integer ranging from between 1 and 20.
(122) In some embodiments, Scaffold is a polyamine (e.g. norspermidine, spermine, and derivatives or analogs thereof; or a polyamine comprising between 2 and 10 amine groups); a heterobifunctional linker (e.g. lysine or lysine derivative); a dendrimer (e.g. polyamidoamine (PAMAM) dendrimers, Janus dendrimers (i.e. dendrimers constituted of two dendrimeric wedges and terminated by two different functionalities), and bis-MPA dendrimers and derivatives thereof); or a polymer. In some embodiments, Scaffold is a bond.
(123) In some embodiments, the click conjugates have the following formulas:
(124) ##STR00040## where Linker and Z are as defined herein,
(125) In some embodiments, the reporter moiety is selected from a chromophore, a fluorophore, an enzyme, a hapten, or a chelator.
(126) Non-limiting examples of suitable haptens include pyrazoles, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenyl ureas, and even more particularly phenyl thioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans, exemplified by Podophyllotoxin and Podophyllotoxin derivatives; and combinations thereof. Further examples of haptens and methods for their synthesis and use are disclosed in U.S. Pat. No. 7,695,929, the disclosure of which is hereby incorporated in its entirety herein by reference.
(127) In some embodiments, suitable haptens include BD (benzodiazepine), BF (benzofurazan), DABSYL (4-(dimethylamino)azobenzene-4-sulfonamide, which has a max of about 436 nm), DCC (7-(diethylamino)coumarin-3-carboxylic acid), DIG (digoxigenin), DNP (dinitrophenyl), HQ (3-hydroxy-2-quinoxalinecarbamide) NCA (nitrocinnamic acid), NP (nitropyrazole), PPT (Podophyllotoxin), Rhod (rhodamine), ROT (rotenone), and TS (thiazolesulfonamide). Other suitable haptens include biotin and fluorescein derivatives (FITC (fluoresceinisothiocyanate), TAMRA (tetramethylrhodamine), Texas Red), and Rhodamine 110 (Rhodamine).
(128) Non-limiting examples of suitable chromophores include coumarin and coumarin derivatives.
(129) Examples of coumarin-based chromophores include DCC and 2,3,6,7-tetrahydro-11-oxo-1H,5H,1 1H-[1]benzopyrano[6,7,8-ij]quinolizine-10-carboxylic acid. Other suitable chromophores include diazo-containing chromogens, such as tartrazine. Yet other suitable chromophores include triarylmethane, including those provided below
(130) ##STR00041##
(131) Other non-limiting examples of suitable chromophores include those provided below:
(132) ##STR00042##
(133) Other suitable chromophores include annulated chromophores, such as those provided below:
(134) ##STR00043## ##STR00044##
(135) Fluorophores belong to several common chemical classes including coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines, resorufins, luminophores and cyanines. Additional examples of fluorescent molecules can be found in Molecular Probes HandbookA Guide to Fluorescent Probes and Labeling Technologies, Molecular Probes, Eugene, OR, TheroFisher Scientific, 11th Edition. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaraine derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent moiety is selected from a CF dye (available from Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY (available from Invitrogen), Alexa Fluor (available from Invitrogen), DyLight Fluor (e.g. DyLight 649) (available from Thermo Scientific, Pierce), Atto and Tracy (available from Sigma Aldrich), FluoProbes (available from Interchim), Abberior Dyes (available from Abberior), DY and MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (available from Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau and Square Dyes (available from SETA BioMedicals), Quasar and Cal Fluor dyes (available from Biosearch Technologies), SureLight Dyes (available from APC, RPEPerCP, Phycobilisomes)(Columbia Biosciences), and APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).
(136) Suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, neuraminidase, -galactosidase, -glucuronidase or -lactamase. In other embodiments, enzymes include oxidoreductases or peroxidases (e.g. HRP, AP). Use of an enzyme as a reporter moiety is further illustrated with reference to
(137) In some embodiments, the reporter moiety is a chelator or chelating agent, which may become chelated in the presence of a lanthanide (e.g. europium). Without wishing to be bound by any particular theory, it is believed that the lanthanide atoms may be detected using Inductively Coupled Plasma Mass Spectral Imaging (ICP-MSI). In addition, the lanthanides may be detected using time-resolved fluorescence microscopy, which take advantage of the relatively long lifetimes of the lanthanide luminescence compared to conventional fluorophores. In order to visualize the lanthanides, an antenna ligand must be present to absorb and transfer energy to the normally poorly-absorbing lanthanides. The reaction product of the DBCO-azide Click reaction may be able to act as the antenna ligand, greatly simplifying the design of these systems.
(138) An example of a compound of Formula (IV) comprising an azide reactive group conjugated to a chelator moiety is shown below:
(139) ##STR00045##
(140) In some specific embodiments, the reporter moiety portion (Z) of the click conjugates of Formula (IV) are selected from:
(141) ##STR00046##
(142) In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVa)
(143) ##STR00047## wherein A is as recited above. While Formula (IVa) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted.
(144) In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVb)
(145) ##STR00048## wherein A is as recited above. While Formula (IVb) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted.
(146) In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVc)
(147) ##STR00049## wherein A is as recited above. While Formula (IVc) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted.
(148) In some embodiments, the compounds of Formula (IV) comprises the formula of Formula (IVd)
(149) ##STR00050## wherein A is as recited above. While Formula (IVd) depicts the compound has comprising a PEG linker, other suitable linkers may be substituted.
(150) Specific non-limiting examples of conjugates of the Formula (IV) include the following:
(151) ##STR00051## ##STR00052##
(152) A non-limiting example of a conjugate of Formula (V) is illustrated below:
(153) ##STR00053##
(154) The reporter moiety compounds of Formula (IV) may be synthesized according to methods known to those of ordinary skill in the art. Example synthetic procedure for the coupling of an NHS ester to an amine. This procedure can be applied to the reaction of any tyramine or quinone methide precursor containing an amine or NHS functionality with a Click partner containing an amine or NHS ester functionality. It can also be applied to the reaction of a reporter group (chromogen, hapten, etc.) containing an amine or NHS functionality with a Click partner containing an amine or NHS ester functionality.
(155) Tyramide-peg5-DBCO. Tyramine (1.1 eq, 110 mg, 0.79 mmol) was dissolved in DMSO (3 mL) followed by addition of triethylamine (5.0 eq, 360 mg, 3.6 mmol). DBCO-peg5-DBCO (1.0 eq, 500 mg, 0.72 mmol) was then added, and the resulting reaction mixture was stirred for 1 hour at room temperature. The reaction mixture was diluted with MeOH (2 mL) and the resulting mixture was purified by preparative RP-HPLC (C18; 40 mL/min; 0.05% TFA in H2O:MeCN 95:5 to 5:95 over 40 minutes) to give Tyramide-peg5-DBCO (450 mg, 87% yield) as a colorless glass after removal of solvents under high vacuum. MS (ESI) m/z (M+H)+ calcd for C40H50N3O9+ 716.4, found 716.6.
(156) ##STR00054##
(157) Coupling of Click Conjugate Pairs
(158) The skilled artisan will recognize that the click conjugates disclosed herein are suitable for coupling to each other to form click adducts. The skilled artisan will also recognize that for one member of a pair of click conjugates to react with another member of the pair of click conjugates, and thus form a covalent bond, the two members of the pair of click conjugates must have reactive functional groups capable of reacting with each other. The table which follows exemplifies different pairs of reactive functional groups that will react with each other to form a covalent bond.
(159) TABLE-US-00001 Reactive Functional Group Reactive Functional Group on a First Member of a on a Second Member of a Pair of Click Conjugates Pair of Click Conjugates DBCO Azide Alkene Tetrazine TCO Tetrazine Maleimide Thiol DBCO 1,3-Nitrone Aldehyde or ketone Hydrazine Aldehyde or ketone Hydroxylamine Azide DBCO Tetrazine TCO Thiol Maleimide 1,3-Nitrone DBCO Hydrazine Aldehyde or ketone Hydroxylamine Aldehyde or ketone Tetrazine Alkene
(160) Specific non-limiting examples of pairs of click conjugates possessing these reactive function groups are illustrated in
(161) In some embodiments, the click conjugates are coupled via strain-promoted azide-alkyne cycloaddition (SPAAC), or TCO-tetrazine ligation (TTL). SPAAC involves the reaction between azides and strained alkynes, whose high energy allows the 1,3-dipolar cycloaddition to occur in the absence of a Cu(I) catalyst (required for traditional azide-alkyne click chemistry). In some embodiments, dibenzocyclooctynes are utilized as the strained cyclooctyne due to their commercial availability and literature precedent. TTL utilizes the reaction between trans-cyclooctene and tetrazine to form a dihydropyridazine bond. These reagents are also commercially available and have been shown to react orthogonally to the SPAAC system.
(162) The schematics which follow further illustrate the coupling of pairs of click conjugates comprising difference reactive functional groups. In the schemes which follow, one member of the pair of click conjugates is provided as an immobilized, tissue-click conjugate complex. As will be described further herein, the immobilized, tissue-click conjugate complex is formed through the reaction of a click conjugate having either Formula (II) or (III) with an appropriate enzyme, and the subsequent coupling of a reactive intermediate generated therefrom with tissue.
(163) For example, Scheme 2 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive azide group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.
(164) ##STR00055##
(165) Likewise, Scheme 3 illustrates the reaction between an immobilized tissue-click conjugate complex having a TCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive tetrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.
(166) ##STR00056##
(167) Scheme 4 again illustrates the reaction between an immobilized tissue-click conjugate complex having a malemide reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive thiol group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.
(168) ##STR00057##
(169) Scheme 5 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive 1,3-nitrone group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.
(170) ##STR00058##
(171) Scheme 6 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde reactive functional group, and a second click conjugate of Formula (IV) a comprising reactive hydrazine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.
(172) ##STR00059##
(173) Scheme 7 illustrates the reaction between an immobilized tissue-click conjugate having an aldehyde reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive hydroxylamine group and at least one reporter moiety Z. In some embodiments, the resulting adduct comprises one reporter moiety. In other embodiments, the resulting adduct comprises at least two reporter moieties, such as joined via a scaffold (e.g. a lysine linker or a dendrimer). In some embodiments, the at least one reporter moiety Z is a chromophore. In some embodiments, the at least one chromophore is selected from TAMRA, Cy5, Dabsyl, and Dabcyl. In some embodiments, the adduct comprises two TAMRA chromophores, such as linked via lysine.
(174) ##STR00060##
(175) Scheme 8 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) comprising a reactive azide group and a chelator as the reporter Z. In some embodiments, a resulting intermediate adduct comprises a chelator which, when a lanthanide is introduced, forms a chelated adduct complex, suitable for detection with MSI.
(176) ##STR00061##
(177) Scheme 9A illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (IV) or Formula (V) comprising a reactive azide group coupled to a dendrimer, the dendrimer coupled to two, four, or eight reporter moieties, as shown. Without wishing to be bound by any particular theory, it is believed that the use of dendrimers allows for the incorporation of a plurality of reporters (which may be the same or different), thus providing significant signal amplification.
(178) ##STR00062##
(179) Scheme 9B illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula (V) comprising a reactive azide group coupled to a dendrimer (PAMAM), the dendrimer coupled to four reporter moieties Z.
(180) ##STR00063##
(181) Scheme 10 illustrates the reaction between an immobilized tissue-click conjugate complex having a DBCO reactive functional group, and a second click conjugate of Formula Formula (V) comprising a reactive azide group coupled to a Z group comprising two chromophores, where the two chromophores are linked via a lysine group. While the chromogens are depicted as being the same, the skilled artisan will recognize that the chromogens linked via the lysine group may be different.
(182) ##STR00064##
(183) Specific examples of immobilized, tissue-click conjugate complexes and their reaction with click conjugates comprising specific reporter moieties are represented in
(184) Methods of Detecting Targets in a Sample Using Click Conjugates
(185) The present disclosure also provides methods of detecting one or more targets within a tissue sample using pairs of any of the click conjugates. While certain disclosed embodiments, examples, or figures herein may refer to the use of the click conjugates in conjunction in an IHC assay, the skilled artisan will appreciate that the click conjugates may also be used in situ hybridization (ISH) assays, or any combination of IHC and ISH assays. The skilled artisan will also appreciate that the click conjugates may be used in both simplex assays and multiplex assays.
(186) The methods described herein refer to pairs of click conjugates suitable for use in biological assays. In those assays, one member (or partner) of a particular pair of click conjugates comprises a conjugate of either Formula (II) or (III), and another member of the pair of click conjugates comprises a conjugate of Formula (IV) or Formula (V). In general, a first member of a pair of click conjugates is covalently deposited onto tissue using QMSA or TSA. Then, a second member of the pair of click conjugates comprising a reporter molecule (i.e. chromophore, fluorophore, enzyme, hapten) is applied to the tissue. The click reaction between the two click partners occurs rapidly, covalently binding the reporter molecules to tissue in the locations dictated by the QMSA or TSA chemistries. Moreover, and as noted herein, the presently disclosed amplification methodology allows for the reporter moiety to be separated from the QMSA or TSA assay conditions, which is believed to enhance signal intensity.
(187) For example,
(188) With reference to
(189)
(190) Similarly, and with reference to
(191)
(192) In some embodiments, the methods of detecting targets in a biological sample comprise the following steps. First, the biological sample is contact with a first detection probe specific to a first target. The first detection probe may be a primary antibody or a nucleic acid probe. Subsequently, the sample is contacted with a first labeling conjugate, the first labeling conjugate comprising a first enzyme. In some embodiments, the first labeling conjugate is a secondary antibody specific for either the primary antibody or to a label conjugated to the nucleic acid probe. Next, the biological sample is contacted with a first member of a pair of click conjugates, the first member of the pair of click conjugates having the structure of any of the compounds of Formulas (II) or (III). As described herein, the first enzyme cleaves the first member of the pair of click conjugates, thereby converting the first member into a reactive intermediate which covalently binds to the biological sample proximally to or directly on the first target. Next, a second member of the pair of click conjugates is introduced, the second member of the pair of click conjugates comprising a first reporter moiety and a second reactive functional group, where the second reactive functional group of the second member of the first pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the pair of click conjugates. The second member of the pair of click conjugates may have the structure as provided in Formula (IV). Finally, signals from the first reporter moiety are detected.
(193) With reference to
(194) In some embodiments, and prior to the introduction of any detection reagents, the tissue samples are pre-treated with an enzyme inactivation composition to substantially or completely inactivate endogenous peroxidase activity. For example, where cells or tissues contain endogenous peroxidase, use of a HRP conjugated antibody may result in high, non-specific background staining. This non-specific background can be reduced by pre-treatment of the sample with an enzyme inactivation composition as disclosed herein. In some embodiments, the samples are pre-treated with hydrogen peroxide only (about 1% to about 3% by weight of an appropriate pre-treatment solution) to reduce endogenous peroxidase activity.
(195) Referring again to
(196) The first specific binding moiety-target complex is subsequently labeled with a first enzyme through the first specific binding moiety (step 110). In some embodiments, the labeling of the target complex may be achieved with a secondary antibody, the secondary antibody being an anti-antibody antibody (e.g. one that is specific to a primary antibody, namely an anti-antibody antibody) or an anti-label antibody (e.g. an anti-label antibody or an anti-hapten antibody), the secondary antibody being conjugated to an enzyme (e.g. HRP, AP, etc.).
(197) The tissue sample is then contacted with a first member of a first pair of click conjugates, where the first member of the first pair of click conjugates comprises a tissue reactive moiety and a first reactive functional group (step 120). The first member of the first pair of click conjugates may have the formula as provided in any of Formulas (II) or (III). The first member of the first pair of click conjugates interacts/reacts with the first enzyme to form a reactive species or intermediate, where the reactive species or intermediate is capable of forming a covalent bond directly or indirectly with the tissue sample either directly on or proximal to the first target. Next, a second member of the first pair of click conjugates is introduced (step 130), the second member of the first pair of click conjugates comprising a first reporter moiety and a second reactive functional group, where the second reactive functional group of the second member of the first pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the first pair of click conjugates. The second member of the first pair of click conjugates may have the structure as provided in Formula (IV). Finally, signals from the first reporter moiety are detected (e.g. brightfield microscopy) (step 140). In some embodiments, the first reporter moiety is a chromophore. In some embodiments, the second member of the first pair of click conjugates is conjugated to at least two chromophores, and where the second member of the first pair of click conjugates has the structure of Formula (V).
(198) The aforementioned process may be repeated for any number of targets within the sample (step 170). In some embodiments, an enzyme inactivation composition may be introduced to substantially or completely inactivate any enzymes from any upstream steps. Then, the tissue sample may be contacted with a second specific binding moiety specific to a second target to provide a second specific binding moiety target complex (step 100). The second specific binding moiety target complex is subsequently labeled with a second enzyme through the second specific binding moiety (step 110). The tissue sample is then contacted with a first member of a second pair of click conjugates, where the first member of the second pair of click conjugates comprises either a quinone methide precursor or a tyramide moiety and a first reactive functional group (step 120). The first member of the second pair of click conjugates interacts with the second enzyme to form a reactive species, where the reactive species is capable of forming a covalent bond either directly on or proximal to the second target. The first member of the first pair of click conjugates may have the formula as provided in any of Formulas (II) or (III). Next, a second member of the second pair of click conjugates is introduced (step 130), the second member of the second pair of click conjugates comprising a second reporter moiety and a second reactive functional group, where the second reactive functional group of the second member of the second pair of click conjugates is capable of reacting with the first reactive functional group of the first member of the second pair of click conjugates. The second member of the second pair of click conjugates may have the structure as provided in Formula (IV). The second reporter moiety is then detected (step 140). The process may be repeated for third, fourth, or nth targets within the tissue sample (step 170).
(199) The skilled artisan will appreciate that the steps illustrated in
(200) Subsequently, a tissue sample having a plurality of enzyme labeled target complexes (steps 100, 110, and 150) may then be contacted with a plurality of click conjugates. First members of pairs of click conjugates may be added simultaneously at step 120 prior to the simultaneous introduction of second members of pairs of click conjugates at step 130. Alternatively, a first member of a first pair of click conjugates may be introduced followed by the introduction of a second member of a first pair of click conjugates, and the sequential introduction of first and second members of pairs of click conjugates may be repeated any number of times (step 160) to introduce a reporter moiety for each of the labeled target complexes.
(201) Advantageously, for the methods just described, the first enzyme and the second enzyme are different enzymes. For example, the first enzyme can be a phosphatase or phosphodiesterase, and the second enzyme can be a peroxidase. In certain embodiments, the first enzyme is alkaline phosphatase and the second enzyme is horseradish peroxidase. Also advantageously, the first enzyme does not interact with the first member of a second pair of click conjugates to deposit a reactive intermediate derived from first member of the second pair of click conjugates proximally to the first target.
(202) Automation
(203) The assays and methods of the present disclosure may be automated and may be combined with a specimen processing apparatus. The specimen processing apparatus can be an automated apparatus, such as the BENCHMARK XT instrument, the SYMPHONY instrument, the BENCHMARK ULTRA instrument sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively, specimens can be manually processed.
(204) The specimen processing apparatus can apply fixatives to the specimen. Fixatives can include cross-linking agents (such as aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone, and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation).
(205) If the specimen is a sample embedded in paraffin, the sample can be deparaffinized with the specimen processing apparatus using appropriate deparaffinizing fluid(s). After the waste remover removes the deparaffinizing fluid(s), any number of substances can be successively applied to the specimen. The substances can be for pretreatment (e.g., protein-crosslinking, expose nucleic acids, etc.), denaturation, hybridization, washing (e.g., stringency wash), detection (e.g., link a visual or marker molecule to a probe), amplifying (e.g., amplifying proteins, genes, etc.), counterstaining, coverslipping, or the like.
(206) The specimen processing apparatus can apply a wide range of substances to the specimen. The substances include, without limitation, stains, probes, reagents, rinses, and/or conditioners. The substances can be fluids (e.g., gases, liquids, or gas/liquid mixtures), or the like. The fluids can be solvents (e.g., polar solvents, non-polar solvents, etc.), solutions (e.g., aqueous solutions or other types of solutions), or the like. Reagents can include, without limitation, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen recovering fluids (e.g., aqueous- or non-aqueous-based antigen retrieval solutions, antigen recovering buffers, etc.), or the like. Probes can be an isolated nucleic acid or an isolated synthetic oligonucleotide, attached to a detectable label. Labels can include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
(207) After the specimens are processed, a user can transport specimen-bearing slides to the imaging apparatus. The imaging apparatus used here is a brightfield imager slide scanner. One brightfield imager is the iScan Coreo brightfield scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the imaging apparatus is a digital pathology device as disclosed in International Patent Application No.: PCT/US2010/002772 (Patent Publication No.: WO/2011/049608) entitled IMAGING SYSTEM AND TECHNIQUES or disclosed in U.S. Patent Application Publication No. 2014/0178169, filed on Feb. 3, 2014, entitled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International Patent Application No. PCT/US2010/002772 and U.S. Patent Application Publication No. 2014/0178169 are incorporated by reference in their entities. In other embodiments, the imaging apparatus includes a digital camera coupled to a microscope.
(208) Counterstaining
(209) Counterstaining is a method of post-treating the samples after they have already been stained with agents to detect one or more targets, such that their structures can be more readily visualized under a microscope. For example, a counterstain is optionally used prior to coverslipping to render the immunohistochemical stain more distinct. Counterstains differ in color from a primary stain. Numerous counterstains are well known, such as hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI (4,6-diamidino-2-phenylindole) is a fluorescent stain that may be used.
(210) In some examples, more than one stain can be mixed together to produce the counterstain. This provides flexibility and the ability to choose stains. For example, a first stain, can be selected for the mixture that has a particular attribute, but yet does not have a different desired attribute. A second stain can be added to the mixture that displays the missing desired attribute. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.
(211) Detection and/or Imaging
(212) Certain aspects, or all, of the disclosed embodiments can be automated, and facilitated by computer analysis and/or image analysis system. In some applications, precise color or fluorescence ratios are measured. In some embodiments, light microscopy is utilized for image analysis. Certain disclosed embodiments involve acquiring digital images. This can be done by coupling a digital camera to a microscope. Digital images obtained of stained samples are analyzed using image analysis software. Color or fluorescence can be measured in several different ways. For example, color can be measured as red, blue, and green values; hue, saturation, and intensity values; and/or by measuring a specific wavelength or range of wavelengths using a spectral imaging camera. The samples also can be evaluated qualitatively and semi-quantitatively. Qualitative assessment includes assessing the staining intensity, identifying the positively-staining cells and the intracellular compartments involved in staining, and evaluating the overall sample or slide quality. Separate evaluations are performed on the test samples and this analysis can include a comparison to known average values to determine if the samples represent an abnormal state.
(213) Kits
(214) In some embodiments, the click conjugates may be utilized as part of a detection kit. In some embodiments, the detection kits comprise at least a first click conjugate in a first container and a second click conjugate in a second container. The first click conjugate is a first member of a pair of click conjugates having a first reactive functional group; and the second click conjugate is a second member of a pair of click conjugates having a second reactive functional group, wherein the first and second reactive functional groups are capable of reacting with each other to form a covalent bond. In some embodiments, the first click conjugate is selected from a compound having the structure of any of Formulas (II) or (III). In some embodiments, the second click conjugate is selected from a compound having the structure of Formula (IV) or Formula (V).
(215) The detection kits may also comprise other reagents including specific binding moieties and secondary antibodies specific to the specific binding moieties, the secondary antibodies conjugated to a detectable label. Of course, any kit may include other agents, including buffers; counterstaining agents; enzyme inactivation compositions; deparaffinization solutions, etc. as needed for manual or automated target detection. The kit may also include instructions for using any of the components of the kit, including methods of applying the kit components to a tissue sample to effect detection of one or more targets therein.
(216) Samples and Targets
(217) Samples include biological components and generally are suspected of including one or more target molecules of interest. Target molecules can be on the surface of cells and the cells can be in a suspension, or in a tissue section. Target molecules can also be intracellular and detected upon cell lysis or penetration of the cell by a probe. One of ordinary skill in the art will appreciate that the method of detecting target molecules in a sample will vary depending upon the type of sample and probe being used. Methods of collecting and preparing samples are known in the art.
(218) Samples for use in the embodiments of the method and with the composition disclosed herein, such as a tissue or other biological sample, can be prepared using any method known in the art by of one of ordinary skill. The samples can be obtained from a subject for routine screening or from a subject that is suspected of having a disorder, such as a genetic abnormality, infection, or a neoplasia. The described embodiments of the disclosed method can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as normal samples. Such normal samples are useful, among other things, as controls for comparison to other samples. The samples can be analyzed for many different purposes. For example, the samples can be used in a scientific study or for the diagnosis of a suspected malady, or as prognostic indicators for treatment success, survival, etc.
(219) Samples can include multiple targets that can be specifically bound by a probe or reporter molecule. The targets can be nucleic acid sequences or proteins. Throughout this disclosure when reference is made to a target protein it is understood that the nucleic acid sequences associated with that protein can also be used as a target. In some examples, the target is a protein or nucleic acid molecule from a pathogen, such as a virus, bacteria, or intracellular parasite, such as from a viral genome. For example, a target protein may be produced from a target nucleic acid sequence associated with (e.g., correlated with, causally implicated in, etc.) a disease.
(220) A target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic acid sequence can have a variable number of nucleic acid residues. For example, a target nucleic acid sequence can have at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues. Similarly, a target polypeptide can vary substantially in size. Without limitation, the target polypeptide will include at least one epitope that binds to a peptide specific antibody, or fragment thereof. In some embodiments that polypeptide can include at least two epitopes that bind to a peptide specific antibody, or fragment thereof.
(221) In specific, non-limiting examples, a target protein is produced by a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) associated with a neoplasm (for example, a cancer). Numerous chromosome abnormalities (including translocations and other rearrangements, amplification or deletion) have been identified in neoplastic cells, especially in cancer cells, such as B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like. Therefore, in some examples, at least a portion of the target molecule is produced by a nucleic acid sequence (e.g., genomic target nucleic acid sequence) amplified or deleted in at least a subset of cells in a sample.
(222) Oncogenes are known to be responsible for several human malignancies. For example, chromosomal rearrangements involving the SYT gene located in the breakpoint region of chromosome 18q11.2 are common among synovial sarcoma soft tissue tumors. The t(18q11.2) translocation can be identified, for example, using probes with different labels: the first probe includes FPC nucleic acid molecules generated from a target nucleic acid sequence that extends distally from the SYT gene, and the second probe includes FPC nucleic acid generated from a target nucleic acid sequence that extends 3 or proximal to the SYT gene. When probes corresponding to these target nucleic acid sequences (e.g., genomic target nucleic acid sequences) are used in an in situ hybridization procedure, normal cells, which lack a t(18q11.2) in the SYT gene region, exhibit two fusion (generated by the two labels in close proximity) signals, reflecting the two intact copies of SYT. Abnormal cells with a t(18q11.2) exhibit a single fusion signal.
(223) In other examples, a target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is selected that is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the p16 region (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), and the pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (that encompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.
(224) The aforementioned examples are provided solely for purpose of illustration and are not intended to be limiting. Numerous other cytogenetic abnormalities that correlate with neoplastic transformation and/or growth are known to those of ordinary skill in the art. Target proteins that are produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have been correlated with neoplastic transformation and which are useful in the disclosed methods, also include the EGFR gene (7p12; e.g., GENBANK Accession No. NC 000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK Accession No. NC 000008, nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK Accession No. NC-000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK Accession No. NC 000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK Accession No. NC 000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK Accession No. NC-000002, complement, nucleotides 151835231-151854620), CHOP (12q13; e.g., GENBANK Accession No. NC 000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK Accession No. NC 000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK Accession No. NC-000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK Accession No. NC-000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCND1 (1113; e.g., GENBANK Accession No. NC-000011, nucleotides 69165054.69178423), BCL2 (18q21.3; e.g., GENBANK Accession No. NC-000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK Accession No. NC-000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK Accession No. NC 000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK Accession No. NC 000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK Accession No. NC-000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK Accession No. NC 000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK Accession No. NC-000007, complement, nucleotides 13897379-13995289), EWS (22q12.2; e.g., GENBANK Accession No. NC 000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK Accession No. NC-000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK Accession No. NC-000002, complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK Accession No. NC 000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK Accession No. NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK Accession No. NC 000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK Accession No. NC 000001, complement, nucleotides 40133685-40140274), REL (2p13-p12; e.g., GENBANK Accession No. NC-000002, nucleotides 60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK Accession No. NC-000005, complement, nucleotides 149413051-149473128).
(225) In other examples, a target protein is selected from a virus or other microorganism associated with a disease or condition. Detection of the virus- or microorganism-derived target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in a cell or tissue sample is indicative of the presence of the organism. For example, the target peptide, polypeptide or protein can be selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite (such as Plasmodium falciparum and other Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).
(226) In some examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from a viral genome. Exemplary viruses and corresponding genomic sequences (GENBANK RefSeq Accession No. in parentheses) include human adenovirus A (NC-001460), human adenovirus B (NC-004001), human adenovirus C(NC 001405), human adenovirus D (NC-002067), human adenovirus E (NC-003266), human adenovirus F (NC-001454), human astrovirus (NC-001943), human BK polyomavirus (V01109; GI:60851) human bocavirus (NC-007455), human coronavirus 229E (NC-002645), human coronavirus HKU1 (NC-006577), human coronavirus NL63 (NC-005831), human coronavirus OC43 (NC-005147), human enterovirus A (NC-001612), human enterovirus B (NC-001472), human enterovirus C(NC-001428), human enterovirus D (NC-001430), human erythrovirus V9 (NC-004295), human foamy virus (NC-001736), human herpesvirus 1 (Herpes simplex virus type 1) (NC-001806), human herpesvirus 2 (Herpes simplex virus type 2) (NC 001798), human herpesvirus 3 (Varicella zoster virus) (NC-001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC-007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC-009334), human herpesvirus 5 strain AD 169 (NC-001347), human herpesvirus 5 strain Merlin Strain (NC-006273), human herpesvirus 6A (NC-001664), human herpesvirus 6B (NC-000898), human herpesvirus 7 (NC-001716), human herpesvirus 8 type M (NC 003409), human herpesvirus 8 type P (NC-009333), human immunodeficiency virus 1 (NC 001802), human immunodeficiency virus 2 (NC-001722), human metapneumovirus (NC 004148), human papillomavirus-1 (NC-001356), human papillomavirus-18 (NC-001357), human papillomavirus-2 (NC-001352), human papillomavirus-54 (NC-001676), human papillomavirus-61 (NC-001694), human papillomavirus-cand90 (NC-004104), human papillomavirus RTRX7 (NC-004761), human papillomavirus type 10 (NC-001576), human papillomavirus type 101 (NC-008189), human papillomavirus type 103 (NC-008188), human papillomavirus type 107 (NC-009239), human papillomavirus type 16 (NC-001526), human papillomavirus type 24 (NC-001683), human papillomavirus type 26 (NC-001583), human papillomavirus type 32 (NC-001586), human papillomavirus type 34 (NC-001587), human papillomavirus type 4 (NC-001457), human papillomavirus type 41 (NC-001354), human papillomavirus type 48 (NC-001690), human papillomavirus type 49 (NC-001591), human papillomavirus type 5 (NC-001531), human papillomavirus type 50 (NC-001691), human papillomavirus type 53 (NC-001593), human papillomavirus type 60 (NC-001693), human papillomavirus type 63 (NC-001458), human papillomavirus type 6b (NC-001355), human papillomavirus type 7 (NC-001595), human papillomavirus type 71 (NC-002644), human papillomavirus type 9 (NC-001596), human papillomavirus type 92 (NC-004500), human papillomavirus type 96 (NC-005134), human parainfluenza virus 1 (NC-003461), human parainfluenza virus 2 (NC-003443), human parainfluenza virus 3 (NC-001796), human parechovirus (NC-001897), human parvovirus 4 (NC-007018), human parvovirus B19 (NC 000883), human respiratory syncytial virus (NC-001781), human rhinovirus A (NC-001617), human rhinovirus B (NC-001490), human spumaretrovirus (NC-001795), human T-lymphotropic virus 1 (NC-001436), human T-lymphotropic virus 2 (NC-001488).
(227) In certain examples, the target protein is produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV18). In other examples, the target protein produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is from a pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).
EXAMPLES
(228) The non-limiting examples presented herein each incorporate the use of at least one pair of click conjugates. Applicants submit that the click conjugates disclosed herein are suitable for use in IHC assays, including multiplex IHC assays, and ISH assays, as demonstrated in the following examples.
(229) General Immunohistochemistry (IHC) Protocol(s)
(230) All IHC staining experiments were carried out on a BenchMark XT automated tissue staining platform and the reagents used in these protocols were from Ventana Medical Systems, Inc. (Tucson, AZ, USA; Ventana) unless otherwise specified. Polyclonal goat anti-rabbit antibodies, polyclonal goat anti-mouse antibodies, horseradish peroxidase (HRP) and alkaline phosphatase (AP) were obtained from Roche Diagnostics (Mannheim, Germany).
(231) The following common steps were performed: (1) deparaffinization with EZ Prep detergent solution (Ventana Medical Systems, Inc. (VMSI), #950-101) (75 C.; 20 minutes); (2) washing with Reaction Buffer (VMSI, #950-300); (3) antigen retrieval in Cell Conditioning 1 (VMSI #950-124) (100 C.; time dependent on antigen of interest); (4) washing (same as step 2); (5) for protocols with subsequent HRP detection steps endogenous peroxidase was inactivated using iVIEW inhibitor (VMSI, E253-2187) (37 C.; 4 minutes); (6) washing (same as step 2); (7) primary antibody incubation (anti-target antibody) was performed at 37 C. for a time dependent on primary antibody ranging from 8-32 minutes; (8) washing (same as step 2); and (9) secondary antibody incubation with a goat polyclonal anti-species antibody conjugated to an enzyme (HRP or AP, 37 C.; 8-12 minutes). All subsequent reagent incubation steps were separated by washing as in step (2). The targets were detected as described in examples 1-6.
Example 1: Click Amplification with Compounds of Formula (II)
(232) Three examples of IHC click amplification with different compounds of Formula (II) are illustrated in
(233) Next, a first member of a pair of click conjugates was introduced and reacted with each AP labeled target. In
(234) In
(235) In
Example 2: Click Amplification with Compounds of Formula (III)
(236) Three examples of IHC click amplification with different compounds of Formula (III) are illustrated in
(237) Next, a first member of a pair of click conjugates was introduced and reacted with each HRP labeled target. In
(238) In
(239) In
Example 3
(240)
Example 4: Comparison of Traditional TSA to Click Amplification in an IHC Assay
(241)
(242) The tissue sample labeled DAB control was stained in an IHC assay utilizing a primary antibody specific for Ki67 and a goat-anti-rabbit antibody conjugated to HRP. The antigen was visualized via a brown precipitate produced by HRP upon the addition of hydrogen peroxide and 3,3-diaminobenzidine (DAB). The DAB hue was toned by the addition of copper sulfate.
(243) The tissue samples identified as being stained with TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl in
(244) The tissue samples identified as being stained with TSA-DBCO:TAMRA-Azide, tyramide-DBCO:Cy5-Azide; and tyramide-DBCO:Dabsyl-Azide in
(245) As compared with the samples stained in the traditional TSA assays, the tissues stained using Cy5 or Dabsyl in click amplification according to the methods described herein showed significant increases in staining intensity, as clearly shown in
Example 5: Comparison of Traditional TSA to Click Amplification in an ISH Assay
(246)
(247) The tissue samples identified as being stained with TSA-TAMRA, TSA-Cy5, and TSA-Dabsyl in
(248) The tissue samples identified as being stained with TSA-DBCO:TAMRA-Azide, tyramide-DBCO:Cy5-Azide; and tyramide-DBCO:Dabsyl-Azide in
(249) As compared with the samples stained in the traditional TSA assays, the tissues stained using Cy5 or Dabsyl in click amplification according to the methods described herein showed significant increases in staining intensity, as clearly shown in
Example 6
(250)
Example 7
(251)
(252) All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
ADDITIONAL EXEMPLARY EMBODIMENTS
(253) The following embodiments are also explicitly disclosed. This is not intended to be an exhaustive list. Embodiment 1. A conjugate having Formula (IIa):
(254) ##STR00065## wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; R.sup.1 is a group selected from phosphate, amide, nitro, urea, sulfate, methyl, ester, beta-lactam, or a sugar; R.sup.2 is a halide; R.sup.3, R.sup.5, and R.sup.6 are independently selected from hydrogen or an aliphatic group having between 1 and 4 carbon atoms; R.sup.4 is a hydrogen, an aliphatic group having between 1 and 4 carbon atoms, or the group CH(R.sup.2)R.sup.7-[Linker]-A; and R.sup.7 is selected from the group consisting of (CH.sub.2).sub.wNH, O(CH.sub.2).sub.wNH, N(H)C(O)(CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2).sub.wNH, (CH.sub.2).sub.wO, O(CH.sub.2).sub.wO, O(CH.sub.2CH.sub.2O).sub.w, N(H)C(O)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2).sub.wO, C(O)N(H)(CH.sub.2CH.sub.2O).sub.w, (CH.sub.2).sub.wS, O(CH.sub.2).sub.wS, N(H)C(O)(CH.sub.2).sub.wS, C(O)N(H)(CH.sub.2).sub.wS, (CH.sub.2).sub.wNH, C(O)N(H)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)(CH.sub.2CH.sub.2O).sub.wCH.sub.2CH.sub.2NH, C(O)N(H)(CH.sub.2)NHC(O)CH(CH.sub.3)(CH.sub.2).sub.wNH, or N(H)(CH2).sub.wNH, where w is an integer ranging from 1 to 12. Embodiment 2. The conjugate of embodiment 1, wherein R.sup.6, R.sup.5, R.sup.4, and R.sup.3 are each hydrogen. Embodiment 3. The conjugate of embodiment 1 or 2, wherein R.sup.1 is a phosphate. Embodiment 4. The conjugate of any of embodiments 1 to 3, wherein R.sup.2 is fluorine. Embodiment 5. The conjugate of embodiment 1, wherein R.sup.1 is a phosphate; R.sup.2 is fluorine; and R.sup.6, R.sup.5, R.sup.4, and R.sup.3 are each hydrogen. Embodiment 6. The conjugate of any of embodiments 1 to 5, wherein Linker has the Formula (Ia):
(255) ##STR00066## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. Embodiment 7. The conjugate of claim 6, wherein R.sup.a and R.sup.b are each hydrogen. Embodiment 8. The conjugate of claim 7, wherein Q is oxygen. Embodiment 9. The conjugate of any of claims 1 to 8, wherein R.sup.7 is C(O)N(H)(CH2).sub.wNH. Embodiment 10. The conjugate of claim 1 or 2, wherein R.sup.1 is a phosphate and R.sup.7 is C(O)N(H)(CH2).sub.wNH, and w ranges from 2 to 10. Embodiment 11. The conjugate of claim 10, wherein R.sup.2 is fluorine; and R.sup.6, R.sup.5, R.sup.4, and R.sup.3 are each hydrogen. Embodiment 12. The conjugate of claim 11, wherein Linker comprises a PEG group. Embodiment 13. A conjugate having Formula (IId):
(256) ##STR00067## wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and w ranges from 1 to 12. Embodiment 14. The conjugate of claim 13, wherein Linker has the Formula (Ia):
(257) ##STR00068## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. Embodiment 15. The conjugate of embodiment 14, wherein w ranges from 1 to 8; and wherein R.sup.a and R.sup.b are each hydrogen. Embodiment 16. The conjugate of embodiment 15, wherein w ranges from 2 to 8, and wherein Q is oxygen. Embodiment 17. The conjugate of embodiment 16, wherein d and e are independently an integer ranging from 2 to 10. Embodiment 18. The conjugate of any of embodiments 13 to 17, wherein A is dibenzocyclooctyne. Embodiment 19. The conjugate of embodiment 18, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group. Embodiment 20. The conjugate of any of embodiments 13 to 17, wherein A is trans-cyclooctene. Embodiment 21. The conjugate of embodiment 20, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group. Embodiment 22. The conjugate of any of embodiments 13 to 17, wherein A is azide. Embodiment 23. The conjugate of embodiment 23, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group. Embodiment 24. The conjugate of any of embodiments 13 to 17, wherein A is tetrazine. Embodiment 25. The conjugate of embodiment 24, wherein w ranges from 2 to 6 and wherein the Linker comprises a PEG group. Embodiment 26. A conjugate having Formula (III):
ALinker
M(III), wherein M is derived from a propionic acid, a cinnamic acid, or a compound of Formula (Ilia),
(258) ##STR00069## wherein each R group is independently selected from hydrogen or lower alkyl group having between 1 and 4 carbon atoms; A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; and Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; provided that when each R is hydrogen, A is selected from the group consisting of an azide, a thiol, a 1,3-nitrone, a hydrazine, or a hydroxylamine. Embodiment 27. The conjugate of embodiment 26, wherein Linker has the formula (Ia)
(259) ##STR00070## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. Embodiment 28. The conjugate of embodiment 27, wherein R.sup.a and R.sup.b are each hydrogen. Embodiment 29. The conjugate of embodiment 27 or 28, wherein Q is oxygen. Embodiment 30. The conjugate of embodiment 27, wherein R.sup.a and R.sup.b are each hydrogen, Q is oxygen, and e ranges from 2 to 10. Embodiment 31. A conjugate having Formula (Id):
(260) ##STR00071## wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and Tissue Reactive Moiety is derived from a compound selected from the group consisting of:
(261) ##STR00072## Embodiment 32. The conjugate of embodiment 31, wherein Linker has the Formula (Ia):
(262) ##STR00073## wherein d and e are integers each independently ranging from 2 to 20; t and u are independently 0 or 1; Q is a bond, O, S, or N(R.sup.c)(R.sup.d); R.sup.a and R.sup.b are independently H, a C.sub.1-C.sub.4 alkyl group, F, Cl, or N(R.sup.c)(R.sup.d); R.sup.c and R.sup.d are independently CH.sub.3 or H; and X and Y are independently a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated group having between 1 and 12 carbon atoms and optionally having one or more O, N, or S heteroatoms. Embodiment 33. The conjugate of embodiment 32, wherein R.sup.a and R.sup.b are each hydrogen. Embodiment 34. The conjugate of embodiment 32 or 33, wherein Q is oxygen. Embodiment 35. The conjugate of embodiment 32, wherein R.sup.a and R.sup.b are each hydrogen, Q is oxygen, and e ranges from 2 to 10. Embodiment 36. A conjugate having Formula (IV):
ALinker
Z(IV), wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator. Embodiment 37. The conjugate of embodiment 36, Z is a chromophore selected from the group consisting of tetramethylrhodamine, Cyanine 5, and Dabsyl. Embodiment 38. The conjugate of embodiment 36, where Z is selected from the group consisting of:
(263) ##STR00074## Embodiment 39. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVa):
(264) ##STR00075## Embodiment 40. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVb):
(265) ##STR00076## Embodiment 41. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVc):
(266) ##STR00077## Embodiment 42. The conjugate of embodiment 36, wherein the conjugate has the structure of Formula (IVd):
(267) ##STR00078## Embodiment 43. The conjugate of embodiment 36, wherein the conjugate is:
(268) ##STR00079## Embodiment 44. The conjugate of embodiment 36, wherein the conjugate is:
(269) ##STR00080## Embodiment 45. The conjugate of embodiment 36, wherein the conjugate is:
(270) ##STR00081## Embodiment 46. The conjugate of embodiment 36, wherein the conjugate is:
(271) ##STR00082## Embodiment 47. The conjugate of embodiment 36, wherein the conjugate is:
(272) ##STR00083## Embodiment 48. The conjugate of embodiment 36, wherein the conjugate is:
(273) ##STR00084## Embodiment 49. The conjugate of embodiment 36, wherein the conjugate is:
(274) ##STR00085## Embodiment 50. The conjugate of embodiment 36, wherein the conjugate is:
(275) ##STR00086## Embodiment 51. A method of detecting a first target in a biological sample, comprising: (i) contacting the biological sample with a first detection probe specific to the first target to form a first detection probe-target complex; (ii) contacting the biological sample with a first labeling conjugate specific for the first detection probe, the first labeling conjugate comprising a first enzyme such that the first-detection probe-target complex becomes labeled with the first enzyme; (iii) contacting the biological sample with a first member of a first pair of click conjugates, the first member of the first pair of click conjugates comprising a tissue reactive moiety, wherein the first enzyme converts the first member of the first pair of click conjugates to a first reactive intermediate which covalently bonds to the biological sample proximally to or directly on the first target to form a first immobilized tissue-click conjugate complex; (iv) contacting the biological sample with a second member of a first pair of click conjugates, the second member of the first pair of click conjugates comprising a second reactive moiety capable of reacting with a first reactive moiety of the first immobilized tissue-click conjugate complex such that a covalent bond is formed between the first immobilized tissue-click conjugate complex and the second member of the first pair of click conjugates to form a first tissue-click conjugate adduct; and (v) detecting signals from a first reporter moiety of first tissue-click conjugate adduct Embodiment 52. The method of embodiment 51, wherein the first member of the first pair of click conjugates comprises the conjugate of any of embodiments 1 to 35. Embodiment 53. The method of embodiment 51 or 52, wherein the second member of the first pair of click conjugates comprises the conjugate of any of embodiments 36 to 50. Embodiment 54. The method of embodiment 51 or 52, wherein the second member of the first pair of click conjugates comprises at least one chromophore. Embodiment 55. The method of embodiment 51, wherein the first member of the first pair of click conjugates comprises a quinone methide precursor moiety; and wherein the second member of the first pair of click conjugates comprises a chromophore. Embodiment 56. The method of embodiment 51, wherein the first member of the first pair of click conjugates comprises a tyramide moiety; and wherein the second member of the first pair of click conjugates comprises a chromophore. Embodiment 57. The method of any of embodiments 51 to 56, wherein the first detection probe is a primary antibody, and wherein the first labeling conjugate comprises an anti-antibody antibody. Embodiment 58. The method of any of embodiments 51 to 57, wherein the first enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, neuraminidase, alpha-lactase and beta-lactase. Embodiment 59. The method of any of embodiments 51 to 58, further comprising detecting a second target in the biological sample, wherein the second target is detected by (i) contacting the biological sample with a second detection probe specific to the second target to form a second detection probe-target complex; (ii) contacting the biological sample with a second labeling conjugate specific for the second detection probe, the second labeling conjugate comprising a second enzyme such that the second-detection probe-target complex becomes labeled with the second enzyme; (iii) contacting the biological sample with a first member of a second pair of click conjugates, the first member of the second pair of click conjugates comprising a tissue reactive moiety, wherein the second enzyme converts the first member of the second pair of click conjugates to a second reactive intermediate which covalently bonds to the biological sample proximally to or directly on the second target to form a second immobilized tissue-click conjugate complex. (iv) contacting the biological sample with a second member of a second pair of click conjugates, the second member of the second pair of click conjugates comprising a second reactive moiety capable of reacting with a first reactive moiety of the second immobilized tissue-click conjugate complex such that a covalent bond is formed between second immobilized tissue-click conjugate complex and the second member of the second pair of click conjugates; and (v) detecting signals from a second reporter moiety of the second tissue-click conjugate adduct, wherein the second reporter moiety is different than the first reporter moiety. Embodiment 60. The method of embodiment 59, wherein the first member of the second pair of click conjugates comprises the conjugate of any of embodiments 1 to 35. Embodiment 61. The method of embodiment 59 or 60, wherein the second member of the second pair of click conjugates comprises the conjugate of any of embodiments 36 to 50. Embodiment 62. The method of embodiment 59 or 60, wherein the second member of the second pair of click conjugates comprises at least one chromophore. Embodiment 63. The method of embodiment 59, wherein the first member of the second pair of click conjugates comprises a quinone methide precursor moiety; and wherein the second member of the second pair of click conjugates comprises a chromophore. Embodiment 64. The method of embodiment 59, wherein the first member of the second pair of click conjugates comprises a tyramide moiety; and wherein the second member of the second pair of click conjugates comprises a chromophore. Embodiment 65. The method of any of embodiments 59 to 64, wherein the second detection probe is a primary antibody, and wherein second first labeling conjugate comprises an anti-antibody antibody. Embodiment 66. The method of any of embodiments 59 to 65, wherein the second enzyme is selected from the group consisting of phosphatase, phosphodiesterase, esterase, lipase, amidase, protease, nitroreductase, urease, sulfatase, cytochrome P450, alpha-glucosidase, beta-glucosidase, beta-lactamase, alpha-glucoronidase, beta-glucoronidase, alpha-5-galactosidase, beta-galactosidase, neuraminidase, alpha-lactase and beta-lactase. Embodiment 67. The method of any of embodiments 51 to 66, wherein one or more of the steps are performed by an automated system. Embodiment 68. An immobilized click-conjugate covalently bonded to a tissue sample, the immobilized click-conjugate comprising a first reactive functional group selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine. Embodiment 69. The immobilized click-conjugate of embodiment 68, wherein the click-conjugate is bonded to the tissue through a tyrosine residue or a nucleophilic species within or on the surface of the tissue sample. Embodiment 70. A detectable tissue-click adduct complex formed by reacting the immobilized click-conjugate of embodiments 68 and 69 with a conjugate having Formula (IV):
ALinker
Z(IV), wherein A is selected from the group consisting of dibenzocyclooctyne, trans-cyclooctene, azide, tetrazine, maleimide, thiol, 1,3-nitrone, aldehyde, ketone, hydrazine, and hydroxylamine; Linker is a branched or unbranched, linear or cyclic, substituted or unsubstituted, saturated or unsaturated, group having between 2 and 80 carbon atoms, and optionally having one or more heteroatoms selected from O, N, or S; and Z is selected from the group consisting of a chromophore, a fluorophore, an enzyme, a hapten, and a chelator; and wherein the conjugate of Formula (IV) comprises an A group capable of reacting with the first reactive functional group of the immobilized click-conjugate. Embodiment 71. The detectable tissue-click adduct complex of embodiment 70, wherein Z is at least one chromophore. Embodiment 72. The detectable tissue-click adduct complex of embodiment 70 or 71, wherein the first reactive functional group is dibenzocyclooctyne and where A of Formula (IV) is selected from the group consisting of an azide or a 1,3-nitrone. Embodiment 73. The detectable tissue-click adduct complex of embodiment 70 or 71, wherein the first reactive functional group is trans-cyclooctene and where A of Formula (IV) is a tetrazine. Embodiment 74. The detectable tissue-click adduct complex of embodiment 70 or 71, wherein the first reactive functional group is an azide and where A of Formula (IV) is a dibenzocyclooctyne. Embodiment 75. The detectable tissue-click adduct complex of embodiment 70 or the detectable tissue-click adduct complex of any of embodiments 72 to 74, wherein Z is a chelator, and wherein a lanthanide is introduced to the formed detectable tissue-click adduct complex.
(276) Although the disclosure herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It is therefore understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present disclosure as defined by the appended claims.