Storage-stable caged hapten

Abstract

Disclosed herein are caged haptens and caged hapten-antibody conjugates that are useful for facilitating detection of targets that are located in close proximity to one another in a sample.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the filing date benefit of U.S. Provisional Patent Application No. 63 / 137,805, filed on 15 January 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Field of Invention The disclosed embodiments relate to detecting targets in a sample, including targets located nearby within the sample. The disclosed embodiments also provide proximity assays for detecting protein dimers in formalin-fixed paraffin-embedded tissue using caged haptens or caged hapten conjugates.

[0003] Description of industrial applicability This disclosure has industrial applicability in the fields of chemistry and diagnostics. [Background technology]

[0004] Background of this disclosure Immunohistochemistry (IHC) refers to a method of detecting, localizing, and / or quantifying antigens, such as proteins, in biological samples using antibodies specific to a particular antigen. IHC offers the significant advantage of precisely identifying where a particular protein is located within a tissue sample. IHC is also an effective method for examining the tissue itself. In-situ hybridization (ISH) refers to a method of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on a variety of biological samples, including tissues (e.g., fresh-frozen, formalin-fixed, paraffin-embedded) and cytological specimens. Target recognition can be detected using a variety of labels (e.g., chromogenic, fluorescent, luminescent, radioactive), regardless of whether the target is a nucleic acid or an antigen. As the ability to reliably detect low-abundance cellular markers for diagnostic purposes becomes increasingly important, amplification of recognition events is desired to reliably detect, localize, and quantify targets in clinical settings. For example, by depositing hundreds or thousands of labeling molecules at a marker site in response to a single antigen detection event, the ability to detect that recognition event is enhanced through amplification.

[0005] Protein-protein interaction networks are a prominent feature of biological systems. These interactions form signaling pathways that regulate all aspects of cellular function in both normal and cancerous cells. While methods have been developed to detect protein-protein interactions, such as transient receptor tyrosine kinase dimerization and complex formation after extracellular growth factor activation, these methods are not specifically designed for use in formalin-fixed, paraffin-embedded (FFPE) tissues.

[0006] The ability to investigate the presence and distribution of specific intermolecular interactions for biomarkers known to be important determinants in cancer biology is of great interest in the context of new diagnostic capabilities and in determining therapeutic efficacy in the context of drug development. The ability to explore and record the distribution of molecular interactions on frozen and paraffin-embedded tissues remains unattainable, and although alternative techniques have been proposed to approach this problem, the solutions have not yet proven effective and reliable in practical use.

[0007] The proximity ligation assay was developed by Olink AB. It is the only known commercially available product for in-situ detection of protein-protein interactions on formalin-fixed, paraffin-embedded tissue. The proximity ligation assay technique utilizes DNA ligase for generating a padlock circular DNA template, as well as Phi29 DNA polymerase for rolling circle amplification. These enzymes are expensive. Furthermore, they are not suitable for use with automated systems and methods. For these reasons, the proximity ligation assay is not generally considered useful for commercial applications. [Overview of the project]

[0008] A brief summary of this disclosure A first aspect of this disclosure is formulas (IA) and (IB): R 2 -R 1 -O-[DIG]-[phosphoryl](IA) R 2 -R 1 -O-[DIG]-PO4H2(IB)

[0009] (In the formula,

[0010] R 1is a bond, or a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N or S,

[0011] R 2 is H or a reactive functional group,

[0012] [DIG] is digoxigenin,

[0013] [phosphoryl] is of the formula: TIFF0007914109000001.tif42170

[0014] (Q 1 is O or S,

[0015] Q 2 is H, -CH3 or -CH2CH3) represented by

[0016] said [phosphoryl] group or said -PO4H2 group may be attached to any position of [DIG]) which is a caged hapten having any one of

[0017] In some embodiments, Q 1 is S.

[0018] In some embodiments, Q 1 is O, and at least one Q 2 is H. In some embodiments, R 2 is selected from amine-reactive groups, thiol-reactive groups and carbonyl-reactive groups. In some embodiments, R 2The group is selected from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups.

[0019] In some embodiments, both Q 2 The base is H. In some embodiments, R 2 R is selected from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups. In some embodiments, R 2 R is selected from the group consisting of dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups. In some embodiments, R 1 Equation (IIIA): TIFF0007914109000002.tif48170

[0020] (In the formula,

[0021] R 8 The bonds are -O-, -S-, -C(R c )(R d ), or -N(R c )- and,

[0022] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0023] R c and R d Each is independently selected from H or -CH3.

[0024] R 9 and R 10Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0025] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0026] t and u are independently 0, 1, or 2, where t+u is at least 1.

[0027] (v is an integer in the range of 1 to 8) It has the structure shown.

[0028] In some embodiments, R a or R b At least one of them is H. In some embodiments, R 8 In some embodiments, R 8 This is a coupling. In some embodiments, R a or R b At least one of them is H. In some embodiments, R a and R b Both are H. In some embodiments, Z is a bond or -CH2-.

[0029] In some embodiments, R 1 Equation (IIIC): TIFF0007914109000003.tif53170

[0030] (In the formula,

[0031] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0032] R c and R dEach is independently selected from H or -CH3.

[0033] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0034] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0035] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0036] (v is an integer in the range of 1 to 8) It has the structure shown.

[0037] In some embodiments, R a or R b At least one of them is H. In some embodiments, Z is a bond or -CH2-. In some embodiments, both Q 2 The base is H. In some embodiments, R 2 The group is selected from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups.

[0038] In some embodiments, R 2 Q is selected from the group consisting of dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups. In some embodiments, Q 1 It is O.

[0039] A second aspect of this disclosure is formula (IIID): TIFF0007914109000004.tif84170

[0040] (In the formula,

[0041] R 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0042] R 2 is H or a reactive functional group,

[0043] R 3 These are H, -CH3, -CH2CH3, -OH, or -O-Me,

[0044] R 4 These are H, -CH3, or -CH2CH3, -OH, or -O-Me,

[0045] R 6 is either H, or a linear or branched, substituted or unsubstituted C1-C6 alkyl group.

[0046] m, n, and o are each independent integers ranging from 0 to 4.

[0047] Y is -CH2-, -C(R 7 )-, -N(H)-, -N(R 7 )-, -O-, or -S-, or -C(O)-, R 7 (A C1-C4 linear or branched alkyl group is either substituted or unsubstituted.) It is a caged hapten that possesses the following properties.

[0048] In some embodiments, R 1 Equation (IIIC): TIFF0007914109000005.tif53170

[0049] (In the formula,

[0050] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0051] R c and R d Each is independently selected from H or -CH3.

[0052] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0053] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0054] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0055] (v is an integer in the range of 1 to 8) It has the structure shown.

[0056] In some embodiments, R a or R b At least one of them is H. In some embodiments, Z is a bond or -CH2-. In some embodiments, R 2 R is selected from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups. In some embodiments, R 2 R is selected from the group consisting of dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups. In some embodiments, R 2R is selected from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups. In some embodiments, R 2 The group is selected from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups.

[0057] In some embodiments, R 1 Equation (IIIA):

[0058] TIFF0007914109000006.tif53170

[0059] (In the formula,

[0060] R 8 The bonds are -O-, -S-, -C(R c )(R d ), or -N(R c )- and,

[0061] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0062] R c and R d Each is independently selected from H or -CH3.

[0063] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0064] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0065] t and u are each independently 0, 1 or 2, provided that t+u is at least 1,

[0066] v is an integer in the range of 1 to 8) having the structure shown in.

[0067] In some embodiments, R a or R b at least one of is H. In some embodiments, R 8 is O. In some embodiments, R 8 is a bond. In some embodiments, R a or R b at least one of is H. In some embodiments, R a and R b both are H. In some embodiments, Z is a bond or -CH2-. In some embodiments, R 3 , R 4 or R 6 at least one of is -CH3. In some embodiments, R 3 and R 4 at least one of is -CH3. In some embodiments, R 6 is H. In some embodiments, R 2 is H. In some embodiments, Y is -C(O)-. In some embodiments, R 2 is H and Y is -C(O)-. In some embodiments, R 1 is of formula (IIIA): TIFF0007914109000007.tif53170

[0068] (wherein,

[0069] R 8 is a bond, -O-, -S-, -C(R c )(R d ), or -N(R c )-,

[0070] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0071] R c and R d Each is independently selected from H or -CH3.

[0072] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0073] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0074] t and u are independently 0, 1, or 2, where t+u is at least 1.

[0075] (v is an integer in the range of 1 to 8) It has the structure shown.

[0076] A third aspect of this disclosure is a conjugate comprising (i) any one of the caged haptens described in the first and second aspects above, and (ii) a primary antibody. In some embodiments, the caged hapten is indirectly conjugated to the primary antibody. In some embodiments, the primary antibody is an intact primary antibody.

[0077] A fourth aspect of this disclosure is a conjugate comprising (i) any one of the caged haptens described in the first and second aspects above, and (ii) a secondary antibody. In some embodiments, the caged hapten is indirectly conjugated to the secondary antibody. In some embodiments, the secondary antibody is an intact secondary antibody.

[0078] A fifth aspect of this disclosure is formulas (IVA) and (IVB):

[0079] [Specific binding entity]-W 1 -W 2 -R 1 -O-[DIG]-[phosphoryl](IVA)

[0080] [Specific binding entity]-W 1 -W 2 -R 1 -O-[DIG]-PO4H2(IVB)

[0081] (In the formula,

[0082] W 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group containing 1 to 10 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S.

[0083] W 2 It originates from a reactive functional group,

[0084] [DIG] is digoxigenin,

[0085] [Phosphoryl] is given by formula: TIFF0007914109000008.tif42170

[0086] (Q 1 is either O or S,

[0087] Q 2 (This is H, -CH3, or -CH2CH3) Represented by,

[0088] [Specific binding entity] is a specific binding entity,

[0089] The group [phosphoryl] or the group -PO4H2 may be attached to any position in [DIG]. It is a conjugate having any one of the following:

[0090] In some embodiments, the [specific binding entity] is an antibody. In some embodiments, the [specific binding entity] is a monoclonal antibody. In some embodiments, the [specific binding entity] is a primary antibody. In some embodiments, the [specific binding entity] is a secondary antibody.

[0091] In some embodiments, the conjugate has formula (IVA) and the [specific binding entity] is a monoclonal antibody. In some embodiments, the conjugate has formula (IVB) and the [specific binding entity] is a monoclonal antibody.

[0092] In some embodiments, Q 1 is O and at least one Q 2 H is H. In some embodiments, W 2 These are derived from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups. In some embodiments, W 2 These are derived from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, or amino groups. In some embodiments, both Q 2 The base is H. In some embodiments, W 2 These are derived from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups. In some embodiments, W 2 These are derived from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, or amino groups.

[0093] In some embodiments, R 1 Equation (IIIA): TIFF0007914109000009.tif53170

[0094] (In the formula,

[0095] R 8 The bonds are -O-, -S-, -C(R c )(R d ), or -N(R c )- and,

[0096] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0097] R c and R d Each is independently selected from H or -CH3.

[0098] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0099] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0100] t and u are independently 0, 1, or 2, where t+u is at least 1.

[0101] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 4.

[0102] In some embodiments, R a And Rb can be independently H, C1-C2 alkyl groups, F, Cl, or -N(R c )(R d) is. In some embodiments, R a and R b Each of these is independently either H or a C1-C2 alkyl group.

[0103] In some embodiments, R a or R b At least one of them is H. In some embodiments, R 8 In some embodiments, R 8 This is a coupling. In some embodiments, R a or R b At least one of them is H. In some embodiments, R a and R b Both are H. In some embodiments, Z is a bond or -CH2-.

[0104] In some embodiments, R 1 Equation (IIIC): TIFF0007914109000010.tif53170

[0105] (In the formula,

[0106] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0107] R c and R d Each is independently selected from H or -CH3.

[0108] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0109] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0110] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0111] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 4.

[0112] In some embodiments, R a And Rb can be independently H, C1-C2 alkyl groups, F, Cl, or -N(R c )(R d ) is. In some embodiments, R a and R b Each of these is independently either H or a C1-C2 alkyl group.

[0113] In some embodiments, R a or R b At least one of them is H. In some embodiments, Z is a bond or -CH2-. In some embodiments, both Q 2 The base is H. In some embodiments, W 2 These are derived from amine-reactive groups, thiol-reactive groups, and carbonyl-reactive groups. In some embodiments, W 2 These are derived from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups. In some embodiments, Q 1 It is O.

[0114] A sixth aspect of the present disclosure is a method for analyzing a sample to determine whether a first target is in proximity to a second target, the method comprising: contacting the sample with a demasking enzyme-antibody conjugate to form a target-demasking enzyme-antibody conjugate complex; contacting the sample with any one of the caged hapten-antibody conjugates described above in relation to the third, fourth and fifth aspects of the present disclosure to form a target-caged hapten-antibody conjugate complex; demasking the caged hapten of the target-caged hapten-antibody conjugate complex to form a target-demasked hapten-antibody conjugate complex; contacting the sample with a first detection reagent to label the first target-demasked hapten-antibody conjugate complex or the first target; and detecting the labeled first target-demasked hapten-antibody conjugate complex or the labeled first target. In some embodiments, the caged hapten-antibody conjugate includes a monoclonal antibody.

[0115] In some embodiments, the first detection reagent comprises (i) a secondary antibody specific to the demastened hapten of the target-demastened hapten-antibody complex, wherein the secondary antibody is conjugated to the first enzyme such that the secondary antibody labels the target-demastened hapten-antibody complex with the first enzyme, and (ii) a first substrate for the first enzyme.

[0116] In some embodiments, the first substrate is a chromogenic substrate or a fluorescent substrate.

[0117] In some embodiments, the first detection reagent includes an amplification component for labeling the demasking enzyme of the target-demasking hapten-antibody conjugate complex with a plurality of first reporter moieties.

[0118] In some embodiments, multiple first reporter portions are haptens.

[0119] In some embodiments, the first detection reagent further comprises a secondary antibody specific to a plurality of first reporter moieties, each of which is conjugated to a second reporter moiety.

[0120] A seventh aspect of the present disclosure is a method for analyzing a sample to determine whether a first target is adjacent to a second target, the method comprising: contacting the sample with a demasking enzyme-antibody conjugate to form a target-demasking enzyme-antibody conjugate complex; contacting the sample with any one of the caged hapten-antibody conjugates described above in relation to the third, fourth and fifth aspects of the present disclosure to form a target-caged hapten-antibody conjugate complex; demasking the caged hapten of the target-caged hapten-antibody conjugate complex to form a target-demasked hapten-antibody conjugate complex; demasking the caged hapten of the target-caged hapten-antibody conjugate complex to form a target-demasked hapten-antibody conjugate complex; performing a signal amplification step to label the target-demasked hapten-antibody conjugate complex with a plurality of reporter moieties; and detecting the plurality of reporter moieties. In some embodiments, the caged hapten-antibody conjugate includes a monoclonal antibody.

[0121] In some embodiments, the reporter portion is a hapten, and the method further comprises introducing a secondary antibody specific to a plurality of first reporter portions, each secondary antibody being conjugated to a second reporter portion. In some embodiments, the second reporter portion is an amplification enzyme, and the method further comprises introducing a chromogenic substrate or a fluorescent substrate to the amplification enzyme. In some embodiments, the method further comprises detecting the total amount of a target in the sample.

[0122] An eighth aspect of the present disclosure is a method for analyzing a sample to determine whether a first target is in proximity to a second target, comprising: contacting the sample with a first detection probe, wherein the first detection probe comprises one of the caged hapten-antibody conjugates or demasking enzyme-antibody conjugates described above in relation to the third, fourth, and fifth aspects of the present disclosure; contacting the sample with a second detection probe, wherein the second detection probe comprises one of the caged hapten-antibody conjugates or the other of the demasking enzyme-antibody conjugates described above in relation to the third, fourth, and fifth aspects of the present disclosure; contacting the sample with at least a first detection reagent to label a demasking hapten-antibody conjugate target complex formed; and detecting a signal from the labeled demasking hapten-antibody conjugate target complex.

[0123] In some embodiments, the method further includes a step of detecting the total amount of target in the sample. In some embodiments, the first detection reagent includes an amplification component for labeling the demasking enzyme of a first target-demasking hapten-antibody conjugate complex with a plurality of first reporter moieties. In some embodiments, the plurality of first reporter moieties are haptens. In some embodiments, the first detection reagent further includes a secondary antibody specific to the plurality of first reporter moieties, each secondary antibody conjugated to a second reporter moiety. In some embodiments, the second reporter moiety is selected from the group consisting of an amplification enzyme or a fluorophore. In some embodiments, the second reporter moiety is an amplification enzyme, and the first detection reagent further includes a first chromogenic substrate or fluorescent substrate for the amplification enzyme. In some embodiments, the method further includes a decaging step. [Brief explanation of the drawing]

[0124] A patent or application file must include at least one drawing made in color. A copy of this patent or patent application publication with color drawings will be provided to the Office upon application and payment of the required fees.

[0125] [Figure 1] Figure 1 shows the carbon numbering of digoxigenin ("DIG"). In this example, the phosphate group is attached to position 12 of digoxigenin.

[0126] [Figure 2] Figure 2 is a schematic diagram illustrating the interaction between a demasking enzyme-antibody conjugate containing alkaline phosphatase (bound to target 2) and a caged hapten-antibody conjugate (bound to target 1), where the demasking enzyme of the demasking enzyme-antibody conjugate reacts with the enzyme substrate portion of the caged hapten-antibody conjugate (due to the close proximity of targets 1 and 2) to provide the respective demasked haptens, which can be detected.

[0127] [Figure 3] Figure 3 is a schematic diagram showing a demasking enzyme-antibody conjugate (bound to target 2) and a caged hapten-antibody conjugate (bound to target 1). The two targets are not in close proximity to each other, and as a result, the demasking enzyme in the demasking enzyme-antibody conjugate does not interact with the enzyme substrate portion of the caged hapten-antibody conjugate. Therefore, the caged hapten remains masked and undetectable.

[0128] [Figure 4] Figure 4 provides a flowchart illustrating the steps for detecting protein dimers and / or total protein in a sample.

[0129] [Figure 5] Figure 5 is a schematic diagram showing one embodiment of an IHC staining protocol in which a single antigen is detected using a secondary antibody labeled with caged DIG.

[0130] [Figure 6] Figure 6 is a schematic diagram showing the decaching (or demasking) of caged DIG, i.e., phosphorylated DIG, to provide a natural-type DIG hapten.

[0131] [Figure 7] Figure 7 is a schematic diagram illustrating the multiple detection of both adjacent proteins (Target 1 and Target 2) and total protein (Target 2).

[0132] [Figure 8] Figure 8 shows the binding of the antibody to the caged hapten, i.e., phosphorylated DIG.

[0133] [Figure 9] Figure 9 shows the hydrolysis of caged nitrophenyl (NP) and caged DIG for the formation of native haptens (NP and DIG).

[0134] [Figure 10] Figure 10 shows an experiment monitoring the amount of caged hapten hydrolyzed (non-enzymatically cleaved by water) to the original hapten, expressed as a percentage of the original material, for two different caged NP molecules and caged DIG.

[0135] [Figure 11A] Figure 11A shows representative images of positive proximity assays for E-cadherin and β-catenin on tonsil tissue using caged NPs.

[0136] [Figure 11B] Figure 11B shows representative images of positive proximity assays for E-cadherin and β-catenin on tonsil tissue using caged DIG. [Modes for carrying out the invention]

[0137] Detailed explanation Caged haptens and methods for synthesizing them are disclosed herein. Caged hapten conjugates are also disclosed herein. As described in more detail herein, caged hapten conjugates may be used to detect proximal antigens in tissue samples. These and other embodiments are described herein.

[0138] definition As used herein, the singular forms “a,” “an,” and “the” include multiple referents unless the context explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context explicitly indicates otherwise. The term “includes” is defined inclusively, so that “includes A or B” means to include A, B, or A and B.

[0139] Where used herein in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as inclusive, that is, including at least one of several elements or lists of elements, but also including several of several elements or lists of elements, and optionally including additional items not listed. Only terms that explicitly indicate the opposite, such as “one of” or “exactly one of” or, when used in claims, “consisting of,” refer to including exactly one element of several elements or lists of elements. In general, where used herein, the term “or” shall be interpreted as indicating an exclusive choice (i.e., “one or the other, but not both”) only when preceded by a term of exclusivity, such as “either,” “one of,” “one of” or “exactly one of.” Where used in claims, “essentially consisting of” shall have the usual meaning as used in the field of patent law.

[0140] As used herein, terms such as “comprises,” “includes,” and “has” are interchangeable and have the same meaning. Similarly, “comprises,” “includes,” and “has” are interchangeable and have the same meaning. Specifically, each of these terms is defined in accordance with the general U.S. Patent Law definition of “comprising” and is therefore interpreted as a non-restrictive term meaning “at least the following,” and 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 including steps a, b, and c” means that the method includes at least steps a, b, and c. Furthermore, while steps and processes may be outlined herein in a particular order, those skilled in the art will recognize that the ordered steps and processes may vary.

[0141] Where used herein in the specification and claims, the phrase “at least one” referring to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of every element specifically listed in the list of elements, nor excluding any combination of elements in the list of elements. This definition also allows for the presence of elements other than those specifically identified in the list of elements referred to by the phrase “at least one,” whether related to or unrelated to those specifically identified elements. Therefore, as a non-restrictive example, “at least one of A and B” (or equivalently, “at least one of A or B,” or equivalently, “at least one of A and / or B”) may, in one embodiment, mean at least one which includes any one or more A and no B (and optionally includes elements other than B); in another embodiment, mean at least one which includes any one or more B and no A (and optionally includes elements other than A); and in yet another embodiment, mean at least one which includes any one or more A and at least one which includes any one or more B (and optionally includes other elements), and so on.

[0142] As used herein, the term "alkaline phosphatase" (AP) refers to an enzyme that removes and transfers a phosphate group organic ester (by hydrolysis) by cleaving the phosphate-oxygen bond and temporarily forming an intermediate enzyme-substrate bond. For example, AP hydrolyzes naphthol phosphate ester (substrate) to a phenol compound and phosphate. The phenol binds to a colorless diazonium salt (pigment source) to produce an insoluble colored azo dye.

[0143] As used herein, terms such as “alkyl,” “aromatic,” “heteroalkyl,” and “cycloalkyl” include both substituted and unsubstituted forms of the radicals shown. In this regard, whenever a group or part is described as “substituted” or “optionally substituted” (or “optionally having” or “optionally containing”), the group may be unsubstituted or substituted with one or more of the substituents shown. Similarly, if a group is described as “substituted or unsubstituted,” and the group is substituted, the substituent may be selected from one or more of the substituents shown. If substituents are not indicated, the indicated "optionally substituted" or "substituted" groups are alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyl, aralkyl, heteroaralkyl, (heteroalicyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thio This means that the group can be substituted with one or more groups individually and independently selected from ocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, ether, amino (e.g., monosubstituted or disubstituted amino groups), and their protected derivatives. Any of the above groups may contain one or more heteroatoms including O, N, or S. For example, if the portion is substituted with an alkyl group, that alkyl group may contain a heteroatom selected from O, N, or S (e.g., -(CH2-CH2-O-CH2-CH3)).

[0144] As used herein, the term “antibody” (Ab) refers to a glycoprotein immunoglobulin or its antigen-binding moiety, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, which specifically bind to an antigen. Each H chain comprises a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions may be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including the first component (C1q) of the classical complement system.

[0145] Immunoglobulins may originate from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to an antibody class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene. The term “antibody” includes, by example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Non-human antibodies may be humanized by recombinant methods to reduce their immunogenicity in humans. Unless expressly stated and the context indicates otherwise, the term “antibody” also includes antigen-binding fragments or parts of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or parts, as well as single-chain antibodies.

[0146] The term “monoclonal antibody” (“mAb”) refers to an unnatural preparation of an antibody molecule with a single molecular composition, i.e., an antibody molecule whose primary sequence is essentially identical and which exhibits a single binding specificity and affinity for a particular epitope. An mAb is an example of an isolated antibody. mAbs can be produced by hybridoma, recombinant, transgenic, or other techniques known to those skilled in the art.

[0147] As used herein, the term “antibody conjugate” refers to an antibody conjugated (directly or indirectly) to one or more labels, the antibody conjugate being specific to a particular target, and the labels being detectable (directly or indirectly) by a secondary antibody (anti-labeled antibody), etc. For example, an antibody conjugate may be conjugated to a hapten via a polymer linker and / or spacer, and the antibody conjugate may be indirectly detected by the hapten. Alternatively, an antibody conjugate may be conjugated to a chromogen via a polymer linker and / or spacer, and the antibody conjugate may be directly detected. Antibody conjugates are further described in U.S. Patent Application Publication No. 2014 / 0147906 and U.S. Patents Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191. As a further example, the term “antibody conjugate” includes antibodies conjugated to an enzyme, such as HRP or AP. In some embodiments, the antibody conjugate includes a monoclonal antibody. In other embodiments, the antibody conjugate includes a polyclonal antibody.

[0148] As used herein, the term “antigen” refers to a compound, composition, or substance that can be specifically bound by antibody molecules or products of specific humoral or cellular immunity, such as T cell receptors. Antigens can be any type of molecule, including, for example, haptens, simple intermediate metabolites, sugars (e.g., oligosaccharides), lipids and hormones, as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins.

[0149] As used herein, the term "aryl" means an aromatic carbocyclic radical or substituted carbocyclic radical, such as phenyl or naphthyl, preferably containing 6 to 10 carbon atoms, which is optionally substituted with at least one substituent selected from the group consisting of phenyl or naphthyl or alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, alkylthio, arylthio, alkylene or -NYY' (where Y and Y' are independently hydrogen, alkyl, aryl, or aralkyl).

[0150] As used herein, the term “biological sample” can be any solid or fluid sample obtained from, excreted by, or secreted by any organism, including, but not limited to, single-celled or multicellular organisms such as bacteria, yeasts, protozoa, and amoebas (including samples from healthy or seemingly healthy human subjects or human patients suffering from a symptom or disease being diagnosed or investigated, such as cancer, such as plants or animals). For example, a biological sample could be, for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid, aqueous humor or vitreous fluid, or any bodily secretions, exudates, or effusions (e.g., fluids obtained from an abscess or any other site of infection or inflammation), or a biological fluid obtained from a joint (e.g., a normal joint or a diseased joint). A biological sample could also be a sample obtained from any organ or tissue (including biopsy or autopsy specimens such as tumor biopsies), or it could include cells (whether primary or cultured) or culture media adapted by any cells, tissue or organ. In some examples, the biological sample is a nuclear extract. In certain examples, the sample is a quality control sample, such as one of the disclosed cell pellet section samples. In other examples, the sample is a test sample. The sample may be prepared using any method known to those skilled in the art. The sample may be obtained from subjects for routine screening or from subjects suspected of having disorders such as genetic abnormalities, infections or neoplasms. The described embodiments of the disclosed method may also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as "normal" samples. The sample may comprise multiple targets that can be specifically bound by one or more detection probes.

[0151] As used herein, "a" and "b" are integers. a ~C b"C1-C4 alkyl" refers to the number of carbon atoms in an alkyl, alkenyl, or alkynyl group, or the number of carbon atoms in a cycloalkyl, cycloalkenyl, cycloalkynyl, or aryl ring, or the total number of carbon atoms and heteroatoms in a heteroalkyl, heterocyclyl, heteroaryl, or heteroalicyl group. That is, an alkyl, alkenyl, alkynyl, cycloalkyl ring, cycloalkenyl ring, cycloalkynyl ring, aryl ring, heteroaryl ring, or heteroalicyl ring can contain "a" to "b" carbon atoms, including "a" and "b". Therefore, for example, a "C1-C4 alkyl" group refers to all alkyl groups having 1 to 4 carbon atoms, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2, CH3CH2CH(CH3)-, and (CH3)3C-. If "a" and "b" are not specified with respect to alkyl, alkenyl, alkynyl, cycloalkylcycloalkenyl, cycloalkynyl, aryl, heteroaryl, or heteroalcyclyl groups, the broadest range described in those definitions is assumed.

[0152] As used herein, the term “conjugate” refers to two or more molecules or parts (including macromolecules or supramolecular molecules) covalently bonded to a larger construct. In some embodiments, the conjugate comprises one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) covalently bonded to one or more other molecular parts. In other embodiments, the conjugate comprises one or more specific binding molecules (such as antibodies) covalently bonded to one or more detectable labels (such as fluorophores, luminophores, fluorescent nanoparticles, haptens, enzymes, and combinations thereof).

[0153] As used herein, the term “contact” is used interchangeably with: being combined with, being added to, being mixed with, passing over, and being incubated, etc.

[0154] As used herein, the terms “join” or “bond” refer to the joining, bonding (e.g., covalent bond) or linking of one molecule or atom to another molecule or atom.

[0155] As used herein, “cycloalkyl” or similar terms (e.g., cyclic alkyl) refer to a monocyclic or polycyclic hydrocarbon ring system that is completely saturated (without double or triple bonds). If composed of two or more rings, the rings may be bonded together in a condensation manner. A cycloalkyl group may contain 3 to 10 atoms in the ring or 3 to 8 atoms in the ring. A cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0156] As used herein, the term “chromophore” refers to the molecule or part of a molecule (e.g., a chromogenic substrate) that produces its color. Color arises when a molecule absorbs visible light of a particular wavelength and transmits or reflects other wavelengths. A molecule having an energy difference between two different molecular orbitals within the visible spectrum absorbs visible light and can therefore be appropriately characterized as a chromophore. Visible light incident on a chromophore is absorbed and can therefore excite electrons from ground-state molecular orbitals to excited-state molecular orbitals.

[0157] As used herein, the term “conjugate” refers to two or more molecules or parts (including macromolecules or supramolecular molecules) that are covalently bonded to a larger construct. In some embodiments, a conjugate includes one or more biomolecules (such as peptides, proteins, enzymes, sugars, polysaccharides, lipids, glycoproteins, and lipoproteins) that are covalently bonded to one or more other molecular parts.

[0158] As used herein, the term “detectable portion” refers to a molecule or material that can produce a detectable signal (visually, electronically, or otherwise, etc.) indicating the presence (i.e., qualitative analysis) and / or concentration (i.e., quantitative analysis) of a label in a sample.

[0159] As used herein, the term “epitope” refers to an antigenic determinant, such as a continuous or discontinuous peptide sequence on a molecule, that is antigenic, i.e., triggers a specific immune response. Antibodies bind to specific antigenic epitopes.

[0160] As used herein, the terms “halogen atom” or “halogen” mean any one of the radiostable atoms in the seventh column of the periodic table, such as fluorine, chlorine, bromine, and iodine.

[0161] As used herein, the term “hapten” refers to a small molecule that can specifically bind to an antibody but is typically substantially incapable of being immunogenic unless bound to a carrier molecule. In some embodiments, haptens include, but are not limited to, pyrazoles (e.g., nitropyrazoles), nitrophenyl compounds, benzofurazans, triterpenes, ureas (e.g., phenylurea), thioureas (e.g., phenylthiourea), rotenones and rotenone derivatives, oxazoles (e.g., oxazolesulfonamides), thiazoles (e.g., thiazolesulfonamides), coumarin derivatives, and cyclolignans. Additional non-limiting examples of haptens include thiazoles, nitroaryls, benzofurans, triperpenes, and cyclolignans. Specific examples of haptens include dinitrophenyl, biotin, digoxigenin, and fluorescein, as well as any derivatives or analogues thereof. Other haptens are described in U.S. Patents 8,846,320, 8,618,265, 7,695,929, 8,481,270 and 9,017,954, which are incorporated herein by reference in their entirety. Haptens themselves may be suitable for direct detection, that is, they may emit signals suitable for detection.

[0162] As used herein, the term “heteroatom” means including boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). In some embodiments, a “heterocycle” may contain one or more heteroatoms. In other embodiments, an aliphatic group may contain one or more heteroatoms or may be substituted with one or more heteroatoms.

[0163] As used herein, horseradish peroxidase (HRP) is an enzyme that can be conjugated to a labeled molecule. When incubated with a suitable substrate, horseradish peroxidase (HRP) produces a colored, fluorescent, or luminescent derivative of the labeled molecule, which can then be detected and quantified. HRP acts in the presence of an electron donor to first form an enzyme-substrate complex, and then oxidize the electron donor. For example, HRP can act on 3,3'-diaminobenzidine tetrahydrochloride (DAB) to produce a detectable color. HRP can also act on labeled tyramide conjugates or tyramide-like reactive conjugates (i.e., ferulate, coumaric acid, caffeic acid, cinnamate, dopamine, etc.) to deposit a colored, fluorescent, or colorless reporter moiety for tyramide signal amplification (TSA).

[0164] As used herein, the term “label” refers to a detectable portion that may be an atom or molecule, or an aggregate of atoms or molecules. A label may provide a detectable optical, electrochemical, magnetic, or electrostatic (e.g., inductive, capacitive) signature.

[0165] As used herein, the terms “multiplexing,” “multiplexed,” or “multiplexed” refer to the simultaneous, substantially simultaneous, or sequential detection of multiple targets in a sample. Multiplexing may include identifying and / or quantifying multiple different nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) individually, and in any combination and all combinations.

[0166] As used herein, the terms “nucleic acid molecule” or “polynucleotide” refer to a polymeric form of a nucleotide of any length, either a deoxyribonucleotide or a ribonucleotide, or an analog thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Unless specifically limited, the term encompasses nucleic acids or polynucleotides including known analogs of native nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a similar manner to naturally occurring nucleotides. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci (one or more) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. Where present, modifications to the nucleotide structure may be conferred before or after the construction of the polymer. Nucleotide sequences can be interrupted by non-nucleotide components. Polynucleotides can be further modified, such as by conjugation with labeling components. Unless otherwise specified, specific nucleic acid sequences also implicitly include not only the explicitly stated sequence but also their conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologues, SNPs, and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0167] As used herein, the term “oligonucleotide” refers to an oligomer of a nucleotide or nucleoside monomer unit, which optionally includes non-nucleotide monomer units and / or other chemical groups attached to the internal and / or external positions of the oligomer. Oligomers may be natural or synthetic and may include naturally occurring oligonucleotides, or oligomers containing bases, sugar moieties, nucleotides having phosphodiester-analogous linkages that are not naturally occurring (or modified), and / or alternative monomer unit chirality and isomer structures (e.g., 5'-to-2' linkages, L-nucleosides, α-anomeric nucleosides, β-anomeric nucleosides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs)).

[0168] As used herein, the term “primary antibody” refers to an antibody that specifically binds to a target protein antigen in a tissue sample. A primary antibody is typically the first antibody used in immunohistochemical techniques. In some embodiments, the primary antibody is a monoclonal antibody.

[0169] As used herein, the terms “reactive group” or “reactive functional group” refer to a functional group that can chemically associate, interact with, hybridize, hydrogen bond, or bond with functional groups of different parts. In some embodiments, “reaction” between two reactive groups or two reactive functional groups may mean the formation of a covalent bond between two reactive groups or two reactive functional groups, or the association, interaction, hybridization, hydrogen bonding between two reactive groups or two reactive functional groups, and so on.

[0170] For example, reactive groups may be amine-reactive groups such as isothiocyanates, acyl azides, NHS esters, acid chlorides such as sulfonyl chlorides, aldehydes and glycols, epoxides and oxiranes, carbonates, arylating agents, imide esters, carbodiimides, anhydrides, and combinations thereof. Suitable thiol-reactive functional groups include haloacetyls and alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide exchange reagents such as pyridyl disulfide, TNB-thiol and disulfide reducing agents, and combinations thereof. Suitable carboxylate-reactive functional groups include diazoalkanes, diazoacetyl compounds, carbonyldiimidazole compounds, and carbodiimides. Suitable hydroxyl-reactive functional groups include epoxides and oxiranes, carbonyl diimidazoles, N,N'-disuccinimidyl carbonates or N-hydroxysuccinimidyl chloroformates, periodic acid oxidation compounds, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone-reactive functional groups include hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and combinations thereof. Active hydrogen-reactive compounds include diazonium derivatives, Mannich condensation products, iodination reaction products, and combinations thereof. Photoreactive chemical functional groups include aryl azides, aryl azides halides, benzophonones, diazo compounds, diazirine derivatives, and combinations thereof.

[0171] As used herein, the term “secondary antibody” refers to an antibody that specifically binds to a primary antibody, thereby forming a crosslink between the primary antibody and the subsequent reagent (e.g., label, enzyme, etc.), if a subsequent reagent is present. A secondary antibody is generally a second antibody used in immunohistochemical techniques.

[0172] As used herein, the term “specific binding entity” refers to a member of a specific binding pair. A specific binding pair is a pair of molecules characterized by binding to each other in a manner that substantially excludes binding to other molecules (for example, a specific binding pair has a binding constant at least 10 times greater than the binding constant of either of the two members of a binding pair with other molecules in a biological sample). -3 M is large, 10 -4 M is large or 10 -5 M may have a large binding constant. Specific examples of specific binding moieties include specific binding proteins (e.g., antibodies, lectins, avidins such as streptavidin, and protein A). Specific binding moieties may also include molecules (or portions thereof) that are specifically bound by such specific binding proteins.

[0173] As used herein, the term “substituted” is intended to include all acceptable substituents of an organic compound. Whenever a group or part is described as “substituted” or “optionally substituted” (or “optionally having” or “optionally containing”), that group may be unsubstituted or substituted with one or more indicated substituents. Similarly, when a group is described as “substituted or unsubstituted” and it is substituted, if the group is substituted, the substituent may be selected from one or more indicated substituents.

[0174] In some embodiments, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of the organic compound. In some embodiments, there may be one or more acceptable substituents for a given organic compound, and they may be identical or different. In some embodiments, where substituents are not shown, the indicated "optionally substituted" or "substituted" groups include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyl, aralkyl, heteroaralkyl, (heteroalicyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarb This means that the group may be substituted with one or more groups individually and independently selected from myl, N-thiocarbamyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamide, amino, ether, amino (e.g., monosubstituted or disubstituted amino groups) and their protected derivatives. Any of the above groups may contain one or more heteroatoms including O, N, or S. For example, if the portion is substituted with an alkyl group, that alkyl group may contain a heteroatom selected from O, N, or S (e.g., -(CH2-CH2-O-CH2-CH2)-).

[0175] As used herein, the term “target” refers to any molecule whose presence, location, and / or concentration are determined or can be determined. Examples of target molecules include proteins, nucleic acid sequences, and haptens such as haptens covalently bound to proteins. Target molecules are typically detected using one or more conjugates of a specific binding molecule and a detectable label.

[0176] As used herein, the terms “Tyramide Signal Amplification” or “TSA” refer to an enzyme-mediated detection method that utilizes the catalytic activity of a peroxidase (such as horseradish peroxidase) to generate high-density labeling of a target molecule (such as a protein or nucleic acid sequence) in situ. TSA typically comprises three basic steps: (1) binding of a specific binding member (e.g., an antibody such as a monoclonal antibody) to the target, followed by secondary detection of the specific binding member by a second peroxidase-labeled specific binding member; (2) activation of multiple copies of a labeled tyramide derivative (e.g., hapten-labeled tyramide) by peroxidase; and (3) covalent bonding of the resulting highly reactive tyramide radical to a residue proximal to the peroxidase-target interaction site (e.g., the phenol moiety of a protein tyrosine residue), thereby depositing the hapten proximal to the target (mediated by diffusion and reactivity). Some examples of TSA involve more or fewer steps. For example, the TSA method can be sequentially repeated to increase the signal. Methods for performing TSA, as well as commercially available kits and reagents for performing TSA, are available (see, for example, Amp Map Detection Kit with TSA®, catalog number 760-121, Ventana Medical Systems, Tucson, Arizona; Invitrogen; TSA kit number T-20911, Invitrogen Corp, Carsbad, Calif.). Other enzyme-catalyzed species, hapten- or signaling-linked species may be available and can be used as alternatives.

[0177] When used in this specification, TIFF0007914109000011.tif7170 indicates the location where one part is joined to another.

[0178] overview

[0179] This disclosure relates to “caged haptens,” conjugates comprising a specific binding entity and a “caged hapten,” and methods for using these to detect one or more targets in a sample (e.g., one or more protein targets in a sample that are in close proximity to each other). As described in more detail herein, the caged haptens or caged hapten-conjugates described herein facilitate the detection of protein dimers or proteins in close proximity to each other.

[0180] A "caged hapten" is a hapten whose structure has been modified so that a suitable anti-hapten antibody no longer recognizes the hapten and a binding event does not occur. For example, a DIG hapten bound to a phosphate group may no longer be recognized by an anti-DIG antibody. In practice, the identity and / or function of the hapten is "masked" or "protected." To achieve this masking or protection, the haptens of this disclosure contain an enzymatically cleavable casing group (also called an enzymatically cleavable masking group).

[0181] Introducing an enzyme that acts on enzymatically cleavable saging or masking groups releases these groups, regenerating a "natural" hapten (also called an "uncaged hapten" or "demasked hapten"). This natural hapten can then be recognized by an anti-hapten antibody. Thus, in the presence of a suitable enzyme, a caged hapten is demasked, and an anti-hapten antibody can freely bind to it. In the example above, when the phosphate group of DIG is cleaved by an alkaline phosphatase enzyme, the natural DIG hapten is exposed, and an anti-DIG hapten can bind to it. Figure 6 shows the demasking of a caged hapten via enzymatic treatment to provide a natural hapten recognizable by an anti-hapten antibody.

[0182] Caged hapten

[0183] In some embodiments, the caged hapten of the present disclosure is of formula (IA) or (IB): R 2 -R 1 -O-[DIG]-[phosphoryl](IA) R 2 -R 1 -O-[DIG]-PO4H2(IB)

[0184] (In the formula,

[0185] R 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0186] R 2 is H or a reactive functional group,

[0187] [DIG] is either digoxigenin or derived from [DIG].

[0188] [Phosphoryl] is given by formula: TIFF0007914109000012.tif42170

[0189] (Q 1 is either O or S,

[0190] Q 2 (This is H, -CH3, or -CH2CH3) It can be represented by,

[0191] The group [phosphoryl] or the group -PO4H2 may be attached to any position in [DIG]. It has any one structure.

[0192] In some embodiments, [phosphoryl] or -PO4H2 is bonded at the carbon 12 position of digoxigenin (see Figure 1). In some embodiments, [phosphoryl] or -PO4H2 is bonded at the carbon 12 position of a derivative or analog of digoxigenin.

[0193] In some embodiments, Q 1 is O. In some embodiments, Q 1 is O and at least one Q 2 H is H. In some embodiments, Q 1 is O, and each Q 2 H is H. In some embodiments, Q 1 is O and at least one Q 2 It is -CH3.

[0194] As described above, in some embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 30 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In other embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 20 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 5 to 15 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 8 to 12 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In some embodiments, R 1 This may include a carbonyl, amine, ester, ether, amide, imine, thion, or thiol group. In other embodiments, R 1It may contain one or more terminal groups selected from amines, carbonyls, esters, ethers, amides, imines, thions, or thiols.

[0195] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 This is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 This is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0196] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1This is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 This is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N.

[0197] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more oxygen heteroatoms. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 This is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 This is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms.

[0198] In some embodiments, R 1 This is a non-branched aliphatic group having 1 to 30 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In other embodiments, R 1 This is a non-branched aliphatic group having 1 to 20 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1This is a non-branched aliphatic group having 1 to 15 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is a non-branched aliphatic group having 1 to 12 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is a non-branched aliphatic group having 1 to 8 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom.

[0199] In some embodiments, R 1 Equation (IIIA): TIFF0007914109000013.tif53170

[0200] (In the formula,

[0201] R 8 The bonds are -O-, -S-, -C(R c )(R d ), or -N(R c )- and,

[0202] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0203] R c and R d Each is independently selected from H or -CH3.

[0204] R 9 and R 10Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0205] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0206] t and u are independently 0, 1, or 2, where t+u is at least 1.

[0207] (v is an integer in the range of 1 to 8) It has the structure shown.

[0208] In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0209] In some embodiments, R 8 is -C(R c )(R d )-, t+u is at least 2, and v is at least 1. In some embodiments, R 8 is -C(R c )(R d )-, t+u is at least 2, and v is at least 2. In some embodiments, R 8 is -C(R c )(R d )-, t+u is at least 2, and v is at least 3.

[0210] In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, and v is at least 1. In some embodiments, R 8 is -C(R c)(R d )- and R c and R d At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, and v is at least 3.

[0211] In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group.

[0212] In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R cand R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group.

[0213] In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R dAt least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group, and both Z groups are different.

[0214] In some embodiments, R 8 is O, t+u is at least 2, and v is at least 1. In some embodiments, R 8 is O, t+u is at least 2, and v is at least 2. In some embodiments, R 8 is O, t+u is at least 2, and v is at least 3.

[0215] In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group.

[0216] In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group, and both Z groups are different.

[0217] In other embodiments, R 1 Equation (IIIB): TIFF0007914109000014.tif53170

[0218] (In the formula,

[0219] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0220] R c and R dEach is independently selected from H or -CH3.

[0221] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0222] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0223] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0224] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0225] In some embodiments, R a and R b At least one of them is H, and t+u is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 3.

[0226] In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and Rb At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, and at least one or R 9 and R 10 It contains an amide group.

[0227] In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group, and both Z groups are different.

[0228] In some embodiments, R a and R b At least one of them is H, and u is 0. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 2. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 4. In some embodiments, R a and Rb At least one of them is H, u is 0, and v is at least 6.

[0229] In some embodiments, R a and R b At least one of them is H, u is 0, and R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 2, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 4, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 6, R 9 and R 10 At least one of them contains an amide group.

[0230] In other embodiments, R 1 Equation (IIIC): TIFF0007914109000015.tif53170

[0231] (In the formula,

[0232] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0233] R c and R d Each is independently selected from H or -CH3.

[0234] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0235] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0236] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0237] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0238] In some embodiments, R a and R b At least one of them is H, and t+u is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 3.

[0239] In some embodiments, R a and R b At least one of them is H, and u is 0. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 2. In some embodiments, R a and R bAt least one of them is H, u is 0, and v is at least 4. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 6.

[0240] In other embodiments, R 1 Equation (IIID): TIFF0007914109000016.tif53170

[0241] (In the formula,

[0242] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0243] R c and R d Each is independently selected from H or -CH3.

[0244] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0245] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0246] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0247] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0248] In some embodiments, R a and R b At least one of them is H, and t+u is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 3.

[0249] In some embodiments, R a and R b At least one of them is H, and u is 0. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 2. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 4. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 6.

[0250] In some embodiments, R a and R b At least one of them is H, u is 0, and R 9 is an amide. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 2, R 9 is an amide. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 4, R 9 is an amide. In some embodiments, R a and R bAt least one of them is H, u is 0, v is at least 6, R 9 It is an amide.

[0251] In other embodiments, R 1 Equation (IIIE): TIFF0007914109000017.tif53170

[0252] (In the formula,

[0253] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0254] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0255] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0256] (v is an integer in the range of 1 to 8) It has the structure shown in [figure name]. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4. In other embodiments, v is an integer in the range of 3 to 6. In yet another embodiment, v is an integer in the range of 4 to 6.

[0257] In some embodiments, R 2 This is a carbonyl reactive group. Suitable carbonyl reactive groups include hydrazine, hydrazine derivatives, and amines.

[0258] In other embodiments, R 2This is an amine-reactive group. Suitable amine-reactive groups include active esters such as NHS or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, and anhydrides.

[0259] In yet another embodiment, R 2 is a thiol-reactive group. Suitable thiol-reactive groups include non-polymerizable Michael acceptors, nucleophilic aromatic groups such as haloacetyl groups (e.g., iodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups, vinyl sulfones, benzoquinones, fluorobenzene groups (e.g., tetrafluorobenzene groups), and disulfide groups such as pyridyl disulfide groups and thiols activated with Ellman reagents.

[0260] In some embodiments, R 2A click chemistry reaction is defined as a functional group or a part containing a functional group that can participate in a click chemistry reaction. "Click chemistry" is a chemical philosophy uniquely defined by the Sharpless and Meldal group, describing a chemistry tuned to rapidly and reliably produce substances by joining small units. "Click chemistry" has been applied to a collection of reliable and autonomous organic reactions (Kolb, HCFinn, MG; Sharpless, KBAngew). Chem. Int. Ed. 2001, 40, 2004-2021. For example, the identification of copper-catalyzed azide-alkyne[3+2] cycloaddition as a reliable molecular bonding method in water (Rostovtsev, VV; et al. Angew. Chem. Int. Ed. 2002, 41, 2596-2599) has been used to reinforce investigations of several types of biomolecular interactions (Wang, Q.; et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Speers, AE; et al. J. Am. Chem. Soc. 2003, 125, 4686-4687; Link, AJ; Tirrell, DAJ Am. Chem. Soc. 2003, 125, 11164-11165; Deiters, A.; et al. J. Am. Chem. Soc. 2003, 125, 11782-11783).Furthermore, our research includes organic synthesis (Lee, LV; et al. J. Am. Chem. Soc. 2003, 125, 9588-9589), drug discovery (Kolb, HC; Sharpless, KBDrug Disc. Today 2003, 8, 1128-1137; Lewis, WG; et al. Angew. Chem. Int. Ed. 2002, 41, 1053-1057), and surface functionalization (Meng, J.-C.; et al. Angew. Chem. Int. Ed. 2004, 43, 1255-1260; Fazio, F.; et al. J. Am. Chem. Soc. 2002, 124, 14397-14402; Collman, JP; et al. Langmuir Applications to Lummerstorfer, T., and Hoffmann (2004, ASAP, in press) have also emerged.

[0261] In some embodiments, R 2 These include dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups.

[0262] In some embodiments, R 1 R is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In other embodiments, R 1 R is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1R is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 This refers to a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0263] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0264] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N.

[0265] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1 R is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group.

[0266] In some embodiments, R 1 Q is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In other embodiments, R 1 Q is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1 Q is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1Q is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1 Q is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group. In further embodiments, R 1 Q is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a thiol-reactive group, a carbonyl-reactive group, or an amine-reactive group.

[0267] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In yet another embodiment, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 R comprises a group or moiety containing a functional group that can participate in a "click chemistry" reaction. Functional groups that can participate in a "click chemistry" reaction are described herein. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 R comprises a group or moiety containing a functional group that can participate in a "click chemistry" reaction. Functional groups that can participate in a "click chemistry" reaction are described herein. In further embodiments, R 1 R is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 2 This includes a group or moiety containing a functional group that can participate in click chemistry reactions. Functional groups that can participate in click chemistry reactions are described herein.

[0268] In some embodiments, R 1 Q is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In other embodiments, R 1 Q is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In yet another embodiment, R 1 Q is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In further embodiments, R 1 Q is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In further embodiments, R 1 Q is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 R includes a group or moiety containing a functional group that can participate in a "click chemistry" reaction. In further embodiments, R 1 Q is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N, 1 is O and at least one Q 2 H is R 2 This includes a group or moiety containing a functional group that can participate in "click chemistry" reactions.

[0269] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more oxygen heteroatoms. In other embodiments, R 1R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms. In a further embodiment, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms.

[0270] In some embodiments, R 1 This is an unbranched aliphatic group having 1 to 30 carbon atoms, optionally containing one or more oxygen heteroatoms, and further comprising at least one substitution on one of the carbon atoms. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms, optionally containing one or more oxygen heteroatoms, and further comprising at least one substitution on one of the carbon atoms. In yet another embodiment, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms, optionally containing one or more oxygen heteroatoms, and further comprising at least one substitution on one of the carbon atoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms, optionally containing one or more oxygen heteroatoms, and further comprising at least one substitution on one of the carbon atoms. In a further embodiment, R 1 This is an unbranched aliphatic group having 1 to 8 carbon atoms, optionally containing one or more oxygen heteroatoms, and further comprising at least one substitution on one of the carbon atoms. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms, optionally containing one or more oxygen heteroatoms, and further containing at least one substitution on one of the carbon atoms.

[0271] In some embodiments, R 1 Q is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more oxygen heteroatoms. 1 is O and at least one Q 2 In another embodiment, R 1 Q is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more oxygen heteroatoms. 1 is O and at least one Q 2 In another embodiment, R 1 Q is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more oxygen heteroatoms. 1 is O and at least one Q 2 is H. In a further embodiment, R 1 Q is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more oxygen heteroatoms. 1 is O and at least one Q 2 is H. In a further embodiment, R 1 Q is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms. 1 is O and at least one Q 2 is H. In a further embodiment, R 1 Q is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms. 1 is O and at least one Q 2 H is H.

[0272] In some embodiments, R 1 It is a bond, R 2 is H. In some embodiments, R 1 It is a bond, R 2 H is Q 1 In some embodiments, R 1 It is a bond, R 2 H is Q 1 is O and at least one Q 2 H is H.

[0273] In some embodiments, the caged hapten of the present disclosure is given by formulas (IIA) to (IIF): TIFF0007914109000018.tif233170TIFF0007914109000019.tif229170

[0274] (In the formula,

[0275] Q 1 is either O or S,

[0276] Q 2 is H, -CH3 or -CH2CH3,

[0277] R 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0278] R 2 is H or a reactive functional group,

[0279] R 3 These are H, -CH3, -CH2CH3, -OH, or -O-Me,

[0280] R 4 These are H, -CH3, or -CH2CH3, -OH, or -O-Me,

[0281] Each R 5 These are independently H, -CH3, -CH2CH3, halogen, or -C(O)H.

[0282] R 6 is either H, or a linear or branched, substituted or unsubstituted C1-C6 alkyl group.

[0283] m, n, and o are each independent integers ranging from 0 to 4.

[0284] p and q are each independent integers in the range of 0 or 1 to 3.

[0285] s is 1 or 2, and

[0286] X and Y are independently -CH2- and -C(R 7 )-, -N(H)-, -N(R 7 )-, -O-, or -S-, or -C(O)-, R 7 (A C1-C4 linear or branched alkyl group is either substituted or unsubstituted.) It has any one structure.

[0287] As described above, in some embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 30 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In other embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 1 to 20 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 5 to 15 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 6 to 12 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R may be a bond, or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having 8 to 12 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. In some embodiments, R 1 This may include a carbonyl, amine, ester, ether, amide, imine, thion, or thiol group. In other embodiments, R 1 It may contain one or more terminal groups selected from amines, carbonyls, esters, ethers, amides, imines, thions, or thiols.

[0288] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O, N, or S.

[0289] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more heteroatoms selected from the group consisting of O or N.

[0290] In some embodiments, R 1 R is an unbranched aliphatic group having 1 to 30 carbon atoms and optionally containing one or more oxygen heteroatoms. In other embodiments, R 1 R is an unbranched aliphatic group having 1 to 20 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 15 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 12 carbon atoms and optionally containing one or more oxygen heteroatoms. In further embodiments, R 1 R is an unbranched aliphatic group having 1 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms. In a further embodiment, R 1This is an unbranched aliphatic group having 4 to 8 carbon atoms and optionally containing one or more oxygen heteroatoms.

[0291] In some embodiments, R 1 This is a non-branched aliphatic group having 1 to 30 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In other embodiments, R 1 This is a non-branched aliphatic group having 1 to 20 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is a non-branched aliphatic group having 1 to 15 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is a non-branched aliphatic group having 1 to 12 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is a non-branched aliphatic group having 1 to 8 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom. In further embodiments, R 1 This is an unbranched aliphatic group having 4 to 8 carbon atoms, optionally containing one or more oxygen heteroatoms, and having at least one substitution on at least one carbon atom.

[0292] In some embodiments, R 1 Equation (IIIA): TIFF0007914109000020.tif53170

[0293] (In the formula,

[0294] R 8 The bonds are -O-, -S-, -C(R c )(R d ), or -N(R c )- and,

[0295] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0296] R c and R d Each is independently selected from H or -CH3.

[0297] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0298] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0299] t and u are independently 0, 1, or 2, where t+u is at least 1.

[0300] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0301] In some embodiments, R 8 is -C(R c )(R d )-, t+u is at least 2, and v is at least 1. In some embodiments, R 8 is -C(R c )(R d )-, t+u is at least 2, and v is at least 2. In some embodiments, R 8 is -C(R c )(R d)-, t+u is at least 2, and v is at least 3.

[0302] In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, and v is at least 1. In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, and v is at least 3.

[0303] In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R 8 is -C(R c )(R d )- and R c and R dAt least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group.

[0304] In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group.

[0305] In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and Rd At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is -C(R c )(R d )- and R c and R d At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group, and both Z groups are different.

[0306] In some embodiments, R 8 is O, t+u is at least 2, and v is at least 1. In some embodiments, R 8 is O, t+u is at least 2, and v is at least 2. In some embodiments, R 8 is O, t+u is at least 2, and v is at least 3.

[0307] In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group.

[0308] In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, and R 8 is O, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group, and both Z groups are different.

[0309] In other embodiments, R 1 Equation (IIIB): TIFF0007914109000021.tif53170

[0310] (In the formula,

[0311] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0312] R c and R d Each is independently selected from H or -CH3.

[0313] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0314] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0315] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0316] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0317] In some embodiments, R a and R b At least one of them is H, and t+u is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R a and R bAt least one of them is H, t+u is at least 2, and v is at least 3.

[0318] In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, and at least one or R 9 and R 10 It contains an amide group.

[0319] In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of the Z groups contains an amide group, and both Z groups are different. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, v is at least 1, R 9 and R 10 At least one of them contains an amide group, and both Z groups are different.

[0320] In some embodiments, R a and R b At least one of them is H, and u is 0. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 2. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 4. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 6.

[0321] In some embodiments, R a and R b At least one of them is H, u is 0, and R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 2, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 4, R 9 and R 10 At least one of them contains an amide group. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 6, R 9 and R 10 At least one of them contains an amide group.

[0322] In other embodiments, R 1 Equation (IIIC): TIFF0007914109000022.tif53170

[0323] (In the formula,

[0324] R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and

[0325] R c and R d Each is independently selected from H or -CH3.

[0326] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0327] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0328] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0329] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0330] In some embodiments, R a and R b At least one of them is H, and t+u is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 3.

[0331] In some embodiments, R a and R b at least one is H, and u is 0. In some embodiments, R a and R b at least one is H, u is 0, and v is at least 2. In some embodiments, R a and R b at least one is H, u is 0, and v is at least 4. In some embodiments, R a and R b at least one is H, u is 0, and v is at least 6.

[0332] In other embodiments, R 1 is represented by formula (IIID): TIFF0007914109000023.tif53170

[0333] (wherein,

[0334] R a and R b are each independently H, a C1~C4 alkyl group, F, Cl or -N(R c )(R d ),

[0335] R c and R d are each independently selected from H or -CH3,

[0336] R 9 and R 10 are each independently a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione and thiol,

[0337] each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0338] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0339] (v is an integer in the range of 1 to 8) It has the structure shown. In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4.

[0340] In some embodiments, R a and R b At least one of them is H, and t+u is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 2. In some embodiments, R a and R b At least one of them is H, t+u is at least 2, and v is at least 3.

[0341] In some embodiments, R a and R b At least one of them is H, and u is 0. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 2. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 4. In some embodiments, R a and R b At least one of them is H, u is 0, and v is at least 6.

[0342] In some embodiments, R a and R b At least one of them is H, u is 0, and R 9 is an amide. In some embodiments, R aand R b At least one of them is H, u is 0, v is at least 2, R 9 is an amide. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 4, R 9 is an amide. In some embodiments, R a and R b At least one of them is H, u is 0, v is at least 6, R 9 It is an amide.

[0343] In other embodiments, R 1 Equation (IIIE): TIFF0007914109000024.tif53170

[0344] (In the formula,

[0345] R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol.

[0346] Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-,

[0347] u and t are independently 0, 1, or 2, where u+t is at least 1.

[0348] (v is an integer in the range of 1 to 8) It has the structure shown.

[0349] In some embodiments, v is in the range of 1 to 6. In other embodiments, v is in the range of 1 to 4. In yet another embodiment, v is in the range of 2 to 6. In yet another embodiment, v is in the range of 2 to 4. In another embodiment, v is an integer in the range of 3 to 6. In yet another embodiment, v is an integer in the range of 4 to 6.

[0350] In some embodiments, R 2 R is a carbonyl reactive group. Suitable carbonyl reactive groups include hydrazine, hydrazine derivatives, and amines. In other embodiments, R 2 R is an amine-reactive group. Suitable amine-reactive groups include active esters such as NHS or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, and anhydrides. In further embodiments, R 2 is a thiol-reactive group. Suitable thiol-reactive groups include non-polymerizable Michael acceptors, nucleophilic aromatic groups such as haloacetyl groups (e.g., iodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups, vinyl sulfones, benzoquinones, fluorobenzene groups (e.g., tetrafluorobenzene groups), and disulfide groups such as pyridyl disulfide groups and thiols activated with Ellman reagents.

[0351] In some embodiments, R 2 R is a functional group or a molar containing a functional group that can participate in click chemistry reactions. In some embodiments, R 2 These include dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups.

[0352] In some embodiments, R3 and R 4 is at least one -CH3. In some embodiments, R 3 and R 4 is at least one -CH3, and R 6 is a C1~C4 alkyl group. In some embodiments, R 3 and R 4 is at least one -CH3, and R 6 is a C1~C2 alkyl group. In some embodiments, R 3 and R 4 is at least one -CH3, and R 6 is H.

[0353] In some embodiments, R 3 and R 4 are both -CH3, and R 6 is a C1~C4 alkyl group. In some embodiments, R 3 and R 4 are both -CH3, and R 6 is a C1~C2 alkyl group. In some embodiments, R 3 and R 4 are both at least -CH3, and R 6 is H.

[0354] In some embodiments, m, n, p and q are each 1, and each R 5 is selected from H or -CH3. In some embodiments, m, n, p and q are each 1, and each R 5 is selected from H or -CH3, and at least one R 3 or R 4 is -CH3. In some embodiments, m, n, p and q are each 1, and each R 5 is selected from H or -CH3, and R 3 and R 4 are both -CH3.

[0355] In some embodiments, m, n, p and q are each 1, and each R5 is selected from H or -CH3, and s is 1. In some embodiments, m, n, p and q are each 1, and each R 5 is selected from H or -CH3, s is 1, and at least one R 3 or R 4 is -CH3. In some embodiments, m, n, p and q are each 1, and each R 5 is selected from H or -CH3, s is 1, R 3 and R 4 Both are -CH3.

[0356] In some embodiments, m, n, p, and q are each 1, and at least one R 5 is -CH3. In some embodiments, m, n, p, and q are each 0. In some embodiments, m, n, p, and q are each 0, and at least one R 3 or R 4 is -CH3. In some embodiments, m, n, p, and q are each 0, and R 3 and R 4 Both are -CH3.

[0357] In some embodiments, X is O. In some embodiments, X is O and Y is -C(O)-. In some embodiments, X is O. In some embodiments, X is O, Y is -C(O)- and s is 1. In some embodiments, X is O, Y is -C(O)-, s is 1 and Q 1 is O. In some embodiments, X is O, Y is -C(O)-, s is 1, and Q 1 is S. In some embodiments, X is O, Y is -C(O)-, s is 1, and Q 1 is O, and each Q 2 H is H.

[0358] Non-limiting examples of compounds of formulas (IIIA) to (IIIF) include: This includes, but is not limited to, TIFF0007914109000025.tif187170.

[0359] Caged Hapten Conjugate

[0360] This disclosure also provides conjugates comprising caged haptens. In some embodiments, the conjugate comprises a specific binding entity and a caged hapten having any one structure of formula (IA), (IB), or (IIA) to (IIF). Methods for conjugating a specific binding entity, such as an antibody, nucleic acid molecule, oligonucleotide, etc., to a caged hapten are described herein.

[0361] In some embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IA). In other embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IB). In other embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IIA). In other embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IIB). In other embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IIC). In other embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IID). In other embodiments, the conjugate includes an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IIE). In other embodiments, the conjugate comprises an antibody (e.g., a primary or secondary antibody) conjugated to a caged hapten having the structure of formula (IIF). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the primary or secondary antibody is a monoclonal antibody.

[0362] Examples of primary antibodies include anti-Her2, anti-Her3, anti-PD-L1, anti-PD-1, anti-E-cadherin, anti-β-catenin, anti-EGFR(Her1), anti-cMET, anti-GRB2, anti-TIGIT, anti-phosphotyrosine, and anti-ubiquitin. Examples of secondary antibodies include anti-rabbit, anti-mouse, anti-rat, anti-goat, anti-camelid, anti-DIG, anti-DNP, and anti-fluorescein.

[0363] The caged haptens of this disclosure may be conjugated to any portion of an antibody or any portion of a monoclonal antibody. Those skilled in the art will understand that antibodies contain three distinct types of functional groups suitable for covalent modification, including (i) amine (-NH2), (ii) thiol (-SH), and (iii) carbohydrate residues. Therefore, any of the caged haptens disclosed herein may be conjugated to an amine residue, a thiol residue, and a carbohydrate residue, or any combination thereof. In some embodiments, the caged hapten is conjugated to the Fc portion of the antibody.

[0364] In some embodiments, the specific binding entity is a nucleic acid molecule or an oligonucleotide. In some embodiments, the nucleic acid molecule contains 5 to about 50 nucleotides. In other embodiments, the nucleic acid molecule contains 5 to about 40 nucleotides. In other embodiments, the nucleic acid molecule contains 5 to about 30 nucleotides. In other embodiments, the nucleic acid molecule contains 5 to about 25 nucleotides. In other embodiments, the nucleic acid molecule contains 5 to about 20 nucleotides. In other embodiments, the nucleic acid molecule contains 5 to about 15 nucleotides.

[0365] In some embodiments, the caged hapten conjugate of the present disclosure is of formula (IVA) or (IVB): [Specific binding entity]-W 1 -W 2 -R 1 -O-[DIG]-[phosphoryl](IVA) [Specific binding entity]-W 1 -W2 -R 1 -O-[DIG]-PO4H2(IVB)

[0366] (In the formula,

[0367] [Specific binding entity] is a specific binding entity,

[0368] W 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group containing 1 to 10 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S.

[0369] W 2 It is either a bond or derived from a reactive functional group.

[0370] "Reactive functional group", R 1 ([DIG] and [phosphoryl] are as described herein.) It has any one structure.

[0371] In some embodiments, the [specific binding entity] is an antibody, for example, a monoclonal antibody. In some embodiments, the [specific binding entity] is a primary antibody (for example, a caged hapten conjugated to an antibody specific to β-catenin). In some embodiments, the [specific binding entity] is a secondary antibody (for example, a caged hapten conjugated to an antibody specific to anti-β-catenin antibody). In some embodiments, the [specific binding entity] is a nucleic acid molecule or oligonucleotide.

[0372] In some embodiments, the caged hapten conjugate is expressed by formula (VA) or (VF): TIFF0007914109000026.tif232170TIFF0007914109000027.tif234170

[0373] (In the formula,

[0374] [Specific binding entity] is a specific binding entity,

[0375] W 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group containing 1 to 10 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S.

[0376] W 2 is either a bond or derived from a reactive functional group (as described herein),

[0377] Q 1 Q 2 , R 1 , R 3 ~R 7 (where m, n, o, p, q, s, X, and Y are as defined herein) It has any one structure.

[0378] In some embodiments, the [specific binding entity] is an antibody, for example, a monoclonal antibody. In some embodiments, the [specific binding entity] is a primary antibody (for example, a caged hapten conjugated to an antibody specific to β-catenin). In some embodiments, the [specific binding entity] is a secondary antibody (for example, a caged hapten conjugated to an antibody specific to anti-β-catenin antibody). In some embodiments, the [specific binding entity] is a nucleic acid molecule or oligonucleotide.

[0379] W 1 and W 2 The following are non-restrictive examples of the base. TIFF0007914109000028.tif199170

[0380] Non-exclusive examples of the conjugates described herein include: TIFF0007914109000029.tif229170

[0381] Synthesis of caged hapten conjugates

[0382] The caged hapten conjugates of this disclosure can be synthesized according to any method known to those skilled in the art. In some embodiments, caged haptens (including any of those of formulas (IA), (IB), and (IIA) to (IIF), as described herein) can be conjugated to the thiol groups of an antibody, for example, the thiol groups of a monoclonal antibody. In some embodiments, the thiol groups are first introduced into the antibody by treating the antibody with a reducing agent such as dithiothreitol (DTT) or dithioerythritol (DTE). For mild reducing agents such as DTE or DTT, concentrations of about 1 mM to about 40 mM (e.g., about 5 mM to about 30 mM or about 15 mM to about 25 mM) are used to introduce a limited number of thiols (about 2 to about 6, etc.) into the antibody while keeping the antibody intact (which can be determined by size exclusion chromatography). After treatment with a reducing agent, an excess of caged haptens having a thiol-reactive group (e.g., a maleimide group) is introduced to form each caged hapten-antibody conjugate. Other methods for introducing one or more thiol groups are described in U.S. Patent Application Publication 2016 / 0187324, the disclosure of which is incorporated herein by reference in whole.

[0383] In other embodiments, a caged hapten may be conjugated to the Fc portion of the antibody. In some embodiments, the Fc portion of the antibody is first oxidized to form an aldehyde, and then the caged hapten is conjugated to the oxidized Fc portion of the antibody through a reactive functional group on the caged hapten (e.g., a carbonyl reactive group such as a hydrazide group).

[0384] In yet another embodiment, the caged hapten may be conjugated to a lysine residue of the antibody, for example, a lysine residue of a monoclonal antibody. In some embodiments, as shown in the following synthesis scheme (Scheme 2), the antibody is first treated with an excess of Trout's reagent (2-iminothiolane hydrochloride) before adding an excess of a well-functionalized caged hapten (e.g., one having a thiol-reactive group such as a maleimide group).

[0385] After the synthesis of caged hapten conjugates, the conjugates can be purified by methods such as size exclusion chromatography (SEC) and then characterized by gel electrophoresis and / or UV-Vis.

[0386] Proximity detection using caged hapten conjugates

[0387] As described in more detail herein, this disclosure facilitates the detection of protein-protein complexes, such as protein dimers or proteins that are in close proximity to each other (e.g., having a proximity of 5000 nm or less). In some embodiments, the assay can detect protein dimers or proteins having a proximity of 4000 nm or less. In other embodiments, the assay can detect protein dimers or proteins having a proximity of 3000 nm or less. In other embodiments, the assay can detect protein dimers or proteins having a proximity of 2500 nm or less. In other embodiments, the assay can detect protein dimers or proteins having a proximity of 2000 nm or less. In other embodiments, the assay can detect protein dimers or proteins having a proximity of 1500 nm or less. In yet another embodiment, the assay can detect protein dimers or proteins having a proximity of 1000 nm or less. In yet another embodiment, the assay can detect protein dimers or proteins having a proximity of 500 nm or less.

[0388] Protein-protein complexes (PPCs) form signaling nodes and hubs in molecular networks during all physiological processes, including cellular disease states and cancer. Reprogrammed cancer-initiated cells acquire and maintain all the characteristics of cancer by acquiring new physical and molecular features and altering molecular signaling pathways, leading to pathological outcomes. PPCs are thought to be involved in transmitting oncogenic signals in those cells. PPCs are also thought to be involved in increasing signaling and evading growth inhibitors, which in turn leads to cancer development and progression. Non-limiting examples of protein-protein complexes include any of the Her1 / 2 / 3 / 4 proteins. Examples include PD-1 and PD-L1; and / or PD-L2, EGFR (Her1) and its associated ligands (AREG, EREG); the adapter protein GRB2 and EGFR, phosphorylated tyrosine proteins such as cMET, Her2, and MUC1; and TIGIT and CD155.

[0389] The applicant believes that PPCs represent a highly promising class of targets for therapeutic development, as well as for functional diagnostics in immunohistochemistry (IHC). Conventional IHC, due to the diffraction limitations of conventional optical microscopy, detects the presence of a single epitope at a resolution limit in the range of 200 nanometers. As a result, it is only possible to describe proteins in terms of co-localization, rather than complexes that exist on the order of tens of nanometers. The ability to investigate the presence and distribution of specific intermolecular complexes on frozen and paraffin-embedded tissues allows IHC to shift from structural to functional diagnostics. Therefore, including PPCs and the molecular networks they represent within the human interactome broadens the definition of biomarkers.

[0390] The disclosed proximity assay is considered more general than simply measuring protein-protein interactions. In fact, the disclosed assay allows for the measurement of proximity of binding sites. In practice, binding sites (e.g., antibodies) can be made against targets where the distance between them is minimal or nonexistent. Examples of this include signaling events such as protein phosphorylation. In this case, if one antibody is made against an epitope on a protein (e.g., HER2) and a second antibody is made against all phosphotyrosine, the proximity signal would represent all phosphorylated HER2 proteins. This type of assay is binary (yes / no) due to the pair of proteins interacting with each other.

[0391] Any of the caged hapten conjugates of this disclosure may be used in either (i) a mono-assay for the detection of protein dimers or protein proximity, or (ii) a multiplex assay for the detection of protein dimers or protein proximity and total protein. "Total protein" refers to the normal IHC visualization of any given protein, whereas proximity signals are the portion of this protein involved in a given interaction. For example, in a PD-1 / PD-L1 assay, proximity signals visualize only the interaction between PD-1 and PD-L1, while total protein signals visualize all PD-1 in the sample. Expressing the score for proximity as the numerator and the score for total protein as the denominator can give the proportion or percentage of PD-1 involved in the interaction. This may be important as a diagnostic method for detecting pharmaceutical active ingredients that interfere with protein-protein interactions when protein expression is less important than the number of interacting proteins. This is thought to also apply to phosphorylation, as mentioned above, where instead of simply receiving an arbitrary score for the phosphorylation signal, it may be possible to quantify what percentage of a given protein is phosphorylated.

[0392] Referring to Figure 4, the detection of protein dimers is performed in two general steps. In the first step 150, the sample is labeled with at least two different antibody conjugates, for example, at least two different monoclonal antibody conjugates. In the second step 160, the sample is brought into contact with a first set of detection reagents (e.g., in a single-component assay) and optionally with a second set of detection reagents (e.g., in a multiple-component assay). After the second step 160, signals from the first and optionally second sets of detection reagents are detected (step 140). The signals may be detected in accordance with methods known to those skilled in the art, such as those described in U.S. Patent No. 10,041,950, and U.S. Patent Application Publication Nos. 2019 / 0204330, 2017 / 0089911, and 2019 / 0187130, and International Publication No. 2014 / 143155, and these disclosures are incorporated herein by reference in their entirety.

[0393] In the first step 150, the sample is contacted with a caged hapten-antibody conjugate specific to the first target to form a first target-caged hapten-antibody conjugate complex (step 100). In some embodiments, the caged hapten-antibody conjugate has any one of the formulas (IVA), (IVB), or (VA) to (VF). As further described herein, the caged hapten portion of the caged hapten-antibody conjugate can be left unmasked to provide the respective demasked haptens, i.e., “natural haptens” or “uncaged haptens”. For example, to provide a natural DIG hapten, a caged DIG hapten can be left unmasked. Similarly, to provide a natural steroid, a caged steroid can be left unmasked.

[0394] Next, in order to form a second target-demasking enzyme-antibody conjugate complex, the sample is first contacted with a demasking enzyme-antibody conjugate specific to the second target (step 110). In some embodiments, the demasking enzyme (e.g., phosphatase, phosphodiesterase, phosphotriesterase) of the demasking enzyme-antibody conjugate is reactive with the enzyme substrate portion of the caged hapten-antibody conjugate introduced in step 100. For example, the demasking enzyme of the demasking enzyme-antibody conjugate may be reactive with the [phosphoryl] group of formula (IVA), the PO4H2 group of formula (IVB), or any one phosphate-containing group of formulas (VA) to (VF).

[0395] In some embodiments, steps 100 and 110 may be performed in any order or simultaneously. In some embodiments, step 100 is performed first, followed by step 110. In other embodiments, step 110 is performed first, followed by step 100.

[0396] In some embodiments, the first step 150 also includes one or more “decaging steps” in which conditions on the slide are modified to enhance enzyme activity. The “decaging steps” include, but are not limited to, one or more washing steps or steps to adjust the pH (e.g., pH in the range of about 7 to about 8.5). In some embodiments, decaging is carried out in Tris buffer at a temperature of about 37°C, at a pH in the range of about 7.4 to about 7.6, for a time in the range of about 4 to about 32 minutes. Each decaging enzyme is thought to have its own optimal conditions (buffers, salts, cofactors, temperature), and the parameters of any decaging step may be selected to enhance enzyme activity and promote “decaging” without interfering with the specific binding of the antibody conjugate.

[0397] In some embodiments, the first step 150 also includes contacting the sample with one or more reversible enzyme inhibitors to prevent the action of the enzyme on the casing group. In some embodiments, one or more reversible enzyme inhibitors are added after the introduction of both the demasking antibody conjugate and the caged hapten antibody conjugate. In relation to alkaline phosphatase (AP), these reversible enzyme inhibitors may include phosphates, phenylalanine, and EDTA, which are thought to reduce enzyme activity by various mechanisms.

[0398] In the second step 160, the sample is then contacted with a first set of detection reagents specific to the native hapten of the caged hapten-antibody conjugate (i.e., the first set of detection reagents is specific to the uncaged form of the hapten of the caged hapten conjugate) (step 120). Optionally, the sample is contacted with a second set of detection reagents specific to the demasking enzyme of the demasking enzyme-antibody conjugate (step 130). In some embodiments, steps 120 and 130 may be performed in any order or simultaneously.

[0399] The protein proximity assays of this disclosure are further illustrated in Figures 2, 3 and 5-7. For example, Figure 2 is a schematic diagram illustrating the interaction between a demasking enzyme-antibody conjugate containing alkaline phosphatase (bound to target 2) and a caged hapten-antibody conjugate (bound to target 1), where the demasking enzyme (e.g., alkaline phosphatase) of the demasking enzyme-antibody conjugate reacts with the enzyme substrate portion (e.g., phosphate group or its derivative) of the caged hapten-antibody conjugate (because targets 1 and 2 are in close proximity to each other) to provide the respective demasked haptens, which can be detected. Similarly, Figure 7 is a schematic diagram illustrating the multiple detection of both proximity proteins (targets 1 and 2) and total protein (target 2).

[0400] Referring to Figures 2 and 7, if the first target 101 is sufficiently close to the second target 102, the caged hapten-antibody conjugate 103A is provided in sufficient close proximity to the demasking enzyme-antibody conjugate 104 (proximity is labeled 105) so that the demasking enzyme of the demasking enzyme-antibody conjugate 104 can react with the enzyme substrate of the caged hapten-antibody conjugate 103A. This subsequently leads to the formation of the first target demasking hapten-antibody conjugate complex (103B). As illustrated in Figures 2, 5 and 7, the first target demasking hapten-antibody conjugate complex (103B) can bind to other specific binding entities (e.g., secondary antibody 106) or can be recognized by other specific binding entities (e.g., secondary antibody 106).

[0401] On the other hand, as shown in Figure 3, if the first target 101 is not sufficiently close to the second target 102, the caged hapten-antibody conjugate 103A will not be provided in close proximity to the demasking enzyme-antibody conjugate 104 (proximity is labeled 108). In this example, the demasking enzyme does not react with the enzyme substrate of the caged hapten-antibody conjugate 103A, and therefore the caged hapten remains masked or protected, i.e., it is unable to bind to other specific binding entities or is not recognized by other specific binding entities.

[0402] Referring again to Figures 2, 5, and 7, after the introduction of the antibody conjugate and any decaging step, the sample is then contacted with a first detection reagent (106) (step 120), the first detection reagent being specific to the demasked hapten (103B) of the demasked hapten-antibody conjugate complex, which is the first target. In some embodiments, the first detection reagent contains a secondary antibody (106) specific to the demasked hapten (103B), i.e., an anti-desmasked hapten antibody. In some embodiments, the anti-desmasked hapten antibody (106) is conjugated to a detectable moiety (for example, in Figures 2 and 7, the detectable moiety is an HRP enzyme, which acts on a substrate such as a silver-chromogenic substrate). In some embodiments, the first detection reagent (106) binds only if the native or demasked hapten (103B) of the first target, the demasked hapten-antibody conjugate complex, is demasked by the demasking enzyme of the demasking enzyme-antibody conjugate (104). Thus, the signal (107) from the detectable portion of the first detection reagent (106) can only be detected in step 140 if the first and second targets (101 and 102), and therefore the antibody conjugates (103A and 104), are in close proximity to each other. Here, the detected signal (107) represents a close protein dimer or protein / target (compare with Figure 3 where the targets were not sufficiently close to each other).

[0403] In some embodiments, an amplification step may be performed to increase the detectable signal. For example, an amplification component may be introduced to further label the demasked hapten of a first target demasked hapten-antibody conjugate with an additional reporter portion, such as an additional hapten or other “detectable portion.” For example, an anti-desmasked hapten antibody conjugated to an amplified hapten (or, in other embodiments, conjugated to an enzyme) may be introduced to label the demasked hapten of a first target demasked hapten-antibody conjugate with multiple amplified haptens. Subsequently, anti-amplified hapten antibodies conjugated to each detectable portion may be introduced. In some embodiments, the anti-amplified hapten antibody is conjugated to an enzyme, which acts on an introduced substrate to generate a signal (e.g., a chromogenic substrate or fluorescent substrate for generating a visual signal). The TSA and QM conjugates described herein, respectively, may be used in any amplification step. In some examples, signal amplification is performed using the OPTIVIEW Amplification Kit (Ventana Medical Systems, Inc., Tucson, Ariz., catalog number 760-099).

[0404] Multiple detection

[0405] In some embodiments, the demasking enzyme in a demasking enzyme-antibody conjugate may perform two functions: (i) demasking or exposing a caged hapten, and (ii) reacting with another substrate (e.g., a chromogenic or fluorescent substrate) so that a signal independent of the signal produced by the demasking hapten (i.e., the demasking hapten-antibody conjugate complex) can be detected. Thus, the systems disclosed herein enable the visualization of proximity between two proteins in the context of total protein staining for one of these proteins. While we do not wish to be bound by any particular theory, the ability to multiplex proximity detection in the context of another protein staining is considered a feature that enables the possibility of having a rapid, guided slide read (i.e., looking only for proximity signals within total protein) or the ability to quantify the percentage of proteins interacting with another protein (a way of scoring proximity assays).

[0406] Referring again to Figures 2, 4, and 7 after the introduction of the first detection reagent (106), a second detection reagent containing a second detectable portion may be optionally introduced into the sample in step 130 so that total protein can be detected. In some embodiments, the second detectable portion gives a signal (112) different from the signal of the first detectable portion (107). In some embodiments, the second detectable portion includes a substrate for a demasking enzyme, for example, a chromogenic substrate that gives a yellow signal (109). In other embodiments, the second detectable portion includes a signal transduction conjugate.

[0407] In some embodiments, biological samples are pretreated with enzyme inactivation compositions to substantially or completely inactivate endogenous peroxidase activity. For example, some cells or tissues contain endogenous peroxidase. The use of HRP conjugate antibodies can result in high nonspecific background staining. This nonspecific background can be reduced by pretreatment of the sample with enzyme inactivation compositions disclosed herein. In some embodiments, to reduce endogenous peroxidase activity, the sample is pretreated with hydrogen peroxide alone (about 1% to about 3% by weight of the appropriate pretreatment solution). Once the endogenous peroxidase activity is reduced or inactivated, a detection kit may be added as described above, and the enzyme present in the detection kit may then be inactivated. The disclosed enzyme inactivation compositions and methods may also be used as methods for inactivating endogenous enzyme peroxidase activity. Further inactivation compositions are described in U.S. Patent Application Publication No. 2018 / 0120202, the disclosure of which is incorporated herein by reference in its entirety.

[0408] In some embodiments, if the specimen is embedded in paraffin, the specimen can be deparaffinized using a suitable deparaffinizing fluid. After a waste liquid removal agent removes the deparaffinizing fluid, any number of substances can be applied to the specimen sequentially. These substances may be for pretreatment (e.g., protein crosslinking, exposing nucleic acids), denaturation, hybridization, washing (e.g., strict washing), detection (e.g., visual or linking of marker molecules to probes), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, coverslips, etc.

[0409] Detection of caged hapten antibody conjugates

[0410] In some embodiments, a detection reagent is used to enable the detection of any of the caged hapten conjugates described herein, or a complex of a caged hapten conjugate with a target, such as a target in a sample. As described herein, in some embodiments, the detection reagent used is specific to the demasked or native hapten corresponding to the caged hapten of any caged hapten-conjugate. For example, if the caged hapten-conjugate is phosphorylated DIG, a detection reagent is used to enable the detection of DIG, which is the demasked or native hapten corresponding to phosphorylated DIG. The detection reagent may also include components designed to increase the signal, such as signal amplification components or signal amplification kits.

[0411] In some embodiments, the detection reagent specific to demasking haptens is a secondary antibody specific to the demasking hapten of a caged hapten conjugate, i.e., an anti-demasking hapten antibody, which is itself conjugated to a detectable moiety. The “detectable moiety” is a molecule or material that can generate a detectable signal (visually, electronically, or otherwise) indicating the presence (i.e., qualitative analysis) and / or concentration (i.e., quantitative analysis) of the caged hapten-antibody conjugate and / or demasking enzyme-antibody conjugate in a sample. The detectable signal may be generated by any known or undiscovered mechanism, including the absorption, emission, and / or scattering of photons (including radio frequency, microwave frequency, infrared frequency, visible frequency, and ultraviolet frequency photons).

[0412] In some embodiments, the detectable portion of an anti-demasked hapten antibody may include chromogenic, fluorescent, phosphorescent, or luminescent molecules and materials; catalysts (such as enzymes) that convert one substance to another (by converting a colorless substance to a colored substance or vice versa, or by producing a precipitate or increasing the turbidity of the sample, etc.) to produce a detectable difference; haptens that can be detected through antibody-hapten binding interactions using additional detectably labeled antibody conjugates; and paramagnetic and magnetic molecules or materials. Of course, it is also possible to detect the detectable portion itself indirectly; for example, if the detectable portion is a hapten, as is known to those skilled in the art, yet another antibody specific to that detectable portion may be used in the detection of the detectable portion.

[0413] In some embodiments, the anti-demasking hapten antibody is Cascade Blue acetyl azide; dapoxylsulfonic acid / carboxylic acid DY-405; Alexa Fluor 405; Cascade Yellow pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue DY-415; 7-hydroxycoumarin-3-carboxylic acid DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; Alexa Fluor 430; Dabcyl NBD chloride / fluoride; QSY35 DY-485XL; Cy2DY-490; Orgeon Green 488; Alexa Fluor 488; BODIPY 493 / 503 C3 DY-480XL; BODIPY FL C3; BODIPY FL C5; BODIPY FL-X DYQ-505; Oregon Green 514 DY-510XL; DY-481XL; 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimidyl ester (JOE); DY-520XL; DY-521XL; BODIPY R6G C3 erythrosine isothiocyanate; 5-carboxy-2',4',5',7'-tetrabromosulfone fluorescein Alexa Fluor 532; 6-carboxy-2',4,4',5',7,7'-hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530 / 550 C3 DY-530; BODIPY TMR-X DY-555; DYQ-1; DY-556; Cy3 DY-547; DY-549; DY-550; Alexa Fluor 555; Alexa Fluor 546 DY-548; BODIPY 558 / 568 C3 Rhodamine Red-X QSY 7; BODIPY 564 / 570 C3; BODIPY 576 / 589 C3 Carboxy-X-Rhodamine (ROX); Alexa Fluor 568 DY-590; BODIPY 581 / 591 C3; DY-591; BODIPY TR-X Alexa Fluor 594 DY-594; Carboxynaphthofluorescein DY-605; DY-610; Alexa Fluor 610 DY-615; BODIPY 630 / 650-X Erioglaucine;Alexa Fluor 633 Alexa Fluor 635 Succinimidyl Ester, ;DY-634;DY-630;DY-631;DY-632;DY-633;DYQ-2;DY-636;BODIPY650 / 665-X DY-635;Cy5 Alexa Fluor 647 DY-647;DY-648;DY-650;DY-654;DY-652;DY-649;DY-651;DYQ-660;DYQ-661;Alexa Fluor 660 Cy5.5 DY-677;DY-675;DY-676;DY-678;Alexa Fluo680 DY-679;DY-680;DY-682;DY-681;DYQ-3;DYQ-700;Alexa Fluor700 The detectable portion is selected from the group consisting of DY-703; DY-701; DY-704; DY-700; DY-730; DY731; DY-732; DY-734; DY-750; Cy7 DY-749; DYQ-4; and Cy7.5.

[0414] Fluorophores belong to several common chemical classes, including coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorphine, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in *Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies*, *Molecular Probes*, Eugene, OR, ThermoFisher Scientific, 11. thThis is described in the Edition. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaline 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 portion may be CF dyes (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), or SureLight. Dyes (available from APC, RPEPerCP, and Phycobilisomes) (Columbia Biosciences), as well as APC, APCXL, RPE, and BPE (available from Phyco-Biotech, Greensea, Prozyme, and Flogen) are selected.

[0415] In other embodiments, the anti-demasking hapten antibody is conjugated to an enzyme. In these embodiments, the final proximity signal can be generated by any enzyme conjugated to the relevant anti-demasking hapten antibody, except for the enzyme used for demasking (e.g., the demasking enzyme of the demasking enzyme-antibody conjugate described further herein). In some embodiments, suitable enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, neuraminidase, β-galactosidase, β-glucuronidase, or β-lactamase. In other embodiments, the enzyme includes oxidoreductase or peroxidase (e.g., HRP). In these embodiments, the enzyme conjugated to the anti-demasking hapten antibody catalyzes the conversion of the chromogenic substrate, covalent hapten, covalent fluorophore, non-covalent chromogen, and non-covalent fluorophore into reactive moieties that label the sample near or directly on the target.

[0416] Specific and non-restrictive examples of chromogenic compounds / substrates include diaminobenzidine (DAB), 4-nitrophenyl phosphate (pNPP), Fast Red, bromochloroindolyl phosphate (BCIP), nitrobluetetrazolium (NBT), BCIP / NBT, AP Orange, AP Blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3-ethylbenzothiazoline sulfonate] (ABTS), o-dianisidine, 4-chloronaphthol (4-CN), and nitrophenyl-β-D-galactopyranoside (ONP G) contains o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal), p-nitrophenyl-α-D-galactopyranoside (PNP), 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, or tetrazolium violet. DAB, when oxidized in the presence of peroxidase and hydrogen peroxide, results in the deposition of a brown, alcohol-insoluble precipitate at the site of enzyme activity.

[0417] In some embodiments, the chromogenic substrate is a signaling conjugate comprising a potentially reactive moiety and a chromogenic moiety. In some embodiments, the potentially reactive moiety of the signaling conjugate is configured to undergo catalytic activation to form reactive species that can covalently bond with a sample or other detection component. Catalytic activation is driven by one or more enzymes (e.g., oxidoreductase enzymes and peroxidase enzymes such as horseradish peroxidase) and results in the formation of reactive species. These reactive species can react with the chromogenic moiety near their formation, i.e., in the vicinity of the enzyme. Specific examples of signaling conjugates are disclosed in U.S. Patent Application Publication No. 2013 / 0260379, which is incorporated herein by reference in its entirety.

[0418] Other substrates are described in U.S. Patent No. 5,583,001, U.S. Patent Application Publication No. 2012 / 0171668, and PCT / EP2015 / 0533556, the disclosures of which are incorporated herein by reference in their entirety. Suitable chromogenic or fluorescent substrates conjugated to a TSA or QM conjugate, as described in the incorporated references above, include N,N'-biscarboxypentyl-5,5'-disulfonate-indodicarbocyanine (Cy5), 4-(dimethylamino)azobenzene-4'-sulfonamide (Dabsyl), tetramethylrhodamine (Tamra), and rhodamine 110 (rhodamine).

[0419] In some embodiments, the chromogenic substrate, fluorescent substrate, or signaling conjugate is selected such that the peak detectable wavelengths of any chromogenic portion do not overlap with each other and are readily detectable by a pathologist or optical detector (e.g., a scanner). In some embodiments, the chromogenic portions are selected such that the peak wavelengths of different chromogenic portions are separated by at least about 50 nm. In other embodiments, the chromogenic portions are selected such that the peak wavelengths of different chromogenic portions are separated by at least about 70 nm. In yet another embodiment, the chromogenic portions are selected such that the peak wavelengths of different chromogenic portions are separated by at least about 100 nm. An example of a suitable detectable portion having a coumarin core is described in U.S. Patent No. 10,041,950, the disclosure of which is incorporated herein by reference in its entirety. Another suitable detectable portion is disclosed in U.S. Provisional Patent Application No. 63 / 071,518, the disclosure of which is incorporated herein by reference in its entirety.

[0420] In further embodiments, the color-developing portions are selected to provide a different color (e.g., yellow, blue, magenta) when introduced into a tissue specimen. In some embodiments, the color-developing portions are selected to give them good contrast to one another, e.g., optically recognizable color separation. In some embodiments, the color-developing portions are selected to give a different signal or color when placed in close proximity to one another than any of the color-developing portions when observed individually.

[0421] kit

[0422] In some embodiments, the caged hapten conjugates of the present disclosure may be used as part of a “detection kit.” Generally, any detection kit may comprise one or more caged hapten conjugates and a detection reagent for detecting one or more caged hapten conjugates. In some embodiments, the kit comprises a caged hapten conjugate of any of the formulas (IVA), (IVB), or (VA) to (VF).

[0423] The detection kit may comprise a first composition containing a caged hapten conjugate and a second composition containing a detection reagent specific to the first composition, so that the caged hapten conjugate can be detected through the detection kit. In some embodiments, the detection kit comprises multiple caged hapten conjugates (e.g., mixed together in a buffer), and the detection kit also comprises detection reagents specific to each of the multiple caged hapten conjugates.

[0424] Of course, any kit may include other active ingredients, such as buffers, counterstains, enzyme inactivation compositions, and deparaffinizing solutions, if required for manual or automated target detection. The kit may also include instructions for using any of the kit components, including how to apply the kit components to a tissue sample to detect one or more targets in the tissue sample.

[0425] automation

[0426] The assays and methods of this disclosure may be automated and may be combined with a sample processing device. The sample processing device may be an automated device such as the BENCHMARK Ultra and DISCOVERY Ultra instruments sold by Ventana Medical Systems, Inc. Ventana Medical Systems, Inc. is the assignee of numerous U.S. patents disclosing systems and methods for performing automated analysis, including U.S. Patents 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. Patent Application Publications 20030211630 and 20040052685, each of which is incorporated herein by reference in whole. Alternatively, the sample may be processed manually.

[0427] The specimen processing device can apply a fixative to the specimen. The fixative may include crosslinking agents (aldehydes, e.g., formaldehyde, paraformaldehyde, and glutaraldehyde, as well as non-aldehyde crosslinking agents), oxidizing agents (e.g., metal ions and complexes such as osmium tetroxide and chromic acid), protein denaturants (e.g., acetic acid, methanol, and ethanol), fixatives of unknown mechanism (e.g., mercury chloride, acetone, and picric acid), compound reagents (e.g., Carnoy's fixative, metacan, Bouin's solution, B5 fixative, Rossmann's liquid, and Gendre's liquid), microwaves, and other fixatives (e.g., excluded volume fixation and vapor fixation).

[0428] If the sample is embedded in paraffin, the sample can be deparaffinized using a sample processing device with an appropriate deparaffinizing solution. After a waste liquid removal agent removes the deparaffinizing solution, any number of substances can be applied to the sample sequentially. These substances may be for pretreatment (e.g., protein crosslinking, nucleic acid exposure), denaturation, hybridization, washing (e.g., strict washing), detection (e.g., visual or marker molecule linking to a probe), amplification (e.g., amplification of proteins, genes, etc.), counterstaining, coverslips, etc.

[0429] Sample processing equipment can coat samples with a wide range of substances. These substances include, but are not limited to, stains, probes, reagents, rinses, and / or conditioners. Substances can be fluids (e.g., gases, liquids, or gas / liquid mixtures). Fluids can be solvents (e.g., polar solvents, nonpolar solvents), solutions (e.g., aqueous solutions or other types of solutions), etc. Reagents may include, but are not limited to, stains, wetting agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies), antigen recovery fluids (e.g., aqueous or non-aqueous-based antigen recovery solutions, antigen recovery buffers, etc.). Probes can be isolated nucleic acids or isolated synthetic oligonucleotides attached to a detectable label. Labels may include radioisotopes, enzyme substrates, cofactors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.

[0430] After the specimen has been processed, the user can transport the slide containing the specimen to an imaging device. The imaging device used here is a bright-field slide scanner. One bright-field scanner is the iScan Coreo® bright-field scanner sold by Ventana Medical Systems, Inc. In an automated embodiment, the imaging device is a digital pathology device as disclosed in International Patent Application No. PCT / US2010 / 002772 (International Publication No. 2011 / 049608) titled "IMAGING SYSTEM AND TECHNIQUES," or in U.S. Patent Application Publication No. 2014 / 0178169, filed on February 3, 2014, titled "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 in their entirety by reference. In other embodiments, the imaging device includes a digital camera coupled to a microscope.

[0431] Counterstaining

[0432] Counterstaining is a post-treatment method used to detect one or more targets in a sample after it has already been stained with an active ingredient, making it easier to visualize the target structure under a microscope. For example, counterstaining is optionally used before covering with a coverslip to make immunohistochemical staining clearer. Counterstaining results in a different color from the primary stain. Numerous counterstains are well known, including hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that can be used.

[0433] In some cases, multiple stains can be mixed together to produce a counterstain. This provides flexibility and the ability to select stains. For example, a first stain may be selected for a mixture that has certain attributes but lacks other desired attributes. A second stain may be added to the mixture to exhibit the missing desired attributes. For example, toluidine blue, DAPI, and pontamine sky blue can be mixed together to form a counterstain.

[0434] Detection and / or imaging

[0435] Some or all aspects of the disclosed embodiments can be automated and facilitated by computer analysis and / or image analysis systems. In some applications, precise color or fluorescence ratios are measured. In some embodiments, optical microscopy is used for image analysis. Certain disclosed embodiments involve the acquisition of digital images, which can be done by linking a digital camera to a microscope. Digital images obtained from stained samples are analyzed using image analysis software. Color or fluorescence can be measured in several different ways; for example, as red, blue, and green values; as hue, saturation, and intensity values; and / or by measuring specific wavelengths or wavelength ranges using a spectral imaging camera. Samples can also be evaluated qualitatively and semi-quantitatively. Qualitative evaluations include evaluating staining intensity, identifying positively stained cells and intracellular compartments involved in staining, and evaluating the quality of the entire sample or slide. Individual evaluations are performed on test samples, and this analysis may include comparison with known mean values ​​to determine whether the sample represents an abnormal condition.

[0436] A suitable detection method is described in International Application No. 2014 / 143155, the disclosure of which is incorporated herein by reference in whole. In some embodiments, a suitable detection system comprises an imaging device, one or more lenses, and a display communicating with the imaging device. The imaging device includes means for sequentially emitting energy and means for capturing images / moving images. In some embodiments, the means for capturing are arranged to capture specimen images, each corresponding to a specimen being exposed to energy. In some embodiments, the means for capturing may include one or more cameras positioned in front of and / or behind a microscope slide carrying a biological specimen. The display means in some embodiments include a monitor or screen. In some embodiments, the means for sequentially emitting energy include a plurality of energy emitters. Each energy emitter may include one or more IR energy emitters, UV energy emitters, LED light emitters, combinations thereof, or other types of energy emission devices. The imaging system may further include means for generating contrast-enhanced color image data based on specimen images captured by the means for capturing. The display means displays the sample based on color image data with enhanced contrast.

[0437] Sample and target

[0438] A sample contains biological components and is generally suspected to contain one or more target molecules of interest. Target molecules can be present on the surface of cells, and cells can be present in suspensions or tissue sections. Target molecules can also be present inside cells and can be detected during cell lysis or cell penetration by a probe. Those skilled in the art will understand that the method for detecting target molecules in a sample varies depending on the type of sample and probe used. Methods for collecting and preparing samples are known in the art.

[0439] Samples for use in embodiments of the methods disclosed herein and with the compositions disclosed herein, such as tissues or other biological specimens, may be prepared using any method known in the art to those skilled in the art. Samples may be obtained from subjects for routine screening or from subjects suspected of having disorders such as genetic abnormalities, infections or neoplasms. Embodiments described of the methods disclosed may also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as “normal” samples. Such normal samples are particularly useful as controls for comparison with other samples. Samples may be analyzed for a variety of purposes. For example, samples may be used in scientific studies, for the diagnosis of suspected diseases, or as prognostic indicators such as treatment success or survival.

[0440] A sample may contain multiple targets that can be specifically bound by a probe or reporter molecule. The targets may be nucleic acid sequences or proteins. Throughout this disclosure, where a target protein is referred to, it is understood that nucleic acid sequences associated with that protein can also be used as targets. In some examples, the targets are proteins or nucleic acid molecules derived from pathogens such as viruses and bacteria, or intracellular parasites such as viral genomes. For example, a target protein may be produced from a disease-related (e.g., correlated, causally related, etc.) target nucleic acid sequence.

[0441] Target nucleic acid sequences can vary substantially in size. A nucleic acid sequence may have a variable number of nucleic acid residues, but is not limited to that number. For example, a target nucleic acid sequence may have at least about 10 nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, or 1000 residues. Similarly, target polypeptides can vary substantially in size. A target polypeptide may include at least one epitope that binds to a peptide-specific antibody or a fragment thereof, but is not limited to that number. In some embodiments, the polypeptide may include at least two epitopes that bind to a peptide-specific antibody or a fragment thereof.

[0442] In certain non-limiting cases, target proteins are produced by target nucleic acid sequences (e.g., genomic target nucleic acid sequences) associated with neoplasms (e.g., cancer). Numerous chromosomal abnormalities (including translocations and other rearrangements, amplifications, or deletions) have been identified in neoplastic cells, particularly cancer cells such as B-cell and T-cell leukemia, lymphoma, breast cancer, colon cancer, and neuroleptic malignancies. Therefore, in some cases, at least a portion of the target molecule is produced by amplified or deleted nucleic acid sequences (e.g., genomic target nucleic acid sequences) in at least a subset of cells in the sample.

[0443] Oncogenes are known to cause 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. A first probe contains an FPC nucleic acid molecule generated from a target nucleic acid sequence extending distally from the SYT gene, and a second probe contains an FPC nucleic acid generated from a target nucleic acid sequence extending 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 lacking t(18q11.2) in the SYT gene region show two fusion signals (generated by two adjacent labels) reflecting two intact copies of SYT. Abnormal cells with t(18q11.2) show a single fusion signal.

[0444] In other examples, target proteins are selected that are produced from nucleic acid sequences (e.g., genomic target nucleic acid sequences) that are tumor suppressor genes deleted (lost) in malignant cells. For example, the p16 region located on chromosome 9p21 (including D9S1749, D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the short arm of chromosome 1 (e.g., including SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322) and the centromere region of chromosome 19 (e.g., 19p13-19q13) (e.g., including MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain types of solid tumors of the central nervous system.

[0445] The examples described above are provided for illustrative purposes only and are not intended to limit the scope of this invention. Many other cytogenetic abnormalities that correlate with neobiotic transformation and / or proliferation are known to those skilled in the art. Target proteins produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences) that correlate with neobiotic transformation and are useful in the disclosed methods include the EGFR gene (7p12; e.g., GENBANK® accession number NC-000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK® accession number NC-000008, nucleotides 128817498-128822856), and D5 S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK® accession number NC-000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK® accession number NC-000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK® accession number NC-000017, complement, nucleotide 75124) 64-7531642), N-MYC (2p24; e.g., GENBANK(trademark) accession number NC-000002, complement, nucleotide 151835231-151854620), CHOP (12q13; e.g., GENBANK(trademark) accession number NC-000012, complement, nucleotide 56196638-56200567), FUS (16p11.2; e.g., GENBANK(trademark) accession number NC-000016, nucleotide 31098954-311 Alongside 10601), FKHR (13p14; e.g., GENBANK(trademark) accession number NC-000013, complement, nucleotide 40027817-40138734), for example, ALK (2p23; e.g., GENBANK(trademark) accession number NC-000002, complement, nucleotide 29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK(trademark) accession number NC-000011, nucleotide 69165054).69178423), BCL2 (18q21.3; e.g., GENBANK(trademark) accession number NC-000018, complement, nucleotide 58941559-59137593), BCL6 (3q27; e.g., GENBANK(trademark) accession number NC-000003, complement, nucleotide 188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK(trademark) accession number NC-000001, complement, nucleotide 59019051-5902 2373), TOP2A (17q21-q22; e.g., GENBANK(trademark) accession number NC-000017, complement, nucleotide 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK(trademark) accession number NC-000021, complement, nucleotide 41758351-41801948), ERG (21q22.3; e.g., GENBANK(trademark) accession number NC-000021, complement, nucleotide 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK(trademark) accession number NC-000007, complement, nucleotide 13897379-13995289), EWS (22q12.2; e.g., GENBANK(trademark) accession number NC-000022, nucleotide 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK(trademark) accession number NC-000011, nucleotide 128069199-128187521), PAX3 (2q35- q37; for example, GENBANK(trademark) accession number NC-000002, complement, nucleotides 222772851-222871944), PAX7(1p36.2-p36.12; for example, GENBANK(trademark) accession number NC-000001, nucleotides 18830087-18935219), PTEN(10q23.3; for example, GENBANK(trademark) accession number NC-000010, nucleotides 89613175-89716382), AKT2(19q13.1-q13.2; For example, GENBANK(trademark) accession number NC-000019, complement, nucleotide 45431556-45483036), MYCL1(1p34.2; for example, GENBANK(trademark) accession number NC-000001, complement, nucleotide 40133685-40140274), REL(2p13-p12; for example, GENBANK(trademark) accession number NC-000002, nucleotide 60962256-61003682), and CSF1R(5q33-q35; for example, GENBANK(trademark) accession number NC-000005, complement, nucleotide 149413051-149473128).

[0446] In other examples, target proteins are selected from viruses or other microorganisms associated with a disease or symptom. Detection of target nucleic acid sequences (e.g., genomic target nucleic acid sequences) of viral or microbial origin in cell or tissue samples indicates the presence of the organism. For example, target peptides, polypeptides, or proteins may be selected from the genomes of oncogenic or pathogenic viruses, bacteria, or intracellular parasites (such as Plasmodium falciparum and other Plasmodium species, Leishmania (species), Cryptosporidium parvum, the protozoan Entamoeba histolytica, and Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).

[0447] In some cases, the target protein is produced from a nucleic acid sequence from the viral genome (e.g., a genome target nucleic acid sequence). Exemplary viruses and their corresponding genome sequences (GENBANK® RefSeq accession number 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), and human BK polyomavirus (V01109; GI:6). 0851) 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-004 295), 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 type 1 (Epstein-Barr virus type 1) (NC-007605), Human herpesvirus type 2 (Epstein-Barr virus type 2) (NC-009334), Human herpesvirus 5 strain AD1 69 (NC-001347), Human Herpesvirus 5 Merlin strain (NC-006273), Human Herpesvirus 6A (NC-001664), Human Herpesvirus 6B (NC-000898), Human Herpesvirus 7 (NC-001716), Human Herpesvirus 8 M (NC-003409), Human Herpesvirus 8 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 papilloma 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- Human papillomavirus (HPV) 001690), Human papillomavirus 49 (NC-001591), Human papillomavirus 5 (NC-001531), Human papillomavirus 50 (NC-001691), Human papillomavirus 53 (NC-001593), Human papillomavirus 60 (NC-001693), Human papillomavirus 63 (NC-001458), Human papillomavirus 6b (NC-001355), Human papillomavirus 7 (NC-001595), Human papillomavirus 71 (NC-002644), Human papillomavirus 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),Examples include human rhinovirus A (NC-001617), human rhinovirus B (NC-001490), human spumaretrovirus (NC-001795), human T lymphotropic virus 1 (NC-001436), and human T lymphotropic virus 2 (NC-001488).

[0448] In certain cases, the target protein is produced from nucleic acid sequences (e.g., genomic target nucleic acid sequences) from oncogenic viruses such as Epstein-Barr virus (EBV) or human papillomavirus (HPV, e.g., HPV16, HPV18). In other cases, the target protein produced from nucleic acid sequences (e.g., genomic target nucleic acid sequences) originates from pathogenic viruses such as respiratory syncytial virus, hepatitis virus (e.g., hepatitis C virus), coronavirus (e.g., SARS virus), adenovirus, polyomavirus, cytomegalovirus (CMV), or herpes simplex virus (HSV). [Examples]

[0449] Examples

[0450] Example 1 - Synthesis of the Compounds of the Disclosure

[0451] MS data was collected using a Waters Acquity QDa (ESI) running Empower 3 (Waters). Analytical HPLC was performed using a Waters XBridge column on a Waters Alliance e2695 (Waters) running Empower 3. Preparative HPLC was performed using a Waters SunFire column (preparative C18 OBD 10 μm, 50 mm × 250 mm) on a Waters 2535 (Waters) running Empower 3. Unless otherwise specified, all chemicals were purchased from commercial suppliers and used as received. TIFF0007914109000030.tif220170 Scheme 1. Synthesis of phosphate-caged digoxigenin (DIG)

[0452] Compound 4 was prepared by acetylation and acid hydrolysis of digoxin 1 to obtain 12-O-acetyldigoxigenin 2, which was converted to 3-ethyloxycarbonylmethyl ether 3 by reaction with ethyl diazoethyl acetate, and subsequently hydrolyzed to digoxigenin-3-carboxymethyl ether 4 as described in the patent [U.S. Patent No. 5198537].

[0453] Compound 6. To a stirred solution of digoxigenin-3-carboxymethyl ether 4 (1.0 equivalent) in THF (6 mL / mmol 4), N-hydroxysuccinimide (1.5 equivalents) and 1 M N,N'-dicyclohexylcarbodiimide (1.5 equivalents) in CH2Cl2 were added. The reaction mixture was kept at room temperature for 20 hours (HPLC was checked to confirm completion of the reaction), filtered, and the solvent was removed under reduced pressure. The residue was diluted with HCl. The resulting solution was filtered again and then washed with brine. The organic layer was dried over MgSO4, and the solvent was removed under reduced pressure to obtain NHS ester 5, which was dissolved in HCl (6 mL / mmol 5), followed by the addition of TEA (1.5 equivalents) and N-Boc-ethylenediamine (1.5 equivalents). The reaction mixture was stirred at room temperature for 1 hour (HPLC was checked to confirm completion of the reaction), diluted with HCl (6 mL / mmol 5), followed by the addition of 1 M HCl (10 mL / mmol 5). The organic layer was separated and subsequently washed with saturated NaHCO3 and brine. The organic layer was dried over MgSO4, and the solvent was removed under reduced pressure to obtain compound 6, which was used without further purification. MS(ESI)m / z(M+H-Boc) + C 31 H 51 The calculated value of N2O6+ was 547.3, and the measured value was also 547.3.

[0454] Compound 7. To a solution of Compound 6 (1.0 equivalent), Ti(Ot-Bu)4 (0.2 equivalents), and TEA (3.5 equivalents) in CH2Cl2 (2 mL / mmol 6), diethyl chlorophosphate (2.5 equivalents) was added. The reaction vessel was sealed and stirred at room temperature for 16 hours (monitored by HPLC to confirm that the reaction was approximately 30% complete). The reaction mixture was diluted with SiO2 (50 mL / mmol 6) and subsequently washed with 0.5 M HCl (50 mL / mmol 6). The organic layer was dried over MgSO4, and the solvent was removed under reduced pressure to obtain a grayish-white foam. The reaction was repeated two more times, at which point HPLC indicated that the reaction was approximately 90% complete compared to the starting material 6. The unrefined oily substance was purified by preparative RP-HPLC (0.05% TFA in H2O:MeCN from 95:5 to 5:95 over 40 minutes; 40 ml / min) to obtain diethyl phosphate 7 as a grayish-white foam (35% yield from 4). MS(ESI)m / z(M+H-Boc) + C 35 H 60 The calculated value of N2O9P+ was 683.4, while the measured value was 683.5.

[0455] Compound 8. Compound 7 (1.0 equivalent) was dissolved in CHCl3 (2 mL / mmol 7), followed by the addition of RINKAN (0.2 equivalents) and TMSBr (3.3 equivalents). The resulting reaction mixture was stirred at room temperature for 18 hours (monitored by HPLC to confirm that the reaction was >95% complete). The solvent was removed under reduced pressure, followed by the addition of MeOH (6 mL / mmol 7). The solvent was again removed under reduced pressure, and the resulting residue was purified by preparative RP-HPLC (0.05% TFA in H2O:MeCN from 95:5 to 5:95 over 40 minutes; 40 ml / min) to obtain phosphate 8 as a white solid (yield 45%). MS(ESI)m / z(M+H) + C 31 H 52 N2O9P + The calculated value was 627.3, while the measured value was 627.4.

[0456] Compound 10. Compound 8 (1.0 equivalent) was suspended in DMF (2 mL / mmol 8), then triethylamine (5 equivalents) was added, followed by 3-maleimidopropionic acid NHS ester 9 (1.1 equivalents). The reaction vessel was sealed, and the reaction mixture was vigorously stirred at room temperature for 4 hours (HPLC was checked to confirm the completion of the reaction). The reaction mixture was then diluted with MeOH and purified directly by preparative RP-HPLC (0.05% TFA in H2O:MeCN from 99:1 to 5:95 over 40 minutes) to obtain compound 10 as a pale yellow solid. MS(ESI)m / z(M+H) + C 38 H 57 N3O 12 P + The calculated value was 778.4, while the measured value was 778.5.

[0457] Example 2 - Preparation of antibody conjugate

[0458] 20 mg of goat anti-rabbit IgG in 2 ml of 1 × PBS (pH 7.2) was added to EDTA to obtain a final concentration of 10 mM, and then 2 mg of Trout's reagent (2-iminothiolane hydrochloride) was added. After keeping the reaction mixture at room temperature for 1 hour, it was purified by size exclusion chromatography using 1 × PBS (pH 7.2) containing 10 mM EDTA (AKTA, Superdex 200 10 / 300GL column). 4.2 mg of compound 10 in 0.2 ml of DMF was added to the combined fraction of thiolated antibodies (6 mg / ml). After keeping the reaction mixture at room temperature for 3 hours, it was purified by size exclusion chromatography using 1 × PBS (pH 7.2) (AKTA, Superdex 200 10 / 300GL column) to obtain caged digoxigenin-modified antibody (3.7 mg / ml). Figure 8 shows the conjugation of the antibody to the caged hapten of this disclosure.

[0459] Example 3 - Stability Test

[0460] To study the hydrolysis stability of caged hapten conjugates having any one of the formulas (IVA), (IVB), or (VA)~(VF) (see Figure 9), model compounds were subjected to high-temperature stability studies.

[0461] The model compounds tested were as follows: As shown in TIFF0007914109000031.tif165170, along with caged DIG, it was an NP hapten having two different casing groups.

[0462] Caged NP and caged DIG samples were stored in 100 mM PBS (pH 7.2) in an oven at 37°C. This buffer and pH were representative of the storage conditions for antibody conjugates. The temperature of 37°C was chosen to stress the samples and accelerate the hydrolysis event. Normal storage conditions for antibody conjugates were considered to be around 4°C. Fixed amounts of the samples were taken at regular intervals and tested by reverse-phase HPLC on a Waters Alliance e2695 (Waters) WatersX Bridge column running Empower 3. HPLC traces of each sample were examined for evidence of decaging or other forms of degradation. After 50 days of storage at 37°C, the first-generation caged NP showed approximately 15.5% hydrolysis, the second-generation caged NP had approximately 6% hydrolysis, and caged DIG had less than 0.5% hydrolysis (see Figure 10). Caged DIG samples were monitored up to 120 days, at which point <0.5% hydrolysis was still observed. It was concluded that the caged haptens of this disclosure exhibit excellent hydrolysis stability.

[0463] Example 4 - Immunohistochemistry

[0464] A typical immunohistochemistry (IHC) protocol.

[0465] All IHC staining experiments were performed on the VENTANA DISCOVERY® Ultra automated tissue staining platform. Unless otherwise specified, the reagents used in these protocols were from Roche Tissue Diagnostics (Tucson, AZ, USA; "RTD").

[0466] General Procedure for Proximity IHC

[0467] All formalin-fixed, paraffin-embedded (FFPE) tissue and cell line samples were placed on Superfrost Plus glass slides (Fisher Scientific, #12-550-15). These were deparaffinized using EZ Prep (RTD, #950-101). Heat-induced epitope retrieval (HIER) or antigen retrieval (AR) was performed using Cell Conditioning 1 (CC1, RTD, #950-124). The general steps after deparaffinization and AR were as follows: (1) Inactivation of endogenous peroxidase with the inhibitor CM (RTD, 760-4307); (2) Co-incubation with primary antibody (approximately 37°C, for a time ranging from approximately 8 to 32 minutes depending on the antibody); (3) Incubation with goat anti-mouse secondary antibody conjugated with alkaline phosphatase (AP); (4) Incubation with goat anti-rabbit secondary antibody conjugated with caged hapten; (5) Incubation with mouse anti-hapten HRP conjugate; (6) Signal amplification with tyramide-HQ and H2O2 (RTD, #760-052); (7) Mouse anti-HQ (8) Incubation with HRP conjugate (RTD, #760-4602); detection with 3,3'-diaminobenzidine (DAB), hydrogen peroxide (H2O2), and copper coloring; (9) counterstaining with hematoxylin II (RTD, #790-2208) and Bluing (RTD, #760-2037) to stain the nucleus; (10) dehydration with alcohol and xylene in a gradient, followed by covering with a coverslip. Between each assay incubation step, the slides were washed with Reaction Buffer (RTD, #950-300).

[0468] Proximity IHC experiment - E-cadherin: Proximity of β-catenin-positive

[0469] FFPE tonsil tissue was deparaffinized and antigens were activated (CC1, 60 min). Rabbit anti-E-cadherin (RTD, 760-4440) and mouse anti-β-catenin (RTD, 760-4242) were co-incubated (approx. 37°C, approx. 32 min). After washing, goat polyclonal anti-mouse antibody conjugated to alkaline phosphatase was applied (approx. 37°C; approx. 12 min). After washing, the samples were incubated with goat polyclonal anti-rabbit antibody conjugated to multiple caged NPs (Figure 11A) or multiple caged digoxigenin (Figure 11B) (approx. 37°C; approx. 12 min). After washing, the samples were incubated with mouse anti-DIG HRP conjugate (approx. 37°C; approx. 12 min). Tyramide amplification was performed using the Amp HQ kit (RTD, 760-052, approx. 37°C, approx. 8 min), followed by incubation with mouse anti-HQ HRP conjugate (RTD, #760-4602, approx. 37°C, approx. 8 min). The signal was visualized with DAB, and then tissue sections were counterstained. Slides were dehydrated with a stepwise ethanol series, clarified with xylene, and covered with coverslips. The results are shown in Figure 11A, which shows positive proximity signals for E-cadherin and β-catenin detected using caged NP, and in Figure 11B, which shows positive proximity signals for E-cadherin and β-catenin detected using caged DIG.

[0470] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in application data sheets are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified to provide further embodiments using various patent, application, and publication concepts as necessary.

[0471] While the disclosures herein are described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. Therefore, it should be understood that numerous modifications can be made to the exemplary embodiments, and that other configurations can be devised without departing from the spirit and scope of the disclosure as defined by the appended claims.

[0472] Additional Embodiments Additional Embodiment 1. A method for analyzing a sample to determine whether a first target is in close proximity to a second target, (a)(a) Contact the sample with a demasking enzyme-antibody conjugate to form a target-demasking enzyme-antibody conjugate complex. (b)(b) Contacting the sample with the caged hapten-antibody conjugate to form a target-caged hapten-antibody conjugate complex, wherein the caged hapten-antibody conjugate is of formulas (IVA) and (IVB): [Specific binding entity]-W 1 -W 2 -R 1 -O-[DIG]-[phosphoryl](IVA) [Specific binding entity]-W 1 -W 2 -R 1 -O-[DIG]-PO4H2(IVB) (In the formula, W 1 This is a bond or a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group containing 1 to 10 carbon atoms and optionally one or more heteroatoms selected from the group consisting of O, N, or S. W 2 It originates from a reactive functional group, [DIG] is digoxigenin, [Phosphoryl] is given by formula: TIFF0007914109000032.tif42170(Q 1 is either O or S, Q 2(This is H, -CH3, or -CH2CH3) Represented by, The [specific binding entity] is an antibody. The group [phosphoryl] or the group -PO4H2 may be attached to any position in [DIG]. To form a target-caged hapten-antibody conjugate complex having any one of the following: (c)(c) Demasking the caged hapten of the target-caged hapten-antibody conjugate complex to form a target-demasked hapten-antibody conjugate complex. (d) Contacting the sample with a first detection reagent to label the first target-demasked hapten-antibody conjugate complex or the first target, and (e)(e)Detecting the labeled first target-demasked hapten-antibody conjugate complex or the labeled first target, Methods that include... Additional Embodiment 2.Q 1 O is O and at least one Q 2 The method according to an additional embodiment 1, wherein H is present. Additional Embodiment 3.W 2 The method according to additional embodiment 2, wherein the group is derived from an amine-reactive group, a thiol-reactive group, and a carbonyl-reactive group. Additional Embodiment 4.W 2 The method according to additional embodiment 2, wherein the product is derived from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, or amino groups. Additional Embodiment 5. Both Q 2 The method according to an additional embodiment 1, wherein the base is H. Additional Embodiment 6.W 2 The method according to additional embodiment 5, wherein the group is derived from an amine-reactive group, a thiol-reactive group, and a carbonyl-reactive group. Additional Embodiment 7.W 2The method according to additional embodiment 5, wherein the product is derived from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, or amino groups. Additional Embodiment 8.R 1 Equation (IIIA): TIFF0007914109000033.tif53170 (in the formula, R 8 The bonds are -O-, -S-, -C(R c )(R d ), or -N(R c )- and, R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and R c and R d Each is independently selected from H or -CH3. R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol. Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-, t and u are independently 0, 1, or 2, where t+u is at least 1. (v is an integer in the range of 1 to 8) The method of an additional embodiment 5, having the structure shown in [figure name]. Additional Embodiment 9.R a or R b The method according to an additional embodiment 8, wherein at least one of is H. Additional Embodiment 10.R 8 The method according to additional embodiment 9, wherein is O. Additional Embodiment 11.R 8The method according to an additional embodiment 9, wherein the combination is a bond. Additional Embodiment 12.R a or R b The method according to an additional embodiment 11, wherein at least one of is H. Additional Embodiment 13. Both R a and R b The method according to an additional embodiment 11, wherein H is present. Additional Embodiment 14. The method of Additional Embodiment 12, wherein Z is bonded or -CH2-. Additional Embodiment 15.R 1 Equation (IIIC): TIFF0007914109000034.tif53170 (in the formula, R a and R b Each of these is independently H, C1-C4 alkyl group, F, Cl, or -N(R) c )(R d ) and R c and R d Each is independently selected from H or -CH3. R 9 and R 10 Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, thione, or thiol. Each Z is independently a bond, -CH2-, -CH2CH2-, or -CH2CH2CH2-, u and t are independently 0, 1, or 2, where u+t is at least 1. (v is an integer in the range of 1 to 8) The method according to an additional embodiment 1, having the structure shown. Additional Embodiment 16.R a or R b The method according to an additional embodiment 15, wherein at least one of is H. Additional Embodiment 17. The method of Additional Embodiment 15, wherein Z is bonded or -CH2-. Additional Embodiment 18. Both Q 2The method according to an additional embodiment 15, wherein the base is H. Additional Embodiment 19.W 2 The method according to an additional embodiment 18, wherein the group is derived from an amine-reactive group, a thiol-reactive group, and a carbonyl-reactive group. Additional Embodiment 20.W 2 The method according to additional embodiment 15, wherein the present invention is derived from dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, ketones, hydrazines, hydroxylamines, and amino groups. Additional Embodiment 21.Q 1 The method according to an additional embodiment 15, wherein is O.

Claims

1. Formula (IIID): (In the formula, R １ Equation (IIIA): (In the formula, R ８ The bond is -O-, -S-, -C(R) ｃ ) (Caution ｄ ), or -N(R ｃ ) - and R ａ and R ｂ are each independently H, C １ to C ４ alkyl, F, Cl or -N(R ｃ )(R ｄ ), R ｃ and R ｄ These are, independently, H or -CH ３ Selected from, R ９ and R １０ Each of these is independently either a bond or a group selected from carbonyl, amide, imide, ester, ether, amine, and thion. Each Z independently forms a bond, -CH ２ -ien-CH ２ CH ２ -, or -CH ２ CH ２ CH ２ - and t and u are independently 0, 1, or 2, provided that t + u is at least 1. v is an integer in the range of 1 to 8. It has the structure shown in, R ２ This is selected from the group consisting of dibenzocyclooctin, trans-cyclooctene, alkynes, alkenes, azides, tetrazines, maleimides, N-hydroxysuccinimide, thiols, 1,3-nitrones, aldehydes, hydrazines, hydroxylamines, and amino groups. R ３ H, -CH ３ ien-CH ２ CH ３ , -OH or -O-Me, R ４ H, -CH ３ ien-CH ２ CH ３ , -OH or -O-Me, R ６ is either H, or linear or branched, or substituted or unsubstituted C １ ~C ６ It is an alkyl group, Y is -CH ２ -, -CH(R ７ )-, -N(H)-, -N(R ７ )-, -O-, -S-, or -C(O)-, R ７ C １ ~C ４ (A linear or branched alkyl group, substituted or unsubstituted) A caged hapten.

2. R ａ or R ｂ The caged hapten according to claim 1, wherein at least one of is H.

3. R ８ A caged hapten according to claim 1, wherein is O.

4. R ８ The caged hapten according to claim 1, wherein the bond is a bond.

5. R ａ or R ｂ The caged hapten according to claim 4, wherein at least one of is H.

6. R ａ and R ｂ The caged hapten according to claim 4, wherein both are H.

7. Z is bonded or -CH ２ - The caged hapten according to claim 6.

8. R ３ , R ４ or R ６ At least one of them is -CH ３ The caged hapten according to claim 1.

9. R ３ and R ４ At least one of them is -CH ３ The caged hapten according to claim 1.

10. R ６ The caged hapten according to claim 9, wherein is H.

11. The caged hapten according to claim 1, wherein Y is -C(O)-.

12. The aforementioned caged hapten is given by formula (III): A caged hapten according to claim 1, having the following characteristics.

13. The aforementioned caged hapten is given by equation (III): A caged hapten according to claim 1, having the characteristics of the present invention.

14. A conjugate comprising a caged hapten and a primary antibody according to any one of claims 1 to 13.

15. The conjugate according to claim 14, wherein the caged hapten is indirectly bound to the primary antibody.

16. The conjugate according to claim 15, wherein the primary antibody is an intact primary antibody.

17. A conjugate comprising a caged hapten and a secondary antibody according to any one of claims 1 to 13.

18. The conjugate according to claim 17, wherein the caged hapten is indirectly bound to the secondary antibody.

19. The conjugate according to claim 17, wherein the secondary antibody is an intact secondary antibody.

20. A method for analyzing a sample to determine whether a first target is in close proximity to a second target, (a) Contacting the sample with a demasking enzyme-antibody conjugate to form a second target-demasking enzyme-antibody conjugate complex, (b) Contacting the sample with the conjugate described in any one of claims 14 to 19 to form a first target-caged hapten-antibody conjugate complex. (c) Demasking the caged hapten of the first target-caged hapten-antibody conjugate complex to form the first target-demasked hapten-antibody conjugate complex. (d) Contacting the sample with the first detection reagent to label the first target-demasked hapten-antibody conjugate complex or the first target, and (e) Detection of a labeled first target-demasked hapten-antibody conjugate complex or a labeled first target. Methods that include...

21. The method according to claim 20, wherein the first detection reagent comprises (i) a secondary antibody specific to the demastened hapten of the first target-demastened hapten-antibody conjugate complex, wherein the secondary antibody is conjugated to the first enzyme such that the secondary antibody labels the first target-demastened hapten-antibody conjugate complex with the first enzyme, and (ii) a first substrate for the first enzyme.

22. The method according to claim 21, wherein the first substrate is a chromogenic substrate or a fluorescent substrate.

23. The method according to claim 20, wherein the first detection reagent includes an amplification component for labeling the demasking enzyme of the first target-demasking hapten-antibody conjugate complex with a plurality of first reporter moieties.

24. The method according to claim 23, wherein the plurality of first reporter portions are haptens.

25. The method according to claim 24, wherein the first detection reagent further comprises secondary antibodies specific to the plurality of first reporter portions, each secondary antibody being conjugated to a second reporter portion.

26. A method for analyzing a sample to determine whether a first target is in close proximity to a second target, (a) Contacting the sample with a demasking enzyme-antibody conjugate to form a second target-demasking enzyme-antibody conjugate complex, (b) Contacting the sample with the conjugate described in any one of claims 14 to 19 to form a first target-caged hapten-antibody conjugate complex. (c) Demasking the caged hapten of the first target-caged hapten-antibody conjugate complex to form the first target-demasked hapten-antibody conjugate complex. (d) Perform a signal amplification step to label the first target-demasked hapten-antibody conjugate complex with multiple reporter moieties, and (e) detecting the plurality of reporter portions Methods that include...

27. The method according to claim 26, wherein the plurality of reporter portions are haptens, and the method further comprises introducing a secondary antibody specific to the plurality of first reporter portions, each secondary antibody being conjugated to a second reporter portion.

28. The method according to claim 27, wherein the second reporter portion is an amplification enzyme, and the method further comprises introducing a chromogenic substrate or a fluorescent substrate to the amplification enzyme.

29. The method according to claim 26, further comprising detecting the total amount of target in the sample.

30. A method for analyzing a sample to determine whether a first target is in close proximity to a second target, (a) Contacting the sample with a first detection probe, wherein the first detection probe comprises one of the conjugate or demasking enzyme-antibody conjugate described in any one of claims 14 to 19, (b) Contacting the sample with a second detection probe, wherein the second detection probe comprises the other of the conjugate described in any one of claims 14 to 19 or the demasking enzyme-antibody conjugate. (c) Contacting the sample with at least the first detection reagent to label the formed demasked hapten-antibody conjugate target complex, and (d) Detecting the signal from the labeled decaszed hapten-antibody conjugate target complex. Methods that include...

31. The method according to claim 30, further comprising the step of detecting the total amount of target in the sample.

32. The method according to claim 30, wherein the first detection reagent includes an amplification component for labeling the demasking enzyme of the first target-demasking hapten-antibody conjugate complex with a plurality of first reporter portions.

33. The method according to claim 32, wherein the plurality of first reporter portions are haptens.

34. The method according to claim 32, wherein the first detection reagent further comprises secondary antibodies specific to the plurality of first reporter portions, each secondary antibody being conjugated to a second reporter portion.

35. The method according to claim 34, wherein the second reporter portion is selected from the group consisting of an amplification enzyme or a fluorophore.

36. The method according to claim 34, wherein the second reporter portion is an amplification enzyme, and the first detection reagent further comprises a first chromogenic substrate or fluorescent substrate for the amplification enzyme.

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